Persistent eNOS activation secondary to caveolin-1 deficiency induces pulmonary hypertension in mice and humans through PKG nitration

Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1^–/– mice were absent in Cav1^–/–Nos3^–/– mice. Further, treatment of Cav1^–/– mice with either MnTMPyP (a superoxide scavenger) or l-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1^–/– lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1^–/– lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration–dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.

Pulmonary hypertension (PH) is characterized by a progressive increase in pulmonary vascular resistance (PVR) and vascular remodeling, which without treatment leads to right-heart failure and death often within 2–3 years of diagnosis (1, 2). There are currently limited options available for the prevention and treatment of progressive PH (3, 4). Increased pulmonary vascular tone and severe structural remodeling of distal pulmonary arteries are the primary determinants of increased PVR. PH types with different etiologies share several common features: increasing severity of pulmonary vasoconstriction, remodeling of pulmonary microvessels, and intravascular thrombosis (1, 5, 6). These changes result in increased medial thickness, microvascular occlusion, and formation of plexiform lesions, all of which contribute to the mechanism of PH (1, 5, 6). Studies have described mutations of bone morphogenetic protein receptor type II in patients with PH (7, 8). As only 10%–15% of these individuals go on to develop the severe form of the disease (8, 9), it is likely that other genetic and environmental factors are also important (10). One such factor may be the dysfunction or injury of pulmonary vascular endothelial cells resulting in aberrant production of endothelium-derived mediators such as endothelin-1 and NO (2, 11), which may have a role in the pathogenesis of PH.

NO is a vasodilator regulating multiple physiological and pathophysiological processes, including host-defense response, neuronal communication, and vascular tone (12). High-output NO production by iNOS is considered detrimental in vascular disorders, whereas low-output eNOS-derived NO in general is regarded as protective (13). The eNOS isoform, predominantly expressed in endothelial cells, regulates basal pulmonary and peripheral vasomotor tone (14). eNOS activity is controlled by fatty acid modification, phosphorylation, and interaction with effector molecules such as caveolin-1 and heat shock protein 90 (hsp90) (15). Binding of caveolin-1 to eNOS has been shown to be a crucial negative regulator of eNOS activity (16, 17). Caveolin-1 also regulates signal transduction by concentrating multiple signaling molecules at its scaffold microdomain (18). Studies have identified an important pathogenic role of caveolin-1 in the development of PH in animal models (19–22). Cav1^–/– mice developed PH and right-ventricular hypertrophy in association with pulmonary vascular remodeling, hypercellularity, and alveolar septal thickening (19–21). Caveolin-1 deficiency was also seen in rat models of PH (22, 23), and the cell-permeable Cav1 inhibitory peptide prevented the development of monocrotaline-induced PH (24). In addition, decreased expression of caveolin-1, predominantly in endothelial cells, was observed in lungs of patients with severe idiopathic pulmonary arterial hypertension (IPAH) (23, 25). In the present study, we addressed the potentially important mechanism of pulmonary vascular remodeling and PH associated with caveolin-1 deficiency. We determined the role of eNOS-derived NO in the pathogenesis of PH using double-knockout mice deficient in Cav1 and eNOS (Nos3) (DKO mice). Our studies showed that persistent eNOS activation secondary to caveolin-1 deficiency induced the nitration of PKG and the resultant impairment of its kinase activity. We demonstrated that this post-translational modification in a mouse model and in lungs of patients with IPAH is a critical determinant of the pathogenesis of PH.

Genetic deletion of Nos3 in Cav1^–/– mice prevents PH. Nos3^–/– mice were bred into the background of Cav1^–/– mice to generate mice with deletions of both Cav1 and Nos3. DKO mice were indistinguishable from WT littermates. We observed that 85% (n = 200) of the DKO mice survived at least 18 months, the same as the life expectancy of WT mice. To eliminate any background effects from either the Nos3^–/– or Cav1^–/– line on the observed phenotype, F₄ or higher generations were used in the present studies. As shown in Figure 1, Cav1^–/– and WT lungs expressed a similar amount of eNOS and its chaperone protein hsp90. However, eNOS association with hsp90 was drastically increased in Cav1^–/– compared with WT lungs (Figure 1C). Basal eNOS-derived NO production as measured by nitrate and nitrite (NO_x) production was 3.5-fold greater in Cav1^–/– than WT lungs (Figure 1D), consistent with the activation of eNOS secondary to loss of caveolin-1 inhibition in vivo. There was little eNOS-derived NO_x produced in either Nos3^–/– lungs or DKO lungs (Figure 1D). Similar expression of iNOS and nNOS was detected in lungs from WT, Cav1 ^–/–, and DKO mice, and the amount of iNOS-derived NO_x in Cav1^–/– lungs was also similar to that in WT lungs (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI33338DS1). Total NO_x production in Cav1^–/– lungs was 1.8-fold greater than in WT (Figure 1E).

