Decreased Plasma Membrane Fluidity Research Articles

Erythrocytes properties in varicose veins patients

Varicose veins (VV) are enlarged veins of the subcutaneous tissue, usually caused by faulty or damaged venous valves leading to impaired blood flow. Blood stasis, excessive clotting disorder and alterations in the vein walls are symptoms of Virchow's triad which may affect the morphotic elements of blood, including erythrocytes.The aim of this study was to investigate alterations in the properties of the erythrocytes taken from varicose veins in comparison to those from antecubital vein of patients with chronic venous disease. The investigation was conducted on whole erythrocytes using spin labeling method in EPR spectroscopy and flow cytometry.The internal viscosity of cells was determined by Tempamine. The conformation state of internal proteins, mainly hemoglobin and membrane proteins was determined by maleimide spin label (MSL, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl). The plasma membrane fluidity was measured using two spin labeled fatty acids (5- and 16-doxylstearic acid), while conformational state of membrane protein was measured using two covalently bound spin labels MSL and ISL [4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl]. The osmotic fragility and the shape and size of the erythrocytes were also determined.A decrease in internal viscosity of the erythrocytes from varicose vein was observed. A significant decrease in lipid membrane fluidity indicated by 5-DS, which is located at the polar region of lipid layer was found in the erythrocytes from varicose vein in comparison to normal vein. A significant decrease in the motion of MSL and ISL attached to erythrocyte membrane proteins from varicose vein was found. Changes in the plasma membrane of the erythrocytes from varicose vein were also confirmed by measuring osmotic fragility. These cells were more sensitive to hemolysis than red blood cells from the peripheral blood vein. Meanwhile, no significant differences in size and shape were observed between the erythrocytes taken from varicose veins and those from peripheral veins.In conclusion, the erythrocytes from varicose veins exhibited decreased intracellular viscosity and decreased plasma membrane fluidity. At the same time, conformational changes of membrane proteins and higher osmotic fragility of these cells were found in comparison to the erythrocytes obtained from peripheral veins in the same patients with chronic venous disease. Our findings strongly suggest that presented abnormalities in the erythrocyte plasma membrane may have significant pathophysiological implications, including shortened cell survival and alterations in the hemorheology of the varicose vein blood.

CIP4-Dependent Defects in Platelet Biogenesis Involve Changes in Membrane Rigidity Independently of WASP

