Caveolin-1 In Endothelial Cells Research Articles

OP30 Endothelial cell-mediated smooth muscle hyperplasia in Crohn’s disease intestinal strictures: Caveolin 1 as a potential therapeutic target

Abstract Background Intestinal stricture is a severe and common complication of Crohn’s disease (CD). Current therapies offer limited success and surgery is often needed. Smooth muscle hyperplasia and hypertrophy play a key role in CD stricture development. This study explores the transcriptomic features of fibrostenotic CD, focusing on the endothelial contribution to smooth muscle changes within intestinal strictures. Methods 9 CD patients undergoing surgery for fibrotic strictures were prospectively enrolled. Dissected samples from the stricture and the adjacent proximal and distal non-strictured areas were obtained from fresh specimens collected directly from the operating theatre. RNA was extracted from all samples for bulk-transcriptomic analysis. Differentially expressed genes (adj. p-value <0.05) were identified and pathway enrichment analysis pinpointed relevant pathways. Subsequently, single-cell RNA sequencing (scRNAseq) analysis was performed using the 10x Chromium Controller® platform. A total of 31.195 cells were sequenced and bioinformatic analysis revealed 13 distinct clusters based on the expression of key lineage target genes. To better characterise the nature of clusters expressing stromal like markers and positioned closely in the UMAP plot, a further subclustering was performed. This resulted in 5 groups, including plasma cells, endothelial cells, fibroblasts, epithelial cells and dendritic like cells. Results Bulk-transcriptomic analysis identified 81 genes differentially expressed in strictures compared to both proximal and distal regions. Notably, genes associated with leukocyte and macrophage recruitment and chemotaxis, namely Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), C-C motif Chemokine Ligand 2 (CCL2), and Endothelin Receptor Type B (EDNRB), resulted overexpressed in strictures. scRNAseq identified a distinct subset of endothelial cells (CD34+, PECAM1+). Caveolin 1 (CAV1), a gene encoding for a structural protein predominantly expressed in smooth muscle cell plasma membrane invaginations, was exclusively up-regulated in endothelial cells of ileal stricture samples. Previous research linked CAV1 to smooth muscle proliferation and fibrosis in various organs. Conclusion This study highlights the pivotal role of endothelial cells in CD strictures. The transcriptomic analysis shows the involvement of inflammatory targets featured genes involved in transendothelial immune cell migration. Furthermore, the up-regulation of CAV1 in endothelial cells within stricture segments suggests a mechanistic connection between endothelial cells and smooth muscle hyperplasia in intestinal strictures. CAV1 holds promise as a potential therapeutic target in fibrostenotic CD.

Clinical evidence for elevated levels of caveolin-1 in circulation of patients with diabetic foot ulcers.

Caveolin-1 directly interacts vascular endothelial growth factor receptor-2 (VEGFR2) and therefore prevents VEGF-induced angiogenesis. In addition, the production of nitric oxide (NO), which is effective in reducing ischemia in diabetic foot ulcers (DFU), is suppressed by caveolin-1 in endothelial cells. The present study was designed to investigate the change of caveolin-1 concentrations in DFU patients. A total of 150 participants were consecutively enrolled, including 40 DFU patients (DFU group), 40 diabetes patients without DFU (type 2 diabetes mellitus [T2DM] group), and 70 participants without diabetes (control group). Significant increased levels of plasma caveolin-1, accompanied with decreased concentration of plasma VEGF-A (vascular endothelial growth factor-A) and NO, were detected in DFU patients. Moreover, Pearson's correlation analysis revealed a negative correlation between plasma caveolin-1 and VEGF-A as well as NO levels in DFU patients. Furthermore, DFU patients had higher expression of caveolin-1 in the popliteal artery, compared to those in control and T2DM groups. Simultaneously, the amounts of eNOS (an enzyme responsible for the production of NO) and VEGFR2 were attenuated in the popliteal artery of DFU patients. Taken together, our study provided clinical evidence for the possible association of elevated caveolin-1 levels and the development of DFU. This may be induced by the suppressed VEGF-A/VEGFR2 and eNOS/NO signalling axis.

EphB1 interaction with caveolin-1 in endothelial cells modulates caveolae biogenesis.

