Cav-1 Levels Research Articles

Selective sphingosine-1-phosphate receptor 1 modulator attenuates blood–brain barrier disruption following traumatic brain injury by inhibiting vesicular transcytosis

BackgroundTraumatic brain injury (TBI) provokes secondary pathological damage, such as damage to the blood–brain barrier (BBB), ischaemia and inflammation. Major facilitator superfamily domain-containing 2a (Mfsd2a) has been demonstrated to be critical in limiting the increase in BBB vesicle transcytosis following brain injury. Recent studies suggest that a novel and selective modulator of the sphingosine-1-phosphate receptor 1 (S1P1), CYM-5442, maintains the integrity of the BBB by restricting vesicle transcytosis during acute ischaemic stroke. In the current study, we investigated whether CYM-5442, evaluated in a short-term study, could protect the brains of mice with acute-stage TBI by reversing the increase in vesicle transport due to reduced Mfsd2a expression after TBI.MethodsWe used the well-characterized model of TBI caused by controlled cortical impact. CYM-5442 (0.3, 1, 3 mg/kg) was intraperitoneally injected 30 min after surgery for 7 consecutive days. To investigate the effect of CYM-5442 on vesicle transcytosis, we downregulated and upregulated Mfsd2a expression using a specific AAV prior to evaluation of the TBI model. MRI scanning, cerebral blood flow, circulating blood counts, ELISA, TEM, WB, and immunostaining evaluations were performed after brain injury.ResultsCYM-5442 significantly attenuated neurological deficits and reduced brain oedema in TBI mice. CYM-5442 transiently suppressed lymphocyte trafficking but did not induce persistent lymphocytopenia. After TBI, the levels of Mfsd2a were decreased significantly, while the levels of CAV-1 and albumin were increased. In addition, Mfsd2a deficiency caused inadequate sphingosine-1-phosphate (S1P) transport in the brain parenchyma, and the regulation of BBB permeability by Mfsd2a after TBI was shown to be related to changes in vesicle transcytosis. Downregulation of Mfsd2a in mice markedly increased the BBB permeability, neurological deficit scores, and brain water contents after TBI. Intervention with CYM-5442 after TBI protected the BBB by significantly reducing the vesicle transcytosis of cerebrovascular endothelial cells.ConclusionIn addition to transiently suppressing lymphocytes, CYM-5442 alleviated the neurological deficits, cerebral edema and protective BBB permeability in TBI mice by reducing the vesicle transcytosis of cerebrovascular endothelial cells.

1279-P: Caveolin 1 rs1997623 Is Associated with Metabolic Syndrome in Arab Adult Individuals from Kuwait

Studies have implicated CAV1 in the pathophysiology of diabetes and obesity. Previously, we demonstrated a potential association between the CAV1 rs1997623 C/A variant and metabolic syndrome (MetS) in Kuwaiti children. In the present study, we substantiate the association of CAV1 with MetS in adult Arab individuals. Method: The CAV1 rs1997623 was genotyped in three cohorts of Arabs (n=479) , South Asians (n=660) and South East Asians (n=362) . MetS status of the individuals was diagnosed using the IDF criteria (i.e., presence of central obesity and of at least two abnormalities of high TG, low HDL, hypertension, or type 2 diabetes) . The quantitative measure of MetS was calculated as siMS=2*WC/Height + FBG/5.6 + TG/1.7 + SBP/130 - HDL/1.02 for males or HDL/1.28 for females. Allelic associations with quantitative and dichotomous traits were assessed using linear and logistic regression, respectively, with and adjustment for age and sex. CAV1 transcripts were quantified in adipose tissues from a cohort of 50 Arab individuals differing in siMS score. Results: The CAV1 variant was significantly associated with MetS status (OR=1.8[1.25-2.61]; P-value=0.0015; Pemp=0.0013) and with siMS (Effect size = 0.206; P-value=0.0035; Pemp=0.0028) in the cohort of Arab individuals. The variant was weakly and insignificantly associated in the cohorts of South Asian and South East Asian individuals (OR= 1.and 1.11; P-values= 0.25 and 0.67, respectively) . siMS scores correlated positively with mRNA levels of CAV1 in adipose tissue. Conclusion: The association of CAV1 rs1997623 C/A with MetS in Arab pediatric is now demonstrated in adults as well. Higher expression levels were seen in individuals poses increasing MetS rates further validates the implication of CAV1 rs1997623 C/A variant in MetS phenotype. Only a weak association signal was seen in the South Asian and South East Asian populations leading us to propose that the CAV1 rs1997623 association with MetS is probably specific to Arab ethnicity. Disclosure A.Al madhoun: None. P.Hebbar: None. R.Nizam: None. D.Haddad: None. M.H.Dashti: None. M.Abu-farha: None. R.Ahmad: None. A.T.Thangavel: None. F.Almulla: None. Funding Kuwait Foundation for the Advancement of Sciences (KFAS) and Dasman Diabetes Institute, Project RA CB-2021-007

