VEGF Protein Levels Research Articles

Chronic restraint stress increases the protein expression of VEGF and its receptor VEGFR-2 in the prefrontal cortex.

In the present study the central and peripheral regulation of VEGF, its cognate receptors, and regulators were examined after acute and chronic restraint stress in rats. After chronic restraint stress (6 h per day for 21 days) the protein levels of VEGF (175 ± 24%) and its receptor VEGFR-2 (169 ± 17%) increased significantly in the prefrontal cortex (A and B). mRNA levels of VEGFR-2 (132 ± 11%) were also significantly increased (D). In the hippocampus no significant changes were observed at the mRNA or protein levels. In serum there was a tendency towards increased VEGF protein expression after both acute and chronic restraint stress (C).

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Objectives Nrf2 is a potent transcription factor that regulates the transcription of a multiple antioxidative-genes. We reported that Nrf2-dependent redox signaling contributes to impaired invasion of the extravillous trophoblast in early onset IUGR associated with preeclampsia. Recent discussion about evidence of a link between Nrf2 and vascular the vascular endothelial growth factor has focused on its downstream target protein, heme oxygenase-1 (HO-1). HO-1 metabolizes heme to biliverdin, iron and carbon monoxide (CO). To assess the interaction between Nrf2 signaling and the angiogenic process, we examined in vitro the effect of Nrf2 activation on the expression of pro- and anti-angiogenic factors, VEGF and the soluble fms-like tyrosine kinase-1 (sFlt-1). Methods The angiogenic effect of Nrf2 activation was tested using BeWo and the primary human umbilical vein endothelial cells (HUVECs). ELISA, scratch- and tube formation-assays were mainly applied in this study. HUVECs migration and tube formation assays were performed in the presence or absence of sulforaphane, a potent inducer of Nrf2 signaling. Results The activation of HO-1 via Nrf2 led to a significant increase in the protein levels of VEGF and decrease in the augmented-sFlt-1 in the supernatant of the treated cells. Up-regulation of HO-1/CO enhanced tube formation and migration of the endothelial cells. And the knock down of Nrf2 expression in the aforementioned cells reduced the angiogenic potential of sulforaphane. Conclusions Our data revealed that the activation of the Nrf2 and its target protein HO-1 induced the expression of VEGF in vitro and exerted positive effects on endothelial functions. We could further demonstrate that the release of sFlt-1 has been reduced after treatment with Nrf2 inducer, sulforaphane. Thus Nrf2 activation during pregnancy may improve the imbalance between the pro-and anti-angiogenic factors mainly described in preeclampsia. Disclosures N. Kweider: None. J. Lambertz: None. T. Goecke: None. A. Ludwig: None. T. Pufe: None. C.J. Wruck: None. W. Rath: None.

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model.

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I–like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Analysis of chemokines and receptors expression profile in the myelin mutant taiep rat.

Taiep rat has a failure in myelination and remyelination processes leading to a state of hypomyelination throughout its life. Chemokines, which are known to play a role in inflammation, are also involved in the remyelination process. We aimed to demonstrate that remyelination-stimulating factors are altered in the brainstem of 1- and 6-month-old taiep rats. We used a Rat RT2 Profiler PCR Array to assess mRNA expression of 84 genes coding for cytokines, chemokines, and their receptors. We also evaluated protein levels of CCL2, CCR1, CCR2, CCL5, CCR5, CCR8, CXCL1, CXCR2, CXCR4, FGF2, and VEGFA by ELISA. Sprague-Dawley rats were used as a control. PCR Array procedure showed that proinflammatory cytokines were not upregulated in the taiep rat. In contrast, some mRNA levels of beta and alpha chemokines were upregulated in 1-month-old rats, but CXCR4 was downregulated at their 6 months of age. ELISA results showed that CXCL1, CCL2, CCR2, CCR5, CCR8, and CXCR4 protein levels were decreased in brainstem at the age of 6 months. These results suggest the presence of a chronic neuroinflammation process with deficiency of remyelination-stimulating factors (CXCL1, CXCR2, and CXCR4), which might account for the demyelination in the taiep rat.

