Promoted Endothelial Progenitor Cells Migration Research Articles

Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

CD146 mediates the anti-apoptotic role of Netrin-1 in endothelial progenitor cells under hypoxic conditions.

Modulating the biological status of endothelial progenitor cells (EPCs), such as function and survival, is essential for therapeutic angiogenesis in ischemic vascular disease environments. This study aimed to explore the role and molecular mechanisms underlying Netrin-1 in the viability and angiogenic function of EPCs. EPCs were isolated from the bone barrow of adult C57/BL6 mice. The apoptosis and various functions of EPCs were analyzed in vitro by manipulating the expression of Netrin-1. The TUNEL assay was performed to detect apoptotic EPCs. Cell migration and tube formation assays were performed to detect EPC function. Trypan blue staining was performed to detect cell viability. Western blot analysis was performed to detect the protein expression levels of Netrin-1, CD146 and apoptotic factors. Quantitative PCR analysis was performed to detect the expression levels of Netrin-1 receptors. The results demonstrated that treatment with exogenous Netrin-1 promoted EPC migration and tube formation, whereas transfection with small interfering (si)RNA targeting Netrin-1 exhibited the opposite effects. Exogenous Netrin-1 protected EPCs from hypoxia-induced apoptosis, whereas the interruption of endogenous Netrin-1 enhancement under hypoxia by Netrin-1-siRNA exacerbated the apoptosis of EPCs. Furthermore, CD146, one of the immunoglobulin receptors activated by Netrin-1, was screened for in the present study. Results demonstrated that CD146 did not participate in Netrin-1-promoted EPC function, but mediated the anti-apoptotic effects of Netrin-1 in EPCs. In conclusion, Netrin-1 enhanced the angiogenic function of EPCs and alleviated hypoxia-induced apoptosis, which was mediated by CD146. This biological function of Netrin-1 may provide a potential therapeutic option to promote EPCs for the treatment of ischemic vascular diseases.

Downregulation of miR-103a-3p Contributes to Endothelial Progenitor Cell Dysfunction in Deep Vein Thrombosis Through PTEN Targeting

Bone-marrow-derived endothelial progenitor cells (EPCs) can accelerate the dissolution of thrombi. However, EPC functions are weakened in deep vein thrombosis (DVT), and miR-130a-3p is downregulated in DVT. As little is known about the function of miR-130a-3p in EPCs, we aimed to explore the effects of miR-130a-3p on EPC functions and the mechanisms of miR-130a-3p regulation of EPCs in DVT. The EPCs were transfected with miR-130a-3p mimics or miR-130a-3p inhibitor. Migration and angiogenesis of EPCs were detected by wound healing, Transwell, and tube formation assays. Dual luciferase assay was used to test the relation of miR-130a-3p and phosphatase and tensin homolog (PTEN). Protein and mRNA levels of associated genes were measured by western blotting (WB) and qRT-PCR. miR-103a-3p could promote EPC migration and angiogenesis, and it was also downregulated in EPCs isolated from DVT patients. Moreover, PTEN was a target of miR-130a-3p. Upregulation of PTEN rescued the auxoaction of miR-130a-3p in EPC function. Downregulation of miR-103a-3p contributes to EPC dysfunction in DVT via targeting PTEN. Thus, miR-130a-3p may be a potential target for DVT treatment.

High-Mobility Group Box 1 (HMGB1) Induces Migration of Endothelial Progenitor Cell via Receptor for Advanced Glycation End-Products (RAGE)-Dependent PI3K/Akt/eNOS Signaling Pathway.

