Vascular Endothelial Growth Factor Synthesis Research Articles

Levels of Vascular Endothelial Growth Factor during First Six Months of Peritoneal Dialysis.

Chronic peritoneal dialysis (PD) up-regulates vascular endothelial growth factor (VEGF) synthesis and VEGF is found in drained dialysate (dd). Aims of this prospective study were to evaluate serum (s) and ddVEGF concentration during the first six months of PD, relationships between these concentrations and demographic and biochemical parameters, presence of diabetes, peritonitis, and the use of medications. The study included 20 patients, with the mean age of 62.9±12.69, 11 of whom were affected by diabetes mellitus. Fasting venous blood samples were taken at the beginning and after six months of PD, in tri-potassium ethylenediaminetetraacetic acid (K3EDTA) vacutainer. After six months of PD, sVEGF concentrations increased significantly, without significant change in ddVEGF. Concentrations of sVEGF at the beginning of chronic PD treatment directly significantly correlated with serum fibrinogen, and after six months with fibrinogen and glycemia. In patients receiving erythropoiesis-stimulating agent (ESA), levels of sVEGF and ddVEGF were lower at baseline, while after six months of PD ddVEGF increased. in patients not receiving ESA, sVEGF increased more prominently, while ddVEGF decreased.The changes were not statistically significant. Patients receiving angiotensin-converting-enzyme inhibitor (ACEi) had sVEGF and ddVEGF levels insignificantly lower than those not using ACEi, however sVEGF significantly increased during six months of PD. After six months of PD, ddVEGF was significantly higher compared to those not using ACEi. Treatment with statins did not significantly influence levels of sVEGF and ddVEGF during the follow-up. Concentrations of sVEGF were continually lower than those of ddVEGF and increased more, while concentrations of ddVEGF were higher in patients using statins. Serum and drained dialysate concentrations of VEGF in PD patients were connected with poorer metabolic profile, while the role of inflammation and treatment agents should be studied further.

Key enzymes of degradation and angiogenesis as factors of tumor progression for squamous-cell cervical carcinoma

Destruction of the connective tissue matrix (CTM) and angiogenesis are the two processes playing a key role in tumor progression. Matrix metalloproteinases (MMPs) play a leading role in processes of tissue destruction. Tissue collagenases MMP-1 and MT1-MMP hydrolyze fibrillar collagens constituting the base of CTM and enable tumor invasion. Gelatinases A and B (MMP-2 and MMP-9) hydrolyze type IV collagen which is the main component of basal membranes and contribute to the development of metastases. Endogenous activators and inhibitors are involved in the regulation of expression and activity of these enzymes. MMP-9 was shown to release vascular endothelial growth factor (VEGF), the principal inductor of angiogenesis, bound to CTM. Angiotensin-converting enzyme (ACE) is involved in the induction of VEGF synthesis and stimulation of endothelial cell proliferation mediated by angiotensin II (AII) and its type 1 receptor (AT1R). Experiments reported in the present article addressed the distinctive features of expression of key degradation and angiogenesis enzymes in squamous cell carcinoma (SCC) of the cervix. MMP-1, MT1-MMP, MMP-2, and MMP-9 and their endogenous regulators TIMP-1 and TIMP-2, as well as ACE, were the objects of research. Experiments were performed with clinical specimens including tumor tissue samples, for which presence or absence of metastasis to regional lymph nodes was taken into account, and morphologically normal tissue samples. Increased expression of MMP-1, MT1-MMP, and MMP-9 and decreased expression of TIMP-1 and TIMP-2 were shown to make the principal contribution to the destructive (invasive) potential of cervical carcinomas; the effect of changes in MMP-2 expression was less pronounced. Dramatically increased expression of MMP-1 and MMP-9 was evident in metastasizing tumors. ACE activity in tumor samples was generally higher than activity in normal tissue. Substantial expression of MMP-1, MMP-2, MMP-9, and ACE was detected in morphologically normal tissue adjacent to the tumor; this can contribute to increased destructive potential of a tumor. The data reported are important for understanding the mechanisms of tumor progression, have prognostic value, and may affect the choice of individual therapeutic strategy for the patients.