The Long Noncoding RNA MEG3 Is Downregulated and Inversely Associated with VEGF Levels in Osteoarthritis.

Osteoarthritis (OA) is becoming a major public health problem in China, especially considering the increase in average life expectancy of the population. Thus, enhanced understanding of the molecular changes associated with OA is urgently needed to develop more effective strategies for the diagnosis and treatment of this debilitating disease. LncRNAs play an important role in the processes of bone and cartilage development. Maternally expressed gene 3 (MEG3) is a maternally expressed lncRNA and may function as a tumor suppressor by inhibiting angiogenesis. OA is closely associated with angiogenesis and the inhibition of angiogenesis presents a novel therapeutic approach to reduce inflammation and pain in OA. In this study, we detected the mRNA expression of MEG3 and VEGF in articular cartilage samples from 20 OA patients and 10 healthy volunteers by real-time RT-PCR. VEGF protein is detected by ELISA in cartilage samples. The results show that human MEG3 is significantly downregulated in OA patients compared to normal cartilage samples. However, higher levels of VEGF mRNA and protein are found in OA compared to the control. Moreover, MEG3 levels are inversely associated with VEGF levels, suggesting that MEG3 may be involved in OA development through the regulation of angiogenesis.

Enhancement of Neurotrophic Factors in Astrocyte for Neuroprotective Effects in Brain Disorders Using Low-intensity Pulsed Ultrasound Stimulation.

Astrocytes play an important role in the growth and survival of developing neurons by secreting neurotrophic factors. The goal of this study was to investigate how low-intensity pulsed ultrasound (LIPUS) stimulation directly affects brain astrocyte function. Here, we report that LIPUS stimulation increased protein levels of BDNF, GDNF, VEGF, and GLUT1 in rat brain astrocytes as measured by western blot analysis. Histological outcomes including demyelination and apoptosis were examined in rats after administration of aluminum chloride (AlCl3). At the mechanistic level, integrin inhibitor (RGD peptide) attenuated the LIPUS-induced neurotrophic factor expression. The data suggest that neurotrophic factor protein levels may be promoted by LIPUS through activation of integrin receptor signaling. In addition, LIPUS stimulation protected cells against aluminum toxicity as demonstrated by an increase in the median lethal dose for AlCl3 from 3.77 to 6.25 mM. In in vivo histological evaluations, LIPUS significantly reduced cerebral damages in terms of myelin loss and apoptosis induced by AlCl3. The results of this study demonstrate that transcranial LIPUS is capable of enhancing the protein levels of neurotrophic factors, which could have neuroprotective effects against neurodegenerative diseases.

Expression of vascular endothelial factor-A, gelatinases (MMP-2, MMP-9) and TIMP-1 in uterine leiomyomas.

Leiomyomas growth involves cellular hypertrophy, modulation of mitotic activity and upregulation of extracellular matrix (ECM). Vascular factors and matrix metalloproteinases (MMPs) play a coordinated role during neoplasia and tissue remodeling. The present study investigates the role of angiogenic factor vascular endothelial growth factor (VEGF)-A with the activity of main gelatinases, MMP-2/MMP-9 and their tissue inhibitor TIMP-1 in patients with leiomyomas. Peripheral blood of 46 women with uterine leiomyomas was obtained prior hysterectomy to assess VEGF-A, MMP-2, -9, TIMP-1 levels by enzyme-linked immunosorbent assay compared to 39 healthy controls. Protein expression levels of VEGF-A, MMP-2 and MMP-9 were evaluated by western immunoblotting and immunohistochemistry in leiomyomas tissue specimens after hysterectomy. Furthermore, the activity of gelatinases in leiomyoma tissue extracts and control myometrium was evaluated by semi-quantitative zymography. Circulating levels of VEGF-A, MMP-2 and TIMP-1 were significantly elevated in leiomyoma patients compared to controls (p<0.001, p=0.004, p=0.003, respectively). A positive correlation was found between VEGF-A and MMP-2 (p=0.021) as well as MMP-9 (p=0.001) peripheral levels in the patient's group. Furthermore, increased VEGF-A protein levels were detected in leiomyoma tissue compared to control myometrium, followed by increased localization of both VEGF-A and MMP-2 in the ECM embedding bundles of smooth muscle cells of leiomyomas. The activity of MMP-2 was significantly higher in leiomyomas than normal myometrium in all investigated tissues. This study demonstrates a possible coordinated role of VEGF-A and MMP-2 during uterine leiomyomas growth and angiogenesis with potential prognostic significance.

