-Chung Cheng

Distinct angiogenesis roles and surface markers of early and late endothelial progenitor cells revealed by functional group analyses

BackgroundEndothelial progenitor cells (EPCs) play a fundamental role in post-natal vascular repair. Currently EPCs are defined as either early and late EPCs based on their biological properties and their time of appearance during in vitro culture. EPCs are rare and therefore optimizing isolation and culture is required before they can be applied as part of clinical therapies.ResultsWe compared the gene profiles of early/late EPCs to their ancestors CD133+ or CD34+ stem cells and to matured endothelial cells pinpointing novel biomarkers and stemness genes. Late EPCs were enriched with proliferation and angiogenesis genes, participating in endothelial tubulogenesis and hence neovascularization. Early EPCs expressed abundant inflammatory cytokines and paracrine angiogenic factors, thereby promoting angiogenesis in a paracrine manner. Transcription factors involved in EPC stemness were pinpointed in early EPCs (MAF/MAFB) and in late EPCs (GATA6/IRF6).ConclusionsThe detailed mRNA expression profiles and functional module analysis for different EPCs will help the development of novel therapeutic modalities targeting cardiovascular disease, tumor angiogenesis and various ischemia-related diseases.

Deficiency of the MicroRNA-31–MicroRNA-720 Pathway in the Plasma and Endothelial Progenitor Cells From Patients With Coronary Artery Disease

Objective—Defects in angiogenesis/vasculogenesis or vessel repair are major complications of coronary artery disease (CAD). Endothelial progenitor cells (EPCs) play a fundamental role in postnatal vascular repair and CAD. The role of microRNAs in CAD pathogenesis and their potential as biomarkers remain to be elucidated. Approach and Results—MicroRNA-31 (miR-31) level in both the plasma and EPCs of patients with CAD is found lower. miR-31 regulates EPC activities by targeting FAT atypical cadherin 4 and thromboxane A2 receptor, which show increased expression in CAD EPCs. Overexpressing miR-31 in CAD EPCs rescued their angiogenic and vasculogenic abilities both in vitro and in vivo. When exploring approaches to restore endogenous miR-31, we found that far-infrared treatment enhanced the expression of not only miR-31, but also miR-720 in CAD EPCs. miR-720, which was also decreased in EPCs and the plasma of patients with CAD, stimulated EPC activity by targeting vasohibin 1. The miR720–vasohibin 1 pair was shown to be downstream of FAT atypical cadherin 4, but not of thromboxane A2 receptor. FAT atypical cadherin 4 inhibited miR-720 expression via repression of the planar cell polarity signaling gene four-jointed box 1 (FJX1), which was required for miR-720 expression through a hypoxia-inducible factor 1, &agr; subunit–dependent mechanism. Restoring miR-720 level strengthened activity of CAD EPCs. The miR-31–miR-720 pathway is shown critical to EPC activation and that downregulation of this pathway contributes to CAD pathogenesis. Circulating levels of miR-31, miR-720, and vasohibin 1 have the potential to allow early diagnosis of CAD and to act as prognosis biomarkers for CAD and other EPC-related diseases. Conclusions—Manipulating the expression of the miR-31–miR-720 pathway in malfunction EPCs should help develop novel therapeutic modalities.

Dysregulated miR-361-5p/VEGF Axis in the Plasma and Endothelial Progenitor Cells of Patients with Coronary Artery Disease

