Paracrine mechanisms of endothelial progenitor cells in vascular repair

Endothelial progenitor cells (EPCs) play an important role in repairing damaged blood vessels and promoting neovascularization. However, the specific mechanism of EPCs promoting vascular repair is still unclear. Currently, there are two different views on the repair of damaged vessels by EPCs, one is that EPCs can directly differentiate into endothelial cells (ECs) and integrate into injured vessels, the other is that EPCs act on cells and blood vessels by releasing paracrine substances. But more evidence now supports the latter. Therefore, the paracrine mechanisms of EPCs are worth further study. This review describes the substances secreted by EPCs, some applications based on paracrine effects of EPCs, and the studies of paracrine mechanisms in cardiovascular diseases--all of these are to support the view that EPCs repair blood vessels through paracrine effects rather than integrating directly into damaged vessels.     

骨髓源性血管内皮前体细胞与出生后生理性和病理性血管形成

Endothelial progenitor cells （EPCs） exist in bone marrow, umbilical cord blood and peripheral blood of adult mammals, including humans. Furthermore, the discovery of EPCs has led to the notion of adult vasculogenesis, in which bone marrow (BM)-derived EPCs home to and incorporate into sites of new blood vessel formation, where they differentiate into endothelial cells, which is consistent with postnatal vasculogenesis. It has become apparent that circulating BM-derived EPCs are involved in promoting physiologic andpathologic neovascularization, such as wound healing and tumor growth. They are of great clinical importance in pro-or anti-angiogenic therapies.     

双特异抗体增效内皮祖细胞移植促进心肌血管新生

目的探讨在双特异抗体(BiAb)的辅助下,内皮祖细胞(EPCs)移植可否更好的定向归巢大鼠缺血心肌促进血管新生。方法体外分离培养鉴定SD大鼠骨髓源性内皮祖细胞;开胸结扎SD大鼠冠状动脉左前降支制备心肌梗死模型;以anti-CD34(能识别内皮祖细胞)和抗肌凝蛋白轻链抗体(AMLCA)(能特异性结合缺血心肌)2种抗体,化学交联法制备BiAb(CD34×AMLCA)。将此BiAb与EPCs经尾静脉输入心肌梗死大鼠(EPCs+BiAb组),另设单纯EPCs移植组、单纯BiAb组、对照组。细胞移植35 d后M型超声心动图检测大鼠左室收缩功能,免疫组织化学法行5-Brdu及VIII因子检测,实时荧光定量PCR及Western blot检测大鼠心肌VEGF mRNA与蛋白表达。结果与其余组相比,EPCs+BiAb组射血分数及短轴缩短率增加,心梗区周围5-BrdU阳性细胞数及微血管密度增加,心肌VEGF mRNA与蛋白表达增加(P<0.05)。结论 CD34×AMLCA双特异抗体可增效大鼠骨髓源性内皮祖细胞定向归巢到大鼠缺血心肌,改善心功能,更好的促进血管新生。     

Beneficial effect of autologous transplantation of bone marrow stromal cells and endothelial progenitor cells on cerebral ischemia in rabbits

We tested the therapeutic effect of autologous transplanted bone marrow stromal cells (BMSCs) and endothelial progenitor cells (EPCs) on cerebral ischemia in rabbits. Rabbit permanent middle cerebral artery occlusion (MCAO) models were intravenously injected with ex vivo expanded autologous BMSCs (n = 8), EPCs (n = 8), or phosphate-buffered saline (n = 6). 14 days after the transplantation, both infusion groups witnessed a functional improvement, a decrease in the number of apoptotic cells and an increase in the microvessel density in the ischemic boundary area, as compared to vehicle-treated control group. The EPCs treated group also exhibited a diminished infarct area in comparison with the control group. Moreover, immunohistochemistry revealed that few transplanted BMSCs expressed markers for astrocytes (GFAP⁺) and neurons (NeuN⁺), and most of EPCs were capable of binding to UEA-1 lectin and were incorporated into capillaries. Our data suggest that both BMSCs and EPCs, despite differences in their action mechanism, can be functional cytoreagents for treatment of cerebral ischemia in rabbits.     

