血管内皮生长因子家族及其受体与肿瘤血管生成研究进展

血管内皮生长因子(vascular endothelial growth factor,VEGF),又名血管通透性因子(vascular permeability factor,VPF)是重要的血管生成正性调节因子,是目前抗癌治疗的研究靶点之一。现已发现的VEGF家族成员包括VEGF—A、VEGF—B、VEGF—C、VEGF—D、VEGF—E和胎盘生长因子(placenta growth factor,PLGF)。VEGF的受体有VEGFR—1(fit—1)、VEGFR-2(flk-1／KDR)、VEGFR-3(fit-4)、neuropilin(NPR1／NPR2)。该家族的成员可以选择性地增强血管和／或淋巴管内皮细胞的有丝分裂,刺激内皮细胞增殖并促进血管生成,提高血管特别是微小血管的通透性,使血浆大分子外渗沉积在血管外的基质中,促进新生毛细血管网的建立,为肿瘤细胞的生长提供营养等。作者对VEGF家族成员及其受体的理化特征、VEGF与肿瘤的关系、VEGF抑制剂的研制作一综述。     

血管内皮生长因子受体--结构与功能

血管内皮生长因子 (VEGF)的生物学效应是通过其特异的膜受体介导实现的。迄今发现VEGF有三种受体 ,受体的结构、功能 ,及VEGF的信号转导途径各不相同 ,也一直是VEGF研究的热点。本文主要综述了这方面的进展。     

VEGF受体功能研究进展

血管内皮生长因子受体（VEGFR）调控心血管系统的发育。VEGFR1对于造血祖细胞的招募及单核巨噬细胞的迁移是必需的;VEGFR2、VEGFR3在调控血管及淋巴管内皮细胞的功能时发挥重要作用,而现在很多研究都聚焦于阻断VEGFR信号通路以达到阻断肿瘤血管生长的目的。     

血管内皮生长因子的功能与应用

血管内皮生长因子是特异作用于血管内皮细胞的一类糖蛋白,以同源二聚体的形成存在,具有强烈的促内皮细胞增殖作用,促进新血管的形成,在血管再狭窄,冠脉缺血和肢体缺血等疾病中,通过加强ＶＥＧＦ的正性治疗作用,达到治疗的目的。而在视网膜新血管形成,类风湿关节炎和肿瘤等疾病时,则通过抑制ＶＥＧＦ的负性治疗作用,减少新血管形成,抑制疾病的发展。     

肾癌患者血清VEGF的水平及其与临床病理分期的关系

目的:探讨肾癌患者血清血管内皮生长因子(VEGF)的水平及其与临床病理分期的关系。方法:选择2013年1月至2015年1月在我院行手术治疗的肾癌患者56例为观察组,选择同期在我院进行健康体检的正常成人50例作为对照,所有患者的诊断均经病理切片证实,对所有研究对象,采集其清晨空腹静脉血,用酶联免疫吸附法(ELISA)检测血清中的VEGF和VEGF受体-1(VEGFR-1)。结果:观察组血清中VEGF和VEGFR-1的浓度分别为(132.75±68.31)ng/mL和(33.76±15.39)ng/mL,均显著高于对照组,差异有统计学差异(均P0.05);不同分期患者血清中VEGF和VEGFR-1浓度差异有统计学意义(均P0.05),病理分期增加,VEGF与VEGFR-1的浓度增加,VEGF与VEGFR-1呈正相关(r=0.625,P0.05)。结论:血清VEGF水平可用于诊断RCC,且对于预判RCC的病例分期具有重要价值。     

血管内皮生长因子受体的结构与功能

血管内皮生长因子(VEGF)受体是存在于血管内皮细胞,介导内皮细胞增殖分化的跨膜受体.研究较多的有两种VEGF特异受体:Flt和KDR.Flt和KDR的基因结构及染色体定位已基本确定,这二者均属RTK Ⅲ型受体,结构相似.细胞外区均有7个类似免疫球蛋白结构,细胞内区催化域均有酪氨酸激酶插入区.当VEGF与受体结合时,引起受体自身的磷酸化,发生细胞内反应.在血管发生与生长、创伤修复、炎症、肿瘤和某些血管疾病中起重要作用.     

