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I 006 Lack of Association Between Inflammatory Parameters and Carotid Intima Media-thickness in Subjects with Spinal Cord Injury
I 005
Focal Adhesion Kinase is Critical for the Expression of Pro-atherogenic Molecules in Vascular Cells Subjected to Mechanical Stress
I 007 1Oliveira
Identification and Characterization of TH17 Response in Human Atherosclerosis RTD, 1Teo FH, 1Silva RM, 1Fornazim MC, 2Souza JRM, RL, 3Altemani AMA, 4Menezes FH, 1Blotta MHSL
Fernandes MN, Cardoso C, Clemente C, Theizen TH, Franchini KG, Nadruz Jr. W
1Mamoni
Laboratório de Biologia Cardiovascular, Universidade Estadual de Campinas – Unicamp, Campinas, SP, Brazil
3Departamento
Introduction: Mechanical stress is an important stimulus for the development of pro-atherogenic phenotype in vascular cells. The aim of this study was to investigate the role of Focal Adhesion Kinase (FAK) on the expression of pro-inflammatory molecules in vascular cells subjected to mechanical stretch in vitro and in vivo. Materials and methods: Rabbit aortic endothelial cells (RAEC) were subjected to cyclical stretch (1 Hz) for up to 6 hours. Western blot analysis evaluated protein expression and functional assays assessed the adhesion of monocytes to RAEC. In vitro inhibition of FAK was performed by treating RAEC with the pharmacological inhibitor PP2 and an oligode- oxynucleotide antisense (ODN-FAK). Moreover, transfection of small interference RNA directed against FAK (siRNA-FAK) was used to block the expression of FAK in vivo and to evaluate the impact of this protein on the expression of adhesion molecules in aorta of rats subjected to the coarctation of this vessel. Results: Mechanical stretch induced a precocious activation of FAK (as early as 10 min) in RAEC, evaluated by its phosphorylation on tyrosine residue 397. Cyclic Stretch for 6 hours promoted a significant increase in the expression of Toll-Like Receptors 2 and 4, E-selectin and VCAM-1, which was inhibited by pre-incubation with PP2 and ODN-FAK. Cyclic stretch also increased the adhesion of monocytes to the RAEC, but pretreatment of RAEC with PP2 or ODN-FAK abrogated this process. In rats, coarctation of aorta promoted an increase in the expression of adhesion molecules in the segment of the vessel proximal to the stenosis. This increase, however, was inhibited by previous treatment of the animals with siRNA-FAK, but not with a control molecule (siRNA-GFP). Conclusions: These data demonstrate that FAK is a critical molecule for the development of pro-atherogenic phenotype induced by mechanical/hemodynamic overload in vascular cells. I 006
Lack of Association Between Inflammatory Parameters and Carotid Intima Media-thickness in Subjects with Spinal Cord Injury
1Departamento
de Patologia Clínica; 2Departamento de Medicina Interna; de Anatomia Patológica; 4Departamento de Cirurgia, Faculdade de Medicina, Universidade Estadual de Campinas – Unicamp, Campinas, SP, Brazil
Introduction: Inflammation plays a central role in initiation and development of atherosclerosis. The migration of CD4+IFN-g+ T cells (TH1 cells) promotes lesion growth and disruption. Recently, a new subtype of IL-17-producing T helper (TH17) lineage of T cells has been described and associated to a variety of inflammatory autoimmune disease. The aim of this study was to verify whether TH17 cells were involved in human atherosclerotic lesion. Methods: Forty two patients (mean: 66.6 years old) with critical stenosis of carotid attended at the Clinics Hospital of the State University of Campinas were evaluated. Carotid specimens (n = 17) were analyzed by real time RT-PCR to detected the expression of mRNA to inflammatory and anti-inflammatory markers. We also investigated the production of IL-17 protein in atherosclerotic plaques stimulated in culture (n = 6) with IL-23, IL-2, PHA or aCD3+aCD28. The presence of TCD3+ and TCD8+ lymphocytes, macrophages and the expression of IL-17 and IFN-g were evaluated by imuno- histochemistry (n = 17) and flow cytometry (n = 2). The results were compared with control artery (n = 3). Results: Large amounts of IFN-g, IL-23p19, EBI3, IL-1b, TNF-a, IL-10, IL-27R, CCR4, FOXP3, RorC, GATA-3, MMP-9, TIMP-1, Granulisina, Granzima A and Perforina mRNA were detected in carotid plaques compared with controls. However, the expression of IL-17 mRNA was rare and detected only in two samples. The analyses of the lesions by immunohistochemistry revealed an inflammatory infiltrate with TCD3+, TCD8+ and macrophages but no IL-17+ or IFN-g+ cells. Flow cytometry analysis revealed rare IL-17+ cells (less than 1%), while cultured of atheroma cells produced IL-17 only after stimulation with aCD3+aCD28 antibodies. Conclusion: Our results showed elevated expression of inflammatory cytokines (IFN-g, IL-1b, TNF-a, IL-23p19, EBI3), chemokine receptors (CCR4), transcription factors (FOXP3, RorC, GATA-3) and cytotoxic molecules (MMP-9, granulysin, granzyme A and perforin) in lesions. However, the rare expression of IL-17 suggests a secondary role of Th17 cells in the development and desestabilization of atherosclerotic plaques.
