- Email: [email protected]
Tu-P7: 154 Laminar flow attenuates cytokine-induced stat activation in endothelial cells
Tues&ty, June 20, 2006: Poster Session P7 Basic science ( lst part)
219
Endothelial dysfunction results in decreased signalling through the NO/cGMP pathway leading to decreased levels of cGMP in diabetes. We investigated whether chronic stimulation of guanylyl cyclase (GC) with the novel activator HMR1766 would improve vascular reactivity in diabetic rats. Metbods and Results: Male Wistar rats received a single high-dose intravenous injection of streptozotocin (STZ)to induce diabetes. 2 weeks after STZ injection, rats were randomized to treatment with either placebo or the GC activator HMR1766 (20 mg/kg/d) for another 2 weeks. Vascular function in the thoracic aorta was assessed. Diabetic rats displayed a highly significant reduction in vascular contraction compared to healthy controls. Treatment with HMR1766 normalized vascular contraction. Endothelium-dependent, NOmediated vasorelaxation was significantly impaired in diabetic placebo-treated animals, which was completely normalized by HMR1766. This was at least partially dependent on reduced smooth muscle sensitivity to NO as demonstrated by endothelium-independent relaxation to DEA-NONOate, which was significantly impaired in diabetic placebo-treated rats and normalized by GC activation. Conclusion: Chronic GC activation in diabetes improves vascular relaxation and contraction. HMR1766 significantly enhanced signaling through the NO/cGMP pathway in diabetes and might be a new potential therapeutic approach for treatment of vascular dysfunction in diabetes.
t o n i d t y by the synthesis and the release of vasoactive substances like the nitric oxide and the hyperpolaxisant factor. Our study analyzed the reactivity of the endothelial vascular wall in a murine model of mesenteric artery under the action of the APL. Eight purified human APL anti-nucleosome IgG were tested (150rag) on CD1 mice. One of these APL presented a different behaviour. Therefore in mice injected with APL 4b7 the contraction was identical with that found in normal mice; but the relaxation was significantly lower (p< 0,0001). These data were confirmed after high purification in order to eliminate the role of the LPS, and with successive re-injections; and it was dose and time dependent. The effect of this antinudeosome antibody seems to be independent of the NOS synthase pathway. In c o n t u s i o n , our study suggests that the reactivity of the endothelial wall is modified by the presence of a antiphospholipid antibody directed agadnst the nucleosome. Complementary studies with m o n o d o n a l APL are necessary before a formal conclusion.
ITu-P7:1541
Objectives: Oxidized phospholipids d e a r l y contribute to the inflammation assodated with atherosclerosis. By investigating the role of oxidized 1-palmitoyl-2-axachidonoyl-sn-3-glycero-phosphorylcholine (OxPAPC) upon gene expression in endothelial cells we seek to identify important genes in the atherosclerosis. Methods: Microaxray analyses identified differentially regulated genes between OxPAPC-treated and control-treated Human Aortic Endothelial Cells (HAECs). We used RT-qPCR to confirm changes in mRNA expression. Western blotting measured protein expression changes upon OxPAPC treatment. Expression changes were measured in dose and time course experiments. Additionally, OxPAPC-treated HAECs were co-treated with human HDL and analyzed for expression of Connexin 37 (Cx37), which has previously been implicated in atherosclerosis. Results: Analysis of expression array data revealed that Cx37 is markedly down-regulated by OxPAPC. Cx37 is a gap junction protein expressed almost exclusively in monocytes and endothelial cells of large arteries. RT-qPCR and Western blotting confirmed Cx37 down-regulation after OxPAPC treatment in HAEC cell culture. Co-treatment with HDL prevents marked down-regulation of Cx37 by OxPAPC. Conclusions: Our results suggest a novel mechanism of regulation of Cx37. Current studies axe aimed at unraveling the pathways by which OxPAPC regulates Cx37. Funding: Vascular Biology Training Grant T32HL69766 and NIH Grant HL30568
I
LAMINAR FLOW ATTENUATES C Y T O K I N E - I N D U C E D STAT A C T I V A T I O N IN ENDOTHELIAL CELLS
L. Wang, Y.-C. Tsai. Inst. of Biomed. Sciences, AccMemia Sinica, Taipei, Taiwan Objective: Endothelial cells (ECs) exposed to steady laminar flow (LF) axe protected from atherosclerosis as compared with those ECs that are exposed to disturbed flow (DF). Our previous studies have shown that LF to ECs suppresses serum- and IL-6- induced STAT3 activation (JBC, 2003 and AJR 2004). Because IFN gamma-inducible CXC chemokines responsible for recruitment of T cells axe associated with atherogenesis, the effect of LF on the IFN-gamma (IFN)-induced responses in ECs was examined. Metbods: Human umbilical vein ECs were exposed to shear stress in a parallel plate flow chamber in the presence of IFN. Results: ECs exposed to IFN triggered JAK/STAT1 activation through the phosphorylation of Tyr701 and Ser727 of STAT1. When LF was applied to IFN-treated ECs, it significantly attenuated IFN-induced Tyr701 phosphorylation, whereas Ser727 phosphorylation was unaffected. Consistently, LF inhibited IFN-induced STAT1 binding to DNA. ECs treated with IFN induced expression of IFN-inducible CXC chemokines that axe specific for T cells: CXCL10, CXCL9 and C X C L l l . IFN induced the expression of CIITA, a transcriptional regulator of major histocompatibility complex class II (MHCII) expression. Application of LF to IFN-treated ECs reduced the expression of IFN-inducible CXC chemokines and of CIITA. Furthermore, the inhibitory effect of LF on the IFN-induced responses was not observed in those ECs that were subjected to DF. The suppression of STAT activation by LF was significantly attenuated if ECs were pretreated with a tyrosine phosphatase inhibitor. Conclusions: LF protects ECs from cytokine-induced STAT activation. Our results provide new insights into the athero-resistant nature of straight segments of the arterial tree, in which cytokine-induced responses are suppressed. F u n d i n g : This work was supported by National Science Council and by the Department of Education (91-B-FA02-2-4), Taiwan.