Flt-1胞外Ⅲ区蛋白的可溶性表达、纯化及活性测定

本研究旨在克隆和表达人血管内皮生长因子受体-1胞外Ⅲ区蛋白,并测定其生物学活性。应用RT-PCR法从人脐静脉内皮细胞中克隆Flt-1胞外Ⅲ区基因片段,经测序鉴定后再克隆到原核表达载体pAYZ中,构建出的表达载体pAYZflt-1Ⅲ转化大肠杆菌16C9后,用低磷培养基诱导表达目的蛋白;采用E-tag亲和层析柱纯化目的蛋白;用SDS-PAGE、Western blotting和BCA法对其进行定性、定量检测鉴定;用ELISA、损伤愈合试验和Transwell法检测靶蛋白生物学活性。克隆的Flt-1胞外Ⅲ区基因经测序鉴定正确。所构建的pAflt-1Ⅲ表达载体经低磷培养基诱导后高表达出可溶性Flt-1胞外Ⅲ区蛋白,产量约为1.1mg/L;ELISA结果显示该蛋白可以结合VEGF165,并表现为剂量依赖性,其与配体结合的解离常数Kd为1.180pmol/L。损伤愈合试验和Transwell结果显示该蛋白可以抑制VEGF165(50ng/ml)和bFGF(100ng/mL)诱导的脐静脉内皮细胞的迁移,并呈剂量依赖性。这将为今后开展人flt-1基因Ⅲ区的功能研究及其单抗研制奠定了实验基础。     

VEGF-C 和VEGFR-3 在胃癌组织中的表达及临床意义

目的:探讨胃癌患者血管内皮生长因子C(vascular endothelial growth factor-C,VEGF-C及血管内皮生长因子受体-3(vascular endothelial growth factor receptor-3,VEGFR-3)在胃癌组织中的表达,从而确定胃癌预后的分子标志物。方法:搜集整理临床资料,采用Real-time PCR及ELISA法检测43例胃癌组织VEGF-C和VEGFR-3的表达。结果:43例胃癌组织中均有不同程度的VEGF-C和VEGFR-3的表达,Real-time PCR结果显示胃癌组织淋巴结转移组和非转移组VEGF-C和VEGFR-3的表达分别为0.07±0.01和0.12±0.01,0.03±0.01和0.06±0.02,与正常对照组相比,差异有显著性(p<0.05)。ELISA检测显示,与正常胃组织中VEGF-C和VEGFR-3的蛋白表达相比,胃癌无淋巴结转移组及胃癌并发淋巴结转移组中VEGF-C和VEGFR-3均明显增加。结论:VEGF-C和VEGFR-3的表达与胃癌淋巴结转移密切相关,提示胃癌标本VEGF-C和VEGFR-3的检测可作为胃癌预后的分子标志物。     

VEGF-C和VEGFR-3在胃癌组织中的表达及临床意义

目的：探讨胃癌患者血管内皮生长因子C（vascular endothelial growth factor-C,VEGF-C及血管内皮生长因子受体-3（vascular endothelial growth factor receptor-3,VEGFR-3）在胃癌组织中的表达,从而确定胃癌预后的分子标志物。方法：搜集整理临床资料,采用Real-time PCR及ELISA法检测43例胃癌组织VEGF-C和VEGFR-3的表达。结果：43例胃癌组织中均有不同程度的VEGF-C和VEGFR-3的表达,Real-time PCR结果显示胃癌组织淋巴结转移组和非转移组VEGF-C和VEGFR-3的表达分别为0.07±0.01和0.12±0.01,0.03±0.01和0.06±0.02,与正常对照组相比,差异有显著性（p〈0.05）。ELISA检测显示,与正常胃组织中VEGF-C和VEGFR-3的蛋白表达相比,胃癌无淋巴结转移组及胃癌并发淋巴结转移组中VEGF-C和VEGFR-3均明显增加。结论：VEGF-C和VEGFR-3的表达与胃癌淋巴结转移密切相关,提示胃癌标本VEGF-C和VEGFR-3的检测可作为胃癌预后的分子标志物。     

