Cholesterol depletion increases membrane stiffness of aortic endothelial cells

This study has investigated the effect of cellular cholesterol on membrane deformability of bovine aortic endothelial cells. Cellular cholesterol content was depleted by exposing the cells to methyl-beta-cyclodextrin or enriched by exposing the cells to methyl-beta-cyclodextrin saturated with cholesterol. Control cells were treated with methyl-beta-cyclodextrin-cholesterol at a molar ratio that had no effect on the level of cellular cholesterol. Mechanical properties of the cells with different cholesterol contents were compared by measuring the degree of membrane deformation in response to a step in negative pressure applied to the membrane by a micropipette. The experiments were performed on substrate-attached cells that maintained normal morphology. The data were analyzed using a standard linear elastic half-space model to calculate Young elastic modulus. Our observations show that, in contrast to the known effect of cholesterol on membrane stiffness of lipid bilayers, cholesterol depletion of bovine aortic endothelial cells resulted in a significant decrease in membrane deformability and a corresponding increase in the value of the elastic coefficient of the membrane, indicating that cholesterol-depleted cells are stiffer than control cells. Repleting the cells with cholesterol reversed the effect. An increase in cellular cholesterol to a level higher than that of normal cells, however, had no effect on the elastic properties of bovine aortic endothelial cells. We also show that although cholesterol depletion had no apparent effect on the intensity of F-actin-specific fluorescence, disrupting F-actin with latrunculin A abrogated the stiffening effect. We suggest that cholesterol depletion increases the stiffness of the membrane by altering the properties of the submembrane F-actin and/or its attachment to the membrane.     

Nuclear Envelope Composition Determines the Ability of Neutrophil-type Cells to Passage through Micron-scale Constrictions

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.     

Plasma membrane cholesterol: a possible barrier to intracellular oxygen in normal and mutant CHO cells defective in cholesterol metabolism

The effect of the cholesterol content of the plasma membrane on the intracellular concentration of oxygen in Chinese hamster ovary (CHO) cells and their mutants was investigated by EPR oximetry. Total and free cholesterol content was significantly higher in 25 RA CHO cells as compared to wild-type and M 19 CHO cells, with most of the free cholesterol in normal and mutant CHO cells located in the plasma membrane. The plasma membrane cholesterol content also was altered by various biochemical means, and the effect on the oxygen gradient was studied. Comparing the three cell lines, the gradient was larger with increased content of cholesterol in the plasma cell membrane. This result also is supported by an additional increase in the oxygen gradients with the incorporation of additional cholesterol in the plasma membrane and a decrease in the oxygen gradient when the cholesterol was depleted from the plasma membrane. The results indicate that the concentration of cholesterol in the plasma membrane can be an important factor for the magnitude of the oxygen gradient observed across the cell membrane.     

Changes in the erythrocyte in liver cirrhosis: role of cholesterol and plasma phospholipids

To investigate the role played by plasmatic lipids in the altered erythrocyte deformability observed in cirrhotic patients we studied 15 patients with liver cirrhosis (histologically diagnosed) and 10 healthy volunteers. Erythrocyte filtration time, plasmatic free and esterified cholesterol and phospholipids were measured in all subjects. The erythrocyte filtration time resulted to be significantly increased in cirrhotic patients (35' +/- 3, 35 M +/- SEM) when compared to control subjects (26' +/- 2, 53: M +/- SEM) (t = 2,078 p less than 0,05). This increase correlated in cirrhotic patients (but not in control subjects) with free/esterified cholesterol ratio (p less than 0,01) as well as free cholesterol/phospholipid ratio (p less than 0,001). Our results confirm that decreased erythrocyte deformability in cirrhotic patients which is accompanied by altered erythrocyte morphology is due, at least in part, to the altered lipids blood levels.     

