Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH.

Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O2, 5% CO2, 93% N(2)) for 24 h followed by reoxygenation (21% O2, 5% CO2, 74% N(2)) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by approximately 30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.

Production of soluble P-selectin by platelets and endothelial cells.

The distribution of a soluble form of a cell adhesion molecule, P-selectin, in human platelets and cultivated endothelial cells has been studied by enzyme-linked immunosorbent assay (ELISA). The concentration of soluble P-selectin in the blood plasma of healthy donors and patients with abnormal platelet count has also been determined. P-selectin was measured in the Triton X-100 lysate of platelets and endothelial cells (total P-selectin), in the 100,000g supernatant obtained after sedimentation of the membrane fraction from the homogenate of sonicated platelets and endothelial cells (intracellular soluble P-selectin), in the supernatant of activated and nonactivated platelets, and in the culture medium of endothelial cells. A soluble form of P-selectin which did not coprecipitate with the membrane fraction was detected in platelets and accounted for approximately 10% of the total P-selectin. Platelet activation by thrombin, ADP, or a thromboxane A2 analog resulted in the secretion of 30-50% of the intracellular soluble P-selectin. Measurements of P-selectin in endothelial cell culture revealed that endothelium from aorta contained about twofold more P-selectin than endothelium from umbilical vein. Intracellular soluble P-selectin was identified in both types of endothelial cells. In endothelial cells from the umbilical vein this form made up approximately 10% of the total P-selectin. Soluble P-selectin was also detected in the medium of cultivated endothelial cells, where its content correlated with the total cellular P-selectin. Concentration of P-selectin in blood plasma strongly correlated with the platelet count in the blood of healthy donors and patients with thrombocytosis and thrombocytopenia. These data indicate that platelets serve as one of the main source of plasma P-selectin. However, the presence of P-selectin in the plasma of patients with severe thrombocytopenia suggests that endothelium can also be involved in plasma P-selectin production. Thus, in vitro experiments as well as measurements of plasma P-selectin have shown that both platelets and endothelial cells can produce a soluble form of the protein. Platelet-derived soluble P-selectin and plasma P-selectin were shown to react with antibodies against the cytoplasmic domain of P-selectin. These data prove that at least part of soluble P-selectin is produced by synthesis employing special mRNA which lacks the sequence encoding the transmembrane domain, but not by the proteolytic shedding of the extracellular portion of membrane P-selectin.

[Interaction of platelets with 125I-labelled collagen type III. The necessity for forming a fibrillary structure]. / Vzaimodeistvie trombotsitov s 125I-mechenym kollagenom III tipa. Neobkhodimost' obrazovaniia fibrilliarnykh struktur.

The interaction of type III collagen (CIII) with washed human platelets was studied, using a CIII preparation from human placenta. CIII was labeled with 125I, and the monomeric and fibrillar forms of 125I-CIII (125I-CIIIm and 125I-CIIIf, respectively) were incubated with the platelets at room temperature. The platelet-associated and free labels were separated by centrifugation through 20% sucrose. The binding of 125I-CIIIf was unsaturable, linearly dependent on the label concentration and made up to 28 +/- 3% of the added protein. In comparison with CIIIf, the binding of 125I-CIIIm was minimal, i.e., only 0.9 +/- 0.2% of the added protein; so it did not significantly increase the background level (label sedimented through 20% sucrose in the absence of platelets). Although the level of 125I-CIIIm was very low, the binding was also unsaturable and linearly dependent on the concentration of the labeled protein. Platelet activation did not influence the level of CIIIf binding, nor did it stimulate the binding of CIIIm. The binding of 125I-CIIIf was not inhibited by the unlabeled CIIIm. The data obtained testify to the absence of high affinity platelet collagen receptors and support the hypothesis on multiple low affinity interactions between collagen fibrils and platelet surface. The binding of CIIIf to platelets was characterized by very fast kinetics; the level of binding reached a plateau within the range of 1 min and was similar in the presence of Ca2+/Mg2+ and EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)