Regulation of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR by estradiol through VEGF in uterus

The induction of vascular endothelial growth factor (VEGF) expression by 17beta-estradiol (E(2)) in many target cells, including epithelial cells, fibroblasts and smooth muscle cells, suggests a role for this hormone in the modulation of angiogenesis and vascular permeability. We have already described a cyclic increase in Flk-1/KDR-expressing capillaries in the human endometrium during the proliferative and mid-secretory phases, strongly suggestive of an E(2) effect on Flk-1/KDR expression in the endometrial capillaries. However, it is unclear whether these processes are due to a direct effect of E(2) on endothelial cells. Using immunohistochemistry, we report an increase in Flk-1/KDR expression in endometrial capillaries of ovariectomized mice treated with E(2), or both E(2) and progesterone. This process is mediated through estrogen receptor (ER) activation. In vitro experiments using quantitative RT-PCR analysis demonstrate that Flk-1/KDR expression was not regulated by E(2) in human endothelial cells from the microcirculation (HMEC-1) or macrocirculation (HUVEC), even in endothelial cells overexpressing ERalpha or ERbeta after ER-mediated adenovirus infection. In contrast, Flk-1/KDR expression was up-regulated by VEGF itself, in a time- and dose-dependent manner, with the maximal response at 10 ng/ml. Thus, we suggest that E(2) up-regulates Flk-1/KDR expression in vivo in endothelial cells mainly through the modulation of VEGF by a paracrine mechanism. It is currently unknown whether or not the endothelial origin might account for differences in the E(2)-modulation of VEGF receptor expression, particularly in relation to the vascular bed of sex steroid-responsive tissues.     

Oroxylin A inhibits angiogenesis through blocking vascular endothelial growth factor-induced KDR/Flk-1 phosphorylation

Purpose

In this study, we examined the antiangiogenic effect of oroxylin A in vitro and in vivo and explored the potential mechanisms for this effect.

Methods

Transwell assay and tube formation assay were used to evaluate the effects of oroxylin A on vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells (HUVECs). Rat aortic ring assay was also employed to assess the effect of oroxylin A on microvessel outgrowth from rat aorta. Human tumor xenografts model in nude mice was further used to investigate the antiangiogenic activity of oroxylin A in vivo. Western blot analysis was used to investigate the related mechanism.

Results

Oroxylin A remarkably suppressed the VEGF-stimulated migration and tube formation of HUVECs. It also inhibited microvessel sprouting from rat aortic ring in vitro. In addition, it suppressed the angiogenesis of xenograft tumor in nude mice, which concurred with the inhibition of tumor growth. Moreover, oroxylin A blocked VEGF-induced phosphorylation of KDR/Flk-1 and related downstream signaling molecules, including p38 mitogen-activated protein kinase, extracellular signal-regulated kinase and Akt.

Conclusion

Oroxylin A possessed antiangiogenic activities in vitro and in vivo, which could be an underlying mechanism of its anticancer effect.     

The vascular endothelial growth factor receptor KDR/Flk-1 is a major regulator of malignant ascites formation in the mouse hepatocellular carcinoma model

The vascular endothelial growth factor-A (VEGF-A), also known as the vascular permeability factor (VPF), has been shown to play an important role in malignant ascites formation. The effects of VEGF-A are mediated through flt-1 and kinase insert domain-containing receptor/fetal liver kinase (KDR/Flk-1) receptors. It has been shown that KDR/Flk-1 is a predominant receptor in solid hepatocellular carcinoma (HCC) development, but the role of this receptor in hepatic ascites formation has not yet been elucidated. In this study, we examined the role of KDR/Flk-1 in the murine MH134 hepatic malignant ascites formation by means of VEGF-A- and KDR/Flk-1-specific neutralizing antibodies (VEGF-A nAb and KDR/Flk-1 nAb, respectively). The mean volume of ascites, number of tumor cells in ascites, and the peritoneal capillary permeability were significantly suppressed by VEGF-A nAb and KDR/Flk-1 nAb treatment. These inhibitory effects of KDR/Flk-1 nAb were more potent than those of VEGF-A nAb. The autophosphorylation of KDR/ Flk-1 in the peritoneal wall was almost completely abolished by KDR/ Flk-1 nAb, whereas a certain level of activation was still shown by VEGF-A nAb treatment. Another VEGF-family, VEGF-C, which also binds KDR/Flk-1, was detected in the ascites. Furthermore, in the therapeutic experiment, although both VEGF-A nAb and KDR/Flk-1 nAb prolonged the survival rate of ascites-bearing mice, the latter showed a more significant impact on the survival of animals. These results suggest that KDR/Flk-1 is a major regulator of malignant hepatic ascites formation, and that in addition to VEGF-A, VEGF-C may also be involved in the malignant ascites formation via KDR/ Flk-1 activation.     