Effects of transforming growth factor-β on the laser-induced choroidal neovascularization in mice

Objective To investigate the effects of transforming growth factor-β (TGF-β) in choroidal neovascularization (CNV) induced by laser in mice.Methods Eighty male C57BL/6J mice at the age of 6-8 weeks old were randomly divided into the normal control,photocoagulation model,photocoagulation with phosphate buffered saline (PBS control group) and photocoagulation with TGF-β receptor inhibitor groups (TGF-β receptor inhibitor group),twenty mice of each group.Fundus argon laser photocoagulation was performed in the photocoagulation model group,PBS control group and TGF-β receptor inhibitor group to induce CNV.One week,two,three and four weeks after the laser procedure,fundus fluorescein angiography (FFA) was carried out in the normal control or photocoagulation model groups to observe CNV formation dynamically.Western blot was used to analyze the expressions of TGF-β in the retina from the mice of normal control or photocoagulation model groups,and VEGF or TNF-α in the retina of normal control,PBS control or TGF-β receptor inhibitor groups.The CNV areas of each group were evaluated by using fluorescein stain on retinal pigment epithelium (RPE)/choroid flat mounts after two weeks of photocoagulation.Results The FFA results showed the retinal vessels centered on the optic disc and arranged radially,while the choroidal vascular present network distribution in the normal control mice.Significant leakage of fluorescein showed discoid strong fluorophore in photocoagulation sites of retina at one week after photocoagulation.The quantitative analysis results of Western blot demonstrated that the TGF-β protein expression levels in retina of photocoagulation model mice gradually increased with time passing.The protein expression levels of TGF-β were significant differences in the photocoagulation model group comparing with the normal control group (F=13.042,P＜0.05).The protein expression levels of TNF-α (F=14.721,17.509) and VEGF (F=18.890,11.251) increased significantly in retina of PBS control or TGF-β receptor inhibitor groups when compared with that of normal control group at one week,two,three and four weeks after photocoagulation,and the differences were both statistically significant (P＜0.05).Compared with PBS control group,the protein levels of TNF-α and VEGF in retina from TGF-β receptor inhibitor group were significantly reduced,the differences was statistically significant (F=21.321,16.160,P＜0.05).Two weeks after laser photocoagulation,a distinct reduction in CNV lesion size in the TGF-β receptor inhibitor group mice when compared to PBS or normal control groups,the differences was statically significant (F=4.482,P＜0.05).Conclusion TGF β may promote CNV formation by up-regulating both TNF α and VEGF protein expressions,the application of its specific inhibitor is able to reduce CNV progression. Key words: Choroidal neovascularization/physiopathology; Transforming growth factor beta/ agonists; Tumor necrosis factor-alpha; Vascular endothelial growth factors; Animal experimentation

Hypoxic conditioned medium from rat cerebral cortical cells enhances the proliferation and differentiation of neural stem cells mainly through PI3-K/Akt pathways.

