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Human Adult Stem Cells Restore Endothelial Migratory Dysfunction in a Hypoxic Environment
816 Abstracts
Spontaneous Dissection with Rupture of the Superior Mesenteric Artery From Segmental Arterial Mediolysis: A Case Report and Review of the Literature Michael N. Tameo, Matthew J. Dougherty, MD, and Keith D. Calligaro, MD, Pennsylvania Hospital, Philadelphia, Pa Background: Spontaneous dissection of the superior mesenteric artery (SMA) is rare. We report a case of rupture of the SMA after spontaneous dissection in a 51-year-old man who presented with acute onset of abdominal pain and hypotension. The patient was diagnosed as having segmental arterial mediolysis (SAM). Case: The patient was initially treated with intravenous fluid resuscitation and endovascular intervention, followed by open surgery. The relevant features of the case as well as SAM are presented. In addition, a review of all available published literature on SAM to date is presented. No identifiable cause for dissection was found. The patient was diagnosed as having SAM. He did well and was discharged home on postoperative day 8. A follow-up CT scan showed additional disease characteristic of SAM in a branch of the inferior mesenteric artery that was treated with coil embolization. Conclusions: SAM is a rare arteriopathy of unknown etiology. Differential diagnosis includes fibromuscular dysplasia and inflammatory arteritis such as polyarteritis nodosa. Differentiation is important, because there is no standard treatment for SAM.
JOURNAL OF VASCULAR SURGERY September 2010
protein expression by Western blotting and immunohistochemistry. Statistical analysis was by ANOVA and t test (P ⬍ .05). Results: Thrombus resolution was 28% and 32% greater (P ⬍ .05) in the stenosis vs the ligation model on postoperative day (POD) 8 and 12 (n ⫽ 5-13). Thrombus resolution induced MT-1 (⬎4-fold vs sham on POD 8 ⫹ 12; P ⬍ .05) but the stenosis model demonstrated a more significant induction early (1.83 vs 1.15, POD4; P ⬍ .05). Thrombus resolution repressed MT-2 expression (0.2-fold, POD4; P ⬍ .05) in both stasis and recanalization models, and initially induced (8-fold, POD4; P ⬍ .05) but then repressed MT-5 and MT-6 expression. MT-4 expression increased late (2.8-fold, POD12; P ⬍ .05) selectively in the stasis model, whereas MT-3 had the opposite induction only in the recanalization model (2-fold; P ⬍ .05). Pro- and active MMP2 protein and gelatinase activity were increased in recanalization ⬎ stasis ⬎ sham. MT1 and MT3 expression localized around areas of recanalization during thrombus resolution. Conclusions: These studies demonstrate that the expression of MTMMP family of genes is differentially induced and repressed during the process of thrombus resolution, further localized in focal areas of recanalization within the thrombus. These findings increase our knowledge of the biology of venous thrombus resolution and identify pathways that may serve as therapeutic targets to accelerate thrombus resolution.
