Enhancement of EPC migration by high-dose lisinopril is superior compared to captopril and ramipril

Background: Angiotensin-converting enzyme (ACE) inhibitors have been shown to promote endothelial progenitor cell (EPC) function. However, the efficacies of different ACE inhibitors in improving the migratory capabilities of ECPs in coronary artery disease (CAD) patients is unclear. This study compared the effectiveness of captopril, lisinopril, and ramipril toward the migration capability of impaired EPCs from CAD patients. Methods: We isolated peripheral blood mononuclear cells (PBMCs), separated EPCs from PBMCs, and divided them into an untreated group (control) and treated groups of captopril, lisinopril, and ramipril at doses of 1mM, 10mM, and 100mM. EPC migration was evaluated using the Boyden chamber assay. Analysis of variance (ANOVA) was performed using SPSS 25.0. Results: This study showed that treatment with captopril, lisinopril, and ramipril starting at the lowest dose (1 mM) increased EPC migration (65,250 ± 6,750 cells; 60,750± 5,030 cells; and 49,500 ± 8,400 cells, respectively) compared to control (43,714 ± 7,216 cells). Increased migration of EPCs was observed by increasing the treatment dose to 10 mM with captopril, lisinopril, and ramipril (90,000 ± 16,837 cells; 79,071 ± 2,043 cells; and 64,285 ± 11,824 cells, respectively). The highest EPC migration was shown for lisinopril 100 mM (150,750 ± 16,380 cells), compared to captopril and ramipril at the same dose (105,750 ± 8112 cells and 86,625 ± 5,845 cells, respectively). Conclusions: Captopril, ramipril, and lisinopril were shown to increase EPC migration in a dose-dependent manner. Low-dose (1 mM) and medium-dose (10 mM) captopril had a larger effect on ECP migration than lisinopril and ramipril. Meanwhile, high-dose lisinopril (100mM) had the highest migration effect, suggesting it may be preferable for promoting EPC migration in CAD patients.


Introduction
Endothelial dysfunction and impaired endothelial regeneration are thought to play an important role in the pathogenesis of arteriosclerosis in coronary arterial disease (CAD) 1 . Endothelial regeneration is not only fulfilled by resident endothelial cells but also repaired by endothelial progenitor cells (ECPs) originating from the bone marrow 2 . ECPs are premature circulating cells, a specific subtype of hematopoietic stem cells that differentiate into endothelial cells in situ and promote neovascularization 3,4 . Several studies have shown that in patients with CAD, there is a significant decrease in the number and migratory function of circulating EPCs, which leads to impaired neovascularization of ischemic tissue 5,6 . Low EPC counts can predict severe endothelial dysfunction, cardiovascular events, and deaths from cardiovascular causes 7,8 . It is suggested that intracellular damage and impaired redox balance in EPCs due to oxidative stress are the predisposes of imbalance in vascular pathology 9,10 .
Angiotensin-converting enzyme (ACE) inhibitors are widely used in cardiovascular disease and have been shown to be associated with beneficial effects on EPCs in several in vitro and clinical studies [11][12][13][14] . An animal study on mice with increased left ventricular pressure showed that ramipril increases the number and improves EPC migration 12 . A clinical study in hypertensive patients showed that enalapril and zofenopril reduce EPC levels and prevent vascular damage and carotid intima-media thickening 13 . In a small clinical trial, administration of ramipril for four weeks in patients with stable CAD augments and increases the functional activity of EPCs, including migration, adhesion, and in vitro capacity of vasculogenesis 15 .
However, no studies have investigated the role of ACE inhibitors of captopril and lisinopril in relation to the EPCs. In addition, the comparison between different types of ACE inhibitors toward the impaired migration function of EPCs remains to be investigated. We aimed to evaluate the effects of captopril, lisinopril, and ramipril on EPCs migration from CAD patients.

Ethical statement
Our study protocol was approved by the Institutional Ethics Committee of Dr. Soetomo General Hospital (945/KEPK/II/ 2019). Informed consent for peripheral blood sampling procedures and participation in research studies was obtained from all patients before the blood was drawn. We have omitted all data that could reveal the identity of the patients.

Study population
In the present study, we used peripheral blood samples from the same participants and performed similar methodology to that described in our previous study 16 . From June 2018 to December 2018, we studied a total of eight patients with stable CAD who underwent coronary angiography. Only patients with the left main coronary artery stenosis of more than 50% or stenosis in other coronary arteries more than 70% were recruited. To prevent the effects of myocardial ischemia on ECP kinetics, we excluded patients with a history of new-onset acute myocardial infarction. In addition, patients with anemia, diabetes, a history of percutaneous coronary intervention, or coronary artery bypass grafting were not included in the study. Physical examination was performed to determine body mass index (BMI) and to assess the vital signs. We also examined the lipid profile and performed echocardiography to assess left ventricular function. The characteristics of the study population are summarized in Table 1.

