Activation of Wnt signaling mitigates blood-brain barrier disruption by inhibiting vesicular transcytosis after traumatic brain injury in mice.

Elevated transport of Caveolin-1 (CAV-1) vesicles within vascular endothelial cells constitutes a significant secondary pathogenic event contributing to the compromise of the blood-brain barrier (BBB) post-traumatic brain injury (TBI). While Wnt/ß-catenin signaling is recognized for its critical involvement in angiogenesis and the maintenance of BBB integrity, its influence on vascular endothelial transcytosis in the aftermath of TBI is not well-defined. This study aims to elucidate the impact of Wnt/ß-catenin signaling on cerebrovascular vesicular transcytosis following TBI. In this experiment, adult male wild-type (WT) C57BL/6 mice underwent various interventions. TBI was induced utilizing the controlled cortical impact technique. Post-TBI, mice were administered either an inhibitor or an agonist of Wnt signaling via intraperitoneal injection. Recombinant adeno-associated virus (rAAV) was administered intracerebroventricularly to modulate the expression of the CAV-1 inhibitory protein, Major facilitator superfamily domain-containing 2a (Mfsd2a). This research utilized Evans blue assay, Western blot analysis, immunofluorescence, transmission electron microscopy, and neurobehavioral assessments. Post-TBI observations revealed substantial increases in macromolecule (Evans blue and albumin) leakage, CAV-1 transport vesicle count, astrocyte end-feet edema, and augmented aquaporin-4 (AQP4) expression, culminating in BBB disruption. The findings indicate that Wnt signaling pathway inhibition escalates CAV-1 transport vesicle activity and aggravates BBB compromise. Conversely, activating this pathway could alleviate BBB damage by curtailing CAV-1 vesicle presence. Post-TBI, there is a diminution in Mfsd2a expression, which is directly influenced by the modulation of WNT signals. Employing a viral approach to regulate Mfsd2a, we established that its down-regulation undermines the protective benefits derived from reducing CAV-1 transport vesicles through WNT signal enhancement. Moreover, we verified that the WNT signaling agonist LiCl notably ameliorates neurological deficits following TBI in mice. Collectively, our data imply that Wnt/ß-catenin signaling presents a potential therapeutic target for safeguarding against BBB damage and enhancing neurological function after TBI.

Caveolin-1's dual impact on endometrioid endometrial carcinoma: a histopathological and immunohistochemical study.

The objectives of this study are to evaluate caveolin-1 expression in endometrioid endometrial cancer and its correlation with clinicopathological parameters. Forty-four cases of endometrioid endometrial carcinomas underwent radical hysterectomy. The archived paraffin sections that were stained for caveolin-1 by immunohistochemistry, caveolin-1 expression were detected in cancerous epithelial cells in 18.2% of the cases, and stromal caveolin-1 was detected in 65.9% of the cases. Caveolin-1 expression in the epithelium showed a significant positive association with the T stage and the FIGO stage. Positive caveolin-1 expression in epithelium has a direct, positive and significant relationship with invasion of other organs and a direct and significant relationship with the advanced FIGO stage. As for caveolin-1 expression in the stroma, it showed a significant negative inversely significant association with myometrial invasion. Also, there is a significant negative association between caveolin-1 expression in the epithelium and its expression in the stroma. We conclude that caveolin-1 expression strongly plays a critical role in endometrioid endometrial carcinoma as a tumor suppressor or promoter of invasion. In early lesions, high stromal levels appear to be protective against progression. While decreased stromal expression and increased epithelial expression were associated with aggressive tumors.

Host caveolin-1 facilitates Zika virus infection via promoting viral RNA replication.

Zika virus (ZIKV) has gained notoriety in recent years without targeted therapies or vaccines available so far. Caveolin-1 (Cav-1) in host cells plays crucial functions in the invasion of many viruses. However, its specific involvement in ZIKV infection has remained unclear. Here, we reveal that depleting Cav-1 leads to a substantial reduction in ZIKV RNA levels, protein expression, and viral particle production, indicating that ZIKV exploits Cav-1 for its infection. By dissecting each stage of the viral life cycle, we unveiled that, unlike its invasion role in many other viruses, Cav-1 depletion selectively impairs ZIKV replication, resulting in altered replication dynamics and reduced strand-specific RNA levels, concurrently without affecting viral entry, maturation and release. These results reveal an unforeseen function of Cav-1 in facilitating ZIKV replication, that provides new insights into the intricate interaction between Cav-1 and ZIKV and underscores Cav-1 as a potential candidate for anti-ZIKV approaches.

Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action.

Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a context-dependent manner. We show here that a positive charge of Ago2 K212, that is preserved by SIR2-mediated Ago2 deacetylation in cancer cells, is responsible for the direct interaction between Ago2 and Caveolin-1 (CAV1). Through this interaction, CAV1 sequesters Ago2 on the plasma membranes and regulates miRNA-mediated translational repression in a compartment-dependent manner. Ago2/CAV1 interaction plays a role in miRNA-mediated mRNA suppression and in miRNA release via extracellular vesicles (EVs) from tumors into the circulation, which can be used as a biomarker of tumor progression. Increased Ago2/CAV1 interaction with tumor progression promotes aggressive cancer behaviors, including metastasis. Ago2/CAV1 interaction acts as a secondary event in miRNA-mediated suppression and increases the complexity of miRNA actions in cancer.

Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

Matrilin-3 supports neuroprotection in ischemic stroke by suppressing astrocyte-mediated neuroinflammation.

In the brain, the role of matrilin-3, an extracellular matrix component in cartilage, is unknown. Here, we identify that matrilin-3 decreased in reactive astrocytes but was unchanged in neurons after ischemic stroke in animals. Importantly, it declined in serum of patients with acute ischemic stroke. Genetic or pharmacological inhibition or supplementation of matrilin-3 aggravates or reduces brain injury, astrocytic cell death, and glial scar, respectively, but has no direct effect on neuronal cell death. RNA sequencing demonstrates that Matn3-/- mice display an increased inflammatory response profile in the ischemic brain, including the nuclear factor κB (NF-κB) signaling pathway. Both endogenous and exogenous matrilin-3 reduce inflammatory mediators. Mechanistically, extracellular matrilin-3 enters astrocytes via caveolin-1-mediated endocytosis. Cytoplasmic matrilin-3 translocates into the nucleus by binding to NF-κB p65, suppressing inflammatory cytokine transcription. Extracellular matrilin-3 binds to BMP-2, blocking the BMP-2/Smads pathway. Thus, matrilin-3 is required for astrocytes to exert neuroprotection, at least partially, by suppressing astrocyte-mediated neuroinflammation.

Caveolin-1 affects early mycobacterial infection and apoptosis in macrophages and mice.

Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the deadliest infections in humans. Because Mycobacterium bovis Bacillus Calmette-Guérin (BCG) share genetic similarities with Mycobacterium tuberculosis, it is often used as a model to elucidate the molecular mechanisms of more severe tuberculosis infection. Caveolin-1 has been implied in many physiological processes and diseases, but it's role in mycobacterial infections has barely been studied. We isolated macrophages from Wildtype or Caveolin-1 deficient mice and analyzed hallmarks of infection, such as internalization, induction of autophagy and apoptosis. For in vivo assays we intravenously injected mice with BCG and investigated tissues for bacterial load with colony-forming unit assays, bioactive lipids with mass spectrometry and changes of protein expressions by Western blotting. Our results revealed that Caveolin-1 was important for early killing of BCG infection in vivo and in vitro, controlled acid sphingomyelinase (Asm)-dependent ceramide formation, apoptosis and inflammatory cytokines upon infection with BCG. In accordance, Caveolin-1 deficient mice and macrophages showed higher bacterial burdens in the livers. The findings indicate that Caveolin-1 plays a role in infection of mice and murine macrophages with BCG, by controlling cellular apoptosis and inflammatory host response. These clues might be useful in the fight against tuberculosis.

Exosomal Prostate-Specific Membrane Antigen (PSMA) and Caveolin-1 as Potential Biomarkers of Prostate Cancer-Evidence from Serbian Population.