Souza JRM, Pithon KR, Oliveira RTD, Cliquet AJ, Blotta MH, Nadruz Jr. W Faculdade de Ciências Médicas, Universidade Estadual de Campinas – Unicamp, Campinas, SP, Brazil Purpose: Subjects with spinal cord injury (SCI) are reported to exhibit increased carotid intima-media thickness (IMT) independent of traditional cardiovascular risk factors. Methods: We evaluated 65 nondiabetic, nonhypertensive, nonsmoker men (34 with SCI and 31 healthy subjects). Results: SCI (18 quadriplegic and 16 paraplegic) subjects had similar age, body mass index, cardiac output, peripheral vascular resistance, glucose and lipid levels but lower systolic blood pressure (109 ± 3 vs 120 ± 2 mmHg; p = 0.009) in comparison to control ones. Injured individuals exhibited higher seric CRP (6.2 ± 1.7 vs 1.1 ± 0.3 mg/mL; p = 0.001), TNF- Receptor I (1.81 ± 0.13 vs 1.48 ± 0.14 ng/mL; p < 0.05) and TNF-Receptor II (3.14 ± 0.05 vs 2.93 ± 0.07 ng/mL; p < 0.05) levels and increased in vitro production of IL-6 (147 ± 18 vs 87 ± 32 ng/mL; p < 0.05) compared to able- bodies ones. Conversely, no differences in ICAM-1, VCAM-1, E-selectin and TGF-beta levels as well as on in vitro release of IL-10, IL-17 and INF-gamma were detected between the studied groups. Carotid IMT was significantly higher in SCI subjects (0.74 ± 0.02 vs 0.52 ± 0.02 mm; p < 0.0001), even after adjusting for CRP levels (p < 0.0001). In addition, quadriplegic subjects exhibited increased IMT (p = 0.002 adjusted for systolic blood pressure and body mass index), but similar levels of inflammatory mediators compared to paraplegic individuals. Conclusion: Subjects with SCI present increased inflammatory status in comparison to able-bodied ones. Nevertheless, these alterations did not seem to explain the higher carotid IMT in the injured group.
XII Brazilian Congress of Atherosclerosis
I 008
Effect of Simvastatin Dose on Inflammation and Endothelial Function after Myocardial Infarction
Camargo N, Santos SN, Silva JCQ, Alvarenga BF, Alexandre A, Silva LP, Sposito AC Universidade de Brasília, Brasília, DF, Brazil; Hospital de Base do Distrito Federal, Brasília, DF, Brazil Although it is extensively proven the benefit of using statins in patients who had a coronary event, it remains unclear if such benefit can be maximized by an early statin prescription in the acute phase of myocardial infarct (MI). C-reactive protein (CRP) was measured daily from the admission to the seventh day after MI in 47 consecutive MI patients. During these 7 days, patients were treated by simvastatin at 20, 40 or 80 mg/day. After the 7th day, the simvastatin dose was changed to 20 mg/day for all patients during additional 3 weeks when brachial artery reactivity was assessed. Significant difference in CRP was observed between the simvastatin doses from the second day of treatment on (p < 0.0001). The area under the curve for this 7 days CRP levels was different between the groups (20 mg:47 21, 40 mg:27 22, 80 mg: 6 5; p < 0.0001). At the thirty day after MI and after three weeks of simvastatin 20mg/day, the ratio between flow-mediated dilation and nitrate- mediated dilation was different between the groups (20 mg:0.19 (0.12-0.33), 40 mg: 0.29 (0.14-0.39), 80 mg: 0.53 (0.40-0.64); p = 0.001). In conclusion, the dose of statin administered during the MI acute phase has a significant effect on in-hospital acute inflammatory response and on post-discharge endothelial function.
