[The effect of Rho-kinase inhibition depends on the nature of factors that modify endothelial permeability].

Endothelium lining the inner surface of all vessels plays barrier role and regulates permeability of vascular walls controling the exchange between circulating blood and tissue fluids. Disturbance of normal functions (endothelial dysfunction) can be caused by both internal, and external factors. Endothelial dysfunction is characterized by increased vascular wall permeability observed in many human diseases. Dysfunction is also a drug side effect of oncological diseases treatment by mitosis-blocking medications. Depolymerization of microtubules is the first step in the cascade of reactions leading to endothelial barrier dysfunction, and this stage is universal, it does not depend upon the nature of a factor provoking dysfunction. To develop the strategy of barrier dysfunction prevention, we are supposed here to find out to what stage the endothelial cell cytoskeleton reaction during the development of barrier dysfunction is universal. It has been found that the cascade stages, which follow the microtubule depolymerization and are connected with Rho-Rho-kinases activity, have the features depending on the factor provoking barrier dysfunction. Under suppression of Rho-kinase activity, the reaction of actin filaments does not depend on what substance caused dysfunction. But the microtubule system responds to the treatment varies depending on the dysfunction-provoking factor. Unlike thrombin, under the conditions of Rho-kinase activity suppression, nocodazole renders more strong effect, as much as possible destroying both dynamic, and stable microtubules. Thus, regardless of the dysfunction provoking factor, the initial stages of dysfunction connected with the depolymerization of microtubules appear to be unalterable. Consequently, endothelial cell defence strategy should be based on cytoplasmatic microtubules protectors application instead of employment of the factors involved in the cascade at later stages as we assumed earlier.

[The microtubule system in endothelial barrier dysfunction: disassembly of peripheral microtubules and microtubules reorganization in internal cytoplasm].

Endothelial cell barrier dysfunction is often associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction and finally gap formation. At present time the role of microtubules in endothelial cell barrier regulation is not fully understood, however a number of observations allow to assume that microtubules reaction is the extremely important part in development of endothelial dysfunction. These observations have been forced us to examine the role of microtubule system reorganization in endothelial cell barrier regulation. In quiescent endothelial cells microtubule density is the highest in the centrosome region and insignificant near the cell margin. The analysis of microtubules distribution after specific antibodies staining using the method of measurement of their fluorescence intensity has shown that in control endothelial cells the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of an exponential regression. The hormone agent, thrombin (25 nM), causes rapid increase of endothelial cell barrier permeability accompanied by fast decrease in quantity of peripheral microtubules and reorganization of microtubule system in internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress already through 10 min after the beginning of the treatment). Both effects are reversible -- through 60 min after the beginning of the treatment the microtubule network does not differ from normal one, so the microtubule system is capable to adapt for influence of a natural regulator thrombin. The microtubules reaction develops more quickly, than reorganization of the actin filaments system, which responsible for the subsequent changes in the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in a circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.

[Free and centrosome-attached microtubules: quantitative analysis and the modeling of two-component system].

In the internal cytoplasm of interphase cells the density of microtubules is the highest in the centrosome area and decreases to the cell periphery. As a rule, the quantity of fluorescent microtubules cannot be counted up in the internal cytoplasm, but it is possible to estimate microtubules quantity using measuring of their optical density. In living 3T3 and CHO cells the microtubules optical density decreased according to different mathematical dependences that apparently reflected the differences of their microtubule system organization. To determine appropriateness that circumscribe the reduction of microtubules optical density from the centrosome region to the direction of cell margin, we modeled cell contours with the certain ratio and interposition of centrosome-attached and free microtubules in vector schedules CorelDraw program. The decrease of optical density was analyzed in MetaMorph program as it was described earlier (Smurova et al., 2002). It was shown that fluorescent microtubules optical density decreased exponentially (y = ae(-bx)) if the system joined only microtubules growing from the centrosome up to the cell margin. The curve became smoother in the case of not all radial centrosome-attached microtubules reached the margin, and adding of free microtubules into the system led to the sharp fall in optical density in the centrosome area and to its gradual decrease at the cell periphery. The increase in free microtubules quantity changed the character of the curve describing the reduction of optical density microtubule system which included free and centrosome-attached microtubules in proportions of 5 : 1 was described by the equation of linear regression (f= k . x + b). Thus, the mathematical dependence describing the microtubules distribution from the centrosome to the cell periphery, depends on the ratio of microtubules and their relative positioning in the cell volume. The data obtained using model systems have coincided with the results of experiments. The graphs which described the increase in microtubules optical density during microtubule repolymerization after nocodazole treatment, corresponded to the graphs for model cells. Thus, the method we used allows to analyze the microtubule system in the cases when the direct observation of individual microtubules is difficult.

[Reorganization of microtubule system in pulmonary endothelial cells in response to thrombin treatment]. / Reorganizatsiia sistemy mikrotrubochek v kletkakh legochnogo éndoteliia v otvet na vozdeistvie trombina.

Thrombin induces rapid and reversible increase of endothelial (EC) barrier permeability associated with actin cytoskeleton remodeling and contraction. The role of microtubules (Mts) in EC barrier regulation compared with actin systems is poorly understood. In this work we studied pathways of Mt and actin regulation in response to thrombin treatment in cultured EC, and the involvement of trimeric G-proteins and in this process. Cells were treated with thrombin, and further analysed using immunofluorescent staining of actin and Mts, digital microscopy and morphometric analysis. In normal cells actin network consists of thin bundles basically located in the cell periphery, Mt density decreases from the cell center to the cell edge. Thrombin (25 nM) induced endothelial dysfunction associated with a rapid (within 5 min) decrease of peripheral Mt network and a slower actin stress fiber formation in the cytoplasm. Pretreatment with Pertussis toxin, which is Gi protein inhibitor, attenuated thrombin-induced stress fiber formation and Mt disassembly. Overexpression of activated G12, G13, Gi and Gq proteins, which are involved in thrombin receptor-mediated signaling, resulted in increasing stress fibers thickness and density and complete Mt disassembly. From the results obtained we suggest that thrombin regulates actin cytoskeleton of EC using local Mt depolymerization at the cell edge.