Distribution Of Caveolin-1 Research Articles

F-45 Early liver steatosis during choline-deficient diet is associated with altered distribution of caveolin-1 (CAV1) within lipid droplets and mitochondria in steatotic rat hepatocytes

F-45 Early liver steatosis during choline-deficient diet is associated with altered distribution of caveolin-1 (CAV1) within lipid droplets and mitochondria in steatotic rat hepatocytes

Altered distribution of caveolin‐1 in early liver steatosis

Caveolin-1, the main structural protein of caveolae, is involved in cholesterol homoeostasis, transcytosis, endocytosis and signal transduction and thought to play an important role in lipidogenesis. Little is known about the pathophysiological role of caveolin-1 in nonalcoholic fatty liver disease (NAFLD), a condition frequently associated with the metabolic syndrome and characterized by abnormal accumulation of intrahepatic triglycerides with a potentially harmful risk of evolution to liver fibrosis, cirrhosis and hepatocellular carcinoma. Liver steatosis (micro/macrovesicular) was induced in adult rats fed a choline-deficient diet for 14days and compared with a control normal diet. The expression and subcellular distribution of caveolin-1 was assessed using light and electron microscopy by immunohistochemical and immunocytochemical techniques and by Western blotting. Caveolin-1 was mainly associated with the hepatocyte basolateral plasma membrane. Fatty hepatocytes were characterized by a significant increase in the expression of caveolin-1 around and within the lipid droplets as well as in the inner membrane of mitochondria. Our data suggest the involvement of caveolin-1 in the case of abnormal lipogenesis and mitochondrial function typical of steatotic hepatocytes in NAFLD. Addressing the role played by caveolin-1 in liver membranes in NAFLD may help future therapeutic choices in a frequent metabolic liver disease.

PTRF–cavin-1 expression decreases the migration of PC3 prostate cancer cells: Role of matrix metalloprotease 9

Caveolae are specialized plasma membrane subdomains with a distinct lipid and protein composition, which play an essential role in cell physiology by performing trafficking and signalling functions. The structure and functions of caveolae have been shown to require caveolin-1, a major protein component of caveolae. Caveolin-1 expression and secretion are increased in metastatic prostate cancer, and caveolin-1 seems to contribute to prostate cancer growth and metastasis. Recently, a cytoplasmic protein named PTRF (Polymerase I and Transcript Release Factor) or cavin-1 was found to be required, in concert with caveolin-1, for the formation and functions of caveolae. Genetic ablation of PTRF results in loss of caveolae while caveolin-1 is still expressed, albeit at reduced level, but associates with flat plasma membrane. In metastatic PC3 prostate cancer cells that express abundant caveolin-1 but no PTRF, heterologous PTRF expression restores caveola formation and caveolin-1 distribution (Hill et al., 2008; Cell 132, 113–124). We now show that PTRF/cavin-1-expressing PC3 cells exhibit decreased migration, and that this effect is mediated by reduced MMP9 production. PTRF/cavin-1, and to a lesser extent, cavin-2, -3, and -4 all decreased MMP9. We further show that the PTRF/cavin-1-mediated reduction of MMP9 production is independent of caveola formation. Taken together, our results suggest that PTRF/cavin-1 expression alters prostate cancer aggressiveness.

Occludin is required for cytokine-induced regulation of tight junction barriers

The function of occludin remains elusive. Proposed roles include maintenance of tight junction barriers, signaling and junction remodeling. To investigate a potential role in mediating cytokine-induced changes in barrier properties, we measured barrier responses to interferon-gamma plus TNFalpha in control, occludin-overexpressing and occludin knockdown MDCK II monolayers. MDCK cells show a complex response to cytokines characterized by a simultaneous increase in the transepithelial electrical resistance and a decrease in the barrier for large solutes. We observed that overexpression of occludin increased and occludin knockdown decreased sensitivity to cytokines as assessed by both these parameters. It is known that caveolin-1 interacts with occludin and is implicated in several models of cytokine-dependent barrier disruption; we found that occludin knockdown altered the subcellular distribution of caveolin-1 and that partitioning of caveolin into detergent-insoluble lipid rafts was influenced by changing occludin levels. Knockdown of caveolin decreased the cytokine-induced flux increase, whereas the increase in the electrical barrier was unaltered; the effect of double knockdown of occludin and caveolin was similar to that of occludin single knockdown, consistent with the possibility that they function in the same pathway. These results demonstrate that occludin is required for cells to transduce cytokine-mediated signals that either increase the electrical barrier or decrease the large solute barrier, possibly by coordinating the functions of caveolin-1.

