Vascular Endothelial Growth Factor mRNA Levels Research Articles

Inhibitory Effect of Intravitreal Injection of Bevacizumab on Nerve Growth Factor

Purpose: To investigate the inhibitory effect of intravitreal injection of bevacizumab on the expression of nerve growth factor (NGF) in rabbit retina.Methods: The right eyes of 40 New Zealand white rabbits were injected with 1.25 mg (0.05 cc) bevacizumab three times monthly; as controls, the left eyes received sham injections. Apoptosis in retinal cells was evaluated by immunohistochemical staining for caspase-3, and by in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) of DNA fragments. NGF and vascular endothelial growth factor (VEGF) proteins in rabbit retinas were measured quantitatively (Enzyme linked immunosorbent assay (ELISA)) and qualitatively (immunohistochemical staining) 1 and 4 months after injection. NGF and VEGF messenger RNAs in rabbit retinas were evaluated by real-time polymerase chain reaction (RT-PCR).Results: As shown by the TUNEL assay and caspase-3 immunostaining, the bevacizumab-injected group had significantly more apoptotic activity than did the control group. Levels of retinal NGF and VEGF proteins in the bevacizumab group were lower than those in the control group (p < 0.05). Immunohistochemical staining of NGF and VEGF was weaker in the bevacizumab group than in the control group. NGF and VEGF mRNA levels in the bevacizumab group were lower than those of the control group (p < 0.05).Conclusions: Findings of the present study suggest that apoptosis in retinal cells after intravitreal bevacizumab injection is increased by down-regulated NGF, caused by VEGF inhibition in rabbits.

Hypoxia‐induced upregulation of pigment epithelium‐derived factor by retinal glial (Müller) cells

Neuronal degeneration and aberrant neovascularization are common problems of ischemic retinopathies. Pigment epithelium-derived factor (PEDF), a neuroprotective protein and an inhibitor of angiogenesis, is produced by retinal glial (Müller) cells and can counterbalance elevated levels of vascular endothelial growth factor (VEGF), the expression of which is regulated primarily by hypoxia-inducible factor (HIF)-1. In an approach to mimic transient ischemia in vitro, primary Müller cells were cultured under transient and strong hypoxia (0.2% O(2) ), followed by reoxygenation at 2.5% O(2) , and molecular mechanisms that might contribute to changes in the intraretinal PEDF level were determined. Hypoxic conditions caused an increasing expression of HIF-1α and led to upregulation of both PEDF and VEGF. Treatment of the cells with synthetic HIF-1α blockers or neutralization of VEGF binding to VEGF receptors (VEGFR-1 and-2) suppressed hypoxia-induced PEDF upregulation. Furthermore, the presence of CoCl(2) (a hypoxia mimetic) induced an accumulation of elevated HIF-1α protein in the nucleus and an upregulation of PEDF expression in Müller cells. Increasing PEDF expression was attenuated when HIF-1α levels were suppressed using HIF-1α small interfering RNA (siRNA). On the other hand, siRNA-mediated depletion of PEDF facilitated HIF-1α upregulation caused by CoCl(2) and resulted in increasing VEGF mRNA and protein levels. These results demonstrate that VEGF and PEDF may be unidirectionally regulated in hypoxia through HIF-1α activation, with upregulation of PEDF, which may occur in a VEGF-dependent manner. However, endogenously produced PEDF seems to be an inherent control element of HIF-1α expression in Müller cells, indicating an important feedback mechanism for limiting upregulation of VEGF.

