Cav-1 Protein Research Articles

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.

The RNA-binding protein HuR regulates intestinal epithelial restitution by modulating Caveolin-1 gene expression.

The integrity of the intestinal mucosal barrier protects hosts against pathological conditions. Early mucosal restitution after wounding refers to epithelial cell migration into a defect. The RNA-binding protein HuR plays an important role in the posttranscriptional regulation of gene expression and is involved in many aspects of cellular physiology. In the present study, we investigated the role of HuR in the regulation of cell migration through the posttranscriptional regulation of Caveolin-1 (Cav-1). Online software was used to identify Cav-1 mRNA as a potential target of HuR. The interaction of HuR with Cav-1 mRNA was investigated via ribonucleoprotein immunoprecipitation (RNP IP) assays and biotin pulldown analysis. HuR was found to bind specifically to the Cav-1 3'-UTR rather than the coding region or 5'-UTR. Transfection of cells with siHuR decreased both HuR protein levels and Cav-1 protein levels; conversely, ectopic overexpression of HuR via infection of cells with an adenoviral vector containing HuR cDNA (AdHuR) increased Cav-1 protein levels without disturbing Cav-1 mRNA levels. Thus, HuR enhanced Cav-1 expression in vitro by stimulating Cav-1 translation. Intestinal epithelium-specific HuR knockout in mice decreased Cav-1 protein levels without changing Cav-1 mRNA levels, consistent with the in vitro results. Decreasing the levels of HuR via siHuR transfection inhibited early epithelial repair, but this effect was reversed by ectopic overexpression of GFP-tagged Cav-1. These results indicate that posttranscriptional regulation of Cav-1 gene expression by HuR plays a critical role in the regulation of rapid epithelial repair after wounding.

Inner retinal injury in experimental glaucoma is prevented upon AAV mediated Shp2 silencing in a caveolin dependent manner.

SH2 domain containing tyrosine phosphatase 2 (Shp2; PTPN11) regulates several intracellular pathways downstream of multiple growth factor receptors. Our studies implicate that Shp2 interacts with Caveolin-1 (Cav-1) protein in retinal ganglion cells (RGCs) and negatively regulates BDNF/TrkB signaling. This study aimed to investigate the mechanisms underlying the protective effects of shp2 silencing in the RGCs in glaucomatous conditions.Methods: Shp2 was silenced in the Cav-1 deficient mice and the age matched wildtype littermates using adeno-associated viral (AAV) constructs. Shp2 expression modulation was performed in an acute and a chronic mouse model of experimental glaucoma. AAV2 expressing Shp2 eGFP-shRNA under a strong synthetic CAG promoter was administered intravitreally in the animals' eyes. The contralateral eye received AAV-eGFP-scramble-shRNA as control. Animals with Shp2 downregulation were subjected to either microbead injections or acute ocular hypertension experimental paradigm. Changes in inner retinal function were evaluated by measuring positive scotopic threshold response (pSTR) while structural and biochemical alterations were evaluated through H&E staining, western blotting and immunohistochemical analysis of the retinal tissues.Results: A greater loss of pSTR amplitudes was observed in the WT mice compared to Cav-1-/- retinas in both the models. Silencing of Shp2 phosphatase imparted protection against inner retinal function loss in chronic glaucoma model in WT mice. The functional rescue also translated to structural preservation of ganglion cell layer in the chronic glaucoma condition in WT mice which was not evident in Cav-1-/- mice retinas.Conclusions: This study indicates that protective effects of Shp2 ablation under chronic experimental glaucoma conditions are dependent on Cav-1 in the retina, suggesting in vivo interactions between the two proteins.

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy

The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n = 3. High-resolution isoelectric focusing liquid chromatography-tandem MS (HiRIEF-LC-MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging.

