Hypoxia regulation of expression and angiogenic effects of vasoactive intestinal peptide (VIP) and VIP receptors in LNCaP prostate cancer cells

Vascular endothelial growth factor (VEGF) is a main factor promoting neovascularization (angiogenesis) of solid tumours as prostate carcinoma. Hypoxia stimulates VEGF gene expression by activating the hypoxia-inducible factor-1 (HIF-1alpha). In the present study, the hypoxia-mimicking agent Ni(2+) induced vasoactive intestinal peptide (VIP) expression at both mRNA and peptide levels but it did not modify the expression of VIP receptors (VPAC(1), VPAC(2) and PAC(1) receptors) in androgen-dependent human LNCaP prostate cancer cells. VIP increased the mRNA levels of VPAC(1) and PAC(1) receptors whereas it decreased VPAC(2) receptor mRNA level. These features support that hypoxia up-regulation of VIP gene expression in prostatic carcinoma may lead to VIP regulation of the expression of its receptors by means of autocrine/paracrine mechanisms. Either VIP or hypoxia mimetics with Ni(2+) increased VEGF expression whereas both conditions together resulted in an additive response. It suggests two independent mechanisms for the observed pro-angiogenic activities of VIP and hypoxia. VIP did not stimulate HIF-1alpha mRNA expression but increased the translocation of HIF-1alpha from the cytosolic compartment to the cell nucleus. Moreover, VIP was unable to modify the expression of the HIF-1alpha inhibitor FIH-1 discarding the possibility of an indirect effect of VIP on HIF-1 transactivation.     

Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis

Hypoxia-inducible factor (HIF) constitutes a target in therapeutic angiogenesis. HIF-1alpha functions as a sensor of hypoxia and induces expression of vascular endothelial growth factor (VEGF), which then induces angiogenesis. To explore the potential of HIF-1alpha gene therapy in stimulating wound healing, we delivered a gene encoding a stabilized form of HIF-1alpha, lacking the oxygen-sensitive degradation domain, namely HIF-1alpha deltaODD, by using a previously characterized peptide-based gene delivery vector in fibrin as a surgical matrix. The peptide vector consisted of multiple domains: (i) A cysteine-flanked lysine hexamer provided DNA interactions that were stable extracellularly but destabilized intracellularly after reduction of the formed disulfide bonds. This DNA-binding domain was fused to either (ii) a fibrin-binding peptide for entrapment within the matrix or (iii) a nuclear localization sequence for efficient nuclear targeting. The HIF-1alpha deltaODD gene was expressed and translocated to the nucleus under normoxic conditions, leading to up-regulation of vascular endothelial growth factor (VEGF)-A165 mRNA and protein levels in vitro. When the peptide-DNA nanoparticles entrapped in fibrin matrices were applied to full-thickness dermal wounds in the mouse (10 microg per wound in 30 microl of fibrin), angiogenesis was increased comparably strongly to that induced by VEGF-A165 protein (1.25 microg per wound in 30 microl of fibrin). However, the maturity of the vessels induced by HIF-1alpha deltaODD was significantly higher than that induced by VEGF-A165 protein, as shown by stabilization of the neovessels with smooth muscle. Nonviral, local administration of this potent angiogenesis-inducing gene by using this peptide vector represents a powerful approach in tissue engineering and therapeutic angiogenesis.     

Expression of peroxisome proliferator-activated receptors in lean and obese Zucker rats

