Angiogenesis Of Endothelial Progenitor Cells Research Articles

Mitochondrial Dysfunction-Mediated Decline in Angiogenic Capacity of Circulating Endothelial Progenitor Cells is Associated with Capillary Rarefaction in Patients with Hypertension

Hypertensive patients exhibit decline in capillary density and endothelial progenitor cells (EPCs). However, whether capillary rarefaction in hypertension is associated with defect angiogenesis of EPCs remains unknown. We hypothesized that impaired mitochondrial function of late EPCs in hypertension is associated with the structural lack of capillary microcirculation via deficient CXCR4/JAK2/SIRT5 signaling. Capillary density of nail fold was significantly reduced in hypertensive patients, which was paralleled to decreased in vitro late EPC function and in vivo angiogenic capacity. Meanwhile the decline of EPC function in hypertension was accompanied by impaired mitochondrial ultrastructure, diminished mitochondrial membrane potential, reduced oxygen consumption, increased ROS generation and NADH level. Furthermore, SIRT5 expression of EPCs in hypertension was markedly reduced, which was correlated to mitochondrial dysfunction. CXCR4 gene transfer enhanced SIRT5 expression, improved mitochondrial functions and augmented angiogenic capacity of EPCs. The beneficial impacts of SIRT5 up-regulation on late EPC-mediated angiogenesis can be abrogated by blockade of CXCR4/JAK2/SIRT5 signaling pathway. Mitochondrial dysfunction-mediated fall in angiogenic capacity due to deficient CXCR4/JAK2/SIRT5 signaling of late EPCs is probably responsible for the capillary rarefaction in hypertension. Our findings provide insight into the potential of EPC mitochondria as a novel target for the treatment of hypertension-related loss of microvascular density. Funding: This work was supported by the National Nature Science Foundation (31530023, 81500205, 81671379) of China, 973 Program (2013CB531200), the Nature Science Foundation (2016A030310184, 2016A020215056) of Guangdong and Science and Technology Planning Project (2017A020215085) of Guangdong. Declaration of Interest: None. Ethical Approval: The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China).

CXCR7 Agonist TC14012 Improves Angiogenic Function of Endothelial Progenitor Cells in Diabetic Limb Ischemia

Stromal cell-derived factor 1 (SDF-1)-chemokine receptors CXCR4/7 axis plays a vital role in diabetic limb ischemia. Our previous studies demonstrated that elevated CXCR7 improves therapeutic efficacy of endothelial progenitor cells (EPCs) in diabetic limb ischemia. The effects of a specific CXCR7 agonist TC14012 on diabetic hind limb ischemia remains untested. We hypothesize that CXCR7 expression is reduced in diabetic EPCs and TC14012 improve angiogenic function of EPCs in diabetic limb ischemia. Early WT-EPCs were transfected with siRNA against CXCR7, and early db/db-EPCs were infected with CXCR7 lentivirus. Tube formation and capillary density of ischemic gastrocnemius muscle (GS) and soleus muscle (SS) at 4 weeks post-ischemic surgery were adopt for indication of angiogenesis. Diabetic hind limb ischemia (HLI) models were established by femoral artery ligation, followed by administration of TC14012 subcutaneously. Total and cell surface expression of CXCR7 were significantly decreased in EPCs from db/db mice compared with those from WT mice, whereas no significant differences of CXCR4 expression were observed. Tube formation by EPCs from db/db mice was also impaired compared with WT-EPCs. siRNA knockdown of CXCR7 was accompanied by impaired tube formation. Conversely, increasing CXCR7 levels in db/db EPCs completely reversed impaired tube formation function. High glucose (HG) dose dependently decreased the expression of CXCR7 in vitro but not that of CXCR4. HG also impaired tube formation of EPC from WT mice. Tube formation function of HUVEC was impaired after treated with HG (25mM) for 24 hours, which could be prevented by TC14012. Most importantly, TC14012 significantly improved blood flow restoration of ischemic hind limb starting from 2 weeks after ligation and promoted angiogenesis of ischemic GS and SS. Diabetes attenuates CXCR7 expression and impairs angiogenesis of EPCs. TC14012 improves blood reperfusion of diabetic hind limb ischemia. Disclosure K. Wang: None. X. Dai: None. J. Chen: None. P.N. Epstein: None. L. Cai: None. K.A. Wintergerst: None. Y. Qian: None. Y. Tan: None.

