Inhibitory Effect Of Cholesterol Research Articles

Ca2+-activated Cl− channel TMEM16A inhibition by cholesterol promotes angiogenesis in endothelial cells

IntroductionCa2+-activated Cl− channel TMEM16A is expressed in endothelial cells, and contributes to many diseases such as hypertension, blood-brain barrier dysfunction, and pulmonary hypertension. It remains unclear whether TMEM16A regulates endothelial angiogenesis, which participates in many physiological and pathological processes. Cholesterol regulates many ion channels including TMEM16A, and high cholesterol levels contribute to endothelial dysfunction. It remains to be determined whether cholesterol regulates TMEM16A expression and function in endothelial cells. ObjectiveThis study aimed to investigate whether cholesterol regulated TMEM16A expression and function in endothelial angiogenesis. MethodsWhole-cell patch clamp techniques were used to record Ca2+-activated Cl− currents in human aortic endothelial cells (HAECs) and HEK293 cells transfected with TMEM16A-overexpressing plasmids. Western blot was used to examine the expression of TMEM16A and DNA methyltransferase 1 (DNMT1) in HAECs. CCK-8 assay, would healing assay, and tube formation assay were used to test endothelial cell proliferation, migration and angiogenesis, respectively. ResultsTMEM16A mediates the Ca2+-activated Cl− channel in HAECs. Cholesterol treatment inhibited TMEM16A expression via upregulation of DNMT1 in HAECs, and the inhibitory effect of cholesterol on TMEM16A expression was blocked by 5-aza, the DNMT1 inhibitor. In addition, direct application of cholesterol inhibited TMEM16A currents in heterologous HEK293 cells with an IC50 of 0.1209 μM. Similarly, cholesterol directly inhibited TMEM16A currents in HAECs. Furthermore, TMEM16A knockdown increased in vitro tube formation, cell migration and proliferation of HAECs, and TMEM16A overexpression produced the opposite effect. ConclusionThis study reveals a novel mechanism of cholesterol-mediated TMEM16A inhibition, by which cholesterol reduces TMEM16A expression via DNMT1-mediated methylation and directly inhibits channel activities. TMEM16A channel inhibition promotes endothelial cell angiogenesis.

LIPOGENESIS IN ADIPOSE TISSUE OF THE LABORATORY ANIMALS AFTER LOADING WITH CHOLESTEROL

<p><em>The intensity of synthesis of different lipids classes was studied in vitro (fatty acids, cholesterol, phospholipids and acylglycerols) in the homogenates of the adipose tissue after a single daily loading of rats and guinea pigs with the cholesterol (300 mg/kg body weight) during 30 days period. For this purpose, the radioactivity of lipid fractions in the homogenates of adipose tissue of the rats and guinea pigs was measured, while homogenates were incubated separately with [6-<sup>14</sup></em><em>C</em><em>] glucose, [2-<sup>14</sup>C] lysine or [1-<sup>14</sup></em><em>C</em><em>] palmitic acid. The inhibitory effect of cholesterol was established by increasing its level in the diet of rats and guinea pigs on all classes of lipid synthesis in adipose tissue in vitro when [6- <sup>14</sup>C] glucose and [2- <sup>14</sup>C] lysine were used as precursors, and on the synthesis of cholesterol when [1-<sup>14</sup>C] palmitic acid was used as precursor.</em></p>

Abstract 120: Inhibitory Effects of Cholesterol on Lysophosphatidylcholine Metabolism in Human Monocytes