Figure 1

Chronic eNOS activation in Cav1^–/– lungs. (A) Western blot analysis of caveolin-1 and eNOS expression in lungs from 2-month-old mice. Lung lysate (20 μg per lane) was loaded and immunoblotted with antibodies against mouse eNOS, caveolin-1, and β-actin (loading control). Neither caveolin-1 nor eNOS was detected in DKO lungs. (B) Similar protein expression of hsp90 in Cav1^–/– and WT lungs. (C) Increased eNOS-hsp90 association in Cav1^–/– lungs. Lung lysates (500 μg) were immunoprecipitated with anti-eNOS and immunoblotted with anti-hsp90. The same blot was immunoblotted with anti-eNOS. (D) Quantitative analysis of eNOS-derived NO_x in mouse lungs. eNOS-derived NO_x was determined using the Griess reagent in the presence of iNOS and nNOS inhibitors but without addition of the eNOS agonist, e.g., calcium ionophore A23187. Data are shown as mean ± SD (n = 4–5). *P < 0.001 versus WT (n = 4–6). Cav1^–/– lungs produced 3.5-fold more eNOS-derived NO_x than WT lungs. (E) Total NO_x production in mouse lungs. Total NO_x production was determined with the Griess reagent. Data are expressed as mean ± SD (n = 4–5). ^†P < 0.05, Cav1^–/– versus WT; **P < 0.01, DKO versus WT; ^#P < 0.05, Nos3^–/– versus WT.

To determine the consequences of chronic activation of eNOS in Cav1^–/– lungs, we measured right-ventricular systolic pressure (RVSP) (to reflect the pulmonary artery systolic pressure). Cav1^–/– mice had significantly increased RVSP compared with WT mice (Figure 2, A and B). However, RVSP in DKO mice was the same as in WT. The weight ratio of right/left ventricle plus septum (RV/LV+S) was normal in DKO mice, in contrast to Cav1^–/– mice, in which the ratio was increased (Figure 2C). PVR in DKO mice was reduced to the WT level (Figure 2D), indicating that PH occurring secondarily to increased PVR in Cav1^–/– mice was the result of chronic eNOS activation.

Figure 2

PH and increased PVR in Cav1^–/– mice were prevented in DKO mice. (A) Representative RVSP tracings (each represents 1 second). (B) Normalized RVSP in DKO mice. RVSP was measured in age- and sex-matched 9-month-old mice. Data are expressed as mean ± SD. *P < 0.01, Cav1^–/– versus WT or DKO (n = 6–8). (C) Restored RV/LV+S weight ratios in DKO mice. Right and left ventricles including septum (S) were dissected free of connective tissues from age- and sex-matched mice and weighed. Data are expressed as mean ± SD. *P < 0.01, Cav1^–/– versus WT (n = 5–7); **P < 0.01, DKO versus Cav1^–/– (n = 6–8). (D) Restored PVR in DKO lungs. Data are expressed as mean ± SD. *P < 0.01, Cav1^–/– versus WT; ^†P < 0.05, Cav1^–/– versus DKO; **P > 0.5, DKO versus WT (n = 4–6).

Rescue of pulmonary vascular pathology in DKO mice. Studies have demonstrated severe lung hypercellularity and thickening of alveolar septa in Cav1^–/– mice (19–21); thus, we addressed the possibility that chronically active eNOS in Cav1^–/– mice was responsible for the lung pathology in these mice. DKO lungs exhibited normal alveolar-capillary structure and vessel wall thickness (Figure 3C) in contrast to Cav1^–/– lungs (Figure 3B). Histological scoring showed normal pulmonary morphology in DKO lungs (Figure 3, E and F). To quantify the number of muscularized distal pulmonary arteries, an underlying feature of pulmonary vascular remodeling in PH, we stained lung sections with anti–α-SMA. Cav1^–/– lungs exhibited a 3-fold increase in muscularized distal arteries (<40 μm in diameter) compared with WT, whereas a similar number of muscularized large vessels was seen in Cav1^–/– and WT lungs (Figure 3, G and H). The number of muscularized distal and large vessels in DKO lungs was similar to that in the WT and Nos3^–/– lungs (Figure 3H).

Figure 3

Prevention of pulmonary vascular remodeling induced by Cav1 deletion in DKO mice. (A–D) Representative micrographs of H&E-stained lung sections from age- and sex-matched WT (A), Cav1^–/– (B), DKO (C), and Nos3^–/– (D) mice. Hypercellularity and medial thickening seen in Cav1^–/– lungs were prevented in DKO lungs. Scale bar: 60 μm. (E) Histology grade score of hypercellularity in lung sections. Cellular hyperplasia was scored from 1 to 5, with 5 as the highest. Data are expressed as mean ± SD. *P < 0.01, Cav1^–/– versus WT, DKO, or eNOS (n = 4–6). (F) Histology grade score of lung vascular hypertrophy. Medial thickness was scored from 1 to 6, with 6 being the highest. Data are expressed as mean ± SD. *P < 0.01 versus WT, DKO, or eNOS (n = 4–6). (G) Representative micrograph of immunostaining of Cav1^–/– lung sections with anti–α-SMA (green). Arrows show muscularized distal pulmonary arterial vessels. Scale bar: 50 μm. (H). Quantification of muscularized pulmonary arterial vessels in mouse lung sections. α-SMA–positive vessels were counted in 20 fields (×200) of each lung section. Data are expressed as mean ± SD. *P < 0.01, Cav1^–/– versus WT, DKO, or eNOS (n = 4–6); **P > 0.5, Cav1^–/– versus WT, DKO, or eNOS. <40 μm, vessels with diameter less than 40 μm; >40 μm, vessels with diameter greater than 40 μm.

