Expression of vascular endothelial growth factor during the development of cardiac hypertrophy in spontaneously hypertensive rats

We have recently demonstrated that lipids, particularly cholesterol, covalently bound to apolipoprotein B (apoB) are a stable marker of low density lipoprotein (LDL) oxidation (Tertov et al. 1995). The present study is an attempt to assess the relationship between the degree of LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intimal smooth muscle cells, i.e. LDL atherogenicity. Native LDL was oxidized in vitro by copper ions, 2,2-azobis-(2-aminopropane hydrochloride), or sodium hypochlorite. Minimum degree of LDL in vitro oxidation necessary to convert LDL into atherogenic one was accompanied by an increase of apoB-bound cholesterol to the level much higher than that usually observed in freshly isolated atherogenic LDL from human blood. Moreover, elimination of LDL aggregates from in vitro oxidized LDL preparations by gel filtration led to loss of its atherogenic properties. Thus, the ability to induce cholesterol accumulation in cells, i.e. the atherogenicity of in vitro oxidized LDL is a result of LDL aggregation but not oxidation. We also studied the relationship between LDL atherogenicity and apoB-bound cholesterol content in LDL freshly isolated from healthy subjects and normo- and hypercholesterolemic patients with coronary atherosclerosis. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.12, n = 90). It is concluded that LDL atherogenicity does not depend on the degree of lipid peroxidation in LDL particle.     

Serum metabolite signature predicts the acute onset of diabetes in spontaneously diabetic congenic BB rats

The clinical presentation of type 1 diabetes is preceded by a prodrome of beta cell autoimmunity. We probed the short period of subtle metabolic abnormalities, which precede the acute onset of diabetes in the spontaneously diabetic BB rat, by analyzing the serum metabolite profile detected with combined gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). We found that the metabolite pattern prior to diabetes included 17 metabolites, which differed between individual diabetes prone (DP) BB rats and their age and sex matched diabetes resistant (DR) littermates. As the metabolite signature at the 40?days of age baseline failed to distinguish DP from DR, there was a brief 10-day period after which the diabetes prediction pattern was observed, that includes fatty acids (e.g. oleamide), phospholipids (e.g. phosphocholines) and amino acids (e.g. isoleucine). It is concluded that distinct changes in the serum metabolite pattern predict type 1 diabetes and precede the appearance of insulitis in spontaneously diabetic BB DP rats. This observation should prove useful to dissect mechanisms of type 1 diabetes.     

Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.     

Role of vascular endothelial growth factor in regulation of physiological angiogenesis

Evidence accumulating over the last decade has established the fundamental role of vascular endothelial growth factor (VEGF) as a key regulator of normal and abnormal angiogenesis. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). The signaling and biological properties of these two receptors are strikingly different. VEGF is essential for early development of the vasculature to the extent that inactivation of even a single allele of the VEGF gene results in embryonic lethality. VEGF is also required for female reproductive functions and endochondral bone formation. Substantial evidence also implicates VEGF as an angiogenic mediator in tumors and intraocular neovascular syndromes, and numerous clinical trials are presently testing the hypothesis that inhibition of VEGF may have therapeutic value.     

Eicosanoid regulation of vascular endothelial growth factor expression and angiogenesis in microvessel endothelial cells

