Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration

Yudi Her Oktaviono; Suryo Ardi Hutomo; Makhyan Jibril Al-Farabi; Angliana Chouw; Ferry Sandra

doi:10.12688/f1000research.23547.2

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Research Article

Revised

Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration

[version 2; peer review: 2 approved]

Yudi Her Oktaviono ¹, Suryo Ardi Hutomo¹, Makhyan Jibril Al-Farabi¹, Angliana Chouw², Ferry Sandra³

Yudi Her Oktaviono ¹, Suryo Ardi Hutomo¹, [...] Makhyan Jibril Al-Farabi¹, Angliana Chouw², Ferry Sandra³

PUBLISHED 10 May 2021

Author details Author details

¹ Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia
² Stem Cell Division, Prodia Laboratory, Jakarta, Indonesia
³ Department of Biochemistry and Molecular Biology, Faculty of Dentistry, Universitas Trisakti, Jakarta, Indonesia

Yudi Her Oktaviono
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Resources, Validation, Writing – Review & Editing

Suryo Ardi Hutomo
Roles: Investigation, Methodology, Project Administration, Visualization, Writing – Original Draft Preparation

Makhyan Jibril Al-Farabi
Roles: Project Administration, Writing – Review & Editing

Angliana Chouw
Roles: Investigation, Software

Ferry Sandra
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Resources, Validation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Cell Migration collection.

Abstract

Background: Human umbilical cord blood-mesenchymal stem cell (hUCB-MSC)-derived secretome is known to be able to promote neovascularization and angiogenesis, so it is also thought to have a capability to modulate endothelial progenitor cell (EPC) functions. Atorvastatin is the cornerstone of coronary artery disease (CAD) treatment which can enhance EPCs proliferation and migration. This study aims to analyze the effect of the hUCB-MSC-derived secretome and its combination with atorvastatin toward EPCs proliferation and migration.
Methods: EPCs were isolated from a CAD patient’s peripheral blood. Cultured EPCs were divided into a control group and treatment group of 2.5 µM atorvastatin, hUCB-MSC-derived secretome (2%, 10%, and 20% concentration) and its combination. EPCs proliferation was evaluated using an MTT cell proliferation assay, and EPC migration was evaluated using a Transwell migration assay kit.
Results: This research showed that hUCB-MSC-derived secretomes significantly increase EPC proliferation and migration in a dose-dependent manner. The high concentration of hUCB-MSC-derived secretome were shown to be superior to atorvastatin in inducing EPC proliferation and migration (p<0.001). A combination of the hUCB-MSC-derived secretome and atorvastatin shown to improve EPCs proliferation and migration compared to hUCB-MSC-derived secretome treatment or atorvastatin alone (p<0.001).
Conclusions: This study concluded that the hUCB-MSC-derived secretome work synergistically with atorvastatin treatment in improving EPCs proliferation and migration.

Keywords

coronary artery disease, endothelial progenitor cells, mesenchymal stem cells, secretome, statins

Corresponding author: Yudi Her Oktaviono

Competing interests: No competing interests were disclosed.

Grant information: This research was funded by the Prodia Education and Research Institute.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Oktaviono YH et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Oktaviono YH, Hutomo SA, Al-Farabi MJ et al. Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration [version 2; peer review: 2 approved]. F1000Research 2021, 9:537 (https://doi.org/10.12688/f1000research.23547.2) First published: 05 Jun 2020, 9:537 (https://doi.org/10.12688/f1000research.23547.1) Latest published: 10 May 2021, 9:537 (https://doi.org/10.12688/f1000research.23547.2)

Revised Amendments from Version 1

The revised version was improved with feedback from both reviewers which are:
-Clear diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries.
-We stated the design of the study which is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.
- Reference has been added for the statement: HUCB-MSCs-derived secretome preparation
- All abbreviations has been spelt out in the beginning or first time used

See the authors' detailed response to the review by John David Symmons and Anwar Tandar
See the authors' detailed response to the review by Anwar Santoso

Introduction

Coronary artery disease (CAD) is the leading cause of mortality and morbidity worldwide¹. It is responsible for the deaths of 7.2 million people or 12.2% of total deaths per year worldwide. Despite advancement in CAD management (e.g. novel antiplatelet therapy, coronary stents, percutaneous coronary intervention techniques and devices, and coronary artery bypass surgery), there are some clinical subsets of CAD which remain untreatable such as ischemic cardiomyopathy, refractory angina, and patients who cannot undergo revascularization due to clinical and anatomical complexity^2,3.

It is already known that CAD is caused by atherosclerosis, which is followed by reduced levels of circulating endothelial progenitor cells (EPCs)⁴. EPCs can differentiate into mature endothelial cells and also promote endothelial repair. Hence, increasing circulating EPC levels is proven to improve endothelial function⁵. EPCs also had a critical role in the stimulation of angiogenesis and vasculogenesis. Hence, increasing EPC proliferation and migration may reduce ischemia and improve myocardial performance^4,6.

Regenerative treatment for CAD using stem cells has been extensively studied in the last decade⁷. However, these treatments faced challenges of low engraftment, poor survival, and low differentiation of the transplanted cells. Despite regenerative treatment shown to be promising in vitro, clinical studies showed unsatisfying results⁸. Hence, the researcher started to shift regenerative treatment from cell based-treatment into cell-free treatment using paracrine stimulation⁹. Nowadays, the usage of cell-free therapeutics as a regenerative therapy in cardiovascular diseases also started to be emerged⁹.

The secretome is the wide array variety of paracrine factors produced by mesenchymal stem cells (MSCs). Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) derived secretome was proven could promote neovascularization, angiogenesis^10–13 and improved cardiac systolic function by protecting myocardial cells from apoptosis¹⁴. However, using this approach to improve neovascularization is yet to be investigated. Hence, it is hypothesized that increasing EPCs proliferation and migration by the hUCB-MSC-derived secretome may be responsible for this effect. Statins, through their pleiotropic effect, are the cornerstone of CAD treatment. Atorvastatin is one of the most prescribed statins, whose ability to modulate EPCs proliferation and migration has already been well studied in both laboratory and clinical settings^15–17. Furthermore, this study aims to compare the effect of the hUCB-MSC-derived secretome, atorvastatin and the two in combination in modulating EPC proliferation and migration.

Methods

This is an experimental laboratory study of atorvastatin and hUCB-MSC-derived secretome and also their combinations on EPCs proliferation and migration. We conducted a controlled, posttest-only group design.

Sample collection

A 50–100 mL peripheral blood sample was obtained from a patient with CAD. The patient was recruited from the outpatient cardiovascular clinic at Pusat Pelayanan Jantung Terpadu, Dr. Soetomo General Hospital, Surabaya, in March 2020. The inclusion criteria were as follows: male, aged 40–59 years old, history of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries¹⁸. The exclusion criteria were as follows: a history of percutaneous coronary intervention procedures or coronary artery bypass grafting surgery, acute coronary syndromes, and anemia.

This study protocol has an ethical clearance from the Health Research Ethics Committee of Dr. Soetomo General Hospital, Surabaya (No.1567/KEPK/X/2019, approved on 8 October 2019). The included subjects provided written informed consent before subject recruitment. All details which include personal information were omitted.

HUCB-MSCs-derived secretome preparation

The HUCB-MSCs-derived secretome was prepared according to the previous study¹⁹. HUCB-MSCs (3H Biomedical AB, Uppsala, Sweden) was cultured in MesenCult^TM MSC Basal medium, supplemented with MesenCult^TM Stimulatory supplement (StemCell Technologies Inc., Vancouver, Canada), and also added with penicillin and streptomycin. Upon reaching 80% confluency, the media was replaced with MesenCult^TM MSC Basal medium (supplement-free media) and incubated for 24 hours. The media was collected and centrifuged. The supernatant was used as a conditioned medium that contained hUCB-MSCs-derived secretome¹⁹.

Isolation and culture of EPCs

Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation of CAD patient’s peripheral blood using Histopaque-1077 (Sigma-Aldrich, USA). After centrifugation of peripheral blood, PBMCs then cultured with STEMLINE-II hematopoietic stem cell expansion medium (Sigma-Aldrich, USA) supplemented with stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6. A total of 5×10⁶ mononuclear cells/ml were seeded into fibronectin-coated 6-well plate dish and cultured at 37°C and 5% CO₂ levels for 5 days. Non-adherent cells were then transferred for the proliferation and migration assay. After five days of culture, EPCs were confirmed using FITC-labeled anti-human CD34 antibody (animal source was mouse, 5 μL antibody was diluted at 500 μL per 1 × 10⁶ cells; catalog number 60013FI, Gene ID: 947, by StemCell Technologies Inc., Vancouver, Canada) staining and examined with immunofluorescence microscopy.

Treatment of EPCs

Cultured EPCs were divided into eight treatment groups for each proliferation and migration assays. Those treatment include control group, 2.5 µM atorvastatin, low (2%), medium (10%) and high (20%) doses of hUCB-MSC-derived secretome, and combination of 2.5 µM atorvastatin with each dose of the hUCB-MSC-derived secretome. There were n=5 replications made from each treatment. To determine the volume of hUCB-MSC-derived secretome given, the concentration was multiplied with total solution given at each treatment.