Effects of hypoxia inducible factor-1 alpha-targeting small interfering RNA on vascular endothelial growth factor gene expression in HaCaT cells

Objective To observe the effects of hypoxia inducible factor-1 alpha (HIF-1α)-targeting small interfering RNA (siRNA) on the expression of HIF-1α and vascular endothelial growth factor (VEGF) in HaCaT ceils under hypoxic conditions. Methods HaCaT cells were cultured and divided into four groups, normal control group (without any treatment), hypoxia group (cultured under hypoxic conditions for 24 hours),liposome control group (transfected with liposome followed by hypoxic culture for 24 hours), RNA interference group (transfected with HIF-1α-targeting siRNA/liposome complexes followed by hypoxic culture for 24 hours). Fluorescence real-time quantitative PCR was utilized to determine HIF-1oα and VEGF mRNA expression in HaCaT cells, and Western blot to detect HIF-1α and VEGF protein expression. Results No significant difference was observed in the mRNA expression of HIF-1α between the hypoxia group and normal control group (0.907 ± 0.032 vs. 0.878 ± 0.034, F =1.108, P ＞ 0.05), while the expression levels of VEGF mRNA,HIF-1α and VEGF proteins were significantly higher in the hypoxia group than in the normal control group (0.935 ± 0.032 vs. 0.652 ± 0.053, 0.813 ± 0.047 vs. 0.236 ± 0.014, 0.791 ± 0.030 vs. 0.316 ± 0.013, all P ＜0.05). A significant decline was noted in the mRNA and protein expression levels of VEGF (0.230 ± 0.044 vs.0.978 ± 0.030, 0.213 ± 0.026 vs. 0.817 ± 0.049, both P ＜ 0.05) and HIF-1α (0.497 ± 0.033 vs. 0.806 ±0.040, 0.249 ± 0.028 vs. 0.833 ± 0.052, both P ＜ 0.05) in the RNA interference group than in the liposome control group. Conclusions Hypoxia may enhance the expression of HIF-1α and VEGF in HaCaT cells, and to inhibit the HIF-1α expression may suppress the expression of VEGF in HaCaT cells under hypoxia. Key words: Keratinocytes; Hypoxia-inducible factor, alpha subunit; RNA interference; Vascular endothelial growth factors

Acacetin inhibits VEGF expression, tumor angiogenesis and growth through AKT/HIF-1α pathway

Acacetin (5,7-dihydroxy-4′-methoxyflavone) is a flavone compound, some of which have anti-cancerous effects. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and tumor growth. In this study, we found that acacetin decreased the steady level of VEGF mRNA level and inhibited VEGF transcriptional activation. To further determine the potential mechanism of acacetin in inhibiting VEGF expression, we showed that acacetin inhibited HIF-1α expression and AKT activation. Over-expression of HIF-1α or AKT restored acacetin-decreasing VEGF transcriptional activation, indicating that AKT and HIF-1 are the essential downstream targets of acacetin for inhibiting VEGF expression in the cells. Moreover, acacetin significantly inhibited ovarian cancer cell-induced angiogenesis and tumor growth in vivo through inhibiting HIF-1α and VEGF expression. Acacetin did not change HIF-1α mRNA level, but inhibited HIF-1α protein level through increasing its degradation and decreasing its stability. These results indicate that acacetin may be a useful natural compound for ovarian cancer prevention and treatment.

Production of VEGF induced by GMCSF via ERK-NF-KB singling 'pathway in human fibroblasts during wound healing

Objective To observe production of vascular endothelial growth factor （VEGF） induced by granulocyte/macrophage colony-stimulating factor （GMCSF） via ERK nerve growth factor （NF） -κB singling pathway in human fibroblasts during wound healing and explore relating mechanism. Methods Human fibroblasts from the injured skin were used for this study and treated with GMCSF. RT-PCR was used for analyzing the protein and mRNA levels of VEGF and Western blotting was employed to determine the phosphorylation of ERK. The fibroblasts were pre-treated with ERK specific inhibitor PD98059 and further treated with GMCSF, then the fibroblasts and the supernatant were collected for detection of protein level of VEGF by means of Western blot. ERK signal pathway was inhibited to detect the activation of NF-κB by means of immunofluorescence staining. Furthermore, the nuclear and cytoplasmic extraction kit was used to separate the cytoplasm and nucleus and Western blot employed for observation of the NF-κB activation. Results The mRNA level and protein level of VEGF were increased significantly with treatment with higher concentration of GMCSF in a dose-dependent manner. VEGF mRNA level was increased two hours after administration with GMCSF and reached peak at 4-6 hours. GMCSF could remarkably activate the ERK phosphorylation. Compared with GMCSF, the ERK specific inhibitor PD98059 inhibited significantly the effect of GMCSF in inducing VEGF expression （ P 〈 0.05 ）. Western blot and immunofluorescence staining analyses showed that the activation of NF-κB was inhibited with reduced production of VEGF after GMCSF treatment. Conclusion GMCSF up-regulates production of VEGF through activating NF-κB via ERK signal pathway in the human fibroblasts. Key words: Wound healing; Fibroblasts; Vascular endothelial growth factor A; Granulocyte/macrophage colony-stimulating factor

