A monoclonal antibody, VM64, reacts with a 130 kDa glycoprotein common to platelets and endothelial cells: heterogeneity in antibody binding to human aortic endothelial cells.

A new monoclonal antibody (mAb), VM64, reacts with a common antigen on the surface of human platelets and vascular endothelial cells (EC). Under nonreduced conditions it recognized in immunoblotting a protein of 130 kDa both in platelets and EC. VM64 precipitated the same 130 kDa protein from the lysate of surface radioiodinated platelets. Electrophoretic mobility of this protein was not altered by reduction and differed from the bands precipitated by reference mAb against platelet glycoproteins (GP) Ia-IIa, Ib, IIb-IIIa and GMP130. VM64 binding to platelets and EC was specific and saturable. The number of binding sites on platelets was 9.9 +/- 3.5 x 10(3) per platelet and on the surface of EC monolayer -2.40 +/- 0.32 x 10(6) per cell. VM64 also binds to platelets from Glanzmann's thrombasthenia patients which lack GPIIb-IIIa. VM64 did not affect platelet aggregation induced by ADP, collagen, thrombin and ristocetin. In the monolayers of EC from umbilical vein and human aorta, VM64 stained the area at the periphery of the cells adjacent to the cell-cell boundaries. In preconfluent cultures preferential staining was observed at the active leading margins of the cells. Unlike EC cultures from umbilical vein, where all cells were positively stained, in aortic EC cultures some unstained or poorly stained cells were constantly present, indicating a heterogeneity of EC population related to the expression of VM64 antigen. The biochemical characteristics of VM64 antigen, its presence both on platelets and EC and typical distribution on the surface of EC suggested that this antigen is identical to PECAM (CD31) protein.

Mechano-chemical control of human endothelium orientation and size.

Human umbilical vein endothelial cells (EC) were grown on elastic silicone membranes subjected to cyclic stretch, simulating arterial wall motion. Stretching conditions (20% amplitude, 52 cycle/min) stimulated stress fiber formation and their orientation transversely to the strain direction. Cell bodies aligned along the same axis after the actin cytoskeleton. EC orientation response was inhibited by the adenylate cyclase activator, forskolin (10(-5) M), which caused stress fiber disassembly and the redistribution of F-actin to the cortical cytoplasm. Preoriented EC depleted of stress fibers by forskolin treatment retained their aligned state. Thus, stress fibers are essential for the process of EC orientation induced by repeated strain, but not for the maintenance of EC orientation. The monolayer formed by EC grown to confluence in conditions of intermittent strain consisted of uniform elongated cells and was resistant to deformation. In contrast, the monolayer assembled in stationary conditions was less compliant and exposed local denudations on initiation of stretching. When stretched in the presence of 10(-5) M forskolin it rapidly (3-4 h) reestablished integrity but gained a heterogeneous appearance since denuded areas were covered by giant cells. The protective effect of forskolin was because of the stimulation of EC spreading. This feature of forskolin was demonstrated while studying its action on EC spreading and repair of a scratched EC monolayer in conventional culture. Thus mechanical deformation and adenylate cyclase activity may be important factors in the control of endothelium morphology in human arteries.

Effects of forskolin and phorbol-myristate-acetate on cytoskeleton, extracellular matrix and protein phosphorylation in human endothelial cells.

The role that the intracellular mediators, cAMP and Ca2+/phosphatidylserine-dependent protein kinase C, play in the regulation of endothelial cell (EC) motility was investigated. The adenylate cyclase activator, forskolin, at 10 microM induced rapid and reversible alterations in the shape of cultured human EC, disappearance of actin bundles and the concentration of F-actin at cell borders. Actin reorganization provoked by forskolin coincide with redistribution of vinculin to the cell periphery and rapid elimination of surface-associated fibronectin. A protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) at 10-100 microM induced no visible alterations of cell shape, but enhanced the effect of forskolin. PMA stimulated formation of "stress fibers" and increased the number of vinculin plaques in central areas of the cell. A decrease in the amount of the surface-associated fibronectin in PMA-treated cells has also been observed, but, this effect was considerably slower than that produced by forskolin. Forskolin, but not PMA stimulated phosphorylation of the major intermediate filament protein, vimentin.

[Reconstruction of a membrane monooxygenase cytochrome P 450-containing system in the liver using detergents in solution]. / Rekonstruktsiia membrannoi monooksigenaznoi soderzhashchei tsitokhrom P-450 sistemy pecheni s pomoshch'iu detergentov v rastvore.

Emulgen 913, Triton N-101 and sodium cholate were compared for their reconstituting action on the dimethylaniline N-demethylation system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The comparison showed that emulgen 913 is the most efficient detergent. The optimum molar ratio of the proteins and emulgen appeared to be equal to 1:1:600. Study on the mechanism of emulgen reconstituting action showed that this effect is due to the mixed complex formation between the cytochrome and reductase, the complexes containing five molecules of the flavoprotein and five molecules of cytochrome P-450. No formation of mixed protein aggregates or reconstitution was observed in the absence of the detergent or at its concentrations exceeding the optimum level.

[Change in the activity of the microsomal system of liver oxidation in rabbits in experimental atherosclerosis]. / Izmenenie aktivnosti mikrosomal'noi sistemy okisleniia pecheni krolikov pri éksperimental'nom ateroskleroze.

Oxidizing ability of rabbit liver tissue was studied in experimental atheromatosis, while the functional activity of microsomal enzymatic system was evaluated by content of cytochromes P-450 and b5 as well by estimation of the reductase activity and the rates of oxidation of aniline, dimethylalanine, p-nitroanisole and amidopyrine with NADN and NADPH as cosubstrates. In rabbits with atheromatous impairment of aorta all the patterns studied were decreased by 30-70%. Severity of the aorta impairment correlated directly with the liver tissue deterioration and the rate of alteration in activity of the microsomal system oxidation constituents. The most distinct changes were found in NADPH-dependent reactions and in cytochrome P-450. The data obtained suggest that experimental atheromatosis of rabbits, produced after cholesterol loading, is responsible for distinct impairment of liver microsomal enzymatic system. The most profound alterations were observed in the components involved in the cholesterol-7 alpha-hydroxylase reaction.