Irina A. Kolosova

Suppression of endotoxin-induced inflammation by taxol

The pathogenesis of acute lung injury includes transendothelial diapedesis of leukocytes into lung tissues and disruption of endothelial/epithelial barriers leading to protein-rich oedema. In vitro studies show that the microtubule network plays a role in the regulation of endothelial permeability as well as in neutrophil locomotion. It was hypothesised that the microtubule-stabilising agent, taxol, might attenuate inflammation and vascular leak associated with acute lung injury in vivo. The effect of intravenously delivered taxol was assessed using a model of murine lung injury induced by intratracheal lipopolysaccharide (LPS) administration. Parameters of lung injury and inflammation were assessed 18 h after treatment. Intravenously delivered taxol significantly reduced inflammatory histological changes in lung parenchyma and parameters of LPS-induced inflammation: infiltration of proteins and inflammatory cells into bronchoalveolar lavage fluid, lung myeloperoxidase activity, and extravasation of Evans blue-labelled albumin into lung tissue. Taxol alone (in the absence of LPS) had no appreciable effect on these parameters. In addition to lung proteins, intravenous taxol reduced accumulation of leukocytes in ascitic fluid in a model of LPS-induced peritonitis. Taken together, the present data demonstrate that microtubule stabilisation with taxol systemically attenuates lipopolysaccharide-induced inflammation and vascular leak.

Signaling Pathways Involved in Adenosine Triphosphate-Induced Endothelial Cell Barrier Enhancement

Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell–cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically G&agr;q and G&agr;i2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of G&agr;q with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein–coupled mechanism and PKA.

The role of caldesmon in the regulation of endothelial cytoskeleton and migration

The actin‐ and myosin‐binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non‐muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l‐CaD cloned from bovine endothelium. Wild‐type CaD (WT‐CaD) and truncated mutants lacking either the N‐terminal myosin‐binding site or the C‐terminal domain 4b (containing actin‐ and calmodulin‐binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N‐terminal myosin‐binding site, resulted in decreased affinity to both the detergent‐insoluble cytoskeleton and soluble actin. Recombinant WT‐CaD co‐localized with acto‐myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin‐binding site are essential for proper localization of CaD in EC. Overexpression of WT‐CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD‐transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h‐CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK‐mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis‐induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS‐induced endothelial hyperpermeability and assessed how the changes in phospho‐MLC distribution affect LPS‐induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS‐induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y‐27632 exacerbated LPS‐induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)‐dependent pathways in LPS‐induced barrier dysfunction. We showed that LPS decreased the level of PKA‐dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz‐cAMP counteracted this effect. Forskolin and bnz‐cAMP also attenuated LPS‐induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009 ), forskolin and bnz‐cAMP provide protection from LPS‐induced barrier dysfunction. We compared the effects of bnz‐cAMP and Y‐27632 on phospho‐MLC distribution and observed that while bnz‐cAMP increased the association of the phospho‐MLC signal with the cortical structures, Y‐27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho‐MLC changes. J. Cell. Physiol. 226: 3132–3146, 2011.

Caldesmon is a cytoskeletal target for PKC in endothelium

We have previously shown that treatment of bovine endothelial cell (EC) monolayers with phorbol myristate acetate (PMA) leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid‐like structure, concomitant with the loss of endothelial barrier function. In the current work, we focused on caldesmon, a cytoskeletal protein, regulating actomyosin interaction. We hypothesized that protein kinase C (PKC) activation by PMA leads to the changes in caldesmon properties such as phosphorylation and cellular localization. We demonstrate here that PMA induces both myosin and caldesmon redistribution from cortical ring into the grid‐like network. However, the initial step of PMA‐induced actin and myosin redistribution is not followed by caldesmon redistribution. Co‐immunoprecipitation experiments revealed that short‐term PMA (5 min) treatment leads to the weakening of caldesmon ability to bind actin and, to the lesser extent, myosin. Prolonged incubation (15–60 min) with PMA, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid‐like actin network formation. PMA stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK‐kinase MEK fails to abolish grid‐like structure formation, although reducing PMA‐induced weakening of the cortical actin ring, whereas inhibition of PKC reverses PMA‐induced cytoskeletal rearrangement. Our results suggest that PKC‐dependent phosphorylation of caldesmon is involved in PMA‐mediated complex cytoskeletal changes leading to the EC barrier compromise. J. Cell. Biochem. 99: 1593–1605, 2006.

Resistin-Like Molecule α Stimulates Proliferation of Mesenchymal Stem Cells While Maintaining Their Multipotency

Resistin-like molecule α (RELMα) is highly upregulated in the lungs of mice subjected to hypoxia. It is secreted from pulmonary epithelium and causes potent mitogenic, angiogenic, and vasoconstrictive effects in the lung vasculature. By using bone marrow transplantation in mice, we previously showed that RELMα is able to increase the number of bone marrow-derived cells in lung tissue, especially in the remodeling pulmonary vasculature. The current study investigated the effect of RELMα on progenitor stem cell content in mouse lung. Hypoxia, while stimulating RELMα expression, caused an increase in the number of Sca1(+)/CD45(-) progenitor cells in lungs of wild-type mice, but not in lungs of RELMα knockout mice. An in vitro study with cultured mesenchymal stem cells (MSCs) showed that RELMα induced a robust proliferative response that was dependent on Phosphatidylinositol 3-kinase/Akt and Erk activation. RELMα treatment of MSCs caused upregulation of a large number of genes involved in cell cycle, mitosis, organelle, and cytoskeleton biogenesis, and DNA metabolism. MSCs cultured in RELMα-supplemented media were able to maintain their differentiation potential into adipogenic, osteogenic, or mesenchymal phenotypes, although adipogenic differentiation was partially inhibited. These results demonstrate that RELMα may be involved in stem cell proliferation in the lung, without affecting differentiation potential.

