Mechanism of Caveolin-1 Degradation

In the present study, we tested the hypothesis that oxidative/nitrosative stress promotes caveolin-1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction in patients with idiopathic pulmonary artery hypertension (IPAH). It has been shown that there is a decrease in Cav-1 in IPAH patient samples, but the mechanism of this loss has not been elucidated. Our work aimed to reveal the mechanism of Cav-1 loss that is seen in several diseases, including IPAH. When we analyzed the human pulmonary artery endothelial cells (PAECs) from patients with IPAH, we observed reduced Cav-1 expression and endothelial nitric oxide synthase (eNOS) hyperphosphorylation. This is consistent with the theory that Cav-1 is a regulator of eNOS. In the IPAH samples, Cav-1 protein levels were decreased despite increased expression of Cav-1 messenger ribonucleic acid (mRNA). In control human lung endothelial cells, tumor necrosis factor alpha (TNF-α)-induced nitric oxide (NO) production and S-nitrosation (SNO) of Cav-1 cysteine-156 (C156) was associated within 5 minutes with Src displacement and activation, Cav-1 tyrosine-14 (Y14) phosphorylation, and destabilization of Cav-1 oligomers, which was blocked by inhibiting either eNOS with the NOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) or Src with 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Prolonged (72 hours) stimulation with the NO donor DETA-NONOate reduced oligomerized and total Cav-1 levels by 40-80%, similar to that observed in IPAH patient samples. Inhibition of the proteasome and Src prevented Cav-1 degradation in cells treated with DETA-NONOate, suggesting that Cav-1 SNO and sustained phosphorylation promote ubiquitination and degradation by the proteasome. In transduced human embryonic kidney (HEK) cells, we observed reduced oligomerization of cysteine 156 to serine (C156S) Cav-1 mutant, indicating this mutation might mimic the effect of nitrosation. Mass spectrometry analysis revealed ubiquitination of Cav-1 on lysine-86 (K86). Thus, reduced Cav-1 expression due to oxidative stress-induced Cav-1 SNO of C156, Src activation, and phosphorylation of Cav-1 Y14, ubiquitination of Cav-1 K86, and ultimately proteosomal degradation of Cav-1 is associated with eNOS hyperactivation and loss of caveolae in PAECs from patients with IPAH. These studies indicate chronic inflammation and sustained oxidative stress may promote endothelial cell dysfunction via degradation of Cav-1

Caveolin-1 Modulates Cardiac Gap Junction Homeostasis and Arrhythmogenecity by Regulating cSrc Tyrosine Kinase

BackgroundGenome-wide association studies have revealed significant association of caveolin-1 (Cav1) gene variants with increased risk of cardiac arrhythmias. Nevertheless, the mechanism for this linkage is unclear.Methods and resultsUsing adult Cav1(-/-) mice, we revealed a marked reduction in the left ventricular 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 and subsequent electric abnormalities. Pharmacological inhibition of cSrc mitigates connexin 43 downregulation, slowed 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, on enhanced cardiac renin-angiotensin system activity, Cav1 dissociated from cSrc because of increased Cav1 S-nitrosation at Cys(156), leading to cSrc activation, connexin 43 reduction, impaired gap junction function, and subsequent increase in the propensity for ventricular arrhythmias and sudden cardiac death. Renin-angiotensin system-induced Cav1 S-nitrosation was associated with increased Cav1-endothelial nitric oxide synthase binding in response to increased mitochondrial reactive oxidative species generation.ConclusionsThe 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 renin-angiotensin system activation

Endothelial barrier protection by local anesthetics: ropivacaine and lidocaine block tumor necrosis factor-α-induced endothelial cell Src activation

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. METHODS: Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed. RESULTS: Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10 M for ropivacaine; IC50 = 5.864 × 10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10 M for ropivacaine; IC50 = 6.377 × 10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated. 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

Nitroglycerin Tolerance in Caveolin-1 Deficient Mice

<div><p>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.</p></div

Schematic representation of GTN-induced Cav-1 degradation and NOS3 dysfunction.

