Mouse ten-m/Odz is a new family of dimeric type II transmembrane proteins expressed in many tissues.
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The Drosophila gene ten-m/odz is the only pair rule gene identified to date which is not a transcription factor. In an attempt to analyze the structure and the function of ten-m/odz in mouse, we isolated four murine ten-m cDNAs which code for proteins of 2,700-2, 800 amino acids. All four proteins (Ten-m1-4) lack signal peptides at the NH2 terminus, but contain a short hydrophobic domain characteristic of transmembrane proteins, 300-400 amino acids after the NH2 terminus. About 200 amino acids COOH-terminal to this hydrophobic region are eight consecutive EGF-like domains. Cell transfection, biochemical, and electronmicroscopic studies suggest that Ten-m1 is a dimeric type II transmembrane protein. Expression of fusion proteins composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene resulted in membrane-associated staining of the alkaline phosphatase. Electronmicroscopic and electrophoretic analysis of a secreted form of the extracellular domain of Ten-m1 showed that Ten-m1 is a disulfide-linked dimer and that the dimerization is mediated by EGF-like modules 2 and 5 which contain an odd number of cysteines. Northern blot and immunohistochemical analyses revealed widespread expression of mouse ten-m genes, with most prominent expression in brain. All four ten-m genes can be expressed in variously spliced mRNA isoforms. The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter bound to specific regions in many tissues which were partially overlapping with the Ten-m1 immunostaining. Far Western assays and electronmicroscopy demonstrated that Ten-m1 can bind to itself. (+info)
Effect of NO, vasodilator prostaglandins, and adenosine on skeletal muscle angiogenic growth factor gene expression.
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Exercise training results in several muscle adaptations, one of which is angiogenesis. Acutely, exercise leads to release of nitric oxide, prostacyclin (PGI2), and adenosine (A) in the skeletal muscles. In this paper, we asked whether any of these locally released vasodilators, as well as other known dilator prostaglandins (PGE1 and PGE2), have the potential to increase angiogenic growth factor gene expression in resting skeletal muscle. Seven groups of 5-7 female Wistar rats (age 8-12 wk, weight 250 +/- 10 g) were anesthetized and instrumented for carotid artery pressure and electromagnetic femoral artery blood flow measurement. One group acted as control while the other groups each received one of the following six agents by constant arterial infusion (dose in microg/min): A (200), nitroprusside (NP, 4.2), acetylcholine (100), PGE1 (1.9), PGE2 (1.7), and PGI2 (1.7). Each agent reduced peripheral vascular resistance to a similar extent (at least twofold). Densitometric mRNA/18S levels for vascular endothelial growth factor (VEGF) were increased 50% by NP and acetylcholine, were unaffected by PGE1 and PGE2, and were reduced 40% by PGI2. For basic fibroblast growth factor, only PGI2 had any effect, reducing mRNA/18S approximately 25%. For transforming growth factor-beta1, A, NP, and PGE1 led to reduced mRNA/18S, whereas PGE2 slightly increased mRNA/18S. For the principal putative angiogenic growth factor, VEGF, these data suggest that naturally secreted vasodilators in contracting skeletal muscle could be involved in regulation of gene expression, namely, nitric oxide in a positive and PGI2 in a negative direction. (+info)
Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: In vivo neovascularization induced by stromal-derived factor-1alpha.
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The contribution of chemokines toward angiogenesis is currently a focus of intensive investigation. Certain members of the CXC chemokine family can induce bovine capillary endothelial cell migration in vitro and corneal angiogenesis in vivo, and apparently act via binding to their receptors CXCR1 and CXCR2. We used an RNAse protection assay that permitted the simultaneous detection of mRNA for various CXC chemokine receptors in resting human umbilical vein endothelial cells (HUVECs) and detected low levels of only CXCR4 mRNA. Stimulation of HUVECs with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) up-regulated levels of only CXCR4 mRNA. CXCR4 specifically binds the chemokine stromal-derived factor-1alpha (SDF-1alpha). Competitive binding studies using 125I-labeled SDF-1alpha with Scatchard analysis indicated that VEGF or bFGF induced an average number of approximately 16,600 CXCR4 molecules per endothelial cell, with a Kd = 1.23 x 10(-9) mol/L. These receptors were functional as HUVECs and human aorta endothelial cells (HAECs) migrated toward SDF-1alpha. Although SDF-1alpha-induced chemotaxis was inhibited by the addition of a neutralizing monoclonal CXCR4 antibody, endothelial chemotaxis toward VEGF was not altered; therefore, the angiogenic effect of VEGF is independent of SDF-1alpha. Furthermore, subcutaneous SDF-1alpha injections into mice induced formation of local small blood vessels that was accompanied by leukocytic infiltrates. To test whether these effects were dependent on circulating leukocytes, we successfully obtained SDF-1alpha-induced neovascularization from cross sections of leukocyte-free rat aorta. Taken together, our data indicate that SDF-1alpha acts as a potent chemoattractant for endothelial cells of different origins bearing CXCR4 and is a participant in angiogenesis that is regulated at the receptor level by VEGF and bFGF. (+info)
Suppression of hypoxia-associated vascular endothelial growth factor gene expression by nitric oxide via cGMP.