Preparation of blood samples and mononuclear cell isolation
We collected 40 ml peripheral blood samples from the median cubital vein following WHO guidelines on drawing blood 17 . From freshly drawn heparinized blood, we isolated peripheral blood mononuclear cells (PBMCs) using Ficoll Histopaque 1077 (Sigma-Aldrich, USA). Briefly, peripheral blood was diluted 1:1 with phosphate buffer saline (PBS) + 2% fetal bovine serum (FBS) to a total volume of 30-35 ml. It was then carefully layered into 20 ml of Ficoll Histopaque 1077 (Sigma-Aldrich, USA) in a 50 ml conical tube. Subsequently, the tube was put into a centrifuge at 300xg for 30 minutes. The PBMC layer was obtained in the form of a buffy coat layer. Using a sterile plastic pipette, the PBMCs were carefully taken and put into another 50 ml conical tube. Furthermore, PBMC was added with PBS + 2% FBS in a ratio of 1: 1, then stirred until homogeneous and centrifuged at 300xg for 7 minutes. This step was repeated with the supernatant removed, 15 ml of PBS + 2% FBS was added to the precipitate formed at the bottom of the tube and centrifuged at 300xg for 7 minutes. Finally, the supernatant was removed, and the sediment was dissolved with a basal medium. Cells were concentrated up to 5×10 6 cells/ml.

Isolation and culture of endothelial progenitor cells
To separate EPCs from PBMCs, we used standard protocols 18 . Briefly, PBMCs isolated from blood samples in a concentration of 5×10 6 cells/mL were collected in Stemline II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich, USA) supplemented with endothelial basal medium (EBM) containing 40 ng/ml of vascular endothelial growth factor (VEGF) and 15% FBS. Then PBMCs were seeded in the six-well plate with fibronectin coating. The cultures were maintained with a humidified atmosphere at 37°C and 5% carbon dioxide. Forty-eight hours after seeding, we separated the medium liquid ontaining the non-adherent cells from the adherent cells attached to the bottom of the plate. All the medium liquid containing the non-adherent cells was collected into one tube, centrifuged with a spin at 300xg for 7 minutes, and the supernatant was discarded. The precipitate formed was dissolved with basal medium and supplement with a concentration of 1×10 6 cells/ml. We confirmed the cells as EPCs through immunofluorescence tests using fluorescein isothiocyanate (FITC) mouse anti-human CD34 monoclonal antibody (cat. no. 343604; Biolegend, USA).

Treatment groups
Isolated EPCs were divided into three treatment groups and one negative control group. The treatment groups were divided into 1) captopril 1 μM, 10 μM, and 100 μM; 2) lisinopril 1 μM, 10 μM, and 100 μM; and 3) ramipril 1 μM, 10 μM, and 100 μM. The cultures were maintained with a humidified atmosphere at 37°C and 5% carbon dioxide for 48 hours. Each data point represented the mean value of quadruplicate cultures.

Migration assay
We used the Boyden chamber migration assay to measure ECP migration. Briefly, migration chambers with 8μm pore-size filters were placed in 24-well plates. Using 1 mmol/L EDTA in PBS, isolated EPCs were detached and then centrifuged at 400xg for ten minutes. EPCs were seeded in the upper chamber (5×10 5 /ml in serum-free medium), and the lower compartment of the Boyden chamber was filled with endothelial basal medium. After 24 hours of incubation at 37°C, we scraped off non-migratory cells on the upper chamber with cotton swabs. The migration chamber was put into a new basal medium and added with 500μL of trypsin + EDTA 0.5% solution. After 10 minutes of incubation, we verified using a light microscope to ensure more than 90% of adhering cells were released from the lower surface of the migration chambers. For quantification, the cells were harvested and stained with trypan blue/Giemsa. Migrated ECPs were counted using an TC20 automated cell counter (Bio-Rad, USA).

Statistical analyses
Continuous data were presented as mean ± SD. Multiple experimental group analysis of total migrated EPCs was performed using analysis of variance (ANOVA). A p-value of less than 0.05 was considered statistically significant. All statistical analysis was completed using SPSS version 25.0 for Windows.

Results
CD34 expression and migration capability of endothelial progenitor cells CD34 is a positive marker for EPCs, and CD34 expression was found in the early to mature culture of EPCs. CD34 expression was characterized by the presence of green luminescence using a fluorescence microscope, indicating the presence of EPCs, as shown in Figure 1 19 . The migration capability of EPCs was evaluated by calculating the number of cells that moved from the upper chamber to the membrane facing the lower chamber with Giemsa staining (Figure 2).

