Increased Vascular Endothelial Growth Factor Expression Research Articles

α‐Tocopheryl phosphate – an activated form of vitamin E important for angiogenesis?

Vitamin E (α‐tocopherol, αT) was originally discovered as a dietary factor essential for reproduction in rats. The molecular basis for reproductive failure during vitamin E deficiency was later described to be associated with impaired formation of an extensive placental vascular network. Recently, small amounts of a novel αT derivative, alpha‐tocopheryl phosphate (αTP), have been detected in plasma and animal tissues as well as in foods. We have now assessed the effects of αT and αTP on gene expression in THP‐1 monocytes using gene arrays. The expression of the vascular endothelial growth factor (VEGF) was induced by αTP at mRNA and protein level leading to enhanced angiogenesis in HUVEC cells. Induction of VEGF by αTP was the consequence of stimulation of the PI3K/Akt signalling pathway. The treatment with αTP, but not with αT, significantly induced a VEGF‐promoter luciferase construct transfected into HEK293 cells. We propose that αT is activated by phosphorylation to αTP which may act as an active cofactor or lipid mediator influencing cellular signaling and gene expression leading, in selected tissues, to increased angiogenesis and most likely vasculogenesis. Increased VEGF expression induced by αTP may explain not only the essential effects of vitamin E on reproduction, but also increased cell survival during ischemia/reperfusion injury in brain and muscle cells. Supported by USDA Contract #58‐1950‐7‐707.

Progesterone is neuroprotective against ischemic brain injury through its effects on the phosphoinositide 3-kinase/protein kinase B signaling pathway

We tested the hypothesis that the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway mediates some of the neuroprotective effects of progesterone (PROG) after ischemic stroke. We examined whether PROG acting through the PI3K/Akt pathway could affect the expression of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Rats underwent permanent focal cerebral ischemia by electrocoagulation and received intraperitoneal injections of PROG (8 mg/kg) or vehicle at 1 h post-occlusion and subcutaneous injections at 6, 24, and 48 h. PAkt/Akt levels, apoptosis and apoptosis-related proteins (phosphorylated Bcl-2-associated death promoter (pBAD), BAD, caspase-3, and cleaved caspase-3) were analyzed by TUNEL assays, Western blotting and immunohistochemistry at 24 h post-pMCAO. VEGF and BDNF were analyzed at 24, 72 h and 14 days post-pMCAO with Western blots. Following pMCAO, PROG treatment significantly (P<0.05) reduced ischemic lesion size and edema. Treatment with PROG significantly (P<0.05) decreased VEGF at 24 and 72 h but increased VEGF expression 14 days after injury. The treatment also increased BDNF, and attenuated apoptosis by increasing Akt phosphorylation compared with vehicle alone. The selective PI3K inhibitor wortmannin compromised PROG-induced neuroprotective effects and reduced the elevation of pAkt levels in the ischemic penumbra. Our findings lead us to suggest that the PI3K/Akt pathway can play a role in mediating the neuroprotective effects of PROG after stroke by altering the expression of trophic factors in the brain.

Effects of HMG on revascularization and follicular survival in heterotopic autotransplants of mouse ovarian tissue

