Skip to main content
NCBI home page
Cell Transplantation logoLink to Cell Transplantation
. 2020 Oct 7;29:0963689720963936. doi: 10.1177/0963689720963936

Clarification of the Role of miR-9 in the Angiogenesis, Migration, and Autophagy of Endothelial Progenitor Cells Through RNA Sequence Analysis

Jian Zhu 1,2,*, Li-Li Sun 1,3, Wen-Dong Li 3,✉, Xiao-Qiang Li 1,3,✉
PMCID: PMC7784562  PMID: 33028108

Abstract

We have previously reported that miR-9 promotes the homing, proliferation, and angiogenesis of endothelial progenitor cells (EPCs) by targeting transient receptor potential melastatin 7 via the AKT autophagy pathway. In this way, miR-9 promotes thrombolysis and recanalization following deep vein thrombosis (DVT). However, the influence of miR-9 on messenger RNA (mRNA) expression profiles of EPCs remains unclear. The current study comprises a comprehensive exploration of the mechanisms underlying the miR-9-regulated angiogenesis of EPCs and highlights potential treatment strategies for DVT. We performed RNA sequence analysis, which revealed that 4068 mRNAs were differentially expressed between EPCs overexpressing miR-9 and the negative control group, of which 1894 were upregulated and 2174 were downregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that these mRNAs were mainly involved in regulating cell proliferation/migration processes/pathways and the autophagy pathway, both of which represent potential EPC-based treatment strategies for DVT. Reverse transcriptase quantitative polymerase chain reaction confirmed the changes in mRNA expression related to EPC angiogenesis, migration, and autophagy. We also demonstrate that miR-9 promotes EPC migration and angiogenesis by regulating FGF5 directly or indirectly. In summary, miR-9 enhances the expression of VEGFA, FGF5, FGF12, MMP2, MMP7, MMP10, MMP11, MMP24, and ATG7, which influences EPC migration, angiogenesis, and autophagy. We provide a comprehensive evaluation of the miR-9-regulated mRNA expression in EPCs and highlight potential targets for the development of new therapeutic interventions for DVT.

Keywords: deep vein thrombosis, miR-9, endothelial progenitor cell, angiogenesis, autophagy

Introduction

Endothelial progenitor cells (EPCs) are pluripotent cells that express specific markers of endothelial cells and differentiate into mature endothelial cells. These cells were first isolated from adult peripheral blood by Asahara et al. in 19971–3 and have been shown to secrete angiogenic growth factors and cytokines4. In the field of regenerative medicine, EPCs have potential therapeutic applications in vascular tissue engineering and cell-based therapy, especially for cardiovascular and vascular-related diseases5. Bone-marrow-derived EPCs are initially released into venous microcirculation; therefore, the concentration of EPCs in the veins may be higher than in the arteries6. Previous studies have demonstrated that EPCs are mobilized from the bone marrow into the lesion created when the intima is damaged7. Circulating EPCs can accelerate thrombus recanalization by restoring damaged or lost endothelium, which enhances neovascularization and prompts recanalization of deep vein thrombosis (DVT)3,8–10. Angiogenesis and thrombolysis are crucial for recanalization following DVT4,11,12, and numerous studies have shown that EPCs are recruited to the thrombus to accelerate thrombus resolution, where they play a vital role in physiological and pathological neovascularization in adults8,9,12,13. Moreover, EPCs can differentiate into endothelial cells to participate in angiogenesis and thus represent a promising therapeutic approach for thrombus resolution, for which there are currently limited treatment options with insufficient success rates12–14. However, the function of EPCs can be impaired by cardiovascular risk factors, ischemic disease, and graft vasculopathy15–18; therefore, exploring methods to improve EPC homing and angiogenesis is important.

MicroRNAs (MiRNAs, MiRs) are small noncoding RNAs comprising approximately 22 nucleotides that influence post-transcriptional gene regulation by targeting the 3′-untranslated regions of messenger RNAs (mRNAs)19–22. These small RNAs play a major role in the regulation of cell migration, proliferation, apoptosis, and angiogenesis, which is essential for the development and progression of vascular disease23. Our previous studies have suggested that miR-9 plays a crucial role in regulating angiogenesis and migration of EPCs by targeting transient receptor potential melastatin 7 (TRPM7) through the AKT autophagy pathway, thereby promoting thrombolysis and recanalization following DVT24. However, the detailed mechanisms underlying regulation of miR-9 in EPCs are poorly understood. The present study aimed to examine the mRNA expression of miR-9 in EPCs using RNA sequence (RNAseq) analysis and to analyze the effects on EPC angiogenesis and migration.