Galectin-1 induces vascular permeability through the neuropilin-1/vascular endothelial growth factor receptor-1 complex

Galectin-1 (Gal-1) is a β-galactoside-binding lectin that regulates endothelial cell migration, proliferation, and adhesion. However, the effect of Gal-1 on vascular permeability and the underlying mechanisms are unclear. We found that high Gal-1 expression was associated with elevated tumor vascular permeability in specimens of oral squamous cell carcinoma. Using transendothelial passage of FITC-dextran and a Miles assay, we demonstrated that Gal-1 increased vascular permeability extracellularly through its carbohydrate recognition domain. Mechanism dissection revealed that the neuropilin (NRP)-1/vascular endothelial growth factor receptor- (VEGFR)-1 complex was required for Gal-1-regulated vascular permeability. Activation of VEGFR-1 triggered activation of Akt which led to a reduction in vascular endothelial-cadherin at cell–cell junctions and resulted in cytoskeletal rearrangement. Both inhibition of Gal-1 secreted from cancer cells and administration of an anti-Gal-1 antibody in the tumor microenvironment suppressed tumor growth and vascular permeability in xenograft models. In conclusion, our results demonstrate a novel function of Gal-1 of increasing vascular permeability through the NRP-1/VEGFR1 and Akt signaling pathway and indicate that targeting Gal-1 by an anti-Gal-1 antibody is a feasible therapy for vascular hyperpermeability and cancer.     

Vascular endothelial growth factor,its receptor KDR/Flk-1, and pituitary tumor transforming gene in pituitary tumors

Pituitary tumorigenesis is a poorly understood process involving dysregulation of the cell cycle, proliferation, and angiogenesis. The novel securin pituitary tumor transforming gene (PTTG) disrupts cell division and stimulates fibroblast growth factor (FGF)-2-mediated angiogenesis. We investigated expression of the angiogenic vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1 in 103 human pituitary tumors, and we assessed functional relationships between these genes in vitro. Nonfunctioning tumors (n = 81) demonstrated markedly raised VEGF mRNA (3.2-fold, P < 0.05) and protein concentrations, compared with normal pituitaries (n = 10). KDR was also highly induced in nonfunctioning tumors (14-fold, P < 0.001, n = 78) as well as in the whole cohort of pituitary tumors, compared with normal pituitary samples (14-fold, P < 0.0001, n = 100). In vitro, PTTG induced VEGF, but not KDR, expression in fetal neuronal NT2 cells (2.7-fold, P < 0.001, n = 8), MCF-7 breast carcinoma cells (1.9-fold, P = 0.03, n = 10), and choriocarcinoma JEG-3 cells (P = 0.0002, n = 8). A mutated PTTG construct that cannot be phosphorylated showed identical VEGF up-regulation (2.9-fold, P < 0.001, n = 8) in NT2 cells, compared with wild-type PTTG, but a further mutated construct with abrogation of the key protein:protein interaction domain of PTTG resulted in a significant reduction in VEGF stimulation, compared with wild-type (0.37-fold reduction, P < 0.001, n = 8). FGF-2 findings mirrored those of VEGF, although antibody depletion of secreted FGF-2 in the cell medium failed to influence VEGF up-regulation by PTTG. Overall, our findings implicate altered VEGF and KDR signaling in pituitary tumorigenesis, and we propose that PTTG stimulation of FGF-2 and VEGF expression in the presence of up-regulated growth factor receptors may account for angiogenic growth and progression of human pituitary tumors.     

Transactivation of KDR/Flk-1 by the B2 receptor induces tube formation in human coronary endothelial cells