Caveolin-1 Promoted Collateral Vessel Formation in Patients With Moyamoya Disease.

BackgroundCaveolin-1 (Cav-1) plays pivotal roles in the endothelial function and angiogenesis postischemia. Moyamoya disease (MMD) is characterized by progressive artery stenosis with unknown etiology. We aim to determine whether serum Cav-1 levels of patients with MMD were associated with collateral vessel formation after bypass surgery.MethodsWe studied serum Cav-1 levels of 130 patients with MMD (16 with RNF213 p.R4810K mutation and 114 without RNF213 p.R4810K mutation), 15 patients with acute stroke, and 33 healthy controls. Cerebral perfusion and collateral circulation were evaluated preoperation and at 6 months after operation using pseudocontinuous arterial spin labeling MRI (pCASL-MRI) and digital subtraction angiography (DSA), respectively. Endothelial expression of Cav-1 was verified in the superficial temporal artery (STA) wall of patients with MMD by immunofluorescence double staining. We also investigated whether overexpression of Cav-1 affects cell migration and tube formation using human microvascular endothelial cells (HMECs).ResultsThe serum Cav-1 level of patients with MMD intermediated between the stroke group and healthy controls and it was enhanced after the bypass surgery (681.87 ± 311.63 vs. 832.91 ± 464.41 pg/ml, p = 0.049). By 6 months after bypass surgery, patients with MMD with better collateral compensation manifested higher postoperative/preoperative Cav-1 ratio (rCav-1) than bad compensation patients. Consistently, cerebral blood flow (CBF) determined by pCASL-MRI (nCBFMCA ratio) was positively in line with rCav-1 ratio (r = 0.8615, p < 0.0001). Cav-1 was expressed in the endothelial cells of the STA vessels of patients with MMD. Overexpression of Cav-1 by plasmid transfection in HMECs promoted tube formation and cell migration.ConclusionThis study indicated that Cav-1 may be a potential driver to promote angiogenesis and collateral formation after bypass surgery in patients with MMD, providing a better understanding of MMD pathophysiology and potential non-surgical targets of MMD.

Cav-1 Protein Levels in Serum and Infarcted Brain Correlate with Hemorrhagic Volume in a Mouse Model of Thromboembolic Stroke, Independently of rt-PA Administration.

Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for acute ischemic stroke. However, its administration is still limited due to the associated increased risk of hemorrhagic transformation (HT). rt-PA may exacerbate blood-brain barrier (BBB) injury by several mechanisms that have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae, has been linked to the endothelial barrier function. The effects of rt-PA on Cav-1 expression remain largely unknown. Here, Cav-1 protein expression after ischemic conditions, with or without rt-PA administration, was analyzed in a murine thromboembolic middle cerebral artery occlusion (MCAO) and in brain microvascular endothelial bEnd.3 cells subjected to oxygen/glucose deprivation (OGD). Our results show that Cav-1 is overexpressed in endothelial cells of infarcted area and in bEnd.3 cell line after ischemia but there is disagreement regarding rt-PA effects on Cav-1 expression between both experimental models. Delayed rt-PA administration significantly reduced Cav-1 total levels from 24 to 72 h after reoxygenation and increased pCav-1/Cav-1 at 72 h in the bEnd.3 cells while it did not modify Cav-1 immunoreactivity in the infarcted area at 24 h post-MCAO. Importantly, tissue Cav-1 positively correlated with Cav-1 serum levels at 24 h post-MCAO and negatively correlated with the volume of hemorrhage after infarction, the latter supporting a protective role of Cav-1 in cerebral ischemia. In addition, the negative association between baseline serum Cav-1 levels and hemorrhagic volume points to a potential usefulness of baseline serum Cav-1 levels to predict hemorrhagic volume, independently of rt-PA administration.

Caveolin-1 negatively regulates inflammation and fibrosis in silicosis.