Caveolae, the cave-like structures abundant in endothelial cells (ECs), are important for multiple signaling processes such as production of nitric oxide and caveolae-mediated intracellular trafficking. Using superresolution microscopy, fluorescence resonance energy transfer, and biochemical analysis, we observed that the EphB1 receptor tyrosine kinase constitutively interacts with caveolin-1 (Cav-1), the key structural protein of caveolae. Activation of EphB1 with its ligand Ephrin B1 induced EphB1 phosphorylation and the uncoupling EphB1 from Cav-1 and thereby promoted phosphorylation of Cav-1 by Src. Deletion of Cav-1 scaffold domain binding (CSD) motif in EphB1 prevented EphB1 binding to Cav-1 as well as Src-dependent Cav-1 phosphorylation, indicating the importance of CSD in the interaction. We also observed that Cav-1 protein expression and caveolae numbers were markedly reduced in ECs from EphB1-deficient (EphB1−/−) mice. The loss of EphB1 binding to Cav-1 promoted Cav-1 ubiquitination and degradation, and hence the loss of Cav-1 was responsible for reducing the caveolae numbers. These studies identify the crucial role of EphB1/Cav-1 interaction in the biogenesis of caveolae and in coordinating the signaling function of Cav-1 in ECs.

Highly glycosylated CD147 promotes hemorrhagic transformation after rt-PA treatment in diabetes: a novel therapeutic target?

BackgroundDiabetes is known to be a main risk factor of post-stroke hemorrhagic transformation following recombinant tissue plasminogen activator (rt-PA) therapy. However, the mechanism through which diabetes exacerbates hemorrhagic transformation is insufficiently understood. We aimed to verify that CD147, the extracellular matrix metalloproteinase (MMP) inducer, played a vital role in the progress.MethodsWe performed middle cerebral artery occlusion on diabetic and non-diabetic rats, with or without rt-PA treatment, and then compared the glycosylation level of CD147, caveolin-1, MMPs activities, and blood-brain barrier (BBB) permeability. In vitro, tunicamycin treatment and genetic tools were used to produce non-glycosylated and lowly glycosylated CD147. An endogenous glucagon-like peptide-1 receptor (GLP-1R) agonist was used to downregulate the glycosylation of CD147 in vivo.ResultsCompared with non-diabetic rats, diabetic rats expressed higher levels of highly glycosylated CD147 in endothelium and astrocytes following rt-PA treatment accompanied by higher activity of MMPs and BBB permeability, in the middle cerebral artery occlusion model. Caveolin-1 was also overexpressed and co-localized with CD147 in astrocytes and endothelium in diabetic rats. In vitro, advanced glycation end products increased the expression of highly glycosylated CD147 in astrocytes and endothelial cells. Downregulating the glycosylation of CD147 lowered the activity of MMPs and promoted the expression of tight junction proteins. The expression of caveolin-1 in endothelial cells and astrocytes was not inhibited by tunicamycin, which revealed that caveolin-1 was an upstream of CD147. In vivo, GLP-1R agonist downregulated the glycosylation of CD147 and further reduced the activity of MMPs and protected the BBB in diabetic rats.ConclusionCD147 is essential for diabetes-associated rt-PA-induced hemorrhagic transformation, and downregulation of CD147 glycosylation is a promising therapy for neurovascular-unit repair after rt-PA treatment of patients with diabetes.

Caveolin-1 is critical for Lymphocyte Trafficking into Central Nervous system during Experimental Autoimmune Encephalomyelitis

Abstract Multiple Sclerosis (MS) is a progressive autoimmune disease in central nervous system (CNS) with its pathogenesis not fully understood. In the present study, we demonstrated that caveolin-1, a major membrane scaffolding protein, plays a critical role in experimental autoimmune encephalomyelitis (EAE), a laboratory murine model of multiple sclerosis (MS). Immunized WT mice have increases of caveolin-1 expressions in serum and CNS tissues associated with disease development. After immunization, Cav-1knockout mice showed remarkable resistance with alleviated incidences and symptoms. With caveolin-1 deficiency, encephalitogenic T lymphocytes processed less efficient trafficking into CNS parenchyma, probably a sequence of declined expressions of adhesion molecule ICAM-1 and VCAM-1 in the white matter of CNS tissues. In agreement with in vivo studies, in vitro silencing of caveolin-1 in endothelial cells prevented the up-regulation of ICAM-1 expression, leading to less trans-endothelial migration of encephalitogenic TH1 and TH17 cells. Taken together, the results indicate caveolin-1 as an active modulator of lymphocyte trafficking into CNS and may be a potential therapeutic target for neuroinflammatory diseases.