Thymosin β4 promotes endothelial progenitor cell angiogenesis via a vascular endothelial growth factor‑dependent mechanism.

Endothelial progenitor cells (EPCs) are a promising cell source for tissue repair and regeneration, predominantly through angiogenesis promotion. Paracrine functions serve a pivotal role in EPC‑mediated angiogenesis, which may be impaired by various cardiovascular risk factors. Therefore, it is important to identify a solution that optimizes the paracrine function of EPCs. Thymosin β4 (Tβ4) is a peptide with the potential to promote tissue regeneration and wound healing. A previous study demonstrated that Tβ4 enhances the EPC‑mediated angiogenesis of the ischemic myocardium. In the present study, whether Tβ4 improved angiogenesis by enhancing the paracrine effects of EPCs was investigated. A tube formation assay was used to assess the effect of angiogenesis, and the paracrine effects were measured using an ELISA kit. The results indicated that Tβ4 improved the paracrine effects of EPCs, evidenced by an increase in the expression of vascular endothelial growth factor (VEGF). EPC‑conditioned medium (EPC‑CM) significantly promoted human umbilical vein endothelial cell angiogenesis invitro, which was further enhanced by pretreatment with Tβ4. The effect of Tβ4 pretreated EPC‑CM on angiogenesis was abolished by VEGF neutralizing antibody invitro, indicating that increased VEGF secretion had a pivotal role in Tβ4‑mediated EPC angiogenesis. Furthermore, transplantation of EPCs pretreated with Tβ4 into infarcted rat hearts resulted in significantly higher VEGF expression in the border zone, compared with EPC transplantation alone. To further investigate whether the Akt/eNOS pathway was involved in Tβ4‑induced VEGF secretion in EPCs, the expression levels of VEGF in EPC‑CM were significantly decreased following knockdown of Akt or eNOS by small interfering RNA transfection. In conclusion, Tβ4 significantly increased angiogenesis by enhancing the paracrine effects of EPCs, evidenced by the increased expression of VEGF. The RAC‑α serine/threonine‑protein kinase/endothelial nitric oxide synthase signal transduction pathway was involved in the regulation of Tβ4‑induced VEGF secretion in EPCs. Further studies are required to investigate the long‑term prognosis of patients with coronary heart disease following Tβ4‑pretreated EPC transplantation.

MiR-503 suppresses hypoxia-induced proliferation, migration and angiogenesis of endothelial progenitor cells by targeting Apelin

Endothelial progenitor cells (EPCs) are of great importance in the process of endogenous blood vessel repair to maintain endothelial integrity and have been applied in a wide range of models of ischemic diseases. MicroRNAs represent a class of non-protein coding endogenous RNAs with 19–24 nucleotides in length and serve an important role in multiple physiological and pathological processes, including angiogenesis. It has been reported that miR-503 reduces angiogenesis in tumorigenesis. However, to our knowledge, the precise role of miR-503 in the regulation of EPCs remains unclear. In the current study, we found that the expression of miR-503 was decreased in mouse bone marrow derived EPCs under the hypoxic condition. Importantly, upregulation of miR-503 suppressed the proliferation, migration and capillary-like tube formation of EPCs induced by hypoxia. Furthermore, a dual luciferase reporter assay showed that Apelin, an endogenous ligand of the G protein-coupled receptor APJ, was a direct target of miR-503 and overexpression of miR-503 significantly inhibited the protein level of Apelin in EPCs. Moreover, hypoxia treatment enhanced the expression of Apelin in EPCs. Meanwhile ectopic expression of Apelin promoted cellular proliferation, migration and tube formation of EPCs in vitro. In summary, our results indicate that miR-503 regulates proliferation, migration and angiogenesis of EPCs by targeting Apelin.