Adipocyte of Obese Breast Cancer Patients Is Characterized by The Overexpression of Caveolin-1 Protein/Mediator the Main Constituent of the Plasma Membrane Vesicles Caveolae That Contain Proteins Contribute to Breast Cancer Progression

Breast cancer (BC) is the second leading mortality cause due to poor survival rates compared to lung cancer all over the world. Recently, lifestyle increased obesity among the population globally. Since, the adipose tissues (AT) are the major contributor to the volume of the breast and adipocytes cells, which constitute AT are one of the major prominent cells play an effective role in cancer progression via releasing different mediators and adipokines. Thus, AT may display a crucial role in BC progression, especially in obese patients compared to non-obese patients, which characterized by increased AT. Interestingly, adipocytes are characterized by expressing caveolin-1 (Cav-1) protein. Cav-1 constitutes the lipid raft of caveola which contains different proteolytic enzymes inducing cancer metastasis. In this regard, the aim of the present study was to explore the level of expression of Cav-1 protein in the tissue specimen of 5 non-obese vs. 15 obese patients using immunohistochemistry (IHC) and immunoblotting techniques. Our finding demonstrates that thelevel ofCav-1expression was statistically significantly low in non-obese compared to obese BC patients (p < 0.05). Herein, our results revealed that the highest expression of Cav-1in obese patients compared to non-obese (control) patients can be considered as a biomarker for BC patients.

Sphingomyelin Effects in Caveolin-1 Mediated Membrane Curvature.

The caveolin-1 (cav-1) protein is an integral component of caveolae and has been reported to colocalize with cholesterol and sphingomyelin-rich curved membrane domains. Here, we analyze the molecular interactions between cav-1 and sphingomyelin containing bilayers using a series of coarse-grain simulations, focusing on lipid clustering and membrane curvature. We considered a palmitoylated-cav-1 construct interacting with phospholipid/cholesterol membranes with asymmetrically distributed sphingomyelin, varying between 5 and 15% in total. We observe that cav-1 binds to the intracellular leaflet and induces a small positive curvature in the leaflet to which it is bound and an opposing negative curvature in the extracellular leaflet. Both cholesterol and sphingomyelin are observed to cluster in cav-1 bound membranes, mainly in the extracellular leaflet. Due to their negative spontaneous curvature, clustering of cholesterol and sphingomyelin facilitates membrane curvature such that the extent of either cholesterol or sphingomyelin clustering is dependent on the curvature induced. Our results suggest that cav-1 binding induces concentration-dependent curvature effects in sphingomyelin-rich membranes. Overall, our work is an important step in understanding the molecular basis of curvature and lipid clustering in cav-1 bound cellular membranes.

Caveolin-1 Regulates CCL5 and PPARγ Expression in Nthy-ori 3-1 Cells: Possible Involvement of Caveolin-1 and CCL5 in the Pathogenesis of Hashimoto's Thyroiditis.

Hashimoto's thyroiditis (HT) is characterized by lymphocytic infiltration of the thyroid parenchyma, which ultimately leads to tissue destruction and loss of function. Caveolin-1 (Cav-1) is an essential structural constituent of lipid rafts in the plasma membrane of cells and is reported to be significantly reduced in thyrocytes from HT patients. However, the mechanism of Cav-1 involvement in HT pathogenesis is still largely unclear. Cav-1 expression in thyroid tissues from HT patients and euthyroid nodular goiter tissues was detected by immunohistochemistry staining. Cav-1 knockdown and overexpression were constructed by lentiviral transfection in the human thyroid follicular epithelial cell (TFC) line of Nthy-ori 3-1. The mRNA expression levels of chemokines in TFCs were determined by quantitative real-time PCR (qPCR). Cav-1 and peroxisome proliferator-activated receptor gamma (PPARγ) levels were analysed by qPCR and Western blot analysis. The migration ability of peripheral blood mononuclear cells (PBMCs) was detected by the Transwell assay. In this study, Cav-1 and PPARγ expression was reduced in the thyroid tissues from HT patients. In vitro experiments showed that the expressions of chemokine (C-C motif) ligand 5 (CCL5) and migration of PBMCs were markedly increased, while the level of PPARγ was significantly decreased after the lentivirus-mediated knockdown of Cav-1 in Nthy-ori 3-1 cells. Interestingly, pioglitazone, a PPARγ agonist, not only upregulated PPARγ and Cav-1 proteins significantly, but also effectively reversed the Cav-1-knockdown-induced upregulation of CCL5 in Nthy-ori 3-1 cells and reduced the infiltration of lymphocytes. The inhibition of Cav-1 upregulated the CCL5 expression and downregulated the PPARγ expression in TFC while pioglitazone, a PPARγ agonist, reversed the detrimental consequence. This outcome might be a potential target for the treatment of lymphocyte infiltration into the thyroid gland and HT development.

