Christiana Dimitropoulou

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

OBJECTIVEnPostmenopausal 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.nnnMETHODSnPatch-clamp and fluorescent microscopy studies were performed on human coronary myocytes in the absence of endothelium.nnnRESULTSnEstrogen 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.nnnCONCLUSIONSnOur 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.

Angiotensin II Relaxes Microvessels Via the AT2 Receptor and Ca2+-Activated K+ (BKCa) Channels

Angiotensin II (Ang II) is one of the most potent vasoconstrictor substances, yet paradoxically, Ang II may dilate certain vascular beds via an undefined mechanism. Ang II–induced vasoconstriction is mediated by the AT1 receptor, whereas the relative expression and functional importance of the AT2 receptor in regulating vascular resistance and blood pressure are unknown. We now report that Ang II induces relaxation of mesenteric microvessels and that this vasodilatory response was unaffected by losartan, an AT1 receptor antagonist, but was inhibited by PD123,319, a selective antagonist of AT2 receptors. In addition, reverse transcriptase–polymerase chain reaction studies revealed high amounts of AT2 receptor mRNA in smooth muscle from these same microvessels. Ang II–induced relaxation was inhibited by either tetraethylammonium or iberiotoxin, suggesting involvement of the large-conductance, calcium- and voltage-activated potassium (BKCa) channel. Subsequent whole-cell and single-channel patch-clamp studies on single myocytes demonstrated that Ang II increases the activity of BKCa channels. As in our tissue studies, the effect of Ang II on BKCa channels was inhibited by PD123,319, but not by losartan. In light of these consistent findings from tissue physiology, molecular studies, and cellular/molecular physiology, we conclude that Ang II relaxes microvessels via stimulation of the AT2 receptor with subsequent opening of BKCa channels, leading to membrane repolarization and vasodilation. These findings provide evidence for a novel endothelium-independent vasodilatory effect of Ang II.

Hydrogen Sulfide Ameliorates Tobacco Smoke-Induced Oxidative Stress and Emphysema in Mice

AIMSnThe mutual interactions between reactive oxygen species, airway inflammation, and alveolar cell death play crucial role in the pathogenesis of chronic obstructive pulmonary disease (COPD). In the present study, we investigated the possibility that hydrogen sulfide (H(2)S) donor sodium hydrosulfide (NaHS) might be a novel option for intervention in COPD.nnnRESULTSnWe used a mouse model of tobacco smoke (TS)-induced emphysema. Mice were injected with H(2)S donor NaHS (50 μmol/kg in 0.25 ml phosphate buffer saline, intraperitoneally) or vehicle daily before exposed to TS for 1 h/day, 5 days/week for 12 and 24 weeks. We found that NaHS ameliorated TS-induced increase in mean linear intercepts, the thickness of bronchial walls, and the numbers of total cell counts as well as neutrophils, monocytes, and tumor necrosis factor α in bronchial alveolar lavage. Moreover, NaHS reduced increases in right ventricular systolic pressure, the thickness of pulmonary vascular walls, and the ratio of RV/LV+S in TS-exposed mice. Further, TS exposure for 12 and 24 weeks reduced the protein contents of cystathionine γ-lyase (CGL), cystathionine β-synthetase (CBS), nuclear erythroid-related factor 2 (Nrf2), P(ser473)-Akt, as well as glutathione/oxidized glutathione ratio in the lungs. TS-exposed lungs exhibited large amounts of 8-hydroxyguanine-positive and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. Treatment with NaHS increased P(ser473)-Akt and attenuated TS-induced reduction of CGL, CBS, and Nrf2 as well as glutathione/oxidized glutathione ratio in the lungs. NaHS also reduced amounts of 8-hydroxyguanine-positive, terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and active caspase-3 in TS-exposed lungs. Additionally, knocking-down Akt protein abolished the protective effects of NaHS against TS-induced apoptosis and downregulation of Nrf2, CGL, and CBS in pulmonary artery endothelial cells.nnnCONCLUSIONnThese results indicate that NaHS protects against TS-induced oxidative stress, airway inflammation, and remodeling and ameliorates the development of emphysema and pulmonary hypertension. H(2)S donors have therapeutic potential for the prevention and treatment of COPD caused by TS.

Afferent Arteriolar Dilation to 11, 12-EET Analogs Involves PP2A Activity and Ca2 +-Activated K+ Channels