OBJECTIVE: Examination of the pattern of expression of peroxisome proliferator-activated receptor (PPAR) isoforms alpha and gamma in a model of obesity. DESIGN: Examination of adipose tissue and primary adipocyte cultures from lean and obese Zucker rats at different ages (28 days and 12 weeks). METHODS: mRNA levels were measured by RNase protection assay.RESULTS: The highest levels of PPARalpha and gamma mRNA were present in brown adipose tissue (BAT), followed by liver and white adipose tissue (WAT) for the alpha and gamma subtypes, respectively, at both ages examined. PPARalpha was expressed 100-fold higher in BAT compared with WAT, and PPARgamma mRNA levels were 2-fold higher in the WAT of obese compared with lean rats. PPARalpha and gamma expression was minimal in m. soleus, although higher levels of PPARgamma were found in the diaphragm. In marked contrast to the findings in vivo, virtually no PPARalpha mRNA could be detected in BAT cultures differentiated in vitro. CONCLUSION: PPARalpha and gamma are most highly expressed in BAT in vivo. However, PPARalpha is undetectable in brown adipose cells in vitro, suggesting that the expression of this receptor is induced by some external stimuli. In addition, the expression of PPARgamma was increased in WAT from young obese animals, compatible with an early adaptive phenomenon. Finally, the presence of PPARgamma mRNA is detectable only in particular muscles, such as the diaphragm, suggesting the possibility of an influence of fiber type on its expression, although exercise did not influence the expression of PPARgamma in other skeletal muscles.     

Cell type-specific regulation of angiogenic growth factor gene expression and induction of angiogenesis in nonischemic tissue by a constitutively active form of hypoxia-inducible factor 1

Understanding molecular mechanisms regulating angiogenesis may lead to novel therapies for ischemic disorders. Hypoxia-inducible factor 1 (HIF-1) activates vascular endothelial growth factor (VEGF) gene expression in hypoxic/ischemic tissue. In this study we demonstrate that exposure of primary cultures of cardiac and vascular cells to hypoxia or AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, modulates the expression of genes encoding the angiogenic factors angiopoietin-1 (ANGPT1), ANGPT2, placental growth factor, and platelet-derived growth factor-B. Loss-of-function effects were also observed in HIF-1alpha-null embryonic stem cells. Depending on the cell type, expression of ANGPT1 and ANGPT2 was either activated or repressed in response to hypoxia or AdCA5. In all cases, there was complete concordance between the effects of hypoxia and AdCA5. Injection of AdCA5 into mouse eyes induced neovascularization in multiple capillary beds, including those not responsive to VEGF alone. Analysis of gene expression revealed increased expression of ANGPT1, ANGPT2, platelet-derived growth factor-B, placental growth factor, and VEGF mRNA in AdCA5-injected eyes. These results indicate that HIF-1 functions as a master regulator of angiogenesis by controlling the expression of multiple angiogenic growth factors and that adenovirus-mediated expression of a constitutively active form of HIF-1alpha is sufficient to induce angiogenesis in nonischemic tissue of an adult animal.     

缺氧对过氧化物酶体增殖物激活受体α表达的影响

目的 研究不同程度缺氧条件下，肺癌A549细胞内过氧化物酶体增殖物激活受体α（PPARα）和缺氧诱导因子1α（HIF-1α）的表达状况，及反义封闭HIF-1α后，对PPARα受体的影响，以了解PPARα和HIF-1α的相关性。方法 首先将A549细胞分为4组：正常对照组（A组）、缺氧24小时组（B组）、缺氧48小时组（C组）、缺氧72小时组（D组）。观察不同缺氧时间对PPARα及 HIF-1α蛋白和mRNA表达水平的影响。为观察HIF-1α对PPARα表达状况的影响，再设计4组：对照组2（E组），HIF-1α反义寡核苷酸组（F组），HIF-1α正义寡核苷酸组（G组），HIF-1α错义寡核苷酸组（H组）。结果 正常对照组，PPARα和HIF-1α蛋白及mRNA均有少量表达；随着缺氧时间的延长，两的表达水平逐渐升高。在缺氧24小时，两mRNA和蛋白开始较高，48小时明显增高，至72小时达到高峰。反义封闭HIF-1α后，PPARα蛋白及mRNA的表达水平明显下降。结论 PPARα及HIF—1α的表达水平随肿瘤缺氧信号的加强而增加，且PPARα受HIF-1α的调控。     

Ontogenic loss of brown adipose tissue sensitivity to beta-adrenergic stimulation in the ovine

