Loss of caveolin-1 and adiponectin induces severe inflammatory lung injury following LPS challenge through excessive oxidative/nitrative stress.

Excessive reactive oxygen/nitrogen species have been associated with the onset, progression, and outcome of sepsis, both in preclinical and clinical studies. However, the signaling pathways regulating oxidative/nitrative stress in the pathogenesis of sepsis-induced acute lung injury and acute respiratory distress syndrome are not fully understood. Employing the novel mouse model with genetic deletions of both caveolin-1 (Cav1) and adiponectin (ADPN) [double knockout (DKO) mice], we have demonstrated the critical role of Cav1 and ADPN signaling cross talk in regulating oxidative/nitrative stress and resulting inflammatory lung injury following LPS challenge. In contrast to the inhibited inflammatory lung injury in Cav1(-/-) mice, we observed severe lung inflammation and markedly increased lung vascular permeability in DKO mice in response to LPS challenge. Accordingly, the DKO mice exhibited an 80% mortality rate following a sublethal dose of LPS challenge. At basal state, loss of Cav1 and ADPN resulted in a drastic increase of oxidative stress and resultant nitrative stress in DKO lungs. Scavenging of superoxide by pretreating the DKO mice with MnTMPYP (a superoxide dismutase mimetic) restored the inflammatory responses to LPS challenge including reduced lung myeloperoxidase activity and vascular permeability. Thus oxidative/nitrative stress collectively modulated by Cav1 and ADPN signalings is a critical determinant of inflammatory lung injury in response to LPS challenge.

Protein kinase G-I deficiency induces pulmonary hypertension through Rho A/Rho kinase activation.

Protein kinase G (PKG) plays an important role in the regulation of vascular smooth cell contractility and is a critical mediator of nitric oxide signaling, which regulates cardiovascular homeostasis. PKG-I-knockout (Prkg1(-/-)) mice exhibit impaired nitric oxide/cGMP-dependent vasorelaxation and systemic hypertension. However, it remains unknown whether PKG-I deficiency induces pulmonary hypertension. In this study, we characterized the hypertensive pulmonary phenotypes in Prkg1(-/-) mice and delineated the underlying molecular basis. We observed a significant increase in right ventricular systolic pressure in Prkg1(-/-) mice in the absence of systemic hypertension and left-sided heart dysfunction. In addition, we observed marked muscularization of distal pulmonary vessels in Prkg1(-/-) mice. Microangiography revealed impaired integrity of the pulmonary vasculature in Prkg1(-/-) mice. Mechanistically, PKG-I-mediated phosphorylation of Rho A Ser188 was markedly decreased, and the resultant Rho A activation was significantly increased in Prkg1(-/-) lung tissues, which resulted in Rho kinase activation. The i.t. administration of fasudil, a Rho kinase inhibitor, reversed the hypertensive pulmonary phenotype in Prkg1(-/-) mice. Taken together, these data show that PKG-I deficiency induces pulmonary hypertension through Rho A/Rho kinase activation-mediated vasoconstriction and pulmonary vascular remodeling.

FoxM1 regulates re-annealing of endothelial adherens junctions through transcriptional control of beta-catenin expression.

Repair of the injured vascular intima requires a series of coordinated events that mediate both endothelial regeneration and reannealing of adherens junctions (AJs) to form a restrictive endothelial barrier. The forkhead transcription factor FoxM1 is essential for endothelial proliferation after vascular injury. However, little is known about mechanisms by which FoxM1 regulates endothelial barrier reannealing. Here, using a mouse model with endothelial cell (EC)-restricted disruption of FoxM1 (FoxM1 CKO) and primary cultures of ECs with small interfering RNA (siRNA)-mediated knockdown of FoxM1, we demonstrate a novel requisite role of FoxM1 in mediating endothelial AJ barrier repair through the transcriptional control of beta-catenin. In the FoxM1 CKO lung vasculature, we observed persistent microvessel leakage characterized by impaired reannealing of endothelial AJs after endothelial injury. We also showed that FoxM1 directly regulated beta-catenin transcription and that reexpression of beta-catenin rescued the defective AJ barrier-reannealing phenotype of FoxM1-deficient ECs. Knockdown of beta-catenin mimicked the phenotype of defective barrier recovery seen in FoxM1-deficient ECs. These data demonstrate that FoxM1 is required for reannealing of endothelial AJs in order to form a restrictive endothelial barrier through transcriptional control of beta-catenin expression. Therefore, means of activating FoxM1-mediated endothelial repair represent a new therapeutic strategy for the treatment of inflammatory vascular diseases associated with persistent vascular barrier leakiness such as acute lung injury.

