Hypoxia-inducible Vascular Endothelial Growth Factor Research Articles

Tumor suppressor protein p53 negatively regulates ischemia-induced angiogenesis and arteriogenesis

ObjectiveThe tumor suppressor protein p53 regulates angiogenesis and is a key regulatory mediator of cellular apoptosis, proliferation, and growth. p53 expression is induced in response to ischemia; however, its role in regulating ischemia-induced angiogenesis and arteriogenesis remains undefined. The objective of this study was to define the role of p53 in regulating ischemia-induced angiogenesis and arteriogenesis and to identify mechanisms by which this regulation occurs in vivo. MethodsSurgically induced hindlimb ischemia or mesenteric artery ligation was performed in wild-type (p53+/+) and p53 knockout (p53−/−) mice. Limb perfusion and revascularization were assessed by laser Doppler perfusion imaging, capillary density, and collateral artery development. Mesenteric collateral artery flow and development were determined by arterial flow measurement and by histologic analysis, respectively. An in vitro aortic ring assay was performed on p53+/+ and p53−/− aortic tissue to evaluate endothelial function. The p53 inhibitor and activator pifithrin-α and quinacrine, respectively, were used to modulate p53 activity in vivo after ischemia. ResultsAbsence of p53 in mice resulted in increased limb perfusion (P < .05), capillary density (P < .05), and collateral artery development (P < .05) after induction of hindlimb ischemia. In the nonischemic mesenteric artery ligation model of arteriogenesis, p53 expression was induced in collateral arteries and increased arterial blood flow in mice lacking p53 (P < .05). Lack of p53 decreased apoptosis in ischemic hindlimb tissue (P < .05) and increased proangiogenic factors hypoxia-inducible factor 1α and vascular endothelial growth factor (VEGF). Endothelial cell outgrowth in vitro increased in the absence of p53 (P < .05). Pharmacologic augmentation of p53 expression after ischemia impaired perfusion and collateral artery formation and decreased VEGF levels (P < .05). Conversely, inhibition of p53 with pifithrin-α augmented limb perfusion (P < .05) and collateral artery formation (P < .05) and increased protein levels of hypoxia-inducible factor 1α and VEGF. Pharmacologic augmentation and inhibition of p53 had no significant effect in mice lacking p53. Conclusionsp53 negatively regulates ischemia-induced angiogenesis and arteriogenesis. Inhibition of p53 increases ischemia-induced arteriogenesis and limb perfusion and thus represents a potential therapeutic strategy for arterial occlusive disease.

Abstract B25: Meformin and LY294002 treatment regulate angiogenesis process through PI3K-MAPK/AKT/mTOR signaling pathways in mammary tumors

Abstract Introduction: The angiogenesis process is regulated by numerous factors, especially the hypoxia-inducible factor 1alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). The HIF-1 is a central regulator of pathophysiologic response of cells to hypoxia conditions, able to activate transcription of the gene that promotes the induction of VEGF, which in turn promotes angiogenesis through its ability to stimulate growth, migration, and invasion of endothelial cells, leading to the formation of new blood vessels and subsequent tumor growth. Metformin has demonstrated its ability to reduce the incidence of cancer in diabetic patients with positive results, especially in breast cancer, inhibiting cell growth by AMPK/Akt/mTOR signaling pathway. Likewise, LY294002, a PI3K signaling pathway inhibitor, has antiangiogenic features. In this context, the aim of this study was to evaluate the effectiveness of metformin and LY294002 inhibitor treatment in angiogenesis as a therapeutic strategy in mammary tumors in an in vitro and in vivo study. Materials and Methods: In the in vitro study, canine mammary tumor cell line (CF41) was cultured in DMEM high-glucose culture medium, at 37°C in 5% CO2. Cell viability was measured by MTT assay after treatment with different concentrations of metformin and LY294002 inhibitor. Once established the concentration of 5 mM for metformin and 5 µM of LY294002, the protein and gene expression of HIF-1alpha and VEGF were detected by immunocytochemistry and real-time PCR, respectively. For an in vivo study, CF41 were injected in nude athymic female mice and treated with metformin (200 mg/kg i.p.) for 4 weeks and LY294002 (7.5 mg/kg intratumorally) every 3 days for three times. At the end, mice were euthanized and the tumors were collected to determine the protein and gene expression of HIF-1alpha and VEGF and microvessel density by immunohistochemistry for CD31. The immunostaining was quantified by optical densitometry technique, slides were analyzed and photographed in Nikon Eclipse E200 microscope at 40X objective, and proteins were quantified by the ImageJ software analysis. For real-time PCR, the relative expression of the genes of interest was determined by DataAssist v3.0 software, using Ct method. Results: There was a significant decrease of cell viability after treatment with different concentrations of metformin and LY294002 in 24 hours. Both HIF-1alpha and VEGFA protein and gene expression significantly decreased after treatment with metformin and LY294002 or in combination. Furthermore, in animals, there was a decrease in tumor size, protein and gene expression of HIF-1alpha and VEGFA, in addition to a decrease of CD31 expression after all treatments. Conclusion: Our results suggest the potential effectiveness of metformin and LY294002 acting in angiogenesis process of mammary tumors. Financial support: FAPESP. Citation Format: Marina Gobbe Moschetta, Camila Leonel, Larissa Bazela Maschio-Signorini, Thaiz Ferraz Borin, Gabriela Bottaro Gelaleti, Lívia Carvalho Ferreira, Bruna Victorasso Jardim-Perassi, Nathália Martins Sonehara, Debora Aparecida Pires de Campos Zuccari. Meformin and LY294002 treatment regulate angiogenesis process through PI3K-MAPK/AKT/mTOR signaling pathways in mammary tumors [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B25.

