Soluble Guanylate Cyclase-α1 Deficiency Selectively Inhibits the Pulmonary Vasodilator Response to Nitric Oxide and Increases the Pulmonary Vascular Remodeling Response to Chronic Hypoxia
Background— Nitric oxide (NO) activates soluble guanylate cyclase (sGC), a heterodimer composed of α- and β-subunits, to produce cGMP. NO reduces pulmonary vascular remodeling, but the role of sGC in vascular responses to acute and chronic hypoxia remains incompletely elucidated. We therefore studied pulmonary vascular responses to acute and chronic hypoxia in wild-type (WT) mice and mice with a nonfunctional α1-subunit (sGCα1−/−).
Methods and Results— sGCα1−/− mice had significantly reduced lung sGC activity and vasodilator-stimulated phosphoprotein phosphorylation. Right ventricular systolic pressure did not differ between genotypes at baseline and increased similarly in WT (22±2 to 34±2 mm Hg) and sGCα1−/− (23±2 to 34±1 mm Hg) mice in response to acute hypoxia. Inhaled NO (40 ppm) blunted the increase in right ventricular systolic pressure in WT mice (22±2 to 24±2 mm Hg, P<0.01 versus hypoxia without NO) but not in sGCα1−/− mice (22±1 to 33±1 mm Hg) and was accompanied by a significant rise in lung cGMP content only in WT mice. In contrast, the NO-donor sodium nitroprusside (1.5 mg/kg) decreased systemic blood pressure similarly in awake WT and sGCα1−/− mice as measured by telemetry (−37±2 versus −42±4 mm Hg). After 3 weeks of hypoxia, the increases in right ventricular systolic pressure, right ventricular hypertrophy, and muscularization of intra-acinar pulmonary vessels were 43%, 135%, and 46% greater, respectively, in sGCα1−/− than in WT mice (P<0.01). Increased remodeling in sGCα1−/− mice was associated with an increased frequency of 5′-bromo-deoxyuridine–positive vessels after 1 and 3 weeks (P<0.01 versus WT).
Conclusions— Deficiency of sGCα1 does not alter hypoxic pulmonary vasoconstriction. sGCα1 is essential for NO-mediated pulmonary vasodilation and limits chronic hypoxia-induced pulmonary vascular remodeling.
Received November 22, 2006; accepted June 26, 2007.
Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance and vascular remodeling.1,2 Inhalation therapy with nitric oxide (NO) can selectively vasodilate pulmonary resistance vessels, reduce right ventricular (RV) afterload, and improve oxygenation in experimental and clinical PAH.3 NO acts, at least in part, via soluble guanylate cyclase (sGC), a heterodimer composed of α- and β-subunits.4 Two sGC isoforms, α1β1 and α2β1, appear able to synthesize cGMP from GTP. The α1β1 heterodimer is thought to be the predominant heterodimer in the vasculature5,6 responsible for smooth muscle relaxation. In contrast, the α2β1 heterodimer is abundant in synaptic membranes7 and may play a role in synaptic transmission.8 Recently, Mergia et al9 reported that the targeted deletion of exon 4 of the sGCα1 subunit results in a >90% reduction in lung NO-stimulated sGC activity.
In normal lungs, NO is produced predominantly by the NO synthase 3 (NOS3) isoform, and studies in mice with targeted deletion of NOS3 (NOS3−/−) have highlighted the importance of NO in regulating pulmonary vascular tone and pulmonary vascular resistance. NOS3−/− mice have pulmonary hypertension at baseline due to increased pulmonary vascular resistance.10,11 Whether or not these mice also have an augmented pulmonary vasoconstrictor response to hypoxia12–14 or increased pulmonary vascular remodeling in response to chronic hypoxia remains controversial.14–16
Among the biological effects of cGMP is its ability to activate the cGMP-dependent protein kinase I (PKGI).17 The activity of PKG can be monitored in blood vessels by assessing the phosphorylation state of vasodilator-stimulated phosphoprotein at serine 239 (P-VASP).18,19 NO can, however, also exert cGMP-independent effects by direct nitrosation of proteins, including ion channels.20 In experimental animal models, strategies aimed at directly activating sGC by the administration of an sGC activator21 or at limiting cGMP breakdown by inhibiting a cGMP-specific phosphodiesterase (PDE)14 can successfully reverse acute pulmonary vasoconstriction. Sildenafil, a PDE5 inhibitor, has also been shown to decrease mean pulmonary arterial pressure in healthy human volunteers breathing low oxygen concentrations14 and in patients with PAH.22,23
To better understand the role of sGC in the pulmonary response to acute and chronic hypoxia, we evaluated NO-mediated vasodilation and pulmonary vascular remodeling in wild-type (WT) mice and in mice lacking a functional sGCα1 subunit (sGCα1−/−). In the present study, we report that NO-mediated pulmonary vasodilation was abrogated in sGCα1−/− mice, whereas the systemic vasodilator response to sodium nitroprusside (SNP) and spermine NONOate (SPER-NO) was preserved. sGCα1 deficiency led to greater smooth muscle cell proliferation and pulmonary vascular remodeling in response to prolonged hypoxia. Our findings suggest that sGCα1 is required for the vasodilator effects of NO in pulmonary vessels and that it protects against pulmonary vascular remodeling.
