Cav-1 Depletion Research Articles

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

Zika virus (ZIKV) has gained notoriety in recent years because there are no 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 unveil 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, but does not affect viral entry, maturation and release. These results reveal an unforeseen function of Cav-1 in facilitating ZIKV replication, which provides new insights into the intricate interaction between Cav-1 and ZIKV and underscores Cav-1 as a potential candidate for anti-ZIKV approaches.

Nutrient scavenging-fueled growth in pancreatic cancer depends on caveolae-mediated endocytosis under nutrient-deprived conditions.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.

Caveolin-1 deficiency alleviates palmitate-induced intracellular lipid accumulation and inflammation in pancreatic β cells.

Lipotoxicity-induced pancreatic β cell damage is a strong predictor of type 2 diabetes mellitus (T2DM). Our previous work showed that Caveolin-1 (Cav-1) depletion decreased β-cell apoptosis and improved β-cell viability. Further microarray analysis indicated significant changes in the expression of genes related to fatty acid metabolism and inflammation. The objective of this study was to explore the role of Cav-1 in intracellular lipid accumulation and inflammation in β cells under lipotoxic conditions. Here, we established a β-cell-specific Cav-1 knockout (β-Cav-1 KO) mouse model and a CAV-1 depleted β cell line (NIT-1). We found that Cav-1 silencing significantly reduced palmitate (PA)-induced intracellular triglyceride (TG) accumulation and decreased proinflammatory factor expression in both the mouse and cell models. Further mechanistic investigation revealed that amelioration of lipid metabolism was achieved through the downregulation of lipogenic markers (SREBP-1c, FAS and ACC) and upregulation of a fatty acid oxidation marker (CPT-1). Meanwhile, decrease of inflammatory cytokines (IL-6, TNF-α, and IL-1β) secretion was found with the involvement of the IKKβ/NF-κB signaling pathways. Our findings suggest that Cav-1 is of considerable importance in regulating lipotoxicity-induced β-cell intracellular lipid accumulation and inflammation.

JNTX-101, a novel albumin-encapsulated gemcitabine prodrug, is efficacious and operates via caveolin-1-mediated endocytosis

Albumin is an attractive candidate carrier for the development of novel therapeutic drugs. Gemcitabine has been FDA approved for the treatment of solid tumors; however, new drugs that optimize gemcitabine delivery are not available for clinical use. The aim of this study was to test the efficacy of a novel albumin-encapsulated gemcitabine prodrug, JNTX-101, and investigate whether Cav-1 expression predicts the therapeutic efficacy of JNTX-101. We first determined the treatment efficacy of JNTX-101 in a panel of pancreatic/lung cancer cell lines and found that increases in Cav-1 expression resulted in higher uptake of albumin, while Cav-1 depletion attenuated the sensitivity of cells to JNTX-101. In addition, decreased Cav-1 expression markedly reduced JNTX-101-induced apoptotic cell death in a panel of cells, particularly in low-serum conditions. Furthermore, we tested the therapeutic efficacy of JNTX-101 in xenograft models and the role of Cav-1 in JNTX-101 sensitivity using a Tet-on-inducible tumor model invivo. Our data suggest that JNTX-101 effectively inhibits cell viability and tumor growth, and that Cav-1 expression dictates optimal sensitivity to JNTX-101. These data indicate that Cav-1 correlates with JNTX-101 sensitivity, especially under nutrient-deprived conditions, and supports a role for Cav-1 as a predictive biomarker for albumin-encapsulated therapeutics such as JNTX-101.

Caveolin-1 in vascular health and glaucoma: A critical vascular regulator and potential therapeutic target.