To address the molecular basis of the hyperplasia and pulmonary vascular remodeling seen in Cav1^–/– lungs, we examined ERK signaling and expression of genes regulating cell-cycle progression. As shown in Figure 4A, ERK signaling was increased in Cav1^–/– lungs compared with either WT or DKO. Quantitative RT-PCR analysis showed that the mRNA level of p21^Cip1, a cyclin-dependent kinase inhibitor, was decreased 5-fold in Cav1^–/– lungs compared with WT, whereas p21^Cip1 expression in DKO lungs was restored (Figure 4B). Interestingly, expression of the growth factors IGF-I and VEGF-A was increased in Cav1^–/– lungs, whereas they were normal in DKO lungs (Figure 4, C and D). Thus, activation of ERK signaling and downregulation of p21^Cip1 in Cav1^–/– lungs may be the result of increased expression of these growth factors in Cav1^–/– lungs.

Figure 4

Normalized ERK signaling and gene expression in DKO lungs. (A) Western blot analysis of ERK signaling. Phosphorylation of p42/44 MAPK was detected with anti–phospho-p42/44 in lung lysates (30 μg per lane). The same blot was immunoblotted with anti-p42/44 for detection of total p42/44 and with anti–β-actin for loading control. (B–D) Quantitative analysis of gene expression in mouse lungs. Total RNA was isolated from lungs collected from 2-month-old mice, and mRNA levels of p21^Cip1 (B), IGF-I (C), and VEGF-A (D) were analyzed with quantitative SYBR Green RT-PCR assay. mRNA levels of cyclophilin were used for normalization. Data are expressed as mean ± SD (n = 3–5). *P < 0.01 versus WT or DKO. Expression of p21^Cip1 and growth factors IGF-I and VEGF-A was normalized in DKO lungs (similar to that in WT lungs).

Tyrosine nitration of PKG secondary to caveolin-1 deficiency impairs PKG activity. NO reacts with superoxide to form the damaging reactive nitrogen species peroxynitrite, which modifies proteins and may interfere with their function through tyrosine nitration (26–28). Immunostaining of nitrotyrosine, a surrogate measure of peroxynitrite, showed that Cav1^–/– lungs had greater nitrotyrosine expression than WT and DKO lungs (Figure 5A), indicating the formation of peroxynitrite in Cav1^–/– lungs. Prominent nitrotyrosine immunostaining was also evident in Cav1^–/– pulmonary vasculature, including muscularized distal arteries (Figure 5A). Western blotting also demonstrated increased tyrosine nitration of proteins in Cav1^–/– lungs (Figure 5B), whereas no difference in S-nitrosylation of proteins was seen in Cav1^–/– and WT lungs (Figure 5C).

Figure 5

Increased tyrosine nitration of proteins in Cav1^–/– mouse lungs. (A) Representative micrographs of immunostaining of lung sections with anti-nitrotyrosine (NT, green) and anti–α-SMA (SMA, red). Nuclei were counterstained with DAPI (blue). Nitrotyrosine was prominent in Cav1^–/– lungs, including large artery (arrowhead) and muscularized distal artery (arrow). Scale bar: 40 μm. (B) Increased protein nitration in Cav1^–/– mouse lungs. Forty micrograms of lung lysates was loaded per lane. Nitrated proteins were directly detected with anti-nitrotyrosine antibody. The same blot was blotted with anti-GAPDH for loading control. We observed primarily 2 groups of proteins at molecular weights of approximately 30 and 70 kDa, respectively, with markedly increased tyrosine nitration in Cav1^–/– lungs. (C) S-nitrosylation of proteins in Cav1^–/– and WT lungs was not different. S-nitrosylation (SNO) in biotin-labeled lung lysates (10 μg per lane) was directly detected by Western blotting with avidin-coupled reagents.