sGCα1−/− mice with a targeted deletion of exon 6 of the α1-subunit gene were generated as described previously.24 Male chimeric animals generated from correctly targeted R1 embryonic stem cell clones were mated to female 129 Sv mice. The F1 progeny was genotyped to identify germline heterozygous offspring, which were intercrossed to obtain WT and sGCα1−/− animals. All experiments were performed in adult mice and were approved by the University of Leuven Institutional Committee on Animal Care and Welfare.
Baseline Invasive Hemodynamic Measurements
WT and sGCα1−/− mice were anesthetized by intraperitoneal injection with urethane (1.4 mg/g body weight; Sigma-Aldrich Chemie, Steinheim, Germany) and allowed to breath spontaneously. A 1.4F Millar catheter (Millar Instruments, Houston, Tex) was inserted via the right jugular vein to measure RV systolic pressure (RVSP) and heart rate. Signals were recorded and analyzed with a PowerLab and Chart 4.1.1 software for Macintosh (ADInstruments, Chalgrove, United Kingdom).
Hemodynamic Measurements During Acute Hypoxia
Adult male mice were anesthetized with urethane, intubated, and mechanically ventilated (250 μL tidal volume and 150 strokes/min) with room air. After baseline pressure recordings, the ventilator was switched to 7% oxygen or a mixture of 7% oxygen with 40 ppm NO. RVSP as an indirect measure of systolic pulmonary artery pressure was recorded at 1, 2, 3, 4, 5, and 10 minutes, as described previously (n=5 to 7 per group).25 In a second set of animals, SPER-NO (2 mg/kg IV) was injected 3 minutes before the start of breathing 7% oxygen (n=3 to 5 per group).
Lung cGMP Concentrations and sGC Activity
Lungs were perfused with ice-cold PBS, immediately frozen in liquid nitrogen, and kept at −80°C until processing for cGMP concentrations with a nonradioactive enzyme immunoassay (Amersham Biosciences, Little Chalfont, United Kingdom) as described previously.26 cGMP concentrations were expressed as pmol/mg protein (n=4 to 5 per group). sGC activity was determined as described previously (n=5 to 9 per group).27
A telemetry device (model TA11PA-C20, Data Sciences International, St. Paul, Minn) was implanted subcutaneously, and the transducer was introduced in the left carotid artery. Mice were allowed to recuperate for at least 7 days before baseline recordings. To evaluate the response to the NO-donor compounds SNP and SPER-NO, male WT and sGCα1−/− mice (n=6 to 12 per group) were injected 2 times with SNP (1.5 mg/kg IP) and SPER-NO (20 mg/kg IP) with an interval of at least 8 hours between injections.
Response to Chronic Hypoxia
Male and female WT and sGCα1−/− mice were exposed for 3 weeks to 10% normobaric hypoxia in a specially designed chamber (3 weeks of Fio2 0.10). Mice were allowed to breathe room air for 30 minutes to 1 hour before administration of anesthesia and measurement of RVSP. Hematocrit was determined with hematocrit capillary tubes (Hirschmann Laborgeräte, Eberstadt, Germany). The RV free wall was separated from the left ventricle (LV+S), and both were dried and weighed to determine the RV/LV+S ratio. The same parameters were also studied in normoxic mice. Additional animals were euthanized at baseline and after 3 weeks of hypoxia and perfused with ice-cold PBS, and lungs were harvested and snap-frozen for mRNA extraction and immunoblot analysis.
