ß-Catenin is markedly induced in a murine model of an arteriovenous fistula: the effect of metalloproteinase inhibition

Neointimal hyperplasia contributes to failure of hemodialysis arteriovenous fistulas (AVFs). Increased expression of matrix metalloproteinase (MMP)-9 occurs in AVFs, and MMP-9 is implicated in neointimal hyperplasia and vascular injury. Recent studies demonstrate that MMP-9, by degrading N-cadherin, leads to increased expression of β-catenin and β-catenin-dependent proliferation of smooth muscle cells. The present study examined this pathway in the venous limb of a murine AVF model. Western analyses demonstrate that, in this model, there is diminished expression of N-cadherin accompanied by increased expression of β-catenin, c-Myc, and proliferating cell nuclear antigen (PCNA). By immunohistochemistry, β-catenin and c-Myc localized to proliferating smooth muscle cells in the venous limb of the AVF. Increased expression of β-catenin was accompanied by augmented expression of phosphorylated (p)-glycogen synthase kinase (GSK)-3β, GSK-3β, and integrin-linked kinase. The administration of doxycycline suppressed MMP-9 expression but did not reduce venous histological injury in the AVF, or increase AVF patency assessed 6 wk after its creation. Doxycycline did not influence expression of β-catenin, c-Myc, GSK-3β, or integrin-linked kinase. Thus, in this vascular injury model, the upregulation of β-catenin cannot be readily attributed to MMP-9 upregulation; increased β-catenin expression may reflect either the upregulation of p-GSK-3β, GSK-3β, or integrin-linked kinase. This study provides the first exploration of β-catenin in an AVF, demonstrating substantial upregulation of this mitogenic signaling molecule and uncovering possible mechanisms that may account for such upregulation.     

Hypoxia-inducible factor-1α mediates oral squamous cell carcinoma invasion via upregulation of α5 integrin and fibronectin

With progressive and rapid growth of malignant tumors, cancer cells in an ischemic condition are expected to develop an increased potential for local invasive growth. To address this hypothesis, we first examined the effect of hypoxia on the invasiveness of oral squamous cell carcinoma (OSCC) cells using the Matrigel invasion assay. We then investigated the effect of hypoxia on the protein and mRNA expression of α5 integrin and fibronectin, which are major factors involved in tumor cell invasion. We showed that (i) hypoxia increased the invasiveness of OSCC cells, (ii) α5 integrin and fibronectin protein and mRNA expression levels were increased in OSCC cells under hypoxic conditions, (iii) hypoxia stimulated autocrine secretion of fibronectin in OSCC cells, (iv) administration of siRNA_HIF-1α caused a significant decrease in α5 integrin and fibronectin protein, confirming that HIF-1α plays a role in their induction, and (v) siRNA_HIF-1α abrogated hypoxia-induced cell invasion. Collectively, these data suggest that hypoxia promotes OSCC cell invasion that is elicited by HIF-1α-dependent α5 integrin and fibronectin induction.     

MicroRNA-22 regulates hypoxia signaling in colon cancer cells

MicroRNAs (MiRNAs) are short, non-coding RNA that regulate a variety of cellular functions by suppressing target protein expression. We hypothesized that a set of microRNA regulate tumor responses to hypoxia by inhibiting components of the hypoxia signaling pathway. We found that miR-22 expression in human colon cancer is lower than in normal colon tissue. We also found that miR-22 controls hypoxia inducible factor 1α (HIF-1α) expression in the HCT116 colon cancer cell line. Over-expression of miR-22 inhibits HIF-1α expression, repressing vascular endothelial growth factor (VEGF) production during hypoxia. Conversely, knockdown of endogenous miR-22 enhances hypoxia induced expression of HIF-1α and VEGF. The conditioned media from cells over-expressing miR-22 contain less VEGF protein than control cells, and also induce less endothelial cell growth and invasion, suggesting miR-22 in adjacent cells influences endothelial cell function. Taken together, our data suggest that miR-22 might have an anti-angiogenic effect in colon cancer.     

