Endothelial Progenitor Cells Tube Formation Research Articles

GroEL of Porphyromonas gingivalis-induced microRNAs accelerate tumor neovascularization by downregulating thrombomodulin expression in endothelial progenitor cells.

We found that GroEL in Porphyromonas gingivalis accelerated tumor growth and increased mortality in tumor-bearing mice; GroEL promoted proangiogenic function, which may be the reason for promoting tumor growth. To understand the regulatory mechanisms by which GroEL increases the proangiogenic function of endothelial progenitor cells (EPCs), we explored in this study. In EPCs, MTT assay, wound-healing assay, and tube formation assay were performed to analyze its activity. Western blot and immunoprecipitation were used to study the protein expression along with next-generation sequencing for miRNA expression. Finally, a murine tumorigenesis animal model was used to confirm the results of in vitro. The results indicated that thrombomodulin (TM) direct interacts with PI3 K/Akt to inhibit the activation of signaling pathways. When the expression of TM is decreased by GroEL stimulation, molecules in the PI3 K/Akt signaling axis are released and activated, resulting in increased migration and tube formation of EPCs. In addition, GroEL inhibits TM mRNA expression by activating miR-1248, miR-1291, and miR-5701. Losing the functions of miR-1248, miR-1291, and miR-5701 can effectively alleviate the GroEL-induced decrease in TM protein levels and inhibit the proangiogenic abilities of EPCs. These results were also confirmed in animal experiments. In conclusion, the intracellular domain of the TM of EPCs plays a negative regulatory role in the proangiogenic capabilities of EPCs, mainly through direct interaction between TM and PI3 K/Akt to inhibit the activation of signaling pathways. The effects of GroEL on tumor growth can be reduced by inhibiting the proangiogenic properties of EPCs through the inhibition of the expression of specific miRNAs.

Additional Azaphilones from the Marine Algae-Derived Fungus Penicillium sclerotiorum with Anti-Angiogenic Activity

Abstract Sixteen azaphilone derivatives, including four new compounds, penicilazaphilones H and I (1 and 2), 11-epi-geumsanol F (3), and 11-epi-geumsanol B (4), together with twelve known analogues (5–16), were isolated from the algae-derived fungus Penicillium sclerotiorum purified from fresh marine macroalgae Grateloupia sp. The planar structures of 1–4 were identified by HRESIMS, IR, NMR, and UV spectroscopic analyses. Their absolute configurations were unambiguously determined by comparing the NMR and ECD spectra. These secondary metabolites were evaluated on the anti-angiogenic effect in human endothelial progenitor cells (EPCs). As a result, compounds 12, 14, and 16 exhibited anti-angiogenic activities by blocking cell growth, migration, and tube formation of EPCs. Additionally, the structure–activity relationships (SAR) for anti-angiogenic effects of isolated azaphilones were deduced.

Nrf2 transcriptional upregulation of IDH2 to tune mitochondrial dynamics and rescue angiogenic function of diabetic EPCs

Endothelial progenitor cells (EPCs) are reduced in number and impaired in function in diabetic patients. Whether and how Nrf2 regulates the function of diabetic EPCs remains unclear. In this study, we found that the expression of Nrf2 and its downstream genes were decreased in EPCs from both diabetic patients and db/db mice. Survival ability and angiogenic function of EPCs from diabetic patients and db/db mice also were impaired. Gain- and loss-of-function studies, respectively, showed that knockdown of Nrf2 increased apoptosis and impaired tube formation in EPCs from healthy donors and wild-type mice, while Nrf2 overexpression decreased apoptosis and rescued tube formation in EPCs from diabetic patients and db/db mice. Additionally, proangiogenic function of Nrf2-manipulated mouse EPCs was validated in db/db mice with hind limb ischemia. Mechanistic studies demonstrated that diabetes induced mitochondrial fragmentation and dysfunction of EPCs by dysregulating the abundance of proteins controlling mitochondrial dynamics; upregulating Nrf2 expression attenuated diabetes-induced mitochondrial fragmentation and dysfunction and rectified the abundance of proteins controlling mitochondrial dynamics. Further RNA-sequencing analysis demonstrated that Nrf2 specifically upregulated the transcription of isocitrate dehydrogenase 2 (IDH2), a key enzyme regulating tricarboxylic acid cycle and mitochondrial function. Overexpression of IDH2 rectified Nrf2 knockdown- or diabetes-induced mitochondrial fragmentation and EPC dysfunction. In a therapeutic approach, supplementation of an Nrf2 activator sulforaphane enhanced angiogenesis and blood perfusion recovery in db/db mice with hind limb ischemia. Collectively, these findings indicate that Nrf2 is a potential therapeutic target for improving diabetic EPC function. Thus, elevating Nrf2 expression enhances EPC resistance to diabetes-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia likely via transcriptional upregulating IDH2 expression and improving mitochondrial function of diabetic EPCs.

