Vascular Endothelial Growth Factor Transcription Research Articles

USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF.

Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).

CREB1 alleviates the apoptosis and potentiates the osteogenic differentiation of zoledronic acid-treated human periodontal ligament stem cells via up-regulating VEGF

Periodontitis represents a severe inflammatory illness in tooth supporting tissue. It has been supported that cAMP response element binding protein 1 (CREB1), a common transcription factor, extensively participates in osteogenic differentiation. Here, the current study was to look into the impacts of CREB1 on the process of periodontitis and its possible action mechanism. After human periodontal ligament stem cells (PDLSCs) were challenged with zoledronic acid (ZA), CREB1 expression was examined with RT-qPCR and western blotting. CCK-8 assay appraised cell activity. Following CREB1 elevation or/and vascular endothelial growth factor (VEGF) silencing in ZA-treated PDLSCs, CCK-8 and TUNEL assays separately estimated cell viability and apoptosis. Western blotting tested the expression of apoptosis- and osteogenic differentiation-associated proteins. ALP staining measured PDLSCs osteogenic ability and ARS staining estimated mineralized nodule formation. JASPAR predicted the potential binding of CREB1 with VEGF promoter, which was then testified by ChIP and luciferase reporter assays. RT-qPCR and western blotting tested VEGF expression. CREB1 expression was declined in ZA-exposed PDLSCs and CREB1 elevation exacerbated the viability and osteogenic differentiation while obstructed the apoptosis of PDLSCs. Additionally, CREB1 bond to VEGF promoter and transcriptionally activated VEGF expression. Further, VEGF absence partially stimulated the apoptosis while suppressed the osteogenic differentiation of CREB1-overexpressing PDLSCs treated by ZA. To be concluded, CREB1 might activate VEGF transcription to obstruct the apoptosis while contribute to the osteogenic differentiation of ZA-treated PDLSCs.

Enhancement of bone regeneration by coadministration of angiogenic and osteogenic factors using messenger RNA

BackgroundBone defects remain a challenge today. In addition to osteogenic activation, the crucial role of angiogenesis has also gained attention. In particular, vascular endothelial growth factor (VEGF) is likely to play a significant role in bone regeneration, not only to restore blood supply but also to be directly involved in the osteogenic differentiation of mesenchymal stem cells. In this study, to produce additive angiogenic-osteogenic effects in the process of bone regeneration, VEGF and Runt-related transcription factor 2 (Runx2), an essential transcription factor for osteogenic differentiation, were coadministered with messenger RNAs (mRNAs) to bone defects in the rat mandible.MethodsThe mRNAs encoding VEGF or Runx2 were prepared via in vitro transcription (IVT). Osteogenic differentiation after mRNA transfection was evaluated using primary osteoblast-like cells, followed by an evaluation of the gene expression levels of osteogenic markers. The mRNAs were then administered to a bone defect prepared in the rat mandible using our original cationic polymer-based carrier, the polyplex nanomicelle. The bone regeneration was evaluated by micro-computerized tomography (μCT) imaging, and histologic analyses.ResultsOsteogenic markers such as osteocalcin (Ocn) and osteopontin (Opn) were significantly upregulated after mRNA transfection. VEGF mRNA was revealed to have a distinct osteoblastic function similar to that of Runx2 mRNA, and the combined use of the two mRNAs resulted in further upregulation of the markers. After in vivo administration into the bone defect, the two mRNAs induced significant enhancement of bone regeneration with increased bone mineralization. Histological analyses using antibodies against the Cluster of Differentiation 31 protein (CD31), alkaline phosphatase (ALP), or OCN revealed that the mRNAs induced the upregulation of osteogenic markers in the defect, together with increased vessel formation, leading to rapid bone formation.ConclusionsThese results demonstrate the feasibility of using mRNA medicines to introduce various therapeutic factors, including transcription factors, into target sites. This study provides valuable information for the development of mRNA therapeutics for tissue engineering.