Abstract 517: Expression of PTRF/cavin-1 in prostate cancer cells reduces migration via decreased MMP9 expression

Abstract Caveolin-1 expression and secretion are increased in metastatic prostate cancer and caveolin-1 seems to contribute to prostate cancer growth and metastasis. Caveolin-1 was long believed to be sufficient for caveolae formation, but a cytoplasmic protein named PTRF (Polymerase I and Transcript Release Factor) or cavin-1 was recently found to be required, in concert with caveolin-1, for the formation and functions of caveolae. We hypothesized that PTRF expression could modulate prostate cancer cell migration. In metastatic PC3 prostate cancer cells that express abundant caveolin-1 but no PTRF, heterologous PTRF expression restores caveolae formation and caveolin-1 distribution. We show that PTRF-expressing PC3 cells exhibit decreased migration, and that this effect is mediated by reduced matrix metalloprotease-9 (MMP9) production. We further show that MMP9 production is affected at the transcriptional level, and independently from caveolae formation. Of all the cavin family members, PTRF-cavin-1 is the only one to dramatically reduce MMP9 expression. Taken together, our results suggest that PTRF-cavin-1 expression alters prostate cancer aggressiveness. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 517.

Gangliosides and β1-Integrin Are Required for Caveolae and Membrane Domains

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin-mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo-glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol ('lipid rafts'), reduced caveolae and caveolin-1 at the plasma membrane by approximately 80%, and blunted activation of beta1-integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo-gangliosides or other sphingolipids) at 10 degrees C, suggesting that sialo-lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Igamma away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin-1 could be recapitulated by beta1-integrin knockdown. These results suggest that both gangliosides and beta1-integrin are required for maintenance of caveolae and plasma membrane domains.

Saphenous Vein Conduits Harvested by Endoscopic Technique Exhibit Structural and Functional Damage

Injury to the saphenous vein endothelium during harvest impacts patency after coronary artery bypass graft surgery. Many centers are adopting endoscopic saphenous vein harvest (ESVH) instead of using the traditional open saphenous vein harvest (OSVH) technique. Our objective was to compare the effects of ESVH and OSVH on the structural and functional viability of saphenous vein endothelium using multiphoton imaging, immunofluorescence, and biochemical techniques. Ten patients scheduled for coronary artery bypass graft surgery were prospectively identified. Each underwent ESVH for one portion and OSVH for another portion of the saphenous vein. A 1-cm segment from each portion was immediately transported to the laboratory for processing. The vessel segments were labeled with fluorescent markers to quantify cell viability (esterase activity), calcium mobilization, and generation of nitric oxide. Samples were also labeled with immunofluorescent antibodies to visualize caveolin, endothelial nitric oxide synthase, von Willebrand factor, and cadherin, and extracted to identify these proteins using Western blot techniques. All labeling, imaging, and image analysis was done in a blinded fashion. Esterase activity was significantly higher in the OSVH group (p < 0.0001). Similarly, calcium mobilization and nitric oxide production were significantly greater in the OSVH group (p = 0.0209, p < 0.0001, respectively). Immunofluoresence and Western blot techniques demonstrated an abnormal alteration in distribution of caveolin and endothelial nitric oxide synthase in the ESVH group. Our study indicates that ESVH has a detrimental effect on the saphenous vein endothelium, which may lead to decreased graft patency and worse patient outcomes.

