Plasma Membrane Cholesterol Content Research Articles

SR-BI regulates the synergistic mast cell response by modulating the plasma membrane-associated cholesterol pool.

The high-affinity IgE receptor FcεRI is the mast cell (MC) receptor responsible for the involvement of MCs in IgE-associated allergic disorders. Activation of the FcεRI is achieved via crosslinking by multivalent antigen (Ag) recognized by IgE resulting in degranulation and proinflammatory cytokine production. In comparison to the T- and B-cell receptor complexes, for which several co-receptors orchestrating the initial signaling events have been described, information is scarce about FcεRI-associated proteins. Additionally, it is unclear how FcεRI signaling synergizes with input from other receptors and how regulators affect this synergistic response. We found that the HDL receptor SR-BI (gene name: Scarb1/SCARB1) is expressed in MCs, functionally associates with FcεRI, and regulates the plasma membrane cholesterol content in cholesterol-rich plasma membrane nanodomains. This impacted the activation of MCs upon co-stimulation of the FcεRI with receptors known to synergize with FcεRI signaling. Amongst them, we investigated the co-activation of the FcεRI with the receptor tyrosine kinase KIT, the IL-33 receptor, and GPCRs activated by adenosine or PGE2. Scarb1-deficient bone marrow-derived MCs showed reduced cytokine secretion upon co-stimulation conditions suggesting a role for plasma membrane-associated cholesterol regulating respective MC activation. Mimicking Scarb1 deficiency by cholesterol depletion employing MβCD, we identified PKB and PLCγ1 as cholesterol-sensitive proteins downstream of FcεRI activation in bone marrow-derived MCs. When MCs were co-stimulated with stem cell factor (SCF) and Ag, PLCγ1 activation was boosted, which could be mitigated by cholesterol depletion and SR-BI inhibition. Similarly, SR-BI inhibition attenuated the synergistic response to PGE2 and anti-IgE in the human ROSAKIT WT MC line, suggesting that SR-BI is a crucial regulator of synergistic MC activation.

Inhibition Mechanism of SARS-CoV-2 Infection by a Cholesterol Derivative, Nat-20(S)-yne.

The coronavirus disease 2019 (COVID-19) is caused by the etiological agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19, with the recurrent epidemics of new variants of SARS-CoV-2, remains a global public health problem, and new antivirals are still required. Some cholesterol derivatives, such as 25-hydroxycholesterol, are known to have antiviral activity against a wide range of enveloped and non-enveloped viruses, including SARS-CoV-2. At the entry step of SARS-CoV-2 infection, the viral envelope fuses with the host membrane dependent of viral spike (S) glycoproteins. From the screening of cholesterol derivatives, we found a new compound 26,27-dinorcholest-5-en-24-yne-3β,20-diol (Nat-20(S)-yne) that inhibited the SARS-CoV-2 S protein-dependent membrane fusion in a syncytium formation assay. Nat-20(S)-yne exhibited the inhibitory activities of SARS-CoV-2 pseudovirus entry and intact SARS-CoV-2 infection in a dose-dependent manner. Among the variants of SARS-CoV-2, inhibition of infection by Nat-20(S)-yne was stronger in delta and Wuhan strains, which predominantly invade into cells via fusion at the plasma membrane, than in omicron strains. The interaction between receptor-binding domain of S proteins and host receptor ACE2 was not affected by Nat-20(S)-yne. Unlike 25-hydroxycholesterol, which regulates various steps of cholesterol metabolism, Nat-20(S)-yne inhibited only de novo cholesterol biosynthesis. As a result, plasma membrane cholesterol content was substantially decreased in Nat-20(S)-yne-treated cells, leading to inhibition of SARS-CoV-2 infection. Nat-20(S)-yne having a new mechanism of action may be a potential therapeutic candidate for COVID-19.

Cholesterol-dependent homeostatic regulation of very long chain sphingolipid synthesis.