MeCP2-mediated epigenetic regulation in senescent endothelial progenitor cells

BackgroundCellular aging may be associated with epigenetics. Methyl-CpG-binding protein 2 (MeCP2) and sirtuin 1 (SIRT1) are two important epigenetic factors. Our former work demonstrated that MeCP2 expression increased and SIRT1 expression decreased in senescent endothelial progenitor cells (EPCs). This article aims to reveal the epigenetic regulation caused by MeCP2 in EPCs and discuss its mechanism.MethodsTube formation assay and cell apoptosis detection were used to evaluate the function of senescent EPCs induced by MeCP2 overexpression. Western blot analysis was used to testify the relative protein expression changed by MeCP2. Bisulfite sequencing methylation assay and chromatin immunoprecipitation assay were used to assess the degree of methylation and the relation of MeCP2 and SIRT1.ResultsMeCP2 reduced angiogenesis of senescent EPCs, promoted apoptosis, and caused senescent EPC dysfunction through SIRT1 promoter hypermethylation and histone modification.ConclusionsMeCP2 mediated senescent EPC dysfunction through epigenetic regulation.

Inhibition of Dll4/Notch1 pathway promotes angiogenesis of Masquelet\u2019s induced membrane in rats

The Masquelet’s induced membrane technique for repairing bone defects has been demonstrated to be a promising treatment strategy. Previous studies have shown that the vessel density of induced membrane is decreased in the late stage of membrane formation, which consequently disrupts the bone healing process. However, relatively little is known about certain mechanisms of vessel degeneration in the induced membrane tissue and whether promotion of angiogenesis in induced membranes can improve bone regeneration. Here, we showed that the Delta-like ligand 4/ Notch homolog 1 (Dll4/Notch1) pathway was relatively activated in the late stage of induced membrane, especially at the subcutaneous site. Then, DAPT, a classical γ-secretase inhibitor, was applied to specifically inhibit Notch1 activation, followed by up-regulation of vascular endothelial growth factor receptor 2 (VEGFR2) and CD31 expression. DAPT-modified induced membranes were further confirmed to contribute to bone regeneration after autogenous bone grafting. Finally, in vitro experiments revealed that knocking down Notch1 contributed to the functional improvement of endothelial progenitor cells (EPCs) and that DAPT-treated induced membrane tissue was more favorable for angiogenesis of EPCs compared with the vehicle group. In conclusion, the present findings demonstrate that Dll4/Notch1 signaling is negatively associated with the vessel density of induced membrane. Pharmacological inhibition of Notch1 attenuated the vessel degeneration of induced membrane both in vitro and in vivo, which consequently improved bone formation at the bone defect site and graft resorption at the subcutaneous site.

Icariin doped bioactive glasses seeded with rat adipose-derived stem cells to promote bone repair via enhanced osteogenic and angiogenic activities

AimsCell communication between mesenchymal stem cells and blood vessel cells are crucial for bone repair. We have previously shown that the phyto-molecule icariin significantly promoted osteogenic differentiation of rat adipose-derived stem cells (ASCs). In the present study, we aimed to investigate the relationship between icariin induced osteogenic differentiation of ASCs and angiogenesis of rat endothelial progenitor cells (EPCs). Besides, we used icariin doped 45S5 Bioglass seeded with ASCs to promote bone healing in rat calvarial bone defect models. Main methodsThe conditioned medium from undifferentiated ASCs (ASCs-CM) and icariin induced ASCs (Icariin-ASCs-CM) was obtained and the vascular endothelial growth factor (VEGF) protein secretion level was measured. The angiogenic capacity and molecular mechanism of ASC-CM and Icariin-ASCs-CM on rat EPCs was analyzed. Rat calvarial bone defect models were established and treated with scaffolds implantation. Micro-CT imaging, histological and immunohistological staining were performed on the isolated specimens at 12 weeks post-surgery. Key findingsVEGF protein expression was significantly increased after icariin treatment with the highest expression in the 10−7 M icariin group. Icariin-ASCs-CM obviously increased the angiogenesis of rat EPCs and this capacity was inhibited by a VEGF/VEGF receptor-specific binding inhibitor bevacizumab. Results of the in vivo investigations showed that all scaffolds promoted bone healing compared to the Control group. Icariin significantly improved the healing capacity of 45S5 Bioglass seeded with ASCs. SignificanceImplantation of Icariin/45S5 Bioglass seeded with rat ASCs could obviously promote both osteogenesis and angiogenesis and therefore represents an ideal candidate bone substitutes for bone repair and regeneration.