MicroRNA-16 inhibits hypoxia-induced vascular endothelial growth factor expression in ARPE-19 cells

Purpose: To investigate the effect of microRNA-16 on hypoxia-induced VEGF expression of ARPE-19 cells.Methods: ARPE-19 cells were cultivated under normoxia and hypoxia state. At 0 h, 12 h, 24 h, and 48 h after cultivation, the supernate of the culture medium was separated to test the VEGF secretion by ELISA, and the cells were purified to measure the expression of VEGF mRNA and microRNA-16 by qRT-PCR; microRNA-16 mimic was then transfected into ARPE-19 cells by the Hiperfect transfection reagent, a liposome transfection system. Scramble group and the non-transfected group were set as the controls. VEGF secretion and the level of VEGF mRNA were measured in these three groups.Results: The VEGF secretion of the hypoxia ARPE cells was significantly higher than the initial state (p < 0.01). Compared with the normoxia cells, the VEGF secretion of the hypoxia cell was significantly increased (p < 0.01), and the division of VEGF secretion between these two groups increased by time. VEGF mRNA of the hypoxia ARPE cell was significantly higher than the initial state (p < 0.01). Compared with the normoxia cells, VEGF mRNA of the hypoxia cells was significantly increased (p < 0.01), and the division of VEGF mRNA between these two groups increased by time. After cultivating under hypoxia, the expression of microRNA-16 in ARPE-19 cells decreased significantly compared with the normal group, and the division of these two groups augmented by time. MicroRNA-16 was successfully transfected into ARPE-19 cells by the Hiperfect transfection system. Twenty four hours and 48 h after the transfection, the VEGF secretion of the miR-16 transfected cell was decreased significantly (p < 0.01) compared with the scramble and the non-transfected group under hypoxia, while VEGF mRNA level had no significant difference among these three groups.Conclusions: Hypoxia can increase the expression of VEGF mRNA and the secretion of VEGF protein of ARPE-19 cells. At the same time, microRNA-16 expression can be down-regulated by hypoxia. Transfection of microRNA-16 exogenously can down-regulate the VEGF protein secretion but cannot affect the expression of VEGF mRNA.

Gene delivery of hypoxia-inducible VEGF targeting collagen effectively improves cardiac function after myocardial infarction

Vascular endothelial growth factor (VEGF) plays important roles in improvement of cardiac function following myocardial infarction (MI). However, the lack of a steerable delivery system of VEGF targeting the infarcted myocardium reduces the therapeutic efficacy and safety. Here, we constructed a series of lentiviral vector systems which could express a fusion protein consisted of a collagen-binding domain (CBD) and hVEGF (CBDhVEGF), under the control of 5HRE-hCMVmp (5HRE), the hypoxia-inducible promoter consists of five copies of the hypoxia-responsive element (HRE) and a human cytomegalovirus minimal promoter (hCMVmp). We demonstrated that 5HRE has the comparable ability to strongly drive CBDhVEGF under hypoxic condition as the ubiquitous CMV promoter, but it can hardly drive target gene under normoxic condition. 5HRE-drived CBDhVEGF specifically bound to type I collagen and significantly promoted the viability of HUVEC cells. Moreover, after injection of lentivirus into heart of mouse with MI, CBDhVEGF was mainly retained in infarcted myocardium where containing rich collagen and significantly improved angiogenesis and cardiac function when compared with hVEGF. Moreover, CBDhVEGF mediated by lentivirus has little leakage from infarcted zone into blood than hVEGF. Taken together, our results indicate that 5HRE-CBDhVEGF lentiviral vector system could improve cardiac function in the collagen-targeting and hypoxia-inducible manners.

