Caveolin-1 Knockout Mice Research Articles

Abstract PO2-28-01: Deleting caveolin-1 in epithelial cells increases doxorubicin sensitivity in advance stage of breast cancer

Abstract Caveolin-1 (Cav-1) is a lipid raft protein with a dual role, serving as a tumor suppressor and a promoter. Studies have unveiled Cav-1’s significance in breast cancer progression, impacting various aspects, including cell proliferation, apoptosis, autophagy, invasion, migration, and metastasis. Previously, our research demonstrated that the knockout (KO) of Cav-1 from epithelial cells reduced lung metastasis originating from primary tumors. In this study, we sought to investigate whether Cav-1 KO in epithelial cells increases sensitivity to doxorubicin (Dox) using both in vitro and in vivo studies. In our in vitro study, we observed reduced cell numbers in doxorubicin-treated Cav-1 KO cells compared to Dox-treated 4T1 cells (wild type). Flow cytometry-based cell cycle assay showed a significant increase in G2M phase arrest in Dox-treated Cav-1 KO cells compared with wild-type, confirming reduced cell growth and proliferation. To validate the increased sensitivity to Dox in vivo, we used a syngeneic tumor model using Cav-1 KO and 4T1 wild-type cells injected into the mammary fat of female Balb/c mice intraperitoneally. Subsequently, after one week of tumor cell injection, both groups received Dox at a dose of 8 mg/kg body weight/week for 3 weeks (a cumulative dose of 24 mg/kg body weight). Primary tumor sizes exhibited a significant reduction in Dox-treated mice, and this was further reduced in Cav-1 KO mice compared to the wild type. To quantify lung metastasis, we explanted the mice’s lungs and plated dissociated single cells in complete RPMI media supplemented with 6-thioguanine (6-GT). The number of 6-TG-resistant 4T1 metastatic cells was significantly lower in the lungs' suspension of Cav-1 KO mice treated with Dox compared to wild-type Dox-treated mice. Our findings confirm that knockout of Cav-1 increased the sensitivity of Dox treatment and inhibited breast cancer lung metastasis in Balb/c mice. Our ongoing analysis of the molecular pathways seeks to elucidate the underlying mechanism. Citation Format: Abhishek Pandit, Dhirendra Singh, Rashmi Pathak, Shilpa Thota, Joseph Francis. Deleting caveolin-1 in epithelial cells increases doxorubicin sensitivity in advance stage of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-28-01.

Arachidonic Acid Metabolism by Cyp4A Modulates Caveolae-Mediated Transcytotic LPS Transport in Mouse Jejunum

Arachidonic acid (AA) is metabolized not only by cyclooxygenases and lipoxygenases, but also by cytochrome P-450 4A family (Cyp4A) which is an androgen-driven enzyme that produces 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is an endogenous ligand for GPR75, which is highly associated with pathogenesis of obesity. Since metabolic endotoxemia is implicated in the induction of obesity, and the gut lumen is the primary source of endotoxin (lipopolysaccharides [LPS]), we hypothesized that AA metabolism may affect LPS entry through the small intestine. Furthermore, we have found that increased LPS transport during long-chain fatty acid (LCFA) absorption is abolished in caveolin-1 (Cav1) knockout (KO) jejunum, suggesting the involvement of caveolae-mediated endocytosis. We thus examined the effects of AA on LPS transport in murine jejunum. FITC-LPS (10 μg/ml) or FD4 (0.1 mM) was applied to the mucosal (m) bath of Ussing chambered muscle-stripped jejunum from male C57Bl6 wild type (WT) and Cav1 KO mice with measurement of the serosal (s) appearance of FITC-LPS or FD4 and transepithelial electrical resistance (TER). We examined the effects of the luminal application (10 mM) of oleic acid (OA), AA, eicosapentaenoic acid (EPA), or docosahexaenoic (DHA) with taurocholic acid (0.1 mM) on these parameters with or without the lipid raft inhibitor methyl-β-cyclodextrin (MβCD, 0.1 mM) or Cyp4A inhibitor HET0016 (10 μM). In another study, male and female WT and Cav1 KO mice were fed a standard diet (SD), or a diet enriched in saturated LCFA fat and sugar (LCD) for 6 wks. Luminal addition of OA and AA, but not EPA nor DHA, increased LPS m-to-s movement in WT jejunal mucosa. AA also increased FD4 m-to-s movement with no effect on TER. AA-induced LPS and FD4 movements were inhibited by luminal application of MβCD or HET0016, suggesting the involvement of lipid rafts and Cyp4A activity. Luminal AA had no effect on LPS and FD4 m-to-s movement in KO jejunum. LPS and FD4 m-to-s movement were also increased by luminal application of 20-HETE (0.3 and 1 μM). Immunostaining revealed that GPR75 was expressed on the plasma membranes of jejunal villous but not crypt cells. LCD induced obesity in WT, not in KO, with increased blood glucose levels, perigonadal fat weight, and liver weight, with changes more evident in male WT than in female WT. LCD increased LPS m-to-s movement in WT male jejunum, not in KO jejunum, which was inhibited by luminal addition of MβCD or HET0016. These results suggest that luminal AA increases 20-HETE production by Cyp4A, then activates Cav1-mediated endocytosis which in turn enhances LPS transport in the small intestine, possibly via GPR75 activation. The AA-Cyp4A-20-HETE-GPR75 system may be a new therapeutic target for metabolic endotoxemia associated with diet-induced obesity, especially in males. VA Merit Review and DoD grants. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High Fat Diet-Induced Constitutive Caveolin-1 Activation in Mouse Jejunum Promotes LPS Transport with Consequent Metabolic Syndrome