Parallel Session II–Basic Science In Vivo Evaluation of a Hand-Held, Battery-Operated Therapeutic Ultrasound Device for the Noninvasive Treatment of Varicose Veins Peter W. Henderson, MD MBA, Allie M. Sohn, BS, Aleid Koppius, BS, George K. Lewis, Jr, BS, William L. Olbricht, PhD, and Jason A. Spector, MD, Weill Cornell Medical College, New York, NY, and Ithaca, NY Objectives: Current treatments for varicose veins and other vascular malformations are to some degree invasive and may therefore be painful and associated with complications such as infection, thrombophlebitis, and bleeding. The development of entirely noninvasive techniques has been hampered by the high cost, large size, and power requirements of candidate technologies, such as high-intensity focused ultrasound (HIFU) imaging. Our group has developed a HIFU device that is hand-held and batteryoperated, and we have previously demonstrated that it is capable of venous ablation ex vivo. The purpose of this study was to determine whether it is capable of transcutaneous venous ablation in vivo. Methods: Our HIFU device weighs 560 grams and has an intensity of 2500 W/cm2 that focuses at a focal length of 3.3 mm. A midline laparotomy was performed on four Sprague-Dawley rats to expose the IVC. The HIFU transducer was covered in a plane 2 mm from the focal point with a piece of previously harvested rat skin, and under direct visualization, the device was applied to the IVC and activated for 60 seconds. Results: Compared with the condition of the IVC before HIFU treatment, the IVC after HIFU was marked by significant contraction and coagulation necrosis. There was no antegrade blood flow from the IVC after transection at the level immediately superior to the treated area, indicating complete venous occlusion. Furthermore, there was no evidence of necrosis in any of the adjacent tissues. Conclusions: In contrast to previous HIFU devices, ours is hand-held, portable, and relatively inexpensive. Although subsequent prototypes will incorporate visualization capabilities, the current device required a modified experimental design that allowed for direct visualization while assessing the ability to effectively ablate veins in a transcutaneous fashion. Our results indicate that this device is capable of successful, targeted, transcutaneous venous ablation in vivo. Because this technique is completely noninvasive, it has the potential to minimize the visible scars and other complications that can be associated with more invasive procedures. Accordingly, we believe that interventions based upon this technology may provide a superior treatment option for varicosities and other vascular malformations.
Regulation of Membrane-Type Matrix Metalloproteinase Expression by Recanalization and Flow During Thrombus Resolution Mohammed Chaudry, MD, Christine Chabasse, PhD, and Rajabrata Sarkar, MD, PhD, University of Maryland, Baltimore, Md Objectives: Expression and activity of matrix metalloproteinase (MMP) enzymes is important in the process of venous thrombus resolution. Expression of the membrane-type MMP family of genes (MT-MMPs) in thrombus resolution, and the regulation of their expression by blood flow (recanalization) remain undefined. We tested the hypothesis that thrombus resolution would activate these genes and that recanalization would further regulate their expression. Methods: CD1 mice underwent surgical inferior vena cava (IVC) ligation (stasis thrombosis), IVC stenosis (thrombosis with recanalization), or a sham procedure. We analyzed thrombus weight over time as a measure of thrombus resolution, and studied mRNA levels of MT-MMP genes by real-time PCR of mRNA extracted from thrombus ⫹ vein wall, as well as
Human Adult Stem Cells Restore Endothelial Migratory Dysfunction in a Hypoxic Environment Sarah Fernandez, MD, Rachel Song, BS, Jason Comeau, MD, Stephen McIlhenny, PhD, Hamid Abdollahi, MD, Ping Zhang, PhD, Thomas N. Tulenko, PhD, and Paul J. DiMuzio, MD, Thomas Jefferson University Hospital, Philadelphia, Pa Objectives: Adipose-derived stem cells (ASCs) injected into the blood stream after an ischemic event promote therapeutic angiogenesis in affected tissues. It has been suggested that the stem cells exert their influence by way of a paracrine effect on native endothelial cells (ECs). Using an in vitro model, we evaluated the effect of ASC coculture on EC function in a hypoxic environment. Methods: Confluent monolayers of human ECs grown on the bottom of transwell plates were wounded to create an even 5-mm defect and cultured in either normoxic (21%) or hypoxic (1%) conditions. Human ASCs were cocultured on the top of the transwell plates to evaluate a paracrine effect. Subsequently, EC migration was determined by measuring the wound size after 3 days. Media samples were collected, and the VEGF concentration was measured by using ELISA. RNA from ASCs incubated in hypoxia for varying time lengths was evaluated using PCR for various growth
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
factors. To confirm mechanism, ECs were treated with recombinant VEGF at various concentrations and migration was measured. Results: Hypoxia inhibited EC migration (0.87 vs 0.78 mm, P ⬍ .05) over 3 days. Coculture of ASC enhanced EC migration in both normoxic (0.87 to 0.93 mm) and hypoxic (0.78 to 1.02 mm; P ⬍ .05) environments. Media from cocultures in hypoxia contained significantly more VEGF (708.3 pg/mL) than normoxic cocultures (311.2 pg/mL) and ECs alone (28.9 pg/mL). The addition of VEGF to wounded EC cultures improved migration, but not to the extent of ASC coculture. Finally, hypoxia markedly increased expression of VEGF and slightly increased expression of bFGF in ASC. Conclusions: These results demonstrate that (1) ASCs restore and enhance EC migratory function in a hypoxic environment, and (2) the effect is primarily due to secretion of VEGF by ASC in response to hypoxia, with other growth factors like bFGF likely playing a minor role. These results suggest that hypoxic preconditioning of ASC might be of value in enhancing their role in therapeutic angiogenesis to treat ischemic heart or limb conditions.
