F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.147482.1 Systematic Review Articles The role of therapeutic MicroRNA in arteriogenesis process in limb ischemia: A systematic review

[version 1; peer review: 1 approved with reservations]

 Ismail Muhamad Taufik Conceptualization Data Curation Formal Analysis Investigation Methodology Project Administration Resources Software Supervision Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-8239-997X a 1 Anggrahini Dyah Wulan Data Curation Investigation Methodology Project Administration Writing – Original Draft Preparation Writing – Review & Editing 1 Haryana Sofia Mubarika Investigation Methodology Project Administration Writing – Original Draft Preparation Writing – Review & Editing 2 Setianto Budi Yuli Methodology Supervision Validation Writing – Original Draft Preparation Writing – Review & Editing 1 Cardiology and Vascular Medicine, Faculty of Medicine, Public Health, and Nursing, Gadjah Mada University, Yogyakarta, Special Region of Yogyakarta, 55281, Indonesia Histology and Cell Biology, Faculty of Medicine, Public Health, and Nursing, Gadjah Mada University, Yogyakarta, Special Region of Yogyakarta, 55281, Indonesia mutaufiq_is@yahoo.com

No competing interests were disclosed.

 9 5 2024 2024 13 470 29 4 2024 Copyright: © 2024 Ismail MT et al. 2024 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

Chronic limb-threatening ischemia (CLTI) is the most advanced stage of peripheral artery disease (PAD) and has poor clinical outcomes. Recently, stimulating arteriogenesis has been proposed to improve clinical outcomes. Several studies have shown that miRNAs have beneficial effects on limb ischemia related to arteriogenesis. This study aimed to review the roles of therapeutic miRNAs in the arteriogenesis of limb ischemia.

 Methods

A systematic search was conducted through July 2021 using the PubMed, Scopus, and ScienceDirect databases. Two authors independently assessed studies that investigated the role of miRNAs in the arteriogenesis of limb ischemia, both in vivo and in clinical studies.

 Results

All selected studies were in vivo studies, with a total of 36 articles and 28 types of miRNAs. miRNAs potentially regulate arteriogenesis by targeting different targets. The following miRNAs were upregulated to enhance arteriogenesis: miRNA-126-3p, -93, -675, -143-3p, -130a, -210, -146b, -21, -let-7g, -132/212, -150, and 155. Meanwhile, microRNAs needed to be downregulated, namely: miRNA-939-5p, -503, -199a-5p, -146a, -92a, -14q32 microRNA gene cluster, -15a/16, -100, -133a, -139-5p, -223, -352, -615-5p, -15b/5p, -124-3p, and 29a. MiRNA-126 was the most studied miRNA, and SPRED1 was the most common target of microRNA. However, the included studies showed high heterogeneity in terms of inducing hindlimb ischemia, the timing of administration, and the method used for evaluating arteriogenesis. Moreover, most studies presented unclear or high-risk bias.

 Conclusion

MicroRNA application in a preclinical model of hindlimb ischemia has beneficial effects on arteriogenesis. This result indicates that miRNAs might be potentially beneficial in patients with CLTI.

 Registration

The review protocol was registered with PROSPERO under registration number CRD42024484988.

 Keywords: microRNA arteriogenesis limb ischemia gene target The author(s) declared that no grants were involved in supporting this work. Introduction

Chronic limb-threatening ischemia (CLTI) is a severe form of peripheral artery disease (PAD) due to the blockage of blood vessels. 1 Its amputation and mortality rates using a conservative approach are still high at 27% and 18% at 12 months after diagnosis, respectively, 2 despite being successfully revascularized. 3 , 4 Patients who present with late-onset and severe degree of tissue damage have the highest risk of amputation. 4 Therefore, other therapeutic approaches need to be developed.

Genetic or cellular-based therapeutic approaches have been developed to increase neovascularization in the form of arteriogenesis and angiogenesis. 1 Unfortunately, angiogenesis is inadequate for replacing occluded or stenotic arteries. Furthermore, the long distance between the stenotic vessel and tissue hypoperfusion makes it more relevant to arteriogenesis than angiogenesis. 5 Therefore, patients with CLTI generally require more arteriogenesis than angiogenesis. 6

Studies on stem cell therapy for CLTI in clinical studies recently has not yet shown promising outcome. 1 It is possibly due to inability of stem cells in the ischemic condition. 6 Similar result occurred with the trials of growth factors administration in CLTI patients. 7 The expectation of increase neovascularization to compensate the ischemic limb, comes into a failure due to angiogenic signaling resistance. Therefore, the findings of miRNA signaling might be a solution for repairing the angiogenic signaling pathway or its resistance. 8

Several miRNAs can improve arteriogenesis through different signaling pathways, either by increasing or inhibiting their expression depending on a specific type of miRNA. 8 Arteriogenesis consists of two phases: an early inflammatory phase followed by an increase in blood vessel diameter, remodeling, and vessel maturation. Furthermore, several processes including proliferation of endothelial cells, smooth muscle cells, and remodeling of tunica adventitia are also essential for arteriogenesis. 9 MicroRNAs can interfere those processes through gene regulation, since several genes are needed, particularly involved in the proliferation of endothelial cells, smooth muscle cells, activation of adhesion molecules, regulation of extracellular matrix, and regulation of growth factors and their receptors. 10 Therefore, this study was aimed to review the role of therapeutic microRNAs in arteriogenesis process of limb ischemia.

 Methods

The review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO) database (registration number: CRD42024484988), however the protocol has yet to be published. We performed this systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.

Search strategy

We searched for basic and clinical studies published in the PubMed, Scopus, and ScienceDirect databases on July 25, 2021. The literature is limited to English. We use the PICO strategy to answer this research question. The following terms were used for the literature search: (peripheral arterial disease OR peripheral artery disease OR chronic limb threatening Ischemia OR critical limb ischemia OR hindlimb Ischemia OR hind-limb ischemia OR femoral artery ligation OR hindlimb) AND (microRNA OR microRNAs OR miRNAs OR “micro RNA” OR miRNA OR “micro RNAs”) AND (angiogenesis OR arteriogenesis OR neovascularization OR collateral).

