Vascular Endothelial Growth Factor Protein Concentration Research Articles

Osteopontin-induced vascular hyperpermeability through tight junction disruption in diabetic retina

Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.

Microglia increase tight-junction permeability in coordination with Müller cells under hypoxic condition in an in vitro model of inner blood-retinal barrier

Microglia and Müller cells (MCs) are believed to be critically involved in hypoxia-induced blood-retinal barrier (BRB) disruption, which is a major pathogenic factor of various retinopathies. However, the underlying mechanism remains poorly defined. The inner BRB (iBRB) is primarily formed of microvascular endothelial cells (ECs) with tight junction (TJ), which are surrounded and supported by retinal glial cells. We developed a novel in vitro iBRB model sheet by sandwiching Transwell membrane with layered mouse brain microvascular ECs (bEnd.3) and mouse retinal MCs (QMMuC-1) on each side of the membrane. Using this model, we tested the hypothesis that under hypoxic condition, activated microglia produce inflammatory cytokines such as interleukin (IL)-1β, which may promote vascular endothelial growth factor (VEGF) production from MCs, leading to TJ disruption. The iBRB model cell sheets were exposed to 1% oxygen for 6 h with or without mouse brain microglia (BV2) or IL-1β. TJ structure and function were examined by zonula occludens (ZO)-1 immunostaining and fluorescein isothiocyanate permeability assay, respectively. Relative gene expression of IL-1β in BV2 under normoxic and hypoxic conditions was examined by real-time reverse transcription-polymerase chain reaction. VEGF protein concentration in QMMuC-1 supernatants was measured by enzyme-linked immunosorbent assay. The bEnd.3 cell sheet incubated with BV2 in hypoxic condition or with IL-1β in normoxic condition showed abnormal localization of ZO-1 and aberrated barrier function. Under normoxic condition, EC-MC iBRB model cell sheet showed lower permeability than bEnd.3 cell sheet. Under hypoxic conditions, the barrier function of EC-MC iBRB model cell sheet was more deteriorated compared to bEnd.3 cell sheet. Under hypoxic condition, incubation of EC-MC iBRB model cell sheet with BV2 cells or IL-1β significantly increased barrier permeability, and hypoxia-treated BV2 cells expressed significantly higher levels of IL-1β mRNA. Incubation of QMMuC-1 with IL-1β increased VEGF production. These results suggest that under hypoxic condition, microglia are activated to release proinflammatory cytokines such as IL-1β that promote VEGF production from MCs, leading to disruption of iBRB function. Modulating microglia and MCs function may be a novel approach to treat hypoxia-induced retinal BRB dysfunction.

Effects of ultrasound on vascular endothelial growth factor in cartilage, synovial fluid, and synovium in rabbit knee osteoarthritis.

Ultrasound is commonly used to treat knee osteoarthritis (KOA), which has unique advantages with regard to relieving pain and inflammation as well as delaying cartilage degeneration, but the underlying mechanisms are less clear. The study aimed to investigate the therapeutic effects of ultrasound on vascular endothelial growth factor (VEGF) expression in cartilage, the synovium, and synovial fluid (SF) in a rabbit model of KOA. Twenty-four New Zealand rabbits were randomly divided into ultrasound (group A), sham ultrasound (group B) and no-ACLT control groups (group C). Six weeks after undergoing anterior cruciate ligament transection (ACLT), group A was treated with ultrasound and group B was treated with sham ultrasound. Two weeks thereafter, the morphology of the synovium and cartilage were observed. Cartilage and synovium were scored using the Mankin scale and Krenn V scores, respectively. VEGF expression in the cartilage, SF, and synovium of ACLT knee joints was analyzed via immunohistochemistry, western blotting, and RT-PCR. Cartilage degeneration and synovitis were the most severe in group B and the least severe in group C. Similarly, Mankin scores and Krenn V scores were highest in group B and lowest in group C (p<0.05). There were also significant differences in the VEGF IOD of cartilage or synovium, VEGF protein content in SF, and VEGF mRNA expression in cartilage or SF (p<0.05). Ultrasound can relieve synovitis and delay cartilage degradation, and the mechanisms of ultrasound for the treatment of KOA may involve inhibition of the expression of VEGF in the synovium, SF, and cartilage.