We determined the activities of soluble guanylyl cyclase (sGC) and PKG, the two major downstream targets of NO signaling (29), to investigate whether their functions were impaired by tyrosine nitration in Cav1^–/– lungs. Baseline sGC activity and maximal activity after stimulation with sodium nitroprusside, a NO donor, were similar in WT, Cav1^–/–, and DKO lungs (Supplemental Figure 2). In sharp contrast, basal PKG activity in Cav1^–/– lungs was 30% less than that in the WT group (Figure 6A) and also following the addition of 2.5 μM cGMP (Figure 6B). PKG activities in DKO lungs were restored to levels similar to those in WT lungs (Figure 6, A and B). PKG dysfunction in Cav1^–/– lungs could not be ascribed to reduced cGMP production, since Cav1^–/– lungs in fact exhibited higher cGMP concentrations (Supplemental Figure 2). The protein levels of PKG-1 were also similar in WT, Cav1^–/–, and DKO lungs (Figure 6C). Quantitative RT-PCR demonstrated similar mRNA expression of PKG-1 and PKG-2 in mouse lungs (Supplemental Figure 3). These data suggest that impaired PKG activity in Cav1^–/– lungs is the result of post-translation modification. Indeed, we observed markedly increased nitrotyrosine modification of PKG-1 in Cav1^–/– lungs as compared with DKO and WT lungs (Figure 6C). In contrast, S-nitrosylation of PKG-1 in Cav1^–/– lungs was minimal and similar to that in WT and DKO lungs (Supplemental Figure 4).

Figure 6

Impaired PKG kinase activity secondary to tyrosine nitration in Cav1^–/– lungs. (A and B) In vitro PKG activity in lung lysates during the basal state (A) and following addition of 2.5 μM cGMP (B). Data are expressed as mean ± SD (n = 3–5). *P < 0.05, Cav1^–/– versus WT or DKO. (C) Prominent tyrosine nitration of PKG detected in lung lysates from Cav1^–/– mice. Lung lysates (150 μg) from WT, DKO, and Cav1^–/– mice were immunoprecipitated with 2 μg anti–PKG-1 antibody overnight, and PKG tyrosine nitration was detected with anti-nitrotyrosine by Western blotting. Protein expression of PKG-1 in lung lysates was also determined directly by Western blotting with anti–PKG-1 antibody. The same blot was reprobed with an antibody against mouse GAPDH as a loading control. (D) PKG-1 tyrosine nitration in cultured human pulmonary artery smooth muscle cells following SIN-1 treatment. Subconfluent primary cultures were treated with SIN-1 at the indicated concentrations for 30 minutes, and each cell lysate (50 μg) was then used for immunoprecipitation and immunoblotting for detection of PKG-1 nitration. Each lysate (15 μg) was also used for direct immunoblotting with anti–PKG-1 and anti-GAPDH. CTL, control. (E and F) SIN-1 treatment resulted in a significant decrease in PKG activity in the basal state (E) and following addition of 2.5 μM cGMP (F). Data are shown as mean ± SD (n = 3). **P < 0.001 versus CTL.

To address the role of peroxynitrite in the mechanism of the observed impairment of PKG activity, we treated human pulmonary artery smooth muscle cells with 3-morpholinosydnonimine (SIN-1), a superoxide and NO donor that forms peroxynitrite simultaneously (30). SIN-1 concentrations as low as 10 μM were shown to cause marked PKG tyrosine nitration (Figure 6D) and decreased PKG activity (Figure 6, E and F). Furthermore, in vitro treatment of purified PKG-1 with peroxynitrite resulted in decreased PKG activity in a concentration-dependent manner, whereas DETA NONOate, a NO donor, even at 100 μM failed to suppress PKG activity (Figure 7A).

Figure 7

Identification of target tyrosine residues responsible for the impairment of PKG kinase activity upon nitration. (A) Dose-dependent impairment of PKG activity by peroxynitrite. Purified PKG-1 was incubated with peroxynitrite at the indicated concentrations for 14 minutes at room temperature or with DETA NONOate (NONOate) for 30 minutes in the dark at room temperature. Kinase activity was then assayed. Data are expressed mean ± SD (n = 3). *P < 0.05 versus control (0 μM). (B) Screening of PKG-1α mutants with in vitro kinase assay. At 48 hours after transfection, myc-tagged WT and PKG-1α mutants were immunoprecipitated with anti-myc beads and aliquoted for incubation with either peroxynitrite (100 μM) or the same amount of 0.1N NaOH only (CTL). In vitro kinase assay was then performed to determine PKG activity. PKG activity is expressed as picomoles/minute/microgram cell lysates. Western blotting was used to detect the protein levels of PKG-1α. (C) Validation of target tyrosine residues. At 48 hours after transfection, myc-tagged WT and PKG-1α mutants were immunoprecipitated for tyrosine nitration and in vitro kinase assay. Kinase activity following peroxynitrite incubation was normalized to that of the respective control. Data are expressed as mean ± SD (n = 3). ^†P < 0.05 versus either PKG-1α mutant. (D) Diminished tyrosine nitration of PKG-1 mutants. After 14 minutes incubation with peroxynitrite (250 μM) at room temperature, the anti-myc immunoprecipitates were used for Western blotting to detect tyrosine nitration. The intensity of each band of PKG-1 tyrosine nitration was normalized to the intensity of the respective PKG-1 band (PKG-NT/PKG).

