Endothelin-mediated increases in lung VEGF content promote vascular leak in young rats exposed to viral infection and hypoxia

Viral respiratory infections increase the susceptibility of young animals to hypoxia-induced pulmonary edema formation. Previous work has shown that increased lung levels of endothelin (ET) contribute to this effect, though the mechanisms by which ET promotes vascular leak remain uncertain. Both in vitro and in vivo evidence suggests that ET can upregulate the production of VEGF, which is known to increase vascular permeability. We hypothesized that increases in lung ET promote increases in lung VEGF, which in turn increases vascular leak in the lung. Weanling rats were exposed to moderate hypoxia for 24 h while recovering from a mild viral respiratory infection, to hypoxia alone, or to viral infection alone. Lung VEGF mRNA and protein content were measured by RT-PCR and Western blotting, respectively. Animals exposed to hypoxia + virus demonstrated significant increases in lung VEGF mRNA and protein content. Immunohistochemical studies showed increased VEGF expression in alveolar septa and small pulmonary vessels in those animals. ET receptor blockade with bosentan prevented this increase in lung VEGF content, suggesting that ET promotes VEGF accumulation in the lung in this setting. Animals exposed to hypoxia + virus also demonstrated substantial increases in lung albumin extravasation, and those increases were blocked by both ET receptor blockade and VEGF antagonism. These findings suggest that ET-driven increases in lung VEGF content can contribute to the formation of pulmonary edema.     

A 40-bp RNA element that mediates stabilization of vascular endothelial growth factor mRNA by HuR

VEGF is a critical mediator of hypoxia-induced angiogenesis in numerous physiological and pathophysiological conditions. The hypoxic induction of VEGF is due in large part to an increase in the stability of its mRNA. We recently demonstrated that the stabilization of VEGF mRNA by hypoxia is dependent upon the RNA-binding protein HuR. This report describes the identification of a 40-bp functional HuR binding site in the VEGF mRNA 3'-untranslated region. This element can confer HuR-mediated stabilization of a heterologous gene in vitro and in vivo. Furthermore, the element is sufficient to confer an increase in the hypoxic induction of a heterologous gene. Deletion of the HuR binding site within this 40-bp element as mapped by RNase T1 and lead footprinting uncouples a stabilizing sequence from a destabilizing sequence, thus providing a novel RNA-protein regulatory model that might be exploited to manipulate VEGF expression and hypoxia-induced angiogenesis.     

p38丝裂原素激活的蛋白激酶在调节低氧诱导人内皮细胞分泌血管内皮生长因子过程中的作用

血管内皮细胞中血管内皮生长因子(vascular endothelial growthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用.然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚.人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12～24 h后分别用实时定量PCR和Western blot的方法来检测VEGF mRNA的表达及ERK1/2和p38激酶的磷酸化水平.分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测.业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制.本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用.p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加.这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用.     

Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO

In this study, an in vitro model of the blood-brain barrier,consisting of porcine brain-derived microvascular endothelial cells(BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMECwas completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxicconditions induced a twofold increase in VEGF binding sites and VEGFreceptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor N^G-monomethyl-L-arginine,suggesting that NO is involved in hypoxia-induced permeability changes,which was confirmed by measurements of the cGMP level. During normoxia,treatment with VEGF (5 ng/ml) increased permeability as well as cGMPcontent in the presence of several antioxidants. These results suggestthat hypoxia-induced permeability in vitro is mediated by the VEGF/VEGFreceptor system in an autocrine manner and is essentially dependent onreducing conditions stabilizing the second messenger NO as the mediatorof changes in barrier function of BMEC.     

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.     

