Vasoconstrictor effect of endothelin-1 on hypertensive pulmonary arterial smooth muscle involves Rho-kinase and protein kinase C

Although one of the common characteristics of pulmonary hypertension is abnormal sustained vasoconstriction, the signaling pathways that mediate this heightened pulmonary vascular response are still not well defined. Protein kinase C (PKC) and Rho-kinase are regulators of smooth muscle contraction induced by G protein-coupled receptor agonists including endothelin-1 (ET-1), which has been implicated as a signaling pathway in pulmonary hypertension. Toward this end, it was hypothesized that both Rho-kinase and PKC mediate the pulmonary vascular response to ET-1 in hypertensive pulmonary arterial smooth muscle, and therefore, the purpose of this study was to determine the role of PKC and Rho-kinase signaling in ET-1-induced vasoconstriction in both normotensive (Sprague-Dawley) and hypertensive (Fawn-Hooded) rat pulmonary arterial smooth muscle. Results indicate that ET-1 caused greater vasoconstriction in hypertensive pulmonary arteries compared with the normal vessels, and treatment with the PKC antagonists chelerythrine, rottlerin, and G? 6983 inhibited the vasoconstrictor response to ET-1 in the hypertensive vessels. In addition, the specific Rho-kinase inhibitor Y-27632 significantly attenuated the effect of ET-1 in both normotensive and hypertensive phenotypes, with greater inhibition occurring in the hypertensive arteries. Furthermore, Western blot analysis revealed that ET-1 increased RhoA expression in both normotensive and hypertensive pulmonary arteries, with expression being greater in the hypertensive state. These results suggest that both PKC and Rho/Rho-kinase mediate the heightened pulmonary vascular response to ET-1 in hypertensive pulmonary arterial smooth muscle.     

RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability

Transforming growth factor (TGF)-beta is a potent inflammatory mediator involved in acute lung injury. TGF-beta directly increases pulmonary endothelial myosin light chain (MLC) phosphorylation, which is associated with increased endothelial stress fiber formation, gap formation, and protein permeability, all hallmarks of pulmonary endothelial responses during acute lung injury. We performed the following experiments in pulmonary endothelial monolayers to determine whether RhoA and Rho-kinase mediate these TGF-beta-induced responses. TGF-beta caused the sustained activation of RhoA 2 h posttreatment associated with increased MLC phosphorylation. Inhibition of either RhoA or Rho-kinase with either C3 exoenzyme or Y-27632 blocked MLC phosphorylation. In addition, both C3 and Y-27632 partially attenuated the maximal TGF-beta-induced increase in permeability but did not affect the initial phase of compromised barrier integrity. Inhibition of Rho-kinase completely blocked the TGF-beta-induced increase in the content of filamentous actin (F-actin) but only partially inhibited TGF-beta-induced changes in actin reorganization. To assess the contribution of Rho-kinase in RhoA-mediated responses independent of additional TGF-beta-induced signals, cells were infected with a constitutively active RhoA adenovirus (RhoAQ63L) with or without Y-27632. RhoAQ63L increased MLC phosphorylation, F-actin content, and permeability. Treatment with Y-27632 blocked these responses, suggesting that Rho-kinase mediates these RhoA-induced effects. Collectively, these data suggest the following: 1) the RhoA/Rho-kinase pathway is an important component of TGF-beta-induced effects on endothelial MLC phosphorylation, cytoskeletal reorganization, and barrier integrity; and 2) additional signaling mechanisms independent of the RhoA/Rho-kinase signaling cascade contribute to TGF-beta-induced changes in cytoskeletal organization and permeability.     

Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.     

Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway

Pulmonary arteries (PA) are resistant to the vasodilator effects of extracellular acidosis in systemic vessels; the mechanism underlying this difference between systemic and pulmonary circulations has not been elucidated. We hypothesized that RhoA/Rho-kinase-mediated Ca2+ sensitization pathway played a greater role in tension development in pulmonary than in systemic vascular smooth muscle and that this pathway was insensitive to acidosis. In arterial rings contracted with the alpha1-agonist phenylephrine (PE), the Rho-kinase inhibitor Y-27632 (< or =3 microM) induced greater relaxation in precontracted PA rings than in aortic rings. In PA rings stimulated by PE, the activation of RhoA was greater than in aorta. Normocapnic acidosis (NA) induced a smaller relaxation in precontracted PA than in aorta. However, in the presence of nifedipine and thapsigargin, when PE-induced contraction was predominantly mediated by Rho-kinase, the relaxant effect of NA was reduced and similar in both vessel types. Furthermore, in the presence of Y-27632, NA induced a greater relaxation in both PA and aorta, which was similar in both vessels. Finally, in alpha-toxin-permeabilized smooth muscle, PE-induced contraction at constant Ca2+ activity was inhibited by Y-27632 and unaffected by acidosis. These results indicate that Ca2+ sensitization induced by the RhoA/Rho-kinase pathway played a greater role in agonist-induced vascular smooth muscle contraction in PA than in aorta and that tension mediated by this pathway was insensitive to acidosis. The predominant role of the RhoA/Rho-kinase pathway in the pulmonary vasculature may account for the resistance of this circulation to the vasodilator effect of acidosis observed in the systemic circulation.     

Relaxin regulates vascular wall remodeling and passive mechanical properties in mice

Administration of recombinant human relaxin (rhRLX) to conscious rats increases global arterial compliance, and small renal arteries (SRA) isolated from these rats demonstrate increased passive compliance. Here we characterize relaxin-induced vascular remodeling and examine its functional relevance. SRA and external iliac arteries (EIA) were examined in rhRLX-treated (1.0 μg/h for 5 days) and relaxin knockout mice. Arterial geometric remodeling and compositional remodeling were quantified using immunohistochemical and biochemical techniques. Vascular mechanical properties were quantified using an ex vivo preparation wherein pressure-diameter data were obtained at various axial lengths. Compared with vehicle-treated mice, SRA from rhRLX-treated mice showed outward geometric remodeling (increased unstressed wall area and wall-to-lumen area ratio), increased smooth muscle cell (SMC) density, reduction in collagen-to-total protein ratio, and unchanged elastin-to-tissue dry weight ratio. Compared with wild-type mice, relaxin knockout mice exhibited the opposite pattern: decreased unstressed wall area and wall-to-lumen area ratio, decreased SMC density, and increased collagen-to-total protein ratio. Although tissue biaxial strain energy of SRA was not different between rhRLX- and vehicle-treated groups at low-to-physiological circumferential and axial strains, it was lower for the rhRLX-treated group at the highest circumferential strain. In contrast to SRA, relaxin administration was not associated with any vascular remodeling or changes in passive mechanics of EIA. Thus relaxin induces both geometric and compositional remodeling in vessel-specific manner. Relaxin-induced geometric remodeling of SRA is responsible for the increase in passive compliance under low-to-physiological levels of circumferential and axial strains, and compositional remodeling becomes functionally relevant only under high circumferential strain.     

Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA

Nitric oxide (NO) acts as a vasoregulatory molecule that inhibits vascular smooth muscle cell (SMC) proliferation. Studies have illustrated that NO inhibits SMC proliferation via the extracellular signal-regulated kinase (ERK) pathway, leading to increased protein levels of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1. The ERK pathway can be pro- or antiproliferative, and it has been demonstrated that the activation status of the small GTPase RhoA determines the proliferative fate of ERK signaling, whereby inactivation of RhoA influences ERK signaling to increase p21^Waf1/Cip1 and inhibit proliferation. The purpose of these investigations was to examine the effect of NO on RhoA activation/S-nitrosation and to test the hypothesis that inhibition of SMC proliferation by NO is dependent on inactivation of RhoA. NO decreases activation of RhoA, as demonstrated by RhoA GTP-binding assays, affinity precipitation, and phalloidin staining of the actin cytoskeleton. Additionally, these effects are independent of cGMP. NO decreases SMC proliferation, and gene transfer of constitutively active RhoA (RhoA^63L) diminished the antiproliferative effects of NO, as determined by thymidine incorporation. Western blots of p21^Waf1/Cip1 correlated with changes in proliferation. S-nitrosation of recombinant RhoA protein and immunoprecipitated RhoA was demonstrated by Western blotting for nitrosocysteine and by measurement of NO release. Furthermore, NO decreases GTP loading of recombinant RhoA protein. These findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation is associated with decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO. cell signaling; posttranslational modification; vascular disease     