To identify the target tyrosine residues responsible for impairment of PKG activity upon nitration, we mutated all tyrosine residues in the catalytic domain of human PKG-1α into phenylalanine and expressed these myc-tagged PKG-1 mutants with either single or double mutations in human lung microvascular endothelial cells. As shown in Figure 7, B and C, mutation of either Tyr345 or Tyr549 resulted in no impairment of PKG activity following peroxynitrite treatment in contrast to other mutations and WT PKG-1. Accordingly, these PKG-1 mutants exhibited diminished tyrosine nitration in contrast to WT PKG-1 following peroxynitrite treatment (Figure 7D). Both Tyr345 and Tyr549 are conserved from zebrafish to human PKG-1 (Supplemental Figure 5). These data demonstrate that peroxynitrite formation impairs PKG activity through nitration at Tyr345 and Tyr549. Decreased kinase activity of PKG-1α mutant with mutation at Tyr524 was likely due to its location next to the activation loop (Figure 7B and Supplemental Figure 5).

Nitrative stress–mediated PKG dysfunction induces PH in Cav1^–/– mice. To determine whether tyrosine nitration–mediated impairment of PKG activity was responsible for the development of PH in Cav1^–/– mice, we carried out a series of experiments that included blocking peroxynitrite formation and overexpressing PKG. We first determined whether scavenging superoxide by manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP, a superoxide dismutase mimetic) (31) could reverse PH in Cav1^–/– mice. In this experiment, 8-month-old Cav1^–/– mice received either saline or MnTMPyP (5 mg/kg, i.p. daily) for 6 weeks. We observed that MnTMPyP treatment reduced RVSP (Figure 8A). We also treated 8-month-old Cav1^–/– mice with either N-nitro-l-arginine methyl ester (l-NAME, a NOS inhibitor) or its inactive analog d-NAME in drinking water (1 mg/ml) for 5 weeks. Inhibition of NO production by l-NAME treatment also reduced RVSP and the number of muscularized distal pulmonary arteries, whereas d-NAME had no effect (Figure 8B and Supplemental Figure 6). These data suggest that high levels of NO per se in Cav1^–/– mice do not cause PH but that superoxide production is also important for its development; i.e., formation of peroxynitrite and resultant protein nitration are required for the development of PH.

Figure 8

Tyrosine nitration–induced impairment of PKG activity mediates PH in Cav1^–/– mice. (A) Reduced RVSP in Cav1^–/– mice treated with MnTMPyP. Cav1^–/– mice were treated with either MnTMPyP (5 mg/kg, i.p., daily) (Cav1-Mn) or saline (Cav1^–/–) for 6 weeks. Data are expressed as mean ± SD (n = 5 per group). *P < 0.01, Cav1^–/– versus WT; **P < 0.05, Cav1-Mn versus Cav1^–/– ; ^†P > 0.05, Cav1-Mn versus WT. (B) Inhibition of NOS with l-NAME reversed PH in Cav1^–/– mice. Cav1^–/– mice received water ad libitum (Cav1^–/–, control) or water with 1 mg/ml l-NAME (Cav-L) or its inactive analog d-NAME (Cav-D) for 5 weeks. Data are expressed as mean ± SD (n = 5–7). *P < 0.01, Cav1^–/– versus WT; ^‡P < 0.05, Cav-L versus Cav1^–/–; ^§P > 0.5, Cav-D versus Cav1^–/–. (C–F) Restoration of PKG-1 activity in Cav1^–/– lungs reversed PH. At 7 days after administration of recombinant adenovirus expressing either human PKG-1 (AdvPKG) or LacZ, lungs were collected for Western blotting (C) and PKG kinase activity assay (D) after measurements of RVSP (E) and PVR (F). PKG activity is expressed as mean ± SD (n = 3–4). ^#P > 0.5 versus WT; ^††P < 0.05 versus WT or Cav1^–/– treated with AdvPKG (D). RVSP is expressed as mean ± SD (n = 4–5). ^‡‡P < 0.05, Cav1^–/–-AdvPKG (PKG) versus Cav1^–/–-AdvLacZ (LacZ) (E). PVR is expressed as mean ± SD (n = 3–4). ^§§P < 0.05, Cav1^–/–-AdvPKG versus Cav1^–/–-AdvLacZ (F).

We further tested the hypothesis that nitrative stress–induced PH in Cav1^–/– mice is mediated by impaired PKG activity. Recombinant adenoviruses overexpressing human PKG-1 or LacZ were administered into lungs of 10-month-old WT and Cav1^–/– mice via an intratracheal microspray device. At 7 days after infection, PKG-1 protein expression was elevated by 50% in AdvPKG-treated Cav1^–/– lungs compared with AdvLacZ-treated control lungs (Figure 8C). PKG activity in AdvPKG-treated Cav1^–/– lungs was restored to a level similar to that in WT lungs (Figure 8D). PKG-mediated phosphorylation of its substrate vasodilator-stimulated phosphoprotein (VASP) at residue Ser239 was also normalized in AdvPKG-treated Cav1^–/– lungs (Supplemental Figure 7). Accordingly, we observed that restoration of PKG-1 activity in Cav1^–/– lungs resulted in a significant decrease in RVSP and PVR, whereas minimal changes were seen in WT mice (Figure 8, E and F).