ITu-P7:1551
I
E F F E C T S OF T H E A N T I P H O S P H O L I P I D ANTIBODIES ON THE ENDOTHELIAL VASCULAR REACTIVITY
C. Behzna "-, V. Richaxd-, A. Larhgue , D. Gilbert 3 , F. Tron 3, H. Levesque ] , C. Thuillez 2. 1 Universi~ Hospital Rouen, Rouen, France: 2bzserm 644, Rouen, France: ~bnmunology Dept Universi~ Hospital Rouen, Rouen, France •
1 ~
•
~
•
3
The antiphospholipid antibodies are associated with severe arterial lesions, which suggest a pro-atherogenic effect. Several physiopathological mechanisms have been suggested in order to explain this effect (Sherer Y Immunobiology. 2003;207:13-6); one of these mechanisms implying the production of nitric oxide and superoxide, and the interference with the activity of the paraoxonase (Delgado J Arthritis Rheum. 2002;46: 2686-94). In physiological conditions the endothelium contributes to the maintenance of the vascular
]
Tu-P7:156
/ J
R E G U L A T I O N OF C O N N E X I N 37 BY O X I D I Z E D P H O S P H O L I P I D S IN A T H E R O S C L E R O S I S
C.E. Culler ] , P.S. Gargalovic ] , T.G. Kirchgessner 2 , A.J. Lusis 1.1Department of Human Genetics, Universi~ of California, Los Angeles, USA: 2Bristol-M~ers Squibb, Pharmaceutical Research bzstitute, Princeton, USA
Tu-P7:157 ] T H E N I T R O P R A V A S T A T I N D E R I V A T I V E , N C X 6550, I M P R O V E S E N D O T H E L I A L D Y S F U N C T I O N IN SPONTANEOUSLY HYPERTENSIVE RATS
C. Presotto, D. Miglietta, R. Olivieri, A. Monopoli. Nicox Research bzstitute, Bresso, Italy Nitric oxide (NO)-releasing statins axe a new class of compounds which exert both statin-like and NO-mediated effects. Here we report the effects of NCX 6550, on the endothelial function in SHR. We studied the effects of repeated administration (7 days) of pravastatin and NCX 6550 at equimolar dose (90uM/kg) given by gavage to both SHR and normotensive Wistar rats. Systolic blood pressure (SBP) was recorded every day, and at sacrifice we determined: (i) plasmatic NOx levels; (ii) vascular responses in aortic rings to ACh and to SNP stimuli. NCX 6550 significantly reduced SBP in SHR vs vehicle, whereas pravastatin showed no effect (192-t-3; 218-t-4 and 210-t-2 mmHg, respectively). SBP was not aYfected by any treatment in normotensive rats. Plasma NOx levels of NCX 6550-treated SHR and Wistar rats were significantly higher than those of pravastatin and vehicle groups (V) (NCX 6550SHR=173+19; VSHR =104-t-8; NCX 6550Wistax=176-t-16; VWistar =63-t-10uM). NCX 6550 improved endothelial dysfunction, as assessed by ACh-induced relaxation in aortic rings compared to pravastatin and vehicle (Emax: vehicle = 46-t-3, pravastatin = 60-t-3 and NCX 6550 = 76-t-5, p < 0.05 for both comparisons). Vascular response in NCX 6550 group was similar to that of normotensive rats (Emax = 84-t-2). The endothelium-independent vasorelaxation induced by SNP was not altered by any treatment.