To prepare lungs for morphometric analysis, WT and sGCα1−/− mice were anesthetized with urethane, a butterfly needle was inserted into the RV, and the lungs were perfused for 5 minutes with PBS at 62.5 cm H2O perfusion pressure. The trachea was then cannulated, and 4% formaldehyde was infused into the trachea at 25 cm H2O perfusion pressure. The left lung was embedded in paraffin, sectioned (5 μm thick), and reacted with Hart’s elastin stain to evaluate the muscularization of small resistance vessels (diameter of 15 to 80 μm). The diameter of each vessel was recorded, and the vessel was classified as nonmuscular, partly muscular (double elastic lamina <75%), or muscular (double elastic lamina >75%) by an observer blinded to the experimental group. At least 50 vessels per animal were analyzed.
WT and sGCα1−/− mice were exposed for 1 week or 3 weeks to 10% normobaric hypoxia (n=5 to 6 per group) and injected with 5′-bromo-deoxyuridine (BrdU, 50 mg/kg IP) 48, 24, and 2 hours before euthanasia. Sections were prepared from paraffin-embedded lungs, and BrdU-positive cells were detected with a monoclonal rat anti-BrdU antibody (Oxford Biotechnology, Kidlington, United Kingdom). Please see the online Data Supplement for details. The number of small resistance vessels (diameter of 15 to 80 μm) with at least 1 BrdU-positive cell was determined and expressed as a percentage of the total number of resistance vessels.
mRNA Expression Levels
Lung sGCα1 (exon 5), sGCα2, sGCβ1, and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA levels were measured in both genotypes at baseline and after 3 weeks of 10% normobaric hypoxia (5 males and 5 females per group) by quantitative reverse-transcription polymerase chain reaction (ABI Prism 7700 Sequence Detection system, Applied Biosystems, Foster City, Calif). Transcript levels of sGCα1, sGCα2, and sGCβ1 were normalized to HPRT levels. For details, see the online Data Supplement.
Protein extracts were prepared, and 50 μg of protein was fractionated on a 10% SDS-PAGE gel. Lung α1- and β1-subunit levels were evaluated by immunoblot analysis with polyclonal rabbit anti-rat sGCα1 antiserum and polyclonal rabbit anti-rat sGCβ1 antisera produced as reported previously.26,28 The anti-rat sGCα2 antibody (α2-82) was a kind gift of Dr S. Behrends (TU Carolo-Wilhemina, Braunschweig, Germany).29 Protein extracts from brain tissue were fractionated and served as positive control for sGCα2. VASP and phosphorylated VASP (Ser239) were detected with commercially available polyclonal antibodies specific for P-VASP at serine 239 (Cell Signaling Technology, #3114; Danvers, Mass) and total VASP (Cell Signaling Technology, #3112).30 Actin immunoblot analysis was performed to confirm equal amounts of protein were loaded in each lane (MAB 1501, Chemicon, Temecula, Calif).
All values are expressed as mean±SEM. A Student t test was used to compare differences between 2 groups, and ANOVA was used to compare multiple groups. A repeated-measures ANOVA was performed to evaluate changes in RVSP after acute hypoxia. Post hoc analysis was performed with a Bonferroni correction. A 2-way ANOVA was used to test for interaction between genotype and the response to hypoxia and between the administration of inhaled NO and the rise in RSVP in response to 7% hypoxia (after 1 and 2 minutes). Results were considered significant if P<0.05.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
sGCα1β1 Is the Predominant sGC Heterodimer in the Lung
To investigate the effect of targeted deletion of sGCα1 on cGMP signaling in the lung, sGC activity was measured. sGC activity was barely detectable at baseline without stimulation. sGC activity after stimulation with DETA-NONOate and BAY 41-2272 in sGCα1−/− mice was less than 1.5% and 0.6%, respectively, of stimulated sGC activity in WT mice (Figure 1A). Similar results were seen in male and female mice, and results for male mice are presented in Figure 1A. Also at baseline, markedly reduced cGMP levels existed in lungs of sGCα1−/− mice (0.14±0.02 versus 0.56±0.13 pmol/mg protein in WT), and again, no differences were observed between male and female mice. These data show that sGC containing α1 is the predominant sGC heterodimer in the lung.
