Absence Of Cav-1 Research Articles

Mechanism for preservation of cone pathways in the absence of CaV1.4 Ca2+ signals

Mechanism for preservation of cone pathways in the absence of CaV1.4 Ca2+ signals

Two zebrafish cacna1s loss-of-function variants provide models of mild and severe CACNA1S-related myopathy.

CACNA1S-related myopathy, due to pathogenic variants in the CACNA1S gene, is a recently described congenital muscle disease. Disease associated variants result in loss of gene expression and/or reduction of Cav1.1 protein stability. There is an incomplete understanding of the underlying disease pathomechanisms and no effective therapies are currently available. A barrier to the study of this myopathy is the lack of a suitable animal model that phenocopies key aspects of the disease. To address this barrier, we generated knockouts of the two zebrafish CACNA1S paralogs, cacna1sa and cacna1sb. Double knockout fish exhibit severe weakness and early death, and are characterized by the absence of Cav1.1 α1 subunit expression, abnormal triad structure, and impaired excitation-contraction coupling, thus mirroring the severe form of human CACNA1S-related myopathy. A double mutant (cacna1sa homozygous, cacna1sb heterozygote) exhibits normal development, but displays reduced body size, abnormal facial structure, and cores on muscle pathologic examination, thus phenocopying the mild form of human CACNA1S-related myopathy. In summary, we generated and characterized the first cacna1s zebrafish loss-of-function mutants, and show them to be faithful models of severe and mild forms of human CACNA1S-related myopathy suitable for future mechanistic studies and therapy development.

Dysfunctional APPL1-Mediated Epigenetic Regulation in Diabetic Vascular Injury.

APN (adiponectin) and APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) are potent vasculoprotective molecules, and their deficiency (eg, hypoadiponectinemia) contributes to diabetic vascular complications. However, the molecular mechanisms that govern their vasculoprotective genes as well as their alteration by diabetes remain unknown. Diabetic medium-cultured rat aortic endothelial cells, mouse aortic endothelial cells from high-fat-diet animals, and diabetic human aortic endothelial cells were used for molecular/cellular investigations. The in vivo concept-prove demonstration was conducted using diabetic vascular injury and diabetic hindlimb ischemia models. In vivo animal experiments showed that APN replenishment caused APPL1 nuclear translocation, resulting in an interaction with HDAC (histone deacetylase) 2, which inhibited HDAC2 activity and increased H3Kac27 levels. Based on transcriptionome pathway-specific real-time polymerase chain reaction profiling and bioinformatics analysis, Angpt1 (angiopoietin 1), Ocln (occludin), and Cav1 (caveolin 1) were found to be the top 3 vasculoprotective genes suppressed by diabetes and rescued by APN in an APPL1-dependent manner. APN reverses diabetes-induced inhibition of Cav1 interaction with APPL1. APN-induced Cav1 expression was not affected by Angpt1 or Ocln deficiency, whereas APN-induced APPL1 nuclear translocation or upregulation of Angpt1/Ocln expression was abolished in the absence of Cav1 both in vivo and in vitro, suggesting Cav1 is upstream molecule of Angpt1/Ocln in response to APN administration. Chromatin immunoprecipitation-qPCR (quantitative polymerase chain reaction) demonstrated that APN caused significant enrichment of H3K27ac in Angpt1 and Ocln promoter region, an effect blocked by APPL1/Cav1 knockdown or HDAC2 overexpression. The protective effects of APN on the vascular system were attenuated by overexpression of HDAC2 and abolished by knocking out APPL1 or Cav1. The double knockdown of ANGPT1/OCLN blunted APN vascular protection both in vitro and in vivo. Furthermore, in diabetic human endothelial cells, HDAC2 activity is increased, H3 acetylation is decreased, and ANGPT1/OCLN expression is reduced, suggesting that the findings have important translational implications. Hypoadiponectinemia and dysregulation of APPL1-mediated epigenetic regulation are novel mechanisms leading to diabetes-induced suppression of vasculoprotective gene expression. Diabetes-induced pathological vascular remodeling may be prevented by interventions promoting APPL1 nuclear translocation and inhibiting HDAC2.