The Effect of Nitric Oxide and Statins on Thrombospondin-1-Induced Chemotaxis in Vascular Smooth Muscle Cells Keri A. Seymour, DO, Xuan Han, Benjamin Sadowitz, MD, Kristopher G. Maier, PhD and Vivian Gahtan, MD, SUNY Upstate Medical University, Department of Veterans Affairs VA Healthcare Network Upstate New York at Syracuse, Syracuse, NY Objective: Vascular smooth muscle cell (VSMC) chemotaxis is important in intimal hyperplasia (IH). Nitric oxide (NO), a diffusible molecule that decreases VSMC chemotaxis to several growth factors, is protective against IH. Thrombospondin-1 (TSP-1), a matricellular glycoprotein that induces VSMC chemotaxis, acts antagonistically to NO in VSMCs. Statins exhibit direct and pleiotropic effects on VSMCs. We showed overnight treatment with lovastatin inhibited TSP-1-induced VSMC chemotaxis by mevalonate pathway inhibition and was Ras dependent. Hypothesis: Shortterm statin treatment will inhibit TSP-1-induced VSMC chemotaxis and NO donors will enhance statin inhibitory effects. Methods: Quiescent VSMCs were pretreated with serum-free media (SFM), hydrophobic fluvastatin (0.1M, 0.2M, 0.3M, 0.5M), or hydrophilic pravastatin (1M, 2M, 3M, 5M) for 20 minutes or 18 hours. Chemotaxis to SFM or TSP-1 (20 g/mL) was determined with a modified Boyden chamber. Also studied was the effect of the NO donors, DETANONOate (0.1M, 10M, 100M, 1000M) or SNAP (0.1M, 10M, 100M, 1000M) on fluvastatin (0.5M) or pravastatin (5M) on TSP-1induced chemotaxis. To determine the effect of acute statin treatment on Ras activation, cells were treated with fluvastatin (0.5M) or pravastatin (5M) and then exposed to TSP-1 or SFM. Active Ras was isolated using Raf-RBD agarose beads, and semiquantitation was performed by Western blot. Assays were performed in triplicate and analyzed by ANOVA. Results: Pravastatin and fluvastatin inhibited TSP-1-induced chemotaxis (20 minute or 18 hour pretreatment, P ⬍ .05). DETA-NONOate and SNAP at high concentrations both impeded statin inhibition of TSP-1induced chemotaxis (P ⬍ .05). Results of cytotoxicity assays were negative. Conclusions: Hydrophilic and hydrophobic statins with acute treatment inhibited TSP-1-induced VSMC chemotaxis. This inhibition is likely a pleiotropic effect independent of HMG-CoA reductase and subsequent Ras prenylation; however, the role of HMG-CoA reductase remains to be determined. Further, high-dose NO reversed statin inhibition of TSP-1induced chemotaxis, indicating NO and statin therapies in combination warrant further study.