HIF-1α and VEGF levels for monitoring hepatocellular carcinoma treatment response to transcatheter arterial chemoembolization

Background: The purpose of the present study was to investigate the use of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) levels to monitor the treatment response to transcatheter arterial chemoembolization (TACE) in hepatocellular carcinoma (HCC). Methods: A total of 41 consecutive HCC patients underwent TACE were enrolled into this study. The serum levels of HIF-1α and VEGF were measured using enzyme-linked immunosorbent assays (ELISAs) 1 day before, and 1, 7 and 28 days after TACE therapy. The overall tumor response was evaluated according to the modified Response Evaluation Criteria in Solid Tumors criteria. Patients with a complete response or partial response comprised the responding group, whereas those with stable disease or progressive disease comprised the non-responding group. The differences in serum HIF-1α and VEGF levels before and after TACE therapy were subjected to analysis of nonparametric test, while correlations between serum HIF-1α and VEGF levels were examined using Pearson’s correlation analysis. Receiver-operating characteristic (ROC) curve was applied to analyze the evaluation value of factors for the response of TACE on the treatment of HCC. P Results: In the present study, the serum levels of HIF-1α correlated positively with the serum levels of VEGF 1 day before TACE (r=0.546, P=0.000). The levels of HIF-1α and VEGF 1 day before, and 1, 7 and 28 days after TACE were significantly different, respectively (χ 2 =90.688, P=0.000 and χ 2 =45.585, P=0.000). The levels of the HIF-1α and VEGF increased markedly on day 1 and 7 after TACE and recovered to the pre-TACE level on day 28 after TACE. The levels of serum VEGF in responder group 28 days after TACE were significantly lower than those in non-responder group (Z=2.774, P=0.006), but the difference of HIF-1α levels between the two groups was not significant (Z=1.905, P=0.057). ROC curve analysis indicated that the sensitivity and specificity were 76.9% and 78.6%, when the threshold value was set at VEGF =254.5 pg/mL for predicting the response of TACE in patients with HCC; the corresponding area under the curve (AUC) was 0.772, respectively. Conclusions: The levels of both HIF-1α and VEGF in patients with HCC after TACE exhibit dynamic changes. However, HIF-1α and VEGF may be insufficient for predicting tumor response to TACE treatment.

BIOMOLECULES MEDIATED TARGETING OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN NEURONAL DYSFUNCTION: AN IN SILICO APPROACH

Objective: Neurodegenerative diseases are a debilitating age-related disorder manifested by memory loss, impaired motor activity, and loss of muscle tone due to the accumulation of toxic metabolites in the brain. Despite the knowledge of factors causing neurodegenerative disorders, it remains irreversible and incurable. Growing evidence have currently advocated the physiological and pathological contribution of hypoxia-induced vascular endothelial growth factor (VEGF) in neuronal loss. The objective of this research report highlights biomolecules mediated targeting of VEGF activity based on in silico approaches that could establish a potential therapeutic window for the treatment of different abnormalities associated with impaired VEGF.Methods: We employed various in silico methods such as drug-likeness parameters, namely, Lipinski filter analysis, Pock Drug tool for active site prediction, AUTODOCK 4.2.1, and LigPlot1.4.5 for molecular docking studiesResults: Three-dimensional structure of VEGF was generated and Ramachandran plot obtained for quality assessment. RAMPAGE displayed 99.5% of residues in the most favored regions, 0.5% residues in additionally allowed, and no residues in disallowed regions in VEGF, showing that stereochemical quality of protein structure is good. Further, initial screenings of the molecules were done based on Lipinski’s rule of five. Finally, we have found Naringenin to be most effective among three biomolecules in modulating VEGF activity based on minimum inhibition constant, Ki, and highest negative free energy of binding with the maximum interacting surface area during docking studies.Conclusion: The present study outlines the novel potential of biomolecules in regulating VEGF activity for the treatment of different abnormalities associated with impaired VEGF.

Abstract 4519: ATM/ATR-SerRS pathway dominates hypoxic angiogenesis in tumor

Abstract Hypoxia-induced angiogenesis is a critical response in multicellular organisms to maintain oxygen homeostasis. It is also an important mechanism to support tumor progression. Hypoxia activates hypoxia-inducible factor (HIF) and HIF-independent pathways to transcriptionally induce the expression of pro-angiogenic genes. However, our knowledge on transcriptional repressors of angiogenesis and how they are regulated during hypoxia are limited. Here we report that seryl-tRNA synthetase (SerRS), a newly identified transcriptional repressor of vascular endothelial growth factor (VEGFA), is a major inhibitor of angiogenesis that is inactivated during hypoxia1-4. Inactivation of SerRS is achieved through its phosphorylation at serine 101 and serine 241 by ataxia telangiectasia mutated (ATM) and the related kinase ATR. Knockdown of HIF is not sufficient to completely block hypoxia-induced VEGFA expression in human cells unless a phosphorylation-deficient form of SerRS (SerRSS101A/S241A) is simultaneously expressed to escape ATM/ATR inactivation. Consistently, in mice, SerRSS101A/S241A can bypass hypoxia response despite HIF activation to strongly inhibit angiogenesis in a matrigel assay and a xenograft model of human breast cancer. These findings reveal ATM/ATR-SerRS pathway as a key negative regulator of angiogenesis related to hypoxia and a new target for cancer therapy.

