Farnaz B.

The NF-κB pathway is central to the regulation of inflammation. Here, we demonstrate that the low-output nitric oxide (NO) synthase 1 (NOS1 or nNOS) plays a critical role in the inflammatory response by promoting the activity of NF-κB. Specifically, NOS1-derived NO production in macrophages leads to proteolysis of suppressor of cytokine signaling 1 (SOCS1), alleviating its repression of NF-κB transcriptional activity. As a result, NOS1-/- mice demonstrate reduced cytokine production, lung… Show more

The NF-κB pathway is central to the regulation of inflammation. Here, we demonstrate that the low-output nitric oxide (NO) synthase 1 (NOS1 or nNOS) plays a critical role in the inflammatory response by promoting the activity of NF-κB. Specifically, NOS1-derived NO production in macrophages leads to proteolysis of suppressor of cytokine signaling 1 (SOCS1), alleviating its repression of NF-κB transcriptional activity. As a result, NOS1-/- mice demonstrate reduced cytokine production, lung injury, and mortality when subjected to two different models of sepsis. Isolated NOS1-/- macrophages demonstrate similar defects in proinflammatory transcription on challenge with Gram-negative bacterial LPS. Consistently, we found that activated NOS1-/- macrophages contain increased SOCS1 protein and decreased levels of p65 protein compared with wild-type cells. NOS1-dependent S-nitrosation of SOCS1 impairs its binding to p65 and targets SOCS1 for proteolysis. Treatment of NOS1-/- cells with exogenous NO rescues both SOCS1 degradation and stabilization of p65 protein. Point mutation analysis demonstrated that both Cys147 and Cys179 on SOCS1 are required for its NO-dependent degradation. These findings demonstrate a fundamental role for NOS1-derived NO in regulating TLR4-mediated inflammatory gene transcription, as well as the intensity and duration of the resulting host immune response. Show less

Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into… Show more

Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into a source of superoxide radicals, peroxynitrite, and oxidative stress. Therefore, we hypothesized that nitrate tolerance induced by persistent GTN treatment results from NOS3 dysfunction and vascular toxicity. Exposure to GTN for 48-72 h resulted in nitrosation and depletion (>50%) of Cav-1, NOS3 uncoupling as measured by an increase in peroxynitrite production (>100%), and endothelial toxicity in cultured cells. In the Cav-1 deficient mice, NOS3 dysfunction was accompanied by GTN tolerance (>50% dilation inhibition at low GTN concentrations). In conclusion, GTN tolerance results from Cav-1 modification and depletion by GTN that causes persistent NOS3 activation and uncoupling, preventing it from participating in GTN-medicated vasodilation. Show less

Genome wide association studies (GWAS) have revealed significant association of caveolin-1 (Cav1) gene variants with increased risk of cardiac arrhythmias. Nevertheless, the mechanism for this linkage is unclear. Using adult Cav1-/- mice, we revealed a marked reduction in the left ventricular (LV) conduction velocity in the absence of myocardial Cav1, which is accompanied with increased inducibility of ventricular arrhythmias. Further studies demonstrated that loss of Cav1 leads to the… Show more

Genome wide association studies (GWAS) have revealed significant association of caveolin-1 (Cav1) gene variants with increased risk of cardiac arrhythmias. Nevertheless, the mechanism for this linkage is unclear. Using adult Cav1-/- mice, we revealed a marked reduction in the left ventricular (LV) conduction velocity in the absence of myocardial Cav1, which is accompanied with increased inducibility of ventricular arrhythmias. Further studies demonstrated that loss of Cav1 leads to the activation of cSrc tyrosine kinase, resulting in the downregulation of connexin 43 (Cx43) and subsequent electrical abnormalities. Pharmacological inhibition of cSrc mitigates Cx43 downregulation, slow conduction and arrhythmia inducibility in Cav1-/- animals. Using a transgenic mouse model with cardiac-specific overexpression of angiotensin converting enzyme (ACE8/8), we demonstrated that, upon enhanced cardiac RAS activity, Cav1 dissociated from cSrc because of increased Cav1 S-nitrosation (SNO) at Cys156, leading to c-Src activation, Cx43 reduction, impaired gap junction function, and subsequent increase in the propensity for ventricular arrhythmias and sudden cardiac death. RAS-induced Cav1 SNO was associated with increased Cav1-eNOS binding in response to increased mitochondrial reactive oxidative species (ROS) generation. The present studies reveal the critical role of Cav1 in modulating cSrc activation, gap junction remodeling and ventricular arrhythmias. These data provide a mechanistic explanation for the observed genetic link between Cav1 and cardiac arrhythmias in humans and suggest that targeted regulation of Cav1 may reduce arrhythmic risk in cardiac diseases associated with RAS activation.
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BACKGROUND: Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory… Show more