Ca2+-stimulated Basal Adenylyl Cyclase Activity Localization in Membrane Lipid Microdomains of Cardiac Sinoatrial Nodal Pacemaker Cells

Spontaneous, rhythmic subsarcolemmal local Ca(2+) releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca(2+) releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca(2+) and cAMP/PKA regulatory components of automaticity are coupled via Ca(2+) activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca(2+)-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca(2+) over the entire physiological Ca(2+) range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca(2+) cycling proteins drive sarcoplasmic reticulum Ca(2+) releases, which, in turn, activate ACs. This feed forward "fail safe" system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.

Lipoprotein binding preference of CD36 is altered by filipin treatment

The class B scavenger receptor CD36 binds multiple ligands, including oxidized and native lipoprotein species. CD36 and the related receptor SR-B1 have been localized to caveolae, domains that participate in cell signaling, transcytosis, and regulation of cellular cholesterol homeostasis. Previous work has indicated that the ligand preference of CD36 may depend on the cell type in which it is expressed. To determine if the presence or absence of caveolae is the determining factor for lipoprotein preference, we treated CHO-CD36 and C32 cells with filipin. Filipin treatment rapidly increased the binding capacity of CD36 for the native lipoproteins HDL and LDL, but did not affect the binding capacity of CD36 for oxidized LDL. Filipin treatment affected the distribution of caveolin and CD36 suggesting that the presence caveolae may modulate the ligand preference of CD36. However, its molecular mechanism how CD36 and caveolin interaction in regulating lipoprotein transport remains to be further studied.

Caveolin-1 polarization in transmigrating endothelial cells requires binding to intermediate filaments

Caveolin-1 influences cell migration through multiple signaling pathways. In a previous report, we have shown that caveolin-1 is polarized in three-dimensional migrating endothelial cells (EC), and that caveolin-1 accumulation at the front of transmigrating cells requires the phosphorylatable Tyr(14) residue of caveolin-1. Immuno-electron microscopy further indicated that caveolin-1 was distributed along cytoskeletal structures in the anterior of transmigrating EC [Parat MO, Anand-Apte B, Fox PL (Mol Biol Cell 14:3156-3168, 2003)]. In the present study, we investigate whether caveolin-1 interacts with intermediate filaments (IF) and whether this interaction is required for caveolin-1 polarization in transmigrating cells. The distribution of vimentin is polarized in cells traversing a filter pore and overlaps with the distribution of caveolin-1, which accumulates in the cell front. In vivo sprouting EC also exhibit an anterior polarization of these two proteins. Furthermore, caveolin-1 co-purifies with intermediate filaments, suggesting an interaction between caveolin-1 and IF. Vimentin-deficient SW13 cells exhibit a dramatically altered polarization of caveolin-1-GFP, which no longer accumulates in the protruding cell extension. In addition, the Tyr(14) residue of caveolin-1 is required for co-purification of the protein with IF. Taken together, our results show that caveolin-1 Tyr(14) is necessary for binding to intermediate filaments, which in turn is required for anterior polarization of caveolin-1 in transmigrating cells.

Distribution and significance of caveolin 2 expression in normal breast and invasive breast cancer: an immunofluorescence and immunohistochemical analysis

The aims of this study were to define the distribution of caveolin 2 (CAV2) in frozen and formalin fixed, paraffin embedded (FFPE) normal breast samples and the significance of CAV2 expression in breast cancer. Caveolin 2 distribution in frozen and paraffin-embedded whole tissue sections of normal breast was evaluated using immunohistochemistry and immunofluorescence, in conjunction with antibodies to define luminal epithelial cells (oestrogen receptor and cytokeratin 8/18) and myoepithelial/ basal cells (cytokeratins 14 and 5/6, p63 and smooth muscle actin). CAV2 expression was also immunohistochemically analysed in two independent cohorts of invasive breast carcinomas (n = 245 and n = 418). In normal breast, CAV2 was expressed in myoepithelial cells, endothelial cells, fibroblasts and adipocytes. Luminal epithelial cells showed no or only negligible staining. CAV2 expression was observed in 9.6% of all breast cancers and was strongly correlated with high histological grade, lack of oestrogen receptor, progesterone receptor and cyclin D1 expression, and positivity for epidermal growth factor receptor, basal markers, p53 expression, and high proliferation index. Furthermore, CAV2 expression was significantly associated with basal-like immunophenotype and proved to be a prognostic factor for breast cancer-specific survival on univariate analysis. Our results demonstrate that CAV2 is preferentially expressed in basal-like cancers and is associated with poor prognosis. Further in vitro studies are required to determine whether CAV2 has oncogenic properties or is only a surrogate marker of basal-like carcinomas.