Levels of the mRNA encoding the mutant sGCα1 in lungs of sGCα1−/− mice were similar to the levels of full-length sGCα1 mRNA in lungs of WT mice, whereas no full-length sGCα1 protein was detected in lungs of sGCα1−/− mice (Figure 1B and 1C). sGCβ1 protein levels were lower in lungs of sGCα1−/− mice than in lungs of WT mice, whereas no difference existed in mRNA expression between genotypes. The targeted deletion of sGCα1 did not result in an upregulation of sGCα2 mRNA and protein expression in lungs of sGCα1−/− mice compared with lungs of WT mice (Figure 1C). When copy numbers were normalized to HPRT, sGCα1 and sGCβ1 mRNA levels were similar, whereas sGCα2 mRNA levels were ≈7-fold lower in both genotypes (Figure 1B).
To examine the effect of sGCα1 deficiency on PKG activity in vivo, we measured the fraction of VASP, which was phosphorylated at serine 239 in lung extracts. Expression levels of total VASP did not differ between genotypes, but phosphorylation of VASP at serine 239 in sGCα1−/− mice (Figure 1D) was only 37±12% of that in WT mice (*P<0.01).
Effect on RV and Systemic Blood Pressure
To evaluate whether or not targeted deletion of sGCα1 results in PAH, mice were anesthetized, and RVSP was measured. RVSP and heart rate were similar in spontaneously breathing male (Figure 2A) and female (data not shown) mice of both genotypes. Using telemetry, we confirmed that systemic blood pressure was greater in awake male sGCα1−/− mice than in WT male mice (Figure 2B), as observed previously by Mergia et al9 and Buys et al24 using tail-cuff measurements.
Response to Acute Hypoxia
To investigate the role of sGC in acute hypoxic pulmonary vasoconstriction, male WT and sGCα1−/− mice were exposed to 7% hypoxia, and RVSP was measured. The RVSP increased similarly in WT and sGCα1−/− mice within 1 minute of hypoxia (Figure 3A). The exposure to 7% hypoxia did not affect lung cGMP content in WT or sGCα1−/− mice (Figure 3B).
Role of sGC in NO-Mediated Vasodilation
To evaluate the pulmonary vasodilator response to inhaled NO, mice were ventilated with a gas mixture containing 7% oxygen and 40 ppm NO. Administration of NO severely blunted the rise in RSVP in WT mice (24±2 mm Hg after 2 minutes, P<0.01 versus without NO; Figure 3A). In sGCα1−/− mice, in contrast, the rise in RVSP and maximum RSVP did not differ with or without NO (33±1 versus 34±1 mm Hg after 2 minutes, respectively). Exposure for 10 minutes to 7% oxygen with 40 ppm NO resulted in a significant rise in lung cGMP content in WT mice but not in sGCα1−/− mice, which indicates that sGCα1 is required for the rise in cGMP induced by breathing NO (Figure 3B).
To confirm the important role of sGCα1 in NO-mediated pulmonary vasodilation, we also measured the pulmonary vascular response to the systemic NO donor SPER-NO, which was injected intravenously 3 minutes before the start of acute hypoxia. Immediately after injection of SPER-NO, RVSP decreased 3 to 4 mm Hg in both WT and sGCα1−/− mice (P<0.05 for both), likely as a result of reduced preload. The subsequent acute hypoxia-induced rise in RVSP was inhibited in WT mice (18±1 to 20±1 mm Hg). In contrast, the ability of SPER-NO to inhibit the hypoxia-induced RVSP rise was abrogated in sGCα1−/− mice (19±2 to 25±1 mm Hg after 2 minutes, P<0.05 versus WT; Data Supplement Figure I).
