Up-regulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2

Platelet-vascular endothelial cell interactions are central to the maintenance of vascular homeostasis. Thromboxane A2 (TXA2) and prostacyclin (prostaglandin (PG)I2) are the major products of cyclooxygenase (COX) metabolism by platelets and the vascular endothelium, respectively. Here we report the effects of platelet-endothelial interactions on human umbilical vein endothelial cells (HUVECs) COX-2 expression and prostanoid synthesis. Co-incubation of platelets with HUVECs resulted in a dose-dependent induction in COX-2 expression. This was accompanied by a relatively small increase in thromboxane B2 synthesis (2 ng) by comparison to the production of 6-keto-PGF1alpha and PGE2, which increased by approximately 14 and 12 ng, respectively. Abrogation of platelet-HUVEC interactions excluded direct cell-cell contact as a required event. Preincubation of HUVECs with SQ29548, a TXA2 receptor antagonist, dose-dependently inhibited platelet-induced COX-2 expression and prostanoid synthesis. Similarly, if platelet TXA2 synthesis was inhibited no induction of COX-2 was observed. Furthermore, a TXA2 analog, carbocyclic TXA2, induced HUVEC COX-2 expression and the synthesis of 6-keto-PGF1alpha and PGE2. This was also associated with an increase in the expression and activity of PGI synthase and PGE synthase but not TX synthase. Platelet co-incubation (or TXA2) also selectively activated the p44/42 mitogen-activated protein kinase pathway to regulate HUVEC COX-2 expression. Thus it seems that platelet-derived TXA2 can act in a paracrine manner to up-regulate endothelial COX-2 expression and PGI2 synthesis. These observations are of particular importance given the recent observations regarding selective COX-2 inhibitors and the suppression of PGI2 synthesis.     

Roles of cyclooxygenase (COX)-1 and COX-2 in prostanoid production by human endothelial cells: selective up-regulation of prostacyclin synthesis by COX-2

The two cyclooxygenase (COX) isoforms, COX-1 and COX-2, both metabolize arachidonic acid to PGH(2), the common substrate for thromboxane A(2) (TXA(2)), prostacyclin (PGI(2)), and PGE(2) synthesis. We characterized the synthesis of these prostanoids in HUVECs in relation to COX-1 and COX-2 activity. Untreated HUVEC expressed only COX-1, whereas addition of IL-1beta caused induction of COX-2. TXA(2) was the predominant COX-1-derived product, and TXA(2) synthesis changed little with up-regulation of COX-2 by IL-1beta (2-fold increase). By contrast, COX-2 up-regulation was associated with large increases in the synthesis of PGI(2) and PGE(2) (54- and 84-fold increases, respectively). Addition of the selective COX-2 inhibitor, NS-398, almost completely abolished PGI(2) and PGE(2) synthesis, but had little effect on TXA(2) synthesis. The up-regulation of COX-2 by IL-1beta was accompanied by specific up-regulation of PGI synthase and PGE synthase, but not TX synthase. An examination of the substrate concentration dependencies showed that the pathway of TXA(2) synthesis was saturated at a 20-fold lower arachidonic acid concentration than that for PGI(2) and PGE(2) synthesis. In conclusion, endothelial prostanoid synthesis appears to be differentially regulated by the induction of COX-2. The apparent PGI(2) and PGE(2) linkage with COX-2 activity may be explained by a temporal increase in total COX activity, together with selective up-regulation of PGI synthase and PGE synthase, and different kinetic characteristics of the terminal synthases. These findings have particular importance with regard to the potential for cardiovascular consequences of COX-2 inhibition.     

Hypoxia inducible factor(HIF) in the tumor microenvironment:friend or foe?

Hypoxia acts as an important regulator of physiological and pathological processes. Hypoxia inducible factors (HIFs) are the central players involved in the cellular adaptation to hypoxia and are regulated by oxygen sensing EGLN prolyl hydroxylases. Hypoxia affects many aspects of cellular growth through both redox effects and through the stabilization of HIFs. The HIF isoforms likely have differential effects on tumor growth via alteration of metabolism, growth, and self-renewal and are likely highly context-dependent. In some tumors such as renal cell carcinoma, the EGLN/HIF axis appears to drive tumorigenesis, while in many others HIF1 and HIF2 may actually have a tumor suppressive role. An emerging role of HIF biology is its effects on the tumor microenvironment. The EGLN/HIF axis plays a key role in regulating the function of the various components of the tumor microenvironment, which include cancer-associated fibroblasts, endothelial cells, immune cells, and the extracellular matrix (ECM). Here, we discuss hypoxia and the diverse roles of HIFs in the setting of tumorigenesis and the maintenance of the tumor microenvironment as well as possible future directions of the field.     

Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.     

Non-hypoxic activation of the negative regulatory feedback loop of prolyl-hydroxylase oxygen sensors

Hypoxia inducible factors (HIF) coordinate cellular responses towards hypoxia. HIFs are mainly regulated by a group of prolyl-hydroxylases (PHDs) that in the presence of oxygen, target the HIFα subunit for degradation. Herein, we studied the role of nitric oxide (NO) in regulating PHD activities under normoxic conditions. In the present study we show that different NO-donors initially inhibited endogenous PHD2 activity which led to accumulation of HIF-1α subsequently to enhance HIF-1 dependent increased PHD2 promoter activity. Consequently PHD2 abundance and activity were strongly induced which caused downregulation of HIF-1α. Interestingly, upregulation of endogenous PHD2 activity by NO was not found in cells that lack an intact pVHL dependent degradation pathway. Recovery of PHD activity required intact cells and was not observed in cell extracts or recombinant PHD2. In conclusion induction of endogenous PHD2 activity by NO is dependent on a feedback loop initiated despite normoxic conditions.     

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen‐induced retinopathy: beneficial effect of the absence of AQP4

Hypoxia‐dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin‐4 (AQP4) is involved in the hypoxia‐dependent VEGF upregulation in the retina of a mouse model of oxygen‐induced retinopathy (OIR). The expression levels of VEGF, the hypoxia‐inducible factor‐1α (HIF‐1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF‐1 binding site (HBS) in the VEGF gene promoter, the binding of HIF‐1α to the HBS, the retinal vascularization and function have been determined in the retina of wild‐type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF‐1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF‐1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF‐1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF‐1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF‐induced retinal damage in response to hypoxia.     

Characterization of three IGFBP mRNAs in Atlantic croaker and their regulation during hypoxic stress: potential mechanisms of their upregulation by hypoxia

Insulin-like growth factor-binding proteins (IGFBPs) play important roles in downregulating IGF activity and growth and development in vertebrates under hypoxic stress. However, the mechanisms of hypoxia regulation of IGFBPs in teleost fishes are unknown. The involvement of reactive oxygen species (ROS) and hypoxia-inducible factors (HIFs) in hypoxia upregulation of IGFBPs in Atlantic croaker were investigated. Three croaker IGFBPs, IGFBP-1, IGFBP-2, and IGFBP-5, were cloned and characterized. Chronic hypoxia exposure [dissolved oxygen (DO): 1.7 mg/l for 2-4 wk] caused significant increases in hepatic and neural IGFBP-1 mRNA expression compared with tissue mRNA levels in fish held under normoxic conditions (6.5 mg DO/l). Moreover, longer-term chronic hypoxia exposure (2-2.7 mg DO/l for 15-20 wk) caused significant increases in mRNA levels of all three IGFBPs in both liver and brain tissues. Hypoxia exposure also markedly increased superoxide radical (O(2)(·-), an index of ROS) production and HIF-1α mRNA and HIF-2α protein expression in croaker livers. Pharmacological treatment with an antioxidant attenuated the hypoxia-induced increases in O(2)(·-) production and HIFα mRNA and protein expression as well as the elevation of IGFBP-1 mRNA levels. These results suggest that the upregulation of IGFBP expression under hypoxia stress is due, in part, to alterations in the antioxidant status, which may involve ROS and HIFs.     

Effects of shear stress on eicosanoid gene expression and metabolite production in vascular endothelium as studied in a novel biomechanical perfusion model

This study investigated the effects of shear stress on gene expression of prostacyclin synthesis-related enzymes cyclooxygenases (COX-1 and COX-2), prostacyclin synthase (PGS), and thromboxane synthase (TXS) and their metabolites prostaglandin (PGI(2)) and thromboxane A(2) (TXA(2)) in endothelium of intact conduit vessels. Paired human umbilical veins were perfused at high/low shear stress (25/<4 dyn/cm(2)) at identical intraluminal pressure (20 mmHg) for 1.5, 3, or 6 hours in a computerized vascular model. High shear perfusion induced a significant, monophasic upregulation of PGS and TXS gene expressions after 6 hours. COX-1 and COX-2 mRNA showed a biphasic response with peaks at 1.5 and 6 hours, with a nadir level at 3 hours. Shear-induced gene expression was associated with a significantly greater accumulation of 6-keto prostaglandin F(1alpha) and TXA(2) in the perfusion medium. Thus, shear stress independently of perfusion pressure alters the expression of prostacyclin synthesis-related enzymes and the biosynthesis of their vasoactive metabolites.     