Materials and Methods

Isolation and Culture of Human EPCs

We isolated and characterized human EPCs as previously described18,25. Peripheral blood was obtained from the veins of recruited subjects, then carried out density gradient centrifugation using Ficoll-Isopaque Plus (Histopaque-1077, Sigma, St. Louis, MO, USA) to isolate mononuclear cells. After washing with phosphate-buffered saline (PBS), isolated mononuclear cells were seeded in fibronectin-coated cell culture flasks, then incubated in complete Endothelial Basal Medium-2 (EBM-2, Lonza, Walkersville, MD, USA) at 37°C and 5% CO2. The culture medium was changed every 4 days. After 14 days of culture, EPCs were detached by the addition of 0.025% trypsin containing 0.02% ethylenediaminetetraacetic acid and harvested for further analysis or transplantation.

Characterization of EPCs

Isolated EPCs were identified using both confocal microscopy and flow cytometry as previously described18. Cells that were positive for DiI-conjugated acetylated low-density lipoproteins and Ulex europaeus agglutinin 1 staining on confocal microscopy and those with antibodies against CD31, CD34, CD45, CD133, and CD309 (all antibodies were purchased from Becton-Dickinson, Franklin Lakes, NJ, USA) on flow cytometry were determined to be EPCs.

Cell Treatment

To detect mRNAs that are regulated by miR-9 in EPCs, recombinant lentiviral particles carrying miR-9 mimics or vehicle controls provided by GenePharma Co. Ltd (Shanghai, China) were used to infect EPCs according to the manufacturer’s instructions. Polybrene (5 µg/ml) was used to enhance infection efficiency. Infected cells were then selected by incubating with 3 µg/ml puromycin (Invitrogen, San Diego, CA, USA) for 1 week to induce stable miR-9 overexpression in EPCs and negative controls (NCs; EPCs are infected with the lentivirus carrying the empty vector).

mRNA Sample Preparation and RNA Sequencing Analysis

Total RNA was extracted from 1 × 107 cells to construct sequencing libraries using RNAiso Plus (TaKaRa Bio, Inc., Ostu, Shiga, Japan). The RNA Integrity Number was measured using an Agilent Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany) to assess RNA quality prior to sequencing.

We performed RNAseq analysis as previously described10. Briefly, the NEBNext® Poly(A) mRNA Magnetic Isolation Module was used to isolate intact poly(A)+ RNA from previously isolated total RNA, and the NEBNext® Ultra RNA Library Prep Kit for Illumina was used to construct the library. Library determination and quantification were performed using the Agilent Bioanalyzer 2100 and Qubit. Sequencing was performed on an Illumina HiSeq 2000 paired-end module (Illumina, San Diego, CA, USA).

Gene Ontological and Pathway Analysis

Gene ontology (GO) analysis was performed to determine processes or functional categories of differential mRNA expression, as described previously26. Genes were considered differentially expressed if |log2 fold change| value was ≥1 between the two groups with a q value of <0.05. Hierarchical clustering with average linkage was performed to identify distinguishable mRNA expression patterns. Differentially expressed mRNAs were classified into three domains: molecular function, cellular component, and biological process. To investigate whether genes share a similar biological function, we searched for overrepresentation in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Enriched genes were identified by hypergeometric distribution, with q value <0.05 used as the selection criterion for enrichment.

Reverse Transcription Quantitative Polymerase Chain Reaction

We selected mRNA transcripts that are closely related to angiogenesis, migration, and autophagy (VEGFA, FGF5, FGF12, MMP2, MMP7, MMP10, MMP11, MMP24, and ATG7) and validated their expression levels by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The RNA remaining after RNAseq was reverse transcribed into complementary DNAs (cDNAs) using the PrimeScript RT reagent kit (TaKaRa Bio, Inc.). Next, RT-qPCR was performed using the SYBR Green quantitative PCR (qPCR) Master Mix (Bio-Rad, CA, USA) according to the manufacturer’s instructions. We determined miRNA levels using the Hairpin-it™ miRNA RT-PCR Quantitation Kit (GenePharma Co. Ltd) according to the manufacturer’s instructions and analyzed mRNA expression levels using mRNA primers (GENEWIZ, Jiangsu, China). Glyceraldehyde 3-phosphate dehydrogenase was used as the endogenous control for mRNAs. Reactions were processed using a High Throughput Quantitative PCR LightCycler480 II (Roche, Basel, Switzerland) and relative expression levels of each candidate gene calculated using the 2ΔΔCT method10. Each experiment was performed independently and in triplicate. The primers used for RT-qPCR are shown in Table 1.

