Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting

The protective genes that mediate endothelial cell (EC) survival during angiogenesis have not been completely characterized. Here, we show that an antisense oligonucleotide to the apoptosis inhibitor survivin suppressed de novo expression of survivin in ECs by vascular endothelial cell growth factor (VEGF). In contrast, the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2 levels in endothelium. When assessed in cell death assays, antisense targeting of survivin abolished the anti-apoptotic function of VEGF against tumor necrosis factor-alpha- or ceramide-induced cell death, enhanced caspase-3 activity, promoted the generation of a approximately 17-kd active caspase-3 subunit, and increased cleavage of the caspase substrate, polyADP ribose polymerase. In contrast, the survivin antisense oligonucleotide had no effect on EC viability in the absence of VEGF. Antisense oligonucleotides to platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), lymphocyte function-associated molecule-3 (LFA-3, CD58), or intercellular adhesion molecule-1 (ICAM-1, CD54) did not reduce the anti-apoptotic function of VEGF in endothelium. When tested on other angiogenic activities mediated by VEGF, survivin antisense treatment induced rapid regression of three-dimensional vascular capillary networks, but did not affect EC migration/chemotaxis. These data suggest that the anti-apoptotic properties of VEGF during angiogenesis are primarily mediated by the induced expression of survivin in ECS: Manipulation of this pathway may increase EC viability in compensatory angiogenesis or facilitate EC apoptosis and promote vascular regression during tumor angiogenesis.     

Enhanced L1CAM expression on pancreatic tumor endothelium mediates selective tumor cell transmigration

L1 cell adhesion molecule (L1CAM) is a transmembrane cell adhesion molecule initially defined as a promigratory molecule in the developing nervous system that appears to be also expressed in some endothelial cells. However, little is known about the functional role of L1CAM on endothelial cells. We observed that L1CAM expression was selectively enhanced on endothelium associated with pancreatic adenocarcinoma in situ and on cultured pancreatic tumor-derived endothelial cells in vitro. L1CAM expression of endothelial cells could be augmented by incubation with immunomodulatory cytokines such as tumor necrosis factor alpha, interferon gamma, or transforming growth factor beta 1. Antibodies to L1CAM and the respective ligand neuropilin-1 blocked tube formation and stromal cell-derived factor 1β induced transmigration of tumor endothelial cells in vitro. L1CAM expression on tumor-derived-endothelial cells enhanced Panc1 carcinoma cell adhesion to endothelial cell monolayers and transendothelial migration. Our data demonstrate a functional role of L1CAM expression on tumor endothelium that could favor metastasis and angiogenesis during tumor progression. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Survivin-dependent angiogenesis in ischemic brain: molecular mechanisms of hypoxia-induced up-regulation

Approaches to regulating angiogenesis in the brain, which may diminish parenchymal damage after stroke, are lacking. Survivin, the inhibitor of apoptosis protein, is up-regulated in vitro in vascular endothelial cells by angiogenic factors, including vascular endothelial cell growth factor (VEGF). To evaluate the in vivo role of survivin in the brain in response to hypoxia/ischemia, we used a mouse model of stroke and show that 2 days after permanent middle cerebral artery occlusion, survivin is uniquely expressed by microvessels that form in the peri-infarct and infarct regions. The extent of vascularization of the infarct is dependent on expression of survivin, since vessel density is significantly reduced in mice with heterozygous deficiency of the survivin gene (survivin+/- mice), even though infarct sizes were not different. Hypoxia alone induces survivin expression in the brain, by cultured endothelial cells and by embryonic stem cells, but this response is at least partially independent of VEGF, hypoxia inducible factor 1alpha, or placental growth factor. Delineating the spatiotemporal pattern of expression of survivin after stroke, and the molecular mechanisms by which this is regulated, may provide novel approaches to therapeutically optimize angiogenesis in a variety of ischemic disorders.     