Sphingomyelin plays a key role in cellular cholesterol homeostasis by binding to and sequestering cholesterol in the plasma membrane. We discovered that synthesis of very long chain (VLC) sphingomyelins is inversely regulated by cellular cholesterol levels; acute cholesterol depletion elicited a rapid induction of VLC-sphingolipid synthesis, increased trafficking to the Golgi apparatus and plasma membrane, while cholesterol loading reduced VLC-sphingolipid synthesis. This sphingolipid-cholesterol metabolic axis is distinct from the sterol responsive element binding protein pathway as it requires ceramide synthase 2 (CerS2) activity, epidermal growth factor receptor signaling, and was unaffected by inhibition of protein translation. Depletion of VLC-ceramides reduced plasma membrane cholesterol content, reduced plasma membrane lipid packing, and unexpectedly resulted in the accumulation of cholesterol in the cytoplasmic leaflet of the lysosome membrane. This study establishes the existence of a cholesterol-sphingolipid regulatory axis that maintains plasma membrane lipid homeostasis via regulation of sphingomyelin synthesis and trafficking.

ABCA1 transporter promotes the motility of human melanoma cells by modulating their plasma membrane organization

BackgroundMelanoma is one of the most aggressive and deadliest skin tumor. Cholesterol content in melanoma cells is elevated, and a portion of it accumulates into lipid rafts. Therefore, the plasma membrane cholesterol and its lateral organization might be directly linked with tumor development. ATP Binding Cassette A1 (ABCA1) transporter modulates physico-chemical properties of the plasma membrane by modifying cholesterol distribution. Several studies linked the activity of the transporter with a different outcome of tumor progression depending on which type. However, no direct link between human melanoma progression and ABCA1 activity has been reported yet.MethodsAn immunohistochemical study on the ABCA1 level in 110 patients-derived melanoma tumors was performed to investigate the potential association of the transporter with melanoma stage of progression and prognosis. Furthermore, proliferation, migration and invasion assays, extracellular-matrix degradation assay, immunochemistry on proteins involved in migration processes and a combination of biophysical microscopy analysis of the plasma membrane organization of Hs294T human melanoma wild type, control (scrambled), ABCA1 Knockout (ABCA1 KO) and ABCA1 chemically inactivated cells were used to study the impact of ABCA1 activity on human melanoma metastasis processes.ResultsThe immunohistochemical analysis of clinical samples showed that high level of ABCA1 transporter in human melanoma is associated with a poor prognosis. Depletion or inhibition of ABCA1 impacts invasion capacities of aggressive melanoma cells. Loss of ABCA1 activity partially prevented cellular motility by affecting active focal adhesions formation via blocking clustering of phosphorylated focal adhesion kinases and active integrin β3. Moreover, ABCA1 activity regulated the lateral organization of the plasma membrane in melanoma cells. Disrupting this organization, by increasing the content of cholesterol, also blocked active focal adhesion formation.ConclusionHuman melanoma cells reorganize their plasma membrane cholesterol content and organization via ABCA1 activity to promote motility processes and aggressiveness potential. Therefore, ABCA1 may contribute to tumor progression and poor prognosis, suggesting ABCA1 to be a potential metastatic marker in melanoma.

An early, reversible cholesterolgenic etiology of diet-induced insulin resistance

ObjectiveA buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1. In this study we aimed to determine whether increased HBP/Sp1 activity represented a preventable cause of insulin resistance. MethodsC57BL/6NJ mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet for 1 week. During this 1-week diet the mice were treated daily with either saline or mithramycin-A (MTM), a specific Sp1/DNA-binding inhibitor. A series of metabolic and tissue analyses were then performed on these mice, as well as on mice with targeted skeletal muscle overexpression of the rate-limiting HBP enzyme glutamine-fructose-6-phosphate-amidotransferase (GFAT) that were maintained on a regular chow diet. ResultsSaline-treated mice fed this HF diet for 1 week did not have an increase in adiposity, lean mass, or body mass while displaying early insulin resistance. Consistent with an HBP/Sp1 cholesterolgenic response, Sp1 displayed increased O-GlcNAcylation and binding to the HMGCR promoter that increased HMGCR expression in skeletal muscle from saline-treated HF-fed mice. Skeletal muscle from these saline-treated HF-fed mice also showed a resultant elevation of PM cholesterol with an accompanying loss of cortical filamentous actin (F-actin) that is essential for insulin-stimulated glucose transport. Treating these mice daily with MTM during the 1-week HF diet fully prevented the diet-induced Sp1 cholesterolgenic response, loss of cortical F-actin, and development of insulin resistance. Similarly, increases in HMGCR expression and cholesterol were measured in muscle from GFAT transgenic mice compared to age- and weight-match wildtype littermate control mice. In the GFAT Tg mice we found that these increases were alleviated by MTM. ConclusionsThese data identify increased HBP/Sp1 activity as an early mechanism of diet-induced insulin resistance. Therapies targeting this mechanism may decelerate T2D development.

