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Simian Immunodeficiency Virus-Like Particle Vaccine Reduces Atherosclerosis in Apolipoprotein E Knockout Mice
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
formation, tensile strength of mesh, surface area and thickness of mesh at variable time points following implantation. We report our results after one month. Methods: Forty eight Sprague-Dawley rats were randomized into two groups, PermacolÔ and StratticeÔ. Each rat had a midline incision, skin flaps raised, and a 3 cm x 3 cm biologic mesh implanted using running 4-0 monofilament, non-absorbable suture. Skin was closed over the mesh. One month following implantation 6 rats in each group were sacrificed. The anterior abdominal wall skin was elevated and a subsequent trap door incision was created. The abdominal wall was reflected cranially and photographed to document adhesions. The thickness of the mesh was measured using a digital caliper. Biologic mesh was then harvested in order to perform tensile testing measurements. A 0.5 cm x 2 cm central portion of the mesh and a 0.5 cm wide portion of mesh/muscle interface were excised and tested to determine the breaking strength of the mesh itself and the mesh/muscle interface using an Instron-Sacks biaxial testing system in uniaxial mode. A 2 cm gauge length and a strain rate of 0.825 mm/min were used for the mesh; the strain rate was increased to 1.625 mm/min for the mesh/muscle interface. Adhesion tenacity,area, and tensile strength were compared using two-tailed t-tests comparing means. Results: At one month, adhesion tenacity was not significantly different between PermacolÔ and StratticeÔ. The failure strengths of PermacolÔ and StratticeÔ meshes were (mean 6 SD) 97.8 6 38.5 N and 140.7 6 61.3 N (p ¼ 0.15), whereas the junction strengths were 3.2 6 1.3 N and 4.2 6 1.7 N (p ¼ 0.29), respectively. The average thickness of both the PermacolÔ and StratticeÔ meshes were 1.1 6 0.1 mm. The surface areas for PermacolÔ and StratticeÔ were 7.3 6 1.1 cm2and 6.7 6 2.1 cm2. Conclusions: StratticeÔ had higher breaking strengths at one month. The biologics had the same thickness and surface area at one month. However, the StratticeÔ material was thicker at the outset, 3.0 mm, and 1.5 mm for PermacolÔ. The crosslinking of the PermacolÔ likely provides protection from biologic degradation by the host collegenase. The 3, 6, and 12 months results evaluating the above parameters including collagen and endothelial ingrowth will reveal which product will be more durable over the life of the hernia repair.
VASCULAR 3: INTIMAL HYPERPLASIA 49.1. Simian Immunodeficiency Virus-Like Particle Vaccine Reduces Atherosclerosis in Apolipoprotein E Knockout Mice. D. Liao, S. Zhang, X. Wang, H. Wang, P. Lin, Q. Yao, C. Chen; Baylor College of Medicine, Houston, TX Introduction: Experimental observations have suggested that modulation of immune responses involved in atherosclerosis may represent a novel approach to the management of atherosclerotic cardiovascular disease. Our previous publications have shown that simian immunodeficiency virus-like particle (SIV-VLP) vaccine induced a unique cytokine profile in splenocytes, such as increasing interleukin-10 (IL-10), which is anti-atherogenic. We therefore hypothesized that SIV-VLP vaccine may reduce atherogenesis though the mechanism of immune modulation. Methods: Insect Sf9 cells were infected with a recombinant baculovirus (rBV) expressing SIVmac239 gag, and SIV-VLPs were produced and purified from the culture medium of the infected cells. The female C56BL/6 J ApoE knockout mice were immunized twice with VLP-emulsified in CFA or with PBS in CFA, and then fed western diet for 2 months (n ¼ 5 for each group). Mouse thoracic and abdominal aortas were dissected and stained with Sudan IV at 5 months of age. The percent surface area occupied by sudan IV-stained lesions viewed en face was measured by using Image J software. Spleen specimens and aorta samples were subjected to immunostaining with mouse anti-CD3, mouse anti-CD68, and rat anti-IL-10 antibody. SIV VLP-specific antibodies in the blood serum were determined by ELISA. Total cholesterol and HDL levels in the mouse serum were measured using Wako kits. Results: Comparison of vascular lesion area in the mouse aorta
between SIV-VLP group and PBS control group revealed a marked difference (9% versus 14% of lesion area in aorta), showing 36% reduction (P < 0.05). SIV-VLP immunization induced a significant increase in the level of anti-SIV IgG antibodies in Apo E-/- mice compared with PBS group (P < 0.05), suggesting the effective immune response. The signal of anti-IL-10 staining in both vessel wall and spleen was much higher in the SIV-VLP group than that in the PBS group. However, the cells with the expression of CD68 and CD3 in either the vessel wall or spleen were similar in both VLP and PBS groups, indicating that macrophage and T lymphocyte contents were not significantly altered by SIV-VLP immunization. There was no significant difference of total cholesterol and HDL levels between two groups. Conclusions: These data demonstrated that SIV-VLP immunization significantly decreased the extent of atherosclerosis and increased IL-10 levels in Apo E-/- mice, independent to changes in blood cholesterol and HDL levels. This study reports a novel strategy to inhibit the atherosclerotic lesion formation, which may have clinical applications.