Mesenchymal stem cells derived from Wharton jelly of the human umbilical cord ameliorate damage to human endometrial stromal cells

To investigate the effect of mesenchymal stem cells isolated from Wharton jelly of umbilical cord (WJ-MSCs) on ameliorating damaged human endometrial stromal cells (ESCs). Experimental study. University-affiliated hospital. Sixteen endometrial tissues were obtained from women undergoing hysterectomy. Eight umbilical cords were obtained from full-term deliveries. ESCs were cultured with mifepristone to get damaged ESCs, then damaged ESCs were co-cultured with WJ-MSCs. The proliferation of ESCs was investigated by Cell Counting Kit 8, and the percentage of apoptosis by annexin-V-fluorescein isothiocyanate binding. The mRNA and protein expression of vascular endothelial growth factor (VEGF) and caspases 3, 8, and 9 were determined by one-step quantitative real-time polymerase chain reaction and Western blot. After exposure to mifepristone, the proliferation of ESCs decreased and the apoptosis percentage increased in a dose- and time-dependent manner. At a certain dose and duration, this damage continued even after the withdrawal of mifepristone at 48 hours. When the damaged ESCs were cocultured with WJ-MSCs, the proliferation of these damaged cells was significantly increased and apoptosis percentage decreased. In addition, the level of VEGF mRNA and protein decreased and that of caspases 3, 8, and 9 increased. WJ-MSCs may serve as a promising treatment approach to ameliorate endometrial damage.

Inhibitory effect of vascular endothelial growth factor siRNA on retinal neovascularization in mouse

Background To suppress vascular endothelial growth factor (VEGF) is a researching hot topic for the treatment and prevention of retinal neovascularization.Some detectable efficacy of VEGF small interference RNA (VEGF siRNA) in anti-tumor neovascularization has been well-known.But relevant study on VEGF siRNA on retinal neovascularization is seldom.Objective Present study was to investigate the inhibiting effect of VEGF siRNA on retinal neovascularization.Methods The 48 clean C57BL/6J mice aged 7-day-old were randomly divided into normoxia group,hypoxia control group,vector group and VEGF siRNA group and 12 mice for each.Hypoxia models were established by raising the pups with mother mice in the airtight oxygen-cabin for 5 days.The lipofectamineTM 2000 (LF2000)-mediated vector plasmids or VEGF siRNA recombinant plasmids were then injected intravitreally in 12 12-day-old pup mice respectively.The animals were sacrificed in 1 week after intravitreal injection,and the numbers of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM) were counted by hematoxylin-eosin stain.The expressions of VEGF protein and mRNA in retina were assayed by immunoinfluorescence technique and RT-PCR.Results The numbers of vascular endothelial cell nuclei extending beyond the ILM were 0.19±0.09,24.89±2.03,23.65±2.15 and 8.83±1.12 in normoxia group,model control group,vector group and VEGF siRNA group separately,showing significant decrease in VEGF siRNA group compared with model control group or vector group (q=5.67,q=4.97,P＜0.01).RT-PCR revealed that VEGF mRNA was faintly expressed in mouse retina in normoxia group.However,in model control group and vector group,the level of VEGF mRNA was 52.3 times and 36.7 times more than that of normoxia group respectively and only 3.5 times in VEGF siRNA group,presenting a inhibitory rate of 43.39% of VEGF siRNA on VEGF.Immunofluorescence showed that the expression of VEGF was weaker in normoxia group and strong positive response in model control group and vector group,but the expression intensity of VEGF protein was significantly weaker in VEGF siRNA group.Conclusion VEGF siRNA recombinant plasmids can efficiently inhibit retinal neovascularization in oxygen-induced retinopathy mouse model through intravitreal injection. Key words: RNA interference; Retina; Neovascularization; Vascular endothelial growth factor; lipofectamine-mediated vector plasmids

