Cav-1 Gene Research Articles

CAV1 Exacerbates Renal Tubular Epithelial Cell Senescence by Suppressing CaMKK2/AMPK-Mediated Autophagy.

Renal proximal tubular epithelial cell (PTEC) senescence and defective autophagy contribute to kidney aging, but the mechanisms remain unclear. Caveolin-1 (CAV1), a crucial component of cell membrane caveolae, regulates autophagy and is associated with cellular senescence. However, its specific role in kidney aging is poorly understood. In this study, we generated Cav1 gene knockout mice and induced kidney aging using D-galactose (D-gal). The results showed that CAV1 expression increased in the renal cortex of the aging mice, which was accompanied by exacerbated renal interstitial fibrosis, elevated levels of senescence-associated proteins γH2AX and p16INK4a, and increased β-galactosidase activity. Moreover, autophagy and AMPK phosphorylation in PTECs were reduced. These phenotypes were partially reversed in D-gal-induced Cav1 knockout mice. Similar results were observed in D-gal-induced human proximal tubular epithelial (HK-2) cells, but these effects were blocked when AMPK activation was inhibited. Additionally, in CaMKK2 knockdown HK-2 cells, siCAV1 failed to promote AMPK phosphorylation, whereas this effect persisted when STK11 was knocked down. Besides, we examined the phosphorylation of CaMKK2 and found that siCAV1 increased its activity. Given that CaMKK2 activity is affected by intracellular Ca2+, we examined Ca2+ levels in HK-2 cells and found that D-gal treatment reduced intracellular Ca2+ concentration, but CAV1 knockdown did not alter these levels. Through GST pull-down assays, we demonstrated a direct interaction between CAV1 and CaMKK2. In conclusion, these findings suggest that CAV1 exacerbates renal tubular epithelial cell senescence by directly interacting with CaMKK2, suppressing its activity and AMPK-mediated autophagy via a Ca2+-independent pathway.

New Insights in Microplastic Cellular Uptake Through a Cell-Based Organotypic Rainbow-Trout (Oncorhynchus mykiss) Intestinal Platform.

Microplastics (MPs) in fish can cross the intestinal barrier and are often bioaccumulated in several tissues, causing adverse effects. While the impacts of MPs on fish are well documented, the mechanisms of their cellular internalization remain unclear. A rainbow-trout (Oncorhynchus mykiss) intestinal platform, comprising proximal and distal intestinal epithelial cells cultured on an Alvetex scaffold, was exposed to 50 mg/L of MPs (size 1-5 µm) for 2, 4, and 6 h. MP uptake was faster in RTpi-MI compared to RTdi-MI. Exposure to microplastics compromised the cellular barrier integrity by disrupting the tight-junction protein zonula occludens-1, inducing significant decreases in the transepithelial-electrical-resistance (TEER) values. Consequently, MPs were internalized by cultured epithelial cells and fibroblasts. The expression of genes related to endocytosis (cltca, cav1), macropinocytosis (rac1), and tight junctions' formation (oclna, cldn3a, ZO-1) was analyzed. No significant differences were observed in cltca, oclna, and cldn3a expression, while an upregulation of cav1, rac1, and ZO-1 genes was detected, suggesting macropinocytosis as the route of internalization, since also cav1 and ZO-1 are indirectly related to this mechanism. The obtained results are consistent with data previously reported in vivo, confirming its validity for identifying MP internalization pathways. This could help to develop strategies to mitigate MP absorption through ingestion.

Exploring the Pharmacological Mechanisms of P-hydroxylcinnamaldehyde for Treating Gastric Cancer: A Pharmacological Perspective with Experimental Confirmation.