血管内皮生长因子与血管生成

本综述了血管内皮生长因子结构特点、体内分布,正常及病理条件下的表达水平变化及其生物学功能,并对血管通透性与血管生成之间的关系进行了评述。     

食管鳞癌VEGF—C mRNA和CD31表达及其意义

To investigate the expression of vascular endothelial growth factor-C (VEGF-C) mRNA and CD 31 in esophageal squamous cell carcinoma (ESCC) and its promotion of lymphatic metastasis. The expression of VEGF-C mRNA was examined in 43 ESCC by in situ hybridization. Intratumoral microvessel density (MVD) was assessed by immunostaining endothelial cells, using anti-CD31 antibody. The positive rate of VEGF-C mRNA expression was 41.86%. The average rank of MVD was 76.36 +/- 20.30/mm2. VEGF-C mRNA expression correlated significantly with lymph node metastasis, TNM stage and depth of invasion (p < 0.05 or p < 0.01) in statistic, but not with histological grade (differentiation) (p > 0.05). MVD correlated significantly with lymph node metastasis and TNM stage (p < 0.05) in statistic, but not with depth of invasion and histological grade (differentiation) (p > 0.05). MVD was significant higher in the VEGF-C positive tumors than negative tumors (p < 0.05). The present study indicated that VEGF-C might play a role in lympatic metastasis via lymphangiogenesis and angiogenesis in ESCC.     

Expression of vascular endothelial growth factor C in human pterygium

Vascular endothelial growth factor C (VEGF-C) and its receptor VEGFR-3 mediate lymphangiogenesis. In this study, we analyzed the expression of VEGF-C and VEGFR-3 as well as lymphatic vessels in the pterygium and normal conjunctiva of humans. Fifteen primary nasal pterygia and three normal bulbar conjunctivas, surgically removed, were examined in this study. The lymphatic vessel density (LVD) and blood vessel density were determined by the immunolabeling of D2-40 and CD31, markers for lymphatic and blood vessels, respectively. VEGF-C and VEGFR-3 expression in pterygial and conjunctival tissue proteins was detected by Western blotting and were evaluated using immunohistochemistry. The LVD was significantly higher in the pterygium than normal conjunctiva (p < 0.05). Western blot demonstrated high-level expression of VEGF-C and VEGFR-3 in the pterygium compared with normal conjunctiva. VEGF-C immunoreactivity was detected in the cytoplasm of pterygial and normal conjunctival epithelial cells. The number of VEGF-C-immunopositive cells in pterygial epithelial cells was significantly higher than in normal conjunctival cells (p < 0.05). VEGFR-3 immunoreactivity was localized in the D2-40-positive lymphatic endothelial cells. The present findings suggest the potential role of VEGF-C in the pathogenesis and development of a pterygium through lymphangiogenesis and the VEGF-C/VEGFR-3 pathway as a novel therapeutic target for the human pterygium.     

Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice

Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.     

Tumor-induced lymphangiogenesis: a target for cancer therapy?

Recent advances in understanding the biology of lymphangiogenesis, the new growth of lymphatic vessels, have cast new light on the molecular basis of metastasis to regional lymph nodes. The receptor tyrosine kinase VEGFR-3 is virtually exclusively expressed on lymphatic but not blood endothelium in the adult, and activation of VEGFR-3 by its ligands VEGF-C and VEGF-D is sufficient to induce lymphangiogenesis. Correlative studies with human tumors and functional studies using animal tumor models show that increased levels of VEGF-C or VEGF-D in tumors lead to enhanced numbers of lymphatic vessels in the vicinity of tumors, which in turn promotes metastasis to regional lymph nodes by providing a greater number of entry sites into the lymphatic system for invading tumor cells. These findings have prompted studies to investigate whether inhibitors of VEGFR-3 activation might represent novel therapeutic agents for the suppression of metastasis. However, a number of points regarding the therapeutic potential of anti-lymphangiogenic treatments in the context of cancer remain to be addressed. The spectrum and relative importance of molecules that induce lymphangiogenesis and the regulation of their expression during tumor progression, the reversibility of tumor-induced lymphangiogenesis, and possible side-effects of anti-lymphangiogenesis-based therapies all need to be investigated. Most importantly, the extent to which lymph node metastases contribute to the formation of metastases in other organs remains to be elucidated. These aspects are the focus of this review, and their investigation should serve as a roadmap to possible translational application.     