The role of fucosylation in the promotion of endothelial progenitor cells in neovascularization and bone repair

Bone marrow-derived endothelial progenitor cells (EPCs) are being tested as a therapy to treat a variety of ischemic diseases. Poor homing to targeted tissues is one of the major factors limiting the therapeutic efficacy of EPCs. Here, we show that human cord blood-derived EPCs expressed little sialyl Lewis X (sLe^x) antigen that is necessary for selectin-mediated cell–cell interactions. Expression of α1,3-fucosyltransferase VI (FucT VI) in the EPCs enhanced sLe^x synthesis, E- and P-selectin-binding, and EPC adhesion to tumor necrosis factor-α-stimulated human umbilical vein endothelial cells in culture. In a mouse model of hind limb ischemia, in which EPCs were injected intravenously, FucT VI expression increased EPC homing, neovascularization, and blood flow in ischemic muscles. In another mouse model of femoral fracture, FucT VI-expressing EPCs were more efficient than control EPCs in targeting to peri-fracture tissues to enhance angiogenesis, blood flow and bone repair. These results indicate that fucosylated EPCs may be used to as an improved cellular source to treat ischemic diseases.     

人外周血来源内皮祖细胞诱导分化为心肌样细胞的实验研究及机制探讨

目的： 研究人外周血来源内皮祖细胞（EPCs）在体外定向分化为心肌样细胞的能力及其可能机制。方法： 分离培养及鉴定健康人群（对照组）及冠心病（CAD）患者（实验组）EPCs。将对照组及实验组EPCs分别分成3组在体外进行分化诱导实验：A组 EPCs与乳鼠心肌共培养；B组 EPCs与多聚甲醛固定后的乳鼠心肌共培养；C组 于EPCs中加入收集到的乳鼠心肌培养液上清诱导培养。观察心肌样细胞形态变化;2周后免疫荧光法检测心肌肌钙蛋白 （cTnT）和结蛋白（desmin）的表达；逆转录-聚合酶链反应（RT-PCR）检测心肌相关基因ANP、cTnT、cTnI和主要组织相容性复合物α链（αMHC）的表达；流式细胞仪检测HLA-DR和人desmin的表达，计数desmin阳性表达细胞。结果： 实验及对照A、B组EPCs均可呈现心肌样细胞形态;ANP、cTnT、cTnI和αMHC基因及cTnT和desmin蛋白均呈阳性表达；流式检测HLA-DR及desmin呈双阳性表达，对照A组分化率为（9±2）％，实验A组分化率约为（4±1）％，差异显著（P<0.01）。实验及对照C组EPCs形态未发生变化，各项检测指标均为阴性表达。结论： 与乳鼠心肌共培养可诱导人外周血来源EPCs分化为心肌样细胞，且其可能机制为细胞间的直接接触。     

Non-invasive monitoring of transplanted endothelial progenitor cells in diabetic ischemic stroke models

Endogenous endothelial progenitor cells (EPCs) are functionally impaired in hyperglycemia through the p38 MAPK signaling pathway. However, the number and function of transplanted exogenous EPCs in diabetic animals remains unclear. The objectives of this study were to establish a non-invasive imaging strategy to monitor the homing of transplanted EPCs in diabetic stroke mice and to assess the effect of RWJ 67657, an inhibitor of p38 MAPK, on the homing ability of exogenous EPCs. Bone marrow-derived EPCs were labeled in vitro with a multi-functional nanoprobe modified with paramagnetic chelators and fluorophores before being infused into stroke mice. The signal of the nanoprobe reached its peak on day 5 in both magnetic resonance imaging and near-infrared fluorescence imaging after EPC transplantation in wild-type stroke models. The signal enhancement of diabetic stroke models was significantly lower than that of wild-type controls. However, the signal intensity of diabetic stroke models significantly increased after oral administration of RWJ 67657, indicating that more transplanted EPCs migrated to the ischemic brain. Furthermore, the increased exogenous EPCs induced remarkably greater angiogenesis after stroke. These results suggest that this dual-modal imaging strategy is feasible for non-invasively monitoring transplanted cells in vivo.     