FOXO3a-dependent up-regulation of Mxi1-0 promotes hypoxia-induced apoptosis in endothelial cells

Endothelial cell apoptosis induced by hypoxia is implicated in the pathogenesis of vascular diseases. However, the underlying mechanism is not clearly elucidated. In this study, we found that hypoxia increased Mxi1-0 expression, and the Mxi1-0 siRNA could inhibit caspase-8 activation and apoptosis in HUVECs induced by hypoxia. In addition, hypoxia induced FOXO3 activation, while Mxi1-0 expression and apoptosis were inhibited by transfection with FOXO3 siRNA. Using ChIP assay, we confirmed that FOXO3a binds to the Mxi1-0 promoter region. Furthermore, hypoxia treatment leads to remarkable production of reactive oxygen species (ROS), while ROS scavenger N-acetyl-L-cysteine (NAC) inhibits hypoxia-induced ROS production, apoptosis and FOXO3a-mediated Mxi1-0 up-regulation. Finally, we found that the HIF-1α siRNA inhibited hypoxia-induced HIF-1α expression and ROS production, as well as FOXO3a/Mxi1-0 activation and apoptosis in HUVECs. Taken together, this study identifies a HIF-1α/FOXO3a/Mxi1-0/caspase-8 signaling pathway in hypoxia-induced endothelial cell apoptosis. These data also indicate that HIF-1α-dependent ROS production is required for FOXO3a-mediated Mxi1-0 up-regulation and apoptosis in hypoxic endothelial cells.     

Intermittent hypoxia changes HIF-1α phosphorylation pattern in endothelial cells: Unravelling of a new PKA-dependent regulation of HIF-1α

Vascularized tumors are exposed to intermittent hypoxia, that is, hypoxia followed by periods of reoxygenation. Abnormal structure and dysfunction of tumor blood vessels are responsible for these conditions. These repeated short periods of hypoxia concern tumor cells as well as endothelial cells. However, the effects of intermittent hypoxia are poorly understood. The aim of this study was to investigate the effects of intermittent hypoxia on endothelial cells and particularly on HIF-1α, a central actor in adaptive response to hypoxia. For that, endothelial cells were exposed to four repeated cycles of 1-h hypoxia followed by 30 min of reoxygenation. We showed that repeated cycles of hypoxia/reoxygenation induced a modification in HIF-lα phosphorylation pattern: a progressive increase in HIF-1α phosphorylated form was observed during the hypoxic periods. Activation of p42/p44, Akt and PKA was observed in parallel. PKA was shown to be involved in the phosphorylation of HIF-lα under intermittent hypoxia, while p42/p44 and Akt were not. As HIF-1 activity is often associated with enhanced cell survival, a better knowledge of the effects of intermittent hypoxia on endothelial cells and the highlight of particular mechanisms induced by intermittent hypoxia are essential to understand the behavior of endothelial cells during neo-angiogenesis.     

Oroxylin A increases the sensitivity of temozolomide on glioma cells by hypoxia-inducible factor 1α/hedgehog pathway under hypoxia

Microenvironmental hypoxia-mediated drug resistance is responsible for the failure of cancer therapy. To date, the role of the hedgehog pathway in resistance to temozolomide (TMZ) under hypoxia has not been investigated. In this study, we discovered that the increasing hypoxia-inducible factor 1α (HIF-1α) activated the hedgehog pathway in hypoxic microenvironment by promoting autocrine secretion of sonic hedgehog protein (Shh), and then upregulating transfer of Gli1 to the nucleus, finally contributed to TMZ resistance in glioma cells. Oroxylin A (C16H12O5), a bioactive flavonoid, could induce HIF-1α degradation via prolyl-hydroxylases–VHL signaling pathway, resulting in the inactivation of the hedgehog. Besides, oroxylin A increased the expression of Sufu, which is a negative regulator of Gli1. By this mechanism, oroxylin A sensitized TMZ on glioma cells. U251 intracranial transplantation model and GL261 xenograft model were used to confirm the reversal effects of oroxylin A in vivo. In conclusion, our results demonstrated that HIF-1α/hedgehog pathway conferred TMZ resistance under hypoxia, and oroxylin A was capable of increasing the sensitivity of TMZ on glioma cells in vitro and in vivo by inhibiting HIF-1α/hedgehog pathway and depressing the activation of Gli1 directly.     

