Different mechanisms of saturated versus polyunsaturated FFA-induced apoptosis in human endothelial cells
2008; Elsevier BV; Volume: 49; Issue: 12 Linguagem: Inglês
10.1194/jlr.m800393-jlr200
ISSN1539-7262
AutoresMichaela Artwohl, Andrea Lindenmair, Veronika Sexl, Christina Maier, Georg Rainer, Angelika Freudenthaler, Nicole Huttary, Michael Wolzt, P. Nowotny, Anton Luger, Sabina Baumgartner‐Parzer,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoApoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 μmol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms. Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 μmol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms. FFAs, elevated in visceral obesity and diabetes, play a vital role in atherogenesis and acute coronary syndromes (1Armstrong K.A. Hiremagalur B. Haluska B.A. Campbell S.B. Hawley C.M. Marks L. Prins J. Johnson D.W. Isbel N.M. Free fatty acids are associated with obesity, insulin resistance, and atherosclerosis in renal transplant recipients.Transplantation. 2005; 80: 937-944Crossref PubMed Scopus (38) Google Scholar, 2Pilz S. Scharnagl H. Tiran B. Seelhorst U. Wellnitz B. Boehm B.O. Schaefer J.R. Marz W. Free fatty acids are independently associated with all-cause and cardiovascular mortality in subjects with coronary artery disease.J. Clin. Endocrinol. Metab. 2006; 91: 2542-2547Crossref PubMed Scopus (142) Google Scholar). In vitro, high FFA concentrations contribute to accelerated apoptosis of the endothelium (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 4Hufnagel B. Dworak M. Soufi M. Mester Z. Zhu Y. Schaefer J.R. Klumpp S. Krieglstein J. Unsaturated fatty acids isolated from human lipoproteins activate protein phosphatase type 2Cβ and induce apoptosis in endothelial cells.Atherosclerosis. 2005; 180: 245-254Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 5Dersch K. Ichijo H. Bhakdi S. Husmann M. Fatty acids liberated from low-density lipoprotein trigger endothelial apoptosis via mitogen-activated protein kinases.Cell Death Differ. 2005; 12: 1107-1114Crossref PubMed Scopus (32) Google Scholar, 6Su J. Liu R. Yang B. Tian H. Effects of glucose and free fatty acids on apoptosis of human vascular endothelial cells.Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2006; 23: 170-174PubMed Google Scholar, 7Staiger K. Staiger H. Weigert C. Haas C. Häring H-U. Kellerer M. Saturated, but not unsaturated, fatty acids induce apoptosis of human coronary artery endothelial cells via nuclear factor-κB activation.Diabetes. 2006; 55: 3121-3126Crossref PubMed Scopus (118) Google Scholar), which ranks among the most endangered target tissues in diabetes. In diabetic retinopathy, endothelial apoptosis occurs before other histopathology is detectable (8Mizutani M. Kern T.S. Lorenzi M. Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy.J. Clin. Invest. 1996; 97: 2883-2890Crossref PubMed Scopus (590) Google Scholar), and procoagulatory apoptotic endothelial cells, topographically associated with microthromboses (9Boeri D. Maiello M. Lorenzi M. Increased prevalence of microthromboses in retinal capillaries of diabetic individuals.Diabetes. 2001; 50: 1432-1439Crossref PubMed Scopus (119) Google Scholar), could contribute to vascular occlusion. In atherosclerosis, in addition to its contribution to initial lesion formation owing to detachment of endothelial cells from the underlying intimal layer, endothelial apoptosis leads to increased vascular permeability, plaque erosion, and plaque rupture (10Rössig L. Dimmeler S. Zeiher A. Apoptosis in the vascular wall and atherosclerosis.Basic Res. Cardiol. 2001; 96: 11-22Crossref PubMed Scopus (133) Google Scholar, 11Choy J.C. Granville D.J. Hunt D.W.C. Manus B.M. Endothelial cell apoptosis: biochemical characteristics and potential implications for atherosclerosis.J. Mol. Cell. Cardiol. 2001; 33: 1673-1690Abstract Full Text PDF PubMed Scopus (398) Google Scholar). At least two observations render obsolete the former assumption that loss of endothelial cells owing to apoptosis is solely accomplished by an increased mitotic response/turnover of endothelial cells located nearby: i) re-endothelialization appears to be more likely attributable to cells migrating over long distances than to local endothelial cells (12Hirsch E.Z. Chisolm III, G.M. White H.M. Reendothelialization and maintenance of endothelial integrity in longitudinal denuded tracks in the thoracic aorta of rats.Atherosclerosis. 