SURZB/Kir6.1通道开放剂纳他卡林对低氧致大鼠主动脉内皮细胞损伤的保护作用

目的:探讨纳他卡林对低氧引起大鼠主动脉内皮细胞损伤的保护作用及其机制。方法:选取大鼠主动脉内皮细胞作为体外低氧损伤的细胞模型,分为正常对照组、低氧模型组、纳他卡林低、中、高剂量组,利用MTT法测定细胞生存率,硝酸还原酶法检测一氧化氮(NO)释放,RT-PCR法检测细胞间粘附因子-1(ICAM-1)、内皮素-1(ET-1)、血管内皮生长因子(VEGF)mRNA水平。结果:纳他卡林三个剂量组均可逆转低氧所致的血管内皮细胞功能改变,包括提高内皮细胞生存活力和NO的释放水平,显著抑制低氧引发的内皮细胞ICAM-1,ET-1,VEGF mRNA表达量的上调。结论:纳他卡林对低氧诱发的血管内皮细胞分泌功能改变、细胞通透性增加及炎性因子的分泌均具有保护作用。     

Identification of DEAD-box RNA Helicase 6 (DDX6) as a Cellular Modulator of Vascular Endothelial Growth Factor Expression under Hypoxia

Vascular endothelial growth factor A (VEGF) is a crucial proangiogenic factor, which regulates blood vessel supply under physiologic and pathologic conditions. The VEGF mRNA 5′-untranslated region (5′-UTR) bears internal ribosome entry sites (IRES), which confer sustained VEGF mRNA translation under hypoxia when 5′-cap-dependent mRNA translation is inhibited. VEGF IRES-mediated initiation of translation requires the modulated interaction of trans-acting factors. To identify trans-acting factors that control VEGF mRNA translation under hypoxic conditions we established an in vitro translation system based on human adenocarcinoma cells (MCF-7). Cytoplasmic extracts of MCF-7 cells grown under hypoxia (1% oxygen) recapitulate VEGF IRES-mediated reporter mRNA translation. Employing the VEGF mRNA 5′-UTR and 3′-UTR in an RNA affinity approach we isolated interacting proteins from translational active MCF-7 extract prepared from cells grown under normoxia or hypoxia. Interestingly, mass spectrometry analysis identified the DEAD-box RNA helicase 6 (DDX6) that interacts with the VEGF mRNA 5′-UTR. Recombinant DDX6 inhibits VEGF IRES-mediated translation in normoxic MCF-7 extract. Under hypoxia the level of DDX6 declines, and its interaction with VEGF mRNA is diminished in vivo. Depletion of DDX6 by RNAi further promotes VEGF expression in MCF-7 cells. Increased secretion of VEGF from DDX6 knockdown cells positively affects vascular tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Our results indicate that the decrease of DDX6 under hypoxia contributes to the activation of VEGF expression and promotes its proangiogenic function.     

VEGF and Bcl-2 Interact Via MAPKs Signaling Pathway in the Response to Hypoxia in Neuroblastoma

Tumor hypoxia has been reported to be a negative prognostic factor in a number of tumor sites, which suggests a positive correlation between tumor hypoxia and increased metastatic efficiency. Evidence shows that vascular endothelial growth factor (VEGF) stimulates angiogenesis in tumor growth and mediates neuroprotection to prevent an apoptotic cell death. Human neuroblastoma cells (CHP126) were exposed to moderate hypoxia for different time spans to explore the molecular stress responses. Apoptotic features as an increase of Bax/Bcl-2 ratio and activation of caspase 3 were observed at early period of exposure time, but these effects were reversed with the extension of hypoxic treatment. Hypoxia also activated MAPKs signaling pathways in a time-relative manner, which were involved in the regulation of hypoxia-related resistance of CHP126 cells. Meanwhile, VEGF and its receptor KDR were found to interact with MAPKs signaling pathways except the effect of hypoxia. Furthermore, rhVEGF₁₆₅ was utilized to discern that VEGF increased Bcl-2 and procaspase 3 expressions, contributing to a synergistic relationship of an angiogenic response with Bcl-2 in hypoxia via a cross talk, while the activation of ERK MAPK is important for both productions. These altered signals may be critical to predict a poor outcome; therefore, our knowledge provides new insight into apoptosis and angiogenesis control of tumor cells and suggests a strategy based on the blockade of hypoxia-induced VEGF signaling under hypoxia in neuroblastoma.     

