The hypoxia-associated proteins (HAPs) are five cellassociated stress proteins (M r 34, 36, 39, 47, and 57) upregulated 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% O 2 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.Tolerance to acute hypoxia and adaptation to chronic hypoxia are crucial to the survival of cells and the organisms they comprise. Among mammalian cells, vascular endothelial cells (EC) 1 offer an excellent example of adaptation to extreme hypoxia. For example, pulmonary arterial EC normally reside in a pO 2 of 35-40 mm Hg and may be exposed to lower oxygen concentrations in disease states. Yet, we and others have shown that bovine and human EC in culture are extremely tolerant to decreases in ambient oxygen tension (1-8). We have speculated that this tolerance may be, in part, acquired. For instance, although exposure to 0% oxygen for 4 -5 days is lethal to bovine pulmonary artery and aortic EC (BPAEC and BAEC, respectively) not acclimatized to hypoxia, BPAEC and BAEC grown in 3% oxygen and then exposed to 0% oxygen survive, proliferate, and maintain characteristic EC appearance for more than 6 days. 2In studying the EC response to hypoxia, we have previously described the hypoxia associated proteins (HAPs), a unique set of five cell-associated stress proteins (M r 34, 36, 39, 47, and 57) up-regulated in a time and oxygen concentration-dependent manner in EC exposed to hypoxia (1-3). The hypoxic upregulation of HAPs in EC occurs in lieu of the stereotypic hypoxic induction of heat-shock and glucose-regulated proteins seen in more hypoxia-sensitive cells and correlates with the ability of EC to tolerate hypoxia. As part of our efforts to elucidate the function of the HAPs in physiologic and hypoxic conditions, we have previously identified the 36-kDa HAP as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (3). In the current study, we report the isolation and identification of HAP47 as non-neuronal enolase (NNE) and examine its hypoxic r...

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.