Title: OxLDL increases endothelial stiffness, force generation, and network formation
Abstract: This study investigates the effect of oxidatively modified low density lipoprotein (OxLDL) on the biomechanical properties of human aortic endothelial cells (HAECs). We show that treatment with OxLDL results in a 90% decrease in the membrane deformability of HAECs, as determined by micropipette aspiration. Furthermore, aortic endothelial cells freshly isolated from hypercholesterolemic pigs were significantly stiffer than cells isolated from healthy animals. Interestingly, OxLDL had no effect on membrane cholesterol of HAECs but caused the disappearance of a lipid raft marker, GM1, from the plasma membrane. Both an increase in membrane stiffness and a disappearance of GM1 were also observed in cells that were cholesterol-depleted by methyl-β-cyclodextrin. Additionally, OxLDL treatment of HAECs embedded within collagen gels resulted in increased gel contraction, indicating an increase in force generation by the cells. This increase in force generation correlated with an increased ability of HAECs to elongate and form networks in a three-dimensional environment. Increased force generation, elongation, and network formation were also observed in cholesterol-depleted cells. We suggest, therefore, that exposure to OxLDL results in the disruption or redistribution of lipid rafts, which in turn induces stiffening of the endothelium, an increase in endothelial force generation, and the potential for network formation. This study investigates the effect of oxidatively modified low density lipoprotein (OxLDL) on the biomechanical properties of human aortic endothelial cells (HAECs). We show that treatment with OxLDL results in a 90% decrease in the membrane deformability of HAECs, as determined by micropipette aspiration. Furthermore, aortic endothelial cells freshly isolated from hypercholesterolemic pigs were significantly stiffer than cells isolated from healthy animals. Interestingly, OxLDL had no effect on membrane cholesterol of HAECs but caused the disappearance of a lipid raft marker, GM1, from the plasma membrane. Both an increase in membrane stiffness and a disappearance of GM1 were also observed in cells that were cholesterol-depleted by methyl-β-cyclodextrin. Additionally, OxLDL treatment of HAECs embedded within collagen gels resulted in increased gel contraction, indicating an increase in force generation by the cells. This increase in force generation correlated with an increased ability of HAECs to elongate and form networks in a three-dimensional environment. Increased force generation, elongation, and network formation were also observed in cholesterol-depleted cells. We suggest, therefore, that exposure to OxLDL results in the disruption or redistribution of lipid rafts, which in turn induces stiffening of the endothelium, an increase in endothelial force generation, and the potential for network formation. Oxidative damage of LDL is associated with an increased risk for coronary artery disease and plaque formation (1Berliner J.A. Heinecke J.W. The role of oxidized lipoproteins in atherogenesis.Free Radic. Biol. Med. 1996; 20: 707-727Crossref PubMed Scopus (1272) Google Scholar, 2Steinberg D. Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime.Nat. Med. 2002; 8: 1211-1217Crossref PubMed Scopus (589) Google Scholar, 3Yla-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar). Earlier studies have demonstrated that oxidized low density lipoprotein (OxLDL) is present in atherosclerotic lesions in human and rabbit arteries (3Yla-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar) and that the level of OxLDL increases dramatically with hypercholesterolemia both in animal models, such as miniature pigs (4Holvoet P. Theilmeier G. Shivalkar B. Flameng W. Collen D. LDL hypercholesterolemia is associated with accumulation of oxidized LDL, atherosclerotic plaque growth, and compensatory vessel enlargement in coronary arteries of miniature pigs.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 415-422Crossref PubMed Scopus (96) Google Scholar) and monkeys (5Hodis H.N. Kramsch D.M. Avogaro P. Bittolo-Bon G. Cazzolato G. Hwang J. Peterson H. Sevanian A. Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL−).J. Lipid Res. 1994; 35: 669-677Abstract Full Text PDF PubMed Google Scholar), and in humans (6Cazzolato G. Avogaro P. Bittolo-Bon G. Characterization of a more electronegatively charged LDL subfraction by ion exchange HPLC.Free Radic. Biol. Med. 1991; 11: 247-253Crossref PubMed Scopus (99) Google Scholar, 7van Tits L.J. van Himbergen T.M. Lemmers H.L. de Graaf J. Stalenhoef A.F. Proportion of oxidized LDL relative to plasma apolipoprotein B does not change during statin therapy in patients with heterozygous familial hypercholesterolemia.Atherosclerosis. July 7, 2005; (Epub ahead of print.)doi:10.1016/j.atherosclersis.2005.06.006Google Scholar). It is also well known that exposure to OxLDL results in endothelial dysfunction, including disruption of the endothelial barrier (8Gardner G. Banka C.L. Roberts K.A. Mullick A.E. Rutledge J.C. Modified LDL-mediated increases in endothelial layer permeability are attenuated with 17β-estradiol.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 854-861Crossref PubMed Scopus (41) Google Scholar), impairment of nitric oxide release (9Blair A. Shaul P.W. Yuhanna I.S. Conrad P.A. Smart E.J. Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation.J. Biol. Chem. 1999; 274: 32512-32519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), and endothelial cell (EC) migration (10Murugesan G. Fox P.L. Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein.J. Clin. Invest. 1996; 97: 2736-2744Crossref PubMed Scopus (98) Google Scholar, 11Wang D.Y. Yang V.C. Chen J.K. Oxidized LDL inhibits vascular endothelial cell morphogenesis in culture.In Vitro Cell. Dev. Biol. Anim. 1997; 33: 248-255Crossref PubMed Scopus (16) Google Scholar). In this study, we focus on the role of OxLDL in the regulation of endothelial biomechanical properties, which play a major role in multiple EC functions, such as endothelial network formation, wound repair, and mechanotransduction. Recent studies have shown that exposure of ECs to OxLDL, rather than enriching the cells with cholesterol, removes cholesterol from cholesterol-rich membrane domains (lipid rafts) and induces the internalization of these domains (9Blair A. Shaul P.W. Yuhanna I.S. Conrad P.A. Smart E.J. Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation.J. Biol. Chem. 1999; 274: 32512-32519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 12Zeng Y. Tao N. Chung K-N. Heuser J.E. Lublin D.M. Endocytosis of oxidized low density lipoprotein through scavenger receptor CD36 utilizes a lipid raft pathway that does not require caveolin-1.J. Biol. Chem. 2003; 278: 45931-45936Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). One of the important remaining questions is how OxLDL-induced changes in endothelial cholesterol affect the biomechanical properties of these cells. Our recent studies have shown that, in contrast to artificial lipid bilayers, in which removal of cholesterol decreases the stiffness of membrane lipid bilayers (13Evans E. Needham D. Physical properties of surfactant bilayer membranes: thermal transition, elasticity, rigidity, cohesion and colloidal interactions.J. Phys. Chem. 1987; 91: 4219-4228Crossref Scopus (718) Google Scholar), ECs become stiffer upon cholesterol depletion (14Byfield F. Aranda-Aspinoza H. Romanenko V.G. Rothblat G.H. Levitan I. Cholesterol depletion constraints mechanical deformation of aortic endothelial cells.in: IASTED International Conference on Biomechanics, Rhodos, Greece, June 30–July 2, 2003. ACTA Press, 2003Google Scholar, 15Byfield F. Aranda-Aspinoza H. Romanenko V.G. Rothblat G.H. Levitan I. Cholesterol depletion increases membrane stiffness of aortic endothelial cells.Biophys. J. 2004; 87: 3336-3343Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Because it is known that cellular stiffness/deformability depends strongly on the submembrane cytoskeleton (16Pourati J. Maniotis A. Spiegel D. Schaffer J.L. Butler J.P. Fredberg J.J. Ingber D.E. Stamenovic D. Wang N. Is cytoskeletal tension a major determinant of cell deformability in adherent endothelial cells?.Am. J. Physiol. Cell Physiol. 1998; 274: C1283-C1289Crossref PubMed Google Scholar, 17Sato M. Theret D.P. Wheeler L.T. Ohshima N. Nerem R.M. Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell viscoelastic properties.J. Biomech. Eng. 1990; 112: 263-268Crossref PubMed Scopus (337) Google Scholar), an increase in cellular stiffness suggests that cholesterol removal alters the biomechanical properties of the membrane-cytoskeleton complex and makes it stiffer. Importantly, recent studies have indicated that increased cellular stiffness is correlated with the magnitude of forces that cells exert on substrates (18Wang N. Tolic-Norrelykke I.M. Chen J. Mijailovich S.M. Butler J.P. Fredberg J.J. Stamenovic D. Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells.Am. J. Physiol. Cell Physiol. 