Aged Rats Have Increased Neointimal Thickening and Ephrin-B2 Expression After Carotid Angioplasty Sammy D. D. Eghbalieh, MD, Jose M. Pimiento, MD, Fabio A. Kudo, MD, Akihito Muto, MD, PhD, Kenneth R. Ziegler, MD, Paraag Chowdhary, MD, Yuka Kondo, MD, PhD, Lynn Model, MD, Xin Li, MD, Yuan Y. Guo, MD, and Alan Dardik, MD, PhD, Yale University School of Medicine, New Haven, Conn Objectives: Although carotid angioplasty is associated with increased adverse events in elderly patients compared with younger patients, some animal models of carotid angioplasty have previously demonstrated only negative remodeling in aged rats compared with younger rats. Therefore, we
Abstracts 817
examined the response to carotid artery angioplasty using a validated animal model of aging. Methods: The right common carotid artery of young adult (6-month) or aged (22 to 24-month) male Fischer 344 rats was injured with a balloon, and the ipsilateral external carotid was ligated. The common carotid arteries were examined by histology and immunohistochemistry after 2 weeks. Results: Aged and young adult rats had similar vessel areas after angioplasty (aged: 0.49 ⫾ 0.02 mm2; young: 0.4 ⫾ 0.02 mm2; n ⫽ 5-6; P ⫽ .2). However, aged rats had a significant reduction in the lumen area (0.18 ⫾ 0.03 vs 0.24 ⫾ 0.01 mm2; P ⫽ .02), corresponding to increased neointimal thickening compared with young adult rats (aged: 0.15 ⫾ 0.04 mm2; young: 0.08 ⫾ 0.03 mm2; P ⫽ .006). Expression of Ephrin-B2, an arterial marker and ligand for Eph receptor signaling, was increased after angioplasty in both aged and young adult rats, but with higher amounts in aged animals (n ⫽ 4). Conclusions: Aged Fischer 344 rats are an accurate model for carotid angioplasty in aged humans, with increased neointimal thickening after carotid angioplasty in aged rats compared with young adult rats. Increased Ephrin-B2 expression after carotid angioplasty suggests additional mechanisms of smooth muscle cell activation that may contribute to the increased neointimal thickening in aged rats and adverse events in aged humans undergoing carotid angioplasty.
Tissue Culture Analysis of an Intact Carotid Plaque: Is Cryoplasty an Option for Carotid Intervention? Paul B. Haser, MD, Daniel I. Fremed, Shaohua Li, MD, Todd R. Vogel, MD, Michael S. Nagar, MD, and Alan M. Graham, MD, UMDNJ-RWJMS, New Brunswick, NJ Objectives: Angioplasty imparts a cellular response to vascular smooth muscle cells (VSMCs) that contributes to intimal hyperplasia (IH). Previous cell culture manipulation has imputed that apoptosis follows rapid freezing, but no direct plaque sampling has confirmed this. An ex vivo carotid plaque model allows procedure-specific histologic and biologic assessment of metabolically active tissue. Methods: An in-line, reconstructed carotid plaque model created after endarterectomy underwent either standard angioplasty (CAS) or dilation with the PolarCath (Boston Scientific) balloon (CRY). VSMCs were grown in tissue culture and harvested for interrogation with histologic staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and cell proliferation studies with bromodeoxyuridine (BrdU). Results: Twelve plaques were successfully treated (6 CAS, 6 CRY). On histologic staining at 5 days, the mean number of mononuclear infiltrate cells (MNC) was 87/mm2 for CRY vs 250/mm2 for CAS (P ⬍ .002). The CAS group showed an initial positive staining for apoptosis at 24 hours, (mean concentration of apoptotic cells, 24/mm2 [SD, 1-3]), which rapidly diminished over the 48 hours and 5 days to nearly zero. The CRY group showed an increased number of apoptotic cells at 24 hours (33/mm2 [SD, 28-32]), and increased slightly over the following 48 hours and 5-day assays (36/ mm2 [SD, 33-38]; and 41/mm2 [SD 38-44], respectively; see Fig). Although positive markers for BrDU were found in each specimen, quantitative analysis of nuclear mitotic activity among the specimens demonstrated no significant difference in cell proliferation between either group. Conclusions: Carotid tissue treated with CRY vs CAS showed lower concentrations of MNC and a higher degree of sustained apoptosis. This suggests CRY may reduce IH lesions associated with endovascular carotid therapy by a mechanism not related to cell proliferation, but associated with apoptosis and inflammatory cell count reduction.