Mechanism-Driven Technology Development for Solving the Intracellular Delivery Problem of Hard-To-Transfect Cells.

The so-called "hard-to-transfect cells" are well-known to present great challenges to intracellular delivery, but detailed understandings of the delivery behaviors are lacking. Recently, we discovered that vesicle trapping is a likely bottleneck of delivery into a type of hard-to-transfect cells, namely, bone-marrow-derived mesenchymal stem cells (BMSCs). Driven by this insight, herein, we screened various vesicle trapping-reducing methods on BMSCs. Most of these methods failed in BMSCs, although they worked well in HeLa cells. In stark contrast, coating nanoparticles with a specific form of poly(disulfide) (called PDS1) nearly completely circumvented vesicle trapping in BMSCs, by direct cell membrane penetration mediated by thiol-disulfide exchange. Further, in BMSCs, PDS1-coated nanoparticles dramatically enhanced the transfection efficiency of plasmids of fluorescent proteins and substantially improved osteoblastic differentiation. In addition, mechanistic studies suggested that higher cholesterol content in plasma membranes of BMSCs might be a molecular-level reason for the greater difficulty of vesicle escape in BMSCs.

Role of Cholesterol in the Regulation of Hydrogen Sulfide Signaling within the Vascular Endothelium.

H2S is a gaseous signaling molecule enzymatically produced in mammals and H2S-producing enzymes are expressed throughout the vascular wall. We previously reported that H2S-induced vasodilation is mediated through transient receptor potential cation channel subfamily V member 4 (TRPV4) and large conductance (BKCa) potassium channels; however, regulators of this pathway have not been defined. Previous reports have shown that membrane cholesterol limits activity of TRPV4 and BKCa potassium channels. The current study examined the ability of endothelial cell (EC) plasma membrane (PM) cholesterol to regulate H2S-induced vasodilation. We hypothesized that EC PM cholesterol hinders H2S-mediated vasodilation in large mesenteric arteries. In pressurized, U46619 pre-constricted mesenteric arteries, decreasing EC PM cholesterol in large arteries using methyl-β-cyclodextrin (MBCD, 100 µM) increased H2S-induced dilation (NaHS 10, 100 µM) but MBCD treatment had no effect in small arteries. Enface fluorescence showed EC PM cholesterol content is higher in large mesenteric arteries than in smaller arteries. The NaHS-induced vasodilation following MBCD treatment in large arteries was blocked by TRPV4 and BKCa channel inhibitors (GSK219384A, 300 nM and iberiotoxin, 100 nM, respectively). Immunohistochemistry of mesenteric artery cross-sections show that TRPV4 and BKCa are both present in EC of large and small arteries. Cholesterol supplementation into EC PM of small arteries abolished NaHS-induced vasodilation but the cholesterol enantiomer, epicholesterol, had no effect. Proximity ligation assay studies did not show a correlation between EC PM cholesterol content and the association of TRPV4 and BK. Collectively, these results demonstrate that EC PM cholesterol limits H2S-induced vasodilation through effects on EC TRPV4 and BKCa channels.

Endothelial cell membrane cholesterol content regulates the contribution of TRPV4 channels in ACh-induced vasodilation in rat gracilis arteries.

Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48 h of chronic hypoxia (CH). CH couples endothelial transient receptor potential subfamily V member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium-dependent hyperpolarization (EDH) pathway does not present in control animals. TRVPV4 channel activity has been shown to be regulated by membrane cholesterol. Hence, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelium-dependent vasodilation. Male Sprague-Dawley rats were exposed to ambient atmospheric (atm.) pressure or 48-h of hypoxia (0.5atm). Vasodilation to acetylcholine (ACh) was determined using pressure myography in gracilis arteries. EC membrane cholesterol was depleted using methyl-β-cyclodextrin (MβCD) and supplemented with MβCD-cholesterol. Inhibiting TRPV4 did not affect ACh-induced vasodilation in normoxic controls. However, TRPV4 inhibition reduced resting diameter in control arteries suggesting basal activity. TRPV4 contributes to ACh-induced vasodilation in these arteries when EC membrane cholesterol is depleted. Inhibiting TRPV4 attenuated ACh-induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion in arteries from CH animals abolished the contribution of TRPV4 to ACh-induced vasodilation. Endothelial cell membrane cholesterol impedes the contribution of TRPV4 channels in EDH-mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.

Hypercholesterolemia Negatively Regulates P2X7-Induced Cellular Function in CD4+ and CD8+ T-Cell Subsets from B6 Mice Fed a High-Fat Diet.

We have previously showed that plasma membrane cholesterol and GM1 ganglioside content are responsible for the opposite sensitivity of mouse leukemic T cells to ATP. We also reported that the sensitivity of CD4+ and CD8+ T cells to ATP depends on their stage of differentiation. Here, we show that CD4+ and CD8+ T cells from B6 mice express different levels of membrane GM1 and P2X7 but similar levels of cholesterol. Thus, in CD4+ T cells, membrane cholesterol content negatively correlated with ATP/P2X7-induced CD62L shedding but positively correlated with pore formation, phosphatidylserine externalization, and cell death. By contrast, in CD8+ T cells, cholesterol, GM1, and P2X7 levels negatively correlated with all these ATP/P2X7-induced cellular responses. The relationship between cholesterol and P2X7-induced cellular responses was confirmed by modulating cholesterol levels either ex vivo or through a high-fat diet. Membrane cholesterol enrichment ex vivo led to a significant reduction in all P2X7-induced cellular responses in T cells. Importantly, diet-induced hypercholesterolemia in B6 mice was also associated with decreased sensitivity to ATP in CD4+ and CD8+ T cells, highlighting the relationship between cholesterol intake and the amplitudes of P2X7-induced cellular responses in T cells.

Alpha-1 Antitrypsin Therapy Modifies Neutrophil Adhesion in Patients with Obstructive Lung Disease.

Alpha-1 antitrypsin deficiency (AATD) is characterized by neutrophil-dominated inflammation resulting in emphysema. The cholesterol-rich neutrophil outer plasma membrane plays a central role in adhesion and subsequent transmigration to underlying tissues. This study aimed to investigate mechanisms of increased neutrophil adhesion in AATD and whether alpha-1 antitrypsin (AAT) augmentation therapy abrogates this effect. Plasma and blood neutrophils were donated by healthy controls (n = 20), AATD (n = 30), and AATD patients after AAT augmentation therapy (n = 6). Neutrophil membrane protein expression was investigated using liquid chromatography-tandem mass spectrometry. The effect of once-weekly intravenous AAT augmentation therapy was assessed by calcium fluorometric, μ-calpain, and cell adhesion assays. Decreased neutrophil plasma membrane cholesterol content (P = 0.03), yet increased abundance of integrin α-M (fold change 1.91), integrin α-L (fold change 3.76), and cytoskeletal adaptor proteins including talin-1 (fold change 4.04) were detected on AATD neutrophil plasma membrane fractions. The described inflammatory induced structural changes were a result of a more than twofold increased cytosolic calcium concentration (P = 0.02), leading to significant calcium-dependent μ-calpain activity (3.5-fold change; P = 0.005), resulting in proteolysis of the membrane cholesterol trafficking protein caveolin-1. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased caveolin-1 and membrane cholesterol content (111.8 ± 15.5 vs. 64.18 ± 7.8 μg/2 × 107 cells before and after treatment, respectively; P = 0.02), with concurrent decreased neutrophil integrin expression and adhesion. Results demonstrate an auxiliary benefit of AAT augmentation therapy, evident by a decrease in circulating inflammation and controlled neutrophil adhesion.