ACE inhibitors increased endothelial progenitor cells migration
The lisinopril at a dose of 100mM had the highest migration effect (p < 0.05) ( Figure 6).

Discussion
In this present study, we demonstrated that captopril, lisinopril, and ramipril therapy in EPC cultures from CAD patients was associated with improved migration of EPCs. This study showed that ACE inhibitor treatment increases EPCs migration in a dose-dependent manner. At the doses of 1 mM and 10 mM, there was no significant difference in EPCs migration between captopril and lisinopril. However, both of them exceeded the results of ramipril at the same dose. Meanwhile, lisinopril at the dose of 100 mM had a superior outcome compared to captopril and ramipril at the same dose.     II receptor antagonists (ARB) 29 . Antioxidative agents with anti-inflammatory properties, such as ginsenoside, salvianolic acids, berberine, Ginkgo biloba, resveratrol, and puerarin, also have been found to increase the number or functional activity of EPCs 30 . ACE inhibitors, which are widely used in cardiovascular therapy, such as for hypertension and congestive heart failure, may have a potential role in restoring the role of EPCs in repair, healing, and neovascularization 11,31 . Several studies have demonstrated the role of ACE inhibitors in increasing the number and function of EPCs in patients with hypertension and stable CAD 13,15 . Each of the ACE inhibitors has a different chemical functional group, which may explain the varying effects of different ACE inhibitor types in several studies, either in vitro or in vivo. Sulfhydryl-containing ACE inhibitors are known to be the most effective compared to other types of ACE inhibitors 31-34 . Captopril has one sulfhydryl group, and zofenopril has two sulfhydryl groups to coordinate the zinc ion of the active side, whereas lisinopril, ramipril, and enalapril do not have sulfhydryl groups 35-38 . Sulfhydrylcontaining ACE inhibitors can reduce oxidative stress and stimulate nitric oxide (NO) activity in human endothelial cells 39 and patients with primary hypertension 40 . In vitro studies have shown that zofenopril is more effective compared to enalapril in preventing foam cell formation and thereby slowing atherosclerosis. In addition, zofenopril can also reduce reactive oxygen species and increase NO production in the endothelium 37, [41][42][43][44] The finding that ACE inhibition therapy augmented the number of circulating EPCs in patients with CAD, and also enhanced EPCs functional activity, may provide a novel strategy to improve neovascularization and reendothelialization after ischemia, thereby providing a therapeutic concept to improve EPC numbers and functions in patients with CAD.

Conclusion
Captopril, ramipril, and lisinopril were shown to increase EPC migration in a dose-dependent manner. Low-dose (1 mM) and medium-dose (10 mM) captopril had a larger effect on ECP migration than lisinopril and ramipril. Meanwhile, high-dose lisinopril (100mM) had the highest migration effect, suggesting it may be preferable for promoting EPC migration in CAD patients.  This study explores the role of ACE inhibitors (captopril, lisinopril, and ramipril) in improving the migratory capabilities of EPCs in coronary artery disease (CAD). Overall, the research published by this author is very interesting. However, several things need to be added to this research report so that readers can understand it better. For example, in the abstract, it is necessary to add the characteristics of the patients included in this study so that the reader can understand the study's outline.

Data availability
In the research method, it is also necessary to explain whether or not the patients included in this study are already on ACE inhibitor therapy. Was the number of patients included in the study (eight patients) sufficient to conclude? What about the effect of other therapies (not ACE inhibitors) that the patient had taken before being included in the study? How to "clean up" the various confounders in the study? Some of these questions may need to be added and explained in this research report.
In addition, there are some inconsistencies in the writing of abbreviations and mistypes that need to be corrected. Here are some points related to this: "However, the efficacies of different ACE inhibitors in improving the migratory capabilities of ECPs in coronary artery disease (CAD) patients is unclear." The singular verb "is" does not appear to agree with the plural subject "patients" or "the efficacies".  The author is inconsistent in writing the abbreviation for endothelial progenitor cells.
○ Sometimes it says "EPC", but in other parts, it says "ECP".
"It is suggested that intracellular damage and impaired redox balance in EPCs due to oxidative stress are the predisposes of imbalance in vascular pathology." The phrase "are the predisposes of" maybe wordy. Consider changing to "predisposes to".
○ "Forty-eight hours after seeding, we separated the medium liquid ontaining the non-adherent cells from…….". It should be "containing".
○ "The precipitate formed was dissolved with basal medium and supplement with……". It should be "supplemented".

© 2021 Hassanpour M.
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