BackgroundHigh-mobility group box1 (HMGB1) is a cytokine that has been demonstrated to have an important role in inducing migration and homing of endothelial progenitor cells (EPCs) in the process of neovascularization during wound healing, but its specific mechanism remains elusive. The aim of this study was to investigate the effects of the HMGB-RAGE axis in EPC migration, as well as the underlying molecular mechanism responsible for these effects.Material/MethodsEPCs were isolated from the mice and identified using flow cytometry and fluorescence staining. The effect of HMGB1 on the activity of EPCs was detected using the Cell Counting Kit-8 (CCK-8). Then, the migration of EPCs was detected by scratch wound-healing and cell migration assay. NO levels were analyzed by ELISA. The expression of p-PI3K, p-Akt, and p-eNOS was determined by Western blot analysis. RAGE expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. F-actin was assessed by fluorescent staining.ResultsThe results showed that HMGB1 induced a concentration-dependent migration of EPCs, and the migration was RAGE-dependent. The migration could be almost completely blocked by PI3K inhibitors and eNOS inhibitor. HMGB1-RAGE upregulated the expression of p-Akt, p-eNOS, and p-ERK. We also demonstrated that the MEK/ERK signaling pathway is not involved in the EPC migration induced by HMGB1-RAGE.ConclusionsThese data demonstrate that HMGB1 activates RAGE and induces PI3K/Akt/eNOS signaling transduction pathway activation to promote EPC migration. Therefore, the HMGB1-RAGE axis plays an important role in the EPC migration process and may become a potential target in wound healing.

Thymosin β4 promotes glucose-impaired endothelial progenitor cell function via Akt/endothelial nitric oxide synthesis signaling pathway.

Circulating endothelial progenitor cells (EPCs) are a subtype of hematopoietic stem cells, which can differentiate into endothelial cells and restore endothelial function. However, high glucose decreases the number and impairs the function of EPCs. A previous study showed that thymosin β4 (Tβ4), a pleiotropic peptide beneficial for multiple functions of various types of cells, could promote EPC migration and dose-dependently upregulate the phosphorylation of Akt and endothelial nitric oxide synthesis signaling (eNOS). In present study, the hypothesis that Tβ4 can improve glucose-suppressed EPC functions via the Akt/eNOS signaling pathway and restores the production of nitric oxide (NO) is investigated. EPCs were isolated from the peripheral blood of healthy volunteers and formed a cobblestone shape after 3–4 weeks of cultivation. Then, EPCs were treated with high concentrations of glucose (25 mM) for 4 days and administrated with Tβ4 for further study. Transwell migration and tube formation assays were performed to access the migratory and angiogenic ability of EPCs. In addition, the quantity of Akt, eNOS and the concentration of nitric oxide (NO) was investigated. Functional studies showed that high concentrations of glucose significantly suppressed EPC function, while this adverse effect was reversed by the administration of Tβ4. In addition, Akt small interfering (si)RNA and eNOS siRNA were demonstrated to reduce the protective effect of Tβ4 against glucose-impaired EPC functions. These findings suggest that Tβ4 improves glucose-impaired EPC functions via the Akt/eNOS signaling pathway.

Effect of heparan sulfate mimetics from Escherichia coli K5 polysaccharide on SDF-1/CXCL12-induced endothelial progenitor cells in vitro

In tumorigenesis, CXCL12 level increases sharply because of tumor tissue hypoxia. As CXCR4 cells, endothelial progenitor cells (EPCs) are mobilized to tumor bed through the CXCL12/CXCR4 axis and are involved in tumor angiogenesis. In this process, either glycosaminoglycan (GAG) or heparan sulfate (HS) carried by membrane proteoglycans is implicated. Exogenous soluble HS mimetics can act as a competitive inhibitor of membranous HS, thereby preventing the formation of a normal signal axis. In this work, the effect of HS mimetics on the CXCL12-induced EPCs in vitro was investigated. HS mimetics, named as K5PSs, were obtained from sulfated Escherichia coli K5 polysaccharide/heparosan, and EPCs were collected from rat bone marrow. Results showed that CXCL12 could promote EPCs viability. This promotion might be related to its regulation of cell cycle and anti-apoptosis activity; it also could promote EPCs migration and secretion of pro-angiogenesis factors. All its functions were obtained by activation of MAPK/ERK pathway, FAK pathway, and PI3k/AKT pathway. However, its effect on EPCs was attenuated by K5PSs, and the existence of sulfate groups both at 2-O-position and N-position in K5PSs is essential to inhibit its effect on EPCs. This work suggested that K5PSs could be applied in anti-tumor treatment through inhibiting tumor angiogenesis.