EPCs proliferation assay

The MTT cell proliferation assay kit (Sigma-Aldrich, St Louis, MO, USA) was used to measure EPCs proliferation as described previously²⁰. Treated EPCs were added with MTT reagent and incubated in a 37°C incubator with 5% CO₂ for 4 hours. Proliferation was determined from the reduction of tetrazolium (MTT) into insoluble formazan product by viable EPCs mitochondria. Absorbance was measured with a microplate reader at 595 nm wavelength. EPCs proliferation was measured as optical density (OD). MTT assay was measured at day 3 after reagent addition.

EPCs migration assay

EPCs migration was evaluated using the 24-mm diameter insert, 3-µm pore size, 6-well Transwell migration assay kit (Corning, USA). A total of 5×10⁵ cultured EPCs were placed in the upper part of the Transwell migration assay kit. Next, 2 mL of EPC media and each treatment were added in the lower chamber compartment and then incubated for 24 hours at 37°C. Non-migratory cells were removed manually. On the receiver plate, the new basal medium was placed and added 500 μL of trypsin + EDTA solution 0.5%, followed by 10 minutes incubation. Then, cells on the bottom surface of the membrane were stained with Giemsa and cell images were obtained on a light microscope and counted manually in n=5 random fields/sample²¹.

Statistical analysis

Statistical analyses were conducted using SPSS Statistics 23.0 to detect significance level at p<0.05. One-way ANOVA was used to compare groups, with Fisher’s least significant difference (LSD) post hoc test. Kruskal-Wallis test was used if there are violations to the assumption of normality and the assumption of homogeneity of variance. Correlation between variables was obtained using Spearman’s correlation followed by a linear regression test.

Results

Baseline characteristics and demography of CAD patient

Clinical examination, blood sampling, electrocardiography, echocardiography and coronary angiography was conducted and evaluated in order to examine the inclusion and exclusion criteria. Our sample had a 1-year history of coronary artery disease, he suffered from refractory chest pain despite the optimum medical therapy. The coronary angiography showed complex lesion (three-vessel disease with chronic total occlusion) which was not amenable to undergo revascularization. The baseline characteristics of the patient are presented in Table 1.

Table 1. Characteristics of the patient.

Variables	Result
Sex	Male
Age	59 years old
Body Mass Index (BMI)	27.3
Blood pressure	140/90
Heart rate	90 beats per minute
Electrocardiography	Sinus rhythm, pathological Q-waves at V-V6 leads.
Laboratory
Total cholesterol (mg/dL)	240
Triglyseride (mg/dL)	131
LDL (mg/dL)	140
HDL (mg/dL)	55
Hemoglobin (mg/dL)	14.2
Blood glucose (mg/dL)	142
Echocardiography
Left ventricle ejection fraction	41% (teich); 36% (biplane)
Left ventricle end-diastolic diameter	5.8 cm
Wall motion	Hypokinesia at anterior, anteroseptal, inferoseptal, other segments kinetic was normal
Valves	mild mitral regurgitation
Coronary Angiography
Left main coronary artery (LMCA)	normal
Left anterior descending artery (LAD)	70% stenosis at osteal, chronic total occlusion (CTO) at distal
Left circumflex coronary artery (LCX)	70% stenosis at proximal, CTO at distal, grade 2 collaterals from LCX to RCA
Right coronary artery (RCA)	CTO at proximal

EPC characteristics

EPCs were successfully isolated and cultured from the CAD patient’s peripheral blood. It was confirmed through light microscopy that displayed a spindle-shape morphology, which is typical for early EPCs and an immunofluorescence assay that showed FITC CD 34+ expression (Figure 1). Raw images are available as Underlying data²². In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research^20,23–25. There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs^26,27

Figure 1. Immunofluorescence characterization of cultured EPCs.

(A) DAPI staining of cultured EPCs showed viable cells through blue fluorescent of cells nuclei. (B) EPCs were confirmed, using FITC-labeled anti-human CD34 expression on immunofluorescence microscope. (C) Merged view of DAPI and FITC stained cells. (D) The light microscope view showed the spindle shape morphology of EPCs. The white bar represents 50 µm.

HUCB-MSCs-derived secretome and atorvastatin increase EPCs proliferation

EPCs were evaluated using the MTT proliferation assay. As shown in Figure 2, both atorvastatin and hUCB-MSCs-derived secretome treatment groups at all doses increase EPCs proliferation compared to the control (p<0.05, ANOVA). hUCB-MSC-derived secretome treatment showed a dose-dependent relationship with EPCs proliferation. At medium (10%) and high (20%) doses, hUCB-MSC-derived secretome was shown to elicit superior EPC proliferation than atorvastatin (OD 1.252±0.104 and 1.585±0.029, respectively, vs 0.738±0.025; p<0.01). Raw absorbance data for MTT assays are available as Underlying data²².

Pearson’s correlation showed a significant and strong correlation between hUCB-MSCs-derived secretome treatment with EPC proliferation (r=0.954; p<0.001). The linear regression test showed an R² of 0.910.

Combination of hUCB-MSCs-derived secretome and atorvastatin increase EPCs proliferation compared with single treatment

Figure 2 showed the combination of atorvastatin and hUCB-MSC-derived secretome at the dose of 2%, 10% and 20% concentration have significantly higher EPCs proliferation compared to atorvastatin alone (OD 0.803±0.046, 1.298±0.075 and 1.761±0.419 vs 0.738±0.025, p<0.05). In addition, combination of hUCB-MSC-derived secretome at dose of 2%, 10% and 20% with atorvastatin showed higher EPCs proliferation compared to hUCB-MSC-derived secretome alone (OD 0.803±0.046 vs 0.713±0.049, 1.298±0.075 vs 1.252±0.104 and 1.761±0.419 vs 1.585±0.029, p<0.05). The combination group showed a significant and very strong correlation with EPC proliferation (r=0.973; p<0.001), Linear regression test showed R² of 0.947.

Figure 2. Comparison of EPC proliferation effects among all treatment groups (see text).

^aSignificant difference compared to the control group (p < 0.001). ^bSignificant difference compared to the 2.5 µM atorvastatin group (p < 0.001). ^cSignificant difference compared to the 2% hUCB-MSC-derived secretome group, (p <0.001). ^dSignificant difference compared to the 10% hUCB-MSC-derived secretome group (p < 0.001). ^eSignificant difference compared to the 20% hUCB-MSC-derived secretome group (p < 0.001), ^fSignificant difference compared to the combination of 2% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001), ^gSignificant difference compared to the combination of 10% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001). ^hSignificant difference compared to the combination of 20% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001)

HUCB-MSCs-derived secretome and atorvastatin increase EPCs migration

EPCs migration from each treatment group was analyzed using the Transwell migration assay. As shown in Figure 3, EPC treatment with atorvastatin and all doses of hUCB-MSCs-derived secretome significantly increase EPC migration compared to the control group (p<0.05, ANOVA). Treatment with 2.5 µM atorvastatin has significantly higher EPCs migration than low (2%) and medium (10%) doses of hUCB-MSC-derived secretome (34.40±3.05 vs 17.20±1.92 and 27.00±4.00, p<0.05). However, high doses (20%) of hUCB-MSC-derived secretome showed significantly higher migrated EPCs than atorvastatin (51.00±5.15 vs 34.40±3.05, p<0.001). Raw cell counts used to assess migration are available as Underlying data²².

Pearson’s correlation showed a significant and very strong correlation between hUCB-MSCs-derived secretome treatment with EPC migration (r=0.968; p<0.001). The linear regression test showed an R² of 0.937.

Combination of hUCB-MSCs-derived secretome and atorvastatin increase EPCs migration compared with single treatment

In Figure 3, EPCs migration was significantly higher in combination treatment groups (atorvastatin and hUCB-MSC-derived secretome) at 2%, 10%, and 20% doses compared to the atorvastatin alone (38.20±3.49, 50.20±5.31 and 76.40±7.50 vs 34.40±3.05, p<0.001).

Figure 3. Comparison of EPCs migration effects among all treatment groups (see text).

^aSignificant difference compared to the control group (p < 0.001). ^bSignificant difference compared to the 2.5 µM atorvastatin group (p < 0.001). ^cSignificant difference compared to the 2% hUCB-MSC-derived secretome group (p < 0.001). ^dSignificant difference compared to the 10% hUCB-MSC-derived secretome group (p < 0.001). ^eSignificant difference compared to the 20% hUCB-MSC-derived secretome group (p < 0.001). ^fSignificant difference compared to the combination of 2% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001). ^gSignificant difference compared to the combination of 10% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001). ^hSignificant difference compared to the combination of 20% hUCB-MSC-derived secretome and atorvastatin group, (p < 0.001)

Combination of hUCB-MSC-derived secretome with atorvastatin also showed higher EPCs migration than the hUCB-MSC-derived secretome-only group at 2%, 10% and 20% concentrations (38.3±3.49 vs 17.20±1.92, 50.20±5.31 vs 27.00±4.00 and 76.40±7.50 vs 51.00±5.15, respectively; all p<0.001). The combination of high-dose (20%) hUCB-MSC-derived secretome and atorvastatin had the highest number of migrated EPC (76.4±7.50 × 10³ cells). The combination group had a significant and very strong correlation with EPC migration (r=0.970; p<0.001). The linear regression test showed an R² of 0.942.