Human ciliary epithelium as a source of synthesis and secretion of vascular endothelial growth factor in neovascular glaucoma.

Retinal ischemia-induced upregulation of vascular endothelial growth factor (VEGF) leads to endothelial proliferation of the anterior segment, resulting in neovascular glaucoma. To investigate the ciliary epithelium as a possible source of VEGF in human eyes enucleated for intractable neovascular glaucoma. In this proof-of-concept, laboratory-based study, 16 human enucleated eyes (8 with neovascular glaucoma and 8 as controls) were investigated. Presence of VEGF by immunohistochemical analysis (VEGF protein) and in situ hybridization (VEGF messenger RNA). In eyes with neovascular glaucoma, strong VEGF immunoreaction in the nonpigmented epithelial cells of the ciliary processes and in the retina was noted. In situ hybridization for VEGF messenger RNA revealed a similar pattern, with positive stain results only in eyes with neovascular glaucoma. A minimal amount of VEGF immunostaining was seen in control eyes. The nonpigmented ciliary epithelium is an important site of VEGF synthesis in patients with neovascular glaucoma. The ciliary epithelium may represent an additional focus of treatment in the management of neovascular glaucoma, especially in eyes that are nonresponsive to panretinal photocoagulation.

Angiotensin type 1 receptor blockage reduces l-dopa-induced dyskinesia in the 6-OHDA model of Parkinson's disease. Involvement of vascular endothelial growth factor and interleukin-1β

Non-neuronal factors such as angiogenesis and neuroinflammation may play a role in l-dopa induced dyskinesias (LID). Vascular endothelial growth factor (VEGF) and proinflammatory cytokines such as interleukin-1β (IL-1β) have been found to be involved in LID. The renin–angiotensin system (RAS) is involved in the inflammatory response and VEGF synthesis via type 1 (AT1) receptors. However, it is not known whether the RAS plays a role in LID and whether AT1 antagonists could constitute a useful therapy against LID. In this study, we investigated whether manipulation of brain RAS is effective in preventing LID. Blocking AT1 receptors with candesartan significantly reduces LID in the 6-OHDA rat model. Chronic dopaminergic denervation induces an increase in striatal levels of VEGF and IL-1β. Dyskinetic animals showed significantly higher levels of VEGF and IL-1β in the lateral striatum and the substantia nigra, as revealed by western blot and real time-PCR analyses. Interestingly, animals treated with both candesartan and l-dopa displayed significantly lower levels of VEGF, IL-1β and dyskinesia than those treated with l-dopa alone. The stimulatory effect of angiotensin II (AII) on VEGF expression was confirmed by the addition of AII to primary mesencephalic cultures and intraventricular administration of AII in rats. The results of the present study reveal for the first time that blockage of AT-1 receptors reduces LID. A candesartan-induced decrease in VEGF and IL-1β may be responsible for the beneficial effects, suggesting the brain RAS as a new target for LID treatment in PD patients.

Antitumor Effect of Angiotensin II Type 1 Receptor Blocker Losartan for Orthotopic Rat Pancreatic Adenocarcinoma

The aim of this study was to investigate the synergistic inhibitory effects of gemcitabine and losartan, angiotensin II type 1 (AT1) receptor blockers, on an orthotopic rat pancreatic cancer model. The rat orthotopic pancreatic cancer model was prepared using DSL-6A/C cells, a rat ductal pancreatic adenocarcinoma cell line. The rats were treated with gemcitabine alone (100 mg/kg per week), losartan alone (100 mg/kg per day), or gemcitabine plus losartan. Survival was significantly improved by treatment with gemcitabine (89.6 ± 21.8 days) or losartan (76.9 ± 18.7 days) alone compared with that in the control group (59.6 ± 13.4 days; P < 0.05). Treatment with gemcitabine plus losartan further prolonged the survival time to 102.6 ± 16.5 days compared with that in the control group (P < 0.0001). Gemcitabine or losartan significantly and dose-dependently reduced the proliferation of DSL-6A/C cells in vitro. Both drugs inhibited pancreatic vascular endothelial growth factor expression compared with that in the control group (P < 0.05). The results of this study indicate that combined treatment with gemcitabine and losartan significantly improved the survival of rats with orthotopic pancreatic cancer by inhibiting vascular endothelial growth factor synthesis and suppressing cancer cell proliferation via AT1 receptor blockade. Thus, an AT1 receptor blocker in combination with gemcitabine might improve the clinical outcomes of patients with advanced pancreatic cancer.