Annexin A6-induced alterations in cholesterol transport and caveolin export from the Golgi complex.

Annexin A6 (AnxA6) belongs to a family of Ca2+-dependent membrane-binding proteins and is involved in the regulation of endocytic and exocytic pathways. We previously demonstrated that AnxA6 regulates receptor-mediated endocytosis and lysosomal targeting of low-density lipoproteins and translocates to cholesterol-enriched late endosomes (LE). As cholesterol modulates the membrane binding and the cellular location of AnxA6, but also affects the intracellular distribution of caveolin, we investigated the localization and trafficking of caveolin in AnxA6-expressing cells. Here, we show that cells expressing high levels of AnxA6 are characterized by an accumulation of caveolin-1 (cav-1) in the Golgi complex. This is associated with a sequestration of cholesterol in the LE and lower levels of cholesterol in the Golgi and the plasma membrane, both likely contributing to retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. Further strengthening these findings, knock down of AnxA6 and the ectopic expression of the Niemann–Pick C1 protein in AnxA6-overexpressing cells restore the cellular distribution of cav-1 and cholesterol, respectively. In summary, this study demonstrates that elevated expression levels of AnxA6 perturb the intracellular distribution of cholesterol, which indirectly inhibits the exit of caveolin from the Golgi complex.

Status of caveolin-1 in various membrane domains of the bovine lens

Recent studies of the distribution and relative concentration of caveolin-1 in fractions of bovine lens epithelial and fiber cells have led to the novel concept that caveolin-1 may largely exist as a peripheral membrane protein in some cells. Caveolin-1 is typically viewed as a scaffolding protein for caveolae in plasma membrane. In this study, membrane from cultured bovine lens epithelial cells and bovine lens fiber cells were divided into urea soluble and insoluble fractions. Cytosolic lipid vesicles were also recovered from the lens epithelial cells. Lipid-raft domains were recovered from fiber cells following treatment with detergents and examined for caveolin and lipid content. Aliquots of all fractions were Western blotted for caveolin-1. Fluorescence microscopy and double immunofluorescence labeling were used to examine the distribution of caveolin-1 in cultured epithelial cells. Electron micrographs revealed an abundance of caveolae in plasma membrane of cultured lens epithelial cells. About 60% of the caveolin-1 in the epithelial-crude membrane was soluble in urea, a characteristic of peripheral membrane proteins. About 30% of the total was urea-insoluble membrane protein that likely supports the structure of caveolae. The remaining caveolin was part of cytosolic lipid vesicles. By contrast, most caveolin in the bovine lens fiber cell membrane was identified as intrinsic protein, being present at relatively low concentrations in caveolae-free lipid raft domains enriched in cholesterol and sphingomyelin. We estimate that these domains occupied 25–30% of the fiber cell membrane surface. Thus, the status of caveolin-1 in lens epithelial cells appears markedly different from that in fiber cells.

Experimental study on the effects of hydrogen peroxide on caveolin in human lens epithelial cells

To observe the expression and distribution of caveolin and phosphorylated caveolin-1 in human lens epithelial cells (HLECs) under H(2)O(2) treatment. HLECs (SRA01/04) were exposed to different concentrations of H(2)O(2) for different periods of time. The distribution of caveolin and phosphorylated caveolin-1 in H(2)O(2) treated cells was observed by laser scanning confocal microscopy and fluorescence microscopy. Western blot was conducted to analyze the protein expression alterations of caveolin and caveolin-1 phosphorylation. HLECs contained abundant caveolin. Immunofluorescence image of caveolin in cytoplasm increased significantly in H(2)O(2) treated cells. One hour after H(2)O(2) treatment, the cells membranes began to break, whereas the immunofluorescence image of caveolin could still be observed. Caveolin-1 was phosphorylated on tyrosine 14 in HLECs after stimulation with H(2)O(2). Western blot analysis revealed that caveolin protein level was down regulated under H(2)O(2) stress. The caveolin is redistributed and the caveolae is destroyed in HLECs when stimulated by H(2)O(2). And the caveolin expression also down regulated by H(2)O(2) stimulation. Caveolae and caveolin are likely to play an important role in the HLECs.