XIV bttetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
219
Endothelial dysfunction results in decreased signalling through the NO/cGMP pathway leading to decreased levels of cGMP in diabetes. We investigated whether chronic stimulation of guanylyl cyclase (GC) with the novel activator HMR1766 would improve vascular reactivity in diabetic rats. Metbods and Results: Male Wistar rats received a single high-dose intravenous injection of streptozotocin (STZ)to induce diabetes. 2 weeks after STZ injection, rats were randomized to treatment with either placebo or the GC activator HMR1766 (20 mg/kg/d) for another 2 weeks. Vascular function in the thoracic aorta was assessed. Diabetic rats displayed a highly significant reduction in vascular contraction compared to healthy controls. Treatment with HMR1766 normalized vascular contraction. Endothelium-dependent, NOmediated vasorelaxation was significantly impaired in diabetic placebo-treated animals, which was completely normalized by HMR1766. This was at least partially dependent on reduced smooth muscle sensitivity to NO as demonstrated by endothelium-independent relaxation to DEA-NONOate, which was significantly impaired in diabetic placebo-treated rats and normalized by GC activation. Conclusion: Chronic GC activation in diabetes improves vascular relaxation and contraction. HMR1766 significantly enhanced signaling through the NO/cGMP pathway in diabetes and might be a new potential therapeutic approach for treatment of vascular dysfunction in diabetes.
t o n i d t y by the synthesis and the release of vasoactive substances like the nitric oxide and the hyperpolaxisant factor. Our study analyzed the reactivity of the endothelial vascular wall in a murine model of mesenteric artery under the action of the APL. Eight purified human APL anti-nucleosome IgG were tested (150rag) on CD1 mice. One of these APL presented a different behaviour. Therefore in mice injected with APL 4b7 the contraction was identical with that found in normal mice; but the relaxation was significantly lower (p< 0,0001). These data were confirmed after high purification in order to eliminate the role of the LPS, and with successive re-injections; and it was dose and time dependent. The effect of this antinudeosome antibody seems to be independent of the NOS synthase pathway. In c o n t u s i o n , our study suggests that the reactivity of the endothelial wall is modified by the presence of a antiphospholipid antibody directed agadnst the nucleosome. Complementary studies with m o n o d o n a l APL are necessary before a formal conclusion.
ITu-P7:1541
Objectives: Oxidized phospholipids d e a r l y contribute to the inflammation assodated with atherosclerosis. By investigating the role of oxidized 1-palmitoyl-2-axachidonoyl-sn-3-glycero-phosphorylcholine (OxPAPC) upon gene expression in endothelial cells we seek to identify important genes in the atherosclerosis. Methods: Microaxray analyses identified differentially regulated genes between OxPAPC-treated and control-treated Human Aortic Endothelial Cells (HAECs). We used RT-qPCR to confirm changes in mRNA expression. Western blotting measured protein expression changes upon OxPAPC treatment. Expression changes were measured in dose and time course experiments. Additionally, OxPAPC-treated HAECs were co-treated with human HDL and analyzed for expression of Connexin 37 (Cx37), which has previously been implicated in atherosclerosis. Results: Analysis of expression array data revealed that Cx37 is markedly down-regulated by OxPAPC. Cx37 is a gap junction protein expressed almost exclusively in monocytes and endothelial cells of large arteries. RT-qPCR and Western blotting confirmed Cx37 down-regulation after OxPAPC treatment in HAEC cell culture. Co-treatment with HDL prevents marked down-regulation of Cx37 by OxPAPC. Conclusions: Our results suggest a novel mechanism of regulation of Cx37. Current studies axe aimed at unraveling the pathways by which OxPAPC regulates Cx37. Funding: Vascular Biology Training Grant T32HL69766 and NIH Grant HL30568
I
LAMINAR FLOW ATTENUATES C Y T O K I N E - I N D U C E D STAT A C T I V A T I O N IN ENDOTHELIAL CELLS
L. Wang, Y.-C. Tsai. Inst. of Biomed. Sciences, AccMemia Sinica, Taipei, Taiwan Objective: Endothelial cells (ECs) exposed to steady laminar flow (LF) axe protected from atherosclerosis as compared with those ECs that are exposed to disturbed flow (DF). Our previous studies have shown that LF to ECs suppresses serum- and IL-6- induced STAT3 activation (JBC, 2003 and AJR 2004). Because IFN gamma-inducible CXC chemokines responsible for recruitment of T cells axe associated with atherogenesis, the effect of LF on the IFN-gamma (IFN)-induced responses in ECs was examined. Metbods: Human umbilical vein ECs were exposed to shear stress in a parallel plate flow chamber in the presence of IFN. Results: ECs exposed to IFN triggered JAK/STAT1 activation through the phosphorylation of Tyr701 and Ser727 of STAT1. When LF was applied to IFN-treated ECs, it significantly attenuated IFN-induced Tyr701 phosphorylation, whereas Ser727 phosphorylation was unaffected. Consistently, LF inhibited IFN-induced STAT1 binding to DNA. ECs treated with IFN induced expression of IFN-inducible CXC chemokines that axe specific for T cells: CXCL10, CXCL9 and C X C L l l . IFN induced the expression of CIITA, a transcriptional regulator of major histocompatibility complex class II (MHCII) expression. Application of LF to IFN-treated ECs reduced the expression of IFN-inducible CXC chemokines and of CIITA. Furthermore, the inhibitory effect of LF on the IFN-induced responses was not observed in those ECs that were subjected to DF. The suppression of STAT activation by LF was significantly attenuated if ECs were pretreated with a tyrosine phosphatase inhibitor. Conclusions: LF protects ECs from cytokine-induced STAT activation. Our results provide new insights into the athero-resistant nature of straight segments of the arterial tree, in which cytokine-induced responses are suppressed. F u n d i n g : This work was supported by National Science Council and by the Department of Education (91-B-FA02-2-4), Taiwan.
