Identification of an oxygen responsive enhancer element in the glyceraldehyde-3-phosphate dehydrogenase gene.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced by hypoxia in endothelial cells (EC). Upregulation occurs primarily at the level of transcription and occurs to a much greater extent in EC than in other cell types. To characterize EC specific hypoxia response elements within the GAPDH gene, we performed transient transfection studies in EC, fibroblasts and smooth muscle cells using portions of the GAPDH promoter linked to a CAT reporter gene. These initial studies identified an EC specific hypoxia responsive region that was further characterized (using SV40-promoter-CAT reporter constructs) as a 19-nucleotide sequence (-130 to -112) containing both an hypoxia inducible factor-1 (HIF-1)-binding site and a novel flanking sequence. Electrophoretic mobility shift assays confirmed inducible EC protein binding to this fragment. Mutation of either the HIF-1-binding site or the flanking sequence resulted in complete loss of function and loss of inducible protein binding. Thus, a single HIF-1-binding site is necessary, but not sufficient, for hypoxic regulation of GAPDH in EC. Furthermore, the novel HIF-1 flanking sequence required for GAPDH upregulation and the protein(s) that bind to it may be EC specific.

Pulmonary edema during acute infusion of epoprostenol in a patient with pulmonary hypertension and limited scleroderma.

Epoprostenol (prostacyclin) is currently approved for treatment of primary pulmonary hypertension; however, it is being evaluated in other forms of pulmonary hypertension, particularly scleroderma. Side effects associated with this medication are usually minor; serious complications are most often due to the delivery system required for continuous infusion. We describe a life threatening side effect of acute epoprostenol infusion (pulmonary edema) in a patient with pulmonary hypertension associated with limited scleroderma and discuss its management and potential etiology. This is the first case where epoprostenol has been successfully reinstituted.

Endothelial cell hypoxic stress proteins.

The vascular endothelium is an important mediator of vascular tone, inflammatory-immune reactions, vascular permeability, angiogenesis, and hemostasis. Endothelial functions may be altered by changes in the local cellular environment, particularly changes in oxygen tension. The mechanisms by which endothelial cells (ECs) respond and adapt to hypoxia are unknown; however, the EC is one of the more hypoxia-tolerant mammalian cell types. Cultured ECs exposed to hypoxia up-regulate a set of stress proteins, termed hypoxia-associated proteins (HAPs), that are distinct from the classically described stress proteins induced by heat shock (heat-shock proteins, HSPs) or glucose deprivation (glucose-regulated proteins, GRPs). Two of these proteins have been identified as the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and non-neuronal enolase (NNE). GAPDH expression during hypoxia is regulated primarily at the level of transcription, while the mechanism of NNE mRNA accumulation remains unclear. GAPDH, NNE, and the other HAPs are up-regulated by transitional metals and deferoxamine; however, unlike the situation with other hypoxia-regulated proteins such as erythropoietin, the up-regulation of GAPDH, NNE, and the other HAPs by hypoxia is not inhibited by carbon monoxide. Subcellular fractionation of hypoxic EC has shown that GAPDH and NNE are up-regulated in the cytoplasmic fraction as would be expected for a glycolytic enzyme; however, a protein corresponding to GAPDH is also up-regulated in the nuclear fraction. This suggests that GAPDH and perhaps NNE have functions aside from their catalytic function in glycolysis. It is unknown whether the up-regulation of GAPDH, NNE, and the other HAPs in ECs is related to the relative ability of ECs to adapt to hypoxia; however, other more-hypoxia-sensitive cells do not up-regulate HAPs.

Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced by hypoxia in endothelial cells (EC). To define the mechanisms by which GAPDH is regulated by hypoxia, EC were exposed to cobalt, other transition metals, carbon monoxide (CO), deferoxamine, or cycloheximide in the presence or absence of hypoxia for 24 h, and GAPDH protein and mRNA levels were measured. GAPDH was induced in cells by the transition metals cobalt, nickel, and manganese and by deferoxamine, and GAPDH mRNA induction by hypoxia was blocked by cycloheximide. GAPDH induction by hypoxia, unlike that of other hypoxia-regulated genes, was not inhibited by CO or by 4,6-dioxoheptanoic acid, an inhibitor of heme synthesis. GAPDH induction was not altered by mediators of protein phosphorylation, a calcium channel blocker, a calcium ionophore, or alterations in redox state. GAPDH induction by hypoxia or transitional metals was partially blocked by sodium nitroprusside but was not altered by the inhibitor of nitric oxide synthase N omega-nitro-L-arginine. These findings suggest that GAPDH induction by hypoxia in EC occurs via mechanisms other than those involved in other hypoxia-responsive systems.

Specificity and uniqueness of endothelial cell stress responses.

The mammalian response to cellular stresses often involves upregulation of certain stress proteins. This response is usually neither cell nor stress specific and sometimes results in cross-protection to other stresses. Endothelial cell (EC) hypoxia-associated proteins (HAP) are a unique set of stress proteins upregulated by exposure to environmental hypoxia. In the present study, the specificity of stress protein upregulation was assessed and any potential cross-protection was evaluated using DNA strand break analysis. EC cultured in 21% or 3% oxygen were exposed to single and combined cellular stresses (0% oxygen, reoxygenation, glucose deprivation, sodium arsenite, heat, or hydrogen peroxide). Although EC can upregulate various stress proteins, the HAP are specifically upregulated only with hypoxia and offer no cross-protection against other cellular stresses. Moreover, induction of other stress proteins does not alter the induction of the HAP or the effects of hypoxia in cultured EC. Thus EC display a unique specificity in regard to the stimulus for upregulation of stress proteins and are distinct from other cell types thus far examined.

Non-neuronal enolase is an endothelial hypoxic stress protein.

The hypoxia-associated proteins (HAPs) are five cell-associated stress proteins (M(r) 34, 36, 39, 47, and 57) up-regulated in cultured vascular endothelial cells (EC) exposed to hypoxia. While hypoxic exposure of other cell types induces heat shock and glucose-regulated proteins, EC preferentially up-regulate HAPs. In order to identify the 47-kDa HAP, protein from hypoxic bovine EC lysates was isolated, digested with trypsin, and sequenced. Significant identity was found with enolase, a glycolytic enzyme. Western analyses confirmed that non-neuronal enolase (NNE) is up-regulated in hypoxic EC. Western analysis of subcellular fractions localized NNE primarily to the cytoplasm and confirmed that it was up-regulated 2.3-fold by hypoxia. Interestingly, NNE also appeared in the nuclear fraction of EC but was unchanged by hypoxia. Northern analyses revealed that NNE mRNA hypoxic up-regulation began at 1-2 h, peaked at 18 h, persisted for 48 h, and returned to base line after return to 21% O2 for 24 h. Hypoxia maximally up-regulated NNE mRNA levels 3.4-fold. While hypoxic up-regulation of NNE may have a protective effect by augmenting anaerobic metabolism, we speculate that enolase may contribute to EC hypoxia tolerance through one or more of its nonglycolytic functions.

Hypoxia-associated proteins.

