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.
BackgroundMicroRNAs (miRNAs) have emerged as master regulators of angiogenesis and other cancer-related events. Discovering new angiogenesis-regulating microRNAs (angiomiRs) will eventually help in developing new therapeutic strategies for tumor angiogenesis and cardiovascular diseases. Kaposi’s sarcoma (KS), which is induced by the etiological infectious agent KS-associated herpesvirus (KSHV), is a peculiar neoplasm that expresses both blood and lymphatic endothelial markers and possesses extensive neovasculature. Using KSHV and its proteins as baits will be an efficient way to discover new angiomiRs in endothelial cells. Kaposin B is one of the latent viral genes and is expressed in all KSHV tumor cells. Since Kaposin B is a nuclear protein with no DNA-binding domain, it may regulate gene expression by incorporating itself into a transcription complex.ResultsWe demonstrated that c-Myc and Kaposin B form a transcription complex and bind to the miR-221/-222 promoter, thereby affecting their expression and anti-angiogenic ability. By small RNA sequencing (smRNA-Seq), we revealed that 72.1 % (173/240) of Kaposin B up-regulated and 46.5 % (113/243) of Kaposin B down-regulated known miRNAs were regulated by c-Myc. We also found that 77 novel miRNA were up-regulated and 28 novel miRNAs were down-regulated in cells expressing both c-Myc and Kaposin B compared with cells expressing Kaposin B only. The result was confirmed by RNA-IP-seq data.ConclusionsOur study identifies known and novel c-Myc-regulated microRNAs and reveals that a c-Myc-oriented program is coordinated by Kaposin B in KSHV-infected cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0242-3) contains supplementary material, which is available to authorized users.
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.
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, α 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. In addition to the number of circulating EPCs present, the regulation of postnatal angiogenesis/vasculogenesis also depends on the activity of mobilized EPCs. 8 In addition to the effects of protein-coding genes, the expression of various different microRNAs (miRNAs, miRs) also plays a critical role in the clinical course of CAD. miRNAs are a class of endogenous, small, noncoding, single-stranded RNAs of ≈22 nucleotides in length. The mature miRNAs negatively regulate gene expression by targeting specific mRNAs for cleavage or translational repression. Detecting the levels of tissue miRNAs, as well as the levels of circulating miRNAs in body fluids, is considered to be a potential approach to identifying new disease biomarkers and novel drug targets. Conclusions-Manipulating9-11 Many proangiogenic and antiangiogenic miRNAs have been pinpointed. Examples such as miR-221 and miR-222, which are transcribed from the same miR-221/222 cluster, have been shown to be able to modulate the angiogenic properties of human umbilical vein endothelial cells by targeting c-Kit and endothelial NO synthase.12,13 miR-221/222 levels in EPCs are significantly higher in patients with CAD.14,15 miR-10a* and miR-21 are also known to modulate EPC senescence via the suppression of high-mobility group A2.16 miR-10b and miR-196b, on the cont...
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.
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.
This nationwide population-based cohort study revealed a significantly higher risk of IBD in patients with CP compared with control group. Clinicians should notice this association to avoid delayed diagnosis of IBD in patients with CP.
Non-invasive far infrared radiation (FIR) has been observed to improve the health of patients with coronary artery disease (CAD). Endothelial colony forming cells (ECFCs) contribute to vascular repair and CAD. The goal of this study was to uncover the role of FIR in ECFCs function and to reveal potential biomarkers for indication of FIR therapy in CAD patients. FIR significantly enhanced in vitro migration (transwell assay) and tube formation (tube length) capacities in a subpopulation of CAD ECFCs. Clinical parameters associated with the responsiveness of ECFCs to FIR include smoking and gender. ECFCs from CAD patients that smoke did not respond to FIR in most cases. In contrast, ECFCs from females showed a higher responsiveness to FIR than ECFCs from males. To decipher the molecular mechanisms by which FIR modulates ECFCs functions, regardless of sex, RNA sequencing analysis was performed in both genders of FIR-responsive and FIR-non/unresponsive ECFCs. Gene Ontology (GO) analysis of FIR up-regulated genes indicated that the pathways enriched in FIR-responsive ECFCs were involved in cell viability, angiogenesis and transcription. Small RNA sequencing illustrated 18 and 14 miRNAs that are up-and down-regulated, respectively, in FIR-responsive CAD ECFCs in both genders. Among the top 5 up-and down-regulated miRNAs, down-regulation of miR-548aq-3p in CAD ECFCs after FIR treatment was observed in FIR-responsive CAD ECFCs by RT-qPCR. Down-regulation of miR-548aq-3p was correlated with the tube formation activity of CAD ECFCs enhanced by FIR. After establishment of the down-regulation of miR-548aq-3p by FIR in CAD ECFCs, we demonstrated through overexpression and knockdown experiments that miR-548aq-3p contributes to the inhibition of the tube formation of ECFCs. This study suggests the down-regulation of miR-548aq-3p by FIR may contribute to the improvement of ECFCs function, and represents a novel biomarker for therapeutic usage of FIR in CAD patients.open Scientific RepoRtS | (2020) 10:6805 | https://doi.org/10.1038/s41598-020-63311-1 www.nature.com/scientificreports www.nature.com/scientificreports/ progenitor cells (EPCs). The classification of EPC has been applied to multiple cell types, including endothelial colony forming cells (ECFCs) 2 . ECFCs exhibit the ability to form colonies after prolonged culture and proliferate after re-plating in an in vitro culture system. These cells are identified as positive for VE-cadherin, VEGFR2 (vascular endothelial growth factor receptor 2), VEGFR1, CD31, CD105, CD146, VWF (von Willebrand factor), CD34, and negative for CD14, CD45, CD115, CD133 3 . ECFCs have the potential to incorporate into the endothelial layer and differentiate into mature ECs, thereby contributing to repair of injured vascular endothelium as well as forming new blood vessels 4 . ECFCs can further enhance vasculogenic properties by releasing trophic factors 5 . With these capacities, ECFCs have been suggested to function in maintaining vascular homeostasis and preventing CAD. In line with this, increas...
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