内皮祖细胞（EPCs）在胶质瘤微血管构筑表型异质性形成中的作用及其机制研究
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摘要
恶性实体瘤(malignant solid tumors)的生长和转移有赖于肿瘤内血管新生(neovascularization)。肿瘤血管新生机制涉及由原血管床成熟内皮芽生的血管生成(angiogenesis)和由骨髓动员的内皮祖细胞(endothelial progenitor cell, EPCs)归巢肿瘤原位分化、增殖而来的血管形成(vasculogenesis)。尽管血管生成机制和抗血管生成一直是近些年肿瘤研究的热点和焦点,但抗肿瘤血管生成治疗效果不显著。其根本原因可能是骨髓EPCs不仅参与血管形成,而且能整合瘤周内芽生血管并形成不同内皮细胞来源的混合性血管。本课题组新近提出肿瘤微血管构筑表型异质性(tumor microvascular architecture phenotype heterogeneity, T-MAPH)的概念,并认为T-MAPH的存在是抗肿瘤血管生成治疗障碍的关键因素。作为参与肿瘤新生血管的关键细胞,很少有人注意到EPCs在T-MAPH形成中的作用及机制。人脑恶性胶质瘤具有高血管生成活性,且微血管密度越高,患者预后越差,被认为是研究T-MAPH的作用及机制的良好模型。
EPCs是从骨髓、脐血或外周血分离并具有CD34、VEGFR2和或CD133阳性标志细胞,能够在趋化因子和生长因子等刺激下募集并归巢于肿瘤血管新生位点,原位分化为内皮细胞,参与肿瘤血管形成。另外,EPCs不但参与肿瘤血管形成,而且还可以促进由原微血管床芽生血管生成。并且越来越多的研究证实,EPCs参与肿瘤的血管新生,在肿瘤生长中具有重要作用。本课题组前期研究发现,T-MAPH不仅表现在与正常血管的不同,而且还表现在不同肿瘤之间、同种肿瘤不同个体之间、同一肿瘤不同区域和发生演进阶段之间,肿瘤组织的微血管之间在密度、形态、结构(组成)、三维分布上的差异性和多样性。因此,作为参与肿瘤新生微血管的关键细胞,EPCs是否或如何与瘤内原血管床的芽生成熟内皮发生连接或吻合参与肿瘤微血管网以及这些血管网是否具有多样性和异质性,尚不清楚。
与成熟内皮细胞相比,EPCs高表达趋化因子受体CXCR4,此受体的表达与活化在EPCs参与血管新生中可能具有重要作用。EPCs中CXCR4被其配体基质细胞衍生因子(stromal-cell derived factor-1, SDF-1)活化后通过G蛋白耦连受体介导EPCs参与血管新生。有研究者证实,SDF-1在募集骨髓niche CXCR4+EPCs进入新生血管活跃区原位分化为内皮细胞参与血管发生中起着主要作用。并且SDF-1诱导内皮细胞增殖、分化、芽生和管型形成及阻止EPCs凋亡。另外,研究者证实淋巴瘤患者尸检淋巴组织整合血管内的EPCs中CXCR4 mRNA的表达高于循环EPCs中CXCR4 mRNA水平,并且EPCs高表达CXCR4 mRNA与肿瘤微血管密度和肿瘤体积成正相关。临床前研究发现拮抗SDF-1/CXCR4信号可有效阻断SDF-1促肿瘤血管新生作用。本课题组前期研究证明,CXCR4活化诱导VEGF和IL-8表达而促进胶质瘤T-MAPH作用。但SDF-1/CXCR4轴活化是否参与介导EPCs参与T-MAPH形成,尚缺乏研究。
为了解CXCR4活化后介导EPCs参与胶质瘤T-MAPH的作用及机制,我们观测了EPCs参与人恶性胶质瘤细胞系U87SCID鼠皮下移植瘤微血管构筑表型,并对EPCs的CXCR4活化是否介导EPCs的迁移及其在胶质瘤T-MAPH形成中的作用进行探索。首先,我们观测了从人脐血分离、鉴定的EPCs生物学特性,研究了在体外标记并经尾静脉移植的EPCs归巢于U87移植瘤内参与T-MAPH形成在微血管密度、形态和三维结构分布等方面的表现;最后,我们探讨了EPCs中CXCR4活化介导EPCs迁移及其在胶质瘤微血管表型形成中的作用,并观测了CXCR4受体抑制剂AMD3100对SDF-1/CXCR4轴介导上述效应的抑制作用。主要结果和结论如下:
1.从人脐血中分离、鉴定的EPCs具有自我更新、可被诱导分化和形成小管样结构的生物学特性。①应用流式细胞术、密度梯度离心联合专用培养基诱导培养结果显示,新鲜分离的CBMCs中存在CD34+/CD133+细胞(1.06%),在添加了生长因子的培养基诱导培养4d后,CD34表达增加,CD133表达减少,CD34+/CD133+减少(0.26%),由祖细胞向成熟细胞分化。②免疫细胞化学显示,获得的单核源性EPCs,可表达CD14 CD34和KDR,吞噬DiI-Ac-LDL,并能连接FITC-UEA-1。另一类是晚期增殖性EPCs,在内皮细胞专用培养液中生长具有形成初级集落和次集集落能力;CD133、CD34、KDR、vWF、CD31,DiI-Ac-LDL和FITC-UEA-1皆呈阳性表达。③Matrigel管样结构形成实验结果显示,上述两亚型的EPCs在Matrigel中均可形成小管样结构。上述结果表明,从人脐血分离单个核细胞(CBMCs)经内皮培养基培养获得的EPCs具有自我更新、诱导分化和形成小管样结构的生物学特性。
2.人源性EPCs特异地整合到胶质瘤细胞异种移植瘤宿主源性新生微血管中,并形成人-鼠嵌合性血管。将膜荧光染料CFSE标记的EPCs经鼠尾静脉植入经60Co亚致死剂量辐射后荷人U87细胞移植瘤的SCID鼠体内,①免疫荧光染色显示,迁移到U87 SCID鼠移植瘤内CFSE+EPCs数量明显高于肝脾组织(p<0.01)。②免疫荧光染色和流式细胞术显示,CFSE+EPCs以两种形式参与血管新生:单个或成簇的EPCs散在分布于鼠源性的血管旁,形成盲端或功能性的血管;或与鼠源性血管连接整合形成人-鼠嵌合性微血管。定量结果显示EPCs参与形成的新生血管占总血管的11.3~18.6%。③免疫荧光染色结果显示,CFSE+/CD31+EPCs与鼠源性CD31阳性的内皮细胞组成的混合性血管形态表现多样:或呈树杆分支样,或呈蛇形蜿蜒,或呈实性条索,或呈血窦状。三维重建结果显示,人源性CFSE+EPCs与鼠源性CD31+内皮细胞相互连接和或吻合构建成杂乱的人-鼠嵌合性新生微血管网,血管分支长短、大小和管壁厚薄不等。人源性CFSE+EPCs连接或聚集于鼠源性血管分支处形成多层内皮细胞的厚壁血管,鼠源性内皮细胞组成薄壁血管。在上述微血管结构中鼠源性内皮细胞主要分布于血管主体,而CFSE+细胞分布于血管盲袢或新生血管分支点。应用IPP图形软件定量分析CFSE+EPCs组成的血管段与人-鼠嵌合性新生微血管网的物理参数,结果显示,CFSE+EPCs组成的血管段像素值约为总血管的(26±7)%,血管长度占总血管的(22±5)%,而血管分支点占总血管的(36±1)%。④人源性EPCs增强耐受骨髓损伤的SCID鼠胶质瘤模型微血管密度(P﹤0.01),增加移植瘤幼稚微血管新生比例(P﹤0.05),且促进了肿瘤生长(P﹤0.05)。以上结果表明,体外植入的EPCs特异地归巢并整合于U87细胞SCID鼠皮下移植瘤新生血管位点,从形态、三维分布和MVD上体现了EPCs参与形成T-MAPH,进而促进移植瘤生长。
3. SDF-1/CXCR4轴活化介导EPCs迁移并促进EPCs参与胶质瘤T-MAPH形成。①间接免疫荧光显示,EPCs表达SDF-1和CXCR4。②MTT结果显示,不同浓度的SDF-1(9、19、37.5、75和150μg/L)在24、48和72 h活化CXCR4后能明显诱导EPCs增殖,各组平均吸光度值均显著高于空白对照组(P<0.05)。CXCR4的小分子抑制剂ADM3100在24、48和72h能有效拮抗CXCR4活化介导的EPCs增殖作用(P<0.05)。③迁移实验结果显示,19μg/L SDF-1活化CXCR4后能显著诱导细胞迁移(P<0.05)。相反,AMD3100能显著拮抗CXCR4活化对EPCs的迁移作用(P<0.05)。④Matrigel管样结构形成实验显示,EPCs表达的CXCR4活化后EPCs形成小管样结构数为16.3±2.2。AMD3100能明显减少EPCs小管样结构形成,平均数为6.4±1.8。⑤植入EPCs的移植瘤组织SDF-1和VEGF表达量显著高于未植入EPCs(即对照组)的移植瘤组织(P﹤0.05)。SDF-1和VEGF表达量与肿瘤微血管密度呈正相关。以上结果表明,EPCs可通过自分泌或旁分泌作用活化CXCR4进而介导EPCs增殖、迁移、管样结构形成并参与T-MAPH形成。而AMD3100具有抑制SDF-1/CXCR4轴介导的上述效应的作用。