Endothelial cells (ECs) are the critical cellular element responsible for postnatal angiogenesis. Vascular endothelial growth factor (VEGF) stimulates angiogenesis via the activation of kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1) in ECs. In addition, transactivation of KDR/Flk-1 by the bradykinin (BK) B2 receptor induces the activation of endothelial nitric oxide synthase (eNOS). These findings indicate that the precise role of BK in angiogenesis is likely to be more complex than initially thought, and it questions the importance of BK in angiogenic processes. Therefore, we examined whether transactivation by BK induced tube formation. We developed an in vitro model of human coronary artery EC (HCEC) tube formation on a matrix gel. We demonstrated that BK dose-dependently induced tube formation. Although a lower concentration of BK and VEGF did not separately induce tube formation, the formation was induced by a combination of lower concentrations of BK and VEGF, suggesting that VEGF and BK had a synergistic effect. The effect was blocked by a B2 receptor antagonist (HOE140) and specific inhibitors of VEGF receptor tyrosine kinases (Tki) and NOS. In addition, BK induced tyrosine phosphorylation of the KDR/Flk-1 receptor, as did VEGF itself. The transactivation was also blocked by HOE140 and Tki. Our results showed that, in HCECs, stimulation of the B2 receptor leads to the transactivation of KDR/Flk-1, as well as to eNOS activation, which induces tube formation. To our knowledge, this is a novel mechanism in which transactivation of KDR/Flk-1 by a G protein-coupled receptor, B2 receptor, may be a potent signal for tube formation.     

Vascular endothelial growth factor and its receptor, Flk-1/KDR, are cytoprotective in the extravascular compartment of the ovarian follicle

Vascular endothelial growth factor (VEGF) is a potent mitogen and cytoprotective factor for vascular endothelial cells. Although VEGF is ubiquitously expressed, its role in nonvascular tissues is poorly understood. VEGF interacts with various cell surface receptors to mediate its cellular effects. It previously has been thought that the VEGF receptor Flk-1/KDR, its main signaling receptor, was expressed exclusively by endothelial cells. However, in the present study using bovine and rodent models, we demonstrate that VEGF and Flk-1/KDR are coexpressed in ovarian granulosa cells. VEGF and Flk-1/KDR mRNA and protein were both detectable in follicle tissue sections and in vitro cultured granulosa cells. Expression of both ligand and receptor increased in healthy follicles throughout follicular development. VEGF treatment of serum-starved and cytokine-exposed granulosa cells resulted in enhanced survival, and this cytoprotection was ameliorated when Flk-1/KDR signaling was inhibited. Reduced expression of Flk-1/KDR was also associated with the onset and progression of follicle atresia, suggesting involvement in follicular health in vivo. The results of this study demonstrate for the first time expression of Flk-1/KDR in ovarian granulosa cells and identify a novel extravascular role for VEGF and its receptor in ovarian function.     

Overexpression of vascular endothelial growth factor and its endothelial cell receptor KDR in type 1 leprosy reaction

The sites of expression of vascular endothelial growth factor (VEGF) and of KDR, its endothelial cell receptor, were investigated in leprosy reaction Type 1, or reversal reaction (RR), by immunohistochemistry and in situ hybridization. In comparison with nonreactional leprosy, overexpression of both VEGF and KDR was seen in granuloma cells, especially epithelioid and foreign body-type giant cells, the epithelium and the vascular endothelium of RR specimens. In granuloma cells, hybridization for VEGF was stronger than immunostaining, a finding that may reflect the rapid turnover of VEGF in an immunologically dynamic situation such as RR. In the epidermis, double immunohistochemistry revealed VEGF overexpression in CDla-positive dendritic cells. The VEGF may not only be relevant for hyperpermeability and mononuclear cell differentiation (the key morphologic features in the acute, clinically evident phase of RR), but it could also be implicated in RR onset, when dendritic cells are activated in response to antigen stimulation.     

布地奈德/福莫特罗对支气管哮喘患者血管内皮生长因子及其受体1表达及气道重塑的调控作用

目的支气管哮喘(简称哮喘)是一种慢性气道炎症疾病,伴随气道高反应性、可逆性气流阻塞和气道重塑。血管生成和微血管重塑在气道慢性炎症过程中可能起到重要的作用。血管内皮生长因子(vascular endothelial growth factor,VEGF)是一种促血管生成因子,其生理作用包括促进内皮细胞存活、增殖和迁移。本研究通过检测哮喘患者气道VEGF和VEGF受体1(VEGFR1)的表达,探讨VEGF和哮喘患者气道重塑的关系以及布地奈德/福莫特罗对哮喘患者气道重塑的调控作用。方法支气管组织来源于2006年4月至11月四川大学华西医院经纤维支气管镜行组织活检。23例为中度哮喘患者,20例为对照组。哮喘患者给予规律吸入布地奈德/福莫特罗4.5/160μg,2次/d,持续半年。VEGF和VEGFR1通过免疫组织化学进行检测。AB-PAS和MassonTrichrome染色用于评估气道重塑程度。结果两组之间年龄和性别差异无统计学意义。而两组之间用力肺活量占预计值%,第一秒用力呼气容积占预计值%,PC20,V75占预计值%,V50占预计值%和V25占预计值%的差异有统计学意义。哮喘组患者气道黏液腺增生、平滑肌增厚、上皮下纤维化以及新生血管增加。与对照组比较,VEGF和VEGFR1阳染细胞数目增多,表达增加。VEGF和VEGFR1表达增加与哮喘患者的气道重塑、气流阻塞和气道高反应性呈正相关。规律吸入布地奈德/福莫特罗6个月后,哮喘患者VEGF和VEGFR1表达减少,气道重塑减轻。结论伴随哮喘患者气道血管生成增多和气道重塑,VEGF和VEGFR1表达增加。规律吸入布地奈德/福莫特罗可以通过减少VEGF和VEGFR1表达而减轻哮喘患者气道重塑。阻断VEGF和VEGFR1可能是治疗哮喘的新策略。     