Podocytes protect glomerular endothelial cells from hypoxic injury via deSUMOylation of HIF-1α signaling

Hypoxia can cause severe tubulointerstitial injury and peritubular capillary loss. However, hypoxia-induced injury in glomerular capillaries is far milder than tubulointerstitium, but the reason for this difference is unclear. We hypothesized that the phenomenon is due to the protective crosstalk among intrinsic glomerular cells. To mimic the microenvironment and investigate the crosstalk process temporally, we established co-culture models of glomerular endothelial cells (GEnCs) with podocytes or with mesangial cells. We found that podocytes rather than mesangial cells prevented GEnCs from injury and hypoxia-induced apoptosis and promoted migration and angiogenesis of GEnCs under hypoxic conditions. We then identified that increased activation of the hypoxia inducible factor 1α (HIF-1α) pathway as the major mechanism enabling podocytes to protect GEnCs against hypoxia. HIF-1α stabilization during hypoxia is known to be dependent on SUMO-specific protease 1 (SENP1)-mediated deSUMOylate modifications. Therefore, we further targeted deSUMOylation, regulated by SENP1, by short hairpin RNA (shRNA) knockdown of SENP1 mRNA in vitro and measured expression of HIF-1α and its downstream gene VEGF in hypoxic podocytes. Our results showed that SENP1 was essential for HIF-1α deSUMOylation in podocytes. The blockade of deSUMOylation by SENP1 shRNA successfully abolished the activation of HIF-1α signaling and consequently suppressed the protective effects of podocytes on GEnCs. In conclusion, we demonstrate for the first time that hypoxia may promote HIF-1α stabilization and activation by increasing SENP1 expression in podocytes, which induce GEnCs survival and angiogenesis to resist hypoxia. Thus, deSUMOylation of HIF-1α signaling is a potentially novel therapeutic target for treating hypoxic renal disorders.     

乏氧诱导因子-1α基因沉默对肺癌细胞乏氧应答相关基因表达的影响

乏氧诱导因子-1α (HIF-1α)是肿瘤细胞适应乏氧微环境的关键调控因子,具有作为治疗靶基因的潜力,以克服乏氧诱导的治疗抗拒等效应.下调其表达可能影响肿瘤细胞内一系列乏氧应答相关基因的表达.本研究采用已构建的HIF-1α RNAi慢病毒载体转导肺腺癌A549细胞,经杀稻瘟素（blasticidin）筛选建立HIF-1α基因稳定沉默的A549细胞株.应用cDNA微阵列技术检测并比较HIF-1α基因沉默A549细胞株和其亲本细胞株在常氧和乏氧状态下的基因表达谱改变. 应用定量RT PCR方法验证部分cDNA芯片差异表达基因的表达改变.HIF-1α基因稳定沉默细胞株A549/HIF-1α,在常氧和乏氧条件下HIF-1αmRNA水平分别较A549细胞下降89.2％和88.1％,HIF-1α蛋白水平分别下降97.2％和88.4％. 在乏氧条件下,cDNA微阵列检测的1 280个基因中,52个基因表达上调,15个基因表达下调. HIF-1α基因沉默显著影响其中27个基因的乏氧诱导效应.定量RT-PCR验证其中ENO2、BCL-2、CXCR4和MMP11的表达水平,与cDNA芯片结果相符合.结果提示,HIF-1α基因沉默能够在一定程度上阻断肺癌细胞的乏氧应答,在克服乏氧导致的肺癌治疗抗拒方面具有潜力.     