Momordica cochinchinensis is a dried and mature seed of Cucurbitaceae plants, which has the effect of dispersing nodules, detumescence, attacking poison, and treating sores, and is used in the treatment of tumors in the clinic. P-hydroxylcinnamaldehyde (CMSP) is an ethanol extract of cochinchina momordica seed (CMS). Our previous studies have found that CMSP is an effective anti-tumor component with good anti-tumor effects on melanoma and esophageal tumors. However, the inhibitory effect of CMSP on gastric cancer (GC) and its potential mechanism remain to be further elucidated. First, we utilized network pharmacology to predict potential targets and mechanisms of action for the treatment of GC. Subsequently, a series of biological function experiments were conducted to assess the effects of CMSP on the proliferation and apoptosis of GC cells in vitro. To elucidate the molecular mechanism of CMSP, bioinformatics and high-efficiency liquid chromatography tandem mass spectrometry (HPLC-MS/MS) were employed for analysis. Additionally, a resistant cell line of the chemotherapy drug paclitaxel for GC was established, and the impact of 10μg/mL CMSP on the sensitivity of GC-resistant cells was examined. The network pharmacology results demonstrated that the active components of CMS exert an anti-GC effect through multi-target and multipathway mechanisms. The main pathways involved included the PI3K/Akt pathway, p53 signaling pathway, multi-species apoptosis pathway, as well as ADRB2 and CAV1 genes. Cell experiments revealed that CMSP can effectively inhibit the proliferation and induce apoptosis of GC cells in vitro. However, it did not show any sensitizing effect on paclitaxel-resistant cells. Importantly, CMSP exhibited no toxic or side effects on normal gastric epithelial cells. Furthermore, differential protein expression patterns following CMSP treatment were elucidated using HPLCMS/ MS and western blot analysis, highlighting its role in regulating apoptosis signaling pathways. Our study presents novel evidence regarding pertinent potential target genes and signaling pathways through which CMSP mediates its anti-GC effects, with a particular emphasis on its role in modulating apoptotic signaling pathways. Collectively, these findings underscore the promising candidacy of CMSP as a therapeutic agent for GC that merits further investigation in clinical contexts.

Abstract 4138340: Cav-1/FGF2 Axis Mediates Endothelial-Mesenchymal Transition Promoting Aortic Valve Calcification

Background: Calcific Aortic Valve Disease is a common cardiac condition with no effective medication available; severe cases require surgery. Endothelial-to-mesenchymal transition is a key pathological process, yet the genetic regulatory mechanisms involved remain unclear. Research Questions: Investigating the mechanism of Cav-1/FGF2 axis-mediated endothelial-mesenchymal transition in aortic valve calcification. Methods: Histopathology, immunohistochemical staining, and western blot analysis were performed on 5 non-calcified and 5 calcified aortic valves. Proteomic sequencing was conducted on these samples. Single-cell RNA sequencing on 3 calcified aortic valves focused on valve endothelial and interstitial cells to explore key genes and functions in EndoMT. In vivo and in vitro experiments, along with single-cell multi-omics sequencing, elucidated the role and mechanism of the Cav-1/FGF2 axis in EndoMT using endothelial cell-specific EndoMT knockout mouse models and cell models. Results: Proteomics and single-cell sequencing results indicated that Cav-1 is involved in the Endothelial-to-mesenchymal transition process in Calcific Aortic Valve Disease. Cav-1 plays a crucial role in the intermediate transition of EndoMT, interacting significantly with FGF2. Immunohistochemical staining showed high expression of Cav-1 in calcified aortic valves. In vitro experiments demonstrated that Cav-1 mediates Endothelial-to-mesenchymal transition in valve endothelial cells, promoting aortic valve calcification. Molecular docking analysis revealed potential direct interaction between Cav-1 and FGF2 proteins. Elevated Cav-1 gene expression increases the number of cell surface caveolae, enhancing cell surface area, which promotes FGF2 binding to its receptor and activates the TGF-beta receptor, both of which facilitate EndoMT through the Smad pathway. Conclusion(s): Activation of the Cav-1/FGF2 axis promotes endothelial-mesenchymal transition, thereby promoting aortic valve calcification.

Differential cellular communication in tumor immune microenvironment during early and advanced stages of lung adenocarcinoma.

Heterogeneous behavior of each cell type and their cross-talks in tumor immune microenvironment (TIME) refers to tumor immunological heterogeneity that emerges during tumor progression and represents formidable challenges for effective anti-tumor immune response and promotes drug resistance. To comprehensively elucidate the heterogeneous behavior of individual cell types and their interactions across different stages of tumor development at system level, a computational framework was devised that integrates cell specific data from single-cell RNASeq into networks illustrating interactions among signaling and metabolic response genes within and between cells in TIME. This study identified stage specific novel markers which remodel the cross-talks, thereby facilitating immune stimulation. Particularly, multicellular knockout of metabolic gene APOE (Apolipoprotein E in mast cell, myeloid cell and fibroblast) combined with signaling gene CAV1 (Caveolin1 in endothelial and epithelial cells) resulted in the activation of T-cell mediated signaling pathways. Additionally, this knockout also initiated intervention of cytotoxic gene regulations during tumor immune cell interactions at the early stage of Lung Adenocarcinoma (LUAD). Furthermore, a unique interaction motif from multiple cells emerged significant in regulating the overall immune response at the advanced stage of LUAD. Most significantly, FCER1G (Fc Fragment of IgE Receptor Ig) was identified as the common regulator in activating the anti-tumor immune response at both stages. Predicted markers exhibited significant association with patient overall survival in patient specific dataset. This study uncovers the significance of signaling and metabolic interplay within TIME and discovers important targets to enhance anti-tumor immune response at each stage of tumor development.

