Attenuation of the exercise-induced increase in skeletal muscle Flt-1 mRNA by nitric oxide synthase inhibition

We investigated the vascular endothelial growth factor (VEGF) receptor [fms-like-tyrosine kinase (Flt-1 and fetal liver kinase-1 (Flk-1)] response to acute exercise. In female Wistar rats, the VEGF receptor messenger RNA (mRNA) response to a single acute exercise bout was examined using semi-quantitative Northern blot from the left gastrocnemius muscles at rest and post-exercise at 0, 1, 2, 4, 8, 16, 24 and 48 h. Exercise altered both Flt-1 and Flk-1 mRNA, with significant increases in Flt-1 mRNA at 1 and 24 h. However, post-hoc analysis was unable to discern the time point where a significant increase in Flk-1 mRNA occurred. To investigate the regulation of Flt-1 mRNA by exercise we examined if nitric oxide synthase (NOS) inhibition alters the Flt-1 mRNA response. Eight groups [Condition: Rest or Exercise; Drug: Saline, 30 mg kg(-1)N(omega)-nitro-L-arginine methyl ester (L-NAME), 300 mg kg(-1) L-NAME or 300 mg kg(-1) D-NAME] were used to determine the effect of NOS inhibition on the Flt-1 mRNA response to exercise. L-NAME, a known NOS inhibitor, attenuated the exercise-induced increase in Flt-1 mRNA by approximately 50%. These findings suggest that: (1) exercise alters Flt-1 and Flk-1 gene expression; and (2) NO is important in the regulation of the Flt-1 gene response to exercise.     

VEGF高表达的胶质瘤细胞C6对共培养微血管内皮细胞表达Flk-1及Flt-1的影响

目的:以正常鼠肝细胞系BRL 3A为对照,研究VEGF高表达的恶性胶质瘤细胞系C6对体外共培养的肺微血管内皮细胞表达Flt-1、Flk-1的影响。方法:建立体外C6,BRL 3A与肺微血管内皮细胞的共培养方法,利用免疫细胞化学方法检测共培养后的微血管内皮细胞上VEGF受体Flt-1、Flk-1蛋白的表达变化,进一步采用RT-PCR和Northernblot分析Flt-1、Flk-1mRNA表达的改变。结果:与胶质瘤细胞C6共培养的微血管内皮细胞Flk-1、Flt-1蛋白表达增加 (P <0.05),而与BRL 3A共培养的内皮细胞Flk-1、Flt-1蛋白表达下降 (P <0.01),RT-PCR和Northernblot检测发现与C6共培养后可上调微血管内皮细胞Flk-1、Flt-1mRNA的表达 (P <0.01),而与BRL 3A共培养下调了Flk-1、Flt-1mRNA的表达 (P <0.01)。结论:VEGF高表达的胶质瘤细胞C6对共培养的微血管内皮细胞表达Flk-1、Flt-1有明显的上调作用,这可能是体内胶质瘤血管新生的重要机制之一.     

Expression of vascular endothelial growth factor receptor Flk-1 in canine mammary tumours

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, and the beginning of angiogenesis, by interacting with specific endothelial receptors termed VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). In this study, Flk-1 expression was evaluated immunohistochemically in 10 benign and 40 malignant canine mammary tumours. There was immunolabelling of endothelial cells located within the neoplastic proliferation and at the infiltrating periphery, and also of neoplastic cells. The number of positive endothelial and neoplastic cells, was higher in malignant than in benign tumours. Moreover, in the malignant tumours, expression of Flk-1 increased from well to less differentiated phenotypes (grade 1-3). The presence of VEGF receptor on neoplastic cells suggests that VEGF has an autocrine function in which neoplastic cells act as both VEGF producers and target cells. Thus, in malignant tumours, VEGF may contribute to neoplastic growth by inducing angiogenesis and by stimulating the proliferation of neoplastic cells.     