Abstract P3011: Nrf2 Transcriptional Upregulation Of Idh2 To Tune Mitochondrial Dynamics And Rescue Angiogenic Function Of Diabetic Epcs

Endothelial progenitor cells (EPCs) are reduced in number and impaired in function in diabetic patients. Whether and how Nrf2 regulates the function of diabetic EPCs remains unclear. In this study, we found that the expression of Nrf2 and its downstream genes were decreased in EPCs from both diabetic patients and db/db mice. Survival ability and angiogenic function of EPCs from diabetic patients and db/db mice also were impaired. Gain- and loss-of-function studies, respectively, showed that knockdown of Nrf2 increased apoptosis and impaired tube formation in EPCs from healthy donors and wild-type mice, while Nrf2 overexpression decreased apoptosis and rescued tube formation in EPCs from diabetic patients and db/db mice. Additionally, proangiogenic function of Nrf2-manipulated mouse EPCs was validated in db/db mice with hind limb ischemia. Mechanistic studies demonstrated that diabetes induced mitochondrial fragmentation and dysfunction of EPCs by dysregulating the abundance of proteins controlling mitochondrial dynamics; upregulating Nrf2 expression attenuated diabetes-induced mitochondrial fragmentation and dysfunction and rectified the abundance of proteins controlling mitochondrial dynamics. Further RNA-sequencing analysis demonstrated that Nrf2 specifically upregulated the transcription of isocitrate dehydrogenase 2 (IDH2), a key enzyme regulating tricarboxylic acid cycle and mitochondrial function. Overexpression of IDH2 rectified Nrf2 knockdown- or diabetes-induced mitochondrial fragmentation and EPC dysfunction. In a therapeutic approach, supplementation of an Nrf2 activator sulforaphane enhanced angiogenesis and blood perfusion recovery in db/db mice with hind limb ischemia. Collectively, these findings indicate that Nrf2 is a potential therapeutic target for improving diabetic EPC function. Thus, elevating Nrf2 expression enhances EPC resistance to diabetes-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia likely via transcriptional upregulation of IDH2 expression and improving mitochondrial function of diabetic EPCs.

CXCR7 Agonist TC14012 Improves Angiogenic Function of Endothelial Progenitor Cells via Activating Akt/eNOS Pathway and Promotes Ischemic Angiogenesis in Diabetic Limb Ischemia.

Endothelial progenitor cells (EPCs) play a critical role in repairing damaged vessels and triggering ischemic angiogenesis, but their number is reduced and function is impaired under diabetic conditions. Improving EPC function has been considered a promising strategy to ameliorate diabetic vascular complications. In the present study, we aim to investigate whether and how CXCR7 agonist TC14012 promotes the angiogenic function of diabetic EPCs. High glucose (HG) treatment was used to mimic the hyperglycemia in diabetes. Tube formation, cell scratch recovery and transwell assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and cleaved-caspase3 expression were used to evaluate the angiogenic capability, cell migration, and apoptosis of EPCs, respectively. Hind limb ischemia (HLI) model was used to appraise the ability of TC14012 in promoting diabetic ischemic angiogenesis in vivo. HG treatment impaired EPC tube formation and migration, and induced EPC apoptosis and oxidative damage, while TC14012 rescued tube formation and migration, and prevented HG-induced apoptosis and oxidative damage of EPCs. Furthermore, these beneficial effects of TC14012 on EPCs were attenuated by specific siRNAs against CXCR7, validating that CXCR7 is a functional target of TC14012 in EPCs. Mechanistic studies demonstrated that HG treatment reduced CXCR7 expression in EPCs, and impaired Akt and endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production; similarly, these signal impairments in HG-exposed EPCs could be rescued by TC14012. However, the protective effects of TC14012 on tube formation and migration, Akt and eNOS phosphorylation, and NO production in HG-treated EPCs were almost completely abolished by siRNAs against CXCR7 or Akt specific inhibitor wortmannin. More importantly, in vivo study showed that TC14012 administration enhanced blood perfusion recovery and angiogenesis in the ischemic hind limb and increased the EPC number in peripheral circulation of db/db mice, demonstrating the capability of TC14012 in promoting EPC mobilization and ischemia angiogenic function. TC14012 can prevent EPCs from HG-induced dysfunction and apoptosis, improve eNOS activity and NO production via CXCR7/Akt signal pathway, and promote EPC mobilization and diabetic ischemia angiogenesis.