In ruminants and other large animals, expression of uncoupling protein-1 (UCP1) in brown adipose tissue (BAT) is confined to the perinatal period when it plays a key role in nonshivering thermogenesis. This study determined whether loss of expression of the BAT phenotype was due to reduced response to a beta-agonist, isoprenaline, and expression of the peroxisome proliferator-activated receptor (PPAR) family [PPARalpha, PPARgamma, PPAR coactivator 1alpha (PGC-1alpha)], which regulates UCP1 gene expression. Perirenal adipose tissue (PAT) was sampled from ovine fetuses, newborn lambs, and lambs on d 1, 5, 7, and 21 of life. UCP1 mRNA and protein in PAT increased from d 123 of fetal life to reach a maximum at birth followed by a rapid decrease over the first 5 d of life. Expression of the coactivator, PGC-1alpha and PPAR alpha, peaked between fetal day 123 and birth, and then declined to undetectable levels in the first days of life. In vivo administration of isoprenaline was able to induce expression of UCP1, PGC-1alpha, and PPARalpha in BAT up to 5 d of age but thereafter was ineffective. In vitro addition of beta-receptor, PPARalpha, and PPARgamma agonists were unable to overcome the suppression of UCP1, PPARalpha, and PPARgamma expression observed in differentiated adipocytes prepared from 30-d-old compared with 1-d-old lambs. These data are consistent with a model in which postnatal loss of UCP1 expression and beta-adrenergic induction of the brown adipocyte phenotype is due to loss of expression of PGC-1alpha and PPARalpha.     

Mitochondrial reactive oxygen species and c-Src play a critical role in hypoxic response in vascular smooth muscle cells

OBJECTIVE: Thickened atherosclerotic plaques are prone to be hypoxic because of poor perfusion. In this study, we tested (a) whether reactive oxygen species (ROS) and c-Src play roles in hypoxic induction of HIF-1alpha protein and PAI-1 gene expression in the rabbit aortic smooth muscle cell line C2/2 cells and primary cultures of rat aortic smooth muscle cells, and (b) how mitochondria act on the hypoxia-induced signaling mechanism. METHODS AND RESULTS: Hypoxic exposure of C2/2 cells increased H2O2 generation, c-Src phosphorylation, HIF-1alpha protein expression, and PAI-1 gene expression. Catalase, a scavenger of H2O2, inhibited the hypoxia-induced ROS generation and PAI-1 gene expression. Src kinase inhibitors PP1 and PP2 inhibited hypoxia-induced HIF-1alpha protein and PAI-1 gene expression. Ablation of mitochondrial respiration by rotenone abolished hypoxia-induced ROS generation, c-Src phosphorylation, HIF-1alpha protein expression, and PAI-1 gene expression. CONCLUSION: Induction of HIF-1alpha protein and PAI-1 gene expression in response to hypoxia was regulated by ROS production and c-Src activation in vascular smooth muscle cells. Mitochondria linked the hypoxic signal to c-Src, which in turn led to HIF-1alpha protein and PAI-1 gene expression. These results provide evidence that hypoxia induces the ROS-mediated and c-Src-dependent signaling cascades which are closely associated with angiogenesis and thrombosis in atherosclerotic vasculature.     

Differential regulation of macrophage peroxisome proliferator-activated receptor expression by glucose : role of peroxisome proliferator-activated receptors in lipoprotein lipase gene expression