Angiogenic Impairment of the Vascular Endothelium

Objective— Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. Approach and Results— We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx -derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated β-galactosidase activity. Mdx -derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell–derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. Conclusions— These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.

Pigment epithelium-derived factor (PEDF) binds to caveolin-1 and inhibits the pro-inflammatory effects of caveolin-1 in endothelial cells

Pigment epithelium-derived factor (PEDF) exerts atheroprotective effects both in cell culture and animal models through its anti-oxidative and anti-inflammatory properties. Caveolin-1 (Cav), a major protein component of caveolae in endothelial cells (ECs), plays a role in the progression of atherosclerosis. However, effects of PEDF on Cav-exposed ECs remain unknown. In this study, we examined whether and how PEDF could inhibit the Cav-induced inflammatory and thrombogenic reactions in human umbilical vein ECs (HUVECs). Surface plasmon resonance revealed that PEDF bound to Cav at the dissociation constant of 7.36×10−7M. Further, one of the major Cav-interacting proteins in human serum was identified as PEDF by peptide mass fingerprinting analysis using BIAcore 1000 combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Exogenously added Cav was taken up into the membrane fraction of HUVECs and dose-dependently increased monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels, all of which were blocked by the simultaneous treatment with 10nM PEDF. Small interfering RNAs directed against Cav decreased endogenous Cav levels and suppressed gene expression of MCP-1, VCAM-1 and PAI-1 in HUVECs. This study indicates that PEDF binds to Cav and could block the inflammatory and thrombogenic reactions in Cav-exposed HUVECs. Our present study suggests that atheroprotective effects of PEDF might be partly ascribed to its Cav-interacting properties.

Duffy Antigen Receptor for Chemokines Mediates Chemokine Endocytosis through a Macropinocytosis-Like Process in Endothelial Cells

BackgroundThe Duffy antigen receptor for chemokines (DARC) shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.Methodology/Principal FindingsWe investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated 125I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. 125I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression. 125I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF) enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.Conclusions/SignificanceThese results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.

Vascular Endothelial Growth Factor Receptor-2 Activates ADP-ribosylation Factor 1 to Promote Endothelial Nitric-oxide Synthase Activation and Nitric Oxide Release from Endothelial Cells

Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates endothelial function via production and release of nitric oxide (NO), an important signaling molecule. The molecular basis leading to NO production involves phosphatidylinositiol-3 kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) activation. In this study, we have examined whether small GTP-binding proteins of the ADP-ribosylation factor (ARF) family act as molecular switches to regulate signaling cascades activated by VEGF in endothelial cells. Our results show that this growth factor can promote the rapid and transient activation of ARF1. In endothelial cells, this GTPase is present on dynamic plasma membrane ruffles. Inhibition of ARF1 expression, using RNA interference, markedly impaired VEGF-dependent eNOS phosphorylation and NO production by preventing the activation of the PI3K/Akt signaling axis. Furthermore, our data indicate that phosphorylation of Tyr(801), on VEGF receptor 2, is essential for activating Src- and ARF1-dependent signaling events leading to NO release from endothelial cells. Lastly, this mediator is known to regulate a broad variety of endothelial cell functions. Depletion of ARF1 markedly inhibits VEGF-dependent increase of vascular permeability as well as capillary tubule formation, a process important for angiogenesis. Taken together, our data indicate that ARF1 is a novel modulator of VEGF-stimulated NO release and signaling in endothelial cells.