Cholesterol depletion inhibits fatty acid uptake without affecting CD36 or caveolin-1 distribution in adipocytes

Caveolin-1 and CD36 are plasma membrane fatty acid binding proteins that participate in adipocyte fatty acid uptake and metabolism. Both are associated with cholesterol-enriched caveolae/lipid rafts in the plasma membrane that are important for long chain fatty acid uptake. Depletion of plasma membrane cholesterol reversibly inhibited oleate uptake by adipocytes without altering the amount or the cell surface distribution of either caveolin-1 or CD36. Cholesterol levels thus regulate fatty acid uptake by adipocytes via a pathway that does not involve altered cell surface localization of caveolin-1 or CD36.

Caveolin‐1 and the Organization of Glycolysis in Astrocytes: Modulation by Ammonia

The organizational role of the membrane protein caveolin-1 (CAV-1), which is responsible for the formation of caveolae was previously examined in cultured vascular smooth muscle cells, and in cultured CAV-1 transfected lymphocytes. The distribution of the glycolytic enzymes aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), and glyceraldehyde phosphate dehydrogenase (GAPDH), was previously determined using immunofluorescence confocal microscopy to validate interaction between proteins. The organizational role of CAV-1 with glycolytic enzymes PK and GAPDH, and with transporters GLUT-1 (for Glucose) and GLT-1 (for Glutamate) with CAV-1 was investigated in mouse cultured astrocytes. We found CAV-1 localizes with multiple glycolytic enzymes and also with GLUT-1 and GLT-1. We then studied the effect of 5 mM NH4Cl on CAV-1 expression in the astrocyte and found CAV-1 expression changes after exposure for 24 hour to 5 mM NH4Cl. The distribution of CAV-1 with PK was not affected after 5 mM NH4Cl incubation neither was the distribution of CAV-1 with GLUT-1 or GLT-1, but rather Metamorph linescan analysis revealed flurophore intensity was enhanced. We conclude that CAV-1 localizes with multiple glycolytic enzymes and may play a role in the formation of a glycolytic compartment in the astrocyte, similarly observed in smooth muscle cells. We propose that CAV-1 may have a role in the organization of glycolysis in astrocytes and that there may be coordinate regulation of the scaffolding protein after exposure to 5 mM NH4Cl. Support by NIH DK 60668.

Distribution of caveolin isoforms in the lemur retina

The distribution of caveolin isoforms was previously evaluated in the retinas of different species, but has not yet been described in the primate retina. In this study, the distribution of caveolins was assessed via immunochemistry using isoform-specific antibodies in the retina of the black-and-white ruffed lemur. Here, we report the presence of a variety of caveolin isoforms in many layers of the lemur retina. As normal human retinas were not available for research and the retinas of primates are fairly similar to those of humans, the lemur retina can be utilized as a model for caveolin distribution in normal humans.

Caveolin 1 Is Overexpressed and Amplified in a Subset of Basal-like and Metaplastic Breast Carcinomas: A Morphologic, Ultrastructural, Immunohistochemical, and In situ Hybridization Analysis

