MAB5382 Sigma-AldrichAnti-Hypoxia Inducible Factor 1 α Antibody, clone H1α67

Accumulating evidence reveals that intrauterine growth retardation (IUGR) can cause varying degrees of pulmonary arterial hypertension (PAH) later in life. Moreover, epigenetics plays an important role in the fetal origin of adult disease. The goal of this study was to investigate the role of epigenetics in the development of PAH following IUGR.The IUGR rats were established by maternal undernutrition during pregnancy. Pulmonary vascular endothelial cells (PVEC) were isolated from the rat lungs by magnetic-activated cell sorting (MACS). We investigated epigenetic regulation of the endothelin-1 (ET-1) gene in PVEC of 1-day and 6-week IUGR rats, and response of IUGR rats to hypoxia.The maternal nutrient restriction increased the histone acetylation and hypoxia inducible factor-1α (HIF-1α) binding levels in the ET-1 gene promoter of PVEC in IUGR newborn rats, and continued up to 6 weeks after birth. These epigenetic changes could result in an IUGR rat being highly sensitive to hypoxia later in life, causing more significant PAH or pulmonary vascular remodeling.These findings suggest that epigenetics is closely associated with the development of hypoxic PAH following IUGR, further providing a new insight for improved prevention and treatment of IUGR-related PAH.

Accurate prognostication of locally advanced nasopharyngeal carcinoma (NPC) will benefit patients for tailored therapy. Here, we addressed this issue by developing a mathematical algorithm based on support vector machine (SVM) through integrating the expression levels of multi-biomarkers.Ninety-seven locally advanced NPC patients in a randomized controlled trial (RCT), consisting of 48 cases serving as training set and 49 cases as testing set of SVM models, with 5-year follow-up were studied. We designed SVM models by selecting the variables from 38 tissue molecular biomarkers, which represent 6 tumorigenesis signaling pathways, and 3 EBV-related serological biomarkers. We designed 3 SVM models to refine prognosis of NPC with 5-year follow-up. The SVM1 displayed highly predictive sensitivity (sensitivity, specificity were 88.0% and 81.9%, respectively) by integrating the expression of 7 molecular biomarkers. The SVM2 model showed highly predictive specificity (sensitivity, specificity were 84.0% and 94.5%, respectively) by grouping the expression level of 12 molecular biomarkers and 3 EBV-related serological biomarkers. The SVM3 model, constructed by combination SVM1 with SVM2, displayed a high predictive capacity (sensitivity, specificity were 88.0% and 90.3%, respectively). We found that 3 SVM models had strong power in classification of prognosis. Moreover, Cox multivariate regression analysis confirmed these 3 SVM models were all the significant independent prognostic model for overall survival in testing set and overall patients.Our SVM prognostic models designed in the RCT displayed strong power in refining patient prognosis for locally advanced NPC, potentially directing future target therapy against the related signaling pathways.

Exogenous delivery of the neurotrophin-3 (NT-3) gene may provide a potential therapeutic strategy for ischemic stroke. To investigate the neuroprotective effects of NT-3 expression controlled by 5HRE after focal cerebral ischemia, we constructed a recombinant retrovirus vector (RV) with five copies of hypoxia-responsive elements (5HRE or 5H) and NT-3 and delivered it to the rat brain. Three groups of rats received RV-5H-NT3, RV-5H-EGFP or saline injection. Three days after gene transfer, the rats underwent 90min of transient middle cerebral artery occlusion (tMCAO), followed by 1-28days of reperfusion. Three days after tMCAO, brain NT-3 expression was significantly increased in the RV-5H-NT3-transduced animals compared with the RV-5H-EGFP or saline group, and brain infarct volume was smaller in the RV-5H-NT3-transduced group than the RV-5H-EGFP or saline group. The percentage of TUNEL-positive cells was reduced in RV-5H-NT3-transduced brains compared with the RV-5H-EGFP or saline group 3 and 7days after tMCAO. Furthermore, the neurological status of RV-5H-NT3-transduced rats was better than that of RV-5H-EGFP- or saline-transduced animals from 1day to 4weeks after tMCAO. Our results demonstrated that 5HRE could modulate NT-3 expression in the ischemic brain environment and that the up-regulated NT-3 could effectively improve neurological status following tMCAO due to decreased initial damage. To avoid unexpected side effects, 5HRE-controlled gene expression might be a useful tool for gene therapy of ischemic disorders in the central nervous system.