Ghrelin induces cell migration through GHSR1a-mediated PI3K/Akt/eNOS/NO signaling pathway in endothelial progenitor cells

ObjectiveThe purpose of this research was to investigate the effects of ghrelin on circulating endothelial progenitor cells (EPC) directional migration and its underlying molecular mechanisms involved in this process. Materials/MethodsEPC were isolated from bone marrow of SD rats by using Percoll density gradient centrifugation, and characterized by double positive for acLDL-Dil uptake and FITC-UEA-1 binding and immunocytochemistry for CD34, CD133, vWF and Flk-1. EPC were treated with different concentrations of ghrelin (10−9~10−6M) with or without GHSR1a inhibitor [D-Lys3]-GHRP-6, PI3K inhibitor LY294002 and endothelial nitric oxide synthase (eNOS) inhibitor L-NAME, migration of EPC was detected by transwell assay, levels of phosphorylated and total Akt and eNOS were determined by Western-blot analysis and Nitric Oxide (NO) production was measured by Griess assay, respectively. ResultsEPC were successfully obtained by Percoll density gradient centrifugation and ghrelin at 10−8M~10−7M promoted EPC migration. Ghrelin-induced EPC migration was accompanied by phosphorylation of Akt and eNOS, as well as an increase in NO production. These biochemical events and EPC directional migration induced by ghrelin were completely inhibited by GHSR-1a blocker [D-Lys3]-GHRP-6. PI3K inhibitor LY294002 attenuated ghrelin-induced EPC migration, phosphorylation of Akt and eNOS, and NO production. eNOS inhibitor L-NAME blocked ghrelin-induced EPC migration, phosphorylation of eNOS, and NO production, but had no effect on Akt phosphorylation. ConclusionsThese findings suggest that ghrelin stimulates EPC directional migration via GHSR1a-mediated PI3K/Akt/eNOS/NO signal pathway. It indicates that ghrelin may be used as a therapeutic strategy to treat ischemic diseases by promoting EPC directional migration.

Globular adiponectin improves high glucose-suppressed endothelial progenitor cell function through endothelial nitric oxide synthase dependent mechanisms

Plasma levels of adiponectin, an adipose-specific protein with putative anti-atherogenic properties, could be down-regulated in obese and diabetic subjects. Recent insights suggest that the injured endothelial monolayer is regenerated by circulating endothelial progenitor cells (EPCs), but high glucose reduces number and functions of EPCs. Here, we tested the hypothesis that globular adiponectin can improve high glucose-suppressed EPC functions by restoration of endothelial nitric oxide synthase (eNOS) activity. Late EPCs isolated from healthy subjects appeared with cobblestone shape at 2–4 weeks. EPCs were incubated with high glucose (25 mM) and treatment with globular adiponectin for functional study. Migration and tube formation assays were used to evaluate the vasculogenetic capacity of EPCs. The activities of eNOS, Akt and concentrations of nitric oxide (NO) were also determined. Administration of globular adiponectin at physiological concentrations promoted EPC migration and tube formation, and dose-dependently upregulated phosphorylation of eNOS, Akt and augmented NO production. Chronic incubation of EPCs in high-glucose medium significantly impaired EPC function and induced cellular senescence, but these suppression effects were reversed by treatment with globular adiponectin. Globular adiponectin reversed high glucose-impaired EPC functions through NO- and p38 MAPK-related mechanisms. In addition, nude mice that received EPCs treated with adiponectin in high glucose medium showed a significant improvement in blood flow than those received normal saline and EPCs incubated in high glucose conditions. The administration of globular adiponectin improved high glucose-impaired EPC functions in vasculogenesis by restoration of eNOS activity. These beneficial effects may provide some novel rational to the vascular protective properties of adiponectin.