Effects of continuous positive airway pressure on exhaled transforming growth factor-β and vascular endothelial growth factor in patients with obstructive sleep apnea.

BackgroundBoth transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF) are master regulators of airway remodeling; however, their pathological roles in obstructive sleep apnea (OSA) remain unclear. The aim of the present study was to evaluate the expression of TGF-β and VEGF protein in the serum and exhaled breath condensate (EBC) before and after continuous positive airway pressure (CPAP) treatment in OSA patients.MethodsForty patients with moderate to severe OSA requiring CPAP and 20 healthy subjects were prospectively recruited. The concentrations of TGF-β and VEGF protein in the serum and EBC were evaluated by enzyme-linked immunosorbent assay. All OSA patients underwent a sleep study that was repeated 3 months after receiving CPAP therapy.ResultsProtein concentrations of TGF-β and VEGF in the serum did not differ between healthy controls and OSA patients before CPAP treatment. There was also no difference in the serum protein concentrations of TGF-β and VEGF of the OSA patients before and after CPAP treatment. However, both the TGF-β and VEGF protein concentrations in the EBC were higher in the OSA patients than those in control subjects, and recovered to normal levels after CPAP.ConclusionsSuccessful treatment of OSA by CPAP can restore the TGF-β and VEGF protein concentrations in the EBC.

Bystander effects of ionizing radiation: conditioned media from X-ray irradiated MCF-7 cells increases the angiogenic ability of endothelial cells

BackgroundNon-targeting effects of radiotherapy have become as clinical concern due to secondary tumorigenesis in the patients receiving radiotherapy. Radiotherapy also affects non-tumoral cells present in the tumor microenvironment and surrounding tissues. As such, the irradiated cells are thought to communicate the signals that promote secondary tumorigenesis by affecting the function and fate of non-irradiated cells in the vicinity including endothelial cells. This may include up-regulation of genes in irradiated cells, secretion of paracrine factors and induction of gene expression in surrounding non-irradiated cells, which favor cell survival and secondary tumorigenesis. In the current study, we aimed to investigate whether the conditioned media from X-ray irradiated MCF-7 cells contribute to induction of gene expression in human umbilical vein endothelial cells (HUVECs) in vitro and modulate their angiogenic capability and migration.MethodsFollowing the co-culturing of X-ray irradiated MCF-7 media with HUVECs, the migration and wound healing rate of HUVECs was monitored using Transwell plate and scratch wound healing assay, respectively. The levels of angiogenic protein i.e. vascular endothelial growth factor (VEGF-A) in the conditioned media of MCF-7 cells was measured using ELISA. Additionally, we quantified mRNA levels of VEGFR-2, HSP-70, Ang-2, and Ang-1 genes in HUVECs by real time-PCR. Tubulogenesis capacity of endothelial cells was measured by growth factor reduced Matrigel matrix, whereas expression of CD34 (a marker of angiogenic tip cells) was detected by flow cytometry.ResultsData showed that VEGF-A protein content of conditioned media of irradiated MCF-7 cells was increased (P < 0.05) with increase in dose. Data showed that irradiated conditioned media from MCF-7 cells, when incubated with HUVECs, significantly enhanced the cell migration and wound healing rate of HUVECs in a dose-dependent manner (P < 0.05). The mRNA levels of VEGFR-2, HSP-70, Ang-2, and Ang-1 were dose-dependently enhanced in HUVECs incubated with irradiated conditioned media (P < 0.05). Importantly, HUVECs treated with irradiated conditioned media showed a marked increase in the tube formation capability as well as in expression of CD34 marker (P < 0.05).ConclusionsOur findings indicate that conditioned media from irradiated MCF-7 cells induce angiogenic responses in endothelial cells in vitro, which could be due to transfer of overexpressed VEGF-A and possibly other factors secreted from irradiated MCF-7 cells to endothelial cells, and induction of intrinsic genes (VEGFR-2, HSP-70, Ang-2, and Ang-1) in endothelial cells.2jWTXz4D4cK47Ck7iekukBVideo abstract.Graphical abstract