Lipopolysaccharides (LPS) entry from the intestinal lumen to the circulation is associated with systemic inflammation. We have found that LPS is transcellularly transported from the gut lumen to the portal vein (PV) in the presence of luminal long-chain fatty acid (LCFA) via CD36- and lipid raft-mediated pathways. Furthermore, LCFA-induced LPS transport is abolished in caveolin-1 (Cav1) knockout (KO) jejunum, suggesting the involvement of caveolae-mediated endocytosis. Here, we examined the effects of diets of varying FA composition on the development of the metabolic syndrome (MetS) and on LPS transport in murine jejunum. Male and female C57Bl6 wild type (WT) and Cav1 KO mice were fed a standard high CHO, high protein, low fat diet (SD), or one of 3 high fat-high sugar diets (34% sucrose, 42% fat) that were enriched in saturated LCFA (LCD), medium-chain FA (MCD) and polyunsaturated FA (PUD) for 6 wks. FITC-LPS was added to the mucosal (m) bath of Ussing-chambered muscle-stripped jejunum. Serosal (s) appearance of FITC-LPS was measured with or without luminal addition of oleic acid (OA, 10 mM)/taurocholic acid (TCA, 0.1 mM). FD4 m-to-s permeability and transepithelial electrical resistance (TER) were also measured. PV LPS levels were measured by a limulus amœbocyte lysate test kit. LCD-induced MetS (increased body weight, blood glucose levels, perigonadal fat weight, and liver weight), predominantly in male, whereas MCD and PUD did not induce MetS in WT. Nevertheless, LCD failed to induce MetS in KO, confirming that Cav1 KO mice are resistant to diet-induced obesity. PV LPS levels were elevated only in male WT LCD with no change in KO. There was no FITC-LPS m-to-s movement in WT SD jejunum, whereas luminal addition of OA/TCA increased LPS m-to-s movement. Interestingly, FITC-LPS m-to-s movement was increased in jejunum from WT LCD with no further increase after OA/TCA challenge, suggesting that LCD constitutively upregulated LPS transport. FD4 permeability was significantly higher in WT LCD than in WT SD, but not in KO. MCD but no other diet somewhat increased TER, compared with WT SD or KO SD. Constitutive LPS transport in WT LCD jejunum was abolished by luminal addition of the lipid raft inhibitor methyl-β-cyclodextrin (0.1 mM), suggesting the involvement of transcytotic rather than paracellular transport mechanisms. No LPS transport was observed in KO SD and KO LCD jejunum, further suggesting that LCD-induced LPS transport is Cav1 dependent. These results suggest that LCD constitutively activates Cav1-mediated endocytotic LPS transport in the small intestine, resulting in metabolic endotoxemia and the MetS. Cav1-mediated LPS transport is a promising therapeutic target for diet-induced, LPS-associated MetS. VA Merit Review and DoD grants. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Buyang Huanwu decoction promotes angiogenesis after cerebral ischemia through modulating caveolin-1-mediated exosome MALAT1/YAP1/HIF-1α axis