Caveolin-1 deficiency dampens Toll-like receptor 4 signaling through eNOS activation.

Caveolin-1 (Cav1), the scaffolding protein of caveolae, has been shown to play an important role in host defense and inflammation. However, the underlying molecular basis for these actions remains elusive. Here, using double mutant mice with genetic deletions of Cav1 and NOS3, we show that chronic endothelial nitric oxide synthase (eNOS) activation secondary to loss of Cav1 serves a crucial immunomodulatory function through tyrosine nitration-mediated impairment of interleukin-1 receptor associated kinase (IRAK)4, a signaling component required for nuclear factor-kappaB activation and innate immunity. We observed an eNOS-dependent decrease in the plasma concentration of pro-inflammatory cytokines and marked improvement of survival in Cav1(-/-) mice following lipopolysaccharide challenge. Activation of eNOS secondary to loss of Cav1 resulted in decreased activation of nuclear factor-kappaB in response to lipopolysaccharide challenge, and thereby protected the animals from lipopolysaccharide-induced lung injury. IRAK4 was prominently nitrated in Cav1-deficient endothelial cells, whereas eNOS deletion in Cav1-deficient endothelial cells resulted in marked decrease of IRAK4 nitration and restored the inflammatory response after lipopolysaccharide challenge. Furthermore, in vitro nitration of IRAK4 resulted in impairment of the kinase activity. Thus, eNOS activation secondary to loss of Cav1 signals dampening of the innate immune response to lipopolysaccharide through IRAK4 nitration and the resultant impairment of kinase activity, and consequently mitigates inflammatory lung injury.

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.

Critical role of Cdc42 in mediating endothelial barrier protection in vivo.

Activation of the Rho GTPase Cdc42 has been shown in endothelial cell monolayers to prevent disassembly of interendothelial junctions and the increase in endothelial permeability. Here, we addressed the in vivo role of Cdc42 activity in mediating endothelial barrier protection in lungs by generating mice expressing the dominant active mutant V12Cdc42 protein in vascular endothelial cells targeted via the VE-cadherin promoter. These mice developed normally and exhibited constitutively active GTP-bound Cdc42. The increase in lung vascular permeability and gain in tissue water content in response to intraperitoneal lipopolysaccharide challenge (7 mg/kg) were markedly attenuated in the transgenic mice. To address the basis of the protective effect, we observed that expression of V12Cdc42 mutant in endothelial monolayers reduced the decrease in transendothelial electrical resistance, a measure of opening of interendothelial junctions, thus indicating that Cdc42 activity preserved junctional integrity. RhoA activity in V12Cdc42-expressing endothelial monolayers was reduced compared with untransfected cells, suggesting that activated Cdc42 functions by counteracting the canonical RhoA-mediated mechanism of endothelial hyperpermeability. Therefore, Cdc42 activity of microvessel endothelial cells is a critical determinant of junctional barrier restrictiveness and may represent a means of therapeutically modulating increased lung vascular permeability and edema formation.

Endothelial cell-restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury.

Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. However, little is known about the molecular mechanisms of endothelial barrier repair following injury. To investigate the functional role of forkhead box M1 (FoxM1) in the mechanism of endothelial repair, we generated endothelial cell-restricted FoxM1-deficient mice (FoxM1 CKO mice). These mutant mice were viable and exhibited no overt phenotype. However, in response to the inflammatory mediator LPS, FoxM1 CKO mice displayed significantly protracted increase in lung vascular permeability and markedly increased mortality. Following LPS-induced vascular injury, FoxM1 CKO lungs demonstrated impaired cell proliferation in association with sustained expression of p27(Kip1) and decreased expression of cyclin B1 and Cdc25C. Endothelial cells isolated from FoxM1 CKO lungs failed to proliferate, and siRNA-mediated suppression of FoxM1 expression in human endothelial cells resulted in defective cell cycle progression. Deletion of FoxM1 in endothelial cells induced decreased expression of cyclins, Cdc2, and Cdc25C, increased p27(Kip1) expression, and decreased Cdk activities. Thus, FoxM1 plays a critical role in the mechanism of the restoration of endothelial barrier function following vascular injury. These data suggest that impairment in FoxM1 activation may be an important determinant of the persistent vascular barrier leakiness and edema formation associated with inflammatory diseases.