Inhalation of crystalline silica causes silicosis, the most common and serious occupational disease, which is characterized by progressive lung inflammation and fibrosis. Recent studies revealed the anti‐inflammatory and anti‐fibrosis role of Caveolin‐1 (Cav‐1) in lung, but this role in silicosis has not been investigated. Thus, this study evaluated Cav‐1 regulatory effects in silicosis. It was found that Cav‐1 levels were significantly reduced in the lung from silicosis patients and silicotic mice. The silicosis models were established in C57BL/6 (wild‐type) and Cav‐1 deficiency (Cav‐1 −/−) mice, and Cav‐1 −/− mice displayed wider alveolar septa, increased collagen deposition and more silicotic nodules. The mice peritoneal‐derived macrophages were used to explore the role of Cav‐1 in silica‐induced inflammation, which plays a central role in mechanism of silicosis. Cav‐1 inhibited silica‐induced infiltration of inflammatory cells and secretion of inflammatory factors in vitro and in vivo, partly by downregulating NF‐κB pathway. Additionally, silica uptake and expression of 4‐hydroxynonenal in silicotic mice were observed, and it was found that Cav‐1 absence triggered excessive silica deposition, causing a stronger oxidative stress response. These findings demonstrate the protective effects of Cav‐1 in silica‐induced lung injury, suggesting its potential therapeutic value in silicosis.

Mechanisms of endosomal dysfunction in APOE4.

Lipid poor apolipoprotein E (ApoE) aggregates are central to the pathogenesis of Alzheimer's disease (AD), promoting cerebral amyloid-β plaques formation, and altering endosomal recycling of apoE complexed proteins. Activating the ATP binding cassette A1 (ABCA1) to lipidate ApoE reduces its aggregation, promotes ApoE recycling and presents an attractive mechanism to rescue AD pathology. ABCA1 was immunoprecipitated using anti-GFP beads (Chromotek) from ApoE3/ApoE4 treated Hela-ABCA1GFP cell lysates. A unbiased proteomics (LC-MS) screen was employed to indentify ABCA1 binding proteins from the immunoprecipitated samples. E3/E4 AD (n=9, cogdx=4) and E3/E3 AD (n=11, cogdx=4) patient inferior frontal cortex from the Religious Order Study and the Memory and Aging Project (ROSMAP) were lysed and analyzed by Western blot for targets from proteomics screen. ApoE4 was associated with lower astrocytic ABCA1 activity. We identified caveolin-1 (Cav-1), an integral membrane protein in plasma membrane lipid rafts, as one of the ABCA1-binding proteins that regulate ABCA1 cell surface level. Using multiple models, we observed elevated Cav-1 in primary astrocytes and brains of ApoE4- targeted replacement (TR) mice. Knockdown Cav-1 through siRNA treatment restored ABCA1 surface expression level, underscoring the importance of Cav-1 in ABCA1 trafficking. In AD patient samples from ROSMAP, Cav-1 protein expression in E3/E4 AD inferior frontal cortex (n=9) demonstrated non-significant increase from 1.2 to 2.6 (normalized to β-actin) compared to E3/E3 AD (n=11). Ongoing efforts are planned to expand the human study sample size. ApoE4 is associated with elevated Cav-1 level and knockdown of Cav-1 can restore ABCA1 surface expression. Our work provides a deeper understanding of the critical mechanisms that define the pathogenicity of ApoE4 and can ultimately identify key pathways that regulate endosomal dysregulation in ApoE4.

Connexin 43 participates in atrial electrical remodelling through colocalization with calcium channels in atrial myocytes.

Atrial fibrillation (AF) is associated with atrial conduction disturbances caused by electrical and/or structural remodelling. In the present study, we hypothesized that connexin might interact with the calcium channel through forming a protein complex and, then, participates in the pathogenesis of AF. Western blot and whole-cell patch clamp showed that protein levels of Cav1.2 and connexin 43 (Cx43) and basal ICa,L were decreased in AF subjects compared to sinus rhythm (SR) controls. In cultured atrium-derived myocytes (HL-1 cells), knocking-down of Cx43 or incubation with 30mmol/L glycyrrhetinic acid significantly inhibited protein levels of Cav1.2 and Cav3.1 and the current density of ICa,L and ICa,T . Incubation with nifedipine or mibefradil decreased the protein level of Cx43 in HL-1 cells. Moreover, Cx43 was colocalized with Cav1.2 and Cav3.1 in atrial myocytes. Therefore, Cx43might regulate the ICa,L and ICa,T through colocalization with calcium channel subunits in atrial myocytes, representing a potential pathogenic mechanism in AF.

