VEGF及其受体基因表达的调控机制

血管内皮细胞生长因子（VEGF）其受体flt-1,flk-1/KDR在血管生成中起重要调控作用。本文对VEGF及其受体基因表达调控机制进行综述。     

048 VEGF及其受体基因表达的调控机制

血管内皮细胞生长因子(VEGF)其受体flt-1、flk-1／KDR在血管生成中起重要调控作用，本文对VEGF及其受体基因表达调控机制进行综述。     

人脑胶质瘤血管内皮细胞生长因子受体表达的研究

目的研究人脑胶质瘤血管内皮细胞生长因子受体(Vascular endot helial growth factor receptors, VEGFR)的表达及意义.方法应用免疫组织化学技术,检测52例手术切除的脑胶质细胞瘤组织中两种VEGFR(flt-1和fl k-1)的表达.结果①胶质细胞瘤组织中有flt-1和flk-1的表达 , 主要表达于肿瘤血管内皮细胞, 同时巨噬细胞的胞浆中也有表达;② 3组胶质细胞瘤中,VEGFR的表达率分别为16.0%、58.8%、90.0%,3组间有差异(p<0.05 );③脑胶质细胞瘤中, flt-1的表达与flk-1 的表达相关(R=0.993,p<0.01).结论正常脑组织中无VEGFR表达,脑胶质细胞瘤中有VEGFR表达,主要表达于血管内皮细胞中,VEGF通过旁分泌途径刺激脑胶质细胞瘤的血管发生; VEGF的促血管发生作用既可通过血管内皮细胞的VEGFR直接产生,又可通过促进巨噬细胞的局灶浸润间接产生;VEGFR表达与脑胶质瘤恶性程度呈正相关.     

血管内皮生长因子及其受体在子宫内膜癌中的表达

目的探讨血管内皮生长因子(VEGF)及其受体fms样酪氨酸受体-1 (flt-1)和含插入区的激酶受体(KDR)在子宫内膜癌血管生成中的作用及其与内膜癌分化程度的关系.方法采用免疫组织化学及原位杂交方法对23例子宫内膜癌及6例正常绝经期子宫内膜中VEGF、flt-1、KDR蛋白质及其mRNA进行检测,并对少数病例行Western印迹分析,以检测VEGF亚型在内膜癌组织的分布,用内皮细胞标志Ⅷ因子标记内膜癌组织中的微血管密度.结果 VEGF、flt-1、KDR蛋白质及其mRNA主要分布在子宫内膜癌组织血管内皮细胞及癌细胞胞质内.VEGF蛋白质在中分化(G2)、低分化(G3)内膜癌血管内皮细胞及癌细胞上的表达高于高分化内膜癌(G1)及正常绝经期子宫内膜(P<0.05), VEGF mRNA在不同分化程度内膜癌组织的表达差异无显著性意义(P>0.05),但均大于正常绝经期子宫内膜(P<0.05);flt-1蛋白质及flt-1mRNA在G3内膜癌血管内皮细胞的表达高于G1、G2及正常绝经期子宫内膜(P<0.05),在癌细胞的表达差异无显著性意义(P>0.05) ,但均高于正常绝经期子宫内膜(P<0.05);KDR蛋白质在子宫内膜癌组织血管内皮细胞及癌细胞上的表达较强,但不随分化程度发生变化,其mRNA在中分化(G2)、低分化(G3)内膜癌血管内皮细胞及癌细胞上的表达高于正常绝经期子宫内膜(P<0.05).G3子宫内膜癌组织的血管密度(48个±12个)高于G1(27个±14个)、G2(26个±16个)及正常绝经期子宫内膜(26个±11个,P<0.05).结论 VEGF、flt-1、KDR及mRNA在子宫内膜癌中的表达形式提示其与癌组织血管生成及血管通透性相关,VEGF及其受体是与子宫内膜癌旺盛生长相关的因子之一.     