The PO(2) within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing PO(2) on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O(2) concentration from 20 to 8% decreased ENaC activity by 40%; increasing O(2) content to 40% increased ENaC activity by 50%. The O(2) effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O(2) medium. Corticosteroids increased ENaC activity at each O(2) concentration; there was no interaction. The pathways for O(2) and steroid regulation of ENaC are different since O(2) did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2-45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O(2) appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O(2) concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O(2) induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O(2); both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.

Nephrin (NPHS1) has been described as an important structural protein of kidney podocytes. Mutations in this gene lead to the Finnish-type congenital nephrotic syndrome. More recently, a role of nephrin as a signalling molecule in kidney podocytes has been identified. Here, we show that nephrin not only has a function in kidney podocytes, but is also required for cardiovascular development. Nephrin is expressed in the epicardium and coronary vessels during human and mouse embryonic development. Nephrin knockout embryos showed abnormal epicardial cell morphology and, at later stages of development, a reduced number of coronary vessels due to increased apoptosis, and in addition, cardiac fibrosis. Connexin 43, which is required for coronary vessel formation, was downregulated in nephrin knockout embryos. Expression of the p75NTR neurotrophin receptor, a known mediator of apoptosis, was increased in mutants. Furthermore, co-immunoprecipitation studies demonstrated a direct interaction of nephrin with p75NTR. Primary nephrin-deficient cardiac cells showed a 5-fold higher rate of apoptosis in response to progenitor of nerve growth factor compared with wild-type cells, which could be rescued by RNAi against p75NTR. Taken together, our data demonstrate that nephrin directly interacts with p75NTR and reveal an important role for nephrin in murine cardiac development by permitting survival of cardiovascular progenitor cells.

Mutations in presenilin-1 (Psen1) cause familial Alzheimer's disease (FAD). Both hypoxia and ischemia have been implicated in the pathological cascade that leads to amyloid deposition in AD. Here we investigated whether Psen1 might regulate hypoxic responses by modulating induction of the transcription factor hypoxia inducible factor 1-α (HIF-1α).In fibroblasts that lack Psen1 induction of HIF-1α was impaired in response to the hypoxia mimetic cobalt chloride, as well as was induction by insulin and calcium chelation. Reintroduction of human Psen1 using a lentiviral vector partially rescued the responsiveness of Psen1-/- fibroblasts to cobalt chloride induction. HIF-1α induction did not require Psen1's associated γ-secretase activity. In addition, the failure of insulin to induce HIF-1α was not explicable on the basis of failed activation of the phosphatidylinositol 3-kinase (PI3K/Akt) pathway which activated normally in Psen1-/- fibroblasts. Rather we found that basal levels of HIF-1α were lower in Psen1-/- fibroblasts and that the basis for lower constitutive levels of HIF-1α was best explained by accelerated HIF-1α degradation. We further found that Psen1 and HIF-1α physically interact suggesting that Psen1 may protect HIF-1α from degradation through the proteasome. In fibroblasts harboring the M146V Psen1 FAD mutation on a mouse Psen1 null background, metabolic induction of HIF-1α by insulin was impaired but not hypoxic induction by cobalt chloride. Unlike Psen1-/- fibroblasts, basal levels of HIF-1α were normal in FAD mutant fibroblasts but activation of the insulin-receptor pathway was impaired. Interestingly, in Psen1-/- primary neuronal cultures HIF-1α was induced normally in response to cobalt chloride but insulin induction of HIF-1α was impaired even though activation of the PI3K/Akt pathway by insulin proceeded normally in Psen1-/- neuronal cultures. Basal levels of HIF-1α were not significantly different in Psen1-/- neurons and HIF-1α levels were normal in Psen1-/- embryos.Collectively these studies show that Psen1 regulates induction of HIF-1α although they indicate that cell type specific differences exist in the effect of Psen1 on induction. They also show that the M146V Psen1 FAD mutation impairs metabolic induction of HIF-1α, an observation that may have pathophysiological significance for AD.

