Immunolocalisation of the VEGF receptors FLT-1, KDR, and FLT-4 in diabetic retinopathy.
(17/1189)
AIM: To determine the spatial and temporal changes in the staining pattern of the VEGF receptors FLT-1, KDR, and the putative receptor FLT-4 during the pathogenesis of diabetic retinopathy. METHODS: Immunohistochemical localisation of VEGF receptors, using antibodies against FLT-1, FLT-4, and KDR, was carried out on specimens of normal human retina (n = 10), diabetic retinas (a) with no overt retinopathy (n = 12), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n = 5), (c) with active proliferative retinopathy (n = 6), and (d) with no residual proliferative retinopathy after scatter photocoagulation therapy (n = 14), and surgically excised diabetic fibrovascular membranes (n = 11). The degree and pattern of immunostaining was recorded. RESULTS: FLT-1 staining was apparent in the retinas from both non-diabetic and diabetic retinas; weak to moderate staining was generally confined to the inner nuclear layer, the ganglion cell layer, and the retinal vessels during all stages of the disease process. Staining of the retinal vessels was raised in diabetic tissue compared with non-diabetic tissue. The preretinal vessels of the diabetic subjects stained moderately to intensely for FLT-1. In contrast with FLT-1 staining minimal immunostaining for KDR was demonstrated in the non-diabetic eyes and the unlasered eyes; however, weak staining for KDR was observed in the inner nuclear layer and the ganglion cell layer of the unlasered eyes with diabetic changes. In those retinas with preretinal neovascularisation KDR immunoreactivity was moderate to intense in the intra- and preretinal vessels. However, in the excised membranes, where the vessels may have been in a quiescent state, the levels of KDR were weak to moderate. After apparently successful laser treatment KDR staining was reduced in the intraretinal vessels. Minimal FLT-4 staining was observed throughout normal eyes while weak to moderate FLT-4 staining was generally confined to the inner nuclear layer and the ganglion cell layer of the unlasered diabetic eyes. Weak to moderate levels of FLT-4 staining were observed in the intraretinal vessels except after apparently successful laser treatment where reduced levels of staining were observed. Weak to moderate staining was observed in the preretinal vessels. CONCLUSIONS: This study supports a role for FLT-1, KDR, and possibly FLT-4 in the pathogenesis of diabetic retinopathy; however, their specific roles in the progression of the disease may differ. (+info)
Constitutive expression of VEGF, VEGFR-1, and VEGFR-2 in normal eyes.
(18/1189)
PURPOSE: The expression of vascular endothelial growth factor (VEGF) and its high-affinity receptors VEGFR-1 and VEGFR-2 was investigated in normal eyes. METHODS: Monkey and rat eyes were surgically removed in animals under deep anesthesia and immediately prepared for study. Ocular VEGF, VEGFR-1, and VEGFR-2 expression was studied using a combination of in situ hybridization, northern blot analysis, immunohistochemistry, immunoassay, and reverse transcription-polymerase chain reaction. RESULTS: Steady state VEGF mRNA levels were detected in the normal vascularized ocular tissues of the monkey: the conjunctiva, iris, retina, and the choroid-retinal pigment epithelial complex. VEGF121 and VEGF165 were the major isoforms identified. VEGF protein was detected in the conjunctiva, retina, and the choroid-retinal pigment epithelial complex. Retinal VEGF mRNA localized to the ganglion, inner nuclear, and retinal pigment epithelial cell layers. VEGF protein localized to these same layers and to the cones of monkey retina. VEGFR-1 and VEGFR-2 mRNAs were detected in normal monkey iris, retina, and the choroid-retinal pigment epithelial complex. In both monkey and rat eyes, VEGFR-1 and VEGFR-2 mRNAs were localized to the inner nuclear layer of the retina. CONCLUSIONS: VEGF, VEGFR-1, and VEGFR-2 are constitutively expressed in the vascularized tissues of normal eyes. (+info)
Endothelial growth factor receptors in human fetal heart.