Background and aims: In hypercholesterolemic conditions blood cells are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PtdCho). While the metabolism of lyso-PtdCho by monocytes has been studied, the effects of high cholesterol, which complexes with lyso-PtdCho, are not well understood. In this study we used a novel assay developed in our laboratory, based on the ability of lyso-PtdCho to solubilize and deliver cholesterol into the cells, to study whether cholesterol affects the metabolism of lyso-PtdCho. Methods: In THP-1 monocytes 14 C-lyso-PtdCho metabolism was analyzed by TLC and cytotoxicity by WST-1 assay. Results: Monocytes incubated with lyso-PtdCho alone metabolize the incorporated lyso-PtdCho by 4h mainly into phosphatidylcholine, with a small percentage into sphingomyelin and phosphocholine. The latter could represent PLase C reaction that would generate mono- and diacyl-glycerols, important signaling molecules. However in cells incubated with cholesterol or cholesteryl ester (CE)/lyso-PtdCho micelles, at 4h the radioactivity remained mainly in the lyso-PtdCho fraction (p<0.01 and p<0.001). Lyso-PtdCho decreased monocyte viability at concentrations over 30μM, but cholesterol and CE inhibited the lyso-PtdCho-induced cytotoxicity (p<0.001). Lyso-PtdCho and CE/lyso-PtdCho, but not cholesterol/lyso-PtdCho, increased the gene expression of the pro-inflammatory chemokine MCP-1 (p<0.05). Peripheral blood monocytes from LDL-R -/- mice on a high fat diet had higher levels of CE than monocytes from mice in a normal diet (n=10, p<0.05). Monocyte CE content in these mice positively correlated with plasma LDL/HDL ratio (p<0.05, R 2 =0.65). Conclusions: We show that cholesterol and CE inhibit the cytotoxic effects of lyso-PtdCho in monocytes, but only cholesterol counteracted the pro-inflammatory action of lyso-PtdCho. Since lyso-PtdCho concentrations in plasma are increased in hypercholesterolemic conditions, and its content is increased during the oxidation of LDL, the current studies might indicate that cholesterol and lyso-Ptdcho could influence each other’s metabolism. This is important as lyso-PtdCho has been suggested to influence plaque vulnerability.

Competitive Binding of Cholesterol and Ergosterol to the Polyene Antibiotic Nystatin. A Fluorescence Study

Competition studies between cholesterol and ergosterol were carried out to gain insight into the binding interactions between nystatin and these sterols. Lipid vesicles were prepared with mixtures of palmitoyloleoylphosphocholine/ergosterol/cholesterol, and both sterol molar ratio and total content were varied. The inhibitory effect of cholesterol toward the ergosterol ability to induce the formation of long-lived fluorescent antibiotic species was used to detect nystatin-cholesterol interactions. It was found that the key factor controlling nystatin photophysical properties in the ternary lipid mixtures was their ergosterol/cholesterol molar ratio and not their overall sterol content. Moreover, permeabilization studies showed that nystatin was able to form pores in all the mixed vesicles, but the initial rate of pore formation was also dependent on the ergosterol/cholesterol molar ratio. Our data show that ergosterol is displaced by competing cholesterol, indirectly confirming cholesterol’s ability to coassemble with nystatin. The distinct spectroscopic properties emphasize the different molecular architecture adopted by nystatin-cholesterol and -ergosterol complexes, and therefore are relevant to understanding the interaction of the antibiotic with membranes.

Characterization of Permeability and Morphological Perturbations Induced by Nisin on Phosphatidylcholine Membranes

Nisin is an antimicrobial peptide used as food preservative. To gain some insights into the hypothesis that its bactericidal activity is due to the perturbation of the lipid fraction of the bacterial plasmic membrane, we have investigated the effect of nisin on model phosphatidylcholine (PC) membranes. We show that nisin affects the PC membrane permeability, and this perturbation is modulated by the lipid composition. Nisin-induced leakage from PC vesicles is inhibited by the presence of cholesterol. This inhibition is associated with the formation of a liquid ordered phase in the presence of cholesterol, which most likely reduces nisin affinity for the membrane. Conversely, phosphatidylglycerol (PG), an anionic lipid, promotes nisin-induced leakage, and this promotion is associated with an increased affinity of the peptide for the bilayer because nisin is a cationic peptide. When the electrostatic interactions are encouraged by the presence of 70 mol% PG in PC, the inhibitory effect of cholesterol is not observed anymore. Nisin drastically modifies the morphology of the dipalmitoyl- sn-glycero-3-phosphatidylcholine (DPPC) multilamellar dispersion without causing a significant change in the gel-to-liquid crystalline phase transition of the lipid. The morphological changes are observed from 31P and 2H NMR and cryo-electron microscopy. From the NMR point of view, the interactions giving rise to a broad signal (quadrupolar interactions and chemical shift anisotropy for 2H NMR and 31P NMR, respectively) are partly averaged out in the presence of nisin. This phenomenon is interpreted by the formation of curved lipid planes that lead to the lipid lateral diffusion occurring in the intermediate motional regime. By cryo-electron microscopy, large amorphous aggregates containing small dense globular particles are observed for samples quenched from 25 and 50°C. Long thread-like structures are also observed in the fluid phase. A structural description of DPPC/nisin complex, consistent with the experimental observation, is proposed. The presence of 30 mol% cholesterol in DPPC completely inhibits the morphological changes induced by nisin. Therefore, it is concluded that nisin can significantly perturb PC bilayers from both the permeability and the structural points of view, and these perturbations are modulated by the lipidic species in the bilayer.

