Vascular endothelial growth factor mRNA expression and arteriovenous balance in response to prolonged,submaximal exercise in humans

Angiogenesis, the growth of new blood vessels from existing ones, occurs in the skeletal muscle as an adaptive response to exercise that satisfies the increased requirement of this tissue for oxygen delivery and metabolic processes. Of the factors that have been identified to regulate this process, the endothelial cell mitogen vascular endothelial growth factor (VEGF) has been proposed to play a key role. The aim of this study was to measure the skeletal muscle VEGF mRNA content and arteriovenous protein balance across the working leg in response to a single bout of prolonged, submaximal exercise. Seven physically active males completed 3 h of two-legged kicking ergometry. Muscle biopsies were collected from the vastus lateralis muscle from both working legs, and blood samples were collected from one femoral artery and femoral vein before, during, and in recovery from exercise. We show that the exercise stimulus elicited a decrease in VEGF protein arteriovenous balance across the exercising leg (P = 0.007), and a ninefold elevation in skeletal muscle VEGF mRNA expression (P < 0.001). The changes in VEGF protein balance and mRNA content were most pronounced 1 h after the cessation of exercise. In conclusion, these findings demonstrate that submaximal exercise, suitable for humans with low CV fitness, induces a decrease in VEGF arteriovenous balance that is likely to be of clinical significance in promoting angiogenic effects.     

Prolonged hypoxia increases vascular endothelial growth factor mRNA and protein in adult mouse brain

Brain hypoxiainduces an increase in brain vascularity, presumably mediated byvascular endothelial growth factor (VEGF), but it is unclear whetherVEGF is required to maintain the increase. In these studies, brain VEGFmRNA and protein levels were measured in adult mice kept in hypobaricchambers at 0.5 atm for 0, 0.5, 1, 2, 4, 7, and 21 days. Hypoxia wasaccompanied by a transient increase of VEGF mRNA expression: twofold by0.5 day and a maximum of fivefold by 2 days; these were followed by adecrease at 4 days and a return to basal levels by 7-21 days. VEGFprotein expression induced by hypoxia was bimodal, initiallyparalleling VEGF mRNA. There was an initial small increase at 12 h thatreached a maximum by day 2, and, aftera transient decrease on day 4, theprotein expression increased again on day7 before it returned to normoxic levels after 21 days.Thus, despite continued hypoxia, both VEGF mRNA and protein levelsreturned to basal after 7 days. These data suggest a metabolicnegative-feedback system for VEGF expression during prolonged hypoxiain the brain.

     

MDM2 regulates vascular endothelial growth factor mRNA stabilization in hypoxia

Expression of vascular endothelial growth factor (VEGF) increases in cancer cells during hypoxia. Herein, we report that the MDM2 oncoprotein plays a role in hypoxia-mediated VEGF upregulation. In studying the characteristics of MDM2 and VEGF expression in neuroblastoma cells, we found that hypoxia induced significantly higher upregulation of both VEGF mRNA and protein in MDM2-positive cells than in the MDM2-negative cells, even in cells without wild-type (wt) p53. We found that hypoxia induced translocation of MDM2 from the nucleus to the cytoplasm, which was associated with increased VEGF expression. Enforcing overexpression of cytoplasmic MDM2 by transfection of the mutant MDM2/166A enhanced expression of VEGF mRNA and protein production, even without hypoxia. The results of mechanistic studies demonstrated that the C-terminal RING domain of the MDM2 protein bound to the AU-rich sequence within the 3' untranslated region (3'UTR) of VEGF mRNA; this binding increased VEGF mRNA stability and translation. In addition, knockdown of MDM2 by small interfering RNA (siRNA) in MDM2-overexpressing cancer cells resulted in inhibition of VEGF protein production, cancer cell survival, and angiogenesis. Our results suggest that MDM2 plays a p53-independent role in the regulation of VEGF, which may promote tumor growth and metastasis.     

Vascular endothelial growth factor in astroglioma stem cell biology and response to therapy

Malignant astrogliomas are among the most aggressive, highly vascular and infiltrating tumours bearing a dismal prognosis, mainly due to their resistance to current radiation treatment and chemotherapy. Efforts to identify and target the mechanisms that underlie astroglioma resistance have recently focused on candidate cancer stem cells, their biological properties, interplay with their local microenvironment or 'niche', and their role in tumour progression and recurrence. Both paracrine and autocrine regulation of astroglioma cell behaviour by locally produced cytokines such as the vascular endothelial growth factor (VEGF) are emerging as key factors that determine astroglioma cell fate. Here, we review these recent rapid advances in astroglioma research, with emphasis on the significance of VEGF in astroglioma stem-like cell biology. Furthermore, we highlight the unique DNA damage checkpoint properties of the CD133-marker-positive astroglioma stem-like cells, discuss their potential involvement in astroglioma radioresistance, and consider the implications of this new knowledge for designing combinatorial, more efficient therapeutic strategies.     

