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Severe pulmonary hypertension (PH) is characterized by a progressive increase in pulmonary vascular resistance and vascular remodeling leading to right heart failure and early death. Our recent studies employing the novel mouse model with genetic deletions of caveolin-1 (Cav1) and eNOS (NOS3) have demonstrated that persistent eNOS activation in Cav1−/− lungs results in tyrosine nitration of protein kinase G (PKG) and impairment of its activity, which thereby induces PH. The finding of eNOS activation and PKG nitration concomitant with Cav1 deficiency was recapitulated in lungs from patients with idiopathic pulmonary arterial hypertension. These data suggest targeting PKG nitration has potential value for the treatment of PH. Here, we will review the current knowledge about Cav1-regulated eNOS activity and its fundamental role in the pathogenesis of PH.
Pulmonary hypertension (PH) is defined as a mean pulmonary arterial pressure greater than 25 mmHg at rest or 30 mm Hg with exercise(Farber and Loscalzo 2004). PH of different etiologies share several features: endothelial dysfunction, worsening vasoconstriction, remodeling of pulmonary microvessels, and intravascular thrombosis (Farber and Loscalzo 2004, Rabinovitch 1997, Rubin 1997). These changes typically result in increased medial thickness, microvascular occlusion, and formation of plexiform lesions, all of which contribute to the mechanism of increased pulmonary vascular resistance (PVR) and PH. As the molecular mechanisms responsible for pulmonary vascular remodeling and vasoconstriction remain elusive, there are limited options available for the prevention and treatment of progressive PH, including idiopathic pulmonary arterial hypertension (IPAH) (Farber 2008; Puri et al. 2007). IPAH is the most severe form of PH, which without treatment leads to right heart failure and premature death (Farber and Loscalzo 2004, Rabinovitch 1997, Rubin 1997, Runo and Loyd 2003). Studies have described mutations of bone morphogenetic protein receptor type II (BMPR-II) in patients with familial PAH and IPAH (Lane et al. 2000; Machado et al. 2006). Given that only 10-15% of these individuals go on to develop severe disease (Humbert et al. 2006, Machado et al. 2006), other genetic and environmental factors are likely to be important. One important factor may be oxidative/nitrative stress (Bowers et al. 2004, Hoshikawa et al. 2001, Nozik-Grayck and Stenmark 2007). Tissue hypoxia, ischemia, and inflammation all contribute to the production of reactive oxygen species (ROS) in the lung tissue of patients with severe PH (Bowers et al. 2004, Hoshikawa et al. 2001). The reaction between ROS and nitric oxide (NO) results in not only low bioavailability of NO but also formation of peroxynitrite and resultant nitrative stress(Beckman et al. 1990, Hurst 2002). Although increased oxidative/nitrative stress has been demonstrated in lung tissues of patients with severe PH, including IPAH (Bowers et al. 2004, Cracowski et al. 2001), the mechanistic role of oxidative/nitrative stress in the pathogenesis of PH has not been fully elucidated. Here, we discuss the critical role of eNOS activation secondary to caveolin-1 (Cav1) deficiency in the pathogenesis of PH through tyrosine nitration-mediated impairment of protein kinase G (PKG) activity.
Caveolae, 50-100 nm vesicular invaginations of the cell plasma membrane, have emerged as the site of the important events at the plasma membrane such as vesicular trafficking as well as signal transduction (Galbiati et al. 2001, Rothberg et al. 1992). Three isoforms of caveolins (Cav1, Cav2 and Cav3), the structural proteins of caveolae, have been identified. Caveolins are shown to act as scaffolding proteins to concentrate and orchestrate many signaling molecules, including eNOS, receptor and non-receptor tyrosine kinase receptors, G protein-coupled receptors, GTPase, and components of the mitogen-activated protein kinases, and regulate their function (Cohen et al. 2004, Galbiati et al. 2001, Rothberg et al. 1992). Cav1, a 22-kDa protein of 178 amino acids, is highly expressed in adipocytes, endothelial cells, fibroblasts and type I pneumocytes. Cav1 expression is sufficient and necessary to drive the formation of morphologically identifiable caveolae in these cells(Cohen et al. 2004). Through its scaffolding domain (residues 82-101), Cav1 acts as an anchor holding various proteins within caveolae and regulates their function (Cohen et al. 2004, Galbiati et al. 2001, Rothberg et al. 1992).