1983; 46: 287-307Abstract Full Text PDF PubMed Scopus (21) Google Scholar); and ii) bone marrow-derived endothelial progenitor cells (EPCs) are presumably responsible for postnatal vasculogenesis in physiological and pathophysiological neovascularization (13Asahara T. Murohara T. Sullivan A. Silver M. van der Zee R. Li T. Witzenbichler B. Schatteman G. Isner J.M. Isolation of putative progenitor endothelial cells for angiogenesis.Science. 1997; 275: 964-967Crossref PubMed Scopus (7666) Google Scholar). Increasing evidence suggests that loss of endothelial integrity, owing to damage/apoptosis induced by atherosclerotic risk factors, might be repaired by circulating EPCs (14Dimmeler S. Zeiher A.M. Vascular repair by circulating endothelial progenitor cells: the missing link in atherosclerosis?.J. Mol. Med. 2004; 82: 671-677Crossref PubMed Scopus (262) Google Scholar, 15Vasa M. Fichtlscherer S. Aicher A. Adler K. Urbich C. Martin H. Zeiher A.M. Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease.Circ. Res. 2001; 89: E1-E7Crossref PubMed Scopus (2092) Google Scholar), which on recruitment are capable of differentiating into endothelial cells, displaying classical morphology and characteristics. Thus, evaluation of the effects of risk factors such as FFAs should not be restricted to damaged target cells (e.g., aortic or retinal endothelial cells), but should also include subsequent "repairing" cells, the EPCs. To date, the pro-apoptotic activity of different FFAs has been examined primarily in immortalized endothelial cell lines (EA.hy926, ECV304) and human umbilical vein endothelial cells (HUVECs) (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 4Hufnagel B. Dworak M. Soufi M. Mester Z. Zhu Y. Schaefer J.R. Klumpp S. Krieglstein J. Unsaturated fatty acids isolated from human lipoproteins activate protein phosphatase type 2Cβ and induce apoptosis in endothelial cells.Atherosclerosis. 2005; 180: 245-254Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 5Dersch K. Ichijo H. Bhakdi S. Husmann M. Fatty acids liberated from low-density lipoprotein trigger endothelial apoptosis via mitogen-activated protein kinases.Cell Death Differ. 2005; 12: 1107-1114Crossref PubMed Scopus (32) Google Scholar, 6Su J. Liu R. Yang B. Tian H. Effects of glucose and free fatty acids on apoptosis of human vascular endothelial cells.Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2006; 23: 170-174PubMed Google Scholar). Studies involving target tissues directly affected by the metabolic syndrome are rare (7Staiger K. Staiger H. Weigert C. Haas C. Häring H-U. Kellerer M. Saturated, but not unsaturated, fatty acids induce apoptosis of human coronary artery endothelial cells via nuclear factor-κB activation.Diabetes. 2006; 55: 3121-3126Crossref PubMed Scopus (118) Google Scholar, 16Chai W. Liu Z. p38 Mitogen-activated protein kinase mediates palmitate-induced apoptosis but not IκB degradation in human coronary artery endothelial cells.Endocrinology. 2007; 148: 1622-1628Crossref PubMed Scopus (56) Google Scholar, 17Eiselein L. Wilson D.W. Lamé M.W. Rutledge J.C. Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis.Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H2745-H2753Crossref PubMed Scopus (84) Google Scholar) and have not been performed in EPCs. Hitherto, FFAs' chain length, degree of saturation, and/or position of double bonds (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 7Staiger K. Staiger H. Weigert C. Haas C. Häring H-U. Kellerer M. Saturated, but not unsaturated, fatty acids induce apoptosis of human coronary artery endothelial cells via nuclear factor-κB activation.Diabetes. 2006; 55: 3121-3126Crossref PubMed Scopus (118) Google Scholar), p38 mitogen-activated protein kinase (p38 MAPK) (5Dersch K. Ichijo H. Bhakdi S. Husmann M. Fatty acids liberated from low-density lipoprotein trigger endothelial apoptosis via mitogen-activated protein kinases.Cell Death Differ. 