Long-Term Experimental Hyperglycemia Does Not Impair Macrovascular Endothelial Barrier Integrity and Function in vitro.

Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.

MLCK and ROCK mutualism in endothelial barrier dysfunction.

Myosin activation contributes to the contractile forces that induce disturbances in the vascular endothelial integrity and promote protein-rich edema of the underlying tissues. Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK) have been reported to phosphorylate myosin regulatory light chains (RLC) for myosin activation. However, the relative contribution and roles of these kinases are debatable and not understood in very detail. In this study, using a combinational inhibitory analysis of MLCK, ROCK, and their antagonist, myosin light chain phosphatase (MLCP), we show that the MLCK-dependent RLC mono-(Ser19)phosphorylation (P-RLC) is sufficient to induce the FITC-dextran hyperpermeability in EA.hy926 endothelial cells (EC) in response to thrombin. However, MLCK relies on the ROCK assistance that attenuates MLCP activity. On the other hand, MLCK supplies P-RLC myosin as an intermediate substrate to ROCK thus adding to a faster accumulation of di-(Thr18/Ser19)phosphorylated RLC (PP-RLC) by the latter kinase. ROCK also produces P-RLC but is solely responsible for the thrombin-induced PP-RLC generation in EA.hy926 EC and other cell types. Still, as a direct myosin activator, ROCK contributes less to endothelial hyperpermeability than MLCK. Our findings are consistent with a concerted complementary mutual interplay between ROCK and MLCK to activate endothelial myosin and elicit the thrombin-mediated EC barrier dysfunction.

Design of peptidase-resistant peptide inhibitors of myosin light chain kinase.

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell-permeant peptide Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys (PIK, Peptide Inhibitor of Kinase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L-PIK in a biological milieu prompts for development of more stable L-PIK analogues for use as experimental tools in basic and drug-oriented biomedical research. Previously, we designed PIK1, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 , that was 2.5-fold more resistant to peptidases in human plasma in vitro than L-PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site-protected peptides based on L-PIK and PIK1 degradation patterns in human plasma as revealed by 1 H-NMR analysis. Implemented modifications increased half-live of the PIK-related peptides in plasma about 10-fold, and these compounds retained 25-100% of L-PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH2 , was identified as the most stable and effective L-PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin-induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell-permeant inhibitors of MLCK in cell culture-based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Thermal transformation of bioactive caffeic acid on fumed silica seen by UV-Vis spectroscopy, thermogravimetric analysis, temperature programmed desorption mass spectrometry and quantum chemical methods.

Thermochemical studies of hydroxycinnamic acid derivatives and their surface complexes are important for the pharmaceutical industry, medicine and for the development of technologies of heterogeneous biomass pyrolysis. In this study, structural and thermal transformations of caffeic acid complexes on silica surfaces were studied by UV-Vis spectroscopy, thermogravimetric analysis, temperature programmed desorption mass spectrometry (TPD MS) and quantum chemical methods. Two types of caffeic acid surface complexes are found to form through phenolic or carboxyl groups. The kinetic parameters of the chemical reactions of caffeic acid on silica surface are calculated. The mechanisms of thermal transformations of the caffeic chemisorbed surface complexes are proposed. Thermal decomposition of caffeic acid complex chemisorbed through grafted ester group proceeds via three parallel reactions, producing ketene, vinyl and acetylene derivatives of 1,2-dihydroxybenzene. Immobilization of phenolic acids on the silica surface improves greatly their thermal stability.

Preparation and Characterization of Silica-Enoxil Nanobiocomposites.

Silica-Enoxil nanobiocomposites with 13 %w of Enoxil were prepared either by mechanical mixing of corresponding powders or by sorptive modification of fumed silica powder with aqueous Enoxil solution under fluidized bed conditions. The interaction of fumed silica with Enoxil and the properties of silica-Enoxil composites have been investigated using IR spectroscopy, thermogravimetric analysis, and quantum chemistry methods, as well as by means of water absorption, Enoxil desorption, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) test. It has been shown that the main biomolecules of Enoxil composition interact with silica involving their hydroxyl groups and surface silanol groups. The water absorption of silica-Enoxil nanocomposites was found to be less than that for the individual components. The Enoxil biomolecules are readily and completely desorbed from silica surface into water, and the antioxidant activity of desorbed Enoxil is practically the same as that for the just dissolved one.