Macrophage migration inhibitory factor is a potential inducer of endothelial progenitor cell mobilization after flap operation

Endothelial progenitor cells (EPCs) promote angiogenesis and play an important role in tissue revascularization and wound healing. Yet, the exact stimuli and mechanisms for the mobilization remain understood poorly. Macrophage migration inhibitory factor (MIF), which is a structurally unique pleiotropic cytokine, has been suggested to play a role in EPC recruitment and thus was a target of this study. This study included 20 patients who underwent flap operation. Subjects were divided into 3 groups according to the pattern of flap applied. The number of circulating EPCs and serum levels of MIF or CXCL12 were determined at different time intervals. In vitro chemotaxis experiments using Transwell devices were carried out to test whether MIF promotes the chemotactic migration of EPCs. To underscore functionally the chemotactic potential of MIF toward EPCs in flap patients, the chemotactic effects of serum samples from all groups were also examined in the presence and absence of monoclonal anti-macrophage migration inhibitory factor and anti-CXCL12 antibodies on EPC recruitment using in vitro migration chambers. In flap patients, the number of circulating EPCs and serum levels of MIF but not CXCL12 serum levels were increased markedly compared with preoperative levels at day 1 after operation, especially in the group of free microvascular flaps. Serum levels of CXCL12 in the flap patients were increased only significantly compared with the healthy control group. An analysis between EPCs and MIF revealed a significant correlation, whereas no correlation was observed for CXCL12. MIF exerted a dose-dependent, prochemotactic effect on isolated human EPCs, and serum samples from all flap patients promoted EPC migration. Importantly, this effect was blocked partially by anti-macrophage migration inhibitory factor and to a weaker extent by anti-CXCL12 antibodies. We conclude that MIF plays an important role in the mobilization of EPCs, which is dependent on the degree of ischemia. Enhancement by MIF of chemotactic EPCs migration in vitro underpins its proposed in vivo function.

Over-expression of hepatocyte growth factor in smooth muscle cells regulates endothelial progenitor cells differentiation, migration and proliferation

Background Endothelial repair is one of key events after vascular injury. The mechanisms by which hepatocyte growth factor (HGF) and endothelial progenitor cells (EPCs) may be responsible for re-endothelialization of injured blood vessel wall are poorly understood. Methods Primary culture SMCs were transfected with pcDNA3.0-HGF followed by G418 selection, one of G418-resistant colonies in well was picked, propagated and used as donor cells for further experiments. HGF and VEGF expression in SMCs were detected with western blot and enzyme linked immunosorbent assays (ELISA). Rat EPCs were cultured in untreated, pcDNA3.0 and pcDNA3.0-HGF transfected SMCs conditioned medium with or without anti-VEGF or exogenous recombinant HGF addition. eNOS, KDR and CD31 expression in EPCs was determined by real-time quantitative polymerase chain reaction (RT-qPCR) or flow cytometry; EPCs migration and proliferation were measured by using a modified Boyden chambers and MTT assay respectively. Results Abundant and stable expression of HGF was found in G418-resistant colony-derived SMCs. VEGF expression significantly increased in HGF transfected SMCs. Exogenous recombinant HGF (rHGF) markedly up-regulated eNOS mRNA expression in EPCs and promoted EPCs migration and proliferation, but no significant changes were found in KDR and CD31 mRNA expression. HGF transfection in SMCs was more effective than exogenous HGF for EPCs differentiation, proliferation and migration. Conclusions Over-expression of HGF in SMCs can be helpful for promoting EPCs differentiation, increasing EPCs migration and proliferation. It may be responsible for angiogenesis of arteriosclerosis lesions and useful for blood vessel tissue engineering.