Increased eNOS activity and PKG tyrosine nitration in lungs of IPAH patients. To address the relevance of these observations in mice to the pathogenesis of IPAH in patients, we determined eNOS activity, PKG tyrosine nitration, and expression of caveolin-1, eNOS, and PKG-1 in lung tissues from IPAH patients. eNOS activities were increased in IPAH lungs compared with normal lungs (Figure 9A and Supplemental Figure 8), whereas eNOS expression was similar to that in normal lungs (Figure 9B). Caveolin-1 expression was decreased in IPAH lungs compared with normal lungs (Figure 9B), consistent with previous reports (23, 25). Moreover, tyrosine nitration of PKG-1 was markedly increased in IPAH lungs (Figure 9, B and C). We also observed that PKG-1 expression was increased 30% in IPAH lungs (Figure 9B; IPAH, 1.3 ± 0.3 versus normal, 1.0 ± 0.07; P = 0.12), suggesting a compensatory PKG protein expression in the face of the decreased PKG activity.

Figure 9

PKG tyrosine nitration in lung tissue from IPAH patients. (A) Quantitative analysis of eNOS-derived NO_x in human lung tissue. Following 20 minutes incubation with selective inhibitors, lung tissues were incubated with 1 mM l-arginine for 3 hours. eNOS-derived NO_x was determined by measuring the concentration of nitrite and nitrate in the medium with the Griess reagent. Bars represent mean. *P < 0.01 versus normal. IPAH lungs produced 2-fold greater eNOS-derived NO_x than normal lungs. (B) PKG tyrosine nitration in IPAH lungs. Each lysate (150 μg) was immunoprecipitated with anti–PKG-1 and immunoblotted with anti-nitrotyrosine for detection of PKG tyrosine nitration. IPAH lungs exhibited prominent PKG-1 tyrosine nitration compared with normal lungs. Protein levels of PKG-1, eNOS, and caveolin-1 were also determined by Western blotting with anti–PKG-1, -eNOS, or –caveolin-1 antibody, respectively. Immunoblot of GAPDH was used as loading control. (C) Densitometric analysis of PKG-1 tyrosine nitration. The intensity of each band of PKG-1 tyrosine nitration (PKG-1–NT) was normalized to the intensity of the respective PKG-1 band. PKG-1 tyrosine nitration increased 2-fold in IPAH lungs compared with normal lungs. Bars represent mean. *P < 0.05 (n = 3–4). (D) Proposed model of PKG nitration–mediated PH. Persistent eNOS activation secondary to caveolin-1 deficiency (Cav1^–/– mice or IPAH patients) leads to formation of peroxynitrite in the pulmonary vasculature and impairment of PKG kinase activity through tyrosine nitration. Impaired PKG signaling induces pulmonary vascular remodeling and vasoconstriction, and thereby PH.

We show here that chronic eNOS-derived NO production secondary to the loss of caveolin-1 inhibition is a critical factor in the mechanism of PH. We have demonstrated that increased production of eNOS-derived NO resulted in the impairment of PKG kinase activity through its tyrosine nitration. The essential observations in mice were recapitulated in lung tissue from IPAH patients. The pulmonary vascular changes seen in Cav1^–/– lungs were prevented in the Cav1^–/–Nos3^–/– DKO mice, and PH was reversed following treatment of Cav1^–/– mice with either superoxide scavenger or NOS inhibitor and by adenovirus-mediated restoration of PKG activity in Cav1^–/– lungs. We also identified Tyr345 and Tyr549 in the catalytic domain of human PKG-1α as responsible for the impairment of PKG activity upon nitration. Thus, the results demonstrate what we believe to be a novel mechanism of PH mediated by the impairment of PKG activity secondary to the tyrosine nitration of the kinase.

Caveolin-1, the scaffolding protein of caveolae, serves a key regulatory signaling function in endothelial cells (32). We observed that eNOS-derived NO_x production in the Cav1^–/– lungs increased 3.5-fold compared with that in WT lungs, whereas eNOS protein expression was similar in the two groups. The activity of other NOS isoforms, iNOS and nNOS, did not differ in WT, Cav1^–/–, and DKO lungs. These data show a fundamental in vivo role of caveolin-1 as a negative regulator of eNOS activity (15–17). eNOS binds with caveolin-1 in the caveolar membrane such that upon agonist activation, hsp90 binds with the eNOS dissociated from caveolin-1 and thereby promotes eNOS activation (33, 34). We demonstrated an increase in eNOS-hsp90 interaction in Cav1^–/– lungs consistent with this model. Decreased eNOS-hsp90 interaction has been suggested to play a role in the pathogenesis of hypoxia-induced PH on the basis of decreased eNOS activity and NO bioavailability (35, 36). However, our data in Cav1^–/– lungs showed that increased eNOS-hsp90 interaction was involved in the mechanism of PH due to persistent activation of eNOS and resultant increased formation of peroxynitrite.

eNOS-derived NO is usually considered to be protective (13). That is, low-output NO produced by eNOS induces vasodilatation and inhibits platelet aggregation, suppresses adhesion of neutrophils and monocytes to endothelial cells, and modulates vascular smooth cell proliferation and migration. In the present study, however, on the basis of genetic evidence from the DKO mice, we show a clear pathogenic role for the persistent production of eNOS-derived NO secondary to caveolin-1 deficiency in the development of PH.