Interleukin-6 inhibits endothelial nitric oxide synthase activation and increases endothelial nitric oxide synthase binding to stabilized caveolin-1 in human vascular endothelial cells

We hypothesized a possible mechanism for atherosclerosis in which interleukin-6 (IL-6) might affect the endothelial nitric oxide synthase (eNOS)-caveolin-1 interaction and result in decreased nitric oxide bioavailability in the setting of low-grade inflammation. Because eNOS and caveolin-1 are crucial for vascular tone control, we studied the effects of IL-6 on the expression and activation of eNOS and caveolin-1 in human vascular endothelial cells. IL-6 inhibited the phosphorylation of eNOS at Ser1177 and the bradykinin-stimulated nitric oxide production; however, eNOS protein expression was not changed. In addition, IL-6 inhibited bradykinin-stimulated Akt phosphorylation at Ser473 and Thr 308 without affecting the Akt protein expression. IL-6 did not alter the mRNA level of caveolin-1; however, the caveolin-1 protein level was significantly increased dose-dependently. The binding of eNOS and caveolin-1 in endothelial cells, as demonstrated by coimmunoprecipitation assay, was increased by IL-6 treatment. IL-6 treatment was found to stabilize caveolin-1 protein and its half-life was estimated to prolong from 7.5 h to longer than 12 h. Furthermore, treatment with PD98059 and short interference RNA of extracellular signal-regulated kinase gene reversed the effects of IL-6 on eNOS and caveolin-1. In addition to decreasing Akt phosphorylation, the results of this study demonstrate, for the first time, the molecular mechanism underlying the effect of IL-6 to decrease the nitric oxide bioavailability by increasing the half-life and, therefore, the protein levels of caveolin-1. The increased caveolin-1 proteins bind more eNOS and consequently decrease eNOS activation by reducing the Ser1177 phosphorylation.

The embryonic blood–CSF barrier has molecular elements for specific glucose transport and for the general transport of molecules via transcellular routes

In vertebrates, early brain development takes place at the expanded anterior end of the neural tube, which is filled with embryonic cerebrospinal fluid (E-CSF). We have recently identified a transient blood–CSF barrier that forms between embryonic days E3 and E4 in chick embryos and that is responsible for the transport of proteins and control of E-CSF homeostasis, including osmolarity. Here we examined the presence of glucose transporter GLUT-1 as well the presence of caveolae-structural protein Caveolin1 (CAV-1) in the embryonic blood-CSF barrier which may be involved in the transport of glucose and of proteins, water and ions respectively across the neuroectoderm. In this paper we demonstrate the presence of GLUT-1 and CAV-1 in endothelial cells of blood vessels as well as in adjacent neuroectodermal cells, located in the embryonic blood–CSF barrier. In blood vessels, these proteins were detected as early as E4 in chick embryos and E12.7 in rat embryos, i.e. the point at which the embryonic blood–CSF barrier acquires this function. In the neuroectoderm of the embryonic blood-CSF barrier, GLUT-1 was also detected at E4 and E12.7 respectively, and CAV-1 was detected shortly thereafter in both experimental models. These experiments contribute to delineating the extent to which the blood–CSF embryonic barrier controls E-CSF composition and homeostasis during early stages of brain development in avians and mammals. Our results suggest the regulation of glucose transport to the E-CSF by means of GLUT-1 and also suggest a mechanism by which proteins are transported via transcellular routes across the neuroectoderm, thus reinforcing the crucial role of E-CSF in brain development.

Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells

Transcytosis via caveolae is critical for maintaining vascular homeostasis by regulating the tissue delivery of macromolecules, hormones, and lipids. In the present study, we test the hypothesis that interactions between F-actin cross-linking protein filamin A and caveolin-1 facilitate the internalization and trafficking of caveolae. Small interfering RNA-mediated knockdown of filamin A, but not filamin B, reduced the uptake and transcytosis of albumin by approximately 35 and 60%, respectively, without altering the actin cytoskeletal structure or cell-cell adherens junctions. Mobility of both intracellular caveolin-1-green fluorescent protein (GFP)-labeled vesicles measured by fluorescence recovery after photobleaching and membrane-associated vesicles measured by total internal reflection-fluorescence microscopy was decreased in cells with reduced filamin A expression. In addition, in melanoma cells that lack filamin A (M2 cells), the majority of caveolin-1-GFP was localized on the plasma membrane, whereas in cells in which filamin A expression was reconstituted (A7 cells and M2 cells transfected with filamin A-RFP), caveolin-1-GFP was concentrated in intracellular vesicles. Filamin A association with caveolin-1 in endothelial cells was confirmed by cofractionation of these proteins in density gradients, as well as by coimmunoprecipitation. Moreover, this interaction was enhanced by Src activation, associated with increased caveolin-1 phosphorylation, and blocked by Src inhibition. Taken together, these data suggest that filamin A association with caveolin-1 promotes caveolae-mediated transport by regulating vesicle internalization, clustering, and trafficking.