Table 1.

Primers Used for RT-qPCR.

Has-VEGFA Forward (5’–3’) AGGGCAGAATCATCACGAAGT
Reverse (5’–3’) AGGGTCTCGATTGGATGGCA
Has-FGF5 Forward (5’–3’) TGTACCGCTATGGTTACACTCG
Reverse (5’–3’) GGCAGGGACAGTTGCTTCT
Has-FGF12 Forward (5’–3’) GGGACCAAGGACGAAAACAG
Reverse (5’–3’) TTGCTGGCGGTACAGTGTG
Has-MMP2 Forward (5’–3’) TGACTTTCTTGGATCGGGTCG
Reverse (5’–3’) AAGCACCACATCAGATGACTG
Has-MMP7 Forward (5’–3’) GAGTGAGCTACAGTGGGAACA
Reverse (5’–3’) CTATGACGCGGGAGTTTAACAT
Has-MMP10 Forward (5’–3’) TGCTCTGCCTATCCTCTGAGT
Reverse (5’–3’) TCACATCCTTTTCGAGGTTGTAG
Has-MMP11 Forward (5’–3’) CCGCAACCGACAGAAGAGG
Reverse (5’–3’) ATCGCTCCATACCTTTAGGGC
Has-MMP24 Forward (5’–3’) GCCGGGCAGAACTGGTTAAA
Reverse (5’–3’) CCCGTAAAACTGCTGCATAGT
Has-ATG7 Forward (5’–3’) CAGTTTGCCCCTTTTAGTAGTGC
Reverse (5’–3’) CCAGCCGATACTCGTTCAGC
Has-GAPDH Forward (5’–3’) CATGAGAAGTATGACAACAGCCT
Reverse (5’–3’) AGTCCTTCCACGATACCAAAGT
Open in a new tab

RT-qPCR: reverse transcription quantitative polymerase chain reaction.

Immunofluorescence

The experiment of immunofluorescence was performed as previously described10. In short, transfected EPCs were grown on cover slides for 24 h, washed with PBS, and then fixed with 4% paraformaldehyde for 10 min at room temperature. After washing three times with PBS, EPCs were blocked with Immunol Staining Blocking Buffer (Beyotime, P0102, Shanghai, China) for 1 h at room temperature. Immunostaining was performed using FGF5 antibody (Abcam, ab168743, Cambridge, UK) to detect the protein expression levels of FGF5 in EPCs. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole. The coverslips were washed with PBS and inverted onto cover slips that had been treated with Fluoromount-GTM seal. For each experiment, images were acquired by confocal microscopy under the same conditions, and no digital image processing was performed.

Tube Formation Assay

Tube formation assay was performed as previously described10. Briefly, NC EPCs and FGF5-overexpressing EPCs (2 × 104 cells) were seeded in Matrigel-coated wells of a 24-well plate to induce tube formation. After 12 h of incubation, tubular structures of EPCs in the Matrigel were examined through an Image Navigator®, and the extent of tube formation assessed by measuring the number and length of tubes using ImageJ software. We examined at least five random fields per well to determine cell-tube formation ability.

Migration Assay

Cell migration was evaluated using 24-well chambers #x0028;8-mm pore size; BD, Franklin Lakes, NJ, USA). Medium containing 10% fetal bovine serum (300 µl) was added to the lower wells and EPCs (5 × 103 cells in 200 µl serum-free EBM-2 medium) were added to the upper transwell chambers. The chambers were incubated for 12 h at 37°C in a 5% CO2 incubator. Migration of cells was evaluated using an inverted microscope and the number of cells in each chamber was quantified using Image J. Three wells were analyzed per group.