Electrochemical detection of Oxaliplatin induced DNA damage in G-quadruplex structures

Oxaliplatin (OXP) is a platinum-based chemotherapeutic agent that induces DNA damage by forming intra- and interstrand crosslinks, mainly at the N7s of adenine (A) and guanine (G) bases. In addition to double-stranded DNA, G-rich G-quadruplex (G4)-forming sequences can also be targeted by OXP. However, high doses of OXP can lead to drug resistance and cause serious adverse effects during treatment. To better understand the targeting of G4 structures by OXP, their interactions as well as the molecular mechanisms underlying OXP resistance and adverse effects, there is a need for a rapid, quantitative, and cost-effective method to detect OXP and the damage it causes. In this study, we successfully fabricated a graphite electrode biosensor modified with gold nanoparticles (AuNPs) to investigate the interactions between OXP and the G4-forming promoter region (Pu22) of Vascular endothelial growth factor (VEGF). The overexpression of VEGF is known to be associated with tumor progression and the stabilization of VEGF G4 by small molecules is shown to suppresses VEGF transcription in different cancer cell lines. Differential pulse voltammetry (DPV) was used to investigate the interactions between OXP and Pu22-G4 DNA by monitoring the decrease in the oxidation signal of guanine with increasing OXP concentration. Under the optimized conditions (37 °C, 1:2 v/v AuNPs/water as electrode surface modifier, and 180 min incubation time) the developed probe showed a linear dynamic range of 1.0–10.0 μM with a detection limit of 0.88 μM and limit of quantification of 2.92 μM. Fluorescence spectroscopy was also used to support the electrochemical studies. We observed a decrease in the fluorescence emission of Thioflavin T in the presence of Pu22 upon addition of OXP. To our knowledge, this is the first electrochemical sensor developed to study OXP-induced damage to G4 DNA structures. Our findings provide new insights into the interactions between VEGF G4 and OXP, which could aid in targeting VEGF G4 structures and the development of new strategies to overcome OXP resistance.

Abstract 4604: Effect of CDK 4/6 inhibition on anti-angiogenesis effect in gastric cancer

Abstract Angiogenesis, a hallmark of cancer, is a phenomenon in which new blood vessels are created from existing blood vessels. As the tumor grows, they secrete ligands such as vascular endothelial growth factor (VEGF). Therefore, anti-tumor effect could be achieved by preventing tumor-associated angiogenesis. In previous reports, CDK4/6 has been acknowledged to regulate VEGF transcription in addition to cell cycle regulation. There are few studies explaining the angiogenesis-related effects of CDK4/6 inhibitors in gastric cancer (GC). In this study, we aim to verify the anti-angiogenesis effect through CDK4/6 inhibition. First, we confirmed proliferation and angiogenesis in endothelial cells. Next, the change in VEGF expression induced by CDK4/6 inhibitor in GC cell lines was investigated. In-vitro angiogenesis research was conducted using human umbilical vein endothelial cells (HUVEC). Abemaciclib was used as a CDK4/6 inhibitor. To determine the efficacy of abemaciclib, HUVEC was treated with abemaciclib for 3 days and cell viability was assessed by CCK-8 assay. IC50 value was determined by the CalcuSyn software. Furthermore, tube formation assay, wound healing assay, cell cycle analysis and SA-β-gal assay were performed to explore effect of abemaciclib on HUVEC. VEGF-A expression was evaluated by ELISA. First, we identified direct anti-angiogenesis effect of abemaciclib on HUVEC. The IC50 value was 1.28 μM. At clinically achievable concentrations of abemaciclib (Cmax, 0.5 μM), HUVEC only showed a 20% growth inhibition. Abemaciclib did not suppress tube formation and migratory potential of HUVECs. However, abemaciclib induced a G1 arrest in HUVEC after 0.5 μM abemaciclib treatment for 24hr. To ensure whether the increase in the number of G1 phase cells is due to senescence, SA-β-gal assay was performed. The levels of SA-β-gal-positive cell staining in abemaciclib-treated group were higher than control, suggesting that abemaciclib induced cell senescence on HUVEC. While our results suggested that CDK4/6 inhibitor did not show direct anti-angiogenic effect on endothelial cells, we studied indirect anti-angiogenesis effect of abemaciclib in GC cell lines through regulation of VEGF transcription. Conditioned media of 49 GC cell lines had a wide range of VEGF secretion ranged from 0.146 to 22.676 ng/mL. (ave. 4.07 ng/mL). We have set the cut off of secreted VEGF as 10 ng/mL, at which angiogenesis can be induced. There were four cell lines (4/49, 8.2%) whose VEGF concentrations were more than the angiogenesis-inducing VEGF concentration (10 ng/mL). Interestingly, 3 of these 4 GC cell lines demonstrated a decrease in VEGF secretion, following 0.5 μM abemaciclib treatment. In conclusion, we did not observe inhibition of angiogenesis in HUVEC. However, CDK4/6 inhibition can cause senescence in HUVEC, inhibit tumor growth, and indirectly induce anti-angiogenic effects in gastric cancer. Citation Format: Sang Woo Cho, Woo Sun Kwon, Tae Soo Kim, Juin Park, Eun Seo Kim, Hyun Cheol Chung, Sun Young Rha. Effect of CDK 4/6 inhibition on anti-angiogenesis effect in gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4604.

