Paracrine mitogenic effect of human endothelial progenitor cells: role of interleukin-8

Endothelial progenitor cells (EPCs) play an important role in repair of vascular injury and neovascularization. Molecular mechanisms underlying vascular effects of EPCs are not fully understood. The present study was designed to test the hypothesis that human EPCs exert a strong paracrine mitogenic effect on mature endothelial cells. Levels of interleukin-8 (IL-8) were significantly higher in conditioned medium (CM) collected from EPCs than in CM derived from mature endothelial cells [umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (CAECs)]. CM of EPCs stimulated proliferation of HUVECs and CAECs. This mitogenic effect was partially inhibited by IL-8-neutralizing antibody. In contrast, CM of HUVECs and CAECs had a weak or no mitogenic effect on mature endothelial cells. Our results demonstrate significantly higher levels of IL-8 secretion by human EPCs than by mature endothelial cells. IL-8 appears to be an important mediator of the paracrine mitogenic effect of EPCs.     

A comparison of the tube forming potentials of early and late endothelial progenitor cells

The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 μm wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.     

重组福安泰-03抑制人脐静脉血管内皮细胞的迁移和增殖

研究重组福安泰-03(recombinant Fuantai-03,rFAT-03)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)迁移和增殖的影响.聚碳酸酯膜小室趋化运动模型(transwell model)检测rFAT-03对HUVECs迁移能力的影响;MTT法检测rFAT-03对HUVECs和多种人癌细胞生长的影响;荧光显微镜观察rFAT-03作用下HUVECs的形态变化;膜联蛋白V-异硫氰酸荧光素(annexin V-fluorescein isothiocyanate,annexinV-FITC)双染检测rFAT-03对HUVECs早期凋亡的影响;流式细胞术分析rFAT-03对HUVECs周期及凋亡的影响;Western印迹检查rFAT-03对HUVECs血管内皮细胞生长因子(VEGF)、Bax和Bcl-2表达的影响.结果显示,rFAT-03明显抑制HUVECs细胞的迁移和增殖,其抑制效果与剂量和作用时间相关.0.20mg/mL恩度(endostar),0.10、0.20mg/mLrFAT-03作用HUVECs24h,对细胞迁移的抑制率分别为32.0%、32.6%、57.1%(P0.01).0.20mg/mL恩度,0.05、0.10、0.20mg/mLrFAT-03作用HUVECs72h,其对细胞生长的抑制率分别为40.9%、63.7%、69.3%、87.0%.但rFAT-03对多种人癌细胞株的生长却无明显影响.rFAT-03处理HUVECs24h,细胞的早期凋亡率增加(P0.05).rFAT-03阻滞HUVECs于G0/G1期,并呈现典型的凋亡峰.终浓度为0.05、0.10、0.20mg/mLrFAT-03作用于HUVECs24h,G0/G1期细胞指数分别为63.4%、67.5%和75.7%(对照组为62.1%),凋亡指数分别为4.2%、8.5%和10.3%.rFAT-03下调HUVECs的VEGF和抑调亡基因Bcl-2的表达,上调促凋亡基因Bax的表达,其效果与剂量相关.结果提示,rFAT-03明显抑制HUVECs细胞的迁移和增殖,诱导其凋亡,它的这些作用与其下调VEGF、Bcl-2表达,上调Bax表达密切相关.     

Hypoxia‐induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non‐cognate ligand of the CXC‐family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine‐like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC‐derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia‐induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non‐classical mechanism and originated from pre‐formed MIF stores. Early hypoxia‐triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia‐inducible factor (HIF)‐1α‐induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF‐1α‐induced de novo synthesis of MIF. To functionally investigate the role of hypoxia‐inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL⁺ KDR⁺ CD31⁺ EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose‐dependent bell‐shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia‐conditioned HUVECs, an effect that was completely abrogated by anti‐MIF‐ or anti‐CXCR4‐antibodies. Thus, hypoxia‐induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia‐induced myocardial damage.     