Overexpression of Axl reverses endothelial cells dysfunction in high glucose and hypoxia

The receptor tyrosine kinase Axl is involved in diabetic vascular disease. This study aims to investigate the effect of high glucose on endothelial cells injury and Axl expression in hypoxia condition in vitro, and we present details of the mechanism associated with overexpression of Axl rescue the high glucose injury. Our results showed that high glucose impaired both human umbilical vein endothelial cells (HUVECs) and EAhy926 cells angiogenesis in hypoxia condition. In addition, high glucose inhibits Axl and hypoxia-inducible factor 1-α (HIF-1α) protein expression in hypoxia condition. Axl overexpression significantly reversed endothelial cells dysfunction in high glucose/hypoxia. Furthermore, Axl overexpression in EAhy926 cells increases HIF-1α protein synthesis through PI3K/Akt/mTOR/p70 ^S6K signal pathway but not Mek/Erk in high glucose/hypoxia condition. This study demonstrates that high glucose can alter Axl signaling and HIF-1α in hypoxia condition. Overexpression of Axl may rescue endothelial cells dysfunction and HIF-1α expression through its downstream signals in high glucose/hypoxia.     

Angiogenesis by transplantation of HIF-1 alpha modified EPCs into ischemic limbs

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. HIF-1 alpha overexpression may be beneficial in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic heart disease. To address this issue, human peripheral blood mononuclear cells (PBMNCs) were induced to differentiate into endothelial progenitor cells (EPCs), and then were transfected with either an HIF-1 alpha-expressing or a control vector and cultured under normoxia or hypoxia. Hypoxia-induced HIF-1 alpha mRNA and protein expression was increased after HIF-1 alpha transfection. This was accompanied by VEGF mRNA induction and increased VEGF secretion. Hypoxia-stimulated VEGF mRNA induction was significantly abrogated by HIF-1 alpha-specific siRNA. Functional studies showed that HIF-1 alpha overexpression further promoted hypoxia-induced EPC differentiation, proliferation and migration. The expressions of endothelial cell markers CD31, VEGFR2 (Flk-1) and eNOS as well as VEGF and NO secretions were also increased. Furthermore, in an in vivo model of hindlimb ischemia, HIF-1 alpha-transfected EPCs homed to the site of ischemia. A higher revascularization potential was also demonstrated by increased capillary density at the injury site. Our results revealed that endothelial progenitor cells ex vivo modification by hypoxia inducible factor-1 alpha gene transfection is feasible and may offer significant advantages in terms of EPC expansion and treatment efficacy.     

Adrenomedullin promotes human endothelial cell proliferation via HIF-1α

Adrenomedullin (ADM) and hypoxia-inducible factor-1α (HIF-1α) are important pro-proliferation genes in response to hypoxic stress. Although it was reported that ADM is a target gene for HIF-1, recent studies also showed that ADM regulates HIF-1 expression and its activity; however, the mechanism of action remains unknown. Two stable human endothelial cell lines with HIF-1α knockdown by hy926-siHIF-1α or HMEC-siHIF-1α were established. mRNA and protein expression of ADM and HIF-1α in EA.hy926 and HMEC1 cells were examined under hypoxic stress. Upon ADM treatment, cell proliferation was investigated and the expression profiles of HIF-1α and its target genes (VEGF, PFKP, PGK1, and AK1) were examined. Furthermore, the proline hydroxylase (PHD) mRNA level and its activity were investigated. We observed that mRNA and protein expression of ADM in hypoxia are earlier events than HIF-1α in EA.hy926 and HMEC1 cells. ADM-promoted cell proliferation of endothelial cells, which was HIF-1α dependent. We also found that ADM up-regulated the mRNA and protein expressions of HIF-1α- and HIF-1-targeted genes, and ADM up-regulated the protein expressions of HIF-1α through down-regulation of PHD mRNA expression and PHD activity.     

Involvement of PI3K and MAPK signaling in bcl-2-induced vascular endothelial growth factor expression in melanoma cells

We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1alpha expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.     

Hypoxia-inducible Factor-1-mediated Regulation of Semaphorin 4D Affects Tumor Growth and Vascularity