Mandibular lateral deviation induces alteration in vascular endothelial growth factor expression and oxidative stress/nitric oxide generation in rat condyle, synovial membrane and masseter muscle

ObjectiveWe aimed to investigate alteration in cellular signaling mediated by vascular endothelial growth factor (VEGF) and parameters of oxidative stress/nitric oxide generation, superoxide dismutase (SOD) and neuronal nitric oxide synthase (nNOS), underlying altered functional mechanical loading of TMJ (temporomandibular joint) during lateral mandibular deviation. DesignThirty-eight 5-week-old male Wistar rats were divided into experimental group, which received acrylic resin appliance that shifted mandible to the left during closure, and control group. Computed tomography and histomorphometry were used for condyle analyses, while samples of condyle, synovial membrane and m. masseter were analyzed with enzyme–linked immunosorbent assay and spectrophotometry to determine VEGF and nNOS protein concentrations, and SOD activity. ResultsExperimental group of rats developed smaller and asymmetrical mandibles. Less of new bone and cartilage formation and larger bone marrow cavities area were found in the experimental group. Higher VEGF expression in condyle and m. masseter as well as higher nNOS expression in m. masseter and synovial membrane were found in the experimental compared to the control group. Alteration of SOD activity was found in m. masseter and synovial membrane in the experimental group. ConclusionsLateral mandibular deviation induces mandibular and condylar morphological changes as well as significant cellular signaling alterations in condyle, synovial membrane and masticatory muscle. Cellular VEGF protein overexpression and oxidative stress/nitric oxide disbalance could be the mechanisms underlying unbalanced functional TMJ loading due to mandibular deviation.

Shear stress promotes differentiation of stem cells from human exfoliated deciduous teeth into endothelial cells via the downstream pathway of VEGF-Notch signaling.

Effects of shear stress on endotheliaxl differentiation of stem cells from human exfoliated deciduous teeth (SHEDs) were investigated. SHEDs were treated with shear stress, then reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to analyse the mRNA expression of arterial markers and western blot analysis was performed to analyse protein expression of angiogenic markers. Additionally, in vitro matrigel angiogenesis assay was performed to evaluate vascular-like structure formation. The secreted protein expression levels of the vascular endothelial growth factor (VEGF) of SHEDs after shear stress was also quantified using corresponding ELISA kits. Untreated SHEDs seeded on Matrigel cannot form vessel-like structures at any time points, whereas groups treated with shear stress formed a few vessel-like structures at 4, 8 and 12 h. When SHEDs were treated with EphrinB2-siRNA for 24, the capability of vessel-like structure formation was suppressed. After being treated with shear stress, the expression of VEGF, VEGFR2, DLL4, Notch1, EphrinB2, Hey1 and Hey2 (arterial markers) gene expression was significantly upregulated, moreover, the protein levels of VEGFR2, EphrinB2, CD31, Notch1, DLL4, Hey1, and Hey2 were also significantly upregulated. Both the mRNA and protein expression levels of EphB4 (venous marker) were downregulated. The average VEGF protein concentration in supernatants secreted by shear stress treated SHEDs groups increased significantly. In conclusion, shear stress was able to induce arterial endothelial differentiation of stem cells from human exfoliated deciduous teeth, and VEGF-DLL4/Notch-EphrinB2 signaling was involved in this process.

Endostar inhibits ascites formation and prolongs survival in mouse models of malignant ascites.

