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MS132 QUANTITATIVE PROTEOMIC APPROACH TO IDENTIFY PROTEINS INVOLVED IN ACUTE CORONARY SYNDROME
136
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
present following release upon stimulation with vascular chemokine IL-8 in cultured PMNs together with lactoferrin± specific granules localized in neutrophil extracellular traps (NETs) formed by extruded DNA. Moreover, extracellular expression of PTX3 protein was observed in thrombi containing abundant PMNs, suggesting the release of PTX3 protein from PMNs. Nevertheless, we did not find any evidence of PTX3 released by foamy M÷s. MS131 THE EXPRESSION OF CYTOKINE GENES IN THE AORTA INFLUENCED BY DIET: THE EFFECT OF MCP-1 DEFICIENCY A. Rull, R. Beltran-Deb ´ on, ´ G. Aragones, ` F. Rodr´ıguez, C. Alonso-Villaverde, ` J. Camps, J. Joven. Centre de Recerca Biomedica, Hospital Universitari Sant Joan de Reus, IISPV, Universitat Rovira i Virgili, Reus, Spain Background: Monocyte chemoattractant protein-1 (MCP-1) facilitates the recruitment of monocytes/macrophages into vascular intima, and it is probably involved in the regulation of other signaling pathways relevant to the pathogenesis of arteriosclerosis and metabolic disturbances. However, chemokines are redundant. Consequently, the protective effect of MCP-1 deficiency may be mediated by changes in other cytokine signals. Methods and Results: Changes in the pattern of gene expression in the aorta were evaluated in LDLr−/− and MCP-1−/− LDLr−/− mice fed either chow or Western-style diet. The effect of high-fat diet was evident. Functional analyses were used to characterize the pathways affected and to identify biological processes in which MCP-1 may play an additional role. Some data suggest that MCP-5 may act as a surrogate for MCP-1 deletion. Arteriosclerosis lesion and plaque composition are associated with enrichment in the cytokine-cytokine receptor interaction pathway. Conclusions: There is a complex network of interactions linking MCP-1 and other cytokines. Particularly, the absence of MCP-1 limits the aortic response to atherogenic stimuli; an effect that it is even more evident when combined with a diet rich in fat and cholesterol. MS132 QUANTITATIVE PROTEOMIC APPROACH TO IDENTIFY PROTEINS INVOLVED IN ACUTE CORONARY SYNDROME F. Gil-Dones1 , C.M. Laborde2 , S. Alonso-Orgaz1 , J. Moreu3 , F. Vivanco4,5 , L.R. Padial1,3 , M.G. Barderas1 . 1 Department of Vascular Pathophysiology, Hospital Nacional de Paraplejicos, 2 Biochemistry, 3 Cardiology Department, ´ Hospital Virgen de la Salud, Toledo, 4 Inmunology Department, Fundacion Jimenez Diaz, 5 Department of Biochemistry and Molecular Biology I, Universidad Complutense de Madrid, Madrid, Spain Introduction: Acute coronary syndrome (ACS) is one of the most important cardiovascular diseases affecting developed countries worldwide. An early endothelium damage in the coronary arteries leads to an endothelial cell barrier dysfunction which allows the lipid storage and monocytes migration into the intima and means the initial stages of atherosclerosis. Thus, due to their strong association with the development of atherosclerosis, the study of plasma and circulating monocytes proteome represents one of the main pathways in the search for potential biomarkers of ACS. Objective: Our goal was to search for differentially expressed proteins in plasma and circulating monocytes using two-dimensional difference gel electrophoresis (2D-DIGE), isobaric tag for relative and absolute quantitation (i-TRAQ) and MALDI-TOF/TOF mass spectrometry. Material and Methods: 20 patients with Non-ST-Segment Elevation ACS recruited from the cardiology service of our hospital and 20 healthy controls with up to two risk factors were used for this study. Depleted plasma samples patients and healthy controls were selected for 2D-DIGE, i-TRAQ and MALDI-TOF/TOF analyses. Results: We found 25 differentially expressed proteins in plasma proteome using 2D-DIGE and MALDI-TOF/TOF, several of which were also identified by i-TRAQ analysis and both results were validated by inmunoblotting. Our ongoing 2D-DIGE and i-TRAQ experiments using circulating monocytes are reporting promising results as well. Conclusions: We expect our simultaneous study of plasma and circulating monocytes proteome allows us to identify potential protein biomarkers for the future development of therapeutic strategies for ACS. MS133 ENDOPLASMIC RETICULUM STRESS INHIBITION PREVENTS THE ABCA-1 REDUCTION IN GLYCATED ALBUMIN-TREATED MACROPHAGES G. Castilho1 , C.H. Sartori1 , A. Machado-Lima1 , E.R. Nakandakare1 , C.X.d.C. Santos2 , F.R. Laurindo2 , M. Passarelli1 . 1 Lipids Laboratory (LIM10), Faculty of Medical Sciences of the University of Sao Paulo, 2 Vascular Biology ˜ Paulo, Sao Paulo, Laboratory, Heart Institute (InCor) − University of Sao Brazil Objectives: In vivo and in vitro glycated albumin (gly-alb) reduces ABCA-1 protein levels, which is not related to alterations in ABCA-1 gene transcription or mRNA levels. We tested in macrophages the in vitro and in vivo effects of gly-alb on the endoplasmic reticulum (ER) stress and on the unfolded protein response (UPR) that trigger protein degradation.
