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Serum amyloid a impairs endothelium-dependent vasorelaxation and increased superoxide anion production in porcine coronary arteries
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
(FGF)-1 [6 nmol/L] for 5 minutes prior to cell lysis. Cell lysates were probed for FAK, FRNK, GFP, and pFAK397 using Western blotting technique. Results: Efficiency of infection of VSMCs was confirmed visually by fluorescent microscopy and molecularly by Western blotting for GFP and FRNK; it was found to be ⬎90% for each virus with similar levels of GFP and GFP-FRNK being expressed in all groups. Using confocal microscopy, FRNK-infected VSMCs had FAK displaced from cellular focal adhesion complexes and replaced by FRNK (punctated green staining in the figure), while GFP-infected cells showed the normal incorporation of FAK into focal adhesion complexes (visualized as red via a rhodamine tagged secondary antibody). FAK expression was similar for all groups, but pFAK397 was not visualized in the FRNK-infected cells. Finally, autophosporylation of FAK397 was not augmented by serum or FGF-1 stimulation (6 nmol/L) in the presence or absence of heparin (5U/mL). Conclusions: FRNK over-expression displaces FAK from focal adhesion complexes and prevents autophosphorylation of FAK in VSMCs. This effect was not recoverable by treating the cells with FGF-1. Localized gene delivery of adv-FRNK may be able to down-regulate FAK-mediated SMC migration, proliferation, and matrix deposition, regardless of exogenous growth factors. This study illustrates a potential mechanism in which FRNK inhibits FAK signalling in VSMCs. Functional activity of these treatment groups will better define the potential clinical benefits that disrupting FAK signalling may have in preventing IH.
199. SERUM AMYLOID A IMPAIRS ENDOTHELIUMDEPENDENT VASORELAXATION AND INCREASED SUPEROXIDE ANION PRODUCTION IN PORCINE CORONARY ARTERIES. Henao EA, Chai H, Lin PH, Lumsden AB, Yao QC, Chen CJ; Baylor College of Medicine Purpose: Serum Amyloid A (SAA), an acute phase reactant, is associated with inflammation. Recent clinical studies have indicated that SAA is a valuable biomarker for cardiovascular disease. However, little is known about the direct biological functions on vascular cells. The objective of this study was to determine the effect of SAA on endothelial functions in porcine coronary arteries. Methods: Porcine coronary arteries were cut into 5-mm rings, which were incubated for 24 hours with DMEM medium for the control, or with a range of clinically relevant concentrations of SAA (1, 10, and 25 g/ml). Myograph tension analysis was performed in response to vasoactive drugs including thromboxane A 2 analog U466419 (contraction), endothelium-dependent vasorelaxation (bradykinin), and endothelium-independent vasorelaxation (sodium nitroprusside). Immunohistochemical staining was performed for endothelial nitric oxide synthase (eNOS). In addition, superoxide anion production was determined by lucigenin-enhanced chemiluminescence. Results: All groups of porcine coronary artery rings showed no difference in maximal contraction after U46619 challenge. However,
endothelium-dependent vasorelaxation in response to 10 -5 mol/L bradykinin was 64% in the 1 g/ml SAA group, 63% in the 10 g/ml SAA group, and 55% in the 25 g/ml SAA group as compared to 71% in the control group. All groups showed no significant difference in endothelium-independent vasorelaxation. In addition, eNOS immunoreactivity was substantially reduced in the SAA group. Furthermore, superoxide anion levels of the endothelial layer of the artery ring were significantly increased by over 70% in vessels treated with SAA (10 and 25 g/ml) as compared to control vessels (P⬍0.05). Conclusion: These data demonstrate that SAA significantly reduces endothelium-dependent vasorelaxation in porcine coronary arteries. In addition, SAA induces superoxide anion production and eNOS down-regulation. This study provides new information for better understanding the molecular mechanisms of SAA in the pathophysiology of cardiovascular disease.