Endostar, a modified recombinant human endostatin, inhibits the growth of a variety of tumors by suppressing neovascularization. Vascular endothelial growth factor (VEGF) has an important role in malignant ascites formation. In order to determine whether Endostar can suppress the formation of ascites and prolong survival times, mouse models of malignant ascites were established using S180 and H22 tumor cells. The experimental mice were randomly divided into four groups: The three treatment groups received different doses of Endostar (4, 8 and 16 mg/kg), and the control group received 0.9% w/v NaCl. The volume of ascites, and the tumor cell, red blood cell (RBC), VEGF protein and mRNA content of the ascites was measured alongside the peritoneal permeability and the mouse survival time. In vitro analysis of cultured Endostar-treated S180 and H22 cells was also performed in order to examine cellular proliferation and the level of VEGF secreted protein and mRNA. The results revealed that Endostar suppressed the ascites volume, decreased the level of tumor cells, RBCs and VEGF in the ascites fluid, and lowered the permeability of the peritoneum. The tumor cells collected from the ascites in the Endostar-treated mice demonstrated a decrease in the expression of VEGF mRNA. The survival rates of the 8 and 16 mg/kg Endostar-treated mice were longer than those of the controls. The in vitro experiments revealed a significant inhibition of VEGF protein secretion and VEGF mRNA by Endostar, but no effect on cellular proliferation. In conclusion, Endostar lowers ascites production by downregulating VEGF expression, and may therefore be effective for the treatment of malignant ascites.

Hypoxia-inducible factor-1 modulates the expression of vascular endothelial growth factor and endothelial nitric oxide synthase induced by eccentric exercise.

The present study investigated the effects of acute and chronic eccentric exercise on the hypoxia-inducible factor (HIF)-1α activation response and the concomitant modulation of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression in rat skeletal muscle. Twenty-four male Wistar rats were randomly assigned to three experimental groups: rested control group, acutely exercised group after an intermittent downhill protocol for 90 min, and acutely exercise group with a previous eccentric training of 8 wk. HIF-1α activation, VEGF and eNOS gene expression, protein content, and promoter activation were assessed in vastus lateralis muscle biopsies. Acute eccentric exercise induced a marked activation of HIF-1α and resulted in increased VEGF and eNOS mRNA level and protein concentration. The binding of HIF-1α to the VEGF and eNOS promoters, measured by a chromatin immunoprecipitation assay, was undetectable in rested rats, whereas it was evident in acutely exercised animals. Acute exercise also increased myeloperoxidase, toll-like receptor-4, tumor necrosis factor-α, and interleukin-1β protein content, suggesting a contribution of proinflammatory stimuli to HIF-1α activation and VEGF overexpression. All of these effects were partially abolished by training. Moreover, training resulted in an increased capillary density. In summary, our findings indicate that eccentric exercise prompts an HIF-1α response in untrained skeletal muscle that contributes to the upregulation of VEGF and eNOS gene expression and is attenuated after an eccentric training program.

VEGF mRNA and protein concentrations in the developing human eye.

BackgroundVascular endothelial growth factor (VEGF), a well-characterized regulator of angiogenesis, has been mechanistically implicated in retinal neovascularization and in the pathogenesis of ROP. However, the ontogeny of VEGF expression in the human fetal retina is not well known. Because retinal vasculature grows with gestational maturation, we hypothesized that VEGF expression also increases in the midgestation human fetal eye as a function of gestational age.MethodsTo identify changes in VEGF gene expression during normal human development, we measured VEGF mRNA by quantitative PCR and measured VEGF protein by ELISA and western blots in 10-24 week gestation fetal vitreous, retina, and serum.ResultsVEGF mRNA expression in the retina increased with gestational age. VEGF isoform A, particularly its VEGF121 splice variant, contributed to this positive correlation. Consistent with these findings, we detected increasing VEGF121 protein concentrations in vitreous humor from fetuses of 10-24 weeks gestation, while VEGF concentrations decreased in fetal serum.ConclusionsVEGF121 mRNA and protein concentrations increase with increasing gestational age in the developing human retina. We speculate that VEGF plays an important role in normal retinal vascular development, and that preterm delivery affects production of this vascular growth factor.