Electric field stimulation unmasks a subtle role for T-type calcium channels in regulating lymphatic contraction

We previously identified two isoforms of T-type, voltage-gated calcium (Cav3) channels (Cav3.1, Cav3.2) that are functionally expressed in murine lymphatic muscle cells; however, contractile tests of lymphatic vessels from single and double Cav3 knock-out (DKO) mice, exhibited nearly identical parameters of spontaneous twitch contractions as wild-type (WT) vessels, suggesting that Cav3 channels play no significant role. Here, we considered the possibility that the contribution of Cav3 channels might be too subtle to detect in standard contraction analyses. We compared the sensitivity of lymphatic vessels from WT and Cav3 DKO mice to the L-type calcium channel (Cav1.2) inhibitor nifedipine and found that the latter vessels were significantly more sensitive to inhibition, suggesting that the contribution of Cav3 channels might normally be masked by Cav1.2 channel activity. We hypothesized that shifting the resting membrane potential (Vm) of lymphatic muscle to a more negative voltage might enhance the contribution of Cav3 channels. Because even slight hyperpolarization is known to completely silence spontaneous contractions, we devised a method to evoke nerve-independent, twitch contractions from mouse lymphatic vessels using single, short pulses of electric field stimulation (EFS). TTX was present throughout to block the potential contributions of voltage-gated Na+ channels in perivascular nerves and lymphatic muscle. In WT vessels, EFS evoked single contractions that were comparable in amplitude and degree of entrainment to those occurring spontaneously. When Cav1.2 channels were blocked or deleted, only small residual EFS-evoked contractions (~ 5% of normal amplitude) were present. These residual, EFS-evoked contractions were enhanced (to 10–15%) by the KATP channel activator pinacidil (PIN) but were absent in Cav3 DKO vessels. Our results point to a subtle contribution of Cav3 channels to lymphatic contractions that can be unmasked in the absence of Cav1.2 channel activity and when the resting Vm is more hyperpolarized than normal.

A caveolin-1 dependent glucose-6-phosphatase trafficking contributes to hepatic glucose production

ObjectiveDeregulation of hepatic glucose production is a key driver in the pathogenesis of diabetes, but its short-term regulation is incompletely deciphered. According to textbooks, glucose is produced in the endoplasmic reticulum by glucose-6-phosphatase (G6Pase) and then exported in the blood by the glucose transporter GLUT2. However, in the absence of GLUT2, glucose can be produced by a cholesterol-dependent vesicular pathway, which remains to be deciphered. Interestingly, a similar mechanism relying on vesicle trafficking controls short-term G6Pase activity. We thus investigated whether Caveolin-1 (Cav1), a master regulator of cholesterol trafficking, might be the mechanistic link between glucose production by G6Pase in the ER and glucose export through a vesicular pathway. MethodsGlucose production from fasted mice lacking Cav1, GLUT2 or both proteins was measured in vitro in primary culture of hepatocytes and in vivo by pyruvate tolerance tests. The cellular localization of Cav1 and the catalytic unit of glucose-6-phosphatase (G6PC1) were studied by western blotting from purified membranes, immunofluorescence on primary hepatocytes and fixed liver sections and by in vivo imaging of chimeric constructs overexpressed in cell lines. G6PC1 trafficking to the plasma membrane was inhibited by a broad inhibitor of vesicular pathways or by an anchoring system retaining G6PC1 specifically to the ER membrane. ResultsHepatocyte glucose production is reduced at the step catalyzed by G6Pase in the absence of Cav1. In the absence of both GLUT2 and Cav1, gluconeogenesis is nearly abolished, indicating that these pathways can be considered as the two major pathways of de novo glucose production. Mechanistically, Cav1 colocalizes but does not interact with G6PC1 and controls its localization in the Golgi complex and at the plasma membrane. The localization of G6PC1 at the plasma membrane is correlated to glucose production. Accordingly, retaining G6PC1 in the ER reduces glucose production by hepatic cells. ConclusionsOur data evidence a pathway of glucose production that relies on Cav1-dependent trafficking of G6PC1 to the plasma membrane. This reveals a new cellular regulation of G6Pase activity that contributes to hepatic glucose production and glucose homeostasis.