Disruption of negative feedback loop between vasohibin-1 and vascular endothelial growth factor decreases portal pressure, angiogenesis, and fibrosis in cirrhotic rats

Pathological angiogenesis represents a critical hallmark for chronic liver diseases. Understanding the mechanisms regulating angiogenesis is essential to develop new therapeutic strategies that specifically target pathological angiogenesis without affecting physiological angiogenesis. Here we investigated the contribution and therapeutic impact of the endogenous angioinhibitor vasohibin-1 in portal hypertension and cirrhosis. The spatiotemporal expression profiling of vasohibin-1 and its relationship with vascular endothelial growth factor (VEGF), angiogenesis, and fibrogenesis was determined through the analysis of human cirrhotic liver specimens, widely accepted in vivo animal models of portal hypertension and cirrhosis, and in vitro angiogenesis assays. Effects of vasohibin-1 overexpression by adenoviral-mediated gene transfer on angiogenesis, fibrogenesis, and portal hypertension-associated hemodynamic alterations were also studied in rats. We found that vasohibin-1 and VEGF are up-regulated, in mesentery and liver, in cirrhotic and precirrhotic portal hypertensive rats and cirrhosis patients. Our results are consistent with vasohibin-1/VEGF cascades being spatially and temporally coordinated through a negative-feedback loop driving pathological angiogenesis. Paradoxically, further overexpression of vasohibin-1 by adenoviral gene transfer exerts multifold beneficial effects in portal hypertension and cirrhosis: reduction of pathologic angiogenesis, attenuation of liver fibrogenesis partly mediated through inhibition of hepatic stellate cell activation, and significant decreases in portocollateralization, splanchnic blood flow, portohepatic resistance, and portal pressure. The explanation for this apparent contradiction is that, unlike endogenous vasohibin-1, the ectopic overexpression is not regulated by VEGF and therefore disrupts the negative-feedback loop, thus generating constant, but lower levels of VEGF synthesis sufficient to maintain vascular homeostasis but not pathological angiogenesis. Our study provides evidence that vasohibin-1 regulates portal hypertension-associated pathological angiogenesis and highlights that increasing vasohibin-1 might be a promising novel therapeutic strategy for portal hypertension and cirrhosis.

Antimuscle atrophy effect of nicotine targets muscle satellite cells partly through an α7 nicotinic receptor in a murine hindlimb ischemia model

We have recently identified that donepezil, an anti-Alzheimer drug, accelerates angiogenesis in a murine hindlimb ischemia (HLI) model. However, the precise mechanisms are yet to be fully elucidated, particularly whether the effects are derived from endothelial cells alone or from other nonvascular cells. Further investigation of the HLI model revealed that nicotine accelerated angiogenesis by activation of vascular endothelial cell growth factor (VEGF) synthesis through nicotinic receptors in myogenic cells, that is, satellite cells, in vivo and upregulated the expression of angiogenic factors, for example, VEGF and fibroblast growth factor 2, in vitro. As a result, nicotine prevented skeletal muscle from ischemia-induced muscle atrophy and upregulated myosin heavy chain expression in vitro. The in vivo anti-atrophy effect of nicotine on muscle was also observed in galantamine, another anti-Alzheimer drug, playing as an allosteric potentiating ligand. Such effects of nicotine were attenuated in α7 nicotinic receptor knockout mice. In contrast, PNU282987, an α7 nicotinic receptor agonist, comparably salvaged skeletal muscle, which was affected by HLI. These results suggest that cholinergic signals also target myogenic cells and have inhibiting roles in muscle loss by ischemia-induced muscle atrophy.