Discussion

This research showed that treatment with hUCB-MSC-derived secretome, atorvastatin and a combination of the two increased the proliferation and migration of EPCs (isolated from CAD patient’s peripheral blood). HUCB-MSC-derived secretome enhances EPCs proliferation and migration in a dose-dependent manner. The combination of hUCB-MSC-derived secretome and atorvastatin was shown to be superior to atorvastatin or hUCB-MSC-derived secretome alone.

In this research, hUCB-MSC-derived secretome treatment increased EPC proliferation in a dose-dependent manner, with the concentrations of 10% and 20% shown to be superior to atorvastatin. Previous studies showed that atorvastatin treatment is superior to other statins at improving EPC proliferation^23,24,28. The HUCB-MSC-derived secretome is also composed of cytokines, chemokines, growth factors, proteins, and extracellular vesicles which may be involved in EPCs proliferation and migration^13,29. Vascular endothelial growth factor (VEGF), stromal-derived factor-1 (SDF-1), insulin-like growth factor (IGF-1) are contained in the hUCB-MSC-derived secretome which may be involved in increasing EPC proliferation³⁰. VEGF has been shown to improve the proliferation and differentiation of EPCs through activation of Ras signaling, and the MAPK/ERK pathway^31–33. SDF-1 and IGF 1 also been shown to increase the EPCs proliferation in response to the PI3K/protein kinase B signaling pathway and promote angiogenesis^34–36. Hence, it is suggested that hUCB-MSC-derived secretome treatment is beneficial to improve EPC proliferation which may involve MAPK/ERK and PI3K/protein kinase B pathway.

HUCB-MSC-derived secretome treatment shown to increase EPCs migration in a dose-dependent manner, with a concentration of 20% shown to be superior to atorvastatin. Similarly, the previous study showed secretome-derived from placental-MSCs is able to significantly increase EPCs migration³⁷. The HUCB-MSC-derived secretome contains pro-angiogenic factors, such as human angiopoietin-1 (Ang-1), hepatocyte growth factor (HGF), insulin-like growth factor I (IGF-I), prostaglandin E2 (PGE2), transforming growth factor-beta 1 (TGF-ß1), vascular cell adhesion protein 1 (VCAM-1) and vascular endothelial growth factor (VEGF)³⁸. MSCs also have immunomodulatory and anti-inflammatory properties, as it contributes to the maintenance of self-renewal capacity through E-Prostanoid 2 (EP2)³⁹ and immune cell activation and maturation, including CD4⁺ helper T cells, B cells, dendritic cells, natural killer cells, monocytes and macrophages⁴⁰. The HUCB-MSC-derived secretome also has a higher anti-inflammatory effect than other MSCs⁴¹ and antioxidant properties, as proven in previous studies conducted in renal injury⁴² and ischemic stroke⁴³. Inflammatory stimuli and oxidative stress are also known to impair EPCs migration⁴⁴. Chemoattractant gradient was an important driving factor to induce EPCs mobilization. Hence, a high concentration of growth factors in the HUCB-MSC-derived secretome increase the gradient between the top and lower parts of the Transwell migration assay may lead into an increase of EPCs migration. taken together, wide array molecules and multiple possible pathways involved in HUCB-MSCs secretome treatment seem to be responsible for its superiority against atorvastatin.

The synergistic effect of the HUCB-MSC-derived secretome with atorvastatin in enhancing EPCs proliferation and migration was demonstrated in this study. These combinations significantly increase EPCs proliferation and migratory activity by up to two-fold. Previously, The combination of MSCs with another compound, including statins, was shown to have beneficial effects in angiogenesis and neovascularization^45–47. Co-culture of MSCs and EPCs have been shown to demonstrate improved EPC proliferation and migration, and enhance their angiogenic capacity^48,49. However, the exact mechanism of these combinations to improve EPCs proliferation and migration is yet to be investigated. It is speculated that the involvement of multiple pathways may be responsible for its superiority against HUCB-MSCs-derived secretome or atorvastatin alone.

A mitogen-activated protein kinase (MAPK) pathway has been known to play a role in increasing EPCs proliferation²⁵. Cell cycle progression through increased Cyclin D1 expression mediated by PI3K/Akt and MAPK pathway also involved in EPCs proliferation⁵⁰. While increasing microRNA 221/222 expression shown to reduce EPCs proliferation capabilities⁵¹. HUCB-MSC-derived secretome treatment was speculated to improve EPCs proliferation through MAPK/ERK and PI3K/protein kinase B pathway. While atorvastatin improves EPCs proliferation through downregulation of microRNA 221/222 expression⁵¹. The involvement of these multiple pathways may result in higher EPCs proliferation in the combination group.

Enhanced growth factor levels through hUCB-MSC-derived secretome treatment will augment the chemoattractant gradient⁴², thus leading to enhanced migration of EPCs. The anti-inflammatory and antioxidant properties of hUCB-MSC-derived secretome also speculated to improve EPCs migration^40,42. While atorvastatin can increase the production of endothelial nitric oxide synthase and nitric oxide, which reduces the oxidative stress that impairs EPCs migration⁴⁷. Statin also could prevent EPCs senescence by upregulating TRF2 of EPCs, hence enhance migratory capacity⁵². Those facts suggested that the combination of hUCB-MSC-derived secretome and atorvastatin will have superior EPCs migration through the involvement of multiple pathways.

In summary, hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. Through those mechanisms, secretome could be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy. However, this research did not measure the exact composition of the hUCB-MSC-derived secretome. The previous study showed that the secretome from another type of MSC can increase EPCs migration but not EPCs proliferation⁵³. Hence, further research should be directed to identify the substance within the hUCB-MSC-derived secretome which is responsible for increasing EPC proliferation and migration, and compare it with other MSC secretomes. Further research should also verify the multiple pathways which may be responsible for the improvement of EPCs proliferation and migration in the combination group.

Conclusions

High dose hUCB-MSC-derived secretome outperforms atorvastatin to improve EPC proliferation and migration. A combination of hUCB-MSC-derived secretome with atorvastatin seems to be beneficial in promoting neovascularization through improved EPCs proliferation and migration effect compared to hUCB-MSC-derived secretome or atorvastatin alone.

Data availability

Underlying data

Figshare: Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration. https://doi.org/10.6084/m9.figshare.12186507.v2²².

This project contains the following underlying data:

RAW Data - F1000 revisi2 by SAH (XLSX). (Raw absorbance data from MTT proliferation assay and cell counts from the Transwell migration assay.)
RAW DATA f1000 (ZIP). (Raw images generated in this study, including images used to generate cell counts and raw immunofluorescence images.)

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgements

The authors are thankful to Professor Djoko Soemantri MD.,SpJP(K), Christian Pramudita, MD., Ilma Alfia Isaridha, MD., Melly Susanti MD., and Dwi Fachrul MD., for invaluable support in this project.