BMP-2 induces angiogenesis by provoking integrin α6 expression in human endothelial progenitor cells

Bone morphogenetic protein-2 (BMP-2) is a multifunctional cytokine, capable of governing several cellular functions, including proliferation, motility, differentiation, and angiogenesis. Circulating endothelial progenitor cells (EPCs) have been shown to facilitate tissue repair, postnatal neovascularization, and tumor associated angiogenesis. Nevertheless, the impact of BMP-2 on angiogenesis of human EPCs has largely remained a mystery. In this study, we found that BMP-2 promoted cell migration and tube formation of EPCs in a concentration-dependent manner, indicating BMP-2 induced in vitro angiogenesis in human EPCs. Furthermore, BMP-2 significantly increased microvessel formation in Matrigel plug assay, and BMP-2 antagonist noggin prevented BMP-2-induced in vivo angiogenesis. Mechanistic investigations showed BMP-2 profoundly induced the expression of Id-1 and integrin α6 as well as EPCs angiogenesis by activating PI3K/Akt and MEK/ERK signaling pathways. Moreover, knockdown of Id-1 and integrin α6 by siRNA transfection obviously attenuated BMP-2-indueced tube formation of EPCs. These results suggest that BMP-2 promotes angiogenesis in human EPCs through the activation of PI3K/Akt, MEK/ERK, and Id-1/integrin α6 signaling cascades. This is the first demonstration that BMP-2 exhibits the angiogenesis property on human EPCs. BMP-2 might serve as the potential therapeutic target for treatment of angiogenesis-related diseases.

Resveratrol Improves Tube Formation in AGE-Induced Late Endothelial Progenitor Cells by Suppressing Syndecan-4 Shedding.

Dysfunction of endothelial progenitor cells (EPCs) contributes to cardiovascular complications in diabetes, and resveratrol has been shown to improve EPC functions. Syndecan-4 (Synd4), a cell surface heparin sulfate proteoglycan, has been shown to promote neovascularization. Thus, the present study was performed to determine whether resveratrol promoted angiogenesis of EPCs by regulating Synd4. Late EPCs were isolated from human peripheral blood and stimulated with AGEs. Western blot showed that AGEs induced Synd4 shedding in a dose- and time-dependent manner. AGE-induced Synd4 shedding was partly reversed by NAC or resveratrol, along with normalized ROS production. Overexpression of Synd4 or pretreatment of resveratrol reversed AGE-impaired tube formation of EPCs and regulated the Akt/eNOS pathway. Furthermore, resveratrol suppressed Synd4 shedding via the inhibition of oxidative stress and improved tube formation of late EPCs via the regulation of the Synd4/Akt/eNOS pathway.

The synergistic effect of bone forming peptide-1 and endothelial progenitor cells to promote vascularization of tissue engineered bone.

