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5-Lipoxygenase Inhibition Attenuates Intestinal Polyposis Through Suppression of Inflammation
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
17.5. NLRP3 Inflammasome Activates Matrix Metalloproteinase-9: Potential Role in Smooth Muscle Cell Dysfunction in Thoracic Aortic Disease. D. Wu,1,2,3 J. C. Choi,1,3 J. Coselli,1,3 Y. H. Shen,1,3 S. A. LeMaire1,2,3; 1Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Houston, TX; 2Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX; 3 Texas Heart Institute At St. Lukes Episcopal Hospital, Houston, TX Introduction: Excessive matrix metalloproteinase (MMP) activity during inflammation disrupts aortic wall homeostasis, causing progression of thoracic aortic aneurysms and dissections (TAAD). The NLRP3-ASCcaspase-1 inflammasome complex is critically involved in the activation and secretion of inflammatory factors. However, the role of this complex in regulating MMP activity remains unknown. We hypothesize that the NLRP3 inflammasome complex activates MMP-9, leading to cleavage of contractile proteins, resulting in thoracic aortic smooth muscle cell (SMC) dysfunction. Methods: Cultured thoracic SMCs were treated with palmitic acid (PA) and NLRP3 siRNA, ASC siRNA, or caspase-1 siRNA. MMP-9 cleavage and activity was assessed by immunoblotting and zymography, respectively.Protein interactions were examined by co-immunoprecipitation of protein lysate from SMCs. Direct cleavage of contractile proteins was determined by incubation of recombinant caspase-1 and recombinant MMP-9 with co-immunoprecipitated proteins. Results: In SMCs treated with PA, there were dose-dependent increases in NLRP3, ASC, and caspase-1 levels. Palmitic acid also increased MMP-9 cleavage to a 67 KDa active fragment. NLRP3 siRNA, ASC siRNA, and caspase-1 siRNA decreased PA-induced MMP-9 cleavage. Co-immunoprecipitation of protein lysate from PA-treated SMCs indicates MMP-9 binds to NLRP3, ASC, and caspase-1. In vitro activity assay showed that recombinant caspase-1 directly cleaved recombinant MMP-9. Addition of recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) decreased caspase-1 cleavage and increased digestion of MMP-9 to an inactive form. To investigate potential intracellular function of MMP-9, we studied the relationship of the NLRP3-ASC-caspase1 inflammasome complex and MMP-9 with contractile proteins. We found that caspase-1 binds to myosin heavy chain, myosin light chain, caldesmon, calmodulin, calponin, and tropomyosin. MMP-9 binds to myosin heavy chain fragment, tropomyosin, and caldesmon. Interestingly, caspase-1 directly cleaved myosin light chain and tropomyosin. Conclusions: In thoracic aortic SMCs, MMP-9 can be cleaved and activated by the NLRP3-ASC-caspase-1 inflammasome complex. Caspase1 and MMP-9 are involved in the cleavage of sarcomere proteins. This study provides a novel mechanism for intracellular MMP-9 activation and SMC dysfunction. In addition to MMP-9’s well-established role in mediating extracellular matrix destruction during the development of TAAD, MMP-9-mediated alterations in SMC function may also contribute to the progression of this disease.
