Caveolin-1 Knockout Mice Research Articles

Cooperative Role of Mineralocorticoid Receptor and Caveolin-1 in Regulating the Vascular Response to Low Nitric Oxide-High Angiotensin II-Induced Cardiovascular Injury.

Aldosterone interacts with mineralocorticoid receptor (MR) to stimulate sodium reabsorption in renal tubules and may also affect the vasculature. Caveolin-1 (cav-1), an anchoring protein in plasmalemmal caveolae, binds steroid receptors and also endothelial nitric oxide synthase, thus limiting its translocation and activation. To test for potential MR/cav-1 interaction in the vasculature, we investigated if MR blockade in cav-1-replete or -deficient states would alter vascular function in a mouse model of low nitric oxide (NO)-high angiotensin II (AngII)-induced cardiovascular injury. Wild-type (WT) and cav-1 knockout mice (cav-1(-/-)) consuming a high salt diet (4% NaCl) received Nω-nitro-l-arginine methyl ester (L-NAME) (0.1-0.2 mg/ml in drinking water at days 1-11) plus AngII (0.7-2.8 mg/kg per day via an osmotic minipump at days 8-11) ± MR antagonist eplerenone (EPL) 100 mg/kg per day in food. In both genotypes, blood pressure increased with L-NAME + AngII. EPL minimally changed blood pressure, although its dose was sufficient to block MR and reverse cardiac expression of the injury markers cluster of differentiation 68 and plasminogen activator inhibitor-1 in L-NAME+AngII treated mice. In aortic rings, phenylephrine and KCl contraction was enhanced with EPL in L-NAME+AngII treated WT mice, but not cav-1(-/-) mice. AngII-induced contraction was not different, and angiotensin type 1 receptor expression was reduced in L-NAME + AngII treated WT and cav-1(-/-) mice. In WT mice, acetylcholine-induced relaxation was enhanced with L-NAME + AngII treatment and reversed with EPL. Acetylcholine relaxation in cav-1(-/-) mice was greater than in WT mice, not modified by L-NAME + AngII or EPL, and blocked by ex vivo L-NAME, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or endothelium removal, suggesting the role of NO-cGMP. Cardiac endothelial NO synthase was increased in cav-1(-/-) versus WT mice, further increased with L-NAME + AngII, and not affected by EPL. Vascular relaxation to the NO donor sodium nitroprusside was increased with L-NAME + AngII in WT mice but not in cav-1(-/-) mice. Plasma aldosterone levels increased and cardiac MR expression decreased in L-NAME + AngII treated WT and cav-1(-/-) mice and did not change with EPL. Thus, during L-NAME + AngII induced hypertension, MR blockade increases contraction and alters vascular relaxation via NO-cGMP, and these changes are absent in cav-1 deficiency states. The data suggest a cooperative role of MR and cav-1 in regulating vascular contraction and NO-cGMP-mediated relaxation during low NO-high AngII-dependent cardiovascular injury.

To be EndMT or not to be, that is the question in pulmonary hypertension.

To be EndMT or not to be, that is the question in pulmonary hypertension.

Endothelial Caveolin-1 regulates the radiation response of epithelial prostate tumors.

The membrane protein caveolin-1 (Cav1) recently emerged as a novel oncogene involved in prostate cancer progression with opposed regulation in epithelial tumor cells and the tumor stroma. Here we examined the role of stromal Cav1 for growth and radiation response of MPR31-4 prostate cancer xenograft tumors using Cav1-deficient C57Bl/6 mice. Syngeneic MPR31-4 tumors grew faster when implanted into Cav1-deficient mice. Increased tumor growth on Cav1-deficient mice was linked to decreased integration of smooth muscle cells into the wall of newly formed blood vessels and thus with a less stabilized vessel phenotype compared with tumors from Cav1 wild-type animals. However, tumor growth delay of MPR31-4 tumors grown on Cav1 knockout mice to a single high-dose irradiation with 20 Gray was more pronounced compared with tumors grown on wild-type mice. Increased radiation-induced tumor growth delay in Cav1-deficient mice was associated with an increased endothelial cell apoptosis. In vitro studies using cultured endothelial cells (ECs) confirmed that the loss of Cav1 expression increases sensitivity of ECs to radiation-induced apoptosis and reduces their clonogenic survival after irradiation. Immunohistochemical analysis of human tissue specimen further revealed that although Cav1 expression is mostly reduced in the tumor stroma of advanced and metastatic prostate cancer, the vascular compartment still expresses high levels of Cav1. In conclusion, the radiation response of MPR31-4 prostate tumors is critically regulated by Cav1 expression in the tumor vasculature. Thus, Cav1 might be a promising therapeutic target for combinatorial therapies to counteract radiation resistance of prostate cancer at the level of the tumor vasculature.