Time to metastatic relapse and breast cancer cells dissemination in bone marrow at metastatic relapse

Breast cancer dissemination can be monitored in patients by detecting circulating and/or disseminated tumor cells. However, bone marrow disseminated tumor cells (BM DTC) may undergo a dormancy during several years before growing (or not) into clinically detectable metastases. We therefore hypothesized that breast cancers which have formed BM DTC in the course of their metastatic growth might exhibit a longer interval before metastatic relapse. We examined the association of DTC detection (cytokeratin 8, 18 or 19 positive epithelial cells with cancerous morphological features), at metastatic relapse, with the metastasis-free interval in breast cancer patients. In the 110 metastatic patients studied, 42% (n = 64/110) were classified as BM DTC-negative. These patients had a significantly shorter metastasis-free interval than BM DTC-positive patients (P = 0.02). In multivariate logistic regression analysis, the metastasis-free interval was an independent predictor of DTC detection (P = 0.02), together with bone metastasis (P = 0.0003) and low tumor grade (grade I or II, P = 0.05). We finally suggest that a faster metastatic process might skip in some patients the BM DTC-associated dormancy step. Dissemination of DTC in other host organ and/or epithelial–mesenchymal transition from cytokeratin-positive to cytokeratin-negative DTC may explain this observation. This work has been presented in part at the 2008 AACR meeting (San Diego, USA).     

Culturing endothelial cells of microvascular origin

There is considerable divergence of opinion on the best methods for the isolation and in vitro culture of microvascular endothelium. Reports have either only described the isolation without mentioning culture conditions or have not fully defined the cell population being cultured. Even at the close of the 20th Century, the isolation and in vitro culture of endothelial cells of microvasculature origin still proves to be technically difficult and many questions remain. These questions need to be addressed by improvements to current methods of isolation and culture of Endothelial cells. A number of more 'high-tech' approaches to this isolation are being explored currently. Use of a more definitive panel of antibodies for immunocytochemical characterization, should enable a more confident characterization of the endothelial cell preparations cultured in vitro. Cell adhesion molecules such as ELAM and VCAM can be used to assist in determining cell population purity.     

A new model of vasculogenesis and angiogenesis in vitro as compared with vascular growth in the avian area vasculosa

In cultures of dissociated quail epiblast the basic constituents of the vascular system, blood cells and endothelial cells can be induced by basic fibroblast growth factor (Flamme and Risau, Development, 116:435–439, 1992). As we show here, in those cultures three types of vascular plexus differentiate spontaneously under different culture conditions: At the 3rd day a vascular plexus appears in situ closely resembling the vascular plexus of the quail area opaca vasculosa (vasculogenesis). Vascular sprouts are formed, extending long filopodia at their tips. Such filopodia are shown to build the first intervascular bridges in the growing vascular plexus of the area vasculosa at embryonic day 3. Connections of filopodia turn out to be precursors of new capillaries interconnecting pre-existing blood vessels (angiogenesis). Two further types of in vitro capillary plexus differentiate in long term endothelial cell cultures derived from induced angioblasts. Whereas one closely resembles so-called angiogenesis in vitro, the third type comprises mainly multinucleated giant endothelial cells lining loop like capillaries and represents a differentiation of aging endothelial cell culture. Thus, the present in vitro model is an approach to the sequence of angioblast induction, vasculogenesis, and angiogenesis. © 1993 Wiley-Liss, Inc.     

Effects of ammonium tetrathiomolybdate,an oncolytic/angiolytic drug on the viability and proliferation of endothelial and tumor cells