Knockout of CaV1.3 L-type calcium channels in a mouse model of retinitis pigmentosa

Retinitis Pigmentosa is a genetically heterogeneous, degenerative retinal disorder characterized by gradual dysfunction and death of photoreceptors, first rods and later cones, and progressive blindness. Studies suggested that application of L-type calcium channel blockers rescues photoreceptors in paradigms related to Ca2+ overflow. To investigate whether Cav1.3 L-type channels have protective effects in the retina, we established a new mouse model by crossing rd10, modeling autosomal-recessive RP, with Cav1.3 deficient mice (rd10/Cav1.3KO). Our immunohistochemical analyses revealed an influence of Cav1.3 channels on the degenerative process of photoreceptors. The absence of Cav1.3 delayed the centre-to-periphery degeneration of rods indicated by a significantly higher number of photoreceptor rows and, consequently, of cones. In accordance with a preserved number of cones we observed a regular row of cone somas in rd10/Cav1.3-KO retinas. Surviving rod photoreceptors maintained synaptic contacts with rod bipolar cells. However, the delay in degeneration was only observed up to postnatal day 45. Although we observed a reduction in the spontaneous oscillatory retinal activity during multielectrode array analyses, measurable functional preservation was lacking in behavioural tests. In conclusion, Cav1.3 channels contribute to photoreceptor degeneration in rd10 retinas but photoreceptor temporary rescue might rather be achieved indirectly through other retinal cell layers.

Caveolin-1 Regulates Perivascular Aquaporin-4 Expression After Cerebral Ischemia.

Edema is a hallmark of many brain disorders including stroke. During vasogenic edema, blood-brain barrier (BBB) permeability increases, contributing to the entry of plasma proteins followed by water. Caveolae and caveolin-1 (Cav-1) are involved in these BBB permeability changes. The expression of the aquaporin-4 (AQP4) water channel relates to brain swelling, however, its regulation is poorly understood. Here we tested whether Cav-1 regulates AQP4 expression in the perivascular region after brain ischemia in mice. We showed that Cav-1 knockout mice had enhanced hemispheric swelling and decreased perivascular AQP4 expression in perilesional and contralateral cortical regions compared to wild-type. Glial fibrillary acidic protein-positive astrocytes displayed less branching and ramification in Cav-1 knockout mice compared to wild-type animals. There was a positive correlation between the area of perivascular AQP4-immunolabelling and branch length of Glial fibrillary acidic protein-positive astrocytes in wild-type mice, not seen in Cav-1 knockout mice. In summary, we show for the first time that loss of Cav-1 results in decreased AQP4 expression and impaired perivascular AQP4 covering after cerebral ischemia associated with altered reactive astrocyte morphology and enhanced brain swelling. Therapeutic approaches targeting Cav-1 may provide new opportunities for improving stroke outcome.Significance StatementSevere brain edema worsens outcome in stroke patients. Available treatments for stroke-related edema are not efficient and molecular and cellular mechanisms are poorly understood. Cellular water channels, aquaporins (AQPs), are mainly expressed in astrocytes in the brain and play a key role in water movements and cerebral edema, while endothelial caveolins have been suggested to play a role in vasogenic edema. Here we used an integrative approach to study possible interaction between AQP4 and caveolin-1 (Cav-1) after stroke. Absence of Cav-1 was associated with perivascular changes in AQP4 expression and enhanced brain swelling at 3 days after cerebral ischemia. The present work indicates a direct or indirect effect of Cav-1 on perivascular AQP4, which may lead to novel edema therapy.

Injury-Induced Shedding of Extracellular Vesicles Depletes Endothelial Cells of Cav-1 (Caveolin-1) and Enables TGF-β (Transforming Growth Factor-β)-Dependent Pulmonary Arterial Hypertension.