血管内皮生长因子(VEGF)对培养内皮细胞VEGF受体表达的影响

目的 :为探讨VEGF对培养内皮细胞 (EC)VEGF受体表达的影响。方法 :将培养的人脐静脉内皮细胞 (HUVEC)随机分为 4组 :( 1)正常对照组 ;( 2 )低氧培养组 ;( 3)VEGF 10ng/ml组 ;( 4)低氧 +VEGF10ng/ml组。HUVEC低氧培养参照Kuwara等介绍的方法并加以改进。HUVECVEGF受体的检测采用免疫组织化学方法。结果 :采用简易低氧培养法 ,48h内培养液氧分压维持在 5 8mmHg ;与对照组相比 ,低氧培养组、VEGF组和低氧 +VEGF组HUVECVEGF受体Flk 1/KDR阳性细胞数增多 ,强度增加 ;但未检测到VEGF受体Flt 1表达。结论 :低氧可使HUVEC表面的VEGF受体Flk 1/KDR表达增加 ,VEGF同源上调其受体Flk 1/KDR ,低氧和VEGF在调节VEGF受体Flk 1/KDR方面有协调作用。     

人脑胶质瘤血管内皮细胞生长因子受体表达的研究

目的　研究人脑胶质瘤血管内皮细胞生长因子受体 (Vascularendothelialgrowthfactorreceptors,VEGFR)的表达及意义 .方法　应用免疫组织化学技术 ,检测 5 2例手术切除的脑胶质细胞瘤组织中两种VEGFR(flt- 1和flk - 1)的表达 .结果　①胶质细胞瘤组织中有flt- 1和flk- 1的表达 ,主要表达于肿瘤血管内皮细胞 ,同时巨噬细胞的胞浆中也有表达 ;② 3组胶质细胞瘤中 ,VEGFR的表达率分别为 16 .0 %、5 8.8%、90 .0 % ,3组间有差异 (p <0 .0 5 ) ;③脑胶质细胞瘤中 ,flt- 1的表达与flk - 1的表达相关 (R =0 .993,p<0 .0 1) .结论　正常脑组织中无VEGFR表达 ,脑胶质细胞瘤中有VEGFR表达 ,主要表达于血管内皮细胞中 ,VEGF通过旁分泌途径刺激脑胶质细胞瘤的血管发生 ;VEGF的促血管发生作用既可通过血管内皮细胞的VEGFR直接产生 ,又可通过促进巨噬细胞的局灶浸润间接产生 ;VEGFR表达与脑胶质瘤恶性程度呈正相关     

胰岛素和葡萄糖对缺氧血管内皮细胞分泌tPA和PAI-1抗原的影响

目的: 观察胰岛素和葡萄糖对缺氧血管内皮细胞分泌组织型纤溶酶原激活物(tPA)及其抑制物-1(PAI-1)的影响。 方法:培养人脐静脉内皮细胞株ECV-304。分组实验:(1)常氧组;(2)在缺氧条件下又分为Ⅰ:缺氧对照组;Ⅱ:低浓度组:胰岛素150 mU/L、葡萄糖5.5 mmol/L;Ⅲ:中浓度组:胰岛素450 mU/L、葡萄糖15 mmol/L;Ⅳ:高浓度组:胰岛素900 mU/L、葡萄糖30 mmol/L;Ⅴ:渗透压对照组:甘露醇24.5 mmol/L。取培养4、8、12 h 3个时点,用ELISA法测定细胞培养上清液tPA、PAI-1抗原。结果:缺氧明显增加tPA和PAI-1抗原分泌,tPA/PAI-1值明显增加(P＜0.01)。胰岛素和葡萄糖能够刺激缺氧内皮细胞分泌tPA和PAI-1抗原,在缺氧8 h以内,tPA/PAI-1比值明显增加(P＜0.05)。随缺氧时间的延长,tPA/PAI-1值逐渐下降。结论:胰岛素和葡萄糖能够刺激内皮细胞分泌tPA和PAI-1抗原,在缺氧8 h以内,tPA/PAI-1值升高,纤溶活性升高,有利于局部自发性纤溶的发生。此作用可能是IGK治疗急性心肌梗死的机制之一。     

