Hypoxic response elements control expression of human vascular endothelial growth factor(165) genes transferred to ischemia myocardium in vivo and in vitro

BACKGROUND: Vascular endothelial growth factor (VEGF) gene transfer with recombinant adeno-associated viral (rAAV) vector for ischemia heart disease therapy is being increasingly studied. However, uncontrolled long-term expression of VEGF may cause some side effects. Therefore, an attempt to develop an effective gene control system for safeguarding against such side effects should be made. Pathphysiologically, an ideal control system for VEGF gene expression is letting it respond to hypoxia. We used nine copies of hypoxic response element (HRE) to regulate expression of hVEGF(165) in the myocardium, and tried to elucidate the feasibility and safety of the application of the HIF-1-HRE system. METHODS: Cardiomyocytes of neonatal Sprague Dawley rats were cultured and incubated with rAAV-9HRE-hVEGF(165), and pig ischemic heart models were established and rAAV-9HRE-hVEGF(165) was injected into ischemia myocardium. RT-PCR, Western blot, ELISA, and immunohistochemistry were used to determine hVEGF(165) expressions of cultured cardiomyocytes and myocardium under hypoxic and reoxygenation conditions. RESULTS: The results of RT-PCR and ELISA determinations revealed that, in cultured cardiomyocytes, expressions of hVEGF(165)mRNA and protein were up-regulated under hypoxic conditions. After 4 h of reoxygenation, hVEGF(165)mNRA expression was decreased, and disappeared following 8 to 12 h of reoxygenation (P < 0.01). RT-PCR and Western blot also showed that, under myocardial ischemia, hVEGF(165) expression was increased significantly (P < 0.01). Following myocardial reperfusion, both hVEGF(165)mRNA and protein expressions were inhibited (P < 0.01). The new vessels in the reperfusion condition were decreased. CONCLUSIONS: This study suggested that 9HRE can effectively control hVEGF(165) gene expression in vivo and in vitro. It has feasibility for using the HIF-1-HRE system for regulation of angiogenic factor expression in ischemia heart.     

Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions

Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions.     

Effect of C-Terminal Sequence on Competitive Semaphorin Binding to Neuropilin-1

Neuropilins (Nrp) are type I transmembrane proteins that function as receptors for vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3) ligand families. Sema3s function as potent endogenous angiogenesis inhibitors but require proteolytically processing by furin to compete with VEGF for Nrp binding. This processing liberates a C-terminal arginine (C_R) that is necessary for binding to the b1 domain of Nrp, a common feature shared by Nrp ligands. The C_R is necessary but not sufficient for potent Nrp inhibition, and the role of upstream residues is unknown. We demonstrate that the second-to-last residue (C-1), immediately upstream of the C_R, plays a significant role in controlling competitive ligand binding by orienting the C-terminus for productive Nrp binding. With the use of a peptide library derived from Sema3F, C-1 residues that preferentially adopt an extended bound-like conformation, including proline and β-branched amino acids, were found to produce the most avid competitors. Consistent with this, analysis of the binding thermodynamics revealed that more favorable entropy is responsible for the observed binding enhancement of C-1 proline. We further tested the effect of the C-1 residue on Sema3F processing by furin and found an inverse relationship between processing and inhibitory potency. Analysis of all Sema3 family members reveals two non-equivalent furin processing sites differentiated by the presence of either a C-1 proline or a C-1 arginine and resulting in up to a 40-fold difference in potency. These data reveal a novel regulatory mechanism of Sema3 activity and define a fundamental mechanism for preferential Nrp binding.     

Hypoxia mediated expression of stem cell markers in VHL-associated hemangioblastomas