Vascular endothelial growth factor modulates skeletal myoblast function

Vascular endothelial growth factor (VEGF) expression is enhanced in ischemic skeletal muscle and is thought to play a key role in the angiogenic response to ischemia. However, it is still unknown whether, in addition to new blood vessel growth, VEGF modulates skeletal muscle cell function. In the present study immunohistochemical analysis showed that, in normoperfused mouse hindlimb, VEGF and its receptors Flk-1 and Flt-1 were expressed mostly in quiescent satellite cells. Unilateral hindlimb ischemia was induced by left femoral artery ligation. At day 3 and day 7 after the induction of ischemia, Flk-1 and Flt-1 were expressed in regenerating muscle fibers and VEGF expression by these fibers was markedly enhanced. Additional in vitro experiments showed that in growing medium both cultured satellite cells and myoblast cell line C2C12 expressed VEGF and its receptors. Under these conditions, Flk-1 receptor exhibited constitutive tyrosine phosphorylation that was increased by VEGF treatment. During myogenic differentiation Flk-1 and Flt-1 were down-regulated. In a modified Boyden Chamber assay, VEGF enhanced C2C12 myoblasts migration approximately fivefold. Moreover, VEGF administration to differentiating C2C12 myoblasts prevented apoptosis, while inhibition of VEGF signaling either with selective VEGF receptor inhibitors (SU1498 and CB676475) or a neutralizing Flk-1 antibody, enhanced cell death approximately 3.5-fold. Finally, adenovirus-mediated VEGF(165) gene transfer inhibited ischemia-induced apoptosis in skeletal muscle. These results support a role for VEGF in myoblast migration and survival, and suggest a novel autocrine role of VEGF in skeletal muscle repair during ischemia.     

Expression of growth factors,growth inhibiting factors,and their receptors in invasive breast cancer. I: An inventory in search of autocrine and paracrine loops

The aim of the present study was to investigate which growth factors, receptors, and growth inhibiting factors are expressed in invasive breast cancer. Five (angiogenic) growth factors and their receptors: platelet-derived growth factor A chain (PDGF-AA) and PDGF receptor alpha (PDGFαR), PDGF-BB and PDGF beta receptor, transforming growth factor alpha (TGFα) and its receptor epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) and its receptors vascular endothelial growth factor receptor I (Flt-1) and vascular endothelial growth factor receptor II (Flk-1/KDR); two growth inhibiting factors: transforming growth factor beta-1 (TGFβ1) and TGFβ2) and their receptor couple transforming growth factor beta receptor I (TGFβR-I) and TGFβR-II; and basic fibroblast growth factor (bFGF) were stained by standard immunohistochemistry on frozen sections in 45 cases of invasive carcinoma of the breast. Staining was scored as negative or positive in tumour epithelium, stroma, and blood vessels. TGFβ1 and TGFβ2 were expressed in the tumour cells in 67 per cent and 76 per cent of cases, respectively, whereas PDGFβR and TGFβR-II were expressed in 0 per cent and 2 per cent, respectively. The other factors showed variable expression in tumour cells. All factors were expressed in the stroma in most cases, except Flt-1, Flk-1/KDR, TGFβ2, and TGFβR-II, which showed variable expression, and EGFR, which showed no expression. The endothelium was in most cases positive for bFGF, PDGF-AA, PDGF-BB, VEGF, PDGFαR, PDGFβR, and TGFβ1 but TGFβ2 was negative in most cases and TGFα, EGFR, Flt-1, Flk-1/KDR, TGFβR-I, and TGFβR-II were variably expressed. The most interesting possible auto/paracrine loops, as demonstrated on serial sections and by fluorescence double staining, were the TGFα/EGFR, TGFβs/TGFβR, VEGF/Flt-1, and the VEGF/Flk-1 combinations. In conclusion, growth factors, growth inhibiting factors, and their receptors are frequently expressed in invasive breast cancer. Indications for some possible auto-and paracrine loops have been found, which should encourage further study on the role of these factors in breast cancer proliferation and angiogenesis. © 1998 John Wiley & Sons, Ltd.     