IMMUNOLOGY
/ 29
Focal Adhesion Kinase is Critical for the Expression of Pro-atherogenic Molecules in Vascular Cells Subjected to Mechanical Stress
I 007 1Oliveira
Identification and Characterization of TH17 Response in Human Atherosclerosis RTD, 1Teo FH, 1Silva RM, 1Fornazim MC, 2Souza JRM, RL, 3Altemani AMA, 4Menezes FH, 1Blotta MHSL
Fernandes MN, Cardoso C, Clemente C, Theizen TH, Franchini KG, Nadruz Jr. W
1Mamoni
Laboratório de Biologia Cardiovascular, Universidade Estadual de Campinas – Unicamp, Campinas, SP, Brazil
3Departamento
Introduction: Mechanical stress is an important stimulus for the development of pro-atherogenic phenotype in vascular cells. The aim of this study was to investigate the role of Focal Adhesion Kinase (FAK) on the expression of pro-inflammatory molecules in vascular cells subjected to mechanical stretch in vitro and in vivo. Materials and methods: Rabbit aortic endothelial cells (RAEC) were subjected to cyclical stretch (1 Hz) for up to 6 hours. Western blot analysis evaluated protein expression and functional assays assessed the adhesion of monocytes to RAEC. In vitro inhibition of FAK was performed by treating RAEC with the pharmacological inhibitor PP2 and an oligode- oxynucleotide antisense (ODN-FAK). Moreover, transfection of small interference RNA directed against FAK (siRNA-FAK) was used to block the expression of FAK in vivo and to evaluate the impact of this protein on the expression of adhesion molecules in aorta of rats subjected to the coarctation of this vessel. Results: Mechanical stretch induced a precocious activation of FAK (as early as 10 min) in RAEC, evaluated by its phosphorylation on tyrosine residue 397. Cyclic Stretch for 6 hours promoted a significant increase in the expression of Toll-Like Receptors 2 and 4, E-selectin and VCAM-1, which was inhibited by pre-incubation with PP2 and ODN-FAK. Cyclic stretch also increased the adhesion of monocytes to the RAEC, but pretreatment of RAEC with PP2 or ODN-FAK abrogated this process. In rats, coarctation of aorta promoted an increase in the expression of adhesion molecules in the segment of the vessel proximal to the stenosis. This increase, however, was inhibited by previous treatment of the animals with siRNA-FAK, but not with a control molecule (siRNA-GFP). Conclusions: These data demonstrate that FAK is a critical molecule for the development of pro-atherogenic phenotype induced by mechanical/hemodynamic overload in vascular cells. I 006
Lack of Association Between Inflammatory Parameters and Carotid Intima Media-thickness in Subjects with Spinal Cord Injury
1Departamento
de Patologia Clínica; 2Departamento de Medicina Interna; de Anatomia Patológica; 4Departamento de Cirurgia, Faculdade de Medicina, Universidade Estadual de Campinas – Unicamp, Campinas, SP, Brazil
Introduction: Inflammation plays a central role in initiation and development of atherosclerosis. The migration of CD4+IFN-g+ T cells (TH1 cells) promotes lesion growth and disruption. Recently, a new subtype of IL-17-producing T helper (TH17) lineage of T cells has been described and associated to a variety of inflammatory autoimmune disease. The aim of this study was to verify whether TH17 cells were involved in human atherosclerotic lesion. Methods: Forty two patients (mean: 66.6 years old) with critical stenosis of carotid attended at the Clinics Hospital of the State University of Campinas were evaluated. Carotid specimens (n = 17) were analyzed by real time RT-PCR to detected the expression of mRNA to inflammatory and anti-inflammatory markers. We also investigated the production of IL-17 protein in atherosclerotic plaques stimulated in culture (n = 6) with IL-23, IL-2, PHA or aCD3+aCD28. The presence of TCD3+ and TCD8+ lymphocytes, macrophages and the expression of IL-17 and IFN-g were evaluated by imuno- histochemistry (n = 17) and flow cytometry (n = 2). The results were compared with control artery (n = 3). Results: Large amounts of IFN-g, IL-23p19, EBI3, IL-1b, TNF-a, IL-10, IL-27R, CCR4, FOXP3, RorC, GATA-3, MMP-9, TIMP-1, Granulisina, Granzima A and Perforina mRNA were detected in carotid plaques compared with controls. However, the expression of IL-17 mRNA was rare and detected only in two samples. The analyses of the lesions by immunohistochemistry revealed an inflammatory infiltrate with TCD3+, TCD8+ and macrophages but no IL-17+ or IFN-g+ cells. Flow cytometry analysis revealed rare IL-17+ cells (less than 1%), while cultured of atheroma cells produced IL-17 only after stimulation with aCD3+aCD28 antibodies. Conclusion: Our results showed elevated expression of inflammatory cytokines (IFN-g, IL-1b, TNF-a, IL-23p19, EBI3), chemokine receptors (CCR4), transcription factors (FOXP3, RorC, GATA-3) and cytotoxic molecules (MMP-9, granulysin, granzyme A and perforin) in lesions. However, the rare expression of IL-17 suggests a secondary role of Th17 cells in the development and desestabilization of atherosclerotic plaques.
