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REGULATION OF MYOFIBROBLAST DIFFERENTIATION IN TISSUE CAPSULES FORMED IN THE PERITONEAL CAVITY
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
P520 MINERAL ION-INDUCED RELEASE OF MINERALIZATION-COMPETENT MATRIX VESICLES FROM HUMAN VASCULAR SMOOTH MUSCLE CELLS. J. Reynolds, D. Proudfoot, J.N. Skepper, R. McNair, A. Johannides, P.L. Weissberg, C.M. Shanahan. University of Cambridge, Division of Cardiovascular Medicine, ACCI Level 6, Box 110, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, U.K., (JNS) Department of Anatomy, Multi-Imaging Centre, Cambridge, U.K. Matrix vesicles (MV) are specialised structures released by budding from the surface of chondrocytes and osteoblasts that nucleate calcium apatite during physiological mineralization. Morphologically similar structures derived from vascular smooth muscle cells (VSMCs) have been observed in association with vascular pathologies including atherosclerosis, Monckeberg’s sclerosis and hypertension, however, little is known about their composition and function. Our previous studies have shown that apoptotic bodies (AB) derived from human VSMCs can undergo calcification and here we report that MV released by viable VSMCs can calcify more readily than AB. Vesicle release from human VSMCs was observed with timelapse videomicroscopy and an increase in vesicle production was observed in the presence of extracellular mineral ions at concentrations similar to those in bone, at sites of inflammation and in the serum of patients with renal disease (1.8 – 5.4 mM calcium, 2.0 – 3.0 mM phosphate). MV produced under these conditions had a dramatically increased calcificationpotential in in vitro calcification assays and EDX-analysis showed that these vesicles contained pre-formed calcium apatite. Mineralization was inhibited when cells were treated with the intracellular calcium ion chelator BAPTA or the calcium channel blockers diltiazem and nickel chloride, suggesting that mineralization was dependent on a rise in intracellular calcium; a phenomenon also observed in chondrocyte release of mineralization-competent MV. In addition, this rise in intracellular calcium was associated with changes in MV components. Alkaline phosphatase activity was altered and changes in the content of mineralization regulating proteins including a decrease in matrix Gla protein and an increase in alpha2-HS glycoprotein/fetuin-A content were observed. Thus, in association with extracellular mineral ion concentrations observed in vascular disease, human VSMCs release mineralization competent vesicles that share many characteristics with chondrocyte MV and therefore provide a nidus for calcification. British Heart Foundation
P521 REGULATION OF MYOFIBROBLAST DIFFERENTIATION IN TISSUE CAPSULES FORMED IN THE PERITONEAL CAVITY. Rolfe BE, Chen Y-C, Le S-J, Wigan M, World CJ, Campbell JH, Campbell GR. Centre for Research in Vascular Biology, University of Queensland, Brisbane, Queensland, Australia. Cells of bone marrow origin recruited to the peritoneal cavity in response to a foreign body form an avascular tissue capsule of myofibroblasts. When subjected to the appropriate environmental cues (e.g., when grafted to a high pressure arterial site) these cells differentiate further into smooth muscle-like cells. The present study investigated 1) apoptosis, proliferation and differentiation of cells within tissue capsules developing in the rat peritoneal cavity and 2) the growth factors that regulate this process. Histological analysis of tissue capsules showed changes in cellular composition, number and density with time. Immunostaining showed that at the early stages of capsule development, the majority of cells are macrophages. Expression of a-smooth muscle actin was highest at 3 weeks, indicating the predominance of myofibroblasts; there was no expression of smooth muscle myosin. At day 6 of capsule development, there was a high proportion of cells undergoing apoptosis (TUNEL assay), matched by a similarly high level of proliferation (PCNA staining). This ‘remodelling’
S183
decreased by 10 days, and remained low until 6 weeks, when there was a second peak of both apoptosis and proliferation. Investigation of growth factor receptor expression showed that TGFb-RI, PDGF-Rb, FGF-RI and LIF-Ra were expressed in tissue at all timepoints. While expression of TGFb-RI remained relatively constant, expression of the other three receptors showed a significant increase with time (p<.05). To test the biological significance of the data, cells from the capsule were cultured with selected growth factors and their effect on proliferation and differentiation examined. Both FGF-2 and PDGF-BB stimulated the proliferation of cells from day 11 tissue capsules, while TGF-b1 inhibited the mitogenic response. Neither TGF-b1, FGF-2 nor PDGF-BB alone was sufficient to induce or maintain cells in a myofibroblast phenotype. Thus waves of apoptosis/proliferation may be an essential part of the tissue remodelling involved in development of the myofibroblast-rich capsule that subsequently differentiates into an artery-like structure when transplanted as a vascular graft. Myofibroblast proliferation/differentiation is likely mediated by a cocktail of growth factors including TGF-b1, PDGFBB, FGF-2 and LIF.