A comprehensive atlas of pig RNA editome across 23 tissues reveals RNA editing affecting interaction mRNA-miRNAs.

RNA editing is a co-transcriptional/post-transcriptional modification that is mediated by the ADAR enzyme family. Profiling of RNA editing is very limited in pigs. In this study, we collated 3813 RNA-seq data from the public repositories across 23 tissues and carried out comprehensive profiling of RNA editing in pigs. In total, 127,927 A-to-I RNA-editing sites were detected. Our analysis showed that 98.2% of RNA-editing sites were located within repeat regions, primarily within the pig-specific SINE retrotransposon PRE-1/Pre0_SS elements. Subsequently, we focused on analyzing specific RNA-editing sites (SESs) in skeletal muscle tissues. Functional enrichment analyses suggested that they were enriched in signaling pathways associated with muscle cell differentiation, including DMD, MYOD1, and CAV1 genes. Furthermore, we discovered that RNA editing event in the 3'UTR of CFLAR mRNA influenced miR-708-5p binding in this region. In this study, the panoramic RNA-editing landscape of different tissues of pigs was systematically mapped, and RNA-editing sites and genes involved in muscle cell differentiation were identified. In summary, we identified modifications to pig RNA-editing sites and provided candidate targets for further validation.

Genetic aspects associated with increased risk of developing atrial fibrillation

Abstract Atrial Fibrillation (AF) is the most common sustained arrhythmia in clinical practice with increasing prevalence and association with increased morbidity and mortality. Studies have suggested a greater contribution of the genetic factor to the etiopathogenesis of arrhythmia, which could indicate up to 40% more risk for the individual who has a family member with the arrhythmia, and later in the form of single nucleotide polymorphisms (SNPs). Objective To verify the association of polymorphisms in the PITX2, HCN4, CAV1 and KCNN3 genes with the occurrence of AF. Method The study is cross-sectional with group comparison, prospective with a quantitative approach. 505 randomly selected subjects were included (357 cases and 148 controls). The real-time PCR methodology was used, using the TAQMAN® system (Thermo scientific, CA, USA). Result The sample was matched by sex and age. The most common type of AF was permanent (69%) followed by paroxysmal (23%). When comparing the groups selected for CAV1, statistical significance was found for AA vs AG genotypes (p 0.003; OR= 2.2; CI 1.29-3.7). For the HCN4 gene allele and genotypes A vs G (p 0.024; OR= 0.66; CI 0.47-0.9) and AA vs AG (p 0.014, OR= 0.57; CI 0.37-0.88), respectively. Regarding the comparison of the dominant and recessive genetic model of each gene, the following models/genes presented statistical significance: in the genotypic model of the CAV1 gene (AA vs AG vs GG) p 0.013, as well as the same model applied to the HCN4 gene (AA vs AG vs GG) (p 0.03); when comparing the dominant models, the HCN4 gene (AA vs AG/GG) presented (p 0.01; OR=0.58; 95% CI 0.38 – 0.88), with the recessive model of the CAV1 gene (AA vs AG/GG) with (p 0.008; OR=2.0; 95% CI: 1.19 – 3.34). Regarding allelic associations between the two groups, a greater chance of susceptibility to developing AF was observed in patients who had the "G" allele (25.6%; p 0.024; OR= 0.66, CI 0.47-0.9) in the HCN4 gene polymorphism. Polymorphisms in the CAV1 (rs3807989) and HCN4 (rs7164883) genes were also included in the multivariate analysis with maintenance of the risk factor for AF. However, only HCN4 rs7164883 showed statistical significan, thus showing it to be an independent variable regarding the probability of the patient with this polymorphism presenting AF. The present study showed a significant association with a greater chance of susceptibility to developing atrial fibrillation in patients with a polymorphism in the CAV1 gene (rs3807989) in a univariate and multivariate model and for patients with the HCN4 gene SNP (rs7164883). Conclusion Therefore, the results revealed an association between the HCN4 gene polymorphism rs7164883 and susceptibility to the development of AF, suggesting that the presence of the G allele may be one of the genetic factors related to the arrhythmia. The CAV1 rs3807989 gene polymorphism also seemed to suggest a risk for the development of AF, also relating the G allele.

