The angiogenic pathway "vascular endothelial growth factor/flk-1(KDR)-receptor" in rheumatoid arthritis and osteoarthritis

Active angiogenesis, together with an up-regulation of angiogenic factors, is evident in the synovium of both rheumatoid arthritis (RA) and osteoarthritis (OA). The present study assessed, by immunohistochemistry, the microvessel density in the synovium of these arthritides and in normal controls, in relation to the expression of the angiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) and the apoptosis-related proteins bcl-2 and p53. More importantly, using the novel 11B5 MAb, the activated "VEGF/flk-1(KDR)-receptor" microvessel density was assessed. VEGF expression in fibroblasts was diffuse in both RA and OA. Diffuse PD-ECGF expression of fibroblasts was noted in all cases of RA, while fibroblast reactivity was focal in the OA material. The standard microvessel density (sMVD), as assessed with the anti-CD31 monoclonal antibody (MAb), was higher in RA (64+/-12) and in OA (65+/-16) than in normal tissues (52+/-8; p=0.008 and 0.0004, respectively). The activated microvessel density (aMVD), assessed with the 11B5 MAb, was significantly higher in RA (29+/-10) than in OA (17+/-4; p<0.0001) and than in normal tissues (14+/-2; p<0.0001). The "activation ratio" (aMVD/sMVD) was statistically higher in RA (0.46+/-0.17) than in OA and normal synovial tissues, the latter two having a similar ratio (0.28+/-0.08 and 0.26+/-0.03, respectively). Cytoplasmic bcl-2 expression was frequent in the synovial cells of OA, but rare in RA. Nuclear p53 protein accumulation was never observed. It is suggested that the angiogenic pathway VEGF/flk-1(KDR) may play an important role in the pathogenesis of RA and OA. Thus, failure of VEGF/flk-1(KDR) activation, in the presence of increased VEGF expression, may indicate a synovium with an impaired capacity to establish a viable vasculature, consistent with the degenerative nature of OA. On the other hand, the activated angiogenesis in RA shows a functional, still pathologically up-regulated VEGF/flk-1(KDR) pathway. Whether restoration of an impaired VEGF/flk-1(KDR) pathway in OA, or inhibition of this in RA, would prove of therapeutic importance requires further investigation.     

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.     

Hepatocyte growth factor increases expression of vascular endothelial growth factor and plasminogen activator inhibitor-1 in human keratinocytes and the vascular endothelial growth factor receptor flk-1 in human endothelial cells

The pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) has been implicated by clinical and experimental studies in repair mechanisms in different organs and tissues. However, no data on the impact of HGF/SF in wound healing in the skin are yet available. Proliferating and migrating keratinocytes play a major role in repair processes in the skin by closing the wound. Recent evidence gathered from studies that used gene-deficient mice has implicated the plasminogen activator (PA)/plasmin system in wound healing, which depends on controlled matrix degradation and deposition during cell migration and proliferation. Furthermore, keratinocytes are an important source of vascular endothelial growth factor (VEGF), which is a potent inducer of angiogenesis. In this study, we show that in human keratinocytes HGF/SF but not the related cytokine macrophage stimulating protein (MSP) significantly increases expression of VEGF and plasminogen activator inhibitor-1 (PAI-1) on the level of protein and mRNA. Furthermore, we demonstrate that HGF/SF increases the expression of the VEGF receptor flk-1 in human endothelial cells and that, in an angiogenesis co-culture assay of endothelial cells and keratinocytes, HGF/SF increases endothelial cell tube formation significantly. Therefore, we propose a role for HGF/SF in wound repair in the skin: HGF/SF--produced by activated fibroblasts--increases in keratinocytes the expression of PAI-1, which leads to increased matrix stability during the repair process and which could also limit activation of HGF/SF by proteases such as urokinase-type PA (u-PA) or tissue-type PA (t-PA). Furthermore HGF/SF also increases the expression of VEGF in these cells, thereby initiating angiogenesis in a paracrine manner. This effect would be enhanced by an increased responsiveness of endothelial cells toward VEGF, resulting from the HGF/SF-induced up-regulation of flk-1 on these cells.     

