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Smooth muscle cell polymeric transfection is an efficient alternative to traditional methods of experimental gene therapy
S160
Surgical Forum Abstracts
CONCLUSIONS: Unmodified siRNA can be delivered throughout the vein graft wall using the intraluminal route with moderate gene silencing. This model can be used to identify appropriate targets and further study the effectiveness of gene silencing in modifying intimal hyperplasia.
Beta-1 integrin is an upstream regulator of Eph-B4-mediated vein graft adaptation Kenneth Richard Ziegler MD, Akihito Muto MD, PhD, Lynn S Model MD, Sammy D Eghbalieh MD, Clinton D Protack MD, Michael J Collins MD, Xin Li MD, Caroline C Jadlowiec MD, Frank J Giordano MD, Alan Dardik MD, PhD, FACS Yale University School of Medicine, New Haven, CT and VA Connecticut Healthcare System, West Haven, CT INTRODUCTION: Decreased expression of Eph-B4, the embryonic venous fate determinant, is associated with successful adaptation of surgical vein grafts to the arterial environment. However, Ephrin-B2, the ligand for Eph-B4, is not expressed in veins; as such, the physiological mediator of Eph-B4 is unknown. We determined if the mechanosensitive integrin-beta-1 is an upstream regulator of Eph-B4 signaling. METHODS: Eph-B4-positive endothelial cells (EC) and Eph-B4negative Cos cells transfected with Eph-B4 were stimulated with collagen and anti-integrin-beta-1 activating antibody (P4G11) and analyzed with Western blotting. Colocalization was determined using immunofluorescence. Unilateral external carotid artery ligation was performed on heterozygous integrin-beta-1 knockout (Het-B1KO) mice; carotid arteries were examined on day 14. Veins from Het-B1-KO mice were placed as aortic interposition grafts into wildtype mice and examined at day 21. RESULTS: EC stimulated with collagen or P4G11 increased Eph-B4 phosphorylation. Eph-B4 and integrin-beta-1 colocalized on both EC and Eph-B4 transfected Cos cells after stimulation with collagen. Het-B1-KO mice that underwent unilateral ligation of the external carotid showed aberrant vascular remodeling. Het-B1-KO vein grafts showed approximately 3-fold increased neointimal thickening compared to wildtype controls. CONCLUSIONS: Integrin-beta-1 is detectable in EC, reacts to hemodynamic forces, induces Eph-B4 signaling, and alters vessel and vein graft remodeling. These results connect hemodynamic forces to vein graft adaptation via extracellular matrix signaling. Integrinbeta-1 is an upstream regulator of Eph-B4 signaling during vein graft adaptation, and may represent a novel therapeutic target to limit neointimal hyperplasia in vein grafts.
Smooth muscle cell polymeric transfection is an efficient alternative to traditional methods of experimental gene therapy Joshua D Arnold MD, Deidra J Mountain PhD, Michael B Freeman MD, FACS, Stacy S Kirkpatrick BS, Scott L Stevens MD, FACS, Mitchell H Goldman MD, FACS,
J Am Coll Surg
Oscar H Grandas MD, FACS University of Tennessee Graduate School of Medicine, Knoxville, TN INTRODUCTION: Gene therapy shows promise in the treatment of vascular disease. However, traditional transfection methods are poorly translatable to in vivo conditions due to the immune response to viral vectors and the technical impracticality of electroporation. Biodegradable polymers have shown promise as a safe alternative, relying on endocytosis of synthetic polymeric carriers bioconjugated to targeted genetic material. To date these methods have been exclusively tested and validated in stem cells. Therefore, we compared the efficiency of polymeric transfection with other transfection agents routinely used in a laboratory setting in cells of vascular origin. METHODS: Human aortic smooth muscle cells (HASMC) were transfected with GAPDH or negative control (NC) siRNAs. Transfection methods included poly(B-amino ester) polymer (StemFECT™) bioconjugates, DharmaFECT2 complexes, or Santa Cruz complexes. GAPDH gene silencing was assayed by qPCR normalized to 18S. RESULTS: Maximum silencing using DharmaFECT2 (81⫾1%; n⫽4) was achieved with a 3l reagent:200pmol siRNA complex. Equivalent silencing (75⫾4%; n⫽4; P⫽NS) was achieved using a comparable StemFECT bioconjugate of 2.6l polymer:200pmol siRNA. By increasing the bioconjugate to 3.9l polymer:200pmol siRNA, silencing was increased to 90⫾1% (n⫽6; P⬍0.05). Santa Cruz reagent complexes were the least efficient, with the maximum achievable silencing at 41⫾5% (n⫽3) using a 8l reagent:1nmol siRNA complex. CONCLUSIONS: HASMCs were efficiently transfected using polymeric bioconjugates in a manner comparable to and exceeding other transfection agents routinely used in vitro. This proof of concept establishes polymeric transfection as a viable method for in vivo investigation that may translate to targeted therapy for vascular disease.
The effects of pharmacotherapy strategies in a high-risk surgical population: A ten-year review of patients presenting with asymptomatic moderate carotid artery stenosis Bryan A Ehlert MD, Christopher A Durham MD, John T Nelson BA, Frank M Parker DO, FACS, William M Bogey MD, FACS, Charles S Powell MD, FACS, Michael C Stoner MD, FACS East Carolina University, Greenville, NC INTRODUCTION: The purpose of this study was to evaluate the optimal pharmacotherapy strategy in a high-risk atherosclerotic patient population referred to a vascular surgery practice for asymptomatic moderate carotid artery stenosis. METHODS: A retrospective review of all new patients referred from July 1998 – December 2001 for asymptomatic moderate carotid artery stenosis was performed. Demographic and clinical variables were collected over a ten year period. Study end points were stroke, mortality, and stroke-death outcomes.