The good,the bad and the ugly: A neuropilin-2 story from normal to tumor-associated lymphangiogenesis

VEGF-C is a crucial player in lymphangiogenesis. Besides VEGFR-2 and VEGFR-3, it also binds NRP2. NRP2 enhances VEGF-C/VEGFR-3 effects in developmental lymphangiogenesis, but its role in adult and tumoral lymphangiogenesis is not known. In their study, Bagri and colleagues demonstrate that blocking NRP2 results in a decrease of metastasis formation, a phenomenon relying on tumoral lymphangiogenesis. Thus, they identified NRP2 as an attractive new target for modulating metastasis.Key words: VEGF-C, NRP2, VEGFR-3, tumoral lymphangiogenesis, metastasisBesides developmental and adult lymphangiogenesis, formation of new lymphatic vessels also occurs during tumor growth. This abnormal lymphangiogenesis enables tumor cells to escape from the solid tumor and to invade the body through the newly formed lymphatics. The resulting metastases are responsible for most cancer-associated deaths. Interestingly, inhibition of VEGF-C/VEGFR-3 axis has been shown to block metastasis formation in preclinical models.¹ However, treatment with VEGFR3_ECD may result in compromise of the normal lymphatic system leading to complications such as lymphedema. VEGF-C belongs to the VEGF family and is particularly involved in lymphangiogenesis. VEGF-C binds VEGFR-2 but mostly associates with high affinity with the third tyrosine kinase VEGF receptor, VEGFR-3.² Recently, it has been proved that VEGF-C also binds a receptor from the neuropilin family, NRP2. NRP2 associates with VEGFR-3, and binding of VEGF-C to NRP2 enhances VEGF-C/VEGFR-3-induced biological effects.³ NRP2 thus modulates developmental lymphangiogenesis, but its significance in adult or tumoral lymphangiogenesis remained unknown until a recent study by Bagri and colleagues who analyzed NRP2 function in tumor cell metastasis.⁴     

Inhibition of Lymphangiogenesis of Endothelial Progenitor Cells with VEGFR-3 siRNA Delivered with PEI-alginate Nanoparticles

Lymphangiogenesis is implicated in lymphatic metastasis of tumor cells. Recently, growing evidences show that endothelial progenitor cells (EPCs) are involved in lymphangiogenesis. This study has investigated effects of VEGF-C/VEGFR-3 (vascular endothelial growth factor receptor-3) signaling pathway on EPC differentiation and effectiveness of inhibiting lymphatic formation of EPCs with VEGFR-3 siRNA delivered in PEI (polyethylenimine)-alginate nanoparticles. CD34⁺VEGFR-3⁺ EPCs were sorted from mononuclear cells of human cord blood. Under induction with VEGF-C, the cells differentiated toward lymphatic endothelial cells. The nanoparticles were formulated with 25 kDa branched PEI and alginate. The size and surface charge of PEI-alginate nanoparticles loading VEGFR-3 siRNA (N/P = 16) are 139.1 nm and 7.56 mV respectively. VEGFR-3 siRNA specifically inhibited expression of VEGFR-3 mRNA in the cells. After treatment with PEI-alginate/siRNA nanocomplexes, EPCs could not differentiate into lymphatic endothelial cells, and proliferation, migration and lymphatic formation of EPC-derived cells were suppressed significantly. These results demonstrate that VEGFR-3 signaling plays an important role in differentiation of CD34⁺VEGFR-3⁺ EPCs. VEGFR-3 siRNA delivered with PEI-alginate nanoparticles can effectively inhibit differentiation and lymphangiogenesis of EPCs. Inhibiting VEGFR-3 signaling with siRNA/nanocomplexes would be a potential therapy for suppression of tumor lymphangiogenesis and lymphatic metastasis.     

