Kinase signaling as a drug target modality for regulation of vascular hyperpermeability: A case for ARDS therapy development.

The endothelial vascular permeability barrier has an important role throughout the body's extensive vasculature, and its disruption leads to vascular hyperpermeability (leakage), which is associated with numerous medical conditions. In the lung, vascular hyperpermeability can lead to pulmonary edema and acute respiratory distress syndrome (ARDS), the most severe and deadly complication of viral and bacterial infections, trauma and radiation exposure. There is currently no pharmacological treatment for ARDS with the only approved options being focused on supportive care.

Plasma 12- and 15-hydroxyeicosanoids are predictors of survival in pulmonary arterial hypertension.

This study aimed to characterize alterations in select eicosanoids in experimental and human pulmonary arterial hypertension (PAH) and to assess their potential utility as predictors of outcome. Using liquid chromatography-mass spectrometry, we performed targeted lipidomic analyses of the lungs and right ventricles (RVs) of chronically hypoxic rats and plasma of consecutive PAH patients and healthy controls. In rat lungs, chronic hypoxia was associated with significantly decreased lung prostacyclin (PGI2)/thromboxane B2 (TXB2) ratio and elevated lung 8-hydroxyeicosanoid (HETE) acid concentrations.

Alpha-Catulin Co-Localizes With Vimentin Intermediate Filaments and Functions in Pulmonary Vascular Endothelial Cell Migration via ROCK.

The ubiquitous α-catulin acts as a scaffold for distinct signalosomes including RhoA/ROCK; however, its function is not well understood. While α-catulin has homology to the cytoskeletal linkers α-catenin and vinculin, it appears to be functionally divergent. Here we further investigated α-catulin function in pulmonary vascular endothelial cells (VEC) on the premise that α-catulin has a unique cytoskeletal role.

Targeted deletion of Tsc1 causes fatal cardiomyocyte hyperplasia independently of afterload.

Despite high expression levels, the role of Tsc1 in cardiovascular tissue is ill defined. We launched this study to examine the role of Tsc1 in cardiac physiology and pathology. Mice in which Tsc1 was deleted in cardiac tissue and vascular smooth muscle (Tsc1c/cSM22cre(+/-)), developed progressive cardiomegaly and hypertension and died early.

Modulating endothelial barrier function by targeting vimentin phosphorylation.

Vimentin is a major intermediate filament protein in vascular endothelial cells which might be involved in their function as a barrier tissue. It is proposed to dynamically maintain integrity of the endothelium as a tightly regulated permeability barrier that is subjected to a variety of shear and contractile forces. The results described in this report demonstrate that vimentin plays that role through mechanisms that are dependent on its phosphorylation state.

Anthrax lethal toxin disrupts the endothelial permeability barrier through blocking p38 signaling.

Exposure to anthrax causes life-threatening disease through the action of the toxin produced by the Bacillus anthracis bacteria. Lethal factor (LF), an anthrax toxin component which causes severe vascular leak and edema, is a protease which specifically degrades MAP kinase kinases (MKK). We have recently shown that p38 MAP kinase activation leading to HSP27 phosphorylation augments the endothelial permeability barrier.

Regulation of vimentin intermediate filaments in endothelial cells by hypoxia.

Hypoxia triggers responses in endothelial cells that play roles in many conditions including high-altitude pulmonary edema and tumor angiogenesis. Signaling pathways activated by hypoxia modify cytoskeletal and contractile proteins and alter the biomechanical properties of endothelial cells. Intermediate filaments are major components of the cytoskeleton whose contribution to endothelial physiology is not well understood.

Modulation of HSP27 alters hypoxia-induced endothelial permeability and related signaling pathways.

This manuscript describes how the permeability of pulmonary artery microvascular endothelial cell (RPMEC) monolayer is elevated by hypoxia and the role played by HSP27 phosphorylation. p38 MAP kinase activation leading to HSP27 phosphorylation was previously shown by our laboratory to alter the actin cytoskeleton and tethering properties of RPMEC. This effect was independent of hypoxia-induced contractility which was ROCK-dependent rather than HSP27-dependent.

Mitogen activated protein kinase activated protein kinase 2 regulates actin polymerization and vascular leak in ventilator associated lung injury.

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury.

Alpha(E)-Catenin induces SRF-dependent transcriptional activity through its C-terminal region and is partly RhoA/ROCK-dependent.

The ubiquitous alpha(E)-catenin is an essential actin cytoskeletal linker. The transcription factor, serum response factor (SRF), induces transcription via binding to the serum response element (SRE) in gene promoters, and in many cases responds to actin dynamics. Here, we report that alpha(E)-catenin expression in HEK293 cells activates the SRE.

Lack of MK2 inhibits myofibroblast formation and exacerbates pulmonary fibrosis.

Fibroblasts play a major role in tissue repair and remodeling. Their differentiation into myofibroblasts, marked by increased expression of smooth muscle-specific alpha-actin (alpha-SMA), is believed to be important in wound healing and fibrosis. We have recently described a role for MK2 in this phenotypic differentiation in culture.

Transforming growth factor-beta1 effects on endothelial monolayer permeability involve focal adhesion kinase/Src.