综上所述,本研究从人脐血中分离、鉴定出的EPCs具有自我更新、诱导分化和形成小管样结构的生物学特性,植入的人源性EPCs能特异地归巢到U87细胞SCID鼠皮下移植瘤内新生血管位点,通过连接或整合瘤内成熟内皮细胞形成人-鼠嵌合性血管,增强耐受骨髓损伤的移植瘤微血管密度;在上述过程中,EPCs所表达的趋化因子受体CXCR4活化后能介导EPCs增殖、迁移、管样结构形成和促进EPCs参与T-MAPH作用,CXCR4受体抑制剂AMD3100具有抑制上述效应的作用。上述研究结果为认识胶质瘤T-MAPH发生机制和针对T-MAPH进行抑制肿瘤血管新生提供了新的实验依据。
Neovascularization is crucial for the growth and metastasis of malignant solid tumors. Tumor neovascularization is now believed to occur via at least two possible mechanisms: the sprouting of pre-existing resident endothelial cells, i.e., angiogenesis, and the recruitment of bone marrow–derived EPCs, known as vasculogenesis. Although the mechanism of neovascularization has been one of the frontiers in cancer research, therapeutic efficacy of antiangiogenesis appears to be quiet. The possible reason is heterogeneity of the newly-formed microvascular network by incorporation of EPCs in the pre-existing endothlieum of tumor vessels. Tumor microvascular architecture phenotype heterogeneity, we nominated it as T-MAPH, has been considered to have a key negative effect on therapeutic efficacy of antiangiogenesis. Thus, it is of great importance and necessary to explore wether and how to EPCs contribute to T-MAPH. Malignant gliomas are highly vascularized tumors and the microvessel densities within the tumors are related to behaviors such as invasiveness and recurrence of gliomas, therefore, malignant gliomas have been considered as useful models for studies of tumor neovacularization and therapeutics.
EPCs derived from either bone marrow, peripheral blood or umbilical cord blood are CD34, VEGFR2, or CD133 antigen-positive cells, which may home to the site of neovascularization and differentiate into endothelial cells in situ. Endothelial cells contribute to tumor angiogenesis, and can originate from sprouting or co-option of neighbouring pre-existing vessels. Emerging evidence indicate that bone marrow-derived circulating EPCs can contribute to tumor angiogenesis. Little is known about how to EPCs contribute to T-MAPH.