Involvement of Caveolin-1-mediated transcytosis in the intratumoral accumulation of liposomes

For achieving efficient cancer treatment, it is important to elucidate the mechanism responsible for the accumulation of nanoparticles in tumor tissue. Recent studies suggest that nanoparticles are not delivered merely through gaps between tumor endothelial cells. We previously reported that the maturation of the vascular structure by the vascular endothelial cell growth factor receptor 2 (VEGFR2) using a previously developed siRNA delivery technology (RGD-MEND) significantly enhanced the accumulation of nanoparticles in types of cancers that area vessel-rich (renal cell carcinoma). This result was completely inconsistent with the generally accepted theory of the enhanced permeability and retention (EPR) effect. We hypothesized that a caveolin-1 (Cav1)-mediated transcellular route would be involved with the penetration of nanoparticles into tumor vasculature. To reveal the exact mechanism responsible for this enhancement, we observed the delivery of long-circulating liposomes (LPs) after Cav1 was co-suppressed by RGD-MEND with VEGFR2. The enhanced delivery of LPs by siRNA against VEGFR2 (siVEGFR2) was accompanied by the elevated expression of the Cav1 protein. In addition, Cav1 knockdown by siRNA against Cav1 (siCav1) canceled the enhanced delivery of LPs by siVEGFR2. The injection of siCav1 had no effect on the formation of alpha smooth muscle actin or vascular endothelial cell adhesion molecules. These results suggest that a Cav1-induced transcellular route and not a paracellular route, at least partially, contributes to the accumulation of nanoparticles in tumors.

Hereditary spastic paraplegia SPG8 mutations impair CAV1-dependent, integrin-mediated cell adhesion.

Mutations in WASHC5 (also known as KIAA0196) cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG8. WASHC5, commonly called strumpellin, is a core component of the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex that activates actin nucleation at endosomes. Although various other cellular roles for strumpellin have also been described, none account for how SPG8-associated mutations lead to HSP. Here, we identified protein interactors of the WASH complex by immunoprecipitation and mass spectrometry and assessed the functions of strumpellin in cultured cells using both overexpression and RNA interference along with cell-spreading assays to investigate cell adhesion. We uncovered a decrease in CAV1 protein abundance as well as endosomal fission defects resulting from pathogenic SPG8 mutations. CAV1, a key component of caveolae, interacted with strumpellin in cells, and strumpellin inhibited the lysosomal degradation of CAV1. SPG8-associated missense mutations in strumpellin did not rescue endosomal tubulation defects, reduction in CAV1 protein abundance, or integrin-mediated cell adhesion in strumpellin-deficient cells. Mechanistically, we demonstrated that the WASH complex maintained CAV1 and integrin protein amounts by inhibiting their lysosomal degradation through its endosomal actin nucleation activity. In addition, the interaction of strumpellin with CAV1 stimulated integrin recycling, thereby promoting cell adhesion. These findings provide a molecular link between WASHC5 mutations and impairment of CAV1- and integrin-mediated cell adhesion, providing insights into the cellular pathogenesis of SPG8.

Loss of Immunohistochemical Reactivity in Association With Handling-Induced Dark Neurons in Mouse Brains.

The handling-induced dark neuron is a histological artifact observed in brain samples handled before fixation with aldehydes. To explore associations between dark neurons and immunohistochemical alterations in mouse brains, we examined protein products encoded by Cav3 (neuronal perikarya/neurites), Rbbp4 (neuronal nuclei), Gfap (astroglia), and Aif1 (microglia) genes in adjacent tissue sections. Here, dark neurons were incidental findings from our prior project, studying the effects of age and high-fat diet on metabolic homeostasis in male C57BL/6N mice. Available were brains from 4 study groups: middle-aged/control diet, middle-aged/high-fat diet, old/control diet, and old/high-fat diet. Young/control diet mice were used as baseline. The hemibrains were immersion-fixed with paraformaldehyde and paraffin-embedded. In the hippocampal formation, we found negative correlations between dark neuron hyperbasophilia and immunoreactivity for CAV3, RBBP4, and glial fibrillary acidic protein (GFAP) using quantitative image analysis. There was no significant difference in dark neuron hyperbasophilia or immunoreactivity for any protein examined among all groups. In contrast, in the hippocampal fimbria, old age seemed to be associated with higher immunoreactivity for GFAP and allograft inflammatory factor-1. Our findings suggest that loss of immunohistochemical reactivity for CAV3, RBBP4, and GFAP in the hippocampal formation is an artifact associated with the occurrence of dark neurons. The unawareness of dark neurons may lead to misinterpretation of immunohistochemical reactivity alterations.