P522 SHEAR STRESS INDUCES THE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO VASCULAR CELLS AND FACILITATES THE FORMATION OF TUBULAR NETWORKS. Kimiko Yamamoto, Shotaro Obi, Makiko Nakamura, Akira Kamiya, Joji Ando. Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Interdisciplinary Science Center, Nihon University. Flk-1+ cells derived from mouse embryonic stem (ES) cells have recently been shown to differentiate into vascular cells, both endothelial cells (ECs) and mural cells (MCs; pericytes and vascular smooth muscle cells), to serve as vascular progenitor cells (VPCs). Vascular endothelial growth factor (VEGF) is also known to promote differentiation into vascular cells that can organize vessel-like structures. However, the exact factors influencing the differentiation of ES cells remain unclear. In this study, we report that shear stress can accelerate the proliferation, differentiation, and capillary-like tube formation of ES cells. ES cells were cultured on a coverslip coated with type IV collagen in culture medium in the absence of leukemia inhibitory factor (LIF). After 4 days of culture, shear stress was applied to the cells using a parallel plate-type flow chamber. Shear stress was applied at an intensity of 1.5, 5.0, or 10.0 dynes/cm2. When exposed to shear stress for 24 hours, the cell density of the ES cells markedly increased, compared to that of ES cells cultured under static conditions, indicating that the mechanical stress increased cell proliferation. Shear stress also markedly increased the expression of two vascular endothelial growth factor receptors (Flk-1 and Flt-1), intercellular adhesion molecule, vascular endothelial-cadherin, and a-smooth muscle actin at both the protein and mRNA levels. These results indicate that ES cell-derived VPCs can simultaneously differentiate into both ECs and MCs. Assays for tube formation in three-dimensional cultures showed that the shear-stressed ES cells formed tube-like structures and developed an extensive tubular network significantly faster than the static controls. These findings suggest that ES cells are sensitive to shear stress and that shear stressed ECs have the potential to undergo vascular tissue engineering.
Vascular Calcification P523 OMEGA-3 FATTY ACIDS REDUCE IN VITRO VASCULAR CELL CALCIFICATION. Moeen Abedin, Yin Tintut, Linda Demer. UCLA, Los Angeles, CA. Population studies have shown increased consumption of fish rich in omega-3 fatty acids is associated with lower incidence of vascular disease.
P520 MINERAL ION-INDUCED RELEASE OF MINERALIZATION-COMPETENT MATRIX VESICLES FROM HUMAN VASCULAR SMOOTH MUSCLE CELLS. J. Reynolds, D. Proudfoot, J.N. Skepper, R. McNair, A. Johannides, P.L. Weissberg, C.M. Shanahan. University of Cambridge, Division of Cardiovascular Medicine, ACCI Level 6, Box 110, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, U.K., (JNS) Department of Anatomy, Multi-Imaging Centre, Cambridge, U.K. Matrix vesicles (MV) are specialised structures released by budding from the surface of chondrocytes and osteoblasts that nucleate calcium apatite during physiological mineralization. Morphologically similar structures derived from vascular smooth muscle cells (VSMCs) have been observed in association with vascular pathologies including atherosclerosis, Monckeberg’s sclerosis and hypertension, however, little is known about their composition and function. Our previous studies have shown that apoptotic bodies (AB) derived from human VSMCs can undergo calcification and here we report that MV released by viable VSMCs can calcify more readily than AB. Vesicle release from human VSMCs was observed with timelapse videomicroscopy and an increase in vesicle production was observed in the presence of extracellular mineral ions at concentrations similar to those in bone, at sites of inflammation and in the serum of patients with renal disease (1.8 – 5.4 mM calcium, 2.0 – 3.0 mM phosphate). MV produced under these conditions had a dramatically increased calcificationpotential in in vitro calcification assays and EDX-analysis showed that these vesicles contained pre-formed calcium apatite. Mineralization was inhibited when cells were treated with the intracellular calcium ion chelator BAPTA or the calcium channel blockers diltiazem and nickel chloride, suggesting that mineralization was dependent on a rise in intracellular calcium; a phenomenon also observed in chondrocyte release of mineralization-competent MV. In addition, this rise in intracellular calcium was associated with changes in MV components. Alkaline phosphatase activity was altered and changes in the content of mineralization regulating proteins including a decrease in matrix Gla protein and an increase in alpha2-HS glycoprotein/fetuin-A content were observed. Thus, in association with extracellular mineral ion concentrations observed in vascular disease, human VSMCs release mineralization competent vesicles that share many characteristics with chondrocyte MV and therefore provide a nidus for calcification. British Heart Foundation