Prostate-specific membrane antigen (PSMA) and caveolin-1 are membrane proteins that are overexpressed in prostate cancer (PCa) and are involved in tumor growth and increase in aggressiveness. The aim of the present study is therefore to evaluate PSMA and caveolin-1 proteins from plasma exosomes as effective liquid biopsy biomarkers for PCa. This study included 39 patients with PCa and 33 with benign prostatic hyperplasia (BPH). The shape and size of the exosomes were confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis. Immunogold analysis showed that PSMA is localized to the membrane of exosomes isolated from the plasma of both groups of participants. The relative protein levels of PSMA and caveolin-1 in the plasma exosomes of PCa and BPH patients were determined by Western blot analysis. The relative level of the analyzed plasma exosomal proteins was compared between PCa and BPH patients and the relevance of the exosomal PSMA and caveoin-1 level to the clinicopathological parameters in PCa was investigated. The analysis performed showed an enrichment of exosomal PSMA in the plasma of PCa patients compared to the exosomes of men with BPH. The level of exosomal caveolin-1 in plasma was significantly higher in PCa patients with high PSA levels, clinical-stage T3 or T4 and in the group of PCa patients with aggressive PCa compared to favorable clinicopathological features or tumor aggressiveness. Plasma exosomes may serve as a suitable object for the identification of potential biomarkers for the early diagnosis and prognosis of PCa as well as carriers of therapeutic agents in precision medicine of PCa treatment.

Caveolin-1 gene expression provides additional prognostic information combined with PAM50 risk of recurrence (ROR) score in breast cancer.

Combining information from the tumor microenvironment (TME) with PAM50 Risk of Recurrence (ROR) score could improve breast cancer prognostication. Caveolin-1 (CAV1) is a marker of an active TME. CAV1 is a membrane protein involved in cell signaling, extracellular matrix organization, and tumor-stroma interactions. We sought to investigate CAV1 gene expression in relation to PAM50 subtypes, ROR score, and their joint prognostic impact. CAV1 expression was compared between PAM50 subtypes and ROR categories in two cohorts (SCAN-B, n = 5326 and METABRIC, n = 1980). CAV1 expression was assessed in relation to clinical outcomes using Cox regression and adjusted for clinicopathological predictors. Effect modifications between CAV1 expression and ROR categories on clinical outcome were investigated using multiplicative and additive two-way interaction analyses. Differential gene expression and gene set enrichment analyses were applied to compare high and low expressing CAV1 tumors. All samples expressed CAV1 with the highest expression in the Normal-like subtype. Gene modules consistent with epithelial-mesenchymal transition (EMT), hypoxia, and stromal activation were associated with high CAV1 expression. CAV1 expression was inversely associated with ROR category. Interactions between CAV1 expression and ROR categories were observed in both cohorts. High expressing CAV1 tumors conferred worse prognosis only within the group classified as ROR high. ROR gave markedly different prognostic information depending on the underlying CAV1 expression. CAV1, a potential mediator between the malignant cells and TME, could be a useful biomarker that enhances and further refines PAM50 ROR risk stratification in patients with ROR high tumors and a potential therapeutic target.

Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

Mechanical stretch leads to increased caveolin-1 content and mineralization potential in extracellular vesicles from vascular smooth muscle cells.

BACKGROUND: Hypertension-induced mechanical stress on vascular smooth muscle cells (VSMCs) is a known risk factor for vascular remodeling, including vascular calcification. Caveolin-1 (Cav-1), an integral structural component of plasma membrane invaginations, is a mechanosensitive protein that is required for the formation of calcifying extracellular vesicles (EVs). However, the role of mechanics in Cav-1-induced EV formation from VSMCs has not been reported. RESULTS: Exposure of VSMCs to 10% mechanical stretch (0.5 Hz) for 72 h resulted in Cav-1 translocation into non-caveolar regions of the plasma membrane and subsequent redistribution of Cav-1 from the VSMCs into EVs. Inhibition of Rho-A kinase (ROCK) in mechanically-stimulated VSMCs exacerbated the liberation of Cav-1 positive EVs from the cells, suggesting a potential involvement of actin stress fibers in this process. The mineralization potential of EVs was measured by incubating the EVs in a high phosphate solution and measuring light scattered by the minerals at 340 nm. EVs released from stretched VSMCs showed higher mineralization potential than the EVs released from non-stretched VSMCs. Culturing VSMCs in pro-calcific media and exposure to mechanical stretch increased tissue non-specific alkaline phosphatase (ALP), an important enzyme in vascular calcification, activity in EVs released from the cells, with cyclic stretch further elevating EV ALP activity compared to non-stretched cells. CONCLUSION: Our data demonstrate that mechanical stretch alters Cav-1 trafficking and EV release, and the released EVs have elevated mineralization potential.