Effects of adipose-derived stromal cells and endothelial progenitor cells on adipose transplant survival and angiogenesis.

BackgroundA paracrine mechanism is thought to mediate the proangiogenic capacity of adipose-derived stromal/stem cells (ASCs). However, the precise mechanism by which ASCs promote the formation of blood vessels by endothelial progenitor cells (EPCs) is unclear.MethodsThe EPCs-ASCs cocultures prepared in different ratios were subjected to tube formations assay to verify whether ASCs could directly participate in the tube genesis. The supernatant from cultured ASCs was used to stimulate EPCs to evaluate the effects on the angiogenic property of EPCs, as well as capacity for migration and invasion. A coculture model with transwell chamber were used to explore the regulation of angiogenesis markers expression in EPCs by ASCs. We then mixed ASCs with EPCs and transplanted them with adipose tissue into nude mice to evaluate the effects on angiogenesis in adipose tissue grafts.ResultsIn the EPCs-ASCs cocultures, the tube formation was significantly decreased as the relative abundance of ASCs increased, while the ASCs was found to migrate and integrated into the agglomerates formed by EPCs. The supernatant from ASCs cultures promoted the migration and invasion of EPCs and the ability to form capillary-like structures. The expression of multiple angiogenesis markers in EPCs were significantly increased when cocultured with ASCs. In vivo, ASCs combined with EPC promoted vascularization in the fat transplant. Immunofluorescence straining of Edu and CD31 indicated that the Edu labeled EPC did not directly participate in the vascularization inside the fat tissue.ConclusionsADSC can participate in the tube formation of EPC although it cannot form canonical capillary structures. Meanwhile, Soluble factors secreted by ASCs promotes the angiogenic potential of EPCs. ASCs paracrine signaling appears to promote angiogenesis by increasing the migration and invasion of EPCs and simultaneously upregulating the expression of angiogenesis markers in EPCs. The results of in vivo experiments showed that ASCs combined with EPCs significantly promote the formation of blood vessels in the fat implant. Remarkably, EPCs may promote angiogenesis by paracrine regulation of endogenous endothelial cells (ECs) rather than direct participation in the formation of blood vessels.

MiRNA-130 Promotes Migration and Angiogenesis of Endothelial Progenitor Cells Through PI3K/AKT/mTOR Pathways

<sec> <title>Aim:</title> In this study, we aimed to investigate the effects and mechanisms of miRNA-130a in human endothelial progenitor cells (EPCs) involved in Deep vein thrombosis (DVT). </sec> <sec> <title>Methods:</title> EPCs were isolated and identified by cell morphology and surface marker detection. The effect of miR-130a on the migration, invasion and angiogenesis of EPCs in vitro were also detected. In addition, whether miR-130a is involved in the MMP-1 expression and Akt/PI3K/mTOR signaling pathway was also demonstrated. </sec> <sec> <title>Results:</title> Results suggested that miRNA-130a promotes migration, invasion, and tube formation of EPCs by positively regulating the expression of MMP-1 through Akt/PI3K/mTOR signaling pathway. </sec> <sec> <title>Conclusion:</title> Thus, as a potential therapeutic target, miRNA-130a may play an important role in the treatment of DVT. </sec>

Exogenous H2S reverses high glucose-induced endothelial progenitor cells dysfunction via regulating autophagy

ABSTRACT This study aims to determine the effect of exogenous hydrogen sulfide (H2S) under high glucose (HG)-induced injury in endothelial progenitor cells (EPCs), and to explore the possible underlying mechanisms. Mononuclear cells were isolated from the peripheral blood of healthy volunteers by density-gradient centrifugation and identified as late EPCs by immunofluorescence and flow cytometry. EPCs were treated with high concentrations of glucose, H2S, Baf-A1, 3-MA or rapamycin. Cell proliferation, cell migration and tube formation were measured using cell counting kit-8, Transwell migration and tube formation assays, respectively. Cellular autophagy flux was detected by RFP-GFP-LC3, and Western blotting was used to examine the protein expression levels of LC3B, P62, and phosphorylated endothelial nitric oxide synthase (eNOS) at Thr495 (p-eNOSThr495). Reactive oxygen species (ROS) levels were measured using a DHE probe. H2S and rapamycin significantly reversed the inhibitory effects of HG on the proliferation, migration, and tube formation of EPCs. Moreover, H2S and rapamycin led to an increase in the number of autophagosomes accompanied by a failure in lysosomal turnover of LC3-II or p62 and p-eNOSThr495 expression and ROS production under the HG condition. However, Baf-A1 and 3-MA reversed the effects of H2S on cell behavior. Collectively, exogenous H2S ameliorated HG-induced EPC dysfunction by promoting autophagic flux and decreasing ROS production by phosphorylating eNOSThr495.