BackgroundAngiogenesis is an effective method for promoting neurological function recovery after cerebral ischemia (CI). Buyang Huanwu decoction (BHD) is a traditional Chinese medicinal recipe that is frequently employed for CI treatment. Previous investigations have validated that it promotes angiogenesis following CI. Nevertheless, the precise mechanism by which it does this has yet to be completely understood. ObjectiveThis study aims to examine the underlying mechanism through which BHD facilitates angiogenesis following CI by regulating the exosomal MALAT1/YAP1/HIF-1α signaling axis, specifically via the involvement of caveolin-1 (Cav1), an endocytosis-associated protein. MethodsA CI model was created using middle cerebral artery occlusion (MCAO). Following the administration of multiple doses of BHD, various parameters, including the neurobehavioral score, pathological damage, and angiogenesis, were assessed in each group of mice to identify the optimal dosage of BHD for treating CI. The molecular processes underlying the angiogenic implications of BHD following CI were investigated exhaustively by employing single-cell sequencing. Finally, the involvement of Cav1 was confirmed in Cav1 knockout mice and Cav1-silenced stably transfected strains to validate the mechanism by which BHD increases angiogenesis following CI. ResultsBHD could promote angiogenesis after CI. Single-cell sequencing results suggested that its potential mechanism of action might be connected with Cav1 and the exosomal MALAT1/YAP1/HIF-1α signaling axis. BHD could promote angiogenesis after CI by regulating the exosomal MALAT1/YAP1/HIF-1α axis through Cav1, as validated in vivo and in vitro experiments. Accordingly, Cav1 may be a key target of BHD in promoting angiogenesis after CI. ConclusionThis investigation represents the initial attempt to comprehensively ascertain the underlying mechanism of action of BHD in treating CI using single-cell sequencing, gene-knockout mice, and stable transfected cell lines, potentially associated with the modulation of the exosomal MALAT1/YAP1/HIF-1α axis by Cav1. Our findings offer novel empirical evidence for unraveling the regulatory pathways through which Cav1 participates in angiogenesis following CI and shed light on the potential mechanisms of BHD.

The Cavin-1/Caveolin-1 interaction attenuates BMP/Smad signaling in pulmonary hypertension by interfering with BMPR2/Caveolin-1 binding

Caveolin-1 (CAV1) and Cavin-1 are components of caveolae, both of which interact with and influence the composition and stabilization of caveolae. CAV1 is associated with pulmonary arterial hypertension (PAH). Bone morphogenetic protein (BMP) type 2 receptor (BMPR2) is localized in caveolae associated with CAV1 and is commonly mutated in PAH. Here, we show that BMP/Smad signaling is suppressed in pulmonary microvascular endothelial cells of CAV1 knockout mice. Moreover, hypoxia enhances the CAV1/Cavin-1 interaction but attenuates the CAV1/BMPR2 interaction and BMPR2 membrane localization in pulmonary artery endothelial cells (PAECs). Both Cavin-1 and BMPR2 are associated with the CAV1 scaffolding domain. Cavin-1 decreases BMPR2 membrane localization by inhibiting the interaction of BMPR2 with CAV1 and reduces Smad signal transduction in PAECs. Furthermore, Cavin-1 knockdown is resistant to CAV1-induced pulmonary hypertension in vivo. We demonstrate that the Cavin-1/Caveolin-1 interaction attenuates BMP/Smad signaling and is a promising target for the treatment of PAH.

Caveolin-1 deficiency alleviates palmitate-induced intracellular lipid accumulation and inflammation in pancreatic β cells.

Lipotoxicity-induced pancreatic β cell damage is a strong predictor of type 2 diabetes mellitus (T2DM). Our previous work showed that Caveolin-1 (Cav-1) depletion decreased β-cell apoptosis and improved β-cell viability. Further microarray analysis indicated significant changes in the expression of genes related to fatty acid metabolism and inflammation. The objective of this study was to explore the role of Cav-1 in intracellular lipid accumulation and inflammation in β cells under lipotoxic conditions. Here, we established a β-cell-specific Cav-1 knockout (β-Cav-1 KO) mouse model and a CAV-1 depleted β cell line (NIT-1). We found that Cav-1 silencing significantly reduced palmitate (PA)-induced intracellular triglyceride (TG) accumulation and decreased proinflammatory factor expression in both the mouse and cell models. Further mechanistic investigation revealed that amelioration of lipid metabolism was achieved through the downregulation of lipogenic markers (SREBP-1c, FAS and ACC) and upregulation of a fatty acid oxidation marker (CPT-1). Meanwhile, decrease of inflammatory cytokines (IL-6, TNF-α, and IL-1β) secretion was found with the involvement of the IKKβ/NF-κB signaling pathways. Our findings suggest that Cav-1 is of considerable importance in regulating lipotoxicity-induced β-cell intracellular lipid accumulation and inflammation.