Evidence for the involvement of SDF-1 and CXCR4 in the disruption of endothelial cell-branching morphogenesis and angiogenesis by TNF-alpha and IFN-gamma

Vigorous inflammatory responses are associated with tissue damage, particularly when toxic levels of inflammatory cytokines are produced. Despite proangiogenic factors being present early at sites of inflammation, vascular repair occurs toward the end of the inflammatory response, suggesting modulation of the proangiogenic response. Endogenous inhibitors of angiogenesis induced during acute inflammation are poorly characterized. Here, we looked for endothelial cell-derived modulators of angiogenesis that may account for delayed neovascularization during inflammation. Gene profiling of endothelial cells showed that the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma) selectively promote expression of the antiangiogenic molecules, IFN-inducible protein-10, monokine induced by IFN-gamma, tryptophanyl-tRNA synthetase, and tissue inhibitor of metalmetalloproteinase-1, and inhibit expression of the proangiogenic molecules, platelet-endothelial cell adhesion molecule-1, vascular endothelial growth factor receptor-2, stromal cell-derived factor-1 (SDF-1), collagen type IV, endothelial cell growth factor-1, and carcinoembryonic antigen-related cell adhesion molecule-1. Reduced endothelial cell expression of SDF-1 protein by TNF-alpha and IFN-gamma disrupts extracellular matrix-dependent endothelial cell tube formation, an in vitro morphogenic process that recapitulates critical steps in angiogenesis. Replacement of SDF-1 onto the endothelial cell surface reconstitutes this morphogenic process. In vivo, TNF-alpha and IFN-gamma inhibit growth factor-induced angiogenesis and SDF-1 expression in endothelial cells. These results demonstrate that SDF-1/CXC chemokine receptor-4 constitutes a TNF-alpha- and IFN-gamma-regulated signaling system that plays a critical role in mediating angiogenesis inhibition by these inflammatory cytokines.     

Angiogenesis in gliomas: biology and molecular pathophysiology

Glioblastoma multiforme (GBM) is characterized by exuberant angiogenesis, a key event in tumor growth and progression. The pathologic mechanisms driving this change and the biological behavior of gliomas remain unclear. One mechanism may involve cooption of native blood vessels by glioma cells inducing expression of angiopoietin-2 by endothelial cells. Subsequently, vascular apoptosis and involution leads to necrosis and hypoxia. This in turn induces angiogenesis that is associated with expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF) in perinecrotic pseudopalisading glioma cells. Here we review the molecular and cellular mechanisms implicated in HIF-1-dependent and HIF-1-independent glioma-associated angiogenesis. In GBMs, both tumor hypoxia and genetic alterations commonly occur and act together to induce the expression of HIF-1. The angiogenic response of the tumor to HIF-1 is mediated by HIF-1-regulated target genes leading to the upregulation of several proangiogenic factors such as VEGF and other adaptive response molecules. Understanding the roles of these regulatory processes in tumor neovascularization, tumor growth and progression, and resistance to therapy will ultimately lead to the development of improved antiangiogenic therapies for GBMs.     

Down-modulation of monocyte transendothelial migration and endothelial adhesion molecule expression by fibroblast growth factor: reversal by the anti-angiogenic agent SU6668

Basic and acidic fibroblast growth factor (bFGF and aFGF, respectively) and vascular endothelial growth factor (VEGF) exert angiogenic actions and have a role in wound healing, inflammation, and tumor growth. Monocytes and endothelial cells are involved in these processes, but the effect of FGF and VEGF on monocyte-endothelial cell interactions has not been defined. We observed that monocyte adhesion to resting or cytokine (tumor necrosis factor-alpha or interleukin-1 alpha)-stimulated human umbilical vein endothelial cells (HUVECs) was markedly inhibited (40 to 65%) by culture (1 to 6 days) of HUVECs with aFGF or bFGF. Monocyte transendothelial migration induced by C5a and chemokines (MCP-1, SDF-1 alpha, RANTES, MIP-1 alpha) was also suppressed (by 50 to 75%) on bFGF-stimulated HUVECs. VEGF did not have these effects at the concentrations used (10 to 20 ng/ml), although like bFGF, it promoted HUVEC proliferation. Culture of HUVECs with bFGF and aFGF significantly down-regulated intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression on resting or tumor necrosis factor-alpha-stimulated HUVECs, but had no influence on platelet endothelial cell adhesion molecule (PECAM)-1 and VE-cadherin expression. bFGF also inhibited MCP-1 production by HUVECs. The inhibitory effects of bFGF on monocyte transendothelial migration and adhesion molecule expression were reversed by SU6668, an anti-angiogenic agent and bFGF receptor tyrosine kinase inhibitor. Our results suggest that bFGF and aFGF may suppress endothelial-dependent monocyte recruitment and thus have an anti-inflammatory action during angiogenesis in chronic inflammation but inhibit the immunoinflammatory tumor defense mechanism. However, SU6668 is an effective agent to prevent this down-regulatory action of bFGF on monocyte-endothelial cell interactions.     