Reduction of a vascular endothelial growth factor receptor, fetal liver kinase-1, by antisense oligonucleotides induces motor neuron death in rat spinal cord exposed to hypoxia

Vascular endothelial growth factor (VEGF) is reported to play a neuroprotective role through a VEGF receptor, fetal liver kinase-1 (Flk-1) in vitro. We investigated whether reduction of Flk-1 could induce motor neuron loss in rat spinal cord by inhibiting the expression of Flk-1 in rat spinal cord using antisense oligodeoxynucleotides (ODNs) against the Flk-1 receptor. Rat spinal cord was repetitively exposed to 12% hypoxia, and the change of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the mitogen-activated protein kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathway was examined. Intrathecal infusion of Flk-1 antisense ODNs for 7 days suppressed almost completely Flk-1 expression in the lumbar segment of the spinal cord and was followed by a hypoxic challenge with 12% oxygen for 1 h that was repeated for 7 more days. In the lumbar segment, we observed that reduced Flk-1 expression and hypoxic challenge for 7 days resulted in approximately 50% loss of motor neurons, in which the activation of Akt and ERK, that is, increased levels of phosphorylated-Akt and of phosphorylated-ERK by hypoxia, was markedly inhibited. In contrast, the reduction of Flk-1 expression alone did not induce motor neuron loss. These results suggest that VEGF exerts its protective effect on motor neurons against hypoxia-induced toxicity by the Flk-1 receptor through the PI3-K/Akt and the MEK/ERK signaling pathways.     

Vascular endothelial growth factor expression,beta-catenin tyrosine phosphorylation,and endothelial proliferative behavior: a pathway for transformation?

The hypothesis that tumor growth is angiogenesis dependent has been documented by a considerable body of direct and indirect experimental data and has generated intense basic and pharmaceutical-related interest. In contrast, the study of endothelial cell tumors has been modest by comparison. Hemangioma is the most common tumor of any kind seen in infancy and also, perhaps, the least understood. We compared a mouse hemangioma-derived cell line (EOMA) and primary human endothelial cells (HUVEC) for their proliferative behavior and molecular alterations. EOMA cells intrinsically expressed vascular endothelial growth factor (VEGF), which acts in an autocrine manner, resulting in an increase in CD1 expression and cell proliferation, both of which were inhibited by anti-VEGF neutralizing antibodies. Such an autocrine loop is supported by constitutive VEGF receptor (Flk-1) tyrosine phosphorylation, Flk-1 and Flt-1 nuclear localization, and mitogen-activated protein kinase activation. beta-catenin was also found to exhibit significant nuclear localization and constitutively associate with Flk-1 and Flt-1 in EOMA cells but much less so in HUVEC, and immunoprecipitated Flk-1 was able to phosphorylate purified beta-catenin in an immune complex kinase assay. EOMA cells were also noted to express reduced levels of N-cadherin and gamma-catenin compared with HUVEC. Interestingly, sequestration of endogenous VEGF in EOMA cultures resulted in a dramatic decrease in nuclear beta-catenin and a reduction in CD1 levels, whereas addition of exogenous VEGF elicited increased nuclear beta-catenin localization and increased CD1 levels in HUVEC. The possible contributions of VEGF signaling pathways, cell junction component expression levels, and phosphorylation states to endothelial cell transformation and proliferation are discussed.     

Time-Dependent Alterations of VEGF and Its Signaling Molecules in Acute Lung Injury in a Rat Model of Sepsis