Pancreatic duct cells as a source of VEGF in mice

Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking. We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo. Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA. Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.

In vivo monitoring of angiogenesis during tendon repair: a novel MRI-based technique in a rat patellar tendon model

Recent in vivo studies were able to show the impairing effect of neoangiogenesis in degenerative tendon diseases. Clinical in vivo monitoring of angiogenesis in injured tendons therefore seems to be crucial for an accurate therapeutic approach. The aim of this study was to develop a novel magnetic resonance imaging (MRI)-based technique for observing angiogenesis during tendon healing in vivo. Tendinopathy was induced by an in situ freezing model of rat patellar tendon and monitored after 7, 14, and 28days. Animals were randomly divided into an imaging and immunohistochemical group. MRI with a 'blood pool' contrast agent was used to determine neoangiogenesis during tendon healing. MRI was compared to histochemical staining and quantification of blood vessels in injured and native tendons. MRI data revealed a peak in changes in the transverse relaxation rate (ΔR 2*), which is proportional to relative blood volume, 7days after surgery and decrease until day 28. Histological microvessel density and vascular endothelial growth factor synthesis were also most evident at day 7 and decreased over time. The current results are demonstrating a time-dependent correlation between microvessel density and ΔR 2*. Thus, MRI-based evaluation of angiogenesis in the tendon might be a new promising technique for in vivo monitoring of angiogenesis and therapy response in the future.

Resveratrol inhibits BMP-4-stimulated VEGF synthesis in osteoblasts: Suppression of S6 kinase

Resveratrol is well known as a natural polyphenol abundantly found in red wine. We previously reported that bone morphogenetic protein-4 (BMP-4) stimulates vascular endothelial growth factor (VEGF) synthesis via p70 S6 kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of resveratrol on the BMP-4-stimulated VEGF synthesis in MC3T3-E1 cells. Resveratrol significantly suppressed BMP-4-stimulated release and expression levels of VEGF mRNA. SRT1720, an activator of SIRT1 with potencies greater than resveratrol, also reduced VEGF release and the mRNA levels. Both resveratrol and SRT1720 markedly attenuated the BMP-4-induced phosphorylation of p70 S6 kinase without affecting the BMP-4-induced phosphorylation of Smad1/5/8. These findings strongly suggest that resveratrol attenuates BMP-4-stimulated VEGF synthesis through suppression of the activation of p70 S6 kinase in osteoblasts, and that the inhibitory effect is mediated at least in part by SIRT1 activation.

Activation of the CXCL12/CXCR4 signaling axis may drive vascularization of the ovine placenta

Early pregnancy, when most embryonic losses occur, is a critical period in which vital placental vascularization is established. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis, and factors that regulate VEGF function, expression, or both may ultimately affect vascularization. Activation of the C-X-C chemokine receptor type 4 (CXCR4) by its cognate ligand, C-X-C chemokine ligand 12 (CXCL12), increases VEGF synthesis and secretion, which in turn stimulates CXCL12 and CXCR4 production and this synergistic regulation may influence placental vascularization. We hypothesized that expression of CXCL12, CXCR4, select angiogenic factors, and their receptors would increase in placental tissues during early pregnancy and that treatment of ovine trophectoderm cells with CXCL12 would increase production of angiogenic factors. To test this hypothesis, maternal caruncle (CAR) and fetal extraembryonic membrane (FM) tissues were collected on days 18, 20, 22, 25, 26, and 30 of pregnancy and on day 10 of the estrous cycle (control, NP) to determine relative mRNA or protein expression of CXCL12 and CXCR4 and selected angiogenic factors. In CAR, expression of mRNA for CXCR4 increased on day 18, 20, 22, and 25 and CXCL12 increased on day 18 and 20 compared with NP ewes. CXCL12 protein followed a similar pattern in CAR tissue, with greater levels on day 20 than in NP tissue. Greater levels of fibroblast growth factor 2 (FGF2) mRNA was observed in CAR on day 20 of gestation than on day 30. In FM, CXCL12, CXCR4, angiopoietin 1, VEGF, and VEGF receptor 1 were enhanced with advancing pregnancy, whereas FGF2 and kinase insert domain receptor (or VEGF receptor 2) peaked on day 25. An increase in protein levels occurred on day 25 compared with day 20 in FM for CXCL12 and CXCR4, as well as a similar tendency for FGF2 protein. Both CXCL12 and CXCR4 are specifically localized to trophoblast cells and to the uterine luminal and glandular epithelium. Treatment of ovine trophectoderm cells with CXCL12 increased mRNA expression for VEGF and FGF2. The relationship between VEGF, FGF2, and the CXCL12/CXCR4 signaling underscores the potential role for this chemokine axis in driving placentation.

