Emerging roles for vascular smooth muscle cell exosomes in calcification and coagulation.

Vascular smooth muscle cell (VSMC) phenotypic conversion from a contractile to 'synthetic' state contributes to vascular pathologies including restenosis, atherosclerosis and vascular calcification. We have recently found that the secretion of exosomes is a feature of 'synthetic' VSMCs and that exosomes are novel players in vascular repair processes as well as pathological vascular thrombosis and calcification. Pro-inflammatory cytokines and growth factors as well as mineral imbalance stimulate exosome secretion by VSMCs, most likely by the activation of sphingomyelin phosphodiesterase 3 (SMPD3) and cytoskeletal remodelling. Calcium stress induces dramatic changes in VSMC exosome composition and accumulation of phosphatidylserine (PS), annexin A6 and matrix metalloproteinase-2, which converts exosomes into a nidus for calcification. In addition, by presenting PS, VSMC exosomes can also provide the catalytic surface for the activation of coagulation factors. Recent data showing that VSMC exosomes are loaded with proteins and miRNA regulating cell adhesion and migration highlight VSMC exosomes as potentially important communication messengers in vascular repair. Thus, the identification of signalling pathways regulating VSMC exosome secretion, including activation of SMPD3 and cytoskeletal rearrangements, opens up novel avenues for a deeper understanding of vascular remodelling processes.

[The effect of hypoxia on the urokinase and adenylate cyclase systems in the culture of endothelial cells of the human umbilical vein].

Hypoxia induces angiogenesis in ischemized tissues by means of pro-angiogenic factor expression. The key role in the growth processes and blood vessel functioning belongs to the matrix metalloproteinases, plasminogen, and its activator systems. Effect of hypoxia on expression of the urokinase activating agent plasminogen and its receptor in endothelium was studied in human umbilical vein endothelial cell model. Incubation of the endothelial cells under the conditions of hypoxia proved to reduce both urokinase formation in these cells and its secreting into the culture medium. The hypoxia-induced reduction of urokinase contents was accompanied by enhancement of expression of the urokinase receptor. The hypoxia also entailed reduction of the adenylate cyclase activity and cAMP contents in the endothelial cells. The data obtained suggest that reduction of the adenylate cyclase activity and cAMP contents under the conditions of hypoxia provide basis for suppression of the urokinase expression by the endothelial cells and, consequently, inhibition of blood vessel formation in the ischemized tissue.

Catalytic activity of NADH-ubiquinone oxidoreductase (complex I) in intact mitochondria. evidence for the slow active/inactive transition.

The mammalian purified dispersed NADH-ubiquinone oxidoreductase (Complex I) and the enzyme in inside-out submitochondrial particles are known to be the slowly equilibrating mixture of the active and de-activated forms (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185). We report here the phenomenon of slow active/de-active transition in intact mitochondria where the enzyme is located within its natural environment being exposed to numerous mitochondrial matrix proteins. A simple procedure for permeabilization of intact mitochondria by channel-forming antibiotic alamethicin was worked out for the "in situ" assay of Complex I activity. Alamethicin-treated mitochondria catalyzed the rotenone-sensitive NADH-quinone reductase reaction with exogenousely added NADH and quinone-acceptor at the rates expected if the enzyme active sites would be freely accessible for the substrates. The matrix proteins were retained in alamethicin-treated mitochondria as judged by their high rotenone-sensitive malate-cytochrome c reductase activity in the presence of added NAD(+). The sensitivity of Complex I to N-ethylmaleimide and to the presence of Mg(2+) was used as the diagnostic tools to detect the presence of the de-activated enzyme. The NADH-quinone reductase activity of alamethicin-treated mitochondria was sensitive to neither N-ethylmaleimide nor Mg(2+). After exposure to elevated temperature (37 degrees C, the conditions known to induce de-activation of Complex I) the enzyme activity became sensitive to the sulfhydryl reagent and/or Mg(2+). The sensitivity to both inhibitors disappeared after brief exposure of the thermally de-activated mitochondria with malate/glutamate, NAD(+), and cytochrome c (the conditions known for the turnover-induced reactivation of the enzyme). We conclude that the slow active/de-active Complex I transition is a characteristic feature of the enzyme in intact mitochondria and discuss its possible physiological significance.