(19/1189)
BACKGROUND: Endothelial receptor tyrosine kinases include 3 members of the vascular endothelial growth factor receptor (VEGFR) family and 2 members of the angiopoietin receptor (Tie) family. In addition, the VEGF(165) isoform binds to neuropilin-1 (NP-1), a receptor for collapsins/semaphorins. The importance of these receptors for vasculogenesis and angiogenesis has been shown in gene-targeted mice, but so far, little is known about their exact expression patterns in the human vasculature. METHODS AND RESULTS: Frozen sections of human fetal heart were stained immunohistochemically with receptor-specific monoclonal (VEGFR, Tie) or polyclonal (NP-1) antibodies. The following patterns were observed: The endocardium was positive for VEGFR-1, VEGFR-2, NP-1, Tie-1, and Tie-2 but negative for VEGFR-3. The coronary vessels were positive for Tie-1, Tie-2, VEGFR-1, and NP-1 and negative for VEGFR-2 and VEGFR-3. Myocardial capillaries and epicardial blood vessels stained for VEGFR-1, VEGFR-2, NP-1, and Tie-1; myocardial capillaries and epicardial veins weakly for Tie-2; and epicardial lymphatic vessels for VEGFR-2 and VEGFR-3, weakly for Tie-1 and Tie-2, but not for VEGFR-1 or NP-1. CONCLUSIONS: The results demonstrate differential expression of the endothelial growth factor receptors in distinct types of vessels in the human heart. This information is useful for the understanding of their roles in physiological and pathological processes and for their diagnostic and therapeutic application in cardiovascular medicine. (+info)
VEGF and VEGF type C play an important role in angiogenesis and lymphangiogenesis in human malignant mesothelioma tumours.
(20/1189)
The vascular endothelial growth factor (VEGF) family is a novel regulator of endothelial cell proliferation. We assessed the mRNA expression of VEGF, VEGF type C (VEGF-C) and their receptors together with the microvessel density (VD) and microlymphatic vessel density (LVD) in pursuit of their connection and prognostic value in malignant pleural mesothelioma (MPM). We used four human MPM cell lines, 54 MPM tumours and five normal pleural tissues. Expression levels for receptors and ligands were assessed by semiquantitative reverse transcriptase polymerase chain reaction analysis. Microvessels were highlighted by immunohistochemical staining for factor VIII. The discrimination of lymphatics was performed by enzyme-histochemistry for 5'-nucleotidase after adequate inhibition of non-specific activity. The expression levels of VEGF, VEGF-C and VEGFRs were high in all MPM cell lines. The percentages of tumours with higher expression compared to the mean values of normal pleural tissues were 31.5% (17/54) for VEGF, 66.7% (36/54) for VEGF-C, 20.4% (11/54) for fms-like tyrosine kinase (flt)-1, 42.6% (23/54) for kinase insert domain-containing recepter (KDR) and 59.3% (32/54) for flt-4. Significant positive correlations were found between VEGF-C and flt-4, VEGF and KDR, VEGF and flt-1 in tumour tissues. The association between LVD and VEGF-C expression level was especially strong (P< 0.0001, r= 0.63). There were also significant correlations between LVD and flt-4, and VD and VEGF. No correlation, however, was found between LVD and nodal metastasis. VD was a negative prognostic indicator in this study. The associations between VEGFNEGF-C and vessel density suggest that these factors play an important role in angiogenesis and lymphangiogenesis in this tumour, and assessment of vascularity may be a useful prognostic indicator for MPM patients. (+info)
Modulation of in vivo growth of thyroid tumor-derived cell lines by sense and antisense vascular endothelial growth factor gene.
(21/1189)
Vascular endothelial growth factor A (VEGF) is a potent mitogen for endothelial cells in vitro and promotes neo-angiogenesis in vivo. VEGF overexpression occurs in most human malignancies including thyroid carcinomas in which elevated VEGF expression is associated with a high tumorigenic potential. To investigate the role of VEGF in angiogenesis associated with development of thyroid carcinomas, we constitutively expressed VEGF121 into a poorly tumorigenic cell line (NPA) expressing minimal levels of endogenous VEGF. Here we report that VEGF overexpressing NPA cells showed the same growth potential as untransfected NPA in vitro but formed well-vascularized tumors when injected subcutaneously into nude mice with markedly reduced latency compared to parental cells. A complementary approach was to suppress VEGF expression in a highly tumorigenic anaplastic cell line (ARO) by the transfection of an antisense construct. Antisense-transfected ARO cells expressed reduced constitutive levels of VEGF, showed the same growth potential as untransfected ARO cells in vitro and formed small tumors characterized by minimal vascularization, extensive necrosis and longer latency compared to parental or vector-transfected ARO cells in vivo. Finally, we investigated the expression of both VEGF tyrosine kinase receptors (Flt-1 and Flk-1/KDR) in tumor specimens by RT - PCR. Expression of (Flt-1 and Flk-1/KDR) was low in tissue specimens derived from NPA tumors, but was found enhanced in NPA VEGF tumors; conversely, the expression of VEGF receptors was high in tissue specimens derived from ARO tumors but was decreased in tumors derived from VEGF depleted ARO cells. These results clearly demonstrate that VEGF indirectly promotes the growth of thyroid tumors by stimulating angiogenesis. (+info)
Vascular endothelial growth factor effect on endothelial cell proliferation, migration, and platelet-activating factor synthesis is Flk-1-dependent.