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.     

Phosphorylated KDR expression in endometrial cancer cells relates to HIF1alpha/VEGF pathway and unfavourable prognosis.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor for many malignant neoplasms exerting its function through activation of specific membrane receptors, that is, KDR/flk-1, residing in endothelial cells. Several recent reports indicate that VEGF receptors are also expressed in cancer cells, suggesting that specific VEGF-originated cancer cell reactions may parallel the endothelial response. Using a novel monoclonal antibody, recognizing the activated (phosphorylated) form of the KDR receptor (pKDR), we assessed the expression of pKDR in normal and malignant endometrium. A strong and consistent cytoplasmic and nuclear pKDR expression was noted in the normally cycling endometrium, including epithelial, stromal and endothelial cells, suggesting a role in the normal menstrual cycle. Approximately, one-third of the 70 stage I endometrioid adenocarcinomas analysed exhibited an intense cytoplasmic and nuclear pKDR expression in both cancer cells and peritumoral vessels. It was noted that such pKDR reactivity in cancer cells was related directly to VEGF, VEGF/KDR complexes and HIF1alpha (hypoxia inducible factor 1alpha) expression. Furthermore, pKDR expression was significantly associated with poor prognosis. It is concluded that the VEGF/KDR pathway is activated in both normally cycling and malignant endometrium, suggestive of an important role in the biology of this tissue. The unfavourable prognosis that VEGF confers to endometrial adenocarcinomas could be attributed to its angiogenic activity, but also to a direct effect on cancer cells through an autocrine VEGF/KDR loop.     

血管内皮生长因子的生物学及其在临床的初步应用

血管内皮生长因子(VEGF)是内皮细胞特异性的有丝分裂原。它诱导内皮细胞增殖、促进内皮细胞迁移，并抑制内皮细胞凋亡，在调节血管和淋巴管新生中起重要作用。VEGF也是胚胎发育、软骨内骨形成、女性生殖系统、以及肿瘤和眼球内血管新生所必需的。另外，VEGF也可诱导血小板粘附于血管内皮细胞而出现高凝状态。目前，有许多临床实验正在评价VEGF在血管新生依赖性疾病的促血管新生作用和抗血管新生药物用于治疗的效果。     

Correlation between tumor vascularity, vascular endothelial growth factor production by tumor cells, serum vascular endothelial growth factor levels, and serum angiogenic activity in patients with breast carcinoma.

Angiogenesis is an important prognostic factor in invasive breast carcinoma. We analyzed sera and tumor samples from 36 patients with primary breast carcinomas to determine the relationship between tumor vascularity, vascular endothelial growth factor (VEGF) production by tumor cells, levels of circulating VEGF (measured by ELISA assay), and levels of endothelial growth factors analyzed by a functional test of human umbilical vein endothelial cells (HUVEC) proliferation. Tumor vascularity was correlated directly with VEGF production by the tumor, indicating that VEGF production is a relevant factor in determining angiogenesis in primary tumor. No correlation was found either between the number of vessels in the tumor or the production of VEGF by tumor cells and the levels of serum angiogenic factors including VEGF. On the contrary, the two serum tests correlated together because a high serum level of VEGF is more frequent in cases with the presence of HUVEC-stimulating growth factors. These data indicate that the principal source of factors stimulating angiogenesis in the primary tumor is the tumor itself. This is an important issue in the context of anti-angiogenic therapeutic approaches, which should be planned to interfere with tumor production of angiogenic factors rather than with circulating angiogenic factors. In conclusion, whereas the vessel count and VEGF production by tumor cells are parameters that give direct information on tumor angiogenesis, long-term follow-up is necessary to determine the clinical significance of the determination of serum HUVEC-stimulating factors in the progression of breast carcinoma.     