EC Membrane Cholesterol Content Regulates the Contribution of TRPV4 Channels in ACh‐induced vasodilation in Rat Gracilis Arteries

Numerous studies show that ion channel activity is sensitive to the level of membrane cholesterol, with cholesterol most often suppressing channel activity. Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48‐hr of exposure to chronic hypoxia (CH). Moreover, CH exposure also couples endothelial Transient Receptor Potential Subfamily V Member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium‐dependent hyperpolarization (EDH)‐pathway not present in control animals. TRPV4 appears to provide activator Ca2+ for endothelial large‐conductance Ca2+‐activation K+ channels in this setting. Taken together, this work suggests that changes in endothelial cell membrane cholesterol mediate this CH‐induced increase in ion channel functionality. Interestingly, both BK and TRPV4 channels contain Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motifs that bind cholesterol through protein‐sterol interactions. It is essential to separate the effects of CH from the influence of EC membrane cholesterol changes related to hypoxic exposure. Thus, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelial‐dependent vasodilation. To test this hypothesis, vasodilation responses to acetylcholine (ACh) were determined in gracilis arteries from male Sprague‐Dawley rats using standard pressure myography. Rats were exposed to either ambient atmospheric (atm.) pressure or 48‐hrs of hypoxia (0.5 atm). Arteries were pretreated with N‐nitro‐L‐arginine and indomethacin. EC membrane cholesterol was depleted using MβCD and supplemented with MβCD‐cholesterol. In arteries from normoxic animals (a replete cholesterol state), inhibition of TRPV4 did not affect ACh‐induced vasodilation. However, TRPV4 channels contribute to ACh‐induced vasodilation in these arteries when EC membrane cholesterol is depleted with MβCD. In contrast, inhibition of TRPV4 channels attenuated ACh‐induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion with MβCD‐cholesterol in arteries from CH animals abolished the contribution of TRPV4 channels to ACh‐induced vasodilation. In conclusion, our results demonstrate that EC membrane cholesterol impedes the contribution of TRPV4 channels in EDH‐mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.

Modulation of Cholesterol Pathways in Human Macrophages Infected by Clinical Isolates of Leishmania infantum.

To increase our understanding of factors contributing to therapeutic failure (TF) in leishmaniasis, we have studied some plasma membrane features of host THP-1 cells infected with clinical isolates of Leishmania infantum from patients with leishmaniasis and TF. The fluorescent probes DPH and TMA-DPH were used to measure changes in membrane fluidity at various depths of the plasma membranes. Steady-state fluorescence anisotropy of DPH embedded in the infected THP-1 membranes showed a significant increase, thereby suggesting a substantial decrease in plasma membrane fluidity relative to controls. Considering that cholesterol affects membrane fluidity and permeability, we determined the cholesterol content in plasma membrane fractions of human macrophages infected with these L. infantum lines and observed a significant increase in cholesterol content that correlates with the measured decrease in plasma membrane fluidity. In order to define the pathways that could explain the increase in cholesterol content, we studied the transcriptomics of the cholesterol-enriched pathways in host THP-1 cells infected with TF clinical isolates by RNA-seq. Specifically, we focused on four enriched Gene Ontology (GO) terms namely cholesterol efflux, cholesterol transport, cholesterol metabolic process and cholesterol storage. Additionally, we analyzed the genes involved in these pathways. Overall, this study shows that these clinical isolates are able to modulate the expression of specific genes in host cells, thereby modifying the cholesterol content in plasma membranes and inducing changes in plasma membrane fluidity that could be associated with the parasite’s ability to survive in the host macrophages, thereby possibly contributing to immune evasion and TF.