Differential regulation of caveolin‐2 phosphorylation at serine 23 and 36 by caveolin‐1 and cell cycle in human endothelial cells.

In the present study, we are focusing on regulation of serine phosphorylation of caveolin‐2 in endothelial cells. We show that phosphorylation of caveolin‐2 at serines 23 and 36 can be differentially regulated by caveolin‐1 and in endothelial cells synchronized in mitosis. To directly address the role of caveolin‐1 in regulating phosphorylation of endogenous caveolin‐2, we have used siRNA approach. The specific knockdown of caveolin‐1 in endothelial cells decreases caveolin‐2 phosphorylation at serine 23 but not serine 36. Furthermore, immunofluorescence microscopy studies determined that serine 23‐phosphorylated caveolin‐2 mostly localizes to plasma membrane caveolae. Thus, upregulation of serine 23 phosphorylation of caveolin‐2 depends on caveolin‐1‐driven targeting to plasma membrane caveolae. Interestingly, although, serine 36 phosphorylation does not seem to be regulated by caveolin‐1, it can be selectively upregulated in endothelial cells synchronized in mitosis. The latter data suggests a possible involvement of serine 36‐phosphorylated caveolin‐2 in modulating endothelial cell cycle, in particular mitosis.

Serine 23 and 36 Phosphorylation of Caveolin-2 Is Differentially Regulated by Targeting to Lipid Raft/Caveolae and in Mitotic Endothelial Cells

In the present study, using a combination of reconstituted systems and endothelial cells endogenously expressing caveolins, we show that phosphorylation of caveolin-2 at serines 23 and 36 can be differentially regulated by caveolin-1 mediated subcellular targeting to lipid raft/caveolae and in endothelial cells synchronized in mitosis. Detergent insolubility and sucrose flotation gradient experiments revealed that serine 23 phosphorylation of caveolin-2 preferably occurs in detergent-resistant membranes (DRMs), while serine 36 phosphorylation takes place in non-DRMs. Furthermore, immunofluorescence microscopy studies determined that in the presence of caveolin-1, serine 23-phosphorylated caveolin-2 mostly localizes to plasma membrane, while serine 36-phosphorylated caveolin-2 primarily resides in intracellular compartments. To directly address the role of caveolin-1 in regulating phosphorylation of endogenous caveolin-2, we have used the siRNA approach. The specific knockdown of caveolin-1 in endothelial cells decreases caveolin-2 phosphorylation at serine 23 but not at serine 36. Thus, upregulation of serine 23 phosphorylation of caveolin-2 depends on caveolin-1-driven targeting to plasma membrane lipid rafts and caveolae. Interestingly, although serine 36 phosphorylation does not seem to be regulated in endothelial cells by caveolin-1, it can be selectively upregulated in endothelial cells synchronized in mitosis. The latter data suggests a possible involvement of serine 36-phosphorylated caveolin-2 in modulating mitosis.

Expression of caveolin‐1 in mucoepidermoid carcinoma of the salivary glands: Correlation with vascular endothelial growth factor, microvessel density, and clinical outcome