The vascular endothelium is an important mediator of vascular tone, angiogenesis, inflammatory-immune reactions, vascular permeability, and hemostasis. Thus, it plays an important role in the pathogenesis of numerous critical care processes, including septic shock, myocardial infarction, the adult respiratory distress syndrome, and acute tubular necrosis. Endothelial functions may be altered by changes in the local cellular environment, particularly changes in PO2. The ability of endothelial cells (EC) to not only sense, but also to adapt to, acute and chronic changes in PO2 is critical to maintaining endothelial metabolic functions and, in turn, to maintaining homeostasis, particularly in the critical care setting. Recent studies have shown that the EC is one of the more hypoxia-tolerant mammalian cell types; however, the mechanisms by which ECs respond and adapt to hypoxia are unknown. Our laboratory has shown that cultured ECs exposed to hypoxia upregulate a set of stress proteins, termed hypoxia-associated proteins (HAPs), that are distinct from the classically described stress proteins induced by heat shock (heat-shock proteins) or glucose deprivation (glucose-regulated proteins). We have recently identified one of these proteins as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Further studies have shown that GAPDH expression is regulated by hypoxia, primarily at the transcriptional level. Subcellular fractionation of hypoxic EC has shown that GAPDH is upregulated in the cytoplasmic fraction as would be expected with a glycolytic enzyme; however, a protein corresponding to GAPDH is also upregulated in the nuclear fraction. This suggests that the upregulation of GAPDH in EC during hypoxia is related to the potential nonglycolytic functions of this enzyme. Furthermore, the upregulation of GAPDH and the other HAPs (that have yet to be identified) may be related to the relative hypoxia tolerance of EC.

Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia.

Exposure of endothelial cells (EC) to hypoxia results in the increased expression of a distinct set of proteins with molecular masses of 56, 47, 39, 36, and 34 kDa. Their induction appears to be unique to EC and the stress of decreased oxygen tension. To understand the mechanism(s) and significance of the up-regulation of these proteins we have identified the 36-kDa protein by limited amino-terminal amino acid sequencing. The 21-amino acid sequence from the bovine protein exhibited 90.5% identity with the human sequence of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Northern blot analysis showed that the time course and extent of EC GAPDH mRNA up-regulation correlated with the increase in 36-kDa protein synthesis. Nuclear runoff analysis demonstrated that this increase in GAPDH expression is regulated, in part, at the transcriptional level; however, the increase in the rate of transcription did not account for the entire mRNA accumulation, suggesting that GAPDH, like other hypoxia-regulated proteins, is posttranscriptionally regulated. Subcellular fractionation of hypoxic EC showed up-regulation of the 36-kDa protein in the cytoplasmic fraction and, to a lesser extent, in the nuclear fraction. The up-regulation of GAPDH in EC may be related to their relative hypoxia tolerance. Alternatively, the up-regulation of GAPDH in EC during hypoxia may be related to the potential nonglycolytic functions of this enzyme.

Endothelial cell hypoxia associated proteins are cell and stress specific.

Vascular endothelial cells (EC) are one of the initial cells exposed to decreases in blood oxygen tension. Bovine EC respond not only by altering secretion of vasoactive, mitogenic, and thrombogenic substances, but also by developing adaptive mechanisms in order to survive acute and chronic hypoxic exposures. EC exposed to hypoxia in vitro upregulate a unique set of stress proteins of Mr 34, 36, 39, 47, and 56 kD. Previous studies have shown that these proteins are cell associated, upregulated in a time and oxygen-concentration dependent manner, and are distinct from heat shock (HSPs) and glucose-regulated proteins (GRPs). To further characterize these hypoxia-associated proteins (HAPs), we investigated their upregulation in human EC from various vascular beds and compared this to possible HAP upregulation in other cell types. Human aortic, pulmonary artery, and microvascular EC upregulated the same set of proteins in response to hypoxia. In comparison, neither lung fibroblasts, pulmonary artery smooth muscle cells, pulmonary alveolar type II cells, nor renal tubular epithelial cells upregulated proteins of these Mr. Instead, most of these cell types induced synthesis of proteins of Mrs corresponding to either HSPs, GRPs, or both. Further studies demonstrated that exposure of EC to related stresses such as cyanide, 2-deoxyglucose, hydrogen peroxide, dithiothreitol, and glucose deprivation did not cause upregulation of HAPs. Evaluation of cellular damage during hypoxia using phase-contrast microscopy, trypan blue exclusion, chromium release, and adherent cell counts showed that EC survived longer with less damage than any of the above cell types. The induction of HAPs, and the lack of induction of HSPs or GRPs, by EC in response to hypoxia may be related to their unique ability to tolerate hypoxia for prolonged periods.