High-fat diet induced alterations in plasma membrane cholesterol content impairs insulin receptor binding and signalling in mouse liver but is ameliorated by atorvastatin

A high-fat diet (HFD) impairs insulin binding and signalling and may contribute to the development of insulin resistance. In addition, in vitro studies have shown that alterations in plasma membrane cholesterol influence ligand binding and downstream signalling for several receptor-tyrosine kinases (RTKs), including the insulin receptor. Using an ex vivo approach, we explored the effects of a HFD on insulin binding and signalling in mouse liver and relate these to observed changes in plasma membrane cholesterol. Mice fed a HFD demonstrated decreased insulin signalling compared to mice fed a normal chow diet (ND), indicated by a 3-fold decrease in insulin binding (P < 0.001) and a similar decrease in insulin receptor phosphorylation (~2.5-fold; P < 0.0001). Interestingly, we also observed a marked decrease in the cholesterol content of liver plasma membranes in the HFD fed mice (P < 0.0001). These effects of the HFD were found to be ameliorated by atorvastatin treatment (P < 0.0001). However, in ND mice, atorvastatin had no influence on membrane cholesterol content or insulin binding and signalling. The influence of membrane cholesterol on insulin binding and signalling was also corroborated in HepG2 cells. To the best of our knowledge, this is the first demonstration of the effects of a HFD and atorvastatin treatment on changes in plasma membrane cholesterol content and the consequent effects on insulin binding and signalling. Collectively, these findings suggest that changes in membrane cholesterol content could be an important underlying reason for the long-known effects of a HFD on the development of insulin resistance.

Hemodynamic Forces, Endothelial Mechanotransduction, and Vascular Diseases.

Cells in the tissues and organs of a living body are subjected to mechanical forces, such as pressure, friction, and tension from their surrounding environment. Cells are equipped with a mechanotransduction mechanism by which they perceive mechanical forces and transmit information into the cell interior, thereby causing physiological or pathogenetic mechano-responses. Endothelial cells (ECs) lining the inner surface of blood vessels are constantly exposed to shear stress caused by blood flow and a cyclic strain caused by intravascular pressure. A number of studies have shown that ECs are sensitive to changes in these hemodynamic forces and alter their morphology and function, sometimes by modifying gene expression. The mechanism of endothelial mechanotransduction has been elucidated, and the plasma membrane has recently been shown to act as a mechanosensor. The lipid order and cholesterol content of plasma membranes change immediately upon the exposure of ECs to hemodynamic forces, resulting in a change in membrane fluidity. These changes in a plasma membrane's physical properties affect the conformation and function of various ion channels, receptors, and microdomains (such as caveolae and primary cilia), thereby activating a wide variety of downstream signaling pathways. Such endothelial mechanotransduction works to maintain circulatory homeostasis; however, errors in endothelial mechanotransduction can cause abnormalities in vascular physiological function, leading to the initiation and progression of various vascular diseases, such as hypertension, thrombosis, aneurysms, and atherosclerosis. Recent advances in detailed imaging technology and computational fluid dynamics analysis have enabled us to evaluate the hemodynamic forces acting on vascular tissue accurately, contributing greatly to our understanding of vascular mechanotransduction and the pathogenesis of vascular diseases, as well as the development of new therapies for vascular diseases.

High-Fat and Resveratrol Supplemented Diets Modulate Adenosine Receptors in the Cerebral Cortex of C57BL/6J and SAMP8 Mice.

Neurodegenerative disorders are devastating diseases in which aging is a major risk factor. High-fat diet (HFD) seems to contribute to cognition deterioration, but the underlying mechanisms are poorly understood. Moreover, resveratrol (RSV) has been reported to counteract the loss of cognition associated with age. Our study aimed to investigate whether the adenosinergic system and plasma membrane cholesterol are modulated by HFD and RSV in the cerebral cortex of C57BL/6J and SAMP8 mice. Results show that HFD induced increased A1R and A2AR densities in C57BL/6J, whereas this remained unchanged in SAMP8. Higher activity of 5′-Nucleotidase was found as a common effect induced by HFD in both mice strains. Furthermore, the effect of HFD and RSV on A2BR density was different depending on the mouse strain. RSV did not clearly counteract the HFD-induced effects on the adenosinergic system. Besides, no changes in free-cholesterol levels were detected in the plasma membrane of cerebral cortex in both strains. Taken together, our data suggest a different modulation of adenosine receptors depending on the mouse strain, not related to changes in plasma membrane cholesterol content.