Objective- To determine whether pulmonary arterial hypertension is associated with endothelial cell (EC)-Cav-1 (caveolin-1) depletion, EC-derived extracellular vesicle cross talk with macrophages, and proliferation of Cav-1 depleted ECs via TGF-β (transforming growth factor-β) signaling. Approach and Results- Pulmonary vascular disease was induced in Sprague-Dawley rats by exposure to a single injection of VEGFRII (vascular endothelial growth factor receptor II) antagonist SU5416 (Su) followed by hypoxia (Hx) plus normoxia (4 weeks each-HxSu model) and in WT (wild type; Tie2.Cre-; Cav1 lox/lox) and EC- Cav1-/- (Tie2.Cre+; Cav1 fl/fl) mice (Hx: 4 weeks). We observed reduced lung Cav-1 expression in the HxSu rat model in association with increased Cav-1+ extracellular vesicle shedding into the circulation. Whereas WT mice exposed to hypoxia exhibited increased right ventricular systolic pressure and pulmonary microvascular thickening compared with the group maintained in normoxia, the remodeling was further increased in EC- Cav1-/- mice indicating EC Cav-1 expression protects against hypoxia-induced pulmonary hypertension. Depletion of EC Cav-1 was associated with reduced BMPRII (bone morphogenetic protein receptor II) expression, increased macrophage-dependent TGF-β production, and activation of pSMAD2/3 signaling in the lung. In vitro, in the absence of Cav-1, eNOS (endothelial NO synthase) dysfunction was implicated in the mechanism of EC phenotype switching. Finally, reduced expression of EC Cav-1 in lung histological sections from human pulmonary arterial hypertension donors was associated with increased plasma concentration of Cav-1, extracellular vesicles, and TGF-β, indicating Cav-1 may be a plasma biomarker of vascular injury and key determinant of TGF-β-induced pulmonary vascular remodeling. Conclusions- EC Cav-1 depletion occurs, in part, via Cav-1+ extracellular vesicle shedding into the circulation, which contributes to increased TGF-β signaling, EC proliferation, vascular remodeling, and pulmonary arterial hypertension.

Caveolin and Endothelial NO Signaling.

Pulmonary vascular diseases are associated with several factors including infection, cigarette smoking, abuse of dietary suppressants and drugs, prolonged exposure to high altitude, and other causes which in part induce significant oxidative stress resulting in endothelial cell injury, apoptosis, hyperproliferation, and vaso-occlusive disease. Maintenance of normal endothelial cell function is a critical role of endothelial nitric oxide synthase (eNOS) activity and physiologic nitric oxide (NO) signaling in the vascular wall. eNOS expression and activity is regulated by the membrane-associated scaffolding protein caveolin-1 (Cav-1), the main protein constituent of caveolae. This chapter summarizes the literature and highlights unanswered questions related to how inflammation-associated oxidative stress affects Cav-1 expression and regulatory functions, and how dysregulated eNOS enzymatic activity promotes endothelial dysfunction. Focus is given to how the conversion of eNOS from a NO-producing enzyme to a transient oxidant-generating system is associated twith Cav-1 depletion, endothelial cell injury, and pulmonary vascular diseases. Importantly, the vascular defects observed in absence of Cav-1 that give rise to injured or hyperproliferative endothelial cells and promote remodeled vasculature can be rescued by "re-coupling," inhibiting, or genetically deleting eNOS, supporting the notion that strict control of Cav-1 expression and eNOS activity and signaling is critical for maintaining pulmonary vascular homeostasis.

Expression of caveolin-1 in the interfollicular but not the follicle-associated epithelial cells in the bursa of fabricius of chickens.

The surface epithelium of the bursa of Fabricius consists of interfollicular (IFE) and follicle-associated epithelium (FAE). The IFE comprises (i) cylindrical-shaped secretory cells (SC) and (ii) cuboidal basal cells (BCs). The FAE provides histological and two-way functional connections between the bursal lumen and medulla of the follicle. We used a carbon solution and anti-caveolin-1 (Cav-1) to study the endocytic activity of FAE. Carbon particles entered the intercellular space of FAE, but the carbon particles were not internalized by the FAE cells. Cav-1 was not detectable in the FAE cells or the medulla of the bursal follicle. The absence of Cav-1 indicates that no caveolin-mediated endocytosis occurs in the FAE cells, B cells, bursal secretory dendritic cells (BSDC), or reticular epithelial cells. Surprisingly, a significant number of Cav-1 positive cells can be found among the SC, which are designated SC II. Cav-1 negative cell are called SC I, and they produce mucin for lubricating the bursal lumen and duct. Occasionally, BCs also express Cav-1, which suggests that BC is a precursor of a SC. Transmission electron microscopy confirmed the existence of type I and II SC. The SC II are highly polarized and have an extensive trans-Golgi network that is rich in different granules and vesicles. Western blot analysis of bursa lysates revealed a 21-23 kDa compound (caveolin) and Filipin fluorescence histochemistry provided evidence for intracellular cholesterol. High amount of cholesterol in the feces shows the cholesterol efflux from SC II. The presence of Cav-1 and cholesterol in SC II indicates, that the bursa is a complex organ in addition to possessing immunological function contributes to the cholesterol homeostasis in the chickens.