Lithium decreases VEGF mRNA expression in leukocytes of healthy subjects and patients with bipolar disorder

Objectives Vascular endothelial growth factor (VEGF) is thought to be involved in the pathophysiology of mood disorders and the target of antidepressants. The aim of this study was to elucidate molecular effects of lithium on VEGF expression by using leukocytes of healthy subjects and patients with bipolar disorder. Methods Eight healthy male subjects participated in the first study. Lithium was prescribed for 2 weeks, enough to reach therapeutic serum concentration. Leukocyte counts and serum lithium concentrations were determined at baseline, at 1- and 2-week medication, and at 2 weeks after stopping medication. VEGF mRNA levels were also examined in nine lithium-treated bipolar patients and healthy controls in the second study. Results In the first study, leukocyte counts were significantly increased at 2 weeks compared with those at baseline and were normalized after 2 weeks. VEGF mRNA levels were significantly decreased at 2 weeks and after 2 weeks compared with those at baseline. Consistent with the first study, VEGF mRNA levels were significantly decreased in the lithium-treated bipolar patients compared with healthy controls. Conclusions Our investigation suggests that VEGF mRNA expression may be useful as a peripheral marker of the effects of lithium. Copyright © 2011 John Wiley & Sons, Ltd.

Vascular Endothelial Growth Factor (VEGF) and the VEGF Soluble Receptor-1 (sFlt-1) in Chorionic Villus Tissue from Chinese Women with Early Recurrent Spontaneous Abortion

This case-control study explored the relationship between early recurrent spontaneous abortion (RSA) and the expression of two genes: VEGFA, the gene encoding vascular endothelial growth factor (VEGF); and fms-related tyrosine kinase 1 (FLT1), the gene encoding the soluble VEGF receptor-1 (sFlt-1). Women experiencing RSA or undergoing induced abortions in the early stage of normal pregnancy were recruited to the study (n = 30 per group). There were no significant between-group differences in maternal age or duration of pregnancy. The levels of VEGF and sFlt-1 mRNA in chorionic villus tissue samples were examined by quanti tative reverse transcription-polymerase chain reaction. Levels of sFlt-1 and VEGF mRNA in the chorionic villus tissue of women with RSA were significantly higher than levels in the control group. This study demonstrated that there is a relationship between early RSA and VEGF and sFlt-1 levels, suggesting that over-expression of the FLT1 and VEGFA genes may be associated with the pathogenesis of RSA.

The effects of acute and intermittent hypoxia on the expressions of HIF-1α and VEGF in the left and right ventricles of the rabbit heart

Hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) are involved in signaling mechanisms of cellular responses to hypoxia. These factors have been investigated in tissue samples by simulating different altitudes by changing the percentage of oxygen. We aimed first to evaluate the effect of normobaric, systemic hypoxia (11% O2) on HIF-1α and VEGF mRNA levels in the heart muscle; secondly, to compare the levels of HIF-1α and VEGF mRNA in the left and right ventricle muscles. In this experimental study, 33 New Zealand male rabbits were assigned to control, acute hypoxia (4 hours) and intermittent hypoxia (4 hours/day for 14 days) groups (n=11/group). Total RNA was isolated from right and left ventricles of the heart. The expressions of HIF-1α and VEGF mRNAs were investigated by using Reverse Transcription Polymerase Chain Reaction (RT-PCR) method. The obtained data were compared by using ANOVA and paired t-test. The results indicated that left ventricle VEGF mRNA expressions in both acute and intermittent hypoxia groups (1.08 ± 0.15 and 1.03 ± 0.19, respectively) were higher than that in the control group (0.88 ± 0.15) (p=0.03). Hypoxia treatments did not significantly alter HIF-1α mRNA in both ventricles (p=0.60 and p=0.51 for left and right ventricles, respectively). Since systemic hypoxia results in induction of VEGF mRNA up-regulation only in left ventricle, it could be related to its higher metabolic activity and oxygen utilization. Hypoxia induced changes in the expression of HIF-1α mRNA may not be the only determining factor for HIF-1/VEGF pathway induction or the observed VEGF induction could be through other hypoxia sensitive pathways.