Expression of caveolin family proteins in serum of patients with systemic lupus erythematosus.

Caveolin family proteins, including caveolin-1 (Cav-1), caveolin-2 (Cav-2), and caveolin-3 (Cav-3), are identified as the principal protein components of caveolae in mammalian cells. Circulating form of caveolin family proteins can be used as a good potential biomarker for predicting disease. To investigate the clinical significance of the serological levels of caveolin family proteins in patients with systemic lupus erythematosus (SLE), we evaluated the soluble serum levels of caveolin family proteins in patients with SLE by enzyme-linked immunosorbent assay (ELISA) and assessed their associations with various known clinical variables. The major findings of our study are as follows: Cav-2 was not detected in serum of SLE patients and normal controls (NCs). Serum Cav-1 and Cav-3 levels were higher in SLE patients compared with NCs. There were no significant correlations between serum Cav-1 and Cav-3 levels and SLE disease activity. Further analysis showed that serum Cav-3 may be more valuable as a marker than serum Cav-1 in SLE patients. Serum levels of Cav-1 and Cav-3 might have a diagnostic role in patients with SLE. However, their predictive and prognostic value was not determined. Further studies are necessary to determine the potential clinical significance of these assays in SLE.

Role of caveolin-1 in chronic postsurgical pain in rats.

Chronic postsurgical pain (CPSP) has a high incidence, but the underlying mechanisms remain elusive. Previous studies have indicated that caveolin-1 (Cav-1) plays a notable role in pain modulation. To study the role of Cav-1 in CPSP in the present study, a rat model of skin/muscle incision and retraction (SMIR) was established. Under anesthesia, skin and superficial muscle of the medial thigh were incised and a small pair of retractors inserted. It was revealed that SMIR increased the expression of Cav-1 in the dorsal root ganglion (DRG) and the injured tissue around the incision. Furthermore, the infiltration of endothelial cells and macrophages in the injured tissue around the incision increased constantly, and the vascular permeability increased due to the destruction of the vascular endothelial barrier function around the injured tissue. Cav-1 was mainly expressed by CD68-positive macrophages and CD34-positive endothelial cells in the injured tissues around the incision, while it was also primarily localized in the medium and large neurofilament 200-positive neurons and a small number of calcitonin gene-related peptide- and isolectin B4-positive small and medium-sized neurons in the DRG. The results demonstrated that the sustained high expression levels of Cav-1 in the injured tissue around the incision could lead to the dysfunction of the vascular endothelial barrier and, thus, could induce the inflammatory response through the lipoprotein transport of endothelial cells, thereby resulting in peripheral sensitization. In addition, the sustained high expression levels of Cav-1 in the DRG could sensitize large-sized neurons and change the transmission mode of noxious stimuli. The findings of the present study indicated that a Cav-1-mediated process could participate in neuronal transmission pathways associated with pain modulation.

Integrated Bioinformatic Analysis of the Expression and Prognosis of Caveolae-Related Genes in Human Breast Cancer.

Caveolae-related genes, including CAVs that encodes caveolins and CAVINs that encodes caveolae-associated proteins cavins, have been identified for playing significant roles in a variety of biological processes including cholesterol transport and signal transduction, but evidences related to tumorigenesis and cancer progression are not abundant to correlate with clinical characteristics and prognosis of patients with cancer. In this study, we investigated the expression of these genes at transcriptional and translational levels in patients with breast cancer using Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), cBioPortal databases, and immunohistochemistry of the patients in our hospital. Prognosis of patients with breast cancer based on the expressions of CAVs and CAVINs was summarized using Kaplan-Meier Plotter with their correlation to different subtyping. The relevant molecular pathways of these genes were further analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database and Gene Set Enrichment Analysis (GSEA). Results elucidated that expression levels of CAV1, CAV2, CAVIN1, CAVIN2, and CAVIN3 were significantly lower in breast cancer tissues than in normal samples, while the expression level of CAVIN2 was correlated with advanced tumor stage. Furthermore, investigations on survival of patients with breast cancer indicated outstanding associations between prognosis and CAVIN2 levels, especially for the patients with estrogen receptor positive (ER+) breast cancer. In conclusion, our investigation indicated CAVIN2 is a potential therapeutic target for patients with ER+ breast cancer, which may relate to functions of cancer cell surface receptors and adhesion molecules.