RhoA and resistance artery remodeling

Resistance arteries are able to adapt to physiological and pathophysiological stimuli to maintain adequate perfusion according to the metabolic demand of the tissue. Although vasomotor control allows rapid adaptation of lumen diameter, vascular remodeling constitutes an active process that occurs in response to long-term alterations of hemodynamic parameters. Unfortunately, this initially adaptive process contributes to the pathology of vascular diseases. Recent studies have demonstrated the participation of Rho protein signaling pathways in several cardiovascular pathologies including hypertension, coronary artery spasm, effort angina, atherosclerosis, and restenosis. Functional analyses have further revealed that RhoA-dependent pathways are involved in excessive contraction, migration, and proliferation associated with arterial diseases. The present review focuses on the role of Rho proteins, in particular RhoA, in vascular smooth muscle cells and the involvement of Rho-dependent signaling pathways in resistance artery remodeling, more particularly in relation to hypertension.     

Involvement of RhoA/Rho kinase signaling in pulmonary hypertension of the fawn-hooded rat.

The fawn-hooded rat (FHR) develops severe pulmonary hypertension (PH) when raised for the first 3-4 wk of life in the mild hypoxia of Denver's altitude (5,280 ft.). The PH is associated with sustained pulmonary vasoconstriction and pulmonary artery remodeling. Furthermore, lung alveolarization and vascularization are reduced in the Denver FHR. We have recently shown that RhoA/Rho kinase signaling is involved in both vasoconstriction and vascular remodeling in animal models of hypoxic PH. In this study, we investigated the role of RhoA/Rho kinase signaling in the PH of Denver FHR. In alpha-toxin permeabilized pulmonary arteries from Denver FHR, the contractile sensitivity to Ca2+ was increased compared with those from sea-level FHR. RhoA activity and Rho kinase I protein expression in pulmonary arteries of Denver FHR (10-wk-old) were higher than in those of sea-level FHR. Acute inhalation of the Rho kinase inhibitor fasudil selectively reduced the elevated pulmonary arterial pressure in Denver FHR in vivo. Chronic fasudil treatment (30 mg.kg-1.day-1, from birth to 10 wk old) markedly reduced the development of PH and improved lung alveolarization and vascularization in Denver FHR. These results suggest that Rho kinase-mediated sustained vasoconstriction, through increased Ca2+ sensitivity, plays an important role in the established PH and that RhoA/Rho kinase signaling contributes significantly to the development of PH and lung dysplasia in mild hypoxia-exposed FHR.     

Inhalation of Stachybotrys chartarum Evokes Pulmonary Arterial Remodeling in Mice, Attenuated by Rho-Kinase Inhibitor

Stachybotrys chartarum, a ubiquitous fungus in our environment, has been suspected of causing respiratory symptoms in humans, such as acute infant pulmonary hemorrhage and asthma. We previously established a mouse model in which repeated inhalation of Stachybotrys chartarum spores caused pulmonary hypertension. To further investigate the model, particularly in the pulmonary circulation, mice were intra-tracheally injected with spores, 18 times over 12 weeks. Severe muscularization was observed in the small- to medium-sized pulmonary arteries. Bronchoalveolar lavage fluid revealed an increase in eosinophils accompanied by high concentrations of Th2-associated cytokines, IL-4, IL-5, but not Th1-associated IFN-γ. The remodeling was temporary, resolving after cessation of spore inhalation. Chronic inhibition of the RhoA/Rho-kinase pathway by fasudil attenuated pulmonary arterial remodeling. These data suggest that Stachybotrys-mediated remodeling is caused by Th2-associated inflammation and can be resolved by Rho-kinase inhibition, either through direct effects on smooth muscle hypertrophy or through indirect effects on vascular inflammation. These data also show that extensive pulmonary vascular remodeling, often thought of as a fixed lesion, will spontaneously resolve in the absence of underlying molecular etiology.     