Molecular mechanisms of sepsis-associated acute lung injury (ALI) are poorly defined. Since vascular endothelial growth factor (VEGF) is a potent vascular permeability and mitogenic factor, it might contribute to the development of ALI in sepsis. Thus, using lipopolysaccharide (LPS)-induced (15 mg/kg, intraperitoneal) endotoxemic rat model, we studied the timeline (1, 3, 6, and 10 h) of pulmonary VEGF expression and its signaling machinery. Levels of pulmonary VEGF and its angiogenic-mediating receptor, Flk-1, were downregulated by LPS in a time-dependent manner; levels of plasma VEGF and its permeability-mediating receptor, Flt-1, in contrast, was upregulated with time. In addition, blockade of Flt-1 could improve the downregulated pulmonary VEGF level and attenuate the elevated plasma and pulmonary levels of TNF-α, followed by improvement of arterial oxygenation and wet-to-dry weight ratio of the lung. Expression of signaling, pro- and or apoptotic factors after LPS administration were as follows: phosphorylated Akt, a downstream molecule was downregulated time dependently; endothelial nitric oxide synthase levels were significantly reduced; pro-apoptotic markers caspase 3 and Bax were upregulated whereas levels of Bcl-2 were downregulated. The present findings show that VEGF may play a role through the expression of Flt-1 in LPS-induced ALI. Moreover, downregulation of VEGF signaling cascade may account for LPS-induced apoptosis and impaired physiological angiogenesis in lung tissues, which in turn may contribute to the development of ALI induced by LPS.     

Flt-1、Flk-1在胶原诱导的关节炎小鼠关节组织中的表达

目的研究血管内皮生长因子(VEGF)受体F lt-1、F lk-1在Ⅱ型胶原诱导的关节炎(C IA)形成期的表达,探讨VEGF在类风湿性关节炎发病中的作用。方法于DBA/1 J小鼠皮下注射Ⅱ型胶原制作小鼠关节炎动物模型并进行关节指数评价,用ELISA法和免疫组织化学技术检测关节组织内VEGF及血管性假性血友病因子(vWF)含量,通过RT-PCR,Southern b lotting技术检测关节组织内VEGF及其特异性受体F lt-1、F lk-1mRNA表达。结果VEGF及vWF水平呈平行变化关系,均在关节炎发生后第四天达到最高水平,并与血管新生程度、关节炎严重程度呈正相关。关节组织内VEGF mRNA分别在279bp和304bp扩增片段有特异性表达、其特异性受体F lt-1、F lk-1 mRNA在377bp、402bp扩增片段有特异性表达。结论VEGF-F lt-F lk系统在关节炎形成早期起着重要作用,影响着实验诱导关节炎血管新生及病程。     

Design of a variant of vascular endothelial growth factor-A (VEGF-A) antagonizing KDR/Flk-1 and Flt-1

Because of its central role in pathological angiogenesis, vascular endothelial growth factor (VEGF) has become a major target for anti-angiogenic therapies. We report here the construction of a heterodimeric antagonistic VEGF variant (HD-VEGF). In this antagonist, binding domains for the VEGF-receptors KDR/Flk-1 and Flt-1 are present at one pole of the dimer, whereas the other pole carries domain swap mutations, which prevent binding to either receptor. As HD-VEGF can only bind to monomeric receptors, it does not lead to signal transduction. Moreover, it antagonizes VEGF and possibly other members of the VEGF family, which are KDR/Flk-1 and Flt-1 ligands. We show here that HD-VEGF is a potent inhibitor of VEGF-mediated proliferation and tissue factor induction in endothelial cell cultures, requiring only a 20-fold and a 4-fold excess, respectively, to block the activity of wtVEGF completely. A 4-fold excess of HD-VEGF over wtVEGF was also sufficient to abrogate vascular permeability as determined in the Miles assay in vivo. Furthermore, HD-VEGF inhibited fetal bone angiogenesis in an ex vivo assay. Thus, HD-VEGF blocks KDR- and Flt-1-mediated VEGF activities that are crucial in the angiogenic process and is therefore a promising, multipotent compound in the treatment of angiogenesis-related diseases.     

