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.     

血管内皮生长因子及其受体mRNA在输卵管妊娠蜕膜组织的表达

目的:检测血管内皮生长因子(VEGF)及其受体(KDR、Flt-1)在输卵管妊娠蜕膜组织的表达,探讨其在输卵管妊娠中的作用。方法:采用半定量RT-PCR方法,检测输卵管妊娠蜕膜组织中的VEGF、KDR、Flt-1 mRNA的表达,并与正常输卵管黏膜及正常宫内早孕子宫蜕膜组织比较。结果:半定量结果表明,VEGF、KDR、Flt-1 mRNA在输卵管妊娠蜕膜组织中的表达强于正常输卵管组织,但弱于正常宫内早孕蜕膜组织,差异均有显著性。结论:输卵管妊娠时,VEGF及其受体mRNA水平的增高是使滞留在输卵管的胚泡着床于输卵管的重要因素。     

Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium

Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6-8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1alpha and CA-IX in the menstrual and early proliferative phases. HIF1alpha was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed by estrogen during the late proliferative phase.     

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.     

Signaling pathways induced by vascular endothelial growth factor (review)

Vasculogenesis and angiogenesis are the mechanisms responsible for the development of the blood vessels. Angiogenesis refers to the formation of capillaries from pre-existing vessels in the embryo and adult organism, while vasculogenesis is the development of new blood vessels from the differentiation of endothelial precursors (angioblasts) in situ. Vascular endothelial growth factor (VEGF) family members are major mediators of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to promote endothelial cell viability, mitogenesis, chemotaxis, and vascular permeability. It mediates its activity mainly via two tyrosine kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR), although other receptors, such as neuropilin-1 and -2, can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (flt-4) binds VEGF-C and VEGF-D and is more important in the development of lymphatic vessels. While the functional effects of VEGF on endothelial cells has been well studied, not as much is known about VEGF signaling. This review summarizes the different pathways known to be involved in VEGF signal transduction and the biological responses triggered by the VEGF signaling cascade.     

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。     

Gene expression of vascular endothelial growth factor and its receptors and angiogenesis in bronchial asthma

BACKGROUND: Angiogenesis is a feature of airway remodeling in bronchial asthma. The mechanism responsible for this angiogenesis is unknown. Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cells, which may contribute to chronic inflammation and angiogenesis. OBJECTIVE: We sought to investigate the molecular mechanisms underlying increased vascularity, and we examined the mRNA expression of VEGF and its receptors (flt-1 and flk-1) within bronchial biopsy specimens from asthmatic patients and normal control subjects. METHODS: Endobronchial biopsy specimens were examined immunocytochemically by staining with anti-type IV collagen mAb to evaluate vessel density by using computer-assisted image analysis. Specimens were also analyzed for the presence of the mRNAs of VEGF and its receptors with in situ hybridization. RESULTS: The extent of airway vascularity was increased in asthmatic subjects compared with that in control subjects (P <.01). Asthmatic subjects exhibited a greater expression of VEGF, flt-1, and flk-1 mRNA(+) cells in the airway mucosa compared with that in control subjects (P <.001 for each comparison). The degree of vascularity was associated with the number of VEGF, flt-1, and flk-1 mRNA(+) cells. Numbers of cells expressing VEGF mRNA inversely correlated with airway caliber (r = -0.83, P <.01) and airway hyperresponsiveness (r = -0.97, P <.001). Colocalization studies showed that macrophages, eosinophils, and CD34(+) cells were the major sources of VEGF; CD34(+) cells, macrophages, and T cells expressed both flt-1 and flk-1. CONCLUSION: These findings provide evidence that VEGF may play an important role in angiogenesis and subsequent airway remodeling in bronchial asthma.     

Expression of vascular endothelial growth factor and its receptors in the developing rat lung

We examined the regulation of the expression of vascular endothelial growth factor (VEGF) and its specific receptors, fetal liver kinase receptor (Flk-1), and fms-like tyrosine kinase receptor (Flt-1) during formation of the capillary network in the developing rat lung. An immunohistochemical study of lung tissue from 19- and 21-d-old fetuses and 1-, 3-, 5-, 7-, and 14-d-old animals revealed that the level of expression of both VEGF and Flk-1 is significantly higher before birth (p < 0.0001) than after. Increased expression of Flt-1 on the first day after birth (p < 0.0001) suggests that this receptor might play an important role in capillary growth in the perinatal period. Immunostaining also revealed the colocalization of VEGF, Flt-1, and Flk-1 in endothelial cells of the lung capillaries at the ultrastructural level. The present studies revealed that VEGF and its two receptors are upregulated during the development of capillaries in the fetal and newborn rat lung.     

Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-Flt1 and VEGF-R2-Flk1/KDR) in non-small cell lung carcinomas (NSCLCs): correlation with angiogenesis and survival

The formation of new vessels (angiogenesis) is essential for primary tumour growth and metastasis and is induced by several angiogenic factors, including vascular endothelial growth factor (VEGF). The microvascular density (MVD) in tumours was assessed and the expression of VEGF and its receptors VEGF-R1-Flt1 and VEGF-R2-KDR/Flk1 was investigated in the different cellular compartments in vivo, in order to establish their interrelationship and their prognostic influence. Immunohistochemical study of 69 stage I–II non-small cell lung carcinomas (NSCLCs) was performed on paraffin sections with CD34 antibody to estimate MVD, using a Chalkley eye-piece graticule and VEGF, VEGF-R1, and VEGF-R2 antibodies. There was strong expression of VEGF and its receptors in tumour cells, endothelial cells, and stromal fibroblasts. In tumour cells, the level of VEGF was correlated with that of VEGF-R1 ( p = 0·018) but not that of VEGF-R2. In fibroblasts, high expression of VEGF was correlated with that of VEGF-R1 ( p = 0·0001) and VEGF-R2 ( p = 0·0001). In endothelial cells, expression of VEGF was correlated with that of VEGF-R1 ( p < 0·0001) and VEGF-R2 ( p = 0·04). The level of VEGF in fibroblasts was correlated with that of VEGF-R1 ( p = 0·0028) and VEGF-R2 ( p = 0·01) in endothelial cells. There was no correlation between the level of MVD and that of VEGF or VEGF-R1 or VEGF-R2. Neither the level of MVD, nor the level of expression of VEGF and VEGF receptors in any compartment influenced the patient's survival. In conclusion, although angiogenesis is essential for tumour growth, this study failed to demonstrate that MVD, VEGF, VEGF-R1, and VEGF-R2 are prognostic markers for stage I–II NSCLC. VEGF, however, might act as a direct autocrine growth factor for tumour cells via VEGF-R1 and angiogenesis could be promoted in a paracrine loop, where VEGF is produced by fibroblasts and tumour cells and then binds to endothelial cells via induced VEGF receptors. VEGF and its receptors thus appear as relevant therapeutic targets in NSCLC. Copyright © 1999 John Wiley & Sons, Ltd.     

Placental vascular endothelial growth factor receptor system mRNA expression in pigs selected for placental efficiency

Selecting Yorkshire breeding stock for increased placental efficiency (PE; piglet weight divided by its placental weight) results in larger litters (i.e. ∼3 more piglets litter⁻¹) and reduced placental sizes. Placental vessel density increases progressively after day 50 of gestation in the pig, and is positively correlated with PE and placental expression of vascular endothelial growth factor (VEGF), a potent angiogenic and permeability factor. To elicit its vascular effects, VEGF must bind to its receptors (R), VEGF-R1 and VEGF-R2. The objective of this study was to compare placental and endometrial blood vessel density and placental VEGF, VEGF-R1 and VEGF-R2 mRNA expression in day 70 and 90 conceptuses from Yorkshire females selected for high PE, low PE, and from unselected controls. A greater ( P < 0.05) PE was observed for conceptuses in the high PE selection group when compared to the low PE selection group. Placental blood vessel density increased ( P < 0.05) from day 70 to 90 (1.8 ± 0.1 versus 2.8 ± 0.2) in association with increases ( P < 0.05) in placental VEGF mRNA expression. No selection group differences were observed in expression of VEGF, VEGF-R1, or VEGF-R2 on day 70. By day 90, however, placentae of conceptuses from the high PE group expressed greater ( P < 0.05) amounts of VEGF and VEGF-R1 mRNA than the unselected controls and the low PE group. These data demonstrate that increased placental expression of the VEGF receptor system is associated with increased placental vascular density observed with the advancement of gestation in the pig. Although placental blood vessel density was not increased in the high PE selection group, elevated levels of the VEGF receptor system suggest an effect on increasing placental and endometrial blood vessel permeability and/or proximity in the high PE group.     