The epoxygenase metabolite, 11, 12‐epoxyeicosatrienoic acid (11, 12‐EET), has renal vascular actions. 11, 12‐EET analogs have been developed to determine the structure activity relationship for 11, 12‐EET and as a tool to investigate signaling mechanisms responsible for afferent arteriolar dilation. We hypothesized that 11, 12‐EET mediated afferent arteriolar dilation involves increased phosphoprotein phosphatase 2A (PP2A) and large‐conductance calcium activated K+ (KCa) channels. We evaluated the chemically and/or metabolically table 11, 12‐EET analogs: 11, 12‐EET‐N‐methylsulfonimide (11, 12‐EET‐SI), 11‐nonyloxy‐undec‐8(Z)‐enoic acid (11, 12‐ether‐EET‐8‐ZE), and 11, 12‐trans‐oxidoeicosa‐8(Z)‐eonoic acid (11, 12‐tetra‐EET‐8‐ZE). Afferent arteriolar responses were assessed. Activation of KCa channels by 11, 12‐EET analogs were established by single cell channel recordings in renal myocytes. Assessment of renal vascular responses revealed that 11, 12‐EET analogs increased afferent arteriolar diameter. Vasodilator responses to 11, 12‐EET analogs were abolished by K+ channel or PP2A inhibition. 11, 12‐EET analogs activated renal myocyte large‐conductance KCa channels. 11, 12‐EET analogs increased cAMP by 2‐fold and PP2A activity increased 3–8 fold in renal myocytes. PP2A inhibition did not significantly affect the 11, 12‐EET analog mediated increase in cAMP and PP2A increased renal myocyte KCa channel activity to a much greater extent than PKA. These data support the concept that 11, 12‐EET utilizes PP2A dependent pathways to activate large‐conductance KCa channels and dilate the afferent arteriole.

NADPH Oxidase 4 Is Expressed in Pulmonary Artery Adventitia and Contributes to Hypertensive Vascular Remodeling

Objective— Pulmonary hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PAs) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PAs and determine the functional relevance of Nox4 in PH. Approach and Results— Elevated expression of Nox4 was detected in hypertensive PAs from 3 rat PH models and human PH using qualititative real-time reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia, and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of reactive oxygen species production. Small-molecule inhibitors of Nox4 reduced adventitial reactive oxygen species generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and noninvasive indices of PA stiffness in monocrotaline-treated rats as determined by morphometric analysis and high-resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PAs. In fibroblasts, Nox4 overexpression stimulated migration and proliferation and was necessary for matrix gene expression. Conclusion— These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling, and development of PH.

Opposing Actions of Heat Shock Protein 90 and 70 Regulate Nicotinamide Adenine Dinucleotide Phosphate Oxidase Stability and Reactive Oxygen Species Production

Objective—Excessive reactive oxygen species contribute to vascular dysfunction. We have previously shown that heat shock protein (Hsp90) inhibitors potently suppress Nox 1 to 3 and 5, and the goals of this study were to identify how molecular chaperones regulate Nox function. Methods and Results—In vitro, protein expression of Nox 1 to 2, 5 was decreased by Hsp90 inhibitors in multiple cell types (human pulmonary artery endothelial cells, neutrophils, macrophages, and human saphenous vein). In mice treated with Hsp90 inhibitors, Nox1 expression was reduced in lung along with reduced reactive oxygen species from leukocytes. Elevated reactive oxygen species production in obese (db/db) aorta was suppressed by Hsp90 inhibition. Hsp90 inhibitors did not alter Nox5 micro RNA levels, and proteasome inhibition prevented Nox2 and 5 protein degradation and increased ubiquitin incorporation. Inhibition of Hsp90 upregulated the expression of Hsp70 and Hsp70-bound Nox2, 5 and promoted degradation. Silencing Hsp70 prevented Hsp90 inhibitor–mediated degradation of Nox5. The Hsp70-regulated ubiquitin ligase, carboxyl terminus of Hsp70-interacting protein (CHIP), also bound Nox5 and promoted increased Nox5 ubiquitination and degradation. The chaperone binding and ubiquitination domains of CHIP were required, and the silencing of CHIP blunted Hsp90 inhibitor–mediated degradation of Nox2 and 5. Conclusion—We conclude that Hsp90 binds to and regulates Nox protein stability. These actions are opposed by Hsp70 and CHIP, which promote the ubiquitination and degradation of Nox proteins and reduce reactive oxygen species production.

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.

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.

SUMO1 Negatively Regulates Reactive Oxygen Species Production From NADPH Oxidases

Objective—Increased protein SUMOylation (small ubiquitin-related modifier [SUMO]) provides protection from cellular stress, including oxidative stress, but the mechanisms involved are incompletely understood. The NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS) and oxidative stress, and thus our goal was to determine whether SUMO regulates NADPH oxidase activity. Methods and Results—Increased expression of SUMO1 potently inhibited the activity of Nox1 to Nox5. In contrast, inhibition of endogenous SUMOylation with small interfering RNA to SUMO1 or ubiquitin conjugating enzyme 9 or with the inhibitor anacardic acid increased ROS production from human embryonic kidney-Nox5 cells, human vascular smooth muscle cells, and neutrophils. The suppression of ROS production was unique to SUMO1, and it required a C-terminal diglycine and the SUMO-specific conjugating enzyme ubiquitin conjugating enzyme 9. SUMO1 did not modify intracellular calcium or Nox5 phosphorylation but reduced ROS output in an isolated enzyme assay, suggesting direct effects of SUMOylation on enzyme activity. However, we could not detect the presence of SUMO1 conjugation on Nox5 using a variety of approaches. Moreover, the mutation of more than 17 predicted and conserved lysine residues on Nox5 did not alter the inhibitory actions of SUMO1. Conclusion—Together, these results suggest that SUMO is an important regulatory mechanism that indirectly represses the production of ROS to ameliorate cellular stress.

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.