Abstract 705Using the Src kinase Lyn as bait in yeast two-hybrid screen, we isolated Cdc42-interacting protein 4 (CIP4). CIP4 is an F-BAR protein implicated in membrane curvature, membrane tubulation, and vesicle formation. CIP4 contains an SH3 domain that interacts with Wiskott-Aldrich Syndrome protein (WASP). We have reported thrombocytopenia in CIP4 knockout (KO) male mice, which was of similar severity as observed in WASP knockout male mice. Because a cardinal feature of Wiskott-Aldrich Syndrome is thrombocytopenia, we hypothesized that the CIP4-dependent thrombocytopenia occurs through the same mechanism as that in WASP-dependent thrombocytopenia. Interestingly, we have just generated a CIP4, WASP double knockout that displays a more severe thrombocytopenia. Neither CFU-MK progenitor studies nor ploidy studies showed a difference between CIP4 KO, WASP KO, double KO, and Wild Type (WT) mice. Thus, we reasoned that the defect lies in the structural production of platelets. We first studied where the proteins were localized and performed immunofluorescence staining and confocal microscopy analysis in cultured mouse megakaryocytes. We found a diffuse staining of CIP4 throughout the cytosol, the nucleus and proplatelets, with accumulation at the plasma membrane. In mouse megakaryocytes WASP staining was diffuse. In the human megakaryocytic CHRF-288-11 cell line, CIP4 staining was also diffuse. Because F-BAR proteins are also known to interact through their SH3 domain with members of the dynamin family, we investigated if CIP4 interacted with dynamin in the megakaryocytic lineage. While we saw diffuse staining for dynamin 3 in mouse megakaryocytes, we found co-localization of dynamin 3 with CIP4 at the cell periphery after exposure to known proplatelet-inducing agents such as phorbol ester (PMA) or fibronectin. We also performed functional assays in CIP4 KO mice. Since we did not find a difference in megakaryocytic progenitor numbers or in ploidy between the CIP4 knockout and the wild type mice, we studied later steps in platelet biogenesis. Preliminary studies were not indicative for decreased platelet lifespan in vivo in CIP4 KO mice. We counted the proplatelet formation in bone marrow-derived megakaryocytes in culture and found that CIP4 knockout megakaryocytes had decreased proplatelet formation compared with wild type (WT: 10.1 ± 1.6%, CIP4 KO: 4.5 ± 1%, p=0.04). We confirmed these findings in the CHRF-288-11 cell line with lentivirally mediated shRNA knockdown of CIP4 by counting the proplatelet-like protrusion formation in response to PMA (control: 15.4± 1.7%, CIP4 knockdown: 8.9 ± 1%, p=0.03). Because of the known direct interaction of CIP4 with the plasma membrane, we hypothesized that CIP4 deficit leads to changes in plasma membrane rigidity. By measuring fluorescence anisotropy, we confirmed decreased plasma membrane fluidity in CHRF-288-11 cells with CIP4 knockdown in response to agents that promote proplatelet-like protrusion formation, PMA and fibronectin. When using PMA, membrane fluidity increased by 4% or more in control samples (similar to the change obtained with known membrane perturbing agent ethanol 10 percent v/v), but did not increase in cells with CIP4 knockdown or N-WASP knockdown. Even more, plasma membrane rigidity increased by 3 ± 1.4% in CHRF-288-11 cells with CIP4 knockdown. Preliminary results showed the same loss of membrane fluidity in splenic B lymphocytes from CIP4 KO mice isolated (a 5% membrane fluidity increase in WT vs. none in CIP4 KO B lymphocytes in response to PMA). After exposure to fibronectin, membrane fluidity increased by 6.4 ± 1.3% in control CHRF-288-11 cells, whereas it did not increase in cells with CIP4 knockdown, and it increased in cells with N-WASP knockdown but only by 3 ± 0.9%. Interestingly, while CHRF-288-11 cells with knockdown of WASP still showed loss of plasma membrane fluidity in response to PMA (increased only by 1.8 ± 0.7%), this was not seen with fibronectin stimulation, where the membrane fluidity increase (5 ± 0.5%) did not appear statistically different from the response in control cells.Altogether our results point to decreased membrane fluidity in CIP4 deficient cells and they suggest that other pathways besides WASP pathways are involved in CIP4 dependent platelet biogenesis. Disclosures:No relevant conflicts of interest to declare.

Differential toxicity of Mn2+ and Mn3+ to rat liver tissues: Oxidative damage, membrane fluidity and histopathological changes

Toxicity due to overexposure to manganese (Mn) is becoming increasingly prevalent. Mn-induced neurodegenerative toxicity has been demonstrated, but little is known concerning the adverse effects of the element on the liver. Under physiological conditions, manganese primarily exists as divalent manganese (Mn(2+)) and trivalent manganese (Mn(3+)). The present study was designed to evaluate and compare the effects of Mn(2+) and Mn(3+) on oxidative hepatic damage, membrane fluidity and histopathological changes in rats. Rats exposed to Mn(2+) or Mn(3+) (2.0mg Mn/kg body weight) showed significant inhibition of superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, as well as decreased levels of glutathione (GSH) and increased levels of malondialdehyde (MDA) in liver tissues. We also showed a significant inhibition of SOD activity and increased MDA levels in hepatocyte nuclei. We also observed reduced Na(+),K(+)-ATPase activity, increased MDA levels and decreased plasma membrane fluidity, which was accompanied by an increase of fluorescence anisotropy (r) values, in hepatic plasma membranes. In addition, Mn(2+) and Mn(3+) both caused histopathological changes, such as mononuclear cell infiltration, congestion, enlargement of the veins and sinusoids, hepatocellular damage, necrotic changes, mitochondrial hyperplasia, swelling and vacuolization, as determined by light and electron microscopy. Taken together, these data suggest that both Mn(2+) and Mn(3+) inhibit the normal physiological functioning of the liver. Under the experimental conditions used, the adverse effects of Mn(2+) were more severe than those of Mn(3+).

Regulation of platelet-activating factor synthesis in human monocytes by dipalmitoyl phosphatidylcholine.