Statistical Analysis

All data are presented as mean ± standard error of the mean. Student’s t test was used to analyze differences between groups. GraphPad Prism software was used for statistical analysis and a P value of <0.05 was considered statistically significant.

Results

Differential Expression Profiles of mRNAs

After processing sequencing data to reduce noise, remove outliers, and normalize, hierarchical clustering revealed systematic variations in mRNA expression between EPCs that overexpressed miR-9 mimics and NC EPCs (Fig. 1A). Differential expression (DE) analysis identified 4068 mRNAs that were significantly altered in miR-9-overexpressing EPCs compared with NC EPCs: 1894 mRNAs were upregulated and 2174 were downregulated (Fig. 1B). The results of volcano plot analysis are shown in Fig. 1C. Because of the high number of DE genes, we sorted the results into mRNAs related to cell migration, angiogenesis, and autophagy to expose meaningful sets of relevant genes, including VEGFA, FGF5, FGF12, MMP2, MMP7, MMP10, MMP11, MMP24, and ATG7 (Table 2).

Figure 1.

Figure 1.

Open in a new tab

Differentially expressed mRNAs identified using RNASeq analysis. (A) Heat maps and hierarchical clustering of mRNAs in the miR-9 mimics groups and the NC groups. Blue and red indicate upregulation and downregulation, respectively. NC indicate NC group and 9M indicate miR-9 mimics group. (B) A total of 4086 mRNAs were differentially expressed in the miR-9 mimics groups (n = 3) compared with the NC groups (n = 3), including 1894 (46.56%) upregulated and 2174 (53.44%) downregulated mRNAs (|log2 fold change| ≥1, q value <0.05). (C) mRNA volcano plots of miR-9-overexpressing EPCs versus NC EPCs (red: upregulated, blue: downregulated, and gray: no significant mRNAs). EPCs: endothelial progenitor cells; mRNA: messenger RNA; NC: negative control; RNAseq: RNA sequence.

Table 2.

RNAseq Analysis Results of the Main Factors Involved in Angiogenesis and Migration Regulated by miR-9 Overexpression.

Gene symbol Log2 fold change test_stat P value P adj Significant
VEGFA 1.80434 2.17582 0.00675 0.037264 Ups
FGF5 3.91977 7.7593 5.00E-05 0.000415 Ups
FGF12 2.06785 3.46969 5.00E-05 0.000415 Ups
MMP2 2.23677 2.10103 0.0001 0.004512 Ups
MMP7 1.15593 1.89401 0.005 0.028731 Ups
MMP10 1.8382 2.62685 5.00E-05 0.000415 Ups
MMP11 1.42101 3.22442 5.00E-05 0.000415 Ups
MMP24 2.96222 5.2389 5.00E-05 0.000415 Ups
ATG7 −1.2249 -2.30742 0.002 0.012854 Down
Open in a new tab

RNAseq analysis was performed after EPC infection with LV3-NC and miR-9 mimics lentivirus, and three groups of LV3-NC and three groups of miR-9 mimics were detected; gene symbol means differentially expressed genes; P value was calculated by t test, and P adj was obtained by P value normalized by Benjamini–Hochberg; significant means the up- or downregulation of the gene expressed in the miR-9 overexpression group compared with the LV3-NC.

EPCs: endothelial progenitor cells; NC: negative control; RNAseq: RNA sequence.

GO and KEGG Pathway Analysis

In the molecular function domain, GO terms included binding, catalytic activity, structural molecular activity, molecular function regulator, signal transducer activity, transcription factor activity, and protein binding. In the cellular component domain, the top five GO terms were cell part, organelle, organelle part, membrane, macromolecular complex, extracellular region part, extracellular region, cell junction, supramolecular complex, and membrane-enclosed lumen. In the biological process domain, the GO terms for differentially expressed mRNAs included structural cellular process, biological regulation, single-organism process, metabolic process, cellular component organization or biogenesis, cell adhesion, cell growth, and so on (Fig. 2A). From KEGG pathway analysis, we identified 30 signaling pathways that were enriched in EPCs regulated by miR-9. These pathways related to cell proliferation, migration, and angiogenesis including the transforming growth factor-β, p53, and FoxO signaling pathways; focal adhesion; cell cycle; and autophagy (Fig. 2B).

Figure 2.

Figure 2.