VEGF in the Development of Ovarian Malignancy

BackgroundIt is well known that the vascular endothelial growth factor (VEGF) system is essential to the growth of tumors, but its possible changes during tumor development are still sparsely elucidated. The current study, therefore, investigated the possible differences of VEGF and VEGFR-1 and VEGFR-2 protein concentration in tissue from patient groups with normal ovaries and those with benign, borderline, and malignant ovarian tumors. Furthermore, we investigated VEGF gene expression of the same groups. Material and MethodsWe analyzed tissue samples from 157 patients undergoing surgery of the ovaries. Tissue VEGF and VEGFR-1 and VEGFR-2 were measured by enzyme-linked immunosorbent assay, and gene expression was analyzed by reverse transcriptase polymerase chain reaction regarding VEGF isotypes 121, 165, and 189. ResultsSignificantly higher VEGF protein concentration was found in malignant tissue compared with normal ovarian tissue (P < 10-6) and benign ovarian tumors (P < 10-6). Similar results were found with respect to VEGFR-1. The gene expression differed among normal ovaries, benign, and malignant tumors for all Three VEGF isotypes (P < 10-6), with significantly higher levels of VEGF mRNA in malignant tumors. Malignant tumors with VEGFR-2 protein levels above the median had decreased progression-free survival (PFS) (P = .0005) and overall survival (P = .002). Median PFS was 26.2 months in the group with low VEGFR-2 levels vs. 7.8 months in the other group. Multivariate analysis confirmed the results (hazard ratio, 1.94; P = .04). ConclusionThe current study showed major changes in the VEGF system during tumor development. A significant difference was found in protein concentration and gene expression among the different patient groups. In addition, VEGFR-2 was correlated with PFS and overall survival.

Bone marrow stromal cells protect myeloma cells from bortezomib induced apoptosis by suppressing microRNA-15a expression

Despite unsurpassed anti-tumor activity of bortezomib for multiple myeloma (MM), drug resistance has emerged as a challenge, especially when MM cells adhere to the stroma. This study aimed to determine whether bone marrow stromal cells (BMSCs) have a role in the development of chemoresistance in MM. Our data demonstrate that the secretion of interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and cell-to-cell contact with microenvironment-derived stromal cells from patients with multiple myeloma (MM-BMSCs) significantly decreased the sensitivity of myeloma cells to bortezomib treatment. Mechanistically, we found that microRNA (miRNA)- 15a expression was up-regulated in U266 and NCI-H929 cells treated by bortezomib, which was inhibited by MM-BMSCs. miRNA-15a transfected myeloma cells were arrested in G1/S checkpoint and secreted less VEGF compared to control transfected cells, although no significant difference was found in VEGF mRNA levels. In conclusion, our data suggest that via suppressing miRNA-15a expression, BMSCs provide survival support and protect myeloma cells from bortezomib induced apoptosis.