Caveolin-1, which has been proposed as a candidate tumor suppressor, plays a regulatory role in several signaling pathways. The importance of caveolin-1 in endothelial cells in angiogenesis has been confirmed. The clinicopathologic significance of caveolin-1 expression and its correlation with angiogenesis remains unknown in mucoepidermoid carcinoma (MEC) of the salivary glands. Based on an immunohistochemical study, the expression levels of caveolin-1 and vascular endothelial growth factor (VEGF) and the intratumoral microvessel density (MVD) (labeled by CD34) in 75 patients with MEC were investigated, and correlations with clinicopathologic variables were evaluated statistically. The expression rates of both caveolin-1 and VEGF were 54.7% (41 of 75 tumors). MVD varied from 9 to 56 (24.45 +/- 10.72)/x 200. Caveolin-1 expression was correlated inversely with duration of tumor, clinical stage, histologic grade, and MVD (P = .027, P = .011, P = .04, and P = .025; respectively). VEGF expression was associated positively with MVD (P = .000). Advanced clinical stage, higher grade, and tumors that originated from minor salivary glands exhibited higher MVD (P = .029, P = .002, and P = .008, respectively). The presence of clinical symptoms, male gender, advanced clinical stage, higher grade, increased MVD, and down-regulated caveolin-1 were correlated significantly with the development of recurrent disease, as indicated by a shorter disease-free interval (P < .05). Both univariate and multivariate analyses indicated that clinical stage, histologic grade, and MVD were independent prognostic factors (P < .05). The presence of clinical symptoms and the down-regulation of caveolin-1 were identified as negative prognostic predictors in the univariate analysis (P < .05) but did not achieve significance in the multivariate analysis (P > .05). The current results suggest that caveolin-1 may function as a tumor suppressor in MEC of the salivary glands. Reduced expression of caveolin-1 and increased MVD may indicate a poor prognosis for certain patients.

Modulation of p‐glycoprotein function by caveolin‐1 phosphorylation

p-glycoprotein (p-gp) is an ATP-binding cassette transporter and its overexpression is responsible for the acquisition of the multidrug resistance phenotype in human tumors. p-gp is localized at the blood-brain barrier and is involved in brain cytoprotection. Our previous work used immunoprecipitation to show that caveolin-1 can interact with p-gp. In this study, we provide evidence that caveolin-1 regulates p-gp transport activity in a rat brain endothelial cell line (RBE4). Down-regulation of caveolin-1 by siRNA reduced the interaction between p-gp and caveolin-1, followed by a decrease in [3H]-Taxol and [3H]-Vinblastine accumulation in RBE4 cells. The latter result showed that down-regulation of caveolin-1 enhanced p-gp transport activity. RBE4 cells were also transfected with Sarcoma in order to modulate caveolin-1 phosphorylation. Overexpression of Sarcoma, a protein tyrosine kinase, stimulated caveolin-1 phosphorylation and increased both [3H]-Taxol and [3H]-Vinblastine accumulation as well as Hoechst 33342 accumulation. Transfection of caveolin-1 inhibits p-gp transport activity. Conversely, transfection of the mutant cavY14F decreased the p-gp/caveolin-1 interaction and reduced accumulation of the two p-gp substrates. Thus, our data show that caveolin-1 regulates p-gp function through the phosphorylation state of caveolin-1 in endothelial cells from the blood-brain barrier.

Altered Endothelial Nitric Oxide Synthase Targeting and Conformation and Caveolin-1 Expression in the Diabetic Kidney

Experimental diabetes is associated with complex changes in renal nitric oxide (NO) bioavailability. We explored the effect of diabetes on renal cortical protein expression of endothelial NO synthase (eNOS) with respect to several determinants of its enzymatic function, such as eNOS expression, membrane localization, phosphorylation, and dimerization, in moderately hyperglycemic streptozotocin-induced diabetic rats compared with nondiabetic control rats and diabetic rats with intensive insulin treatment to achieve near-normal metabolic control. We studied renal cortical expression and localization of caveolin-1 (CAV-1), an endogenous modulator of eNOS function. Despite similar whole-cell eNOS expression in all groups, eNOS monomer and dimer in membrane fractions were reduced in moderately hyperglycemic diabetic rats compared with control rats; the opposite trend was apparent in the cytosol. Stimulatory phosphorylation of eNOS (Ser1177) was also reduced in moderately hyperglycemic diabetic rats. eNOS colocalized and interacted with CAV-1 in endothelial cells throughout the renal vascular tree both in control and moderately hyperglycemic diabetic rats. However, the abundance of membrane-localized CAV-1 was decreased in diabetic kidneys. Intensive insulin treatment reversed the effects of diabetes on each of these parameters. In summary, we observed diabetes-mediated alterations in eNOS and CAV-1 expression that are consistent with the view of decreased bioavailability of renal eNOS-derived NO.

Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells.

During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the ‘on-rate’ of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

Palmitoylation of Caveolin-1 in Endothelial Cells Is Post-translational but Irreversible

Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents subsequent repalmitoylation.