Effect of astroglial cells on hypoxia-induced permeability in PBMEC cells

An in vitro model of the blood-brain barrier (BBB),consisting of porcine brain-derived microvascular endothelial cells(PBMEC), was used to evaluate the effect of astrocytes in theBBB disruption during hypoxia. Hypoxia-induced hyperpermeability wasdecreased significantly in a coculture model of astroglia cells, either astrocytes or C6 glioma cells, with PBMEC and, to the same extent, whenglia cell-conditioned medium was used. Corresponding to effects onhypoxia-induced hyperpermeability, astrocyte- and C6 cell-conditioned medium diminished hypoxia-induced vascular endothelial growth factor(VEGF) mRNA and protein expression, which recently was shown to beresponsible for hypoxia-induced permeability changes in vitro. Theeffect on hypoxia-induced hyperpermeability and VEGF expression wasspecific for astroglia cells because conditioned medium from bovinesmooth muscle cells (BSMC) did not show any effect. Immunocytochemistryrevealed that 24 h of hypoxia disrupted the continuity of thetight junction protein, zonula occludens-1 (ZO-1), which lines thecytoplasmic face of intact tight junctions. These changes wereprevented when hypoxia was performed in glia cell-conditioned medium.Results suggest that astrocytes protect the BBB from hypoxia-inducedparacellular permeability changes by decreasing hypoxia-induced VEGFexpression in microvascular endothelial cells.

     

Anethole prevents hydrogen peroxide-induced apoptosis and collagen metabolism alterations in human skin fibroblasts

Thyroid hormone stimulates erythropoietic differentiation. However, severe anemia is sometimes seen in patients with hyperthyroidism, and the mechanisms have not been fully elucidated. Bone marrow is comprised about 2–8 % oxygen, and the characteristics of hematopoietic stem cells have been shown to be influenced under hypoxia. Hypoxia-inducible factor-1 is a critical mediator of cellular responses to hypoxia and an important mediator in signal transduction of thyroid hormone [triiodothyronine (T3)]. The aim of this study was to investigate the effect of T3 on erythropoiesis under hypoxia mimicking physiological conditions in the bone marrow. We maintained human erythroleukemia K562 cells under hypoxic atmosphere (2 % O₂) and examined their cellular characteristics. Compared to that under normal atmospheric conditions, cells under hypoxia showed a reduction in the proliferation rate and increase in the hemoglobin content or benzidine-positive rate, indicating promotion of erythroid differentiation. T3 had no effect on hypoxia-induced erythroid differentiation, but significantly inhibited activin A/erythroid differentiation factor-induced erythroid differentiation. Moreover, GATA2 mRNA expression was suppressed in association with erythroid differentiation, while T3 significantly diminished that suppression. These results suggest that T3 has a direct suppressive effect on erythroid differentiation under hypoxic conditions.     

Exploring the limits of benzimidazole DNA-binding oligomers for the hypoxia inducible factor (HIF) site

The vascular endothelial growth factor (VEGF) and its receptors have been implicated as key-factors in tumor angiogenesis and are major targets in cancer therapy. New oligomers which mimic the architecture of DNA-binding polyamides have been designed to target the hypoxia inducible factor (HIF-1) binding site on the promoter of VEGF gene. These oligomers incorporate an increasing number of six–five fused rings such as hydroxybenzimidazole–imidazole, benzimidazole–pyrrole, benzimidazole–chlorothiophene, and imidazopyridine–pyrrole, and bind the VEGF hypoxia response element (HRE) 5′-TACGT-3′ with high affinity and selectivity.     

Regulation of Vascular Endothelial Growth Factor Expression by cAMP in Rat Aortic Smooth Muscle Cells

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10⁻⁵M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.