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Urokinase-induced smooth muscle cell migration involves the mammalian target of rapamycin
234
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
growth factor (EGF) restores barrier integrity, yet the mechanisms involved remain undefined. We now hypothesize that EGF enhances gap junction function through post-translational modification of Cx43 and sought to determine the mechanisms involved. Methods: IEC-6 enterocytes were treated with EGF (500ng/mL, 1-12h) and the expression and phosphorylation of Cx43 were assessed by SDSPAGE. Gap junction function was determined using fluorescence recovery after photobleaching with the tracer calcein. To assess ubiquitination of Cx43, IEC-6 lysates which had first been immunoprecipitated with anti-ubiquitin antibodies were immunoblotted against Cx43. To assess in vivo relevance, 3-week old male SwissWebster mice were injected with EGF (500 ng/mL, 4h or 12h) and Cx43 expression in the intestinal mucosa was assessed by SDSPAGE. Results: EGF treatment resulted in an initial increase (1-3h) then a decrease (6-12h) in Cx43 phosphorylation and function in IEC-6 cells. EGF caused a time-and dose-dependent increase in the ubiquitination of Cx43, leading to degradation of the protein by 12 hrs. To determine the in vivo relevance of these findings, administration of EGF to mice also led to an initial increase and subsequent decrease in the expression of Cx43 in the intestinal mucosa, consistent with changes in Cx43 ubiquitination. Strikingly, pre-incubation of IEC-6 cells with the ubiquitination inhibitor MG-132 (10uM) led to a restoration of Cx43 expression and phosphorylation, and a restoration of gap junction function in enterocytes over time. Conclusions: These findings indicate that EGF modifies gap junction connectivity through changes in the phosphorylation and ubiquitination of Cx43 both in vitro and in vivo. We propose that modulation of Cx43 ubiquitination may enhance the therapeutic use of EGF to restore barrier function in diseases like NEC.
VASCULAR IV-Cytokine and Apoptosis 194. UROKINASE-INDUCED SMOOTH MUSCLE CELL MIGRATION INVOLVES THE MAMMALIAN TARGET OF RAPAMYCIN. C. Potack, E. Roztocil, S. M. Nicholl, M. G. Davies; University of Rochester, Rochester, NY. Purpose: To determine a role for the mammalian target of rapamycin (mTOR) in urokinase (uPA)-induced smooth muscle cell (SMC) migration, and to examine specific kinase pathways that may act downstream of mTOR to influence the response of SMCs to uPA. Background: Vascular SMC migration is an important component of the development of intimal hyperplasia. During tissue remodeling, uPA is one of the key serine proteases involved and stimulates SMC migration in vitro. Rapamycin is an antifungal and immunosuppressant that inhibits mTOR, which regulates p70S6 kinase (p70S6K) activity. We examined the effect of rapamycin on uPA-induced SMC migration, the activation of mTOR and p70S6K and its effects on MMP-2 expression. Methods: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of uPA (1-100 nM) with and without rapamycin (1-10 nM). Additional studies were performed in ˆ M). Westthe presence of the PI3-Kinase inhibitor LY294002 (10A ern blotting was performed for phosphorylated and total mTOR and p70S6K after stimulation with uPA (10nM) with and without rapamycin. MMP-2 activity determined by zymography. Results: uPA stimulated migration of SMCs in both the linear wound assay (p⬍0.01) and in the Boyden chamber (p⬍0.01); these response were inhibited completely by rapamycin in a concentration dependent manner. uPA stimulated phosphorylation of mTOR and p70S6K (2-fold increase over control for both, p⬍0.05). These responses were inhibited by the presence of the PI3-K inhibitor (p⬍0.01). Rapamycin completely inhibited both mTOR and p70S6K phosphorylation. uPA induced expression and activity of MMP-2 was markedly inhibited by the presence of rapamycin. Conclusions: uPA-induced SMC migration is mTOR dependent. Activation of mTOR and p70S6K is PI3-K dependent. Inhibition of mTOR and p70S6K resulted in a decrease in MMP-2 expression and activity. Understanding the basic mecha-