Chemokine receptor CXCR4 is involved in the regulation of tumor vascularization via EPCs. It has been reported that CXCR4 contribute to tumor vascularization by mobilizing, recruiting, inducing differentiation and tubular formation of EPCS. We found that CXCR4 promote T-MAPH by inducing the expression of VEGF and IL-8. However, exact role and detailed mechanism of CXCR4 in EPCs contributing to T-MAPH remain unclear.
For a better understanding of the possible role and mechanism of CXCR4 in the genesis of malignant glioma microvascular architecture phenotype via EPCs, in this study, we investigated contribution of EPCs to T-MAPH. Firstly, we isolated and identified EPCs from human umbilical cord blood. Then, we explored EPCs contribution to T-MAPH. Finally, we studied the functional expression of CXCR4 by EPCs promoting T-MAPH and AMD3100 abolished the effects induced by activation of CXCR4. The main results and conclusions are as follows:
1. Two types of EPCs isolated and identified from human umbilical cord blood were found to have potentials of renewal, differentiation and tubulogenesis. (1) By flow cytometry assay, the percentage of CD34+/CD133+ in mononuclear cells isolated from human umbilical cord blood decreased from1.06% to 0.26%. (2) By immunostaning assay, we identified that two types of EPCs, referred to as early EPCs and late outgrow EPCs. The early EPCs showed to be spindle-shaped with a tendency to form colonies which weakly potential, and were positive for CD14, CD34 and KDR. In addition, cells took up DiI-Ac-LDL and bound FITC-UEA-1.The late outgrowth EPCs demonstrated colony forming cells with typical cobblestone morphology, showed robust proliferative potential and gave rise to secondary colonies. During the 8 and 15 days, cells continued to be positively expressed CD34/CD133 and CD34/KDR. In addition, cells took up DiI-Ac-LDL and bound FITC-UEA-1.Those cells became positive for vWF and CD31 after 40 days of culture. (3)By three-diamensional in vitro Matrigel assays, we found that two types EPCs are capable of in vitro tubulogenesis.
2. By transplantation of EPCs into the impaired bone-marrow glioma model we evaluated incorporation of EPCs into the vascular architecture of xenograft and role of EPCs in T-MAPH. Our data suggested that: (1) by immunostaning assay, we found that CFSE+EPCs expressed human CD31 preferentially home to tumor angiogenic site, not to liver and spleen organs, p<0.01.(2) by immunostaning assay, we found that EPCs contributed to tumor vascularization via two possible pathways, including the incorporation into the vascular architecture of U87 cell xenograft and chimeric vascular network formation or formation of functional blood vessels by singe cell and multicellular clusters localized in the vicinity of mouse-derived endothelia. With quantitative immunofluorescence and flow cytometry analysis, the percentage of EPCs-derived the endothelial population ranged from 11.3% to 18.6%. (3) By immunostaning labeled three-dimensional reconstruction, we found that chimeric vascular network was chaos with different structures, such as a sinusoid structure or a branching pattern. EPCs linked mouse ECs in branches to form thickened vessels of multicellular clusters. (4) EPCs enhanced MVD of glioma in impaired bone-marrow mouse and progressed tumor growth.
3. We examined the effect of functional CXCR4 expressed by EPCs on T-MAPH. (1) Immunostaning assay was used to evaluate expression of CXCR4 and SDF-1 in EPCs. MTT, chemotaxis and three-diamensional in vitro Matrigel assays were used to explore proliferation, migration and in vitro tubulogenesis of EPCs, respectively. When activation or inactivation of SDF-1/CXCR4 axis with CXCR4 ligand SDF-1 or CXCR4 inhibitor AMD3100,expressed high levels of CXCR4 on EPCs were found to induce proliferation, migration and tubulogenesis of EPCs in response to the CXCR4 ligand SDF-1. However, AMD3100 abolished the effects induced by SDF-1. (2) In xenografts with EPCs, xenografts expressed higher levels of SDF-1, VEGF localized in or near the newly-forming microvessel and higher microvessel densities labeled by CD31.