12(R)-Hydroxy-5,8,14-eicosatrienoic acid (HETrE) is a potent inflammatory and angiogenic eicosanoid in ocular and dermal tissues. Previous studies suggested that 12(R)-HETrE activates microvessel endothelial cells via a high affinity binding site; however, the cellular mechanisms underlying 12(R)-HETrE angiogenic activity are unexplored. Because the synthesis of 12(R)-HETrE is induced in response to hypoxic injury, we examined its interactions with vascular endothelial growth factor (VEGF) in rabbit limbal microvessel endothelial cells. Addition of 12(R)-HETrE (0.1 nm) to the cells increased VEGF mRNA levels with maximum 5-fold increase at 45 min. The increase in VEGF mRNA was followed by an increase in immunoreactive VEGF protein. 12(R)-HETrE (0.1 nm) rapidly activated the extracellular signal-regulated kinases (ERKs) ERK1 and ERK2. Moreover, preincubation of cells with PD98059, a selective inhibitor of MEK-1, inhibited 12(R)-HETrE-induced VEGF mRNA. Addition of VEGF antibody to cells grown in Matrigel-coated culture plates inhibited 12(R)-HETrE-induced capillary tube-like formation, suggesting that VEGF mediates, at least in part, the angiogenic response to 12(R)-HETrE. The results indicate that in microvessel endothelial cells, 12(R)-HETrE induces VEGF expression via activation of ERK1/2 and that VEGF mediates, at least in part, the angiogenic activity of 12(R)-HETrE. Given the fact that both VEGF and 12(R)-HETrE are produced in the cornea after hypoxic injury, their interaction may be an important determinant in the development of neovascularized tissues.     

Constitutive and inducible expression and regulation of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF), which was originally discovered as vascular permeability factor, is critical to human cancer angiogenesis through its potent functions as a stimulator of endothelial cell survival, mitogenesis, migration, differentiation and self-assembly, as well as vascular permeability, immunosuppression and mobilization of endothelial progenitor cells from the bone marrow into the peripheral circulation. Genetic alterations and a chaotic tumor microenvironment, such as hypoxia, acidosis, free radicals, and cytokines, are clearly attributed to numerous abnormalities in the expression and signaling of VEGF and its receptors. These perturbations confer a tremendous survival and growth advantage to vascular endothelial cells as manifested by exuberant tumor angiogenesis and a consequent malignant phenotype. Understanding the regulatory mechanisms of both inducible and constitutive VEGF expression will be crucial in designing effective therapeutic strategies targeting VEGF to control tumor growth and metastasis. In this review, molecular regulation of VEGF expression in tumor cells is discussed.     

MicroRNA-9 inhibits retinal neovascularization in rats with diabetic retinopathy by targeting vascular endothelial growth factor A

Diabetic retinopathy (DR) is a leading cause of adult visual impairment and loss. This study aims to explore the effects of microRNA-9 (miR-9) on retinal neovascularization during DR by targeting the vascular endothelial growth factor A (VEGFA). DR rat models were successfully established. Retinal microvascular endothelial cells (RMECs) of DR rats were isolated and treated with miR-9 mimic, miR-9 inhibitor or small interfering RNA (siRNA)-VEGFA. The expressions of miR-9, VEGFA, and cluster of differentiation 31 (CD31) of the rats’ tissues and cells were examined. The targeting relationship between miR-9 and VEGFA was testified. The tubule formation, the cell proliferation and the periodic distribution and apoptosis were evaluated after transfection. In the retinal tissues of DR rats, miR-9 expression decreased while the expression of VEGFA and CD31 increased. Notably, miR-9 targeted and inhibited VEGFA expression. In response to the treatment of miR-9 mimic and siRNA-VEGFA, a reduction was identified in CD31 expression, tubule formation, and proliferation of RMECs and cell ratio in the S phase, but an increase was observed in apoptosis rate of RMECs. The treatment of miR-9 inhibitor reversed the manifestations. Our study demonstrated that miR-9 could inhibit retinal neovascularization of DR and tubule formation, and promote apoptosis in RMECs by targeting VEGFA.     

Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms.