Ovarian tissue transplantation is now considered as a procedure to preserve the fertility of young women patients undergoing cancer therapy. The present study investigated the effects and mechanism of human menopausal gonadotrophin (HMG) intervention on vascular remoulding in ovarian heterotopic autotransplantation. Ovaries of 8-week-old mice were cultured in vitro with different concentrations of HMG for 3h for measuring the expression of vascular endothelial growth factor (VEGF). The cultured ovaries were implanted under the kidney capsule and removed 24, 36, 48h or 1month after transplantation. Revascularization, fluid exudation and the number of surviving ovarian follicles were observed. The results showed that VEGF was increased 1.6–6.5 times in the HMG intervention groups. Revascularization appeared 24–36h after transplantation and was earlier than that of the control. Fluid exudation increased incrementally with increasing HMG concentrations. The total number of surviving ovarian follicles was increased by 1.2–1.5 times in the HMG 0.15IU/ml group as compared with the other groups 1month after transplantation. It is concluded that intervention with HMG in vitro before transplantation could improve the blood supply reconstruction and survival of the autotransplanted ovarian follicles, which might be associated with increased VEGF expression.Early diagnosis and effective therapy have been improving the long-term survival rate of children, adolescents and young women suffering from malignant tumours. Chemotherapy and/or radiation therapy commonly compromise the ovary functions. Cryopreservation of cortical fragments of ovarian tissue before cancer therapy, followed by later transplantation, is an emerging technique, but ischaemia after transplantation appears to be one of the main obstacles to successful transplantation. The aim of the present study was to investigate the effects of human menopausal gonadotrophin (HMG) on revascularization and follicular survival of autotransplanted ovary. Half ovaries of 8-week-old mice were cultured in vitro with different concentrations of HMG (0.00, 0.15, 0.30 and 0.60IU/ml) for 3h and transplanted back to the mice. The expression of vascular endothelial growth factor (VEGF), blood vessel repatency and survival ovarian follicles were observed. We found that the in vitro intervention of HMG could improve expression of VEGF in ovarian tissue, shorten the blood supply reconstruction time and increase the number of survival ovarian follicles. The starting date of the oestrous cycle was significantly earlier in the HMG 0.60IU/ml treatment group than the HMG 0.00IU/ml group. We also found that treatment with HMG 0.15IU/ml was better in preserving ovarian follicle numbers than HMG 0.30 or 0.60IU/ml. We concluded the intervention of HMG in vitro before transplantation could improve the blood supply reconstruction and survival of the autotransplanted ovarian follicles, which might be associated with increased VEGF expression. This method may have the potential for clinical usage.

Analysis of vascular endothelial growth factor gene expression in the tissues of patients with chronic venous insufficiency

Immunohistochemical techniques have revealed the presence of vascular endothelial growth factor (VEGF) in the epidermis of patients with chronic venous disease (CVD). Our objective was to perform a quantitative analysis of the VEGF gene transcription in tissues that are potential sources of this factor (skin, varicose veins [VV] and great saphenous vein [GSV]) in patients with CVD. In all, 212 skin and venous tissue samples were collected from patients diagnosed with CVD and controls. The VEGF gene expression was analysed using quantitative realtime polymerase chain reaction (PCR). The skin VEGF expression was lower in the CVD group than in the control group (P = 0.04). There were no significant differences between the insufficient GSV of the CVD group and the control healthy vein (P = 0.22). There was a greater expression of VEGF in the VV of the CVD group than in the control healthy vein (P = 0.03). Comparison of the VEGF expression between the different tissue types in the CVD group revealed significant differences between the skin and GSV (P = 0.02) and between the skin and the VV (P = 0.004), and between the VV and the GSV (P = 0.02). The results of the present study show an over-expression of VEGF gene in the VV tissue of patients with CVD. Based on the data in patients with C2 disease, the VVs appear to be the source of increased VEGF expression.

Role of shear-stress-induced VEGF expression in endothelial cell survival

Vascular endothelial growth factor (VEGF) plays a crucial role in developmental and pathological angiogenesis. Expression of VEGF in quiescent adult tissue suggests a potential role in the maintenance of mature blood vessels. We demonstrate, using a Vegf-lacZ reporter mouse model, that VEGF is expressed by arterial but not by venous or capillary endothelial cells (ECs) in vivo. Using an in vitro model, we show that arterial shear stress of human umbilical vein ECs (HUVECs) decreases apoptosis and increases VEGF expression, which is mediated by the induction of Krüppel-like factor 2 (KLF2). Additionally, shear stress stimulates the expression of VEGF receptor 2 (VEGFR2) and is associated with its activation. Knockdown of VEGF in shear stressed HUVECs blocks the protective effect of shear stress, resulting in EC apoptosis equivalent to that in control ECs cultured under static conditions. Similarly, treatment of ECs subjected to arterial shear stress with the VEGF receptor tyrosine kinase inhibitor SU1498, or VEGFR2 neutralizing antiserum, led to increased apoptosis, demonstrating that the mechanoprotection from increased shear is mediated by VEGFR2. Taken together, these studies suggest that arterial flow induces VEGF-VEGFR2 autocrine-juxtacrine signaling, which is a previously unidentified mechanism for vascular EC survival in adult arterial blood vessels.