Caveolin-1 forms a complex with P2X7 receptor and tunes P2X7-mediated ATP signaling in mouse bone marrow-derived macrophages.

The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger proinflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca2+] and sustained decrease in cytosolic [K+]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca2+] increase and cytosolic [K+] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca2+ influx and K+ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macropore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1β secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these datasets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1β secretion and pyroptosis.NEW & NOTEWORTHY In bone marrow-derived macrophages, Cav-1 suppresses the macropore formation of P2X7 receptors through their direct or indirect interactions, resulting in reduced membrane permeability of cations (Ca2+ and K+) and large cell-impermeable dye (TO-PRO3) induced by ATP. Cav-1 also inhibits ATP-induced IL-1β secretion, ROS production, GSDMD cleavage, and pyroptosis. Cav-1 contributes to the maintenance of proper immune responses by finely tuning IL-1β secretion and cell death in macrophages.

Caveolin-1 is critical for hepatic iron storage capacity in the development of nonalcoholic fatty liver disease

BackgroundNonalcoholic fatty liver disease (NAFLD) is associated with disordered lipid and iron metabolism. Our previous study has substantiated the pivotal role of Caveolin-1 (Cav-1) in protecting hepatocytes and mediating iron metabolism in the liver. This study aimed to explore the specific mechanisms underlying the regulation of iron metabolism by Cav-1 in NAFLD.MethodsHepatocyte-specific Cav-1 overexpression mice and knockout mice were used in this study. Cav-1-knockdown of RAW264.7 cells and mouse primary hepatocytes were performed to verify the changes in vitro. Moreover, a high-fat diet and palmitic acid plus oleic acid treatment were utilized to construct a NAFLD model in vivo and in vitro, respectively, while a high-iron diet was used to construct an in vivo iron overload model. Besides, iron concentration, the expression of Cav-1 and iron metabolism-related proteins in liver tissue or serum were detected using iron assay kit, Prussian blue staining, Western blotting, immunofluorescence staining, immunohistochemical staining and ELISA. The related indicators of lipid metabolism and oxidative stress were evaluated by the corresponding reagent kit and staining.ResultsSignificant disorder of lipid and iron metabolism occurred in NAFLD. The expression of Cav-1 was decreased in NAFLD hepatocytes (P < 0.05), accompanied by iron metabolism disorder. Cav-1 enhanced the iron storage capacity of hepatocytes by activating the ferritin light chain/ferritin heavy chain pathway in NAFLD, subsequently alleviating the oxidative stress induced by excess ferrous ions in the liver. Further, CD68+CD163+ macrophages expressing Cav-1 were found to accelerate iron accumulation in the liver, which was contrary to the effect of Cav-1 in hepatocytes. Positive correlations were also observed between the serum Cav-1 concentration and the serum iron-related protein levels in NAFLD patients and healthy volunteers (P < 0.05).ConclusionsThese findings confirm that Cav-1 is an essential target protein that regulates iron and lipid metabolic homeostasis. It is a pivotal molecule for predicting and protecting against the development of NAFLD.

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-β-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

Buyang Huanwu Decoction alleviates cerebral ischemic injury through modulating caveolin-1-mediated mitochondrial quality control.

Introduction: Mitochondrial quality control (MQC) is an important mechanism of neural repair after cerebral ischemia (CI). Recent studies have shown that caveolin-1 (Cav-1) is an important signaling molecule in the process of CI injury, but its mechanism of regulating MQC after CI is still unclear. Buyang Huanwu Decoction (BHD) is a classic traditional Chinese medicine formula that is often used to treat CI. Unfortunately, its mechanism of action is still obscure. Methods: In this study, we tested the hypothesis that BHD can regulate MQC through Cav-1 and exert an anti-cerebral ischemia injury effect. We used Cav-1 knockout mice and their homologous wild-type mice, replicated middle cerebral artery occlusion (MCAO) model and BHD intervention. Neurobehavioral scores and pathological detection were used to evaluate neurological function and neuron damage, transmission electron microscopy and enzymology detection of mitochondrial damage. Finally, western blot and RT-qPCR expression of MQC-related molecules were tested. Results: After CI, mice showed neurologic impairment, neuronal damage, and significant destruction of mitochondrial morphology and function, and MQC was imbalanced. Cav-1 deletion aggravated the damage to neurological function, neurons, mitochondrial morphology and mitochondrial function after CI, aggravated the imbalance of mitochondrial dynamics, and inhibited mitophagy and biosynthesis. BHD can maintain MQC homeostasis after CI through Cav-1 and improve CI injury. Discussion: Cav-1 can affect CI injury by regulating MQC, and this mechanism may be another target of BHD for anti-cerebral ischemia injury.