(22/1189)
Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell (EC) proliferation and migration in vitro as well as inflammation in vivo. We showed recently that VEGF effect on vascular permeability was dependent on the synthesis of platelet-activating factor (PAF) by EC. Consequently, we sought to evaluate by antisense knockdown of gene expression the contribution of VEGF receptors (Flt-1 and Flk-1) on these events. VEGF (10(-11) to 10(-8) M) elicited a dose-dependent increase of bovine aortic EC proliferation, migration, and PAF synthesis by up to 2.05-, 1.31- and 35.9-fold above basal levels, respectively. A treatment with two modified antisense oligomers (1-5 x 10(-7) M) directed against Flk-1 mRNA blocked by 100, 91, and 85% the proliferation, migration, and PAF synthesis mediated by VEGF, respectively. A treatment with two antisense oligomers directed against Flt-1 mRNA failed to modulate these activities. The use of placenta growth factor (up to 10(-8) M), an Flt-1-specific agonist, induced only a slight increase (0.6-fold) of PAF synthesis. These data illustrate the crucial role of Flk-1 in EC stimulation by VEGF. The capacity to inhibit the protein synthesis of Flt-1 and Flk-1 by antisense oligonucleotides provides a new approach to block VEGF pathological effects in vivo. (+info)
Expression of the Ets-1 transcription factor in human astrocytomas is associated with Fms-like tyrosine kinase-1 (Flt-1)/vascular endothelial growth factor receptor-1 synthesis and neoangiogenesis.
(23/1189)
Marked neovascularization and vascular endothelial proliferation are characteristic features of malignant gliomas. Vascular endothelial growth factor (VEGF), an angiogenic protein secreted by glioma cells, appears to play a crucial role for induction of neoangiogenesis. The VEGF receptors fms-like tyrosine kinase-1 (Flt-1)/VEGFR-1 and kinase insert domain-containing receptor (KDR)/ VEGFR-2 are up-regulated on the surface of endothelial cells (ECs) in gliomas. Both receptor genes contain an Ets-responsible element in their promoters. The proto-oncogene ets-1 encodes a transcription factor that has been associated with blood vessel formation in vivo under physiological and pathophysiological conditions including tumor neovascularization. Ets-1 is induced by VEGF in cultured ECs. In vitro data also point to a role of Ets-1 as a transcriptional activator of Flt-1. These properties prompted us to investigate Ets-1 expression in 32 human astroglial tumors of WHO grades I-IV and to correlate the data with the expression pattern of VEGF, Flt-1, and KDR. By in situ hybridization, high ets-1 mRNA levels were found in the glioma microvasculature with particularly prominent signals in glomeruloid vascular endothelial proliferations of glioblastomas (WHO grade IV). Semiquantitative reverse transcription-PCR identified the full-length ets-1 transcript but none of three known splice variants encoding isoforms with different functional domains. Immunohistochemical staining demonstrated Ets-1 protein preferentially in the nucleus of those ECs with an epithelioid morphology consistent with an activated state, whereas quiescent flat-shaped ECs predominantly displayed cytosolic immunoreactivity. This observation proposes nuclear translocation of Ets-1 during neoangiogenesis. VEGF synthesis by glioma cells was accompanied by Ets-1 expression in adjacent microvascular ECs. Furthermore, a highly significant correlation was observed between Ets-1 and Flt-1 (but not KDR) expression in ECs of the glioma microvasculature. Our data suggest that VEGF secreted by glioma cells induces Ets-1 in adjacent microvascular ECs, which subsequently transactivates the VEGF receptor Flt-1. This cascade may crucially promote neoangiogenesis in human gliomas. (+info)
VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain.
(24/1189)
VEGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8-12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion-related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders. (+info)