BACKGROUND: Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase-Akt-nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability.

CONCLUSIONS:Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory "side-effect" of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease. Show less

In the present study, we tested the hypothesis that chronic inflammation and oxidative/nitrosative stress induce caveolin 1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction and pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). We observed reduced Cav-1 protein despite increased Cav-1 messenger RNA expression and also endothelial nitric oxide synthase (eNOS) hyperphosphorylation in human pulmonary… Show more

In the present study, we tested the hypothesis that chronic inflammation and oxidative/nitrosative stress induce caveolin 1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction and pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). We observed reduced Cav-1 protein despite increased Cav-1 messenger RNA expression and also endothelial nitric oxide synthase (eNOS) hyperphosphorylation in human pulmonary artery endothelial cells (PAECs) from patients with IPAH. In control human lung endothelial cell cultures, tumor necrosis factor α-induced nitric oxide (NO) production and S-nitrosation (SNO) of Cav-1 Cys-156 were associated with Src displacement and activation, Cav-1 Tyr-14 phosphorylation, and destabilization of Cav-1 oligomers within 5 minutes that could be blocked by eNOS or Src inhibition. Prolonged stimulation (72 hours) with NO donor DETANONOate reduced oligomerized and total Cav-1 levels by 40%-80%, similar to that observed in IPAH patient-derived PAECs. NO donor stimulation of endothelial cells for >72 hours, which was associated with sustained Src activation and Cav-1 phosphorylation, ubiquitination, and degradation, was blocked by NOS inhibitor L-NAME, Src inhibitor PP2, and proteosomal inhibitor MG132. Thus, chronic inflammation, sustained eNOS and Src signaling, and Cav-1 degradation may be important causal factors in the development of IPAH by promoting PAEC dysfunction/activation via sustained oxidative/nitrosative stress. Show less

von Willebrand factor (vWF) secretion by endothelial cells (EC) is essential for hemostasis and thrombosis, however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13-/-;Gα12-/- mice that could be normalized by infusion of human vWF. Blood from Gα12-/- mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation indicating Gα12 plays a prominent role in EC vWF secretion required for… Show more

von Willebrand factor (vWF) secretion by endothelial cells (EC) is essential for hemostasis and thrombosis, however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13-/-;Gα12-/- mice that could be normalized by infusion of human vWF. Blood from Gα12-/- mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation indicating Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12-/- whereas thrombin-induced vWF secretion was defective in both EC-Gαq-/-;Gα11-/- and Gα12-/- mice. Using siRNA in cultured HUVEC and HPAEC, depletion of Gα12 and α-SNAP (but not Gα13) inhibited both basal and thrombin-induced vWF secretion while over-expression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted HUVEC, thrombin-induced vWF secretion was reduced by 40% whereas basal secretion was unchanged. Finally, in vitro binding assays revealed Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs. evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease. Show less

Prolonged treatment with nitroglycerin (GTN) has been shown to induce nitrate tolerance and cross-tolerance. However, much of the mechanism of how GTN leads to tolerance remains unknown. Recent evidence indicated that pharmacological doses (<50 nM) of GTN induce endothelial nitric oxide synthase (eNOS)-dependent vasodilation. Thus, we hypothesized that eNOS dysfunction consequential to continuous GTN exposure could underlie nitrate tolerance and endothelial dysfunction secondary to GTN… Show more