Identification of Metabolism-Related Prognostic Biomarkers and Immune Features of Head and Neck Squamous Cell Carcinoma.

We aimed to identify an effective metabolic subtype and risk score to predict survival and immunotherapy response in head and neck squamous cell carcinoma (HNSCC). Data were obtained from an online database. We screened significant prognostic metabolism-related genes between the normal and tumor groups using a series of bioinformatics methods. Based on the selected prognostic genes, we conducted a subtype analysis to identify significantly different subtypes in HNSCC. We then investigated survival, immune features, and hallmark differences among different subtypes. LASSO was utilized to identify optimal genes for the risk score model construction. Finally, distribution of the risk score samples was analyzed for different subtypes. A total of 32 significantly prognostic metabolism-related genes were screened, and all samples were grouped into two subtypes: cluster 1 and cluster 2. Cluster 1 had worse survival. Different immune cell infiltration (CD8 T cells, macrophages, and regulatory T cells) and immune checkpoint gene expression (PD-1 and CLAT-4) were observed between the two clusters. Twelve optimal genes were involved in risk score model, and high-risk group had poorer survival. Cluster 1 contained more high-risk samples (60%). Finally, four genes CAV1, GGT6, PYGL, and HS3ST1 were identified as significantly related to immune cells, and these genes were differentially expressed in the normal oral epithelial cells and HNSCC cells. The subtypes and risk score model in the study provide a promising biomarker for prognosis and immunotherapy response.

Gene expression in calcium triggered healthy primary limabal epithelial cells, in vitro

Aims/Purpose: Calcium supplementation can induce differentiation of several cell types. The tear film has an important role in maintenance of the ocular surface homeostasis with an ionic calcium concentration of 0.4 mM‐1.0 mM. The effect of calcium concentration on healthy primary limbal epithelial cells (LECs) has not been analysed, yet. Our purpose was to investigate the effect of calcium supplementation on gene expression of primary limbal epithelial cells (LECs) of healthy donor corneas, in vitro.Methods: Primary LECs were isolated from corneoscleral rims of healthy donors (n = 6) and were cultured in serum free low calcium (0.06 mM Ca+2) Keratocyte growth medium 3 (KGM3). The primary LECs were then supplemented by 0.3 mM‐2.0 mM Ca+2 concentrations for 72 h. Control LEC cultures were supplemented by low calcium (0.06 mM Ca+2) KGM3 medium. After cell harvesting, RNA and protein were extracted from treated and control LECs to perform qPCR and western blot analysis.Results: The calcium supplementation had no significant effect on PAX6 mRNA expression after 72 h. The differentiation marker and adhesion protein DSG1 and the cornea specific keratin K3 expression did not change significantly through Ca+2 supplementation (p ≥ 0.5). The retinoic acid signalling component fatty acid binding protein FABP5 expression (p ≥ 0.5) the wound healing and vascularization marker CAV1 gene expression (p ≥ 0.5) did not change significantly through Ca+2 supplementation, after 72 h.Conclusions: Calcium supplementation for 72 h did not change PAX6 expression and did not trigger differentiation of healthy primary limbal epithelial cells. We plan to analyse the effect of longer incubation time in the future in order to trigger a full differentiated status of limbal epithelial cells, using calcium supplementation.

The Influence of Adiponectin on Transport of Low-Density Lipoproteins through Human Endothelial Cell Monolayer In Vitro.

