Published online before print August 28, 2003, doi: 10.1161/01.RES.0000093220.90027.D9
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© 2003 American Heart Association, Inc.
Integrative Physiology |
Sildenafil Prevents Change in RhoA Expression Induced by Chronic Hypoxia in Rat Pulmonary Artery
From Inserm U-533, Faculté des Sciences (V.S., M.R.D., G.L., P.P.), Nantes, France, and Inserm U-541, Hôpital Lariboisière (S.L.), Paris, France.
Correspondence to Pierre Pacaud, Laboratoire de Physiologie Cellulaire et Moléculaire, Inserm U-533, Faculté des Sciences, 2 rue de la Houssinière, BP 92208 44322, Nantes Cedex 3, France. E-mail pacaud{at}svt.univ-nantes.fr
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Abstract |
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Exposure to chronic hypoxia (CH) induces a sustained pulmonary hypertension associated with structural and functional changes in the pulmonary arterial bed, including alterations of contractile properties. The small G-protein RhoA and its effector Rho kinase play a major role in the sustained rise in tension induced by vasoconstrictors. The aim of this study was to analyze the effect of CH on the RhoA/Rho kinase signaling pathway in the rat pulmonary artery. Maximal contraction of pulmonary artery rings to endothelin-1, noradrenaline, and the thromboxane A2 analog U46619 was markedly decreased in rats exposed to CH (10% O2, 2 weeks). This CH-induced decrease response to agonists was attributable to the abolition of RhoA-mediated Ca2+ sensitization of the contraction. Real-time reverse transcriptase–polymerase chain reaction and Western blot analysis revealed a decrease in RhoA mRNA (79.4±6.0%, n=4) and RhoA (81.1±8.0%, n=4) expression in the main pulmonary artery from CH rats, whereas RhoA expression was not modified in arterial smooth muscle cells and arteries exposed to hypoxia and high intraluminal pressure, respectively. Treatment of rats with sildenafil (25 mg/kg per day) throughout 2 weeks of exposure to CH prevented CH-induced downregulation of RhoA, reduction of contraction, and pulmonary artery remodeling. These findings indicate that CH-induced downregulation of RhoA expression, leading to the abolition of RhoA/Rho kinase–mediated Ca2+ sensitization of contraction, is responsible for the decreased responses to contracting agonists in the pulmonary artery of CH rats. These alterations are prevented by sildenafil, indicating a major role of the NO/cyclic GMP pathway in CH-induced altered RhoA signaling in the pulmonary artery.
Key Words: smooth muscle • contraction • G proteins • pulmonary hypertension
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Introduction |
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Pulmonary hypertension (PHT) is characterized by high pulmonary blood pressure, vascular remodeling, and right ventricular hypertrophy. Whereas primary PHT results from unknown causes and genetic predisposition, secondary PHT is a common complication of pulmonary obstructive disease, lung diseases, and heart failure. The pathogenesis of secondary pulmonary hypertension is not fully understood, but hypoxia is considered a major factor. In many mammalian species, including humans, acute hypoxia causes a selective pulmonary arteriolar vasoconstriction and increases pulmonary blood pressure. Exposure to chronic hypoxia (CH) induces a sustained PHT associated with marked structural and functional changes in the pulmonary arterial bed.1,2 The remodeling of the pulmonary artery wall includes smooth muscle cell proliferation and abnormal extracellular matrix protein deposition that decrease the lumen of the vessels and reduce the elasticity of the arterial wall.3,4 These structural changes also contribute to functional alterations, notably to modifications of contractile properties.