Caveolin-1 facilitates cell migration by upregulating nuclear receptor 4A2/retinoid X receptor α-mediated β-galactoside α2,6-sialyltransferase I expression in human hepatocarcinoma cells.

It has been reported that caveolin-1 (Cav-1) acts as a tumor promoter in hepatocellular carcinoma (HCC). Our previous studies showed that Cav-1 promoted mouse hepatocarcinoma cell adhesion to fibronectin by upregulating β-galactoside α2,6-sialyltransferase I (ST6Gal-I) expression. However, the detailed mechanism by which Cav-1 regulates ST6Gal-I is not fully understood. In this study, we found that the expression levels of Cav-1 and ST6Gal-I were increased in HCC tissues and correlated with poor prognosis. Cav-1 upregulated ST6Gal-I expression to promote the migration and invasion of HCC cells by inducing epithelial-to-mesenchymal transition. Importantly, the binding of the transcription factor nuclear receptor 4A2/retinoid X receptor alpha (NR4A2/RXRα) to the -550/-200 region of the ST6GAL1 promoter was critical for Cav-1-induced ST6GAL1 gene expression. Furthermore, Cav-1 expression activated the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway, followed by upregulation of NR4A2 expression and phosphorylation of RXRα, which facilitated the complex of NR4A2 and phosphorylated RXRα forming and binding to the ST6GAL1 promoter region to induce its transcription. Finally, in the diethylnitrosamine (DEN)-induced HCC murine model, the expression levels of NR4A2, p-RXRα, ST6Gal-I, and α2,6-linked sialic acid decreased in parallel in Cav-1-/- mice compared with Cav-1+/+ mice, which was consistent with the above in vitro results. These findings provide insight into the mechanism of ST6GAL1 gene transcription mediated by Cav-1, which may lead to the development of novel therapeutic strategies targeting metastasis in HCC.

Caveolin‐1 increases glycolysis in pancreatic cancer cells and triggers cachectic states

In pancreatic cancer, autocrine insulin-like growth factor-1 (IGF-1) and paracrine insulin stimulate both IGF-1 receptor (IGF1R) and insulin receptor (IR) to increase tumor growth and glycolysis. In pancreatic cancer patients, cancer-induced glycolysis increases hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis and, thereby, causes cancer cachexia. As a protein coexisting with IGF1R and IR, caveolin-1 (cav-1) may be involved in pancreatic cancer-induced cachexia. We undertook the present study to test this hypothesis. Out of wild-type MiaPaCa2 and AsPC1 human pancreatic cancer cell lines, we created their stable sub-lines whose cav-1 expression was diminished with RNA interference or increased with transgene expression. When these cells were studied in vitro, we found that cav-1 regulated IGF1R/IR expression and activation and also regulated cellular glycolysis. We transplanted the different types of MiaPaCa2 cells in growing athymic mice for 8 weeks, using intact athymic mice as tumor-free controls. We found that cav-1 levels in tumor grafts were correlated with expression levels of the enzymes that regulated hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis in the respective tissues. When the tumors had original or increased cav-1, their carriers’ body weight gain was less than the tumor-free reference. When cav-1 was diminished in tumors, the tumor carriers’ body weight gain was not changed significantly, compared to the tumor-free reference. In conclusion, cav-1 in pancreatic cancer cells stimulated IGF1R/IR and glycolysis in the cancer cells and triggered cachectic states in the tumor carrier.

When multiple caveolins make the difference: Cav1 partly compensates Cav3 alterations and rescues ion channels expression