The influence of adiponectin, a protein secreted by adipocytes, on the activation of transendothelial LDL transport, the initial event of atherogenesis, was studied. The addition of adiponectin to the cultured endothelial hybridoma EA.hy926 cells did not affect both basal and TNF-stimulated transendothelial transport of LDL. In addition, adiponectin affects neither expression levels of CAV1, SCARB1, and ACVRL1 genes encoding proteins involved in transendothelial LDL transport, nor the MMP secretion by the EA.hy926cells. At the same time, adiponectin suppressed the TNF-stimulated IL-8 production and expression of the adhesion molecule gene ICAM1 in these cells. Thus, adiponectin reduces proinflammatory activation of EA.hy926 cells, which is not accompanied by changes in the transendothelial LDL transport. We speculate that anti-inflammatory action of adiponectin is the base for the influence of this adipokine on atherogenesis.

A genetic polymorphism on chromosome 4q25 (rs17570669_T) may predict atrial fibrillation recurrence after successful electrical cardioversion

Abstract Background Direct current electrical cardioversion (DCCV), is an effective treatment method in patients with symptomatic persistent atrial fibrillation (AF). However, AF recurrence may occur following successful DCCV in nearly half of the patients. There is not enough data investigating the association between single nucleotide polymorphisms (SNP) and AF recurrence after DCCV. Purpose We aimed to investigate whether 11 SNPs previously associated with AF (rs2200733, rs10033464, rs6838973, rs3853445 and rs17570669 SNPs in PITX2 gene; rs2106261 SNP in ZFHX3 gene; rs751141 SNP on chromosome 8q21; rs3807989 SNP in CAV1 gene; rs10570248 SNP in TBX5 gene; rs1800469 SNP in TGF-1 gene; and rs6795970 SNP in SCN10A gene) were predictive for recurrence after successful DCCV in patients with persistent AF in Turkish society. Methods We included 75 patients with sinus rhythm after direct current electrical cardioversion (DCCV). Baseline clinical features and 11 AF-related SNPs were compared between patients with and without AF recurrence during the follow-up. The association between SNPs and AF recurrence was evaluated with the additive model (wild type vs heterozygous variant vs homozygous variant), the dominant model (wild type/heterozygous vs homozygous variant), and the recessive model (wild type vs heterozygous variant/homozygous variant). Results The mean age of the patients was 61.9 ± 11.5 years. At a mean follow-up of 17.0 (11.0-25.0) months, 38 patients (50.7%) developed AF recurrence. The frequency of the female gender was higher (55.3% vs. 32.4%), AF duration before DCCV was longer [9.2 (5.5-11.6) vs 5.0 (2.7-11.0) months], and left atrial volume index before DCCV was higher (43.6 ± 8.8 ml/m2 vs. 38.2 ± 12.9 ml/m2) in patients who developed AF recurrence than those without it (p=0.046, 0.029 and 0.036, respectively) (Figure 1a). In the additive model, a SNP at the PITX2 gene (rs17570669_T: OR 9.00, 95% CI 1.28-63.02, p=0.027) and a SNP at the ZFHX3 gene (rs2106261_T: OR 8.96, 95% CI 1.03-77.66, p=0.047) were significantly associated with AF recurrence. Female gender (HR: 1.99, 95% CI: 1.05-3.79, p=0.035) and the rs17570669_T SNP (HR: 4.16, 95% CI: 1.22-14.11, p=0.022) were predictors for AF recurrence in univariate analysis. A multivariate Cox regression analysis showed that the rs17570669_T SNP was an independent predictor of AF recurrence following successful DCCV in the additive model (HR: 3.59, 95% CI: 1.05-12.21, p=0.04) (Figure 1b). A Kaplan-Meier curve showed that AF-free survival was significantly lower in patients who had rs17570669_T SNP in the additive model (Figure 2). Conclusion A SNP at the PITX2 locus (rs17570669_T) may predict AF recurrence after successful DCCV in patients with persistent AF in Turkish society. Such individuals may be closely monitored for the development of AF recurrence after DCCV. In addition, reversible risk factors associated with AF recurrence may be more tightly controlled in such patients.Baseline features and Cox analysisKaplan-Meier curve

Molecular pathogenesis, mechanism and therapy of Cav1 in prostate cancer.

Prostate cancer is the second incidence of malignant tumors in men worldwide. Its incidence and mortality are increasing year by year. Enhanced expression of Cav1 in prostate cancer has been linked to both proliferation and metastasis of cancer cells, influencing disease progression. Dysregulation of the Cav1 gene shows a notable association with prostate cancer. Nevertheless, there is no systematicreview to report about molecular signal mechanism of Cav1 and drug treatment in prostate cancer. This article reviews the structure, physiological and pathological functions of Cav1, the pathogenic signaling pathways involved in prostate cancer, and the current drug treatment of prostate cancer. Cav1 mainly affects the occurrence of prostate cancer through AKT/mTOR, H-RAS/PLCε, CD147/MMPs and other pathways, as well as substance metabolism including lipid metabolism and aerobic glycolysis. Baicalein, simvastatin, triptolide and other drugs can effectively inhibit the growth of prostate cancer. As a biomarker of prostate cancer, Cav1 may provide a potential therapeutic target for the treatment of prostate cancer.