Peroxisome proliferator-activated receptors (PPARs) are implicated in several metabolic disorders with altered glucose and lipid metabolism, including atherosclerosis and diabetes. In the present study, we evaluated the in vitro and ex vivo effects of high glucose concentrations on macrophage PPAR mRNA expression. Exposition of monocyte-derived macrophages isolated from healthy donors to a high glucose environment led to an increase in PPARalpha and PPARbeta mRNA expression. In contrast, this treatment significantly decreased human macrophage PPARgamma mRNA expression. Overexpression of PPARalpha and PPARbeta mRNA and inhibition of PPARgamma mRNA expression were also observed in monocyte-derived macrophages isolated from patients with type 2 diabetes. Because high glucose and PPARalpha agonists increase lipoprotein lipase (LPL) gene expression, the role of PPARalpha in the glucose-mediated upregulation of macrophage LPL gene expression was next evaluated. Incubation of murine J774 macrophages with high glucose concentrations increased the expression of PPARalpha at the mRNA and protein levels and enhanced nuclear protein binding to the peroxisome proliferator responsive element of the LPL promoter. Incubation of nuclear extracts in the presence of anti-PPARalpha and anti-PPARbeta antibodies decreased glucose-stimulated nuclear protein binding to the peroxisome proliferator responsive element. These results demonstrate that glucose is an important regulator of macrophage PPAR expression and suggest a role of PPARalpha and PPARbeta in the upregulation of macrophage LPL by glucose. Dysregulation of macrophage PPAR expression in type 2 diabetes may contribute, by altering arterial lipid metabolism and inflammatory response, to the accelerated atherosclerosis associated with diabetes.     

Peroxisome proliferator-activated receptor alpha (PPARalpha) potentiates, whereas PPARgamma attenuates, glucose-stimulated insulin secretion in pancreatic beta-cells

Fatty acids (FAs) are known to be important regulators of insulin secretion from pancreatic beta-cells. FA-coenzyme A esters have been shown to directly stimulate the secretion process, whereas long-term exposure of beta-cells to FAs compromises glucose-stimulated insulin secretion (GSIS) by mechanisms unknown to date. It has been speculated that some of these long-term effects are mediated by members of the peroxisome proliferator-activated receptor (PPAR) family via an induction of uncoupling protein-2 (UCP2). In this study we show that adenoviral coexpression of PPARalpha and retinoid X receptor alpha (RXRalpha) in INS-1E beta-cells synergistically and in a dose- and ligand-dependent manner increases the expression of known PPARalpha target genes and enhances FA uptake and beta-oxidation. In contrast, ectopic expression of PPARgamma/RXRalpha increases FA uptake and deposition as triacylglycerides. Although the expression of PPARalpha/RXRalpha leads to the induction of UCP2 mRNA and protein, this is not accompanied by reduced hyperpolarization of the mitochondrial membrane, indicating that under these conditions, increased UCP2 expression is insufficient for dissipation of the mitochondrial proton gradient. Importantly, whereas expression of PPARgamma/RXRalpha attenuates GSIS, the expression of PPARalpha/RXRalpha potentiates GSIS in rat islets and INS-1E cells without affecting the mitochondrial membrane potential. These results show a strong subtype specificity of the two PPAR subtypes alpha and gamma on lipid partitioning and insulin secretion when systematically compared in a beta-cell context.     

Peroxisome proliferator-activated receptor alpha down-regulation is associated with enhanced ceramide levels in age-associated cardiac hypertrophy

We used an experimental murine model of accelerated aging, the senescence-accelerated mouse (SAM), to examine the effect of age-associated cardiac hypertrophy on peroxisome proliferator-activated receptor alpha (PPARalpha) expression and activity in the heart. Senescence-accelerated prone mice (SAM-P8) showed cardiac hypertrophy compared with senescence-accelerated resistant mice (SAM-R1). Furthermore, a decrease in PPARalpha messenger RNA (mRNA; 28% reduction, p<.001) and protein (47%, p<.05) levels and in PPAR DNA-binding activity was observed in SAM-P8 hearts. Increased protein-protein interaction between PPARalpha and the p65 subunit of nuclear factor-kappaB (NF-kappaB) was found, suggesting that this mechanism may prevent PPARalpha from binding to its response elements. The mRNA levels of PPARalpha target genes involved in fatty acid use were strongly suppressed in SAM-P8, which was consistent with the accumulation of ceramide in SAM-P8 hearts (2.5-fold induction, p<.05). These findings suggest that NF-kappaB activation in SAM-P8 heart prevents PPARalpha from binding to its response elements leading to changes in gene expression that may lead to ceramide accumulation in the aged heart.