Vascular endothelial growth factor, vascular endothelial growth factor receptor-3 and cyclooxygenase-2 expression in psoriasis

OBJECTIVE: To investigate expression patterns and relationship of vascular endothelial growth factor (VEGF), vascular endothelial receptor-3 (VEGF-R3) (FLT-4) and cyclooxygenase-2 (COX-2) in psoriasis. STUDY DESIGN: Forty-three patients were included in this study. The clinical severity of psoriasis was assessed using the psoriasis area and severity index (PASI). Punch biopsy samples both from psoriatic and nonlesional skin were taken and VEGF, VEGF-R3 and COX-2 expressions determined. RESULTS: VEGF, VEGF-R3 and COX-2 expressions were detected in 90.9%, 78.0% and 86.4% of psoriatic and 84.1%, 71.8%, and 84.1% of nonlesional skin, respectively. Epidermal VEGF, VEGF-R3 and COX-2 expressions were detected in 56.8%, 77.8% and 34.1 of psoriatic and 75%, 78.1% and 65.9% of nonlesional skin, respectively. In dermis, VEGF, VEGF-R3 and COX-2 expression was observed in 88.6%, 77.5% and 84.1% of psoriatic and 81.8%, 64.1% and 77.3% of nonlesional skin, respectively. Among the PASI subgroups no statistically significant differences were detected for VEGF, VEGF-R3 and COX-2 expression. CONCLUSION: Our study demonstrated that VEGF, VEGF-R3 and COX-2 expression in psoriatic and nonlesional skin is significantly high in epidermis and dermis. Although there was significant concordance between VEGF and VEGF-R3 expressions in psoriatic lesions, there seems to be no concordance between the others.     

Expression and functional activity of pro-oxidants and antioxidants in murine heart exposed to acute hypobaric hypoxia

Under hypobaric hypoxia, antioxidant defenses of the heart are stressed by the enhanced production of ROS. Mammalian heart acclimatizes to hypoxia through altered gene expression, which we studied in murine heart exposed to 10h of acute hypobaric hypoxia (AHH), equivalent to 15000ft, using cDNA arrays. Functional classification of genes with a > or =2-fold change revealed a number of pro-oxidants like Cyba, Xdh, Txnip, Ppp1r15b and antioxidants like Cat, Gpx1, Mt1, Mgst1. Interestingly, the protein level of Cyba, a subunit of NADPH oxidase, was markedly decreased in AHH exposed heart, suggesting the involvement of some stress response pathways. The AHH exposure also caused a significant reduction (50%) in the level of GSH (P<0.05). The present study provides a retrospective insight on the cellular antioxidant defense mechanisms under AHH.     

Vascular endothelial growth factor stimulates differential signaling pathways in in vivo microcirculation

Vascular endothelial growth factor (VEGF) induces mild vasodilation and strong increases in microvascular permeability. Using intravital microscopy and digital integrated optical intensity image analysis, we tested, in the hamster cheek pouch microcirculation, the hypothesis that differential signaling pathways in arterioles and venules represent an in vivo regulatory mechanism in the control of vascular diameter and permeability. The experimental design involved blocking specific signaling molecules and simultaneously assessing VEGF-induced changes in arteriolar diameter and microvascular transport of FITC-Dextran 150. Inhibition of Akt [indirectly via phosphatidylinositol 3-kinase with LY-294002 or wortmannin] or PKC (with bisindolylmaleimide) reduced VEGF-induced hyperpermeability. However, phosphatidylinositol 3-kinase/Akt inhibition enhanced the early phase and attenuated the late phase of VEGF-induced vasodilation, whereas blocking PKC had no effect. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 (with PD-98059 or AG-126) also reduced VEGF-induced hyperpermeability but did not block VEGF-induced vasodilation. Blockade of endothelial nitric oxide synthase (with N(omega)-monomethyl-l-arginine) inhibited VEGF-induced changes in both permeability and diameter. Furthermore, immunofluorescence studies with human umbilical vein endothelial cells revealed that bisindolylmaleimide, PD-98059, and l-NMMA attenuate VEGF-induced reorganization of vascular endothelial cadherin. Our data demonstrate that 1) endothelial nitric oxide synthase is a common convergence pathway for VEGF-induced changes in arteriolar diameter and microvascular permeability; 2) PKC and ERK-1/2 do not play a major role in VEGF-induced vasodilation in the hamster cheek pouch microcirculation; and 3) Akt, PKC, and ERK-1/2 are elements of the signaling cascade that regulates VEGF-stimulated microvascular hyperpermeability. Our data provide evidence for differential signaling as a regulatory step in VEGF-stimulated microvascular dynamics.     