Activation of the AT1R/HIF-1 α /ACE axis mediates angiotensin II-induced VEGF synthesis in mesenchymal stem cells.

A local renin-angiotensin system (RAS) is expressed in mesenchymal stem cells (MSCs) and regulates stem cell function. The local RAS influences the survival and tissue repairing ability of transplanted stem cells. We have previously reported that angiotensin II (Ang II) pretreatment can significantly increase vascular endothelial growth factor (VEGF) synthesis in MSCs through the ERK1/2 and Akt pathways via the Ang II receptor type 1 (AT1R). However, the role of angiotensin-converting enzyme (ACE) has not been clarified. Furthermore, whether Ang II pretreatment activates hypoxia-inducible factor-1α (HIF-1α) in MSCs has not been elucidated. Our data show that both ACE and HIF-1α are involved in promoting VEGF expression in MSCs, and that both are upregulated by Ang II stimulation. The upregulation of ACE appeared after the rapid degradation of exogenous Ang II, and led to the formation of endogenous Ang II. On the other hand, the ACE inhibitor, captopril, attenuated Ang II-enhanced HIF-1α upregulation, while HIF-1α suppression markedly attenuated ACE expression. This interesting finding suggests an interaction between ACE and HIF-1α. We conclude that Ang II pretreatment, as a trigger, activated the AT1R/HIF-1α/ACE axis that then mediated Ang II-induced VEGF synthesis in MSCs.

Vascular Endothelial Growth Factor in Peritoneal Dialysis Patients

Vascular endothelial growth factor (VEGF) is a glycoprotein which exerts mitogenic effects on endo thelial cells, enhances neoangiogenesis and microvascular permeability, influences leukocyte kinetics when upreg ulated by hypoxia and high-glucose concentration in experimental conditions and in human pathology. Peritoneal synthesis of VEGF has been demonstrated in patients on peritoneal dialysis (PD) treated with glucose-based dialy sate solutions. The aim of the study was to determine the serum and peritoneal effluent VEGF concentrations in patients on chronic PD and to assess the relationship between age, gender, comorbidities, dialysis modality and vintage, therapy with erythropoiesis stimulating agents (ESA), angiotensin-converting enzyme inhibitors (ACEi) and statins and VEGF concentrations. Data on the use of ACEi, ESA, and statins were collected from patients' medical histories. VEGF was measured in serum and peritoneal effluent using the quantitative sandwich enzyme immunoassay (ELISA) kits (Quantikine® Human VEGF, R&D Systems, USA & Canada). Complete blood count and standard biochemical analyses (serum glucose, urea, creatinine, total protein, albumin, cholesterol, triglycerides, sodium, potassium, chloride, iron, total iron-binding capacity, ferritin, fibrinogen, C-reactive protein, and intact parathyroid hormone) were performed in fasting venous blood samples. Dialysis and residual components of Kt/V and normalized weekly creatinine clearance were calculated based on 24-hour urine and effluent collections. Peritoneal transport type was determined using the peritoneal equilibration test. Samples from 63 PD patients (39 males and 24 females, average age 61.97 ± 11.01 years) were analyzed. The average serum and effluent VEGF concentrations (231.84 ± 173.91 pg/mL and 38.39 ± 49.38 pg/mL, respectively) correlated significantly (p = 0.002). No significant difference was found in serum and effluent VEGF concentrations in relation to demographic characteristics, comorbidities, dialysis modality, therapy with ESA, ACEi, and statins. Patients treated with PD longer than 5 years had significantly higher serum VEGF levels (p < 0.05). Correlation analysis showed a statistically significant relationship between statin therapy and lower effluent VEGF concentration (p = 0.030). Serum VEGF concentration significantly correlated with fibrinogen serum concentration (p = 0.034) and glycemia (p = 0.004). Effluent VEGF concentration significantly correlated with cholesterolemia (p = 0.004). Serum VEGF concentrations were significantly higher in long term PD patients, and peritoneal effluent VEGF concentrations were significantly lower in patients receiving statins, suggesting a protective effect of those drugs on peritoneal membrane.