AT1-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers

BackgroundBlockers of angiotensin II type 1 receptor (AT1R) and the voltage gated calcium channel 1.2 (CaV1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT1R signaling and whether there is a reciprocal regulation of AT1R signaling by CaV1.2.MethodsTo elucidate these questions, we have studied the Ca2+ signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of CaV1.2 blockers. Semi-quantitative imaging methods were used to assess the plasma membrane expression of AT1R and G-protein activation.ResultsRepeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca2+ response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca2+ response. The up-regulation of the Ca2+ response to repeated 1 nM AngII doses and the down-regulation of the Ca2+ response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT1R plasma membrane expression. The Ca2+ response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the CaV1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT1R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT1R is sensitive to calcium influx.ConclusionsOur findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca2+ channel blockers as mono-therapy in hypertension.

Phosphorylated CAV1 activates autophagy through an interaction with BECN1 under oxidative stress

CAV1/Caveolin1, an integral membrane protein, is involved in caveolae function and cellular signaling pathways. Here, we report that CAV1 is a positive regulator of autophagy under oxidative stress and cerebral ischemic injury. Treatment with hydrogen peroxide enhanced autophagy flux and caused the localization of BECN1 to the mitochondria, whereas these changes were impaired in the absence of CAV1. Among many autophagy signals, only LC3 foci formation in response to hydrogen peroxide was abolished by CAV1 deficiency. Under oxidative stress, CAV1 interacted with a complex of BECN1/VPS34 through its scaffolding domain, and this interaction facilitated autophagosome formation. Interestingly, the phosphorylation of CAV1 at tyrosine-14 was essential for the interaction with BECN1 and their localization to the mitochondria, and the activation of autophagy in response to hydrogen peroxide. In addition, the expression of a phosphatase PTPN1 reduced the phosphorylation of CAV1 and inhibited autophagy. Further, compared to that in wild-type mice, autophagy was impaired and cerebral infarct damage was aggravated in the brain of Cav1 knockout mice. These results suggest that the phosphorylated CAV1 functions to activate autophagy through binding to the BECN1/VPS34 complex under oxidative stress and to protect against ischemic damage.

Atheroprotective role of Caveolin-1 and eNOS in an innovative transplantation model is mainly mediated by local effects

Endothelial dysfunction is crucial in the initiation of atherosclerosis, which is associated with a lack of nitric oxide. The endothelial NO synthase (eNOS) is responsible for constitutive synthesis of NO and inhibited by caveolin-1 (Cav1). In the current study, we examined the influence on intima formation through single and combined deletion of eNOS and Cav1 with a focus on differentiation of local and systemic effects.A sex-mismatch transplantation of denudated aortae from female C57BL/6n (WT), Cav1−/−, eNOS−/− and Cav1−/−/eNOS−/− (C/e−−/−−) mice in common carotid artery of male WT mice was performed. After six weeks on Western-type diet, the aortae were explanted and intimal lesions were quantified by determining the intima-media-ratio (IMR). Significantly larger plaques were observed in all knockout mice compared to WT. The highest IMR was detected in Cav1−/− arteries associated with an increased expression of α-smooth muscle actin (αSMA) and the proliferating cell nuclear antigen (PCNA). Both were reduced in aortae from C/e−−/−−. Galectin-3 (Gal3) immunostaining revealed only small infiltrations of macrophages. Systemic cell invasion was detected by Y chromosome fluorescence in situ hybridization (Y-FISH), which showed only small numbers of systemic cells and no differences between the genotypes.Loss of Cav1 increased vascular lesion by enhancing neointimal proliferation. The combined loss of Cav1 and eNOS, compared to Cav1−/−, lowered intima formation, suggesting an increasing effect of eNOS in the absence of Cav1 on vascular lesion. Furthermore, these effects seem to be mediated by local cells rather than by systemically invaded ones.