Spontaneous Dissection with Rupture of the Superior Mesenteric Artery From Segmental Arterial Mediolysis: A Case Report and Review of the Literature Michael N. Tameo, Matthew J. Dougherty, MD, and Keith D. Calligaro, MD, Pennsylvania Hospital, Philadelphia, Pa Background: Spontaneous dissection of the superior mesenteric artery (SMA) is rare. We report a case of rupture of the SMA after spontaneous dissection in a 51-year-old man who presented with acute onset of abdominal pain and hypotension. The patient was diagnosed as having segmental arterial mediolysis (SAM). Case: The patient was initially treated with intravenous fluid resuscitation and endovascular intervention, followed by open surgery. The relevant features of the case as well as SAM are presented. In addition, a review of all available published literature on SAM to date is presented. No identifiable cause for dissection was found. The patient was diagnosed as having SAM. He did well and was discharged home on postoperative day 8. A follow-up CT scan showed additional disease characteristic of SAM in a branch of the inferior mesenteric artery that was treated with coil embolization. Conclusions: SAM is a rare arteriopathy of unknown etiology. Differential diagnosis includes fibromuscular dysplasia and inflammatory arteritis such as polyarteritis nodosa. Differentiation is important, because there is no standard treatment for SAM.
JOURNAL OF VASCULAR SURGERY September 2010
protein expression by Western blotting and immunohistochemistry. Statistical analysis was by ANOVA and t test (P ⬍ .05). Results: Thrombus resolution was 28% and 32% greater (P ⬍ .05) in the stenosis vs the ligation model on postoperative day (POD) 8 and 12 (n ⫽ 5-13). Thrombus resolution induced MT-1 (⬎4-fold vs sham on POD 8 ⫹ 12; P ⬍ .05) but the stenosis model demonstrated a more significant induction early (1.83 vs 1.15, POD4; P ⬍ .05). Thrombus resolution repressed MT-2 expression (0.2-fold, POD4; P ⬍ .05) in both stasis and recanalization models, and initially induced (8-fold, POD4; P ⬍ .05) but then repressed MT-5 and MT-6 expression. MT-4 expression increased late (2.8-fold, POD12; P ⬍ .05) selectively in the stasis model, whereas MT-3 had the opposite induction only in the recanalization model (2-fold; P ⬍ .05). Pro- and active MMP2 protein and gelatinase activity were increased in recanalization ⬎ stasis ⬎ sham. MT1 and MT3 expression localized around areas of recanalization during thrombus resolution. Conclusions: These studies demonstrate that the expression of MTMMP family of genes is differentially induced and repressed during the process of thrombus resolution, further localized in focal areas of recanalization within the thrombus. These findings increase our knowledge of the biology of venous thrombus resolution and identify pathways that may serve as therapeutic targets to accelerate thrombus resolution.
Parallel Session II–Basic Science In Vivo Evaluation of a Hand-Held, Battery-Operated Therapeutic Ultrasound Device for the Noninvasive Treatment of Varicose Veins Peter W. Henderson, MD MBA, Allie M. Sohn, BS, Aleid Koppius, BS, George K. Lewis, Jr, BS, William L. Olbricht, PhD, and Jason A. Spector, MD, Weill Cornell Medical College, New York, NY, and Ithaca, NY Objectives: Current treatments for varicose veins and other vascular malformations are to some degree invasive and may therefore be painful and associated with complications such as infection, thrombophlebitis, and bleeding. The development of entirely noninvasive techniques has been hampered by the high cost, large size, and power requirements of candidate technologies, such as high-intensity focused ultrasound (HIFU) imaging. Our group has developed a HIFU device that is hand-held and batteryoperated, and we have previously demonstrated that it is capable of venous ablation ex vivo. The purpose of this study was to determine whether it is capable of transcutaneous venous ablation in vivo. Methods: Our HIFU device weighs 560 grams and has an intensity of 2500 W/cm2 that focuses at a focal length of 3.3 mm. A midline laparotomy was performed on four Sprague-Dawley rats to expose the IVC. The HIFU transducer was covered in a plane 2 mm from the focal point with a piece of previously harvested rat skin, and under direct visualization, the device was applied to the IVC and activated for 60 seconds. Results: Compared with the condition of the IVC before HIFU treatment, the IVC after HIFU was marked by significant contraction and coagulation necrosis. There was no antegrade blood flow from the IVC after transection at the level immediately superior to the treated area, indicating complete venous occlusion. Furthermore, there was no evidence of necrosis in any of the adjacent tissues. Conclusions: In contrast to previous HIFU devices, ours is hand-held, portable, and relatively inexpensive. Although subsequent prototypes will incorporate visualization capabilities, the current device required a modified experimental design that allowed for direct visualization while assessing the ability to effectively ablate veins in a transcutaneous fashion. Our results indicate that this device is capable of successful, targeted, transcutaneous venous ablation in vivo. Because this technique is completely noninvasive, it has the potential to minimize the visible scars and other complications that can be associated with more invasive procedures. Accordingly, we believe that interventions based upon this technology may provide a superior treatment option for varicosities and other vascular malformations.