Caveolin-1 (Cav-1) is an integral scaffolding membrane protein found in most cell types. Cav-1 has been found to contribute significantly to ocular function, with mutations of Cav-1 being associated with a genetic risk of glaucoma development. Raised intraocular pressure (IOP) is a major modifiable risk factor for glaucoma. Cav-1 may be involved in both IOP-dependent and independent mechanisms involving vascular dysregulation. Systemic vascular diseases including hypertension, diabetes and hyperlipidaemia, have been shown to be associated with glaucoma development. Cav-1 is closely interlinked with endothelial nitric oxide synthase pathways that mediate vascular function and prevent cardiovascular diseases. Endothelial nitric oxide synthase and endothelin-1 are key vasoactive molecules expressed in retinal blood vessels that function to autoregulate ocular blood flow (OBF). Disruptions in the homeostasis of OBF have led to a growing concept of impaired neurovascular coupling in glaucoma. The imbalance between perfusion and neuronal stimulation arising from Cav-1 depletion may result in relative ischemia of the optic nerve head and glaucomatous injury. OBF is also governed by circadian variation in IOP and systemic blood pressure (BP). Cav-1 has been shown to influence central BP variability and other circadian rhythms such as the diurnal phagolysosomal digestion of photoreceptor fragments and toxic substrates to maintain ocular health. Overall, the vast implications of Cav-1 on various ocular mechanisms leading to glaucoma suggest a potential for new therapeutics to enhance Cav-1 expression, which has seen success in other neurodegenerative diseases.

Abstract 15005: Tlr4/py14-Caveolin-1-Mediated Endothelial C-iap2 Expression During Schistosomiasis-Associated Pulmonary Arterial Hypertension

Introduction: During schistosomiasis-associated pulmonary arterial hypertension (Sch-PAH), hyperproliferation of abnormal endothelial cells (ECs) contributes to development of severe vascular lesions within the lungs; a life-threatening phenomenon that remains with no target therapy. Hypothesis: Here, we found that antigenic molecules from the major etiological agent of Sch-PAH, i.e., S. mansoni , amplify the secretion of the antiapoptotic protein c-IAP2 via TLR4-pY14Cav-1 signaling pathway, which we hypothesized to contribute to generation of an EC memory of survival and severity of PAH. Methods/Results: Cav-1 deficiency via shedding of extracellular vesicles (EVs) leads to survival of an apoptosis-resistant EC phenotype during idiopathic PAH. Similarly, S. mansoni egg antigens also reduced lung EC-Cav-1 expression, suggesting that Cav-1 also plays a role during pathogen-associated PAH. Cav-1 depletion via EVs precedes its phosphorylation at residue Tyr14 (pY14Cav-1), which in turn, contributes to EV loading. Among apoptotic regulators, IAPs have been found as a cargo of EVs in different conditions, including in TLR4-activated cells. Accordingly, our data indicates that S. mansoni egg antigen p40 also activated TLR4/NFkB-mediated signaling, inducing c-IAP2 expression and a time-dependent phosphorylation of Cav-1 in human lung microvascular EC (HMVEC-L: 228.2 +/- 38.28% of control; p<0.05; n=4). Moreover, although Sm-p40 alone was unable to induce shedding of EVs, combination with NFkB-associated proinflammatory mediators (TNF-α, IL-6, and ATP), significantly increased EV shedding by HMVEC-L, suggesting that S. mansoni may epigenetically prime ECs, but co-factors are required to induce EV shedding. Finally, Sch-PAH model using EC- Cav1 -/- mice ( End-scl.Cre ERT2 ;Rosa mt/mg ;Cav1 fl/fl ) revealed that Cav-1 absence led to accumulation of eggs within the lungs and loss of eGFP+ cells, indicating that EC-Cav-1 phosphorylation may be required to development of Sch-PAH. Conclusions: Our data suggest that S. mansoni -induced pY14Cav-1 is associated to expression of the anti-apoptotic c-IAP2 protein, which may be critical for prolonged survival of a pathogenic EC phenotype during the development of Sch-PAH.

Abstract 4070: Caveolin-1 functions as a potential predictive biomarker for JNTX-101, an albumin encapsulated gemcitabine prodrug