Dysfunction and reduction of circulating endothelial progenitor cell (EPC) is correlated with the onset of cardiovascular disorders including coronary artery disease (CAD). VEGF is a known mitogen for EPC to migrate out of bone marrow to possess angiogenic activities, and the plasma levels of VEGF are inversely correlated to the progression of CAD. Circulating microRNAs (miRNAs) in patient body fluids have recently been considered to hold the potential of being novel disease biomarkers and drug targets. However, how miRNAs and VEGF cooperate to regulate CAD progression is still unclear. Through the small RNA sequencing (smRNA-seq), we deciphered the miRNome patterns of EPCs with different angiogenic activities, hypothesizing that miRNAs targeting VEGF must be more abundant in EPCs with lower angiogenic activities. Candidates of anti-VEGF miRNAs, including miR-361-5p and miR-484, were enriched in not only diseased EPCs but also the plasma of CAD patients. However, we found out only miR-361-5p, but not miR-484, was able to suppress VEGF expression and EPC activities. Reporter assays confirmed the direct binding and repression of miR-361-5p to the 3′-UTR of VEGF mRNA. Knock down of miR-361-5p not only restored VEGF levels and angiogenic activities of diseased EPCs in vitro, but further promoted blood flow recovery in ischemic limbs of mice. Collectively, we discovered a miR-361-5p/VEGF-dependent regulation that could help to develop new therapeutic modalities not only for ischemia-related diseases but also for tumor angiogenesis.

miRNome traits analysis on endothelial lineage cells discloses biomarker potential circulating microRNAs which affect progenitor activities

BackgroundEndothelial progenitor cells (EPCs) play a fundamental role in not only blood vessel development but also post-natal vascular repair. Currently EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Both EPC types assist angiogenesis and have been linked to ischemia-related disorders, including coronary artery disease (CAD).ResultsWe found late EPCs are more mobile than early EPCs and matured endothelial cells (ECs). To pinpoint the mechanism, microRNA profiles of early EPCs late EPCs, and ECs were deciphered by small RNA sequencing. Obtained signatures made up of both novel and known microRNAs, in which anti-angiogenic microRNAs such as miR-221 and miR-222 are more abundant in matured ECs than in late EPCs. Overexpression of miR-221 and miR-222 resulted in the reduction of genes involved in hypoxia response, metabolism, TGF-beta signalling, and cell motion. Not only hamper late EPC activities in vitro, both microRNAs (especially miR-222) also hindered in vivo vasculogenesis in a zebrafish model. Reporter assays showed that miR-222, but not miR-221, targets the angiogenic factor ETS1. In contrast, PIK3R1 is the target of miR-221, but not miR-222 in late EPCs. Clinically, both miR-221-PIK3R1 and miR-222-ETS1 pairs are deregulated in late EPCs of CAD patients.ConclusionsOur results illustrate EPCs and ECs exploit unique miRNA modalities to regulate angiogenic features, and explain why late EPC levels and activities are reduced in CAD patients. These data will further help to develop new plasma biomarkers and therapeutic approaches for ischemia-related diseases or tumor angiogenesis.

Genetic module and miRNome trait analyses reflect the distinct biological features of endothelial progenitor cells from different anatomic locations

BackgroundEndothelial progenitor cells (EPCs) play a fundamental role in post-natal vascular repair, yet EPCs from different anatomic locations possess unique biological properties. The underlying mechanisms are unclear.ResultsEPCs from CB expressed abundant genes involved in cell cycle, hypoxia signalling and blood vessel development, correlating with the phenotypes that CB-EPCs proliferated more rapidly, migrated faster, and formed tubule structure more efficiently. smRNA-seq further deciphered miRNome patterns in EPCs isolated from CB or PB: 54 miRNAs were enriched in CB-EPCs, while another 50 in PB-EPCs. Specifically, CB-EPCs expressed more angiogenic miRNAs such as miR-31, while PB-EPCs possessed more tumor suppressive miRNAs including miR-10a. Knocking down miR-31 levels in CB-EPCs suppressed cell migration and microtubule formation, while overexpressing miR-31 in PB-EPCs helped to recapitulate some of CB-EPC functions.ConclusionsOur results show the foundation for a more detailed understanding of EPCs from different anatomic sources. Stimulating the expression of angiogenic microRNAs or genes in EPCs of low activity (such as those from patients with cardiovascular diseases) might allow the development of novel therapeutic strategies.