Vascular endothelial growth factor (VEGF) and placental growth factor (PLGF) directly correlate with ovarian follicle size in women undergoing in vitro fertilization (IVF)

To assess VEGF and PlGF concentrations in FF from women undergoing IVF, stratified according to follicle size, and to correlate these with VEGF and PlGF mRNA expression in corresponding luteinized granulosa cells (GC), as a follow-up of our previous demonstration that these genes are expressed in GC(1). Prospective sample collection for in vitro studies. 18 consenting subjects with normal ovarian function (IVF being undertaken due to male factor) were recruited prior to oocyte retrieval (mean age of the women = 31 years). Only blood-free FF obtained with the first puncture of each ovary was collected, without the use of flushing media to avoid dilution of the FF contents. All specimens were categorized and analyzed as FF derived from small follicles (8-14 mm diameter) or large follicles (>14 mm diameter). VEGF and PlGF concentrations in the FF were measured using validated commercial ELISAs (RayBio®). Levels of FF estradiol (E2) and progesterone (P4) were determined by RIA. VEGF and PlGF mRNA expression was evaluated by real time RT-PCR, using cultured GC derived from the small and large follicles as indicated above. We also evaluated the expression of other relevant ovarian genes. FF VEGF concentrations from large follicles were markedly higher (6788 ± 2863 pg/ml) compared to those in small follicles (1942 ± 879 pg/ml, p = 0.0003). PlGF concentrations in FF from large follicles also was higher (2004 ± 816 pg/ml) than in small follicles (1144 ± 898 pg/ml, p =0.002). By RT-PCR, the expression of VEGF mRNA was ∼2.5 fold greater in GC from large follicles than from small follicles. However, we observed no significant statistical differences in PlGF mRNA expression relative to follicle size. As expected, expression of mRNAs encoding steroidogenic acute regulatory protein (StAR), anti-mullerian hormone (AMH) and aromatase P450 (CYP19) were all increased in cells from large follicles as compared with those from small follicles. VEGF and PlGF protein concentrations in FF are correlated with follicle size; this relationship also applied to E2 concentration and StAR, AMH and CYP19 expression. Moreover, VEGF and PlGF levels were significantly higher in FF than in matched serum. Similar trends were noted for VEGF mRNA, but not PlGF mRNA expression from cultured GC. We conclude that local VEGF and PlGF gene expression are likely to mediate ovarian follicle angiogenesis and may contribute to oocyte development, maturation and selection of dominant follicle.

Platelet Vascular Endothelial Growth Factor is a Potential Mediator of Transfusion-Related Acute Lung Injury.

ObjectiveThe occurrence of non-hemolytic transfusion reactions is highest with platelet and plasma administration. Some of these reactions are characterized by endothelial leak, especially transfusion related acute lung injury (TRALI). Elevated concentrations of inflammatory mediators secreted by contaminating leukocytes during blood product storage may contribute to such reactions, but platelet-secreted mediators may also contribute. We hypothesized that platelet storage leads to accumulation of the endothelial permeability mediator vascular endothelial growth factor (VEGF), and that intravascular administration of exogenous VEGF leads to extensive binding to its lung receptors.MethodsSingle donor, leukocyte-reduced apheresis platelet units were sampled over 5 days of storage. VEGF protein content of the centrifuged supernatant was determined by ELISA, and the potential contribution of VEGF from contaminating leukocytes was quantified. Isolated-perfused rat lungs were used to study the uptake of radiolabeled VEGF administered intravascularly, and the effect of unlabeled VEGF on lung leak.ResultsThere was a time-dependent release of VEGF into the plasma fraction of the platelet concentrates (62 ± 9 pg/ml on day one, 149 ± 23 pg/ml on day 5; mean ± SEM, p<0.01, n=8) and a contribution by contaminating leukocytes was excluded. Exogenous 125I-VEGF bound avidly and specifically to the lung vasculature, and unlabeled VEGF in the lung perfusate caused vascular leak.ConclusionRising concentrations of VEGF occur during storage of single donor platelet concentrates due to platelet secretion or disintegration, but not due to leukocyte contamination. Exogenous VEGF at these concentrations rapidly binds to its receptors in the lung vessels. At higher VEGF concentrations, VEGF causes vascular leak in uninjured lungs. These data provide further evidence that VEGF may contribute to the increased lung permeability seen in TRALI associated with platelet products.