Faculty Opinions recommended

References

1. Benjamin EJ, Muntner P, Alonso A, et al.: Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. Circulation. 2019; 139(10): e56–e528. PubMed Abstract | Publisher Full Text
2. Sainsbury PA, Fisher M, de Silva R: Alternative interventions for refractory angina. Heart. 2017; 103(23): 1911–1922. PubMed Abstract | Publisher Full Text
3. Cheng K, Sainsbury P, Fisher M, et al.: Management of Refractory Angina Pectoris. Eur Cardiol. 2016; 11(2): 69–76. PubMed Abstract | Publisher Full Text | Free Full Text
4. Siddique A, Shantsila E, Lip GY, et al.: Endothelial progenitor cells: what use for the cardiologist? J Angiogenes Res. 2010; 2(1): 6. PubMed Abstract | Publisher Full Text | Free Full Text
5. Wils J, Favre J, Bellien J: Modulating putative endothelial progenitor cells for the treatment of endothelial dysfunction and cardiovascular complications in diabetes. Pharmacol Ther. 2017; 170: 98–115. PubMed Abstract | Publisher Full Text
6. George AL, Bangalore-Prakash P, Rajoria S, et al.: Endothelial progenitor cell biology in disease and tissue regeneration. J Hematol Oncol. 2011; 4: 24. PubMed Abstract | Publisher Full Text | Free Full Text
7. Fisher SA, Doree C, Mathur A, et al.: Stem cell therapy for chronic ischaemic heart disease and congestive heart failure. Cochrane Database Syst Rev. 2016; 12: CD007888. PubMed Abstract | Publisher Full Text | Free Full Text
8. Madonna R, Van Laake LW, Davidson SM, et al.: Position Paper of the European Society of Cardiology Working Group Cellular Biology of the Heart: cell-based therapies for myocardial repair and regeneration in ischemic heart disease and heart failure. Eur Heart J. 2016; 37(23): 1789–1798. PubMed Abstract | Publisher Full Text | Free Full Text
9. Gnecchi M, Zhang Z, Ni A, et al.: Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res. 2008; 103(11): 1204–1219. PubMed Abstract | Publisher Full Text | Free Full Text
10. Vasa M, Fichtlscherer S, Adler K, et al.: Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation. 2001; 103(24): 2885–90. PubMed Abstract | Publisher Full Text
11. Llevadot J, Murasawa S, Kureishi Y, et al.: HMG-CoA reductase inhibitor mobilizes bone marrow--derived endothelial progenitor cells. J Clin Invest. 2001; 108(3): 399–405. PubMed Abstract | Publisher Full Text | Free Full Text
12. Dimmeler S, Aicher A, Vasa M, et al.: HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J Clin Invest. 2001; 108(3): 391–7. PubMed Abstract | Publisher Full Text | Free Full Text
13. Gallina C, Turinetto V, Giachino C: A New Paradigm in Cardiac Regeneration: The Mesenchymal Stem Cell Secretome. Stem Cells Int. 2015; 2015: 765846. PubMed Abstract | Publisher Full Text | Free Full Text
14. Konala VB, Mamidi MK, Bhonde R, et al.: The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration. Cytotherapy. 2016; 18(1): 13–24. PubMed Abstract | Publisher Full Text | Free Full Text
15. Vizoso FJ, Eiro N, Cid S, et al.: Mesenchymal Stem Cell Secretome: Toward Cell-Free Therapeutic Strategies in Regenerative Medicine. Int J Mol Sci. 2017; 18(9): pii: E1852. PubMed Abstract | Publisher Full Text | Free Full Text
16. Phelps J, Sanati-Nezhad A, Ungrin M, et al.: Bioprocessing of Mesenchymal Stem Cells and Their Derivatives: Toward Cell-Free Therapeutics. Stem Cells Int. 2018; 2018: 9415367. PubMed Abstract | Publisher Full Text | Free Full Text
17. Zhao Y, Sun X, Cao W, et al.: Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Relieve Acute Myocardial Ischemic Injury. Stem Cells Int. 2015; 2015: 761643. PubMed Abstract | Publisher Full Text | Free Full Text
18. Fox K, Alonso Garcia MA, Ardissino D, et al.: Guidelines on the management of stable angina pectoris: executive summary: The Task Force on the Management of Stable Angina Pectoris of the European Society of Cardiology. Eur Heart J. 2006; 27(11): 1341–1381. PubMed Abstract | Publisher Full Text
19. Sandra F, Sudiono J, Sidharta EA, et al.: Conditioned Media of Human Umbilical Cord Blood Mesenchymal Stem Cell-derived Secretome Induced Apoptosis and Inhibited Growth of HeLa Cells. Indones Biomed J. 2014; 6(1): 57. Publisher Full Text
20. Oktaviono YH, Al-Farabi MJ, Oliva L, et al.: Preliminary Study: Purple Sweet Potato Extract Seems to Be Superior to Increase the Migration of Impaired Endothelial Progenitor Cells Compared to l -Ascorbic Acid. Sci Pharm. 2017; 87(3): 16. Publisher Full Text
21. Sherman H: Cell Migration , Chemotaxis and Invasion Assay Using Staining. Corning Protoc.
22. Al Farabi MJHuman umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12186507.v2
23. Meuthia F, Oktaviono YH, Soemantri D: Effects of Statins on Endothelial Progenitor Cell Proliferation from Peripheral Blood of Stable Coronary Artery Disease Patient. J Kardiol Indons. 2017; 38(1): 6–12. Publisher Full Text
24. Oktaviono YH, Savitri TVR, Soemantri D: Rosuvastatin is Superior Compared to Simvastatin and Atorvastatin to Induce Endothelial Progenitor Cells Migration. J Clin Diagnostic Res. 2019; 1077: 5–8. Publisher Full Text
25. Sandra F, Oktaviono YH, Widodo MA: Endothelial progenitor cells proliferated via MEK-dependent p42 MAPK signaling pathway. Mol Cell Biochem. 2015; 400(1–2): 201–6. PubMed Abstract | Publisher Full Text
26. Hur J, Yoon CH, Kim HS, et al.: Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol. 2004; 24(2): 288–293. PubMed Abstract | Publisher Full Text
27. Chopra H, Hung, M K, Kwong DL, et al.: Insights into Endothelial Progenitor Cells: Origin, Classification, Potentials, and Prospects. Stem Cells Int. 2018; 2018: 9847015. PubMed Abstract | Publisher Full Text | Free Full Text
28. Rawlings R, Nohria A, Liu PY, et al.: Comparison of effects of rosuvastatin (10 mg) versus atorvastatin (40 mg) on rho kinase activity in caucasian men with a previous atherosclerotic event. Am J Cardiol. 2009; 103(4): 437–441. PubMed Abstract | Publisher Full Text | Free Full Text
29. Eleuteri S, Fierabracci A: Insights Into the Secretome of Mesenchymal Stem Cells and Its Potential Applications. Int J Mol Sci. 2019; 20(18): 4597. PubMed Abstract | Publisher Full Text | Free Full Text
30. Kwon HM, Hur SM, Park KY, et al.: Multiple paracrine factors secreted by mesenchymal stem cells contribute to angiogenesis. Vascul Pharmacol. 2014; 63(1): 19–28. PubMed Abstract | Publisher Full Text
31. Xu J, Liu X, Jiang Y, et al.: MAPK/ERK signalling mediates VEGF-induced bone marrow stem cell differentiation into endothelial cell. J Cell Mol Med. 2008; 12(6A): 2395–2406. PubMed Abstract | Publisher Full Text | Free Full Text
32. Ge Q, Zhang H, Hou J, et al.: VEGF secreted by mesenchymal stem cells mediates the differentiation of endothelial progenitor cells into endothelial cells via paracrine mechanisms. Mol Med Rep. 2018; 17(1): 1667–1675. PubMed Abstract | Publisher Full Text | Free Full Text
33. Arutyunyan I, Fatkhudinov T, Kananykhina E, et al.: Role of VEGF-A in angiogenesis promoted by umbilical cord-derived mesenchymal stromal/stem cells: in vitro study. Stem Cell Res Ther. 2016; 7: 46. PubMed Abstract | Publisher Full Text | Free Full Text
34. Chen SL, Sheiban I, Xu BO, et al.: Impact of the complexity of bifurcation lesions treated with drug-eluting stents: the DEFINITION study (Definitions and impact of complEx biFurcation lesIons on clinical outcomes after percutaNeous coronary IntervenTIOn using drug-eluting steNts). JACC Cardiovasc Interv. 2014; 7(11): 1266–1276. PubMed Abstract | Publisher Full Text
35. Yu H, Feng Y: The potential of statin and stromal cell-derived factor-1 to promote angiogenesis. Cell Adh Migr. 2008; 2(4): 254–257. Publisher Full Text
36. Hou J, Peng X, Wang J: Mesenchymal stem cells promote endothelial progenitor cell proliferation by secreting insulin-like growth factor-1. Mol Med Rep. 2017; 16(2): 1502–1508. PubMed Abstract | Publisher Full Text
37. Kamprom W, Kheolamai P, U-Pratya Y: Endothelial Progenitor Cell Migration-Enhancing Factors in the Secretome of Placental-Derived Mesenchymal Stem Cells. Stem Cells Int. 2016; 2016: 2514326. PubMed Abstract | Publisher Full Text | Free Full Text
38. Wu M, Zhang R, Zou Q, et al.: Comparison of the Biological Characteristics of Mesenchymal Stem Cells Derived from the Human Placenta and Umbilical Cord. Sci Rep. 2018; 8(1): 5014. PubMed Abstract | Publisher Full Text | Free Full Text
39. Lee BC, Kim HS, Shin TH, et al.: PGE 2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact. Sci Rep. 2016; 6: 26298. PubMed Abstract | Publisher Full Text | Free Full Text
40. Murphy MB, Moncivais K, Caplan AI: Mesenchymal stem cells: Environmentally responsive therapeutics for regenerative medicine. Exp Mol Med. 2013; 45(11): e54. PubMed Abstract | Publisher Full Text | Free Full Text
41. Jin HJ, Bae YK, Kim M, et al.: Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy. Int J Mol Sci. 2013; 14(9): 17986–18001. PubMed Abstract | Publisher Full Text | Free Full Text
42. Lee KH, Tseng WC, Yang CY, et al.: The Anti-Inflammatory, Anti-Oxidative, and Anti-Apoptotic Benefits of Stem Cells in Acute Ischemic Kidney Injury. Int J Mol Sci. 2019; 20(14): 3529. PubMed Abstract | Publisher Full Text | Free Full Text
43. Cunningham CJ, Redondo-castro E, Allan SM: The therapeutic potential of the mesenchymal stem cell secretome in ischaemic stroke. J Cereb Blood Flow Metab. 2018; 38(8): 1276–1292. PubMed Abstract | Publisher Full Text | Free Full Text
44. Tousoulis D, Andreou I, Antoniades C, et al.: Role of inflammation and oxidative stress in endothelial progenitor cell function and mobilization: Therapeutic implications for cardiovascular diseases. Atherosclerosis. 2008; 201(2): 236–247. PubMed Abstract | Publisher Full Text
45. Luo R, Lu Y, Liu J, et al.: Enhancement of the efficacy of mesenchymal stem cells in the treatment of ischemic diseases. Biomed Pharmacother. 2019; 109(1): 2022–2034. PubMed Abstract | Publisher Full Text
46. Kwon YW, Heo SC, Jeong GO, et al.: Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis. Biochim Biophys Acta. 2013; 1832(12): 2136–2144. PubMed Abstract | Publisher Full Text
47. Song L, Yang YJ, Dong QT, et al.: Atorvastatin enhance efficacy of mesenchymal stem cells treatment for swine myocardial infarction via activation of nitric oxide synthase. PLoS One. 2013; 8(5): 1–12. PubMed Abstract | Publisher Full Text | Free Full Text
48. Liang T, Zhu L, Gao W, et al.: Coculture of endothelial progenitor cells and mesenchymal stem cells enhanced their proliferation and angiogenesis through PDGF and Notch signaling. FEBS Open Bio. 2017; 7: 1722–1736. PubMed Abstract | Publisher Full Text | Free Full Text
49. Li Z, Yang A, Yin X, et al.: Mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating CXCR2-Src-PKL/Vav2-Rac1. FASEB J. 2014; 400(1–2): 201–206. PubMed Abstract | Publisher Full Text
50. Qiu C, Xie Q, Zhang D, et al.: GM-CSF induces cyclin D1 expression and proliferation of endothelial progenitor cells via PI3K and MAPK signaling. Cell Physiol Biochem. 2014; 63(1): 19–28. PubMed Abstract | Publisher Full Text
51. Minami Y, Satoh M, Maesawa C, et al.: Effect of atorvastatin on microRNA 221 / 222 expression in endothelial progenitor cells obtained from patients with coronary artery disease. Eur J Clin Invest. 2009; 39(5): 359–67. PubMed Abstract | Publisher Full Text
52. Assmus B, Urbich C, Aicher A, et al.: HMG-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes. Circ Res. 2003; 92(9): 1049–55. PubMed Abstract | Publisher Full Text
53. Kamprom W, Kheolamai P, U-Pratya Y, et al.: Effects of mesenchymal stem cell-derived cytokines on the functional properties of endothelial progenitor cells. Eur J Cell Biol. 2016; 95(3–5): 153–63. PubMed Abstract | Publisher Full Text