Distribution of Flk-1 and Flt-1 receptors in neonatal and adult rat brains

Double-fluorescence staining was combined with confocal laser scanning microscopy to localize fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1) in the neonatal rat brain. The results showed that Flk-1 and Flt-1 immunostaining was observed in the cells with neuron-specific enolase, a neuronal marker, and with factor VIII (F VIII), an endothelium marker, but not in cells with glial fibrillary acidic protein (GFAP), a glial marker, of brain sections from rats on postnatal day 7 (P7). This indicates that both vascular endothelial growth factor (VEGF) receptors were distributed in the neurons and the vascular endothelium. A regional analysis showed that Flt-1 was distributed most densely in the hippocampus, followed by the retrosplenial agranular cortex and the striatum, and Flk-1 was evenly distributed throughout the brain. In a comparison of the density of immunopositive staining neurons, Flt-1 was much higher than Flk-1 in most of the brain regions. A time-course analysis showed that both Flt-1 and Flk-1 were highly expressed in the cerebral vessel of rats on P1, P7, and P14, and then declined in adults, consistent with the development of angiogenesis in neonates. In the neurons, Flt-1 was highest in the cerebral cortex and hippocampus of P1-P14 rats, and then gradually decreased, whereas Flk-1 abruptly increased and reached its highest level in adults. The results suggest that Flt-1 and Flk-1 are expressed in the neurons with their individual time-dependent manners and regional distribution in the brain. However, the significance of the neuronal distribution of Flt-1 and Flk-1 remains to be determined.     

Vascular endothelial growth factor and its receptor Flk-1 are expressed in the hippocampus following entorhinal deafferentation

Vascular endothelial growth factor (VEGF) has been thought of as a mitogen that promotes proliferation of endothelial cells and as a neurotrophic factor that stimulates neurogenesis and axonal growth in both peripheral and central nervous systems. To investigate the potential involvement of VEGF in the lesion-induced reorganization in the brain, the expression changes of VEGF and its receptor Flk-1 were analyzed in the mouse hippocampus after transections of the entorhinal afferents. In situ hybridization and immunohistochemistry showed the time-dependent expression upregulation of VEGF mRNA and protein in the entorhinally denervated hippocampal stratum lacunosum-moleculare and dentate outer molecular layer, which initiated by 3 days postlesion, reached its maximum at 7-15 days postlesion, still persisted by 30 days postlesion for protein, and recovered to the normal levels at 30 days postlesion for mRNA and at 60 days postlesion for protein. Double labeling of VEGF and glial fibrillary acidic protein revealed that VEGF-expressing cells in the denervated areas were reactive astrocytes. Semi-quantitative RT-PCR analysis showed that VEGF receptor Flk-1 mRNA was also time-dependently upregulated in the deafferented hippocampus with its maximal elevation at 7-15 days postlesion while the Flt-1 mRNA levels remained unchanged at any time point we examined. Immunohistochemistry analysis also displayed the upregulation of Flk-1 protein in the denervated stratum lacunosum-moleculare and outer molecular layer with a time course similar to that of VEGF mRNA upregulation. Flk-1 receptors were found to be expressed not only by reactive astrocytes but also by neurites, which most likely belong to sprouting axons by 7 days postlesion and regrowing dendrites by 15-30 days postlesion. From these data we suggest that the spatiotemporal upregulation of VEGF and Flk-1 in the hippocampus is induced by entorhinal deafferentation and that VEGF may be involved in the structural reorganization in the deafferented hippocampus via directly or indirectly promoting neurite growth.     

Regulation of embryonic lung vascular development by vascular endothelial growth factor receptors,Flk‐1 and Flt‐1