The vascular endothelial growth factor (VEGF) family encompasses four polypeptides that result from alternative splicing of mRNA. We have previously demonstrated differences in the secretion pattern of these polypeptides. Stable cell lines expressing VEGFs were established in human embryonic kidney CEN4 cells. VEGF121, the shortest form, was secreted and freely soluble in tissue culture medium. VEGF189 was secreted, but was almost entirely bound to the cell surface or extracellular matrix. VEGF165 displayed an intermediary behavior. Suramin induced the release of VEGF189, permitting its characterization as a more basic protein with higher affinity for heparin than VEGF165 or VEGF121, but with similar endothelial cell mitogenic activity. Heparin, heparan sulfate, and heparinase all induced the release of VEGF165 and VEGF189, suggesting heparin-containing proteoglycans as candidate VEGF-binding sites. Finally, VEGF165 and VEGF189 were released from their bound states by treatment with plasmin. The released 34-kDa dimeric species are active as endothelial cell mitogens and as vascular permeability agents. We conclude that the bioavailability of VEGF may be regulated at the genetic level by alternative splicing that determines whether VEGF will be soluble or incorporated into a biological reservoir and also through proteolysis following plasminogen activation.     

Effects of nitric oxide donors on vascular endothelial growth factor gene induction

Nitric oxide (NO) has been reported to modulate the vascular endothelial growth factor (VEGF) gene by accumulating hypoxia-inducible factor-1alpha (HIF-1alpha) protein, but there is a contradiction among effects of various NO donors. The effects of NO donors including S-nitroso-N-acetyl-penicillamine (SNAP), S-nitroso-glutathione (GSNO), 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), 3-[(+/-)-(E)-ethyl-2(')-[(E)-hydroxyimino]-5-nitro-3-hexenecarbamoyl]-pyridine (NOR4), 3-morpholinosydnonimine (SIN-1), and nitroprusside (SNP) on the VEGF reporter gene were examined. SNAP, GSNO, NOC18, and NOR4 enhanced the VEGF reporter activity under normoxia and modulated the hypoxic induction. In contrast, SNP had only an inhibitory effect. An NO scavenger attenuated the reporter activation by NO donors except NOR4, but did not ameliorate the inhibitory effect of SNP. A reducing compound dithiothreitol suppressed NO-induced activation of the VEGF reporter gene. SNAP, GSNO, and NOC18 induced the accumulation of HIF-1alpha protein, while others did not. These results suggest that SNAP, GSNO, and NOC compounds are suitable for pharmacological studies in HIF-1-mediated VEGF gene activation by NO.     

Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc

The induction of vascular endothelial growth factor (VEGF) is an essential feature of tumor angiogenesis. Hypoxia is a potent stimulator of VEGF expression, and hypoxia-inducible factor-1 (HIF-1) is considered to be critical for this induction. However, we have previously demonstrated that induction of VEGF by hypoxia was preserved when HIF-1alpha was silenced. We sought to better define the molecular basis of this HIF-1-independent regulation. In colon cancer cells, hypoxia stimulated multiple K-ras effector pathways including phosphatidylinositol 3-kinase. VEGF promoter deletion studies identified a novel promoter region between -418 and -223 bp that was responsive to hypoxia in a PI3K/Rho/ROCK-dependent manner. Electrophoretic mobility shift assays identified a fragment between -300 and -251 bp that demonstrated a unique shift only in hypoxic conditions. Inhibition of PI3K or ROCK blocked the formation of this complex. A binding site for c-Myc, a target of ROCK, was identified at -271 bp. A role for c-Myc in the hypoxic induction of VEGF was demonstrated by site-directed mutagenesis of the VEGF promoter and silencing of c-Myc by small interfering RNA. Collectively, these findings suggest an alternative mechanism for the hypoxic induction of VEGF in colon cancer that does not depend upon HIF-1alpha but instead requires the activation of PI3K/Rho/ROCK and c-Myc.     

Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells

Vascular endothelial growth factor (VEGF) is a potent endothelial cell growth and permeability factor highly expressed in rodent alveolar epithelium after injury and repair. To investigate VEGF synthesis in human lung epithelial cells, we examined VEGF expression by cultured cells under basal conditions and after cytokine treatment or oxidative stress. Basal VEGF expression was detected in transformed human epithelial cell lines (A549 and 1HAEo-) and in primary human bronchial epithelial cells with RT-PCR, Western blot, and immunocytochemistry. Among the cytokines tested, only transforming growth factor-beta1 increased the levels of excreted VEGF(165) as measured by ELISA. Under hypoxia (0% O(2) for 24 h), the VEGF(165) level increased fivefold, and this effect was O(2) concentration dependent. VEGF concentrations in the medium of all the cell types studied reached values similar to those found in bronchoalveolar lavage fluids from normal patients. Endothelial cells (human umbilical vein endothelial cells) exposed to conditioned medium from primary bronchial epithelial cell cultures showed an increased growth rate, which was inhibited in the presence of a specific neutralizing antibody to VEGF. These results suggest that lung epithelial cells participate in the endothelial repair and angiogenesis that follow lung injury through the synthesis of VEGF.     

Synergistic induction of endothelial tissue factor by tumor necrosis factor and vascular endothelial growth factor: functional analysis of the tumor necrosis factor receptors

Tissue factor expression on the surface of endothelial cells can be induced by tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) in a synergistic manner. We have investigated the role of the two different TNF receptors for this synergy. Firstly, stimulation of the 60 kDa TNF receptor (TNFR60) by a mutant of TNF specific for TNFR60 induced responses comparable to wild-type TNF. In contrast, stimulation of TNFR80 by a TNFR80-specific TNF mutein did not result in enhancement of tissue factor expression even in the presence of a suboptimal TNFR60 triggering. Secondly, we tested neutralizing TNF receptor antibodies for inhibition of tissue factor synthesis induced by VEGF and TNF. A TNFR60-specific antibody inhibited tissue factor production over a broad range of TNF concentrations, indicating an essential role of TNFR60 in the TNF/VEGF synergy. In contrast, blocking of TNF binding to TNFR80 strongly inhibited TNF-induced tissue factor expression at low, but less pronounced at high, TNF concentrations. In conclusion, these data are in agreement with a model in which TNFR80 participates in the synergy between VEGF and low concentrations of soluble TNF by passing the ligand to the signalling TNFR60.     

Glomerular function in spontaneously diabetic rats.

This study was undertaken to determine whether hyperfiltration exists at the single nephron level and whether albumin excretion is increased early in the course of diabetes in Biobreeding rats. Diabetic rats were studied at 8-12 weeks after the onset of diabetes. Control animals were age-matched, diabetes-resistant rats. Urinary and tubular fluid albumin concentrations were measured by polyacrylamide gel electrophoresis. Clearance and micropuncture techniques were used to determine whole kidney and single nephron glomerular filtration rate, renal blood flow, and glomerular capillary pressure. The urinary albumin excretion rate (1.3 +/- 0.1 mg/24 hr) and the tubular fluid albumin concentration (4.7 +/- 0.7 mg/dl) in the diabetic group were significantly elevated when compared with urinary albumin excretion (0.9 +/- 0.1 mg/24 hr) and tubular fluid albumin concentration (2.5 +/- 0.5 mg/dl) in the control group. There were no significant differences in glomerular hemodynamics (whole kidney or single nephron glomerular filtration rate or glomerular capillary pressure) between diabetic and control rats. The kidney weight and kidney weight to body weight ratio were significantly higher in diabetic rats when compared with control rats. Early diabetes in Biobreeding rats is characterized by mild albuminuria and increased kidney size, but not glomerular hyperfiltration.     

重组血管内皮细胞生长因子包涵体复性条件研究

利用大肠杆菌ＢＬ２１（ＤＥ３） 表达血管内皮细胞生长因子１６５（ ＶＥＧＦ１６５） ,表达蛋白以包涵体形式存在。为了获得大量有生物活性的蛋白质,我们对影响复性的参数：氧化型、还原型谷胱甘肽比例,复性液的ｐＨ 值,复性时间,精氨酸浓度以及血管内皮细胞生长因子（ ＶＥＧＦ１６５） 包涵体的起始浓度进行了较为系统的研究,初步获得了具有一定生物活性的ＶＥＧＦ１６５ 二聚体蛋白。