PurposeTo investigate the effects of hypoxic conditioned media from rat cerebral cortical cells on the proliferation and differentiation of neural stem cells (NSCs) in vitro, and to study the roles of PI3-K/Akt and JNK signal transduction pathways in these processes.MethodsCerebral cortical cells from neonatal Sprague–Dawley rat were cultured under hypoxic and normoxic conditions; the supernatant was collected and named ‘hypoxic conditioned medium’ (HCM) and ‘normoxic conditioned medium’ (NCM), respectively. We detected the protein levels (by ELISA) of VEGF and BDNF in the conditioned media and mRNA levels (by RT-PCR) in cerebral cortical cells. The proliferation (number and size of neurospheres) and differentiation (proportion of neurons and astrocytes over total cells) of NSCs was assessed. LY294002 and SP600125, inhibitors of PI3-K/Akt and JNK, respectively, were applied, and the phosphorylation levels of PI3-K, Akt and JNK were measured by western blot.ResultsThe protein levels and mRNA expressions of VEGF and BDNF in 4% HCM and 1% HCM were both higher than that of those in NCM. The efficiency and speed of NSCs proliferation was enhanced in 4% HCM compared with 1% HCM. The highest percentage of neurons and lowest percentage of astrocytes was found in 4% HCM. However, the enhancement of NSCs proliferation and differentiation into neurons accelerated by 4% HCM was inhibited by LY294002 and SP600125, with LY294002 having a stronger inhibitory effect. The increased phosphorylation levels of PI3-K, Akt and JNK in 4% HCM were blocked by LY294002 and SP600125.Conclusions4%HCM could promote NSCs proliferation and differentiation into high percentage of neurons, these processes may be mainly through PI3-K/Akt pathways.

Arginase inhibition enhances angiogenesis in endothelial cells exposed to hypoxia

Hypoxia-induced arginase elevation plays an essential role in several vascular diseases but influence of arginase on hypoxia-mediated angiogenesis is completely unknown. In this study, in vitro network formation in bovine aortic endothelial cells (BAEC) was examined after exposure to hypoxia for 24h with or without arginase inhibition. Arginase activity, protein levels of the two arginase isoforms, eNOS, and VEGF as well as production of NO and ROS were examined to determine the involvement of arginase in hypoxia-mediated angiogenesis. Hypoxia elevated arginase activity and arginase 2 expression but reduced active p-eNOSSer1177 and NO levels in BAEC. In addition, both VEGF protein levels and endothelial elongation and network formation were reduced with continued hypoxia, whereas ROS levels increased and NO levels decreased. Arginase inhibition limited ROS, restored NO formation and VEGF expression, and prevented the reduction of angiogenesis. These results suggest a fundamental role of arginase activity in regulating angiogenic function.

TNF-α and LPS activate angiogenesis via VEGF and SIRT1 signalling in human dental pulp cells.

To assess whether SIRT1 and VEGF are responsible for tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS)-induced angiogenesis and to examine the molecular mechanism(s) of action in human dental pulp cells (HDPCs). Immortalized HDPCs obtained from Prof. Takashi Takata (Hiroshima University, Japan) were treated with LPS (1 μg mL(-1) ) and TNF-α (10 ng mL(-1) ) for 24 h. mRNA and protein levels were examined by RT-PCR and Western blotting, respectively. Migration and tube formation were examined in human umbilical vein endothelial cells (HUVECs). The data were analysed by one-way anova. Statistical analysis was performed at α = 0.05. LPS and TNF-α upregulated VEGF and SIRT1 mRNA and protein levels. Inhibition of SIRT1 activity by sirtinol and SIRT1 siRNA or inhibition of the VEGF receptor by CBO-P11 significantly attenuated LPS + TNF-α-stimulated MMPs production in HDPCs, as well as migration and tube formation in HUVECs (P < 0.05). Furthermore, sirtinol, SIRT1 siRNA and CBO-P11 attenuated phosphorylation of Akt, extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) and the nuclear translocation of NF-κB p65. Pre-treatment with inhibitors of p38, ERK, JNK, PI3K and NF-κB decreased LPS + TNF-α-induced VEGF and SIRT1 expression, MMPs activity in HDPCs and angiogenesis (P < 0.05) in HUVECs. TNF-α and LPS led to upregulation of VEGF and SIRT1, and subsequent upregulation of MMP-2 and MMP-9 production, and promote angiogenesis via pathways involving PI3K, p38, ERK, JNK and NF-κB. The results suggest that inhibition of SIRT1 and VEGF might attenuate pro-inflammatory mediator-induced pulpal disease.