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in the chick embryo area vasculosa

Vascular endothelial growth factor is an angiogenic factor in vivo and in vitro that plays a crucial role in the control of blood vessel development and in pathological angiogenesis. The vascularized extraembryonic membranes of the chick embryo include the area vasculosa and the chorioallantoic membrane. In this study, we investigated the expression of vascular endothelial growth factor and of its receptor-2, specifically expressed by the endothelial cells, in the chick area vasculosa at days 6, 10 and 14 of incubation. Our results indicate that, in all the three developmental stages examined, vascular endothelial growth factor is clearly expressed in the endodermal cells immediately adjacent to the mesodermal endothelial cells which, in turn, expressed vascular endothelial growth factor receptor-2. These observations suggest that during the development of the vascular system, endodermal cells, expressing vascular endothelial growth factor, initiate angiogenesis by stimulating directly mesodermal cells, which express vascular endothelial growth factor receptor-2. Moreover, our data demonstrate that vascular endothelial growth factor receptor-2 expression is also maintained by endothelial cells in the later stages of development, until day 14 of incubation. In accord with other literature data, this suggests that vascular endothelial growth factor is required not only for proliferation, but also for the survival of endothelial cells.     

Role and differential expression of calpastatin mRNA and protein in cultured cardiomyocytes exposed to hypoxic stress

This study investigated the beneficial effects and mechanism of action of the juice of Momordica charantia in streptozotocin (STZ)-induced diabetes mellitus in rats. Diabetes mellitus was associated with significant (p < 0.01) time course reductions in body weight, plasma insulin and the number of insulin positive cells per islet and significant (p < 0.01) time course elevation in blood glucose and osmolarity and systolic blood pressure compared to age-matched healthy controls. Oral intake of M. charantia juice by STZ-induced diabetic rats partially reversed all the diabetes-induced effects measured. Daily oral administration of M. charantia juice to STZ-induced diabetic rates significantly (p < 0.01) reduced the Na+- and K+-dependent absorptions of glucose by the brush border membrane vesicles of the jejunum compared to the responses obtained in STZ-induced diabetic rat. Either insulin (100 MM) or the fruit juice lyophilised extract (5 microg x ml(-1)) can stimulate 14C-D-glucose uptake in L6 myotubes. These effects were completely blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. High concentrations (10-200 microg x ml(-1)) of M. charantia juice extract inhibited 14C-D-glucose uptake in L6 myotubes compared to the control response. The effect of M. charantia treatment was also investigated on myelinated fibre abnormalities in the tibial nerve of STZ-induced diabetic and control rats. The results show that diabetes was associated with significant (p < 0.05) reduction in the mean cross-sectional myelinated nerve fibres, axonal area, myelin area and maximal fibre area compared to end controls. Treatment of STZ-induced diabetic rats with M. charantia juice normalised the structural abnormalities of peripheral nerves. The results indicate that M. charantia can exert marked beneficial effects in diabetic rats, and moreover, it can regulate glucose uptake into jejunum membrane brush border vesicles and stimulate glucose uptake into skeletal muscle cells similar to the response obtained with insulin.     

Vascular endothelial growth factor expression and regulation of murine collagen-induced arthritis

We have examined the expression and function of the angiogenic factor, vascular endothelial growth factor (VEGF) during the evolution of type II collagen-induced arthritis (CIA). Biologically active VEGF was expressed along a time course that paralleled the expression of two specific VEGF receptors, Flk-1 and Flt-1, and the progression of joint disease. Moreover, levels of VEGF expression correlated with the degree of neovascularization, as defined by vWF levels, and arthritis severity. Macrophage- and fibroblast-like cells, which infiltrated inflamed sites and were then activated by other inflammatory mediators, are probably important sources of VEGF and may thus regulate angiogenesis during the development of CIA. Administration of anti-VEGF antiserum to CIA mice before the onset of arthritis delayed the onset, reduced the severity, and diminished the vWF content of arthritic joints. By contrast, administration of anti-VEGF antiserum after the onset of the disease had no effect on the progression or ultimate severity of the arthritis. These data suggest that VEGF plays a crucial role during an early stage of arthritis development, affecting both neovascularization and the progression of experimentally induced synovitis.     

Excitability changes of cortical neurons during the postnatal period in rats exposed to prenatal hypobaric hypoxia

Pregnant rats were exposed to intermittent hypobaric hypoxia (at a simulated altitude of 7000 m or 5000 m) and the excitability of cortical neurons of their pups was tested. Stimulation of the sensorimotor cortex of rats prenatally exposed to hypoxia shortened the duration of cortical afterdischarges in 12-day-old rats, but did not change the excitability in 25-day-old animals. Shortening of the first afterdischarge in 35-day-old rats but the prolongation of the first afterdischarge in adult rats (as compared to the duration of cortical afterdischarges in rats not exposed to prenatal hypoxia) were registered. The possible mechanisms of different excitability of cortical neurons in rats prenatally exposed to hypobaric hypoxia are discussed.     