Abstract LB-035: Caveolin-1 loss alters matrix protein deposition and duct formation in murine mammary glands

Abstract Breast cancer is responsible for 28.5% of all newly diagnosed cancers in women, but has only a 20% 5-year survival rate. The microenvironment, which is comprised of fibroblasts, extracellular matrix (ECM), immune cells and endothelial cells, is abnormal in malignant mammary glands in comparison to healthy tissues. The complex and ill-understood changes which take place in the ECM, a supportive stromal tissue comprised of collagens glycoproteins and proteoglycans, and the subsequent changes in its physical composition have been reported to act as a contributing factor for the development of the disease. Of particular interest is the composition and density of the ECM and cross-linking of these proteins which contributes to the increased mechanical stiffness of the tumor. The aberrant ECM associated with breast tumors is in part mediated by reduced expression of caveolin-1 (cav-1), a scaffolding protein of caveolae, which participates in endocytosis and cellular signaling cascades, and has been reported to act as a tumor suppressor. Decreased expression of cav-1 has been observed in patients with breast cancer and has been linked to metastasis and decreased patient survival. In particular, decreased expression of cav-1 has been reported in fibroblasts, mesenchymal cells which synthesize ECM, in patients with breast cancer. To determine whether the absence of cav-1 results in an abnormal mammary gland architecture, the expression of several ECM proteins in addition to changes in duct structure were assessed in the mammary glands of cav-1 deficient (cav-1 -/-) and wild type (cav-1 +/+) mice. The ECM proteins fibronectin, tenascin-c and collagen-I were analyzed given their atypical expression in breast cancers. Overall, expression of fibronectin and collagen-1 were elevated in the mammary glands of cav-1 -/- mice in comparison to mammary glands of cav-1 +/+ mice. Conversely, tenascin-c appeared to be downregulated in the mammary glands of cav-1 -/- mice when compared to the mammary glands of cav-1 +/+ mice. Utilizing α smooth muscle actin as a marker of the myoepithelial cells surrounding the ducts, the number of mammary lactiferous ducts in histological sections was counted. Results demonstrated a significant increase in the number of ducts in cav-1 -/- mice compared to cav-1 +/+ mice, averaging 7.8 and 4.6 ducts per 9.4 x105 μm2 of tissue respectively (p<0.0001). In addition, significant increases were observed in average duct circumference, 280.8μm and 203.8μm (p = 0.0273), and stromal area, 11,166μm2 and 5,391μm2 (p = 0.0109), in cav-1 -/- and cav-1 +/+ mice, respectively. Patent ducts were compared to determine the total openness of duct lumens independent of total stromal tissue. This was calculated by dividing the luminal area by the stromal area and multiplying by 100 to yield a percentage of openness. Despite increases in stromal area and protein deposition, cav-1 -/- ducts had increased patency at 14.6% openness compared to 9.5% for cav-1 +/+ (p = 0.0307). These data demonstrate that cav-1 expression is directly linked to increased expression of ECM proteins and aberrant development of lactiferous ducts and suggest a potential role of the loss of cav-1 in prompting tumor initiation and progression. Citation Format: Christopher M. Thompson, Abigail Hielscher. Caveolin-1 loss alters matrix protein deposition and duct formation in murine mammary glands. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-035.

Caveolin-1 regulates the anti-atherogenic properties of macrophages

Atherosclerosis is a complex disease initiated by the vascular accumulation of lipoproteins in the sub-endothelial space, followed by the infiltration of monocytes into the arterial intima. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and of various signaling pathways. In order to study specifically the role of macrophage Cav-1 in atherosclerosis, we used Cav-1 (-/-) Apoe (-/-) mice and transplanted them with bone marrow (BM) cells obtained from Cav-1 (+/+) Apoe (-/-) or Cav-1 (-/-) Apoe (-/-) mice and vice versa. We found that Cav-1 (+/+) mice harboring Cav-1 (-/-) BM-derived macrophages developed significantly larger lesions than Cav-1 (+/+) mice harboring Cav-1 (+/+) BM-derived macrophages. Cav-1 (-/-) macrophages were more susceptible to apoptosis and more prone to induce inflammation. The present study provides clear evidence that the absence of Cav-1 in macrophage is pro-atherogenic, whereas its absence in endothelial cells protects against atherosclerotic lesion formation. These findings demonstrate the cell-specific role of Cav-1 during the development of this disease.