Tumor progression and metastasis depend on the ability of cancer cells to initiate angiogenesis to ensure delivery of oxygen, nutrients, and growth factors to tumor cells and provide access to the systemic circulation. Hypoxia-inducible factor-1 (HIF-1) can activate expression of a broad range of genes that mediate many of the adaptive responses to decreased oxygen concentration, such as enhanced glucose uptake and formation of new blood vessels. Acting through Plexin-B1 on endothelial cells, Semaphorin 4D (Sema4D) has been shown to promote angiogenesis and enhance invasive growth and proliferation in some tumors. Here we show that the gene for Sema4D, the product of which is elevated in head and neck squamous cell carcinoma (HNSCC) cells, contains upstream hypoxia response elements (HRE) and is strongly induced in hypoxia in a HIF-1-dependent manner. Knocking down Sema4D expression with short hairpin (sh) RNA reduces in vitro endothelial cell migration and growth and vascularity of HNSCC xenografts expressing a degradation resistant HIF-1α subunit. We also demonstrate a correlation between HIF-1 activity and Sema4D expression in HNSCC specimens. These findings indicate that Sema4D is induced by hypoxia in a HIF-1-dependent manner and influences endothelial cell migration and tumor vascularity. Expression of Sema4D may be a strategy by which carcinomas promote angiogenesis and therefore could represent a therapeutic target for these malignancies.     

HIF-1α在骨组织细胞代谢及骨疾病中的调控作用

低氧诱导因子-1α(HIF-1α)是调节细胞对低氧应答的关键因子,可在氧含量降低时被激活,能够调节氧代谢、糖酵解等多种生理活动。骨代谢主要包括骨形成和骨吸收作用,均受到氧浓度等多种因素的调控。HIF-1α在细胞代谢、骨组织生理及病理过程的调控中起着重要的作用,能够增加骨组织的低氧耐受能力,调节骨形成和矿化过程。该文主要综述了HIF-1α对成骨细胞、破骨细胞、骨髓间充质干细胞、软骨细胞等骨组织细胞的调控,对骨血管形成过程的影响,以及对肿瘤骨转移、股骨头坏死、异位骨化等病理过程的调节作用,为探讨HIF-1α对骨代谢的调控和相关疾病的治疗提供参考。     

Macrophages Aggravate Hypoxia-Induced Cardiac Microvascular Endothelial Cell Injury via Peroxynitrite: Protection by Tongxinluo

Abstract

Activated macrophages contribute to endothelial dysfunction; however, it is unclear how peroxynitrite contributes to macrophage-mediated human cardiac microvascular endothelial cell (HCMEC) injury in hypoxia. In macrophage-HCMEC co-cultures subjected to hypoxia, there was an increase in hypoxia-inducible factor (HIF)-1α, HIF-2α, inducible nitric oxide synthase (iNOS), endothelin-converting enzyme (ECE)-1 and cyclooxygenase-2 (COX-2), and concomitant decrease in prostacyclin synthase (PGIS). This was mimicked by a peroxynitrite donor and attenuated by its decomposition catalyst. Tongxinluo (TXL) could decrease HIF-2α, iNOS, ECE-1 and COX-2 and increase PGIS in a dose-dependent manner, with increase of vascular endothelial growth factor. The protein alterations verified the remarkably affected mRNAs, indicating that the effects of TXL were similar to but better than that of peroxynitrite decomposition catalyst. Furthermore, TXL inhibited macrophage-mediated nitrotyrosine accumulation and attenuated HCMEC injury. The results suggest that peroxynitrite contributes to macrophage-mediated HCMEC injury in hypoxia, and TXL attenuates HCMEC injury mainly by inhibiting peroxynitrite.     

A new anti-angiogenic small molecule,G0811, inhibits angiogenesis via targeting hypoxia inducible factor (HIF)-1α signal transduction

Regulation of hypoxia inducible factor (HIF)-1α stabilization, which in turn contributes to adaptation of tumor cells to hypoxia has been highlighted as a promising therapeutic target in angiogenesis-related diseases. We have identified a new small molecule, G0811, as a potent angiogenesis inhibitor that targets HIF-1α signal transduction. G0811 suppressed HIF-1α stability in cancer cells and inhibited in vitro and in vivo angiogenesis, as validated by tube formation, chemoinvasion, and chorioallantoic membrane (CAM) assays. In addition, G0811 effectively decreased the expression of vascular endothelial growth factor (VEGF), which is one of target genes of HIF-1α. However, G0811 did not exhibit anti-proliferative activities or toxicity in human umbilical vein endothelial cells (HUVECs) at effective doses. These results demonstrate that G0811 could be a new angiogenesis inhibitor that acts by targeting HIF-1α signal transduction pathway.