Up-regulated miR-204-5p promoted the migration, invasion, and angiogenesis of endothelial progenitor cells to enhance the thrombolysis of rats with deep venous thrombosis by targeting SPRED1

Deep venous thrombosis (DVT) endangers human health. Endothelial progenitor cells (EPCs) were proven to promote thrombolysis and miR-204-5p was discovered to be low-expressed in DVT patients. This study concentrated on exploring whether miR-204-5p had a regulatory effect on EPCs and DVT. Concretely, the expression of miR-204-5p in DVT patients’ blood was detected by qRT-PCR. The target of miR-204-5p was predicted by bioinformatics and verified by dual-luciferase reporter assay. After rat EPCs were isolated, identified, and transfected with miR-204-5p agomiR, antagomiR, or SPRED1 plasmids, the viability, migration, invasion, and tube formation of EPCs were detected by MTT, wound healing, Transwell, and tube formation assays, respectively. MiR-204-5p, SPRED1, p-PI3K, PI3K, p-AKT, AKT, VEGFA, and Ang1 expressions in EPCs were measured by qRT-PCR or Western blot. EPCs transfected with miR-204-5p overexpression lentivirus plasmid were injected into the DVT rat model. The histopathology of the thrombus and the homing of EPCs to thrombus in the DVT rats were observed by hematoxylin-eosin staining and confocal microscopy, respectively. We found that miR-204-5p was low-expressed in DVT patients and SPRED1 was a target gene of miR-204-5p. MiR-204-5p agomiR promoted the viability, migration, invasion, and tube formation of EPCs, the levels of VEGFA and Ang1 and the activation of PI3K/AKT pathway in EPCs, while miR-204-5p antagomiR and SPRED1 worked oppositely. SPRED1 reversed the effect of miR-204-5p agomiR on EPCs. Up-regulated miR-204-5p inhibited thrombosis and promoted EPCs homing to thrombus in DVT rats. Collectively, up-regulated miR-204-5p enhanced the angiogenesis of EPCs and thrombolysis in DVT rats by targeting SPRED1.

Pharmaceutical Activation of Nrf2 Accelerates Diabetic Wound Healing by Exosomes from Bone Marrow Mesenchymal Stem Cells.

Background and ObjectivesDespite advances in wound treatments, chronic diabetic wounds remain a significant medical challenge. Exosomes from mesenchymal stem cells (MSCs) and small molecule activators of nuclear factor erythroid 2–related factor 2 (Nrf2) have emerged as potential therapies for nonhealing diabetic wounds. This study aimed to evaluate the effects of exosomes from bone marrow-derived MSCs (BMSCs) alone, or in combination with a small molecule activator of Nrf2 on diabetic wound healing.Methods and ResultsBMSCs and endothelial progenitor cells (EPCs) were isolated from the femur and tibia bone marrow of Sprague-Dawley (SD) rats and culture-expanded. Exosomes were harvested from the BMSC culture supernatants through ultracentrifugation. The effects of the exosomes and Nrf2 knockdown, alone or in combination, on EPC tube formation were evaluated. Streptozotocin-induced diabetic rats bearing a fresh full-thickness round wound were treated with the exosomes alone, or in combination with a lentiviral shRNA targeting Nrf2 (Lenti-sh-Nrf2) or tert-butylhydroquinone (tBHQ), a small molecule activator of Nrf2. Two weeks later, wound closure, re-epithelization, collagen deposition, neovascularization, and local inflammation were evaluated. BMSC exosomes promoted while Nrf2 knockdown inhibited EPC tube formation. BMSC exosomes accelerated wound closure, re-epithelization, collagen deposition, and neovascularization, and reduced wound inflammation in diabetic rats. These regenerative and anti-inflammatory effects of the exosomes were inhibited by Lenti-sh-Nrf2 but enhanced by tBHQ administration.ConclusionsBMSC exosomes in combination with a small molecule Nrf2 activator hold promise as a new therapeutic option for chronic diabetic wounds.

S1P Increases VEGF Production in Osteoblasts and Facilitates Endothelial Progenitor Cell Angiogenesis by Inhibiting miR-16-5p Expression via the c-Src/FAK Signaling Pathway in Rheumatoid Arthritis.