Caveolin-1 deletion exacerbates cardiac interstitial fibrosis by promoting M2 macrophage activation in mice after myocardial infarction

Adverse remodeling following myocardial infarction (MI) leading to heart failure is driven by an imbalanced resolution of inflammation. The macrophage cell is an important control of post-MI inflammation, as macrophage subtypes secrete mediators to either promote inflammation and extend injury (M1 phenotype) or suppress inflammation and promote scar formation (M2 phenotype). We have previously shown that the absence of caveolin-1 (Cav1), a membrane scaffolding protein, is associated with adverse cardiac remodeling in mice, but the mechanisms responsible remain to be elucidated. We explore here the role of Cav1 in the activation of macrophages using wild type C57BL6/J (WT) and Cav1tm1Mls/J (Cav1−/−) mice. By echocardiography, cardiac function was comparable between WT and Cav1−/− mice at 3days post-MI. In the absence of Cav1, there were a surprisingly higher percentage of M2 macrophages (arginase-1 positive) detected in the infarcted zone. Conversely, restoring Cav1 function after MI in WT mice by adding back the Cav1 scaffolding domain reduced the M2 activation profile. Further, adoptive transfer of Cav1 null macrophages into WT mice on d3 post-MI exacerbated adverse cardiac remodeling at d14 post-MI. In vitro studies revealed that Cav1 null macrophages had a more pronounced M2 profile activation in response to IL-4 stimulation. In conclusion, Cav1 deletion promotes an array of maladaptive repair processes after MI, including increased TGF-β signaling, increased M2 macrophage infiltration and dysregulation of the M1/M2 balance. Our data also suggest that cardiac remodeling can be improved by therapeutic intervention regulating Cav1 function during the inflammatory response phase.

Caveolin-1 limits human influenza A virus (H1N1) propagation in mouse embryo-derived fibroblasts

Caveolin expression supports the multiplication of retro-, ortho- and paramyxoviruses in susceptible cells. However, human influenza A virus (IAV), an orthomyxovirus, does not multiply efficiently in mouse embryo fibroblasts (MEFs), which are abundant in caveolin-1 (Cav-1). Surprisingly, the absence of Cav-1 in a MEF cell line removed the block for IAV replication and raised the infectious titer 250-fold, whereas the re-introduction of Cav-1 reversed the effect. The monitoring of cellular pathways revealed that Cav-1 loss considerably increased activities of p53. Furthermore, infection of MEF Cav-1 (−/−) induced reactive oxygen species (ROS) and pronounced apoptosis in the late phase of viral multiplication, but no type I IFN response. Strikingly, pharmacological inactivation showed that the elevated levels of ROS together with apoptosis caused the increase of virus yield. Thus, Cav-1 represents a new negative regulator of IAV infection in MEF that diminishes IAV infectious titer by controlling virus-supportive pathways.

Airway hyperresponsiveness is associated with airway remodeling but not inflammation in aging Cav1 -/- mice