Platelet-activating factor (PAF) has a major role in inflammatory responses within the lung. This study investigates the effect of pulmonary surfactant on the synthesis of PAF in human monocytic cells. The pulmonary surfactant preparation Curosurf significantly inhibited lipopolysaccharide (LPS)-stimulated PAF biosynthesis (P<0.01) in a human monocytic cell line, Mono mac-6 (MM6), as determined by (3)H PAF scintillation-proximity assay. The inhibitory properties of surfactant were determined to be associated, at least in part, with the 1,2-dipalmitoyl phosphatidylcholine (DPPC) component of surfactant. DPPC alone also inhibited LPS-stimulated PAF biosynthesis in human peripheral blood monocytes. DPPC treatment did not affect LPS-stimulated phospholipase A(2) activity in MM6 cell lysates. However, DPPC significantly inhibited LPS-stimulated coenzyme A (CoA)-independent transacylase and acetyl CoA:lyso-PAF acetyltransferase activity. DPPC treatment of MM6 cells decreased plasma membrane fluidity as demonstrated by electron paramagnetic resonance spectroscopy coupled with spin labeling. Taken together, these findings indicate that pulmonary surfactant, particularly the DPPC component, can inhibit LPS-stimulated PAF production via perturbation of the cell membrane, which inhibits the activity of specific membrane-associated enzymes involved in PAF biosynthesis.

The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: studies on Langmuir-Blodgett monolayers, liposomes and J774 macrophages.

The macrolide antibiotic azithromycin was shown to markedly inhibit endocytosis. Here we investigate the interaction of azithromycin with biomembranes and its effects on membrane biophysics in relation to endocytosis. Equilibrium dialysis and 31P NMR revealed that azithromycin binds to lipidic model membranes and decreases the mobility of phospholipid phosphate heads. In contrast, azithromycin had no effect deeper in the bilayer, based on fluorescence polarization of TMA-DPH and DPH, compounds that, respectively, explore the interfacial and hydrophobic domains of bilayers, and it did not induce membrane fusion, a key event of vesicular trafficking. Atomic force microscopy showed that azithromycin perturbed lateral phase separation in Langmuir-Blodgett monolayers, indicating a perturbation of membrane organization in lateral domains. The consequence of azithromycin/ phospholipid interaction on membrane endocytosis was next evaluated in J774 macrophages by using three tracers with different insertion preferences inside the biological membranes and intracellular trafficking: C6-NBD-SM, TMA-DPH and N-Rh-PE. Azithromycin differentially altered their insertion into the plasma membrane, slowed down membrane trafficking towards lysosomes, as evaluated by the rate of N-Rh-PE self-quenching relief, but did not affect bulk membrane internalization of C6-NBD-SM and TMA-DPH. Azithromycin also decreased plasma membrane fluidity, as shown by TMA-DPH fluorescence polarization and confocal microscopy after labeling by fluorescent concanavalin A. We conclude that azithromycin directly interacts with phospholipids, modifies biophysical properties of membrane and affects membrane dynamics in living cells. This antibiotic may therefore help to elucidate the physico-chemical properties underlying endocytosis.

Abnormal plasma membrane properties and functions in prion-infected cell lines.

A long trail of evidence indicates that the formation of PrPSc or its accumulation causes the neuronal dysfunction and clinical features of prion diseases. The results of our current line of studies argue that the main neuropathological and clinical features of prion diseases are explained by altered ion channel function secondary to decreased plasma membrane fluidity. This kind of mechanism has the potential to functionally disconnect neuronal networks and cause neuronal vacuolation. Our laboratory is currently focusing its investigations on pathogenic mechanisms that have the potential to link the formation of PrPSc with plasma membrane abnormalities in prion diseases. In summary, the first hypothesis suggests that the conversion of PrPC to PrPSc affects plasma membrane fluidity directly, which secondarily alters the properties and functions of its components. In contrast, the second hypothesis argues that PrPSc accumulation alters the ability of chaperones to correctly fold plasma-membrane proteins during their synthesis, which directly affects the properties of nascent proteins and secondarily affects membrane fluidity. Our current investigations are attempting to determine which of these mechanisms are plausible and, then, which is primary.