Thromboxane-induced actin polymerization in hypoxic pulmonary artery is independent of Rho

Actin polymerization (APM), regulated by Rho GTPases, promotes myocyte force generation. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes. We compared basal and agonist-induced APM in myocytes from PA and descending aorta (Ao), under hypoxic and normoxic conditions. We also examined effects of thromboxane challenge on force generation and cytoskeletal assembly in resistance PA and renal arteries from neonatal swine with persistent pulmonary hypertension (PPHN) induced by 72-h normobaric hypoxia, compared with age-matched controls. Synthetic and contractile phenotype myocytes from neonatal porcine PA or Ao were grown in hypoxia (10% O(2)) or normoxia (21% O(2)) for 7 days, then challenged with 10(-6) M thromboxane mimetic U46619. F/G actin ratio was quantified by laser-scanning cytometry and by cytoskeletal fractionation. Thromboxane receptor (TP) G protein coupling was measured by immunoprecipitation and probing for Gαq, G12, or G13, RhoA activation by Rhotekin-RBD affinity precipitation, and LIM kinase (LIMK) and cofilin phosphorylation by Western blot. Isometric force to serial concentrations of U46619 was measured in muscular pulmonary and renal arteries from PPHN and control swine; APM was quantified in fixed contracted vessels. Contractile PA myocytes exhibit marked Rho-dependent APM in hypoxia, with increased active RhoA and LIMK phosphorylation. Their additional APM response to U46619 challenge is independent of RhoA, reflecting decreased TP association with G12/13 in favor of Gαq. In contrast, hypoxic contractile Ao myocytes polymerize actin modestly and depolymerize to U46619. Both basal APM and the APM response to U46619 are increased in PPHN PA. APM corresponds with increased force generation to U46619 challenge in PPHN PA but not renal arteries.     

Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms

Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr(696) and Thr(850) resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly.     

Vascular smooth muscle contraction evoked by cell volume modulation: role of the cytoskeleton network.

Previously, we reported that hyposmotic swelling evoked transient vascular smooth muscle cell (SMC) contraction that was completely abolished by L-type Ca(2+) channel blockers. In contrast, sustained contraction revealed in hyper- and isoosmotically-shrunken SMCs was insensitive to L-type channel blockers and was diminished in Ca(2+)-free medium by only 30-50%. Several research groups reported cell volume-dependent cytoskeleton network rearrangements. This study examines the role of cytoskeleton proteins in cell volume-dependent contraction of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat thoracic aorta. Hyperosmotic shrinkage and hyposmotic swelling were triggered by modulation of medium osmolality; isosmotic shrinkage was induced by VSMR transfer from hypo- to isosmotic medium. The relative content of globular (G) and fibrillar (F) actin was estimated by fluorescence microscopy. Hyperosmotic shrinkage and hyposmotic swelling led to elevation of the F-actin/G-actin ratio by 2.5- and 1.8-fold respectively. Contraction of shrunken and swollen VSMR was insensitive to modulators of microtubules such as vinblastine, colchicine and docetaxel. Microfilament disassembly by cytochalasin B resulted in dramatic attenuation of the maximal amplitude of contraction of hyperosmotically-shrunken and hyposmotically-swollen VSMR, and almost completely abolished the contraction triggered by isosmotic shrinkage. These data suggest that both L-type Ca(2+) channel-mediated contraction of swollen vascular SMC and Ca(2+)(o)-insensitive contractions of shrunken cells are triggered by reorganization of the microfilament network caused by elevation of the F-actin/G-actin ratio.     