胰岛素对牛胸主动脉内皮细胞血管内皮生长因子及其受体表达的影响

目的:评价胰岛素对培养的牛胸主动脉内皮细胞血管内皮生长因子(VEGF)及其受体表达的影响。方法: 取新生的小牛胸主动脉,做血管内皮细胞原代及传代培养,取4-6代培养细胞分组,应用不同浓度的胰岛素(30 mU/L、300 mU/L、3 000 mU/L)干预培养过程,48 h后应用免疫组化法测定内皮细胞VEGF及其受体(flt-1、flk-1/KDR)的表达水平。结果: 低浓度胰岛素组(30 mU/L、300 mU/L)内皮细胞VEGF表达明显高于不用胰岛素组(P<0.01);高浓度组(3 000 mU/L)内皮细胞VEGF表达明显低于不用胰岛素组(P＜0.05);各组内皮细胞VEGF受体(flt-1及flk-1/KDR)的表达无明显差异(P＞0.05)。结论: 低浓度胰岛素促进小牛主动脉血管内皮细胞VEGF表达;高浓度胰岛素可抑制血管内皮细胞VEGF表达;胰岛素对小牛主动脉血管内皮细胞VEGF受体(flt-1、flk-1/KDR)的表达无直接影响。     

肝细胞癌中血管内皮生长因子和cNOS与血管生成及细胞增殖的关系

目的　观察血管内皮生长因子 (VEGF)及含激酶插入区受体 (KDR)在原发性肝癌中的表达情况及其与cNOS表达的相关性 ,探讨它们在肝癌肿瘤性血管生成、肿瘤细胞增殖和转移过程中的作用。　方法　收集手术切除的 80例原发性肝癌、4 0例肝硬化、2 0例正常肝组织标本 ,应用免疫组织化学和原位杂交的方法观察VEGF及其KDR、cNOS在肝细胞癌中的表达情况 ,分析VEGF及其受体KDR、cNOS与微血管密度 (MVD)、肿瘤细胞增殖指数和转移的关系。　结果　肝癌组织中癌细胞VEGF的表达与MVD、细胞增殖指数明显相关 (P <0 0 1和P <0 0 5 ) ,与肝癌内皮细胞中cNOSmRNA的表达之间也有相关性 (Pearson列联系数 =0 2 984 ,P <0 0 5 )。内皮细胞中cNOSmRNA与VEGF均阳性者微血管密度、细胞增殖指数均明显高于cNOSmRNA阴性和VEGF阳性者 (P <0 0 1) ,也明显高于两者均阴性者 (P <0 0 1)。　结论　肝细胞癌中癌细胞VEGF的表达与血管生成、细胞增殖和肝癌转移密切相关 ,且内皮细胞cNOSmRNA的表达可能参与VEGF的促血管生成作用。     

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.     

Induction of a potential paracrine angiogenic loop between human THP-1 macrophages and human microvascular endothelial cells during Bartonella henselae infection

Bartonella henselae is responsible for various disease syndromes that loosely correlate with the immune status of the host. In the immunocompromised individual, B. henselae-induced angiogenesis, or bacillary angiomatosis, is characterized by vascular proliferative lesions similar to those in Kaposi's sarcoma. We hypothesize that B. henselae-mediated interaction with immune cells, namely, macrophages, induces potential angiogenic growth factors and cytokines which contribute in a paracrine manner to the proliferation of endothelial cells. Vascular endothelial growth factor (VEGF), a direct inducer of angiogenesis, and interleukin-1beta (IL-1beta), a potentiator of VEGF, were detected within 12 and 6 h, respectively, in supernatants from phorbol 12-myristate 13-acetate-differentiated human THP-1 macrophages exposed to live B. henselae. Pretreatment of macrophages with cytochalasin D, a phagocytosis inhibitor, yielded comparable results, suggesting that bacterium-cell attachment is sufficient for VEGF and IL-1beta induction. IL-8, an angiogenic cytokine with chemotactic properties, was induced in human microvascular endothelial cells (HMEC-1) within 6 h of infection, whereas no IL-8 induction was observed in infected THP-1 cells. In addition, conditioned medium from infected macrophages induced the proliferation of HMEC-1, thus demonstrating angiogenic potential. These data suggest that Bartonella modulation of host or target cell cytokines and growth factors, rather than a direct role of the bacterium as an endothelial cell mitogen, is the predominant mechanism responsible for angiogenesis. B. henselae induction of VEGF, IL-1beta, and IL-8 outlines a broader potential paracrine angiogenic loop whereby macrophages play the predominant role as the effector cell and endothelial cells are the final target cell, resulting in their proliferation.     