Caveolin‐1‐Dependent Regulation of Myocardin Expression and Contractile Phenotype in Colonic Circular Smooth Muscle

The signaling molecules in smooth muscle are compartmentalized by binding to caveolin‐1, a key protein in the formation of caveolae. Our recent studies showed that carbachol‐induced Rho kinase activity and colonic smooth muscle contraction, and colonic propulsion were decreased in caveolin‐1 knockout (Cav‐1‐/‐) mice. The aim of this study is to test the hypothesis that caveolin‐1 acts as a pro‐contractile and anti‐proliferative regulatory molecule that promotes smooth muscle function. Expression of myocardin, a co‐activator of the transcription factor serum response factor, and microRNA‐144 that regulate smooth muscle phenotype was decreased in colonic circular smooth muscle of Cav‐1‐/‐ mice, and by silencing caveolin‐1 expression in control cultured muscle cells. Decrease in myocardin in Cav‐1‐/‐ mice was accompanied by a decrease in smooth muscle myosin heavy chain (SM‐MHC) expression and increase in EGF receptor phosphorylation and ERK1/2 activity. Treatment of muscle cells with carbachol for 24 h increased myocardin expression; the increase in expression was blocked by inhibitors of Rho kinase (Y27632) and actin polymerization (cytochalasin D), implying that changes in expression of myocardin in Cav‐1‐/‐ mice could be a consequence of the decrease in Rho kinase activity in these mice. We conclude that caveolin‐1 promotes myocardin expression and contractile phenotype via a Rho/Rho kinase pathway.

Pulmonary Vascular Inflammation Promotes Caveolin‐1 Degradation, eNOS Uncoupling, and De‐differentiation of Endothelial Cells

Introduction Caveolin-1 (Cav-1) knockout mice exhibit vascular abnormalities such as endothelial cell (EC) hypercellularity and fibrosis. We and others observed that lung ECs from Cav-1-/- mice exhibit a mesenchymal-like phenotype in culture suggesting Cav-1 is required to maintain differentiation of ECs. We also showed reduced Cav-1 expression in lung sections and HPAECs from patients with PAH suggesting EC Cav-1 degradation may play a role in pulmonary remodeling. The aim of the study was to determine whether chronic inflammatory lung injury (ALI) induces Cav-1 degradation and de-differentiation of ECs, and whether EC-specific Cav-1-/- mice exhibit EC hyperplasia and vascular remodeling. Methods: ALI was induced in wild-type (WT) and EC-specific Tie2.Cre;Cav-1-/- mice by exposure to nebulized E. coli LPS (10 mg/1 hr). After 96 hr, Cav-1 and eNOS expression and phosphorylation were quantified in lung lysates and histological sections. Results: LPS exposure of WT mice reduced Cav-1 expression compared to untreated WT mice, and the extent of Cav-1 depletion was similar to that observed in EC-Cav-1-/- (P ˂ 0.05; n= 3). Moreover, EC Cav-1 depletion in WT and EC-Cav-1-/- mouse lungs was associated with reduced eNOS dimerization and an increase in pSer1179-eNOS. Conclusion ALI reduced Cav-1 expression and promoted eNOS uncoupling suggesting Cav-1 degradation may play a significant role in the onset of inflammatory pulmonary vascular disease. Funding support: CNPq Fellowship - CsF/Brazil; P01 HL60678; R01EY019494.

The potential protective role of caveolin-1 in intestinal inflammation in TNBS-induced murine colitis.

BackgroundCaveolin-1 (Cav-1) is a multifunctional scaffolding protein serving as a platform for the cell’s signal-transduction and playing an important role in inflammation. However, its role in inflammatory bowel disease is not clear. A recent study showed that Cav-1 is increased and mediates angiogenesis in dextran sodium sulphate-induced colitis, which are contradictory to our pilot findings in 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. In the present study, we further clarified the role of Cav-1 in TNBS-induced colitis.MethodsIn BALB/c mice, acute colitis was induced by intra-rectal administration of one dose TNBS, while chronic colitis was induced by administration of TNBS once a week for 7 weeks. To assess the effects of complete loss of Cav-1, Cav-1 knockout (Cav-1−/−) and control wild-type C57 mice received one TNBS administration. Body weight and clinical scores were monitored. Colon Cav-1 and pro-inflammatory cytokine levels were quantified through ELISAs. Inflammation was evaluated through histological analysis.ResultsColon Cav-1 levels were significantly decreased in TNBS-induced colitis mice when compared to normal mice and also inversely correlated with colon inflammation scores and proinflammatory cytokine levels (IL-17, IFN-γ and TNF) significantly. Furthermore, after administration of TNBS, Cav-1−/− mice showed significantly increased clinical and colon inflammatory scores and body weight loss when compared with control mice.Conclusions and SignificanceCav-1 may play a protective role in the development of TNBS-induced colitis. Our findings raise an important issue in the evaluation of specific molecules in animal models that different models may exhibit opposite results because of the different mechanisms involved.