200. THE ROLE OF SURVIVIN IN REGULATION OF CYTOKINE/GROWTH FACTOR SIGNALING IN VASCULAR SMOOTH MUSCLE CELLS. R. A. Bafford 1, X. Sui 2, G. J. Wang 3, M. S. Conte 2; 1Harvard-Longwood Research Fellow/Brigham and Women’s Hospital, Boston, MA, 2Brigham and Women’s Hospital, Boston, MA, 3Harvard-Longwood Research Fellow/Massachusetts General Hospital, Boston, MA. Introduction: Survivin (SVV) is a unique member of the inhibitor of apoptosis (IAP) family of proteins which functions as both an inhibitor of apoptosis and a regulator of mitosis, and is upregulated in many cancers. Recent work from our lab suggests that SVV plays a critical role in the vascular injury response via mechanisms that depend, at least in part, on platelet-derived growth factor (PDGF) signaling. We speculate that SVV is a central point of convergence of multiple signaling pathways in vascular injury, and that it also regulates the local amplification of these pathways in the vessel wall. Methods: Primary cultures of vascular smooth muscle cells (VSMC) were obtained from human greater saphenous vein. Quiescent cells were treated with angiotensin II (Ang II; 1 M) or tumor necrosis factor-␣ (TNF-␣; 10 ng/ml) and then harvested for RNA. We examined the expression of SVV, the transcription factor KLF-5, and its known downstream target PDGF-A by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). An adenoviral construct expressing SVV was also employed in select experiments. A rabbit carotid interposition vein graft model (N⫽ 14; 1-51 days) was used to assess SVV and KLF5 expression in-vivo. Results: Stimulation of VSMC by Ang II led to a rapid upregulation of KLF5 and PDGF-A expression (peak induction at 2-6 hours), and a later increase in SVV expression at 24 hours (2.1-fold induction relative to serum-free (SF) control; p⬍0.05). TNF-␣ stimulation also induced a significant increase in SVV transcripts with a similar temporal pattern (2.5-fold induction at 24 hrs; p⬍0.05). Conversely, overexpression of SVV in VSMC led to an early and persistent induction of KLF5 beginning at 8 hours after adenoviral exposure. Analogous to the in vitro data, KLF5 and SVV were both upregulated in rabbit vein grafts with KLF5 induced at an earlier time after grafting (1 day) compared to survivin (⬍7 days). Conclusions: Complementing prior data which demonstrated induction of SVV in VSMC by PDGF stimulation, we now show that both Ang II and TNF-␣, other cytokines known to be expressed at sites of vascular injury, also induce SVV. The data further suggest a potential positive feedback loop in which SVV itself amplifies further growth factor signaling, perhaps via KLF-5. KLF5 and SVV are upregulated sequentially in the rabbit vein graft model. SVV may play a central role in autocrine/paracrine signaling in the healing vein graft.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
(FGF)-1 [6 nmol/L] for 5 minutes prior to cell lysis. Cell lysates were probed for FAK, FRNK, GFP, and pFAK397 using Western blotting technique. Results: Efficiency of infection of VSMCs was confirmed visually by fluorescent microscopy and molecularly by Western blotting for GFP and FRNK; it was found to be ⬎90% for each virus with similar levels of GFP and GFP-FRNK being expressed in all groups. Using confocal microscopy, FRNK-infected VSMCs had FAK displaced from cellular focal adhesion complexes and replaced by FRNK (punctated green staining in the figure), while GFP-infected cells showed the normal incorporation of FAK into focal adhesion complexes (visualized as red via a rhodamine tagged secondary antibody). FAK expression was similar for all groups, but pFAK397 was not visualized in the FRNK-infected cells. Finally, autophosporylation of FAK397 was not augmented by serum or FGF-1 stimulation (6 nmol/L) in the presence or absence of heparin (5U/mL). Conclusions: FRNK over-expression displaces FAK from focal adhesion complexes and prevents autophosphorylation of FAK in VSMCs. This effect was not recoverable by treating the cells with FGF-1. Localized gene delivery of adv-FRNK may be able to down-regulate FAK-mediated SMC migration, proliferation, and matrix deposition, regardless of exogenous growth factors. This study illustrates a potential mechanism in which FRNK inhibits FAK signalling in VSMCs. Functional activity of these treatment groups will better define the potential clinical benefits that disrupting FAK signalling may have in preventing IH.