Regulation of Membrane-Type Matrix Metalloproteinase Expression by Recanalization and Flow During Thrombus Resolution Mohammed Chaudry, MD, Christine Chabasse, PhD, and Rajabrata Sarkar, MD, PhD, University of Maryland, Baltimore, Md Objectives: Expression and activity of matrix metalloproteinase (MMP) enzymes is important in the process of venous thrombus resolution. Expression of the membrane-type MMP family of genes (MT-MMPs) in thrombus resolution, and the regulation of their expression by blood flow (recanalization) remain undefined. We tested the hypothesis that thrombus resolution would activate these genes and that recanalization would further regulate their expression. Methods: CD1 mice underwent surgical inferior vena cava (IVC) ligation (stasis thrombosis), IVC stenosis (thrombosis with recanalization), or a sham procedure. We analyzed thrombus weight over time as a measure of thrombus resolution, and studied mRNA levels of MT-MMP genes by real-time PCR of mRNA extracted from thrombus ⫹ vein wall, as well as
Human Adult Stem Cells Restore Endothelial Migratory Dysfunction in a Hypoxic Environment Sarah Fernandez, MD, Rachel Song, BS, Jason Comeau, MD, Stephen McIlhenny, PhD, Hamid Abdollahi, MD, Ping Zhang, PhD, Thomas N. Tulenko, PhD, and Paul J. DiMuzio, MD, Thomas Jefferson University Hospital, Philadelphia, Pa Objectives: Adipose-derived stem cells (ASCs) injected into the blood stream after an ischemic event promote therapeutic angiogenesis in affected tissues. It has been suggested that the stem cells exert their influence by way of a paracrine effect on native endothelial cells (ECs). Using an in vitro model, we evaluated the effect of ASC coculture on EC function in a hypoxic environment. Methods: Confluent monolayers of human ECs grown on the bottom of transwell plates were wounded to create an even 5-mm defect and cultured in either normoxic (21%) or hypoxic (1%) conditions. Human ASCs were cocultured on the top of the transwell plates to evaluate a paracrine effect. Subsequently, EC migration was determined by measuring the wound size after 3 days. Media samples were collected, and the VEGF concentration was measured by using ELISA. RNA from ASCs incubated in hypoxia for varying time lengths was evaluated using PCR for various growth
JOURNAL OF VASCULAR SURGERY Volume 52, Number 3
factors. To confirm mechanism, ECs were treated with recombinant VEGF at various concentrations and migration was measured. Results: Hypoxia inhibited EC migration (0.87 vs 0.78 mm, P ⬍ .05) over 3 days. Coculture of ASC enhanced EC migration in both normoxic (0.87 to 0.93 mm) and hypoxic (0.78 to 1.02 mm; P ⬍ .05) environments. Media from cocultures in hypoxia contained significantly more VEGF (708.3 pg/mL) than normoxic cocultures (311.2 pg/mL) and ECs alone (28.9 pg/mL). The addition of VEGF to wounded EC cultures improved migration, but not to the extent of ASC coculture. Finally, hypoxia markedly increased expression of VEGF and slightly increased expression of bFGF in ASC. Conclusions: These results demonstrate that (1) ASCs restore and enhance EC migratory function in a hypoxic environment, and (2) the effect is primarily due to secretion of VEGF by ASC in response to hypoxia, with other growth factors like bFGF likely playing a minor role. These results suggest that hypoxic preconditioning of ASC might be of value in enhancing their role in therapeutic angiogenesis to treat ischemic heart or limb conditions.