MANNosylation of Mesoporous Silica Nanoparticles Modifies TLR4 Localization and NF-κB Translocation in T24 Bladder Cancer Cells.

D-mannose is widely used as non-antibiotic treatment for bacterial urinary tract infections. This application is based on a well-studied mechanism of binding to the type 1 bacterial pili and, therefore, blocking bacteria adhesion to the uroepithelial cells. To implement D-mannose into carrier systems, the mechanism of action of the sugar in the bladder environment is also relevant and requires investigation. Herein, two different MANNosylation strategies using mesoporous silica nanoparticles (MSNs) are described. The impact of different chemical linkers on bacterial adhesion and bladder cell response is studied via confocal microscopy imaging of the MSN interactions with the respective organisms. Cytotoxicity is assessed and the expression of Toll-like receptor 4 (TLR4) and caveolin-1 (CAV-1), in the presence or absence of simulated infection with bacterial lipopolysaccharide (LPS), is evaluated using the human urinary bladder cancer cell line T24. Further, localisation of the transcription factor NF-κB due to the MANNosylated materials is examined over time. The results show that MANNosylation modifies bacterial adhesion to the nanomaterials and significantly affects TLR4, caveolin-1, and NF-κB in bladder cells. These elements are essential components of the inflammatory cascade/pathogens response during urinary tract infections. These findings demonstrate that MANNosylation is a versatile tool to design hybrid nanocarriers for targeted biomedical applications.

Caveolin-1, a Determinant of the Fate of MCF-7 Breast Cancer Cells.

Background: The scaffolding protein, caveolin-1 (Cav-1), participates in multiple cellular functions including promotion of sodium excretion from the kidney. Loss of expression of Cav-1 is associated with tumorigenesis of various types of cancer. We have shown the potential link between hypertension and breast cancer via abnormal function of the G protein-coupled receptor kinase type 4 (GRK4). Objective: The current studies tested the hypothesis that Cav-1 acts as a tumor-suppressive factor in breast cancer cells and enhances the sensitivity to the inhibitory effect of the type 1 dopaminergic receptor (D1R). Methods: Michigan Cancer Foundation (MCF) MCF-7 cells stably expressing a Cav-1/mCherry fusion protein or mCherry alone were used as models to examine the effect of Cav-1 on cell growth, apoptosis, and senescence. Cell proliferation was determined by cell counting, cell cycle analysis (flow cytometry), and BrdU incorporation. Apoptosis was determined using the Cell Death Detection ELISA kit from Roche Diagnosis. Senescence was determined using the senescence associated beta galactosidase (SA-ß-gal) assay. Reactive oxygen species (ROS) was measured using 2',7'-dichlorodihydrofluorescein diacetate. Western blot analysis was used to measure activation of signaling pathway molecules. All statistical analyses were conducted with Microsoft Excel. Results: Overexpression of Cav-1 in MCF-7 cells reduced cellular growth rate. Both inhibition of proliferation and induction of cell death are contributing factors. Multiple signaling pathways were activated in Cav-1-expressing MCF-7 cells. Activation of Akt was prominent. In MCF-7-expressing Cav-1 (MCF-7 Cav-1) cells, the levels of phosphorylated Akt at S473 and T308 were increased 28- and 8.7-fold, respectively. Instead of protecting cells from apoptosis, extremely high levels of activated Akt resulted in increased levels of ROS which led to apoptosis and senescence. The tumor-suppressive effect plus downregulation of GRK4 makes Cav-1-expressing MCF-7 cells significantly more sensitive to the inhibitory effect of the D1R agonist, SKF38393. Conclusion: Caveolin-1 acts as a tumor-suppressing factor via extreme activation of Akt and down regulation of survival factors such as GRK4, survivin, and cyclin D1.

DHCR24 Insufficiency Promotes Vascular Endothelial Cell Senescence and Endothelial Dysfunction via Inhibition of Caveolin-1/ERK Signaling.