2002; 282: C606-C616Crossref PubMed Scopus (561) Google Scholar). Cell-derived forces in turn result in matrix compaction and remodeling (19Barocas V.H. Tranquillo R.T. An anisotropic biphasic theory of tissue-equivalent mechanics: the interplay among cell traction, fibrillar network deformation, fibril alignment, and cell contact guidance.J. Biomech. Eng. 1997; 119: 137-145Crossref PubMed Scopus (306) Google Scholar). Furthermore, it was shown that across different human EC lines (large vessel, microvascular, and blood-derived), force generation correlated with the extent to which the cells formed three-dimensional networks in collagen gels (20Sieminski A.L. Hebbel R.P. Gooch K.J. The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro.Exp. Cell Res. 2004; 297: 574-584Crossref PubMed Scopus (241) Google Scholar, 21Sieminski A.L. Hebbel R.P. Gooch K.J. Improved microvascular network in vitro by human blood outgrowth endothelial cells relative to vessel-derived endothelial cells.Tissue Eng. 2005; 11: 1332-1345Crossref PubMed Scopus (58) Google Scholar), an established in vitro model of EC morphogenesis. Many in vivo processes can be recapitulated in this system, including endothelial cell elongation, lumen formation, and cell-cell interactions, all suggestive of increased angiogenic potential. This system has been used to investigate multiple endothelial functions, including the mechanisms of action of angiogenic growth factors and angiogenesis inhibitors (22Park H.J. Kong D. Iruela-Arispe L. Begley U. Tang D. Galper J.B. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of RhoA.Circ. Res. 2002; 91: 143-150Crossref PubMed Scopus (254) Google Scholar, 23Yang S. Graham J. Jeanne W.K. Schwartz E.A. Gerritsen M.E. Functional roles for PECAM-1 (CD31) and VE-cadherin (CD144) in tube assembly and lumen formation in three-dimensional collagen gels.Am. J. Pathol. 1999; 155: 887-895Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Our study shows that exposure to OxLDL increases both the stiffness and the forces exerted by the cells on the substrate and facilitates EC network formation. Human aortic endothelial cells (HAECs; BioWhittaker, Rutherford, NJ) were maintained between passages three and five in 2% fetal bovine serum endothelium growth medium-2 (EGM-2; Cambrex), as described previously (24Fang Y. Schram G. Romanenko V. Shi C. Conti L. Vandenberg C.A. Davies P.F. Nattel S. Levitan I. Functional expression of Kir2.x in human aortic endothelial cells: the dominant role of Kir2.2.Am. J. Physiol. Cell Physiol. 2005; 289: C1134-C1144Crossref PubMed Scopus (55) Google Scholar). Pig aortic endothelial cells (PAECs) were isolated from aortas of normal and hypercholesterolemic Yorkshire pigs. Briefly, the pigs were fed either standard low-cholesterol chow (control group) or high-cholesterol chow supplemented with 10% lard and 0.5% cholesterol for 3–6 months. The high-cholesterol diet resulted in a significant increase in the level of plasma cholesterol (∼300 mg/dl), whereas in control pigs cholesterol remained at <100 mg/dl. PAECs were isolated by gentle mechanical scraping on the inner surface of aortas immediately after sacrifice, as described previously (25Passerini A.G. Polacek D.C. Shi C. Francesco N.M. Manduchi E. Grant G.R. Pritchard W.F. Powell S. Chang G.Y. Stoeckert C.J.J. et al.Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta.Proc. Natl. Acad. Sci. USA. 2004; 101: 2482-2487Crossref PubMed Scopus (312) Google Scholar). The purity of endothelial cells was verified by staining the cells with two endothelial markers: platelet endothelial cell adhesion molecule (PECAM) and von Willebrand factor (>95% of cells were positive for both markers and negative for smooth muscle actin). The protocol was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. The University of Pennsylvania is Association for Assessment of Laboratory Animal Care (AALAC)-accredited. LDL and OxLDL (Biomedical Technologies, Stoughton, MA) were dissolved in EGM-2 supplemented with 0.2% FBS to a final concentration of 10–50 μg/ml. Thiobarbituric acid-reactive (TBAR) substances were assayed as a measure of oxidative lipid modification. Methyl-β-cyclodextrin (MβCD; Sigma Chemical, St. Louis, MO) was also dissolved in 0.2% or serum-free EGM-2. Micropipette aspiration of substrate-attached endothelial cells was performed as described in our earlier study (15Byfield F. Aranda-Aspinoza H. Romanenko V.G. Rothblat G.H. Levitan I. Cholesterol depletion increases membrane stiffness of aortic endothelial cells.Biophys. J. 2004; 87: 3336-3343Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Briefly, the membranes were visualized with a fluorescent membrane dye, carbocyanide DiIC18 (Molecular Probes, Eugene, OR), and then aspirated using micropipettes with 3–5 μm outer diameter pulled from borosilicate glass capillaries (SG10 glass; Richland Glass, Richland, NJ). Negative pressure was applied to a pipette by a pneumatic transducer tester (BioTek Instruments, Winooski, VT). Cholesterol was measured with the Amplex Red cholesterol assay kit (Molecular Probes) according to the manufacturer's specifications. EC membrane fractions were isolated using a detergent-free method, as described (26Blank N. A fast, simple and sensitive method for the detection and quantification of detergent-resistant membranes.J. Immunol. Methods. 