Phosphofructokinase 1 Platelet Isoform Enhances VEGF Expression in Part Through HIF-1α Up-regulation in Breast Cancer.

Phosphofructokinase 1 platelet isoform (PFKP) catalyzes a rate-limiting reaction in glycolysis. It is highly expressed in several tumors, including breast cancer (BC). It can regulate tumor progression through metabolic reprogramming and gene transcription. In addition, overexpression of vascular endothelial growth factor (VEGF) is commonly observed in BC, which is associated with poor prognosis. However, whether PFKP regulates VEGF expression in BC remains unknown. Thus, the aim of this study was to investigate whether PFKP could regulate VEGF expression in BC. We designed an in vitro study to investigate the role of PFKP in VEGF expression and angiogenesis using several experiments, including shRNA-mediated PFKP knock-down, RNAi-resistant PFKP restoration, qPCR, immunoblotting, luciferase reporter assay and tube formation assay. The clinical relationship between PFKP and VEGF was analyzed using The Cancer Genome Atlas (TCGA) database. PFKP expression was associated with VEGF expression in BC patients from the TCGA database. Importantly, PFKP played an essential role in the EGFR activation-induced VEGF expression in BC cells. Mechanistically, EGFR-phosphorylated PFKP Y64 played a critical role in AKT-mediated transcriptional expression of HIF-1α and subsequent VEGF transcription. Hence, PFKP expression played a role in human umbilical vein endothelial cells (HUVECs) tube formation by regulating VEGF expression in BC cells. These findings highlight a novel mechanism underlying the non-metabolic function of PFKP in VEGF expression in BC and provide a therapeutic potential of targeting PFKP in BC patients.

Mutual regulation between phosphofructokinase 1 platelet isoform and VEGF promotes glioblastoma tumor growth

Glioblastoma (GBM) is a highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, whether PFKP and VEGF are reciprocally regulated during GBM tumor growth remains unknown. Here, we show that PFKP can promote EGFR activation-induced VEGF expression in HIF-1α-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in both AKT/SP1-mediated transcriptional expression of HIF-1α and in the AKT-mediated β-catenin S552 phosphorylation, to fully enhance VEGF transcription, subsequently promoting blood vessel formation and brain tumor growth. Levels of PFKP Y64 phosphorylation in human GBM specimens are positively correlated with HIF-1α expression, β-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth and also suggest that targeting the PFKP/VEGF regulatory loop might show therapeutic potential for treating GBM patients.

Bladder Cancer-Derived Small Extracellular Vesicles Promote Tumor Angiogenesis by Inducing HBP-Related Metabolic Reprogramming and SerRS O-GlcNAcylation in Endothelial Cells.