Hypoxia induces tumor and endothelial cell migration in a Semaphorin 3F- and VEGF-dependent manner via transcriptional repression of their common receptor Neuropilin 2

Neuropilin-2 (NRP2) is a receptor expressed by tumor cells and endothelial cells (EC) that binds both semaphorin 3F (SEMA3F), a potent inhibitor of tumor angiogenesis and metastasis, and vascular endothelial growth factor (VEGF), a potent stimulator of tumor angiogenesis. It was found that glioblastoma and melanoma cells repressed NRP2 expression when maintained under hypoxic conditions and after treatment with the hypoxia-mimetic agent desferrioxamine (DFO), at both the mRNA and protein levels. Silencing of HIF1-α, the hypoxia-induced subunit of the hypoxia inducible factor (HIF), abrogated DFO-induced NRP2 repression. Conversely, ectopic expression of HIF1-α directly repressed NRP2 promoter activity and expression. NRP2 is the sole receptor for SEMA3F. Loss of NRP2 expression in tumor cells inhibited SEMA3F-dependent activities, such as inactivation of RhoA, depolymerization of F-actin, and inhibition of tumor cell migration. On the other hand, loss of NRP2 expression in tumor cells increased VEGF protein levels in conditioned media, with no effects on VEGF mRNA levels. This increase in VEGF protein levels promoted paracrine activation of EC, including VEGF receptor-2 phosphorylation, and activation of downstream signaling proteins such as p44/42 MAPK and p38 MAPK. In addition, the elevated VEGF levels induced EC migration and sprouting, two key steps of tumor angiogenesis in vivo. It was concluded that hypoxia regulates VEGF and SEMA3F activities through transcriptional repression of their common receptor NRP2, providing a novel mechanism by which hypoxia induces tumor angiogenesis, growth and metastasis.

Lack of Keratinocyte Growth Factor Retards Angiogenesis in Cutaneous Wounds

This study investigated the effects of keratinocyte growth factor (KGF) on wound healing. Full-thickness excisional dorsal wounds were created on KGF knockout mice (KGF KO, n = 12) and wild-type C57BL/6 mice (WT, n = 12), and wound closure rates were measured. Immunohistochemical staining was used to investigate cell proliferation and blood vessel density by assessing Ki67 and CD31 protein levels, respectively, and real-time reverse transcription-polymerase chain reaction was used to measure vascular endothelial growth factor (VEGF) mRNA levels. No differences in the rate of wound closure were found between KGF KO and WT mice, however the KGF KO mice showed decreased proliferation of keratinocytes, angiogenesis and VEGF mRNA levels in vivo. These results suggest that KGF may play an important role in the regulation of VEGF gene expression and angiogenesis during wound healing.

Expression of Aquaporin 4 and Kir4.1 in Diabetic Rat Retina: Treatment with Minocycline

This study examined aquaporin 4 (AQP4) and Kir4.1 (a potassium channel subunit) in normal and diabetic adult Sprague-Dawley rats, and determined the effect of minocycline treatment. Retinal expression of the AQP4 and Kir4.1 genes was examined using double immuno fluorescence, Western blot analysis, and real-time reverse transcription-polymerase chain reaction. Retinal levels of vascular endothelial growth factor (VEGF), ionized calcium-binding adaptor molecule (Iba)-1 and interleukin (IL)-1β were also ascertained. The blood-retinal barrier (BRB) and retinal oedema were assessed using rhodamine isothiocyanate. AQP4, VEGF, Iba-1, and IL-1β mRNA and protein levels increased, and Kir4.1 mRNA and protein levels decreased, in diabetic rat retinas. Both BRB disruption and retinal oedema were also observed in these retinas. In diabetic rats, minocycline treatment decreased AQP4, VEGF, Iba-1 and IL-1β levels and retinal oedema, and increased Kir4.1 levels. These findings suggest that minocycline might be beneficial for retinal fluid clearance and reduction of retinal oedema in diabetic retinopathy.