Besides extensive studies focused on the mechanisms involved in the pulmonary vasoconstrictor response to acute hypoxia,5,6 there is still no consensus regarding the alterations of contractile properties of pulmonary artery induced by CH and the mechanisms involved. Although impairment of the endothelium-dependent regulation of pulmonary vascular tone is consistently reported, the analysis of the role of nitric oxide (NO) and cyclic GMP (cGMP) signaling pathway in CH-induced PHT has yielded conflicting data, with both increase and decrease of endothelial NO synthase having been described.7–11 Regarding the reactivity of pulmonary artery smooth muscle to vasoconstrictors, inconsistent data have been reported depending on the agonists used, the duration of exposure to CH, the portion of the pulmonary vascular bed examined, and the animal species used. It has been described recently that the maximal contraction to endothelin (ET)-1 and angiotensin II was decreased in the main pulmonary artery from CH rats, whereas 5-hydroxytryptamine–mediated contraction was enhanced both in the first branch and small muscular pulmonary arteries.12,13 Such agonists that bind to G-protein–coupled receptors produced contraction by increasing both the cytosolic Ca2+ concentration and the Ca2+ sensitivity of the contractile apparatus.14 The contribution of altered Ca2+ signaling to CH-induced change in pulmonary arterial reactivity has been investigated,12,15 but the involvement of CH-induced alteration in Ca2+ sensitization has not been analyzed.
Ca2+ sensitization mediated by the small G-protein RhoA and its target Rho kinase constitutes the major component of the sustained rise in tension induced by vasoconstrictors and contributes to arterial blood pressure regulation.16–18 Data are now accumulating regarding the involvement of Rho proteins and Rho kinase in arterial disorders associated with arterial wall remodeling, altered cell contractility, and cell migration, such as hypertension, atherosclerosis, and restenosis.16,19–22 Although a better knowledge of the effect of CH on the signaling mechanisms involved in the regulation of the contractile properties would help in understanding the pathophysiology of the pulmonary circulation, there is no data regarding the RhoA/Rho kinase pathway in this context.
The present study was thus designed to analyze the effect of CH on agonist-induced contraction and on RhoA/Rho kinase signaling in the rat pulmonary artery. We show that CH induced a downregulation of RhoA expression and RhoA/Rho kinase–mediated Ca2+ sensitization of the contraction, responsible for a decreased response to contracting agonists. These alterations are completely prevented by the oral administration of sildenafil to rats exposed to CH, indicating a major role of the NO/cGMP pathway in the CH-induced alteration of RhoA signaling in the pulmonary artery.
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Materials and Methods |
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Animals
The normoxic rats were housed in room air at a normal atmospheric pressure (760 mm Hg). The hypoxic rats were housed in a hypobaric chamber (Vacucell 111 L, Medcenter) for 15 days. At completion of the exposure, tissues were prepared as indicated for contraction measurements, Western blot analysis, RNA extraction, or morphological analysis.
NOx- Assay
Left ventricular plasma NOx- concentration was determined by spectrophotometric analysis, as described previously.23
Tension Measurements in Intact Fibers
The aorta and extralobar pulmonary artery rings were suspended under isometric conditions and connected to a force transducer (Pioden Controls Ltd). Cumulative concentration-response curves were constructed in response to KCl, endothelin (ET)-1, noradrenaline (NA), and the thromboxane A2 receptor agonist U46619. Amplitude of the contraction was expressed as a percentage of the maximal KCl-induced contraction. The amplitude of relaxation was expressed as percentage of the maximal amplitude of contraction induced by phenylephrine recorded before carbachol (CCH) or sodium nitroprusside (SNP) application.
Isometric Tension Measurement in Skinned Fibers
Small muscle strips isolated from the media of extralobar pulmonary arteries were connected to a force transducer (AE 801, SensoNor) and permeabilized with ß-escin (50 to 70 µmol/L) for 35 minutes at 25°C, as previously described.24 Tension developed by permeabilized muscle strips was measured in activating solutions, containing 10 mmol/L EGTA and a specified amount of CaCl2 to give a desired concentration of free Ca2+.24
Western Blot Analysis
Expression of RhoA and Rho kinase was analyzed using mouse monoclonal anti-RhoA antibody or with goat polyclonal anti-Rho kinase I antibody, respectively. Immunoreactive bands were visualized using horseradish peroxidase–conjugated secondary antibodies and subsequent ECL detection (Amersham Pharmacia).
