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061 Extracellular matrix remodeling by granzyme B in aging tissues of apolipoprotein E knockout mice
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proliferation and migration from the medial layer into the neointimal layer of the vessel. Our lab has recently shown that after loss of contact with the endothelial cell layer, smooth muscle cells attain planar cell polarity, triggering the specific intracellular localization of signaling molecules, and the reorganization of the cytoskeleton to allow for directed cell migration from the medial to the intimal layer. In addition, the medial cells must degrade the internal elastic lamina and other matrix molecules to invade through the vessel wall. We now report that smooth muscle cells accomplish this using invadopodia, which are similar to structures formed in invading cancer cells. Invadopodia are actin- and cortactin-containing podosome-like structures which localize the degradative machinery onto discrete points in the extracellular matrix. METHODS: Primary cultures of medial vascular smooth muscle cells were obtained by isolating rat carotid arteries and gently denuding the endothelial layer. Cells were plated on Alexa-488 labeled fibronectin which was layered on type I collagen-coated glass bottom dishes. Some cultures were stimulated with the phorbol ester PDBU (10 M), a known inducer of invadopodia, and also treated with the src kinase inhibitor, SU6656 (10M) or the MMP inhibitor, GM6001 (25 M). Live cultures were imaged using time-lapse confocal microscopy. RESULTS: Vascular smooth muscle cells grown on the fibronectin-collagen substrate spontaneously formed structures that contained actin and cortactin, two well-known markers of invadopodia. Matrix metalloproteinase 14 was also evident at these structures. Degradation and clearance of the Alexa-488 labeled fibronectin layer was observed in association with these structures, along with phagocytosis of the fibronectin. This process was stimulated by the addition of PDBU, as evidenced by an increased area of substrate clearance. The addition of SU6656 greatly diminished the area of cleared Alexa-488 labeled fibronectin. A similar inhibition was observed after treatment with GM6001, the MMP inhibitor. CONCLUSION: Medial smooth muscle cells, when removed from their contact with endothelial cells, spontaneously develop invasive structures which aid in the digestion of the matrix, and ultimately lead to smooth muscle cell migration and formation of neointima. By understanding novel mechanisms that promote vascular smooth muscle cell migration and invasion, potential therapeutic targets aimed at inhibiting these processes may be realized. Heart and Stroke Foundation 061 EXTRACELLULAR MATRIX REMODELING BY GRANZYME B IN AGING TISSUES OF APOLIPOPROTEIN E KNOCKOUT MICE P Hiebert, W Boivin, T Abraham, S Pazooki, H Zhao, D Granville Vancouver, British Columbia
Recent aging theories have identified molecular inflammation as an important mechanism contributing to the accumulation of damage and tissue deterioration that occurs with age. Granzyme B (GrB) is a serine protease that can be released into the extracellular spaces by inflammatory cells during chronic inflammation. This can have pathological consequences influencing aging and
BACKGROUND:
Canadian Journal of Cardiology Volume 27 2011
disease of the vessels due to the ability of GrB to degrade several components of the extracellular matrix (ECM) including decorin, a proteoglycan that closely associates with collagen and is vital for proper collagen organization and structural integrity. The objective of this work is to use GrB deficient mice to examine the role of GrB in ECM degradation that occurs in the tissues of apolipoprotein E knockout (apoE-KO) mice, and how this might accelerate vascular aging and disease. METHODS: Because apoE-KO mice fed a high fat diet exhibit chronic inflammation in their skin, we used the skin from these mice as a model to examine immune-mediated mechanisms of aging and disease. Specifically, GrB-mediated ECM degradation in the context of aging and disease was examined. C57BL/6 wild type, apoE-KO and GrB/apoE double knockout (DKO) mice were fed either a high fat or regular chow diet for 0, 5, 15 and 30 weeks. Hematoxylin & eosin, picrosirius red and luna’s elastin stains were used to assess morphology, collagen and elastin respectively, in formalin fixed tissue sections. Multi-photon microscopy was also used on fresh ex vivo tissue samples to examine collagen organization and density. Immunohistochemistry was also performed on fixed sections to examine decorin and GrB. RESULTS: ApoE-KO mice fed a high fat diet demonstrated several premature aging characteristics including frailty, increased morbidity, skin thinning and a loss of dermal collagen density and organization that increased in severity over time. DKO mice showed protection against these deleterious changes. Abnormal elastin morphology was also observed in apoE-KO mice. High fat diet fed apoE-KO mice also showed reduced levels of the proteoglycan, decorin, while DKO mice showed increased decorin which corresponded to better collagen organization and structural integrity. GrB staining was also observed in the skin of apoE-KO mice, particularly in and around mast cells and areas of ECM remodeling. CONCLUSIONS: ApoE-KO mice fed a high fat diet display features of premature aging, a phenotype that is reduced when knocking out GrB suggesting that GrB plays a role in agerelated tissue damage such as collagen disorganization, possibly through decorin degradation. 062 C-TYPE NATRIURETIC PEPTIDE IS INVOLVED IN PROTEOGLYCAN-RICH LEAFLET THICKENING IN EARLY AORTIC VALVE DISEASE MC Blaser, KL Sider, Y Zhou, M Henkelman, CA Simmons Toronto, Ontario BACKGROUND: Calcific aortic valve disease (CAVD) is a cell-mediated pathology without effective pharmacotherapy. Previous microarray studies have found that C-type natriuretic peptide (CNP) is differentially expressed between the aortic (disease-prone) and ventricular (protected) sides of porcine aortic valves. We investigated the effects of a mildly atherogenic diet on early CAVD development, and the involvement of CNP in this process. METHODS: Male WT C57Bl/6J mice were fed a control diet or BioServ F3282, a high-fat, high-carbohydrate diet (HF/HC) with 58.7%