CTRP3 protects against uric acid-induced endothelial injury by inhibiting inflammation and oxidase stress in rats

Hyperuricemia, which contributes to vascular endothelial damage, plays a key role in multiple cardiovascular diseases. This study was designed to investigate whether C1q/tumor necrosis factor (TNF)-related protein 3 (CTRP3) has a protective effect on endothelial damage induced by uric acid and its underlying mechanisms. Animal models of hyperuricemia were established in Sprague-Dawley (SD) rats through the consumption of 10% fructose water for 12 weeks. Then, the rats were given a single injection of Ad-CTRP3 or Ad-GFP. The animal experiments were ended two weeks later. In vitro, human umbilical vein endothelial cells (HUVECs) were first infected with Ad-CTRP3 or Ad-GFP. Then, the cells were stimulated with 10 mg/dL uric acid for 48 h after pretreatment with or without a Toll-like receptor 4 (TLR4)-specific inhibitor. Hyperuricemic rats showed disorganized intimal structures, increased endothelial apoptosis rates, increased inflammatory responses and oxidative stress, which were accompanied by reduced CTRP3 and elevated TLR4 protein levels in the thoracic aorta. In contrast, CTRP3 overexpression decreased TLR4 protein levels and ameliorated inflammatory responses and oxidative stress, thereby improving the morphology and apoptosis of the aortic endothelium in rats with hyperuricemia. Similarly, CTRP3 overexpression decreased TLR4-mediated inflammation, reduced oxidative stress, and rescued endothelial damage induced by uric acid in HUVECs. In conclusion, CTRP3 ameliorates uric acid-induced inflammation and oxidative stress, which in turn protects against endothelial injury, possibly by inhibiting TLR4-mediated inflammation and downregulating oxidative stress.     

Synthesis of an endogeneous lectin, galectin-1, by human endothelial cells is up-regulated by endothelial cell activation

The pattern of expression of an endogenous lectin, galectin-1, was examined in human lymphoid tissue. Galectin-1 was detected in the endothelial cells lining specialized vessels, termed high endothelial venules, in activated lymphoid tissue, but not in a resting lymph node. Cultured endothelial cells (human aortic and umbilical vein endothelial cells (HAECs and HUVECs)) expressed galectin-1. Activation of the cultured endothelial cells increased the level of galectin-1 expression, as determined by ELISA, Northern blot analysis and high throughput cDNA sequencing. These results suggest that galectin-1 expressed by endothelial cells may bind to and affect the trafficking of cells emigrating from blood into tissues.     

Chemokine receptor CXCR7 mediates human endothelial progenitor cells survival, angiogenesis, but not proliferation

Stromal cell-derived factor 1 (SDF-1) is a critical regulator of endothelial progenitor cells (EPCs) mediated physiological and pathologic angiogenesis. It was considered to act via its unique receptor CXCR4 for a long time. CXCR7 is a second, recently identified receptor for SDF-1, and its role in human EPCs is unclear. In present study, CXCR7 was found to be scarcely expressed on the surface of human EPCs derived from cord blood, but considerable intracellular CXCR7 was detected, which differs from that on EPCs derived from rat bone marrow. CXCR7 failed to support SDF-1 induced human EPCs migration, proliferation, or nitric oxide (NO) production, but mediated human EPCs survival exclusively. Besides that, CXCR7 mediated EPCs tube formation along with CXCR4. Blocking CXCR7 with its antagonist CCX733 impaired SDF-1/CXCR4 induced EPCs adhesion to active HUVECs and trans-endothelial migration. Those results suggested that CXCR7 plays an important role in human cord blood derived EPCs in response to SDF-1.     