2002; 271: 25-35Crossref PubMed Scopus (32) Google Scholar). Localization of GM1 was determined by incubating HAECs with Alexa 488-conjugated cholera toxin (Molecular Probes) at a concentration of 50 μg/ml in 0.1% BSA for 40 min on ice. Cells were then washed, mounted, and viewed using a Zeiss Axiovert 100TV microscope (Zeiss, Jena, Germany). For analysis, 20 images were taken per experimental condition, and the average fluorescence intensity was determined using Meta-View software. Collagen gels were prepared according to the manufacturer's instructions to a final collagen concentration of 1.5 mg/ml (Becton Dickinson, Franklin Lanes, NJ). HAECs were seeded into gel mixtures at 1 × 106/ml, and gels were allowed to polymerize for 45 min at 37°C in well plates. Thereafter, the gels were mechanically loosened from the sides of the wells, and growth medium supplemented with vascular endothelial growth factor, basic fibroblast growth factor (R&D Systems, Minneapolis, MN), and phorbol myristate acetate (Sigma Chemical) at concentrations of 50 μg/ml each was added. Gels were cultured for 48 h and imaged with a Nikon Coolpix 4500 digital camera, and gel contraction was quantified using Scion Image (Las Vegas, NV). To visualize the cells, gels were fixed in 4% paraformaldehyde for 40 min, stained for 5 min in 0.1% toluidine blue, which stains cells darkly and extracellular matrix faintly, or treated for 40 min in 1 μM rhodamine-phalloidin (Molecular Probes). Images of toluidine blue staining were obtained at 10× magnification to observe EC networks, whereas actin images were obtained at 63× magnification to observe cell-cell connections (Zeiss Axiophot microscope; Zeiss, Thornwood, NY). For network quantification, five images were taken per gel from three gels per experimental condition, threshholded, made binary, and skeletonized using built-in Scion Image functions as described previously (20Sieminski A.L. Hebbel R.P. Gooch K.J. The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro.Exp. Cell Res. 2004; 297: 574-584Crossref PubMed Scopus (241) Google Scholar). For lumen visualization, gels were fixed, dehydrated, embedded in paraffin, sectioned, and stained with Masson's trichrome blue (Richard-Allen Scientific, Kalamazoo, MI). Lumens were defined as collagen-free areas surrounded by cellular bodies. Ten micrometer sections were cut from each gel, and 15 images containing one or more lumens were taken per experimental condition using 10× magnification. HAECs were exposed to 10–50 μg/ml OxLDL, levels similar to the circulating levels of OxLDL in human plasma (7–35 μg/ml) (27Holvoet P. Mertens A. Verhamme P. Bogaerts K. Beyens G. Verhaeghe R. Collen D. Muls E. Van de Werf F. Circulating oxidized LDL is a useful marker for identifying patients with coronary artery disease.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 844-848Crossref PubMed Scopus (444) Google Scholar). Similar to earlier studies (9Blair A. Shaul P.W. Yuhanna I.S. Conrad P.A. Smart E.J. Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation.J. Biol. Chem. 1999; 274: 32512-32519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 11Wang D.Y. Yang V.C. Chen J.K. Oxidized LDL inhibits vascular endothelial cell morphogenesis in culture.In Vitro Cell. Dev. Biol. Anim. 1997; 33: 248-255Crossref PubMed Scopus (16) Google Scholar), the oxidation state of LDL was 10–15 nmol/mg protein TBAR, consistent with the LDL oxidation level reported for atherosclerotic lesions (11 nmol/mg protein TBAR) (3Yla-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar). Two types of controls were used in this study: exposure to the same levels of nonoxidized LDL, and exposure to the growing medium alone. Representative images and the time courses of membrane deformation show that membrane projections in OxLDL-treated cells were significantly shorter than in cells treated with nonoxidized LDL or in cells that were exposed to medium alone, indicating that