Circulating levels of free and total vascular endothelial growth factor (VEGF)-A, soluble VEGF receptors-1 and -2, and angiogenin during ovarian stimulation in non-human primates and women

BACKGROUND: In a prospective study we measured circulating levels of vasoactive factors and their soluble receptors in women undergoing controlled ovarian stimulation (COS) for IVF who were at risk for ovarian hyperstimulation syndrome (OHSS), and compared them to those in a primate model, the rhesus monkey. METHODS: A total of 23 women were enrolled in the study and serum vascular endothelial growth factor (VEGF)-A (free and total), soluble (s)VEGF-R1 and -R2, and angiogenin levels were compared in pregnant and non-pregnant women, and in monkeys, during follicular stimulation, the luteal phase and early pregnancy. RESULTS: VEGF levels were similar during the period of follicular stimulation in pregnant and non-pregnant women, but a significant rise in both free and total VEGF occurred in pregnant women during the luteal phase (P < 0.05). The level of sVEGF-R1 (but not -R2) increased (P < 0.05) following implantation, and the rise in sVEGF-R1 corresponded to an abrupt fall in free (but not total) VEGF. In contrast, total VEGF levels remained similar to those observed on the day of hCG injection. Angiogenin levels tended to decline during follicular stimulation, then increased marginally during the luteal phase and were unchanged in early pregnancy. In contrast to women, free VEGF levels were non-detectable and total levels remained constant through the natural menstrual cycle and COS protocols in monkeys. CONCLUSIONS: The levels of circulating angiogenic factors and soluble receptors demonstrate significant changes during COS cycles and early pregnancy in women. Thus, the systemic effect of these agents is influenced by ligand-receptor protein-binding interactions, and these relationships may exhibit dynamic changes during COS cycles and early pregnancy, and could contribute to the development of OHSS.     

Expression of vascular endothelial growth factor (VEGF) and its receptors (VEGF-R1 [Flt-1] and VEGF-R2 [KDR/Flk-1]) in tumorlets and in neuroendocrine cell hyperplasia of the lung

Pulmonary tumorlets and neuroendocrine (NE) cell hyperplasia are part of a continuous spectrum of NE-cell hyperplasia, going from NE hyperplasia to carcinoid. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen that has been shown to be increased in hypoxic lung. We hypothesized that tumorlets and NE-cell hyperplasia, which occur frequently in this context, were partly responsible for VEGF secretion. Immunohistochemical analysis of VEGF and both VEGF-R1 and VEGF-R2 was performed on paraffin sections of 12 lung tissues containing tumorlets and NE-cell hyperplasia in parallel with a control group of 11 lung specimens. VEGF and its receptor expressions were compared in bronchial epithelial cells and endothelial cells in both groups. VEGF and its receptors were consistently expressed in tumorlets and in NE-cell hyperplasia. When compared with control group lungs, the staining score for VEGF in lung bearing tumorlets was significantly higher in endothelial cells, but was not different in bronchial epithelial cells. VEGF-R1 expression was significantly increased both on bronchial epithelial cells (P = 0.001) and endothelial cells (P = 0.006), and VEGF-R2 expression was significantly increased on endothelial cell (P = 0.044). There was a significant positive correlation between the level of expression of VEGF and VEGF-R1 (P = 0.04) in both control groups and lung bearing tumorlets, but there was no significant correlation between VEGF and VEGF-R2 expression (P = 0.1). We concluded that VEGF is highly expressed in localized NE cell proliferations without potential of malignancy and might participate in local development of fibrosis.     

Spinal vascular endothelial growth factor (VEGF) and erythropoietin (EPO) induced phrenic motor facilitation after repetitive acute intermittent hypoxia

Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) exert neurotrophic and neuroprotective effects in the CNS. We recently demonstrated that VEGF, EPO and their receptors (VEGF-R2, EPO-R) are expressed in phrenic motor neurons, and that cervical spinal VEGF-R2 and EPO-R activation elicit long-lasting phrenic motor facilitation (pMF). Since VEGF, VEGF-R, EPO, and EPO-R are hypoxia-regulated genes, and repetitive exposure to acute intermittent hypoxia (rAIH) up-regulates these molecules in phrenic motor neurons, we tested the hypothesis that 4 weeks of rAIH (10 episodes per day, 3 days per week) enhances VEGF- or EPO-induced pMF. We confirm that cervical spinal VEGF and EPO injections elicit pMF. However, neither VEGF- nor EPO-induced pMF was affected by rAIH pre-conditioning (4 wks). Although our data confirm that spinal VEGF and EPO may play an important role in respiratory plasticity, we provide no evidence that rAIH amplifies their impact. Further experiments with more robust protocols are warranted.     

Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor, is a secreted protein implicated in tumor-associated microvascular hyperpermeability and angiogenesis. Tumor cells in 11 of 12 renal cell carcinomas expressed high levels of VPF messenger RNA (mRNA) by in situ hybridization, the only exception being a case of the relatively avascular papillary variant. Expression was further accentuated adjacent to areas of necrosis. Both tumor cells and endothelial cells in small vessels adjacent to tumor stained strongly for VPF protein by immunohistochemistry. Endothelial cells did not express detectable VPF mRNA, but did express high levels of mRNA for the VPF receptors flt-1 and KDR indicating that the endothelial cell staining likely reflects binding of VPF secreted by adjacent tumor cells. Three transitional cell carcinomas also labeled strongly for VPF mRNA. These data suggest an important role for VPF in the vascular biology of these two common human malignancies.     

Overexpression of vascular endothelial growth factor 189 in breast cancer cells leads to delayed tumor uptake with dilated intratumoral vessels

Vascular endothelial growth factor (VEGF) is essential for breast cancer progression and is a relevant target in anti-angiogenesis. Although VEGF121 and VEGF165, the fully or partially secreted isoforms, respectively, have been the focus of intense studies, the role of the cell-associated VEGF189 isoform is not understood. To clarify the contribution of VEGF189 to human mammary carcinogenesis, we established several clones of MDA-MB-231 cells stably overexpressing VEGF189 (V189) and VEGF165 (V165). V189 and V165 clones increased tumor growth and angiogenesis in vivo. Remarkably, V165 induced the most rapid tumor uptake, whereas V189 increased vasodilation. In vitro overexpression of VEGF165 and VEGF189 increases the proliferation and chemokinesis of these cancer cells. Interestingly, overexpression of VEGF189 increased cell adhesion on fibronectin (1.9-fold) and vitronectin (1.6-fold), as compared to VEGF165, through alpha5beta1 and alphavbeta5 integrins. Using the BIACore system we demonstrated for the first time that VEGF189 binds directly to neuropilin-1, which is strongly expressed in MDA-MB-231 cells. In contrast, VEGF-R2 was not significantly expressed and VEGF-R1 was expressed at low level. Our in vitro results suggest an autocrine effect of VEGF189 on breast cancer cells, probably through neuropilin-1. In conclusion, our data indicate that VEGF189 participates in mammary tumor growth through both angiogenesis and nonangiogenic functions. Whether VEGF189 overexpression is correlated to prognosis in human breast tumors remains to be established.     

Vascular endothelial growth factor inhibits outward delayed-rectifier potassium currents in acutely isolated hippocampal neurons

In the present study, whole-cell patch-clamp recording was used to study whether vascular endothelial growth factor (VEGF) had a regulatory effect on the potassium-channel currents. The outward delayed-rectifier potassium currents (I(K)) were recorded in acutely isolated hippocampal neurons from 14-day-old rat brains. A local application of VEGF at the concentrations from 50 ng/ml to 200 ng/ml dose-dependently inhibited I(K). Administration of VEGF (100 ng/ml) to the neurons only for seconds could significantly reduce I(K) in 26 of 39 recorded cells. The currents could recover to 82.8+/-3.7% of the control level at 60 s after removing VEGF in the buffer. In the I-V curve analysis, VEGF negatively shifted the I-V curve of I(K); the inhibition was gradually enhanced as the membrane potential increased from -40 mV to 50 mV in 13 cells. Thus, the results reveal that VEGF inhibits I(K) in acute, reversible and voltage-dependent manners. Double staining combined with confocal laser scanning microscopy was used to simultaneously detect the distribution of VEGF receptors (flt-1 and flk-1) in the hippocampal section and isolated neuron. Results showed that flt-1-positive staining, but not flk-1, could be observed on the membrane of the hippocampal neuron in both preparations, suggesting the presence of neuronal membrane VEGF flt-1 receptors in the hippocampus. To investigate if the inhibition by VEGF on I(K) is related to the presence of flt-1 receptors, we further did flt-1-receptor immunostaining for the recorded neurons, which was labeled with Lucifer Yellow during the recording. Among nine recorded cells, five showing the inhibition by VEGF had detectable signals for flt-1 receptors on their membrane, whereas the other four showing no inhibition had no flt-1 receptors either. The results suggest that VEGF can acutely inhibit I(K) in the hippocampal neurons probably related to the presence of membrane flt-1 receptors in the neurons.