VEGE.flt-1及KDR/flk-1与子宫内膜预后价值的研究

有学者认为肿瘤血管形成是在宿主微环境和介质影响下的高度可调空过程。通过对乳腺癌 ,卵巢癌及宫颈癌的研究 ,表明了新生血管形成与肿瘤转移和复发有关。血管内皮因子 (VEGF)是子宫内膜血管形成的重要调控者 ,其受体flt- 1、 KDR/ flk- 1也是血管形成的良好标志。为研究 VEGE及其受体 flt- 1、KDR/ flt- 1与子宫内膜癌预后的关系进行了下述研究。47例全子宫切除标本用福尔马林液固定 ,石蜡包埋 ,在肿瘤分化最差 ,浸润最严重处取 4um切片 ,常规染色行组织学分类 ,并作免疫组化染色。其中浸润性内膜腺癌 35例 ( 期 2 2例 , 期 4例 , 期…     

血管内皮生长因子及其受体在糖尿病小鼠视网膜的表达

目的：观察血管内皮生长因子（Vascular Endothelial Growth Factor,VEGF)及其受体flk-1在糖尿病视网膜细胞的表达,以期阐明VEGF与糖尿病性盲的关系。方法：应用免疫组织化学这及发病组视网膜VEGF和flk-1阳性细胞,并计算单位面积视网膜中神经节细胞VEGF阳性率和flk-1阳性细胞数,结果：发病组视网膜神经节细胞VEGF阳性率和flk-1阳性神经节细胞数从3月龄时开始增多（P＜0．05）,并随着病程延长有上升趋势,VEGF在6月龄后增多更显著,flk-1在8月龄后增加也更为明显。结论：VEGF和flk-1在糖尿病视网膜神经节细胞表达量增加,VEGF通过与flk-1相互作用,不仅使血管内皮细胞增殖,导致视网膜新生网管形成,而且还可能通过改变神经节细胞及Muller细胞膜上的受体数量而对神经节细胞功能活动产生影响。     

血管内皮生长因子及其受体在肝癌细胞中的表达及意义

目的 探讨人肝癌细胞株血管内皮生长因子（VEGF）及其受体的表达,进一步认识VEGF在肝癌血管形成中的作用机制,方法 以人脐静脉血管内皮细胞系ECV304和小鼠成纤维细胞系L929作为对照,采用免疫组化染色及RT-PCR,检测体外培养的人肝细胞肝癌细胞系SMMC7721、HHCC和HepG2中VEGF及其受体的表达。结果 SMMC7721、HHCC和HepG2细胞均有VEGF的表达。同时VEGF受体1（Flt-1)在SMMC7721细胞中也有表达;而HHCC和HepG2细胞则表达VEGF的受体2（KDR）。结论 在肝癌的血管形成中可能存在VEGF的自分泌机制。     

Non-endothelial KDR/flk-1 expression is associated with increased survival of patients with urothelial bladder carcinomas

AIMS: To investigate the immunohistochemical expression of KDR/flk-1 in a series of 114 urothelial bladder carcinomas in relation to clinicopathological parameters, Ki67, p53 and Bcl-2 protein expression and patient survival. KDR/flk-1 is a high-affinity tyrosine kinase receptor for vascular endothelial growth factor (VEGF), on vascular endothelium. However, there is increasing evidence that KDR/flk-1 is also expressed by normal non-endothelial and tumour cells. METHODS AND RESULTS: Immunohistochemistry was performed on paraffin sections using monoclonal and polyclonal antibodies. Statistical analysis was univariate (chi2 log rank test) and multivariate (Cox's model). KDR/flk-1 expression was observed in the cytoplasm of cancerous cells in 68.4% of cases. No statistically significant associations were observed between KDR/flk-1 expression and grade or stage of urothelial carcinomas, Ki67, p53 or Bcl-2 expression. In contrast, widespread KDR/flk-1 expression in more than 50% of cancerous cells was associated with increased survival, on univariate and multivariate analysis (P = 0.0119 and P = 0.042, respectively). CONCLUSIONS: Although the biological significance of non-endothelial KDR/flk-1 expression has not yet been elucidated, its association with better patient survival may be related to the failure of non-endothelial KDR/flk-1 to mediate angiogenic and mitogenic effects.     