A malformed tumour vascular network provokes the nutrient‐deprived tumour microenvironment (TME), which conversely activates endothelial cell (EC) functions and stimulates neovascularization. Emerging evidence suggests that the flexible metabolic adaptability of tumour cells helps to establish a metabolic symbiosis among various cell subpopulations in the fluctuating TME. In this study, the authors propose a novel metabolic link between bladder cancer (BCa) cells and ECs in the nutrient‐scarce TME, in which BCa‐secreted glutamine‐fructose‐6‐phosphate aminotransferase 1 (GFAT1) via small extracellular vesicles (sEVs) reprograms glucose metabolism by increasing hexosamine biosynthesis pathway flux in ECs and thus enhances O‐GlcNAcylation. Moreover, seryl‐tRNA synthetase (SerRS) O‐GlcNAcylation at serine 101 in ECs promotes its degradation by ubiquitination and impeded importin α5‐mediated nuclear translocation. Intranuclear SerRS attenuates vascular endothelial growth factor transcription by competitively binding to the GC‐rich region of the proximal promotor. Additionally, GFAT1 knockout in tumour cells blocks SerRS O‐GlcNAcylation in ECs and attenuates angiogenesis both in vitro and in vivo. However, administration of GFAT1‐overexpressing BCa cells‐derived sEVs increase the angiogenetic activity in the ECs of GFAT1‐knockout mice. In summary, this study suggests that inhibiting sEV‐mediated GFAT1 secretion from BCa cells and targeting SerRS O‐GlcNAcylation in ECs may serve as novel strategies for BCa antiangiogenetic therapy.

Influence of cilostazol on thromboangiitis obliterans in rats and the mechanism involved

Purpose: To study the effect of Cilostazol on rat thromboangiitis obliterans (TAO), and the mechanism of action involved.
 Methods: Rats (N = 45) were injected with sodium laurate to establish the model of TAO, and then divided into Sham group (n = 15), TAO group (n = 15) and TAO + cilostazol group (n = 15). After administration of cilostazol, TAO lesions in the rats were graded, and the femoral arteries were stained by hematoxylin-eosin (H&E) to determine the degree of vascular lesions. The status of vascular endothelial cells was determined using transmission electron microscopy. Furthermore, the expression and transcription levels of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) proteins were evaluated by Western blotting and real-time polymerase chain reaction (RT-PCR) respectively.
 Results: In contrast to Sham group, TAO group exhibited symptoms such as changes in skin temperature and color, and limb swelling and thanatosis, while in the TAO + cilostazol group, the damage was reversed, vascular and vascular endothelial cell lesions were significantly ameliorated (p < 0.05), and the transcription and translation levels of HIF-1α and VEGF significantly suppressed (p < 0.05).
 Conclusion: Cilostazol alleviates sodium laurate-induced TAO lesions in rats via HIF-1α/VEGF pathway. This study may provide new insights for the treatment of TAO.
 Keywords: Cilostazol; Hypoxia-inducible factor/vascular endothelial growth factor pathway; Thromboangiitis obliterans; Limb swelling; Thanatosis

Low oxygen concentration improves yak oocyte maturation and inhibits apoptosis through HIF-1 and VEGF.

The gas-phase environment of in vitro culture system plays an important role in the development of oocytes, and oxygen concentration is one of the important factors. In the present study, we aimed to explore the effect of different oxygen concentrations (20%, 10%, 5% or 1% O2 ) in yak oocyte maturation and to detect the expression of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF) and cell apoptosis in yak COCs. First, the maturation rate of oocytes, cleavage rate and blastocysts rate following parthenogenetic activation in the group with 5% oxygen concentration were significantly higher (p<.05) than the other groups. Then, TUNEL analysis showed that the 5% oxygen concentration group significantly inhibited apoptosis of cumulus-oocyte complexes (COCs) compared to the other group, and the transcription and protein levels of pro-apoptotic factor Bax, HIF-1α and VEGF in yak COCs significantly reduced, while anti-apoptotic factor Bcl-2 significantly increased. Furthermore, immunohistochemical staining results indicated that HIF-1α protein was mainly located in theca follicle interna, mural follicular stratum granulosum, corona radiata and ovarian stroma in the follicular ovarian tissue, while VEGF protein was mainly located in the granulosa and theca cell layers. In summary, our findings demonstrate that 5% oxygen concentration may promote maturation and inhibit apoptosis of oocytes through HIF-1α-mediated VEGF expression.