Memory Stick Presentations
Methods: Gly-alb was made by incubating bovine albumin with 10mM glycolaldehyde (4 days, 37ºC), and control albumin (C-alb) with PBS alone. In vivo gly-alb (DM-alb) was isolated from uncontrolled diabetes mellitus patients serum by FPLC (HbA1c>10%). Mouse peritoneal macrophages (MPM) were incubated with C-alb or gly-alb along time and ER stress markers assessed by immunoblot. ABCA-1 content was determined by immunocytochemistry in MPM treated with gly-alb in the absence or presence of the proteasomal inhibitor (MG132, 1mM), or the ER stress inhibitor 4-phenylbutyric acid (PBA, 5mM). Results: As compared to C-alb, gly-alb induced a time-dependent increase in Grp78, Grp94, eIf2a, ATF6 and ubiquitin indicating ER stress. No difference was observed in CHOP expression which indicates lack of apoptotic signalling. DM-alb induced greater expression of both PDI and ubiquitin comparing to albumin from non diabetics indicating cell redox imbalance and proteasomal activation, respectively. Nonetheless, PBA, but not MG132, was able to recover the ABCA-1 content in gly-alb-treated MPM. Conclusion: Glycated albumin induces ER stress and triggers UPR adaptive pathways leading to ABCA-1 reduction in macrophages. Inhibition of the ER stress recovers the ABCA-1 content thus improving the macrophage reverse cholesterol transport in diabetes mellitus. Funding: FAPESP (Brazil) MS134 HISTONE ACETYLATION MODIFIERS BUTYRATE AND TRICHOSTATIN A MODULATE EXPRESSION OF HISTONE DEACETYLASES (HDACS) IN VASCULAR SMOOTH MUSCLE CELLS (VSMC) K. Ranganna1 , F. Yatsu2 , O. Mathew1 . 1 Pharmaceutical Sciences, Texas Southern University, 2 Neurology, University of Texas Health Science Center at Houston, Houston, TX, USA Background: Besides genetic components epigenetic events are equally responsible for the etiology of many diseases including atherosclerosis and restenosis. Histone acetylation, a key epigenetic modification that controls chromatin structure and through it, gene expression is regulated by the counterbalancing activities of HDACs and histone acetyltransferases (HATs). Inhibition of HDAC activity has been shown to arrest proliferation, a key factor in atherogenesis, and induce differentiation or apoptosis by modulating gene transcription. Aim: To assess effect of butyrate and trichostatin A (TSA) on the expression of HDACs to determine whether altered expression of HDACs also contributes to arrest of VSMC proliferation. Methods: Cell proliferation was assessed by cell counting. Expression of HDACs and histone H3 (H3) modifications were determined by Western analysis, immunocytofluorescence, and immunoprecipitation. Results: Butyrate and TSA treatment arrested VSMC proliferation and caused almost similar effects on the expression of HDACs and H3 modifications. However, contrary to untreated VSMC, butyrate and TSA treated VSMC exhibited downregulation of HDAC2 and HDAC5, which was confirmed by immunoprecipitation and immunocytofluorescence. Conversely, no significant difference in HDAC1 expression was observed between untreated, and butyrate and TSA treated VSMC. Moreover, acetylation of lysine9 and lysine14 of H3 was significantly increased with butyrate and TSA treatment. Conclusions: Overall both HDAC inhibitory activity and altered expression of certain HDACs appear to contribute to induction of histone H3 acetylation by butyrate and TSA. This butyrate and TSA induced H3 acetylation appears to contribute to arrest of VSMC proliferation through the transcriptional modulation of cell cycle regulatory proteins. (NCRR/NIH/RCMI) MS135 ADIPOPHILIN LOW EXPRESSION REDUCE CELLULAR LIPID Z. Yuan, X. Liu, Q. Liu, Z. Liu, C. Tang, Z. Wang, G. Yi, L. Liu, Z. Jiang, Y. Yang. Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Human Province, University of South China, Hengyang, China Objective: To explore whether adipophilin affects the expression of ACAT1 mediated by PKC signal pathway and illuminate the lipid-accumulation mechanism mediated by adipophilin. Method: Making recombinant retroviral vector of pSuper-retro-adipophilin siRNA, transfecting it into packaging cell PA317 and getting the retrovirus. The retrovirus were infected RAW264.7 cell and got adipophilin gene low expression RAW264.7 cell line after screening with Puromycin for two weeks. The level of adipohilin and ACAT1 was determined by RT-PCR and Western Blot. Cellular lipid accumulation was determined by Oil Red O staining. The cells were incubated with atorvastatin or PKC inhibitor calphostin C for determining the mechanism. Result: With adipophilin siRNA, RT-PCR and Western Blot showed adipophilin mRNA and protein both down-regulated significantly in RAW264.7 cells. It also made the lipid droplets decreasing, but both the blank and negative control not. As negative control, the pSuper-retro-scramble siRNA transfection could slightly down-regulated the expression of ACAT1, and the pSuper transfection had no effect on ACAT1 expression. But adipophilin siRNA significantly decreased ACAT1 expression. Adipophilin siRNA also declined the expression of ACAT1
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
present following release upon stimulation with vascular chemokine IL-8 in cultured PMNs together with lactoferrin± specific granules localized in neutrophil extracellular traps (NETs) formed by extruded DNA. Moreover, extracellular expression of PTX3 protein was observed in thrombi containing abundant PMNs, suggesting the release of PTX3 protein from PMNs. Nevertheless, we did not find any evidence of PTX3 released by foamy M÷s. MS131 THE EXPRESSION OF CYTOKINE GENES IN THE AORTA INFLUENCED BY DIET: THE EFFECT OF MCP-1 DEFICIENCY A. Rull, R. Beltran-Deb ´ on, ´ G. Aragones, ` F. Rodr´ıguez, C. Alonso-Villaverde, ` J. Camps, J. Joven. Centre de Recerca Biomedica, Hospital Universitari Sant Joan de Reus, IISPV, Universitat Rovira i Virgili, Reus, Spain Background: Monocyte chemoattractant protein-1 (MCP-1) facilitates the recruitment of monocytes/macrophages into vascular intima, and it is probably involved in the regulation of other signaling pathways relevant to the pathogenesis of arteriosclerosis and metabolic disturbances. However, chemokines are redundant. Consequently, the protective effect of MCP-1 deficiency may be mediated by changes in other cytokine signals. Methods and Results: Changes in the pattern of gene expression in the aorta were evaluated in LDLr−/− and MCP-1−/− LDLr−/− mice fed either chow or Western-style diet. The effect of high-fat diet was evident. Functional analyses were used to characterize the pathways affected and to identify biological processes in which MCP-1 may play an additional role. Some data suggest that MCP-5 may act as a surrogate for MCP-1 deletion. Arteriosclerosis