BackgroundAirway inflammation and airway remodeling are the key contributors to airway hyperresponsiveness (AHR), a characteristic feature of asthma. Both processes are regulated by Transforming Growth Factor (TGF)-β. Caveolin 1 (Cav1) is a membrane bound protein that binds to a variety of receptor and signaling proteins, including the TGF-β receptors. We hypothesized that caveolin-1 deficiency promotes structural alterations of the airways that develop with age will predispose to an increased response to allergen challenge.MethodsAHR was measured in Cav1-deficient and wild-type (WT) mice 1 to 12 months of age to examine the role of Cav1 in AHR and the relative contribution of inflammation and airway remodeling. AHR was then measured in Cav1-/- and WT mice after an ovalbumin-allergen challenge performed at either 2 months of age, when remodeling in Cav1-/- and WT mice was equivalent, and at 6 months of age, when the Cav1-/- mice had established airway remodeling.ResultsCav1-/- mice developed increased thickness of the subepithelial layer and a correspondingly increased AHR as they aged. In addition, allergen-challenged Cav1-/- mice had an increase in AHR greater than WT mice that was largely independent of inflammation. Cav1-/- mice challenged at 6 months of age have decreased AHR compared to those challenged at 2 months with correspondingly decreased BAL IL-4 and IL-5 levels, inflammatory cell counts and percentage of eosinophils. In addition, in response to OVA challenge, the number of goblet cells and α-SMA positive cells in the airways were reduced with age in response to OVA challenge in contrast to an increased collagen deposition further enhanced in absence of Cav1.ConclusionA lack of Cav1 contributed to the thickness of the subepithelial layer in mice as they aged resulting in an increase in AHR independent of inflammation, demonstrating the important contribution of airway structural changes to AHR. In addition, age in the Cav1-/- mice is a contributing factor to airway remodeling in the response to allergen challenge.

In MMTV-Her-2/neu transgenic mammary tumors the absence of caveolin-1−/− alters PTEN and NHERF1 but not β-catenin expression

In a recent study, we have shown that in mammary tumors from mice lacking the Cav-1 gene, there are alterations in specific heat shock proteins as well as in tumor development. With this in mind, we have now investigated other proteins in the same mammary mouse tumor model (Her-2/neu expressing mammary tumors from Cav-1 wild type and Cav-1 null mice), to further comprehend the complex tumor-stroma mechanisms involved in regulating stress responses during tumor development. In this tumor model the cancer cells always lacked of Cav-1, so the KO influenced the Cav-1 in the stroma. By immunohistochemistry, we have found a striking co-expression of β-catenin and Her-2/neu in the tumor cells. The absence of Cav-1 in the tumor stroma had no effect on expression or localization of β-catenin and Her-2/neu. Both proteins appeared co-localized at the cell surface during tumor development and progression. Since Her-2/neu activation induces MTA1, we next evaluated MTA1 in the mouse tumors. Although this protein was found in numerous nuclei, the absence of Cav-1 did not alter its expression level. In contrast, significantly more PTEN protein was noted in the tumors lacking Cav-1 in the stroma, with the protein localized mainly in the nuclei. P-Akt levels were relatively low in tumors from both Cav-1 WT and Cav-1 KO mice. There was also an increase in nuclear NHERF1 expression levels in the tumors arising from Cav-1 KO mice. The data obtained in the MMTV-neu model are consistent with a role for Cav-1 in adjacent breast cancer stromal cells in modulating the expression and localization of important proteins implicated in tumor cell behavior.

Neurochemical characterization of the striatum and the nucleus accumbens in L-type Cav1.3 channels knockout mice

L-type Cav1.3 channels control the autonomous pacemaking of the substantia nigra (SN) dopamine (DA) neurons, which maintains the sustained release of DA in the striatum, its target structure. The persistent engagement of L-type channels during pacemaking might lead to increased vulnerability to environmental stressors or degenerative processes, providing a mechanism for the development of Parkinson’s disease (PD). Interestingly, L-type channels are not necessary for pacemaking, opening the possible use of calcium channel antagonists as neuroprotective agents for PD without disturbing normal DA function. In this study we aimed to evaluate the consequences of Cav1.3 channels deletion at the neurochemical level. For this purpose, tissue concentrations of DA and their respective metabolites were measured using high performance liquid chromatography (HPLC) in the striatum and the nucleus accumbens (NAcc) of mice lacking the gene for the Cav1.3 channel subunit (CACNA1D) and compared to those in wild-type mice. Striatal DA level did not differ between the two groups. In contrast, the level of serotonin, glutamate, GABA, and taurine were increased by more than 50% in the striatum of Cav1.3 null mice. Neurotransmitters levels in the NAcc did not differ between the different groups. In conclusion, our results neurochemically corroborate the robustness of the nigrostriatal DA neurons in the absence of Cav1.3 channels, but suggest that complete deletion of this channel affected a variety of other transmitter systems.