MiR-18b suppresses high-glucose-induced proliferation in HRECs by targeting IGF-1/IGF1R signaling pathways

MicroRNAs (miRNAs) are important for the proliferation of endothelial cells and have been shown to be involved in diabetic retinopathy (DR). In previous study, we found that miRNAs might play a critical role in hyperglycemia-induced endothelial cell proliferation based on miRNA expression profiling. Here, the roles of microRNA-18b (miR-18b) in the proliferation of human retinal endothelial cells (HRECs) were investigated in an in vitro model of HRECs grown in high glucose. We identified that levels of miR-18b were decreased in high-glucose-induced HRECs, compared with those in cells incubated in normal glucose. However, the reduction of miR-18b up-regulated vascular endothelial growth factor (VEGF) secretion and promoted effects on in vitro proliferation of HRECs. Mechanistically, insulin growth factor-1 (IGF-1) was identified as a target of miR-18b. IGF-1 simulation could antagonize the effect induced by miR-18b up-regulation, promoting cell proliferation and increasing VEGF production. In contrast, the opposite results were observed with silencing IGF-1, which was consistent with the effects of miR-18b overexpression. MiR-18b exerted its function on VEGF synthesis and cell proliferation by suppressing the IGF-1/insulin growth factor-1 receptor (IGF1R) pathway, consequently inhibiting the downstream phosphorylation of Akt, MEK, and ERK. Hence, this may provide a new insight into understanding the mechanism of DR pathogenesis, as well as a potential therapeutic target for proliferative DR.     

Exosomes derived from acute myeloid leukemia cells promote chemoresistance by enhancing glycolysis-mediated vascular remodeling

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. AML cells secrete angiogenic factors to remodel vasculature and acquire chemoresistance; however, antiangiogenic drugs are often ineffective in AML treatment. Cancer cell-derived exosomes can induce angiogenesis, but their role in vascular remodeling during AML is unclear. Here, we found that exosomes secreted by AML cells promoted proliferation and migration and tube-forming activity of human umbilical vein endothelial cells (HUVECs), whereas HUVECs conferred chemoresistance to AML cells. AML cell-derived exosomes contained vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) messenger RNA and induced VEGFR expression in HUVECs. Furthermore, they enhanced glycolysis, which correlated with HUVEC proliferation, tube formation, and resistance to apoptosis. Thus, AML cells secrete VEGF/VEGFR-containing exosomes that induce glycolysis in HUVECs leading to vascular remodeling and acquisition of chemoresistance. These findings may contribute to the development of novel therapeutic strategies targeting exosomes in AML.     

2, 3, 7, 8‐Tetrachlorodibenzo‐p‐dioxin promotes endothelial cell apoptosis through activation of EP3/p38MAPK/Bcl‐2 pathway

Endothelial injury or dysfunction is an early event in the pathogenesis of atherosclerosis. Epidemiological and animal studies have shown that 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) exposure increases morbidity and mortality from chronic cardiovascular diseases, including atherosclerosis. However, whether or how TCDD exposure causes endothelial injury or dysfunction remains largely unknown. Cultured human umbilical vein endothelial cells (HUVECs) were exposed to different doses of TCDD, and cell apoptosis was examined. We found that TCDD treatment increased caspase 3 activity and apoptosis in HUVECs in a dose‐dependent manner,at doses from 10 to 40 nM. TCDD increased cyclooxygenase enzymes (COX)‐2 expression and its downstream prostaglandin (PG) production (mainly PGE₂ and 6‐keto‐PGF_1α) in HUVECs. Interestingly, inhibition of COX‐2, but not COX‐1, markedly attenuated TCDD‐triggered apoptosis in HUVECs. Pharmacological inhibition or gene silencing of the PGE₂ receptor subtype 3 (EP3) suppressed the augmented apoptosis in TCDD‐treated HUVECs. Activation of the EP3 receptor enhanced p38 MAPK phosphorylation and decreased Bcl‐2 expression following TCDD treatment. Both p38 MAPK suppression and Bcl‐2 overexpression attenuated the apoptosis in TCDD‐treated HUVECs. TCDD increased EP3‐dependent Rho activity and subsequently promoted p38MAPK/Bcl‐2 pathway‐mediated apoptosis in HUVECs. In addition, TCDD promoted apoptosis in vascular endothelium and delayed re‐endothelialization after femoral artery injury in wild‐type (WT) mice, but not in EP3^?/? mice. In summary, TCDD promotes endothelial apoptosis through the COX‐2/PGE₂/EP3/p38MAPK/Bcl‐2 pathway. Given the cardiovascular hazard of a COX‐2 inhibitor, our findings indicate that the EP3 receptor and its downstream pathways may be potential targets for prevention of TCDD‐associated cardiovascular diseases.     