The Effect of Nitric Oxide and Statins on Thrombospondin-1-Induced Chemotaxis in Vascular Smooth Muscle Cells Keri A. Seymour, DO, Xuan Han, Benjamin Sadowitz, MD, Kristopher G. Maier, PhD and Vivian Gahtan, MD, SUNY Upstate Medical University, Department of Veterans Affairs VA Healthcare Network Upstate New York at Syracuse, Syracuse, NY Objective: Vascular smooth muscle cell (VSMC) chemotaxis is important in intimal hyperplasia (IH). Nitric oxide (NO), a diffusible molecule that decreases VSMC chemotaxis to several growth factors, is protective against IH. Thrombospondin-1 (TSP-1), a matricellular glycoprotein that induces VSMC chemotaxis, acts antagonistically to NO in VSMCs. Statins exhibit direct and pleiotropic effects on VSMCs. We showed overnight treatment with lovastatin inhibited TSP-1-induced VSMC chemotaxis by mevalonate pathway inhibition and was Ras dependent. Hypothesis: Shortterm statin treatment will inhibit TSP-1-induced VSMC chemotaxis and NO donors will enhance statin inhibitory effects. Methods: Quiescent VSMCs were pretreated with serum-free media (SFM), hydrophobic fluvastatin (0.1M, 0.2M, 0.3M, 0.5M), or hydrophilic pravastatin (1M, 2M, 3M, 5M) for 20 minutes or 18 hours. Chemotaxis to SFM or TSP-1 (20 g/mL) was determined with a modified Boyden chamber. Also studied was the effect of the NO donors, DETANONOate (0.1M, 10M, 100M, 1000M) or SNAP (0.1M, 10M, 100M, 1000M) on fluvastatin (0.5M) or pravastatin (5M) on TSP-1induced chemotaxis. To determine the effect of acute statin treatment on Ras activation, cells were treated with fluvastatin (0.5M) or pravastatin (5M) and then exposed to TSP-1 or SFM. Active Ras was isolated using Raf-RBD agarose beads, and semiquantitation was performed by Western blot. Assays were performed in triplicate and analyzed by ANOVA. Results: Pravastatin and fluvastatin inhibited TSP-1-induced chemotaxis (20 minute or 18 hour pretreatment, P ⬍ .05). DETA-NONOate and SNAP at high concentrations both impeded statin inhibition of TSP-1induced chemotaxis (P ⬍ .05). Results of cytotoxicity assays were negative. Conclusions: Hydrophilic and hydrophobic statins with acute treatment inhibited TSP-1-induced VSMC chemotaxis. This inhibition is likely a pleiotropic effect independent of HMG-CoA reductase and subsequent Ras prenylation; however, the role of HMG-CoA reductase remains to be determined. Further, high-dose NO reversed statin inhibition of TSP-1induced chemotaxis, indicating NO and statin therapies in combination warrant further study.