In summary, the present results suggest that (1) EPCs isolated and identified from human umbilical cord blood have characteristics of renewal, differentiation and tubulogenesis. Implantation of EPCs formed chimeric vascular network incorporated of EPCs into the vascular architecture and enhanced MVD of U87 cell xenograft of impaired bone-marrow mouse. (2) CXCR4 of EPCs induced proliferation, migration and tubulogenesis of EPCs and promoted T-MAPH via EPCs, which might be of significant importance in antiangiogenic therapy for cancer.
EPCs是从骨髓、脐血或外周血分离并具有CD34、VEGFR2和或CD133阳性标志细胞,能够在趋化因子和生长因子等刺激下募集并归巢于肿瘤血管新生位点,原位分化为内皮细胞,参与肿瘤血管形成。另外,EPCs不但参与肿瘤血管形成,而且还可以促进由原微血管床芽生血管生成。并且越来越多的研究证实,EPCs参与肿瘤的血管新生,在肿瘤生长中具有重要作用。本课题组前期研究发现,T-MAPH不仅表现在与正常血管的不同,而且还表现在不同肿瘤之间、同种肿瘤不同个体之间、同一肿瘤不同区域和发生演进阶段之间,肿瘤组织的微血管之间在密度、形态、结构(组成)、三维分布上的差异性和多样性。因此,作为参与肿瘤新生微血管的关键细胞,EPCs是否或如何与瘤内原血管床的芽生成熟内皮发生连接或吻合参与肿瘤微血管网以及这些血管网是否具有多样性和异质性,尚不清楚。
与成熟内皮细胞相比,EPCs高表达趋化因子受体CXCR4,此受体的表达与活化在EPCs参与血管新生中可能具有重要作用。EPCs中CXCR4被其配体基质细胞衍生因子(stromal-cell derived factor-1, SDF-1)活化后通过G蛋白耦连受体介导EPCs参与血管新生。有研究者证实,SDF-1在募集骨髓niche CXCR4+EPCs进入新生血管活跃区原位分化为内皮细胞参与血管发生中起着主要作用。并且SDF-1诱导内皮细胞增殖、分化、芽生和管型形成及阻止EPCs凋亡。另外,研究者证实淋巴瘤患者尸检淋巴组织整合血管内的EPCs中CXCR4 mRNA的表达高于循环EPCs中CXCR4 mRNA水平,并且EPCs高表达CXCR4 mRNA与肿瘤微血管密度和肿瘤体积成正相关。临床前研究发现拮抗SDF-1/CXCR4信号可有效阻断SDF-1促肿瘤血管新生作用。本课题组前期研究证明,CXCR4活化诱导VEGF和IL-8表达而促进胶质瘤T-MAPH作用。但SDF-1/CXCR4轴活化是否参与介导EPCs参与T-MAPH形成,尚缺乏研究。
为了解CXCR4活化后介导EPCs参与胶质瘤T-MAPH的作用及机制,我们观测了EPCs参与人恶性胶质瘤细胞系U87SCID鼠皮下移植瘤微血管构筑表型,并对EPCs的CXCR4活化是否介导EPCs的迁移及其在胶质瘤T-MAPH形成中的作用进行探索。首先,我们观测了从人脐血分离、鉴定的EPCs生物学特性,研究了在体外标记并经尾静脉移植的EPCs归巢于U87移植瘤内参与T-MAPH形成在微血管密度、形态和三维结构分布等方面的表现;最后,我们探讨了EPCs中CXCR4活化介导EPCs迁移及其在胶质瘤微血管表型形成中的作用,并观测了CXCR4受体抑制剂AMD3100对SDF-1/CXCR4轴介导上述效应的抑制作用。主要结果和结论如下:
1.从人脐血中分离、鉴定的EPCs具有自我更新、可被诱导分化和形成小管样结构的生物学特性。①应用流式细胞术、密度梯度离心联合专用培养基诱导培养结果显示,新鲜分离的CBMCs中存在CD34+/CD133+细胞(1.06%),在添加了生长因子的培养基诱导培养4d后,CD34表达增加,CD133表达减少,CD34+/CD133+减少(0.26%),由祖细胞向成熟细胞分化。②免疫细胞化学显示,获得的单核源性EPCs,可表达CD14 CD34和KDR,吞噬DiI-Ac-LDL,并能连接FITC-UEA-1。另一类是晚期增殖性EPCs,在内皮细胞专用培养液中生长具有形成初级集落和次集集落能力;CD133、CD34、KDR、vWF、CD31,DiI-Ac-LDL和FITC-UEA-1皆呈阳性表达。③Matrigel管样结构形成实验结果显示,上述两亚型的EPCs在Matrigel中均可形成小管样结构。上述结果表明,从人脐血分离单个核细胞(CBMCs)经内皮培养基培养获得的EPCs具有自我更新、诱导分化和形成小管样结构的生物学特性。