Biologically inhibitory effects of VEGF siRNA on endometrial carcinoma cells

To study the biological effects of small inhibitory RNA (siRNA) on endometrial carcinoma cells through disrupting the mRNA of Vascular endothelial growth factor (VEGF), and its inhibitory functions on tumor cells' proliferation. The sequence-specific siRNA of VEGF165 and the mock siRNA were designed, they were transfected into the endometrial carcinoma cell line, Ishikawa cells. Total cell RNAs were extracted from the transfected cells. The mRNA levels of VEGF were then analyzed utilizing real-time fluorescence quantitative RT-PCR (FQ-PCR) post-transfection at 12, 24, 48, 72, and 168 h, respectively. Simultaneously its protein levels were also determined by western blot. Cell proliferations were monitored by MTT assay and cytopathological effects (CPE) were determined by Multispect Imaging System. The mRNA and protein levels of VEGF declined post-transfection at 12, 24, 48, 72 h, respectively, with a maximum decrease post-transfection at 48 h. The proliferation of Ishikawa cells was significantly inhibited in a similar manner during this time window. All the data were repeated at least three independent times and similar results were achieved. The small siRNA of VEGF165 can effectively down-regulate its target mRNA and protein levels; consequently proliferation of Ishikawa cells was inhibited. The data strongly imply that VEGF may be involved in the genesis of endometrial carcinoma, which provides a new pathway to treat human endometrial caners.

Fibulin-5 Is Up-regulated by Hypoxia in Endothelial Cells through a Hypoxia-inducible Factor-1 (HIF-1α)-dependent Mechanism

Hypoxia modulates gene expression and affects multiple aspects of endothelial cell biology. Fibulin-5 (FBLN5) is an extracellular matrix protein essential for elastic fiber assembly and vasculogenesis that participates in vascular remodeling and controls endothelial cell adhesion, motility, and proliferation. In this context, we aimed to analyze FBLN5 regulation by hypoxia in endothelial cells. Hypoxia (1% O(2)) increased FBLN5 mRNA levels in endothelial cells in a time-dependent manner. Maximal induction (∼2.5-fold) was achieved after 24 h of hypoxia. This effect paralleled an increase in both intracellular and extracellular FBLN5 protein levels. The increase in FBLN5 mRNA levels observed in hypoxic cells was blocked by inhibitors of the PI3K/Akt/mTOR pathway (LY294002 and rapamycin) and mimicked by dimethyl oxal glycine, which prevents proline hydroxylase-mediated degradation of HIF-1α. Silencing of HIF-1α completely prevented hypoxia-induced FBLN5 up-regulation. Accordingly, both hypoxia and HIF-1α overexpression increased FBLN5 transcriptional activity. Serial promoter deletion and mutagenesis studies revealed the involvement of a putative hypoxia response element (HRE) located at -78 bp. In fact, EMSA and ChIP assays demonstrated increased HIF-1 binding to this site in hypoxic cells. Interestingly, the rate of endothelial cells undergoing apoptosis in cultures exposed to hypoxia increased in FBLN5 knockdown cells, suggesting that hypoxia-induced FBLN5 expression contributes to preserve cell survival. These results provide evidence that HIF-1 signaling underlies the increase of FBLN5 expression elicited by hypoxia in endothelial cells and suggest that FBLN5 induction could be involved in the adaptive survival response of endothelial cells to hypoxia.

OX-LDL Up-Regulates the Vascular Endothelial Growth Factor-to-Pigment Epithelium-Derived Factor Ratio in Human Retinal Pigment Epithelial Cells

Native and oxidized (OX) low-density lipoprotein (LDL) may contribute to the pathogenesis of age related macular degeneration (AMD). In this study, we investigated the effects of lipoproteins, including LDL and ox-LDL, on cell viability, apoptosis, and vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) expression in cultured human retinal pigment epithelial (RPE) cells. ARPE-19 cells were incubated with 10-100 mg/ml n-LDL, ox-LDL for 24 hr. Cell viability was assessed using the Cell Titer 96 Aqueous One Solution cell proliferation assay. The apoptosis of RPE was measured with TUNEL. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF and PEDF mRNA in RPE cells. The expression of VEGF and PEDF protein was measured by western blotting. To examine the role of MAPK signal transduction in LDL- and OX-LDL-induced VEGF and PEDF protein expression, ARPE-19 cells were pretreated with one of several MAPK inhibitors for 2 hr and then incubated with native LDL or OX-LDL for 24 hr. One-way analysis of variance was used to compare the differences. OX-LDL treatment decreased ARPE-19 cell viability in a dose-dependent manner, whereas native LDL had no effect. Incubation of ARPE-19 cells with 10 mg/mL OX-LDL induced marked apoptosis, compared with untreated control cells. OX-LDL also increased VEGF expression and decreased PEDF expression, whereas native LDL had no significant effect. The VEGF-to-PEDF ratio was elevated after OX-LDL treatment. OX-LDL-induced VEGF protein synthesis was partly abolished by inhibiting p38 and JNK, while inhibiting ERK did not show a significant effect. OX-LDL treatment induced cellular changes in ARPE-19 cells that appeared to reflect pathogenic events in neovascular AMD, providing potential insight into the roles of OX-LDL in human RPE cells and its potential role in the pathogenesis of AMD.