Differential influences of bFGF and VEGF on the expression of vascular cell adhesion molecule-1 on human umbilical vein endothelial cells]

A fundamental feature of inflammation includes angiogenesis, adhesion of leukocytes to vascular endothelium, and entry of leukocytes into inflamed tissues. Recent studies have suggested that angiogenesis and cellular adhesion may be mutually linked processes. Both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have been shown to facilitate angiogenesis. However, their roles in the expression of adhesion molecules on the endothelial cells have not been clarified. The current studies therefore examined the effect of bFGF and VEGF on the expression of vascular cell adhesion molecule-1 (VCAM-1) on human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor alpha (TNF-alpha). HUVEC (1 x 10(4)/well) were incubated in a 96 well microtiter plate with culture medium containing endothelial cell growth supplement (ECGS) for 24 h. After the incubation, culture medium was replaced by ECGS free culture medium with or without TNF-alpha (10 ng/ml), bFGF (10 ng/ml) and VEGF (10 ng/ml), and the culture was further carried out for additional 24 h. The expression of VCAM-1, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) was measured by cell ELISA and the proliferation of HUVEC was measured by MTT colorimetric assay. Soluble VCAM-1 (sVCAM-1) in the supernatants were assessed by ELISA. Although, both bFGF and VEGF supported the proliferation of HUVEC, bFGF, but not VEGF, selectively suppressed the expression of VCAM-1 on HUVEC stimulated with TNF-alpha. The expression of ICAM-1 and E-selectin induced by TNF-alpha was not inhibited by either bFGF or VEGF. In addition, bFGF also decreased the levels of sVCAM-1 in the supernatants of TNF-alpha stimulated HUVEC. The data indicate that bFGF, but not VEGF, suppresses the production of VCAM-1 by HUVEC under stimulation with TNF-alpha. These results therefore suggest that angiogenic cytokines bFGF and VEGF play different roles in the regulation of the expression of adhesion molecules on endothelial cells under inflammation.     

Cyclooxygenase inhibitors suppress angiogenesis and reduce tumor growth in vivo

Angiogenesis plays a key role in the development of malignant tumors. To clarify the roles of cyclooxygenase (COX) in malignant tumor development and angiogenesis, we investigated the effects of COX inhibitors on two kinds of gastrointestinal cancer xenograft, one of which overexpresses COX-2 and the other expresses no COX, in nude mice in vivo. There was a positive correlation between tumor volume and angiogenesis within the xenograft. Oral administration with a specific COX-2 or a nonspecific COX inhibitors lowered the expression of potent angiogenic factors; vascular endothelial growth factor and basic fibroblast growth factor, reduced angiogenesis and growth, induced apoptosis, and suppressed cell replication of the COX-2 overexpressing cancer xenografts in a dose-dependent manner. A nonspecific COX inhibitor, not a specific COX-2 inhibitor, reduced growth and angiogenesis of non-COX expressing cancer xenograft by inhibition of COX-1 in vascular endothelial cells. These results demonstrate that COX inhibitors suppress angiogenesis and tumor growth by inhibiting expression of angiogenic factors and vascular endothelial cell growth. They support the hypothesis that COX plays an important role in cancer growth via angiogenesis. These findings offer a new strategy against cancer using COX inhibitors (nonsteroidal anti-inflammatory drugs).     