2.人源性EPCs特异地整合到胶质瘤细胞异种移植瘤宿主源性新生微血管中,并形成人-鼠嵌合性血管。将膜荧光染料CFSE标记的EPCs经鼠尾静脉植入经60Co亚致死剂量辐射后荷人U87细胞移植瘤的SCID鼠体内,①免疫荧光染色显示,迁移到U87 SCID鼠移植瘤内CFSE+EPCs数量明显高于肝脾组织(p<0.01)。②免疫荧光染色和流式细胞术显示,CFSE+EPCs以两种形式参与血管新生:单个或成簇的EPCs散在分布于鼠源性的血管旁,形成盲端或功能性的血管;或与鼠源性血管连接整合形成人-鼠嵌合性微血管。定量结果显示EPCs参与形成的新生血管占总血管的11.3~18.6%。③免疫荧光染色结果显示,CFSE+/CD31+EPCs与鼠源性CD31阳性的内皮细胞组成的混合性血管形态表现多样:或呈树杆分支样,或呈蛇形蜿蜒,或呈实性条索,或呈血窦状。三维重建结果显示,人源性CFSE+EPCs与鼠源性CD31+内皮细胞相互连接和或吻合构建成杂乱的人-鼠嵌合性新生微血管网,血管分支长短、大小和管壁厚薄不等。人源性CFSE+EPCs连接或聚集于鼠源性血管分支处形成多层内皮细胞的厚壁血管,鼠源性内皮细胞组成薄壁血管。在上述微血管结构中鼠源性内皮细胞主要分布于血管主体,而CFSE+细胞分布于血管盲袢或新生血管分支点。应用IPP图形软件定量分析CFSE+EPCs组成的血管段与人-鼠嵌合性新生微血管网的物理参数,结果显示,CFSE+EPCs组成的血管段像素值约为总血管的(26±7)%,血管长度占总血管的(22±5)%,而血管分支点占总血管的(36±1)%。④人源性EPCs增强耐受骨髓损伤的SCID鼠胶质瘤模型微血管密度(P﹤0.01),增加移植瘤幼稚微血管新生比例(P﹤0.05),且促进了肿瘤生长(P﹤0.05)。以上结果表明,体外植入的EPCs特异地归巢并整合于U87细胞SCID鼠皮下移植瘤新生血管位点,从形态、三维分布和MVD上体现了EPCs参与形成T-MAPH,进而促进移植瘤生长。
3. SDF-1/CXCR4轴活化介导EPCs迁移并促进EPCs参与胶质瘤T-MAPH形成。①间接免疫荧光显示,EPCs表达SDF-1和CXCR4。②MTT结果显示,不同浓度的SDF-1(9、19、37.5、75和150μg/L)在24、48和72 h活化CXCR4后能明显诱导EPCs增殖,各组平均吸光度值均显著高于空白对照组(P<0.05)。CXCR4的小分子抑制剂ADM3100在24、48和72h能有效拮抗CXCR4活化介导的EPCs增殖作用(P<0.05)。③迁移实验结果显示,19μg/L SDF-1活化CXCR4后能显著诱导细胞迁移(P<0.05)。相反,AMD3100能显著拮抗CXCR4活化对EPCs的迁移作用(P<0.05)。④Matrigel管样结构形成实验显示,EPCs表达的CXCR4活化后EPCs形成小管样结构数为16.3±2.2。AMD3100能明显减少EPCs小管样结构形成,平均数为6.4±1.8。⑤植入EPCs的移植瘤组织SDF-1和VEGF表达量显著高于未植入EPCs(即对照组)的移植瘤组织(P﹤0.05)。SDF-1和VEGF表达量与肿瘤微血管密度呈正相关。以上结果表明,EPCs可通过自分泌或旁分泌作用活化CXCR4进而介导EPCs增殖、迁移、管样结构形成并参与T-MAPH形成。而AMD3100具有抑制SDF-1/CXCR4轴介导的上述效应的作用。
综上所述,本研究从人脐血中分离、鉴定出的EPCs具有自我更新、诱导分化和形成小管样结构的生物学特性,植入的人源性EPCs能特异地归巢到U87细胞SCID鼠皮下移植瘤内新生血管位点,通过连接或整合瘤内成熟内皮细胞形成人-鼠嵌合性血管,增强耐受骨髓损伤的移植瘤微血管密度;在上述过程中,EPCs所表达的趋化因子受体CXCR4活化后能介导EPCs增殖、迁移、管样结构形成和促进EPCs参与T-MAPH作用,CXCR4受体抑制剂AMD3100具有抑制上述效应的作用。上述研究结果为认识胶质瘤T-MAPH发生机制和针对T-MAPH进行抑制肿瘤血管新生提供了新的实验依据。