Caveolin-1 protein expression as a prognostic biomarker of gastrointestinal tumours: A systematic review and meta-analysis.

Gastrointestinal (GI) cancers remain a major threat worldwide, accounting for over 30% of cancer deaths. The identification of novel prognostic biomarkers remains a challenge despite significant advances in the field. The CAV1 gene, encoding the caveolin-1 protein, remains enigmatic in cancer and carcinogenesis, as it has been proposed to act as both a tumour promoter and a tumour suppressor. To analyse the differential role of caveolin-1 expression in both tumour cells and stroma in relation to prognosis in GI tumours, we performed a systematic review and meta-analysis according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines; PROSPERO registration number: CRD42022299148. Our analysis showed that high levels of caveolin-1 in tumour cells were associated with poor prognosis and inferior overall survival (OS) in oesophageal and pancreatic cancer and hepatocellular carcinoma (HCC), but not in gastric and colorectal cancer. Importantly, our study showed that higher stromal caveolin-1 expression was associated with significantly longer OS and disease-free survival in colorectal cancer. Analysis of stromal caveolin-1 expression in the remaining tumours showed a similar trend, although it did not reach statistical significance. The data suggest that caveolin-1 expression in the tumour cells of oesophageal, pancreatic cancer and HCC and in the stroma of colorectal cancer may be an important novel predictive biomarker for the clinical management of these diseases in a curative setting. However, the main conclusion of our analysis is that caveolin-1 expression should always be assessed separately in stroma and tumour cells.

Integration of clinical and spatial data to explore lipid metabolism-related genes for predicting prognosis and immune microenvironment in gliomas.

Lipid metabolism is crucial to tumor growth and immune microenvironment as well as drug sensitivity in glioma. Identifying prognostic indicators of glioma and elucidating the mechanisms of glioma progression are criticalfor improving the prognosis ofglioma patients. In this study, we investigated the role and prognostic value of metabolism-related genes in glioma by integrative analysis of datasets fromGEO, CGGA, and TCGA. Based on clinical data and transcriptome data, we found that the expression pattern of three major pathways related to lipid metabolism is fatty acidhigh-phospholipidhigh-triglyceridelow, which is associated with better prognosis and immune infiltration. The genes involved inthese three pathwayswere used to generate a prognostic model, which showed highstability and efficiency in the test set and validation set. The spatial transcriptome of glioma patientsrevealed that the microenvironment of the regions with high expression of risk genes CAV1 and SCD is in a state of hypoxia, EMT, and cell cycle arrest, and thus can be used as markers of metabolic reprogramming in the tumor microenvironment. In the high-risk group, M0 macrophages and M1 macrophages were significantly enriched, and the risk score was significantly correlated with gene mutation and methylation of risk genes. We further performed drug sensitivity screening corresponding to different risk genes. This study provided novel insights into the differential immune microenvironment with different expression patternsof metablism-related genes and highlighted the spatial and temporal synergy of tumor progression and metabolic reprogramming.

Large-scale analyses of CAV1 and CAV2 suggest their expression is higher in post-mortem ALS brain tissue and affects survival.