Reduced endothelial caveolin-1 underlies deficits in brain insulin signalling in type 2 diabetes.

Patients with type 2 diabetes exhibit severe impairments in insulin signalling in the brain and are five times more likely to develop Alzheimer's disease. However, what leads to these impairments is not fully understood. Here, we show reduced expression of endothelial cell caveolin-1 (Cav-1) in the db/db (Leprdb) mouse model of type 2 diabetes. This reduction correlated with alterations in insulin receptor expression and signalling in brain microvessels as well as brain parenchyma. These findings were recapitulated in the brains of endothelial cell-specific Cav-1 knock-out (Tie2Cre; Cav-1fl/fl) mice. Lack of Cav-1 in endothelial cells led to reduced response to insulin as well as reduced insulin uptake. Furthermore, we observed that Cav-1 was necessary for the stabilization of insulin receptors in lipid rafts. Interactome analysis revealed that insulin receptor interacts with Cav-1 and caveolae-associated proteins, insulin-degrading enzyme and the tight junction protein Zonula Occludence-1 in brain endothelial cells. Restoration of Cav-1 in Cav-1 knock-out brain endothelial cells rescued insulin receptor expression and localization. Overall, these results suggest that Cav-1 regulates insulin signalling and uptake by brain endothelial cells by modulating IR-α and IR-β localization and function in lipid rafts. Furthermore, depletion of endothelial cell-specific Cav-1 and the resulting impairment in insulin transport leads to alteration in insulin signalling in the brain parenchyma of type 2 diabetics.

Critical role of caveolin-1 in intestinal ischemia reperfusion by inhibiting protein kinase C βII.

Intestinal ischemia reperfusion (I/R) is a common clinical pathological process. We previously reported that pharmacological inhibition of protein kinase C (PKC) βII with a specific inhibitor attenuated gut I/R injury. However, the endogenous regulatory mechanism of PKCβII inactivation is still unclear. Here, we explored the critical role of caveolin-1 (Cav1) in protecting against intestinal I/R injury by regulating PKCβII inactivation. PKCβII translocated to caveolae and bound with Cav1 after intestinal I/R. Cav1 was highly expressed in the intestine of mice with I/R and IEC-6cells stimulated with hypoxia/reoxygenation (H/R). Cav1-knockout (KO) mice suffered from worse intestinal injury after I/R than wild-type (WT) mice and showed extremely low survival due to exacerbated systemic inflammatory response syndrome (SIRS) and remote organ (lung and liver) injury. Cav1 deficiency resulted in excessive PKCβII activation and increased oxidative stress and apoptosis after intestinal I/R. Full-length Cav1 scaffolding domain peptide (CSP) suppressed excessive PKCβII activation and protected the gut against oxidative stress and apoptosis due to I/R injury. In summary, Cav1 could regulate PKCβII endogenous inactivation to alleviate intestinal I/R injury. This finding may represent a novel therapeutic strategy for the prevention and treatment of intestinal I/R injury.

Increased TMEM16A-Mediated Ca2+-Activated Cl- Currents in Portal Vein Smooth Muscle Cells of Caveolin 1-Deficient Mice.

Ca2+-activated Cl- (ClCa) channels regulate membrane excitability and myogenic tone in vascular smooth muscles. TMEM16A-coding proteins are mainly responsible for functional ClCa channels in vascular smooth muscles, including portal vein smooth muscles (PVSMs). Caveolae are cholesterol-rich and Ω-shaped invaginations on the plasma membrane that structurally contributes to effective signal transduction. Caveolin 1 (Cav1) accumulates in caveolae to form functional complexes among receptors, ion channels, and kinases. The present study examined the functional roles of Cav1 in the expression and activity of ClCa channels in the portal vein smooth muscle cells (PVSMCs) of wild-type (WT) and Cav1-knockout (KO) mice. Contractile experiments revealed that the amplitude of spontaneous PVSM contractions was larger in Cav1-KO mice than WT mice. Under whole-cell patch-clamp configurations, ClCa currents were markedly inhibited by 1 µM Ani9 (a selective TMEM16A ClCa channel blocker) in WT and Cav1-KO PVSMCs. However, Ani9-sensitive ClCa currents were significantly larger in Cav1-KO PVSMCs than in WT PVSMCs. Expression analyses showed that TMEM16A expression levels were higher in Cav1-KO PVSMs than in WT PVSMs. Therefore, the caveolar structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated ClCa channels in vascular smooth muscle cells.