Cholesterol-mediated changes of neutral cholesterol esterase activity in macrophages. Mechanism for mobilization of cholesteryl esters in lipid droplets by HDL.

Cholesteryl esters (CE) in lipid droplets undergo a continual cycle of hydrolysis and reesterification by neutral cholesterol esterase (N-CEase) and acyl CoA:cholesterol acyltransferase (ACAT), respectively. The mechanism by which HDL mobilizes CE from lipid droplets in J774 A.1 cells was investigated, focusing on N-CEase activity. We asked whether HDL enhances the activity and, if so, what signals induce the change of the activity. An incubation of cells with HDL enhanced the decline of cholesteryl-[l-14C]-oleate in foam cells and increased N-CEase activity in the supernatant of cell homogenate in a concentration-dependent manner, whereas incubation with LDL decreased the activity. In addition, N-CEase activity was fivefold higher when cells were cultured in 10% lipoprotein-deficient serum (LPDS) medium (2 micrograms cholesterol/mL) than when cultured in 10% fetal calf serum medium (31 micrograms cholesterol/mL), suggesting that changes in N-CEase activity are mediated by cholesterol. An addition of cholesterol (0 to 30 micrograms/mL) in LPDS medium markedly inhibited N-CEase activity with a concomitant increase in cellular cholesterol concentration. This inhibitory effect of cholesterol was also observed in mouse peritoneal macrophages. In vitro addition of cholesterol did not affect N-CEase activity. Treatment of cells with HMG-CoA reductase inhibitors enhanced N-CEase activity, whereas ACAT inhibitor decreased the activity. Northern blot analysis of N-CEase mRNA showed that the expression was not altered by the presence of cholesterol in LPDS medium. These results suggest that cholesterol downregulates N-CEase activity, probably through cholesterol-dependent appearance of some factors.

Effect of pulmonary surfactant protein A (SP-A) and calcium on the adsorption of cholesterol and film stability

The effect of exogenous cholesterol on the stability of surface films at 37°C from various surfactants was studied with the pulsating bubble surfactometer. Addition of cholesterol (5%, w/w) to bovine lipid extract surfactant (bLES) or mixtures of dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoyl-phosphatidylglycerol/SP-B (7:3:1%) dispersed in 1.5 mM CaCl 2/0.9% NaCl resulted in unstable surface films. Although 10% cholesterol only partially impaired the surface activity of bLES, it virtually abolished that of the reconstituted surfactant. The inhibitory effects of cholesterol were significantly repressed by SP-A (10%, w/w of lipid) and 3 mM CaCl 2 or 5 mM CaCl 2 without SP-A. Adsorption of cholesterol from various surfactants into the air/water interface was examined by measuring the surface radioactivity of [ 14C]cholesterol. Cholesterol alone dispersed in 1.5 mM CaCl 2/0.9% NaCl could not adsorb to the interface, but it adsorbed readily when mixed with bLES. Cholesterol adsorption was markedly suppressed by SP-A in 3 mM CaCl 2/0.9% NaCl or 5 mM CaCl 2/0.9% NaCl without SP-A. Electron microscopy revealed striking ultrastructural differences between bLES/5% cholesterol/10% SP-A in 3 mM CaCl 2/0.9% NaCl and bLES/5% cholesterol in 3 or 5 mM CaCl 2/0.9% NaCl. The former exhibited large multilayer and small unilamellar vesicles, while the latter displayed condensed patches of aggregates. Adsorption studies showed aggregated patches adsorbed more rapidly than vesicles but attained lower equilibrium surface pressures. These results indicate SP-A and calcium limit the adsorption of surfactant cholesterol to the air-water interface.

Inhibitory effect of cholesterol on interaction between cytoplasmic actin and liposomes, and restorative effect of high osmotic pressure.