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. Vascular endothelial growth factor (VEGF) stimulation of endothelial progenitor cells (EPCs) facilitates angiogenesis and the progression of RA. Phosphorylation of sphingosine kinase 1 (SphK1) produces sphingosine-1-phosphate (S1P), which increases inflammatory cytokine production, although the role of S1P in RA angiogenesis is unclear. In this study, we evaluated the impact of S1P treatment on VEGF-dependent angiogenesis in osteoblast-like cells (MG-63 cells) and the significance of SphK1 short hairpin RNA (shRNA) on S1P production in an in vivo model. We found significantly higher levels of S1P and VEGF expression in synovial fluid from RA patients compared with those with osteoarthritis by ELISA analysis. Treating MG-63 cells with S1P increased VEGF production, while focal adhesion kinase (FAK) and Src siRNAs and inhibitors decreased VEGF production in S1P-treated MG-63 cells. Conditioned medium from S1P-treated osteoblasts significantly increased EPC tube formation and migration by inhibiting miR-16-5p synthesis via proto-oncogene tyrosine-protein kinase src (c-Src) and FAK signaling in chick chorioallantoic membrane (CAM) and Matrigel plug assays. Infection with SphK1 shRNA reduced angiogenesis, articular swelling and cartilage erosion in the ankle joints of mice with collagen-induced arthritis (CIA). S1P appears to have therapeutic potential in RA treatment.

Gleevec and Rapamycin Synergistically Reduce Cell Viability and Inhibit Proliferation and Angiogenic Function of Mouse Bone Marrow-Derived Endothelial Progenitor Cells

Objective: This study investigates the synergistic effects of Gleevec (imatinib) and rapamycin on the proliferative and angiogenic properties of mouse bone marrow-derived endothelial progenitor cells (EPCs). Materials and Methods: EPCs were isolated from mouse bone marrow and treated with different concentrations of Gleevec or rapamycin individually or in combination. The cell viability and proliferation were examined using the MTT assay. An analysis of cell cycle and apoptosis was performed using flow cytometry. Formation of capillary-like tubes was examined in vitro, and the protein expression of cell differentiation markers was determined using Western blot analysis. Results: Gleevec significantly reduced cell viability, cell proliferation, and induced cell apoptosis in EPCs. Rapamycin had similar effects on EPCs, but it did not induce cell apoptosis. The combination of Gleevec and rapamycin reduced the cell proliferation but increased cell apoptosis. Although rapamycin had no demonstratable effect on tube formation, the combined therapy of Gleevec and rapamycin significantly reduced tube formation when compared with Gleevec alone. Mechanistically, Gleevec, but not rapamycin, induced a significant elevation in caspase-3 activity in EPCs, and it attenuated the expression of the endothelial protein marker platelet-derived growth factor receptor α. Functionally, rapamycin, but not Gleevec, significantly enhanced the expression of endothelial differentiation marker proteins, while attenuating the expression of mammalian target of rapamycin signaling-related proteins. Conclusions: Gleevec and rapamycin synergistically suppress cell proliferation and tube formation of EPCs by inducing cell apoptosis and endothelial differentiation. Mechanistically, it is likely that rapamycin enhances the proapoptotic and antiangiogenic effects of Gleevec by promoting the endothelial differentiation of EPCs. Given that EPCs are involved in the pathogenesis of some cardiovascular diseases and critical to angiogenesis, pharmacological inhibition of EPC proliferation by combined Gleevec and rapamycin therapy may be a promising approach for suppressing cardiovascular disease pathologies associated with angiogenesis.

Additional alkaloids from Zoanthus vietnamensis with neuroprotective and anti-angiogenic effects

A chemical investigation of the zoantharian Zoanthus vietnamensis, collected off Taiwan, yielded eleven new alkaloids, 7α-hydroxykuroshine J (1), 18β-hydroxykuroshine J (2), 5α-hydroxyzoanthenamine (3), 5β-hydroxyzoanthenamine (4), 14α-hydroxyzoanthenamine (5), 30-hydroxyzoanthenamine (6), 11-dehydroxy-18-epi-kuroshine A (7), 5α-hydroxykuroshine A (8), 7β-hydroxykuroshine A (9), 11-keto-oxyzoanthamine (10), and 30-hydroxyzoanthamine (11), along with eight known compounds (12–19). The structures of these compounds were identified by detailed spectroscopic data, including HRESIMS, IR, NMR, and UV spectra. All secondary metabolites isolated from Z. vietnamensis were investigated for the anti-angiogenic effect in human endothelial progenitor cells (EPCs). Compounds 6, 7, 11, and 13 exhibited mild anti-angiogenic effect by blocking cell growth and tube formation of EPCs. The neuroprotective potential of four major compounds 12, 14, 15, and 19 against paclitaxel-induced neurotoxicity was evaluated. Pretreatment of 14 and 15 protected paclitaxel-damaged neurite outgrowth of dorsal root ganglion (DRG) neurons, without interfering the cytotoxic activity of paclitaxel on cervical cancer SiHa cells.