Real-Time RT-PCR
Total RNA was extracted using TRIzol Reagent (Life Technologies), and reverse transcription was performed according to standard techniques. Quantitative real-time polymerase chain reaction (PCR) assays were carried out with sequence-specific primers pairs on the iCycler iQ system (BioRad) using intercalation of Sybr Green as fluorescent probe. The Sybr Green Kit (Perkin Elmer Applied Biosystems) was used for real-time monitoring of amplification. Results were evaluated by iCycler iQ Real Time Detection System software (BioRad). The expression of GAPDH mRNA was used to normalize the expression of RhoA mRNA.
Pressurized Arteries
Removed rat carotid artery segments were maintained in the organ culture system for 1 or 3 days, pressurized at normal (80 mm Hg) or hypertensive (150 mm Hg) levels. The device used for application of intraluminal pressure to vessel segments in organ culture has been described previously.25
Morphological Analysis
Pulmonary arteries and aortas from control and hypoxic rats were collected and fixed for 1 hour with paraformaldehyde (4% wt/vol). Transverse sections (6 µm thick) were stained with Sirius red (Sigma) for collagen fibers and eosin-hematoxylin (Sigma) for nuclei and then analyzed using Metamorph software (Universal Imaging Co).
Chemicals and Drugs
Mouse monoclonal RhoA antibody (26C4) and rabbit polyclonal Rho kinase antibody (C9) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Sildenafil was purchased from Pfizer (Sandwich, UK), and the Rho kinase inhibitor Y-27632, synthesized by Lesieur (Lille University), was a gift from Institut International de Recherche Servier (Courbevoie, France). All other reagents were purchased from Sigma (Saint Quentin Fallavier).
Statistics
All results are expressed as mean±SEM, and n is the sample size. In experiments with comparison of two conditions, a nonpaired Student’s t test was used. Differences among multiple groups were tested with ANOVA (one-way ANOVA, Fisher’s test). Data were considered statistically significant when P<0.05. Concentration-response curves were fitted to a logistic equation using Origin software (Dipsi).
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
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Results |
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Chronic Hypoxia-Induced Histological Remodeling of the Pulmonary Artery
Hematocrit was increased in rats maintained in the hypobaric chamber for 15 days, attesting the hypoxic condition, and the rise in the RV/LV+S ratio in CH rats was consistent with the development of PHT (Table 1). Extralobar pulmonary arteries of rats maintained in CH for 15 days underwent major morphological alterations attested by a significant increase in the media thickness and area in the absence of change in the thoracic aorta (Table 2). These alterations are associated with histological remodeling, including an increase in the elastin and collagen content without modification of the elastin to collagen ratio (Table 2). Remodeling also affected small pulmonary arteries, which showed medial thickening similar to that classically described in response to CH.26,27
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Chronic Hypoxia Alters the Contractile Properties of the Pulmonary Artery
Concentration-dependent contractions were recorded in response to KCl, ET-1, NA, and U46619 in aorta and pulmonary artery rings from normoxic and CH rats. In aorta rings, the parameters of contractile responses were identical in normoxic and CH rats for each agonist tested (not shown). In pulmonary artery rings, contractions induced by KCl in CH rats were similar to those obtained in controls (maximal amplitude, 780±39 and 794±45 mg, respectively; n=10, P>0.1; Figure 1A). A slight decrease in the response to ET-1 was observed in pulmonary artery rings from CH rats, attested by a reduction of the maximal amplitude of the contraction (97.3±3.6% versus 114.6±2.7% in control, n=4, P<0.001), associated with a slight increase in the EC50 (11.8±0.9 versus 4.3±0.8 nmol/L in control, n=4, P<0.005; Figure 1B). Concentration-response curve to NA obtained in pulmonary artery rings from CH rats was significantly desensitized compared with control curve; maximal contraction response was decreased from 68.7±4.6% to 44.2±3.2% (n=8, P<0.01), and the EC50 was increased from 6.1±3.5 to 78.9±8.1 nmol/L (n=8, P<0.005) (Figure 1C). The maximal contractile response to the thromboxane A2 analog U46619 was also strongly reduced in pulmonary arteries from CH rats (40.8±5.9% versus 132.0±3.1% in control, n=4, P<0.001) without significant change in the EC50 (99.2±10.6 versus 117.0±18.7 nmol/L in control, n=4, P>0.05; Figure 1D). These results indicate that alteration of the contractile properties of the pulmonary artery induced by CH depended on the agonist used. Contractions induced by KCl that depend on rise in intracellular Ca2+ concentration were not affected by CH. Contractions induced by ET-1 and NA that involve both increase in intracellular Ca2+ and Ca2+ sensitization of contractile proteins14 were reduced by 15% and 37% by CH, respectively. Contractile response induced by U46619 that nearly exclusively involves an increase in Ca2+ sensitivity of the contractile apparatus14 was inhibited by 70% by CH. It could thus be hypothesized that CH alters the contractile properties of pulmonary arteries through the inhibition of agonist-mediated Ca2+-sensitizing mechanisms.