17.6. Organ Tissue Engineering Using Autologous Decellularized Microcirculatory Beds AS Vascularized Bioscaffolds. L. H. Evers, W. Liu, D. Simons, M. Sorkin, M. T. Longaker, G. C. Gurtner; Stanford University School of Medicine Department of Surgery, Stanford, California Introduction: Advances in tissue engineering are hampered by the inability of artificial scaffolds to recapitulate complex microvascular structures. However, functional microvascular beds exist throughout the body, they are explantable and readily re-integrated into the systemic circulation (i.e. microvascular free flaps). In the current study we utilize these explantable microvascular beds (EMBs) as scaffolds for progenitor cell seeding as a novel approach to organ-level tissue engineering. Methods: Superficial inferior epigastric artery flaps (SIEA) were harvested from Sprague-Dawley rats and maintained on a perfusion bioreactor. After decellularization, EMBs were seeded with Luciferase positive Rat adipose-de-
rived stem cells (ASCs). After 24 h the ASC seeded-EMBs were microsurgically re-anastomosed into the femoral vessels of the rat. Vascular integrity was confirmed using SEM and immunohistochemical staining for matrix-specific components. Cell viability and integration were followed using bioluminescent imaging. Results: SIEA/EMB tissue flaps were effectively decellularized and maintained on the bioreactor for 24 hours. SEM and immunohistochemical staining for collagen IV and laminin confirmed vascular integrity. Seeding of ASCs onto EMBs was successfully achieved with perfusion methods. ASCs seeded onto EMBs demonstrated excellent viability and engraftment in vitro. Transplanted EMB/ASC constructs were viably sustained in vivo based on bioluminescence imaging and histology. Conclusions: Autologous vascular beds are ideal bioscaffolds that can be effectively decellularized and seeded with ASCs. Reintegration of seeded EMBs with longterm viability is feasible. Together, these successful studies suggest that progenitor cell-seeded vascularized scaffolds are a promising approach to fabricate complex organ-level constructs.
17.7. 5-Lipoxygenase Inhibition Attenuates Intestinal Polyposis Through Suppression of Inflammation. M. J. Heiferman,1,2 J. R. Heiferman,1,2 N. R. Blatner,2 J. D. Phillips,1,2 E. C. Cheon,1,2 L. M. Knab,1,2 E. A. Wayne,1,2 K. Khazaie,2 P. J. Grippo,1,2 D. J. Bentrem1,2,3; 1Northwestern University Department of Surgery, Chicago, IL; 2Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL; 3Jesse Brown VA Medical Center, Chicago, IL Introduction: Inflammation is known to play a key role in the pathogenesis of colorectal cancer. Patients with inflammatory bowel disease such as Crohn’s disease or ulcerative colitis have an increased risk of developing colorectal cancer. Our lab has demonstrated that 5-lipoxygenase (5LO), an enzyme in the inflammatory arachidonic acid pathway, is critical for the development of adenomatous polyps, which are known precursor lesions to colorectal cancer. We previously used the APCD468 mouse model, which develops a severe intestinal polyposis at four months of age, and genetically knocked out 5LO in this model resulting in a dramatic decrease in intestinal polyps. In this study, we hypothesize that an oral 5LO inhibitor, zileution, will attenuate inflammation and polyp growth. Methods: Pharmacological inhibition of 5LO was achieved using zileuton, a 5LO specific inhibitor presently used to treat chronic asthma. Four week old APCD468 mice were fed a chow containing 1200 parts per million of zileuton, while control mice were fed the same chow without the drug. Mice were sacrificed at four months of age and evaluated by polyp count, body and spleen mass, serum cytokine levels, and local inflammatory cell infiltration. Results: We found the zileuton fed APCD468 mice had a 60% decrease in polyp number compared to APCD468 mice on the control diet (P < 0.05). Furthermore, the zileuton fed mice weighed 28% more (P < 0.01) and had 64% lighter spleens (P < 0.05) compared to the untreated control mice. A panel of cytokines was measured