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Version 2

VERSION 2 PUBLISHED 05 Jun 2020

Author details Author details

Yudi Her Oktaviono
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Resources, Validation, Writing – Review & Editing

Suryo Ardi Hutomo
Roles: Investigation, Methodology, Project Administration, Visualization, Writing – Original Draft Preparation

Makhyan Jibril Al-Farabi
Roles: Project Administration, Writing – Review & Editing

Angliana Chouw
Roles: Investigation, Software

Ferry Sandra
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Resources, Validation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This research was funded by the Prodia Education and Research Institute.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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https://doi.org/10.12688/f1000research.23547.2

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Oktaviono YH, Hutomo SA, Al-Farabi MJ et al. Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration [version 2; peer review: 2 approved]. F1000Research 2021, 9:537 (https://doi.org/10.12688/f1000research.23547.2)

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Reviewer Report 11 Aug 2021

Anwar Santoso, Department of Cardiology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia; National Cardiovascular Centre, Harapan Kita Hospital, Jakarta, Indonesia

Approved

https://doi.org/10.5256/f1000research.56385.r85035

I have re-reviewed this interesting manuscript on secretome and I have no further comments on that.
... Continue reading

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Reviewer Report 12 May 2021

Anwar Tandar, Division of Cardiovascular Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA

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https://doi.org/10.5256/f1000research.56385.r85034

Concerns were appropriately addressed. ... Continue reading

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Reviewer Report 03 Sep 2020

John David Symmons, University of Utah School of Medicine, Salt Lake City, UT, USA

Anwar Tandar, Division of Cardiovascular Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.25984.r64523

This is an interesting research involving the evaluation of EPCs in CAD and association with statin.

Several concerns should be addressed:

It is derived from one patient. The conclusion and validity need to

This is an interesting research involving the evaluation of EPCs in CAD and association with statin.

Several concerns should be addressed:

It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria
Typgraphical error in the Introduction... refractory angina, and patients
Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5.4.
All abbreviations need to be spelled out in the beginning or first time used
Similar to the rational for EPCs treatment, in EPC migrations assay section: Please describe the rationale for 5 fields and describe the sizes of the field.
This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
Other statins should also be investigated as they are being used widely in real world.
The exact composition of the hUCB-MSc derived secretome should be described.

In summary, this research needs more diligent work especially with the number of samples from more robust patients population.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Anwar Tandar, MD: Clinician, Interventional Cardiologist. John D. Symmons, PhD: Basic Scientist

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

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Author Response 10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

10 May 2021

Author Response

Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
... Continue reading Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria.
A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Q: Typographical error in the Introduction... refractory angina, and patients
A: Thank you for the feedback, we will revise accordingly

Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
A: We have added the reference, thank you

Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5
A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.

Q: All abbreviations need to be spelt out in the beginning or first time used
A: Thank you for the feedback, we will revise accordingly

Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field.
A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.

Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.

Q: Other statins should also be investigated as they are being used widely in real world.
A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.

Q: The exact composition of the hUCB-MSc derived secretome should be described.
A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.

Again, many thanks for the review,,

Best Regards
Yudi Her
Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria.
A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Q: Typographical error in the Introduction... refractory angina, and patients
A: Thank you for the feedback, we will revise accordingly

Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
A: We have added the reference, thank you

Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5
A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.

Q: All abbreviations need to be spelt out in the beginning or first time used
A: Thank you for the feedback, we will revise accordingly

Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field.
A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.

Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.

Q: Other statins should also be investigated as they are being used widely in real world.
A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.

Q: The exact composition of the hUCB-MSc derived secretome should be described.
A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.

Again, many thanks for the review,,

Best Regards
Yudi Her
Competing Interests: No competing interests were disclosed. Close
Report a concern
Author Response 10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

10 May 2021

Author Response

Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
... Continue reading Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria.
A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Q: Typographical error in the Introduction... refractory angina, and patients
A: Thank you for the feedback, we will revise accordingly

Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
A: We have added the reference, thank you

Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5
A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.

Q: All abbreviations need to be spelt out in the beginning or first time used
A: Thank you for the feedback, we will revise accordingly

Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field.
A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.

Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.

Q: Other statins should also be investigated as they are being used widely in real world.
A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.

Q: The exact composition of the hUCB-MSc derived secretome should be described.
A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.

Again, many thanks for the review,,

Best Regards
Yudi Her
Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria.
A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Q: Typographical error in the Introduction... refractory angina, and patients
A: Thank you for the feedback, we will revise accordingly

Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
A: We have added the reference, thank you

Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5
A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.

Q: All abbreviations need to be spelt out in the beginning or first time used
A: Thank you for the feedback, we will revise accordingly

Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field.
A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.

Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.

Q: Other statins should also be investigated as they are being used widely in real world.
A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.

Q: The exact composition of the hUCB-MSc derived secretome should be described.
A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.

Again, many thanks for the review,,

Best Regards
Yudi Her
Competing Interests: Authors declare no competing Interest Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

10 May 2021

Author Response

Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
... Continue reading Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria.
A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Q: Typographical error in the Introduction... refractory angina, and patients
A: Thank you for the feedback, we will revise accordingly

Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
A: We have added the reference, thank you

Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5
A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.

Q: All abbreviations need to be spelt out in the beginning or first time used
A: Thank you for the feedback, we will revise accordingly

Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field.
A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.

Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.

Q: Other statins should also be investigated as they are being used widely in real world.
A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.

Q: The exact composition of the hUCB-MSc derived secretome should be described.
A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.

Again, many thanks for the review,,

Best Regards
Yudi Her
Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria.
A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Q: Typographical error in the Introduction... refractory angina, and patients
A: Thank you for the feedback, we will revise accordingly

Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
A: We have added the reference, thank you

Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5
A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.

Q: All abbreviations need to be spelt out in the beginning or first time used
A: Thank you for the feedback, we will revise accordingly

Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field.
A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.

Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.

Q: Other statins should also be investigated as they are being used widely in real world.
A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.

Q: The exact composition of the hUCB-MSc derived secretome should be described.
A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.

Again, many thanks for the review,,

Best Regards
Yudi Her
Competing Interests: No competing interests were disclosed. Close
Report a concern
Author Response 10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

10 May 2021

Author Response

Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
... Continue reading Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria.
A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Q: Typographical error in the Introduction... refractory angina, and patients
A: Thank you for the feedback, we will revise accordingly

Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
A: We have added the reference, thank you

Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5
A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.

Q: All abbreviations need to be spelt out in the beginning or first time used
A: Thank you for the feedback, we will revise accordingly

Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field.
A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.

Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.

Q: Other statins should also be investigated as they are being used widely in real world.
A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.

Q: The exact composition of the hUCB-MSc derived secretome should be described.
A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.

Again, many thanks for the review,,

Best Regards
Yudi Her
Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria.
A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Q: Typographical error in the Introduction... refractory angina, and patients
A: Thank you for the feedback, we will revise accordingly

Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
A: We have added the reference, thank you

Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5
A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.

Q: All abbreviations need to be spelt out in the beginning or first time used
A: Thank you for the feedback, we will revise accordingly

Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field.
A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.

Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.

Q: Other statins should also be investigated as they are being used widely in real world.
A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.

Q: The exact composition of the hUCB-MSc derived secretome should be described.
A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.