 Eligibility criteria

Two authors (MT and DA) independently assessed the eligibility of the studies. The inclusion criteria were as follows: 1) Explaining the role of miRNAs in the arteriogenesis process of limb ischemia, either in vivo or clinical studies; 2) if in vivo study, inducing limb ischemia in animal models must be clearly described; 3) articles published in full-text journals; 4) Articles in English; and 5) articles assessing arteriogenesis using imaging or staining methods. The exclusion criteria were as follows: 1) the study involved only an in vitro study, 2) there was no control group in an in vivo study, and 3) the study was not conducted as primary research.

 Data extraction

Two authors independently extracted the data from the included studies (MT and DA). The extracted data included the author’s name, publication year, animal models (type of animal, strain, age, how to induce limb ischemia, concomitant conditions, and if any animal gender was also included), type of microRNA, administration method in intervention and control groups, and techniques for evaluating arteriogenesis, including time points of assessment and outcomes. Any disagreements between the authors were resolved through discussion. Owing to the high heterogeneity of the included studies, a meta-analysis could not be conducted. We developed ‘Characteristics of study’ tables to summarize and compare the data above.

 Risk of bias assessment

The risk of bias was evaluated by two authors (MT and DA) using the SYRCLE’s risk of bias tool for animal studies. Disagreements were resolved through discussion.

 Results Search results

A systematic search of several databases identified 1317 relevant papers for this study (PRISMA Flowchart for The Systematic Review). 59 A total of 130 articles remained after removing duplicate publications and screening titles and abstracts. Furthermore, 3 articles with no full text available and 91 studies were not included because there was no analysis of miRNA in arteriogenesis. Therefore, 36 articles with 28 types of miRNAs were eligible for further analysis in this systematic review. The selected articles showed substantial heterogeneity, including animal models, intervention approaches, control groups, timing of administration, and methodology used to assess arteriogenesis.

 Study characteristics

All the selected studies in Characteristics of Study ( Table 1) 59 show that strain, animal models, concomitant conditions, and techniques used to induce limb ischemia varied among studies. Almost all of the studies (n=35, 97,2%) used a rodent as a model, of which 33 studies used mice and 2 studies used rats. BALB/c and C57Bl/6 were the most commonly used mouse strains. The two strains used in this study were Fischer and Adult Sprague-Dawley rats. Furthermore, only one study used New Zealand White rabbits. Regarding concomitant conditions, diabetic mice were used in five studies and immunocompromised mice were used in two studies, 2 studies used hypercholesterolemic mice model, and 1 study used aging mice. Three studies used genetically modified mice compared to wild-type mice to determine the effect of a specific type of microRNA on arteriogenesis. Moreover, the techniques used to create hindlimb ischemia vary widely varied. 59

MicroRNAs involved in arteriogenesis.
MiRNA Expression (Therapeutic Purpose) Details Target
let-7g ↑

let-7g enhanced expression of VEGF-A and VEGFR-2 through targeting HIF-3α and TP53. Moreover, let-7g influenced the splicing factor SC35, which consequently increased the alternative splicing of VEGF-A, from VEGF-A 165b (anti-angiogenic isoform) to VEGF-A 164a (pro-angiogenic isoform). 11

HIF-3α and TP53.
15a/16 ↓

Inhibition of miR-15a/16 enhanced arteriogenesis and angiogenesis in a mouse model of limb ischemia. 12

Overexpression of miR-15a or-16 decreased Tie2 at the protein level. 13

VEGF-A and AKT3

Tie2

14q32 microRNA gene cluster ↓

Inhibition of miR-329, miR-487b, miR-494, and miR-495 led to arteriogenesis and angiogenesis. 10

-
15b/5p ↓

Regulating arteriogenesis and angiogenesis via targeting AKT3/eNOS pathway. 14

AKT3
21 ↑

Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) contained miR-21 which repaired neovascularization by increasing the activity of HIF-1α by targeting CHIP. 15

CHIP
29a ↓

Downregulation of MiR29a and upregulation of ADAM12 in ischemia with hyperglycemia state was impaired. Knocking down miR29a was adequate for fixing ADAM12 upregulation in ischemia with a hyperglycemia state. Hence, improving perfusion and angiogenesis even when vascular endothelial growth factor signaling was impaired. 16

ADAM12
92a ↓

Inhibition of miR-92a contributed to enhancing angiogenesis and arteriogenesis. 17

mRNA encoding integrin subunit alpha5
93 ↑

miR-93 impeded interferon regulatory factor-9 to reduce the production of IRG1–itaconic acid, therefore it stimulated M2-like polarization in ischemic muscle. Consequently, it increased angiogenesis, arteriogenesis, and blood perfusion. 18 Another study showed that curcumin improved angiogenesis and arteriogenesis by promoting the expression of miR-93. 19

miR-93 increased arteriogenesis and angiogenesis by regulating the expression of multiple genes that coordinated the pathway of apoptosis and cell proliferation. 20

miR-93 inhibited CDKN1A expression at the mRNA and protein expression levels. Hence, promoting arteriogenesis and angiogenesis. 21

interferon regulatory factor-9 (IRF9) multiple genes

Cyclin dependent kinase inhibitor 1A (CDKN1A) gene

100 ↓ mTOR expression was down-regulated in endothelial cells and vascular smooth muscle cells by overexpression of miR-100, hence it decreased cellular proliferation. 22 mTOR
124-3p ↓ Overexpression of miR-124-3p reduced STAT3 protein expression levels. Hence, mir 124-3p impaired perfusion and angiogenesis. 23 STAT3
126 ↑

CD34Exo contained high level of miR-126-3p which enhanced arteriogenesis and angiogenesis by suppressing the expression of SPRED1. Hence, modulating severals genes expression (VEGF, angiopoietin 1, angiopoietin 2, MMP9, thrombospondin 1, etc). 24