Breast Cancer Cells Adapt Contractile Forces to Overcome Steric Hindrance

Cell migration through the extracellular matrix is governed by the interplay between cell-generated propulsion forces, adhesion forces, and resisting forces arising from the steric hindrance of the matrix. Steric hindrance in turn depends on matrix porosity, matrix deformability, cell size, and cell deformability. In this study, we investigate how cells respond to changes in steric hindrance that arise from altered cell mechanical properties. Specifically, we measure traction forces, cell morphology, and invasiveness of MDA-MB 231 breast cancer cells in three-dimensional collagen gels. To modulate cell mechanical properties, we either decrease nuclear deformability by twofold overexpression of the nuclear protein lamin A or we introduce into the cells stiff polystyrene beads with a diameter larger than the average matrix pore size. Despite this increase of steric hindrance, we find that cell invasion is only marginally inhibited, as measured by the fraction of motile cells and the mean invasion depth. To compensate for increased steric hindrance, cells employ two alternative strategies. Cells with higher nuclear stiffness increase their force polarity, whereas cells with large beads increase their net contractility. Under both conditions, the collagen matrix surrounding the cells stiffens dramatically and carries increased strain energy, suggesting that increased force polarity and increased net contractility are functionally equivalent strategies for overcoming an increased steric hindrance.     

Induction of monocyte differentiation and foam cell formation in vitro by 7-ketocholesterol.

Oxidized LDL (OxLDL) is composed of many potentially proatherogenic molecules, including oxysterols. Of the oxysterols, 7-ketocholesterol (7-KC) is found in relatively large abundance in OxLDL, as well as in atherosclerotic plaque and foam cells in vivo. Although there is evidence that 7-KC activates endothelial cells, its effect on monocytes is unknown. We tested the hypothesis that 7-KC may induce monocyte differentiation and promote foam cell formation. THP-1 cells were used as a monocyte model system and were treated with 7-KC over a range of concentrations from 0.5 to 10 microg/ml. Changes in cell adhesion properties, cell morphology, and expression of antigens characteristic of differentiated macrophages were monitored over a 7-day period. 7-KC promoted cells to firmly adhere and display morphologic features of differentiated macrophages; this effect was time and dose dependent and was markedly more potent than cholesterol treatment (45% of cells became adherent after 7 days of treatment with 7-KC at 10 microg/ml vs. less then 5% for control cells, P < 0.01). Similar effects were obtained when LDL enriched with 7-KC or OxLDL were added to THP-1 cells. 7-KC-differentiated cells expressed CD11b, CD36, and CD68, phagocytized latex beads, and formed lipid-laden foam cells after exposure to acetylated LDL or OxLDL. In contrast to 7-KC, oxysterols with known cell regulatory effects such as 25-hydroxycholesterol, 7beta-hydroxycholesterol, and (22R)-hydroxycholesterol did not effectively promote THP-1 differentiation.In conclusion, these results demonstrate for the first time that 7-KC, a prominent oxysterol formed in OxLDL by peroxidation of cholesterol, may play an important role in promoting monocyte differentiation and foam cell formation. These studies also suggest that 7-KC induces monocyte differentiation through a sterol-mediated regulatory pathway that remains to be characterized.     

Asymmetric requirement for cholesterol in receptor-bearing but not envelope-bearing membranes for fusion mediated by ecotropic murine leukemia virus

We show that fusion mediated by ecotropic murine leukemia virus envelope is dependent on cholesterol in receptor-bearing membranes. The effect is >10 times larger in insect cells than mammalian cells, probably because the former can be more extensively depleted of cholesterol. The fact that cholesterol is apparently not needed in envelope-bearing membranes suggests that it plays a role in an asymmetric step in membrane fusion and argues against a class of models in which cholesterol is important in symmetric fusion intermediates. The insect cell system has promise for clarifying the role of membrane rafts in other aspects of cell physiology.     

3D environment controls H3K4 methylation and the mechanical response of the nucleus in acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, and the infiltration of leukemic cells is critical for disease progression and relapse. Nuclear deformability plays a critical role in cancer cell invasion through confined spaces; however, the direct impact of epigenetic changes on the nuclear deformability of leukemic cells remains unclear. Here, we characterized how 3D collagen matrix conditions induced H3K4 methylation in ALL cell lines and clinical samples. We used specific shRNA and chemical inhibitors to target WDR5 (a core subunit involved in H3K4 methylation) and determined that targeting WDR5 reduced the H3K4 methylation induced by the 3D environment and the invasiveness of ALL cells in vitro and in vivo. Intriguingly, targeting WDR5 did not reduce the adhesion or the chemotactic response of leukemia cells, suggesting a different mechanism by which H3K4 methylation might govern ALL cell invasiveness. Finally, we conducted biochemical, and biophysical experiments to determine that 3D environments promoted the alteration of the chromatin, the morphology, and the mechanical behavior of the nucleus in ALL cells. Collectively, our data suggest that 3D environments control an upregulation of H3K4 methylation in ALL cells, and targeting WDR5 might serve as a promising therapeutic target against ALL invasiveness in vivo.     

