Connie Snead

Endothelium-independent effect of estrogen on Ca2+-activated K+ channels in human coronary artery smooth muscle cells

OBJECTIVE Postmenopausal estrogen replacement therapy lowers the incidence of cardiovascular disease, suggesting that estrogens support cardiovascular function. Estrogens dilate coronary arteries; however, little is known about the molecular basis of how estrogen affects the human coronary circulation. The cellular/molecular effects of estrogen action on human coronary smooth muscle were investigated in the present study. METHODS Patch-clamp and fluorescent microscopy studies were performed on human coronary myocytes in the absence of endothelium. RESULTS Estrogen increased whole-cell currents over a range of membrane potentials, and further studies indicated that the large-conductance (186.5 +/- 3 pS), calcium- and voltage-activated potassium (BK(Ca)) channel was the target of estrogen action. Channel activity was stimulated approximately 15-fold by nanomolar concentrations of 17 beta-estradiol, and this stimulation was reversed >90% by inhibiting cGMP-dependent protein kinase activity with 300 nM KT5823. 17 beta-Estradiol increased the level of cGMP and nitric oxide in human myocytes, and the stimulatory effect of estrogen on channel activity and NO production was reversed by inhibiting NO synthase with 10 microM N(G)-monomethyl-L-arginine. CONCLUSIONS Our cellular and molecular studies identify the BK(Ca) channel as a target of estrogen action in human coronary artery smooth muscle. This response to estrogen involves cGMP-dependent phosphorylation of the BK(Ca) channel or a closely associated regulatory molecule, and further evidence suggests involvement of the NO/cGMP signaling system in coronary smooth muscle. These findings are the first to provide direct evidence for a molecular mechanism that can account for endothelium-independent effects of estrogen on human arteries, and may also help explain why estrogens reduce myocardial ischemia and stimulate coronary blood flow in patients with diseased coronary arteries.

Heat shock protein 90 inhibitors attenuate LPS-induced endothelial hyperpermeability

Endothelial hyperperme ability leading to vascular leak is an important consequence of sepsis and sepsis-induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis-induced lung injury. We now examine the effects of hsp90 inhibitors on LPS-mediated endothelial hyperpermeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary arterial endothelial cells (BPAEC). Vehicle-pretreated cells exposed to endotoxin exhibited a concentration-dependent decrease in TER, activation of pp60(Src), phosphorylation of the focal adhesion protein paxillin, and reduced expression of the adherens junction proteins, vascular endothelial (VE)-cadherin and beta-catenin. Pretreatment with the hsp90 inhibitor, radicicol, prevented the decrease in TER, maintained VE-cadherin and beta-catenin expression, and inhibited activation of pp60(Src) and phosphorylation of paxillin. Similarly, when BPAEC hyperpermeability was induced by endotoxin-activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil-induced decrease in TER. Increased paxillin phosphorylation and decreased expression of beta-catenin and VE-cadherin were also observed in mouse lungs 12 h after intraperitoneal endotoxin and attenuated in mice pretreated with radicicol. These results suggest that hsp90 plays an important role in sepsis-associated endothelial barrier dysfunction.

Effect of PPARγ inhibition on pulmonary endothelial cell gene expression: gene profiling in pulmonary hypertension

Peroxisome proliferator-activated receptor type gamma (PPARgamma) is a subgroup of the PPAR transcription factor family. Recent studies indicate that loss of PPARgamma is associated with the development of pulmonary hypertension (PH). We hypothesized that the endothelial dysfunction associated with PPARgamma inhibition may play an important role in the disease process by altering cellular gene expression and signaling cascades. We utilized microarray analysis to determine if PPARgamma inhibition induced changes in gene expression in pulmonary arterial endothelial cells (PAEC). We identified 100 genes and expressed sequence tags (ESTs) that were upregulated by >1.5-fold and 21 genes and ESTs that were downregulated by >1.3-fold (P < 0.05) by PPARgamma inhibition. The upregulated genes can be broadly classified into four functional groups: cell cycle, angiogenesis, ubiquitin system, and zinc finger proteins. The genes with the highest fold change in expression: hyaluronan-mediated motility receptor (HMMR), VEGF receptor 2 (Flk-1), endothelial PAS domain protein 1 (EPAS1), basic fibroblast growth factor (FGF-2), and caveolin-1 in PAEC were validated by real time RT-PCR. We further validated the upregulation of HMMR, Flk-1, FGF2, and caveolin-1 by Western blot analysis. In keeping with the microarray results, PPARgamma inhibition led to re-entry of cell cycle at G(1)/S phase and cyclin C upregulation. PPARgamma inhibition also exacerbated VEGF-induced endothelial barrier disruption. Finally we confirmed the downregulation of PPARgamma and the upregulation of HMMR, Flk-1, FGF2, and Cav-1 proteins in the peripheral lung tissues of an ovine model of PH. In conclusion, we have identified an array of endothelial genes modulated by attenuated PPARgamma signaling that may play important roles in the development of PH.