In the systemic circulation, however, the reduction in systolic blood pressure after injection of SNP (1.5 mg/kg IP) was similar in WT and sGCα1−/− mice (−37±2 versus −42±4 mm Hg, P=NS). Similar observations were made with SPER-NO (data not shown), which suggests that sGCα1 is required for the pulmonary but not the systemic vasodilator response to NO.
sGC Expression and sGC Activity After 3 Weeks of Chronic Hypoxia
NO has been shown to limit the pulmonary vascular remodeling response to chronic hypoxia, but the extent to which its intracellular receptor, sGC, is involved has not been fully defined. We investigated the effect of 3 weeks of 10% normobaric hypoxia on pulmonary sGC subunit mRNA and protein levels, as well as sGC activity. We observed that prolonged hypoxia increased sGCα1 and sGCβ1 mRNA and protein levels in WT mice (Figure 1B; supplemental Figure II), as has been reported previously by Li et al,31 whereas no increase occurred in sGC activity (supplemental Figure III). In sGCα1−/− mice, we also observed an increase in sGCβ1 mRNA and protein levels with prolonged hypoxia. However, sGCβ1 protein levels after 3 weeks of hypoxia were lower in sGCα1−/− mice than in WT mice (supplemental Figure II), whereas transcript levels were not different between genotypes (Figure 1B). A persistent decrease was observed in sGC activity in sGCα1−/− mice under chronic hypoxic conditions (supplemental Figure III). Finally, no upregulation of sGCα2 mRNA or protein levels occurred in response to chronic hypoxia in either genotype (Figure 1B; supplemental Figure III). No gender-specific differences were observed in either genotype.
Increased RVSP and RV Hypertrophy After 3 Weeks of Hypoxia
To investigate the effect of the targeted deletion of sGCα1 on the response to chronic hypoxia, we measured RVSP, RV/LV+S, RV/body weight, and hematocrit in both genotypes. The increase in RVSP and RV hypertrophy in response to 3 weeks of hypoxia was significantly greater in sGCα1−/− mice than in WT mice (P<0.01; Table; Figure 4A and 4B). The hypoxia-induced decrease in body weight was similar in WT and sGCα1−/− mice (5±1 versus 7±2 g). Results were similar in male and female mice.
sGC and Hypoxia-Induced Vascular Remodeling
To evaluate whether or not genotype-dependent changes in NO responsiveness were associated with altered pulmonary vascular remodeling, we assessed the degree of muscularization of intra-acinar vessels from animals exposed to normoxia and chronic hypoxia (Figure 5A and 5B). In normoxic animals of both genotypes, <10% of intra-acinar vessels were muscularized (8±2% in WT versus 9±1% in sGCα1−/− mice, n=4 each). Chronic hypoxia induced remodeling in 23±1% of intra-acinar vessels in WT mice and in 33±2% of intra-acinar vessels in sGCα1−/− mice (P<0.01 for the difference in muscularized vessels between genotypes; Figure 5C). No difference existed in the percentage of partially muscularized vessels after chronic hypoxia in WT and sGCα1−/− (Figure 5C).
We observed a greater proportion of muscularized vessels with a diameter of 25 to 50 μm in sGCα1−/− mice than in WT mice (46±4% versus 27±3%, P<0.01). In contrast, in vessels with a diameter of 15 to 25 μm, fewer than 5% of the vessels were muscularized (3±1% in WT versus 1±1% in sGCα1−/− mice, P=NS). Most vessels with a diameter of >50 μm were fully muscularized (83±5% in WT versus 91±4% in sGCα1−/− mice, P=NS). Pulmonary vascular remodeling was similar in male and female mice.
Increased BrdU Incorporation After Chronic Hypoxia
The enhanced hypoxia-induced pulmonary vascular remodeling in sGCα1−/− mice was associated with increased smooth muscle cell proliferation in pulmonary resistance vessels. We observed a greater percentage of BrdU-positive pulmonary resistance vessels in sGCα1−/− mice after 1 week of hypoxia (80±3% versus 62±2% in WT vessels, P<0.01; Figure 5D through 5F). The difference in DNA turnover between genotypes persisted after 3 weeks of hypoxia (78±2% BrdU-positive vessels in sGCα1−/− versus 58±4% in WT mice, P<0.01; Figure 5D). The number of BrdU-positive cells per vessel was also significantly higher in sGCα1−/− mice after 1 week (2.2±0.2 versus 1.7±0.1 in WT mice, P<0.05) and 3 weeks of hypoxia (2.5±0.2 versus 1.9±0.1 in WT mice, P<0.05).