Possible Role of Raf-1 Kinase in the Development of Cerebral Vasospasm and Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats.

This study aims to clarify the potential role of Raf-1 kinase in cerebral vasospasm (CVS) and early brain injury (EBI) after subarachnoid hemorrhage (SAH). Two experimental SAH models in rats, including cisterna magna double injection model for CVS study and prechiasmatic cistern single injection model for EBI study, were performed in this research. As a specific inhibitor of Raf-1, BAY 43-9006 was used in this study. In CVS study, time course study showed that the basilar artery exhibited vasospasm after SAH and became most severe at day 5, and the phosphorylation of Raf-1 had the same trends, while both vasospasm and the phosphorylation of Raf-1 induced by SAH were inhibited by BAY 43-9006 treatment. In addition, BAY 43-9006 treatment significantly reversed the phosphorylation of ERK1/2 and the activation of NF-κB induced by SAH and decreased the messenger RNA (mRNA) levels of IL-6 and IL-1β. In EBI study, BAY 43-9006 treatment significantly suppressed the brain injury induced by SAH. Besides, BAY 43-9006 inhibited the phosphorylation of Raf-1 and ERK1/2; decreased the protein levels of COX-2, VEGF, and MMP-9; and reversed the activation of NF-κB induced by SAH. These results demonstrate that Raf-1 kinase contributes to CVS and EBI after SAH by enhancing the activation of the Raf-1/ERK1/2 and Raf-1/NF-κB signaling pathways, and that the inhibition of these pathways might offer new treatment strategies for CVS and EBI.

Evaluation of transforming growth factor-β1 suppress Pokemon/epithelial-mesenchymal transition expression in human bladder cancer cells.

Transforming growth factor-β1 (TGF-β1) plays a dual role in apoptosis and in proapoptotic responses in the support of survival in a variety of cells. The aim of this study was to determine the function of TGF-β1 in bladder cancer cells and the relationship with POK erythroid myeloid ontogenic factor (Pokemon). TGF-β1 and its receptors mediate several tumorigenic cascades that regulate cell proliferation, migration, and survival of bladder cancer cells. Bladder cancer cells T24 were treated with different levels of TGF-β1. Levels of Pokemon, E-cadherin, Snail, MMP2, MMP9, Twist, VEGF, and β-catenin messenger RNA (mRNA) and protein were examined by real-time quantitative fluorescent PCR and Western blot analysis, respectively. The effects of TGF-β1 on epithelial-mesenchymal transition of T24 cells were evaluated with wound-healing assay, proliferation of T24 was evaluated with reference to growth curves with MTT assay, and cell invasive ability was investigated by Transwell assay. Data show that Pokemon was inhibited by TGF-β1 treatment; the gene and protein of E-cadherin and β-catenin expression level showed decreased markedly after TGF-β1 treatment (P < 0.05). While the bladder cancer cell after TGF-β1 treatment showed a significantly reduced wound-closing efficiency at 6, 12, and 24h, mechanistic analyses demonstrated that different levels of TGF-β1 promotes tumor cell growth, migration, and invasion in bladder cancer cells (P < 0.01, P < 0.05, respectively). In summary, our findings suggest that TGF-β1 may inhibit the expression of Pokemon, β-catenin, and E-cadherin. The high expression of TGF-β1 leads to an increase in the phenotype and apical-base polarity of epithelial cells. These changes of cells may result in the recurrence and progression of bladder cancer at last. Related mechanism is worthy of further investigation.