Scutellaria barbata inhibits angiogenesis through downregulation of HIF‐1 α in lung tumor

Hypoxia, a hallmark of many solid tumors, is associated with angiogenesis and tumor progression. Hypoxia-inducible factor-1 (HIF-1) plays a significant role in tumor angiogenesis. In this study, the authors constructed a selective platform to screen the traditional Chinese medicine as anti-angiogenic agent. The authors examined the molecular mechanism by which Scutellaria barbata regulates HIF-1-dependent expression of vascular endothelial growth factor (VEGF), which is an important angiogenic factor. Hypoxia promotes angiogenesis by increasing VEGF expression and secretion. Herein, the expression of VEGF was decreased by treatment with S. barbata in tumor cells. Meanwhile, S. barbata reduced the migration and proliferation of endothelial cells under hypoxic condition. S. barbata inhibited the expression of HIF-1α, as well as phosphorylated their upstream signal mediators AKT. S. barbata significantly inhibited the tumor growth in vivo and immunohistochemical studies in the tumors revealed decreased intratumoral microvessel density. These results suggest that the traditional Chinese medicine therapy using S. barbata, which exerts anti-angiogenic activities, represents a promising strategy for the treatment of tumors.

Effects of 17β-estradiol on the oxygen-induced retinal neovasularization in rat

Objective To investigate the effects and mechanism of 17β-estradiol on the retinal neovasularization in rats with oxygen-induced retinopathy (OIR).Methods A total of 48 SD rats were randomly divided into control group A,control group B,experimental group A and experimental group B with 12 rats in each group.The rats in control group A and experimental group A received a hypodermic injection of 0.1 ml PBS,and the rats in control group B and experimental group B group received an a hypodermic injection of 0.1 ml 17β-estradiol.At postnatal day 7 (P7) and P14,the mRNA expression of vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor (HIF) -1α in the retina were measured by real-time polymerase chain reaction (RT-PCR). At P14,endothelial cell nuclei breaking through the internal limiting membrane were counted after staining with hematoxylin and eosin (HE),and the protein expression of VEGF was measured after immunohistochemical staining.The changes of retinal ultrastructure were observed by transmission electron microscopy.Results At P14,the difference of the number of endothelial cell nuclei among four groups was statistically significant (F=10.7,P＜0.05).The number of endothelial cell nuclei in experimental group A was increased greater than that in control group A (P＜0.05) and experimental group B(q=5.16,P＜0.05).But there was no difference between control group A and experimental group B (q=0.25,P＞0.05).The difference of VEGF protein expression among the four groups was statistically significant (P＜0.05).Comparing experimental group A with control group A,B and experimental group B,the difference was statistically significant (P＜0.05).In experimental group A there was ganglion cell swelling,pale staining cytoplasm and mitochondria vacuolizationin,while these were normal in other three groups.At P7 and P14,the differences of VEGF and HIF-1 mRNA expression among four groups were statistically significant (F=14.7,16.1,13.4,17.5； P=0.001,0.005,0.003,0.009).At P7,the VEGF mRNA expression in control group B was more than that in control group A (q=5.22,P＜0.05).The VEGF mRNA expression in experimental group B was more than that in experimental group A (q=4.32,P＜0.05).At P14,the VEGF mRNA expression in control group B was more than that in control group A (q=3.72,P＜0.05),but there was no difference of HIF-1 mRNA expression between two groups.The VEGF and HIF-1 mRNA expression in experimental group B were both decreased more than those in experimental group A (q =5.12,4.08 ； P＜ 0.05).Conelusions 17β-estradiol has the effect of two-way regulation in VEGF mRNA,which increases VEGF expression in retina under hyperoxic conditions so as to develop the vascular system； which reduces VEGF and HIF-1αexpression so as to prevent pathologic neovascularization under hypoxic conditions. It provides some protection from the damage of retinal neovascularization. Key words: Retinal neovascularization/drug therapy; Estradiol; Vascular endothelial growth factors; Hypoxia-inducible factor 1, alpha subunit

Correlation between heparanase and vascular endothelial growth factor in human retinal microvascular endothelial cells induced by hypoxia