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), CH rats (
), and CH rats treated by sildenafil (25 mg/kg per day) (
). Contraction is expressed as a percentage of the maximal KCl-induced contraction. Data shown are the mean of 4 to 10 independent experiments. pCa-tension relationships (E and F) measured under control condition (
) 10 µmol/L Y-27632 in ß-escin–permeabilized pulmonary artery smooth muscle from control (E) and CH (F) rats. Data shown are the mean of 6 to 8 independent experiments.
Chronic Hypoxia Abolishes Rho Kinase–Mediated Ca2+ Sensitization
Ca2+-dependent contractions and Ca2+ sensitization of contractile proteins could be independently evoked in ß-escin–permeabilized smooth muscle strips. Ca2+-dependent contractions were induced by gradual increase in Ca2+ concentration (submaximal pCa 8 to maximal pCa 4.5), and Ca2+ sensitization was evoked by addition of GTP
S. The Ca2+ sensitization appears as a leftward shift of the pCa-tension relationship. In ß-escin–permeabilized pulmonary artery strips from normoxic rats, GTPS induced an increase in the Ca2+ sensitivity of contractile proteins, illustrated by an increase in the pCa50 from 6.10±0.01 (n=8) to 6.68±0.06 (P<0.001, n=8) (Figure 1E). This shift in the pCa-tension relationship was completely abolished in the presence of the Rho kinase inhibitor Y-27632 (10 µmol/L), indicating that the GTPS-induced Ca2+ sensitization in pulmonary artery was exclusively mediated by the RhoA/Rho kinase pathway (Figure 1E). The pCa-tension relationship in permeabilized pulmonary artery strips from CH rats was similar to that of controls (pCa50, 6.09±0.01; P>0.1; n=6) (Figure 1F). However, the GTPS-induced Ca2+ sensitization was completely lost in pulmonary arteries from CH rats (pCa50, 6.11±0.02; n=6; P>0.1 compared with CH in the absence of GTPS), and Y-27632 had no effect (Figure 1F). These results are consistent with an inhibitory effect of CH on Ca2+ sensitization in pulmonary artery smooth muscle and suggest that CH mediates inhibition of the RhoA/Rho kinase signaling pathway.