Cholesterol Disrupts H 2 S‐Mediated Vasodilation in Large Arteries

Small resistance size arteries are key players in vascular resistance and overall blood pressure control. We previously demonstrated that within endothelial cells (EC), H2S-mediated vasodilation involves activation of transient receptor potential cation channel subfamily V member 4 (TRPV4)-dependent Ca2+ influx to activate nearby large-conductance Ca2+-activated potassium (BK) channel in resistance-size arteries. Comparison of vasodilatory responses in large and small (resistance-size) arteries show that small arteries dilate at lower concentrations of H2S donors vs large arteries. The importance of endogenous H2S in endothelium-dependent vasodilatory responses in small resistance size arteries highlights a role for H2S in regulating blood pressure and flow. However, little is known of the differences in H2S signaling in large and small arteries and how these differences modulate vasodilation. Based on previous reports that cell membrane cholesterol negatively regulates TRPV4 mobility and BK activity, we hypothesized that H2S-mediated vasodilation is disrupted by elevated EC membrane cholesterol in large arteries. We first verified EC plasma membrane cholesterol content was higher in large vs small arteries by immunofluorescence using the cholesterol stain, filipin III (20 µg/ml) in EC labeled with the glycocalyx marker tomato lectin (20 µg/ml) and the nuclear stain SYTOX green (large 145.20 ± 13.67, small: 59.85 ± 5.50, p = 0.0004, n = 5 animals/group). Next, we examined the effect of cholesterol depletion using methyl β cyclodextrin (MBCD, 100 μM), on dilatory responses to H2S in large (300-380 μm) and small (60-130 μm) mesenteric arteries using the H2S donor, NaHS (1, 10 or 100 μM). In large arteries, MBCD pretreatment significantly enhanced H2S-mediated vasodilation (10 μM +vehicle: 0.72% ± 2.17, +MBCD: 9.71% ± 2.710, p = 0.026), (100 μM +vehicle: 2.25% ± 5.74, +MBCD: 20.55% ± 7.687, p < 0.0001) n = 5 animals/group. Although NaHS dilated small arteries more than large arteries, there was no effect of cholesterol depletion in small arteries (1 μM +vehicle: 16.25% ± 2.36, +MBCD: 16.29% ± 4.44, NS), (10 μM +vehicle: 33.22% ± 7.10, +MBCD: 39.00% ± 5.85, NS), (100 μM +vehicle: 59.84% ±5.98, +MBCD: 63.18% ± 4.639, NS) n = 3 animals/group. Additionally, immunofluorescence studies of EC TRPV4 expression showed no difference between large and small arteries (large 39.6 ± 12.9, small 41.8 ± 14.1, NS) n =3 animals/group. These studies suggest that membrane cholesterol disrupts H2S-mediated vasodilation and contributes to the relative differences in sensitivity to H2S-mediated vasodilation in large and small arteries.

ORP2, a cholesterol transporter, regulates angiogenic signaling in endothelial cells.

Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P2 countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P2 content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P2 distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P2 coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knock-out mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, and a putative disturbance of Notch signaling. Our data identifies ORP2 as a novel regulator of EC cholesterol and PI(4,5)P2 homeostasis and cholesterol-dependent angiogenic signaling.

High-Fat Diet-Induced Obesity Modulates Colonic Lgr5 + Stem Cell Homeostasis by Dysregulating Plasma Membrane Organization