Hemangioblastomas of the retina, central nervous system, and kidney are observed in patients with mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. Mutations in the VHL lead to constitutive activation of hypoxia-inducible-factor (HIF) pathway. HIF-mediated expression of pro-angiogenic genes causes extensive pathological neovascularization in hemangioblastomas. A number of studies have shown coexistence of pro-angiogenic and stem cell markers in ‘tumorlet-like stromal cells’ in the retinal and optic nerve hemangioblastomas, leading to suggestions that hemangioblastomas originate from developmentally arrested stem cells or embryonic progenitors. Since recent studies have shown that the HIF pathway also plays a role in the maintenance/de-differentiation of normal and cancerous stem cells, we evaluated the role of the HIF pathway in the expression of stem cell markers in VHL−/− renal cell carcinoma cells under normoxia or VHL+/+ retinal pigment epithelial cells under hypoxia. Here we show that the expression of stem cell markers in hemangioblastomas is due to activation of the HIF pathway. Further, we show that honokiol, digoxin, and doxorubicin, three recently identified HIF inhibitors from natural sources, blocks the expression of stem cell markers. Our results show the mechanism for the cytological origin of neoplastic stromal cells in hemangioblastomas, and suggest that inhibition of the HIF pathway is an attractive strategy for the treatment of hemangioblastomas.     

Characterization of neuritin as a novel angiogenic factor

Neuritin (NRN1), a neurotrophic factor, plays an important role in neurite growth and neuronal survival. In this study, we identify a new function of neuritin as a novel angiogenic factor in vitro and in vivo. Recombinant neuritin protein had no effect on the proliferation and adhesion of human umbilical vein endothelial cells (HUVEC), but it dose-dependently increased endothelial cell migration. Furthermore, overexpression of neuritin significantly promoted tumor angiogenesis, and surprisingly, it inhibited tumor growth in a xenograft tumor model. Thus, our results indicate that neuritin may act as an important angiogenic factor and serve as a potential target for cancer therapy.     

Structure-activity relationships study of neolamellarin A and its analogues as hypoxia inducible factor-1 (HIF-1) inhibitors

The novel marine pyrrole alkaloid neolamellarin A derived from sponge has been shown to inhibit hypoxia-induced HIF-1 activity. In this work, we designed and synthesized neolamellarin A and its series of derivatives by a convergent synthetic strategy. The HIF-1 inhibitory activity and cytotoxicity of these compounds were evaluated in Hela cells by dual-luciferase reporter gene assay and MTT assay, respectively. The results showed that neolamellarin A 1 (IC₅₀ = 10.8 ± 1.0 μM) and derivative 2b (IC₅₀ = 11.9 ± 3.6 μM) had the best HIF-1 inhibitory activity and low cytotoxicity. Our SAR research focused on the effects of key regions aliphatic carbon chain length, aromatic ring substituents and C-7 substituent on biological activity, providing a basis for the subsequent research on the development of novel pyrrole alkaloids as HIF-1 inhibitors and design of small molecule probes for target protein identification.     

Nerve growth factor stimulates regeneration of perivascular nerve,and induces the maturation of microvessels around the injured artery

An immature vasa vasorum in the adventitia of arteries has been implicated in induction of the formation of unstable atherosclerotic plaques. Normalization/maturation of the vasa vasorum may be an attractive therapeutic approach for arteriosclerotic diseases. Nerve growth factor (NGF) is a pleotropic molecule with angiogenic activity in addition to neural growth effects. However, whether NGF affects the formation of microvessels in addition to innervation during pathological angiogenesis is unclear. In the present study, we show a new role for NGF in neovessels around injured arterial walls using a novel in vivo angiogenesis assay.     

In vivo antivascular endothelial growth factor treatment induces corneal endothelium apoptosis in rabbits through changes in p75NTR–proNGF pathway

Intravitreal injection (IVT) of antivascular endothelial growth factor (anti‐VEGF) agents is widely used for the treatment of retinal vascular diseases. Recently, the injection of anti‐VEGF agents in the ocular anterior chamber has been proposed for the treatment of neovascular glaucoma and potential side effects on the corneal structures have been investigated with contrasting results. Increasing evidence has demonstrated that VEGF inhibition is associated with cellular apoptotic changes and that this effect may be mediated by alterations in nerve growth factor (NGF) pathway. In this study, we demonstrated that anterior chamber injection (IC), but not IVT injection of two different anti‐VEGF agents, aflibercept and ranibizumab, affects rabbit corneal endothelium in terms of survival and apoptosis and is associated with changes in endothelial expression of NGF precursor (proNGF) and p75 neurotrophin receptor (p75NTR) receptor. We observed an increase in corneal endothelial cell incorporation of trypan blue and expression of cleaved‐caspase 3 (c‐Casp3), p75NTR, and RhoA after IC injection of both anti‐VEGF drugs when compared with the vehicle. Our results showed that apoptosis induction by aflibercept was more pronounced when compared with that of ranibizumab. Aflibercept also mediated a significant increase in endothelial expression of proNGF when compared with the vehicle. In line with these data, IC administration of both anti‐VEGF agents induced the activation of apoptotic signals in endothelial cells, including an increase in c‐Casp3, decrease in Bad Ser 112 phosphorylation, and unbalance of AKT phosphorylation. These results demonstrated that administration of anti‐VEGF in the anterior chamber of rabbit affects endothelial cell survival by inducing apoptosis through alteration of NGF pathway.     