TWIST1在人脐静脉内皮细胞增殖、迁移及血管形成中的作用

探讨TWIST1在原代人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）增殖、迁移及体外血管生成中的作用。用有靶向人TWIST1基因shRNA（pLL3.7-shTwist1-GFP）的慢病毒液感染试验组细胞,同时以携带Scramble shRNA的慢病毒液（pLL3.7-shCtrl-GFP）感染对照组细胞,用流式细胞术测定细胞感染效率,实时荧光定量PCR（real-time fluorescent quantitative PCR,qRT-PCR）检测shRNA的基因沉默效率。通过制作细胞生长曲线、Annexin V/7AAD染色流式细胞术、细胞划痕实验、小管形成实验、qRT-PCR检测TWIST1表达降低对HUVECs的增殖、凋亡、迁移、血管形成能力以及血管生长因子受体2（vascular endothelial growth factor receptor 2,VEGFR2）基因表达的影响。试验组TWIST1基因表达下降为对照组的30%,表明shTWIST1能有效降低TWIST1基因的表达。与对照组相比,敲降TWIST1能明显抑制HUVECs的增殖（P<0.01）,诱导细胞凋亡（P<0.05）。试验组HUVECs划痕愈合率、体外生成的血管样结构数目和总小管分支长度均显著低于对照组（P<0.01）;与对照组相比,试验组HUVECs中VEGFR2的表达显著降低（P<0.01）。通过探究HUVECs表达的TWIST1在内皮细胞增殖、存活、迁移和毛细血管样结构的形成中的作用,为TWIST1作为抑制肿瘤血管新生治疗的新靶点提供一定的理论依据。     

Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid-based implantation model

Objectives:  Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co-implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular-derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC).
Materials and methods:  EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice.
Results:  EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE-cadherin and vascular endothelial growth factor-R2, and in their ability to endocytose acetylated low-density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro , as assessed by in vitro Matrigel sprouting assay. However in vivo , a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions.
Conclusion:  Our results indicate that vascular-derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.     

AREL1 resists the apoptosis induced by TGF-β by inhibiting SMAC in vascular endothelial cells

Abnormal apoptosis of vascular endothelial cells is an important feature of arteriosclerosis (AS). Here, we induced apoptosis in human umbilical vein endothelial cells (HUVECs) using transforming growth factor-β (TGF-β), and investigated the role of antiapoptotic E3 ubiquitin ligase (AREL1) in the apoptosis of vascular endothelial cells. We proved that AREL1 is downregulated in TGF-β treated HUVECs. The overexpression of AREL1 inhibits the activation of Caspase-3 and Caspase-9 and attenuates cell apoptosis induced by TGF-β. According to the result of coimmunoprecipitation, AREL1 interacts with the proapoptotic proteins the second mitochondria-derived activator of caspases (SMAC) in TGF-β treated HUVECs. In addition, miR-320b inhibits the expression of AREL1, and the overexpression of AREL1 attenuates the apoptosis induced by miR-320b mimics in HUVECs. In conclusion, AREL1 is downregulated by miR-320b. AREL1 overexpression inhibits TGF-β induced apoptosis through downregulating SMAC in vascular endothelial cells. Our study explores pathogenesis regulation mechanism and new biological therapeutic targets for vascular disease.     

Activation of ERK pathway is required for 15-HETE-induced angiogenesis in human umbilical vascular endothelial cells