Differentially regulated splice variants and systems biology analysis of Kaposi's sarcoma-associated herpesvirus-infected lymphatic endothelial cells

Alternative RNA splicing greatly increases proteome diversity, and the possibility of studying genome-wide alternative splicing (AS) events becomes available with the advent of high-throughput genomics tools devoted to this issue. Kaposis sarcoma associated herpesvirus (KSHV) is the etiological agent of KS, a tumor of lymphatic endothelial cell (LEC) lineage, but little is known about the AS variations induced by KSHV. We analyzed KSHV-controlled AS using high-density microarrays capable of detecting all exons in the human genome. Splicing variants and altered exon–intron usage in infected LEC were found, and these correlated with protein domain modification. The different 3′-UTR used in new transcripts also help isoforms to escape microRNA-mediated surveillance. Exome-level analysis further revealed information that cannot be disclosed using classical gene-level profiling: a significant exon usage difference existed between LEC and CD34+ precursor cells, and KSHV infection resulted in LEC-to-precursor, dedifferentiation-like exon level reprogramming. Our results demonstrate the application of exon arrays in systems biology research, and suggest the regulatory effects of AS in endothelial cells are far more complex than previously observed. This extra layer of molecular diversity helps to account for various aspects of endothelial biology, KSHV life cycle and disease pathogenesis that until now have been unexplored.

MicroRNA-134 Contributes to Glucose-Induced Endothelial Cell Dysfunction and This Effect Can Be Reversed by Far-Infrared Irradiation

Diabetes mellitus (DM) is a metabolic disease that is increasing worldwide. Furthermore, it is associated with the deregulation of vascular-related functions, which can develop into major complications among DM patients. Endothelial colony forming cells (ECFCs) have the potential to bring about medical repairs because of their post-natal angiogenic activities; however, such activities are impaired by high glucose- (HG) and the DM-associated conditions. Far-infrared radiation (FIR) transfers energy as heat that is perceived by the thermoreceptors in human skin. Several studies have revealed that FIR improves vascular endothelial functioning and boost angiogenesis. FIR has been used as anti-inflammatory therapy and as a clinical treatment for peripheral circulation improvement. In addition to vascular repair, there is increasing evidence to show that FIR can be applied to a variety of diseases, including cardiovascular disorders, hypertension and arthritis. Yet mechanism of action of FIR and the biomarkers that indicate FIR effects remain unclear. MicroRNA-134 (miR-134-5p) was identified by small RNA sequencing as being increased in high glucose (HG) treated dfECFCs (HG-dfECFCs). Highly expressed miR-134 was also validated in dmECFCs by RT-qPCR and it is associated with impaired angiogenic activities of ECFCs. The functioning of ECFCs is improved by FIR treatment and this occurs via a reduction in the level of miR-134 and an increase in the NRIP1 transcript, a direct target of miR-134. Using a mouse ischemic hindlimb model, the recovery of impaired blood flow in the presence of HG-dfECFCs was improved by FIR pretreatment and this enhanced functionality was decreased when there was miR-134 overexpression in the FIR pretreated HG-dfECFCs. In conclusion, our results reveal that the deregulation of miR-134 is involved in angiogenic defects found in DM patients. FIR treatment improves the angiogenic activity of HG-dfECFCs and dmECFCs and FIR has potential as a treatment for DM. Detection of miR-134 expression in FIR-treated ECFCs should help us to explore further the effectiveness of FIR therapy.