P521 REGULATION OF MYOFIBROBLAST DIFFERENTIATION IN TISSUE CAPSULES FORMED IN THE PERITONEAL CAVITY. Rolfe BE, Chen Y-C, Le S-J, Wigan M, World CJ, Campbell JH, Campbell GR. Centre for Research in Vascular Biology, University of Queensland, Brisbane, Queensland, Australia. Cells of bone marrow origin recruited to the peritoneal cavity in response to a foreign body form an avascular tissue capsule of myofibroblasts. When subjected to the appropriate environmental cues (e.g., when grafted to a high pressure arterial site) these cells differentiate further into smooth muscle-like cells. The present study investigated 1) apoptosis, proliferation and differentiation of cells within tissue capsules developing in the rat peritoneal cavity and 2) the growth factors that regulate this process. Histological analysis of tissue capsules showed changes in cellular composition, number and density with time. Immunostaining showed that at the early stages of capsule development, the majority of cells are macrophages. Expression of a-smooth muscle actin was highest at 3 weeks, indicating the predominance of myofibroblasts; there was no expression of smooth muscle myosin. At day 6 of capsule development, there was a high proportion of cells undergoing apoptosis (TUNEL assay), matched by a similarly high level of proliferation (PCNA staining). This ‘remodelling’
S183
decreased by 10 days, and remained low until 6 weeks, when there was a second peak of both apoptosis and proliferation. Investigation of growth factor receptor expression showed that TGFb-RI, PDGF-Rb, FGF-RI and LIF-Ra were expressed in tissue at all timepoints. While expression of TGFb-RI remained relatively constant, expression of the other three receptors showed a significant increase with time (p<.05). To test the biological significance of the data, cells from the capsule were cultured with selected growth factors and their effect on proliferation and differentiation examined. Both FGF-2 and PDGF-BB stimulated the proliferation of cells from day 11 tissue capsules, while TGF-b1 inhibited the mitogenic response. Neither TGF-b1, FGF-2 nor PDGF-BB alone was sufficient to induce or maintain cells in a myofibroblast phenotype. Thus waves of apoptosis/proliferation may be an essential part of the tissue remodelling involved in development of the myofibroblast-rich capsule that subsequently differentiates into an artery-like structure when transplanted as a vascular graft. Myofibroblast proliferation/differentiation is likely mediated by a cocktail of growth factors including TGF-b1, PDGFBB, FGF-2 and LIF.
P522 SHEAR STRESS INDUCES THE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO VASCULAR CELLS AND FACILITATES THE FORMATION OF TUBULAR NETWORKS. Kimiko Yamamoto, Shotaro Obi, Makiko Nakamura, Akira Kamiya, Joji Ando. Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Interdisciplinary Science Center, Nihon University. Flk-1+ cells derived from mouse embryonic stem (ES) cells have recently been shown to differentiate into vascular cells, both endothelial cells (ECs) and mural cells (MCs; pericytes and vascular smooth muscle cells), to serve as vascular progenitor cells (VPCs). Vascular endothelial growth factor (VEGF) is also known to promote differentiation into vascular cells that can organize vessel-like structures. However, the exact factors influencing the differentiation of ES cells remain unclear. In this study, we report that shear stress can accelerate the proliferation, differentiation, and capillary-like tube formation of ES cells. ES cells were cultured on a coverslip coated with type IV collagen in culture medium in the absence of leukemia inhibitory factor (LIF). After 4 days of culture, shear stress was applied to the cells using a parallel plate-type flow chamber. Shear stress was applied at an intensity of 1.5, 5.0, or 10.0 dynes/cm2. When exposed to shear stress for 24 hours, the cell density of the ES cells markedly increased, compared to that of ES cells cultured under static conditions, indicating that the mechanical stress increased cell proliferation. Shear stress also markedly increased the expression of two vascular endothelial growth factor receptors (Flk-1 and Flt-1), intercellular adhesion molecule, vascular endothelial-cadherin, and a-smooth muscle actin at both the protein and mRNA levels. These results indicate that ES cell-derived VPCs can simultaneously differentiate into both ECs and MCs. Assays for tube formation in three-dimensional cultures showed that the shear-stressed ES cells formed tube-like structures and developed an extensive tubular network significantly faster than the static controls. These findings suggest that ES cells are sensitive to shear stress and that shear stressed ECs have the potential to undergo vascular tissue engineering.
Vascular Calcification P523 OMEGA-3 FATTY ACIDS REDUCE IN VITRO VASCULAR CELL CALCIFICATION. Moeen Abedin, Yin Tintut, Linda Demer. UCLA, Los Angeles, CA. Population studies have shown increased consumption of fish rich in omega-3 fatty acids is associated with lower incidence of vascular disease.