Transcriptional Regulation of Thrombin-Induced Endothelial VEGF Induction and Proangiogenic Response

Thrombin, the ligand of the protease-activated receptor 1 (PAR1), is a well-known stimulator of proangiogenic responses in vascular endothelial cells (ECs), which are mediated through the induction of vascular endothelial growth factor (VEGF). However, the transcriptional events underlying this thrombin-induced VEGF induction and angiogenic response are less well understood at present. As reported here, we conducted detailed promotor activation and signal transduction pathway studies in human microvascular ECs, to decipher the transcription factors and the intracellular signaling events underlying the thrombin and PAR-1-induced endothelial VEGF induction. We found that c-FOS is a key transcription factor controlling thrombin-induced EC VEGF synthesis and angiogenesis. Upon the binding and internalization of its G-protein-coupled PAR-1 receptor, thrombin triggers ERK1/2 signaling and activation of the nuclear AP-1/c-FOS transcription factor complex, which then leads to VEGF transcription, extracellular secretion, and concomitant proangiogenic responses of ECs. In conclusion, exposure of human microvascular ECs to thrombin triggers signaling through the PAR-1–ERK1/2–AP-1/c-FOS axis to control VEGF gene transcription and VEGF-induced angiogenesis. These observations offer a greater understanding of endothelial responses to thromboinflammation, which may help to interpret the results of clinical trials tackling the conditions associated with endothelial injury and thrombosis.

PRMT4 drives post-ischemic angiogenesis via YB1/VEGF signaling.

Angiogenesis is an integral process in many ischemic disorders, and vascular endothelial growth factor (VEGF) plays an important role in it. Protein arginine methyltransferase 4 (PRMT4), a member of the type I PRMT family, is involved in various biological activities, but its role in endothelial cell (EC) remains elusive. Here, we aimed to investigate the role of PRMT4 in ischemic angiogenesis and explore the possible underlying mechanism. We found that PRMT4 was upregulated in ischemic muscles, and VEGF treatment potentiated its expression in ECs. In vitro, adenovirus-mediated PRMT4 overexpression promoted, while its gene disruption inhibited, EC proliferation, migration, and tube formation. In an in vivo hindlimb ischemia model, forced expression of PRMT4 in ECs showed accelerated blood flow recovery and increased capillary density, whereas its knockdown exhibited the opposite effect. Mechanistically, PRMT4 activated the transcription of VEGF via the interaction with Y-box binding protein-1 (YB1), leading to accelerated angiogenesis. Interestingly, the loss of YB1 partially abolished PRMT4-mediated angiogenesis in vitro. Collectively, our data revealed that PRMT4 promoted angiogenesis through interacting with YB1 and the consequential VEGF upregulation, suggesting that PRMT4 may present as a potential therapeutic target in ischemic angiogenesis. KEY MESSAGES: •PRMT4 is induced by VEGF and upregulated in a hindlimb ischemia model. •PRMT4 promotes angiogenesis both in vitro and in vivo. •PRMT4 regulates VEGF expression through interacting with YB1. •YB1 knockdown retards PRMT4-mediated angiogenic effects in vitro.

Effect of low VEGF on lung development and function.