MiR-222-3p downregulation prompted the migration, invasion and recruitment of endothelial progenitor cells via ADIPOR1 expression increase-induced AMKP activation

BackgroundClinical data show that aneurysm rupture causes high mortality in aged men. MicroRNAs (miRNAs) were reported to regulate endothelial progenitor cells (EPCs) which play a vital role in repairing endothelial damage and maintaining vascular integrity. This study identified a novel miRNA regulator for the functions of EPCs in aneurysm repair. MethodsAbdominal aortic aneurysm (AAA) model was established on Sprague-Dawley rats which later underwent antagomiR-222 treatment. The histopathological changes of AAA rats were examined by hematoxylin-eosin staining. Flow cytometry was performed to quantify EPCs in peripheral blood and identify EPCs isolated from the rat femur. The potential target of miR-222-3p was predicted by TargetScan v7.2 and validated by Dual-luciferase reporter assay. The effects of miR-222-3p and ADIPOR1 on the migration, invasion and tube formation of EPCs were evaluated by wound healing, Transwell and tube formation assays. The expressions of miR-222-3p and ADIPOR1 in aortic aneurysm tissues and EPCs were assessed by qRT-PCR or Western blot. ResultsAAA exhibited histopathological abnormality, a decreased number of EPCs in the peripheral blood and an increased miR-222-3p expression. AntagomiR-222 injection reversed all these phenomena in AAA rats. Upregulating miR-222-3p expression inhibited the migration, invasion, and tube formation of EPCs, and the expressions of ADIPOR1 and phosphorylated-AMKP, while downregulating miR-222-3p expression exerted opposite effects in EPCs. ADIPOR1 was identified as a target gene of miR-222-3p. Overexpressing ADIPOR1 abrogated the effects of miR-222-3p upregulation on EPCs. ConclusionDownregulated miR-222-3p prompted the migration, invasion and recruitment of EPCs by targeting ADIPOR1-induced AMKP activation.

MiR-17-5p promotes the endothelialization of endothelial progenitor cells to facilitate the vascular repair of aneurysm by regulating PTEN-mediated PI3K/AKT/VEGFA pathway

ABSTRACT The endothelialization of endothelial progenitor cells (EPCs) was proven to facilitate the vascular repair of aneurysm. MiR-17-5p regulated angiogenesis in various cancers. This research focused on exploring the effect of miR-17-5p on EPCs and the vascular repair of aneurysm. In vivo study: the aneurysm rat model was established and treated with AgomiR-17-5p; the histopathology of aneurysm tissues was examined by hematoxylin-eosin staining; and the level of EPCs in the aneurysm tissues and peripheral blood of rats were evaluated by immunofluorescence and flow cytometry, respectively. In vitro study: EPCs were cultured and identified using flow cytometry; the target of miR-17-5p was proven by dual-luciferase reporter assay; after transfection, the viability, migration, and tube formation of the EPCs were detected by MTT, wound healing, and tube formation assays, respectively; the expressions of VEGFA and factors related to PTEN-mediated PI3K/AKT pathway were detected by ELISA, qPCR, or Western blot as needed. In vivo study: miR-17-5p overexpression promoted the vascular repair in aneurysm rats and increased the level of EPCs in the aneurysm tissues and peripheral blood of the rats. In vitro study: miR-17-5p overexpression promoted the viability, migration, and tube formation of EPCs, up-regulated the expressions of VEGFA, p-PI3K, and p-AKT, and down-regulated the PTEN expression in EPCs; miR-17-5p silencing did the opposite; PTEN was targeted by miR-17-3p and further abrogated the effects of miR-17-5p overexpression on EPCs. MiR-17-5p promoted the endothelialization of EPCs to facilitate the vascular repair of aneurysm by regulating PTEN-mediated PI3K/AKT/VEGFA pathway.