ITu-P7:1551
I
E F F E C T S OF T H E A N T I P H O S P H O L I P I D ANTIBODIES ON THE ENDOTHELIAL VASCULAR REACTIVITY
C. Behzna "-, V. Richaxd-, A. Larhgue , D. Gilbert 3 , F. Tron 3, H. Levesque ] , C. Thuillez 2. 1 Universi~ Hospital Rouen, Rouen, France: 2bzserm 644, Rouen, France: ~bnmunology Dept Universi~ Hospital Rouen, Rouen, France •
1 ~
•
~
•
3
The antiphospholipid antibodies are associated with severe arterial lesions, which suggest a pro-atherogenic effect. Several physiopathological mechanisms have been suggested in order to explain this effect (Sherer Y Immunobiology. 2003;207:13-6); one of these mechanisms implying the production of nitric oxide and superoxide, and the interference with the activity of the paraoxonase (Delgado J Arthritis Rheum. 2002;46: 2686-94). In physiological conditions the endothelium contributes to the maintenance of the vascular
]
Tu-P7:156
/ J
R E G U L A T I O N OF C O N N E X I N 37 BY O X I D I Z E D P H O S P H O L I P I D S IN A T H E R O S C L E R O S I S
C.E. Culler ] , P.S. Gargalovic ] , T.G. Kirchgessner 2 , A.J. Lusis 1.1Department of Human Genetics, Universi~ of California, Los Angeles, USA: 2Bristol-M~ers Squibb, Pharmaceutical Research bzstitute, Princeton, USA
Tu-P7:157 ] T H E N I T R O P R A V A S T A T I N D E R I V A T I V E , N C X 6550, I M P R O V E S E N D O T H E L I A L D Y S F U N C T I O N IN SPONTANEOUSLY HYPERTENSIVE RATS
C. Presotto, D. Miglietta, R. Olivieri, A. Monopoli. Nicox Research bzstitute, Bresso, Italy Nitric oxide (NO)-releasing statins axe a new class of compounds which exert both statin-like and NO-mediated effects. Here we report the effects of NCX 6550, on the endothelial function in SHR. We studied the effects of repeated administration (7 days) of pravastatin and NCX 6550 at equimolar dose (90uM/kg) given by gavage to both SHR and normotensive Wistar rats. Systolic blood pressure (SBP) was recorded every day, and at sacrifice we determined: (i) plasmatic NOx levels; (ii) vascular responses in aortic rings to ACh and to SNP stimuli. NCX 6550 significantly reduced SBP in SHR vs vehicle, whereas pravastatin showed no effect (192-t-3; 218-t-4 and 210-t-2 mmHg, respectively). SBP was not aYfected by any treatment in normotensive rats. Plasma NOx levels of NCX 6550-treated SHR and Wistar rats were significantly higher than those of pravastatin and vehicle groups (V) (NCX 6550SHR=173+19; VSHR =104-t-8; NCX 6550Wistax=176-t-16; VWistar =63-t-10uM). NCX 6550 improved endothelial dysfunction, as assessed by ACh-induced relaxation in aortic rings compared to pravastatin and vehicle (Emax: vehicle = 46-t-3, pravastatin = 60-t-3 and NCX 6550 = 76-t-5, p < 0.05 for both comparisons). Vascular response in NCX 6550 group was similar to that of normotensive rats (Emax = 84-t-2). The endothelium-independent vasorelaxation induced by SNP was not altered by any treatment.
XIV bttetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006