Although cholesterol is one of the major components of plasma membranes in eukaryotic cells, very little is known about its role in biological membranes. We reported previously (Okimasu et al., Cell Struct. Funct. 11, 273-283, 1986) that introduction of cholesterol into the liposomal membrane caused a decrease in membrane permeability, especially by the binding of cytoplasmic proteins to the liposomal membrane. The present study was carried out to further clarify the biochemical function of cholesterol in the membrane-protein interactions, especially under high osmotic pressure. The association of membranes with cytoplasmic proteins and their permeability were decreased by the introduction of cholesterol, but its effects were diminished in a hypertonic medium. The protein species associated with cholesterol-containing liposomes vary depending on the sort of hypertonic condition. It was suggested that since the degree of lipid packing by the cholesterol was reduced by the locally increased curvature in the lipid bilayer under high osmotic pressure, some cytoplasmic proteins can penetrate into the liposomal membrane.

Inhibitory effect of cholesterol on the uptake of liposomes by liver and spleen

The effect of cholesterol content of small unilamellar (SUV) and reverse phase (REV) liposomes on blood clearance and tissue distribution has been studied. [ 14C]Inulin has been used as an aqueous marker of liposomes to represent the uptake of intact liposomes in tissues. The blood clearance of the intravenously-injected SUV and REV liposomes depends on the cholesterol content of liposomes. The cholesterol-free (0 mol%) liposomes are cleared more readily from the circulation than the cholesterol-poor liposomes (20 mol%) and the cholesterol-poor are cleared more rapidly than the cholesterol-rich (46.6 mol%) liposomes. This clearance pattern of liposomes from the circulation is not attributed to the change of size of liposomes due to the increase in cholesterol content of liposomes. However, poor stability of cholesterol-free or cholesterol-poor liposomes in the circulation is partly responsible, but the predominant factor responsible for the observed blood clearance pattern is the inhibitory effect of cholesterol on the uptake of liposomes by reticuloendothelial-rich tissues liver and spleen. Uptake of liposomes by these organs is decreased with increasing cholesterol content of vesicles. It is suggested that to produce liposome preparations with a long circulating half life in vivo it is necessary to inhibit their uptake by liver and spleen.

Effect of alterations in vitro and in vivo of the cholesterol content in rat liver microsomes on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase.

3-Hydroxy-3-methylglutaryl-CoA reductase, the regulatory enzyme of cholesterol synthesis in liver, is bound to the microsomal fraction. Lipoprotein-bound cholesterol (from human serum) in vitro inhibits the microsomal bound 3-hydroxy-3-methylglutaryl-CoA reductase. The solubilized enzyme, however, is not inhibited. Immunotitration of the microsomal enzyme with a monospecific antibody reveals that the loss in enzyme activity caused by cholesterol corresponds well with the lowering of the equivalence points. In contrast, the equivalence points of the solubilized enzyme remain unchanged. This indicates that the inhibitory effect of cholesterol is restricted to the microsomal bound enzyme. In vivo different physiological conditions lead to relatively small changes in the cholesterol content in the microsomes while drastic changes in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase are observed. When microsomes from these rats are incubated with exogenous cholesterol, the activity of the enzyme is always found to be decreased to the same extent independent of the physiological condition of the animals. The findings suggest that 3-hydroxy-3-methylglutaryl-CoA reductase may be "masked" by a cholesterol-mediated modification of the microsomal membrane.

239) - Inhibitory effects of cholesterol on changes in membrane permeability and potential induced with lysolecithin in red blood cells

239) - Inhibitory effects of cholesterol on changes in membrane permeability and potential induced with lysolecithin in red blood cells

Lysolecithnによる赤血球の膜電位変化と膜透過性充進に対するCholestero1の抑制作用

Membrane stabilizing effects of cholesterol on the permeability of K+ and change in membrane potential induced with lysolecithin were investigated. Cholesterol inhibited K+ release from rabbit red blood cells treated with lysolecithin. 3.3 X 10(-6)M of cholesterol was the optimum concentration required to inhibit K+ release from rabbit red blood cells treated with lysolecithin (1.25 microgram/ml) at the level of 100 percent. Change in membrane potential was evident with lysolecithin by the method of fluorescent dye and, cholesterol inhibited the change which was dose dependent. These inhibitory effects of cholesterol on the K+ release and changes in membrane potential served as the membrane stabilizing action on red blood cell membrane. It is assumed that cholesterol acts as an inhibitor to increase membrane fluidity and permeability of malignant transformed cells such as tumor and lymphoid cells.