Neovascularization is crucial for the growth and metastasis of malignant solid tumors. Tumor neovascularization is now believed to occur via at least two possible mechanisms: the sprouting of pre-existing resident endothelial cells, i.e., angiogenesis, and the recruitment of bone marrow–derived EPCs, known as vasculogenesis. Although the mechanism of neovascularization has been one of the frontiers in cancer research, therapeutic efficacy of antiangiogenesis appears to be quiet. The possible reason is heterogeneity of the newly-formed microvascular network by incorporation of EPCs in the pre-existing endothlieum of tumor vessels. Tumor microvascular architecture phenotype heterogeneity, we nominated it as T-MAPH, has been considered to have a key negative effect on therapeutic efficacy of antiangiogenesis. Thus, it is of great importance and necessary to explore wether and how to EPCs contribute to T-MAPH. Malignant gliomas are highly vascularized tumors and the microvessel densities within the tumors are related to behaviors such as invasiveness and recurrence of gliomas, therefore, malignant gliomas have been considered as useful models for studies of tumor neovacularization and therapeutics.
EPCs derived from either bone marrow, peripheral blood or umbilical cord blood are CD34, VEGFR2, or CD133 antigen-positive cells, which may home to the site of neovascularization and differentiate into endothelial cells in situ. Endothelial cells contribute to tumor angiogenesis, and can originate from sprouting or co-option of neighbouring pre-existing vessels. Emerging evidence indicate that bone marrow-derived circulating EPCs can contribute to tumor angiogenesis. Little is known about how to EPCs contribute to T-MAPH.
Chemokine receptor CXCR4 is involved in the regulation of tumor vascularization via EPCs. It has been reported that CXCR4 contribute to tumor vascularization by mobilizing, recruiting, inducing differentiation and tubular formation of EPCS. We found that CXCR4 promote T-MAPH by inducing the expression of VEGF and IL-8. However, exact role and detailed mechanism of CXCR4 in EPCs contributing to T-MAPH remain unclear.