New Insights into the Pathogenesis of Preeclampsia - The Role of Nrf2 Activators and their Potential Therapeutic Impact.

Preeclampsia (PE), characterized by proteinuric hypertension and occurring in 2-3 % of all pregnancies, is one of the leading causes of maternal, fetal and neonatal morbidity and mortality. The etiology of PE still remains unclear and current treatments for this devastating disorder are still limited to symptomatic therapies. Placental oxidative stress may be a key intermediate step in the pathogenesis of PE; it has been related to excessive secretion of multiple antiangiogenic factors, mainly soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng). The nuclear factor-erythroid 2-like 2 (Nrf2) pathway is one ofthe most important systems that enhance cellular protection against oxidative stress. Nrf2 serves as a master transcriptional regulator of the basal and inducible expression of a multitude of genes encoding detoxification enzymes and antioxidative proteins. Evidence for a link between Nrf2 and restoring the balance between pro- and antiangiogenic factors mainly through its downstream target protein heme oxygenase-1 (HO-1) has lately been discussed. HO-1 metabolizes heme to biliverdin, iron and carbon monoxide (CO). CO enhances vascular endothelial growth factor (VEGF) synthesis in vascular smooth muscle and promotes its relaxation and hence vasodilatation. In addition, HO-1 has been shown in vitro to inhibit the production of sFlt-1. A recent animal study demonstrated that the induction of HO-1 in a mouse model of PE attenuates the induced hypertension in pregnant mice. This provides compelling evidence for the protective role of Nrf2/HO-1 in pregnancy and identifies this pathway as a target to treat women with PE. We summarize the recent findings on the involvement of Nrf2 in the pathogenesis of PE, and provide an overview of the possible beneficial effects of Nrf2 inducers in PE.

Two New Phenolic Compounds from the Heartwood of Caesalpinia sappan L.

Two new phenolic compounds, epicaesalpin J and 7,10,11-trihydroxydracaenone, were isolated from the heartwood of Caesalpinia sappan L. Their structures were identified by spectroscopic analysis methods, such as 1D and 2D NMR, along with the high resolution mass spectral data. The NO inhibition activities of two new compounds and six known compounds were tested.

Inhibition of rat brain glioma proliferation resulting from recombinant adeno-associated virus with nitricoxide synthaser of antisense gene

Objective To establish the rat brain glioma model and explore the proliferation of brain glioma and the expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) gene influenced by the recombinant adeno-associated virus (rAAV-AsiNOS) in vivo.Methods C6 cells were injected into the right caudate nucleus areas of SD rats by stereotactic technology.After 7 days,rats were divided into 4 groups:control,rAAV-LacZ,rAAV-AsiNOS and blank.The neurological deficit score of all rats was separately acquired before the establishment of rat model,7 days after the establishment of rat model and 7 days after transfection.Seven days after transfection,the weight and volume of brain glioma were calculated by plain and enhanced MR scan.The expression of iNOS and VEGF was detected by using semi-quantified analysis of reverse transcriptase-polymerase chain reaction (RT-PCR).Resuits The neurological deficit score in rAAV-iNOS group was increased,but that in control and rAAVLacZ groups was reduced.MR images showed that brain tumors in control and rAAV-LacZ groups at 7th day after transfection were larger than before transfection,but the brain tumors in rAAV-iNOS group were obviously reduced.RT-PCR showed that mean expression levels of iNOS mRNA in control,rAAV-LacZ and rAAV-iNOS groups were separately 0.67 ±0.04,0.72 ±0.07 and 0.21 ±0.02,and mean expression levels of VEGF mRNA in control,rAAV-LacZ and rAAV-iNOS groups were separately 0.71 ± 0.08,0.76 ± 0.07 and 0.29 ± 0.02.The expression of iNOS and VEGF mRNA in rAAV-iNOS group was lower than that in control and rAAV-LacZ groups (P ＜0.05).Conclusion rAAV-iNOS can inhibit the expression of iNOS,regulate the synthesis of VEGF,reduce glioma angiogenesis and perform the mechanism of anti-glioma in vivo. Key words: Glioma; Inducible nitric oxide synthase; Vascular endothelial growth factor; Adeno-associated virus ; Angiogenesis