Vascular endothelial growth factor (VEGF) is important for lung development and function but ideal mouse models are limited to decipher the quantitative relationship between VEGF expression levels and its proper development and pathogenesis. Human SPC promoter has been used to faithfully express genes or cDNAs in the pulmonary epithelium in many transgenic mouse models. In the study, a mouse model of lung-specific and reversible VEGF repression (hspc-rtTRtg/+/VegftetO/tetO) was generated. Human SPC promoter was used to drive lung-specific rtTR expression, a cDNA coding for doxycycline-regulated transcription repression protein. By crossing with VegftetO/tetO mice, that has tetracycline operator sequences insertion in 5'-UTR region, it allows us to reversibly inhibit lung VEGF transcription from its endogenous level through doxycycline food, water or injection. The tissue-specific inhibition of VEGF is used to mimic abnormal expression levels of VEGF in lung. Reduced VEGF expression in lung is confirmed by quantitative real time PCR and immunoblotting. Lung development and structure was analyzed by histology analysis and found significantly affected under low VEGF. The pulmonary epithelium and alveolarization are found abnormal with swelling alveolar septum and enlargement of air space. Genome-wide gene expression analysis identified that immune activities are involved in the VEGF-regulated lung functions. The transgenic mouse model can be used to mimic human pulmonary diseases. The mouse model confirms the important regulatory roles of epithelial expressed VEGF in lung development and function. This mouse model is valuable for studying VEGF-regulated lung development, pathogenesis and drug screening under low VEGF expression.

Inhibitory Effects of Pectic Polysaccharide Isolated from Diospyros kaki Leaves on Tumor Cell Angiogenesis via VEGF and MMP-9 Regulation.

Persimmon leaves are an attractive source of phytochemicals with potential health benefits. However, there are only a few reports on the chemical properties and biological activity of the polysaccharide fractions (PLE-I–III) of persimmon leaves. We evaluated the angiogenesis-inhibiting ability of pectic-polysaccharides. The molecular weight of PLEs was determined using a high-performance size-exclusion chromatography system. Tube formation assay of human umbilical vein endothelial cells (HUVECs) was performed using Matrigel-coated 96-well plates. Matrix metalloproteinase (MMP-9), vascular endothelial growth factor (VEGF), PI3K, Akt, and p38 phosphorylation levels were determined using Western blotting; VEGF and MMP-9 transcript levels were measured using SYBR Green qRT-PCR. PLE-I–III significantly inhibited HUVEC tube formation at 12.5 and 25 μg/mL. Among them, PLE-II showed the strongest anti-tube formation activity, and the mRNA/protein expression of angiogenesis-related factors (VEGF/MMP-9) was significantly reduced by PLE-II. PLE-II also suppressed the phosphorylation of PI3K/AKT and p38, JNK, and NF-κB p65 in HUVECs. These results suggest that the polysaccharide PLE-II isolated from persimmon leaves inhibited VEGF and MMP-9 expression in HUVECs via regulation of PI3K/AKT, p38, JNK, and NF-κB p65 signaling pathways.

Signaling in TNFSF15-mediated Suppression of VEGF Production in Endothelial Cells.

Vascular endothelial growth factor (VEGF) plays a pivotal role in promoting neovascularization. Tumor necrosis factor superfamily 15 (TNFSF15) is an antiangiogenic cytokine prominently produced by endothelial cells in a normal vasculature. In this study, Western blot, quantitative polymerase chain reaction (qPCR), and dual luciferase reporter gene assay were used to validate the mechanisms of TNFSF15-mediated suppression of VEGF production in endothelial cells. We report that TNFSF15 inhibits VEGF production via microRNA-29b (miR-29b) targeting the 3'-UTR of VEGF transcript in mouse endothelial cell line bEnd.3. Neutralizing antibody against TNFSF15, 4-3H, inhibits the level of miR-29b and reinvigorates VEGF. In addition, TNFSF15 activates the JNK signaling pathway as well as the transcription factor GATA3, resulting in enhanced miR-29b production. SP600125, an inhibitor of JNK, eradicates TNFSF15-induced GATA3 expression. Moreover, GATA3 siRNA suppressed TNFSF15-induced miR-29b expression. Together, this study provides evidence and method of activation of the JNK-GATA3 signaling pathway by TNFSF15 that suppresses VEGF gene expression, which gives rise to upregulation of miR-29b.