proliferation and migration from the medial layer into the neointimal layer of the vessel. Our lab has recently shown that after loss of contact with the endothelial cell layer, smooth muscle cells attain planar cell polarity, triggering the specific intracellular localization of signaling molecules, and the reorganization of the cytoskeleton to allow for directed cell migration from the medial to the intimal layer. In addition, the medial cells must degrade the internal elastic lamina and other matrix molecules to invade through the vessel wall. We now report that smooth muscle cells accomplish this using invadopodia, which are similar to structures formed in invading cancer cells. Invadopodia are actin- and cortactin-containing podosome-like structures which localize the degradative machinery onto discrete points in the extracellular matrix. METHODS: Primary cultures of medial vascular smooth muscle cells were obtained by isolating rat carotid arteries and gently denuding the endothelial layer. Cells were plated on Alexa-488 labeled fibronectin which was layered on type I collagen-coated glass bottom dishes. Some cultures were stimulated with the phorbol ester PDBU (10 M), a known inducer of invadopodia, and also treated with the src kinase inhibitor, SU6656 (10M) or the MMP inhibitor, GM6001 (25 M). Live cultures were imaged using time-lapse confocal microscopy. RESULTS: Vascular smooth muscle cells grown on the fibronectin-collagen substrate spontaneously formed structures that contained actin and cortactin, two well-known markers of invadopodia. Matrix metalloproteinase 14 was also evident at these structures. Degradation and clearance of the Alexa-488 labeled fibronectin layer was observed in association with these structures, along with phagocytosis of the fibronectin. This process was stimulated by the addition of PDBU, as evidenced by an increased area of substrate clearance. The addition of SU6656 greatly diminished the area of cleared Alexa-488 labeled fibronectin. A similar inhibition was observed after treatment with GM6001, the MMP inhibitor. CONCLUSION: Medial smooth muscle cells, when removed from their contact with endothelial cells, spontaneously develop invasive structures which aid in the digestion of the matrix, and ultimately lead to smooth muscle cell migration and formation of neointima. By understanding novel mechanisms that promote vascular smooth muscle cell migration and invasion, potential therapeutic targets aimed at inhibiting these processes may be realized. Heart and Stroke Foundation 061 EXTRACELLULAR MATRIX REMODELING BY GRANZYME B IN AGING TISSUES OF APOLIPOPROTEIN E KNOCKOUT MICE P Hiebert, W Boivin, T Abraham, S Pazooki, H Zhao, D Granville Vancouver, British Columbia
Recent aging theories have identified molecular inflammation as an important mechanism contributing to the accumulation of damage and tissue deterioration that occurs with age. Granzyme B (GrB) is a serine protease that can be released into the extracellular spaces by inflammatory cells during chronic inflammation. This can have pathological consequences influencing aging and
BACKGROUND:
Canadian Journal of Cardiology Volume 27 2011
disease of the vessels due to the ability of GrB to degrade several components of the extracellular matrix (ECM) including decorin, a proteoglycan that closely associates with collagen and is vital for proper collagen organization and structural integrity. The objective of this work is to use GrB deficient mice to examine the role of GrB in ECM degradation that occurs in the tissues of apolipoprotein E knockout (apoE-KO) mice, and how this might accelerate vascular aging and disease. METHODS: Because apoE-KO mice fed a high fat diet exhibit chronic inflammation in their skin, we used the skin from these mice as a model to examine immune-mediated mechanisms of aging and disease. Specifically, GrB-mediated ECM degradation in the context of aging and disease was examined. C57BL/6 wild type, apoE-KO and GrB/apoE double knockout (DKO) mice were fed either a high fat or regular chow diet for 0, 5, 15 and 30 weeks. Hematoxylin & eosin, picrosirius red and luna’s elastin stains were used to assess morphology, collagen and elastin respectively, in formalin fixed tissue sections. Multi-photon microscopy was also used on fresh ex vivo tissue samples to examine collagen organization and density. Immunohistochemistry was also performed on fixed sections to examine decorin and GrB. RESULTS: ApoE-KO mice fed a high fat diet demonstrated several premature aging characteristics including frailty, increased morbidity, skin thinning and a loss of dermal collagen density and organization that increased in severity over time. DKO mice showed protection against these deleterious changes. Abnormal elastin morphology was also observed in apoE-KO mice. High fat diet fed apoE-KO mice also showed reduced levels of the proteoglycan, decorin, while DKO mice showed increased decorin which corresponded to better collagen organization and structural integrity. GrB staining was also observed in the skin of apoE-KO mice, particularly in and around mast cells and areas of ECM remodeling. CONCLUSIONS: ApoE-KO mice fed a high fat diet display features of premature aging, a phenotype that is reduced when knocking out GrB suggesting that GrB plays a role in agerelated tissue damage such as collagen disorganization, possibly through decorin degradation. 062 C-TYPE NATRIURETIC PEPTIDE IS INVOLVED IN PROTEOGLYCAN-RICH LEAFLET THICKENING IN EARLY AORTIC VALVE DISEASE MC Blaser, KL Sider, Y Zhou, M Henkelman, CA Simmons Toronto, Ontario BACKGROUND: Calcific aortic valve disease (CAVD) is a cell-mediated pathology without effective pharmacotherapy. Previous microarray studies have found that C-type natriuretic peptide (CNP) is differentially expressed between the aortic (disease-prone) and ventricular (protected) sides of porcine aortic valves. We investigated the effects of a mildly atherogenic diet on early CAVD development, and the involvement of CNP in this process. METHODS: Male WT C57Bl/6J mice were fed a control diet or BioServ F3282, a high-fat, high-carbohydrate diet (HF/HC) with 58.7%