Droloxifene (3-hydroxytamoxifen) has membrane antioxidant ability: potential relevance to its mechanism of therapeutic action in breast cancer

Droloxifene (3-hydroxytamoxifen), is a triphenylethylene derivative recently developed for the treatment of breast cancer. Droloxifene was found to exhibit a membrane antioxidant ability in that it inhibited Fe(III)-ascorbate dependent lipid peroxidation in rat liver microsomes and ox-brain phospholipid liposomes. It also inhibited microsomal lipid peroxidation induced by Fe(III)-ADP/NADPH. Droloxifene was a better inhibitor of lipid peroxidation than tamoxifen, but was less effective than 17β-oestradiol in the two microsomal systems and in the preformed liposomal system. When introduced into oxbrain phospholipid liposomes, droloxifene inhibited Fe(III)-ascorbate induced lipid peroxidation to approximately the same extent as similarly introduced cholesterol and tamoxifen, although to a lesser extent than 17β-oestradiol. This inhibition of lipid peroxidation by droloxifene may result from a membrane stabilization that could be associated in cancer cells with decreased plasma membrane fluidity. This mechanism may be related to the clinically important antiproliferative action of droloxifene on cancer cells.

Effect of membrane fatty acyl composition on LDL metabolism in Hep G2 hepatocytes

The mechanism by which dietary cis-unsaturated fatty acids lower plasma levels of low-density lipoprotein (LDL) cholesterol is unknown. Since plasma membrane incorporation of dietary cis-unsaturated fatty acids is known to alter the function of plasma membrane associated proteins, perhaps by increasing membrane fluidity, we examined LDL receptor function in Hep G2 hepatocytes that were unmodified, enriched with the cis-unsaturated fatty acids oleate or linoleate, or enriched with the saturated fatty acids stearate or palmitate. Hepatocytes enriched in cis-unsaturated fatty acids exhibited augmented LDL binding, uptake, and degradation in comparison to unmodified cells. In contrast, Hep G2 hepatocytes enriched in saturated fatty acids had decreased LDL binding, uptake, and degradation. Enrichment with oleate or linoleate resulted in a decrease in the calculated fatty acyl mole-weighted melting point of the plasma membrane and an increase in plasma membrane fluidity, as measured by the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into the plasma membrane. Conversely, stearate or palmitate enrichment resulted in an increased plasma membrane fatty acyl mole-weighted melting point and decreased plasma membrane fluidity. LDL binding, uptake, and degradation varied with plasma membrane fluidity in a highly correlated manner. Thus, one mechanism by which dietary cis-unsaturated fatty acids lower LDL cholesterol may possibly involve an alteration in membrane lipid composition or membrane fluidity that promotes enhanced LDL receptor function, thereby leading to increased hepatic clearance of LDL.

Decreased plasma membrane fluidity of peripheral blood lymphocytes after diethyldithiocarbamate (DTC) therapy in HIV-infected patients

In seeking the putative mechanism of action of diethyldithiocarbamate (DTC) on the immune status of HIV infected patients, the plasma membrane fluidity of peripheral blood lymphocytes (PBL) from DTC-treated and untreated patients (CDC III-IVc1) was determined. Anisotropy values of the fluorescent probe 6-(9-anthroyloxy) stearic acid were increased in DTC-treated patients (0.175 vs 0.161), indicating decreased PBL plasma membrane fluidity. The membrane rigidifying effect was significantly greater 4 h after i.v. drug administration (0.185 in treated patients). As the membrane fluidity and the function of membrane embedded antigen are interdependent, it is possible that alterations in biophysical and/or biochemical properties of membranes may account for the beneficial effect of DTC on the immune function and clinical status of HIV infected patients.

Decreased plasma membrane fluidity in the development of atherosclerosis in cholesterol-fed rabbits

Rabbits were fed a diet supplemented with 3% peanut oil or 3% peanut oil plus 0.5% cholesterol. Aortas from rabbits fed the cholesterol supplemented diet for 2 weeks were free of grossly visible lesions; however, aortas from rabbits fed this diet for 10 weeks exhibited extensive lesion development. Lesions were not observed in aortas of rabbits fed the high-fat supplemented diet. The fluorescence anisotropy of DPH was significantly ( P = 0.0001) increased in plasma membranes isolated from the aortas of rabbits fed the high-fat plus cholesterol vs. high-fat supplemented diet; the increase in fluorescence anisotropy observed after only 2 weeks on diet (0.201 ± 0.002 vs. 0.144 ± 0.002) was similar to that observed after 10 weeks on diet (0.244 ± 0.002 vs. 0.193 ± 0.001). The data support the hypothesis that plasma membrane fluidity [is decreased in atherosclerosis and indicate the decrease in membrane fluidity] occurs early in the development of the disease.