Objective: This study examines the target specificity of tetrathiomolybdate (ATM), an anti-angiogenic, anti-tumor agent against the viability / proliferation of arterial, venous, capillary endothelial and tumor cells. Materials and Methods: Cells were seeded at 10 or 100% density (to measure viability / proliferation respectively) in medium ± 0–250 uM ATM. Viability and proliferation were measured by metabolic labeling-colorimetry. One-way ANOVA + Bonferroni testing examined base metabolism; Dunnett’s testing was used for viability/proliferation. Results: Venous proliferation showed high ATM sensitivity (50% reduction ~ ≥5 uM ATM, p<0.01), capillary proliferation was inhibited ≥10 uM (p<0.05). Arterial endothelium were less sensitive to ATM, (50% inhibition ~ ≥ 20 uM, p<0.01). YPEN-1 were inhibited >50 uM ATM. Capillary viability was inhibited ≥20 μM ATM (p<0.01); venous, arterial and tumor viability show less ATM sensitivity. Conclusions: Our data suggest that venous and capillary endothelial proliferation are important targets in ATM therapy, but that other vascular segments and tumor cells may be less influenced. Received 13 February 2007; returned for revision 10 April 2007; returned for final revision 1 June 2007; accepted by A. Falus 13 July 2007     

白藜芦醇对于外周血内皮祖细胞体外增殖分化的影响

目的:观察白藜芦醇对内皮祖细胞体外扩增分化过程中内皮系基因的表达及粘附、迁移功能的影响。方法:密度梯度离心法获得人外周血单个核细胞,在向内皮祖细胞诱导分化的同时,加入不同浓度的白藜芦醇(1、5、15、60μM)干预7天,随后采用流式细胞术观察CD34、CD31、KDR基因的表达;用RT-PCR和ELISA方法检测干预后内皮型一氧化氮合成酶的表达;同时测定干预后细胞的粘附和迁移功能。结果:低浓度白藜芦醇(5μM)不同程度促进内皮祖细胞增殖分化中CD34、CD31、KDR的表达,上调eNOS的mRNA和蛋白水平表达,并促进内皮祖细胞粘附和迁移功能;而高浓度白藜芦醇(60μM)则不同程度下调CD34、CD31、KDR、eNOS的表达和抑制粘附、迁移功能。结论:低浓度白藜芦醇可促进外周血内皮祖细胞内皮系基因的表达,并且促进其粘附和迁移,而高浓度则产生相反的作用。     

白藜芦醇抑制叔丁基过氧化物诱导的外周血内皮祖细胞损伤

目的:观察白藜芦醇对叔丁基过氧化物诱导的人外周血内皮祖细胞(EPC)凋亡的影响,探讨其可能机制。方法:密度梯度离心法获取人外周血单个核细胞,培养4 d后,收集贴壁细胞。实验分为对照组、叔丁基过氧化物诱导组和白藜芦醇+叔丁基过氧化物组。采用二苯基四氮唑溴盐(MTT)比色法和5-溴脱氧尿嘧啶核苷(Brd U)ELISA法检测EPC增殖能力;采用改良Boyden小室法检测EPC增殖及迁移能力;Annexin V-FITC/PI染色及流式细胞术检测EPC凋亡率;比色法检测caspase-3活性;荧光探针H_2DCF-DA法检测细胞内活性氧簇(ROS)水平;Western blot检测cleaved caspase-3、Bax及Bcl-2蛋白的表达。结果:白藜芦醇能抑制叔丁基过氧化物诱导的EPC凋亡,并增加EPC增殖及迁移能力;白藜芦醇降低EPC细胞内ROS水平,抑制caspase-3的活性及cleaved caspase-3的蛋白水平,同时能促进Bcl-2及抑制Bax蛋白表达。结论:白藜芦醇对叔丁基过氧化物诱导的EPC凋亡有抑制作用,其机制可能与抗氧化应激有关。     