Large segmental bone defect repair remains a challenge in orthopedic surgeries. The tissue engineered bone graft will be a promising approach if vascularization of the graft is realized. In this study, beta-tricalcium phosphate (β-TCP) scaffold incorporated with bone forming peptide-1 (BFP-1) was fabricated. Endothelial progenitor cells (EPCs) were introduced as well. We investigated the effect of BFP-1 on the proliferation, differentiation, and angiogenic functions of EPCs. Additionally, segmental femur bone defect was created in rabbits. Prevascularized β-TCP scaffold was constructed and implanted into the bone defect. The vascularization and bone formation were evaluated after 4 and 12 weeks. The results showed that BFP-1 promoted the angiogenesis of EPCs through activating the activin receptor-like kinase-1/Smad pathway. The prevascularized tissue engineered bone graft enhanced capillary vessel in-growth and new bone formation. Significantly higher values of vascularization and radiographic grading scores were observed in groups involving EPCs and BFP-1, compared to β-TCP scaffold alone. In conclusion, the synergy between EPCs and BFP-1 improved the vascularization and new bone regeneration, which has great potentials in clinical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1008-1021, 2018.

Graphene-Based MicroRNA Transfection Blocks Preosteoclast Fusion to Increase Bone Formation and Vascularization.

The objective of this study is to design a graphene‐based miRNA transfection drug delivery system for antiresorptive therapy. An efficient nonviral gene delivery system is developed using polyethylenimine (PEI) functionalized graphene oxide (GO) complex loaded with miR‐7b overexpression plasmid. GO‐PEI complex exhibits excellent transfection efficiency within the acceptable range of cytotoxicity. The overexpression of miR‐7b after GO‐PEI‐miR‐7b transfection significantly abrogates osteoclast (OC) fusion and bone resorption activity by hampering the expression of an essential fusogenic molecule dendritic cell‐specific transmembrane protein. However, osteoclastogenesis occurs without cell–cell fusion and preosteoclast (POC) is preserved. Through preservation of POC, GO‐PEI‐miR‐7b transfection promotes mesenchymal stem cell osteogenesis and endothelial progenitor cells angiogenesis in the coculture system. Platelet‐derived growth factor‐BB secreted by POC is increased by GO‐PEI‐miR‐7b both in vitro and in vivo. In treating osteoporotic ovariectomized mice, GO‐PEI‐miR‐7b significantly enhances bone mineral density, bone volume as well as bone vascularization through increasing CD31hiEmcnhi cell number. This study provides a cell–cell fusion targeted miRNA transfection drug delivery strategy in treating bone disorders with excessive osteoclastic bone resorption.

Tanshinone IIA inhibits angiogenesis in human endothelial progenitor cells in vitro and in vivo.

Accumulating evidence reports that bone marrow-derived endothelial progenitor cells (EPCs) regulate angiogenesis, postnatal neovascularization and tumor metastasis. It has been suggested that understanding the molecular targets and pharmacological functions of natural products is important for novel drug discovery. Tanshinone IIA is a major diterpene quinone compound isolated from Danshen (Salvia miltiorrhiza) and is widely used in traditional Chinese medicine (TCM). Evidence indicates that tanshinone IIA modulates angiogenic functions in human umbilical vein endothelial cells. However, the anti-angiogenic activity of tanshinone IIA in human EPCs has not been addressed. Here, we report that tanshinone IIA dramatically suppresses vascular endothelial growth factor (VEGF)-promoted migration and tube formation of human EPCs, without cytotoxic effects. We also show that tanshinone IIA markedly inhibits VEGF-induced angiogenesis in the chick embryo chorioallantoic membrane (CAM) model. Importantly, tanshinone IIA significantly attenuated microvessel formation and the expression of EPC-specific markers in the in vivo Matrigel plug assay in mice. Further, we found that tanshinone IIA inhibits EPC angiogenesis through the PLC, Akt and JNK signaling pathways. Our report is the first to reveal that tanshinone IIA reduces EPC angiogenesis both in vitro and in vivo. Tanshinone IIA is a promising natural product worthy of further development for the treatment of cancer and other angiogenesis-related pathologies.

High glucose and free fatty acids induce endothelial progenitor cell senescence via PGC-1α/SIRT1 signaling pathway.