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Cariplo Caveolae are small-membrane invagination that contribute both to buffering excessive contraction-dependent membrane strain and to initiation of membrane repair. Moreover, they constitute micro-domains where receptors and ion channels are clustered, favouring their functional interaction. Caveolin-3 (Cav3) is the key structural component of muscular caveolae. Mutations in Cav3 gene are associated with alterations of the skeletal muscle architecture leading to some rare forms of hereditary skeletal myopathies and/or cardiomyopathies called caveolinopathies. Notably, skeletal muscle dysfunctions usually precede cardiac dysfunctions, even though the mutated Cav3 is expressed in both cell types. An important difference between skeletal fibers and cardiomyocytes is that in the latter, caveolin-1 (Cav1) participates with Cav3 to form caveolae; skeletal myotubes instead do not express Cav1.The delay or lack of onset of cardiac alterations in caveolinopathies may depend on a preserved micro-domains organization in the heart compared to skeletal muscle, due to Cav1 expression. We decided to focus on a specific mutation T78K found in heterozygous in a patient with Ripple muscle disease and hyperCKemia. We have characterized human cardiomyocytes (CM) differentiated from induced pluripotent stem cells (iPSC) derived from this patient and one healthy control. In particular, we have investigated which caveolin isoforms are expressed at day 30 of differentiation, finding both Cav1 and Cav3, with a significant decrease of Cav3 isoform in T78K-CM. Their different expressions significantly increase T78K membrane resistance (3.27 ± 0.6 GΩ versus 1.64 ± 0.4 GΩ in the CTRL-CM), and consequently membrane excitability. The T78K_CM showed an increase spontaneous beating rate compared to CTRL (1,75± 0,08 Hz and 0,89 ± 0,4 Hz, respectively). Previous laboratory analysis conducted in caveolin-free MEF cells, co-transfected with WT and T78K Cav3 mutation, revealed that the T78K mutant is dominant, inducing the retention of WT Cav3 in the perinuclear areas and causes significant reduction in current density of three ion channels (HCN4, Kv1.5 and Kir2.1) known to interact with caveolins. The dominant decreased in cav3 expression is in line with previous data in skeletal muscle biopsy, however, electrophysiological data would be likely incompatible with life. For this reason, we decided to compare the impact of this mutation in CHO cells that exhibit high levels of Cav1, and in cav-1 expressing MEF line. In these systems, the membrane localization of Cav3 T78K is rescued both in heterozygous and homozygous conditions. In line with caveolin membrane expression, HCN4, Kv1.5 and Kir2.1 density is also rescued to normal levels. These results constitute the first evidence of a possible role of Cav1 in compensating membrane disorganization and disfunction due to Cav3 mutations in the heart, making this organ less susceptible to caveolinopathies.

Low Pre-Transplant Caveolin-1 Serum Concentrations Are Associated with Acute Cellular Tubulointerstitial Rejection in Kidney Transplantation.

Acute and chronic transplant rejections due to alloreactivity are essential contributors to graft loss. However, the strength of alloreactivity is biased by non-immunological factors such as ischemia reperfusion injury (IRI). Accordingly, protection from IRI could be favorable in terms of limiting graft rejection. Caveolin-1 (Cav-1) is part of the cell membrane and an important regulator of intracellular signaling. Cav-1 has been demonstrated to limit IRI and to promote the survival of a variety of cell types including renal cells under stress conditions. Accordingly, Cav-1 could also play a role in limiting anti-graft immune responses. Here, we evaluated a possible association between pre-transplant serum concentrations of Cav-1 and the occurrence of rejection during follow-up in a pilot study. Therefore, Cav-1-serum concentrations were analyzed in 91 patients at the time of kidney transplantation and compared to the incidence of acute and chronic rejection. Higher Cav-1 levels were associated with lower occurrence of acute cellular tubulointerstitial rejection episodes.

Hypoxia-Induced Suppression of Antiapoptotic Bcl-2 Expression in Human Bladder Tumor Cells Is Regulated by Caveolin-1-Dependent Adenosine Monophosphate-Activated Protein Kinase Activity.

PurposeAdenosine monophosphate-activated protein kinase (AMPK) is thought to inhibit cell proliferation or promote cell death, but the details remain unclear. In this study, we propose that AMPK inhibits the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) by relying on the hypoxia-inducible factor 1 alpha (HIF-1α)-induced caveolin-1 (Cav-1) expression pathway in noninvasive human bladder tumor (RT4) cells.MethodsIn cells exposed to a hypoxic environment (0.5% oxygen), the levels of expression and phospho-activity of the relevant signaling enzymes were examined via Western blots and reverse transcription-polymerase chain reaction. Cell proliferation was assessed using a Cell Counting Kit-8 assay.ResultsThe level of expression of Cav-1 was very low or undetectable in RT4 cells. Hypoxia was associated with significantly decreased cell growth, along with marked induction of HIF-1α and Cav-1 expression; additionally, it suppressed the expression of the antiapoptotic marker Bcl-2 while leaving AMPK activity unchanged. Under hypoxic conditions, HIF-1α acts as a transcription factor for Cav-1 mRNA gene expression. The cell growth and Bcl-2 expression suppressed under hypoxia were reversed along with decreases in the induced HIF-1α and Cav-1 levels by AMPK activation with metformin (1mM) or phenformin (0.1mM). In addition, pretreatment with AMPK small interfering RNA not only increased the hypoxia-induced expression of HIF-1α and Cav-1, but also reversed the suppression of Bcl-2 expression. These results suggest that HIF-1α and Cav-1 expression in hypoxic environments is regulated by basal AMPK activity; therefore, the inhibition of Bcl-2 expression cannot be expected when AMPK activity is suppressed, even if Cav-1 expression is elevated.ConclusionsFor the first time, we find that AMPK activation can regulate HIF-1α induction as well as HIF-1α-induced Cav1 expression, and the hypoxia-induced inhibitory effect on the antiapoptotic pathway in RT4 cells is due to Cav-1-dependent AMPK activity.