Abstract Purpose: Human serum albumin (HSA) is an attractive candidate as a carrier in developing novel albumin-bound nanoparticle therapeutic drugs. Gemcitabine has been used in the treatment of solid tumors, including non-small cell lung, pancreatic, bladder, and breast cancers. Our previous work in pancreatic and lung cancer models demonstrates that caveolin-1 (Cav-1) enhances uptake of albumin and albumin-conjugated drugs. The aim of this study is to investigate whether the presence of Cav-1 expression alters therapeutic efficacy of JNTX-101, a novel albumin encapsulated gemcitabine prodrug. Experimental design: We first determined the treatment efficacy of JNTX-101 in a panel of 4 pancreatic/lung cancer cell lines (MIAPaCa-2, PANC-1, H23, and HCC15) by alamarBlue cell viability assay. We further established 4 pairs of Cav-1 isogenic cells stably expressing shRNA targeting Cav-1 (shCav-1) or scrambled control shRNA (shCtrl). We investigated whether the absence of Cav-1 expression alters sensitivity of JNTX-101 using alamarBlue cytotoxicity assay and western blotting in both high and low serum conditions. In addition, we tested whether JNTX-101 treatment can alter expression and cellular distribution of Cav-1 as well as other caveolae resident proteins, such as Cav-2 and Cavin-1. Results: Differences in Cav-1 expression resulted in different uptake of albumin prodrug conjugate and Cav-1 depletion attenuated sensitivity of the cells to JNTX-101. Decreased Cav-1 expression markedly reduced JNTX-101-induced apoptosis in a panel of cells in both high and low serum conditions, but particularly in low serum conditions. In addition, our data showed both serum starvation and JNTX-101 independently increased Cav-1 expression in a time-dependent manner (24hrs, 48hrs, 72hrs) similar to gemcitabine, with peak level at 48 hrs post treatment, while other caveolae resident proteins were minimally affected. Conclusions: Our preliminary results suggest that JNTX-101 effectively inhibits cell viability in pancreatic/lung cancer cells and Cav-1 expression dictates sensitivity to JNTX-101, at least partially through reduced apoptotic cell death. These data indicate that Cav-1 may be a potential predictive biomarker of JNTX-101 sensitivity, especially in nutrient deprived conditions. Further study will be performed to explore the impact of Cav-1 expression on JNTX-101 sensitivity in mouse models of pancreatic/lung cancer. Citation Format: Tiantian Cui, Sergio Corrales-Guerrero, Dan Maneval, Curtis Monnig, Patrick Kearney, Sam Ellis, Terence M. Williams. Caveolin-1 functions as a potential predictive biomarker for JNTX-101, an albumin encapsulated gemcitabine prodrug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4070.

Abstract 388: Role Of The Antiapoptotic C-iap2 On The Generation Of An Endothelial Memory In Schistosomiasis-associated Pulmonary Arterial Hypertension

Introduction: Schistosomiasis-associated Pulmonary Arterial Hypertension (Sch-PAH) is a life-threatening complication of chronic S. mansoni infection, which can evolve to heart failure and death. During PAH, hyperproliferation of apoptosis-resistant endothelial cells (ECs) has been extensively reported, but therapeutic approaches to reverse this pathological phenotype remain clinically challenging. Hypothesis: Thus, our hypothesis is that antigenic molecules from S. mansoni eggs amplify the secretion of inhibitor of apoptosis protein 2 (c-IAP2), leading to generation of an abnormal EC memory of survival and the development of PAH. Methods/Results: Previously, we showed that depletion of the anti-apoptotic protein Caveolin-1 (Cav-1) via shedding of extracellular vesicles (EVs) leads to the survival of an abnormal EC phenotype through an unclear mechanism. Analysis of survival-associated genes in isolated murine lung ECs from Flk1 +/GFP ;Cav1 -/- mice, revealed high expression of the anti-apoptotic genes BIRC3 and BIRC5 (which encodes cIAP2 and survivin, respectively) when compared to control ECs. Interesting, exposure to 1 μg/mL of the major S. mansoni egg antigen Sm-p40 induced time-dependent phosphorylation of Cav-1 at residue Tyr14 in human lung microvascular EC (HMVEC-L: 228.2 +/- 38.28% of control; p<0.05; n=4), culminating in c-IAP2 expression with no difference on the constitutive expression of c-IAP1. Furthermore, Sm-p40 treatment of HMVEC-L in the presence of pro-inflammatory mediators IL-6, TNF-α, and ATP induced a significant release of EVs containing c-IAP2. Finally, in vivo analysis of lungs from S. mansoni -infected mice confirmed a significant decrease in Cav-1 expression unique to the egg-dependent granuloma area, indicating that local inflammatory response due to egg-derived antigenic molecules is essential for reprogramming and survival of an abnormal EC phenotype during Sch-PAH. Conclusions: Our data suggest that S. mansoni -induced Cav-1 depletion promotes the secretion of the anti-apoptotic c-IAP2 protein, which may be critical for prolonged survival of a pathogenic EC phenotype during the development of Sch-PAH.