Expression and prognostic significance of caveolin-1 and forkhead box protein A1 in gastric adenocarcinoma

Background Gastric cancer is one of the commonest malignant tumors worldwide. Caveolin-1 (Cav-1) has been involved in variable processes, including migration, cellular transformation, signal transduction, and carcinogenesis. Forkhead box protein A1 (FOXA1) is a transcription factor that regulates chromatin structure and recruits other transcription factors to enhance transcription of downstream molecules. Materials and methods The present work was conducted to study the immunohistochemical expression of Cav-1 and Forkhead box protein A1 (FOXA1) in 60 formalin-fixed paraffin-embedded tissue blocks from gastric adenocarcinoma. Results High nuclear expression of Cav-1 and FOXA1 was detected in 37 (61.7%) cases and 35 (58.3%) cases (61.7% and 58.3%), respectively. A statistically significant relationship was detected between their immunoexpression and tumor grade, lymph node metastasis, TNM stage, and overall survival. Cav-1 and FOXA1 immunoexpression showed a moderate positive correlation in gastric adenocarcinoma. Conclusion Cav-1 and FOXA1 immunoexpression was significantly associated with poor prognostic factors and thus could be used as prognostic indicators for patients with gastric carcinoma.

Abstract B60: Optimization of biologic scheduling of gemcitabine and abraxane improves treatment response compared to the standard concurrent regimen in preclinical models of pancreatic cancer

Abstract Introduction: Gemcitabine (gem) and Abraxane (albumin-bound paclitaxel, abx) combination chemotherapy delivered on the same day is now one of the preferred chemotherapy regimens for metastatic and locally advanced pancreatic ductal adenocarcinoma (PDAC) based on results of the phase III MPACT clinical trial that showed improved overall survival of gem+abx compared to gem alone in the metastatic/advanced setting. Our previous published works revealed that caveolin-1 (Cav-1), the principal structural component of caveolae and mediator of endocytosis, is highly expressed in PDAC cells and associated with enhanced tumor progression and resistance to gem in PDAC preclinical models. We also found that loss of Cav-1 through genetic knockdown inhibited both PDAC cell uptake of albumin and response to albumin-based chemotherapies, i.e., abx. In addition, gem treatment resulted in a time-dependent increase in Cav-1 protein expression. Taken together, we hypothesized pretreatment with gem would increase the efficacy of the gem/abx combination via increased Cav-1 dependent albumin-bound chemotherapy uptake. Results: ASPC1 and HPAFII PDAC cell lines were exposed to gem (25-100nM) for 24-48 hours followed by a 1-hour albumin pulse and displayed a time-dependent increase in intracellular albumin via Western blotting. We next tested PDAC cell proliferation and cell survival via alamarBlue and colony formation assays with various different gem/abx scheduling combinations. HPAFII and ASPC1 cells were treated for 24 hours with the following schedules: (1) vehicle, (2) gem alone, (3) abx alone, (4) gem+abx on same day (d1gem+abx), (5) gem on day 1 and abx day 2 (d1gem_d2abx), or (6) gem on day 1, no drug on day 2 and abx on day 3 (d1gem_d3abx). Both alternative schedules of d1gem_d2abx and d1gem_d3abx had greater inhibition of proliferation and colony formation compared to the current standard of care d1gem+abx, and of all the combinations, d1gem_d3abx was the most efficacious (p&amp;lt;0.01). Genetic knockdown of Cav-1 through short-hairpin loop RNA (shRNA) in Mia-Paca2 cells resulted in reversal of the observed altered scheduling effect on cell proliferation and survival. In vivo, we compared tumor growth rates with altering gem-abx scheduling following subcutaneous injection into athymic nude mice of ASPC1 or HPAFII cells. Similar to our in vitro studies, mice were treated with either the control-vehicle or three cycles delivered over 9 days of the following gem-abx combinations: 1) d1gem+abx, 2) d1gem_d2abx, or 3) d1gem_d3abx. The d1gem_d3abx scheduling had the most significant delay in tumor growth and tumor doubling time compared to the other gem-abx schedules (p&amp;lt;0.001). Conclusion: We found 24- to 48-hour gem treatment increased albumin uptake, leading to maximal treatment efficacy with an optimized schedule of gem delivered on day 1 and abx on day 3 compared to the standard gem+abx administered on the same day. These findings support further testing of an altered and biologically optimized scheduling of gemcitabine and Abraxane. Citation Format: Adam R. Wolfe, Ryan Robb, Ahmad Hegazi, Duan-Liang Shyu, Laith Abushahin, Terence M. Williams. Optimization of biologic scheduling of gemcitabine and abraxane improves treatment response compared to the standard concurrent regimen in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B60.