Endothelial cell-derived nitric oxide (NO) is a critical mediator of cardiovascular homeostasis regulating multiple physiological and pathophysiological processes including vascular tone, vascular remodeling, platelet aggregation, and angiogenesis (Ignarro et al. 1999). The enzyme activity of eNOS isoform (also called NOS3, predominantly expressed in endothelial cells) is regulated by fatty acid modification, post-translational phosphorylation, and interaction with effector molecules including Cav1 and heat shock protein 90 (Hsp90). eNOS is mostly targeted to caveolae in the plasma membrane through interaction with Cav1 in endothelial cells (Feron et al. 1996), which is dynamically regulated by Ca2+-calmodulin (Michel et al. 1997). The primary binding region of Cav1 with eNOS is within the scaffolding domain (amino acids 60-101) (Ju et al. 1997), Amount evidences have demonstrated that Cav1 binding to eNOS is a crucial negative regulator of eNOS activity. In vitro studies have shown that interaction of eNOS with GST-Cav1 fusion proteins significantly inhibits its enzyme activity (Ju et al. 1997). A membrane permeable chimeric peptide containing a cellular internalization sequence fused to the Cav1 scaffolding domain (residues 82-101) has been demonstrated as a potent inhibitor of eNOS in mice and reduces inflammation (Bucci et al. 2000). Furthermore, genetic deletion of Cav1 in mice results in a marked increase in plasma NO levels (Zhao et al. 2002) and enhanced eNOS activation in vessels from Cav1−/− mice (Drab et al. 2001). We recently also demonstrate that loss of Cav1 results in 3-fold increase of eNOS-derived NO production in lung tissues from Cav1−/− mice (Zhao et al. 2009). However, genetic deletion of eNOS completely blocks the increase of total NO production induced by Cav1 deficiency in lung tissues (Zhao et al. 2009). Consistent with previous studies, the interaction of eNOS with Hsp90, a positive regulator of eNOS activity is enhanced in lung tissues from Cav1−/− mice (Zhao et al. 2009). In addition, abnormal Cav1-eNOS interaction is associated with various pathophysiological conditions such as atherosclerosis. It has been shown that hypercholesterolemia-induced decrease in NO production is ascribed to enhanced interaction of Cav1 and eNOS (Feron et al. 1999). These data clearly demonstrate Cav1 as a crucial negative regulator of eNOS activity.
The critical role of Cav1 deficiency in the pathogenesis of PH was first identified by Zhao and colleagues (Zhao et al. 2002). Cav1-deficient mice exhibit pulmonary hypertension and right ventricle hypertrophy. Hemodynamic measurements also reveal a significant increase of right ventricular contractility and diastolic function at baseline in Cav1−/− mice compared to age- and gender-matched wild-type mice. Our recent studies further demonstrate that Cav1−/− lungs exhibit increased pulmonary vascular resistance associated with pulmonary vascular remodeling including increased medial thickness and muscularization of distal pulmonary vessels (an underlying feature of pulmonary vascular remodeling in PH) (Zhao et al. 2009). Consistent with the role of Cav1 in regulating cell proliferation, Cav1−/− lungs exhibit hypercellularity and alveolar septal thickening (Drab et al. 2001, Razani et al. 2001, Zhao et al. 2009). Maniatis et al. also identify a marked decrease of pulmonary artery density and defective pulmonary artery filling in Cav1−/− lungs (Maniatis et al. 2008). These studies with Cav1−/− mice provide direct evidence of the critical role of Cav1 deficiency in the pathogenesis of PH.
Cav1 deficiency is also seen in rat models of PH including monocrotaline-induced PH (Mathew et al. 2004) and SU5416/hypoxia-induced PH (Achcar et al. 2006). Cav1 (mainly the endothelial cell-restricted isoform Cav1α) expression is decreased in rat lungs as early as 48 h and reaches nadir (70% reduction) at 2 weeks post-monocrotaline challenge (Mathew et al. 2004). Intriguingly, the progressive reduction of Cav1 expression is mainly identified in the intimal layer of the pulmonary arteries, not the pulmonary veins. Decreased Cav1 expression and associated hyperactivation of STAT3 and ERK1/2 signaling and cell proliferation are confirmed in pulmonary endothelial cell cultures treated with monocrotaline-pyrrole (Mathew et al. 2004). In the rat model of severe PH induced by a single s.c. injection of the VEGF receptor inhibitor SU5416 and subsequent 3 weeks exposure of chronic hypoxia, Cav1 expression is selectively decreased or diminished in the complex cellular arterial lesions (Achcar et al. 2006). Interestingly, the cell-permeable Cav1 inhibitory peptide has been shown effective to prevent the development of monocrotaline-induced PH in rats (Jasmin et al. 2006).