Caveolin-1 Is Essential for the Improvement of Insulin Sensitivity through AKT Activation during Glargine Treatment on Diabetic Mice.

Insulin treatment was confirmed to reduce insulin resistance, but the underlying mechanism remains unknown. Caveolin-1 (Cav-1) is a functional protein of the membrane lipid rafts, known as caveolae, and is widely expressed in mammalian adipose tissue. There is increasing evidence that show the involvement of Cav-1 in the AKT activation, which is responsible for insulin sensitivity. Our aim was to investigate the effect of Cav-1 depletion on insulin sensitivity and AKT activation in glargine-treated type 2 diabetic mice. Mice were exposed to a high-fat diet and subject to intraperitoneal injection of streptozotocin to induce diabetes. Next, glargine was administered to treat T2DM mice for 3 weeks (insulin group). The expression of Cav-1 was then silenced by injecting lentiviral-vectored short hairpin RNA (shRNA) through the tail vein of glargine-treated T2DM mice (CAV1-shRNA group), while scramble virus injection was used as a negative control (Ctrl-shRNA group). The results showed that glargine was able to upregulate the expression of PI3K and activate serine phosphorylation of AKT through the upregulation of Cav-1 expression in paraepididymal adipose tissue of the insulin group. However, glargine treatment could not activate AKT pathway in Cav-1 silenced diabetic mice. These results suggest that Cav-1 is essential for the activation of AKT and improving insulin sensitivity in type 2 diabetic mice during glargine treatment.

Loss of Caveolin-1 Impairs Light Flicker-Induced Neurovascular Coupling at the Optic Nerve Head.

Glaucoma is a neurodegenerative disease, which results in characteristic visual field defects. Intraocular pressure (IOP) remains the main risk factor for this leading cause of blindness. Recent studies suggest that disturbances in neurovascular coupling (NVC) may be associated with glaucoma. The resultant imbalance between vascular perfusion and neuronal stimulation in the eye may precede retinal ganglion cell (RGC) loss and increase the susceptibility of the eye to raised IOP and glaucomatous degeneration. Caveolin-1 (Cav-1) is an integral scaffolding membrane protein found abundantly in retinal glial and vascular tissues, with possible involvement in regulating the neurovascular coupling response. Mutations in Cav-1 have been identified as a major genetic risk factor for glaucoma. Therefore, we aim to evaluate the effects of Cav-1 depletion on neurovascular coupling, retinal vessel characteristics, RGC density and the positive scotopic threshold response (pSTR) in Cav-1 knockout (KO) versus wild type C57/Bl6 mice (WT). Following light flicker stimulation of the retina, Cav-1 KO mice showed a smaller increase in perfusion at the optic nerve head and peripapillary arteries, suggesting defective neurovascular coupling. Evaluation of the superficial capillary plexus in Cav-1 KO mice also revealed significant differences in vascular morphology with higher vessel density, junction density and decreased average vessel length. Cav-1 KO mice exhibited higher IOP and lower pSTR amplitude. However, there was no significant difference in RGC density between Cav-1 KO and wild type mice. These findings highlight the role of Cav-1 in regulating neurovascular coupling and IOP and suggest that the loss of Cav-1 may predispose to vascular dysfunction and decreased RGC signaling in the absence of structural loss. Current treatment for glaucoma relies heavily on IOP-lowering drugs, however, there is an immense potential for new therapeutic strategies that increase Cav-1 expression or augment its downstream signaling in order to avert vascular dysfunction and glaucomatous change.