Objective To investigate the effects of heparanase and vascular endothelial growth factor (VEGF) and their correlation in CoCl2-induced human retinal microvascular endothelial cells (HRECs) in an hypoxia model.Methods Human eyes were selected to establish CoCl2-induced HRECs hypoxia model in this study.Four experimental groups were studied:normal control group,hypoxia group (CoCl2 100 μmol/L,48 hours),PI-88 group (specific competitive inhibitor of heparanase: phosphomannopentaose sulfate,PI-88,5 μg/ml,combined with CoCl2 100 μmol/L,48 hours) and PBS control group.Heparanase,VEGF and Pol Ⅱ expression in HRECs of normal and hypoxia group were analyzed with immunofluorescence.Western blot was used to evaluate the expression of heparanase and VEGF in HRECs of normal,hypoxia,PI-88 and PBS control groups. Results Immunofluorescence studies showed that the expression of heparanase and VEGF in cytoplasm was intense in hypoxia HRECs,but faint in normal group.Heparanase was also observed in the nucleus of hypoxia HRECs.Western blot results showed that the expression of Hpa and VEGF protein was increased significantly in hypoxia group compared with normal group (Hpa:F=- 4.005,P ＜ 0.05 ; VEGF:F =- 4.063,P ＜ 0.05),and VEGF was decreased in HRECs treated with PI-88 (F=5.963,P＜0.05).Conclusions Heparanase is up-regulated that resulted in increase of VEGF expression,therefore enhanced angiogenesis in CoCl2-induced hypoxia HRECs. Key words: Retinal neovascularization/pathophysiology; Vascular endothelial growth factors; Anoxia; Retinal vessels; Endothelial cells

Etiology of OHSS and use of dopamine agonists

Vascular endothelial growth factor is the factor that causes increased capillary permeability and therefore the most serious manifestations of ovarian hyperstimulation syndrome. Dopamine agonists can both prevent and treat ovarian hyperstimulation syndrome by blocking expression of the vascular endothelial growth factor receptor.

Hepatitis C virus core protein upregulates the expression of vascular endothelial growth factor via the nuclear factor‐κB/hypoxia‐inducible factor‐1α axis under hypoxic conditions

Hepatitis C virus (HCV) core protein critically contributes to hepatocarcinogenesis, which is often observed in liver cirrhosis. Since the liver cirrhosis microenvironment is affected by hypoxia, we focused on the possible driving force of HCV core protein on signal relay from hypoxia-inducible factor (HIF)-1α to vascular endothelial growth factor (VEGF). Human hepatocellular carcinoma cells stably overexpressing HCV core (Core cells) and NS5A (NS5A cells) were established; empty vector-transfected (EV) cells were used as controls. Hypoxia was induced by oxygen deprivation or by using cobalt chloride (CoCl(2) ). YC-1 was used to inhibit HIF-1α expression. Protein analyses for cultured cells and liver tissues obtained from CoCl(2) -treated HCV core-transgenic (Core-Tg) mice were performed by western blot and/or immunocytochemistry. Cellular mRNA levels were evaluated by quantitative real-time reverse transcription-polymerase chain reaction. Under hypoxia, the sustained expression of HIF-1α, but not HIF-2α, was profoundly observed in Core cells but, was faint in EV and NS5A cells. Immunocytochemistry revealed increased HIF-1α in the nucleus. HIF-1α mRNA levels were significantly higher in Core cells than in EV cells under both normoxia and hypoxia. The HIF-1α-targeted VEGF and Bcl-xL expressions were increased in Core cells under hypoxia and abolished by YC-1 treatment. Hypoxic liver samples of Core-Tg mice indicated significant increases in both HIF-1α and VEGF expression compared with the wild type. Hepatitis C virus core protein has the distinct potential to transcriptionally upregulate and sustain HIF-1α expression under hypoxia, thereby contributing to increased VEGF expression, a key regulator in the hypoxic milieu of liver cirrhosis.

Ras homolog gene family, member A promotes p53 degradation and vascular endothelial growth factor‐dependent angiogenesis through an interaction with murine double minute 2 under hypoxic conditions