Vasopressin-induced mouse urethral contraction is modulated by caveolin-1

Caveolae are 50–100nm large invaginations in the cell membrane that are considered to play roles in receptor signaling. Here we aimed to investigate the expression and distribution of the arginine-vasopressin (AVP) V1a receptor and its functional dependence on caveolin-1 (Cav1) in the mouse urethra. Female Cav1 knockout (KO) and wild type (WT) mice were used, and urethral preparations were micro-dissected for mechanical experiments. Methyl-β-cyclodextrin (mβcd) was used to deplete cholesterol and to disrupt caveolae. Protein expression and localization was determined using immunofluorescence and western blotting and transcript expression was determined by qRT-PCR. We found that Cav1 and AVP V1a receptors were expressed in urethral smooth muscle cells with apparent co-localization at the cell membrane. AVP caused urethral contraction that was inhibited by the V1a receptor antagonist SR49059. Concentration–response curves for AVP were right-shifted and maximal contraction was reduced in Cav1 KO mice and after mβcd treatment. In addition to caveolin-1 we also detected caveolin-2, cavin-1 and cavin-3 in the mouse urethra by western blotting. Caveolin-2, cavin-1 and cavin-3 as well as V1a receptor expression was reduced in KO urethra. We conclude that AVP regulates urethral contractility via the V1a receptor through a Cav1-dependent mechanism involving, in part, altered V1a receptor expression.

Caveolin-1 prevents sustained angiotensin II-induced resistance artery constriction and obesity-induced high blood pressure.

The type 1 angiotensin II (ANG II) receptor (AT1R) undergoes internalization following stimulation by ANG II. Internalization reduces cell surface AT1Rs, and it is required for AT1R resensitization. In this process AT1R may interact with caveolin-1 (Cav1), the main scaffolding protein of caveolae. We hypothesized that the interaction between Cav1 and AT1R delays AT1R resensitization and thereby prevents sustained ANG II-induced resistance artery (RA) constriction under normal conditions and in experimental obesity. In rat and mouse skeletal muscle RA (diameter: ∼90-120 μm) ANG II-induced constrictions were reduced upon repeated (30-min apart) administrations. Upon disruption of caveolae with methyl-β-cyclodextrin or in RA of Cav1 knockout mice, repeated ANG II applications resulted in essentially maintained constrictions. In vascular smooth muscle cells, AT1R interacted with Cav1, and the degree of cell surface interactions was reduced by long-term (15-min), but not short-term (2-min), exposure to ANG II. When Cav1 was silenced, the amount of membrane-associated AT1R was significantly reduced by a short-term ANG II exposure. Moreover, Cav1 knockout mice fed a high-fat diet exhibited augmented and sustained RA constriction to ANG II and had elevated systemic blood pressure, when compared with normal or high-fat fed wild-type mice. Thus, Cav1, through a direct interaction, delays internalization and subsequent resensitization of AT1R. We suggest that this mechanism prevents sustained ANG II-induced RA constriction and elevated systemic blood pressure in diet-induced obesity.

Absence of Caveloin-1 Leads to Delayed Development of Chronic Lymphocytic Leukemia in Eµ-TCL1 Mouse Model