Caveolin-1 is a primary determinant of endothelial stiffening associated with dyslipidemia, disturbed flow, and ageing

Endothelial stiffness is emerging as a major determinant in endothelial function. Here, we analyzed the role of caveolin-1 (Cav-1) in determining the stiffness of endothelial cells (EC) exposed to oxidized low density lipoprotein (oxLDL) under static and hemodynamic conditions in vitro and of aortic endothelium in vivo in mouse models of dyslipidemia and ageing. Elastic moduli of cultured ECs and of the endothelial monolayer of freshly isolated mouse aortas were measured using atomic force microscopy (AFM). We found that a loss of Cav-1 abrogates the uptake of oxLDL and oxLDL-induced endothelial stiffening, as well as endothelial stiffening induced by disturbed flow (DF), which was also oxLDL dependent. Mechanistically, Cav-1 is required for the expression of CD36 (cluster of differentiation 36) scavenger receptor. Genetic deletion of Cav-1 abrogated endothelial stiffening observed in the DF region of the aortic arch, and induced by a high fat diet (4–6 weeks) and significantly blunted endothelial stiffening that develops with advanced age. This effect was independent of stiffening of the sub-endothelium layer. Additionally, Cav-1 expression significantly increased with age. No differences in elastic modulus were observed between the sexes in advanced aged wild type and Cav-1 knockout mice. Taken together, this study demonstrates that Cav-1 plays a critical role in endothelial stiffening induced by oxLDL in vitro and by dyslipidemia, disturbed flow and ageing in vivo.

Abstract 122: Effect Of Tie2-cre Endothelial Caveolin-1 Deficiency Mice On Murine Cardiovascular And Metabolic Phenotypes

In humans, caveolin-1 (cav-1) gene polymorphisms confer a metabolic syndrome (MetS) phenotype and higher aldosterone (Aldo) levels. Further, global cav-1 knock out (KO) mice recapitulate the MetS phenotype, with both cardiovascular (CV) and metabolic dysfunction. We have shown that vascular dysfunction in the cav-1 KO is dissociated from the metabolic phenotype and is mediated by an endothelial crosstalk between cav-1 and the Aldo-mineralocorticoid receptor (MR) signaling pathway. Hence, we hypothesized endothelial cav-1 deficiency in mice is a major contributor to CV (but not metabolic) dysfunction.To assess this hypothesis, we generated Tie-2 mediated endothelium-specific cav-1 KO (ECKO) and appropriate genetic wild type (WT) control mice and studied their metabolic and CV traits in response to 1) changes in dietary salt intake (0.03% (LS) vs. 1.6% Na + (HS)) and 2) 3-weeks of Aldo infusion (200μg/kg/day) during sodium loading. Our results showed no difference in glucose, lipids and HOMA-IR in ECKO vs. WT mice, irrespective of the dietary intervention. However, on a HS (but not a LS) diet, ECKO vs. WT mice displayed similar CV dysfunction as previously shown in the full cav-1 KO: increased Aldo levels (73.7±11.1 vs 47.8±4.7ng/dl, p&lt;0.05) and systolic BP (116.4±1.5 vs111±1.4 mmHg, p&lt;0.015). Further, ECKO vs. WT mice had increased cardiac MR, eNOS, PAI-1 and CD68 expression (p&lt;0.05 for each) as well as left ventricular hypertrophy (echocardiogram, p&lt;0.05). In addition, 3-week HS diet led to higher albuminuria (p&lt;0.05) and kidney/body weight ratios in ECKO vs. WT (7.2±0.2 vs 6.3±0.1). Surprisingly, as compared to placebo, Aldo infusion induced significant CV and renal damage only in the WT, but not in the ECKO. However, as compared to WT, Aldo infused ECKO had increased cardiac hypertrophy (5.9±0.2 vs 5.2±0.1) and molecular markers noted above (p&lt;0.05 for each).In conclusion, lack of endothelial cav-1 in the mouse segregated the metabolic and CV phenotypes, and the associated CV phenotype resembles that described for the global cav-1 KO. Further, the CV and renal damage in the ECKO at baseline cannot be further enhanced by Aldo infusion. These data confirm our hypothesis that endothelial cav-1 is a major player in CV but not metabolic homeostasis.

Local Ca2+ Signals within Caveolae Cause Nuclear Translocation of CaMK1α in Mouse Vascular Smooth Muscle Cells.