In the present study, we report that sGCα1 is essential for the pulmonary vasodilator response to inhaled NO during acute hypoxia and limits pulmonary vascular remodeling and RV hypertrophy associated with chronic hypoxia-induced pulmonary hypertension. The deletion of sGCα1, which markedly inhibited lung sGC activity and VASP phosphorylation, did not affect baseline RVSP or the acute vasoconstrictor response to hypoxia, which indicates that unlike NOS3, sGCα1 does not modulate baseline pulmonary vascular tone. In the systemic circulation, however, the deletion of sGCα1 was associated with hypertension but did not affect the vasodilator response to NO-donor compounds. The present results suggest that sGCα1 has a critical role in NO-mediated signal transduction in the pulmonary circulation.
To investigate the role of sGC in hypoxic pulmonary vasoconstriction, we subjected sGCα1−/− and WT mice to an acute hypoxic challenge as described previously.25 Acute hypoxia did not alter lung cGMP concentrations, and we did not observe any difference in the pulmonary vasoconstrictor response to acute hypoxia between WT and sGCα1−/− mice. We did, however, observe a difference in the pulmonary vasodilator response to inhaled NO between genotypes when the pulmonary circulation was constricted during hypoxia. Although simultaneous administration of NO almost abolished the acute hypoxia–induced increase in RSVP in WT mice, it failed to do so in sGCα1−/− mice. At the same time, NO inhalation increased lung cGMP concentrations in WT mice but not in sGCα1−/− mice. The important role of sGCα1 in NO-mediated pulmonary vasodilation was further supported by similar observations in which intravenous administration of SPER-NO blunted the pulmonary vasoconstrictor response to hypoxia in WT but not in sGCα1−/− mice. Interestingly and in distinct contrast to the pulmonary circulation, the vasodilator response to SNP and SPER-NO in the systemic circulation did not differ in WT and sGCα1−/− mice. These results indicate that sGCα1 is required for the pulmonary but not the systemic vasodilator response to NO.
The underlying mechanisms that account for the different sensitivity to NO in pulmonary versus systemic resistance vessels of sGCα1−/− mice are unknown. In a recent study, Mergia et al9 showed that NO-mediated vasorelaxation was preserved in isolated aortic rings of sGCα1−/− mice, although NO-stimulated sGC activity was reduced by 94%. These authors concluded that the majority of NO-sensitive sGC activity is not required for cGMP synthesis and that sGCα2β1 was responsible for the residual NO-mediated relaxation in isolated aortic rings. The present mRNA and protein expression data do not suggest a compensatory increase in sGCα2 gene function in lungs of sGCα1−/− mice. Another possible explanation is that alternative cGMP-independent signaling pathways, including endothelium-derived hyperpolarizing factor or voltage-gated potassium channels, might mediate the effects of NO on systemic blood pressure.20,32 The present report is the first to show a selective role for sGCα1 in NO-mediated pulmonary vasodilation and supports the use of direct sGC stimulators33 in the treatment of PAH.
Pulmonary vascular remodeling and RV hypertrophy are characteristic pathophysiological changes that determine the course of a variety of cardiopulmonary diseases associated with PAH.34 Some of the pathological changes in patients with PAH (neomuscularization of pulmonary arteries, increased RVSP, and RV hypertrophy) are mimicked in rats31 and mice14,15,35 exposed to chronic hypoxia. To evaluate the role of sGC in pulmonary vascular remodeling, we investigated the effects of sGCα1 deficiency on the response to 3 weeks of chronic hypoxia. After prolonged hypoxia, we observed that RVSP, RV/LV+S ratio, and the fraction of muscularized pulmonary arterioles increased to a greater extent in sGCα1−/− than in WT mice. To further study the underlying molecular mechanisms that mediate hypoxia-induced pulmonary vascular remodeling, we investigated the proliferative smooth muscle cell response in precapillary resistance vessels of WT and sGCα1−/− mice exposed for 1 week to 10% oxygen. After 1 week of hypoxia, the percentage of BrdU-positive vessels, an index of smooth muscle cell turnover, and the number of BrdU-positive cells per vessel were higher in sGCα1−/− than in WT mice. This difference in DNA turnover between genotypes persisted after 3 weeks of hypoxia. These observations suggest that sGC serves to limit hypoxia-induced pulmonary vascular remodeling, at least in part, by inhibiting smooth muscle cell proliferation.