Background: Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the United States. Clinical heterogeneity, a characteristic feature of CLL is a major problem in the clinical management of this currently incurable leukemia. We and others have demonstrated that the tissue microenvironment, specifically the lymph node (LN), influence the biological and clinical behavior including the clinical heterogeneity of CLL. Using gene expression profiling of CLL cells from peripheral blood (PB), bone marrow (BM) and LNs, we identified Cav-1 a member of the Tolerogenic Signature (genes associated with host immune tolerance) as one of the candidate genes which might be involved in the pathogenesis of CLL. We found that Cav-1 levels were significantly elevated (11 fold) in CLL cells from LNs compared to BM and PB. Cav-1 is the major element of caveolae, which are flask-shaped membrane invaginations. Cav-1 is involved in multiple cellular processes like the regulation and transportation of cellular cholesterol and lipids, clathrin independent endocytosis and signal transduction leading to oncogenesis or tumor suppression. We have previously shown that knock down of Cav-1 results in a significant decrease in cell migration and proliferation of primary human CLL cells in vitro. We have also demonstrated that knock down of Cav-1 prevents CLL cells from forming immune synapses. These immune synapses are important for the interaction between the CLL cells and their tumor microenvironment. These results suggest that Cav-1 protect CLL cells from undergoing apoptosis and enhances their migration in vitro.Objectives and Methodology: To understand the precise role of Cav-1 in leukemic progression in vivo, we crossed Cav-1-/- mice to Eµ-TCL1 mice, which is a well-established transgenic murine model for CLL. The offspring were observed and evaluated for the development of CLL. These mice were sacrificed at the age of 12, 24, 36 and 40+ weeks and peripheral blood, bone marrow and spleen and were examined for the presence of CD5+B220+CD19+ CLL cells using flow cytometry. Spleen, lymph nodes, liver, lungs and kidney were evaluated for the presence of CLL cells using H&E staining of histologic slides.Results: To study the role of Cav-1 in Eµ-TCL1, we isolated splenic B cells and measured the expression of Cav-1. We observed a gradual increase in the expression of Cav-1 in splenic B cells from Eµ-TCL1 mice at age of 12, 24 and 36 weeks when compared with wild type mice. This suggested that Cav-1 might be playing a role in CLL progression in Eµ-TCL1 mice. Therefore, to study the role of Cav-1 in CLL disease progression we decreased the expression of Cav-1 in vivo by breeding Eµ-TCL1 with Cav1 knockout mice. We generated Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice to study the effect of Cav-1 knock down in aggressiveness of CLL in vivo. We have shown that Cav-1 is overexpressed in CLL cells from patients with poorer clinical outcome and protects CLL cells from undergoing apoptosis. Therefore, we analyze the number of CLL cells in Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice. We observed a significant reduction in the number of B220+CD5+ CLL cells population in bone marrow and spleen of Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice when compared with Eµ-TCL1-Cav1wt/wt mice. We have previously shown that Cav-1 is important for CLL cells migration in vitro. Therefore, to study its effect in vivo we analyzed infiltration of CLL cells in spleen, lymph nodes, liver, kidney and lungs in these mice. There was no or significant decrease in tumor infiltration of CLL cells in spleen, lymph nodes, liver, lungs and kidney in Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice when compared with Eµ-TCL1-Cav1wt/wt alone. Next, we wanted to examine the effect of Cav-1 knock down on splenomegaly and hepatomegaly. We found that there was a significant decrease in splenomegaly and hepatomegaly in Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice. The spleen and liver size of Eµ-TCL1-Cav1-/+ and Eµ-TCL1-Cav1-/- mice was significantly reduced when compared with Eµ-TCL1 mice. Together these results suggest that high expression of Cav-1 in CLL cells leads to enhance proliferation and promotes disease progression in Eµ-TCL1 mice. DisclosuresNo relevant conflicts of interest to declare.

Vascular smooth muscle cell proliferation depends on caveolin-1-regulated polyamine uptake.

Much evidence highlights the importance of polyamines for VSMC (vascular smooth muscle cell) proliferation and migration. Cav-1 (caveolin-1) was recently reported to regulate polyamine uptake in intestinal epithelial cells. The aim of the present study was to assess the importance of Cav-1 for VSMC polyamine uptake and its impact on cell proliferation and migration. Cav-1 KO (knockout) mouse aortic cells showed increased polyamine uptake and elevated proliferation and migration compared with WT (wild-type) cells. Both Cav-1 KO and WT cells expressed the smooth muscle differentiation markers SM22 and calponin. Cell-cycle phase distribution analysis revealed a higher proportion of Cav-1 KO than WT cells in the S phase. Cav-1 KO cells were hyper-proliferative in the presence but not in the absence of extracellular polyamines, and, moreover, supplementation with exogenous polyamines promoted proliferation in Cav-1 KO but not in WT cells. Expression of the solute carrier transporters Slc7a1 and Slc43a1 was higher in Cav-1 KO than in WT cells. ODC (ornithine decarboxylase) protein and mRNA expression as well as ODC activity were similar in Cav-1 KO and WT cells showing unaltered synthesis of polyamines in Cav-1 KO cells. Cav-1 was reduced in migrating cells in vitro and in carotid lesions in vivo. Our data show that Cav-1 negatively regulates VSMC polyamine uptake and that the proliferative advantage of Cav-1 KO cells is critically dependent on polyamine uptake. We provide proof-of-principle for targeting Cav-1-regulated polyamine uptake as a strategy to fight unwanted VSMC proliferation as observed in restenosis.

Potential of caveolae in the therapy of cardiovascular and neurological diseases.

Caveolae are membrane micro-domains enriched in cholesterol, sphingolipids and caveolins, which are transmembrane proteins with a hairpin-like structure. Caveolae participate in receptor-mediated trafficking of cell surface receptors and receptor-mediated signaling. Furthermore, caveolae participate in clathrin-independent endocytosis of membrane receptors. On the one hand, caveolins are involved in vascular and cardiac dysfunction. Also, neurological abnormalities in caveolin-1 knockout mice and a link between caveolin-1 gene haplotypes and neurodegenerative diseases have been reported. The aim of this article is to present the rationale for considering caveolae as potential targets in cardiovascular and neurological diseases.

Silencing of Kir2 channels by caveolin-1: cross-talk with cholesterol.