Surgical Forum Abstracts
CONCLUSIONS: Unmodified siRNA can be delivered throughout the vein graft wall using the intraluminal route with moderate gene silencing. This model can be used to identify appropriate targets and further study the effectiveness of gene silencing in modifying intimal hyperplasia.
Beta-1 integrin is an upstream regulator of Eph-B4-mediated vein graft adaptation Kenneth Richard Ziegler MD, Akihito Muto MD, PhD, Lynn S Model MD, Sammy D Eghbalieh MD, Clinton D Protack MD, Michael J Collins MD, Xin Li MD, Caroline C Jadlowiec MD, Frank J Giordano MD, Alan Dardik MD, PhD, FACS Yale University School of Medicine, New Haven, CT and VA Connecticut Healthcare System, West Haven, CT INTRODUCTION: Decreased expression of Eph-B4, the embryonic venous fate determinant, is associated with successful adaptation of surgical vein grafts to the arterial environment. However, Ephrin-B2, the ligand for Eph-B4, is not expressed in veins; as such, the physiological mediator of Eph-B4 is unknown. We determined if the mechanosensitive integrin-beta-1 is an upstream regulator of Eph-B4 signaling. METHODS: Eph-B4-positive endothelial cells (EC) and Eph-B4negative Cos cells transfected with Eph-B4 were stimulated with collagen and anti-integrin-beta-1 activating antibody (P4G11) and analyzed with Western blotting. Colocalization was determined using immunofluorescence. Unilateral external carotid artery ligation was performed on heterozygous integrin-beta-1 knockout (Het-B1KO) mice; carotid arteries were examined on day 14. Veins from Het-B1-KO mice were placed as aortic interposition grafts into wildtype mice and examined at day 21. RESULTS: EC stimulated with collagen or P4G11 increased Eph-B4 phosphorylation. Eph-B4 and integrin-beta-1 colocalized on both EC and Eph-B4 transfected Cos cells after stimulation with collagen. Het-B1-KO mice that underwent unilateral ligation of the external carotid showed aberrant vascular remodeling. Het-B1-KO vein grafts showed approximately 3-fold increased neointimal thickening compared to wildtype controls. CONCLUSIONS: Integrin-beta-1 is detectable in EC, reacts to hemodynamic forces, induces Eph-B4 signaling, and alters vessel and vein graft remodeling. These results connect hemodynamic forces to vein graft adaptation via extracellular matrix signaling. Integrinbeta-1 is an upstream regulator of Eph-B4 signaling during vein graft adaptation, and may represent a novel therapeutic target to limit neointimal hyperplasia in vein grafts.
Smooth muscle cell polymeric transfection is an efficient alternative to traditional methods of experimental gene therapy Joshua D Arnold MD, Deidra J Mountain PhD, Michael B Freeman MD, FACS, Stacy S Kirkpatrick BS, Scott L Stevens MD, FACS, Mitchell H Goldman MD, FACS,
J Am Coll Surg
Oscar H Grandas MD, FACS University of Tennessee Graduate School of Medicine, Knoxville, TN INTRODUCTION: Gene therapy shows promise in the treatment of vascular disease. However, traditional transfection methods are poorly translatable to in vivo conditions due to the immune response to viral vectors and the technical impracticality of electroporation. Biodegradable polymers have shown promise as a safe alternative, relying on endocytosis of synthetic polymeric carriers bioconjugated to targeted genetic material. To date these methods have been exclusively tested and validated in stem cells. Therefore, we compared the efficiency of polymeric transfection with other transfection agents routinely used in a laboratory setting in cells of vascular origin. METHODS: Human aortic smooth muscle cells (HASMC) were transfected with GAPDH or negative control (NC) siRNAs. Transfection methods included poly(B-amino ester) polymer (StemFECT™) bioconjugates, DharmaFECT2 complexes, or Santa Cruz complexes. GAPDH gene silencing was assayed by qPCR normalized to 18S. RESULTS: Maximum silencing using DharmaFECT2 (81⫾1%; n⫽4) was achieved with a 3l reagent:200pmol siRNA complex. Equivalent silencing (75⫾4%; n⫽4; P⫽NS) was achieved using a comparable StemFECT bioconjugate of 2.6l polymer:200pmol siRNA. By increasing the bioconjugate to 3.9l polymer:200pmol siRNA, silencing was increased to 90⫾1% (n⫽6; P⬍0.05). Santa Cruz reagent complexes were the least efficient, with the maximum achievable silencing at 41⫾5% (n⫽3) using a 8l reagent:1nmol siRNA complex. CONCLUSIONS: HASMCs were efficiently transfected using polymeric bioconjugates in a manner comparable to and exceeding other transfection agents routinely used in vitro. This proof of concept establishes polymeric transfection as a viable method for in vivo investigation that may translate to targeted therapy for vascular disease.
The effects of pharmacotherapy strategies in a high-risk surgical population: A ten-year review of patients presenting with asymptomatic moderate carotid artery stenosis Bryan A Ehlert MD, Christopher A Durham MD, John T Nelson BA, Frank M Parker DO, FACS, William M Bogey MD, FACS, Charles S Powell MD, FACS, Michael C Stoner MD, FACS East Carolina University, Greenville, NC INTRODUCTION: The purpose of this study was to evaluate the optimal pharmacotherapy strategy in a high-risk atherosclerotic patient population referred to a vascular surgery practice for asymptomatic moderate carotid artery stenosis. METHODS: A retrospective review of all new patients referred from July 1998 – December 2001 for asymptomatic moderate carotid artery stenosis was performed. Demographic and clinical variables were collected over a ten year period. Study end points were stroke, mortality, and stroke-death outcomes.