Open in a new tab

GO and KEGG pathway analysis. (A) GO analysis assigns high-level terms from each of the following three major GO terms: biological processes, cellular components, and molecular functions. (B) KEGG analysis of the differential genes. The top 30 enriched pathways were shown. The vertical axis represents the name of the pathway, the horizontal axis represents the rich factor, the size of the dots indicates the number of differentially expressed genes in the pathway, and the color of the dots corresponds to different q value ranges. GO: gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Validation of the Microarray Results by RT-qPCR

We selected nine mRNAs with high DES scores and analyzed further with RT-qPCR to evaluate the consistency of the results of mRNA DE from sequencing. Expression trends of these transcripts confirmed the results of RNAseq (Fig. 3). Our data show that mRNA expression of VEGFA, FGF5, FGF12, MMP2, MMP7, MMP10, MMP11, and MMP24 was upregulated, while ATG7 was downregulated in miR-9-mimic-expressing EPCs compared with NC EPCs. All DE was in the expected direction.

Figure 3.

Figure 3.

Open in a new tab

RT-qPCR validation of selected mRNAs in EPCs. *P < 0.05, **P < 0.01 compared to the NC group. Data are represented as mean ± standard error of the mean. EPCs: endothelial progenitor cells; mRNA: messenger RNA; NC: negative control; RNAseq: RNA sequence; RT-qPCR: reverse transcription quantitative polymerase chain reaction.

MiR-9 Enhances the Protein Expression of FGF in EPCs

To examine the effect of miR-9 on the protein expression of FGF5 in EPCs, immunofluorescence technology was performed. As shown in Fig. 4, the results of immunofluorescence showed that miR-9 significantly upregulates FGF5 protein expression.

Figure 4.

Figure 4.

Open in a new tab

The expression levels of FGF5 protein in EPCs. Immunofluorescence results showed that miR-9 significantly upregulated FGF5 protein expression. *P < 0.05. EPCs were immunostained with an antibody against FGF5 (green). Nuclei were stained with DAPI (blue). Results are representative of ≥3 independent experiments. DAPI: 4’,6-diamidino-2-phenylindole; EPCs: endothelial progenitor cells.

Effect of FGF5 on Migration and Angiogenesis of EPCs

Tube formation and transwell analyses revealed that FGF5 significantly enhances EPC angiogenesis and migration (Fig. 5).

Figure 5.

Figure 5.

Open in a new tab

Effect of FGF5 on angiogenesis and migration of EPCs. (A) FGF5 significantly improves the angiogenic capacity of EPCs. (B) FGF5 enhances the EPC migration. *P < 0.05, **P < 0.01 compared to the NC group. Data are represented as mean ± standard error of the mean. EPCs: endothelial progenitor cells; NC: negative control.

Discussion

DVT is a common and difficult clinical problem, and complications associated with the condition—such as pulmonary embolism and pulmonary vein thrombosis—can be life-threatening10. The current treatment for venous thromboembolism is primary anticoagulation; however, this causes impairment of hemostasis, and many patients who present with DVT have already progressed past the point where anticoagulants are effective27. Thus, identifying new biomolecular treatment therapies for DVT could have great clinical value10. There is increasing evidence of the critical role of EPCs in the angiogenesis and resolution of DVT. However, the limited capacities of homing and angiogenesis of EPCs impair their therapeutic effects. Intriguingly, our previous study demonstrated that miR-9 prevents apoptosis but promotes proliferation, migration, and angiogenesis of EPCs by targeting TRPM7, which enhances recanalization following venous thrombosis24. Although we identified the PI3K/Akt/autophagy pathway to be involved in the process, the effects of miR-9 on mRNA expression profiles of EPCs remain unclear.

The present study involved a comprehensive exploration of the mechanisms underlying the influence of miR-9 on the migration and angiogenesis of EPCs to highlight potential treatment strategies based on miR-9 targets. We determined the mRNA expression profiles of miR-9-overexpressing EPCs and NC EPCs and demonstrate for the first time, to the best of our knowledge, that a total of 4068 mRNAs are differentially expressed between these two cell lines. The results of GO and KEGG analyses indicated the downregulated and upregulated mRNAs to be mainly involved in cell adhesion, angiogenesis, and cell differentiation, which suggests that differentially expressed mRNAs might regulate EPC migration, angiogenesis, and differentiation by influencing the expression of related genes.