Silencing of CCR7 inhibits the growth,invasion and migration of prostate cancer cells induced by VEGFC

Early in prostate cancer development, tumor cells express vascular endothelial growth factor C (VEGF-C), a secreted molecule that is important in angiogenesis progression. CC-chemokine receptor 7 (CCR7), another protein involved in angiogenesis, is strongly expressed in most human cancers, where it activated promotes tumor growth as well as favoring tumor cell invasion and migration. The present study aimed to investigate the effect of down-regulating CCR7 expression on the growth of human prostate cancer cells stimulated by VEGFC. The CCR7-specific small interfering RNA (siRNA) plasmid vector was constructed and then transfected into prostate cancer cells. The expression of CCR7 mRNA and protein was detected by quantitative polymerase chain reaction and western blot analysis, respectively. Cell proliferation, apoptosis, cell cycle distribution and cell migration were assessed following knockdown of CCR7 by RNA interference (RNAi). Western blot analysis was used to identify differentially expressed angiogenesis- and cell cycle-associated proteins in cells with silenced CCR7. The expression levels of CCR7 in prostate cancer cells transfected with siRNA were decreased, leading to a significant inhibition of prostate cancer cell proliferation, migration and invasion induced by VEGFC. Western blot analysis revealed that silencing of CCR7 may inhibit vascular endothelial growth factor, matrix metalloproteinase (MMP)-2 and MMP-9 protein expression. In conclusion, the present study demonstrated that RNAi can effectively silence CCR7 gene expression and inhibit the growth of prostate cancer cells, which indicates that there is a potential of targeting CCR7 as a novel gene therapy approach for the treatment of prostate cancer.     

Naked DNA immunization as an approach to target the generic tumor antigen survivin induces humoral and cellular immune responses in mice

Survivin, a 16.5 kDa tumor associated antigen, is the smallest member of the inhibitor of apoptosis family that is abundantly expressed during development but essentially absent in normal adult tissues. Interestingly, survivin expression is up-regulated in virtually all types of cancers studied, as well as in vascular endothelial cells during tumor associated angiogenesis. Survivin links apoptosis to cell cycle progression and plays a pivotal role in regulation of cell proliferation. These characteristics make survivin a potentially promising generic target for cancer immunotherapy. Hence, a genetic immunization strategy to induce tumor-specific immune responses against human survivin in a pre-clinical animal model was developed. In initial studies, BALB/c mice were immunized by intramuscular injection with DNA coding for human survivin (pcDNA3.1/hSurv). In addition, a construct encoding a secreted version of survivin (pSecTag2B/hSurv) was designed. A plasmid coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was co-injected in both cases as a molecular adjuvant. Expression of survivin following transfection in mouse cells was corroborated. Humoral responses against human survivin were detected in mice sera using two immunization protocols (injections at 2- or 3-week intervals). The humoral response was markedly improved by secretion of survivin and co-expression of GM-CSF. The predominant antibody subclass detected in responsive mice was IgG2a, suggesting that a Th1-CD4+ cellular response had been induced. Furthermore, DNA immunization with survivin encoding vectors generated an effective CD8+ T cell response measured as an increase of cytotoxic Interferon-gamma (IFN-gamma) secreting CD8+ T cells. In conclusion, intramuscular genetic immunization of mice with human survivin encoding plasmids induced a survivin-specific humoral as well as cellular immune response in recipient mice. Secretion of survivin and co-injection of GM-CSF as a genetic adjuvant appear to be more important in generating an humoral than a cellular immune response.     

Vascular endothelial growth factor expression, angiogenesis, and necrosis in renal cell carcinomas

Rapidly growing tumors often develop necrosis. In the present study the expression of vascular endothelial growth factor (VEGF) was investigated and compared to microvessel density and necrosis of renal cell carcinomas. In the tumor-host interface the microvessel density was significantly increased compared to central tumor areas. Tumor necrosis was associated with a decrease of microvessel density and an increase of the VEGF protein expression within the perinecrotic rim. VEGF protein was focally upregulated in vital tumor tissue. An increase of the apoptotic rate of endothelia and vital tumor tissue in tumors with necrosis could not be detected. VEGF(121,165) mRNA was decreased in proliferatively active carcinomas compared to less proliferative tumors. Multicellular renal cell cancer spheroids as a model of chronic hypoxia developed central apoptosis but no necrosis. VEGF was upregulated in the spheroid. Tumor microvessels expressed matrix metalloproteinase -2 and -9 and an incomplete pericyte covering in comparison to tumor-free tissue indicating immature active angiogenesis. We conclude that highly proliferative renal cell carcinomas outgrow their vascular supply and develop chronic hypoxia inducing a decrease of proliferation and an increase of VEGF expression. However, chronic hypoxia does not cause significant necrosis or apoptosis. Tumor necrosis is more likely induced by acute hypoxia due to immature microvessels. Furthermore, VEGF expression associated with concomitant tumor necrosis may help identify renal cell carcinomas susceptible to antiangiogenic therapy.     