HS-1793, a resveratrol analogue, downregulates the expression of hypoxia-induced HIF-1 and VEGF and inhibits tumor growth of human breast cancer cells in a nude mouse xenograft model.

A synthetic analogue of resveratrol, 4-(6-hydroxy-2-naphtyl)-1,3-benzenediol(HS-1793), with improved photosensitivity and stability profiles, has been recently reported to exert anticancer activity on various cancer cells. However, the molecular mechanism of action and invivo efficacy of HS-1793 in breast cancer cells have not been fully investigated. In the present study, we evaluated the effect of HS-1793 on hypoxia-inducible factor-1α(HIF-1α), which drives angiogenesis and the growth of solid tumors, in addition to the invivo therapeutic effects of HS-1793 on breast cancer cells. HS-1793 was found to inhibit hypoxia (1.0% oxygen)-induced HIF-1α expression at the protein level, and its inhibitory effect was more potent than that of resveratrol in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, HS-1793 reduced the secretion and mRNA expression of vascular endothelial growth factor(VEGF), a key mediator of HIF-1-driven angiogenesis, without affecting cell viability. To evaluate the anticancer effects of HS-1793 invivo, triple-negative MDA-MB-231 breast cancer xenografts were established in nude mice. HS-1793 significantly suppressed the growth of breast cancer tumor xenografts, without any apparent toxicity. Additionally, decreases in Ki-67, a proliferation index marker, and CD31, a biomarker of microvessel density, were observed in the tumor tissue. Expression of HIF-1 and VEGF was also downregulated in xenograft tumors treated with HS-1793. These invivo results reinforce the improved anticancer activity of HS-1793 when compared with that of resveratrol. Overall, the present study suggests that the synthetic resveratrol analogue HS-1793 is a potent antitumor agent that inhibits tumor growth via the regulation of HIF-1, and demonstrates significant therapeutic potential for solid cancers.

Hypoxia and inflammation in the release of VEGF and interleukins from human retinal pigment epithelial cells.

Retinal diseases are closely associated with both decreased oxygenation and increased inflammation. It is not known if hypoxia-induced vascular endothelial growth factor (VEGF) expression in the retina itself evokes inflammation, or whether inflammation is a prerequisite for the development of neovascularization. Human ARPE-19 cell line and primary human retinal pigment epithelium (RPE) cells were used. ARPE-19 cells were kept either under normoxic (24h or 48h) or hypoxic conditions (1% O2, 24h). Part of the cells were re-oxygenated (24h). Some ARPE-19 cells were additionally pre-treated with bacterial lipopolysaccharide (LPS). The levels of IL-6, IL-8, IL-1β, and IL-18 were determined from medium samples by an enzyme-linked immunosorbent assay (ELISA) method. Primary human RPE cells were exposed to hypoxia for 24h, and the subsequent release of IL-6 and IL-8 was measured with ELISA. VEGF secretion from ARPE-19 cells was determined up to 24h. Hypoxia induced significant (P<0.01) increases in the levels of both IL-6 and IL-8 in ARPE-19 cells, and LPS pre-treatment further enhanced these responses. Hypoxia exposure did not affect the IL-1β or IL-18 release irrespective of LPS pre-treatment. If primary RPE cells were incubated for 4h in hypoxic conditions, IL-6 and IL-8 concentrations were increased by 7 and 8-fold respectively. Hypoxia increased the VEGF secretion from ARPE-19 cells in a similar manner with or without pre-treatment with LPS. Hypoxia causes an inflammatory reaction in RPE cells that is potentiated by pre-treatment with the Toll-like receptor-activating agent, LPS. The secretion of VEGF from these cells is regulated directly by hypoxia and is not mediated by inflammation.

Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and is involved in pathological angiogenesis associated with chronic liver diseases. However, the precise mechanisms underlying PlGF signalling contributing to liver fibrosis and angiogenesis remain largely unexplored. This study aimed to assess the effect of reducing PlGF expression using small interfering RNA (siRNA) on experimental liver fibrosis and angiogenesis, and to elucidate the underlying molecular mechanisms. Fibrosis was induced in mice by carbon tetrachloride (CCl4) for 8 weeks, and mice were treated with PlGF siRNA or non‐targeting control siRNA starting two weeks after initiating CCl4 injections. The results showed that PlGF was highly expressed in cirrhotic human and mice livers; which mainly distributed in activated hepatic stellate cells (HSCs). PlGF silencing robustly reduced liver inflammation, fibrosis, intrahepatic macrophage recruitment, and inhibited the activation of HSCs in vivo. Moreover, PlGF siRNA‐treated fibrotic mice showed diminished hepatic microvessel density and angiogenic factors, such as hypoxia‐inducible factor‐1α (HIF‐1α), VEGF and VEGF receptor‐1. Moreover, down‐regulation of PlGF with siRNA in HSCs inhibited the activation and proliferation of HSCs. Mechanistically, overexpression of PlGF in activated HSCs was induced by hypoxia dependent on HIF‐1α, and PlGF induces HSC activation and proliferation via activation the phosphatidylinositol 3‐kinase (PI3K)/Akt signalling pathways. These findings indicate that PlGF plays an important role in liver fibrosis‐associated angiogenesis and that blockage of PlGF could be an effective strategy for chronic liver disease.

Establishment and characterization of a squamous cell carcinoma cell line, designated hZK-1, derived from a metastatic lymph node tumor of the tongue.

The hZK-1 cell line was successfully established from the metastatic foci of a lymph node of an 82-year-old Japanese woman with squamous cell carcinoma of the tongue. The pathological diagnosis of the tumor was moderately to well-differentiated squamous cell carcinoma. The hZK-1 cells were angular in shape, and had neoplastic and pleomorphic features. Adjacent hZK-1 cells were joined by desmosomes and well-developed microvilli, and many free ribosomes were observed in the cytoplasm. The doubling time of the hZK-1 cells was approximately 36, 33, and 29h at the 10th, 20th, and 30th passages, respectively. The cell line was shown to be triploid, with a chromosomal distribution of 75-80. Immunocytochemical staining of the hZK-1 cells revealed cytokeratin (CK) 17-, Ki67-, and p53-positive staining, and negative staining for CK13. The hZK-1 cells were negative for human papillomavirus (HPV)-16 or-18 infection. Grafting was not successful when the hZK-1 cells were transplanted into the subcutis of SCID mice. The hZK-1 cells (2×106 cells/3ml of growth medium) secreted vascular endothelial growth factor (VEGF) that reached a concentration of 2.6ng/ml media after 3days of culture. Hypoxia enhanced cellular HIF-1α expression and VEGF secretion in hZK-1 cells. The HIF-1α inhibitor YC-1 partially inhibited hypoxia-induced VEGF secretion in ZK-1 cells. The reverse transcription-polymerase chain reaction (RT-PCR) results revealed that the expression of CK17, Ki67, and p53 was elevated in the hZK-1 cells. hZK-1 cells were not sensitive to CDDP, TXT, 5-FU, or a mixture of these three anti-tumor agents.

Correlating oxidative stress-related factors with bone metabolic markers in older adult male patients exhibiting degenerative osteoporosis in the high-altitude hypoxic area of China: study protocol for a non-randomized controlled trial

Background and objectives: Hypoxia is an important factor that affects bone formation and regulates bone growth. Therefore, many older adult patients living in high-altitude hypoxic areas exhibit osteoporosis. Oxidative stress-related hypoxia-inducible factors can induce abnormal expression of various factors including vascular endothelial growth factor (VEGF), insulin-like growth factor, and endothelin. However, it remains unclear whether these factors influence changes in bone metabolic markers. This study protocol aimed to investigate the correlation between oxidative stress-related factors and bone metabolic markers in older adult male patients with degenerative osteoporosis who reside in the high-altitude hypoxic area of China. Design: A prospective, single-center, non-randomized, controlled trial. Methods: One hundred and twenty older adult male patients with degenerative osteoporosis residing in the high-altitude area of China who receive treatment at the Affiliated Hospital of Qinghai University of China between January 2015 and February 2018 are being included in the osteoporosis group. One hundred and twenty healthy older adult males who concurrently received physical examination are being included in the control group. One day after admission, serum levels of hypoxia-inducible factor 1-alpha (HIF-1α), HIF-2α, VEGF, osteocalcin, and tartrate-resistant acid phosphatase 5b (TRACP 5b) were measured using an enzyme-linked immunosorbent assay. Bone mineral density in L1–4 segments, right femoral neck, and the greater trochanter of the femur was detected using dual-energy X-ray absorptiometry. Outcome measures: The primary outcome measure of this study is serum HIF-1α levels at 1 day after admission. Secondary outcome measures include serum levels of HIF-1α, HIF-2α, VEGF, osteocalcin, and TRACP 5b at 1 day after admission, as well as the correlation between serum levels of oxidative stress indicators (HIF-1α, HIF-2α, and VEGF) and bone metabolic markers (osteocalcin and TRACP 5b) at 1 day after admission. Discussion: Findings from this study aim to validate the correlation between oxidative stress-related factors and bone metabolic markers in older adult male patients with degenerative osteoporosis who reside in the high-altitude area of China. Ethics and dissemination: This study was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University of China (approval No. QHY1402G). The study was performed in accordance with the Declaration of Helsinki. Participants are informed of the study protocol and procedures, and sign an informed consent. Participant recruitment, blood sampling, and data collection began in January 2015 and will be ended in February 2018. Outcome measure analysis and trial completion will be in March 2018. Results will be disseminated through presentations at scientific meetings and/or by publication in peer-reviewed journals. Trial registration: This trial was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR-ROC-17012848).