Again, many thanks for the review,,

Best Regards
Yudi Her
Competing Interests: Authors declare no competing Interest Close
Report a concern

Views

Reviewer Report 08 Jul 2020

Anwar Santoso, Department of Cardiology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia; National Cardiovascular Centre, Harapan Kita Hospital, Jakarta, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.25984.r64524

The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal

The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behavior and decreased gliosis, this study was measured in other target disorders¹.
The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression.
In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.
Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].
There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.
In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?
In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?³.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

References

1. Zhang X, Zhang Q, Li W, Nie D, et al.: Therapeutic effect of human umbilical cord mesenchymal stem cells on neonatal rat hypoxic-ischemic encephalopathy.J Neurosci Res. 2014; 92 (1): 35-45 PubMed Abstract | Publisher Full Text
2. Kamprom W, Kheolamai P, U-Pratya Y, Supokawej A, et al.: Endothelial Progenitor Cell Migration-Enhancing Factors in the Secretome of Placental-Derived Mesenchymal Stem Cells.Stem Cells Int. 2016; 2016: 2514326 PubMed Abstract | Publisher Full Text
3. Broughton K, Wang B, Firouzi F, Khalafalla F, et al.: Mechanisms of Cardiac Repair and Regeneration. Circulation Research. 2018; 122 (8): 1151-1163 Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: 1. Cardiovascular disease (particularly ischemic heart disease) 2. Lipidology and diabetes mellitus 3. Hypertension 4. Stem cell and regenerative medicine

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

Report a concern

Author Response 10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

10 May 2021

Author Response

1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in ... Continue reading 1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behavior and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as other research which did similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression.I
A. ndeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.
1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behavior and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as other research which did similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression.I
A. ndeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Author Response 10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

10 May 2021

Author Response

1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in ... Continue reading 1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behaviour and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as for other research which did a similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression
A. Indeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.
1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behaviour and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as for other research which did a similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression
A. Indeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.
Competing Interests: There are no competing interest Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

10 May 2021

Author Response

1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in ... Continue reading 1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behavior and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as other research which did similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression.I
A. ndeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.
1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behavior and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as other research which did similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression.I
A. ndeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Author Response 10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

10 May 2021

Author Response

1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in ... Continue reading 1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behaviour and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as for other research which did a similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression
A. Indeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.
1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behaviour and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as for other research which did a similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression
A. Indeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.
Competing Interests: There are no competing interest Close
Report a concern

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Version 2

VERSION 2 PUBLISHED 05 Jun 2020

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Anwar Santoso, University of Indonesia, Jakarta, Indonesia; Harapan Kita Hospital, Jakarta, Indonesia
Anwar Tandar, University of Utah School of Medicine, Salt Lake City, USA

John David Symmons, University of Utah School of Medicine, Salt Lake City, USA

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11 Aug 2021 | for Version 2

Anwar Santoso, Department of Cardiology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia; National Cardiovascular Centre, Harapan Kita Hospital, Jakarta, Indonesia

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Approved

I have re-reviewed this interesting manuscript on secretome and I have no further comments on that.

Certainly, I would be happy to recommend that this wonderful paper would be indexed.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

1. Cardiovascular disease (particularly ischemic heart disease) 2. Lipidology and diabetes mellitus 3. Hypertension 4. Stem cell and regenerative medicine

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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12 May 2021 | for Version 2

Anwar Tandar, Division of Cardiovascular Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA

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Approved

Concerns were appropriately addressed. Now acceptable for indexing.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Clinician, Interventional Cardiologist

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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03 Sep 2020 | for Version 1

John David Symmons, University of Utah School of Medicine, Salt Lake City, UT, USA

Anwar Tandar, Division of Cardiovascular Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA

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Approved With Reservations

This is an interesting research involving the evaluation of EPCs in CAD and association with statin.

Several concerns should be addressed:

It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria
Typgraphical error in the Introduction... refractory angina, and patients
Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5.4.
All abbreviations need to be spelled out in the beginning or first time used
Similar to the rational for EPCs treatment, in EPC migrations assay section: Please describe the rationale for 5 fields and describe the sizes of the field.
This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
Other statins should also be investigated as they are being used widely in real world.
The exact composition of the hUCB-MSc derived secretome should be described.

In summary, this research needs more diligent work especially with the number of samples from more robust patients population.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Anwar Tandar, MD: Clinician, Interventional Cardiologist. John D. Symmons, PhD: Basic Scientist

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Author Response

10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

Dear Dr Anwar Tandar

Thank you very much for your valuable feedback
I am truly honoured with your feedback

In this opportunity, please let me answer the question
Q: It is derived from one patient. The conclusion and validity need to be addressed seriously when there is sample from one patient. In addition, since there is only one patient, why is there even a need for inclusion and exclusion criteria.
A: In this experimental laboratory research, the sample used was EPCs derived from single patient with coronary artery disease. We have ensured the diagnosis of coronary artery disease from the ECG, echocardiography and coronary angiography. The purpose of the single sample being used in this research is to ensure the homogeneity of the sample while inclusion and exclusion criteria was used to identify the best EPCs quality that represent patient with CAD. Mixing EPCs from the various patient may cause EPCs failed to grow efficiently due to incompatibility with other source of EPCs. Inclusion and exclusion criteria is still relevant because we want to ensure that the patient was really diagnosed with coronary artery disease with specific criteria without any other significant disease. Without clear inclusion and exclusion criteria, we may select patient with multiple disease which EPCs function may differ from patient with coronary artery disease only.

Q: Typographical error in the Introduction... refractory angina, and patients
A: Thank you for the feedback, we will revise accordingly

Q: Reference needed for the statement: HUCB-MSCs-derived secretome preparation. The media was collected......(reference 3).
A: We have added the reference, thank you

Q: Treatment of EPCs...Please describe the rationale for 5 replications. Please describe the rationale behind the selected number 5
A: The 5 replication was based on Federer's formula: (t-1)(n-1) ≥ 15, where t is the number of treatments and n is the number of replication.

Q: All abbreviations need to be spelt out in the beginning or first time used
A: Thank you for the feedback, we will revise accordingly

Q: Similar to the rationale for EPCs treatment, in EPC migrations essay section: Please describe the rationale for 5 fields and describe the sizes of the field.
A: The usage of the 5 fields was the standard protocol of cell migration calculation using transwell migration assay based on the Transwell protocol. In this research, we used 0.04mm in diameter as the sizes of the field.

Q: This interesting research should investigate more patients to allow a stronger interpretation and to allow more sound conclusion
A: Thank you very much for your genuine feedback. This research was part of our preliminary research which explore the possibilities of secretome as an alternative regenerative treatment other than cell-based regeneration treatment for the patient with coronary artery disease. In this early research we used limited patient but we will expand further the research with involving more samples and aim to have clinical trials should we achieve satisfying results. We also consider to combine the secretome treatment with existing coronary artery disease treatment, to ensure the usage of secretome combined with current medical treatment will benefit synergistically.

Q: Other statins should also be investigated as they are being used widely in real world.
A: Thank you for the feedback, we will consider that as our future research suggestion. However, we first prefer atorvastatin since it is readily available in our country, Indonesia. Previously, we have compared the effect of atorvastatin, rosuvastatin and simvastatin effect on the EPCs (without secretome) and conclude that atorvastatin was the most superior in inducing EPCs migration. Thus, this research prefers to use atorvastatin and combine it with secretome.

Q: The exact composition of the hUCB-MSc derived secretome should be described.
A: Thanks, we would like to admit that the limitation of this research was the inability to exactly describe the molecule in the hUCB-MSc derived secretome. Further research is required through proteomic analysis to determine the molecule in the hUCB-MSc derived secretome. This will also help to standardize the composition of hUCB-MSc derived secretome.

Again, many thanks for the review,,

Best Regards
Yudi Her

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Competing Interests

No competing interests were disclosed.

Author Response

10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

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Competing Interests

Authors declare no competing Interest

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Reviewer Report

63 Views

08 Jul 2020 | for Version 1

Anwar Santoso, Department of Cardiology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia; National Cardiovascular Centre, Harapan Kita Hospital, Jakarta, Indonesia

63 Views Cite this report Responses(2)

Approved With Reservations

The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behavior and decreased gliosis, this study was measured in other target disorders¹.
The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression.
In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.
Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].
There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.
In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?
In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?³.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

References

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

1. Cardiovascular disease (particularly ischemic heart disease) 2. Lipidology and diabetes mellitus 3. Hypertension 4. Stem cell and regenerative medicine

Respond to this report

Responses (2)

Author Response

10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behavior and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as other research which did similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression.I
A. ndeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.

View more View less

Competing Interests

No competing interests were disclosed.

Author Response

10 May 2021

Yudi Her Oktaviono, Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Airlangga, Soetomo General Academic Hospital, Surabaya, Indonesia

1.
Q The study aims to compare the effect of hUCB-MSC-derived secretome, atorvastatin, and the two combinations in modulating EPC proliferation and migration. The study addresses the novel issues in refractory angina, whether the atorvastatin and secretome derived mesenchymal stem cells improve EPC expression. There are similar studies available addressing these issues. Zhang X et al. demonstrated that intravenous transplantation of huC-MSCs at an early stage could improve hypoxic-ischemic rats' behaviour and decreased gliosis, this study was measured in other target disorders¹.