Chrysin Inhibits High Glucose-Induced Migration on Chorioretinal Endothelial Cells via VEGF and VEGFR Down-Regulation

Background: Diabetes mellitus (DM) is a chronic inflammatory disease, which causes multiple complications. Diabetic retinopathy (DR) is among these complications and is a dominant cause of vision loss for diabetic patients. Numerous studies have shown that chrysin, a flavonoid, has many biological activities such as anti-oxidation and anti-inflammation. However, it is rarely used in ocular diseases. In this study, we examined the inhibitory effects of flavonoid on high glucose induced migration of chorioretinal endothelial cells (RF/6A cells) and its mechanism. Materials and methods: The viability of RF/6A cells treated with chrysin was examined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The migration of RF/6A cells was assessed by the transwell migration and scratch wound assays. The expression of AKT, ERK, vascular endothelial growth factor (VEGF), HIF−1α and MMP-2 were determined by western blotting. To observe the mRNA expression of VEGF receptor (VEGFR), qRT-PCR, was utilized. Results: The results showed that chrysin can dose-dependently inhibit the RF/6A cell migration in vitro transwell and the scratch wound assays which are induced by high glucose. After pretreatment of RF/6A cells with different concentrations of chrysin, they did not produce any cytotoxicity in MTT assay. Moreover, chrysin down-regulated both phosphorylated AKT and ERK, as well as attenuated the expression levels of MMP-2. It also decreased the expression of the VEGF transcription factor and VEGF. Furthermore, it was shown that chrysin could suppress the protein and mRNA expression levels of VEGFR. Conclusion: The results indicate that chrysin could down-regulate the phosphorylation of AKT, ERK and MMP-2 and reduce the effects of VEGF and VEGFR in a high glucose environment. It further inhibits the high glucose-induced migration of RE/6A cells. Therefore, chrysin may have the potential for visual protection.

Ginsenoside Re Attenuates High Glucose-Induced RF/6A Injury via Regulating PI3K/AKT Inhibited HIF-1α/VEGF Signaling Pathway.

Hyperglycaemia-induced retinal microvascular endothelial cell apoptosis is a critical and principle event in diabetic retinopathy (DR), which involves a series of complex processes such as mitochondrial dysfunction and oxidative stress. Ginsenoside Re (Re), a key ingredients of ginseng, is considered to have various pharmacologic functions, such as antioxidative, inhibition of inflammation and anti-apoptotic properties. However, the effects of Re in DR and the related mechanisms of endothelial cell injury induced by high glucose (HG) exposure remain unclear. The present study was designed to investigate and evaluate the ability of Re to ameliorate HG-induced retinal endothelial RF/6A cell injury and the potential mechanisms involved in the hypoxia-inducible factor-1-alpha (HIF-1α)/vascular endothelial growth factor (VEGF) signaling regulated by phosphoinositide 3-kinase (PI3K)/AKT pathway. Our results showed that preincubation with Re exerted cytoprotective effects by reversing the HG-induced decrease in RF/6A cell viability, downregulation of apoptosis rate and inhibition of oxidative-related enzymes, thereby reducing the excess intracellular reactive oxygen species (ROS) and HG-triggered RF/6A cell injury. In addition, Western blot analysis results showed ginsenoside Re significantly increased HIF-1α expression in the cytoplasm but decreased its expression in the nucleus, suggesting that it reduced the translocation of HIF-1α from the cytoplasm to the nucleus, and downregulated VEGF level. Moreover, this effect is involved in the activation of the PI3K/Akt pathway. LY294002, a PI3K inhibitor, was used to block the Akt pathway. Afterwards, the effects of Re on the regulation of apoptotic related proteins, VEGF and HIF-1α nuclear transcription was partially reversed. These findings suggested the exerting protective effects of ginsenoside Re were associated with regulating of PI3K/AKT and HIF-1α/VEGF signaling pathway, which indicates that ginsenoside Re may ameliorates HG-induced retinal angiogenesis and suggests the potential for the development of Re as a therapeutic for DR.