in the serum of zileuton treated APCD468 mice, untreated control APCD468 mice, and untreated healthy B6 mice. As expected, the B6 mice had low levels of IL1b, TNFa, IL10, and IL17 while the untreated APCD468 mice had elevated inflammatory cytokines. The zileuton treated APCD468 mice had serum cytokine concentrations similar to the B6 mice that were significantly lower compared to untreated APCD468 mice. Finally, we observed a significant decrease in mast cell, macrophage, and myeloid derived suppressor cell infiltration into the tumor microenvironment of the zileuton treated mice compared to the APCD468 controls. Conclusions: This study demonstrates that oral pharmacological inhibition of 5LO in the APCD468 mouse model results in a decreased inflammatory response and a dramatic reduction of polyp burden. These results are clinically significant because zileuton is already an FDA approved medication. Our study provides pre-clinical evidence for the expansion of zileuton as a possible anti-cancer therapy, however, further studies are needed.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
17.5. NLRP3 Inflammasome Activates Matrix Metalloproteinase-9: Potential Role in Smooth Muscle Cell Dysfunction in Thoracic Aortic Disease. D. Wu,1,2,3 J. C. Choi,1,3 J. Coselli,1,3 Y. H. Shen,1,3 S. A. LeMaire1,2,3; 1Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Houston, TX; 2Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX; 3 Texas Heart Institute At St. Lukes Episcopal Hospital, Houston, TX Introduction: Excessive matrix metalloproteinase (MMP) activity during inflammation disrupts aortic wall homeostasis, causing progression of thoracic aortic aneurysms and dissections (TAAD). The NLRP3-ASCcaspase-1 inflammasome complex is critically involved in the activation and secretion of inflammatory factors. However, the role of this complex in regulating MMP activity remains unknown. We hypothesize that the NLRP3 inflammasome complex activates MMP-9, leading to cleavage of contractile proteins, resulting in thoracic aortic smooth muscle cell (SMC) dysfunction. Methods: Cultured thoracic SMCs were treated with palmitic acid (PA) and NLRP3 siRNA, ASC siRNA, or caspase-1 siRNA. MMP-9 cleavage and activity was assessed by immunoblotting and zymography, respectively.Protein interactions were examined by co-immunoprecipitation of protein lysate from SMCs. Direct cleavage of contractile proteins was determined by incubation of recombinant caspase-1 and recombinant MMP-9 with co-immunoprecipitated proteins. Results: In SMCs treated with PA, there were dose-dependent increases in NLRP3, ASC, and caspase-1 levels. Palmitic acid also increased MMP-9 cleavage to a 67 KDa active fragment. NLRP3 siRNA, ASC siRNA, and caspase-1 siRNA decreased PA-induced MMP-9 cleavage. Co-immunoprecipitation of protein lysate from PA-treated SMCs indicates MMP-9 binds to NLRP3, ASC, and caspase-1. In vitro activity assay showed that recombinant caspase-1 directly cleaved recombinant MMP-9. Addition of recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) decreased caspase-1 cleavage and increased digestion of MMP-9 to an inactive form. To investigate potential intracellular function of MMP-9, we studied the relationship of the NLRP3-ASC-caspase1 inflammasome complex and MMP-9 with contractile proteins. We found that caspase-1 binds to myosin heavy chain, myosin light chain, caldesmon, calmodulin, calponin, and tropomyosin. MMP-9 binds to myosin heavy chain fragment, tropomyosin, and caldesmon. Interestingly, caspase-1 directly cleaved myosin light chain and tropomyosin. Conclusions: In thoracic aortic SMCs, MMP-9 can be cleaved and activated by the NLRP3-ASC-caspase-1 inflammasome complex. Caspase1 and MMP-9 are involved in the cleavage of sarcomere proteins. This study provides a novel mechanism for intracellular MMP-9 activation and SMC dysfunction. In addition to MMP-9’s well-established role in mediating extracellular matrix destruction during the development of TAAD, MMP-9-mediated alterations in SMC function may also contribute to the progression of this disease.