The sustained release of miR-126 loaded with poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) enhanced perfusion and angiogenesis by suppressing the expression of SPRED1. 25 Another study showed that miR-126 loaded with Bubble liposomes had the same result. 26

Adipose-Derived Stem Cell Therapeutic Factor Concentrate had a high level of miR-126, which increased perfusion and angiogenesis. 27

MiR-126-3p repaired perfusion and angiogenesis by suppressing the expression of SPRED1 and PIK3R2. 28

SPRED1, PIK3R2
130a ↑ Overexpression of miR-130a decreased the expression of MEOX2 and HOXA5 (antiangiogenic genes). Those genes were up-regulated in ischemic muscles of aging mice. Hence, miR-130a improved perfusion and angiogenesis in ischemia related to aging. 29 MEOX2 and HOXA5
132/212 ↑ MiR-132/212 enhanced the Ras-Mitogen-activated protein kinase MAPK signaling pathway through direct inhibition of Rasa1 and Spred1. The miR-132/212 cluster could enhance arteriogenesis by regulating the Ras-MAPK signaling pathway via direct targeting of the inhibitors Rasa1 and Spred1. 30 RASA1, SPRY1, SPRED1
133a ↓ In models of occlusive artery disease with diabetes, overexpression of miR-133a inhibited NO- CGMP angiogenic pathway via targeting GCH1. 31 GCH1
139-5p ↓ The diabetic condition led to overexpression of miR-139-5p in endothelial cells, thereby it inhibited the c-jun pathway and downregulated the expression of VEGF and PDGF-β. Hence, contributing in the reduction of ECFC migration, proliferation, and tube formation, which consequently diminished endothelial cell survival. 32 c-jun pathway
143-3p ↑ In vivo study showed that increased expression of microRNA 143-3p in response to fluid shear stress could play a role in the reorganization of the extracellular matrix which was important in the process of vascular remodeling through inhibition of V-á2 collagen biosynthesis. 33 V-á2 collagen
146b ↑ miR-146b promoted angiogenesis and arteriogenesis through targets TRAF6 and impeded oxLDL-induced TRAF6-TNFa inflammatory pathway. Therefore, decreased miR-146b expression led to impair arteriogenesis and angiogenesis in hypercholesterolemic conditions. 34 TRAF6
146a ↓ Anti microRNA 146a in vivo with femoral artery ligation model showed a 22% increase in collateral artery diameter compared to controls. However, there was no beneficial effect in the process of angiogenesis or muscle regeneration. These effects occurred by upregulation of pro-inflammatory (i.e., NFκB) endothelial cell activation. In addition, arteriogenesis was also possibly mediated via pericollateral Ly6C hi monocyte proliferation and/or migration. 35 NFκB
150 ↑ Hypercholesterolemia and exposure to oxidized LDL were associated with increased expression of SRC kinase signaling inhibitor 1 and decreased Src activity. MiR-150 mimics could reduce the expression of SRC kinase signaling inhibitor 1 and restored Src activity and Akt (protein kinase B) activity. Hypercholesterolemia was associated with decreased miR-150 expression, impaired Src signaling pathway, and inefficient neovascularization in response to ischemia. Administration of miR-150 using miR mimics might be a new therapeutic strategy to enhance ischemia-induced neovascularization in atherosclerotic conditions. 36 SRCIN-1
155 ↑ MicroRNA 155 had the opposite effect as proarteriogenic and antiangiogenic. Its expression in endothelial cells and bone marrow–derived cells was important for arteriogenesis in response to ischemia. MiRNA-155 directly suppressed SOCS1 in monocytes or macrophages, which was a negative feedback regulator of JAK-STAT signaling. Hence, miRNA-155 enhanced monocyte/macrophage rolling, adhesion, migration, and the production of pro-arteriogenic cytokine. 37 SOCS1
199a-5p ↓ miR-199a-5p suppressed gene expression of pro-arteriogenic (IKKb, Cav1) and inhibited pericollateral macrophage recruitment. In addition, overexpression of miR-199a reduced collateral artery growth by ±25% (±2.4-fold decrease in conductance) and inhibition of miR-199a increased arteriogenesis by 36% (>3.4-fold conductance increase). 38 IKKb, Cav1
210 ↑ miR-210 diminished ROS in endothelial cells and ischemic muscle tissue through downregulated P4HB protein levels in EC under a hypoxic environment. 39 P4HB
223 ↓ miR-223 deterred endothelial cell proliferation by targeting β1 integrin. 40 β1 integrin
352 ↓ Anti miR-352 increased the expression of insulin-like growth factor II receptor (IGF2R), which may contribute in the complex pathway for arterial growth. Furthermore, infusion of antagomir miR-352 could enhance collateral vessel growth. 41 IGF2R
503 ↓ Inhibition of miR-503 in ischemic adductor muscle ameliorated post-ischemic blood flow restoration in a mouse model which involved the CDC25A target. 42 CDC25A
615-5p ↓ miR-615-5p impeded the VEGF-AKT/eNOS signaling pathway in endothelial cells by directly reduced the level of Ras-associating domain family member 2 (RASSF2) and insulin-like growth factor 2 (IGF2). 43 RASSF-2 dan IGF-2
675 ↑ Incorporating miR-675 into exosomes encapsulated in silk fibroin hydrogel promoted blood perfusion by inhibiting the TGF-β1/p21 signaling pathway. 44 TGF-β1/p21 pathway
939-5p ↓ miR-939-5p in exosomes from patients with myocardial ischemia (isc-Exo) inhibited the expression and activity of iNOS, therefore it reduced NO production in endothel. Consequently, it impaired angiogenesis and arteriogenesis. 45 iNOS

The therapeutic and timing of administration varied among the included studies. Intramuscular injection was the most commonly used (n=21), followed by tail vein (n=5), intra-arterial (n=2), oral (n=1), retro-orbital (n=1), and not clearly explained (n=1). Meanwhile, there were 3 studies that did not use therapeutic agents (owing to genetic modification). Regarding the timing of administration, 11 studies performed repeated administration at different time points: 10 studies administered agent immediately after ligation, 2 studies administered drug one day before ligation, one study delivered agent 30 min before ligation, one study administered agent on day 1 after ligation, one study gave agent on day 7 after ligation. Unfortunately, 7 studies did not clearly state when the agent was administered.