The distribution and significance of caveolin 1 (CAV1) expression in different breast cell types and role in breast carcinogenesis remain poorly understood. Both tumor-suppressive and oncogenic roles have been proposed for this protein. The aims of this study were to characterize the distribution of CAV1 in normal breast, benign breast lesions, breast cancer precursors, and metaplastic breast carcinomas; to assess the prognostic significance of CAV1 expression in invasive breast carcinomas; and to define whether CAV1 gene amplification is the underlying genetic mechanism driving CAV1 overexpression in breast carcinomas. CAV1 distribution in frozen and paraffin-embedded whole tissue sections of normal breast was evaluated using immunohistochemistry, immunofluorescence, and immunoelectron microscopy. CAV1 expression was immunohistochemically analyzed in benign lesions, breast cancer precursors, and metaplastic breast carcinomas and in a cohort of 245 invasive breast carcinomas from patients treated with surgery followed by anthracycline-based chemotherapy. In 25 cases, CAV1 gene amplification was assessed by chromogenic in situ hybridization. In normal breast, CAV1 was expressed in myoepithelial cells, endothelial cells, and a subset of fibroblasts. Luminal epithelial cells showed negligible staining. CAV1 was expressed in 90% of 39 metaplastic breast carcinomas and in 9.4% of 245 invasive breast cancers. In the later cohort, CAV1 expression was significantly associated with 'basal-like' immunophenotype and with shorter disease-free and overall survival on univariate analysis. CAV1 gene amplification was found in 13% of cases with strong CAV1 expression. The concurrent CAV1 amplification and overexpression call into question its tumor-suppressive effects in basal-like breast carcinomas.

The high heart performance in mako shark is related to increased caveolin expression in buoyant fraction

Mammalian hearts have a developed sympathetic system coupled to complex intracellular signaling pathways to markedly increase heart rate and contractility. Caveolae are sub-cellular structures enriched in caveolin proteins and are essential for t-tubules formation, signaling, and calcium homeostatsis. In general, species from this clade, such as mako shark, can attain mammalian-like cardiac function. This study examined the plausible traits in the design of a high performance heart of the regional endothermic mako sharks to the ectothermic blue sharks and rats by comparing caveolin distribution and myocyte ultrastructure. Caveolin 1/3 distribution was determined by sucrose density fractionation of heart homogenates. Electron microscopy (EM) was performed to assess the presence of caveolae and t-tubule. Following fractionation, caveolin was predominantly detected in buoyant fraction (BF) with relatively little heavy fraction (HF) in both sharks, findings akin to rat vascular smooth muscle cells (VSMC, Figure), which lack t-tubules. In rats, caveolin was present in both BF and HF, the latter indicative of t-tubule in mammals. EM analyses showed no t-tubules in both shark hearts, but were found in rats. Compared to blue sharks, the preponderance in BF of Cav1/3 (i.e., more caveolae) in the mako is one of the traits in achieving higher heart performance.

A role for neutral sphingomyelinase activation in the inhibition of LPS action by phospholipid oxidation products

Previous studies from our laboratory and others presented evidence that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine can inhibit lipopolysaccharide (LPS)-mediated induction of interleukin-8 (IL-8) in endothelial cells. Using synthetic derivatives of phosphatidylethanolamine, we now demonstrate that phospholipid oxidation products containing alpha,beta-unsaturated carboxylic acids are the most active inhibitors we examined. 5-Keto-6-octendioic acid ester of 2-phosphatidylcholine (KOdiA-PC) was 500-fold more inhibitory than OxPAPC, being active in the nanomolar range. Our studies in human aortic endothelial cells identify one important mechanism of the inhibitory response as involving the activation of neutral sphingomyelinase. There is evidence that Toll-like receptor-4 and other members of the LPS receptor complex must be colocalized to the caveolar/lipid raft region of the cell, where sphingomyelin is enriched, for effective LPS signaling. Previous work from our laboratory suggested that OxPAPC could disrupt this caveolar fraction. These studies present evidence that OxPAPC activates sphingomyelinase, increasing the levels of 16:0, 22:0, and 24:0 ceramide and that the neutral sphingomyelinase inhibitor GW4869 reduces the inhibitory effect of OxPAPC and KOdiA-PC. We also show that cell-permeant C6 ceramide, like OxPAPC, causes the inhibition of LPS-induced IL-8 synthesis and alters caveolin distribution similar to OxPAPC. Together, these data identify a new pathway by which oxidized phospholipids inhibit LPS action involving the activation of neutral sphingomyelinase, resulting in a change in caveolin distribution. Furthermore, we identify specific oxidized phospholipids responsible for this inhibition.