17.6. Organ Tissue Engineering Using Autologous Decellularized Microcirculatory Beds AS Vascularized Bioscaffolds. L. H. Evers, W. Liu, D. Simons, M. Sorkin, M. T. Longaker, G. C. Gurtner; Stanford University School of Medicine Department of Surgery, Stanford, California Introduction: Advances in tissue engineering are hampered by the inability of artificial scaffolds to recapitulate complex microvascular structures. However, functional microvascular beds exist throughout the body, they are explantable and readily re-integrated into the systemic circulation (i.e. microvascular free flaps). In the current study we utilize these explantable microvascular beds (EMBs) as scaffolds for progenitor cell seeding as a novel approach to organ-level tissue engineering. Methods: Superficial inferior epigastric artery flaps (SIEA) were harvested from Sprague-Dawley rats and maintained on a perfusion bioreactor. After decellularization, EMBs were seeded with Luciferase positive Rat adipose-de-
rived stem cells (ASCs). After 24 h the ASC seeded-EMBs were microsurgically re-anastomosed into the femoral vessels of the rat. Vascular integrity was confirmed using SEM and immunohistochemical staining for matrix-specific components. Cell viability and integration were followed using bioluminescent imaging. Results: SIEA/EMB tissue flaps were effectively decellularized and maintained on the bioreactor for 24 hours. SEM and immunohistochemical staining for collagen IV and laminin confirmed vascular integrity. Seeding of ASCs onto EMBs was successfully achieved with perfusion methods. ASCs seeded onto EMBs demonstrated excellent viability and engraftment in vitro. Transplanted EMB/ASC constructs were viably sustained in vivo based on bioluminescence imaging and histology. Conclusions: Autologous vascular beds are ideal bioscaffolds that can be effectively decellularized and seeded with ASCs. Reintegration of seeded EMBs with longterm viability is feasible. Together, these successful studies suggest that progenitor cell-seeded vascularized scaffolds are a promising approach to fabricate complex organ-level constructs.
17.7. 5-Lipoxygenase Inhibition Attenuates Intestinal Polyposis Through Suppression of Inflammation. M. J. Heiferman,1,2 J. R. Heiferman,1,2 N. R. Blatner,2 J. D. Phillips,1,2 E. C. Cheon,1,2 L. M. Knab,1,2 E. A. Wayne,1,2 K. Khazaie,2 P. J. Grippo,1,2 D. J. Bentrem1,2,3; 1Northwestern University Department of Surgery, Chicago, IL; 2Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL; 3Jesse Brown VA Medical Center, Chicago, IL Introduction: Inflammation is known to play a key role in the pathogenesis of colorectal cancer. Patients with inflammatory bowel disease such as Crohn’s disease or ulcerative colitis have an increased risk of developing colorectal cancer. Our lab has demonstrated that 5-lipoxygenase (5LO), an enzyme in the inflammatory arachidonic acid pathway, is critical for the development of adenomatous polyps, which are known precursor lesions to colorectal cancer. We previously used the APCD468 mouse model, which develops a severe intestinal polyposis at four months of age, and genetically knocked out 5LO in this model resulting in a dramatic decrease in intestinal polyps. In this study, we hypothesize that an oral 5LO inhibitor, zileution, will attenuate inflammation and polyp growth. Methods: Pharmacological inhibition of 5LO was achieved using zileuton, a 5LO specific inhibitor presently used to treat chronic asthma. Four week old APCD468 mice were fed a chow containing 1200 parts per million of zileuton, while control mice were fed the same chow without the drug. Mice were sacrificed at four months of age and evaluated by polyp count, body and spleen mass, serum cytokine levels, and local inflammatory cell infiltration. Results: We found the zileuton fed APCD468 mice had a 60% decrease in polyp number compared to APCD468 mice on the control diet (P < 0.05). Furthermore, the zileuton fed mice weighed 28% more (P < 0.01) and had 64% lighter spleens (P < 0.05) compared to the untreated control mice. A panel of cytokines was measured in the serum of zileuton treated APCD468 mice, untreated control APCD468 mice, and untreated healthy B6 mice. As expected, the B6 mice had low levels of IL1b, TNFa, IL10, and IL17 while the untreated APCD468 mice had elevated inflammatory cytokines. The zileuton treated APCD468 mice had serum cytokine concentrations similar to the B6 mice that were significantly lower compared to untreated APCD468 mice. Finally, we observed a significant decrease in mast cell, macrophage, and myeloid derived suppressor cell infiltration into the tumor microenvironment of the zileuton treated mice compared to the APCD468 controls. Conclusions: This study demonstrates that oral pharmacological inhibition of 5LO in the APCD468 mouse model results in a decreased inflammatory response and a dramatic reduction of polyp burden. These results are clinically significant because zileuton is already an FDA approved medication. Our study provides pre-clinical evidence for the expansion of zileuton as a possible anti-cancer therapy, however, further studies are needed.