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PURPOSE: To investigate the suppressive effect of nitric oxide (NO) on vascular endothelial growth factor (VEGF) gene expression and to elucidate its mechanism of action. METHODS: Immortalized human retinal epithelial (RPE) cells, H-ras-transfected murine capillary endothelial cells, and nuclear factor-kappaB (NF-kappaB) RelA knockout 3T3 fibroblasts had VEGF gene expression stimulated by hypoxia, TPA (phorbol ester 12-O-tetradecanoylphorbol-13 acetate), and ras-transfection. The dose response and time course of inhibition of VEGF gene expression by NO were characterized by northern blot analysis, ribonuclease protection assay, and enzyme-linked immunosorbent assay. The effects of NF-kappaB and cGMP in the NO-induced suppression of VEGF gene expression were quantitated. cGMP production was inhibited by LY 83583 (6-anilino-5,8-quinolinedione), a specific inhibitor of guanylate cyclase production, and cGMP accumulation was quantitated by immunoassay. RelA knockout 3T3 fibroblasts were used to assess the contribution of NF-kappaB to the downregulation of VEGF by NO. RESULTS: The NO donor sodium nitroprusside (SNP) decreased hypoxia-induced VEGF gene expression in a dose- and time-dependent manner. One hundred fifty micromolar SNP completely suppressed hypoxia-induced VEGF mRNA levels for at least 24 hours. Constitutive VEGF expression was not altered by SNP. The SNP-mediated decreases in VEGF expression were associated with increases in intracellular cGMP and were blocked by LY 83583. Sodium nitroprusside was able to decrease hypoxia-induced VEGF mRNA increases in fibroblasts deficient in the RelA subunit of NF-kappaB. Nitric oxide was also effective at suppressing increased VEGF expression secondan, to mutant ras and TPA. CONCLUSIONS: These data indicate that NO decreases hypoxia-induced VEGF via a cGMP-dependent mechanism and suggest that NO may serve as an endogenous inhibitor of both hypoxia- and non- hypoxia-enhanced VEGF expression in vivo. (+info)
Correlation of VEGF expression by leukocytes with the growth and regression of blood vessels in the rat cornea.
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PURPOSE: To determine the temporal and spatial relationships between neovascularization and basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) mRNA and protein expression in the rat cornea after cautery with silver nitrate. METHODS: In female Sprague-Dawley rats, a silver nitrate applicator was placed on the central cornea to elicit circumferential angiogenesis, and blood vessel growth was quantified by digital image analysis of corneal flat-mounts. Total RNA or protein was extracted from whole corneas until 1 week after cautery, and bFGF and VEGF mRNA and protein levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). To localize VEGF mRNA and protein, paraformaldehyde-fixed and paraffin-embedded histologic cross sections of corneas were examined by in situ hybridization and immunohistochemistry. Macrophages were identified by ED2 immunohistochemistry. To examine the regulation of VEGF, rats were treated with dexamethasone (0.5 mg/kg per day) and hyperoxia (70% O2). RESULTS: The neovascular response progresses in three phases: (1) a nonproliferative phase preceding vessel growth (< or = 48 hours after cautery); (2) a proliferative phase with maximal growth rate between 3 and 4 days; and (3) a regressive phase (day 7) with a decrease in vessel density accompanying the completion of vessel elongation. In corneas after cautery, bFGF mRNA expression was unchanged, and bFGF protein concentration decreaseed by 97% after 24 hours and returned to control levels by day 7. In contrast, VEGF164 and VEGF188 mRNA splice variants and protein peaked 48 hours after cautery, remained elevated 4 days after cautery, and decreased to near baseline by day 7. The peak concentration of VEGF in the cornea at 48 hours was calculated to be 720 pM, which is sufficient to evoke a functional response. In situ hybridization and immunohistochemistry showed VEGF expressed initially in neutrophils (24 - 48 hours) and subsequently in macrophages (4 days) adjacent to the cautery site. Treatment with either dexamethasone or systemic hyperoxia inhibited both neovascularization and the increase in VEGF expression. Dexamethasone inhibited 27% of cautery-induced VEGF upregulation at 24 hours and 23% at 48 hours, hyperoxia inhibited 32% at 24 hours and 43% at 48 hours, and combined treatment with both dexamethasone and hyperoxia had an additive effect (56% inhibition at 24 hours). CONCLUSIONS: VEGF production by leukocytes correlates temporally and spatially with cautery-induced angiogenesis in the rat cornea. Both inflammatory products and hypoxia appear to sufficiently increase VEGF expression near the cautery lesion to increase vascular permeability of limbal vessels and induce endothelial cell migration and proliferation. (+info)
Targetting VEGF in anti-angiogenic and anti-tumour therapy: where are we now?