Tumor neovascularization (TNV) is a common pathologic basis for malignant growth and metastasis. However, the mechanism of TNV pathogenesis is not fully understood. Ras homolog gene family, member A (RhoA), a Rho guanosine triphosphatase (GTPase) family member, may be involved in a hypoxia-induced vascular endothelial growth factor (VEGF) pathway that regulates TNV angiogenesis through an unclear mechanism. The regulation of RhoA on p53, the p53 binding protein homolog murine double minute 2 (MDM2), and VEGF was analyzed in hypoxic MCF-7 cells using Western blot analysis, real-time polymerase chain reaction (PCR) analysis, coimmunoprecipitation, and immunofluorescence staining assays. Changes in proliferation, invasion, migration, stress fiber formation, and tube formation were detected in an MCF-7 human umbilical vein endothelial cell (HUVEC) coculture system. Correlations of RhoA expression with MDM2, wild-type p53 (wt-p53), and VEGF expression in breast cancer tissues and relations between RhoA and breast cancer clinical features were analyzed by immunohistochemistry. Activated RhoA down-regulated p53 protein, which increased VEGF expression in hypoxic MCF-7 cells; whereas p53 messenger RNA levels were not altered. In addition, the ubiquitin-mediated degradation of p53 was enhanced by active RhoA. RhoA and MDM2 colocalized in the cytoplasm of hypoxic MCF-7 cells and interacted with each other physically. Furthermore, nutlin-3, a specific MDM2 inhibitor, was capable of reducing activated RhoA-induced p53 protein stability and attenuating VEGF accumulation. In an MCF-7-HUVEC coculture system, nutlin-3 effectively inhibited HUVEC proliferation, invasion, migration, stress fiber formation, and tube formation mediated by activated RhoA under hypoxic conditions. Data from 129 clinical breast cancer specimens with wt-p53 revealed that high RhoA expression was correlated with high MDM2 expression, low wt-p53 expression, and high VEGF expression. The current data suggested that activated RhoA promotes VEGF expression and hypoxia-induced angiogenesis through the up-regulation of MDM2 to decrease p53 stability.

α‐Tocopheryl phosphate—An activated form of vitamin E important for angiogenesis and vasculogenesis?

Vitamin E was originally discovered as a dietary factor essential for reproduction in rats. Since then, vitamin E has revealed many important molecular properties such as the scavenging of reactive oxygen and nitrogen species or the modulation of signal transduction and gene expression in antioxidant and nonantioxidant manners. A congenital disease, ataxia with vitamin E deficiency, which is characterized by impaired enrichment of α-tocopherol (αT) in plasma due to mutations in the α-tocopherol transfer protein gene, has been discovered. An effect of vitamin E on angiogenesis and vasculogenesis has been observed in several studies, and recently, it has been demonstrated in the placenta of pregnant ewes, possibly involving the stimulation of vascular endothelial growth factor (VEGF) expression. We recently observed that the phosphorylated form of αT, α-tocopheryl phosphate (αTP), increases the expression of VEGF. We propose that the stimulatory effect of αT on angiogenesis and vasculogenesis is potentiated by phosphorylation to αTP, which may act as a cofactor or active lipid mediator increasing VEGF expression. Increased VEGF expression and consequent enhanced angiogenesis and vasculogenesis induced by αTP may explain not only the essential roles of vitamin E on reproduction, but also its beneficial effects against pre-eclampsia, ischemia/reperfusion injury, and during wound healing. It may also serve as a survival factor for brain and muscle cells. The finding that αTP may regulate vasculogenesis may indicate potential, important pathophysiological implications.

Identification of colorectal cancer metastasis markers by an angiogenesis-related cytokine-antibody array

To investigate the angiogenesis-related protein expression profile characterizing metastatic colorectal cancer (mCRC) with the aim of identifying prognostic markers. The expression of 44 angiogenesis-secreted factors was measured by a novel cytokine antibody array methodology. The study evaluated vascular endothelial growth factor (VEGF) and its soluble vascular endothelial growth factor receptor (sVEGFR)-1 protein levels by enzyme immunoassay (EIA) in a panel of 16 CRC cell lines. mRNA VEGF and VEGF-A isoforms were quantified by quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) and vascular endothelial growth factor receptor (VEGFR)-2 expression was analyzed by flow cytometry. Metastasis-derived CRC cell lines expressed a distinctive molecular profile as compared with those isolated from a primary tumor site. Metastatic CRC cell lines were characterized by higher expression of angiogenin-2 (Ang-2), macrophage chemoattractant proteins-3/4 (MCP-3/4), matrix metalloproteinase-1 (MMP-1), and the chemokines interferon γ inducible T cell α chemoattractant protein (I-TAC), monocyte chemoattractant protein I-309, and interleukins interleukin (IL)-2 and IL-1α, as compared to primary tumor cell lines. In contrast, primary CRC cell lines expressed higher levels of interferon γ (IFN-γ), insulin-like growth factor-1 (IGF-1), IL-6, leptin, epidermal growth factor (EGF), placental growth factor (PlGF), thrombopoietin, transforming growth factor β1 (TGF-β1) and VEGF-D, as compared with the metastatic cell lines. VEGF expression does not significantly differ according to the CRC cellular origin in normoxia. Severe hypoxia induced VEGF expression up-regulation but contrary to expectations, metastatic CRC cell lines did not respond as much as primary cell lines to the hypoxic stimulus. In CRC primary-derived cell lines, we observed a two-fold increase in VEGF expression between normoxia and hypoxia as compared to metastatic cell lines. CRC cell lines express a similar pattern of VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅ and VEGF₁₈₉) despite variability in VEGF expression, where the major transcript was VEGF₁₂₁. No relevant expression of VEGFR-2 was found in CRC cell lines, as compared to that of human umbilical vein endothelial cells and sVEGFR-1 expression did not depend on the CRC cellular origin. A distinct angiogenesis-related expression pattern characterizes metastatic CRC cell lines. Factors other than VEGF appear as prognostic markers and intervention targets in the metastatic CRC setting.