Differential regulation of prostaglandin E2 and thromboxane A2 production in human monocytes: implications for the use of cyclooxygenase inhibitors

There is an autocrine relationship between eicosanoid and cytokine synthesis, with the ratio of prostaglandin E2 (PGE2)/thromboxane A2 (TXA2) being one of the determinants of the level of cytokine synthesis. In monocytes, cyclooxygenase type 1 (COX-1) activity appears to favor TXA2 production and COX-2 activity appears to favor PGE2 production. This has led to speculation regarding possible linkage of COX isozymes with PGE and TXA synthase. We have studied the kinetics of PGE2 and TXA2 synthesis under conditions that rely on COX-1 or -2 activity. With small amounts of endogenously generated prostaglandin H2 (PGH2), TXA2 synthesis was greater than PGE2. With greater amounts of endogenously generated PGH2, PGE2 synthesis was greater than TXA2. Also, TXA synthase was saturated at lower substrate concentrations than PGE synthase. This pattern was observed irrespective of whether PGH2 was produced by COX-1 or COX-2 or whether it was added directly. Furthermore, the inhibition of eicosanoid production by the action of nonsteroidal anti-inflammatory drugs or by the prevention of COX-2 induction with the p38 mitogen-activated protein kinase inhibitor SKF86002 was greater for PGE2 than for TXA2. It is proposed that different kinetics of PGE synthase and TXA synthase account for the patterns of production of these eicosanoids in monocytes under a variety of experimental conditions. These properties provide an alternative explanation to notional linkage or compartmentalization of COX-1 or -2 with the respective terminal synthases and that therapeutically induced changes in eicosanoid ratios toward predominance of TXA2 may have unwanted effects in long-term anti-inflammatory and anti-arthritic therapy.     

Histone acetyltransferase 1 (HAT1) acetylates hypoxia-inducible factor 2 alpha (HIF2A) to execute hypoxia response

Hypoxic response to low oxygen levels is characteristic of most solid cancers. Hypoxia-inducible factors (HIFs) regulate cellular metabolism, survival, proliferation, and cancer stem cell growth during hypoxia. The genome-wide analysis identified HAT1, a type B histone acetyltransferase, as an upregulated and essential gene in glioblastoma (GBM). GSEA analysis of differentially regulated genes in HAT1 silenced cells identified significant depletion of “hypoxia” gene sets. Hypoxia conditions induced HIF2A, not HIF1A protein levels in glioma cells in a HAT1-dependent manner. HAT1 and HIF2A interacted with each other and occupied the promoter of VEGFA, a bonafide HIF1A/HIF2A target. Acetylation of K512 and K596 residues by HAT1 is essential for HIF2A stabilization under normoxia and hypoxia as HIF2A carrying acetylation mimic mutations at either of these residues (H512Q or K596Q) showed stable expression in HAT1 silenced cells under normoxia and hypoxia conditions. Finally, we demonstrate that the HAT1-HIF2A axis is essential for hypoxia-promoted cancer stem cell maintenance and reprogramming. Thus, our study identifies that the HAT1-dependent acetylation of HIF2A is vital to executing the hypoxia-induced cell survival and cancer stem cell growth, therefore proposing the HAT1-HIF2A axis as a potential therapeutic target.     

Visfatin in adipocytes is upregulated by hypoxia through HIF1alpha-dependent mechanism

Obesity is associated with metabolic disorders, such as insulin resistance. Visfatin is an adipose-derived secretory factor to exert insulin-mimetic effects. Plasma visfatin levels and mRNA levels of visfatin in adipose tissues are increased in obesity. However, the mechanism that mediates induction of visfatin mRNA in adipose tissue of obesity remains unknown. Recent studies have reported that fat tissue is hypoxia in obesity. In this study, we investigated the effects of hypoxia on mRNA expression of visfatin in adipocytes. Hypoxia increased visfatin mRNA expression. Desferoxamine and Cobaltous chloride, two hypoxia mimetic compounds, also increased visfatin mRNA levels. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1alpha (HIF1alpha), mimicked the hypoxia-mediated upregulation of visfatin, and YC1, an inhibitor of HIF1 cancelled the hypoxia-induced upregulation of visfatin mRNA. We identified two functional hypoxia responsive elements (HRE) in mouse visfatin promoter. Hypoxic treatment and overexpression of HIF1alpha increased the promoter activity, and mutation of the HRE blunted hypoxia-induced activation of visfatin promoter. Our results suggest that visfatin mRNA expression is upregulated in the fat tissue of obesity through the activation of HIF1alpha pathway due to hypoxia.