A monoclonal antibody that blocks VEGF binding to VEGFR2 (KDR/Flk-1) inhibits vascular expression of Flk-1 and tumor growth in an orthotopic human breast cancer model

Vascular endothelial growth factor (VEGF) is a primary stimulant of tumor angiogenesis. We previously raised a neutralizing anti-VEGF monoclonal antibody 2C3 that blocks the interaction of VEGF with VEGFR2 (KDR/Flk-1) but not with VEGFR1 (FLT-1/flt-1). Here, we describe the therapeutic effects of 2C3 on tumor growth in an orthotopic model of MDA-MB-231 human breast carcinoma implanted in the mammary fat pads (MFP) of nude mice. Administration of 2C3 to mice with 100–150 mm³ tumors inhibited tumor growth by 75%, as compared to recipients of the isotype-matched irrelevant control IgG, C44. Treatment with 2C3 also inhibited the establishment of tumor colonies and reduced tumor burden in the lungs of mice injected intravenously with MDA-MB-231 cells. No toxicity was observed in these studies. The mean microvascular density (MVD) of tumors in 2C3-treated mice was 55 ± 5 per mm², as compared to 188 ± 5 per mm² in the C44-treated control group. The decrease in MVD closely correlated with the degree of inhibition of tumor growth. Treated tumors mostly contained mid-size and large vessels. Microvessels were mainly confined to the peripheral layer of tumor that bordered on the normal MFP epithelium. Tumor vessels had decreased expression of VEGFR2, indicating that neutralization of tumor-derived VEGF by 2C3 down-regulates the expression of VEGFR2 on tumor vasculature. This, in turn, may limit re-initiation of angiogenesis by either tumor-derived or stromal VEGF. These findings suggest that 2C3 is a candidate for treating primary cancer and for preventing the outgrowth of tumor metastases in cancer patients.     

Interleukin-1 induces the expression of vascular endothelial growth factor in human pericardial mesothelial cells

Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells. We have studied the production of VEGF by human pericardial mesothelial cells. Mesothelial cells were separated by scraping the pericardial surface during cardiac surgery and cultured. When stimulated with interleukin (IL)-1α, pericardial mesothelial cells expressed VEGF mRNA and protein in concentration- and time-dependent manners. Hypoxia was also found to enhance mesothelial VEGF mRNA expression. The cells expressed mRNA for Flt-1 (VEGF receptor 1) and Flk-1 (VEGF receptor 2), and exogenous VEGF was found to have migration-promoting activity on cultured cells. We conclude that pericardial mesothelial cells express VEGF, which may serve as an autocrine growth-regulatory mechanism.     