Effect of exercise on HIF-1 and VEGF signaling

This review summarizes the literature describing the significance of various conditions, such as hypoxia, oxidative stress, and, above all, physical exercise, in the hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) signaling pathway mainly in skeletal muscle. HIF-1α acts as a master regulator for the expression of genes involved in the hypoxia response of most mammalian cells. Namely, HIF-1α initiates transcription of various hypoxia-adaptive genes, such as angiogenesis, glycolysis, and erythropoiesis, after the formation of heterodimer with HIF-1β. Among them, VEGF is the most potent endothelial specific mitogen, which recruits endothelial cells into hypoxic foci and avascular area and stimulates their proliferation. The study on acute exercise shows that several components of the HIF-1 pathway, involving VEGF and erythropoietin, are activated in response to acute changes in oxygen demand in human skeletal muscle, suggesting that oxygen sensitive pathways could be relevant for adaptaion to physical activity by increasing capillary growth. Also, the effects of endurance training on the activity of the HIF pathway in human skeletal muscle under hypoxic conditions appear to be definitely higher than those under normoxic conditions, indicating that combining hypoxia with exercise training appears to improve some aspects of muscle O2 transport and/or metabolism. On the other hand, increased levels of reactive oxygen species (ROS) due to physical exercise induce the expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which regulates mitochondrial biogenesis in multiple cell types, resulting in increases in VEGF expression and subsequent angiogenesis, strongly suggesting HIF-1α-independent regulation of VEGF and angiogenesis. Thus, the precise relationship among exercise, the HIF-1 pathway including VEGF, PGC-1α, and ROS needs further study.

Chronic Deficiency of Nitric Oxide Affects Hypoxia Inducible Factor-1α (HIF-1α) Stability and Migration in Human Endothelial Cells

BackgroundEndothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated.ResultsEndothelial nitric oxide synthase (eNOS) was chronically inhibited either by N G-Nitro-l-arginine methyl ester (l-NAME) treatment or its expression was down-regulated by RNA interference. After long-term l-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1α (HIF-1α). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O2 consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1α accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells.ConclusionBased on our results, we propose that basal release of NO may act as a negative controller of HIF-1α levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.

Psoriasin (S100A7) increases the expression of ROS and VEGF and acts through RAGE to promote endothelial cell proliferation

Psoriasin (S100A7), originally identified in psoriasis, is a calcium-binding protein belonging to the multigenic S100 family. In high-grade ductal carcinoma in situ, psoriasin was identified as one of the most abundant transcripts. We have previously shown that psoriasin was induced by reactive oxygen species (ROS). Moreover, the downregulation of psoriasin by short hairpin RNA (shRNA) led to the reduced expression of vascular endothelial growth factor (VEGF) and inhibited tumor growth in vivo. The aim of the present study was to investigate whether psoriasin could have direct effects on endothelial cells. In this study we demonstrated that psoriasin increased VEGF expression in mammary epithelial cells. The treatment of endothelial cells with recombinant psoriasin increased proliferation comparable to that of recombinant VEGF protein. No change in proliferation was seen when endothelial cells were infected with psoriasin-expressing adenoviruses, suggesting that the proliferative effect of psoriasin was mediated by a specific receptor. Treatment with sRAGE, targeting the receptor for advanced glycation end products (RAGE), thus inhibited endothelial cell proliferation and tube formation enhanced by recombinant psoriasin. We showed that VEGF expression was not induced by hydrogen peroxide, when psoriasin was silenced by shRNA, which led to the hypothesis that psoriasin induces ROS. Indeed, psoriasin was shown to induce ROS in both endothelial and epithelial cells. Moreover, sRAGE inhibited the psoriasin-dependent generation of ROS in endothelial cells. Finally, treatment with antioxidant Bcl-2 protein abolished the effect of psoriasin on endothelial cell proliferation. Our data suggest that psoriasin expression in mammary epithelial cells leads to increased endothelial cell proliferation in a paracrine manner through RAGE. Psoriasin may therefore play a role in breast cancer progression by promoting oxidative stress response and angiogenesis.