Multimodal, in Situ Imaging of Ex Vivo Human Skin Reveals Decrease of Cholesterol Sulfate in the Neoepithelium during Acute Wound Healing.

Skin repair is a significant aspect of human health. While the makeup of healthy stratum corneum and epidermis is generally understood, the mobilization of molecular components during skin repair remains largely unknown. In the present work, we utilize multimodal, in situ, mass spectrometry, and immunofluorescence imaging for the characterization of newly formed epidermis, following an initial acute wound for the first 96 h of epithelization. In particular, TOF-SIMS and confirmatory MALDI FT-ICR MS (/MS) analysis permitted the mapping of several lipid classes, including phospholipids, neutral lipids, cholesterol, ceramides, and free fatty acids. Endogenous lipid species were localized in discrete epidermal skin layers, including the stratum corneum (SC), stratum granulosum (SG), stratum basale (SB), and dermis. Experiments revealed that healthy re-epithelializing skin is characterized by diminished cholesterol sulfate signal along the stratum corneum toward the migrating epithelial tongue. The spatial distribution and relative abundances of cholesterol sulfate are reported and correlated with the healing time. The multimodal imaging approach enabled in situ high-confidence chemical mapping based on accurate mass and fragmentation pattern of molecular components. The use of postanalysis immunofluorescence imaging from the same tissue confirmed the localization of endogenous lipid species and Filaggrin and Cav-1 proteins at high spatial resolution (approximately a few microns).

Lifelong n-3 Polyunsaturated Fatty Acid Exposure Modulates Size of Mammary Epithelial Cell Populations and Expression of Caveolae Resident Proteins in Fat-1 Mice.

Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been associated with reduced breast cancer risk; however, the exact mechanism remains elusive. Female wildtype (WT) and fat-1 mice were fed a 10% safflower diet until 6 weeks of age. Mammary gland epithelial cells (EC) were isolated and EC populations were determined by CD24 surface expression. Fat-1 mice expressed 65%, 20%, and 15% while WT mice expressed 65%, 26% and 9% for non-, myo- and luminal ECs, respectively. The luminal EC population was significantly greater in fat-1 mice (p ≤ 0.05), while the total number of mammary ECs were similar between groups (p = 0.79). Caveolae was isolated from ECs and Her-2/neu, ER-α and cav-1 protein expression was determined by Western blotting. Fat-1 mice had a two-fold greater ER-α (p ≤ 0.05) and a 1.5-fold greater cav-1 (p ≤ 0.05) expression than WT with a similar amount of Her-2/neu protein (p = 0.990) between groups. Overall, this study provides novel mechanistic evidence by which n-3 PUFA modifies early mammary gland development that may potentially reduce breast cancer risk later in life.

Caveolae-Mediated Activation of Mechanosensitive Chloride Channels in Pulmonary Veins Triggers Atrial Arrhythmogenesis.