Effects of pressure on cell morphology and cell division of lactic acid bacteria

The effect of pressure and temperature on the growth of the mesophilic lactic acid bacteria Lactococcus lactis and Lactobacillus sanfranciscensis was studied. Both strains were piezosensitive. Lb. sanfranciscensis failed to grow at 50 MPa and the growth rate of Lc. lactis at 50 MPa was less than 30% of that at atmospheric pressure. An increase of growth temperature did not improve the piezotolerance of either organism. During growth under high-pressure conditions, the cell morphology was changed, and the cells were elongated as cell division was inhibited. At atmospheric pressure, temperatures above the optimal temperature for growth caused a similar effect on cell morphology and cell division in both bacteria as that observed under high-pressure conditions. The segregation and condensation of chromosomal DNA were observed by DAPI staining and occurred normally at high-pressure conditions independent of changes in cell morphology. Immunofluorescence microscopy of Lc. lactis cells demonstrated an inhibitory effect of high pressure on the formation of the FtsZ ring and this inhibition of the FtsZ ring formation is suggested to contribute to the altered cell morphology and growth inhibition induced by high pressure.Communicated by K. Horikoshi     

Gene expression profiling in response to the histone deacetylase inhibitor BL1521 in neuroblastoma

Neuroblastoma is a childhood tumor with a poor survival in advanced stage disease despite intensive chemotherapeutic regimes. The new histone deacetylase (HDAC) inhibitor BL1521 has shown promising results in neuroblastoma. Inhibition of HDAC resulted in a decrease in proliferation and metabolic activity, induction of apoptosis and differentiation of neuroblastoma cells. In order to elucidate the mechanism mediating the effects of BL1521 on neuroblastoma cells, we investigated the gene expression profile of an MYCN single copy (SKNAS) and an MYCN amplified (IMR32) neuroblastoma cell line after treatment with BL1521 using the Affymetrix oligonucleotide array U133A. An altered expression of 255 genes was observed in both neuroblastoma cell lines. The majority of these genes were involved in gene expression, cellular metabolism, and cell signaling. We observed changes in the expression of vital genes belonging to the cell cycle (cyclin D1 and CDK4) and apoptosis (BNIP3, BID, and BCL2) pathway in response to BL1521. The expression of 37 genes was altered by both BL1521 and Trichostatin A, which could indicate a common gene set regulated by different HDAC inhibitors. BL1521 treatment changed the expression of a number of MYCN-associated genes. Several genes in the Wnt and the Delta/Notch pathways were changed in response to BL1521 treatment, suggesting that BL1521 is able to induce the differentiation of neuroblastoma cells into a more mature phenotype.     

The effect of gamma-tocopherol on proliferation, integrin expression, adhesion, and migration of human glioma cells

The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. gamma-tocopherol at 50 microM concentration exerted more inhibitory effect than alpha-tocopherol at the same concentration on glioma cell proliferation. Integrin alpha5 and beta1 protein levels were increased upon both alpha- and gamma-tocopherol treatments. In parallel, an increase in the alpha5beta1 heterodimer cell surface expression was observed. The tocopherols inhibited glioma cell-binding to fibronectin where gamma-tocopherol treatment induced glioma cell migration. Taken together, the data reported here are consistent with the notion that the inhibition of glioma cell proliferation induced by tocopherols may be mediated, at least in part, by an increase in integrin alpha5 and beta1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the alpha5beta1 heterodimer. Cell migration is stimulated by gamma-tocopherol. It is concluded that alpha5 and beta1 integrin expression and surface appearance are not sufficient to explain all the observations and that other integrins or in general other factors may be associated with these events.     

Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.     

A computational study of leukocyte adhesion and its effect on flow pattern in microvessels

Three-dimensional computational modeling and simulation are presented on the adhesive rolling of deformable leukocytes over a P-selectin coated surface in parabolic shear flow in microchannels. The computational model is based on the immersed boundary method for cell deformation and Monte Carlo simulation for receptor/ligand interaction. The simulations are continued for at least 1 s of leukocyte rolling during which the instantaneous quantities such as cell deformation index, cell/substrate contact area, and fluid drag remain statistically stationary. The characteristic ‘stop-and-go’ motion of rolling leukocytes, and the ‘tear-drop’ shape of adherent leukocytes as observed in experiments are reproduced by the simulations. We first consider the role of cell deformation and cell concentration on rolling characteristics. We observe that compliant cells roll slower and more stably than rigid cells. Our simulations agree with previous in vivo observation that the hydrodynamic interactions between nearby leukocytes affect cell rolling, and that the rolling velocity decreases inversely with the separation distance, irrespective of cell deformability. We also find that cell deformation decreases, and the cells roll more stably with reduced velocity fluctuation, as the cell concentration is increased. However, the effect of nearby cells on the rolling characteristics is found to be more significant for rigid cells than compliant cells. We then address the effect of cell deformability and rolling velocity on the flow resistance due to, and the fluid drag on, adherent leukocytes. While several earlier computational works have addressed this problem, two key features of leukocyte adhesion, such as cell deformation and rolling, were often neglected. Our results suggest that neglecting cell deformability and rolling velocity may significantly overpredict the flow resistance and drag force. Increasing the cell concentration is shown to increase the flow resistance and reduce the fluid drag. The reduced drag then results in slower and more stable rolling of the leukocytes with longer pause time and shorter step distance. But the increase/decrease in the flow resistance/fluid drag due to the increase in the cell concentration is observed to be more significant in case of rigid cells than compliant cells.     

Lectin-induced cell agglutination and membrane cholesterol levels

The membranes of human and guinea pig erythrocytes were enriched with, or depleted of cholesterol. Ehrlich ascites carcinoma cells were also enriched with cholesterol and the extra slerol shown to be present in the plasma membrane. Enrichment of the cells with sterol made them less susceptible to agglutination by concanavalin A (ConA) or phytohemagglutinin (PHA), while removal of sterol from the erythrocytes increased their susceptibilily to agglutination. It is suggested that following changes in surface membrane sterol levels there are changes both in short-range movement of individual receptor molecules and in cell shape and deformability which control the agglutinability of the cells.     

A requirement for cholesterol for phorbol ester-induced adhesion of a human monocyte-like cell line

The human monocyte/macrophage-like cell line U937, which is a cholesterol auxotroph, is nonadherent. However, it becomes adherent after treatment with phorbol 12-myristate 13-acetate (phorbol ester). We investigated the effects of cellular cholesterol depletion and repletion on the effectiveness of phorbol ester to induce adhesion to substratum. Almost 70% of cellular cholesterol is depleted by incubation of the cells for 24 hrs in the growth medium in which delipidated fetal calf serum is substituted for fetal calf serum without affecting viability or the rate of growth. The use of delipidated fetal calf serum inhibited phorbol ester-induced adhesion by 40%. If the cells were preincubated in the medium containing delipidated fetal calf serum 6 hrs prior to addition of phorbol ester, adhesion was inhibited by 90%. Addition of cholesterol to the medium containing delipidated fetal calf serum, which replenishes cellular cholesterol, restored the ability of phorbol ester to induce adhesion to levels seen in cells cultured in the medium containing fetal calf serum. Epicholesterol was not as effective as cholesterol in supporting adhesion. Cholesterol depletion did not inhibit phorbol ester stimulation of superoxide anion production. These observations indicate a function for cholesterol in phorbol ester-induced adhesion that is independent of phorbol ester-induced superoxide anion production. It is proposed that cholesterol is required for synthesis and/or proper orientation and distribution, in the plasma membrane, of macromolecule(s) that mediate phorbol ester-induced adhesion.     