Decreased expression of Cav1 has also been demonstrated in lungs of patients with severe PH. Achcar et al. showed absent or decreased expression of Cav1 in plexiform lesions and in some muscularized precapillary arterioles in lung tissues from patients with severe PH although Cav1 expression in total lung lysates is not significantly changed (Achcar et al. 2006). Patel et al. identify decreased expression of Cav1 in total lung lysates and pulmonary vascular endothelial cells in lungs tissues from IPAH patients. In contrast, Cav1 expression is, however, increased in pulmonary vascular smooth muscle cells in lung tissues from IPAH patients (Patel et al. 2007). Our recent studies also demonstrate a marked decrease of Cav1 expression in total lung lysates from IPAH patients (Zhao et al. 2009). Taken together, these data strongly support the concept that Cav1 is a critical regulator of the pulmonary vascular function in animals and humans.
To determine the consequences of chronic activation of eNOS in Cav1−/− lungs, our recent studies have employed a mouse model with genetic deletions of both Cav1 and NOS3 (DKO) (Zhao et al. 2009). Surprisingly, DKO mice don't develop pulmonary hypertension. In contrast to Cav1−/− mice, DKO mice have the same right ventricular systolic pressure (RVSP) as WT mice. The ratio of right weight /left ventricle plus septum weight is normal in DKO mice and PVR in DKO mice is reduced to the WT level. The defects in pulmonary vasculatures in Cav1−/− mice are also corrected in DKO mice. DKO lungs exhibit normal alveolar-capillary structure and vessel wall thickness in contrast to Cav1−/− lungs. Muscularization, an indicator of vascular remodeling, of distal pulmonary arteries is inhibited in DKO lungs compared to Cav1−/− lungs. In Cav1−/− lungs, pulmonary vascular remodeling is associated with hyperactivation of ERK signaling and downregulation of p21Cip1, a cyclin-dependent kinase inhibitor. These cell cycle progression-related signaling events are normalized in DKO lung tissues as seen in WT lungs. These data provide unequivocal evidence that chronic activation of eNOS secondary to Cav1 deficiency is a critical factor in the mechanism of PH. Consistent with our observations, Wunderlich et al. also show that L-NAME inhibition of NOS prevents adverse lung remodeling and PH in Cav1−/− mice (Wunderlich et al. 2008). However, previous studies have shown that increased production of NO in eNOS transgenic mice prevents the increase in RVSP, lung vascular remodeling, and right ventricular hypertrophy induced by chronic hypoxia (Ozaki et al. 2001), and cell-based eNOS gene transfer also inhibits monocrotaline-induced PH in rats (Zhao et al. 2005).
Our recent studies provide additional insights into the molecular basis of eNOS activation-induced PH in Cav1−/− mice. Given that NO reacts with superoxide to form the damaging reactive oxygen species peroxynitrite, which modifies proteins and may interfere with their function through tyrosine nitration, we have detected a significant increase in nitrotyrosine expression, a surrogate measure of peroxynitrite in Cav1−/− lungs. Prominent nitrotyrosine immunostaining is also evident in Cav1−/− pulmonary vasculature, indicating peroxynitrite formation in Cav1−/− lungs. Accordingly, PKG, the downstream target of NO signaling responsible for vasorexalation is tyrosine-nitrated by peroxynitrite and its kinase activity is impaired in Cav1−/− lungs. However, genetic deletion of eNOS blocks peroxynitrite formation and resultant PKG nitration, and thereby restores PKG activity in DKO lungs. In primary culture of human pulmonary arterial smooth muscle cells, treatment with 3-morpholinosydnonimine (SIN-1), a superoxide and NO donor that forms peroxynitrite simultaneously, induces PKG nitration and impairs its kinase activity. Site-directed mutagenesis and in vitro PKG kinase activity assays have identified tyrosine residues 345 and 549 in the kinase domain of human PKG-1α as the potential target tyrosines responsible for impaired PKG activity following nitration by peroxynitrite in Cav1−/− lungs. PKG nitration and resultant impairment of its kinase activity in the pulmonary vasculature under hypoxia condition have also been reported by other investigators (Negash et al. 2007). In contrast to our observation in Cav1−/− lungs, the hypoxia-induced tyrosine nitration of PKG in these studies is eNOS-independent.