Endothelial cells (ECs) senescence is critical for vascular dysfunction, which leads to age-related disease. DHCR24, a 3ß-hydroxysterol δ 24 reductase with multiple functions other than enzymatic activity, has been involved in age-related disease. However, little is known about the relationship between DHCR24 and vascular ECs senescence. We revealed that DHCR24 expression is chronologically decreased in senescent human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. ECs senescence in endothelium-specific DHCR24 knockout mice was characterized by increased P16 and senescence-associated secretory phenotype, decreased SIRT1 and cell proliferation, impaired endothelium-dependent relaxation, and elevated blood pressure. In vitro, DHCR24 knockdown in young HUVECs resulted in a similar senescence phenotype. DHCR24 deficiency impaired endothelial migration and tube formation and reduced nitric oxide (NO) levels. DHCR24 suppression also inhibited the caveolin-1/ERK signaling, probably responsible for increased reactive oxygen species production and decreased eNOS/NO. Conversely, DHCR24 overexpression enhanced this signaling pathway, blunted the senescence phenotype, and improved cellular function in senescent cells, effectively blocked by the ERK inhibitor U0126. Moreover, desmosterol accumulation induced by DHCR24 deficiency promoted HUVECs senescence and inhibited caveolin-1/ERK signaling. Our findings demonstrate that DHCR24 is essential in ECs senescence.

Caveolin-1 mediates blood-brain barrier permeability, neuroinflammation, and cognitive impairment in SARS-CoV-2 infection.

Blood-brain barrier (BBB) permeability can cause neuroinflammation and cognitive impairment. Caveolin-1 (Cav-1) critically regulates BBB permeability, but its influence on the BBB and consequent neurological outcomes in respiratory viral infections is unknown. We used Cav-1-deficient mice with genetically encoded fluorescent endothelial tight junctions to determine how Cav-1 influences BBB permeability, neuroinflammation, and cognitive impairment following respiratory infection with mouse adapted (MA10) SARS-CoV-2 as a model for COVID-19. We found that SARS-CoV-2 infection increased brain endothelial Cav-1 and increased transcellular BBB permeability to albumin, decreased paracellular BBB Claudin-5 tight junctions, and caused T lymphocyte infiltration in the hippocampus, a region important for learning and memory. Concordantly, we observed learning and memory deficits in SARS-CoV-2 infected mice. Importantly, genetic deficiency in Cav-1 attenuated transcellular BBB permeability and paracellular BBB tight junction losses, T lymphocyte infiltration, and gliosis induced by SARS-CoV-2 infection. Moreover, Cav-1 KO mice were protected from the learning and memory deficits caused by SARS-CoV-2 infection. These results establish the contribution of Cav-1 to BBB permeability and behavioral dysfunction induced by SARS-CoV-2 neuroinflammation.

Ginsenoside Rb1 prevents age-related endothelial senescence by modulating SIRT1/caveolin-1/enos signaling pathway.

Background: Advancing age is one of the independent risk factors for cardiovascular disorders. The Compendium of Materia Medica, a classic book on traditional Chinese medicine, states that ginseng "harmonizes the five internal organs, calming the spirit and prolonging the years of life." Considered one of the primary bioactive compounds derived from Panax ginseng, ginsenoside Rb1 (g-Rb1) has been scientifically suggested to possess anti-senescence efficacy. More research is needed to explore the vascular pharmacological activity and potential clinical application value of g-Rb1. Aims of the study: Our previous study demonstrated that g-Rb1 could mitigate cellular senescence via the SIRT1/eNOS pathway. This study was performed to explore the exact mechanisms by which g-Rb1 modulates the SIRT1/eNOS pathway. Materials and methods: We used human primary umbilical vein endothelial cells (HUVECs) to establish a replicative ageing model. Real-time (RT‒PCR), western blotting, small interfering RNA (siRNA), and immunoprecipitation were conducted to detect the effect of g-Rb1 on the SIRT1/caveolin-1/eNOS axis. Results: G-Rb1 increased NO production and alleviated replicative senescence of HUVECs. The application of g-Rb1 elevated the mRNA and protein abundance of both SIRT1 and eNOS while concomitantly suppressing the expression of caveolin-1. Inhibition of SIRT1 and eNOS by siRNAs suppressed the anti-senescence function of g-Rb1, while caveolin-1 siRNA could enhance it. G-Rb1 decreased the acetylation level of caveolin-1 and increased NO production, which was suppressed by SIRT1 siRNA. Both g-Rb1 and caveolin-1 siRNA could reduce the acetylation level of eNOS and increase NO production. Conclusion: G-Rb1 prevents age-related endothelial senescence by modulating the SIRT1/caveolin-1/eNOS signaling pathway.

Extracellular Vesicles Maintain Blood-Brain Barrier Integrity by the Suppression of Caveolin-1/CD147/VEGFR2/MMP Pathway After Ischemic Stroke.