Changes in gene expression patterns in postmortem human myocardial infarction

In murine models, the expression of inducible nitric oxide synthase (iNOS) in myocardial infarction (MI) has been reported to be the result of tissue injury and inflammation. In the present study, mRNA expression of iNOS, hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF) was investigated in postmortem human infarction hearts. Since HIF-1α is the inducible subunit of the transcription factor HIF-1, which regulates transcription of iNOS and VEGF, the interrelation between the three genes was observed, to examine the molecular processes during the emergence of MI. iNOS and VEGF mRNAs were found to be significantly upregulated in the affected regions of MI hearts in comparison to healthy controls. Upregulation of HIF-1α was also present but not significant. Correlation analysis of the three genes indicated a stronger and significant correlation between HIF-1α and iNOS mRNAs than between HIF-1α and VEGF. The results of the study revealed differences in the expression patterns of HIF-1 downstream targets. The stronger transcription of iNOS by HIF-1 in the affected regions of MI hearts may represent a pathological process, since no correlation of iNOS and HIF-1α mRNA was found in non-affected areas of MI hearts. Oxidative stress is considered to cause molecular changes in MI, leading to increased iNOS expression. Therefore, it may also represent a forensic marker for detection of early changes in heart tissue.

CCAAT/Enhancer-Binding Protein β Mediates Oxygen-Induced Retinal Neovascularization via Retinal Vascular Damage and Vascular Endothelial Growth Factor.

Objective To evaluate the role of CCAAT/enhancer-binding protein β (C/EBP β (C/EBP Methods Rats with OIR were exposed to alternating hypoxic and hyperopic conditions for 14 days. Then, the rats with OIR were assigned randomly to groups that received intravitreal injections of either shRNA lentiviral particles targeting C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP Results In OIR rats, the expression levels of C/EBP β (C/EBP P < 0.01). The p-C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP β (C/EBP P < 0.01). The p-C/EBP β (C/EBP β (C/EBP β (C/EBP P < 0.01). The p-C/EBP Conclusions C/EBP β shRNA inhibits RNV in OIR. A potential mechanism may be that the activity of C/EBP β increases with its overexpression, which in turn aggravates the amount of the retinal vascular damage and promotes transcription of VEGF. C/EBP β might be a new therapeutic target for preventing RNV.β (C/EBP β (C/EBP β (C/EBP

Neem Leaf Glycoprotein Restrains VEGF Production by Direct Modulation of HIF1α-Linked Upstream and Downstream Cascades.

Neem Leaf Glycoprotein (NLGP) is a natural immunomodulator, have shown sustained tumor growth restriction as well as angiogenic normalization chiefly by activating CD8+ T cells. Here, we have investigated the direct role of NLGP as a regulator of tumor microenvironmental hypoxia and associated vascular endothelial growth factor (VEGF) production. We observed a significant reduction in VEGF level in both in vivo murine tumor and in vitro cancer cells (B16Mel, LLC) and macrophages after NLGP treatment. Interestingly, NLGP mediated VEGF downregulation in tumor cells or macrophages within hypoxic chamber was found at an early 4 h and again at late 24 h in mRNA level. Our data suggested that NLGP prevented hypoxia-induced strong binding of HIF1α with its co-factors, CBP/p300 and Sp3, but not with Sp1, which eventually limit the binding of HIF1α-transcriptional complex to hypoxia responsive element of VEGF promoter and results in restricted early VEGF transcription. On the otherhand, suppressed phosphorylation of Stat3 by NLGP results reduction of HIF1α at 24 h of hypoxia that further support sustained VEGF down-regulation. However, NLGP fails to regulate VHL activity as observed by both in vivo and in vitro studies. Therefore, this study for the first time reveals a mechanistic insight of NLGP mediated inhibition of angiogenesis by suppressing VEGF, which might help in vascular normalization to influence better drug delivery.