Effects of acute exercise, exercise training, and diabetes on the expression of lymphangiogenic growth factors and lymphatic vessels in skeletal muscle

Blood and lymphatic vessels together form the circulatory system, allowing the passage of fluids and molecules within the body. Recently we showed that lymphatic capillaries are also found in the capillary bed of skeletal muscle. Exercise is known to induce angiogenesis in skeletal muscle, but it is not known whether exercise has effects on lymphangiogenesis or lymphangiogenic growth factors. We studied lymphatic vessel density and expression of the main lymphangiogenic growth factors VEGF-C and VEGF-D and their receptor VEGFR-3 in response to acute running exercise and endurance exercise training in the skeletal muscle of healthy and diabetic mice. VEGF-C mRNA expression increased after the acute exercise bout (P < 0.05) in healthy muscles, but there was no change in diabetic muscles. VEGF-C levels were not changed either in healthy or in diabetic muscle after the exercise training. Neither acute exercise nor exercise training had an effect on the mRNA expression of VEGF-D or VEGFR-3 in healthy or diabetic muscles. Lymphatic vessel density was similar in sedentary and trained mice and was >10-fold smaller than blood capillary density. Diabetes increased the mRNA expression of VEGF-D (P < 0.01). Increased immunohistochemical staining of VEGF-D was found in degenerative muscle fibers in the diabetic mice. In conclusion, the results suggest that acute exercise or exercise training does not significantly affect lymphangiogenesis in skeletal muscle. Diabetes increased the expression of VEGF-D in skeletal muscle, and this increase may be related to muscle fiber damage.     

Differential mRNA and tissue expression of lymphangiogenic growth factors (VEGF-C and -D) and their receptor (VEGFR-3) during tail regeneration in a gecko

Lymphangiogenesis, the growth of new lymph vessels, has important roles in both normal and pathological lymphatic function. Despite recent advances, the precise molecular mechanisms behind the lymphangiogenic process remain unclear. The Australian marbled gecko, Christinus marmoratus, voluntarily drops its tail (autotomy) as a predator avoidance strategy. Following autotomy a new tail is regenerated including lymphatic drainage pathways. We examined the molecular control of lymphangiogenesis within the unique model of the regenerating gecko tail. Partial sequences were obtained of the gecko lymphangiogenic growth factors, vascular endothelial growth factor C (VEGF-C) and VEGF-D along with their receptor VEGFR-3. These were highly homologous to other vertebrates. Quantitative real-time polymerase chain reaction (PCR) demonstrated up-regulation of VEGF-C, VEGF-D and VEGFR-3 mRNA expression during the early and middle stages of tail regeneration (between 4 and 9 weeks following autotomy), in late regeneration (12 weeks) and during mid-regeneration (7 and 9 weeks), respectively. VEGF-C and VEGF-D immunostaining was observed lining some lymphatic-like and blood vessels in early–mid tail regeneration, indicating possible associations of the proteins with VEGFRs on endothelia. Keratinocytes and fibroblasts also showed positive staining of VEGF-C and VEGF-D in early–mid tail regeneration. Additionally, VEGF-C was localised in adipose tissue in all tail states examined. This work suggests that specific timings exist for the expression of the lymphangiogenic growth factors, VEGF-C and VEGF-D, and their receptor, VEGF-R3, throughout the regeneration of a functional lymphatic network. Along with localisation data, this suggests potential functions for the growth factors in lymphangiogenesis and angiogenesis throughout tail regeneration.     