Heat Shock Protein 90 Inhibitors Protect and Restore Pulmonary Endothelial Barrier Function

Heat shock protein 90 (hsp90) inhibitors inactivate and/or degrade various client proteins, including many involved in inflammation. Increased vascular permeability is a hallmark of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Thus, we tested the hypothesis that hsp90 inhibitors may prevent and/or restore endothelial cell (EC) permeability after injury. Exposure of confluent bovine pulmonary arterial endothelial cell (BPAEC) monolayer to TGF-beta1, thrombin, bacterial lipopolysaccharide (LPS), or vascular endothelial growth factor (VEGF) increased BPAEC permeability, as revealed by decreased transendothelial electrical resistance (TER). Treatment of injured endothelium with hsp90 inhibitors completely restored TER of BPAEC. Similarly, preincubation of BPAEC with hsp90 inhibitors prevented the decline in TER induced by the exposure to thrombin, LPS, VEGF, or TGF-beta1. In addition, hsp90 inhibitors restored the EC barrier function after PMA or nocodazole-induced hyperpermeability. These effects of the hsp90 inhibitors were associated with the restoration of TGF-beta1- or nocodazole-induced decrease in VE-cadherin and beta-catenin expression at EC junctions. The protective effect of hsp90 inhibitors on TGF-beta1-induced hyperpermeability was critically dependent upon preservation of F-actin cytoskeleton and was associated with the inhibition of agonist-induced myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) phosphorylation, F-actin stress fibers formation, microtubule disassembly, increase in hsp27 phosphorylation, and association of hsp90 with hsp27, but independent of p38MAPK activity. We conclude that hsp90 inhibitors exert barrier protective effects on BPAEC, at least in part, via inhibition of hsp27-mediated, agonist-induced cytoskeletal rearrangement, and therefore may have useful therapeutic value in ALI, ARDS, and other pulmonary inflammatory disease.

Estrogen Replacement Therapy Prevents Airway Dysfunction in a Murine Model of Allergen-Induced Asthma

We previously reported that 17β-estradiol (E2) prevents hyperresponsiveness to carbachol of murine asthmatic tracheal rings in vitro. We now investigated whether E2 is similarly effective in reducing airway hyperreactivity in a murine model of allergic asthma in vivo. Female ovariectomized BALB/c mice were rendered asthmatic by a 25-day protocol of sensitization to ovalbumin. Under positive-pressure ventilation, anesthetized asthmatic mice exhibited a dramatic increase in airway responsiveness to increasing doses of inhaled methacholine compared to PBS-sensitized controls, as reflected in decreased dynamic compliance of the respiratory system and increased tissue damping, tissue elastance, and airway resistance. Furthermore, asthmatic mice exhibited hypercellularity and increased protein concentration in the bronchoalveolar lavage, strong signs of peribronchial cuffing with inflammatory cells and increased goblet cell activity. To test the effects of estrogen, three additional groups of mice were implanted subcutaneously with different amounts of slow-release E2 pellets at the time of ovariectomy and rendered asthmatic as before. E2 dose-dependently inhibited airway hyperresponsiveness to methacholine, reduced bronchoalveolar lavage hypercellularity, and virtually eliminated histologic signs of inflammation and goblet cell hyperactivity. The inflammation and airway hyperactivity in asthmatic mice was associated with an increase in bronchoalveolar lavage levels of TGFβ1, which was completely abolished in E2-treated asthmatic mice. We conclude that estrogen replacement therapy effectively ameliorates the pathologic profile of murine allergic asthma.

The green tea polyphenol epigallocatechin-3-gallate improves systemic hemodynamics and survival in rodent models of polymicrobial sepsis.

Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid found in green tea. Recent in vitro studies have suggested that EGCG inhibits activation of the nuclear factor-&kgr;B (NF-&kgr;B) pathway. The NF-&kgr;B is a transcriptional factor required for gene expression of many inflammatory mediators, including the inducible isoform of nitric oxide synthase (NOS2). Excessive NO production by NOS2 is directly linked to the vasoplegia, shock, and mortality associated with sepsis. Accordingly, we hypothesized that EGCG administration would inhibit NOS2 gene expression and thereby improve survival in a rodent model of polymicrobial sepsis. Polymicrobial sepsis was induced in male Sprague-Dawley rats (hemodynamic study) and C57BL6 mice (mortality study) via cecal ligation and double puncture (CL2P). Rodents were treated with either EGCG (10 mg/kg intraperitoneally) or vehicle at 1 and 6 h after CL2P and every 12 h thereafter. In the hemodynamic study, mean arterial blood pressure was monitored for 18 h, and rats were killed at 3, 6, and 18 h after CL2P. In the mortality study, survival was monitored for 72 h after CL2P in mice. In vehicle-treated rodents, CL2P was associated with profound hypotension and greater than 80% mortality rate. Epigallocatechin-3-gallate treatment significantly improved both the hypotension and survival. In vitro experiments further showed that EGCG inhibited activation of NF-&kgr;B and subsequent NOS2 gene expression in a primary culture of rat aortic smooth muscle cells. Epigallocatechin-3-gallate may therefore represent a potential nutritional supplement or pharmacologic agent in patients with sepsis.

Mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: role of asymmetric dimethylarginine.