2005; 12: 1107-1114Crossref PubMed Scopus (32) Google Scholar, 16Chai W. Liu Z. p38 Mitogen-activated protein kinase mediates palmitate-induced apoptosis but not IκB degradation in human coronary artery endothelial cells.Endocrinology. 2007; 148: 1622-1628Crossref PubMed Scopus (56) Google Scholar), NF-κB (7Staiger K. Staiger H. Weigert C. Haas C. Häring H-U. Kellerer M. Saturated, but not unsaturated, fatty acids induce apoptosis of human coronary artery endothelial cells via nuclear factor-κB activation.Diabetes. 2006; 55: 3121-3126Crossref PubMed Scopus (118) Google Scholar, 16Chai W. Liu Z. p38 Mitogen-activated protein kinase mediates palmitate-induced apoptosis but not IκB degradation in human coronary artery endothelial cells.Endocrinology. 2007; 148: 1622-1628Crossref PubMed Scopus (56) Google Scholar), bcl-2 family members (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 4Hufnagel B. Dworak M. Soufi M. Mester Z. Zhu Y. Schaefer J.R. Klumpp S. Krieglstein J. Unsaturated fatty acids isolated from human lipoproteins activate protein phosphatase type 2Cβ and induce apoptosis in endothelial cells.Atherosclerosis. 2005; 180: 245-254Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and executioner caspases, i.e., caspase-3 and/or -7 (5Dersch K. Ichijo H. Bhakdi S. Husmann M. Fatty acids liberated from low-density lipoprotein trigger endothelial apoptosis via mitogen-activated protein kinases.Cell Death Differ. 2005; 12: 1107-1114Crossref PubMed Scopus (32) Google Scholar, 17Eiselein L. Wilson D.W. Lamé M.W. Rutledge J.C. Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis.Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H2745-H2753Crossref PubMed Scopus (84) Google Scholar, 18Artwohl M. Hölzenbein T. Fürnsinn C. Freudenthaler A. Huttary N. Waldhäusl W.K. Baumgartner-Parzer S.M. Thiazolidinediones inhibit apoptosis and heat shock protein 60 expression in human vascular endothelial cells.Thromb. Haemost. 2005; 93: 810-815Crossref PubMed Scopus (26) Google Scholar) have been reported to be involved in the pro-apoptotic action of selected FFAs. Characterization of underlying mechanisms for a broad spectrum of both selected nutritional FFAs and different human endothelial cell types is missing as yet. The present study therefore evaluated the effects of a broad spectrum of nutritional FFAs (saturated/monounsaturated/polyunsaturated; ω3/ω6/ω9) on endothelial apoptosis in target tissues of late diabetic vascular complications, i.e., human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs) as well as in human EPCs, which presumably have a role in vascular repair. Our study shows that FFA-induced apoptosis depends on the vascular origin of endothelial cells, the growth/proliferation status of the cells, and the FFA structure. Whereas the pro-apoptotic activity of saturated stearic acid is apparently related to the membrane rigidity of the cells, PUFA-induced apoptosis is mediated via c-myc/E2F-1/XRCC1/caspase-8. HUVECs, HAECs, and HRECs were isolated, cultured, and identified as described (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 18Artwohl M. Hölzenbein T. Fürnsinn C. Freudenthaler A. Huttary N. Waldhäusl W.K. Baumgartner-Parzer S.M. Thiazolidinediones inhibit apoptosis and heat shock protein 60 expression in human vascular endothelial cells.Thromb. Haemost. 2005; 93: 810-815Crossref PubMed Scopus (26) Google Scholar, 19Artwohl M. Fürnsinn C. Waldhäusl W. Hölzenbein T. Rainer G. Freudenthaler A. Roden M. Baumgartner-Parzer S.M. Thiazolidinediones inhibit proliferation of micro- and macrovascular endothelial cells. Evidence for a PPARγ-independent mitochondrial mechanism.Diabetologia. 2005; 48: 586-594Crossref PubMed Scopus (31) Google Scholar, 20Artwohl M. Graier W. Roden M. Bischof M. Freudenthaler A. Waldhäusl W. Baumgartner-Parzer S.M. Diabetic LDL triggers apoptosis in vascular endothelial cells.Diabetes. 