Differential Expression of Distinct Surface Markers in Early Endothelial Progenitor Cells and Monocyte-Derived Macrophages

Bone marrow-derived endothelial progenitor cells (EPCs) play a fundamental role in postnatal angiogenesis. Currently, EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Reports have shown that early EPCs share common properties and surface markers with adherent blood cells, especially CD14+ monocytes. Distinguishing early EPCs from circulating monocytes or monocyte-derived macrophages (MDMs) is therefore crucial to obtaining pure endothelial populations before they can be applied as part of clinical therapies. We compared the gene expression profiles of early EPCs, blood cells (including peripheral blood mononuclear cells, monocytes, and MDMs), and various endothelial lineage cells (including mature endothelial cells, late EPCs, and CD133+ stem cells). We found that early EPCs expressed an mRNA profile that showed the greatest similarity to MDMs than any other cell type tested. The functional significance of this molecular profiling data was explored by Gene Ontology database search. Novel plasma membrane genes that might potentially be novel isolation biomarkers were also pinpointed. Specifically, expression of CLEC5A was high in MDMs, whereas early EPCs expressed abundant SIGLEC8 and KCNE1. These detailed mRNA expression profiles and the identified functional modules will help to develop novel cell isolation approaches that will allow EPCs to be purified; these can then be used to target cardiovascular disease, tumor angiogenesis, and various ischemia-related diseases.

Dysregulation of Vascular Endothelial Growth Factor Receptor-2 by Multiple miRNAs in Endothelial Colony-Forming Cells of Coronary Artery Disease

Background/Aims: Endothelial colony-forming cells (ECFCs) have the potential to be used in regenerative medicine. Dysfunction of ECFCs is correlated with the onset of cardiovascular disorders, especially coronary artery disease (CAD). Binding of vascular endothelial growth factor A (VEGFA) to vascular endothelial growth factor receptor-2 (VEGFR2) triggers cell motility and angiogenesis of ECFCs, which are crucial to vascular repair. Methods: To identify the miRNA-VEGFR2-dependent regulation of ECFC functions, ECFCs isolated from peripheral blood of disease-free and CAD individuals were subjected to small RNA sequencing for identification of anti-VEGFR2 miRNAs. The angiogenic activities of the miRNAs were determined in both in vitro and in vivo mice models. Results: Three miRNAs, namely miR-410-3p, miR-497-5p, and miR-2355-5p, were identified to be upregulated in CAD-ECFCs, and VEGFR2 was their common target gene. Knockdown of these miRNAs not only restored the expression of VEGFR2 and increased angiogenic activities of CAD-ECFCs in vitro, but also promoted blood flow recovery in ischemic limbs in vivo. miR-410-3p, miR-497-5p, and miR-2355-5p could serve as potential biomarkers for CAD detection as they are highly expressed in the plasma of CAD patients. Conclusions: This modulation could help develop new therapeutic modalities for cardiovascular diseases and other vascular dysregulated diseases, especially tumor angiogenesis.

Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients

Functional impairment of endothelial colony-forming cells (ECFCs), a specific cell lineage of endothelial progenitor cells (EPCs) is highly associated with the severity of coronary artery disease (CAD), the most common type of cardiovascular disease (CVD). Emerging evidence show that circulating microRNAs (miRNAs) in CAD patients’ body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq), we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson’s correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up-regulation compared to healthy control. Knockdown of miR-146a-5p or miR-146b-5p in CAD ECFCs enhanced migration and tube formation activity in diseased ECFCs. Contrarily, overexpression of miR-146a-5p or miR-146b-5p in healthy ECFC repressed migration and tube formation in ECFCs. TargetScan analysis showed that miR-146a-5p and miR-146b-5p target many of the angiogenesis-related genes that were down-regulated in CAD ECFCs. Knockdown of miR-146a-5p or miR-146b-5p restores CAV1 and RHOJ levels in CAD ECFCs. Reporter assays confirmed the direct binding and repression of miR-146a-5p and miR-146b-5p to the 3’-UTR of mRNA of RHOJ, a positive regulator of angiogenic potential in endothelial cells. Consistently, RHOJ knockdown inhibited the migration and tube formation ability in ECFCs. Collectively, we discovered the dysregulation of miR-146a-5p/RHOJ and miR-146b-5p/RHOJ axis in the plasma and ECFCs of CAD patients that could be used as biomarkers or therapeutic targets for CAD and other angiogenesis-related diseases.