49.2. Nitrotyrosine Reduces the Expression and Function of Endothelial Nitric Oxide Synthase in Human Coronary Artery Endothelial Cells. J. Jiang, P. Lin, Q. Yao, C. Chen; Baylor College of Medicine, Houston, TX Introduction: Recent studies demonstrate that the serum nitrotyrosine level is a new biomarker for inflammatory disease including coronary artery atherosclerosis and other cardiovascular diseases. However, it is not clear whether nitrotyrosine could directly affect vascular cells and contribute to the vascular lesion formation. In this study, we determined the effects and potential mechanisms of nitrotyrosine on the expression and function of endothelial nitric oxide (eNOS) in human coronary artery endothelial cells (HCAECs) and porcine coronary arteries. Methods: HCAECs were treated with clinically relevant concentrations (0.3, 3, 30 nM) for 6, 24 or 48 hrs. Tyrosine (30 nM) was used as a control. The expression of eNOS at both mRNA and protein levels was determined by real-time PCR and Western blot analysis, respectively. Activation status of signal transduction molecules including mitogen-activated protein kinases (MAPKs) and IkBa was studied using BioPlex immunoassay. In addition, porcine coronary artery rings were cultured with nitrotyrosine (30 nM) for 24 hrs. Vasomotor reactivity in response to vascular active drugs was studied using a myograph tension system. Antioxidants seleno-L-methionine (SeMet) and ginsenoside Rb1 were included. Results: HCAECs were treated with three different concentrations of nitrotyrosine (0.3, 3 and 30 nM) for 24 hrs, and eNOS mRNA was significantly reduced by 38% in the 30 nM nitrotyrosine treated group (P < 0.05), compared with the tyrosine-treated group (control). Time course study showed that eNOS mRNA levels in 30 nM nitrotyrosine-treated HCAECs started to decrease from 6 hrs and further decreased at 24 and 48 hrs. The eNOS protein levels were also significantly decreased with nitrotyrosine (30 nM) treatment in HCAECs at 6, 24 and 48 hrs as compared with tyrosine controls (P < 0.05). Decrease in the eNOS protein level was more sensitive to nitrotyrosine treatment than the eNOS mRNA level, suggesting the posttranslational regulation may be more affected. Antioxidant SeMet effectively blocked nitrotyrosine-induced downregulation of eNOS at both mRNA and protein levels in HCAECs. The phosphorylation levels of JNK, ERK2 and IkBa, but not p38, were substantially increased at 15 min after 30 nM nitrotyrosine treatment. In porcine coronary artery ring cultures, treatment of nitrotyrosine (30 nM) for 24 hrs significantly reduced endothelium-dependent vasorelaxation by 37% in response to bradykinin (106 M) as compared with tyrosine controls (P < 0.05), while ginsenoside Rb1 (10 mM) effectively blocked nitrotyrosine’s effects in porcine coronary artery rings. Conclusions: These data demonstrate that clinically relevant concentration of nitrotyrosine significantly reduces eNOS expression at both mRNA and protein levels in HCAECs. This effect may be mediated by oxidative stress and signal transduction pathway with JNK, ERK1/2 and NF-kB.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
formation, tensile strength of mesh, surface area and thickness of mesh at variable time points following implantation. We report our results after one month. Methods: Forty eight Sprague-Dawley rats were randomized into two groups, PermacolÔ and StratticeÔ. Each rat had a midline incision, skin flaps raised, and a 3 cm x 3 cm biologic mesh implanted using running 4-0 monofilament, non-absorbable suture. Skin was closed over the mesh. One month following implantation 6 rats in each group were sacrificed. The anterior abdominal wall skin was elevated and a subsequent trap door incision was created. The abdominal wall was reflected cranially and photographed to document adhesions. The thickness of the mesh was measured using a digital caliper. Biologic mesh was then harvested in order to perform tensile testing measurements. A 0.5 cm x 2 cm central portion of the mesh and a 0.5 cm wide portion of mesh/muscle interface were excised and tested to determine the breaking strength of the mesh itself and the mesh/muscle interface