Angiogenic response to passive movement and active exercise in individuals with peripheral arterial disease

Peripheral arterial disease (PAD) is caused by atherosclerosis and is associated with microcirculatory impairments in skeletal muscle. The present study evaluated the angiogenic response to exercise and passive movement in skeletal muscle of PAD patients compared with healthy control subjects. Twenty-one PAD patients and 17 aged control subjects were randomly assigned to either a passive movement or an active exercise study. Interstitial fluid microdialysate and tissue samples were obtained from the thigh skeletal muscle. Muscle dialysate vascular endothelial growth factor (VEGF) levels were modestly increased in response to either passive movement or active exercise in both subject groups. The basal muscle dialysate level of the angiostatic factor thrombospondin-1 protein was markedly higher (P < 0.05) in PAD patients compared with the control subjects, whereas soluble VEGF receptor-1 dialysate levels were similar in the two groups. The basal VEGF protein content in the muscle tissue samples was ∼27% lower (P < 0.05) in the PAD patients compared with the control subjects. Analysis of mRNA expression for a range of angiogenic and angiostatic factors revealed a modest change with active exercise and passive movement in both groups, except for an increase (P < 0.05) in the ratio of angiopoietin-2 to angiopoietin-1 mRNA in the PAD group with both interventions. PAD patients and aged individuals showed a similar limited angiogenic response to active exercise and passive movement. The limited increase in muscle extracellular VEGF combined with an elevated basal level of thrombospondin-1 in muscle extracellular fluid of PAD patients may restrict capillary growth in these patients.

Effects of tyrosine kinase receptor B-brain-derived neurotrophic factor signal pathway on the secretion of vascular endothelial growth factor and matrix metalloproteinase-9 of neuroblastoma

Objective To study the effects of tyrosine kinase receptor B-brain-derived neurotrophic factor (TrkB-BDNF) signal pathway on the secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-9(MMP-9) of neuroblastoma.Methods We used all-trans retinoic acid (ATRA) to induce the high expression of TrkB in the SH-SY5Y cell line,and then added the ectogenid BDNF to activate the TrkB-BDNF and its three downstream signal pathways.TrkB-BDNF signal pathway was inhibited by specific tyrosine kinase inhibitor K252a.The three downstream signal pathway was respectively inhibited by LY294002 (the phosphatidylinositol 3-hydroxy kinase (PI3 K) pathway inhibitor)、U73122 (the phospholipase C pathway inhibitor) 、U0126(the mitogen activated protein kinase pathway inhibitor).Enzyme linked immunosorbent assay was used to detect the concentration of VEGF and MMP-9 protein in the SY5Y cell culture supernatants.Results VEGF [(485.89 ± 109.99) pg/ml] and MMP-9 [(15.73 ± 1.72) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA + BDNF were significantly higher than that of the control group and ATRA alone group(P ＜0.05).VEGF [(272.42 ±86.33) pg/ml]and MMP-9 [(5.25 ± 1.44) pg/ml] protein levels in the group of ATRA + BDNF + K252a were significantly lower than those of the ATRA + BDNF group(P ＜ 0.05) and had no significant difference compared with the control group and the ATRA alone group(P ＞0.05).VEGF [(314.12 ±24.68) pg/ml] and MMP-9 [(4.91 ± 1.08) pg/ml] protein levels in the group of ATRA + BDNF + LY294002 were significantly lower than those of the ATRA + BDNF group(P ＜ 0.05) and had no significant difference compared with the control group and the ATRA alone group(P ＞0.05).VEGF [(444.08 ±64.49) pg/ml] and MMP-9 [(13.28 ±3.38) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA +BDNF + U73122 had no significant difference compared with the ATRA + BDNF group(P ＞ 0.05).VEGF [(429.97 ± 19.95) pg/ml] and MMP-9 [(13.96 ± 4.45) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA + BDNF + U0126 had no significant difference compared with the ATRA + BDNF group(P ＞ 0.05).Conclusion Activation of TrkB-BDNF signal pathway can increase the synthesis and secretion of VEGF and MMP-9 in human neuroblastoma cells.TrkB-BDNF signal pathway may be through activating its downstream PI3K pathway to increase the synthesis and secretion of VEGF and MMP-9 in human neuroblastoma cells.The synthesis and secretion of VEGF and MMP-9 can be inhibited by blocking the TrkB-BDNF signal pathway with K252a or blocking its downstream signal pathway PI3 K with LY294002. Key words: Neuroblastoma; TrkB-BDNF signal pathway; K252a; LY294002; Vascular endothelial growth factor; Matrix metalloproteinases-9