Expression Pattern of HIF-1α and VEGF Supports Circumferential Application of Scatter Laser for Proliferative Sickle Retinopathy.

PurposeRetinal vascular occlusions in sickle cell anemia patients cause tissue ischemia and the release of angiogenic mediators that promote the development of retinal neovascularization, initiating proliferative sickle retinopathy (PSR). Laser photocoagulation (LPC) has emerged as the most common treatment for PSR. Nonetheless, only two randomized controlled clinical trials have evaluated the use of LPC for PSR, and both failed to definitively demonstrate efficacy of this approach. This may be due to a lack of knowledge regarding the appropriate location for placement of laser coagulations in PSR eyes. To help address this question, we examined the expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in PSR eyes.MethodsThe expression pattern of HIF-1α and VEGF in PSR (n = 5) and control (n = 3) eyes was examined by immunohistochemistry in different retinal regions defined by the presence or absence of retinal vessels.ResultsHypoxia-inducible factor 1α and VEGF were expressed in the inner retina of 5/5 untreated PSR eyes adjacent to retinal neovascularization; expression of HIF-1α was not detected (and VEGF only lightly detected) in normal retinal and choroidal vasculature of 3/3 control eyes. Hypoxia-inducible factor 1α and VEGF were strongly expressed in retinal cells within avascular (nonperfused) retina, anterior to the boundary between perfused and nonperfused retina, as well as in posterior ischemic retina in the presence or absence of neovascular sea fans.ConclusionsIf the goal of LPC in PSR is to quench the expression of HIF-1–driven angiogenic mediators, our results support broad application of peripheral laser for its treatment.

Tristetraprolin regulates the decay of the hypoxia-induced vascular endothelial growth factor mRNA in ARPE-19 cells.

The aim of the present study was to investigate the effects of tristetraprolin (TTP) on the vascular endothelial growth factor (VEGF) mRNA and protein expression levels in retinal pigment epithelial cells under hypoxic conditions, and to consider the possibility of using TTP as a novel treatment tool for neovascular age‑related macular degeneration (AMD). Overexpression of TTP reduced the expression and secretion levels of VEGF in ARPE‑19 cells under hypoxic conditions. TTP destabilized the VEGF mRNA by binding to adenosine and uridine‑rich elements regions in its 3'‑untranslated region. Furthermore, conditioned medium (CM) from TTP‑overexpressing ARPE‑19 cells suppressed the tube formation in human umbilical vein endothelial cells compared with hypoxic CM. These findings indicate that regulation of TTP expression may be a promising therapeutic tool for neovascular AMD, however, further research is required.

Simultaneous regulation of apoptotic gene silencing and angiogenic gene expression for myocardial infarction therapy: Single-carrier delivery of SHP-1 siRNA and VEGF-expressing pDNA

Gene therapy is aimed at selectively knocking up or knocking down the target genes involved in the development of diseases. In many human diseases, dysregulation of disease-associated genes is occurred concurrently: some genes are abnormally turned up and some are turned down. In the field of non-viral gene therapy, plasmid DNA (pDNA) and small interfering RNA (siRNA) are suggested as representative regulation tools for activating and silencing the expression of genes of interest, representatively. Herein, we simultaneously loaded both siRNA (Src homology region 2 domain-containing tyrosine phosphatase-1 siRNA, siSHP-1) for anti-apoptosis and pDNA (hypoxia-inducible vascular endothelial growth factor expression vector, pHI-VEGF) for angiogenesis in a single polymeric nanocarrier and used to synergistically attenuate ischemia-reperfusion (IR)-induced myocardial infarction, which is mainly caused by dysregulating of cardiac apoptosis and angiogenesis. For dual-modality cardiac gene delivery, siSHP-1 and pHI-VEGF were sequentially incorporated into a stable nanocomplex by using deoxycholic acid-modified polyethylenimine (DA-PEI). The resulting DA-PEI/siSHP-1/pHI-VEGF complexes exhibited the high structural stability against polyanion competition and the improved resistance to digestion by nucleases. The cardiac administration of DA-PEI/siSHP-1/pHI-VEGF reduced cardiomyocyte apoptosis and enhanced cardiac microvessel formation, thereby reducing infarct size in rat ischemia-reperfusion model. The simultaneous anti-apoptotic and angiogenic gene therapies synergized the cardioprotective effects of each strategy; thus our dual-modal single-carrier gene delivery system can be considered as a promising candidate for treating ischemic heart diseases.