An increase in intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+-sensitive enzymes such as Ca2+/calmodulin-dependent kinases (CaMK) and induces gene transcription in various types of cells. This signaling pathway is called excitation-transcription (E-T) coupling. Recently, we have revealed that a L-type Ca2+ channel/CaMK kinase (CaMKK) 2/CaMK1α complex located within caveolae in vascular smooth muscle cells (SMCs) can convert [Ca2+]i changes to gene transcription profiles that are related to chemotaxis. Although CaMK1α is expected to be the key molecular identity that can transport Ca2+ signals originated within caveolae to the nucleus, data sets directly proving this scheme are lacking. In this study, multicolor fluorescence imaging methods were utilized to address this question. Live cell imaging using mouse primary aortic SMCs revealed that CaMK1α can translocate from the cytosol to the nucleus; and that this movement was blocked by nifedipine or a CaMKK inhibitor, STO609. Experiments using two types of Ca2+ chelators, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), combined with caveolin-1 knockout (cav1-KO) mice showed that local Ca2+ events within caveolae are required to trigger this CaMK1α nuclear translocation. Importantly, overexpression of cav1 in isolated cav1-KO myocytes recovered the CaMK1α translocation. In SMCs freshly isolated from mesenteric arteries, CaMK1α was localized mainly within caveolae in the resting state. Membrane depolarization induced both nuclear translocation and phosphorylation of CaMK1α. These responses were inhibited by nifedipine, STO609, cav1-KO, or BAPTA. These new findings strongly suggest that CaMK1α can transduce Ca2+ signaling generated within or very near caveolae to the nucleus and thus, promote E-T coupling.

Fyn-kinase and caveolin-1 in the alveolar epithelial junctional adherence complex contribute to the early stages of pulmonary fibrosis

Current pathophysiological findings indicate that damage to the alveolar epithelium plays a decisive role in the development of idiopathic pulmonary fibrosis (IPF). The available pharmacological interventions (i.e., oral pirfenidone and nintedanib) only slow down progression of the disease, but do not offer a cure. In order to develop new drug candidates, the pathophysiology of IPF needs to be better understood on a molecular level. It has previously been reported that a loss of caveolin-1 (Cav-1) contributes to profibrotic processes by causing reduced alveolar barrier function and fibrosis-like alterations of the lung-parenchyma. Conversely, overexpression of caveolin-1 appears to counteract the development of fibrosis by inhibiting the inflammasome NLRP3 and the associated expression of interleukin-1β. In this study, the interaction between Fyn-kinase and caveolin-1 in the alveolar epithelium of various bleomycin (BLM)/TGF-β damage models using precision-cut lung slices (PCLS), wildtype (WT) and caveolin-1 knockout (KO) mice as well as the human NCI-H441 cell line, were investigated. In WT mouse lung tissues, strong signals for Fyn-kinase were detected in alveolar epithelial type I cells, whereas in caveolin-1 KO animals, expression shifted to alveolar epithelial type II cells. Caveolin-1 and Fyn-kinase were found to be co-localized in isolated lipid rafts of NCI-H441 cell membrane fractions. These findings were corroborated by co-immunoprecipitation studies in which a co-localization of Cav-1 and Fyn-kinase was detected in the cell membrane of the alveolar epithelium. After TGF-β and BLM-induced damage to the alveolar epithelium both in PCLS and cell culture experiments, a decrease in caveolin-1 and Fyn-kinase was found. Furthermore, TEER (transepithelial electrical resistance) measurements indicated that TGF-β and BLM have a damaging effect on cell-cell contacts and thus impair the barrier function in NCI-H441 cell monolayers. This effect was attenuated after co-incubation with the Fyn-kinase inhibitor, PP-2. Our data suggest an involvement of Fyn-kinase and caveolin-1 in TGF-β/bleomycin-induced impairment of alveolar barrier function and thus a possible role in the early stages of pulmonary fibrosis. Fyn-kinase and/or its complex with caveolin-1 might, therefore, be novel therapeutic targets in IPF.