lesion and plaque composition are associated with enrichment in the cytokine-cytokine receptor interaction pathway. Conclusions: There is a complex network of interactions linking MCP-1 and other cytokines. Particularly, the absence of MCP-1 limits the aortic response to atherogenic stimuli; an effect that it is even more evident when combined with a diet rich in fat and cholesterol. MS132 QUANTITATIVE PROTEOMIC APPROACH TO IDENTIFY PROTEINS INVOLVED IN ACUTE CORONARY SYNDROME F. Gil-Dones1 , C.M. Laborde2 , S. Alonso-Orgaz1 , J. Moreu3 , F. Vivanco4,5 , L.R. Padial1,3 , M.G. Barderas1 . 1 Department of Vascular Pathophysiology, Hospital Nacional de Paraplejicos, 2 Biochemistry, 3 Cardiology Department, ´ Hospital Virgen de la Salud, Toledo, 4 Inmunology Department, Fundacion Jimenez Diaz, 5 Department of Biochemistry and Molecular Biology I, Universidad Complutense de Madrid, Madrid, Spain Introduction: Acute coronary syndrome (ACS) is one of the most important cardiovascular diseases affecting developed countries worldwide. An early endothelium damage in the coronary arteries leads to an endothelial cell barrier dysfunction which allows the lipid storage and monocytes migration into the intima and means the initial stages of atherosclerosis. Thus, due to their strong association with the development of atherosclerosis, the study of plasma and circulating monocytes proteome represents one of the main pathways in the search for potential biomarkers of ACS. Objective: Our goal was to search for differentially expressed proteins in plasma and circulating monocytes using two-dimensional difference gel electrophoresis (2D-DIGE), isobaric tag for relative and absolute quantitation (i-TRAQ) and MALDI-TOF/TOF mass spectrometry. Material and Methods: 20 patients with Non-ST-Segment Elevation ACS recruited from the cardiology service of our hospital and 20 healthy controls with up to two risk factors were used for this study. Depleted plasma samples patients and healthy controls were selected for 2D-DIGE, i-TRAQ and MALDI-TOF/TOF analyses. Results: We found 25 differentially expressed proteins in plasma proteome using 2D-DIGE and MALDI-TOF/TOF, several of which were also identified by i-TRAQ analysis and both results were validated by inmunoblotting. Our ongoing 2D-DIGE and i-TRAQ experiments using circulating monocytes are reporting promising results as well. Conclusions: We expect our simultaneous study of plasma and circulating monocytes proteome allows us to identify potential protein biomarkers for the future development of therapeutic strategies for ACS. MS133 ENDOPLASMIC RETICULUM STRESS INHIBITION PREVENTS THE ABCA-1 REDUCTION IN GLYCATED ALBUMIN-TREATED MACROPHAGES G. Castilho1 , C.H. Sartori1 , A. Machado-Lima1 , E.R. Nakandakare1 , C.X.d.C. Santos2 , F.R. Laurindo2 , M. Passarelli1 . 1 Lipids Laboratory (LIM10), Faculty of Medical Sciences of the University of Sao Paulo, 2 Vascular Biology ˜ Paulo, Sao Paulo, Laboratory, Heart Institute (InCor) − University of Sao Brazil Objectives: In vivo and in vitro glycated albumin (gly-alb) reduces ABCA-1 protein levels, which is not related to alterations in ABCA-1 gene transcription or mRNA levels. We tested in macrophages the in vitro and in vivo effects of gly-alb on the endoplasmic reticulum (ER) stress and on the unfolded protein response (UPR) that trigger protein degradation.