Vascular endothelial growth factor gene polymorphisms and renal cell carcinoma: A systematic review and meta-analysis

Renal cell carcinoma (RCC) accounts for 3% of all cancer-related mortalities in adults. The risk factors for the development of RCC remain under investigation. Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and is crucial for the development and metastasis of tumors, including RCC. VEGF gene polymorphisms may alter VEGF protein concentrations, affect the process of angiogenesis and may be involved in inter-individual variation in carcinogenesis. In the present study, a systematic review and meta-analysis were performed based on published case-control studies in order to estimate the association between VEGF gene polymorphisms and the susceptibility to RCC. A total of five studies that involved eight polymorphisms and were published between January 2000 and December 2012 were identified from PubMed. The results of this systematic review and meta-analysis indicate that the VEGF 936C/T, 1612G/A, −1154G/A, −2549I/D, −460T/C and 405G/C gene polymorphisms are not associated with the risk of RCC. There was no polymorphism in 702C/T and RCC and the −2578C/A gene polymorphism may be associated with an increased risk of RCC. However, due to the limitations of the present study, further high quality case-control studies are warranted to confirm these findings.

Effects of Ginkgo biloba Extract on Cultured Human Retinal Pigment Epithelial Cells under Chemical Hypoxia

Purpose: To investigate the effects of Ginkgo biloba extract (GBE) on expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells under chemical hypoxia.Materials and Methods: RPE cells (ARPE-19) were cultured in either untreated media (control group), media treated with 200 μM cobalt chloride (hypoxia group) or media treated with both 200 μM cobalt chloride and 100 μg/ml GBE (hypoxia + GBE group) for various amounts of time. HIF-1α and VEGF expression were compared between groups. HIF-1α and VEGF messenger RNA (mRNA) were quantified using real-time polymerase chain reaction (PCR). HIF-1α of extracted nuclei and VEGF of the media were quantified using enzyme-linked immunosorbent assay (ELISA). The expression of HIF-1α was also assessed with Western blot and immunocytochemistry.Results: HIF-1α mRNA, VEGF mRNA, HIF-1α and VEGF levels were higher in the hypoxia group compared with the control group; however, real-time PCR revealed decreased expression of HIF-1α mRNA and VEGF mRNA in the hypoxia + GBE group compared with the hypoxia group. In the ELISA, the HIF-1α and VEGF protein concentrations also decreased with GBE treatment. Western blot and immunostaining showed that the intensity of HIF-1α signals in the hypoxia + GBE group was lower than that of the hypoxia group.Conclusion: GBE reduced HIF-1α and VEGF expression in RPE cells cultured under chemical hypoxia in vitro.

Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells

Given the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability. Varying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass. Lipofectamine LTX 5 μL and Plus reagent 4 μL with 2 μg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per μg DNA) through at least day 12 (888 ± 386 pg/mL per μg DNA) after transfection. Plasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors.