LPS transport during fat absorption is inhibited in caveolin‐1 knockout murine jejunum

Lipopolysaccharides (LPS) entry from the intestinal lumen to the circulation is associated with systemic inflammation. We have found that LPS is transcellularly transported to portal vein during long‐chain fatty acid (LCFA) exposure via CD36‐ and lipid raft‐mediated pathways in rats, suggesting the involvement of caveolae‐mediated endocytosis. We thus examined LPS transport in caveolin‐1 (Cav1) knockout (KO) murine jejunum.FITC‐LPS was applied to the mucosal bath of Ussing chambered muscle‐stripped jejunal mucosa‐submucosa preparations of Cav1 KO and wild type (WT) mice. Serosal appearance of FITC‐LPS was measured with or without luminal application of oleic acid (OA, 30 mM) with taurocholic acid (TCA, 0.1 mM), or medium‐chain fatty acid sodium caprate (C10, 10 mM).Luminal application of OA/TCA increased FITC‐LPS m‐to‐s transport in WT jejunum, inhibited by the CD36 inhibitor sulfosuccinimidyl oleate (SSO, 0.1 mM) or the lipid raft inhibitor methyl‐β‐cyclodextrin (MβCD, 0.1 mM), not by the clathrin inhibitor chlorpromazine (0.1 mM) or Pitstop2 (30 µM), suggesting that LCFA‐induced LPS transport is mediated by caveolae‐mediated endocytosis, not by clathrin‐mediated endocytosis. In contrast, OA/TCA‐induced FITC‐LPS transport was abolished in Cav1 KO jejunum. Nevertheless, luminal application of C10 increased FITC‐LPS transport both in WT and Cav1 KO jejuna without transepithelial electrical resistance changes, suggesting that C10 enhances transcellular LPS transport via caveolin‐independent endocytosis in the jejunum.These results suggest that LPS transport during LCFA exposure is mediated by Cav1‐mediated endocytosis, whereas MCFA‐induced LPS transport is likely via clathrin‐mediated endocytosis. Modulation of epithelial endocytosis may be a new therapeutic target for LPS‐associated diseases mediated by dietary fatty acids, including metabolic syndrome.

Genetic deletion of endothelial Caveolin‐1 is protective against metabolic disease

In metabolic disease, the endothelium is exposed to a substantial increase in fatty acid concentrations creating a pathological role for endothelial cells (ECs) as lipid metabolizing cells. One mechanism whereby lipids may enter ECs is by caveolin‐1 (Cav1)‐mediated endocytosis. Because both ECs and adipocytes both have abundant Cav1 and both can readily take up lipids, we wanted to differentiate the role of Cav1 between the two cell types. For these reasons, we used an inducible EC‐specific Cav1 knockout mouse (Cav1fl/fl/Cdh5‐CreERT2+). Using these mice, we induced Cav1 genetic deletion from endothelium at 6 weeks, and began a high fat diet (HFD; 60% fat) to induce metabolic disease at 8 weeks. Normal chow fed littermates were used as controls. All mice were examined at 20 weeks (i.e., 12 weeks of HFD). Thus, we had four cohorts: Cre+ HFD, Cre‐ HFD, Cre+ normal chow, and Cre‐ normal chow all injected with tamoxifen. All mice regardless of genotype had comparable weight gain within diet groups, and gonadal fat pad mass. Food consumption was similar between groups, and electron microscopy was used to confirm presence of caveola in adipocytes, but not endothelium. Initial experiments examining plasma lipids found HFD Cre+ mice had both increased serum cholesterol (175mg/dL) and LDL (75mg/dL) compared to HFD Cre‐ controls (cholesterol, 125mg/dL; LDL, 60mg/dL). When these mice were challenged with a bolus of insulin, the blood glucose of Cre+ HFD mice dropped significantly resembling an insulin tolerance curve from normal chow fed controls. Similarly, Cre+ HFD mice were protected from hyperglycemia after bolus glucose injections, resulting in a glucose tolerance phenotype similar to that of a normal chow fed mouse (HFD Cre+ AUC 3000 vs. HFD Cre‐ AUC 60000). There was no difference in Cre+ or Cre‐ mice on normal chow diets with serum lipids or after challenge with insulin or glucose. It isn’t clear why the insulin and glucose protection correspond with significantly increased lipids in the Cre+ HFD mice. For these reasons, we began investigating the possible mechanisms with primary cultures of Human Adipose Microvascular Endothelial Cells (HAMECs) and knocked down Cav1 with siRNA. In monolayers of HAMEC without any other cell type present, we did not observe altered lipid uptake after addition of 50µM of exogenous fatty acids with a 70% knockdown of Cav1 protein. Additionally, HAMECs seahorse assays were used to determine if there were changes in metabolic function when Cav1 was knocked down. This work provides initial evidence for the unique metabolic roles of endothelial Cav1 during periods of high lipid circulation.

A molecular complex of Cav1.2/CaMKK2/CaMK1a in caveolae is responsible for vascular remodeling via excitation–transcription coupling

Elevation of intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitation–transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.