Paradoxical effects of polyphenolic compounds from Clusiaceae on angiogenesis

Clusiaceae plants display high contents of xanthones and coumarins, the effects of which on endothelium, more particularly on angiogenesis, have not been assessed yet. We screened the capacity of six molecules from Clusiaceae – belonging to xanthones, coumarins and acid chromanes classes – to induce endothelium-dependent relaxation on mice aortic rings. Endothelial nitric oxide (NO) production was assessed in endothelial cell line using electron paramagnetic resonance technique. Then, the capacity of these molecules to induce capillary-like structures of endothelial cells was assessed. Cellular processes implicated in angiogenesis (adhesion, migration and proliferation) and Western blot analyses were then investigated. Among the tested molecules, isocalolongic acid (IA) and 2-deprenylrheediaxanthone (DRX) induced an endothelium-dependent relaxation of the aorta associated with an increase of NO production in endothelial cells. Using in vitro and ex vivo angiogenesis assays, it was shown that IA treatment promoted the formation of capillary-like network. In contrast, DRX prevented the ability of vascular endothelial growth factor (VEGF) to increase the formation of capillary-like network. IA increased endothelial cell proliferation while DRX decreased all cellular processes of angiogenesis. Western blot analysis showed that IA increased VEGF expression whereas DRX decreased ICAM-1 expression. Altogether, these data allowed identifying isolated molecules from Clusiaceae that exhibit a potential activity towards the modulation of endothelium-dependent relaxation involving NO release. Interestingly, they also highlighted paradoxical effects of the two compounds on cellular angiogenic processes, IA being pro-angiogenic and DRX anti-angiogenic.

The biphasic nature of hypoxia‐induced directional migration of activated human hepatic stellate cells

Liver fibrogenesis is sustained by pro-fibrogenic myofibroblast-like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)fibroblasts, and is favoured by hypoxia-dependent angiogenesis. Human HSC/MFs were reported to express vascular-endothelial growth factor (VEGF) and VEGF-receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia-induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non-oriented and oriented migration were evaluated by using wound healing assay and the modified Boyden's chamber assay, respectively. The data indicate that hypoxia-induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (15 min) and mitochondria-related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF-1α-dependent increase in VEGF expression (facilitated by ROS) and its progressive, time-dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV-related fibrotic/cirrhotic livers revealed HIF-2α and haem-oxygenase-1 positivity in hepatocytes and α-SMA-positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia-induced migration of HSC/MFs involves an early, mitochondrial-dependent ROS-mediated activation of ERK and JNK, followed by a delayed- and HIF-1α-dependent up-regulation and release of VEGF.

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.

Demineralised human dentine matrix stimulates the expression of VEGF and accelerates the bone repair in tooth sockets of rats

ObjectiveIn this study we investigated the possible use of human demineralised dentine matrix (DHDM), obtained from the extracted teeth, as bone graft material and evaluated the expression of vascular endothelial growth factor (VEGF) induced by this material in the healing process of tooth sockets of rats. DesignTo evaluate bone regeneration and expression of VEGF induced by DHDM, thirty-two male Wistar rats weighing approximately 200g were used. After maxillary second molar extraction, the left sockets were filled with DHDM and the right sockets were naturally filled by blood clot (control). The animals were sacrificed at 3, 7, 14 and 21 days after surgery and upper maxillaries were processed for histological, morphometric and immunohistochemical analyses. DHDM was used to evaluate the mechanical effect of bone graft material into sockets. Expression of VEGF was determined by immunohistochemistry in all groups. ResultsOur results demonstrated a significant increase in the newly formed bone tissue in sockets of 7, 14 and 21 days and a significant increase in VEGF expression at days 7 and 14 on treated sockets. ConclusionsOur results showed that DHDM increases the expression of VEGF and accelerates the healing process in rats tooth sockets, by stimulating bone deposition and also vessels formation. These results suggest that DHDM has osteoinductive/osteoconductive potential and may represent an efficient grafting material on guided bone regeneration.