BackgroundAtrial fibrillation often occurs in the setting of hypertension and associated atrial dilation with pathologically increased cardiomyocyte stretch. In the setting of atrial dilation, mechanoelectric feedback has been linked to the development of ectopic beats that trigger paroxysmal atrial fibrillation mainly originating from pulmonary veins (PVs). However, the precise mechanisms remain poorly understood.Methods and ResultsWe identify mechanosensitive, swelling‐activated chloride ion channels (IC l,swell) as a crucial component of the caveolar mechanosensitive complex in rat and human cardiomyocytes. In vitro optical mapping of rat PV, single rat PV, and human cardiomyocyte patch clamp studies showed that stretch‐induced activation of I Cl,swell leads to membrane depolarization and decreased action potential amplitude, which trigger conduction discontinuities and both ectopic and reentrant activities within the PV. Reverse transcription quantitative polymerase chain reaction, immunofluorescence, and coimmunoprecipitation studies showed that I Cl,swell likely consists of at least 2 components produced by mechanosensitive ClC‐3 (chloride channel‐3) and SWELL1 (also known as LRRC8A [leucine rich repeat containing protein 8A]) chloride channels, which form a macromolecular complex with caveolar scaffolding protein Cav3 (caveolin 3). Downregulation of Cav3 protein expression and disruption of caveolae structures during chronic hypertension in spontaneously hypertensive rats facilitates activation of I Cl,swell and increases PV sensitivity to stretch 10‐ to 50‐fold, promoting the development of atrial fibrillation.ConclusionsOur findings identify caveolae‐mediated activation of mechanosensitive I Cl,swell as a critical cause of PV ectopic beats that can initiate atrial arrhythmias including atrial fibrillation. This mechanism is exacerbated in the setting of chronically elevated blood pressures.

Intact caveolae are required for proper extravillous trophoblast migration and differentiation.

Caveolae constitute membrane domains critical for the organization and synchronization of different signaling molecules related to numerous cell processes such as cell migration, invasion, and differentiation. Caveolin-1 (Cav-1) is the main integral membrane protein of these domains. Recently, it was found that a normal expression of aquaporin-3 (AQP3) is required for extravillous trophoblast (EVT) cell migration. Our aim was to investigate the role of caveolae in the migration, invasion, and endovascular differentiation of human EVT cells during placentation and its interaction with AQP3. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's modified Eagle's medium-nutrient mixture F12 and treated with 5 mM methyl-β-cyclodextrin (MβCD) to disrupt caveolae. We found that after MβCD treatment, Cav-1 protein was undetectable. In this condition, the ability of the cells to migrate was significantly decreased compared with the control cells, while no differences were observed in the number of invading cells and the metalloproteinases activity between control and MβCD-treated cells. Surprisingly, the disruption of caveolae significantly enhanced EVT endovascular differentiation. On the contrary, the silencing of AQP3, negatively affected tube-like formation. The theoretical analysis of the primary sequence of AQP3 protein revealed a putative Cav-1-binding site. In addition, immunoprecipitation and double immunofluorescence assays showed that AQP3 colocalized with Cav-1. These results showed that during placentation an intact caveola in EVT cells may be necessary for AQP3 and Cav-1 interaction and any perturbations might result in serious pregnancy disorders.

Caveolae and scaffold detection from single molecule localization microscopy data using deep learning.

Caveolae are plasma membrane invaginations whose formation requires caveolin-1 (Cav1), the adaptor protein polymerase I, and the transcript release factor (PTRF or CAVIN1). Caveolae have an important role in cell functioning, signaling, and disease. In the absence of CAVIN1/PTRF, Cav1 forms non-caveolar membrane domains called scaffolds. In this work, we train machine learning models to automatically distinguish between caveolae and scaffolds from single molecule localization microscopy (SMLM) data. We apply machine learning algorithms to discriminate biological structures from SMLM data. Our work is the first that is leveraging machine learning approaches (including deep learning models) to automatically identifying biological structures from SMLM data. In particular, we develop and compare three binary classification methods to identify whether or not a given 3D cluster of Cav1 proteins is a caveolae. The first uses a random forest classifier applied to 28 hand-crafted/designed features, the second uses a convolutional neural net (CNN) applied to a projection of the point clouds onto three planes, and the third uses a PointNet model, a recent development that can directly take point clouds as its input. We validate our methods on a dataset of super-resolution microscopy images of PC3 prostate cancer cells labeled for Cav1. Specifically, we have images from two cell populations: 10 PC3 and 10 CAVIN1/PTRF-transfected PC3 cells (PC3-PTRF cells) that form caveolae. We obtained a balanced set of 1714 different cellular structures. Our results show that both the random forest on hand-designed features and the deep learning approach achieve high accuracy in distinguishing the intrinsic features of the caveolae and non-caveolae biological structures. More specifically, both random forest and deep CNN classifiers achieve classification accuracy reaching 94% on our test set, while the PointNet model only reached 83% accuracy. We also discuss the pros and cons of the different approaches.