卵巢上皮性癌中MMP-7、VEGF—C的表达和微淋巴管密度变化的相关性及其意义

目的观察基质金属蛋白酶-7（matrix metalloproteinase-7,MMP-7）、血管内皮生长因子-C（vascular endothelial growth factor—C,VEGF—C）和D2—40在上皮性卵巢癌中的表达,探讨MMP-7、VEGF-C表达以及D2—40标记的微淋巴管密度（microlymphatic vessel density,MLVD）与卵巢癌临床病理生物学行为的关系。方法应用免疫组化对80例EOC及20例卵巢良性上皮性肿瘤组织进行MMP-7、VEGF-C和D2—40的检测,并进行MLVD测定。结果MMP-7、VEGF—C蛋白的表达阳性率及MLVD计数在EOC组分别为60．0％、57．5％以及13．06±6．16,均分别高于良性肿瘤组25．0％、25．0％及9．25±4．33（P〈0．05）。EOC中,MMP-7、VEGF-C蛋白的阳性表达率随肿瘤组织学分级的增加而升高,分别为29．4％、56．7％、78．8％和11．8％、60．0％、78．8％（P〈0．05）,腹腔脏器及淋巴结有转移者高于无转移者,分别为86．8％＆35．7％及86．8％＆27．1％（P〈0．05）,两个指标在PTNMⅢ-Ⅳ期阳性表达率分别为83．8％、83．8％,均显著高于PTNMⅠ-Ⅱ期的阳性表达率39．5％、34．9％（P〈o．05）;D2—40标记的MLVD计数在EOC中随组织学分级的增加而增高,分别为7．29±2．85、12．63±6．25、16．48±4．94,腹腔脏器及淋巴结有转移者高于无转移者16．50±4．31＆10．00±5．99（P〈0．05）,PTNMⅢ-Ⅳ期显著高于PTNMⅠ-Ⅱ期16．65±4．15＆10．02±5．99（P〈0．05）。EOC中,MMP-7、VEGF-C的表达分别与MLVD呈正相关性（r分别为0．510,0．455,均PG0．01）;MMP-7、VEGF-C的表达分别与EOC患者腹腔器官及淋巴结转移亦呈正相关（r分别为0．521,0．565,均P〈0．01）。结论MMP-7、VEGF-C的表达可以促进EOC中淋巴管的形成及淋巴道转移,联合检测MMP-7、VEGF-C及MLVD可能作为预测EOC患者侵袭、转移等生物学行为和评价预后的重要指标。     

VEGF—C和VEGFR-3在大肠癌中的表达及与淋巴结转移的关系

目的研究大肠癌患者癌组织中VEGF—C和VEGFR-3的表达及其相互之间的关系,以探讨VEGF—C和VEGFR-33大肠癌发生发展中的作用及与临床预后的关系。方法运用原位杂交方法及免疫组化SP法检测69例大肠癌组织中VEGF—CmRNA和VEGF—C的表达,同时运用免疫组化法分析相同组织中VEGFR-3表达状况。结果VEGF—CmRNA及VEGF—C在大肠癌组织中的表达阳性率分别为52．17％（36／69）和49．27％（34／69）,VEGF—C基因和蛋白表达基本相符合。VEGFR-3表达阳性率为43．48％（30／69）,正常黏膜未见VEGFR-3表达（P〈0．05）;大肠癌组织中VEGF—C与VEGFR-3蛋白表达之间存在显著的相关性（P〈0．01）。在伴有淋巴结转移的大肠腺癌中,VEGF—C与VEGFR～3的阳性表达率分别为67．74％（21／31）和64．52％（20／31）,转移组与非转移组相比较均有显著性差异（P〈0．01）;VEGF—C及VEGFR-3表达与Duke’s分期呈负相关（P〈0．05）,与组织分化、性别、年龄、肿瘤大小等临床病理因素无关（P〉0．05）。结论VEGF—C与VEGFR-3表达可增加大肠癌淋巴道转移和侵袭能力,其检测有助于大肠癌恶性程度评价及预后的判断,两者在大肠癌的发展中起重要作用。