<p>Figure summarizes main findings in this study and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104101#pone.0104101-Bakhshi1" target="_blank">[30]</a> that together indicate that S-nitrosated Cav-1 is degraded by the proteasome promoting eNOS dysfunction and a transition from the NO synthase activity to that of a NADPH oxidase.</p

GTN provokes the proteolytic degradation of Cav-1.

<p>(A) Partial restoration of Cav-1 by proteasome inhibitor. MECs were treated with GTN (20 µM) for indicated time (24, 48 and 72 h). MG132 (5 µM) was added to the media and incubated over night (12 h) before lysing the cells. Cav-1 and Actin levels were analyzed by western blot. Results are representative of four independent experiments. (B) mRNA level (n = 3, mean ± SD) of cav-1 in MECs treated with GTN for 0, 24, 48 and 72 hours measured by nested PCR. GAPDH was used as internal standard for quantification. (C) Biotin switch assay of Cav-1 nitrosation. WT MECs were exposed to the nitrosation reagent, S-Nitroso-N-Acetylpenicillamine (SNAP, 1 mM) for 1 h or GTN (20 µM) for 72 h. S-nitrosated proteins were labeled with Biotin and pull-down with Streptavidin conjugated agarose beads. Whole cell lysates and eluted samples were analyzed for Cav-1 in western blot. Images are representative of three independent experiments. (D) Ubiquitination of immunoprecipitated Cav-1 in MECs. Cells were treated with GTN or vehicle for 72 hours, and incubated with MG132 (25 µM) 4 hours prior to lysis and immunoprecipitation. Ubiquitination levels in immunoprecipitated Cav-1 were examined using western blot. Actin and Cav-1 were analyzed as references of total input. Results are representative of three independent experiments. (E) Cav-1 oligomer/monomer distribution in tolerant cells assessed by non-reducing SDS-Page. MECs were treated with GTN or vehicle for 72 h. Cells lysates were subjected to native SDS-PAGE without addition of reducing reagent and boiling. Results shown are representative of three independent experiments.</p

Continuous GTN-exposure leads to Cav-1 loss, NOS3 hyperactivation and dysfunction.

<p>Representative western blot image of at least three independent experiments showing GTN-induced cav-1 depletion in (A) human umbilical vein endothelial cell (HUVEC) and (B) primary mouse lung microvascular endothelial cells (MEC). Cells were treated with 20 µM GTN or vehicle control of indicated time. Cav-1 levels were examined by western blot. (C) Representative western blot of GTN-induction of cav-1 depletion paralleled by NOS3 activation in mouse aorta. Mice were exposed to GTN in the form of ointment at 2% concentration continuously for 72 h. Controls were exposed to vehicle-white petrolatum base paste. Bar graph shows the relative amounts of cav-1 compared with control group (mean ± SD), n = 3, * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001</p

Caveolin-1 knockdown recapitulate NOS3 dysfunction and leads to nitrate tolerance.

<p>(A) Representative measurement of basal NO and peroxynitrite level in WT and Cav-1 KO MECs. n = 6, ** <i>p<</i>0.01, *** <i>p</i><0.001 (B) Representative measurement of NO and peroxynitrite in WT and Cav-1 KO MEC stimulated with A23187 (10 µM) for 1 h. (C) Basal NO production in WT, Cav-1 knockout and Cav-1/NOS3 double knockout (DKO) MECs, n = 3 to 5, <i>* p</i><0.05, ** <i>p</i><0.01. (D) Basal Peroxynitrite production in WT, Cav-1 knockout and Cav-1/NOS3 double knockout MECs, n = 3 to 5, ** <i>p</i><0.01.</p

Caveolin-1 knockout mice are tolerant to GTN.

<p>Vasoreactivity experiments were performed in WT and Cav-1 -/- mouse mesenteric arteries. Results revealed that Cav-1 KO mice are resistant to both Acetylcholine (Ach, left panel) and low concentrations of GTN (middle panel). This indicates resistance is not due to defective signaling downstream of NO as shown by the normal responses of Cav-1 KO to sodium nitroprusside (SNP, right panel), a direct NO donor.</p