nisms of cell migration will allow therapeutic intervention for restenosis. 195. HISTOLOGICAL ANALYSIS OF ANGIOJET RHEOLYTIC PHARMACOMECHANICAL THROMBECTOMY VERSUS ANGIOJET RHEOLYTIC THROMBECTOMY IN A PORCINE PERIPHERAL ARTERIAL MODEL. Mussa FF, Hedayati N, Naoum J, Chen C, Bush RL,Zhou W, Lumsden AB, Lin PH; Baylor College of Medicine Introduction: Rheolytic thrombectomy using the AngioJet catheter in arterial thrombosis has been shown to be effective in restoring blood flow. Additional infusion of thrombolytic agents via the AngioJet catheter results in a combined rheolytic pharmacomechanical thrombolysis (PMT) which further enhances thrombectomy efficacy. However, the histological response of the rheolytic PMT therapy remains unclear. This study compares the acute and chronic vessel wall response of conventional AngioJet thrombectomy (AT) and AngioJet PMT in porcine peripheral arterial model. Methods: Fourteen juvenile pigs were divided into acute and chronic groups. In the acute group, bilateral common carotid, femoral, and iliac arteries ranging from 3 to 6 mm in diameter were randomized to either control AT (n⫽21 arteries) or PMT (n⫽21 arteries) therapy. Vessels were analyzed at 4 days following interventions. In the chronic group, bilateral common carotid, femoral, and iliac arteries ranging from 3 to 6 mm in diameter were randomized to either control AT (n⫽21 arteries) or PMT (n⫽21 arteries) therapy. Vessels were analyzed at 30 days following interventions. Results: In the acute group, similar histological injury grade were noted between the AT and PMT treated femoral and iliac vessels. Endothelial denudation in the AT and PMT vessels were 43% and 39% (NS), respectively. Vessels with intact internal elastic lamina (IEL) in the AT and PMT groups were 54% and 57% (NS), respectively. In vessels less than 4mm in diameter, fractured IEL in the AT and PMT groups occurred in 23% and 27% (NS), respectively. The degrees of smooth muscle cell (SMC) loss were similar between the AT and PMT treated vessels, which were 45% and 40% (NS), respectively. In the chronic group, no differences were seen between the AT and PMT groups with respect to endothelial denudation, IEL fracture rate or SMC loss. Similar degrees of medial thickening or intimal hyperplasia was noted in the AT and PMT groups, which was 49% and 43% (NS), respectively. Conclusions: AngioJet rheolytic pharmacomechanical thrombectomy treatment incurs equivalent safety profile in medium caliber peripheral arteries when compared to rheolytic thrombectomy treatment. The observed clinical efficacy of rheolytic pharmacomechanical thrombectomy does not result in untoward vessel injury compared to the conventional rheolytic thrombectomy therapy. 196. CD39 ENZYMOSOMES INHIBIT PLATELET ACTIVATION IN VITRO AND IN VIVO. E. L. Chaikof 1, C. A. Haller 1, W. Cui 1, J. Wen 1, S. C. Robson 2; 1Emory University, Atlanta, GA, 2Beth Israel-Deaconess Medical Center, Boston, MA. Objective: CD39 (NTPDase-1) is expressed on the luminal surface of endothelial cells, rapidly metabolizes ATP and ADP to AMP, and reduces platelet reactivity to most agonists. Optimization of CD39 enzymatic activity appears dependent upon the expression of both of its transmembrane domains. Thus, motivation exists to examine therapeutic anti-platelet ‘enzymosome’ formulations that consist of CD39 integrated within liposome lipid bilayers. Methods: The full length human CD39 was produced using a yeast expression system, purified, and reconstituted within lipid vesicles. The catalytic efficiency (kcat/Km) of CD39 with respect to the dephosphorylation of ADP and ATP was determined both for detergent solubilized CD39 and when reconstituted within a lipid membrane. The capacity of CD39 containing lipid vesicles to inhibit platelet activation induced by ADP, collagen, or thrombin was defined in vitro by platelet aggregometry. A murine model of thromboplastin induced thromboem-