A. Thank you for the references and comparison, we will note this feedback and put it on the paper as for other research which did a similar intervention

2.
Q. The rationale and scientific background of this manuscript were justified, to disclose the role of secretomes and paracrine stimulation on EPC expression
A. Indeed, that was our research aim

3.
Q. In the #Method section, the authors mentioned the inclusion criteria: male, aged 40-59 years old, history of chronic ischemic heart disease as proven by CAG (coronary angiography). The authors should quote the diagnostic criterion. Additionally, the authors should clearly state this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

A. We have added the diagnostic criteria of chronic ischemic heart disease as proven by coronary angiography results that showed >50% stenosis of left main coronary artery or >70% of other coronary arteries. We also explain that this is an experimental study of atorvastatin and hUCB-MSC-derived secretome and their combinations on EPC proliferation and migration.

4.
Q. Umbilical cord blood-derived EPC established in a previous study displayed cobblestone-like morphology; this is a typical feature of the EPC. The authors did not state this in the manuscript, except they confirmed using FITC-labelled anti-human CD 34+ expression. The authors should clearly explain it[ref-2].

A. Thank you very much for reminding us, We did evaluate the EPCs cobblestone-like morphology before staining the cells with CD34 antibody. We already put these information on the method section.

5.
Q. There was another immunophenotype of EPC as determined by flow cytometry, VEGFR2-PE, vWF-FITC, and CD31-PE². Is there any reason why the authors only demonstrated with CD34+ expression. The authors should provide their ideas on it.

A. In this study, the use of FITC CD34+ only to confirmed the EPCs are sufficient, as the same EPCs culture method was used in authors previous research ( which mentioned on references number 19, 21-22,45). There was also uncertainty about the use of another immunophenotype of EPC as determined by flow cytometry (VEGFR2-PE, vWF-FITC, and CD31-PE), caused by heterogenous types of EPCs (mentioned on the references of 51,52)

6.
Q. In the #EPCs proliferation assay, the authors explain EPCs proliferation measured using OD; there was no explanation of how OD transferred in a measurement scale in Figure 2 (Y-ordinate)?
A. EPCs proliferation can be determined through various method, In the previous study, proliferation can be determined relatively through OD (references no 21-22,45). Thus, we use OD as proliferation measurement scale in Figure 2 (Y-ordinate).

7.
Q.How did the authors determine the percentage of hUCB-MSC-derived secretome? Is there any control over the measurement?

A. The percentage of hUCB-MSC-derived secretome was determined from the dilution level of hUCB-MSC-derived secretome. For example, 1 mL of hUCB-MSC-derived secretome with 49 mL of the phosphate buffer saline (PBS) plus 2% fetal bovine serum (FBS). However, no control over measurement.

8.
Q. In the #Table 1 – Characteristics of the patient, the authors should explain "Left ventricle internal diameter" = 5.8 cm; I wonder whether that is either "end-systolic dimension or end-diastolic dimension"?
A. It should be Left ventricle end-diastolic dimension, thanks for correcting this phrase

9.
Q. In #summary, the authors should open the opportunity on the horizon, whether the cell-based therapy or cell-free measures that win the future game?

A. Cardiac cell-based therapy has emerged as a novel therapeutic option for patients dealing with untreatable refractory angina (RA). However, after more than a decade of controlled studies, no definitive consensus has been reached regarding clinical efficacy. While in this research hUCB-MSC-derived secretome may be developed and combined with atorvastatin treatment in CAD patients to improve EPCs proliferation and migration. In this early study, we can conclude that secretome-based treatment may be a game changer in refractory angina therapeutic options and outperforms the previous cell-based therapy.

Again, I would express my appreciation to all authors to address these evolving issues in regenerative medicine.

View more View less

Competing Interests

There are no competing interest

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] 1. Benjamin EJ, Muntner P, Alonso A, et al.: Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. Circulation. 2019; 139(10): e56–e528. PubMed Abstract | Publisher Full Text

[2] 2. Sainsbury PA, Fisher M, de Silva R: Alternative interventions for refractory angina. Heart. 2017; 103(23): 1911–1922. PubMed Abstract | Publisher Full Text

[3] 3. Cheng K, Sainsbury P, Fisher M, et al.: Management of Refractory Angina Pectoris. Eur Cardiol. 2016; 11(2): 69–76. PubMed Abstract | Publisher Full Text | Free Full Text

[4] 4. Siddique A, Shantsila E, Lip GY, et al.: Endothelial progenitor cells: what use for the cardiologist? J Angiogenes Res. 2010; 2(1): 6. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Wils J, Favre J, Bellien J: Modulating putative endothelial progenitor cells for the treatment of endothelial dysfunction and cardiovascular complications in diabetes. Pharmacol Ther. 2017; 170: 98–115. PubMed Abstract | Publisher Full Text

[6] 6. George AL, Bangalore-Prakash P, Rajoria S, et al.: Endothelial progenitor cell biology in disease and tissue regeneration. J Hematol Oncol. 2011; 4: 24. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Fisher SA, Doree C, Mathur A, et al.: Stem cell therapy for chronic ischaemic heart disease and congestive heart failure. Cochrane Database Syst Rev. 2016; 12: CD007888. PubMed Abstract | Publisher Full Text | Free Full Text

[8] 8. Madonna R, Van Laake LW, Davidson SM, et al.: Position Paper of the European Society of Cardiology Working Group Cellular Biology of the Heart: cell-based therapies for myocardial repair and regeneration in ischemic heart disease and heart failure. Eur Heart J. 2016; 37(23): 1789–1798. PubMed Abstract | Publisher Full Text | Free Full Text

[9] 9. Gnecchi M, Zhang Z, Ni A, et al.: Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res. 2008; 103(11): 1204–1219. PubMed Abstract | Publisher Full Text | Free Full Text

[10] 10. Vasa M, Fichtlscherer S, Adler K, et al.: Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation. 2001; 103(24): 2885–90. PubMed Abstract | Publisher Full Text

[11] 11. Llevadot J, Murasawa S, Kureishi Y, et al.: HMG-CoA reductase inhibitor mobilizes bone marrow--derived endothelial progenitor cells. J Clin Invest. 2001; 108(3): 399–405. PubMed Abstract | Publisher Full Text | Free Full Text

[12] 12. Dimmeler S, Aicher A, Vasa M, et al.: HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J Clin Invest. 2001; 108(3): 391–7. PubMed Abstract | Publisher Full Text | Free Full Text

[13] 13. Gallina C, Turinetto V, Giachino C: A New Paradigm in Cardiac Regeneration: The Mesenchymal Stem Cell Secretome. Stem Cells Int. 2015; 2015: 765846. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Konala VB, Mamidi MK, Bhonde R, et al.: The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration. Cytotherapy. 2016; 18(1): 13–24. PubMed Abstract | Publisher Full Text | Free Full Text

[15] 15. Vizoso FJ, Eiro N, Cid S, et al.: Mesenchymal Stem Cell Secretome: Toward Cell-Free Therapeutic Strategies in Regenerative Medicine. Int J Mol Sci. 2017; 18(9): pii: E1852. PubMed Abstract | Publisher Full Text | Free Full Text

[16] 16. Phelps J, Sanati-Nezhad A, Ungrin M, et al.: Bioprocessing of Mesenchymal Stem Cells and Their Derivatives: Toward Cell-Free Therapeutics. Stem Cells Int. 2018; 2018: 9415367. PubMed Abstract | Publisher Full Text | Free Full Text

[17] 17. Zhao Y, Sun X, Cao W, et al.: Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Relieve Acute Myocardial Ischemic Injury. Stem Cells Int. 2015; 2015: 761643. PubMed Abstract | Publisher Full Text | Free Full Text

[18] 18. Fox K, Alonso Garcia MA, Ardissino D, et al.: Guidelines on the management of stable angina pectoris: executive summary: The Task Force on the Management of Stable Angina Pectoris of the European Society of Cardiology. Eur Heart J. 2006; 27(11): 1341–1381. PubMed Abstract | Publisher Full Text

[19] 19. Sandra F, Sudiono J, Sidharta EA, et al.: Conditioned Media of Human Umbilical Cord Blood Mesenchymal Stem Cell-derived Secretome Induced Apoptosis and Inhibited Growth of HeLa Cells. Indones Biomed J. 2014; 6(1): 57. Publisher Full Text

[20] 20. Oktaviono YH, Al-Farabi MJ, Oliva L, et al.: Preliminary Study: Purple Sweet Potato Extract Seems to Be Superior to Increase the Migration of Impaired Endothelial Progenitor Cells Compared to l -Ascorbic Acid. Sci Pharm. 2017; 87(3): 16. Publisher Full Text

[21] 21. Sherman H: Cell Migration , Chemotaxis and Invasion Assay Using Staining. Corning Protoc.