OxLDL-treated cells were less deformable than the other two experimental cell populations (Fig. 1). Membrane deformation was analyzed at 2–5 mm Hg negative pressure because in HAECs, membrane projections typically started to develop at −2 mm Hg and could be maintained at −5 mm Hg, whereas higher pressures resulted in breakage of the membrane. Significant differences between the lengths of membrane projections were observed at both pressures. Noteworthy, the stiffening was observed after 1 h of OxLDL exposure, and no further effect developed after prolonging the exposure to 6 h (Fig. 2). No difference was observed between 10 and 50 μg/ml OxLDL (data not shown).Fig. 2Prolonged exposure to OxLDL has no further effect on membrane deformation. A: Representative images of membrane deformation for control cells and cells treated with OxLDL for 1 and 6 h. Images show the maximal deformation at −5 mm Hg. Arrows indicate the positions of the aspirated projections. Bar = 30 μm. B: Average time courses of aspirated lengths for control and OxLDL-treated cells at −2 and −5 mm Hg. The graphs show means + SEM (n = 7, 14, and 6 for control, 1 h, and 6 h).View Large Image Figure ViewerDownload Hi-res image Download (PPT) There is a general consensus that OxLDL is one of the major factors in hypercholesterolemia-induced cellular dysfunction and that the levels of OxLDL increase in hypercholesterolemic plasma. Specifically, strong increases in OxLDL were reported in plaques of diet-induced atherosclerotic miniature pigs (high-cholesterol diet for 3–6 months) (4Holvoet P. Theilmeier G. Shivalkar B. Flameng W. Collen D. LDL hypercholesterolemia is associated with accumulation of oxidized LDL, atherosclerotic plaque growth, and compensatory vessel enlargement in coronary arteries of miniature pigs.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 415-422Crossref PubMed Scopus (96) Google Scholar) and hypercholesterolemic rabbits (3Yla-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar) as well as in plasma of hypercholesterolemic monkeys maintained on a high-cholesterol diet for 32 months (5Hodis H.N. Kramsch D.M. Avogaro P. Bittolo-Bon G. Cazzolato G. Hwang J. Peterson H. Sevanian A. Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL−).J. Lipid Res. 1994; 35: 669-677Abstract Full Text PDF PubMed Google Scholar). Therefore, we compared the stiffness of endothelial cells freshly isolated from the aortas of control and hypercholesterolemic pigs within 24 h after isolation. Representative images and time courses of membrane deformation of cells isolated from normal and hypercholesterolemic pigs are shown in Fig. 3. Cells appear rounded as they were aspirated within 24 h of isolation. In general, PAECs were stiffer than HAECs, so that the membrane could be deformed by applying at least −10 mm Hg, but the effect of plasma hypercholesterolemia on cell stiffness was similar to that of OxLDL. An increase in endothelial stiffness in cells freshly isolated from hypercholesterolemic pigs indicates that hypercholesterolemia induces the stiffening of endothelial cells in vivo. To test further the mechanism of OxLDL-induced endothelial stiffening, we investigated the relationship between OxLDL-induced stiffening and the level of cellular cholesterol. We have shown previously that endothelial stiffening is observed in bovine aortic endothelium when the cells are depleted of cholesterol but not when the cells are cholesterol-enriched (15Byfield F. Aranda-Aspinoza H. Romanenko V.G. Rothblat G.H. Levitan I. Cholesterol depletion increases membrane stiffness of aortic endothelial cells.Biophys. J. 2004; 87: 3336-3343Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Furthermore, it was shown that exposure to OxLDL in vitro and hypercholesterolemia in vivo result in depleting cholesterol from endothelial caveolae (9Blair A. Shaul P.W. Yuhanna I.S. Conrad P.A. Smart E.J. Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation.J. Biol. Chem. 1999; 274: 32512-32519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 28Kincer J.F. Uittenbogaard A. Dressman J. Guerin T.M. Febbraio M. Guo L. Smart E.J. Hypercholesterolemia promotes a CD36-dependent and endothelial nitric-oxide synthase-mediated vascular dysfunction.J. Biol. Chem. 2002; 277: 23525-23533Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We hypothesized, therefore, that OxLDL may increase endothelial stiffness by depleting endothelial cholesterol. To address this hypothesis, we tested 1) whether OxLDL results in cholesterol depletion and/or redistribution of lipid rafts in HAECs, and 2) whether the effect of