Angiogenesis after plasmid VEGF165 gene transfer in an animal model

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Skóra J, Barć P, Dawiskiba T, Baczyńska D, Mastalerz-Migas A. Angiogenesis after plasmid VEGF165 gene transfer in an animal model. Central European Journal of Immunology. 2013;38(3):305-309. doi:10.5114/ceji.2013.37751. APA Skóra, J., Barć, P., Dawiskiba, T., Baczyńska, D., & Mastalerz-Migas, A. (2013). Angiogenesis after plasmid VEGF165 gene transfer in an animal model. Central European Journal of Immunology, 38(3), 305-309. https://doi.org/10.5114/ceji.2013.37751 Chicago Skóra, Jan, Piotr Barć, Tomasz Dawiskiba, Dagmara Baczyńska, and Agnieszka Mastalerz-Migas. 2013. "Angiogenesis after plasmid VEGF165 gene transfer in an animal model". Central European Journal of Immunology 38 (3): 305-309. doi:10.5114/ceji.2013.37751. Harvard Skóra, J., Barć, P., Dawiskiba, T., Baczyńska, D., and Mastalerz-Migas, A. (2013). Angiogenesis after plasmid VEGF165 gene transfer in an animal model. Central European Journal of Immunology, 38(3), pp.305-309. https://doi.org/10.5114/ceji.2013.37751 MLA Skóra, Jan et al. "Angiogenesis after plasmid VEGF165 gene transfer in an animal model." Central European Journal of Immunology, vol. 38, no. 3, 2013, pp. 305-309. doi:10.5114/ceji.2013.37751. Vancouver Skóra J, Barć P, Dawiskiba T, Baczyńska D, Mastalerz-Migas A. Angiogenesis after plasmid VEGF165 gene transfer in an animal model. Central European Journal of Immunology. 2013;38(3):305-309. doi:10.5114/ceji.2013.37751.

Involvement of COX‐2/PGE2 signalling in hypoxia‐induced angiogenic response in endothelial cells

To evaluate the impact of hypoxia on the angiogenic capability of endothelial cells (ECs), and further investigate whether the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) signalling is involved in the angiogenic response of ECs to hypoxia. We explored the impact of various periods (1, 3, 6, 12, 24 hrs) of hypoxia (2% O2) on human umbilical vein endothelial cells (HUVECs) in vitro. We observed cell viability, migration, tube formation, analysed COX-2, vascular endothelial growth factor (VEGF), AQP1 mRNA transcription, protein expression and measured PGE2, VEGF protein concentration in cell supernatants. Then we treated HUVECs with COX-2 selective inhibitor NS398, EP1/2 combined antagonist AH6809 and exogenous PGE2 to investigate the role of COX-2/PGE2 signalling in the angiogenic response of ECs to hypoxia. The results demonstrated that short-term hypoxic treatment enhanced HUVECs proliferation, migration, tube formation, significantly up-regulated COX-2, VEGF, AQP1 mRNA level, protein expression and promoted PGE2, VEGF release. The pharmacological inhibition study revealed that exposure of HUVEC to NS398 and AH6809 under hypoxia impaired the biological responses of ECs to hypoxia. Exogenous PGE2 augments the effects of hypoxia on HUVECs, and partially reversed the inhibitory effects of NS398 on HUVECs proliferation and angiogenic capability. Short-term hypoxic treatment enhanced angiogenic capability of ECs, and COX-2/PGE2 signalling may play a critical role in the biological response of ECs to hypoxia.

Evaluation of Plasma and Tissue S100A4 Protein and mRNA Levels as Potential Markers of Metastasis and Prognosis in Clear Cell Renal Cell Carcinoma

To investigate levels of S100A4 protein in plasma and S100A4 mRNA in tumours from patients with clear cell renal cell carcinoma (CCRCC), and correlate these with metastasis, survival and levels of vascular endothelial growth factor (VEGF). Plasma S100A4 and VEGF protein concentrations were measured using enzyme-linked immuno sorbent assays in 39 healthy subjects and 68 consecutive patients with untreated CCRCC. Levels of S100A4 and VEGF mRNA in tumour and matched control (healthy) tissue samples were measured using realtime quantitative reverse transcription- polymerase chain reaction. Findings were analysed with respect to clinico pathological characteristics. Plasma VEGF concentrations were higher in patients with CCRCC than in healthy subjects. S100A4 and VEGF mRNA levels were up-regulated in CCRCC tumour tissue compared with control tissue samples. Logistic regression analysis revealed that up-regulated tumour S100A4 and VEGF mRNA levels were independent risk factors for the presence of invasion and/or metastasis. S100A4 and VEGF are associated with tumour invasion and metastasis, and may be useful prognostic markers in patients with CCRCC. S100A4 and VEGF may represent potential targets for therapeutic intervention.