The results of RT-qPCR supported RNAseq analysis and confirm that miR-9 significantly increased the expression levels of eight miR-9 mimics in EPCs but decreased the mRNA expression of ATG7. It has been reported that VEGFA regulates multiple functions and induces angiogenesis in vascular endothelial cells; previous studies have shown it to be a prominent proangiogenic factor that enhances angiogenesis in vitro and in vivo by inducing EPC proliferation, migration, and tube formation28,29. Additionally, VEGF promotes the recruitment of EPCs to ischemic sites and contributes to the growth of new blood vessels30. The matrix metalloproteinase (MMP) superfamily comprising metalloproteases that degrade the extracellular matrix to facilitate cell migration, invasion, and angiogenesis10,31. Among the members of this superfamily, MMP2 plays an essential role in vascular disease and promotes EPC migration and angiogenesis3,32. Autophagy has attracted attention as a potential therapeutic target for vascular disease, and the process plays an important role in vascular biology and homeostasis and is implicated in many physiological and pathological processes involving cell angiogenesis and migration33. Our previous studies have shown miR-9 to promote EPC migration, proliferation, and angiogenesis by regulating autophagy and MMP2 protein expression, thereby promoting thrombolysis and vascularization in DVT24. However, tissue inhibitor of matrix metalloproteinases (TIMPs), specific inhibitor of MMPs, had no significant differences in our RNAseq results. Therefore, miR-9 may upregulate the expression levels of MMPs in EPCs through other targets and pathways. In fact, MMPs can be upregulated through a variety of pathways, such as the PI3K-AKT and autophagy pathway24,33. Our previous studies also showed that miR-9 can upregulate the PI3K-AKT pathway24, which is consistent with this result. There may be other detailed mechanisms and pathways that need further study. Additionally, previous studies have shown that FGF5 promotes angiogenesis of human aortic endothelial cells and increases blood flow and function34,35. However, the role of FGF5 in regulating the migration and angiogenesis of EPCs has remained unclear until now. The present study demonstrates that FGF5 significantly enhances EPC migration and angiogenesis and identifies the major mRNAs that promote angiogenesis and migration, thus highlighting new targets for DVT therapy.

In summary, we have determined comprehensive mRNA profiles of EPCs regulated by miR-9. DE of VEGFA, FGF5, FGF12, MMP2, MMP7, MMP10, MMP11, MMP24, and ATG7 may participate in the migration, angiogenesis, and autophagy of EPCs, and may contribute to the use of EPCs for the treatment of DVT.

Acknowledgments

We are very grateful to all the members involved in the literature search and the authors of numerous original studies for clarifying the data and providing information.

Footnotes

Ethical Approval: The present study was approved by the Ethics Committee of Soochow University, Suzhou, Jiangsu Province, China. All research involving the human was approved by the institutional review committee of Soochow University.

Statement of Human and Animal Rights: All experiments were performed with relevant laws and guidelines, and following institutional guidelines of Soochow University. Human blood samples were obtained using protocols approved by the Ethics Committee of Soochow University. This article does not contain any studies with animals performed by any of the authors.

Statement of Informed Consent: Human EPCs were extracted and isolated from the venous blood of recruited subjects.

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was sponsored by grants from the National Natural Science Foundation of China (No. 81770483, 81800418), the Natural Science Foundation of Jiangsu Province (No. BK20180125), and the Key Project supported by Medical Science and Technology Development Foundation (No. YKK18063, ZKX18021).