Souza JRM, Pithon KR, Oliveira RTD, Cliquet AJ, Blotta MH, Nadruz Jr. W Faculdade de Ciências Médicas, Universidade Estadual de Campinas – Unicamp, Campinas, SP, Brazil Purpose: Subjects with spinal cord injury (SCI) are reported to exhibit increased carotid intima-media thickness (IMT) independent of traditional cardiovascular risk factors. Methods: We evaluated 65 nondiabetic, nonhypertensive, nonsmoker men (34 with SCI and 31 healthy subjects). Results: SCI (18 quadriplegic and 16 paraplegic) subjects had similar age, body mass index, cardiac output, peripheral vascular resistance, glucose and lipid levels but lower systolic blood pressure (109 ± 3 vs 120 ± 2 mmHg; p = 0.009) in comparison to control ones. Injured individuals exhibited higher seric CRP (6.2 ± 1.7 vs 1.1 ± 0.3 mg/mL; p = 0.001), TNF- Receptor I (1.81 ± 0.13 vs 1.48 ± 0.14 ng/mL; p < 0.05) and TNF-Receptor II (3.14 ± 0.05 vs 2.93 ± 0.07 ng/mL; p < 0.05) levels and increased in vitro production of IL-6 (147 ± 18 vs 87 ± 32 ng/mL; p < 0.05) compared to able- bodies ones. Conversely, no differences in ICAM-1, VCAM-1, E-selectin and TGF-beta levels as well as on in vitro release of IL-10, IL-17 and INF-gamma were detected between the studied groups. Carotid IMT was significantly higher in SCI subjects (0.74 ± 0.02 vs 0.52 ± 0.02 mm; p < 0.0001), even after adjusting for CRP levels (p < 0.0001). In addition, quadriplegic subjects exhibited increased IMT (p = 0.002 adjusted for systolic blood pressure and body mass index), but similar levels of inflammatory mediators compared to paraplegic individuals. Conclusion: Subjects with SCI present increased inflammatory status in comparison to able-bodied ones. Nevertheless, these alterations did not seem to explain the higher carotid IMT in the injured group.
XII Brazilian Congress of Atherosclerosis
I 008
Effect of Simvastatin Dose on Inflammation and Endothelial Function after Myocardial Infarction
Camargo N, Santos SN, Silva JCQ, Alvarenga BF, Alexandre A, Silva LP, Sposito AC Universidade de Brasília, Brasília, DF, Brazil; Hospital de Base do Distrito Federal, Brasília, DF, Brazil Although it is extensively proven the benefit of using statins in patients who had a coronary event, it remains unclear if such benefit can be maximized by an early statin prescription in the acute phase of myocardial infarct (MI). C-reactive protein (CRP) was measured daily from the admission to the seventh day after MI in 47 consecutive MI patients. During these 7 days, patients were treated by simvastatin at 20, 40 or 80 mg/day. After the 7th day, the simvastatin dose was changed to 20 mg/day for all patients during additional 3 weeks when brachial artery reactivity was assessed. Significant difference in CRP was observed between the simvastatin doses from the second day of treatment on (p < 0.0001). The area under the curve for this 7 days CRP levels was different between the groups (20 mg:47 21, 40 mg:27 22, 80 mg: 6 5; p < 0.0001). At the thirty day after MI and after three weeks of simvastatin 20mg/day, the ratio between flow-mediated dilation and nitrate- mediated dilation was different between the groups (20 mg:0.19 (0.12-0.33), 40 mg: 0.29 (0.14-0.39), 80 mg: 0.53 (0.40-0.64); p = 0.001). In conclusion, the dose of statin administered during the MI acute phase has a significant effect on in-hospital acute inflammatory response and on post-discharge endothelial function.
IMMUNOLOGY
/ 29