Intraocular expression of thymosin β4 in proliferative diabetic retinopathy

To examine an association between thymosin β4 as potentially angioproliferative factor and proliferative diabetic retinopathy. The clinical study part included 62 patients with proliferative diabetic retinopathy (PDR) (study group) and 24 patients with non-diabetic pre-retinal membranes (control group). All patients underwent pars plana vitrectomy. We examined the thymosin β4 concentration in vitreous and plasma; and the expression of thymosin β4, glial fibrillary acidic protein (GFAP) and CD31 (PECAM-1 or Platelet Endothelial Cell Adhesion Molecule) and the levels of thymosin β4 mRNA and vascular endothelial growth factor (VEGF) mRNA in the excised membranes. The experimental study part consisted of 24 Sprague--Dawley rats with streptozotocin-induced diabetes mellitus and 24 age-matched control animals without diabetes. We determined the mRNA concentrations of thymosin β4, VEGF and GFAP in the rat retinas. In the clinical study part, the vitreal and plasma thymosin β4 concentrations were significantly higher in the study group than control group (p =0.04 and p=0.01, respectively), and were significantly (p=0.028) correlated with each other. Co-expression of thymosin β4 and CD31 was observed in the diabetic fibrovascular membranes. Thymosin β4 mRNA and VEGF mRNA levels were significantly (p<0.01) higher in diabetic membranes than in non-diabetic membranes. In the experimental study part, the diabetic retinas showed co-localization of thymosin β4 and GFAP. The mRNA levels of thymosin β4, VEGF and GFAP were significantly (p<0.01) higher in diabetic rats than in control animals. Thymosin β4 was produced in intraocular fibrovascular membranes of patients with PDR and in rats with experimental diabetes mellitus. Thymosin β4 may play a role in diabetic retinal neovascularization.

Effects of JAK2/STAT3 signaling pathway on angiogenesis in non-small cell lung cancer

To investigate the relationship between janus kinase2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway and angiogenesis in non-small cell lung cancer (NSCLC) and explore the effects on the mRNA expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) by blocking JAK2/STAT3 signaling pathway. Immunohistochemistry was used to determine the expression of P-JAK2, P-STAT3 and microvessel density (MVD) in 68 NSCLC tissues and 27 normal lung tissues. And the relationship with their clinical pathological features was analyzed. Human lung cancer A549 cells were treated with different concentrations of AG490. Cell proliferation was measured by MTT assay. Western blot was performed to detect the activation of JAK2/STAT3 signaling pathway. The mRNA expressions of VEGF and bFGF were determined by RT-PCR (reverse transcription-polymerase chain reaction). A549 cells were transfected with STAT3 siRNA. And the protein of STAT3, Phos-STAT3 (P-STAT3) and mRNA levels of VEGF and bFGF were detected. The activation of JAK2/STAT3 signaling pathway was closely related to MVD in NSCLC. AG490 and STAT3 siRNA could block the JAK2/STAT3 signaling pathway and down-regulated the mRNA expressions of VEGF and bFGF in lung cancer cells. JAK2/STAT3 signaling pathway plays an important role in the angiogenesis of NSCLC. Blocking this pathway may inhibit the expression of angiogenic cytokines. JAK2/STAT3 signaling pathway may be a critical therapeutic target for the treatment of angiogenesis in NSCLC.