2003; 52: 1240-1247Crossref PubMed Scopus (53) Google Scholar) and used in the 1st, 5th, and 2nd subcultures, respectively. Circulating EPCs from peripheral blood of healthy human volunteers were obtained with the approval of the local ethics board from the Department of Clinical Pharmacology and were isolated by density-gradient centrifugation (400 g for 30 min) using Histopaque-1077 (Sigma; St. Louis, MO) overlaid with an equal volume of fresh anti-coagulated whole blood at room temperature. The fraction containing the peripheral blood mononuclear cells was then transferred into a fresh vial, washed with PBS (Hyclone; Logan, UT), and cultured on fibronectin- (Sigma) coated culture dishes in Medium 199 (M199; Sigma) supplemented with 20% fetal calf serum (Hyclone), 5 U/ml heparin (Biochrom; Berlin, Germany), 100 U/ml penicillin, 100 μg/ml streptomycin, and 500 ng/ml fungizone (Hyclone). EPCs grew out from colonies and differentiated into endothelial cells of >90% purity, as characterized by triple staining showing the following expression pattern: CD31+/CD144+/CD14−, representing positive staining for the endothelial marker proteins CD31 (platelet-endothelial cell adhesion molecule-1; BD Pharmingen, San José, CA) and CD144 (vascular endothelial-cadherin; Bender MedSystems, Vienna, Austria), but negative staining for the monocyte marker CD14 (BD Pharmingen). Additionally, the isolated EPCs expressed the endothelial marker proteins CD146 (S-endo; BD Pharmingen) and endocan (endothelial specific molecule-1; R and D Systems, Minneapolis, MN). Growing HUVECs, HAECs, HRECs, and EPCs were exposed either to FFAs (100–300 μmol/l; palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1ω9), linoleic acid (C18:2ω6), α-linolenic acid (C18:3ω3), arachidonic acid (C20:4ω6) (Sigma), or to the respective ethanol concentration (1–3 ppm; used as solvent control) in M199 supplemented with 20% delipidated FBS (Sigma; final concentration of albumin, 0.4%) and antibiotics. Solely for analysis of c-myc dependence of apoptosis, confluent HUVECs were, as well, exposed to 300 μmol/l of palmitic acid, stearic acid, oleic acid, linoleic acid, and α-linolenic acid. [3H]thymidine- (1 μCi/ml) labeled endothelial cells, trypsinized and seeded into 24-well culture plates, were exposed to FFAs for 24 h (HUVECs, HAECs, EPCs, HRECs) and for 48 h (HRECs), and experimental as well as intra-individual control cultures (exposed to ethanol) were tested for apoptosis (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 18Artwohl M. Hölzenbein T. Fürnsinn C. Freudenthaler A. Huttary N. Waldhäusl W.K. Baumgartner-Parzer S.M. Thiazolidinediones inhibit apoptosis and heat shock protein 60 expression in human vascular endothelial cells.Thromb. Haemost. 2005; 93: 810-815Crossref PubMed Scopus (26) Google Scholar, 20Artwohl M. Graier W. Roden M. Bischof M. Freudenthaler A. Waldhäusl W. Baumgartner-Parzer S.M. Diabetic LDL triggers apoptosis in vascular endothelial cells.Diabetes. 2003; 52: 1240-1247Crossref PubMed Scopus (53) Google Scholar). In brief, the cells were lysed (20 mmol/l Tris-HCl, pH 7.5, 0.4% Triton X-100; 10 min on ice), and fragmented (apoptotic) DNA was separated by centrifugation (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 18Artwohl M. Hölzenbein T. Fürnsinn C. Freudenthaler A. Huttary N. Waldhäusl W.K. Baumgartner-Parzer S.M. Thiazolidinediones inhibit apoptosis and heat shock protein 60 expression in human vascular endothelial cells.Thromb. Haemost. 2005; 93: 810-815Crossref PubMed Scopus (26) Google Scholar, 20Artwohl M. Graier W. Roden M. Bischof M. Freudenthaler A. Waldhäusl W. Baumgartner-Parzer S.M. Diabetic LDL triggers apoptosis in vascular endothelial cells.Diabetes. 2003; 52: 1240-1247Crossref PubMed Scopus (53) Google Scholar). Radioactivity of fragmented versus total DNA (digested with 180 μg/ml DNase I; Boehringer Mannheim, Germany) was quantified using a Tri-Carb 3100 TR liquid scintillation analyzer (Packard Instruments; Meriden, CT). Experiments were performed in triplicate. In examination of FFA-induced endothelial apoptosis, we have already shown that these assays lead to results comparable to those of different commercially available kits for apoptosis detection (i.e., TdT-FragEL™, Oncogene, Boston, MA; MitoCapture™, BioVision, Palo Alto, CA) (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 18Artwohl M. Hölzenbein T. Fürnsinn C. Freudenthaler A. Huttary N. Waldhäusl W.K. Baumgartner-Parzer S.M. Thiazolidinediones inhibit apoptosis and heat shock protein 60 expression in human vascular endothelial cells.Thromb. Haemost. 