Jagged1过表达促进老龄大鼠来源的内皮祖细胞向成熟内皮细胞分化

目的：探讨上调Jagged1表达对内皮培养条件下老龄大鼠来源的内皮祖细胞（EPC）向内皮细胞分化的影响。方法：脱臼处死1～2月龄和19～26月龄SD大鼠,PBS冲洗股骨和胫骨骨髓,Ficoll密度梯度离心分离单个核细胞,应用含10%FBS的DMEM/F12培养基以差速贴壁法进行体外培养,DiI-ac-LDL与FITC-UEA-1荧光双染进行EPC特性鉴定。实验分为4组：对照组、PIRES2-EGFP转染组、PIRES2-EGFP-Jagged1转染组和未转染的年轻大鼠来源EPC组。荧光显微镜下计数GFP阳性细胞数并计算转染效率;免疫荧光、RT-PCR和Westernblotting检测Jagged1mRNA和蛋白、vonWillebrand因子（vWF）及血管内皮生长因子激酶插入区受体（KDR）mRNA表达,体外血管生成实验检测EPC的血管形成能力。结果：转染后Jagged1在EGFP-Jagged1组表达较对照组显著增强（P<0.01）;Jagged1过表达显著促进老龄大鼠EPCvWF与KDRmRNA表达（P<0.01）和体外血管生成能力（P<0.01）;vWF与KDRmRNA表达以及体外血管生成能力在Jagged1转染组与年轻大鼠EPC组间未见有显著差别。结论：Jagged1过表达促进内皮培养条件下老龄大鼠来源EPC向成熟内皮细胞分化。     

Mobilization of circulating endothelial progenitor cells after endovascular therapy for ruptured cerebral aneurysms

Emerging evidence shows that circulating endothelial progenitor cells (EPCs) promote regeneration of the endothelium at sites of vessel injury. This study was designed to test the hypothesis that EPCs are mobilized in patients who had ruptured cerebral aneurysm (CA) and underwent endovascular therapy. Fourteen patients with ruptured CAs were recruited and blood samples were analyzed after coil embolization surgery. Blood samples were also obtained from 18 healthy control subjects who had no evidence of CAs and did not undergo endovascular surgery. We measured the numbers of circulating EPCs, levels of plasma vascular endothelial growth factor (VEGF) and platelet counts. EPCs significantly increased in patients with ruptured CAs and underwent surgical coil embolization, reaching a peak level on day 14 post operation. The levels of plasma VEGF and platelet counts also significantly increased in parallel with the increase in EPCs, leading to significant positive correlations of circulating EPCs with VEGF in plasma (r = 0.636, P < 0.01) and platelet counts (r = 0.721, P < 0.001), respectively. The finding suggests that EPCs are mobilized upon surgery and may play a critical role in repairing injured vascular endothelium. Levels of EPCs in peripheral blood could also serve as a prognostic marker for the outcomes of ruptured cerebral aneurysms after surgical repair.     

人脐带内皮祖细胞的分离、鉴定及转染EGFP质粒的实验研究

目的 从脐带中分离内皮祖细胞(EPCs),考察其体外增殖、基因转染绿色荧光蛋白质粒的行为.方法 以酶消化法从脐带中分离出内皮祖细胞,并通过流式细胞仪和共聚焦显微镜对内皮祖细胞进行鉴定,以Lipofectamine 2000为转染试剂考察了内皮祖细胞转染绿色荧光蛋白质粒的行为.结果 从脐带中分离培养的内皮祖细胞在第9天形成了典型的内皮细胞集落,流式细胞仪分析结果显示CD133和激酶插入区受体(KDR)的含量均有所提高,并具有内皮祖细胞结合异硫氰酸荧光素(FITC)标记的荆豆凝聚素1(FITC-UEA-1)和吞噬DiI标记的低密度脂蛋白(DiI-ac-LDL)的功能,能较好地表达绿色荧光蛋白.结论 从脐带中分离的内皮祖细胞体外在适当的培养条件下,可增殖、诱导分化为内皮细胞,并能较好地表达外源基因,是基因与细胞治疗理想的载体.     

Tissue engineering a surrogate niche for metastatic cancer cells

In breast and prostate cancer patients, the bone marrow is a preferred site of metastasis. We hypothesized that we could use tissue-engineering strategies to lure metastasizing cancer cells to tissue-engineered bone marrow. First, we generated highly porous 3D silk scaffolds that were biocompatible and amenable to bone morphogenetic protein 2 functionalization. Control and functionalized silk scaffolds were subcutaneously implanted in mice and bone marrow development was followed. Only functionalized scaffolds developed cancellous bone and red bone marrow, which appeared as early as two weeks post-implantation and further developed over the 16-week study period. This tissue-engineered bone marrow microenvironment could be readily manipulated in situ to understand the biology of bone metastasis. To test the ability of functionalized scaffolds to serve as a surrogate niche for metastasis, human breast cancer cells were injected into the mammary fat pads of mice. The treatment of animals with scaffolds had no significant effect on primary tumor growth. However, extensive metastasis was observed in functionalized scaffolds, and the highest levels for scaffolds that were in situ manipulated with receptor activator of nuclear factor kappa-B ligand (RANKL). We also applied this tissue-engineered bone marrow model in a prostate cancer and experimental metastasis setting. In summary, we were able to use tissue-engineered bone marrow to serve as a target or “trap” for metastasizing cancer cells.     

