Hypoxia induces endothelial cell synthesis of membrane-associated proteins.

Hypoxemia is associated with a prothrombotic tendency. In this study we report the purification and partial characterization of an activator of a central coagulation component, factor X, induced in endothelium by exposure to hypoxia (hypoxia-induced factor X activator or Xact). Expression of Xact occurred in a reversible manner when endothelial cell cultures were exposed to hypoxia or sodium azide but not in response to a variety of other alterations in the cellular milieu, such as heat shock or glucose deprivation. The activity of Xact, which was not detected in normoxic endothelial cells, was maximal under acidic conditions, pH 6.0-6.8, which often coexist with hypoxia in an ischemic milieu. By sequential isoelectric focusing and preparative SDS/PAGE of endothelial membrane-rich fractions, Xact was purified approximately 19,000-fold and found to be a single-chain, approximately 100-kDa polypeptide with pI approximately 5.0. Activation of factor X by purified Xact was not affected by blocking antibodies to other coagulation proteins or by phenylmethylsulfonyl fluoride or leupeptin but was prevented by mercury chloride or iodoacetamide. In addition to the induction of Xact, two-dimensional gel analysis of membrane fractions from metabolically labeled hypoxic endothelial cultures revealed two groups of approximately 10 additional spots: (i) a group for which expression was maximal after 24 hr and (ii) a group for which expression continued to increase up to 48 hr. The pattern of hypoxia-mediated modulation of protein expression was distinct from that seen with other cellular stimuli but could be duplicated, in part, by sodium azide. These results indicate that hypoxia elicits a specific biosynthetic response, including the expression of endothelial cell-surface molecules that can alter cellular function and may potentially serve as markers of hypoxemic vessel-wall injury.

Macrovascular and microvascular endothelium during long-term hypoxia: alterations in cell growth, monolayer permeability, and cell surface coagulant properties.

In bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose-dependent manner at lower oxygen concentrations, as progression into S phase from G1 was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a approximately 3-fold increase in the total number of high-affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia-mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the monolayers consisted of larger cells with altered assembly of the actin-based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monolayers to macromolecules and lower molecular weight solutes was increased. Cell surface coagulant properties were also perturbed: the anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that micro- and macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endothelium exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vascular lesions.

Modulation of endothelial function by hypoxia: perturbation of barrier and anticoagulant function, and induction of a novel factor X activator.

Exposure of the vessel wall to hypoxemia is a central feature of ischemic cardiovascular disease. This led us to examine the perturbation of endothelial cell properties under hypoxia. An atmosphere of pO2 of 12 mmHg is not lethal to the endothelial cells for up to five days, but barrier function was impaired. Increased passage of macromolecule tracers were observed in time- and dose-dependent manner and electron microscopy demonstrated small gaps (0.5-1.0 micron) between cells. Expression of the anticoagulant cofactor thrombomodulin was also perturbed: thrombomodulin activity and antigen decreased in parallel. Northern blots showed almost complete suppression of thrombomodulin in hypoxic culture. Furthermore, synthesis of other proteins, such as fibronectin, was slightly enhanced under hypoxia. In addition to the suppression of these anticoagulant cofactor, hypoxic endothelial cell displayed a noval procoagulant activity distinct from tissue factor. Further study revealed that hypoxic endothelial cultures directly activated Factor X, as assessed by functional assays and SDS-PAGE. In addition to this no activation of Factor IX or prothrombin was observed. The hypoxia-induced Factor X activator was membrane-associated, required calcium to form Factor Xa, was inhibited by HgCl2 but not by PMSF, and had Km approximately 25 micrograms/ml. Co-incubation of hypoxic cultures with cycloheximide prevented the expression of this activity, suggesting that protein synthesis is required for its expression. These functional perturbations of endothelial cells were reversible following reoxygenation. These data indicate that hypoxia imposes a selective perturbation on endothelial cell function, suggesting the possible contribution of hypoxemia to vascular dysfunction in ischemia.