Cyclosporine (CsA) is among the most widely used immunosuppressants for preventing graft rejection and autoimmune diseases. However, its clinical use is hampered by its significant nephrotoxicity and effects as a cause of hypertension. The proximal tubular Na+-H+ exchanger (NHE3) is responsible for transcellular reabsorption of 30%-60% of the sodium filtered by the glomerulus. CsA induces a reduction of absolute sodium reabsorption, and this effect is, most probably, correlated with the decrease of NHE3 activity. In Henles loop, in physiological conditions, the Na+-K+-2Cl- cotransporter (NKCC2) reabsorbs approximately 20% of the filtered Na+ and Cl-. CsA increases the NKCC2 activity in cultured bovine renal NBL-1 cells. In the collecting duct, CsA may cause hypertension by stimulating the epithelial Na+ channel (ENaC) through a pathway associated with inhibition of ABCA1 and consequent elevation of cholesterol in the cells. It is still unclear whether CsA regulates the Na+-Cl- cotransporter in the distal tubule and ENaC in the collecting duct. Aside from this, there is evidence suggesting the possible involvement of free radicals during the development of CsA-induced hypertension. The hypertensive effect is, most probably, correlated with higher levels of superoxide (O2-) that decreases glomerular filtration rate and may affect fluid reabsorption along the nephron.

BACKGROUND: Lactate-dehydrogenase-5 (LDH-5) is an important isoenzyme converting pyruvate to lactate under hypoxic conditions and might play an important role in the development and progression of malignancies. However, the role of LDH-5 in gastric cancer is still unclear. In this study, we investigated the clinical significance of LDH-5 expression in gastric carcinoma. METHODS: LDH-5 expression in 152 patients with different grade and stage gastric carcinoma was analyzed by immunohistochemistry. In addition, hypoxia-inducible factor 1alpha (HIF-1alpha) as a marker of tumor hypoxia, as well as vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) as angiogenesis parameters were also assessed in this study. Correlations between the expression of investigated proteins and various clinicopathological factors including survival were determined. RESULTS: There were 94 cases (61.8%) showing high LDH-5 expression, and 95 patients (62.5%) had high HIF-1alpha expression. Positive correlation was found between LDH-5 expression and HIF-1alpha, VEGF, and COX-2. The overexpression of LDH-5 was more prevalent in advanced tumors having positive vessel invasion. Patients with overexpression of LDH-5 showed far lower disease-free (63.5% vs 82.7%) and overall (56.3% vs 78.4%) survival rates compared with patients with low LDH-5 expression. HIF-1alpha expression was shown to have no significance on survival. In multivariate analysis, high LDH-5 expression kept its independence as a negative prognostic indicator. CONCLUSION: The results of the current study show that LDH-5 expression may be a useful prognostic factor for patients with gastric carcinoma.

We investigate the relationship between the fate and healing effect of transplanted mesenchymal stromal cells (MSCs) in a rat diabetic skin wound model. Rats are treated with streptozotocin to induce diabetic conditions. A full-thickness skin defect is surgically made on the head of diabetic rats, and covered with an artificial dermis impregnated with either bone marrow cells (BMCs) or bone-marrow-derived MSCs from firefly luciferase (luc) transgenic (Tg) rats. Wound healing is evaluated using planimetry and immunohistochemistry, and the fate of transplanted MSCs is determined using in-vivo luminescent imaging. The diabetic wound treated with MSCs-impregnated artificial dermis is significantly smaller than that treated with artificial dermis alone at 1 week postoperation. Photons of luc+ MSCs are detected at the transplanted site during healing (3 weeks), whereas those of luc+ MSCs are depleted only after 1 week postimplantation. Immunohistochemistry at the healing site treated with MSCs demonstrates that CD31+ vessels increase with expression of vascular endothelial growth factor, suggesting that MSCs accelerate angiogenesis. These findings suggest that transplanted MSCs could be retained at wound sites during the healing process in a diabetic rat model, and subsequently promote wound healing through angiogenesis.