Background: Ischemic stroke (IS) causes tragic death and disability worldwide. However, effective therapeutic interventions are finite. After IS, blood-brain barrier (BBB) integrity is disrupted, resulting in deteriorating neurological function. As a novel therapeutic, extracellular vesicles (EVs) have shown ideal restorative effects on BBB integrity post-stroke; however, the definite mechanisms remain ambiguous. In the present study, we investigated the curative effects and the mechanisms of EVs derived from bone marrow mesenchymal stem cells and brain endothelial cells (BMSC-EVs and BEC-EVs) on BBB integrity after acute IS. Methods: EVs were isolated from BMSCs and BECs, and we investigated the therapeutic effect in vitro oxygen-glucose deprivation (OGD) insulted BECs model and in vivo rat middle cerebral artery occlusion (MCAo) model. The cell monolayer leakage, tight junction expression, and metalloproteinase (MMP) activity were evaluated, and rat brain infarct volume and neurological function were also analyzed. Results: The administration of two kinds of EVs not only enhanced ZO-1 and Occludin expressions but also reduced the permeability and the activity of MMP-2/9 in OGD-insulted BECs. The amelioration of the cerebral infarction, BBB leakage, neurological function deficits, and the increasing ZO-1 and Occludin levels, as well as MMP activity inhibition was observed in MCAo rats. Additionally, the increased levels of Caveolin-1, CD147, vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor A (VEGFA) in isolated brain microvessels were downregulated after EVs treatment. In vitro, the employment of Caveolin-1 and CD147 siRNA partly suppressed the expressions of VEGFR2, VEGFA and MMP-2/9 activity and reduced the leakage of OGD insulted BECs and enhanced ZO-1 and Occludin expressions. Conclusion: Our study firstly demonstrates that BEC and BMSC-EVs administrations maintain BBB integrity via the suppression of Caveolin-1/CD147/VEGFR2/MMP pathway after IS, and the efficacy of BMSC-EVs is superior to that of BEC-EVs.

The anti-neoplastic effects of metformin modulate the acquired phenotype of fibroblast cells in the breast cancer-normal fibroblast co-culture system.

Intracellular communications between breast cancer and fibroblast cells were reported to be involved in cancer proliferation, growth, and therapy resistance. The hallmarks of cancer-fibroblast interactions, consisting of caveolin 1 (Cav1) and mono-carboxylate transporter 4 (MCT4) (metabolic coupling markers), along with IL-6, TGFß, and lactate secretion, are considered robust biomarkers predicting recurrence and metastasis. In order to promote a novel phenotype in normal fibroblasts, we predicted that breast cancer cells could be able to cause loss of Cav1 and increase of MCT4, as well as elevate IL-6 and TGFß in nearby normal fibroblasts. We created a co-culture model using breast cancer (4T1) and normal fibroblast (NIH3T3) cell lines cultured under specific experimental conditions in order to directly test our theory. Moreover, we show that long-term co-culture of breast cancer cells and normal fibroblasts promotes loss of Cav1 and gain of MCT4 in adjacent fibroblasts and increase lactate secretion. These results were validated using the monoculture of each group separately as a control. In this system, we show that metformin inhibits IL-6 and TGFß secretion and re-expresses Cav1 in both cells. However, MCT4 and lactate stayed high after treatment with metformin. In conclusion, our work shows that co-culture with breast cancer cells may cause significant alterations in the phenotype and secretion of normal fibroblasts. Metformin, however, may change this state and affect fibroblasts' acquired phenotypes. Moreover, mitochondrial inhibition by metformin after 8 days of treatment, significantly hinders tumor growth in mouse model of breast cancer.

Caveolin-3 and Caveolin-1 Interaction Decreases Channel Dysfunction Due to Caveolin-3 Mutations.

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.

Interleukin 17A Contributes to Blood-Brain Barrier Disruption of Hypothalamic Paraventricular Nucleus in Rats With Myocardial Infarction.

BACKGROUND: Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood-brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction-induced HF. METHODS AND RESULTS: Male Sprague-Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno-associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate-dextran 10 kDa+rhodamine-dextran 70 kDa). Compared with sham-operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate-dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin-1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin-1 and tight junction-associated proteins in the PVN. CONCLUSIONS: Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.