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Since the recognition of the importance of the vascular bed for growth and metastasis of solid tumours, many researchers have investigated the approach of attacking the tumour vascular bed instead of the tumour cells themselves in anti-cancer therapy. Such approaches have become possible with the increasing knowledge of the angiogenic process and the factors that regulate it. Especially the potent angiogenic factor VEGF has been the subject of extensive study in this regard. A number of studies showed that inactivation of this factor or its receptors led to a profound negative effect on the development of experimental tumours. However, despite the encouraging results obtained in animal studies, it remains to be established whether human tumours, which might be in a state of relative quiescence, are as sensitive to anti-VEGF treatment as the fast-growing tumours that are generally used in animal studies. If so, anti-VEGF treatment might certainly represent a powerful tool in anti-cancer therapy, either or not in combination with other blockers of angiogenesis. (+info)
Human albumin enhances expression of vascular endothelial growth factor in cultured human luteinizing granulosa cells: importance in ovarian hyperstimulation syndrome.
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Ovarian hyperstimulation syndrome (OHSS) is a severe complication of ovarian stimulation for assisted reproductive techniques. Clinical manifestations are massive extravascular fluid accumulation and haemoconcentration. Vascular endothelial growth factor (VEGF) has been demonstrated to mediate the development of OHSS. Intravenous albumin at the time of oocyte aspiration has been suggested as an effective prophylactic treatment against the occurrence of severe OHSS. Here it is reported that in cultured human luteinizing granulosa cells, VEGF mRNA expression was enhanced by human albumin and maximum expression was observed in cultured granulosa cells obtained from patients with serum oestradiol concentrations >2000 pg/ml on the day of human chorionic gonadotrophin injection (P < 0. 05). (+info)
Vascular endothelial growth factor is bound in amniotic fluid and maternal serum.
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To study vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) concentrations and their possible binders, serum from 22 non-pregnant and 55 pregnant women (15 at weeks 10-13; 40 at term), umbilical vein (n = 24) and artery (n = 13) and amniotic fluid (a pool of 50 at weeks 15-17; 11 at term) were assessed for VEGF and PlGF by an enzyme-linked immunosorbent assay. In amniotic fluid and maternal serum VEGF concentrations were <16 ng/ml and added VEGF was not recovered. VEGF was detected in serum from mothers post-partum (137 +/- 142 ng/l, mean +/- SD), umbilical artery (421 +/- 288 ng/l) and vein (502 +/- 339 ng/l) and non-pregnant controls (182 +/- 147 ng/l), and added VEGF was fully recovered. PlGF was detected in pregnancy serum (52 +/- 23 ng/l early pregnancy; 439 +/- 217 ng/l term pregnancy) and in amniotic fluid (early pregnancy 56 ng/l; term pregnancy 30 +/- 18 ng/l). PlGF was fully recovered in all samples. Gel filtration and isoelectric focusing revealed that in maternal serum and amniotic fluid [125I]VEGF was bound to a protein with an Mr of 400-700 kDa and an isoelectric point of approximately 8. This protein was not identical with alpha-2-macroglobulin (by an immunofluorometric assay), pregnancy zone protein or pregnancy associated plasma protein-A (by immunodiffusion). In conclusion, VEGF-binding activity is present in amniotic fluid and maternal blood. It disappears after delivery and is not detectable in fetal or non-pregnant serum. (+info)