Rho-kinase inhibition alleviates pulmonary hypertension in transgenic mice expressing a dominant-negative type II bone morphogenetic protein receptor gene

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by a sustained elevation in the pulmonary artery pressure and subsequent right heart failure. The activation of Rho/Rho-kinase activity and the beneficial effect of Rho-kinase inhibition have been demonstrated in several experimental models of pulmonary hypertension. However, it remains unclear whether Rho-kinase inhibitors can also be used against pulmonary hypertension associated with mutations in the type II bone morphogenetic protein receptor (BMPRII) gene. Transgenic mice expressing a dominant-negative BMPRII gene (with an arginine to termination mutation at amino acid 899) in smooth muscle by a tetracycline-gene switch system (SM22-tet-BMPR2(R899X) mice) were examined. They developed an elevated right ventricular systolic pressure (RVSP), right ventricular (RV) hypertrophy, muscularization of small pulmonary arteries, and an associated disturbed blood flow in their lungs. The Rho/Rho-kinase activity and Smad activity were determined by a Western blot analysis by detecting GTP-RhoA and the phosphorylation of myosin phosphatase target subunit 1, Smad1, and Smad2. In the lungs of SM22-tet-BMPR2(R899X) mice, the Rho/Rho-kinase activity was elevated significantly, whereas the Smad activity was almost unchanged. Fasudil, a Rho-kinase inhibitor, significantly decreased RVSP, alleviated RV hypertrophy and muscularization of small pulmonary arteries, and improved blood flow in SM22-tet-BMPR2(R899X) mice, although it did not alter Smad signaling. Our study demonstrates that Rho/Rho-kinase signaling is activated via a Smad-independent pathway in an animal model of pulmonary hypertension with a BMPRII mutation in the cytoplasmic tail domain. Rho-kinase inhibition is therefore a possible therapeutic approach for the treatment of PAH associated with genetic mutation.     

Cyclic AMP-Rap1A signaling activates RhoA to induce α2c-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells

Intracellular signaling by the second messenger cyclic AMP (cAMP) activates the Ras-related small GTPase Rap1 through the guanine exchange factor Epac. This activation leads to effector protein interactions, activation, and biological responses in the vasculature, including vasorelaxation. In vascular smooth muscle cells derived from human dermal arterioles (microVSM), Rap1 selectively regulates expression of G protein-coupled α(2C)-adrenoceptors (α(2C)-ARs) through JNK-c-jun nuclear signaling. The α(2C)-ARs are generally retained in the trans-Golgi compartment and mobilize to the cell surface and elicit vasoconstriction in response to cellular stress. The present study used human microVSM to examine the role of Rap1 in receptor localization. Complementary approaches included murine microVSM derived from tail arteries of C57BL6 mice that express functional α(2C)-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 μM) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2'-O-Me-cAMP (100 μM) activated RhoA, increased α(2C)-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α(2C)-ARs mobilized from the perinuclear region to intracellular filamentous structures and to the plasma membrane. Similar results were obtained in murine wild-type microVSM, coupling Rap1-Rho-actin dynamics to receptor relocalization. This signaling was impaired in Rap1A-null murine microVSM and was rescued by delivery of constitutively active (CA) mutant of Rap1A. When tested in heterologous HEK293 cells, Rap1A-CA or Rho-kinase (ROCK-CA) caused translocation of functional α(2C)-ARs to the cell surface (～4- to 6-fold increase, respectively). Together, these studies support vascular bed-specific physiological role of Rap1 and suggest a role in vasoconstriction in microVSM.     