using an Instron-Sacks biaxial testing system in uniaxial mode. A 2 cm gauge length and a strain rate of 0.825 mm/min were used for the mesh; the strain rate was increased to 1.625 mm/min for the mesh/muscle interface. Adhesion tenacity,area, and tensile strength were compared using two-tailed t-tests comparing means. Results: At one month, adhesion tenacity was not significantly different between PermacolÔ and StratticeÔ. The failure strengths of PermacolÔ and StratticeÔ meshes were (mean 6 SD) 97.8 6 38.5 N and 140.7 6 61.3 N (p ¼ 0.15), whereas the junction strengths were 3.2 6 1.3 N and 4.2 6 1.7 N (p ¼ 0.29), respectively. The average thickness of both the PermacolÔ and StratticeÔ meshes were 1.1 6 0.1 mm. The surface areas for PermacolÔ and StratticeÔ were 7.3 6 1.1 cm2and 6.7 6 2.1 cm2. Conclusions: StratticeÔ had higher breaking strengths at one month. The biologics had the same thickness and surface area at one month. However, the StratticeÔ material was thicker at the outset, 3.0 mm, and 1.5 mm for PermacolÔ. The crosslinking of the PermacolÔ likely provides protection from biologic degradation by the host collegenase. The 3, 6, and 12 months results evaluating the above parameters including collagen and endothelial ingrowth will reveal which product will be more durable over the life of the hernia repair.
VASCULAR 3: INTIMAL HYPERPLASIA 49.1. Simian Immunodeficiency Virus-Like Particle Vaccine Reduces Atherosclerosis in Apolipoprotein E Knockout Mice. D. Liao, S. Zhang, X. Wang, H. Wang, P. Lin, Q. Yao, C. Chen; Baylor College of Medicine, Houston, TX Introduction: Experimental observations have suggested that modulation of immune responses involved in atherosclerosis may represent a novel approach to the management of atherosclerotic cardiovascular disease. Our previous publications have shown that simian immunodeficiency virus-like particle (SIV-VLP) vaccine induced a unique cytokine profile in splenocytes, such as increasing interleukin-10 (IL-10), which is anti-atherogenic. We therefore hypothesized that SIV-VLP vaccine may reduce atherogenesis though the mechanism of immune modulation. Methods: Insect Sf9 cells were infected with a recombinant baculovirus (rBV) expressing SIVmac239 gag, and SIV-VLPs were produced and purified from the culture medium of the infected cells. The female C56BL/6 J ApoE knockout mice were immunized twice with VLP-emulsified in CFA or with PBS in CFA, and then fed western diet for 2 months (n ¼ 5 for each group). Mouse thoracic and abdominal aortas were dissected and stained with Sudan IV at 5 months of age. The percent surface area occupied by sudan IV-stained lesions viewed en face was measured by using Image J software. Spleen specimens and aorta samples were subjected to immunostaining with mouse anti-CD3, mouse anti-CD68, and rat anti-IL-10 antibody. SIV VLP-specific antibodies in the blood serum were determined by ELISA. Total cholesterol and HDL levels in the mouse serum were measured using Wako kits. Results: Comparison of vascular lesion area in the mouse aorta
between SIV-VLP group and PBS control group revealed a marked difference (9% versus 14% of lesion area in aorta), showing 36% reduction (P < 0.05). SIV-VLP immunization induced a significant increase in the level of anti-SIV IgG antibodies in Apo E-/- mice compared with PBS group (P < 0.05), suggesting the effective immune response. The signal of anti-IL-10 staining in both vessel wall and spleen was much higher in the SIV-VLP group than that in the PBS group. However, the cells with the expression of CD68 and CD3 in either the vessel wall or spleen were similar in both VLP and PBS groups, indicating that macrophage and T lymphocyte contents were not significantly altered by SIV-VLP immunization. There was no significant difference of total cholesterol and HDL levels between two groups. Conclusions: These data demonstrated that SIV-VLP immunization significantly decreased the extent of atherosclerosis and increased IL-10 levels in Apo E-/- mice, independent to changes in blood cholesterol and HDL levels. This study reports a novel strategy to inhibit the atherosclerotic lesion formation, which may have clinical applications.