Chronic Hypoxia Induces a Loss of RhoA in the Pulmonary Artery
The expression of RhoA in pulmonary artery from rats exposed to CH for 4 to 15 days was analyzed by immunoblotting. CH induced a time-dependent loss of RhoA expression, which was already significant after 4 days and almost complete after 15 days (Figure 2A). Reduction of RhoA expression at 15 days was also detected in CH rat lung protein extracts (68±7% of control, n=3), suggesting that the downregulation of RhoA was not restricted to extralobar arteries but also affected small pulmonary arteries. Examination of the abundance of RhoA mRNA by quantitative RT-PCR in the pulmonary artery revealed that the CH-induced decrease in RhoA expression was associated with reduction in RhoA mRNA level (Figure 6A). In contrast to that observed in pulmonary artery, RhoA expression in aorta remained unchanged during exposure to CH (Figure 2B). Expression of Rho kinase, which is the RhoA target responsible for RhoA-mediated Ca2+ sensitization of smooth muscle contraction,14 was not modified by CH, either in the pulmonary artery or in the aorta (Figure 2C). These results suggest that CH-induced inhibition of RhoA-dependent Ca2+ sensitization of pulmonary artery smooth muscle was attributable to CH-induced loss of RhoA expression. However, direct exposure of pulmonary artery smooth muscle cells to hypoxia (2% O2) did not decrease RhoA mRNA level (102.7±1.8% and 101.6±1.1% of control [n=4] after 8 and 15 hours of hypoxia, respectively). This result suggests that the decrease in RhoA expression in the pulmonary artery from rats exposed to CH was not directly attributable to hypoxia but involved a secondary mechanism that was absent in cultured cells.
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Elevated Intraluminal Pressure Does Not Affect RhoA Expression
Exposure to CH induces a sustained PHT. We therefore assessed whether the decrease in RhoA expression in the pulmonary artery of rats exposed to CH in vivo could be a consequence of the rise in blood pressure. RhoA expression was analyzed in arterial segments pressurized at normal or hypertensive levels for 1 to 3 days. As shown in Figure 3, RhoA expression was not affected by the increase in intraluminal pressure and remained similar to that detected in arteries freshly removed from animals, used as a reference for RhoA protein content in vivo. Thus, the decrease in RhoA expression observed in the pulmonary artery of rats exposed to CH could not be attributed to the rise in pulmonary artery pressure.
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Sildenafil Prevents Chronic Hypoxia-Induced Loss of RhoA and RhoA-Mediated Ca2+ Sensitization
CH results in an impaired production/biodisponibility of NO in pulmonary arteries.28 Altered NO signaling was confirmed by the reduction of the NOx- concentration detected in the plasma from the left ventricle of rat exposed to CH for 15 days compared with normoxic rats (Table 1). This change was associated with a decrease in the maximal endothelium-dependent cholinergic relaxation induced by CCH in the pulmonary artery of CH rats (Figure 4A) without alteration of the EC50 (16.9±1.3 µmol/L in CH versus 19.4±2.1 µmol/L in controls; n=5, P>0.05) (Figure 4A). Endothelium-independent relaxation to SNP was not significantly different between the two groups of rats (Figure 4C). None of these changes were found in thoracic aorta, indicating that CH, or indirectly the resulting pulmonary hypertension, specifically altered the NO/cGMP pathway in pulmonary artery (Figures 4B and 4D).
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We next assessed whether the effect of CH on RhoA expression and RhoA-dependent Ca2+ sensitization in pulmonary arteries was related to alteration of the NO/cGMP signaling pathway. For this purpose, we used sildenafil, an orally active, potent, and selective inhibitor of the type 5 phosphodiesterase. Rats treated orally with sildenafil (25 mg/kg per day) throughout 15 days of exposure to CH exhibited a significant reduction in right ventricular hypertrophy (RV/LV+S ratio, Table 1) and an inhibition of pulmonary vascular remodeling (Figure 5A; Table 2) despite a reduction in NOx- concentration similar to that of nontreated CH rats (Table 1). The maximal endothelium-dependent cholinergic relaxation in pulmonary artery of sildenafil-treated CH rats remained reduced similarly to that of nontreated CH rats (27.3±3.1%; n=4, P>0.05). Sildenafil had no effect in the aorta and pulmonary arteries of normoxic rats and in the aorta of CH rats (Table 2). Our results are thus in agreement with previous observations showing that sildenafil reduced pulmonary arterial pressure in patients with PHT and pulmonary vascular remodeling in mice exposed to CH.29,30
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Concentration-response curves to contracting agonists show that sildenafil prevented the decrease in reactivity induced by CH in pulmonary artery (Figure 1). Indeed, the maximal amplitude of the responses to U46619 obtained in sildenafil-treated CH rats (153.8±3.5%, n=4) was significantly higher (P<0.001) than that recorded in controls (Figure 1D).