Astrocyte-derived fatty acid-binding protein 7 protects blood-brain barrier integrity through a caveolin-1/MMP signaling pathway following traumatic brain injury

The astrocyte-endothelial cell interaction is crucial for normal brain homeostasis and blood-brain barrier (BBB) disruption in pathological conditions. However, the mechanism by which astrocytes control BBB integrity, especially after traumatic brain injury (TBI), remains unclear. Here, we present evidence that astrocyte-derived fatty acid-binding protein 7 (FABP7), a differentiation- and migration-associated molecule, may function as a modulator of BBB permeability in a rat weight-drop model of TBI. Immunohistochemical analysis revealed that TBI induced increased expression of FABP7 in astrocytes, accompanied by caveolin-1 (Cav-1) upregulation in endothelial cells. Administration of recombinant FABP7 significantly ameliorated TBI-induced neurological deficits, brain edema, and BBB permeability, concomitant with upregulation of endothelial Cav-1 and tight junction protein expression, while FABP7 knockdown resulted in the opposite effects. Furthermore, pretreatment with daidzein, a specific inhibitor of Cav-1, reversed the inhibitory effects of recombinant FABP7 on matrix metalloproteinase (MMP)-2/9 expression and abolished its BBB protection after TBI. Altogether, these findings suggest that astrocyte-derived FABP7 upregulation may represent an endogenous protective response to BBB disruption partly mediated through a Cav-1/MMP signaling pathway following TBI.

KBR develops new propane dehydrogenation technology

The astrocyte-endothelial cell interaction is crucial for normal brain homeostasis and blood-brain barrier (BBB) disruption in pathological conditions. However, the mechanism by which astrocytes control BBB integrity, especially after traumatic brain injury (TBI), remains unclear. Here, we present evidence that astrocyte-derived fatty acid-binding protein 7 (FABP7), a differentiation- and migration-associated molecule, may function as a modulator of BBB permeability in a rat weight-drop model of TBI. Immunohistochemical analysis revealed that TBI induced increased expression of FABP7 in astrocytes, accompanied by caveolin-1 (Cav-1) upregulation in endothelial cells. Administration of recombinant FABP7 significantly ameliorated TBI-induced neurological deficits, brain edema, and BBB permeability, concomitant with upregulation of endothelial Cav-1 and tight junction protein expression, while FABP7 knockdown resulted in the opposite effects. Furthermore, pretreatment with daidzein, a specific inhibitor of Cav-1, reversed the inhibitory effects of recombinant FABP7 on matrix metalloproteinase (MMP)-2/9 expression and abolished its BBB protection after TBI. Altogether, these findings suggest that astrocyte-derived FABP7 upregulation may represent an endogenous protective response to BBB disruption partly mediated through a Cav-1/MMP signaling pathway following TBI.

Altered expression of caveolin-1 in the colon of patients with Hirschsprung's disease.

The pathogenesis of Hirschsprung's disease-associated enterocolitis (HAEC) is unclear. Caveolin-1 (Cav-1) regulates the functions of different nitric oxide synthase (NOS) isoforms, which play critical roles in inflammation and intestinal epithelial barrier function. We designed this study to investigate the hypothesis that Cav-1 expression is altered in the bowel of patients with Hirschsprung's disease (HSCR). HSCR tissue specimens (n = 10) were collected at the time of pull-through surgery and control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 10). qRT-PCR analysis was undertaken to quantify Cav-1 gene expression, and Western blot analysis was undertaken to determine Cav-1 protein quantification. Immunolabelling of Cav-1 proteins was visualized using confocal microscopy. qRT-PCR and Western blot analysis revealed that Cav-1 was significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR compared to controls (p < 0.01). Confocal microscopy revealed a markedly decreased expression of Cav-1 in colonic epithelium of aganglionic and ganglionic bowel of patients with HSCR compared to controls. To our knowledge, this is the first report of significantly decreased Cav-1 expression in patients with HSCR. Decreased expression of Cav-1 in the bowel of HSCR may increase susceptibility to HAEC in HSCR.