Abstract P138: Schistosoma Mansoni- Induced Shedding Of Extracellular Vesicles From Lung Microvascular Endothelial Cells

Introduction: Schistosomiasis-associated Pulmonary Arterial Hypertension (Sch-PAH) is a life-threatening complication of chronic Schistosoma mansoni infection. Sch-PAH is marked by obliteration and remodeling of the pulmonary vasculature in response to S. mansoni eggs. Remodeled and occluded vessels increase vascular resistance and can lead to heart failure and death. Currently, there are no target therapies for the treatment of Sch-PAH. Hypothesis: S. mansoni egg-derived antigens alter pulmonary endothelial cell (EC) Caveolin-1 (Cav-1) expression and phosphorylation contributing to the loading and shedding of extracellular vesicles (EVs), which in turn stimulates abnormal EC survival leading to Sch-PAH. Methods/Results: Immunohistology and western blot analysis of lungs from S. mansoni -infected mice revealed a significant decrease in Cav-1 expression in the egg-dependent granuloma area when compared to uninfected control animals, indicating that local S. mansoni -associated molecules and inflammatory mediators contribute to Cav-1 depletion. Previously we showed that upon vascular injury, Cav-1 deficiency leads to the survival of an abnormal EC phenotype through an unclear mechanism. Analysis of survival-associated genes in isolated lung ECs from Flk1 +/GFP ; Cav1 -/- mice, revealed a high expression of the anti-apoptotic BIRC2 and BIRC5 (known as c-IAP2 and surviving, respectively) when compared to control ECs. Interesting, in vitro exposure to the major S. mansoni egg antigen, Sm-p40 (1 μg/mL), induced time-dependent phosphorylation of Cav-1 Tyr14 in human lung microvascular ECs (HMVEC-L; 228.2 +/- 38.28% of control; p<0.05; n=4), which culminated in c-IAP2 expression. Furthermore, Sm-p40 treatment of HMVEC-L increased ERK1/2 phosphorylation, which in presence of pro-inflammatory mediators IL-6, TNF-a, and ATP, significantly increased the shedding of EVs implicating P-ERK1/2 in the mechanism of shedding of Cav-1-EVs. Conclusions: This work is uncovering the biological role and potential translational relevance of pathogen-induced EC-Cav-1 depletion via shedding of EVs during Sch-PAH.

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

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

Caveolin-1 Deficiency Inhibits Palmitate-Induced Intracellular Lipid Accumulation and Enhances Autophagy in Pancreatic ß Cells via AMPK/mTOR Pathway

Lipotoxicity induced intracellular lipid accumulation in pancreatic β cells, one of the strongest predictors for T2DM. The objective of this study is to explore the role of Cav-1 in intracellular lipid accumulation of β cells under lipotoxicity condition. Our data showed that Cav-1 silencing significantly reduced PA-induced intracellular triglyceride (TG) accumulation, enhanced autophagy and insulin secretion in NIT-1 cells and murine pancreatic islets. Further investigation found that Cav-1 depletion facilitated AMPK phosphorylation, decreased the expression of downstream lipogenic makers (SREBP-1c, FAS and ACC) and increased the expression of fatty acid oxidation maker (CPT-1), leading to the ultimate enhancement of lipid metabolism. Meanwhile, Cav-1 depletion also suppressed mTOR phosphorylation, decreased the expression of p62 protein and increased the expression of LC3-II proteins, resulting in the ultimate increase of autophagy. Subsequent inhibition of AMPK and mTOR pathways abolished Cav-1 depletion mediated autophagy enhancement and TG content reduction, leading to the reduction of insulin secretion. Our findings suggest the potential application of the Cav-1 molecule as a target for effective T2DM treatment through preservation of lipotoxicity-induced β-cell intracellular lipid accumulation and dysfunction. Disclosure W. Zeng: None. K. Liu: None. J. Tang: None. H. Peng: None. C. Lin: None. S. Lin: None. K. Lin: None. Y. Jiang: None. L. Zeng: None.