For a better understanding of the possible role and mechanism of CXCR4 in the genesis of malignant glioma microvascular architecture phenotype via EPCs, in this study, we investigated contribution of EPCs to T-MAPH. Firstly, we isolated and identified EPCs from human umbilical cord blood. Then, we explored EPCs contribution to T-MAPH. Finally, we studied the functional expression of CXCR4 by EPCs promoting T-MAPH and AMD3100 abolished the effects induced by activation of CXCR4. The main results and conclusions are as follows:
1. Two types of EPCs isolated and identified from human umbilical cord blood were found to have potentials of renewal, differentiation and tubulogenesis. (1) By flow cytometry assay, the percentage of CD34+/CD133+ in mononuclear cells isolated from human umbilical cord blood decreased from1.06% to 0.26%. (2) By immunostaning assay, we identified that two types of EPCs, referred to as early EPCs and late outgrow EPCs. The early EPCs showed to be spindle-shaped with a tendency to form colonies which weakly potential, and were positive for CD14, CD34 and KDR. In addition, cells took up DiI-Ac-LDL and bound FITC-UEA-1.The late outgrowth EPCs demonstrated colony forming cells with typical cobblestone morphology, showed robust proliferative potential and gave rise to secondary colonies. During the 8 and 15 days, cells continued to be positively expressed CD34/CD133 and CD34/KDR. In addition, cells took up DiI-Ac-LDL and bound FITC-UEA-1.Those cells became positive for vWF and CD31 after 40 days of culture. (3)By three-diamensional in vitro Matrigel assays, we found that two types EPCs are capable of in vitro tubulogenesis.
2. By transplantation of EPCs into the impaired bone-marrow glioma model we evaluated incorporation of EPCs into the vascular architecture of xenograft and role of EPCs in T-MAPH. Our data suggested that: (1) by immunostaning assay, we found that CFSE+EPCs expressed human CD31 preferentially home to tumor angiogenic site, not to liver and spleen organs, p<0.01.(2) by immunostaning assay, we found that EPCs contributed to tumor vascularization via two possible pathways, including the incorporation into the vascular architecture of U87 cell xenograft and chimeric vascular network formation or formation of functional blood vessels by singe cell and multicellular clusters localized in the vicinity of mouse-derived endothelia. With quantitative immunofluorescence and flow cytometry analysis, the percentage of EPCs-derived the endothelial population ranged from 11.3% to 18.6%. (3) By immunostaning labeled three-dimensional reconstruction, we found that chimeric vascular network was chaos with different structures, such as a sinusoid structure or a branching pattern. EPCs linked mouse ECs in branches to form thickened vessels of multicellular clusters. (4) EPCs enhanced MVD of glioma in impaired bone-marrow mouse and progressed tumor growth.
3. We examined the effect of functional CXCR4 expressed by EPCs on T-MAPH. (1) Immunostaning assay was used to evaluate expression of CXCR4 and SDF-1 in EPCs. MTT, chemotaxis and three-diamensional in vitro Matrigel assays were used to explore proliferation, migration and in vitro tubulogenesis of EPCs, respectively. When activation or inactivation of SDF-1/CXCR4 axis with CXCR4 ligand SDF-1 or CXCR4 inhibitor AMD3100,expressed high levels of CXCR4 on EPCs were found to induce proliferation, migration and tubulogenesis of EPCs in response to the CXCR4 ligand SDF-1. However, AMD3100 abolished the effects induced by SDF-1. (2) In xenografts with EPCs, xenografts expressed higher levels of SDF-1, VEGF localized in or near the newly-forming microvessel and higher microvessel densities labeled by CD31.
In summary, the present results suggest that (1) EPCs isolated and identified from human umbilical cord blood have characteristics of renewal, differentiation and tubulogenesis. Implantation of EPCs formed chimeric vascular network incorporated of EPCs into the vascular architecture and enhanced MVD of U87 cell xenograft of impaired bone-marrow mouse. (2) CXCR4 of EPCs induced proliferation, migration and tubulogenesis of EPCs and promoted T-MAPH via EPCs, which might be of significant importance in antiangiogenic therapy for cancer.
引文
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