Resistin-Like Molecule α in Allergen-Induced Pulmonary Vascular Remodeling

Resistin-like molecule α (RELMα) has mitogenic, angiogenic, vasoconstrictive, and chemokine-like properties and is highly relevant in lung pathology. Here, we used RELMα knockout (Retnla(-/-)) mice to investigate the role of RELMα in pulmonary vascular remodeling after intermittent ovalbumin (OVA) challenge. We compared saline- and OVA-exposed wild-type (WT) mice and found that OVA induced significant increases in right ventricular systolic pressure, cardiac hypertrophy, pulmonary vascular remodeling of intra-alveolar arteries, goblet cell hyperplasia in airway epithelium, and intensive lung inflammation, especially perivascular inflammation. Genetic ablation of Retnla prevented the OVA-induced increase in pulmonary pressure and cardiac hypertrophy seen in WT mice. Histological analysis showed that Retnla(-/-) mice exhibited less vessel muscularization, less perivascular inflammation, reduced medial thickness of intra-alveolar vessels, and fewer goblet cells in upper airway epithelium (250-600 μm) than did WT animals after OVA challenge. Gene expression profiles showed that genes associated with vascular remodeling, including those related to muscle protein, contractile fibers, and actin cytoskeleton, were expressed at a lower level in OVA-challenged Retnla(-/-) mice than in similarly treated WT mice. In addition, bronchoalveolar lavage from OVA-challenged Retnla(-/-) mice had lower levels of cytokines, such as IL-1β, -1 receptor antagonist, and -16, chemokine (C-X-C motif) ligand 1, -2, -9, -10, and -13, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, TIMP metallopeptidase inhibitor-1, and triggering receptor expressed on myeloid cells-1, than did that from WT mice when analyzed by cytokine array dot blots. Retnla knockout inhibited the OVA-induced T helper 17 response but not the T helper 2 response. Altogether, our results suggest that RELMα is involved in immune response-induced pulmonary vascular remodeling and the associated increase in inflammation typically observed after OVA challenge.

Putative protein partners for the human CPI-17 protein revealed by bacterial two-hybrid screening

We have previously demonstrated that PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI-17) is expressed in lung endothelium. CPI-17, a specific inhibitor of myosin light chain phosphatase (MLCP), is involved in the endothelial cytoskeletal and barrier regulation. In this paper, we report the identification of fourteen putative CPI-17 interacting proteins in the lung using BacterioMatch Two-Hybrid System. Five of them: plectin 1 isoform 1, alpha II spectrin, OK/SW-CL.16, gelsolin isoform a, and junction plakoglobin are involved in actin cytoskeleton organization and cell adhesion, suggesting possible significance of these binding partners in CPI-17-mediated cytoskeletal reorganization of endothelial cells. Furthermore, we confirmed the specific interaction between plakoglobin and CPI-17, which is affected by the phosphorylation status of CPI-17 in human lung microvascular endothelial cells.

Toll like receptors signaling pathways as a target for therapeutic interventions

This review summarizes the key role of Toll-Like Receptor (TLRs) molecules for igniting the immune system. Activated by a broad spectrum of pathogens, cytokines or other specific molecules, TLRs trigger innate immune responses. Published data demonstrate that the targeting and suppression of TLRs and TLR-related proteins with particular inhibitors may provide pivotal treatments for patients with cancer, asthma, sepsis, Crohns disease and thrombosis. Many drugs that target cytokines act in the late phases of the activated pathways, after the final peptides, proteins or glycoproteins are formed in the cell environment. TLR activity occurs in the early activation of cellular pathways; consequently inhibiting them might be most beneficial in the treatment of human diseases.

12 PHORBOL ESTER-INDUCED ENDOTHELIAL CYTOSKELETAL REMODELING.

We have previously shown that treatment of bovine endothelial cell monolayers with phorbol myristate acetate leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. Here we demonstrate that phorbol myristate acetate induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of actin and myosin redistribution is not followed by caldesmon. Coimmunoprecipitation experiments revealed that short-term (5 minutes) treatment with phorbol ester leads to the weakening of caldesmon ability to bind actin and myosin. Prolonged incubation with phorbol myristate acetate, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. Phorbol ester stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing weakening of the cortical actin ring, whereas inhibition of protein kinase C reverses phorbol ester-induced cytoskeletal rearrangement. Our results suggest that protein kinase C-dependent phosphorylation of caldesmon is involved in phorbol ester-mediated complex cytoskeletal changes leading to the endothelial cell barrier compromise.