Hypoxic stress, brain vascular system, and β-amyloid: A primary cell culture study

This study stresses the hypothesis whether hypoxic events contribute to formation and deposition of β-amyloid (Aβ) in cerebral blood vessels by affecting the processing of endothelial amyloid precursor protein (APP). Therefore, cerebral endothelial cells (ECs) derived from transgenic Tg2576 mouse brain, were subjected to short periods of hypoxic stress, followed by assessment of formation and secretion of APP cleavage products sAPPα, sAPPβ, and Aβ as well as the expression of endothelial APP. Hypoxic stress of EC leads to enhanced secretion of sAPPβ into the culture medium as compared to normoxic controls, which is accompanied by increased APP expression, induction of vascular endothelial growth factor (VEGF) synthesis, nitric oxide production, and differential changes in endothelial p42/44 (ERK1/2) expression. The hypoxia-mediated up-regulation of p42/44 at a particular time of incubation was accompanied by a corresponding down-regulation of the phosphorylated form of p42/44. To reveal any role of hypoxia-induced VEGF in endothelial APP processing, ECs were exposed by VEGF. VEGF hardly affected the amount of sAPPβ and Aβ(1–40) secreted into the culture medium, whereas the suppression of the VEGF receptor action by SU-5416 resulted in decreased release of sAPPβ and Aβ(1–40) in comparison to control incubations, suggesting a role of VEGF in controlling the activity of γ-secretase, presumably via the VEGF receptor-associated tyrosine kinase. The data suggest that hypoxic stress represents a mayor risk factor in causing Aβ deposition in the brain vascular system by favoring the amyloidogenic route of endothelial APP processing. The hypoxic-stress-induced changes in β-secretase activity are presumably mediated by altering the phosphorylation status of p42/44, whereas the stress-induced up-regulation of VEGF appears to play a counteracting role by maintaining the balance of physiological APP processing.

Nitric Oxide Donors Augment Interleukin-1β-Induced Vascular Endothelial Growth Factor in Airway Smooth Muscle Cells

Angiogenesis and microvascular leakage are features of chronic inflammatory diseases of which molecular mechanisms are poorly understood. We investigated the effects of interleukin-1β (IL-1β) on the expression and secretion of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in porcine airway smooth muscle cells (PASMC) in relation to a nitric oxide (NO) pathway. Serum-deprived (48h) PASMC were stimulated with IL-1β alone or with NO donor, L-arginine and/or NO synthase inhibitor L-NAME for 4 and 24h. IL-1β did not affect PlGF release, but augmented VEGF release (2.4-fold) after 24h. VEGF release was inhibited by L-NAME (531.8±52pg/ml), but restored and further elevated by L-arginine (1,529±287pg/ml). IL-1β up-regulated VEGF mRNA (1.8-fold) and this response was attenuated by L-NAME (1.1-fold) and augmented by L-arginine (3.8-fold) at 4h. Restoration of a NO pathway by L-arginine in L-NAME-treated cells resulted in elevated VEGF mRNA levels (2.2-fold). [(3)H]Thymidine incorporation assay revealed enhanced porcine pulmonary artery endothelial cell proliferation in response to IL-1β, VEGF and PlGF, and this mitogenic effect was not influenced via the NO pathway. Our results suggest that a NO pathway modulates VEGF synthesis during inflammation contributing to bronchial angiogenesis and vascular leakage.