Tumor specific activation of the VEGF/KDR angiogenic pathway in a subset of locally advanced squamous cell head and neck carcinomas

Vascular endothelial growth factor (VEGF) and its receptors, Flt-1 and flk-1(KDR), constitute an important angiogenic pathway which, under hypoxic conditions, is up-regulated in many solid tumours. We used the monoclonal antibody 11B5, specific for recognizing VEGF expression and the `VEGF/flk-1(KDR) complex' on tumour endothelium, to assess free VEGF protein expression and VEGF/receptor activated microvessel density (aMVD) in a series of 104 inoperable locally advanced squamous cell carcinomas of the head and neck, treated with chemo-radiotherapy. High VEGF expression in cancer cells was strongly associated with high VEGF/receptor expression in the vasculature. The high VEGF expression and the aMVD were not associated with the standard microvessel density (sMVD), as assessed with the monoclonal antibody anti-CD31 and, were not detected in normal tissue. An increased sMVD, however, was significantly related with the expression thymidine phosphorylase (TP), and also with the nuclear accumulation of the oncoprotein p53, but neither p53 nor TP was associated with VEGF expression by cancer cells or VEGF/receptor complex aMVD. In 35% of cancer cases examined, more than 20% of the microvessels assessed with anti-CD31 also expressed the VEGF/KDR complex. The vasculature of the normal head and neck mucosa did not express the VEGF/KDR complex. There was no association between VEGF expression or VEGF/receptor complex aMVD and response to chemo-radiotherapy or patient's survival. It is concluded that activation of the angiogenic pathway VEGF/flk-1(KDR) is tumor specific in a subgroup of locally advanced squamous cell carcinomas of the head and neck. Selective destruction of this type of vasculature, using immunoconjugates directed against the VEGF/receptor complex, may prove therapeutically useful for patients with a high tumoral VEGF/flk-1(KDR) activated microvessel fraction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of Vascular Endothelial Growth Factor (VEGF) and a receptor subtype (KDR/flk-1) in the liver of rats exposed to riddelliine: a potential role in the development of hemangiosarcoma

Riddelliine alters hepatocellular and endothelial cell kinetics and function including stimulating an increase in hepatocytic vascular endothelial growth factor (VEGF) in the absence of increased serological levels of VEGF (Nyska etal. 2002). The objective of this study was to further assess hepatic VEGF and KDR/flk-1 synthesis and expression by hepatic cells under riddelliine treatment conditions. Forty-two male F344/N rats were dosed by gavage with riddelliine (0, 1.0, and 2.5 mg/kg/day) for 6 weeks. Seven animals/group were sacrificed after 8 consecutive daily doses; remaining rats were terminated after 30 daily doses, excluding weekends. Hepatic tissues were evaluated by immunohistochemistry and in situ hybridization. The results showed that VEGF mRNA expression was observed in control and treated animals; however, qualitative differences were noted. Treated animals exhibited VEGF mRNA in clustered, focal hepatocytes and bile duct epithelium, whereas VEGF mRNA in hepatocytes from vehicle control rats was distributed evenly across all hepatocytes. Results evaluating the distribution of the VEGF cognate receptor, KDR/flk-1 showed that randomly distributed, rare sinusoidal endothelium, including those demonstrating karyomegaly and cytomegaly expressed KDR/flk-1. Phosphorylation of KDR/flk-1 at pTyr996 and pTyr1054/1059, but not pTyr951, was also detected, evidence that endothelial cell KDR/flk-1 was activated. These results suggest that both hepatocytes and endothelial cells are targets of riddelliine-induced injury. We speculate that damage to both populations of cells may lead to dysregulated VEGF synthesis by hepatocytes and activation of KDR/flk-1 by endothelium leading to the induction of sustained endothelial cell proliferation, culminating in the development of hepatic hemangiosarcoma.     

The vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR-1) modulates Flk-1 (VEGFR-2) signaling during blood vessel formation

Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 (VEGFR-1) die from vascular overgrowth, caused primarily by aberrant endothelial cell division (Kearney JB, Ambler CA, Monaco KA, Johnson N, Rapoport RG, Bautch VL: Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood 2002, 99:2397-2407). Because a second high-affinity VEGF receptor, flk-1, produces a positive endothelial proliferation signal, it was logical to ask whether flt-1 affects developmental blood vessel formation by modulating signaling through flk-1. Differentiated embryonic stem cell cultures lacking flt-1 (flt-1-/-) had increased flk-1 tyrosine phosphorylation, indicating that flk-1 signaling is up-regulated in the mutant background. The selective flk-1 inhibitor SU5416 partially rescued the flt-1-/- mutant phenotype, and this rescue was accompanied by a decrease in the relative amount of flk-1 tyrosine phosphorylation. Thus reduced flk-1 signal transduction can partially compensate for the lack of flt-1. The flt-1-/- mutant phenotype was also partially rescued by Flt-1/Fc, a truncated flt-1 that binds and sequesters the VEGF ligand. Taken together, these data show that down-regulation of flk-1 signaling by two different strategies partially rescues the developmental vascular overgrowth seen in the absence of flt-1, and they support a model whereby flt-1 modulates the flk-1 signal at an early point in the pathway.     

Strong expression of kinase insert domain-containing receptor, a vascular permeability factor/vascular endothelial growth factor receptor in AIDS-associated Kaposi's sarcoma and cutaneous angiosarcoma.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the angiogenesis associated with the growth of many human and animal tumors. VPF/VEGF stimulates endothelial cell growth and increases microvascular permeability by interacting with two endothelial cell tyrosine kinase receptors, KDR and flt-1. We studied 16 cases of AIDS-associated Kaposi's sarcoma (KS), 2 cases of cutaneous angiosarcoma, and 6 cases of capillary hemangioma by in situ hybridization for expression of VPF/VEGF, KDR, and flt-1 mRNAs. We also performed immunohistochemical staining for VPF/VEGF protein in 15 cases. Tumor cells in KS and angiosarcoma strongly expressed KDR but not flt-1 mRNA. Endothelial cells in small stromal vessels in and around these tumors strongly expressed both KDR and flt-1 mRNAs. Tumor cells expressed VPF/VEGF mRNA strongly in only one case of KS, adjacent to an area of necrosis. This was also the only case in which the tumor cells stained substantially for VPF/VEGF protein. VPF/VEGF mRNA and protein were, however, strongly expressed by squamous epithelium in areas of hyperplasia and near areas of ulceration overlying tumors. VPF/VEGF mRNA was also expressed focally at lower levels by infiltrating inflammatory cells, probably macrophages. The strong expression of both KDR and flt-1 in small stromal vessels in and around tumors suggests that VPF/VEGF may be an important regulator of the edema and angiogenesis seen in these tumors. The strong expression of KDR by tumor cells in KS and angiosarcoma implies that VPF/VEGF may also have a direct effect on tumor cells. Tumor cells in four of six capillary hemangiomas strongly expressed both KDR and flt-1 mRNAs in contrast to the high level expression of only KDR observed in the malignant vascular tumors studied. Neither VPF/VEGF mRNA or protein were strongly expressed in capillary hemangiomas. VPF/VEGF and its receptors may play an important but as yet incompletely understood role in the pathogenesis of both benign and malignant vascular tumors.     