A growing number of studies show that different types of ion channels localize in caveolae and are regulated by the level of membrane cholesterol. Furthermore, it has been proposed that cholesterol-induced regulation of ion channels might be attributed to partitioning into caveolae and association with caveolin-1 (Cav-1). We tested, therefore, whether Cav-1 regulates the function of inwardly rectifying potassium channels Kir2.1 that play major roles in the regulation of membrane potentials of numerous mammalian cells. Our earlier studies demonstrated that Kir2.1 channels are cholesterol sensitive. In this study, we show that Kir2.1 channels co-immunoprecipitate with Cav-1 and that co-expression of Kir2.1 channels with Cav-1 in HEK293 cells results in suppression of Kir2 current indicating that Cav-1 is a negative regulator of Kir2 function. These observations are confirmed by comparing Kir currents in bone marrow-derived macrophages isolated from Cav-1(-/-) and wild-type animals. We also show, however, that Kir2 channels maintain their sensitivity to cholesterol in HEK293 cells that have very low levels of endogenous Cav-1 and in bone marrow-derived macrophages isolated from Cav-1(-/-) knockout mice. Thus, these studies indicate that Cav-1 and/or intact caveolae are not required for cholesterol sensitivity of Kir channels. Moreover, a single point mutation of Kir2.1, L222I that abrogates the sensitivity of the channels to cholesterol also abolishes their sensitivity to Cav-1 suggesting that the two modulators regulate Kir2 channels via a common mechanism.

Abstract 518: Loss of Caveolin-1 in Mouse, Rat and Human Proximal Tubule Cells Increases GRK4 Activity and Decreases D1-Like Receptor Cell Surface Levels

Normal dopaminergic signaling in the renal proximal tubule under conditions of sodium loading leads to natriuresis without the need to increase blood pressure. Low caveolin-1 (CAV1) expression in the kidney is associated with dopaminergic signaling defects and salt sensitive hypertension in both mice and rats. We and others have shown that SNPs in human CAV1 are associated with hypertension. We hypothesize that low CAV1 expression causes increased GRK4 kinase activity due to lack of physical interaction and inhibition of GRK4; thus overactive GRK4 would inactivate the dopamine-1 receptor (D 1 R). We have previously shown that CAV1 reduction, knockdown or knockout in mouse, rat and human renal proximal tubule cells (RPTC) leads to decreased dopamine D 1 -like receptor binding at the proximal tubule cell surface and that re-expression of CAV1 alpha isoform is necessary to rescue this dopaminergic defect. Here we extend those studies by showing that defects in D 1 R cell surface expression due to low CAV1 expression can also be rescued by inhibiting GRK4 in human RPTC using GRK4 siRNA or by introducing heparin (1 μmol/L, 24 hrs) in mouse and rat RPTC. GRK4 siRNA lowered GRK4 expression by greater than 80% in all four human RPTC cell lines and partially restored fluorescent D 1 -like receptor antagonist bodipy630 SKF83566 cell surface binding (scrambled siRNA 66,231±7,286 RFU, GRK4 siRNA 96,206±11,035 RFU, P<0.05, N=28). In RPTC from CAV1 knockout mice, electroporation of heparin restored bodipy630 SKF83566 cell surface binding (vehicle 21,346±3,219 RFU, heparin 58,380±8,032 RFU, P<0.05, N=7). In rat SHR RPTC, CAV1 expression is very low, and the stable transfection of CAV1 partially restored their D 1 -like receptor surface expression. Electroporation of heparin in these CAV1 transfected cells (SHR CAV1) restored bodipy630 SKF83566 cell surface binding back to levels found in WKY RPTC (SHR CAV1 35,241±4,280 RFU, SHR CAV1 heparin 54,218±6,564 RFU, WKY 56,821± 3,546 RFU, P<0.05, N=7). In summary, we show that dopamine D 1 -like receptor defects caused by loss of CAV1 are likely due to increased GRK4 activity and can be restored by either increasing CAV1 expression or decreasing GRK4 expression.

Caveolin-1 increases proinflammatory chemoattractants and blood-retinal barrier breakdown but decreases leukocyte recruitment in inflammation.