Aged Rats Have Increased Neointimal Thickening and Ephrin-B2 Expression After Carotid Angioplasty Sammy D. D. Eghbalieh, MD, Jose M. Pimiento, MD, Fabio A. Kudo, MD, Akihito Muto, MD, PhD, Kenneth R. Ziegler, MD, Paraag Chowdhary, MD, Yuka Kondo, MD, PhD, Lynn Model, MD, Xin Li, MD, Yuan Y. Guo, MD, and Alan Dardik, MD, PhD, Yale University School of Medicine, New Haven, Conn Objectives: Although carotid angioplasty is associated with increased adverse events in elderly patients compared with younger patients, some animal models of carotid angioplasty have previously demonstrated only negative remodeling in aged rats compared with younger rats. Therefore, we
Abstracts 817
examined the response to carotid artery angioplasty using a validated animal model of aging. Methods: The right common carotid artery of young adult (6-month) or aged (22 to 24-month) male Fischer 344 rats was injured with a balloon, and the ipsilateral external carotid was ligated. The common carotid arteries were examined by histology and immunohistochemistry after 2 weeks. Results: Aged and young adult rats had similar vessel areas after angioplasty (aged: 0.49 ⫾ 0.02 mm2; young: 0.4 ⫾ 0.02 mm2; n ⫽ 5-6; P ⫽ .2). However, aged rats had a significant reduction in the lumen area (0.18 ⫾ 0.03 vs 0.24 ⫾ 0.01 mm2; P ⫽ .02), corresponding to increased neointimal thickening compared with young adult rats (aged: 0.15 ⫾ 0.04 mm2; young: 0.08 ⫾ 0.03 mm2; P ⫽ .006). Expression of Ephrin-B2, an arterial marker and ligand for Eph receptor signaling, was increased after angioplasty in both aged and young adult rats, but with higher amounts in aged animals (n ⫽ 4). Conclusions: Aged Fischer 344 rats are an accurate model for carotid angioplasty in aged humans, with increased neointimal thickening after carotid angioplasty in aged rats compared with young adult rats. Increased Ephrin-B2 expression after carotid angioplasty suggests additional mechanisms of smooth muscle cell activation that may contribute to the increased neointimal thickening in aged rats and adverse events in aged humans undergoing carotid angioplasty.
Tissue Culture Analysis of an Intact Carotid Plaque: Is Cryoplasty an Option for Carotid Intervention? Paul B. Haser, MD, Daniel I. Fremed, Shaohua Li, MD, Todd R. Vogel, MD, Michael S. Nagar, MD, and Alan M. Graham, MD, UMDNJ-RWJMS, New Brunswick, NJ Objectives: Angioplasty imparts a cellular response to vascular smooth muscle cells (VSMCs) that contributes to intimal hyperplasia (IH). Previous cell culture manipulation has imputed that apoptosis follows rapid freezing, but no direct plaque sampling has confirmed this. An ex vivo carotid plaque model allows procedure-specific histologic and biologic assessment of metabolically active tissue. Methods: An in-line, reconstructed carotid plaque model created after endarterectomy underwent either standard angioplasty (CAS) or dilation with the PolarCath (Boston Scientific) balloon (CRY). VSMCs were grown in tissue culture and harvested for interrogation with histologic staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and cell proliferation studies with bromodeoxyuridine (BrdU). Results: Twelve plaques were successfully treated (6 CAS, 6 CRY). On histologic staining at 5 days, the mean number of mononuclear infiltrate cells (MNC) was 87/mm2 for CRY vs 250/mm2 for CAS (P ⬍ .002). The CAS group showed an initial positive staining for apoptosis at 24 hours, (mean concentration of apoptotic cells, 24/mm2 [SD, 1-3]), which rapidly diminished over the 48 hours and 5 days to nearly zero. The CRY group showed an increased number of apoptotic cells at 24 hours (33/mm2 [SD, 28-32]), and increased slightly over the following 48 hours and 5-day assays (36/ mm2 [SD, 33-38]; and 41/mm2 [SD 38-44], respectively; see Fig). Although positive markers for BrDU were found in each specimen, quantitative analysis of nuclear mitotic activity among the specimens demonstrated no significant difference in cell proliferation between either group. Conclusions: Carotid tissue treated with CRY vs CAS showed lower concentrations of MNC and a higher degree of sustained apoptosis. This suggests CRY may reduce IH lesions associated with endovascular carotid therapy by a mechanism not related to cell proliferation, but associated with apoptosis and inflammatory cell count reduction.