Abstract 494: Tumor ascorbate content is associated with extended disease-free survival and decreased hypoxia-inducible factor-1 activation in patients with colorectal cancer

Abstract Ascorbate (vitamin C) is a specific cofactor for a range of dioxygenase enzymes, including those involved in histone and DNA demethylation and also the enzymes that ‘switch off’ hypoxia-inducible factor (HIF)-1. These processes have to potential to influence tumor biology, where inadequate intracellular ascorbate levels may contribute to tumor progression and an over-active HIF-1 survival response. We previously demonstrated an association between low ascorbate levels and high HIF-1 activation in clinical samples from patients with endometrial cancer. In order to determine whether this is a universal phenomenon, we have now proceeded to a second cancer type, colorectal cancer. Clinical, pathological and follow-up data, as well as samples of tumor and adjacent normal tissue, were collected from 50 consenting colorectal cancer patients. Protein levels of HIF-1α and three pro-survival target gene products, Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), glucose transporter 1 (GLUT-1) and VEGF, were measured by Western blot or ELISA and related to the tumor ascorbate content. HIF-1α, BNIP3 and VEGF protein levels were all elevated in tumor compared to adjacent normal tissue (p&amp;lt;0.01), while ascorbate levels were lower in tumor tissue (p&amp;lt;0.001). There was a significant inverse relationship between the tumor ascorbate content and HIF-1 pathway activation (p=0.002) and tumor size (p=0.018). High grade tumors had up to 40% less ascorbate than adjacent normal tissue and also compared to low grade tumors. Patients with tumors containing low ascorbate had significantly shorter disease-free survival than patients with high levels of ascorbate in their tumors (p=0.04). The ability of tumors to accumulate ascorbate, according to the ratio of ascorbate in tumor compared to paired normal tissue, also tended to be associated with disease-free survival (p=0.08). Levels of ascorbate in normal tissue were not associated with disease-free survival (p=0.614). This is the first report demonstrating a relationship between ascorbate levels in human tumors and patient survival. This relationship may rely on the ability of ascorbate to promote dioxygenase activity and dampen the HIF survival response, as this is the second tumor type where low cellular ascorbate levels were associated with increased HIF-1 activation. An intervention clinical trial is now warranted to determine if tumor levels of ascorbate can be manipulated. Citation Format: Caroline Kuiper, Gabi Dachs, Margaret Currie, John Pearson, Delwyn Munn, Margreet Vissers. Tumor ascorbate content is associated with extended disease-free survival and decreased hypoxia-inducible factor-1 activation in patients with colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 494. doi:10.1158/1538-7445.AM2014-494

Abstract 5219: MicroRNA-874 impedes angiogenesis in non-small cell lung cancer

Abstract MicroRNAs (miRs) are short (20-24nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in eukaryotes by affecting both the stability and translation of mRNAs. MicroRNAs play crucial roles in normal biological processes and are commonly dysregulated in human diseases. Thus far, miRs have been shown to act as either tumor suppressor genes or oncogenes in driving critical gene expression pathways in cancer stem cells in a wide range of human malignancies, including non-small cell lung cancer (NSCLC).We previously found that miR-874 could suppress tumor growth and metastasis of NSCLC. Since angiogenesis is important for tumor growth and metastasis, in this study, we further investigated the possible roles of miR-874 in tumor angiogenesis. Attenuation of miR-874 expression in histological sections of NSCLC tumors and the CD133 positive population of NSCLC cells (NSCLC-initiating cells; NSCLC-ICs), as compared to its normal counterparts, was observed by in situ hybridization and real time quantitative PCR, respectively. Tumor-conditioned medium from NSCLC-ICs induced significantly high levels of endothelial cell capillary network and neovascularization in vivo; whereas, re-expression of miR-874 in NSCLC-ICs inhibited this capillary network formation ability by suppressing the protein levels of VEGF, Stat3, MMP-2, uPA and PI3K. Further, miR-874-treatment decreased in vivo angiogenesis and pre-established orthotopic tumor growth in nude mice as compared to mock- and control-miR-treatments. Immunohistochemical analysis revealed a significant decrease in the expression of VEGF, Stat3 and microvessel density (CD31 levels) in xenograft tumor sections from miR-874-treated mice. In conclusion, our study highlights the possible anti-angiogenic role of miR-874 in NSCLC-initiating cells and presents a new, hopeful therapeutic target and prognostic marker for NSCLC. Citation Format: Chandramu Chetty, Dilip R. Maddirela, Venkateswara R. Gogineni, Subramanyam Chittivelu, Jasti S. Rao, Bhagavatula Moorthy. MicroRNA-874 impedes angiogenesis in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5219. doi:10.1158/1538-7445.AM2014-5219