Angiogenesis plays a critical role in the progression of cardiovascular disease, retinal ischemia, or tumorigenesis. The imbalance of endothelial cell proliferation and apoptosis disturbs the establishment of the vasculogenesis, which is affected by several arachidonic acid metabolites. 15-Hydroxyeicosatetraenoic acid (15-HETE) is one of the metabolites. However, the underlying mechanisms of angiogenesis induced by 15-HETE in human umbilical vascular endothelial cells (HUVECs) are still poorly understood. Since extracellular signal-regulated kinase (ERK) is a critical regulator of cell proliferation, there may be a crosstalk between 15-HETE-regulating angiogenic process and ERK-proliferative effect in HUVECs. To test this hypothesis, we study the effect of 15-HETE on cell proliferation, angiogenesis, and apoptosis using cell viability measurement, cell cycle analysis, western blot, scratch–wound, tube formation assay, and nuclear morphology determination. We found that 15-HETE promoted HUVEC angiogenesis, which were mediated by ERK. Moreover, 15-HETE-induced proliferation and cell cycle transition from the G₀/G₁ phase to the G₂/M?+?S phase. All these effects were reversed after blocking ERK with PD98059 (an ERK inhibitor). In addition, HUVEC apoptosis was relieved by 15-HETE through the ERK pathway. Thus, ERK is necessary for the effects of 15-HETE in the regulation of HUVEC angiogenesis, which may be a novel potential target for the treatment of angiogenesis-related diseases.     

VEGF和SDF-1 的协同作用对内皮祖细胞增殖与迁移的影响

目的:研究血管内皮生长因子(VEGF)和基质衍生因子-1(SDF-1)的协同作用对高血压脑出血患者内皮祖细胞(EPCs)增殖迁移能力的影响。方法:采集急性期高血压脑出血患者与健康对照的外周静脉血,分离培养外周血中的EPCs。用分别含有不同VEGF与SDF-1的培养基处理患者外周血分离的EPCs,Western blot检测各组细胞以及患者和对照中VEGF和SDF-1的蛋白表达。MTT法检测细胞增值能力,Transwell法检测细胞迁移能力,分别转染VEGF-si RNA与SDF-1-si RNA至各组细胞观察抑制VEGF和SDF-1对EPCs增殖迁移能力的影响。结果:VEGF和SDF-1在患者中的表达显著高于健康对照组。VEGF和SDF-1对EPCs的增殖和迁移能力有促进作用,且在其协同作用下效果显著。抑制VEGF或SDF-1显著降低VEGF和SDF-1对EPCs增殖迁移能力的促进作用。结论:VEGF和SDF-1的协同作可促进急性期高血压脑出血患者EPCs的细胞增殖能力和迁移能力,对患者血管修复提供新的治疗方向。     

RGD-peptide presents anti-adhesive effect, but not direct pro-apoptotic effect on endothelial progenitor cells

Circulating endothelial progenitor cells (EPCs) contribute to neovascularization in tumor or ischemic tissues by multi-step events, including adhesion, migration, chemoattraction, and differentiation to endothelial cells. Anti-angiogenic RGD-peptides have been shown to directly induce apoptosis in human umbilical vein endothelial cells (HUVECs) and T cells. Here, we examined the effects of RGD-peptides on EPCs in terms of adhesive differentiation and apoptosis. When mononuclear cells (MNCs) isolated from human cord blood were cultured on fibronectin-coated plates for 7 days, RGD-peptide treatment decreased dose-dependently the number of adherent cells double positive for DiI-ac-LDL uptake and UEA-1 binding. The cells treated with RGD peptide were also stained less strongly by vWF or KDR antibody by immunofluorescence staining. Immobilization of the RGD-peptide promoted cell adhesion, but resulted in a deficiency in the development of ability of ac-LDL uptake and UEA-1 binding, showing an antagonistic effect. Accordingly, ex vivo-cultivated EPCs expressed integrin alpha5, alphav, beta1, beta3, and beta5, and antibodies to integrins alpha5, alphav, and beta1 decreased the number of adherent cells. However, viability of total MNCs containing early EPCs was not affected by RGD-peptide. In addition, neither an increase in apoptotic cell death nor a direct activation of caspase-3 by RGD-peptide was detected in ex vivo-cultivated EPCs, unlike in HUVECs. Interestingly, RGD-peptide rather enhanced Bcl-2 expression in ex vivo-cultivated EPCs and the EPCs themselves with a high Bcl-2/Bax ratio are comparatively resistant to apoptosis. Therefore, these results suggest that RGD-peptides may inhibit EPC differentiation by anti-adhesive effect, but not by a direct pro-apoptotic effect.