Prolonged treatment with nitroglycerin (GTN) has been shown to induce nitrate tolerance and cross-tolerance. However, much of the mechanism of how GTN leads to tolerance remains unknown. Recent evidence indicated that pharmacological doses (<50 nM) of GTN induce endothelial nitric oxide synthase (eNOS)-dependent vasodilation. Thus, we hypothesized that eNOS dysfunction consequential to continuous GTN exposure could underlie nitrate tolerance and endothelial dysfunction secondary to GTN. Caveolin-1 (CAV-1) is known to interact with eNOS and tightly regulate eNOS function, warranting temporal and quantitative NO synthesis. Loss of CAV-1 results in eNOS dysfunction. In this study we investigated the roles of CAV-1 and eNOS in tolerance. We report that when endothelial cells are continuously exposed to GTN for extended periods of time, eNOS becomes a source of peroxynitrite secondary to CAV-1 depletion. We demonstrate that CAV-1 is degraded in both GTN treated endothelial cells and animal tissues which parallels a marked increase in eNOS phosphorylation (Ser 1177) and accumulation of monomeric eNOS. We also demonstrate in endothelial cells that although CAV-1 mRNA level is not changed, ubiquitination on the protein is increased with GTN, and that treatment with the proteasome inhibitor MG132 partially restores CAV-1 levels, indicating that GTN-induced CAV-1 loss is proteolytic and not transcriptional. We also confirmed that eNOS is dysfunctional in a CAV-1 deficient mouse model, which is naturally tolerant to pharmacologic relevant doses of GTN. Moreover, we show that the elevated production of peroxynitrite from dysregulated eNOS is antagonized by CAV-1 transfection. Taken together, these results imply that GTN-induced CAV-1 depletion and consequential eNOS dysfunction contribute to tolerance. Show less

Endothelial nitric oxide synthase (eNOS)–mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1–mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses… Show more

Endothelial nitric oxide synthase (eNOS)–mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1–mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca2 ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor l-NG-nitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca2 -induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1–Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition. Show less

We assessed the cooperative roles of C-terminal Src kinase (Csk) binding protein (Cbp) and Caveolin-1 (Cav-1) in the mechanism of Src family tyrosine kinase (SFK) inhibition by Csk. SFKs are inactivated by phosphorylation of their C-terminal tyrosine by Csk. Whereas SFKs are membrane-associated, Csk is a cytoplasmic protein and therefore requires membrane adaptors such as Cbp or Cav-1 for recruitment to the plasma membrane to mediate SFK inhibition. To determine the specific role of Cav-1 and… Show more

We assessed the cooperative roles of C-terminal Src kinase (Csk) binding protein (Cbp) and Caveolin-1 (Cav-1) in the mechanism of Src family tyrosine kinase (SFK) inhibition by Csk. SFKs are inactivated by phosphorylation of their C-terminal tyrosine by Csk. Whereas SFKs are membrane-associated, Csk is a cytoplasmic protein and therefore requires membrane adaptors such as Cbp or Cav-1 for recruitment to the plasma membrane to mediate SFK inhibition. To determine the specific role of Cav-1 and Cbp in SFK inhibition, we measured c-Src activity in the absence of each membrane adaptor. It is noteworthy that in lungs and fibroblasts from Cav-1(-/-) mice, we observed increased expression of Cbp compared with wild-type (WT) controls. However, both c-Src activity and Csk localization at the membrane were similar between Cav-1(-/-) fibroblasts and WT cells. Likewise, Cbp depletion by small interfering RNA (siRNA) treatment of WT cells had no effect on basal c-Src activity, but it increased the phosphorylation state of Cav-1. Immunoprecipitation then confirmed increased association of Csk with phosphomimicking Cav-1. Knockdown of Cbp by siRNA in Cav-1(-/-) cells revealed increased basal c-Src activity, and re-expression of WT Cav-1 in the same cells reduced basal c-Src activity. Taken together, these results indicate that Cav-1 and Cbp cooperatively regulate c-Src activity by recruiting Csk to the membrane where it phosphorylates c-Src inhibitory tyrosine 529. Furthermore, when either Cav-1 or Cbp expression is reduced or absent, there is a compensatory increase in the phosphorylation state or expression level of the other membrane-associated Csk adaptor to maintain SFK inhibition Show less