In previous studies, increased production of NO in eNOS transgenic mice prevented the increase in RVSP, lung vascular remodeling, and right-ventricular hypertrophy induced by chronic hypoxia (37), and cell-based eNOS gene transfer also inhibited the development of PH induced by monocrotaline treatment in rats (38). We found in Cav1^–/– mice, contrary to these observations, that increased eNOS activity was required for the development of PH. Genetic deletion of eNOS prevented the PH in Cav1^–/– mice, while pharmacological inhibition of NO production by l-NAME reversed the PH in adult Cav1^–/– mice. We observed that inhibition of superoxide production by the superoxide scavenger MnTMPyP also reversed the PH in adult Cav1^–/– mice. Thus, a high concentration of NO itself in Cav1^–/– mice did not cause PH, but NO coupled to superoxide production is critically important for the development of PH. The caveolin-1 deficiency–induced production of superoxide and eNOS-derived NO reacted to form peroxynitrite and induced PH in Cav1^–/– lungs. Studies have shown that NO alone was incapable of inducing tissue injury even at high concentrations (39), whereas NO in the presence of superoxide produced peroxynitrite, thereby causing severe nitrative stress and tissue injury (26–28), findings consistent with our observations. Taken together, our data show the fundamental role of caveolin-1 in the pathogenesis of PH such that disruption of the negative regulation of eNOS by caveolin-1 leads to pulmonary vascular remodeling and PH.

Studies have suggested that eNOS uncoupling may be sufficient to increase the production of superoxide in Cav1^–/– lungs and may thus mediate PH (40). Treatment of neonatal Cav1^–/– mice with tetrahydrobiopterin, an essential cofactor for all 3 NOS isoforms (41, 42), prevented PH (40). However, in other studies, eNOS uncoupling alone did not cause PH (37). eNOS transgenic mice failed to develop PH but were resistant to hypoxia-induced PH (37), even though these mice exhibited eNOS uncoupling (43). l-NAME inhibition of NOS in neonatal Cav1^–/– mice (44) and restoration of caveolin-1 expression in endothelial cells of Cav1^–/– mice (45) prevented PH in Cav1^–/– mice. Since none of these studies directly examined eNOS activation, they provide circumstantial evidence concerning the role of eNOS activation secondary to caveolin-1 deficiency in the pathogenesis of PH. It is well known that tetrahydrobiopterin activates all 3 isoforms of NOS (41, 42), whereas l-NAME inhibits all isoforms. Studies in a different ex vivo mouse lung model show that inhibition of NO production by l-NAME paradoxically increased PVR, with the effect being more pronounced in Cav1^–/– compared with WT lungs (46). These results may be ascribed to differences inherent in the ex vivo lung model compared with the intact animal. Our studies using Cav1^–/–Nos3^–/– DKO mice have demonstrated the essential role of eNOS-derived NO production and peroxynitrite formation in the mechanism of PH in Cav1^–/– mice.

NO mediates vasodilatation through the activation of the sGC/cGMP/PKG signaling pathway (29). We observed that PKG activity was impaired in Cav1^–/– lungs even in the face of high NO concentrations, whereas sGC activity was not affected. There was markedly increased peroxynitrite production in Cav1^–/– lungs, as evidenced by the increased nitrotyrosine immunostaining and protein tyrosine nitration. In contrast, DKO and WT lungs produced little peroxynitrite. It is known that peroxynitrite affects protein function through tyrosine nitration (26–28). We observed that PKG was in fact tyrosine-nitrated in Cav1^–/– lungs. Decreased PKG activity was probably the result of PKG tyrosine nitration, since PKG expression in Cav1^–/– lungs was the same as in WT and DKO lungs and cGMP levels in Cav1^–/– lungs did not decrease compared with WT and DKO lungs. Importantly, treatment with either superoxide scavenger or NOS inhibitor reversed the PH of Cav1^–/– mice, as did restoration of PKG activity through overexpression of PKG-1 in Cav1^–/– lungs. In further support of the pathogenic role of PKG nitration, we identified the target tyrosine residues responsible for the impairment of PKG kinase activity following nitration. PKG-1α mutant with mutations at Tyr345 or Tyr549 located at the catalytic domain was resistant to peroxynitrite-induced tyrosine nitration and resultant impairment of PKG kinase activity. Both Tyr345 and Tyr549 are conserved from zebrafish to human PKG-1. Previous studies suggest that tyrosine nitration is enhanced by the close proximity of acidic residues (47–49). Consistent with these observations, the two target tyrosine residues are also next to an acidic residue.