ORCID iD: Xiao-Qiang Li Inline graphic https://orcid.org/0000-0001-5279-9158

References

  • 1. Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC, Hicklin DJ, Witte L, Moore MAS, Rafii S. Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood. 2000;95(3):952–958. [PubMed] [Google Scholar]
  • 2. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302):964–966. [DOI] [PubMed] [Google Scholar]
  • 3. Sun LL, Xiao L, Du XL, Hong L, Li CL, Jiao J, Li WD, Li XQ. MiR-205 promotes endothelial progenitor cell angiogenesis and deep vein thrombosis recanalization and resolution by targeting PTEN to regulate Akt/autophagy pathway and MMP2 expression. J Cell Mol Med. 2019;23(12):8493–8504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Li WD, Li XQ. Endothelial progenitor cells accelerate the resolution of deep vein thrombosis. Vascul Pharmacol. 2016;83:10–16. [DOI] [PubMed] [Google Scholar]
  • 5. Asahara T, Kawamoto A, Masuda H. Concise review: circulating endothelial progenitor cells for vascular medicine. Stem Cells. 2011;29(11):1650–1655. [DOI] [PubMed] [Google Scholar]
  • 6. Moldovan NI, Asahara T. Role of blood mononuclear cells in recanalization and vascularization of thrombipast, present, and future. Trends Cardiovasc Med. 2003;13(7):265–269. [DOI] [PubMed] [Google Scholar]
  • 7. Yan J, Bao H, Fan YJ, Jiang ZL, Qi YX, Han Y. Platelet-derived microvesicles promote endothelial progenitor cell proliferation in intimal injury by delivering TGF-beta1. FEBS J. 2020. doi:10.1111/febs.15293. [DOI] [PubMed] [Google Scholar]
  • 8. Modarai B, Burnand KG, Sawyer B, Smith A. Endothelial progenitor cells are recruited into resolving venous thrombi. Circulation. 2005;111(20):2645–2653. [DOI] [PubMed] [Google Scholar]
  • 9. Modarai B, Burnand KG, Humphries J, Waltham M, Smith A. The role of neovascularisation in the resolution of venous thrombus. Thromb Haemost. 2005;93(5):801–809. [DOI] [PubMed] [Google Scholar]
  • 10. Sun LL, Lei FR, Jiang XD, Du XL, Xiao L, Li WD, Li XQ. LncRNA GUSBP5-AS promotes EPC migration and angiogenesis and deep vein thrombosis resolution by regulating FGF2 and MMP2/9 through the miR-223-3p/FOXO1/Akt pathway. Aging. 2020;12(5):4506–4526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Sun J, Zhang Z, Ma T, Yang Z, Zhang J, Liu X, Lu D, Shen Z, Yang J, Meng Q. Endothelial progenitor cell-derived exosomes, loaded with miR-126, promoted deep vein thrombosis resolution and recanalization. Stem Cell Res Ther. 2018;9(1):223. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 12. Di Santo S, Tepper OM, Wyler von Ballmoos M, Diehm N, Völzmann J, Baumgartner I, Kalka C. Cell-based therapy facilitates venous thrombus resolution. Thromb Haemost. 2009;101(03):460–464. [PubMed] [Google Scholar]
  • 13. Mo J, Zhang D, Yang R. MicroRNA-195 regulates proliferation, migration, angiogenesis and autophagy of endothelial progenitor cells by targeting GABARAPL1. Biosci Rep. 2016;36(5):e00396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Miller-Kasprzak E, Jagodzinski PP. Endothelial progenitor cells as a new agent contributing to vascular repair. Arch Immunol Ther Exp (Warsz). 2007;55(4):247–259. [DOI] [PubMed] [Google Scholar]
  • 15. Werner N, Nickenig G. Influence of cardiovascular risk factors on endothelial progenitor cells: limitations for therapy? Arterioscler Thromb Vasc Biol. 2006;26(2):257–266. [DOI] [PubMed] [Google Scholar]
  • 16. Al Mheid I, Hayek SS, Ko YA, Akbik F, Li Q, Ghasemzadeh N, Martin GS, Long Q, Hammadah M, Maziar Zafari A, Vaccarino V, et al. Age and human regenerative capacity impact of cardiovascular risk factors. Circ Res. 2016;119(7):801–809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003;348(7):593–600. [DOI] [PubMed] [Google Scholar]
  • 18. Li WD, Zhou DM, Sun LL, Xiao L, Liu Z, Zhou M, Wang WB, Li XQ. LncRNA WTAPP1 promotes migration and angiogenesis of endothelial progenitor cells via MMP1 through MicroRNA 3120 and Akt/PI3K/autophagy pathways. Stem Cells. 2018;36(12):1863–1874. [DOI] [PubMed] [Google Scholar]