The objective of the research was to investigate the function of endothelial progenitor cells (EPCs) in the conditions of high glucose and lipids, which has been widely used to mimic the metabolic disorder that occurs in type 2 diabetic mellitus, and further to verify the role of PGC-1α and SIRT1, cellular energy metabolism regulators, in the process of senescence of EPCs with these combined stimuli. Circulating EPCs were incubated in absence or presence of high glucose (25 mM), FFA (200 µM) or both. EPCs senescence was assessed by β-galactosidase staining, EPCs telomerase activity was measured by telomeric repeat ampli-fication protocol assay, in vitro angiogenesis assay and MTT assays were performed to assess angiogenesis and proliferation ability of EPCs. The results showed that combined stimuli inhibited EPCs reendothelialization ability in vitro, accelerated EPCs senescence and decreased the telomerase activity. Meanwhile, with combined stimuli, the expression of PGC-1α increased whereas SIRT1 expression decreased in EPCs accompanied by activation of P53/P21 signaling pathway. Conversely, transfection of EPCs with PGC-1α-siRNA rescued EPCs premature senescence and up-regulated SIRT1 and decreased P53/P21 expression, correlating closely with the down-regulation of PGC-1α itself. In addition, the combined stimuli induced up-regulation of PGC-1α expression was partly mediated by ROS and P38 signaling pathway. Overall, the data presented here identify PGC-1α as a potent negative regulator of EPCs' senescence under combined stimuli, which is partly mediated by SIRT1/P53/P21 signaling pathway.

Toll‑like receptor 4 is expressed and functional in late endothelial progenitor cells.

It has been previously demonstrated that lipopolysaccharides (LPS) inhibit the viability, migration, adhesion and invitro angiogenesis of late endothelial progenitor cells (EPCs). However, the mechanisms underlying this LPS‑induced impairment of late EPC functional activity are unknown. The aim of the present study was to investigate whether Toll‑like receptor 4 (TLR4) is expressed and functional on late EPCs, using late EPCs of 3‑5 passages. Cells were deprived of serum for 24h prior to experiments and incubated with 10µg/ml LPS for 24h with or without pretreatment with 2µg/ml TLR4 signaling inhibitor CLI‑095 for 30min. The viability, migration, adhesion and invitro angiogenesis, as well as the expression of silent information regulator1 (SIRT1), in late EPCs were evaluated. Treatment with 10µg/ml LPS decreased the viability, migration and adhesion abilities, and invitro angiogenesis of late EPCs. Pretreatment with the TLR4 signaling inhibitor reversed this LPS‑induced dysfunction of late EPCs. LPS downregulated the expression of SIRT1 protein, however, blocking TLR4 attenuated the effect of LPS on SIRT1 expression. Therefore, the results of the present study indicate that LPS impaired the functional activity of late EPCs via TLR4, which may be associated with decreased SIRT1 expression.

Curcumin induces therapeutic angiogenesis in a diabetic mouse hindlimb ischemia model via modulating the function of endothelial progenitor cells

BackgroundNeovascularization is impaired in diabetes mellitus, which leads to the development of peripheral arterial disease and is mainly attributed to the dysfunction of endothelial progenitor cells (EPCs). Previous studies proved the promotional effect of curcumin on neovascularization in wound healing of diabetes. Thus, we hypothesize that curcumin could promote neovascularization at sites of hindlimb ischemia in diabetes and might take effect via modulating the function of EPCs.MethodsStreptozotocin-induced type 1 diabetic mice and nondiabetic mice both received unilateral hindlimb ischemic surgery. Curcumin was then administrated to the mice by lavage for 14 days consecutively. Laser Doppler perfusion imaging was conducted to demonstrate the blood flow reperfusion. Capillary density was measured in the ischemic gastrocnemius muscle. In addition, angiogenesis, migration, proliferation abilities, and senescence were determined in EPCs isolated from diabetic and nondiabetic mice. Quantitative PCR was then used to determine the mRNA expression of vascular endothelial growth factor (VEGF) and angiopoetin-1 (Ang-1) in EPCs.ResultsCurcumin application to type 1 diabetic mice significantly improved blood reperfusion and increased the capillary density in ischemic hindlimbs. The in-vitro study also revealed that the angiogenesis, migration, and proliferation abilities of EPCs and the number of senescent EPCs were reversed by curcumin application. Quantitative PCR confirmed the overexpression of VEGF-A and Ang-1 in EPCs after curcumin treatment.ConclusionCurcumin could enhance neovascularization via promoting the function of EPCs in a diabetic mouse hindlimb ischemia model.