Caveolin-1 (Cav-1), the signature protein of caveolae, modulates inflammatory responses, and innate immunity. However, Cav-1's role in retinal inflammation has not been rigorously tested. In this study, we examined the effect of Cav-1 ablation on the sensitivity of the retina to inflammation. Cav-1 knockout (KO) mice were challenged by intravitreal injection of lipopolysaccharide (LPS) and inflammatory cell recruitment was assessed by flow cytometry and immunohistochemistry. Leukostasis was assessed in retinal flatmounts after perfusion with FITC-labeled Concanavalin A (FITC-ConA). Chemoattractants were measured by multiplex immunoassays. Blood-retinal barrier (BRB) breakdown was assessed quantitatively by a FITC-dextran permeability assay. The ratio of extravascular to total immune cells was determined by CD45 immunohistochemistry of retinal flatmounts. Inflammatory challenge resulted in significant blunting of proinflammatory cytokine (monocyte chemoattractant protein-1 [MCP-1/CCL2], CXCL1/KC, IL-6, and IL-1β) responses as well as reduced inflammatory BRB breakdown in Cav-1 KO retinas. Paradoxically, Cav-1 deficiency resulted in significantly increased recruitment of immune cells compared with controls as well as increased leukostasis. A similar ratio of extravascular/total leukocytes were found in Cav-1 KO and wild-type (WT) retinas suggesting that Cav-1 deficient leukocytes were as competent to extravasate as those from WT mice. We found increased levels of circulating immune cells in naïve (not challenged with LPS) Cav-1 KO mice compared with controls. Caveolin-1 paradoxically modulates inflammatory signaling and leukocyte infiltration through distinct mechanisms. We hypothesize that Cav-1 expression may enhance inflammatory signaling while at the same time supporting the physical properties of the BRB.

Massive formation of square array junctions dramatically alters cell shape but does not cause lens opacity in the cav1-KO mice

The wavy square array junctions are composed of truncated aquaporin-0 (AQP0) proteins typically distributed in the deep cortical and nuclear fibers in wild-type lenses. These junctions may help maintain the narrowed extracellular spaces between fiber cells to minimize light scattering. Herein, we investigate the impact of the cell shape changes, due to abnormal formation of extensive square array junctions, on the lens opacification in the caveolin-1 knockout mice. The cav1-KO and wild-type mice at age 1–22 months were used. By light microscopy examinations, cav1-KO lenses at age 1–18 months were transparent in both cortical and nuclear regions, whereas some lenses older than 18 months old exhibited nuclear cataracts. Scanning EM consistently observed the massive formation of ridge-and-valley membrane surfaces in young fibers at approximately 150 μm deep in all cav1-KO lenses studied. In contrast, the typical ridge-and-valleys were only seen in mature fibers deeper than 400 μm in wild-type lenses. The resulting extensive ridge-and-valleys dramatically altered the overall cell shape in cav1-KO lenses. Remarkably, despite dramatic shape changes, these deformed fiber cells remained intact and made close contact with their neighboring cells. By freeze-fracture TEM, ridge-and-valleys exhibited the typical orthogonal arrangement of 6.6 nm square array intramembrane particles and displayed the narrowed extracellular spaces. Immunofluorescence analysis showed that AQP0 C-terminus labeling was significantly decreased in outer cortical fibers in cav1-KO lenses. However, freeze-fracture immunogold labeling showed that the AQP0 C-terminus antibody was sparsely distributed on the wavy square array junctions, suggesting that the cleavage of AQP0 C-termini might not yet be complete. The cav1-KO lenses with nuclear cataracts showed complete cellular breakdown and large globule formation in the lens nucleus. This study suggests that despite dramatic cell shape changes, the massive formation of wavy square array junctions in intact fibers may provide additional adhesive support for maintaining the narrowed extracellular spaces that are crucial for the transparency of cav1-KO lenses.

Peroxynitrite disrupts endothelial caveolae leading to eNOS uncoupling and diminished flow-mediated dilation in coronary arterioles of diabetic patients.

Peroxynitrite (ONOO−) contributes to coronary microvascular dysfunction in diabetes mellitus (DM). We hypothesized that in DM, ONOO− interferes with the function of coronary endothelial caveolae, which plays an important role in nitric oxide (NO)-dependent vasomotor regulation. Flow-mediated dilation (FMD) of coronary arterioles was investigated in DM (n = 41) and non-DM (n = 37) patients undergoing heart surgery. NO-mediated coronary FMD was significantly reduced in DM patients, which was restored by ONOO− scavenger, iron-(III)-tetrakis(N-methyl-4'pyridyl)porphyrin-pentachloride, or uric acid, whereas exogenous ONOO− reduced FMD in non-DM subjects. Immunoelectron microscopy demonstrated an increased 3-nitrotyrosine formation (ONOO−-specific protein nitration) in endothelial plasma membrane in DM, which colocalized with caveolin-1 (Cav-1), the key structural protein of caveolae. The membrane-localized Cav-1 was significantly reduced in DM and also in high glucose–exposed coronary endothelial cells. We also found that DM patients exhibited a decreased number of endothelial caveolae, whereas exogenous ONOO− reduced caveolae number. Correspondingly, pharmacological (methyl-β-cyclodextrin) or genetic disruption of caveolae (Cav-1 knockout mice) abolished coronary FMD, which was rescued by sepiapterin, the stable precursor of NO synthase (NOS) cofactor, tetrahydrobiopterin. Sepiapterin also restored coronary FMD in DM patients. Thus, we propose that ONOO− selectively targets and disrupts endothelial caveolae, which contributes to NOS uncoupling, and, hence, reduced NO-mediated coronary vasodilation in DM patients.