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
growth factor (EGF) restores barrier integrity, yet the mechanisms involved remain undefined. We now hypothesize that EGF enhances gap junction function through post-translational modification of Cx43 and sought to determine the mechanisms involved. Methods: IEC-6 enterocytes were treated with EGF (500ng/mL, 1-12h) and the expression and phosphorylation of Cx43 were assessed by SDSPAGE. Gap junction function was determined using fluorescence recovery after photobleaching with the tracer calcein. To assess ubiquitination of Cx43, IEC-6 lysates which had first been immunoprecipitated with anti-ubiquitin antibodies were immunoblotted against Cx43. To assess in vivo relevance, 3-week old male SwissWebster mice were injected with EGF (500 ng/mL, 4h or 12h) and Cx43 expression in the intestinal mucosa was assessed by SDSPAGE. Results: EGF treatment resulted in an initial increase (1-3h) then a decrease (6-12h) in Cx43 phosphorylation and function in IEC-6 cells. EGF caused a time-and dose-dependent increase in the ubiquitination of Cx43, leading to degradation of the protein by 12 hrs. To determine the in vivo relevance of these findings, administration of EGF to mice also led to an initial increase and subsequent decrease in the expression of Cx43 in the intestinal mucosa, consistent with changes in Cx43 ubiquitination. Strikingly, pre-incubation of IEC-6 cells with the ubiquitination inhibitor MG-132 (10uM) led to a restoration of Cx43 expression and phosphorylation, and a restoration of gap junction function in enterocytes over time. Conclusions: These findings indicate that EGF modifies gap junction connectivity through changes in the phosphorylation and ubiquitination of Cx43 both in vitro and in vivo. We propose that modulation of Cx43 ubiquitination may enhance the therapeutic use of EGF to restore barrier function in diseases like NEC.
VASCULAR IV-Cytokine and Apoptosis 194. UROKINASE-INDUCED SMOOTH MUSCLE CELL MIGRATION INVOLVES THE MAMMALIAN TARGET OF RAPAMYCIN. C. Potack, E. Roztocil, S. M. Nicholl, M. G. Davies; University of Rochester, Rochester, NY. Purpose: To determine a role for the mammalian target of rapamycin (mTOR) in urokinase (uPA)-induced smooth muscle cell (SMC) migration, and to examine specific kinase pathways that may act downstream of mTOR to influence the response of SMCs to uPA. Background: Vascular SMC migration is an important component of the development of intimal hyperplasia. During tissue remodeling, uPA is one of the key serine proteases involved and stimulates SMC migration in vitro. Rapamycin is an antifungal and immunosuppressant that inhibits mTOR, which regulates p70S6 kinase (p70S6K) activity. We examined the effect of rapamycin on uPA-induced SMC migration, the activation of mTOR and p70S6K and its effects on MMP-2 expression. Methods: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of uPA (1-100 nM) with and without rapamycin (1-10 nM). Additional studies were performed in ˆ M). Westthe presence of the PI3-Kinase inhibitor LY294002 (10A ern blotting was performed for phosphorylated and total mTOR and p70S6K after stimulation with uPA (10nM) with and without rapamycin. MMP-2 activity determined by zymography. Results: uPA stimulated migration of SMCs in both the linear wound assay (p⬍0.01) and in the Boyden chamber (p⬍0.01); these response were inhibited completely by rapamycin in a concentration dependent manner. uPA stimulated phosphorylation of mTOR and p70S6K (2-fold increase over control for both, p⬍0.05). These responses were inhibited by the presence of the PI3-K inhibitor (p⬍0.01). Rapamycin completely inhibited both mTOR and p70S6K phosphorylation. uPA induced expression and activity of MMP-2 was markedly inhibited by the presence of rapamycin. Conclusions: uPA-induced SMC migration is mTOR dependent. Activation of mTOR and p70S6K is PI3-K dependent. Inhibition of mTOR and p70S6K resulted in a decrease in MMP-2 expression and activity. Understanding the basic mecha-