YKL-40-Induced Inhibition of miR-590-3p Promotes Interleukin-18 Expression and Angiogenesis of Endothelial Progenitor Cells.

YKL-40, also known as human cartilage glycoprotein-39 or chitinase-3-like-1, is a pro-inflammatory protein that is highly expressed in rheumatoid arthritis (RA) patients. Angiogenesis is a critical step in the pathogenesis of RA, promoting the infiltration of inflammatory cells into joints and providing oxygen and nutrients to RA pannus. In this study, we examined the effects of YKL-40 in the production of the pro-inflammatory cytokine interleukin-18 (IL-18), and the stimulation of angiogenesis and accumulation of osteoblasts. We observed that YKL-40 induces IL-18 production in osteoblasts and thereby stimulates angiogenesis of endothelial progenitor cells (EPCs). We found that this process occurs through the suppression of miR-590-3p via the focal adhesion kinase (FAK)/PI3K/Akt signaling pathway. YKL-40 inhibition reduced angiogenesis in in vivo models of angiogenesis: the chick embryo chorioallantoic membrane (CAM) and Matrigel plug models. We report that YKL-40 stimulates IL-18 expression in osteoblasts and facilitates EPC angiogenesis.

WISP-3 inhibition of miR-452 promotes VEGF-A expression in chondrosarcoma cells and induces endothelial progenitor cells angiogenesis.

Chondrosarcoma is the second most prevalent general primary tumor of bone following osteosarcoma. Chondrosarcoma development may be linked to angiogenesis, which is principally elicited by vascular endothelial growth factor-A (VEGF-A). VEGF-A level has been recognized as a prognostic marker in angiogenesis. WNT1-inducible signaling pathway protein-3 (WISP)-3/CCN6 belongs to the CCN family and is involved in regulating several cellular functions, including cell proliferation, differentiation, and migration. Nevertheless, the effect of WISP-3 on VEGF-A production and angiogenesis in human chondrosarcoma remains largely unknown. This current study shows that WISP-3 promoted VEGF-A production and induced angiogenesis of human endothelial progenitor cells. Moreover, WISP-3-enhanced VEGF-A expression and angiogenesis involved the c-Src and p38 signaling pathways, while miR-452 expression was negatively affected by WISP-3 via the c-Src and p38 pathways. Our results illustrate the clinical significance of WISP-3, VEGF-A and miR-452 in human chondrosarcoma patients. WISP-3 may illustrate a novel therapeutic target in the metastasis and angiogenesis of chondrosarcoma.

Thymosin β4 promotes the survival and angiogenesis of transplanted endothelial progenitor cells in the infarcted myocardium.

The survival of transplanted stem cells in ischemic tissue is poor. In the present study, the effects of thymosin β4 (Tβ4) on the survival and angiogenesis of endothelial progenitor cells (EPCs) and improvement in cardiac functions after transplantation of Tβ4-treated EPCs in the infarcted myocardium were investigated. EPCs were isolated from bone marrow of adult male rats and incubated in Endothelial Basal Medium-2. Then the cells were treated with Tβ4 at different concentrations (0.05, 0.1 and 0.2 µM), and cells incubated with DMEM were set as controls. MTT assay, Transwell assay and tube formation in Matrigel were used to detect cell viability, migration and angiogenesis, respectively. For examining the protective effect of Tβ4 on EPCs, the cells were also incubated in the condition of hypoxia and serum deprivation. p-Akt expression was also examined using western blot analysis. Rat models of myocardial infarction (MI) were established by ligation of the anterior descending branch of the left coronary artery. At four weeks after intramyocardial injection of Tβ4-treated EPCs, the changes in cardiac functions, size of the scar tissue and density of microvessels were examined by echocardiography, Masson's trichrome staining, immunohistochemistry and fluorescence in situ hybridization (FISH) for the Y-chromosome. Tβ4 enhanced EPC viability, protected the cells from apoptosis in hypoxia and serum deprivation, and promoted the proliferation and migration of the cells and formation of capillary-like structures in the cells. Moreover, Tβ4 increased p-Akt expression in the cells. The cytoprotective and proangiogenic effects of Tβ4 were in a dose-dependent manner. Tβ4-treated EPCs improved cardiac function, enhanced the repair of the infarcted myocardium, and promoted angiogenesis after transplantation in the infarcted myocardium. In conclusion, pretreatment of EPCs with Tβ4 is a novel strategy for the repair of ischemic tissue after transplantation in MI.