Traumatic brain injury enhances neuroinflammation and lesion volume in caveolin deficient mice

BackgroundTraumatic brain injury (TBI) enhances pro-inflammatory responses, neuronal loss and long-term behavioral deficits. Caveolins (Cavs) are regulators of neuronal and glial survival signaling. Previously we showed that astrocyte and microglial activation is increased in Cav-1 knock-out (KO) mice and that Cav-1 and Cav-3 modulate microglial morphology. We hypothesized that Cavs may regulate cytokine production after TBI.MethodsControlled cortical impact (CCI) model of TBI (3 m/second; 1.0 mm depth; parietal cortex) was performed on wild-type (WT; C57Bl/6), Cav-1 KO, and Cav-3 KO mice. Histology and immunofluorescence microscopy (lesion volume, glia activation), behavioral tests (open field, balance beam, wire grip, T-maze), electrophysiology, electron paramagnetic resonance, membrane fractionation, and multiplex assays were performed. Data were analyzed by unpaired t tests or analysis of variance (ANOVA) with post-hoc Bonferroni’s multiple comparison.ResultsCCI increased cortical and hippocampal injury and decreased expression of MLR-localized synaptic proteins (24 hours), enhanced NADPH oxidase (Nox) activity (24 hours and 1 week), enhanced polysynaptic responses (1 week), and caused hippocampal-dependent learning deficits (3 months). CCI increased brain lesion volume in both Cav-3 and Cav-1 KO mice after 24 hours (P < 0.0001, n = 4; one-way ANOVA). Multiplex array revealed a significant increase in expression of IL-1β, IL-9, IL-10, KC (keratinocyte chemoattractant), and monocyte chemoattractant protein 1 (MCP-1) in ipsilateral hemisphere and IL-9, IL-10, IL-17, and macrophage inflammatory protein 1 alpha (MIP-1α) in contralateral hemisphere of WT mice after 4 hours. CCI increased IL-2, IL-6, KC and MCP-1 in ipsilateral and IL-6, IL-9, IL-17 and KC in contralateral hemispheres in Cav-1 KO and increased all 10 cytokines/chemokines in both hemispheres except for IL-17 (ipsilateral) and MIP-1α (contralateral) in Cav-3 KO (versus WT CCI). Cav-3 KO CCI showed increased IL-1β, IL-9, KC, MCP-1, MIP-1α, and granulocyte-macrophage colony-stimulating factor in ipsilateral and IL-1β, IL-2, IL-9, IL-10, and IL-17 in contralateral hemispheres (P = 0.0005, n = 6; two-way ANOVA) compared to Cav-1 KO CCI.ConclusionCCI caused astrocyte and microglial activation and hippocampal neuronal injury. Cav-1 and Cav-3 KO exhibited enhanced lesion volume and cytokine/chemokine production after CCI. These findings suggest that Cav isoforms may regulate neuroinflammatory responses and neuroprotection following TBI.

Loss of Caveolin-1 Causes Blood–Retinal Barrier Breakdown, Venous Enlargement, and Mural Cell Alteration

Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma.

Abstract 18096: Novel Role of Caveolin-1 in Endothelial Function by Regulating Extracellular SOD Activity and Cu Transporter ATP7A Function

Caveolin (Cav)-enriched lipid raft (C/LR) microdomains play an important role for regulating insulin signaling and endothelial function; however underlying mechanisms is incompletely understood. Vascular extracellular superoxide dismutase (ecSOD), a secretory copper (Cu) enzyme synthesized from vascular smooth muscle cells (VSMCs), preserves nitric oxide (NO)-mediated endothelial function by reducing extracellular superoxide anion (O2•-) levels. We previously demonstrated that Cu transporter ATP7A is required for full activity of ecSOD and that both ecSOD and ATP7A are found in C/LR. However, the functional significance of ATP7A-ecSOD pathway in insulin signaling and C/LR remains unknown. Here we show that in organoid culture of mouse aorta, insulin stimulation increases ecSOD specific activity (47%) and ATP7A expression. In cultured VSMCs, insulin stimulation promotes ATP7A translocation to the C/LR (1.7 fold) where ecSOD is activated by ATP7A through their binding. Furthermore, insulin stimulation promotes ATP7A association with Cav1 as shown by immunofluorescence and co-immunoprecipitation assay. Specific activity of ecSOD, but not Cu/Zn SOD, is significantly decreased (60%) in aorta of Cav1 knockout (KO) mice, indicating that Cav1 or C/LR is required for full ecSOD activity. Of note, ATP7A expression is decreased in aorta from Cav1 KO mice or Cav1-depleted VSMCs with siRNA, which is due to enhanced ATP7A ubiquitination. These results suggest that Cav1 binding to ATP7A at C/LR protects ATP7A from proteosomal degradation, thereby increasing ecSOD activity. Functionally, NO-dependent vasorelaxation induced by acetylcholine, but not NO-independent relaxation induced by sodium nitroprusside, is impaired in mesenteric arteries of Cav1 KO mice vs. wild type mice (P&lt;0.05), which is associated with overproduction of O2•- in the vessels and rescued by gene transfer of ecSOD. In summary, Cav1 plays an essential role for insulin-induced full ecSOD activation by regulating ATP7A translocation and expression. Our studies will provide novel insights into the Cav1-ATP7A-ecSOD axis as a potential therapeutic target for promoting endothelial function against oxidative stress-dependent cardiovascular and metabolic diseases.