Leptin receptor-deficient obese Zucker rats reduce their food intake in response to hypobaric hypoxia

Exposure to hypoxia induces anorexia in humans and rodents, but the role of leptin remains under discussion and that of orexigenic and anorexigenic hypothalamic neuropeptides remains unknown. The present study was designed to address this issue by using obese (Lepr(fa)/Lepr(fa)) Zucker rats, a rat model of genetic leptin receptor deficiency. Homozygous lean (Lepr(FA)/Lepr(FA)) and obese (Lepr(fa)/Lepr(fa)) rats were randomly assigned to two groups, either kept at ambient pressure or exposed to hypobaric hypoxia for 1, 2, or 4 days (barometric pressure, 505 hPa). Food intake and body weight were recorded throughout the experiment. The expression of leptin and vascular endothelial growth factor (VEGF) genes was studied in adipose tissue with real-time quantitative PCR and that of selected orexigenic and anorexigenic neuropeptides was measured in the hypothalamus. Lean and obese rats exhibited a similar hypophagia (38 and 67% of initial values at day 1, respectively, P < 0.01) and initial decrease in body weight during hypoxia exposure. Hypoxia led to increased plasma leptin levels only in obese rats. This resulted from increased leptin gene expression in adipose tissue in response to hypoxia, in association with enhanced VEGF gene expression. Increased hypothalamic neuropeptide Y levels in lean rats 2 days after hypoxia exposure contributed to accounting for the enhanced food consumption. No significant changes occurred in the expression of other hypothalamic neuropeptides involved in the control of food intake. This study demonstrates unequivocally that altitude-induced anorexia cannot be ascribed to anorectic signals triggered by enhanced leptin production or alterations of hypothalamic neuropeptides involved in anabolic or catabolic pathways.     

Proteomic analysis of differential protein expression in response to epidermal growth factor in neonatal porcine pancreatic cell monolayers

We have proposed that porcine neonatal pancreatic cell clusters (NPCCs) may be a useful alternative source of cells for islet transplantation, and that monolayer cultures might provide an opportunity to manipulate the cells before transplantation. In addition we previously identified 10 genes up-regulated by epidermal growth factor (EGF) in cultured porcine NPCC monolayers. We have now analyzed the intracellular signaling pathways activated by EGF and searched for proteins differentially expressed following EGF treatment of the monolayers, using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). EGF treatment resulted in phosphorylation of both Erk 1/2 and Akt, as well as increased cell proliferation. Five unknown and 13 previously identified proteins were differentially expressed in response to EGF. EGF treatment increased the expression of several structural proteins of epithelial cells, such as cytokeratin 19 and plakoglobin, whereas vimentin, the intermediate filament protein of mesenchymal cells, and non-muscle myosin alkali chain isoform 1, decreased. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 factor, which promotes epithelial cell proliferation, and hemoglobin alpha I & II also increased, whereas cyclin A1, immunoglobulin heavy chain, apolipoprotein A1, 5,10-ethylenetetrahydrofolated reductase (5,10-MTHFR), angiotensin-converting enzyme 2 (ACE2), co-lipase II precursor, and NAD+ isocitrate dehydrogenase (NAD+ IDH) alpha chain proteins decreased. Our results show that EGF stimulates proliferation of pancreatic epithelial cells by simultaneously activating the MAPK and PI-3K pathways. HnRNP A2/B1, hemoglobin, cyclin A1, and ACE2 may play roles in the proliferation of epithelial cells in response to EGF.     

Vascular endothelial growth factor: biology and therapeutic applications

While the development of anti-angiogenic therapy, as it pertains to cancer treatment, may still be in its infancy relative to well-established modalities such as chemotherapy, radiation, and surgery, major strides made in the past several decades have allowed translation of basic science discoveries in this field into clinical reality. The discovery of key molecular modulators of angiogenesis, notably vascular endothelial growth factor (VEGF), has catalyzed the development of numerous neutralizing therapeutic agents. The validity of VEGF inhibition as a therapeutic strategy has been well supported in randomized clinical trials, as well as U.S. Food and Drug Administration approval of the VEGF antagonists bevacizumab, sunitinib malate, sorafenib, pegaptinib and ranibizumab. Accordingly, this review will (1) briefly review the basic molecular biology of VEGF and (2) summarize recent progress in targeting the VEGF molecular pathway as therapy for angiogenic diseases such as cancer and age-related macular degeneration.