OxLDL on HAEC stiffness can be simulated by cholesterol depletion. To determine the effect of OxLDL on the level of membrane cholesterol in raft and nonraft membrane domains, membrane fractions were prepared using a nondetergent separation method based on the differential buoyancy of cholesterol-rich and cholesterol-poor membrane domains. As expected, membrane cholesterol shows a clear double peak distribution between the fractions, with the major peak in high-buoyancy fractions (lipid rafts) and a minor peak in low-buoyancy fractions (nonraft). Indeed, also as expected, the high-buoyancy fractions were enriched in caveolin (data not shown). Exposure of HAECs to OxLDL, however, had no effect on the level of membrane cholesterol either in raft or nonraft membrane fractions (Fig. 4A). The same results were obtained in four independent experiments. Importantly, exposure to OxLDL resulted in a decrease of surface expression of GM1 (Fig. 4B), a major lipid raft marker (29Parton R.G. Ultrastructural localization of gangliosides: Gm1 is concentrated in caveolae.J. Histochem. Cytochem. 1994; 42: 155-166Crossref PubMed Scopus (452) Google Scholar), suggesting that OxLDL induces the internalization of caveolae/lipid rafts. Depleting cholesterol with MβCD resulted in a similar effect on GM1. This is consistent with earlier studies demonstrating the internalization of caveolae/lipid rafts in OxLDL (9Blair A. Shaul P.W. Yuhanna I.S. Conrad P.A. Smart E.J. Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation.J. Biol. Chem. 1999; 274: 32512-32519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 12Zeng Y. Tao N. Chung K-N. Heuser J.E. Lublin D.M. Endocytosis of oxidized low density lipoprotein through scavenger receptor CD36 utilizes a lipid raft pathway that does not require caveolin-1.J. Biol. Chem. 2003; 278: 45931-45936Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and in MβCD-treated cells (30del Pozo M.A. Alderson N.B. Kiosses W.B. Chiang H.H. Anderson R.G. Schwartz M.A. Integrins regulate rac targeting by internalization of membrane domains.Science. 2004; 303: 839-842Crossref PubMed Scopus (456) Google Scholar). To address the second question, the level of cellular cholesterol in HAECs was either chronically depleted by serum starvation for 24 h or acutely depleted by exposure to MβCD, resulting in an ∼40% or 80% decrease in cholesterol, respectively (Fig. 5, top). A decrease in cellular cholesterol resulted in a proportional increase in cell stiffness, as demonstrated by the representative images of control, serum-starved, and MβCD-treated cells (Fig. 5A) and by the average time courses of membrane deformation of the same three cell populations (Fig. 5B). In this series of experiments, cell stiffness was compared only at −5 mm Hg, because no aspiration was observed in MβCD-treated cells at lower pressures. Thus, our results show that although OxLDL had no significant effect on cholesterol levels of both raft and nonraft membrane fractions, its effect on HAEC stiffness could be simulated by both chronic and acute cholesterol depletion. Furthermore, when the cells were simultaneously exposed to OxLDL and depleted of cholesterol (by 24 h serum starvation), no additional stiffening was observed (data not shown), suggesting that OxLDL and cholesterol depletion affect endothelial stiffness through a common pathway. The correlation between HAECs' stiffness and their ability to generate force on the cell-substrate interface was tested by measuring gel contraction by HAECs, as described previously (19Barocas V.H. Tranquillo R.T. An anisotropic biphasic theory of tissue-equivalent mechanics: the interplay among cell traction, fibrillar network deformation, fibril alignment, and cell contact guidance.J. Biomech. Eng. 1997; 119: 137-145Crossref PubMed Scopus (306) Google Scholar, 20Sieminski A.L. Hebbel R.P. Gooch K.J. The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro.Exp. Cell Res. 2004; 297: 574-584Crossref PubMed Scopus (241) Google Scholar). As expected, seeding the cells into gels resulted in gel contraction under all experimental conditions (Fig. 6A;compare the gel that contains no cells with the rest of the gels). Importantly, however, cells that were pretreated with OxLDL or MβCD showed significantly greater gel contraction, as indicated by a decrease in gel area (Fig. 6A, B). As the same number of cells were seeded into each gel, these observations suggest that the contractile forces that OxLDL-treated and MβCD-treated cells applied to their substrates were stronger than the forces applied by control cells. Similar to other types of