NO-elicited S-nitrosylation has been demonstrated as an important mechanism regulating protein function (50). eNOS-mediated S-nitrosylation in mice that received a high dose of N-acetylcysteine was found to be involved in the pathogenesis of PH (51). However, our data demonstrated that there was no difference in total S-nitrosylation of proteins in Cav1^–/– lungs compared with WT lungs, and the basal low levels of S-nitrosylation of PKG-1 in Cav1^–/– lungs were similar to those in WT and DKO lungs. In addition, in vitro incubation with peroxynitrite but not DETA NONOate, a NO donor, resulted in impairment of PKG kinase activity. PKG kinase activity was in fact increased by DETA NONOate in intact vessels (52). High levels of NO itself (Cav1^–/– mice treated with ROS scavenger) failed to promote the progression of PH but reversed PH in Cav1^–/– mice. Taken together, these data strongly support the hypothesis that nitrative stress–induced PH in Cav1^–/– mice is mediated by impaired PKG kinase activity through the tyrosine nitration.

Hypoxia has also been shown to impair PKG activity and PKG-mediated relaxation in ovine fetal intrapulmonary veins through both downregulation of PKG expression and tyrosine nitration of PKG (53), thus suggesting a role for PKG nitration in regulating hypoxic venoconstriction in utero. In contrast to our findings in Cav1^–/– lungs, the hypoxia-induced tyrosine nitration of PKG in these studies was eNOS independent (53). Other studies showed that pulmonary vascular PKG expression was increased in chronically hypoxic rats, although the vasodilatory responses to exogenous NO and cGMP analog were attenuated (54). These data suggested that hypoxia-induced PKG nitration plays a role in the pathogenesis of hypoxia-induced PH. The present study provides direct mechanistic evidence of the critical role of the hypoxia-independent PKG nitration and resultant impairment of its kinase activity in mediating PH.

The balance between the protective and adverse effects of NO is determined by the relative amounts of NO and reactive oxygen species in the pathogenesis of PH (55). Here we show that PKG nitration resulting from eNOS activation secondary to caveolin-1 deficiency induces PH. Our data support the model outlined in Figure 9D according to which caveolin-1 deficiency results in persistent eNOS activation and hypoxia-independent production of reactive oxygen species and induces formation of peroxynitrite, which post-translationally modifies PKG through tyrosine nitration and impairs its kinase activity. Impaired PKG kinase activity in turn causes vasoconstriction and pulmonary vascular remodeling and thereby PH.

Previous studies showed a reduction in CAV1 mRNA (56) and protein expression in lungs of patients with IPAH (23, 25). Decreased caveolin-1 expression in IPAH lungs was predominant in endothelial cells (25). Additionally, eNOS was robustly expressed in the plexiform lesions of IPAH lungs (57). To address the pathophysiological relevance of caveolin-1–regulated eNOS activity in the mechanism of increased PVR and vascular remodeling, we used lung tissue obtained from patients with IPAH. We observed decreased caveolin-1 expression and increased eNOS activity as well as increased PKG tyrosine nitration in IPAH lungs compared with normal lungs in the absence of marked changes in eNOS expression. These findings are in agreement with the studies in Cav1^–/– mouse lungs described above.

In summary, we have demonstrated the crucial role of chronic activation of eNOS induced by loss of caveolin-1 in the pathogenesis of PH and provide evidence that hypoxia-independent PKG nitration induces PH. The results help to explain the mechanism of PH seen in patients with caveolin-1 deficiency (24, 25). Downregulation of caveolin-1 in lungs of IPAH patients occurred predominantly in endothelial cells (25), supporting the role of persistent eNOS activation induced by caveolin-1 deficiency in the mechanism of PH. Our results suggest the potential benefit of targeting increased eNOS activity and preventing nitration of PKG as novel therapy against IPAH.

View Supplemental data

We thank X-P. Du of the University of Illinois and K.D. Bloch of Massachusetts General Hospital for their generosity in providing anti–PKG-1 antibody and recombinant AdvPKG, respectively; and R.A. Skidgel for critical comments concerning the manuscript. We also thank Jun Yuan, Liquan Cai, and Weijuan Yao for their expert technical support. This work was supported by NIH grants P01 HL060678 to A.B. Malik and R01 HL085462 to Y.-Y. Zhao.

Address correspondence to: You-Yang Zhao, Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, 835 South Wolcott Avenue, Mail Code 868, Chicago, Illinois 60612, USA. Phone: (312) 355-0238; Fax: (312) 996-1225; E-mail: yyzhao@uic.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: hsp90, heat shock protein 90; IPAH, idiopathic pulmonary arterial hypertension; l-NAME, N-nitro-l-arginine methyl ester; MnTMPyP, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride; NO_x, nitrate/nitrite; PH, pulmonary hypertension; PVR, pulmonary vascular resistance; RVSP, right-ventricular systolic pressure; sGC, soluble guanylyl cyclase; SIN-1, 3-morpholinosydnonimine; VASP, vasodilator-stimulated phosphoprotein.

Reference information: J. Clin. Invest.119:2009–2018 (2009). doi:10.1172/JCI33338

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