  • 19. Thamotharan S, Chu A, Kempf K, Janzen C, Grogan T, Elashoff DA, Devaskar SU. Differential microRNA expression in human placentas of term intra-uterine growth restriction that regulates target genes mediating angiogenesis and amino acid transport. PLoS One. 2017;12(5):e0176493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Lucas T, Schafer F, Muller P, Eming SA, Heckel A, Dimmeler S. Light-inducible antimiR-92a as a therapeutic strategy to promote skin repair in healing-impaired diabetic mice. Nat Commun. 2017;8:15162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Siomi H, Siomi MC. Posttranscriptional regulation of microRNA biogenesis in animals. Mol Cell. 2010;38(3):323–332. [DOI] [PubMed] [Google Scholar]
  • 22. Karreth FA, Tay Y, Perna D, Ala U, Tan SM, Rust AG, DeNicola G, Webster KA, Weiss D, Perez-Mancera PA, Krauthammer M, et al. In vivo identification of tumor- suppressive PTEN ceRNAs in an oncogenic BRAF-induced mouse model of melanoma. Cell. 2011;147(2):382–395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Sun L-L, Li W-D, Lei F-R, Li X-Q. The regulatory role of microRNAs in angiogenesis-related diseases. J Cell Mol Med. 2018;22(10):4568–4587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Zhou DM, Sun LL, Zhu J, Chen B, Li XQ, Li WD. MiR-9 promotes angiogenesis of endothelial progenitor cell to facilitate thrombi recanalization via targeting TRPM7 through PI3K/Akt/autophagy pathway. J Cell Mol Med. 2020;24(8):4624–4632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA. 2000;97(7):3422–3427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Speed T, T. TP GOstat: find statistically overrepresented gene ontologies within a group of genes. J Bioinformatics. 2004;20(9):1464–1465. [DOI] [PubMed] [Google Scholar]
  • 27. Nicklas JM, Gordon AE, Henke PK. Resolution of deep venous thrombosis: proposed immune paradigms. Int J Mol Sci. 2020;21(6):2080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Chen CY, Su CM, Hsu CJ, Huang CC, Wang SW, Liu SC, Chen WC, Fuh LJ, Tang CH. CCN1 promotes VEGF production in osteoblasts and induces endothelial progenitor cell angiogenesis by inhibiting miR-126 expression in rheumatoid arthritis. J Bone Miner Res. 2017;32(1):34–45. [DOI] [PubMed] [Google Scholar]
  • 29. Kanzler I, Tuchscheerer N, Steffens G, Simsekyilmaz S, Konschalla S, Kroh A, Simons D, Asare Y, Schober A, Bucala R, Weber C, et al. Differential roles of angiogenic chemokines in endothelial progenitor cell-induced angiogenesis. Basic Res Cardiol. 2013;108(1):310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Anderson EM, Kwee BJ, Lewin SA, Raimondo T, Mehta M, Mooney DJ. Local delivery of VEGF and SDF enhances endothelial progenitor cell recruitment and resultant recovery from ischemia. Tissue Eng Part A. 2015;21(7-8):1217–1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Beate H, Koichi H, Sergio D, Matthias F, Barbara F, Hackett NR, Crystal RG, Peter B, David L, Moore MAS. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell. 2002;109(5):625–637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Starke RD, Ferraro F, Paschalaki KE, Dryden NH, Mckinnon TA, Sutton RE, Payne EM, Haskard DO, Hughes AD, Cutler DF. Endothelial von Willebrand factor regulates angiogenesis. Blood. 2011;117(3):1071–1080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Li WD, Hu N, Lei FR, Wei S, Rong JJ, Zhuang H, Li XQ. Autophagy inhibits endothelial progenitor cells migration via the regulation of MMP2, MMP9 and uPA under normoxia condition. Biochem Biophys Res Commun. 2015;466(3):376–380. [DOI] [PubMed] [Google Scholar]
  • 34. Seo HR, Jeong HE, Joo HJ, Choi SC, Park CY, Kim JH, Choi JH, Cui LH, Hong SJ, Chung S, Lim DS. Intrinsic FGF2 and FGF5 promotes angiogenesis of human aortic endothelial cells in 3D microfluidic angiogenesis system. Sci Rep. 2016;6:28832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Giordano FJ, Ping P, Mckirnan MD, Nozaki S, Demaria AN, Dillmann WH, Mathieu-Costello O, Hammond HK. Intracoronary gene transfer of fibroblast growth factor–5 increases blood flow and contractile function in an ischemic region of the heart. J Nature Medicine. 1996;2(5):534–539. [DOI] [PubMed] [Google Scholar]

Articles from Cell Transplantation are provided here courtesy of SAGE Publications

RESOURCES