49.2. Nitrotyrosine Reduces the Expression and Function of Endothelial Nitric Oxide Synthase in Human Coronary Artery Endothelial Cells. J. Jiang, P. Lin, Q. Yao, C. Chen; Baylor College of Medicine, Houston, TX Introduction: Recent studies demonstrate that the serum nitrotyrosine level is a new biomarker for inflammatory disease including coronary artery atherosclerosis and other cardiovascular diseases. However, it is not clear whether nitrotyrosine could directly affect vascular cells and contribute to the vascular lesion formation. In this study, we determined the effects and potential mechanisms of nitrotyrosine on the expression and function of endothelial nitric oxide (eNOS) in human coronary artery endothelial cells (HCAECs) and porcine coronary arteries. Methods: HCAECs were treated with clinically relevant concentrations (0.3, 3, 30 nM) for 6, 24 or 48 hrs. Tyrosine (30 nM) was used as a control. The expression of eNOS at both mRNA and protein levels was determined by real-time PCR and Western blot analysis, respectively. Activation status of signal transduction molecules including mitogen-activated protein kinases (MAPKs) and IkBa was studied using BioPlex immunoassay. In addition, porcine coronary artery rings were cultured with nitrotyrosine (30 nM) for 24 hrs. Vasomotor reactivity in response to vascular active drugs was studied using a myograph tension system. Antioxidants seleno-L-methionine (SeMet) and ginsenoside Rb1 were included. Results: HCAECs were treated with three different concentrations of nitrotyrosine (0.3, 3 and 30 nM) for 24 hrs, and eNOS mRNA was significantly reduced by 38% in the 30 nM nitrotyrosine treated group (P < 0.05), compared with the tyrosine-treated group (control). Time course study showed that eNOS mRNA levels in 30 nM nitrotyrosine-treated HCAECs started to decrease from 6 hrs and further decreased at 24 and 48 hrs. The eNOS protein levels were also significantly decreased with nitrotyrosine (30 nM) treatment in HCAECs at 6, 24 and 48 hrs as compared with tyrosine controls (P < 0.05). Decrease in the eNOS protein level was more sensitive to nitrotyrosine treatment than the eNOS mRNA level, suggesting the posttranslational regulation may be more affected. Antioxidant SeMet effectively blocked nitrotyrosine-induced downregulation of eNOS at both mRNA and protein levels in HCAECs. The phosphorylation levels of JNK, ERK2 and IkBa, but not p38, were substantially increased at 15 min after 30 nM nitrotyrosine treatment. In porcine coronary artery ring cultures, treatment of nitrotyrosine (30 nM) for 24 hrs significantly reduced endothelium-dependent vasorelaxation by 37% in response to bradykinin (106 M) as compared with tyrosine controls (P < 0.05), while ginsenoside Rb1 (10 mM) effectively blocked nitrotyrosine’s effects in porcine coronary artery rings. Conclusions: These data demonstrate that clinically relevant concentration of nitrotyrosine significantly reduces eNOS expression at both mRNA and protein levels in HCAECs. This effect may be mediated by oxidative stress and signal transduction pathway with JNK, ERK1/2 and NF-kB.