Introduction: Caveolin-1 and Caveolin-2 (CAV1 and CAV2) are proteins associated with intercellular neurotrophic signalling. There is converging evidence that CAV1 and CAV2 (CAV1/2) genes have a role in amyotrophic lateral sclerosis (ALS). Disease-associated variants have been identified within CAV1/2 enhancers, which reduce gene expression and lead to disruption of membrane lipid rafts. Methods: Using large ALS whole-genome sequencing and post-mortem RNA sequencing datasets (5,987 and 365 tissue samples, respectively), and iPSC-derived motor neurons from 55 individuals, we investigated the role of CAV1/2 expression and enhancer variants in the ALS phenotype. Results: We report a differential expression analysis between ALS cases and controls for CAV1 and CAV2 genes across various post-mortem brain tissues and three independent datasets. CAV1 and CAV2 expression was consistently higher in ALS patients compared to controls, with significant results across the primary motor cortex, lateral motor cortex, and cerebellum. We also identify increased survival among carriers of CAV1/2 enhancer mutations compared to non-carriers within Project MinE and slower progression as measured by the ALSFRS. Carriers showed a median increase in survival of 345 days. Discussion: These results add to an increasing body of evidence linking CAV1 and CAV2 genes to ALS. We propose that carriers of CAV1/2 enhancer mutations may be conceptualised as an ALS subtype who present a less severe ALS phenotype with a longer survival duration and slower progression. Upregulation of CAV1/2 genes in ALS cases may indicate a causal pathway or a compensatory mechanism. Given prior research supporting the beneficial role of CAV1/2 expression in ALS patients, we consider a compensatory mechanism to better fit the available evidence, although further investigation into the biological pathways associated with CAV1/2 is needed to support this conclusion.

A truncated and catalytically inactive isoform of KDM5B histone demethylase accumulates in breast cancer cells and regulates H3K4 tri-methylation and gene expression

KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncogenesis, depending on the specific context. This ambivalence could be explained by the expression of KDM5B protein isoforms with diverse functional roles, which could be present at different levels in various cancer cell lines. We show here that one of these isoforms, namely KDM5B-NTT, accumulates in breast cancer cell lines due to remarkable protein stability relative to the canonical PLU-1 isoform, which shows a much faster turnover. This isoform is the truncated and catalytically inactive product of an mRNA with a transcription start site downstream of the PLU-1 isoform, and the consequent usage of an alternative ATG for translation initiation. It also differs from the PLU-1 transcript in the inclusion of an additional exon (exon-6), previously attributed to other putative isoforms. Overexpression of this isoform in MCF7 cells leads to an increase in bulk H3K4 methylation and induces derepression of a gene cluster, including the tumor suppressor Cav1 and several genes involved in the interferon-alpha and -gamma response. We discuss the relevance of this finding considering the hypothesis that KDM5B may possess regulatory roles independent of its catalytic activity.

Characteristics and Resistance to Cisplatin of Human Neuroblastoma Cells Co-Cultivated with Immune and Stromal Cells.

We investigated the features of the morphology and cytokine profiles of neuroblastoma SH-SY5Y cells, bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs), and peripheral blood mononuclear cells (PBMCs) in double (BM-MSCs + SH-SY5Y cells) and triple (BM-MSCs + SH-SY5Y cells + PBMCs) co-cultures incubated on plastic and Matrigel. Cells in the co-cultures communicated by vesicular transport and by exchanging membrane and cytoplasmic components. The cytokine profile of double and triple co-cultures incubated on Matrigel and plastic had differences and showed the highest concentration of a number of chemokines/cytokines, such as CXCL8/IL-8, I-TAC/CXCL11, IP10/CXCL10, MDC/CCL22, MIP-1α/CCL3, IL-1β, ENA-78/CXCL5, Gro-α/CXCL1, MCP-1/CCL2, TERC/CCL25, CXCL8/IL-8, and IL-6. High concentrations of inflammatory chemokines/cytokines in the conditioned medium of triple co-culture form a chronic inflammation, which brings the presented co-cultivation system closer to a natural tumor. Triple co-cultures were more resistant to cisplatin (CDDP) than the double- and monoculture of SH-SY5Y. The mRNA levels of BCL2, BCL2L1, RAC1, CAV1, CASP3, and BAX genes were changed in cells after co-culturing and CDDP treatment in double and triple co-cultures. The expression of the BCL2, BAX, CAV1, and CASP3 proteins in SH-SY5Y cells after the triple co-culture and CAV1 and BAX protein expression in SH-SY5Y cells after the double co-culture were determined. This study demonstrated the nature of the cellular interactions between components of tumor niche and the intercellular influence on chemoresistance observed in our tumor model, which should enable the development of novel test systems for anti-tumor agents.

PSUN81 A novel case of congenital generalized lipodystrophy (CGL) with no genetic abnormalites in the AGPAT2, BSCL2, CAV1, and CAVIN1 genes.