CO2 laser increases the regenerative capacity of human adipose-derived stem cells by a mechanism involving the redox state and enhanced secretion of pro-angiogenic molecules.

CO2 laser has a beneficial effect on stem cells by mechanisms that are not clearly elucidated. We hypothesize that the effect of fractional CO2 laser on human adipose-derived stem cells (ADSC) could be due to changes in redox homeostasis and secretion of factors contributing to cellular proliferation and angiogenic potential. ADSC incubated in medium containing 0.5 or 10% FBS were exposed to a single irradiation of a 10,600-nm fractional CO2 laser; non-irradiated ADSC were used as control. Viability/proliferation of ADSC was assessed by MTT assay; the intracellular reactive oxygen species (ROS) levels and the mitochondrial membrane potential (∆Ψm) were determined with DCFH-DA and JC-1 fluorescent probes, respectively. Molecules secreted by ADSC in the medium were determined by ELISA assay, and their capacity to support endothelial tube-like formation by the Matrigel assay. The results showed that compared to controls, ADSC kept in low FBS medium and irradiated with CO2 laser at 9W exhibited: (a) increased proliferation (∼20%), (b) transient increase of mitochondrial ROS and the capacity to restore Δψm after rotenone induced depolarization, and (c) augmented secretion in the conditioned medium of MMP-2 (twofold), MMP-9 (eightfold), VEGF (twofold), and adiponectin (∼50%) that have the capacity to support angiogenesis of endothelial progenitor cells. In conclusion, the mechanisms underlying the benefic effect of CO2 laser on ADSC are the activation of the redox pathways which increases cell proliferation and enhances secretion of angiogenic molecules. These results explain, in part, the mechanisms involved in the increased regenerative potential of CO2 laser-exposed ADSC that could be exploited for clinical applications.

CCN1 Promotes VEGF Production in Osteoblasts and Induces Endothelial Progenitor Cell Angiogenesis by Inhibiting miR-126 Expression in Rheumatoid Arthritis.

Angiogenesis is the formation of new capillaries from preexisting vasculature. The perpetuation of angiogenesis plays a critical role in the pathogenesis of various disease states including rheumatoid arthritis (RA). Cysteine-rich 61 (Cyr61 or CCN1) is an important proinflammatory cytokine in RA. Here, we investigated the role of CCN1 in angiogenesis associated with vascular endothelial growth factor (VEGF) production and osteoblasts. We found higher expression of CCN1 and VEGF in synovial fluid from RA patients compared with healthy controls. CCN1 induced VEGF expression in osteoblasts and increased endothelial progenitor cells (EPCs) angiogenesis by inhibiting miR-126 via the protein kinase C-alpha (PKC-α) signaling pathway. CCN1 knockdown inhibited angiogenesis in both in vitro and in vivo models. Inhibition of CCN1 expression with lentiviral vectors expressing short hairpin RNA (shRNA) ameliorated articular swelling, cartilage erosion, and angiogenesis in the ankle joint of mice with collagen-induced arthritis (CIA). Our study is the first to describe how CCN1 promotes VEGF expression in osteoblasts and increased EPCs angiogenesis in RA disease. CCN1 may serve as a potential target for RA treatment. © 2016 American Society for Bone and Mineral Research.