Transforming growth factor (TGF)-beta1 activity has been shown to increase vascular endothelial barrier permeability, which is believed to precede several pathologic conditions, including pulmonary edema and vessel inflammation. In endothelial monolayers, TGF-beta1 increases permeability, and a number of studies have demonstrated the alteration of cell-cell contacts by TGF-beta1. We hypothesized that focal adhesion complexes also likely contribute to alterations in endothelial permeability.

Smooth muscle alpha-actin expression and myofibroblast differentiation by TGFbeta are dependent upon MK2.

Fibroblasts play a major role in processes such as wound repair, scarring, and fibrosis. Differentiation into myofibroblasts, characterized by upregulation of smooth muscle alpha-actin (smalpha) in response to profibrotic agents such as TGFbeta is believed to be an important step in fibrosis. Therefore, elucidating mechanisms of myofibroblast differentiation might reveal novel targets in treating diseases such as idiopathic pulmonary fibrosis (IPF).

Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways.

Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined.

Hypoxia alters biophysical properties of endothelial cells via p38 MAPK- and Rho kinase-dependent pathways.

Hypoxia alters the barrier function of the endothelial cells that line the pulmonary vasculature, but underlying biophysical mechanisms remain unclear. Using rat pulmonary microvascular endothelial cells (RPMEC) in culture, we report herein changes in biophysical properties, both in space and in time, that occur in response to hypoxia. We address also the molecular basis of these changes.

5-HT activates ERK MAP kinase in cultured-human peripheral blood mononuclear cells via 5-HT1A receptors.

In the present work, we tested the hypothesis that serotonin (5-hydroxytryptamine = 5-HT) might activate the extracellular signal-regulated kinase (ERK) pathway in human peripheral blood mononuclear cells (PBMC). PBMC were maintained in culture for 72 hrs at 37 degrees C prior to the addition of 5-HT. Our results showed an increase in ERK activation by 5-HT with a peak effect at 30 min and maximal stimulation with 5-HT at 1microM.

Hypoxia decreases expression of soluble guanylate cyclase in cultured rat pulmonary artery smooth muscle cells.

Nitric oxide (NO) has an important role in modulating the pulmonary vascular tone. NO acts, in part, by stimulating soluble guanylate cyclase (sGC) to synthesize the intracellular second messenger cyclic GMP. In vascular smooth muscle cells, sGC is a heterodimer composed of alpha1 and beta1 subunits.

Role of heat shock protein 27 in cytoskeletal remodeling of the airway smooth muscle cell.

Remodeling of the airway smooth muscle (ASM) cell has been proposed to play an important role in airway hyperresponsiveness. Using a functional assay, we have assessed remodeling of the cultured rat ASM cell and the role of heat shock protein (HSP) 27 in that process. To probe remodeling dynamics, we measured spontaneous motions of an individual Arg-Gly-Asp-coated microbead that was anchored to the cytoskeleton.

Estrogen modulates xanthine dehydrogenase/xanthine oxidase activity by a receptor-independent mechanism.

Hypoxia causes up-regulation and activation of xanthine dehydrogenase/xanthine oxidase (XDH/XO) in vitro and in the lungs in vivo. This up-regulation, and the likely corresponding production of reactive oxygen species, may underlie the pathogenesis of an array of disorders. Thus, compounds that prevent hypoxia-induced increase in XDH/XO activity may provide a therapeutic strategy in such disorders.

Upregulation of xanthine oxidase by tobacco smoke condensate in pulmonary endothelial cells.

Tobacco smoking has been causally linked to the development of chronic obstructive pulmonary disease. It has been reported that the reactive oxygen species (ROS)- generating enzyme xanthine dehydrogenase/oxidase (XO) is increased in smoking-related stomach ulcers and that gastric mucosal damage caused by tobacco smoke can be blocked by the XO inhibitor allopurinol. In order to test the hypothesis that tobacco may cause the upregulation of XO in the lung, cultured rat pulmonary microvascular endothelial cells were exposed to tobacco smoke condensate (TSC).

Cytoskeletal changes in hypoxic pulmonary endothelial cells are dependent on MAPK-activated protein kinase MK2.

Exposure to hypoxia causes structural changes in the endothelial cell layer that alter its permeability and its interaction with leukocytes and platelets. One of the well characterized cytoskeletal changes in response to stress involves the reorganization of the actin cytoskeleton and the formation of stress fibers. This report describes cytoskeletal changes in pulmonary microvascular endothelial cells in response to hypoxia and potential mechanisms involved in this process.

Modulation of inducible nitric oxide synthase by hypoxia in pulmonary artery endothelial cells.

The effects of hypoxia on the regulation of inducible nitric oxide synthase (NOS) 2 expression were examined in cultured rat pulmonary microvascular endothelial cells (EC). EC did not express NOS 2 mRNA or protein when exposed to normoxia or hypoxia unless they were pretreated with interleukin (IL)-1beta and/or tumor necrosis factor (TNF)-alpha for 24 h. Induction of NOS 2 by IL-1beta+TNF-alpha was significantly attenuated by concomitant exposure of EC to hypoxia or treatment of EC with antioxidants such as tiron, diphenyliodonium, and catalase, suggesting that NOS 2 expression is dependent on the production of reactive oxygen species.