Abstract Objectives The emerging obesity epidemic and its co-morbid conditions, such as colorectal cancer, pose a great challenge to global health and healthcare cost. This reinforces the urgent need to elucidate the underlying mechanisms contributing to the promotion of colon cancer in individuals with obesity. Extensive epidemiological studies have demonstrated a consistent and compelling association between obesity and the risk of cancer development and progression. Furthermore, our previous studies demonstrate that obesity induced by high-fat diet results in the expansion of the colonic stem cell pool during cancer initiation. However, the mechanistic link between obesity and the initiation and development of colon cancer remains elusive. Since the biophysical properties of the plasma membrane contribute to cellular signaling, we hypothesized that obesity alters colonic stem cell homeostasis via modulation of the biophysical properties of the plasma membrane, subsequently altering cell signaling events. Methods Our studies utilized complementary in vivo and ex vivo mouse models including colonic organoids. Plasma membrane biophysical properties and cholesterol content were determined utilizing the physico-chemical sensitive dye, Di-4-ANEPPDHQ and Filipin III, respectively. Super-resolution stochastic optical reconstruction microscopy (STORM) was utilized to determine the nanoscale clustering of Wnt receptor, LRP6, and epidermal growth factor receptor (EGFR). Effects of nanoclustering on the activation status of downstream signaling targets was subsequently assessed. Results High-fat diet induced obesity increased plasma membrane free cholesterol and rigidity of colonic crypts and Lgr5+ cells. The increase in rigidity was associated with an increase in the nanoclustering of key stem cell regulating membrane-bound receptors, LRP6 and EGFR. These biophysical perturbations were associated with changes in stem cell signaling and organoid-forming efficiency, a measure of stemness. Conclusions Our results indicate that obesity alters the biophysical properties of the plasma membrane which influences the clustering and downstream signaling of membrane receptors. These findings are noteworthy because they may explain in part how obesity drives colon cancer progression. Funding Sources This work was supported by NIH grant R35CA197707.

Effect of Electrical Parameters and Cholesterol Concentration on Giant Unilamellar Vesicles Electroformation.

Giant unilamellar vesicles (GUVs) are used extensively as models that mimic cell membranes. The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. Thus, a methodological paper pertaining to preparations of model lipid bilayer membranes with high Chol content would significantly help the study of properties of these membranes. Lipid solutions containing 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and Chol were fluorescently labeled with phospholipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC18(3)) and spin-coated to produce thin lipid films. GUVs were formed from these films using the electroformation method and the results were obtained using fluorescent microscopy. Electroformation outcomes were examined for different electrical parameters and different Chol concentrations. A wide range of field frequency-field strength (ff-fs) combinations was explored: 10-10,000 Hz and 0.625-9.375 V/mm peak-to-peak. Optimal values for GUVs preparation were found to be 10-100 Hz and 1.25-6.25 V/mm, with largest vesicles occurring for 10 Hz and 3.75 V/mm. Chol:POPC mixing ratios (expressed as a molar ratio) ranged from 0 to 3.5. We show that increasing the Chol concentration decreases the GUVs size, but this effect can be reduced by choosing the appropriate ff-fs combination.

Confocal Microscopy Confirmed that in Phosphatidylcholine Giant Unilamellar Vesicles with very High Cholesterol Content Pure Cholesterol Bilayer Domains Form.

The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. This high Chol content forms pure Chol bilayer domains (CBDs) and Chol crystals in model membranes and membranes formed from the total lipid extracts from human lenses. CBDs have been detected using electron paramagnetic resonance (EPR) spin-labeling approaches. Here, we confirm the presence of CBDs in giant unilamellar vesicles prepared using the electroformation method from Chol/1-palmitoyl-2-oleoylphosphocholine and Chol/distearoylphosphatidylcholine mixtures. Confocal microscopy experiments using phospholipid (PL) analog (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid) and cholesterol analog fluorescent probes (23-(dipyrrometheneboron difluoride)-24-norcholesterol) were performed, allowing us to make three major conclusions: (1) In all membranes with a Chol/PL mixing ratio (expressed as a molar ratio) >2, pure CBDs were formed within the bulk PL bilayer saturated with Chol. (2) CBDs were present as the pure Chol bilayer and not as separate patches of Chol monolayers in each leaflet of the PL bilayer. (3) CBDs, presented as single large domains, were always located at the top of giant unilamellar vesicles, independent of the change in sample orientation (right-side-up/upside-down). Results obtained with confocal microscopy and fluorescent Chol and PL analogs, combined with those obtained using EPR and spin-labeled Chol and PL analogs, contribute to the understanding of the organization of lipids in the fiber cell plasma membranes of the human eye lens.