Memory Stick Presentations
Methods: Gly-alb was made by incubating bovine albumin with 10mM glycolaldehyde (4 days, 37ºC), and control albumin (C-alb) with PBS alone. In vivo gly-alb (DM-alb) was isolated from uncontrolled diabetes mellitus patients serum by FPLC (HbA1c>10%). Mouse peritoneal macrophages (MPM) were incubated with C-alb or gly-alb along time and ER stress markers assessed by immunoblot. ABCA-1 content was determined by immunocytochemistry in MPM treated with gly-alb in the absence or presence of the proteasomal inhibitor (MG132, 1mM), or the ER stress inhibitor 4-phenylbutyric acid (PBA, 5mM). Results: As compared to C-alb, gly-alb induced a time-dependent increase in Grp78, Grp94, eIf2a, ATF6 and ubiquitin indicating ER stress. No difference was observed in CHOP expression which indicates lack of apoptotic signalling. DM-alb induced greater expression of both PDI and ubiquitin comparing to albumin from non diabetics indicating cell redox imbalance and proteasomal activation, respectively. Nonetheless, PBA, but not MG132, was able to recover the ABCA-1 content in gly-alb-treated MPM. Conclusion: Glycated albumin induces ER stress and triggers UPR adaptive pathways leading to ABCA-1 reduction in macrophages. Inhibition of the ER stress recovers the ABCA-1 content thus improving the macrophage reverse cholesterol transport in diabetes mellitus. Funding: FAPESP (Brazil) MS134 HISTONE ACETYLATION MODIFIERS BUTYRATE AND TRICHOSTATIN A MODULATE EXPRESSION OF HISTONE DEACETYLASES (HDACS) IN VASCULAR SMOOTH MUSCLE CELLS (VSMC) K. Ranganna1 , F. Yatsu2 , O. Mathew1 . 1 Pharmaceutical Sciences, Texas Southern University, 2 Neurology, University of Texas Health Science Center at Houston, Houston, TX, USA Background: Besides genetic components epigenetic events are equally responsible for the etiology of many diseases including atherosclerosis and restenosis. Histone acetylation, a key epigenetic modification that controls chromatin structure and through it, gene expression is regulated by the counterbalancing activities of HDACs and histone acetyltransferases (HATs). Inhibition of HDAC activity has been shown to arrest proliferation, a key factor in atherogenesis, and induce differentiation or apoptosis by modulating gene transcription. Aim: To assess effect of butyrate and trichostatin A (TSA) on the expression of HDACs to determine whether altered expression of HDACs also contributes to arrest of VSMC proliferation. Methods: Cell proliferation was assessed by cell counting. Expression of HDACs and histone H3 (H3) modifications were determined by Western analysis, immunocytofluorescence, and immunoprecipitation. Results: Butyrate and TSA treatment arrested VSMC proliferation and caused almost similar effects on the expression of HDACs and H3 modifications. However, contrary to untreated VSMC, butyrate and TSA treated VSMC exhibited downregulation of HDAC2 and HDAC5, which was confirmed by immunoprecipitation and immunocytofluorescence. Conversely, no significant difference in HDAC1 expression was observed between untreated, and butyrate and TSA treated VSMC. Moreover, acetylation of lysine9 and lysine14 of H3 was significantly increased with butyrate and TSA treatment. Conclusions: Overall both HDAC inhibitory activity and altered expression of certain HDACs appear to contribute to induction of histone H3 acetylation by butyrate and TSA. This butyrate and TSA induced H3 acetylation appears to contribute to arrest of VSMC proliferation through the transcriptional modulation of cell cycle regulatory proteins. (NCRR/NIH/RCMI) MS135 ADIPOPHILIN LOW EXPRESSION REDUCE CELLULAR LIPID Z. Yuan, X. Liu, Q. Liu, Z. Liu, C. Tang, Z. Wang, G. Yi, L. Liu, Z. Jiang, Y. Yang. Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Human Province, University of South China, Hengyang, China Objective: To explore whether adipophilin affects the expression of ACAT1 mediated by PKC signal pathway and illuminate the lipid-accumulation mechanism mediated by adipophilin. Method: Making recombinant retroviral vector of pSuper-retro-adipophilin siRNA, transfecting it into packaging cell PA317 and getting the retrovirus. The retrovirus were infected RAW264.7 cell and got adipophilin gene low expression RAW264.7 cell line after screening with Puromycin for two weeks. The level of adipohilin and ACAT1 was determined by RT-PCR and Western Blot. Cellular lipid accumulation was determined by Oil Red O staining. The cells were incubated with atorvastatin or PKC inhibitor calphostin C for determining the mechanism. Result: With adipophilin siRNA, RT-PCR and Western Blot showed adipophilin mRNA and protein both down-regulated significantly in RAW264.7 cells. It also made the lipid droplets decreasing, but both the blank and negative control not. As negative control, the pSuper-retro-scramble siRNA transfection could slightly down-regulated the expression of ACAT1, and the pSuper transfection had no effect on ACAT1 expression. But adipophilin siRNA significantly decreased ACAT1 expression. Adipophilin siRNA also declined the expression of ACAT1