Abstract Congenital generalized lipodystrophy (CGL) is a genetic disorder charaterized by total body fat loss. The extent of fat loss is usually associated with the severity of the disease and patients typically develop metabolic complications such as hypertriglyceridemia, hepatic steatosis, insulin resistance, and diabetes mellitus. Mutations in the AGPAT2, BSCL2, CAV1, and CAVIN1 genes are known to cause CGL. We report a new case of CGL but where no genetic aetiology has yet been identified in the known genes that cause CGL. The patient is a female child born at 36 weeks gestation with a birth weight os 2.57 kg. Soon after birth she was diagnosed clinically with CGL with both the DEXA and MRI scans showing severely diminished adipose tissue and 0% body fat respectively. She had severe failure to thive and developed diabetes mellitus, hyperlipidaemia, hypertrophic cardiomyopathy, severe scalp seborrheic dermatitis and hepatic steotosis. At 2 years of age her weight was only 3.7kg and she died from an upper respiratory tract infection. Whole exome sequencing and trio analysis did not find any mutations or copy number variants (CNV) in the AGPAT2, BSCL2, CAV1, and CAVIN1 genes associated with CGL. De-novo variants were found in the genes PCNT, PDE4DIP and STAT3 which need further functional validation. This case suggests that other possibly novel genetic mechanisms may be responsible for the CGL observed in this patient. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Targeting Neurofibromin 2 (NF2) prevents endothelial dysfunction in obesity: a study in mice and humans

Abstract Introduction The mechanisms underlying endothelial dysfunction (ED) in obesity are poorly understood. Neurofibromin 2 (NF2) is a scaffold-like protein involved in cell growth and survival. However, its role in the vascular endothelium is unknown. Purpose To investigate NF2 function in obesity-related ED. Methods Human aortic endothelial cells (HAECs) were exposed to palmitic acid (PA, 200 uM) or vehicle for 48 hours. Gene silencing of NF2 was performed by small interfering RNA (siRNA). Gene and protein expression were assessed by real time PCR and Western blot, respectively. The interaction of NF2 with endothelial proteins was investigated by co-immunoprecipitation. A constitutive active mutant form of NF2 (Ala518) was employed to study the effects of NF2 gain-of-function. To specifically investigate NF2 role in the vascular endothelium, we generated mice with endothelium-specific deletion of NF2 (NF2 ECKO) by crossing NF2flox/flox mice with tamoxifen-inducible endothelial-specific Cre mice [Cdh5(PAC)-CreERT2]. Endothelium-dependent relaxations to acetylcholine (Ach) were assessed in aortas from NF2 ECKO and wild type (WT) littermates, fed a control and a high fat diet (60 kcal% fat) for 20 weeks. NF2 signalling and endothelial function were also assessed in small visceral fat arteries (VFA) isolated from 18 obese and 18 age-matched healthy subjects undergoing bariatric surgery and cholecystectomy, respectively. Gene in silencing of NF2 by siRNA was performed in VFA from obese patients. Results In HAECs, PA promoted NF2 activation by decreasing its phosphorylation at Ser518. Akt and MYPT-1 were responsible for NF2 dephosphorylation. In PA-treated HAECs, NF2 was mainly found in the plasma membrane as compared to other cell fractions. Among different membrane proteins implicated in endothelial homeostasis, NF2 binds and activates Caveolin 1 (Cav-1), a pivotal repressor of endothelial NO synthase (eNOS). NF2 knockdown in PA-treated HAECs prevented eNOS–Cav-1 interaction, thus preserving eNOS activity and NO levels. By contrast, HAECs expressing the constitutive active mutant form of NF2 displayed reduced eNOS activity. In aortas from obese mice, we found that NF2-Cav-1 interaction was responsible for impaired eNOS activity and ED. Cav-1 gene silencing in NF2-overexpressing aortas prevented ED, thus confirming the direct involvement of Cav-1 in NF2-induced ED. Interestingly, Ach-dependent vasorelaxation was preserved in obese NF2 ECKO mice as compared to WT littermates. Moreover, NO bioavailability was preserved in aortas from NF2 ECKO mice. In VFA from obese patients, NF2 was upregulated, and its activity negatively correlated with Ach-dependent vasorelaxation. Of note, NF2 gene silencing in VFA from obese patients rescued ED. Conclusions In human endothelial cells, mice with endothelium-specific deletion of NF2 and VFA from obese patients, we show that NF2 drives ED by repressing Cav-1. Targeting NF2 may prevent ED in obese patients. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Holcim Stiftung

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

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