The methods used to evaluate arteriogenesis also varied among the included studies. Imaging techniques were the most commonly used among the studies, from which 31 studies used laser Doppler perfusion imaging, one study used high-resolution laser color Doppler imaging, one study used laser speckle perfusion imaging, one study used contrast-enhanced ultrasound (CEU), one study used periCam perfusion speckle imager, two studies used whole-mount vascular casting, one study used postmortem vascular casting, and one study used postmortem angiogram. Staining methods were also used, and α-smooth muscle actin (α-SMA) staining was the most commonly used method, either by immunohistochemistry or immunofluorescence (n=12). Some of the techniques were combined, of which the most common combination was laser Doppler perfusion imaging and α-smooth muscle actin (α-SMA) staining (n=11). The duration of outcome observation varied among the studies from 7 days to 48 days. The observation time points varied during the observation period.

Table 1 shows that, depending on the specific type of miRNA, some miRNAs need to be upregulated or downregulated to enhance arteriogenesis and vice versa. Some of microRNAs need to be upregulated, namely, miRNA-126-3p, -93, -675, -143-3p, -130a, -210, -146b, -21, -let-7g, -132/212, -150, and 155. In contrast, several microRNAs needed to be downregulated, namely: miRNA-939-5p, -503, -199a-5p, -146a, -92a, -14q32 microRNA gene cluster, -15a/16, -100, -133a, -139-5p, -223, -352, -615-5p, -15b/5p, -124-3p, and 29a. Several microRNAs were loaded when administered to hindlimb ischemia models, namely microRNA-126 was naturally contained in CD34Exo and adipose-derived stem cell therapeutic factor concentrate. MicroRNA-939-5p was abundant in exosomes from patients with myocardial ischemia (isc-Exo). Furthermore, microRNA-21 is abundant in umbilical cord blood-derived mesenchymal stem cells (UCBMSCs). Finally, curcumin contained a high level of microRNA-93. Table 1 also shows that miR-126 and miR-93 are the most commonly assessed miRNAs in hindlimb ischemia. Regarding the therapeutic target of microRNAs, SPRED1 was the most common, namely miR-126 and -132/212.

 Risk of bias assessment

SYRCLE’s risk of bias tool for animal studies was used to assess the risk of bias, and the outcomes are shown in Risk of Bias Assessment. 59 In most studies, the risk of bias fell into two categories: “unclear” or “high.” In particular, the majority of studies did not specify the adequacy of the generation and application of the allocation sequence, the concealment of allocation to different groups during the experiment, or the random housing or selection of animals for outcome assessment, whether investigators were blinded to the interventions received by each animal, or whether the outcome assessor was blinded.

 Discussion

This study aimed to systematically search and review the literature on the therapeutic potential of microRNAs in limb ischemia. To date, only in vivo studies have evaluated the potential role of miRNAs in arteriogenesis. Depending on the specific type, the expression could lead to enhanced or decreased arteriogenesis. The following miRNAs were upregulated to enhance arteriogenesis: miRNA-126-3p, -93, -675, -143-3p, -130a, -210, -146b, -21, -let-7g, -132/212, -150, and 155. Meanwhile, microRNAs needed to be downregulated, namely: miRNA-939-5p, -503, -199a-5p, -146a, -92a, -14q32 microRNA gene cluster, -15a/16, -100, -133a, -139-5p, -223, -352, -615-5p, -15b/5p, -124-3p, and 29a. These miRNAs have different target sites in arteriogenesis, with miRNA-126 being the most studied ( Figure 1). The high heterogeneity among selected studies in terms of microRNA used, techniques for inducing limb ischemia, administration methods in intervention and control groups, and techniques for evaluating arteriogenesis as well as time-points of outcome assessment, hindered the performance of a meta-analysis.

Proposed mechanism of several microRNAs in arteriogenesis process.

In this study, mice were the most commonly used limb ischemia models because of their practical aspects and their similarity to humans in terms of neovascularization patterns and genetic aspect. 46 – 48 Moreover the availability of several transgenic mice might be the reason for preferring the use of mice, as shown in these studies. Unfortunately, only two studies were performed in immunocompromised mice, since miRNAs might stimulate immune responses, including macrophages and T cells, and thus might affect the outcome of arteriogenesis. 46 , 49 The technical approach for inducing limb ischemia was also widely varied in this study, which might affect the arteriogenesis outcome. Double ligation of the iliac and femoral arteries causes severe ischemia rather than single ligation. Moreover, the ligation approach is better than electrocoagulation since recoil will not occur. 46 Concurrent ligation and excision methods will increase the severity level of ischemia. 50 Regarding to sex animal models, most of the selected studies used male due to hinder the effects of estrogen on vascular regrowth. 51 Unfortunately, several studies did not clearly declare animal sex, and two studies in rabbits and mice used female sex.

Imaging techniques can assess arteriogenesis anatomically or functionally. 52 In this study, most studies used a functional approach such as laser doppler perfusion imaging which assesses restoration of tissue perfusion, equivalent to the sign of effective arteriogenesis. 52 The anatomical approaches used in this study were whole mount vascular casting, postmortem vascular casting, and postmortem angiogram. A combination of anatomical and functional approaches can provide more insight into both the visual and quantitative extent of circulation formation and its functional effectiveness. 52 In this study, miR-199a-5p and miR-146 were assessed using both methods. In addition to imaging methods, histological staining has also been used to assess arteriogenesis. 53 Unfortunately, none of these studies used a combination of anatomical, functional imaging, and histological staining. Such a combination may yield more reliable results.