2005; 93: 810-815Crossref PubMed Scopus (26) Google Scholar). In some experiments, apoptosis was determined in HUVECs, HAECs, EPCs, and HRECs coincubated with the different FFAs along with caspase inhibitors (30 μmol/l z-VAD.fmk or 20 μmol/l z-IETD.fmk; Calbiochem, Palo Alto, CA) or eicosapentaenoic acid (EPA, 5–20 μmol/l; Sigma). HUVECs, HAECs, EPCs, and HRECs were exposed (24 h) to FFAs (300 μmol/l) followed by cell membrane preparation. In brief, the cells were lysed on ice in hypotonic buffer (final concentrations: 5 mmol/l MgCl2, 10 mmol/l HEPES, pH 7.4, 40 mmol/l KCl) followed by shearing the lysate 10 times through a 30 G needle. After centrifugation (10 min, 200 g, 4°C), the supernatant was subjected to an ultracentrifugation step (30 min., 28,000 rpm, 4°C, rotor: TLA 55), and the pelleted cell membranes were resuspended in PBS, followed by lyophilization. Lyophilized cell membrane preparations were reconstituted in distilled water, and an internal standard (C17:0, heptadecanoic acid; Riedel de Haën, Seelze, Germany) was added to each sample and to an FFA standard mixture (Altech, Deerfield, IL). The samples were then extracted and transesterified to methyl esters by a one-step reaction as follows. After addition of the reaction mixture [2-propanol-n-heptane-H2SO4 (0.25 mol/l) 40:10:1; all Merck, Darmstadt, Germany], samples were mixed vigorously before extraction with n-heptane and distilled water. After centrifugation (10 min, 1,000 g, 4°C), the resulting upper layer was transferred into reaction vials (Pyrex; Bibby Sterilin Ltd, Staffordshire, UK) and evaporated under nitrogen. Methanolysis was performed by addition of methanol-benzene (4:1) and acetyl chloride (Fluka; Buchs, Switzerland) at 100°C for 75 min under continuous stirring. The reaction was stopped by addition of 6% K2CO3, and the organic phase was collected, followed by extraction with benzene. Fatty acid methyl esters were analyzed using a Hewlett-Packard (Boise, ID) GC-MSD 5973 system equipped with a 30 m, 0.12 μm DB23 fused silica column, inner diameter 0.25 mm·(J and W Scientific, Folsom, CA). Gas chromatography was operated at 50°C for 2 min, rising to 180°C at 10°C/min, followed by a 5 min hold, rising to 240°C at 5°C/min, followed by a 2 min hold and rising at least to 250°C at 3°C/min under constant flow (1.1 ml/min) of helium as a carrier gas. The above-mentioned standards (internal standard and FFA standard mixture) were detected by electronic impact ionization mass spectrometry using total ion current and their extracted ionized total mass peak (M+) values to calculate quantification factors in relation to the intensity of the internal standard. FFAs incorporated in the cellular membranes were identified by their retention times and were quantified in relation to the intensity of the internal standard. HUVECs were cultured on a poly-d-lysine-coated LabTek chambered coverglass and then exposed to stearic acid ± EPA or to arachidonic acid (used as PUFA control) versus ethanol (solvent). Cells were then incubated (20 min) with 10 μmol/l of the lipophilic membrane marker 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO; Molecular Probes, Eugene, OR) in a nonfluorescent binding buffer (21Maier C. Rünzler D. Schindelar J. Grabner G. Waldhäusl W. Köhler G. Luger A. G-protein-coupled glucocorticoid receptors on the pituitary cell membrane.J. Cell Sci. 2005; 118: 3353-3361Crossref PubMed Scopus (55) Google Scholar). Fluorescence correlation spectroscopy (FCS) was used to evaluate the diffusion behavior of the fluorescent marker DiO in the cell membrane. Details of the method have been published elsewhere (21Maier C. Rünzler D. Schindelar J. Grabner G. Waldhäusl W. Köhler G. Luger A. G-protein-coupled glucocorticoid receptors on the pituitary cell membrane.J. Cell Sci. 2005; 118: 3353-3361Crossref PubMed Scopus (55) Google Scholar). Briefly, FCS measurements were carried out on a Confocor spectrofluorimeter (Carl Zeiss-Evotec, Jena, Germany). The pinhole diameter was set to 45 μm, resulting in a confocal volume element of 0.17 μm in the radial dimension and 2.4 μm in the axial dimension. The confocal volume was positioned in the cells using an x-y