The hypothesis that NO/cGMP signal transduction protects against the pathophysiological changes associated with hypoxia-induced PAH is further supported by the observation that long-term administration of the PDE5 inhibitor sildenafil reduces RV hypertrophy and vascular remodeling in response to chronic hypoxia in WT mice but not in NOS3−/− mice.14 Recently, Dumitrascu et al36 reported that administration of direct sGC activators could reverse the hemodynamic and structural changes associated with hypoxia-induced pulmonary hypertension in mice. These preclinical findings suggest that long-term administration of the PDE5 inhibitors and direct sGC stimulators are attractive new therapeutic strategies for patients with pulmonary hypertension.
Several limitations of the present study need to be considered with respect to the experimental model and the analysis of pulmonary cGMP signal transduction. First, the effect of sGCα1 deficiency on pulmonary vascular remodeling has only been studied in chronic hypoxia, and we have used RVSP as an indirect evaluation of changes in pulmonary vascular tone. Second, cGMP levels in whole lung extracts do not exclusively reflect changes in pulmonary vessels. Finally, to what extent residual sGCα2 gene function or altered PDE gene function in mice in the present study modulates pulmonary vasomotor and remodeling responses remains to be determined.
In summary, sGCα1 is essential for NO-mediated pulmonary but not systemic vasodilation and limits chronic hypoxia-induced pulmonary vascular remodeling by inhibiting smooth muscle cell proliferation. sGCα1 does not appear to regulate baseline pulmonary vascular tone or the acute response to hypoxia. The present results support a potential future role for sGC sensitizers or sGC stimulators in pulmonary hypertension and pulmonary vascular remodeling.
Sources of Funding
This work was supported by a grant of the Fund for Scientific Research–Flanders and the Flemish Institute for Biotechnology (VIB; to Dr Janssens) and National Heart, Lung, and Blood Institute grant HL70896 (to Dr Bloch). Dr Vermeersch is a research assistant of the Fund for Scientific Research–Flanders. Dr Buys was supported by an award from the Northeast Affiliate Research Committee of the American Heart Association. Dr Janssens is a Clinical Investigator of the Fund for Scientific Research-Flanders and holder of a chair supported by AstraZeneca.
↵*Dr Vermeersch and Dr Buys contributed equally to this article.
The online-only Data Supplement, consisting of Methods and figures, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.677245/DC1.
Ichinose F, Roberts JD Jr, Zapol WM. Inhaled nitric oxide: a selective pulmonary vasodilator: current uses and therapeutic potential. Circulation. 2004; 109: 3106–3111.
Hofmann F. The biology of cyclic GMP-dependent protein kinases. J Biol Chem. 2005; 280: 1–4.
Russwurm M, Koesling D. Guanylyl cyclase: NO hits its target. Biochem Soc Symp. 2004; 51–63.
Russwurm M, Wittau N, Koesling D. Guanylyl cyclase/PSD-95 interaction: targeting of the nitric oxide-sensitive alpha2beta1 guanylyl cyclase to synaptic membranes. J Biol Chem. 2001; 276: 44647–44652.
Steudel W, Ichinose F, Huang PL, Hurford WE, Jones RC, Bevan JA, Fishman MC, Zapol WM. Pulmonary vasoconstriction and hypertension in mice with targeted disruption of the endothelial nitric oxide synthase (NOS 3) gene. Circ Res. 1997; 81: 34–41.
Fagan KA, Oka M, Bauer NR, Gebb SA, Ivy DD, Morris KG, McMurtry IF. Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase. Am J Physiol Lung Cell Mol Physiol. 2004; 287: L656–L664.
Liu R, Evgenov OV, Ichinose F. NOS3 deficiency augments hypoxic pulmonary vasoconstriction and enhances systemic oxygenation during one-lung ventilation in mice. J Appl Physiol. 2005; 98: 748–752.
Zhao L, Mason NA, Morrell NW, Kojonazarov B, Sadykov A, Maripov A, Mirrakhimov MM, Aldashev A, Wilkins MR. Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation. 2001; 104: 424–428.
Steudel W, Scherrer-Crosbie M, Bloch KD, Weimann J, Huang PL, Jones RC, Picard MH, Zapol WM. Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3. J Clin Invest. 1998; 101: 2468–2477.