In general, the arteriogenesis process requires endothelial and smooth muscle cell proliferation, in which growth factor is crucial to proliferate smooth muscle cells. In the later process, adventitia remodeling through activation and proliferation of fibroblast caused enlargement of lumen vessels. 9 Nevertheless, the exact molecular mechanisms involved in all multifactorial arteriogenesis process is still not fully understood. 54 However, recent evidence showed that microRNA is involved in signaling pathways related to arteriogenesis. 8 As shown in this study, microRNAs affect several pathways related to arteriogenesis, such as Ras-MAPK, TGF-β1/p21, AKT3/eNOS, and JAK-STAT signaling pathway. Table 1 also shows that miRNAs can lead to arteriogenesis by directly targeting several genes and proteins.

VEGF-A is essential for endothelial proliferation by binding to VEGFR-2 to stimulate intracellular signaling process. 55 That evidence is supported in this study, in which promoting mir126 and letting 7 g as well as inhibiting mir 15a/16 activate VEGF, leading to arteriogenesis. Another study showed that an increase in miR-139-5p expression in diabetic mice led to downregulation of VEGF, consequently decreasing arteriogenesis. Moreover, miR-223, 100, and 93 also affect cell proliferation by binding to their respective targets, ultimately affecting arteriogenesis.

Macrophage recruitment is essential for arteriogenesis. Activated macrophages produce TNF-α, growth factor, and chemokines and recruit more monocytes, thereby promoting arteriogenesis. 9 Those results were supported by the results of the present study, in which miRNAs 199a-5p, 146a, and 155 had a role in macrophage recruitment. Moreover, macrophage polarization to an anti-inflammatory macrophage type 2 in ischemic muscle, shown by miR-93, is essential during post-ischemic vascular injury and repair, since M2 macrophages are pro-angiogenic. 56

In this study, the delivery approach varied; however, most of them used intramuscular injections. However, all delivery approaches, including tail vein, intravenous injection, oral, retro-orbital, and intra-arterial, showed positive results related to arteriogenesis. These results suggested that miRNAs have systemic effects. Depending on the specific type of microRNA, all studies that used concomitant conditions such as diabetic and hypercholesterolemic mice showed positive results in arteriogenesis. These results might suitably explain the use of microRNAs in humans, since CLTI is strongly associated with cardiovascular risk factors, such as diabetes mellitus and dyslipidemia. 57 Aging mice might also be analogous to patients with CLTI due to the increasing number of CLTI in the older population. 58

The application of SYRCLE’s risk of bias tool to assess animal studies revealed a prevalent “unclear” or “high” risk of bias across the majority of cases. These studies exhibited deficiencies in essential aspects, such as the generation and application of allocation sequences, allocation concealment, randomization of housing, selection for outcome assessment, and blinding of investigators and outcome assessors. Therefore, these results should be cautiously interpreted.

Our systematic review had several limitations. First, we reviewed only studies published in English. Second, all research conducted in animal models had high heterogeneity related to inducing hindlimb ischemia, intervention, and outcome assessment methods. Therefore, clinical studies in humans are required to verify the effect of miRNA modulation using mimic and/or inhibitor miRNAs in patients with chronic limb-threatening ischemia.

 Conclusion

Taken together, our review identifies several miRNAs in preclinical models that show potential for therapeutic purposes to enhance arteriogenesis in patients with chronic limb-threatening ischemia. Hence, a study involving patients with chronic limb-threatening ischemia is required.

 Data availability Underlying data

All data underlying the results are available as part of the article and no additional source data are required.

 Extended data

Figshare: The Role of Therapeutic MicroRNA in Arteriogenesis Process in Limb Ischemia: A Systematic Review. DOI: https://doi.org/10.6084/m9.figshare.25036163.v3. 59

This project contains the following extended data:

PRISMA Flowchart for The Systematic Review

Table 1. Characteristics of Study

Risk of Bias Assessment

Completed PRISMA checklist

Data are available under the terms of the Creative Commons Zero CC0.

 Acknowledgements

We thank Hariadi, Yohanes William, Tinanda Tarigan, Afif Ramadhan, and Jessica Eve from Department of Cardiology and Vascular Medicine, Faculty of Medicine, Public Health and Nursing Gadjah Mada University, Sleman, Indonesia for their technical support, all of whom approved their inclusion.