Cholesterol controls lipid endocytosis through Rab11

Cellular cholesterol increases when cells reach confluency in Chinese hamster ovary (CHO) cells. We examined the endocytosis of several lipid probes in subconfluent and confluent CHO cells. In subconfluent cells, fluorescent lipid probes including poly(ethylene glycol)derivatized cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol, and fluorescent sphingomyelin analogs were internalized to pericentriolar recycling endosomes. This accumulation was not observed in confluent cells. Internalization of fluorescent lactosylceramide was not affected by cell confluency, suggesting that the endocytosis of specific membrane components is affected by cell confluency. The crucial role of cellular cholesterol in cell confluency-dependent endocytosis was suggested by the observation that the fluorescent sphingomyelin was transported to recycling endosomes when cellular cholesterol was depleted in confluent cells. To understand the molecular mechanism(s) of cell confluency- and cholesterol-dependent endocytosis, we examined intracellular distribution of rab small GTPases. Our results indicate that rab11 but not rab4, altered intracellular localization in a cell confluency-associated manner, and this alteration was dependent on cell cholesterol. In addition, the expression of a constitutive active mutant of rab11 changed the endocytic route of lipid probes from early to recycling endosomes. These results thus suggest that cholesterol controls endocytic routes of a subset of membrane lipids through rab11.     

The effect of lipocortin 1 on neutrophil deformability

Lipocortn 1 (Lc1) is an anti-inflammatory protein, which, given systemically, inhibits polymorphonuclear neutrophil (PMN) emigration from the circulation to sites of inflammation; delivery of Lc1 to the inflamed site is ineffective. We have examined the effect of Lc1 on changes in PMN deformability, and observed a consistent improvement in the deformability of unstimulated PMN; N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated cell deformability was unaltered. A Lc1-induced increase in cell deformability may reduce PMN sequestration so contributing to the anti-migratory effects of systemic Lc1 previously demonstrated in vivo.     

Influence of metabolic equilibrium on erythrocyte deformability in diabetes mellitus

Red blood cell filtration test (Reid's test) was performed in 23 diabetic patients and in 10 normal subjects and it was related to metabolic equilibrium. Results showed an increase of filtration time in diabetics when compared to controls (35.1' +/- 2.3; M +/- SEM vs 22.2' +/- 0.7, p less than 0.001) and a significant correlation to cholesterol (178.7 mg% +/- 8.9, r = 0.40, p less than 0.05), triglycerides (131.3 mg% +/- 20.6, r = 0.72, p less than 0.001) and to glycosylated hemoglobin (10.7% +/- 0.5, r = 0.60, p less than 0.01) in diabetic patients. No correlation was observed in control subjects. The values of red blood cells filtration time observed in diabetics suggest that an altered erythrocyte deformability in diabetic patients can play an important role in peripheral hypoxia and therefore in diabetic microangiopathy.     

Coxiella burnetii inhabits a cholesterol-rich vacuole and influences cellular cholesterol metabolism

Coxiella burnetii directs the synthesis of a large parasitophorous vacuole (PV) required for replication. While some lysosomal characteristics of the PV have been described, the origin and composition of the PV membrane is largely undefined. Cholesterol is an essential component of mammalian cell membranes where it plays important regulatory and structural roles. Here we investigated the role of host cholesterol in biogenesis and maintenance of the C. burnetii PV in Vero cells. The C. burnetii PV membrane stained with filipin and was positive for the lipid raft protein flotillin-1, suggesting PV membranes are enriched in cholesterol and contain lipid raft microdomains. C. burnetii infection increased host cell cholesterol content by 1.75-fold with a coincident upregulation of host genes involved in cholesterol metabolism. Treatment with U18666A, lovastatin, or 25-hydroxycholesterol, pharmacological agents that inhibit cholesterol uptake and/or biosynthesis, altered PV morphology and partially inhibited C. burnetii replication. Complete inhibition of C. burnetii PV development and replication was observed when infected cells were treated with imipramine or ketoconazole, inhibitors of cholesterol uptake and biosynthesis respectively. We conclude that C. burnetii infection perturbs host cell cholesterol metabolism and that free access to host cholesterol stores is required for optimal C. burnetii replication.