Striatin Is a Novel Regulator Of Aldosterone-Mediated Endothelial Cell Activation

Activation of the minerolocorticoid receptor (MR) by aldosterone (ALDO) has been shown to play an important role in inflammatory vascular responses in addition to its well described effects on sodium homeostasis. Steroid responses are mediated by well-known genomic and less known rapid/nongenomic responses. However, characterization of the mechanisms underlying ALDO's rapid/nongenomic actions have been difficult to study and are not clearly understood. We recently reported that in vivo and in vitro activation of MR leads to increases of striatin levels in endothelial cells, aortas and heart tissue (Pojoga, Amer J Hypertens, 2012) and that MR forms a complex with caveolin-1 and striatin within caveolae in endothelial cells. We hypothesized that striatin is a critical intermediary of the rapid effects of ALDO and that striatin serves as a novel link for MR regulation in endothelial cells activation. Endothelial cell activation promotes, among other factors, increased levels of reactive oxygen species (ROS) and protein disulfide isomerase (PDI), a redox modifying enzyme that catalyze disulfide interchange reactions. We studied EA.hy926 cells (EA), a human endothelial cell line that expresses MR, striatin and maintains its caveolae while in culture. We incubated EA cells with ALDO (10–9–10–7M) for 60 min and observed a dose-dependent rise in ROS production (P<0.001, n=4) using the oxidative fluorescent indicator dye 5-6-chloromethyl-2ʼ,7ʼ-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) that peaked at around 10-8M ALDO, an event that was blocked by pre-incubation of EA cells for 30 mins with 1μM canrenoic acid (CA), an MR antagonist (P<0.03, n=3). Time course analyses showed ALDO stimulated ROS responses that increased for up to 3 hours following the addition of ALDO. As there are no known inhibitors for striatin we then used siRNA technology to down regulate striatin in these cells. EA cells were transfected with striatin siRNA and subsequently stimulated with ALDO and ROS production measured. The transfection process itself did not modify baseline levels of ROS significantly, as assessed in cells transfected with scrambled siRNA and non-transfected cells, which had nearly identical ROS levels, basally and in response to ALDO. In addition, in the presence of lower levels of striatin protein the ALDO-stimulated ROS response was abrogated, supporting the concept that striatin is necessary for the rapid effects of ALDO. We also measured phosphorylated ERK-1/2 (pERK) levels that peaked within 10 minutes in EA as estimated by western blot analyses. Consistent with these observations pre-incubation of EA cells with 10-6M PD0325901, a selective MEK-1/2 inhibitor was associated with greater than 90% reduction of the ALDO-stimulated ROS responses (7244.3±497 vs 4386.6±586 RFU, P<0.02, n=3). Qualitatively similar responses were observed using another MEK inhibitor, U0126 [10-5M] (P<0.01, n=3). We then tested the effects of ALDO on PDI secretion. Incubation of EA cells with ALDO (10-7M) led to PDI increases when compared to vehicle treated cells (P<0.01, n=3). We also tested the effects of low levels of striatin using siRNA on PDI activity in EA cells. We found that PDI secretion was reduced by 62% in striatin knockdown conditions. We then tested the effects of Methyl-β-cyclodextrin to disrupt caveolae in these cells and observed a blunted 10nM ALDO–stimulated PDI response (530±117 to 215±99 RFU/mg protein, n=3, P<0.01). We then isolated early cultures of mouse aortic endothelial cells (MAEC) from endothelial-specific caveolin-1 knockout mice and measured PDI activity following 24 hrs of incubation in 0.4% fetal bovine serum. Our results show that MAEC from caveolin-1 knockout mice had lower PDI secretion when compared to cells from WT mice (99.4±16 vs 129.9±35, n=5, P<0.03). These results suggest that striatin is a novel mediator for ALDO's rapid effects on PDI and ROS, thereby suggesting a unique level of interaction between the MR and striatin in endothelial cell activation. Supported by NIH R01HL090632 (AR), R01HL104032 (LHP) and R01HL096518 (JRR). Disclosures:No relevant conflicts of interest to declare.