Hypoxia and cobalt stimulate vascular endothelial growth factor receptor gene expression in rats

 This study aimed to examine the influence of acute tissue hypo-oxygenation on the expression of the vascular endothelial growth factor (VEGF) receptor genes. To this end male Sprague-Dawley rats were exposed to different hypoxic conditions such as 10% or 8% oxygen, 0.1% carbon monoxide and cobalt chloride (60 mg/kg) for 6 h and the abundance of flt-1, flt-4 and flk-1 mRNA in lungs and livers was determined by RNase protection assay. The relative proportions of flt-1, flt-4 and flk-1 were 10 : 2.5 : 1 and 10 : 10 : 2 in normoxic lungs and livers, respectively. It was found that 8% but not 10% oxygen increased flt-1 mRNA two- to threefold in both organs, whilst flt-4 and flk-1 mRNA were not changed by acute inspiratory hypoxia. Carbon monoxide inhalation also increased flt-1 mRNA but not flt-4 or flk-1 mRNA in both organs. Subcutaneous cobalt administration increased flt-1 mRNA in the livers only, whilst flt-4 and flk-1 mRNA remained unchanged. These findings show that acute tissue hypo-oxygenation is a rather selective stimulus for flt-1 gene expression. The efficiency of the different manoeuvres applied to stimulate flt-1 gene expression is rather similar to the stimulation of erythropoietin gene expression. It is not unreasonable to assume, therefore, that the oxygen-dependent regulation of both genes at the cellular level has significant similarities.     

Vasoactive intestinal peptide in rats with focal cerebral ischemia enhances angiogenesis

We studied the effect of vasoactive intestinal peptide (VIP) on angiogenesis in the ischemic boundary area after focal cerebral ischemia. Adult male Sprague–Dawley rats underwent middle cerebral artery occlusion for 2 h. A single dose of VIP was given via i.c.v. injection at the beginning of reperfusion. Immunohistochemistry and Western blotting were performed to assay angiogenesis and brain levels of vascular endothelial growth factor (VEGF) protein, respectively. In addition, the expression of VEGF and its receptors (flt-1 and flk-1), as well as endothelial proliferation, was measured using rat brain microvascular endothelial cells. Immunohistochemical analyses revealed significant (P<0.05) increases in the numbers of bromodeoxyuridine (BrdU) positive endothelial cells and microvessels at the boundary of the ischemic lesion in rats treated with VIP compared with rats treated with saline. Western blotting analysis showed that treatment with VIP significantly (P<0.05) raised VEGF levels in the ischemic hemisphere. In addition, treatment with VIP increased flt-1 and flk-1 immunoreactivity in endothelial cells. In vitro, incubation with VIP significantly (P<0.01) increased the proliferation of endothelial cells and induced the expression of VEGF, flt-1 and flk-1 in endothelial cells. The stimulatory effect of VIP on the proliferation of endothelial cells was significantly (P<0.01) inhibited by SU5416, a selective inhibitor of VEGF receptor tyrosine kinase. Our data suggest that treatment with VIP enhances angiogenesis in the ischemic brain, and this effect may be mediated by increases in levels of VEGF and its receptors.     

The role of estrogen receptors,erbB receptors,vascular endothelial growth factor and its receptors,and vascular endothelial growth inhibitor in the development of the rat mammary gland

We identified the localization and distribution of cell-specific epidermal growth factor receptors (EGFRs: erbB-1, erbB-2, erbB-3, erbB-4), vascular endothelial growth factor (VEGF), VEGF receptors [VEGFRs: VEGF-R1 (flt-1), VEGF-R2 (flk-1/KDR), VEGF-R3 (flt-4)], vascular endothelial growth inhibitor (VEGI), and estrogen receptor (ER), and determined whether or not these growth factors in rat mammary glands are functional. Thirty-five adult female Spraque-Dawley rats were randomly divided into five groups, each of which were at the 7th, 14th, and 21st day of pregnancy; 7th day post-delivery; and 7th day after weaning. It was determined that erbB, VEGF and its receptors, VEGI, and ER stained at different intensities. Intense staining was observed, in particular, in erbB receptors during pregnancy and involution, and also in VEGF and its receptors during lactation, while ER stained during the last periods of pregnancy and lactation. In conclusion, the expression of erbB, VEGF and its receptors, and ER were determined at varying intensities at different sites of the mammary gland during pregnancy, lactation, and involution periods.