Inhibitory effects of cholesterol on cell division in mouse skin: Physicochemical and biological aspects(The problem of male pattern baldness)

In vivo studies show that a mixture of cholesterol, the precursor of all natural steroids, and a detergent of the Tween 60 type, when applied to a mouse skin, causes a statistically highly significant (P < 0.001) decrease in the mitotic activity of the epidermal cells and consequently in the number of the daughter cells. Electron microscopy and in vitro experiments show that an excess quantity of cholesterol at the surface membrane increases surface viscosity and rigidity.

Role of cholesterol in membranes effects on phospholipid-protein interactions, membrane permeability and enzymatic activity

The effect of cholesterol on the interaction of proteins with phospholipid membranes was studied using three independent techniques: effects on vesicle permeability, monolayer expansion, and phospholipid-dependent (Na + + K +)-ATPase activity. The proteins studied were: cytochrome c, albumin, hemoglobin, lysozyme, myelin basic protein, myelin proteolipid apoprotein, and the polypeptide gramicidin A. The results were as follows: 1. 1. All the proteins in this study produced a large increase in the permeability of phospholipid vesicles to Na +. When cholesterol was mixed with phospholipid in equimolar proportions, most proteins produced only a comparatively small increase in permeability. This inhibitory effect of cholesterol on the permeability of phospholipid-protein membranes, was 38-fold for cytochrome c and 10-fold for hemoglobin. The only protein that was not affected by cholesterol was the myelin proteolipid apoprotein. Experiments with cytochrome c indicated that the above effects were unlikely to be due to inhibition of its binding to phosphatidylserine vesicles. 2. 2. The presence of cholesterol in phosphatidylserine monolayers inhibits the area expansion produced by the addition of cytochrome c to the bulk phase. The inhibition of monolayer expansion by cholesterol was shown to be considerably larger than that obtained by equivalent dilution of the surface charges of phosphatidylserine by phosphatidylcholine. 3. 3. The presence of cholesterol inhibits the ability of phospholipids to activate a delipidated preparation of (Na + + K +)-ATPase. The degree of inhibition produced by nearly equimolar mixtures of cholesterol with brain phosphatidylserine and dioleylphosphoatidylglycerol was approximately 60% while in mixtures with dipalmitoylphosphatidylglycerol, it was more than 90%. 4. 4. The biological significance of these data are discussed in relation to the possible effects of increased cholesterol levels in cell membranes during the process of aging and the development of atherosclerosis.

The role of lysolecitbin and cholesterol in icterus neonatorum

Two groups of newborn infants were studied, one with icterus neonatorum and one without. Cord blood serum and fourth-day blood serum were examined for free and esterified cholesterol, bilirubin (which was always indirect), and enzymatic formation of lysolecithin. There was a marked rise in free cholesterol and bilirubin in the fourth-day blood sera of the icteric group, compared with those of the infants in the nonicteric group. Formation of lysolecithin, a strong hemolytic substance, was demonstrated in 16 of 18 cases of the icteric group and in only 1 of 10 nonicteric infants. It is suggested that icterus neonatorum is caused by a combination of two factors: (1) hemolysis and (2) immaturity of the liver, each factor complementing the other. The enzymatic formation of lysolecithin is suggested as a causative factor of the hemolysis. An inhibitory effect of cholesterol on the hemolytic action of lysolecithin was demonstrated. The hemolytic process is probably self-controlled by the action of cholesterol, liberated from the destroyed erythrocytes. Two groups of newborn infants were studied, one with icterus neonatorum and one without. Cord blood serum and fourth-day blood serum were examined for free and esterified cholesterol, bilirubin (which was always indirect), and enzymatic formation of lysolecithin. There was a marked rise in free cholesterol and bilirubin in the fourth-day blood sera of the icteric group, compared with those of the infants in the nonicteric group. Formation of lysolecithin, a strong hemolytic substance, was demonstrated in 16 of 18 cases of the icteric group and in only 1 of 10 nonicteric infants. It is suggested that icterus neonatorum is caused by a combination of two factors: (1) hemolysis and (2) immaturity of the liver, each factor complementing the other. The enzymatic formation of lysolecithin is suggested as a causative factor of the hemolysis. An inhibitory effect of cholesterol on the hemolytic action of lysolecithin was demonstrated. The hemolytic process is probably self-controlled by the action of cholesterol, liberated from the destroyed erythrocytes.