Acute lung injury (ALI) is associated with severe alterations in lung structure and function and is characterized by hypoxemia, pulmonary edema, low lung compliance and widespread capillary leakage. Asymmetric dimethylarginine (ADMA), a known cardiovascular risk factor, has been linked to endothelial dysfunction and the pathogenesis of a number of cardiovascular diseases. However, the role of ADMA in the pathogenesis of ALI is less clear. ADMA is metabolized via hydrolytic degradation to l-citrulline and dimethylamine by the enzyme, dimethylarginine dimethylaminohydrolase (DDAH). Recent studies suggest that lipopolysaccharide (LPS) markedly increases the level of ADMA and decreases DDAH activity in endothelial cells. Thus, the purpose of this study was to determine if alterations in the ADMA/DDAH pathway contribute to the development of ALI initiated by LPS-exposure in mice. Our data demonstrate that LPS exposure significantly increases ADMA levels and this correlates with a decrease in DDAH activity but not protein levels of either DDAH I or DDAH II isoforms. Further, we found that the increase in ADMA levels cause an early decrease in nitric oxide (NO(x)) and a significant increase in both NO synthase (NOS)-derived superoxide and total nitrated lung proteins. Finally, we found that decreasing peroxynitrite levels with either uric acid or Manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTymPyp) significantly attenuated the lung leak associated with LPS-exposure in mice suggesting a key role for protein nitration in the progression of ALI. In conclusion, this is the first study that suggests a role of the ADMA/DDAH pathway during the development of ALI in mice and that ADMA may be a novel therapeutic biomarker to ascertain the risk for development of ALI.

Heat Shock Protein 90 Inhibitors Prevent LPS-Induced Endothelial Barrier Dysfunction by Disrupting RhoA Signaling

Permeability of the endothelial monolayer is increased when exposed to the bacterial endotoxin LPS. Our previous studies have shown that heat shock protein (Hsp) 90 inhibitors protect and restore LPS-mediated hyperpermeability in bovine pulmonary arterial endothelial cells. In this study, we assessed the effect of Hsp90 inhibition against LPS-mediated hyperpermeability in cultured human lung microvascular endothelial cells (HLMVECs) and delineated the underlying molecular mechanisms. We demonstrate that Hsp90 inhibition is critical in the early phase, to prevent LPS-mediated hyperpermeability, and also in the later phase, to restore LPS-mediated hyperpermeability in HLMVECs. Because RhoA is a well known mediator of endothelial hyperpermeability, we investigated the effect of Hsp90 inhibition on LPS-mediated RhoA signaling. RhoA nitration and activity were increased by LPS in HLMVECs and suppressed when pretreated with the Hsp90 inhibitor, 17-allylamino-17 demethoxy-geldanamycin (17-AAG). In addition, inhibition of Rho kinase, a downstream effector of RhoA, protected HLMVECs from LPS-mediated hyperpermeability and abolished LPS-induced myosin light chain (MLC) phosphorylation, a target of Rho kinase. In agreement with these findings, 17-AAG or dominant-negative RhoA attenuated LPS-induced MLC phosphorylation. MLC phosphorylation induced by constitutively active RhoA was also suppressed by 17-AAG, suggesting a role for Hsp90 downstream of RhoA. Inhibition of Src family kinases also suppressed RhoA activity and MLC phosphorylation. Together, these data indicate that Hsp90 inhibition prevents and repairs LPS-induced lung endothelial barrier dysfunction by suppressing Src-mediated RhoA activity and signaling.

Harvesting, identification and barrier function of human lung microvascular endothelial cells

Endothelial barrier dysfunction is an important contributor to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Even though approaches that target the prevention and repair of endothelial barrier dysfunction are clearly needed, our understanding of the molecular regulation of pulmonary microvascular endothelial permeability remains incomplete. Cultured pulmonary microvascular endothelial cells represent an attractive paradigm for the study of barrier function. Here, we describe a method for the harvest, identification and culture of human lung microvascular endothelial cells (HLMVEC). HLMVEC thus obtained, grow as a monolayer, exhibit contact inhibition and have the typical cobblestone appearance. They express endothelial proteins, such as von Willebrand factor and endothelial nitric oxide synthase and take up an acetylated LDL. Furthermore, HLMVEC respond predictably and with superior sensitivity to the barrier disruptive effects of Gram positive and Gram negative bacterial products, thrombin, vascular endothelial growth factor and microtubule disrupting agents. These HLMVEC present an in-house-derived alternative to commercially available human cells for the study of mechanisms contributing to ALI and ARDS.

Lipopolysaccharide-induced lung injury involves the nitration-mediated activation of RhoA

Background: The activation of RhoA is a critical event in acute lung injury (ALI), but the role of nitration in this process is unresolved. Results: The nitration of RhoA at Tyr34 produced GEF-like conformational changes that stimulate RhoA by decreasing GDP binding. Conclusion: We have identified a new mechanism of RhoA activation. Significance: Preventing RhoA nitration may be useful for the management of ALI. Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr34 in RhoA. Tyr34 is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr34 against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr34 nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of “shielding” peptides to prevent RhoA nitration in the management of ALI.