 References Conte MS Bradbury AW Kolh P : Global vascular guidelines on the management of chronic limb-threatening ischemia. Eur. J. Vasc. Endovasc. Surg. 2019;58(1):S1–S109.e33. 31182334 10.1016/j.ejvs.2019.05.006 PMC8369495 Reijen NS van Hensing T Santema TKB : Outcomes of conservative treatment in patients with chronic limb threatening ischaemia: A systematic review and meta-analysis. Eur. J. Vasc. Endovasc. Surg. 2021;62(2):214–224. 33674157 10.1016/j.ejvs.2021.01.005 Fridh EB Andersson M Thuresson M : Amputation rates, mortality, and pre-operative comorbidities in patients revascularised for intermittent claudication or critical limb ischaemia: A population based study. Eur. J. Vasc. Endovasc. Surg. 2017;54(4):480–486. 10.1016/j.ejvs.2017.07.005 Reinecke H Unrath M Freisinger E : Peripheral arterial disease and critical limb ischaemia: Still poor outcomes and lack of guideline adherence. Eur. Heart J. 2015;36(15):932–938. 25650396 10.1093/eurheartj/ehv006 Scholz D Cai WJ Schaper W : Arteriogenesis, a new concept of vascular adaptation in occlusive disease. Angiogenesis. 2001;4(4):247–257. 12197469 10.1023/a:1016094004084 Caicedo D Devesa P Arce VM : Chronic limb-threatening ischemia could benefit from growth hormone therapy for wound healing and limb salvage. Ther. Adv. Cardiovasc. Dis. 2018;12(2):53–72. 29271292 10.1177/1753944717745494 PMC5772430 Iyer SR Annex BH : Therapeutic angiogenesis for peripheral artery disease: Lessons learned in translational science. JACC Basic Transl. Sci. 2017;2(5):503–512. 29430558 10.1016/j.jacbts.2017.07.012 PMC5802410 Pérez-Cremades D Cheng HS Feinberg MW : Noncoding RNAs in critical limb ischemia. Arterioscler. Thromb. Vasc. Biol. 2019;40:523–533. 31893949 10.1161/ATVBAHA.119.312860 PMC7047615 Hutchings G Kruszyna Ł Nawrocki MJ : Molecular mechanisms associated with ros-dependent angiogenesis in lower extremity artery disease. Antioxidants. 2021;10(5). 34066926 10.3390/antiox10050735 PMC8148529 Welten SMJ Bastiaansen AJNM Jong RCM de : Inhibition of 14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 increases neovascularization and blood flow recovery after ischemia. Circ. Res. 2014;115(8):696–708. 25085941 10.1161/CIRCRESAHA.114.304747 Hsu PY Hsi E Wang TM : MicroRNA let-7g possesses a therapeutic potential for peripheral artery disease. J. Cell. Mol. Med. 2017;21(3):519–529. 27696675 10.1111/jcmm.12997 PMC5323674 Spinetti G Fortunato O Caporali A : MicroRNA-15a and microRNA-16 impair human circulating proangiogenic cell functions and are increased in the proangiogenic cells and serum of patients with critical limb ischemia. Circ. Res. 2013;112(2):335–346. 23233752 10.1161/CIRCRESAHA.111.300418 PMC3616367 Besnier M Shantikumar S Anwar M : miR-15a/-16 inhibit angiogenesis by targeting the tie2 coding sequence: Therapeutic potential of a miR-15a/16 decoy system in limb ischemia. Mol. Ther. Nucleic Acids. 2019;17:49–62. 31220779 10.1016/j.omtn.2019.05.002 PMC6586592 Zhu LP Zhou JP Zhang JX : MiR-15b-5p regulates collateral artery formation by targeting AKT3 (Protein Kinase B-3). Arterioscler. Thromb. Vasc. Biol. 2017;37(5):957–968. 28254819 10.1161/ATVBAHA.116.308905 Zhou Y Zhu Y Zhang L : Human stem cells overexpressing MIR-21 promote angiogenesis in critical limb ischemia by targeting CHIP to enhance HIF-1α activity. Stem Cells. 2016;34(4):924–934. 26841045 10.1002/stem.2321 Chen L Okeke E Ayalew D : Modulation of miR29a improves impaired post-ischemic angiogenesis in hyperglycemia. Exp. Biol. Med. 2017;242(14):1432–1443. 28637396 10.1177/1535370217716424 PMC5544167 Bonauer A Carmona G Iwasaki M : MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science. 2009;324(5935):1710–1713. 19460962 10.1126/science.1174381 Ganta VC Choi MH Kutateladze A : A microRNA93-interferon regulatory factor-9-immunoresponsive gene-1-itaconic acid pathway modulates M2-like macrophage polarization to revascularize ischemic muscle. Circulation. 2017;135(24):2403–2425. 28356443 10.1161/CIRCULATIONAHA.116.025490 PMC5503157 Zhang J Wang Q Rao G : Curcumin improves perfusion recovery in experimental peripheral arterial disease by upregulating microRNA-93 expression. Exp. Ther. Med. 2019;17(1):798–802. 30651865 10.3892/etm.2018.7000 PMC6307414 Hazarika S Farber CR Dokun AO : MicroRNA-93 controls perfusion recovery after hindlimb ischemia by modulating expression of multiple genes in the cell cycle pathway. Circulation. 2013;127(17):1818–1828. 23559675 10.1161/CIRCULATIONAHA.112.000860 PMC3856290 Shu X Mao Y Li Z : MicroRNA-93 regulates angiogenesis in peripheral arterial disease by targeting CDKN1A. Mol. Med. Rep. 2019;19(6):5195–5202. 31059098 10.3892/mmr.2019.10196 PMC6522868 Grundmann S Hans FP Kinniry S : MicroRNA-100 regulates neovascularization by suppression of mammalian target of rapamycin in endothelial and vascular smooth muscle cells. Circulation. 2011;123(9):999–1009. 21339483 10.1161/CIRCULATIONAHA.110.000323 Shi Y Xu X Luan P : miR-124-3p regulates angiogenesis in peripheral arterial disease by targeting STAT3. Mol. Med. Rep. 2020;22(6):4890–4898. 33174610 10.3892/mmr.2020.11538 PMC7646933 Mathiyalagan P Liang Y Kim D : Angiogenic mechanisms of human CD34+ stem cell exosomes in the repair of ischemic hindlimb. Circ. Res. 2017;120(9):1466–1476. 28298297 10.1161/CIRCRESAHA.116.310557 PMC5420547 Tsumaru S Masumoto H Minakata K : Therapeutic angiogenesis by local sustained release of microRNA-126 using poly lactic-co-glycolic acid nanoparticles in murine hindlimb ischemia. J. Vasc. Surg. 2018;68(4):1209–1215. 29242072 10.1016/j.jvs.2017.08.097 Endo-Takahashi Y Negishi Y Nakamura A : Systemic delivery of miR-126 by miRNA-loaded Bubble liposomes for the treatment of hindlimb ischemia. Sci. Rep. 2014;4:3883. 24457599 10.1038/srep03883 PMC3900923 Procházka V Jurčíková J Vítková K : The role of miR-126 in critical limb ischemia treatment using adipose-derived stem cell therapeutic factor concentrate and extracellular matrix microparticles. Med. Sci. Monit. 2018;24:511–522. 29371587 10.12659/MSM.905442 PMC5795917 Cao WJ Rosenblat JD Roth NC : Therapeutic angiogenesis by ultrasound-mediated microRNA-126-3p delivery. Arterioscler. Thromb. Vasc. Biol. 2015;35(11):2401–2411. 26381870 10.1161/ATVBAHA.115.306506 Dhahri W Dussault S Légaré É : Reduced expression of microRNA-130a promotes endothelial cell senescence and age-dependent impairment of neovascularization. Aging (Albany NY). 