Caveolin-1 Deficiency Protects Pancreatic ß Cells against Palmitate-Induced Dysfunction and Apoptosis

The regeneration of β-cell mass and preservation of its endocrine function under lipotoxicity are long-sought goals in diabetes research. Membrane protein caveolin-1 (Cav-1) is related to β-cell apoptosis and insulin secretion, however, the underlying mechanisms still remains unclear. Our objective is to explore whether Cav-1 depletion protects pancreatic β cells from lipotoxicity and investigate the related mechanisms. Here, we found that Cav-1 silencing significantly promoted β-cell proliferation, inhibited palmitate (PA)-induced apoptosis and enhanced insulin production and secretion. These effects were associated with enhanced phosphorylation of Akt and ERK1/2 protein, which then downregulated the expression of cell cycle inhibitors (FOXO1, GSK3β, P21, P27 and P53) and upregulated Cyclin D2 and Cyclin D3 expression. Subsequent inhibition of PI3K/Akt and ERK/MAPK pathways abolished Cav-1 depletion induced β-cell mass protection. Furthermore,under PA enhanced endoplasmic reticulum (ER) stress, Cav-1 silencing significantly reduced eIF2α phosphorylation and expression of ER stress-responsive markers BiP and CHOP, which mediated the sensitization to lipotoxicity. Our findings suggest the potential application of the Cav-1 molecule as a target for effective T2DM treatment through preservation of lipotoxicity-induced β-cell dysfunction and mass reduction. Disclosure W. Zeng: None. K. Liu: None. J. Tang: None. H. Li: None. H. Xu: None. H. Lu: None. H. Peng: None. C. Lin: None. R. Gao: None. S. Lin: None. K. Lin: None. Y. Jiang: None. L. Zeng: None.

Caveolin-1 Was Required for Glargine to Improve Insulin Sensitivity and Activate PI3K/AKT Pathway in Visceral Adipose Tissue of Type 2 Diabetic Mice

Insulin treatment improves insulin sensitivity, but the underlying mechanism remains unknown. Caveolin-1(Cav-1) konckout mice present insulin resistance.Our aim is to investigate the effect of Cav-1 depletion on insulin sensitivity and PI3K/AKT activation in glargine treatment of type 2 diabetic mice. C57BL/6J mice were divided into five groups(n=5 for each group).We firstly used high-fat high-carbonhydrate diet with intraperitoneal injection of streptozotocin(40mg/kg for 3 days) to induce type 2 diabetic(T2DM group) mice. Next, glargine was started at a dose of 0.4u/day to T2DM mice for 3 weeks(Insulin group).Lastly, Cav-1 expression was silenced by lentiviral-vectored short hairpin RNA (shRNA) through tail vein injection in glargine treated T2DM mice(CAV1-shRNA group) with scramble virus injection as a negative control(Ctrl-shRNA group). As a result, improvement of insulin sensitivity and PI3K/AKT activation with upregulation of Cav-1 were observed in visceral adipose tissue of Insulin group compared with T2DM group (P<0.05). In CAV1-shRNA group,impairment of insulin tolerance and PI3K/AKT activation couldn’t be improved by glargine compared with Ctrl-shRNA group (P<0.05). Our findings suggest that Cav-1 is required for insulin to improve insulin sensitivity and PI3K/AKT activation. More studies are needed to elucidate the mechanism. Disclosure H. Peng: None. H. Li: None. S. Lin: None. W. Zeng: None. C. Lin: None. K. Lin: None. L. Zeng: None.

Endothelial Caveolin‐1 Positive Microvesicles and Apoptotic Bodies as Early Biomarkers of Acute Lung Injury and Possible Mediators of TGF‐β‐mediated Repair/Remodeling