[22] 22. Al Farabi MJHuman umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12186507.v2

[23] 23. Meuthia F, Oktaviono YH, Soemantri D: Effects of Statins on Endothelial Progenitor Cell Proliferation from Peripheral Blood of Stable Coronary Artery Disease Patient. J Kardiol Indons. 2017; 38(1): 6–12. Publisher Full Text

[24] 24. Oktaviono YH, Savitri TVR, Soemantri D: Rosuvastatin is Superior Compared to Simvastatin and Atorvastatin to Induce Endothelial Progenitor Cells Migration. J Clin Diagnostic Res. 2019; 1077: 5–8. Publisher Full Text

[25] 25. Sandra F, Oktaviono YH, Widodo MA: Endothelial progenitor cells proliferated via MEK-dependent p42 MAPK signaling pathway. Mol Cell Biochem. 2015; 400(1–2): 201–6. PubMed Abstract | Publisher Full Text

[26] 26. Hur J, Yoon CH, Kim HS, et al.: Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol. 2004; 24(2): 288–293. PubMed Abstract | Publisher Full Text

[27] 27. Chopra H, Hung, M K, Kwong DL, et al.: Insights into Endothelial Progenitor Cells: Origin, Classification, Potentials, and Prospects. Stem Cells Int. 2018; 2018: 9847015. PubMed Abstract | Publisher Full Text | Free Full Text

[28] 28. Rawlings R, Nohria A, Liu PY, et al.: Comparison of effects of rosuvastatin (10 mg) versus atorvastatin (40 mg) on rho kinase activity in caucasian men with a previous atherosclerotic event. Am J Cardiol. 2009; 103(4): 437–441. PubMed Abstract | Publisher Full Text | Free Full Text

[29] 29. Eleuteri S, Fierabracci A: Insights Into the Secretome of Mesenchymal Stem Cells and Its Potential Applications. Int J Mol Sci. 2019; 20(18): 4597. PubMed Abstract | Publisher Full Text | Free Full Text

[30] 30. Kwon HM, Hur SM, Park KY, et al.: Multiple paracrine factors secreted by mesenchymal stem cells contribute to angiogenesis. Vascul Pharmacol. 2014; 63(1): 19–28. PubMed Abstract | Publisher Full Text

[31] 31. Xu J, Liu X, Jiang Y, et al.: MAPK/ERK signalling mediates VEGF-induced bone marrow stem cell differentiation into endothelial cell. J Cell Mol Med. 2008; 12(6A): 2395–2406. PubMed Abstract | Publisher Full Text | Free Full Text

[32] 32. Ge Q, Zhang H, Hou J, et al.: VEGF secreted by mesenchymal stem cells mediates the differentiation of endothelial progenitor cells into endothelial cells via paracrine mechanisms. Mol Med Rep. 2018; 17(1): 1667–1675. PubMed Abstract | Publisher Full Text | Free Full Text

[33] 33. Arutyunyan I, Fatkhudinov T, Kananykhina E, et al.: Role of VEGF-A in angiogenesis promoted by umbilical cord-derived mesenchymal stromal/stem cells: in vitro study. Stem Cell Res Ther. 2016; 7: 46. PubMed Abstract | Publisher Full Text | Free Full Text

[34] 34. Chen SL, Sheiban I, Xu BO, et al.: Impact of the complexity of bifurcation lesions treated with drug-eluting stents: the DEFINITION study (Definitions and impact of complEx biFurcation lesIons on clinical outcomes after percutaNeous coronary IntervenTIOn using drug-eluting steNts). JACC Cardiovasc Interv. 2014; 7(11): 1266–1276. PubMed Abstract | Publisher Full Text

[35] 35. Yu H, Feng Y: The potential of statin and stromal cell-derived factor-1 to promote angiogenesis. Cell Adh Migr. 2008; 2(4): 254–257. Publisher Full Text

[36] 36. Hou J, Peng X, Wang J: Mesenchymal stem cells promote endothelial progenitor cell proliferation by secreting insulin-like growth factor-1. Mol Med Rep. 2017; 16(2): 1502–1508. PubMed Abstract | Publisher Full Text

[37] 37. Kamprom W, Kheolamai P, U-Pratya Y: Endothelial Progenitor Cell Migration-Enhancing Factors in the Secretome of Placental-Derived Mesenchymal Stem Cells. Stem Cells Int. 2016; 2016: 2514326. PubMed Abstract | Publisher Full Text | Free Full Text

[38] 38. Wu M, Zhang R, Zou Q, et al.: Comparison of the Biological Characteristics of Mesenchymal Stem Cells Derived from the Human Placenta and Umbilical Cord. Sci Rep. 2018; 8(1): 5014. PubMed Abstract | Publisher Full Text | Free Full Text

[39] 39. Lee BC, Kim HS, Shin TH, et al.: PGE 2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact. Sci Rep. 2016; 6: 26298. PubMed Abstract | Publisher Full Text | Free Full Text

[40] 40. Murphy MB, Moncivais K, Caplan AI: Mesenchymal stem cells: Environmentally responsive therapeutics for regenerative medicine. Exp Mol Med. 2013; 45(11): e54. PubMed Abstract | Publisher Full Text | Free Full Text

[41] 41. Jin HJ, Bae YK, Kim M, et al.: Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy. Int J Mol Sci. 2013; 14(9): 17986–18001. PubMed Abstract | Publisher Full Text | Free Full Text

[42] 42. Lee KH, Tseng WC, Yang CY, et al.: The Anti-Inflammatory, Anti-Oxidative, and Anti-Apoptotic Benefits of Stem Cells in Acute Ischemic Kidney Injury. Int J Mol Sci. 2019; 20(14): 3529. PubMed Abstract | Publisher Full Text | Free Full Text

[43] 43. Cunningham CJ, Redondo-castro E, Allan SM: The therapeutic potential of the mesenchymal stem cell secretome in ischaemic stroke. J Cereb Blood Flow Metab. 2018; 38(8): 1276–1292. PubMed Abstract | Publisher Full Text | Free Full Text

[44] 44. Tousoulis D, Andreou I, Antoniades C, et al.: Role of inflammation and oxidative stress in endothelial progenitor cell function and mobilization: Therapeutic implications for cardiovascular diseases. Atherosclerosis. 2008; 201(2): 236–247. PubMed Abstract | Publisher Full Text

[45] 45. Luo R, Lu Y, Liu J, et al.: Enhancement of the efficacy of mesenchymal stem cells in the treatment of ischemic diseases. Biomed Pharmacother. 2019; 109(1): 2022–2034. PubMed Abstract | Publisher Full Text

[46] 46. Kwon YW, Heo SC, Jeong GO, et al.: Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis. Biochim Biophys Acta. 2013; 1832(12): 2136–2144. PubMed Abstract | Publisher Full Text

[47] 47. Song L, Yang YJ, Dong QT, et al.: Atorvastatin enhance efficacy of mesenchymal stem cells treatment for swine myocardial infarction via activation of nitric oxide synthase. PLoS One. 2013; 8(5): 1–12. PubMed Abstract | Publisher Full Text | Free Full Text

[48] 48. Liang T, Zhu L, Gao W, et al.: Coculture of endothelial progenitor cells and mesenchymal stem cells enhanced their proliferation and angiogenesis through PDGF and Notch signaling. FEBS Open Bio. 2017; 7: 1722–1736. PubMed Abstract | Publisher Full Text | Free Full Text

[49] 49. Li Z, Yang A, Yin X, et al.: Mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating CXCR2-Src-PKL/Vav2-Rac1. FASEB J. 2014; 400(1–2): 201–206. PubMed Abstract | Publisher Full Text

[50] 50. Qiu C, Xie Q, Zhang D, et al.: GM-CSF induces cyclin D1 expression and proliferation of endothelial progenitor cells via PI3K and MAPK signaling. Cell Physiol Biochem. 2014; 63(1): 19–28. PubMed Abstract | Publisher Full Text

[51] 51. Minami Y, Satoh M, Maesawa C, et al.: Effect of atorvastatin on microRNA 221 / 222 expression in endothelial progenitor cells obtained from patients with coronary artery disease. Eur J Clin Invest. 2009; 39(5): 359–67. PubMed Abstract | Publisher Full Text

[52] 52. Assmus B, Urbich C, Aicher A, et al.: HMG-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes. Circ Res. 2003; 92(9): 1049–55. PubMed Abstract | Publisher Full Text

[53] 53. Kamprom W, Kheolamai P, U-Pratya Y, et al.: Effects of mesenchymal stem cell-derived cytokines on the functional properties of endothelial progenitor cells. Eur J Cell Biol. 2016; 95(3–5): 153–63. PubMed Abstract | Publisher Full Text

Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Methods

Sample collection

HUCB-MSCs-derived secretome preparation

Isolation and culture of EPCs

Treatment of EPCs

EPCs proliferation assay

EPCs migration assay

Statistical analysis

Results

Baseline characteristics and demography of CAD patient

Table 1. Characteristics of the patient.

EPC characteristics

Figure 1. Immunofluorescence characterization of cultured EPCs.

HUCB-MSCs-derived secretome and atorvastatin increase EPCs proliferation

Combination of hUCB-MSCs-derived secretome and atorvastatin increase EPCs proliferation compared with single treatment

Figure 2. Comparison of EPC proliferation effects among all treatment groups (see text).

HUCB-MSCs-derived secretome and atorvastatin increase EPCs migration

Combination of hUCB-MSCs-derived secretome and atorvastatin increase EPCs migration compared with single treatment

Figure 3. Comparison of EPCs migration effects among all treatment groups (see text).

Discussion

Conclusions

Data availability

Underlying data

Acknowledgements

References

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