Regulation of tumor angiogenesis by microRNAs: State of the art

MicroRNAs (miRNAs, miRs) are small (21–25 nucleotides) endogenous and noncoding RNAs involved in many cellular processes such as apoptosis, development, proliferation, and differentiation via binding to the 3′-untranslated region of the target mRNA and inhibiting its translation. Angiogenesis is a hallmark of cancer, which provides oxygen and nutrition for tumor growth while removing deposits and wastes from the tumor microenvironment. There are many angiogenesis stimulators, among which vascular endothelial growth factor (VEGF) is the most well known. VEGF has three tyrosine kinase receptors, which, following VEGF binding, initiate proliferation, invasion, migration, and angiogenesis of endothelial cells in the tumor environment. One of the tumor microenvironment conditions that induce angiogenesis through increasing VEGF and its receptors expression is hypoxia. Several miRNAs have been identified that affect different targets in the tumor angiogenesis pathway. Most of these miRNAs affect VEGF and its tyrosine kinase receptors expression downstream of the hypoxia-inducible Factor 1 (HIF-1). This review focuses on tumor angiogenesis regulation by miRNAs and the mechanism underlying this regulation.     

高原鼢鼠血管内皮生长因子基因编码和mRNA的表达以及微血管密度:与其它鼠类的比较

动物组织微血管密度(microvessel density,MVD)的大小与其对低氧的适应能力有关.为进一步探讨高原鼢鼠对严重低氧、高CO2洞道环境的适应机制,本文就高原鼢鼠脑组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)的mRNA表达水平及MVD与其它鼠类进行...     

Hypoxia-inducible factor 1-mediated regulation of PPP1R3C promotes glycogen accumulation in human MCF-7 cells under hypoxia

Hundreds of genes can be regulated by hypoxia-inducible factor 1 (HIF1) under hypoxia. Here we demonstrated a HIF1-mediated induction of protein phosphatase 1, regulatory subunit 3C gene (PPP1R3C) in human MCF7 cells under hypoxia. By mutation analysis we confirmed the presence of a functional hypoxia response element that is located 229 bp upstream from the PPP1R3C gene. PPP1R3C induction correlates with a significant glycogen accumulation in MCF7 cells under hypoxia. Knockdown of either HIF1α or PPP1R3C attenuated hypoxia-induced glycogen accumulation significantly. Knockdown of HIF2α reduced hypoxia-induced glycogen accumulation slightly (but not significantly). Our results demonstrated that HIF1 promotes glycogen accumulation through regulating PPP1R3C expression under hypoxia, which revealed a novel metabolic adaptation of cells to hypoxia.     

Role of pigment epithelium-derived factor in the involution of hemangioma: Autocrine growth inhibition of hemangioma-derived endothelial cells

Hemangioma is a benign tumor derived from abnormal blood vessel growth. Unlike other vascular tumor counterparts, a hemangioma is known to proliferate during its early stage but it is followed by a stage of involution where regression of the tumor occurs. The critical onset leading to the involution of hemangioma is currently not well understood. This study focused on the molecular identities of the involution of hemangioma. We demonstrated that a soluble factor released from the involuting phase of hemangioma-derived endothelial cells (HemECs) and identified pigment epithelium-derived factor (PEDF) as an anti-angiogenic factor that was associated with the growth inhibition of the involuting HemECs. The growth inhibition of the involuting HemECs was reversed by suppression of PEDF in the involuting HemECs. Furthermore, we found that PEDF was more up-regulated in the involuting phase of hemangioma tissues than in the proliferating or the involuted. Taken together, we propose that PEDF accelerates the involution of hemangioma by growth inhibition of HemECs in an autocrine manner. The regulatory mechanism of PEDF expression could be a potential therapeutic target to treat hemangiomas.