Endothelial cell injury is a hallmark of Acute Lung Injury and Acute Respiratory Distress Syndrome (ALI/ARDS). We and others have shown endothelial cell caveolin‐1 (Cav‐1) expression is critical for maintaining vascular homeostasis. The aim of this study was to determine how Cav‐1 is depleted during ALI/ARDS and how this may lead to defective vascular repair. ALI was induced in C57BL6 WT mice (male; 2.5–3 months old) by nebulized Escherichia coli lipopolysaccharide (LPS; 10 mg over 1 hr daily for 1 or 4 days). Lung pathology and Cav‐1+ EVs in platelet‐free plasma were assessed by immunohistochemistry, quantification of immune cell number in bronchoalveolar lavage fluid, and western blot. In vitro, human pulmonary artery endothelial cells (HPAECs) and isolated bone marrow‐derived macrophages (BMDMs) were treated with LPS or EVs from control or LPS‐treated animals. At 24 hs, LPS‐induced ALI increased the total number of EVs in murine plasma by 50%. Characterization of isolated EVs indicated Cav‐1 is abundantly expressed in large microvesicles and apoptotic bodies when compared with smaller vesicles, suggesting Cav1+ EVs may be an endothelial cell damage‐associated factor. By western blot, we observed EV‐Cav‐1 expression was increased by 38% after 24 hrs of LPS exposure (0.61 ± 0.05 to 0.98 ± 0.16 a.u.) in control and LPS‐treated mice, respectively (n= 8–10 mice; P<0.05) and decreased below basal level after 96 hrs indicating endothelial Cav‐1 depletion may be partially due to release Cav‐1+ EVs into the circulation. In vitro, LPS‐treated HPAECs released Cav1+ EVs in a concentration‐dependent manner and EVs‐derived from LPS‐treated mice induced TGF‐β release from BMDMs. Thus, EC‐Cav‐1 depletion may be due in part to EV shedding as a result of EC damage which in vivo may be a mechanism of macrophage‐dependent TGF‐β‐induced vascular repair/remodeling. These novel observations may provide an important link to new ways of preventing the onset or progression of ALI/ARDS.Support or Funding InformationCNPq Fellowship‐CsF/Brazil (SDO) and HL125356 and DOD W911NF‐15‐R‐002 (RDM, MGB).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Caveolin-1 deficiency protects pancreatic β cells against palmitate-induced dysfunction and apoptosis

Lipotoxicity leads to insulin secretion deficiency, which is among the important causes for the onset of type 2 diabetes mellitus. Thus, the restoration of β-cell mass and preservation of its endocrine function are long-sought goals in diabetes research. Previous studies have suggested that the membrane protein caveolin-1 (Cav-1) is implicated in β-cell apoptosis and insulin secretion, however, the underlying mechanisms still remains unclear. Our objective is to explore whether Cav-1 depletion protects pancreatic β cells from lipotoxicity and what are the underlying mechanisms. In this study, we found that Cav-1 silencing significantly promoted β-cell proliferation, inhibited palmitate (PA)-induced pancreatic β-cell apoptosis and enhanced insulin production and secretion. These effects were associated with enhanced activities of Akt and ERK1/2, which in turn downregulated the expression of cell cycle inhibitors (FOXO1, GSK3β, P21, P27 and P53) and upregulated the expression of Cyclin D2 and Cyclin D3. Subsequent inhibition of PI3K/Akt and ERK/MAPK pathways abolished Cav-1 depletion induced β-cell mass protection. Furthermore, under PA induced endoplasmic reticulum (ER) stress, Cav-1 silencing significantly reduced eIF2α phosphorylation and the expression of ER stress-responsive markers BiP and CHOP, which are among the known sensitizers of lipotoxicity. Our findings suggest Cav-1 as potential target molecule in T2DM treatment via the preservation of lipotoxicity-induced β-cell mass reduction and the attenuation of insulin secretion dysfunction.

Caveolin and Endothelial NO Signaling.

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

Inflammation-induced caveolin-1 and BMPRII depletion promotes endothelial dysfunction and TGF-β-driven pulmonary vascular remodeling.

Endothelial cell (EC) activation and vascular injury are hallmark features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Caveolin-1 (Cav-1) is highly expressed in pulmonary microvascular ECs and plays a key role in maintaining vascular homeostasis. The aim of this study was to determine if the lung inflammatory response to Escherichia coli lipopolysaccharide (LPS) promotes priming of ECs via Cav-1 depletion and if this contributes to the onset of pulmonary vascular remodeling. To test the hypothesis that depletion of Cav-1 primes ECs to respond to profibrotic signals, C57BL6 wild-type (WT) mice (Tie2.Cre-;Cav1fl/fl ) were exposed to nebulized LPS (10 mg; 1 h daily for 4 days) and compared with EC-specific Cav1-/- (Tie2.Cre+;Cav1fl/fl ). After 96 h of LPS exposure, total lung Cav-1 and bone morphogenetic protein receptor type II (BMPRII) expression were reduced in WT mice. Moreover, plasma albumin leakage, infiltration of immune cells, and levels of IL-6/IL-6R and transforming growth factor-β (TGF-β) were elevated in both LPS-treated WT and EC-Cav1-/- mice. Finally, EC-Cav1-/- mice exhibited a modest increase in microvascular thickness basally and even more so on exposure to LPS (96 h). EC-Cav1-/- mice and LPS-treated WT mice exhibited reduced BMPRII expression and endothelial nitric oxide synthase uncoupling, which along with increased TGF-β promoted TGFβRI-dependent SMAD-2/3 phosphorylation. Finally, human lung sections from patients with ARDS displayed reduced EC Cav-1 expression, elevated TGF-β levels, and severe pulmonary vascular remodeling. Thus EC Cav-1 depletion, oxidative stress-mediated reduction in BMPRII expression, and enhanced TGF-β-driven SMAD-2/3 signaling promote pulmonary vascular remodeling in inflamed lungs.