The pCa-tension relationship in permeabilized pulmonary artery strips from CH rats treated with sildenafil was similar to that of control CH rats (pCa50, 6.09±0.02; n=6, P>0.1). However, similarly to that observed in pulmonary arteries from normoxic rats, GTPS shifted the pCa-tension relationship toward higher pCa in pulmonary arteries from sildenafil-treated CH rats, indicating that the GTPS-induced Ca2+ sensitization was completely restored (pCa50, 6.64±0.02; P>0.1 compared with normoxic rats in the presence of GTPS; n=6). The GTPS-induced Ca2+ sensitization in sildenafil-treated CH rats was inhibited in the presence of Y-27632, suggesting that it was mediated by RhoA/Rho kinase activation (Figure 5B). Consistently, quantitative RT-PCR analysis and Western blotting demonstrated that the restoration of RhoA-mediated Ca2+ sensitization in sildenafil-treated rats was associated with a complete prevention of the loss of both RhoA mRNA and protein expression in the pulmonary artery from CH rats (Figure 6).
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Discussion |
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The results of the present study show that CH induces downregulation of RhoA expression and RhoA/Rho kinase–mediated Ca2+ sensitization in the pulmonary artery, which in turn is responsible for a decreased response to contracting agonists. These changes of RhoA expression and RhoA-dependent functions are completely prevented by the oral administration of sildenafil, indicating a major role of the NO/cGMP pathway in CH-induced alteration of RhoA signaling in the pulmonary artery.
Although surprising, the evidence for a decrease in the responses of pulmonary arteries to contracting agonists has been previously shown to occur in animal models of PHT. Decreased reactivity to angiotensin I and II has been described in main and intralobar arteries from rats exposed to hypoxia for 1 week.31 Similarly, exposure to hypoxia for 2 weeks leads to reduction in the main pulmonary artery contractile response to ET-1 and angiotensin II.12 In addition, in a rat model of PHT induced by left ventricular infarction, a marked decrease in contractile response to NA occurred in the pulmonary artery, associated with a decreased production of NO.32 The mechanisms leading to this reduced reactivity had not been investigated. However, because this effect was not restricted to a specific type of agonist or membrane receptor, it has been suggested that altered regulation of the Ca2+ sensitivity of the contractile apparatus might be involved.12 Ca2+ sensitization mediated by RhoA and its target Rho kinase is recognized as the major determinant of the sustained rise in tension induced by vasoconstrictors in arterial smooth muscle.16–18 In this study, we demonstrate that RhoA/Rho kinase–mediated Ca2+ sensitization in the pulmonary artery is abolished by CH. We observed that the reduced contractility induced by CH varied quantitatively between agonists used, in accordance with the relative contribution of Rho kinase–mediated Ca2+ sensitization to agonist-mediated contractions.14 This correlation suggests that inhibition of the RhoA/Rho kinase–mediated Ca2+ sensitization is the key process whereby agonist-mediated contraction is reduced by CH in the pulmonary artery. However, we cannot rule out the involvement of other accessory mechanisms, including changes in Ca2+ signaling and ion channel expression or modification of receptor density.