Hypoxia-induced vascular endothelial growth factor secretion by retinal pigment epithelial cells is inhibited by melatonin via decreased accumulation of hypoxia-inducible factors-1α protein.

Hypoxia is the most important stimulus leading to up-regulation of vascular endothelial growth factor (VEGF) in the retina via elevation of hypoxia-inducible factors-1α (HIF-1α) protein. The purpose of this study was to test the effects of melatonin on the expression of VEGF and HIF-1α in the cultured human retinal pigment epithelial (RPE) cells under normoxia and hypoxia. An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2 ) to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without melatonin under normoxia and hypoxia. The protein and RNA levels of VEGF and HIF-1α were measured by ELISA kits and RT-PCR, respectively. Hypoxia induced a significant increase of expression and secretion of VEGF and accumulation of HIF-1α protein in RPE cells (P < 0.05). Melatonin at 10-5 to 10-8 M significantly inhibited hypoxia-induced expression, the secretion of VEGF and the accumulation of HIF-1α protein (P < 0.05), but not affected expression of VEGF and HIF-1α under normoxia (P > 0.05). This study suggests that melatonin may have potential value in the prevention and treatment of various retinal diseases associated with increase of VEGF, vascular leakage and angiogenesis.

Floroquinone induces apoptosis and inhibits carcinoma growth in mouse tumor xenograft model of anaplastic thyroid cancer

&lt;p class="Abstract"&gt;In the present study, effects of floroquinone on anaplastic thyroid cancer cell lines and mouse tumor xenografts were investigated. Increase in the concentration of floroquinone from 10 to 100 µM reduced the cell growth from 98 to 17% after 48 hours in HOTHC cells. Similarly, in FRO cells growth rate was found to be 96 and 21%, respectively at 10 and 100 µM concentra-tions of floroquinone. Western blot analysis showed a marked reduction in Bcl-2 expression and increased in Bax, caspase-3 and cleaved PARP expre-ssion in HOTHC cell lines on treatment with floroquinone. Floroquinone treatment of the HOTHC cells led to inhibition of the cobalt chloride-induced increase in the hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor expression. In HOTHC cells, floroquinone treatment inhibited the tube formation and migration potential significantly compared to control cells. Treatment of the mouse bearing HOTHC tumor xenograft with 50 and 100 mg/kg doses of floroquinone alternatively for one month reduced the tumor volume to 48 and 17%, respectively compared to the control. Thus, floroquinone effectively inhibits growth of thyroid cancer and can be used for its treatment.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Video Clip&lt;/strong&gt;:&lt;/p&gt;&lt;p&gt;&lt;a href="https://www.youtube.com/v/sub0JQlrffc"&gt;Migration assay&lt;/a&gt;: 1 min 09 sec&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Abstract 5261: Novel resveratrol analogue, HS-1793, inhibits hypoxia-induced HIF-1 and VEGF expressions and inhibits tumor growth of human breast cancer cells in a nude mouse xenograft model

Abstract Taking phytochemicals provide various benefits for human health. Among them, resveratrol, from the blueberry, is well known for its beneficial effects for human. However, resveratrol has photosensitive and easily decomposition by digestive enzyme. Thus we synthesized several synthetic analogues of resveratrol with potent activity. One of them, 4-(6-hydroxy-2-naphtyl)-1, 3-benzenediol (HS-1793) treatment showed potent antitumor activity in various human cancer cells. In this study, we investigated the effects of a novel analogue of resveratrol, HS-1793 on the expressions of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in MCF-7 with MDA-MB-231 human breast cancer cells. We found that HS-1793 inhibited hypoxia (1.0% oxygen) induced HIF-1α protein level and its downstream target, vascular endothelial growth factor (VEGF) expression dose-dependently in both cell lines. Decreased HIF-1α by HS-1793 modulated through 26S proteasome pathway. We also found that HS-1793 suppressed tumor growth in tumor xenograft mice model. Treatment with 10 mg/kg of HS-1793 suppressed Ki-67 and CD31 expressions in tumor tissues. Above all, HS-1793 shows more powerful effects than resveratrol. Taken together, these results implied that HS-1793, a novel analogue of resveratrol might be a new potent chemopreventive agent and anticancer therapy in human breast cancer. Citation Format: Dong Hwan Kim, Min Jeong Kim, Bokyung Sung, Yong Jung Kang, Seong Yeon Hwang, Na Lam Hwang, Nam Deuk Kim. Novel resveratrol analogue, HS-1793, inhibits hypoxia-induced HIF-1 and VEGF expressions and inhibits tumor growth of human breast cancer cells in a nude mouse xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5261.