Oxymatrine improves blood-brain barrier integrity after cerebral ischemia-reperfusion injury by downregulating CAV1 and MMP9 expression

BackgroundIschemic stroke (IS) is a major neurological condition associated with extremely high morbidity and mortality worldwide. Oxymatrine (OMT), a quinolizidine alkaloid extracted from the root of Sophora flavescens, has neuroprotective properties and protects against IS. However, whether its protective effect involves alterations in the integrity of the blood-brain barrier (BBB) is unknown. PurposeHere, we used in vivo and in vitro models of IS to evaluate the protective effects of OMT and to establish whether its effects are mediated via the modulation of the BBB function. MethodsWe assessed the effects of OMT by using neurological function scores, triphenyltetrazolium chloride staining, Nissl staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling. ResultsOMT significantly prevented cellular damage, improved neurological function, and reduced BBB permeability in a mouse model of cerebral ischemia-reperfusion. Additionally, OMT protected the function of the tight junctions of bEend.3 cells against the consequences of oxygen-glucose deprivation. Furthermore, intracranial lentivirus injection of short hairpin RNA targeting Cav1 decreased caveolin-1 expression and inhibited the neuroprotective effects of OMT. ConclusionsOMT attenuated ischemia-reperfusion injury-induced damage to the BBB, and this neuroprotective action was at least partially dependent on the expression levels of CAV1 and MMP9 proteins. Therefore, OMT may offer effective protection against BBB injury induced by ischemia-reperfusion episodes.

Azelnidipine treatment reduces the expression of Cav1.2 protein

AimsAzelnidipine, a third-generation dihydropyridine calcium channel blocker (DHP CCB), has a characteristic hypotensive effect that persists even after it has disappeared from the plasma, which is thought to be due to its high hydrophobicity. However, because azelnidipine is unique, it might have other unknown effects on L-type Cav1.2 channels that result in the long-lasting decrease of blood pressure. The aim of this study was to investigate the potential quantitative modification of Cav1.2 by azelnidipine. Main methodsHEK293 cells were used to express Cav1.2 channels. Immunocytochemical analysis was performed to detect changes in the surface expression of the pore-forming subunit of the Cav1.2 channel, Cav1.2α1c. Western blotting analysis was performed to evaluate changes in expression levels of total Cav1.2α1c and Cavβ2c. Key findingsThe surface expression of Cav1.2α1c was markedly reduced by treatment with azelnidipine, but not with other DHP CCBs (amlodipine and nicardipine). Results obtained with a dynamin inhibitor and an early endosome marker suggested that the reduction of surface Cav1.2α1c was not likely caused by internalization. Azelnidipine reduced the total amount of Cav1.2α1c protein in HEK293 cells and rat pulmonary artery smooth muscle cells. The reduction of Cav1.2α1c was rescued by inhibiting proteasome activity. In contrast, azelnidipine did not affect the amount of auxiliary Cavβ2c subunits that function as a chaperone of Cav1.2. SignificanceThis study is the first to demonstrate that azelnidipine reduces the expression of Cav1.2α1c, which might partly explain its long-lasting hypotensive effect.