Effect of Flt-1 gene on the expression of VEGF and proliferation in BT325 cells

Objective To study the Effects of Flt-1 gene on the expression of VEGF and proliferation in BT325 cells.Methods The total RNA was extracted from human umbilical vein endothelial cells as the template and Flt-1 gene was amplified by reverse transcription polymerase chain reaction(RT-PCR).By using gene recombination technique,the Flt-1 cDNA was inserted into eukaryotic expression vector pEGFP-C1.The recombinant plasmid was identified by restriction enzyme digestion and sequence analysis.It was infected into BT325 cells.The quantity of VEGF protein in supernatant was detected by ELISA.The proliferation abilities were measured by MTT and FCM.Results The Flt-1 gene could be obtained by RT-PCR.The restriction enzyme digestion and PCR suggested that the recombinant plasmids were constructed successfully.72 hours after transfection,the VEGF protein levels were decreased (transfection group (56.3 ±41.2) pg/ml,untransfection group (95.5 ±35.3) pg/ml(P ＜0.05) and the proliferation rate of cells was inhibited transfection group (42.6 ± 3.7) ％ and untransfection group (92.8 ± 6.6) ％ (P ＜ 0.05).Conclusions Flt-1 gene could inhibit the expression of VEGF and BT325 cells proliferation and induce their apoptosis in vitro. Key words: Flt-1 gene; Gene recombination; BT325 cell line

Leptin increases VEGF expression and enhances angiogenesis in human chondrosarcoma cells

BackgroundLeptin, 16kDa product of obese gene, is adipocytokine playing critical role in regulation of body weight. In recent years, leptin is also defined as potent angiogenic factor involving in tumorigenesis, angiogenesis, and metastasis. However, it is unknown whether leptin regulates VEGF production in human chondrosarcoma and contributing the tumor-associated angiogenesis. MethodsWe analyzed protein level of leptin and VEGF in human chondrosarcoma tissues. Effects of leptin on chondrosarcoma cells were examined by in vitro and in vivo assays. In addition, intracellular signal pathways were investigated by pharmacological and genetic approaches. ResultsWe found that both leptin and VEGF are highly expressed in human chondrosarcoma tissues, and positively correlated with tumor stage. Leptin increases VEGF production by activating OBRl receptor and MAPKs (p38, ERK, and JNK), which in turn enhances binding of AP-1 transcription factor to VEGF promoter, resulting in the transactivation of VEGF expression and subsequently promoting migration and tube formation in endothelial progenitor cells (EPCs). In vivo, knockdown leptin significantly reduces angiogenesis and tumor growth. ConclusionLeptin may be a therapeutic target of angiogenesis and metastasis in chondrosarcoma. General significanceThese findings provide better understanding of pathogenesis of chondrosarcoma and can utilize this knowledge to design new therapeutic strategy.

Superpulsed (Ga-As, 904 nm) low-level laser therapy (LLLT) attenuates inflammatory response and enhances healing of burn wounds.

Low-level laser therapy (LLLT) using superpulsed near-infrared light can penetrate deeper in the injured tissue and could allow non-pharmacological treatment for chronic wound healing. This study investigated the effects of superpulsed laser (Ga-As 904 nm, 200 ns pulse width; 100 Hz; 0.7 mW mean output power; 0.4 mW/cm(2) average irradiance; 0.2 J/cm(2) total fluence) on the healing of burn wounds in rats, and further explored the probable associated mechanisms of action. Irradiated group exhibited enhanced DNA, total protein, hydroxyproline and hexosamine contents compared to the control and silver sulfadiazine (reference care) treated groups. LLLT exhibited decreased TNF-α level and NF-kB, and up-regulated protein levels of VEGF, FGFR-1, HSP-60, HSP-90, HIF-1α and matrix metalloproteinases-2 and 9 compared to the controls. In conclusion, LLLT using superpulsed 904 nm laser reduced the inflammatory response and was able to enhance cellular proliferation, collagen deposition and wound contraction in the repair process of burn wounds. Photomicrographs showing no, absence inflammation and faster wound contraction in LLLT superpulsed (904 nm) laser treated burn wounds as compared to the non-irradiated control and silver sulfadiazine (SSD) ointment (reference care) treated wounds.