Genetically modified human myoblasts with eNOS may improve regenerative ability of myogenic stem cells to infarcted heart

Modern therapies of post infarcted heart failure are focused on perfusion improvement of the injured myocardium. This effect can be achieved by, among other means, implanting stem cells which could be genetically modified with factors inducing the formation of new blood vessels in the post infarction scar area. Combined stem cell and gene therapy seems to be a promising strategy to heal an impaired myocardium. The creation of new blood vessels can be indirectly stimulated via factors inducing vascular endothelial growth factor synthesis, for example endothelial nitric oxide synthase (eNOS). The product of this enzyme, nitric oxide, is a molecule that can influence numerous physiological activities; it can contribute to vasodilation, stimulation of endothelial cell growth, prevention of platelet aggregation and leukocyte adhesion to the endothelium. To verify the pro-angiogenic and regenerative potential of human primary myoblasts and murine myoblast cell line C2C12 transiently transfected with eNOS gene. Stem cells (either human or murine) were maintained in standard in vitro conditions. Next, both types of myoblasts were modified using electroporation and lipofection (human and murine cells), respectively. The efficacy of the transfection method was evaluated using flow cytometry. The concentration of eNOS protein was measured by ELISA immunoassay. The biological properties of modified cells were assessed using an MTT proliferation test and DAPI cell cycle analysis. To verify the influence of oxidative stress on myoblasts, cytometric tests using Annexin V and propidium iodide were applied. To check possible alterations in myogenic gene expression of stem cells transduced by genetic modification, the myogenic regulatory factors were evaluated by real-time PCR. The function of genetic modification was confirmed by a HUVEC capillary sprouting test using myoblasts supernatants. Electroporation turned out to be an efficient transfection method. High amounts of secreted protein were obtained (in the range 2,000 pg/mL) in both cell types studied. Moreover, the functionality of gene overexpression product was confirmed in capillary development assay. Human myoblasts did not exhibit any changes in cell cycle; however, eNOS transfected murine myoblasts revealed a statistically significant reduction in cell cycle ratio compared to controls (p < 0.001). In the case of myogenic gene expression, a decrease in Myogenin level was only detected in the human transfected myoblast population (p < 0.05). The results of our study may suggest that transplantation of myoblasts overexpressing eNOS could be promising for cell therapy in regenerating the post infarction heart.

Oleic Acid Increases Synthesis and Secretion of VEGF in Rat Vascular Smooth Muscle Cells: Role of Oxidative Stress and Impairment in Obesity

Obesity is characterized by poor collateral vessel formation, a process involving vascular endothelial growth factor (VEGF) action on vascular smooth muscle cells (VSMC). Free fatty acids are involved in the pathogenesis of obesity vascular complications, and we have aimed to clarify whether oleic acid (OA) enhances VEGF synthesis/secretion in VSMC, and whether this effect is impaired in obesity. In cultured aortic VSMC from lean and obese Zucker rats (LZR and OZR, respectively) we measured the influence of OA on VEGF-A synthesis/secretion, signaling molecules and reactive oxygen species (ROS). In VSMC from LZR we found the following: (a) OA increases VEGF-A synthesis/secretion by a mechanism blunted by inhibitors of Akt, mTOR, ERK-1/2, PKC-beta, NADPH-oxidase and mitochondrial electron transport chain complex; (b) OA activates the above mentioned signaling pathways and increases ROS; (c) OA-induced activation of PKC-beta enhances oxidative stress, which activates signaling pathways responsible for the increased VEGF synthesis/secretion. In VSMC from OZR, which present enhanced baseline oxidative stress, the above mentioned actions of OA on VEGF-A, signaling pathways and ROS are impaired: this impairment is reproduced in VSMC from LZR by incubation with hydrogen peroxide. Thus, in OZR chronically elevated oxidative stress causes a resistance to the action on VEGF that OA exerts in LZR by increasing ROS.