Mir199a-3p and 5p Influence Fibrotic Disease States, And Increase In Expression After Radiation

The mir199a family, composed of 3p and 5p splice variants, are associated with fibrotic cardiac, lung and renal diseases of different etiologies. Mir199a-3p and 5p target vital negative regulators of fibrosis-inducing pathways including JAK/STAT via targeting of SOCS7, and caveolin-dependent internalization pathway through targeting of CAV1. The objective of this study was to investigate if mir199a 3p and 5p expression increase after irradiation (RT) and regulate radiation-induce fibrosis via the similar pathways. C57BL/6 were treated with focal 14Gy renal irradiation and kidneys and overlaying skin harvested after 24hrs, 10wks, or 20wks. Untreated mouse kidneys and skin served as age-matched controls. RNA was isolated from the skin and kidney tissue using a trizol extraction method, and quantitative PCR analysis was performed to assess gene expression changes in mir199a-3p and 5p and critical components of the JAK/STAT and caveolin pathways. Both mir199a-3p and 5p expression increased in kidneys at the 20wks, with increases over untreated controls of 2.1-fold (p<0.001) and 2.4-fold (p<0.001) respectively. A 10-fold increase in expression of mir199a precursor RNA was seen for skin at 20 wks, and this translated into a 3.1-fold increase of mir199a-3p (p = 0.046) but without a measurable increase in the mir199a-5p variant. SOCS7 expression levels decreased at 20wk consistent with mir199a targeting, although not significantly when comparing to untreated controls. Levels of CAV1 increased significantly over untreated controls at the 10wk (1.8-fold, p = 0.04). At 20 weeks, transcript levels of CAV1 decreased to a level no longer significantly different than in age-matched controls, consistent with mir199a targeting. We show that mir199a family members are elevated in the kidneys and overlaying skin after 14Gy of radiation and mechanistically-associated with fibrosis. On-going studies are evaluating mir199a-3p and 5p after more clinically-relevant doses of RT.

Expression of caveolin-1 in tooth germ, ameloblastoma and ameloblastic carcinoma.

Background The caveolin-1 protein (structural component of membrane caveolae) plays important roles in several biological functions, such as endocytosis, cell adhesion, and cell signaling. However, this protein has been associated with mechanisms of tumorigenesis in several neoplasms. The expression patterns and roles of caveolin-1 in the oral epithelium and in embryonic and odontogenic tumor tissues are still unclear. Material and Methods The expression of caveolin-1 was evaluated in samples of the normal gingival epithelium (n=7), human tooth germ (TG) (n=12), ameloblastoma (AM) (n=83), and ameloblastic carcinoma (AC) (n=9) by immunohistochemistry. Additionally, AM samples were analyzed by qRT-PCR and Western blot. Results Most TG (91.7%), AM (73.5%) and AC (100%) samples showed diverse patterns of immunohistochemical positivity for caveolin-1, while only one gingival sample was positive. The transcript levels of cav-1 were significantly upregulated by 14.9-fold in AM tissue (P = 0.0014) compared to those in normal gingival epithelial tissue, as shown by qRT-PCR. Presence of caveolin-1 protein was confirmed by Western blot analysis. The caveolin-1 immunoexpression patterns throughout the stages of TG show its importance during odontogenesis. Conclusions The overexpression of caveolin-1 in AM and AC compared to its expression in normal gingival epithelium (adult tissue) suggests a possible role of caveolin-1 in protumoral events, but due to the similar immunoexpression observed in AM and AC, caveolin-1 may not necessarily participate in the malignant transformation process. However, future studies are needed to clarify and confirm these hypotheses. Key words:Ameloblastoma, ameloblastic carcinoma, caveolin-1, immunohistochemistry, real-time polymerase chain reaction.

Tunicamycin induced endoplasmic reticulum stress in the small intestine

ABSTRACT Because the small intestine is exposed to variety of foreign substances, it participates in host immune response. We investigated whether the expression levels of intestinal MAdCAM-1, PECAM-1 (CD31) and CAV-1 are affected by endoplasmic reticulum (ER) stress following brief treatment with tunicamycin (TN). We administered a single dose of TN intraperitoneally. Twenty-four hours later, MAdCAM-1, PECAM-1 and CAV-1 expression levels in Peyer’s patches and villi were examined using immunohistochemistry (IHC), immunofluorescence (IF) and western blotting. Immunostaining of MAdCAM-1 and CAV-1 in control and TN treated Peyer’s patches and villi exhibited similar staining patterns. The immunoreactivity of PECAM-1 was similar for the control and TN treated Payer’s patches, whereas staining was decreased significantly in TN treated villi. Our findings suggest that short term TN treatment did not affect leukocyte movement to lymphoid compartments of the small intestine, but it altered villus architecture due to decreased PECAM-1 expression.