stage with 1 μm resolution, whereas the correct focus positioning on the cell membrane was ascertained by a scanning procedure in 1 μm steps. Autocorrelation curves were best-fitted to the two-component model, with component τ1 corresponding to the diffusion time of DiO in solution and component τ2 representing DiO diffusion on the cell membrane derived from the fitting procedure. Autocorrelation curves (n = 20–34) taken at the membrane position from six individual cells were evaluated for each experimental subgroup. Associated protein expression was determined by Western blot analyses as described (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 19Artwohl M. Fürnsinn C. Waldhäusl W. Hölzenbein T. Rainer G. Freudenthaler A. Roden M. Baumgartner-Parzer S.M. Thiazolidinediones inhibit proliferation of micro- and macrovascular endothelial cells. Evidence for a PPARγ-independent mitochondrial mechanism.Diabetologia. 2005; 48: 586-594Crossref PubMed Scopus (31) Google Scholar) in growing HUVECs with/without FFAs after 24 h exposure. In brief, endothelial cells were lysed in cold Weinberg buffer (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 19Artwohl M. Fürnsinn C. Waldhäusl W. Hölzenbein T. Rainer G. Freudenthaler A. Roden M. Baumgartner-Parzer S.M. Thiazolidinediones inhibit proliferation of micro- and macrovascular endothelial cells. Evidence for a PPARγ-independent mitochondrial mechanism.Diabetologia. 2005; 48: 586-594Crossref PubMed Scopus (31) Google Scholar) and total protein was measured using a BCA protein assay (Pierce; Rockford, IL) according the manufacturer's instructions. An aliquot containing exactly 10 μg total protein was loaded in each lane and subsequently subjected to SDS-PAGE, followed by transfer onto nitrocellulose membranes (Schleicher and Schüll; Kassel, Germany). After controlling for homogenous sample loading by staining the blots with Ponceau S (Sigma), blocking of unspecific binding sites with nonfat dry milk (5% in TBS) containing 0.05% Tween-20 (Bio Rad; Hercules, CA) was followed by incubation with primary antibodies (mad, 1:2,000, PharMingen, San Diego, CA; c-myc, 1:200, BioSource, Camarillo, CA; XRCC1, 1:500, Kamiya Biomedical Co., Seattle, WA; E2F-1, 1:200, NeoMarkers, Fremont, CA) and detection with HRP-conjugated anti-mouse or anti-rabbit IgG (Amersham Pharmacia; Buckinghamshire, UK) using Super Signal Substrate (Pierce). Results are expressed in relation to intra-individual control cells (set to 100%). Confluent monolayers of primary HUVECs were transiently transfected (4 h) with 2 μg/ml In373-Myc (generously provided by V. Sexl, Vienna) or with the empty vector (pSRαMSV) using polyethyleneimine 25,000 (pH 7.0, Aldrich; Milwaukee, WI) (22Mahato R.I. Lee M. Han S. Maheshwari A. Kim S.W. Intratumoral delivery of p2CMVmIL-12 using water-soluble lipopolymers.Mol. Ther. 2001; 4: 130-138Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) in serum-free M199. After overnight equilibration in full growth medium, HUVECs were trypsinized and seeded into 35 mm culture plates (2 × 107 cells/plate). After adherence (6 h) and exposure (24 h) to the different FFAs versus intra-individual control cells (incubated with ethanol as solvent), the cells were fixed (75% ice-cold ethanol) and DNA fragmentation was determined after staining with propidium iodide (1 μg/ml) by fluorescence-activated cell sorting analyses using an FACS Calibur (Becton Dickinson; Heidelberg, Germany) as described previously (3Artwohl M. Roden M. Waldhäusl W. Freudenthaler A. Baumgartner-Parzer S.M. Free fatty acids trigger apoptosis and inhibit cell cycle progression in human vascular endothelial cells.FASEB J. 2004; 18: 146-148Crossref PubMed Scopus (132) Google Scholar, 19Artwohl M. Fürnsinn C. Waldhäusl W. Hölzenbein T. Rainer G. Freudenthaler A. Roden M. Baumgartner-Parzer S.M. Thiazolidinediones inhibit proliferation of micro- and macrovascular endothelial cells. Evidence for a PPARγ-independent mitochondrial mechanism.Diabetologia. 2005; 48: 586-594Crossref PubMed Scopus (31) Google Scholar). Transfection efficiency was controlled by immunocytochemical determination of cotransfected CD8. Da
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