nisms of cell migration will allow therapeutic intervention for restenosis. 195. HISTOLOGICAL ANALYSIS OF ANGIOJET RHEOLYTIC PHARMACOMECHANICAL THROMBECTOMY VERSUS ANGIOJET RHEOLYTIC THROMBECTOMY IN A PORCINE PERIPHERAL ARTERIAL MODEL. Mussa FF, Hedayati N, Naoum J, Chen C, Bush RL,Zhou W, Lumsden AB, Lin PH; Baylor College of Medicine Introduction: Rheolytic thrombectomy using the AngioJet catheter in arterial thrombosis has been shown to be effective in restoring blood flow. Additional infusion of thrombolytic agents via the AngioJet catheter results in a combined rheolytic pharmacomechanical thrombolysis (PMT) which further enhances thrombectomy efficacy. However, the histological response of the rheolytic PMT therapy remains unclear. This study compares the acute and chronic vessel wall response of conventional AngioJet thrombectomy (AT) and AngioJet PMT in porcine peripheral arterial model. Methods: Fourteen juvenile pigs were divided into acute and chronic groups. In the acute group, bilateral common carotid, femoral, and iliac arteries ranging from 3 to 6 mm in diameter were randomized to either control AT (n⫽21 arteries) or PMT (n⫽21 arteries) therapy. Vessels were analyzed at 4 days following interventions. In the chronic group, bilateral common carotid, femoral, and iliac arteries ranging from 3 to 6 mm in diameter were randomized to either control AT (n⫽21 arteries) or PMT (n⫽21 arteries) therapy. Vessels were analyzed at 30 days following interventions. Results: In the acute group, similar histological injury grade were noted between the AT and PMT treated femoral and iliac vessels. Endothelial denudation in the AT and PMT vessels were 43% and 39% (NS), respectively. Vessels with intact internal elastic lamina (IEL) in the AT and PMT groups were 54% and 57% (NS), respectively. In vessels less than 4mm in diameter, fractured IEL in the AT and PMT groups occurred in 23% and 27% (NS), respectively. The degrees of smooth muscle cell (SMC) loss were similar between the AT and PMT treated vessels, which were 45% and 40% (NS), respectively. In the chronic group, no differences were seen between the AT and PMT groups with respect to endothelial denudation, IEL fracture rate or SMC loss. Similar degrees of medial thickening or intimal hyperplasia was noted in the AT and PMT groups, which was 49% and 43% (NS), respectively. Conclusions: AngioJet rheolytic pharmacomechanical thrombectomy treatment incurs equivalent safety profile in medium caliber peripheral arteries when compared to rheolytic thrombectomy treatment. The observed clinical efficacy of rheolytic pharmacomechanical thrombectomy does not result in untoward vessel injury compared to the conventional rheolytic thrombectomy therapy. 196. CD39 ENZYMOSOMES INHIBIT PLATELET ACTIVATION IN VITRO AND IN VIVO. E. L. Chaikof 1, C. A. Haller 1, W. Cui 1, J. Wen 1, S. C. Robson 2; 1Emory University, Atlanta, GA, 2Beth Israel-Deaconess Medical Center, Boston, MA. Objective: CD39 (NTPDase-1) is expressed on the luminal surface of endothelial cells, rapidly metabolizes ATP and ADP to AMP, and reduces platelet reactivity to most agonists. Optimization of CD39 enzymatic activity appears dependent upon the expression of both of its transmembrane domains. Thus, motivation exists to examine therapeutic anti-platelet ‘enzymosome’ formulations that consist of CD39 integrated within liposome lipid bilayers. Methods: The full length human CD39 was produced using a yeast expression system, purified, and reconstituted within lipid vesicles. The catalytic efficiency (kcat/Km) of CD39 with respect to the dephosphorylation of ADP and ATP was determined both for detergent solubilized CD39 and when reconstituted within a lipid membrane. The capacity of CD39 containing lipid vesicles to inhibit platelet activation induced by ADP, collagen, or thrombin was defined in vitro by platelet aggregometry. A murine model of thromboplastin induced thromboem-