2020;12(11):10180–10193. 32457253 10.18632/aging.103340 PMC7346016 Lei Z Mil A van Brandt MM : MicroRNA-132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras-MAPK pathway. J. Cell. Mol. Med. 2015;19(8):1994–2005. 25945589 10.1111/jcmm.12586 PMC4549050 Chen L Liu C Sun D : MicroRNA-133a impairs perfusion recovery after hindlimb ischemia in diabetic mice. Biosci. Rep. 2018;38(4). 29789398 10.1042/BSR20180346 PMC6028757 Luo YF Wan XX Zhao LL : MicroRNA-139-5p upregulation is associated with diabetic endothelial cell dysfunction by targeting c-jun. Aging (Albany NY). 2021;13(1):1186–1211. 33293476 10.18632/aging.202257 PMC7835005 Troidl K Hammerschick T Albarran-Juarez J : Shear stress-induced miR-143-3p in collateral arteries contributes to outward vessel growth by targeting collagen V-α2. Arterioscler. Thromb. Vasc. Biol. 2020;40(5):e126–e137. 32188276 10.1161/ATVBAHA.120.313316 Desjarlais M Dussault S Rivard F : Forced expression of microRNA-146b reduces TRAF6-dependent inflammation and improves ischemia-induced neovascularization in hypercholesterolemic conditions. Atherosclerosis. 2019;289:73–84. 31479774 10.1016/j.atherosclerosis.2019.08.010 Heuslein JL McDonnell SP Song J : MicroRNA-146a regulates perfusion recovery in response to arterial occlusion via arteriogenesis. Front. Bioeng. Biotechnol. 2018;6:1. 29404323 10.3389/fbioe.2018.00001 PMC5786509 Desjarlais M Dussault S Dhahri W : MicroRNA-150 modulates ischemia-induced neovascularization in atherosclerotic conditions. Arterioscler. Thromb. Vasc. Biol. 2017;37(5):900–908. 28254813 10.1161/ATVBAHA.117.309189 Pankratz F Bemtgen X Zeiser R : MicroRNA-155 exerts cell-specific antiangiogenic but proarteriogenic effects during adaptive neovascularization. Circulation. 2015;131(18):1575–1589. 25850724 10.1161/CIRCULATIONAHA.114.014579 Heuslein JL Gorick CM McDonnell SP : Exposure of endothelium to biomimetic flow waveforms yields identification of miR-199a-5p as a potent regulator of arteriogenesis. Mol. Ther. Nucleic Acids. 2018;12:829–844. 30153567 10.1016/j.omtn.2018.08.001 PMC6118158 Zhang J Rao G Qiu J : MicroRNA-210 improves perfusion recovery following hindlimb ischemia via suppressing reactive oxygen species. Exp. Ther. Med. 2020;20(6):231–236. 33149789 10.3892/etm.2020.9366 PMC7604736 Shi L Fisslthaler B Zippel N : MicroRNA-223 antagonizes angiogenesis by targeting β1 integrin and preventing growth factor signaling in endothelial cells. Circ. Res. 2013;113(12):1320–1330. 24044949 10.1161/CIRCRESAHA.113.301824 Guan Y Cai B Wu X : MicroRNA-352 regulates collateral vessel growth induced by elevated fluid shear stress in the rat hind limb. Sci. Rep. 2017;7(1):6643. 28751690 10.1038/s41598-017-06910-9 PMC5532297 Caporali A Meloni M Völlenkle C : Deregulation of microRNA-503 contributes to diabetes mellitus-induced impairment of endothelial function and reparative angiogenesis after limb ischemia. Circulation. 2011 January 25;123(3):282–291. 21220732 10.1161/CIRCULATIONAHA.110.952325 Icli B Wu W Ozdemir D : MicroRNA-615-5p regulates angiogenesis and tissue repair by targeting Akt/eNOS (protein kinase B/endothelial nitric oxide synthase) signaling in endothelial cells. Arterioscler. Thromb. Vasc. Biol. 2019;39(7):1458–1474. 31092013 10.1161/ATVBAHA.119.312726 PMC6594892 Han C Zhou J Liu B : Delivery of miR-675 by stem cell-derived exosomes encapsulated in silk fibroin hydrogel prevents aging-induced vascular dysfunction in mouse hindlimb. Mater. Sci. Eng. C. 2019;99:322–332. 30889706 10.1016/j.msec.2019.01.122 Li H Liao Y Gao L : Coronary serum exosomes derived from patients with myocardial ischemia regulate angiogenesis through the miR-939-mediated nitric oxide signaling pathway. Theranostics. 2018;8(8):2079–2093. 29721064 10.7150/thno.21895 PMC5928872 Aref Z Vries MR de Quax PHA : Variations in surgical procedures for inducing hind limb ischemia in mice and the impact of these variations on neovascularization assessment. Int. J. Mol. Sci. 2019;20(15):3704. 31362356 10.3390/ijms20153704 PMC6696155 Weel V van Deckers MML Grimbergen JM : Vascular endothelial growth factor overexpression in ischemic skeletal muscle enhances myoglobin expression in vivo. Circ. Res. 2004;95(1):58–66. 15155530 10.1161/01.RES.0000133247.69803.c3 Vandamme TF : Use of rodents as models of human diseases. J. Pharm. Bioallied Sci. 2014;6(1):2–9. 24459397 10.4103/0975-7406.124301 PMC3895289 Lee SWL Paoletti C Campisi M : MicroRNA delivery through nanoparticles. J. Control. Release. 2019;313:80–95. 31622695 10.1016/j.jconrel.2019.10.007 PMC6900258 Lotfi S Patel AS Mattock K : Towards a more relevant hind limb model of muscle ischaemia. Atherosclerosis. 2013;227(1):1–8. 23177969 10.1016/j.atherosclerosis.2012.10.060 Barnabas O Wang H Gao XM : Role of estrogen in angiogenesis in cardiovascular diseases. J. Geriatr. Cardiol. 2013;10(4):377–382. 24454332 10.3969/j.issn.1671-5411.2013.04.008 PMC3888921 Simons M : Chapter 14. Assessment of arteriogenesis. Methods Enzymol. 2008;445(08):331–342. 19022066 10.1016/S0076-6879(08)03014-0 Limbourg A Korff T Napp LC : Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Nat. Protoc. 2009;4(12):1737–1748. 19893509 10.1038/nprot.2009.185 Deindl E Quax PHA : Arteriogenesis and therapeutic angiogenesis in its multiple aspects. Cells. 2020;9(6). 32531915 10.3390/cells9061439 PMC7349222 Simons M Eichmann A : Molecular controls of arterial morphogenesis. Circ. Res. 2015;116(10):1712–1724. 25953926 10.1161/CIRCRESAHA.116.302953 PMC4509635 Tidball JG : Regulation of muscle growth and regeneration by the immune system. Nat. Rev. Immunol. 2017 March;17(3):165–178. 28163303 10.1038/nri.2016.150 PMC5452982 Davies MG : Criticial limb ischemia: epidemiology. Methodist Debakey Cardiovasc. J. 2012;8(4):10–14. 23342182 10.14797/mdcj-8-4-10 PMC3549644 Hiatt WR Fowkes FGR Heizer G : Ticagrelor versus clopidogrel in symptomatic peripheral artery disease. N. Engl. J. Med. 2017;376(1):32–40. 27959717 10.1056/nejmoa1611688 Ismail MT Anggrahini DA Haryana SM Setianto BY : Data from: The role of therapeutic microRNA in arteriogenesis process in limb ischemia: A systematic review. figshare. 2024. 10.6084/m9.figshare.25036163.v3 10.5256/f1000research.161682.r326630 Reviewer response for version 1 Annex Brian H 1 Referee https://orcid.org/0000-0001-6538-8243 Medical College of Georgia at Augusta University, Augusta, Georgia, USA