199. SERUM AMYLOID A IMPAIRS ENDOTHELIUMDEPENDENT VASORELAXATION AND INCREASED SUPEROXIDE ANION PRODUCTION IN PORCINE CORONARY ARTERIES. Henao EA, Chai H, Lin PH, Lumsden AB, Yao QC, Chen CJ; Baylor College of Medicine Purpose: Serum Amyloid A (SAA), an acute phase reactant, is associated with inflammation. Recent clinical studies have indicated that SAA is a valuable biomarker for cardiovascular disease. However, little is known about the direct biological functions on vascular cells. The objective of this study was to determine the effect of SAA on endothelial functions in porcine coronary arteries. Methods: Porcine coronary arteries were cut into 5-mm rings, which were incubated for 24 hours with DMEM medium for the control, or with a range of clinically relevant concentrations of SAA (1, 10, and 25 g/ml). Myograph tension analysis was performed in response to vasoactive drugs including thromboxane A 2 analog U466419 (contraction), endothelium-dependent vasorelaxation (bradykinin), and endothelium-independent vasorelaxation (sodium nitroprusside). Immunohistochemical staining was performed for endothelial nitric oxide synthase (eNOS). In addition, superoxide anion production was determined by lucigenin-enhanced chemiluminescence. Results: All groups of porcine coronary artery rings showed no difference in maximal contraction after U46619 challenge. However,
endothelium-dependent vasorelaxation in response to 10 -5 mol/L bradykinin was 64% in the 1 g/ml SAA group, 63% in the 10 g/ml SAA group, and 55% in the 25 g/ml SAA group as compared to 71% in the control group. All groups showed no significant difference in endothelium-independent vasorelaxation. In addition, eNOS immunoreactivity was substantially reduced in the SAA group. Furthermore, superoxide anion levels of the endothelial layer of the artery ring were significantly increased by over 70% in vessels treated with SAA (10 and 25 g/ml) as compared to control vessels (P⬍0.05). Conclusion: These data demonstrate that SAA significantly reduces endothelium-dependent vasorelaxation in porcine coronary arteries. In addition, SAA induces superoxide anion production and eNOS down-regulation. This study provides new information for better understanding the molecular mechanisms of SAA in the pathophysiology of cardiovascular disease.
200. THE ROLE OF SURVIVIN IN REGULATION OF CYTOKINE/GROWTH FACTOR SIGNALING IN VASCULAR SMOOTH MUSCLE CELLS. R. A. Bafford 1, X. Sui 2, G. J. Wang 3, M. S. Conte 2; 1Harvard-Longwood Research Fellow/Brigham and Women’s Hospital, Boston, MA, 2Brigham and Women’s Hospital, Boston, MA, 3Harvard-Longwood Research Fellow/Massachusetts General Hospital, Boston, MA. Introduction: Survivin (SVV) is a unique member of the inhibitor of apoptosis (IAP) family of proteins which functions as both an inhibitor of apoptosis and a regulator of mitosis, and is upregulated in many cancers. Recent work from our lab suggests that SVV plays a critical role in the vascular injury response via mechanisms that depend, at least in part, on platelet-derived growth factor (PDGF) signaling. We speculate that SVV is a central point of convergence of multiple signaling pathways in vascular injury, and that it also regulates the local amplification of these pathways in the vessel wall. Methods: Primary cultures of vascular smooth muscle cells (VSMC) were obtained from human greater saphenous vein. Quiescent cells were treated with angiotensin II (Ang II; 1 M) or tumor necrosis factor-␣ (TNF-␣; 10 ng/ml) and then harvested for RNA. We examined the expression of SVV, the transcription factor KLF-5, and its known downstream target PDGF-A by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). An adenoviral construct expressing SVV was also employed in select experiments. A rabbit carotid interposition vein graft model (N⫽ 14; 1-51 days) was used to assess SVV and KLF5 expression in-vivo. Results: Stimulation of VSMC by Ang II led to a rapid upregulation of KLF5 and PDGF-A expression (peak induction at 2-6 hours), and a later increase in SVV expression at 24 hours (2.1-fold induction relative to serum-free (SF) control; p⬍0.05). TNF-␣ stimulation also induced a significant increase in SVV transcripts with a similar temporal pattern (2.5-fold induction at 24 hrs; p⬍0.05). Conversely, overexpression of SVV in VSMC led to an early and persistent induction of KLF5 beginning at 8 hours after adenoviral exposure. Analogous to the in vitro data, KLF5 and SVV were both upregulated in rabbit vein grafts with KLF5 induced at an earlier time after grafting (1 day) compared to survivin (⬍7 days). Conclusions: Complementing prior data which demonstrated induction of SVV in VSMC by PDGF stimulation, we now show that both Ang II and TNF-␣, other cytokines known to be expressed at sites of vascular injury, also induce SVV. The data further suggest a potential positive feedback loop in which SVV itself amplifies further growth factor signaling, perhaps via KLF-5. KLF5 and SVV are upregulated sequentially in the rabbit vein graft model. SVV may play a central role in autocrine/paracrine signaling in the healing vein graft.