Cyclic Stretch Induces Vascular Smooth Muscle Cells to Secrete Connective Tissue Growth Factor and Promote Endothelial Progenitor Cell Differentiation and Angiogenesis

Endothelial progenitor cells (EPCs) play a vital role in endothelial repair following vascular injury by maintaining the integrity of endothelium. As EPCs home to endothelial injury sites, they may communicate with exposed vascular smooth muscle cells (VSMCs), which are subjected to cyclic stretch generated by blood flow. In this study, the synergistic effect of cyclic stretch and communication with neighboring VSMCs on EPC function during vascular repair was investigated. In vivo study revealed that EPCs adhered to the injury site and were contacted to VSMCs in the Sprague–Dawley (SD) rat carotid artery injury model. In vitro, EPCs were cocultured with VSMCs, which were exposed to cyclic stretch at a magnitude of 5% (which mimics physiological stretch) and a constant frequency of 1.25 Hz for 12 h. The results indicated that stretched VSMCs modulated EPC differentiation into mature endothelial cells (ECs) and promoted angiogenesis. Meanwhile, cyclic stretch upregulated the mRNA expression and secretion level of connective tissue growth factor (CTGF) in VSMCs. Recombinant CTGF (r-CTGF) treatment promoted endothelial differentiation of EPCs and angiogenesis, and increased their protein levels of FZD8 and β-catenin. CTGF knockdown in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of the FZD8/β-catenin signaling pathway. FZD8 knockdown repressed endothelial differentiation of EPCs and their angiogenic activity. Wnt signaling inhibitor decreased the endothelial differentiation and angiogenetic ability of EPCs cocultured with stretched VSMCs. Consistently, an in vivo Matrigel plug assay demonstrated that r-CTGF-treated EPCs exhibited enhanced angiogenesis; similarly, stretched VSMCs also induced cocultured EPC differentiation toward ECs. In a rat vascular injury model, r-CTGF improved EPC reendothelialization capacity. The present results indicate that cyclic stretch induces VSMC-derived CTGF secretion, which, in turn, activates FZD8 and β-catenin to promote both differentiation of cocultured EPCs into the EC lineage and angiogenesis, suggesting that CTGF acts as a key intercellular mediator and a potential therapeutic target for vascular repair.

MiR-143-3p targets ATG2B to inhibit autophagy and promote endothelial progenitor cells tube formation in deep vein thrombosis

Deep vein thrombosis (DVT) is a common disease in vascular surgery. In recent study, microRNA (miRNA) plays a regulatory role in function of Endothelial progenitor cells (EPCs), which showed promising therapeutic choice for DVT. However, the function of miR-143-3p in EPCs remains incomplete. Flow cytometry was used to identify EPCs surface markers. Cell viability, migration, invasion and tube formation of EPCs were detected by 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium broide (MTT), wound healing, transwell and tube formation assay, respectively. TargetScan was used to predict miR-143-3p targeting genes. Dual-luciferase report assay was used to verify the interactions between miR-143-3p and autophagy-related 2B (ATG2B). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to examine the mRNA expression levels of ATG2B and miR-143-3p. Western blot was used to examine the protein expression levels of ATG2B, LC3 and p62. The cultured EPCs showed cobblestone morphology and were identified by cell surface markers. Overexpression of miR-143-3p enhanced the viability, migration, invasion and tube formation of EPCs, but low expression of miR-143-3p obtained the reverse results. ATG2B directly bound to miR-143-3p. Overexpression of miR-143-3p reduced the expression of ATG2B, but low expression of miR-143-3p increased. Overexpression of miR-143-3p decreased the expression of LC3I/II, but increased the expression of p62. Overexpression of ATG2B reversed the above-mentioned effects of EPCs which regulated by overexpression of miR-143-3p. MiR-143-3p targets ATG2B to modulate the function of EPCs and recanalization and resolution of DVT.

Down-regulation of miR-361-5p promotes the viability, migration and tube formation of endothelial progenitor cells via targeting FGF1

Transplantion of bone marrow-derived endothelial progenitor cells (EPCs) may be a novel treatment for deep venous thrombosis (DVT). The present study probed into the role of microRNA (miR)-361-5p in EPCs and DVT recanalization. EPCs were isolated from male Sprague–Dawley (SD) rats and identified using confocal microscopy and flow cytometry. The viability, migration and tube formation of EPCs were examined using MTT assay, wound-healing assay and tube formation assay, respectively. Target gene and potential binding sites between miR-361-5p and fibroblast growth factor 1 (FGF1) were predicted by StarBase and confirmed by dual-luciferase reporter assay. Relative expressions of miR-361-5p and FGF1 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. A DVT model in SD rats was established to investigate the role of EPC with miR-361-5p antagomir in DVT by Hematoxylin–Eosin (H&E) staining. EPC was identified as 87.1% positive for cluster of difference (CD)31, 2.17% positive for CD133, 85.6% positive for von Willebrand factor (vWF) and 94.8% positive for vascular endothelial growth factor receptor-2 (VEGFR2). MiR-361-5p antagomir promoted proliferation, migration and tube formation of EPCs and up-regulated FGF1 expression, thereby dissolving thrombus in the vein of DVT rats. FGF1 was the target of miR-361-5p, and overexpressed FGF1 reversed the effects of up-regulating miR-361-5p on suppressing EPCs. Down-regulation of miR-361-5p enhanced thrombus resolution in vivo and promoted EPC viability, migration and angiogenesis in vitro through targeting FGF1. Therefore, miR-361-5p may be a potential therapeutic target for DVT recanalization.