Involvement of bone marrow-derived endothelial progenitor cells in glomerular capillary repair in habu snake venom-induced glomerulonephritis

Neovasculogenesis is essential in tissue remodeling. Endothelial progenitor cells (EPCs) mobilize from bone marrow (BM) and participate in neovasculogenesis. This study examined the role of EPCs in a model of reversible glomerulonephritis induced by habu snake venom (HSV). Lethally irradiated FVB/N wild-type mice were transplanted with BM cells from donor transgenic mice expressing beta-galactosidase gene under the control of endothelial-specific tie-2 promoter. HSV or saline was injected intravenously after BM transplantation (BMT). The kidneys were removed before injection and at days 1, 7, 28, and 56 after injection. beta-Galactosidase-expressing cells were identified by X-gal staining. The expressions of CD31 (endothelial cell marker) and vascular endothelial cell growth factor (VEGF) in renal tissues were examined by immunohistochemistry. In BMT mice injected with saline, few X-gal-positive cells were detected in glomeruli. In HSV-injected mice, X-gal-positive EPCs were increased in damaged glomeruli, reaching maximum at day 28. Recovery of glomeruli was observed at day 56 in association with reduction of X-gal-positive EPCs. VEGF overexpression was detected in glomerular epithelial and endothelial cells, mesangial cells, and EPCs. Our results indicated that EPCs were mobilized into the damaged glomeruli, suggesting EPCs participation in glomerular capillary repair of damaged glomeruli in HSV-induced glomerulonephritis.     

雌激素对孕妇外周血内皮祖细胞动员作用的体外研究

目的探讨雌激素对孕妇外周血内皮祖细胞（EPC）的动员作用。方法取孕期为10、20、30周的健康、单胎孕妇外周静脉血，采用Ficoll密度梯度离心法分离单个核细胞并进行体外培养。采用免疫荧光细胞化学染色方法进行EPC鉴定，取培养4d的细胞，加入DiI标记的乙酰化低密度脂蛋白（acLDL）和FITC标记的荆豆凝集素（UEA—I），用激光共聚焦扫描显微镜观察和记录图像。取培养7d的细胞，在相差显微镜下计数不同孕期孕妇外周血EPC集落（≥50个细胞）形成数（CFU）。分别通过跨膜迁移和增殖实验观察雌激素对EPC的动员和促增殖作用，随机选取培养7d的细胞，分别加入浓度为1×10^-10、1×10^-9、1×10^-8mol／L的雌二醇（雌二醇作用组），在相差显微镜下分别计数培养24h后EPC的跨膜迁移数和培养48h后EPC的增殖数，以加入PBS作为对照组，实验均重复3次。结果免疫荧光细胞化学染色显示，平均85％以上的细胞摄取DiI-acLDL并结合FITC—UEA—I，可以鉴定为EPC。孕期为10、20、30周孕妇外周血EPC的CFU分别为1．67±0．33、4．83±0．60、7．50±0．67，组间差异均有统计学意义（P〈0．05）。浓度为1×10^-10、1×10^-9、1×10^-8mol／L雌二醇作用组EPC跨膜迁移数（×10 ^3个／膜）分别为1．50±0，09、1．61±0．06、1．90±0．07，均明显高于对照组（1．03±0．06，P〈0．01）：EPC增殖数（×10^5个）分别为1．07±0．05、1．33±0．05、1．19±0．03，均明显高于对照组（1．00±0．03，P〈0．01）。结论孕妇外周血EPC的功能状态可能与雌激素水平有关，雌激素对EPC的动员具有重要作用。