Cytokine gene expression and protein production in West Nile virus‐infected retinal pigment epithelium

AbstractPurpose To investigate if West Nile virus (WNV) infection of human retinal pigment epithelium (RPE) cells induces differential expression of mRNA and proteins for cytokines important for leucocyte recruitment (e.g. CCL5) and integrity of the outer blood retinal barrier (BRB) (e.g. TNF).Methods Primary human RPE cells (P3‐5) were cultured until a complete monolayer was observed, and then infected with 1, 5 or 10 plaque‐forming units (p.f.u.) of WNV (Sarafend) for 24, 48 and 72hrs. Supernatants were collected for Luminex‐based cytokine assay. RNA was prepared from cells and processed for real‐time (RT)‐PCR. Pro‐inflammatory, leucocyte recruitment and viral defense cytokines and chemokines were studied.Results CCL5, IL8, IL6 (pro‐inflammatory) and IFNB1 (anti‐viral) showed increases in mRNA expression from 24 h post‐infection (p.i.) and protein levels from 48h p.i. However in WNV‐infected RPE, protein levels of VEGF and TNF were similar to mock‐infected cells, despite significant increases in mRNA (TNF 24 h p.i., p &lt;0.05; VEGF 48 h, p&lt;0.01). Interestingly, CCL2 mRNA and protein showed no change in expression post‐WNV infection at 24, 48 and 72hConclusion We previously observed that WNV induced transepithelial electrical resistance (TEER) changes in human RPE (outer BRB) after 48 hours, although the mechanisms(s) remain unclear. Studies in mice show that both TNF and CCL2 are important for blood‐brain barrier integrity and leucocyte recruitment following WNV infection. Surprisingly we find little or no effect on TNF or CCL2 expression in WNV‐infected RPE, suggesting alternate mechanisms operate in RPE post‐viral infection. Funding‐ NFMRI and NHMRC

Development of new anti‐VEGF drug by SRPK1 inhibitor screening

AbstractPurpose AMD is a major cause of blindness. Current therapy aims to suppress VEGF levels in order to prevent CNV, which causes AMD. Recently we found that SRPIN340, a specific inhibitor of SR protein kinase 1 (SRPK1) which phosphorylates Serine‐arginine‐rich (SR) proteins and regulates splicing and transcription, suppressed the expression of VEGF and CNV. Here we screened for more active SRPK1 inhibitors which suppressed CNV effectively than SRPIN340.Methods In‐silico screening; Structure‐based virtual screening (SBVS) was applied against the general chemical library, which include 71955 compounds. In vitro kinase assay; The reaction mixture was incubated at 30 °C for 15 min and detected the residual ATP was measured by Kinase Glow following the manufacturer’s instruction. VEGF ELISA; We added 10μM compounds or control to ARPE‐19 cells. The medium was collected 72 hours after the addition of the compounds. We evaluated the VEGF protein level in the culture medium by ELISA with manufacturer’s instruction. In vivo examination; The compound was intravitreally injected to laser CNV model mice immediately after laser photocoagulation. Seven days after laser injury, the mice were perfused with 1mL of 0.5% FITC‐labeled dextran. Flat mounts of RPE‐choroid complex were obtained and the area of CNV was measured by fluorescence microscopy.Results 11994 compounds were selected by SBVS, and we were able to obtain 9535 compounds among them. Next we screened by in vitro kinase assay and selected four compunds, which were named SRPIN801‐804. SRPIN(03 decreased the VEGF protein levels most effectively as measured by VEGF ELISA, and suppressed CNV growth in a dose‐dependent manner.Conclusion We obtained a new anti‐VEGF drug, which inhibits SRPK1 kinase acitivity.