19,20-EpDPE, a bioactive CYP450 metabolite of DHA monoacyglyceride, decreases Ca²⁺ sensitivity in human pulmonary arteries

The aim of this study was to investigate the effect of docosahexaenoic acid monoacylglyceride (MAG-DHA) on human pulmonary arterial tone. Tension measurements on pulmonary arterial tissues demonstrated that MAG-DHA reduced U-46619-induced tone, which is highly sensitive to the H-1152 inhibitor. Results also showed that MAG-DHA treatments decreased RhoA activity levels, which in turn inactivated the Rho-kinase pathway, leading to a reduction in U-46619-induced Ca(2+) sensitivity of permeabilized pulmonary artery smooth muscle cells. According to the mechanical responses assessing U-46619-induced Ca(2+) sensitivity in the absence or presence of 3 μM MAG-DHA, MAG-DHA plus 1 μM N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH, a cytochrome P-450 epoxygenase inhibitor) and 300 nM 19,20-epoxydocosapentaenoic acid (a cytochrome P-450 epoxygenase-dependent DHA metabolite), our data suggest that the MAG-DHA is metabolized in a bioactive epoxymetabolite. This epoxyeicosanoid in turn decreases active tone and Ca(2+) sensitivity of smooth muscles cells through an inhibition of the Rho-kinase pathway. Together, these data provide primary evidence regarding the mode of action of MAG-DHA in human pulmonary arteries and suggest that this compound may be of pharmacological interest in patients with pulmonary hypertension to generate intracellular bioactive metabolites.     

Chronic hypoxia augments protein kinase G-mediated Ca2+ desensitization in pulmonary vascular smooth muscle through inhibition of RhoA/Rho kinase signaling

Pulmonary vascular smooth muscle (VSM) sensitivity to nitric oxide (NO) is enhanced in pulmonary arteries from rats exposed to chronic hypoxia (CH) compared with controls. Furthermore, in contrast to control arteries, relaxation to NO following CH is not reliant on a decrease in VSM intracellular free calcium ([Ca(2+)](i)). We hypothesized that enhanced NO-dependent pulmonary vasodilation following CH is a function of VSM myofilament Ca(2+) desensitization via inhibition of the RhoA/Rho kinase (ROK) pathway. To test this hypothesis, we compared the ability of the NO donor, spermine NONOate, to reverse VSM tone generated by UTP, the ROK agonist sphingosylphosphorylcholine, or the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate in Ca(2+)-permeabilized, endothelium-denuded pulmonary arteries (150- to 300-microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca(2+)](i). We further examined effects of NO on levels of GTP-bound RhoA and ROK membrane translocation as indexes of enzyme activity in arteries from each group. We found that spermine NONOate reversed Y-27632-sensitive Ca(2+) sensitization and inhibited both RhoA and ROK activity in vessels from CH rats but not control animals. In contrast, spermine NONOate was without effect on PKC-mediated vasoconstriction in either group. We conclude that CH mediates a shift in NO signaling to promote pulmonary VSM Ca(2+) desensitization through inhibition of RhoA/ROK.     

Animal models of pulmonary hypertension: Rho kinase inhibition

Pulmonary Hypertension is a terminology encompassing a range of etiologically different pulmonary vascular diseases. The most common is that termed pulmonary arterial hypertension or PAH; a rare but often fatal disease characterized by a mean pulmonary arterial pressure of >25?mmHg. PAH is associated with a complex etiology highlighted by core characteristics of increased pulmonary vascular resistance and elevation of mean pulmonary artery pressure. When sustained, pulmonary vascular remodeling occurs and eventually patients pass away due to right heart failure. Hypoxic pulmonary vasoconstriction is an early event occurring in pulmonary hypertension due to chronic exposure to hypoxia. While the underlying mechanisms of hypoxic pulmonary vasoconstriction may be controversial, a role for RhoA/Rho kinase mediated regulation of intracellular Ca(2+) has been recently identified. Further study suggests that RhoA may have an integral role in other pathophysiological processes such as cell proliferation and migration occurring in all forms of PH. Indeed Rho proteins are known to play essential roles in actin cytoskeleton organization in all eukaryotic cells and thus Rho and Rho-GTPases are implicated in fundamental cellular processes such as cellular proliferation, migration, adhesion, apoptosis and gene expression. This review focuses on providing an overview of the role of RhoA/Rho kinase in currently available animal models of pulmonary hypertension.