Exendin-4 reverses high glucose-induced endothelial progenitor cell dysfunction via SDF-1β/CXCR7-AMPK/p38-MAPK/IL-6 axis.

Exendin-4, a glucagon-like peptide-1 (GLP-1) analog, has been used for treating diabetes mellitus (DM). However, its effects on improving the dysfunction of high glucose (HG)-induced endothelial progenitor cells (EPCs) remain unclear. The present study explored the effects of Exendin-4 on improving dysfunction of EPCs and the underlying mechanism. EPCs were isolated from SD rats and identified by flow cytometry. Next, the EPCs were treated by HG and high or low concentration of Exendin-4, and cell viability, migration and tube formation were, respectively, examined by performing MTT assay, wound-healing assay and tube formation assay. Interleukin-6 (IL-6) secretion was measured by enzyme-linked immunosorbent assay (ELISA). The protein expressions of relative stromal-derived growth factor-1β (SDF-1β), C-X-C chemokine receptor type 7 (CXCR7), AMP-activated protein kinase (AMPK), p38 and expressions of CXCR7 and IL-6 in EPCs were measured by Western blot. The cell behaviors of EPCs treated by HG and Exendin-4 with or without silencing of CXCR7 and IL-6 were detected. Exendin-4 reversed the inhibitory effects of HG on viability, migration and tube formation of EPCs and on SDF-1β/CXCR7-AMPK pathway in EPCs in a dose-dependent manner. Moreover, Exendin-4 promoted the effects of HG on IL-6 level in EPCs through the promotion of p38-MAPK phosphorylation and reduction of cleaved caspase-3 protein expressions in EPCs. However, silencing of CXCR7 and IL-6 reversed the effects of Exendin-4 on cell behaviors, inactivated SDF-1β/CXCR7-AMPK pathway and increased cleaved caspase-3 expression in EPCs. Exendin-4 could ameliorate HG-induced EPC dysfunction through regulating the production of IL-6 via SDF-1β/CXCR7-AMPK/p38-MAPK axis.

Interleukin-1β augments the angiogenesis of endothelial progenitor cells in an NF-κB/CXCR7-dependent manner.

Endothelial progenitor cells (EPCs) are able to trigger angiogenesis, and pro‐inflammatory cytokines have beneficial effects on angiogenesis under physiological and pathological conditions. C‐X‐C chemokine receptor type 7 (CXCR‐7), receptor for stromal cell‐derived factor‐1, plays a critical role in enhancing EPC angiogenic function. Here, we examined whether CXCR7 mediates the pro‐angiogenic effects of the inflammatory cytokine interleukin‐1β (IL‐1β) in EPCs. EPCs were isolated by density gradient centrifugation and angiogenic capability was evaluated in vitro by Matrigel capillary formation assay and fibrin gel bead assay. IL‐1β elevated CXCR7 expression at both the transcriptional and translational levels in a dose‐ and time‐dependent manner, and blockade of the nuclear translocation of NF‐κB dramatically attenuated the IL‐1β‐mediated up‐regulation of CXCR7 expression. IL‐1β stimulation significantly promoted EPCs tube formation and this effect was largely impaired by CXCR7‐siRNA transfection. IL‐1β treatment stimulated extracellular signal‐regulated kinase 1/2 (Erk1/2) phosphorylation, and inhibition of Erk1/2 phosphorylation partially impaired IL‐1β‐induced tube formation of EPCs but without significant effects on CXCR7 expression. Moreover, blocking NF‐κB had no significant effects on IL‐1β‐stimulated Erk1/2 phosphorylation. These findings indicate that CXCR7 plays an important role in the IL‐1β‐enhanced angiogenic capability of EPCs and antagonizing CXCR7 is a potential strategy for inhibiting angiogenesis under inflammatory conditions.