Hypoxia on the Expression of Hepatoma Upregulated Protein in Prostate Cancer Cells.

Hepatoma upregulated protein (HURP) is a multifunctional protein with clinical promise. This protein has been demonstrated to be a predictive marker for the outcome in high-risk prostate cancer (PCa) patients, besides being a resistance factor in PCa. Although changes in oxygen tension (pO2) are associated with PCa aggressiveness, the role of hypoxia in the regulation of tumor progression genes such as HURP has not yet been described. We hypothesized that pO2 alteration is involved in the regulation of HURP expression in PCa cells. In the present study, PCa cells were incubated at 2% O2 (hypoxia) and 20% O2 (normoxia) conditions. Hypoxia reduced cell growth rate of PCa cells, when compared to the growth rate of cells cultured under normoxia (p < 0.05). The decrease in cell viability was accompanied by fivefold (p < 0.05) elevated rate of vascular endothelial growth factor (VEGF) release. The expression of VEGF and the hypoxia-inducible metabolic enzyme carbonic anhydrase 9 were elevated maximally nearly 61-fold and 200-fold, respectively (p < 0.05). Noted in two cell lines (LNCaP and C4-2B) and independent of the oxygen levels, HURP expression assessed at both mRNA and protein levels was reduced. However, the decrease was more pronounced in cells cultured under hypoxia (p < 0.05). Interestingly, the analysis of patients’ specimens by Western blot revealed a marked increase of HURP protein (fivefold), when compared to control (cystoprostatectomy) tissue (p < 0.05). Immunohistochemistry analysis showed an increase in the immunostaining intensity of HURP and the hypoxia-sensitive molecules, hypoxia-inducible factor 1-alpha (HIF-1α), VEGF, and heat-shock protein 60 (HSP60) in association with tumor grade. The data also suggested a redistribution of subcellular localization for HURP and HIF-1α from the nucleus to the cytoplasmic compartment in relation to increasing tumor grade. Analysis of HURP Promoter for HIF-1-binding sites revealed presence of four putative HIF binding sites on the promoter of DLGAP5/HURP gene in the non-translated region upstream from the start codon, suggesting association between HIF-1α and the regulation of HURP protein. Taken together, our findings suggest a modulatory role of hypoxia on the expression of HURP. Additionally our results provide basis for utilization of tumor-associated molecules as predictors of aggressive PCa.

Multifunctional Polymeric Nanosystems for Dual-Targeted Combinatorial Chemo/Angiogenesis Therapy of Tumors.

Combination of antiangiogenesis and chemotherapy holds vast promise for effective inhibition of tumor proliferation and invasion. Herein, a multifunctional self-assembled nanosystem consisting of amphiphilic c(RGDyK)-functionalized low-molecular-weight heparin-gambogic acid conjugate (cRHG) is developed, using c(RGDyK) peptide as αv β3 integrin targeting moiety to realize a double-targeted delivery to both tumor cells and angiogenic vasculature. cRHG with a markedly decreased anticoagulant activity can self-assemble into nanosized particles (around 150 nm). cRHG nanoparticles effectively inhibit vascular endothelial growth factor (VEGF)-triggered tube-like formation of HUVEC and neovascularization of subcutaneous Matrigel plugs. More importantly, the targeting ability of cRHG nanoparticles is revealed by their efficient internalization by αv β3 integrin-positive cells (U87MG and HUVEC) in vitro and obvious accumulation of Cy7/cRHG in the tumor site with strong fluorescence signals in both cancer cells and neovasculature. Besides, cRHG maintains the in vitro cytotoxic activity of gambogic acid, while achieving the highest in vivo antitumor activity in U87MG mouse xenograft model and displaying better safety profile than free drugs solutions. Mechanistic investigations reveal the substantial inhibition of hypoxia-inducible factor-1 alpha, VEGF, and CD31 expression by cRHG nanoparticles with remarkable down-regulation of VEGFR2 phosphorylation. These results suggest that cRHG nanoparticles provide a versatile nanoplatform for efficacious combinatorial tumor therapy.