Potential autocrine function of vascular endothelial growth factor in head and neck cancer via vascular endothelial growth factor receptor-2.

Vascular endothelial growth factor is a peptide with well-defined actions on the vasculature and fundamental role in tumor angiogenesis. Its action in vascular endothelium is exerted in a paracrine manner. The immunohistochemical expression of this protein by cancer cells in head and neck squamous cell carcinoma was correlated with increased tumor aggressiveness and poor survival in previous studies. In the past years, an increasing amount of studies demonstrated potential autocrine action of vascular endothelial growth factor in various neoplasms. However, the existence and the impact of such autocrine action in head and neck cancer have not been demonstrated yet. In this retrospective study, we evaluated the expression of vascular endothelial growth factor and its receptors in neoplastic cells, in a cohort of patients with head and neck squamous cell carcinoma, and compared this expression with tumor aggressiveness, clinicopathologic parameters and outcome. High expression of vascular endothelial growth factor was strongly correlated with high expression of vascular endothelial growth factor receptor-2 (but not vascular endothelial growth factor receptor-1) on the cancer cells (P<0.001). The co-overexpression of both the protein and vascular endothelial growth factor receptor-2 was associated with higher tumor proliferation rate (P<0.001). The above co-overexpression also correlated with worse survival (log rank P<0.05) in patients with oral-larynx squamous cell carcinoma. Our results suggest that an autocrine vascular endothelial growth factor loop, mediated via vascular endothelial growth factor receptor-2, probably exists in head and neck squamous cell carcinoma. These observations support the hypothesis that the use of vascular endothelial growth factor receptor-2 inhibitors as adjuvant antiangiogenic therapy might have beneficial effects for these patients, by disrupting both paracrine (endothelial-dependent) and autocrine actions of vascular endothelial growth factor.     

Sustained expression of homeobox D10 inhibits angiogenesis

Homeobox (Hox) genes are master regulatory genes that direct organogenesis and maintain differentiated tissue function. As HoxD3 and HoxB3 promote angiogenesis, we investigated whether endothelial cells use other Hox genes to maintain a mature quiescent phenotype. HoxD10 expression was higher in quiescent as compared to tumor-associated angiogenic endothelium. Microarray analysis of HoxD10-overexpressing endothelial cells revealed a pattern of gene expression consistent with a nonangiogenic phenotype. Moreover, sustained expression of HoxD10 impaired endothelial cell migration and blocked angiogenesis induced by basic fibroblast growth factor and vascular endothelial growth factor in the chick chorioallantoic membrane in vivo. HoxD10-overexpressing human endothelial cells also failed to form new vessels when implanted into immunocompromised mice. These results indicate a role for HoxD10 in maintaining a nonangiogenic state in the endothelium.     

Cell Type-Specific Expression of Angiopoietin-1 and Angiopoietin-2 Suggests a Role in Glioblastoma Angiogenesis