Abstract 405: Caveolin-1 expression mediates response to albumin-bound paclitaxel

Abstract BACKGROUND: Albumin-bound chemotherapies such as nab-paclitaxel are approved to treat pancreatic cancer, non-small cell lung cancer (NSCLC), and breast cancer. Predictive biomarkers to select patients who may benefit most from nab-paclitaxel are lacking. Nab-paclitaxel is thought to enter cells through a caveolae-gp60 endocytic mechanism. Caveolin-1 (Cav-1) is a principal structural component of caveolae, and Cav-1 has been shown to be important for albumin uptake in endothelial cells. Cav-1 is known to be up-regulated in multiple tumor types, including pancreatic cancer, and certain subtypes of non-small cell lung cancer, and breast cancer. We hypothesize that Cav-1 expression may predict response to nab-paclitaxel therapy. METHODS: We correlated Cav-1 expression with nab-paclitaxel sensitivity in a panel of NSCLC and pancreatic cancer cell lines. We also assessed albumin uptake in tumor and non-tumor cell lines. We genetically depleted Cav-1 by shRNA in cells and performed cytotoxicity assays. In addition, we measured how Cav-1 levels affected albumin and nab-paclitaxel uptake into tumor cells by immunofluorescence, immunoblotting, and mass spectrometry. Annexin V flow cytometry analysis and immunoblotting for apoptosis pathway intermediates were also performed. Nab-paclitaxel resistant cell lines were created by culturing cells with increasing doses of nab-paclitaxel for extended periods of time. The role of Cav-1 expression in mediating response to nab-paclitaxel in vivo was assessed using xenograft models. RESULTS: H23 and MIA-PaCa2 tumor cells uptake more albumin compared to FHs74Int and HBEC3KT non-tumor cell lines. Higher Cav-1 expression in a panel of pancreatic cancer and NSCLC cell lines was correlated with lower IC50 for nab-paclitaxel. Cav-1 depletion resulted in reduced albumin and nab-paclitaxel uptake by tumor cells as measured by immunofluorescence, immunoblotting, and mass spectrometry. Loss of Cav-1 resulted in resistance to nab-paclitaxel but no change in sensitivity to free paclitaxel in vitro. Cav-1 down-regulation resulted in protection from nab-paclitaxel-induced apoptosis. Conversely, re-expression of Cav-1 in low-Cav-1 endogenously expressing cell lines AsPC-1 and HPAFII resulted in increased nab-paclitaxel uptake and sensitization through apoptosis. Furthermore, nab-paclitaxel resistant cells generated by prolonged exposure demonstrated downregulation of Cav-1 levels and reduced albumin uptake. Finally, genetic depletion of Cav-1 rendered tumor cells resistant to nab-paclitaxel in xenograft models, with concomitant reduced albumin uptake and activation of apoptosis. CONCLUSIONS: Our data suggest that Cav-1 expression and caveolae are critical determinants of response to nab-paclitaxel. This data supports further testing of Cav-1 as a potential predictive biomarker of response to nab-paclitaxel and potentially other albumin-bound chemotherapies. Citation Format: Terence M. Williams, Moumita Chatterjee, Ryan Robb, Marally Vedaie, Star Seum, Thirumoorthy Krishnan, Palanichamy Kamalakanan, Edgar Ben-Josef, Arnab Chakravarti. Caveolin-1 expression mediates response to albumin-bound paclitaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 405.