Data are now accumulating regarding the involvement of altered Rho protein expression or activity in arterial disorders such as hypertension, atherosclerosis, and restenosis.16,19–22 Although Rho protein expression or activity has not been directly investigated, in vitro pharmacological studies using the Rho kinase inhibitor Y-27632 have suggested that in response to acute hypoxia, pulmonary vasoconstriction recorded in isolated rat lung or isolated small pulmonary arteries and myosin light chain phosphorylation measured in cultured pulmonary artery smooth muscle cells involved Rho kinase.33,34 Our results show that the amount of RhoA is markedly decreased in pulmonary arteries and lungs of CH rats, inferring that the downregulation of Rho kinase–mediated Ca2+ sensitization induced by CH is attributable to the loss of RhoA expression. Therefore, contrary to that observed in acute response to hypoxia, our data do not support a role for Rho kinase–dependent vasoconstriction in the sustained PHT induced by CH. This interpretation is in agreement with the absence of any effect of Y-27632 (30 mg/kg per day), administered throughout the entire duration of exposure to CH, on pulmonary artery remodeling or reactivity and on right ventricular hypertrophy (not shown). In contrast, sildenafil treatment, which strongly reduced right ventricular hypertrophy and pulmonary artery remodeling in CH rats, prevented the CH-induced decrease of RhoA mRNA and protein expression and preserved RhoA/Rho kinase–mediated Ca2+ sensitization. CH-induced PHT is reduced by sildenafil treatment,35 raising the possibility that the observed changes in RhoA expression in CH- and sildenafil-treated CH rats could be secondary to modifications of vascular intraluminal pressure. However, our present results showing that increased intraluminal pressure alone has no effect on RhoA expression suggest that the downregulation of RhoA in pulmonary artery of CH rats was not attributable to the development of PHT. This observation is in agreement with a recent study reporting that arterial expression of RhoA in different hypertensive rat models remained at normotensive levels.36
Taken together, the present data demonstrate a major role of the NO/cGMP pathway in the modulation of RhoA expression by CH. This agrees with our recent work demonstrating that cGMP-dependent kinase positively regulates RhoA expression through mechanisms involving both cGMP-dependent kinase–mediated regulation of RhoA protein stability and cGMP-dependent kinase–mediated rhoA transcription.37 Also, in N-
-nitro-L-arginine–treated rats, chronic inhibition of NO synthesis induced a strong decrease in RhoA mRNA and protein expression in aorta and pulmonary artery, associated with inhibition of RhoA-mediated Ca2+ sensitization.37 Modulation of RhoA expression thus seems to be a regulatory mechanism that controls the capability of cells to respond to external stimuli. Indeed, the reduction of RhoA/Rho kinase–dependent Ca2+ sensitization associated with the downregulation of RhoA expression indicates that the level of RhoA expression is a limiting factor of RhoA-dependent functions. In addition to contraction, RhoA regulates several processes in vascular smooth muscle cells, including migration, proliferation, gene transcription, and differentiation.38 Consequently, a change in RhoA expression in pulmonary artery smooth muscle may lead to phenotype modulation, abnormal gene expression, and defective response to external stimuli and so participate in the pulmonary arterial wall remodeling induced by CH. The NO-dependent regulation of RhoA expression therefore might thus represent a crucial component of the determinant action of NO on the structure and function of the vessel wall in normal and pathological conditions, where NO biodisponibility is altered.28
In summary, the data presented herein indicate that CH induces the loss of RhoA expression and RhoA/Rho kinase–mediated Ca2+ sensitization of contraction in the pulmonary artery. These changes are completely prevented by sildenafil, indicating a major role for the NO/cGMP pathway in the CH-induced alteration of RhoA signaling. Therefore, paradoxically, stimulation of the cGMP pathway restores normal contractile properties of pulmonary artery smooth muscle. Taken together, our results also suggest that the sustained PHT induced by CH is more likely to be attributable to pulmonary artery remodeling than to increased vascular reactivity and that the beneficial effect of sildenafil is probably attributable more to its effect on the vessel wall structure than to a direct vasodilator action. Additional studies are now required to understand the consequences of CH-induced loss of RhoA expression in pulmonary artery smooth muscle and their roles in the development of PHT associated with CH.
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Acknowledgments |
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This work was supported by grants from INSERM, Institut International de Recherche Servier, and Action Molécules et Cibles Thérapeutiques (INSERM-CNRS). M.R.-D. was supported by a grant from Société Française d’Athérosclérose.
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Footnotes |
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Original received January 30, 2003; resubmission received July 7, 2003; revised resubmission received August 19, 2003; accepted August 19, 2003.
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References |
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