VEGF反义寡核苷酸对胆囊癌细胞VEGF,Flt-1及KDRmRNA表达和VEGF蛋白分泌的影响

目的:体外研究Oligofectamine介导的VEGF反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)转染对人胆囊癌GBC-SD细胞VEGF,Flt-1及KDR mRNA表达和VEGF蛋白分泌的影响.方法:运用Oligofectamine介导的VEGF反义寡核苷酸(ASODN)和错义寡核苷酸(Scrambled Oligodeoxynucleotide,SODN)转染人胆囊癌细胞GBC-SD,半定量RT-PCR检测转染后各组细胞不同时间的VEGF,Flt-1及KDR mRNA表达变化,ELISA测定转染后各组细胞培养上清液VEGF蛋白浓度.结果:半定量RT-PCR发现ASODN组及ASODN Oligofectamine组24,48,72,96 h VEGF(ASODN组VEGF165:0.686±0.033,0.569±0.049,0.489±0.036,0.716±0.017;ASODN组VEGF121:0.462±0.046,0.338±0.034,0.219±0.022,0.471±0.038;ASODN Oligofectamine组VEGF165:0.601±0.021,0.465±0.042,0.416±0.023,0.662±0.035:ASODN Oligofectamine组VEGF121:0.408±0.014,0.286±0.019,0.157±0.021,0.418±0.037)、Flt-1(ASODN组:0.694±0.019,0.562±0.045,0.435±0.042,0.724±0.026;ASODN Oligofectamine组:0.609±0.018,0.442±0.049,0.314±0.015,0.614±0.029)及KDR(ASODN组:0.667±0.063,0.490±0.033,0.301±0.029,0.665±0.068;ASODN Oligofectamine组:0.523±0.048,0.432±0.027,0.218±0.036,0.524±0.037) mRNA的表达显著低于Control组(P<0.05),且ASODN Oligofectamine的抑制作用比ASODN强(P>0.05).ELISA测定结果显示ASODN组(281.26±18.62,526.44±34.95,791.13±20.99)及ASODN Oligofectamine组(250.7±14.57,506.09±19.14,711.79±19.91)24,48,72 h VEGF蛋白的分泌浓度均显著低于Control组(394.23±16.26,711.6±26.21,933.85±28.65)(P<0.05),且ASODN Oligofectamine的抑制作用比ASODN强(P>0.05).结论:Oligofectamine介导的VEGF ASODN能抑制GBC-SD细胞VEGF,Flt-1及KDR mRNA表达和VEGF蛋白分泌.     

KDR/Flk-1 is a major regulator of vascular endothelial growth factor-induced tumor development and angiogenesis in murine hepatocellular carcinoma cells.

Vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors, has been shown to play a pivotal role in tumor angiogenesis, including hepatocellular carcinoma (HCC). The effects of VEGF are mediated mainly through two distinct receptors, flt-1 and KDR/Flk-1. It has been suggested that KDR/Flk-1 plays an important role in tumor development. However, the role of KDR/Flk-1 in HCC has not been examined. We previously reported that VEGF tightly regulated murine HCC development, based on the results of a study using a retroviral tetracycline-regulated (Retro-Tet) gene expression system. This system allows VEGF gene expression to be manipulated in vivo by providing tetracycline in the drinking water. In the present study, we combined the KDR/Flk-1-specific neutralizing monoclonal antibody (KDR/Flk-1mAb) and the Retro-Tet system to elucidate the role of KDR/Flk-1 in VEGF-induced tumor development and angiogenesis in a murine HCC experimental model. In a xenograft study, tumor augmentation induced by VEGF overexpression was almost abolished by means of KDR/Flk-1mAb treatment, with accompanying inhibition of angiogenesis, KDR/Flk-1 autophosphorylation, but not interference of flt-1 activation. This inhibitory effect was achieved even on established tumors and regardless of whether the tumor size was small or large. On the contrary, KDR/Flk-1mAb treatment significantly increased the apoptosis in the tumor. With orthotopic transplantation, KDR/Flk-1mAb also inhibited HCC development in the liver. These results suggest that KDR/Flk-1 is a major regulator of VEGF-mediated HCC development and angiogenesis not only at the initial stage, but also after the tumor has fully developed.     

Expression and function of vascular endothelial growth factor receptors (Flt-1 and Flk-1) in vascular adventitial fibroblasts

Vascular endothelial growth factor receptors (VEGFRs) are previously considered to exist exclusively in endothelial cells. However, little is known if the receptors are expressed in other non-endothelial cells. In this study, we measured activation of two VEGFRs, Flk-1 and Flt-1, and their biological functions in cultured adventitial fibroblasts and injured rat carotid injury arteries induced by balloon angioplasty. Our results indicated that Flt-1, but not Flk-1, existed in adventitial fibroblasts. Angiotensin II increased Flt-1 protein expression in a time- and concentration-dependent manner. Adventitial fibroblast migration stimulated by vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) required Flt-1 expression. The Flt-1-induced adventitial fibroblast migration was blocked by anti-Flt-1 neutralizing antibody and soluble VEGFR1 protein (sFlt-1). However, Flt-1 activation did not enhance cell proliferation. In addition, Flt-1 expression was significantly increased in the neointima and adventitia in injured rat carotid arteries. We concluded that functional expression of Flt-1 in adventitial fibroblasts might be an important mediator in the pathogenesis of vascular remodeling after arterial injury.