Vascular endothelial growth factor-mediated autocrine stimulation of prostate tumor cells coincides with progression to a malignant phenotype

Vascular endothelial growth factor (VEGF), which is often produced at high levels by tumor cells, is a well-known mediator of tumor angiogenesis. VEGF receptor tyrosine kinases, KDR/Flk-1 and Flt-1, have been thought to be expressed exclusively by endothelial cells. In this study, we have used a prostate tumor progression series comprised of a differentiated rat prostate epithelial cell line, NbE-1, and its highly motile clonal derivative, FB2. Injection of NbE-1 cells into the inferior vena cava of syngeneic rats indicated that these cells are nontumorigenic. Using the same model, FB2 cells generated rapidly growing and well-vascularized tumors in the lungs. NbE-1 expressed marginal levels of VEGF, whereas high levels of VEGF protein were detected in FB2-conditioned medium and in FB2 tumors in vivo. Analysis of (125)I-VEGF(165) binding to NbE-1 and FB2 cells indicated that only motile FB2 cells expressed the VEGF receptor Flt-1. Consistent with this finding, physiological concentrations of VEGF induced chemotactic migration in FB2 but not in NbE-1 cells. This is the first documentation of a functional Flt-1 receptor in prostate tumor cells. Our results suggest two roles for VEGF in tumor progression: a paracrine role as an angiogenic factor and a previously undescribed role as an autocrine mediator of tumor cell motility.     

Vascular endothelial growth factor confers a growth advantage in vitro and in vivo to stromal cells cultured from neonatal hemangiomas.

Neonatal hemangioma is a common benign proliferation of unorganized structures containing stromal and capillary endothelial cells. We tested the hypothesis that such cell proliferation might result from the release by stromal cells of endothelial cell mitogens. Stromal cells cultured from biopsies of surgically removed life-threatening hemangiomas released an endothelial cell mitogen in vitro that was indistinguishable from vascular endothelial growth factor (VEGF) based on independent criteria such as affinity chromatography for heparin or anti-VEGF IgG and radioreceptor assay. A functional product of the KDR gene encoding a cognate VEGF receptor was also expressed by these stromal cells. Transient transfection with antisense oligonucleotides targeted on the translation initiation codon of KDR abolished its tyrosine phosphorylation and mitogenic response of neonatal hemangioma cells to VEGF, confirming the existence of an autocrine loop of proliferation. When grafted in nude mice, these stromal cells elicited an angiogenic response that was blocked by neutralizing anti-VEGF IgG. These results might provide a clue to the importance of stromal cells in the pathogeny of neonatal hemangiomas.     

Novel highly efficient intrabody mediates complete inhibition of cell surface expression of the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR)

The human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) and its ligand vascular endothelial growth factor (VEGF) play an essential role in tumor angiogenesis and in haematological malignancies. To inhibit VEGF induced signalling, intrabodies derived from two scFv fragments recognizing the VEGF receptor were generated. When these intrabodies were expressed in endothelial cells, they blocked the transport of KDR to the cell surface. We developed a cell culture model using porcine aortic endothelial cells overexpressing KDR for testing the efficiency of anti-KDR intrabodies. The two intrabodies were targeted to the ER and colocalised with the KDR receptor in an intracellular compartment. No degradation of the receptor was observed. An immature incomplete glycosylated protein of 195 kDa was detected, suggesting that the intrabodies affect the maturation of the receptor. Despite the presence of significant amounts of receptor protein, the inactivation by one of the two intrabodies was highly effective, resulting in complete functional inhibition of KDR and inhibition of in vitro angiogenesis. The new intrabody appears to be a powerful tool with which to inhibit KDR function.