To address the causal role of tyrosine nitration-mediated impairment of PKG activity in the development of PH in Cav1−/− mice, a series of experiments including blocking peroxynitrite formation and overexpressing PKG have been performed (Zhao et al. 2009). To block peroxynitrite formation, Cav1−/− mice are treated with either L-NAME to inhibit NOS or manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP, a superoxide dismutase mimetic) to scavenge superoxide. Following 5 to 6 weeks treatment of either L-NAME or MnTMPyP, the pulmonary hypertensive phenotypes in Cav1−/− lungs including increased RVSP and pulmonary vascular remodeling are reversed. These data suggest that formation of peroxynitrite and resultant protein nitration are required for the development of PH in Cav1−/− mice. To determine nitrative stress-induced PH in Cav1−/− mice is mediated by tyrosine nitration-induced impairment of PKG activity, PKG-1 is overexpressed in Cav1−/− lungs through recombinant adenovirus-mediated transduction. Restoration of PKG activity results in reduction of RVSP and PVR in Cav1−/− mice. Taken together, these data strongly support the concept that nitrative stress-induced PH in Cav1−/− mice is mediated by impaired PKG kinase activity through tyrosine nitration.
To address the pathophysiological relevance of Cav1-regulated eNOS activity in the mechanism of increased PVR and pulmonary vascular remodeling in patients with PH, we have examined Cav1 expression, eNOS activity and PKG nitration in lung tissues from IPAH patients (Zhao et al. 2009). We observed increased eNOS activity and PKG nitration concomitant with decreased Cav1 expression in lung tissues from IPAH patients compared with normal lungs in the absence of marked changes of eNOS and PKG expression. These findings are in agreement with the studies in Cav1−/− mouse lungs. Previous observations that decreased Cav1 expression in IPAH lungs is mainly in the plexiform lesions (Achcar et al. 2006) and selectively in endothelial cells (Patel et al. 2007), and that eNOS is robustly expressed in the plexiform lesions of IPAH lungs (Mason et al. 1998) support the concept that eNOS (predominantly expressed in endothelial cells) activation secondary to Cav1 deficiency plays a causal role in the mechanism of PH in mice and humans through PKG nitration and resultant impairment of its kinase activity (Figure 1).
Severe PH (including IPAH) is a fatal disease characterized by a progressive increase in PVR and vascular remodeling leading to right heart failure and early death. Despite recent advances in our understanding of the mechanisms and genetic determinants of PH, there are limited options available for the prevention and treatment of severe PH. Prominent oxidative/nitrative stress is a hallmark of the pathology of severe PH. Our recent studies have now provided a mechanistic insight into the critical role of oxidative/nitrative stress in the pathogenesis of PH in mice. We have demonstrated the crucial role of chronic eNOS activation secondary to Cav1 deficiency in the mechanism of PH and provide evidence that PKG nitration induces PH. These key observations in mice were recapitulated in lung tissue from IPAH patients. To further establish the causal role of PKG nitration in the pathogenesis of PH, it would be interesting to determine whether overexpression of PKG mutants resistant to tyrosine nitration in the pulmonary vasculature will be more efficient to reverse the hypertensive pulmonary phenotypes in Cav1−/− mice and in other experimental animal models of PH. Second, it remains unclear what are the source(s) of superoxide in Cav1−/− lungs. Although Wunderlich et al. have shown evidence of eNOS uncoupling as a potential source of ROS production in Cav1−/− lungs, eNOS uncoupling alone doesn't cause PH in other studies. In addition, it also remains unknown in which cell type(s) Cav1 deficiency induces PH. Future studies directed to address these questions will strengthen our understanding of the mechanism of progressive PH. Importantly, future efforts may be directed to identify signaling pathways regulating oxidative/nitrative stress and determine the correlation of oxidative/nitrative stress and the severity of PH. These new studies will provide us with a comprehensive understanding of the molecular basis of oxidative/nitrative stress-induced progression of PH, and thereby identify potential novel therapeutic approaches targeting the underlying signaling pathways for the prevention and treatment of progressive PH. Recently, homologous transplantation of stem/progenitor cells has shown efficacy in treatment of severe PH. Stem/progenitor cells-based eNOS overexpression is currently under clinical trial for treatment of PH. On the basis of our recent studies, it would be interesting to see whether stem/progenitor cells-based gene therapy targeting oxidative/nitrative stress is a more efficacious approach for the treatment of severe PH.
We apologize to the many authors whose significant works were not cited owing to the space limitations. This work was supported by NIH grants R01 HL085462 to Y.Y. Zhao, and P01 HL060678 to A.B. Malik.
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