The biological effects of vascular endothelial growth factor A (VEGF‐A) are mediated by fetal liver kinase‐1 (Flk‐1) and fms‐like tyrosine kinase‐1 (Flt‐1). In lung tissue, VEGF‐A is diffusely expressed throughout the embryonic stages, whereas the development of vascular endothelial cells is not uniform. Noting the signaling properties of the two receptors, we hypothesized that Flk‐1 and Flt‐1 regulate the embryonic development of lung vasculature. We herein show the spatiotemporal expression and experimental inhibition of Flk‐1 and Flt‐1 of embryonic mouse lung tissue. When Flk‐1 was predominantly expressed (embryonic day [E] 9.5–E13.5), then vascular endothelial cells actively proliferated. When Flt‐1 was enhanced (E14.5–E16.5), these cells less actively proliferated, thereby constituting organized networks. The treatment of cultured lung buds (E11.5) with antisense oligonucleotides complementary to Flk‐1 inhibited branching of capillaries and proliferation of endothelial cells. In contrast, the inhibition of Flt‐1 promoted the branching of capillaries and enhanced proliferation of endothelial cells. Of interest, inhibition of Flt‐1 promoted Flk‐1 expression. These results suggest that the two VEGF‐A receptors regulate pulmonary vascular development by modulating the VEGF‐A signaling. Anat Rec, 290:958–973, 2007. © 2007 Wiley‐Liss, Inc.     

Angiogenesis is involved in the pathogenesis of nonrheumatic aortic valve stenosis

Angiogenesis is an essential biological process not only in embryogenesis, but also in the progression of several major diseases, including cancer, diabetes, and inflammation. Excessive vascularization can also contribute to some cardiovascular pathologies, such as atherosclerosis, but contradictory reports still prevail regarding its impact on aortic stenosis. Using immunohistochemical techniques, we assessed the vascular density and distribution of angiogenesis (FVIII) and vascular endothelial growth factor (VEGF) expression as well as the expression of 2 VEGF receptors, Flt-1 and Flk-1, in 55 nonrheumatic and 6 control aortic valves. In the light of the fact that the angiogenic effect of VEGF is mediated by sustained formation of nitric oxide, the samples were also immunostained with 3 nitric oxide synthase (eNOS, iNOS, and nNOS) antibodies. The immunohistochemical findings of VEGF and its receptors were verified by immunoblotting techniques. Vascular density was highest in the cases with moderate valve stenosis, and the mean number of FVIII-positive blood vessels was 1.7 +/- 1.9 vessels/mm(2) in the diseased valves, whereas the normal valves contained no blood vessels. Vascular density was significantly higher in the cases showing chronic inflammation (P = 0.007). Interestingly, the patients receiving statin therapy had significantly lower vascular densities than those not receiving such therapy (P = 0.001). Diseased valves showed distinct VEGF, Flt-1, Flk-1, and eNOS positivity of activated endothelial, stromal fusiform myofibroblastic, and histocytic cells. In contrast, immunoreactivity for iNOS and nNOS was seen only in nonendothelial stromal cells, and their expression was weaker. Enhanced vascular density was significantly associated with increased expression of Flk-1 (P = 0.028 for endothelial and P = 0.009 for stromal cells) and with endothelial eNOS expression (P = 0.024). A similar tendency was also observed for VEGF, but not for Flt-1. Our results show a distinct angiogenic response and the presence of angiogenic factors in nonrheumatic aortic valve stenosis, suggesting that angiogenesis may influence on the evolution of this disease.     

Role of signals from the dorsal root ganglion in neuropathic pain in a rat model

Vascular endothelial growth factor (VEGF), known as an endothelial cell-specific mitogen, has been reported to be linked also to the NO/cGMP-pathway, which has been notified in the inner ear. Up to now, VEGF has not yet been described in the inner ear. We performed immunohistochemical analysis using specific antibodies to VEGF and to both known VEGF-receptors Flt-1 and KDR/Flk-1 on paraffin-sections of temporal bones from guinea pigs (n=5). Immunoreactivity of VEGF, Flt-1 and KDR/Flk-1 was detectable in a subpopulation of vestibular ganglion cells. VEGF could be found also in the endothelium of blood vessels, in fibrocytes of the lamina propria and in the neuroepithelium. Strong immuno-labelling to Flt-1 was evident in nerve fibres, vascular endothelium and in the neuroepithelium. Fibrocytes, endothelium of blood vessels, supporting cells and calyces in the sensory epithelium revealed immunoreactivity to KDR/Flk-1. These findings give evidence that VEGF, Flt-1 and KDR/Flk-1 are constitutively expressed in the vestibule.