Glioblastomas are highly vascular tumors which overexpress the angiogenesis factor vascular endothelial growth factor (VEGF). VEGF and its receptors, VEGF-R1 and VEGF-R2, have been shown to be necessary for embryonic angiogenesis as well as for tumor angiogenesis. Recently, the angiopoietin/Tie2 receptor system has been shown to exert functions in the cardiovascular system that are distinct from VEGF but are also critical for normal vascular development. To assess the potential role of Tie2 and its ligands angiopoietin-1 and angiopoietin-2 in tumor vascularization, we analyzed their expression pattern in human gliomas. Tie-2 was up-regulated in tumor endothelium compared to normal human brain tissue. We further observed cell type-specific up-regulation of the message for both angiopoietin-1 and angiopoietin-2 in gliomas. Whereas Ang-1 mRNA was expressed in tumor cells, Ang-2 mRNA was detected in endothelial cells of a subset of glioblastoma blood vessels. Small capillaries with few periendothelial support cells showed strong expression of Angiopoietin-2, whereas larger glioblastoma vessels with many periendothelial support cells showed little or no expression. Although the function of Tie2 and its ligands in tumor angiogenesis remains a subject of speculation, our findings are in agreement with a recently proposed hypothesis that in the presence of VEGF, local production of Ang-2 might promote angiogenesis.     

Angiopoietin-2 is implicated in the regulation of tumor angiogenesis

We addressed the effect of angiopoietin expression on tumor growth and metastasis. Overexpression of angiopoietin-2 (Ang-2) in Lewis lung carcinoma and TA3 mammary carcinoma cells inhibited their ability to form metastatic tumors and prolonged the survival of mice injected with the corresponding transfectants. In contrast, angiopoietin-1 (Ang-1) overexpression had no detectable effect on the ability of either tumor type to disseminate. Tumors derived from Ang-2-overexpressing cells displayed aberrant angiogenic vessels that took the form of vascular cords or aggregated vascular endothelial cells with few associated smooth muscle cells. These vascular cords or aggregates were accompanied by endothelial and tumor cell apoptosis, suggesting that an imbalance in Ang-2 expression with respect to Ang-1 and vascular endothelial growth factor may disrupt angiogenesis and tumor survival in vivo. Our observations suggest that Ang-2 may play an important role in regulating tumor angiogenesis.     

肿瘤坏死因子a促进人冠状动脉内皮细胞的凋亡

背景：研究表明冠状动脉内皮细胞凋亡参与了动脉粥样硬化的发生、发展过程。 目的：观察肿瘤坏死因子a对人冠状动脉内皮细胞的诱导损伤作用。 方法：取对数生长期的人冠状动脉内皮细胞c-12221,分别加入含0(对照),200,400,600 mg/L肿瘤坏死因子a的培养液,采用MTT检测细胞增殖率变化,Hoechst 33258/PI双染观察凋亡细胞形态变化,Annexin V-FITC和PI双染流式检测细胞凋亡率变化,高内涵活细胞成像系统检测细胞线粒体膜电位变化。 结果与结论：肿瘤坏死因子a呈剂量依赖性抑制人冠状动脉内皮细胞的增殖。Hoechst 33258/PI染色观察可见凋亡细胞染色质凝集、细胞核碎裂成碎片等典型细胞凋亡的特征性变化,不同质量浓度肿瘤坏死因子a组细胞凋亡率高于对照组(P < 0.05),线粒体膜电位低于对照组(P < 0.05),且呈剂量依赖性。表明肿瘤坏死因子a呈剂量依赖性促进人冠状动脉内皮细胞凋亡,抑制其增殖,作用机制与线粒体凋亡通路有关。     

血管内皮生长因子165转染骨髓间充质干细胞绿色荧光蛋白的表达

背景：血管内皮生长因子是一种有效的血管形成和通透性诱导因子,其中在体内以血管内皮生长因子165和血管内皮生长因子121表达为主,具有强烈的促血管新生作用。 目的：观察以血管内皮生长因子165基因转染骨髓间充质干细胞,继而分化为血管内皮细胞的可行性。 方法：分离提取50 g SD大鼠骨髓间充质干细胞,采用流式细胞仪鉴定,将携有血管内皮生长因子165基因的质粒pGLV-EF1a,采用慢病毒转染骨髓间充质干细胞,转染后于荧光显微镜下观察绿色荧光蛋白表达情况。 结果与结论：转染后12 h可见细胞内有绿色荧光蛋白表达,48 h 后表达增多,72 h后达到高峰,其后部分细胞荧光开始减退。结果证明血管内皮生长因子165基因转染骨髓间充质干细胞后,骨髓间充质干细胞内有绿色荧光蛋白表达,提示细胞转染成功,骨髓间充质干细胞定向分化为血管内皮细胞具有可行性。