Competing interests: I am founder of start-up company focusing on miR-93 for PAD.  I have founder shares in this start-up company.

 29 10 2024 Copyright: © 2024 Annex BH 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The manuscript by Asmail et al. is a systematic/literature review of micro-RNA reported to be related to peripheral arterial disease.  In general, the limitations of such reviews are well known but the benefits are in providing a single go-to source of information.  Before this reviewer lists a series of relatively minor comments/areas that require clarification, one request for additional (new) data will be listed.

1) Often in a study that focuses on a microRNA, other microRNAs are also examined.  If a publication focused on miR-A (letter intentionally used to avoid confusion) were other miRs that ultimately ended in this report also studied?  For example, if in the study of miR-A, miR-B and miR-C (where miR-B and miR-C ultimately made this report) were also found to differentially regulated, that would provide a validation.  If several reports of course even better.  It of course could turn out that study of the regulation miR-B in teh report on miR-A could be different.  That would be valuable as well.  Likely, in many, this may not be possible but this could expand the value of the review.

More minor comments:

1) Introduction, paragraph 2, last sentence.  Given that no agent has definitely driven angiogiogenesis in CLTI, this sentence may not be completely accurate.   To make the statement one would need an agent that promoted angiogenesis, not arteriogenesis, and had no therapeuitc effect.

2) Introduction, paragraph 3, miRs do not signal unless the authors are referring to miR-protein interactions.

3) Methods, when the two "selectors" disagreed initially on inclusion -- how often was the agreement for and how often against inclusion.

4) Curcumin was mentioned.  Was this referring to the dietary supplement?  Would a miR be stable through the GI track?

Are the rationale for, and objectives of, the Systematic Review clearly stated?

Partly

Is the statistical analysis and its interpretation appropriate?

Partly

If this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.)

No

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

Yes

Are the conclusions drawn adequately supported by the results presented in the review?

Partly

Reviewer Expertise:

Human and experimental peripheral arterial disease including but not limited to non-coding RNAs.

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.

Ismail Muhamad Taufik Cardiology and Vascular, Gadjah Mada University, Yogyakarta, Special Region of Yogyakarta, Indonesia

Competing interests: We confirm that there are no competing interests that could be construed to influence our assessment of the article’s or the peer review report’s validity or significance.

 11 11 2024

Dear Brian H Annex, thank you for your time and insightful comments. In our study, of the 36 studies reviewed, 35 directly employed specific miRs or agents containing/enriched with/affecting specific miRs, and therefore did not assess other miRs. One study by Li did examine additional miRs using microarray technology; however, only the miRs with a significant role in arteriogenesis were reported. Some studies validate each other due to shared regulatory pathways, such as with miR-93 and miR-126. No studies presented conflicting findings.

Here is the response for addressing minor comments:

1. Our aim was to emphasize the general importance of arteriogenesis over angiogenesis in CLTI, as highlighted by Caicedo et al. According to the 2019 ESVS guidelines on CLTI, agents such as stem cells have been shown in clinical trials to enhance angiogenesis, however, they have yet to yield clinical benefits in terms of reducing amputation rates or mortality. This finding underscores the need to prioritize arteriogenesis rather than angiogenesis in this context.

2. We sincerely appreciate your insightful comments and suggestions for revising the paper. We have revised the sentence to clarify the role of miRNAs in influencing signaling pathways rather than directly "signaling." This revision aims to reflect the regulatory role of miRNAs in modulating pathway activity without implying direct signaling.

3. Thank you for your question. In cases where the two reviewers (MT and DA) initially disagreed on inclusion, an additional review by senior reviewers (BY and SM) was conducted to reach a consensus. In 80% of the journals, both reviewers shared the same inclusion criteria, while in 20% of cases, differing opinions required a final decision from the senior reviewers.

4. Based on the article, curcumin is categorized as a dietary supplement because it is a naturally occurring compound derived from turmeric and was administered orally, similar to the typical consumption of dietary supplements. In this study, curcumin is used to investigate its therapeutic effects, showing that while curcumin does not contain miRNAs necessary for arteriogenesis, it modulates the expression of endogenous miRNAs to impact molecular pathways, specifically targeting miRNA pathways within cells. I will incorporate these points in revising the manuscript. It is well-established that many naked miRNAs degrade in the gastrointestinal (GI) tract due to exposure to enzymes, stomach acidity, and intestinal nucleases.