Quinlan TR, Li D, Laubach VE, Shesely EG, Zhou N, Johns RA. eNOS-deficient mice show reduced pulmonary vascular proliferation and remodeling to chronic hypoxia. Am J Physiol Lung Cell Mol Physiol. 2000; 279: L641–L650.
Lincoln TM, Dey N, Sellak H. Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol. 2001; 91: 1421–1430.
Oelze M, Mollnau H, Hoffmann N, Warnholtz A, Bodenschatz M, Smolenski A, Walter U, Skatchkov M, Meinertz T, Munzel T. Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a sensitive monitor of defective nitric oxide/cGMP signaling and endothelial dysfunction. Circ Res. 2000; 87: 999–1005.
Schulz E, Tsilimingas N, Rinze R, Reiter B, Wendt M, Oelze M, Woelken-Weckmuller S, Walter U, Reichenspurner H, Meinertz T, Munzel T. Functional and biochemical analysis of endothelial (dys)-function and NO/cGMP signaling in human blood vessels with and without nitroglycerin pretreatment. Circulation. 2002; 105: 1170–1175.
Evgenov OV, Ichinose F, Evgenov NV, Gnoth MJ, Falkowski GE, Chang Y, Bloch KD, Zapol WM. Soluble guanylate cyclase activator reverses acute pulmonary hypertension and augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs. Circulation. 2004; 110: 2253–2259.
Michelakis E, Tymchak W, Lien D, Webster L, Hashimoto K, Archer S. Oral sildenafil is an effective and specific pulmonary vasodilator in patients with pulmonary arterial hypertension: comparison with inhaled nitric oxide. Circulation. 2002; 105: 2398–2403.
Buys E, Sips P, Rogge E, Dewerchin M, Brouckaert P. Gender-specific hypertension in mice deficient in the a1 subunit of soluble guanylate cyclase. Circulation. 2004; 110 (suppl III): III-307. Abstract.
Pokreisz P, Fleming I, Kiss L, Barbosa-Sicard E, Fisslthaler B, Falck JR, Hammock BD, Kim IH, Szelid Z, Vermeersch P, Gillijns H, Pellens M, Grimminger F, van Zonneveld AJ, Collen D, Busse R, Janssens S. Cytochrome P450 epoxygenase gene function in hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling. Hypertension. 2006; 47: 762–770.
Sinnaeve P, Chiche JD, Nong Z, Varenne O, Van Pelt N, Gillijns H, Collen D, Bloch KD, Janssens S. Soluble guanylate cyclase alpha(1) and beta(1) gene transfer increases NO responsiveness and reduces neointima formation after balloon injury in rats via antiproliferative and antimigratory effects. Circ Res. 2001; 88: 103–109.
Smolenski A, Bachmann C, Reinhard K, Honig-Liedl P, Jarchau T, Hoschuetzky H, Walter U. Analysis and regulation of vasodilator-stimulated phosphoprotein serine 239 phosphorylation in vitro and in intact cells using a phosphospecific monoclonal antibody. J Biol Chem. 1998; 273: 20029–20035.
Scotland RS, Madhani M, Chauhan S, Moncada S, Andresen J, Nilsson H, Hobbs AJ, Ahluwalia A. Investigation of vascular responses in endothelial nitric oxide synthase/cyclooxygenase-1 double-knockout mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo. Circulation. 2005; 111: 796–803.
Stasch JP, Schmidt P, Alonso-Alija C, Apeler H, Dembowsky K, Haerter M, Heil M, Minuth T, Perzborn E, Pleiss U, Schramm M, Schroeder W, Schroder H, Stahl E, Steinke W, Wunder F. NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle. Br J Pharmacol. 2002; 136: 773–783.
Li D, Laubach VE, Johns RA. Upregulation of lung soluble guanylate cyclase during chronic hypoxia is prevented by deletion of eNOS. Am J Physiol Lung Cell Mol Physiol. 2001; 281: L369–L376.
Dumitrascu R, Weissmann N, Ghofrani HA, Dony E, Beuerlein K, Schmidt H, Stasch JP, Gnoth MJ, Seeger W, Grimminger F, Schermuly RT. Activation of soluble guanylate cyclase reverses experimental pulmonary hypertension and vascular remodeling. Circulation. 2006; 113: 286–295.