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Localization and characterization of the response of chondrocytes to mechanical load using in-situ hybridization
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Fifth International Conference on the Molecular Biology and Pathology of Matrix
Angiotensin II Stimulates Collagen Synthesis and Expression of Collagen Type I Gene in Adult Rat Cardiac Fibroblasts
Locahzation and Characterization of the Response of Chondrocytes to Mechanical Load Using In-Situ Hybridization
T. Liu, J. Kyle, S. A. Jimenez and R. I. Bashey
A.K. Macfarland, S. Ewen, J.A. Hoyland, T. Aigner, G. Keenan, D. Cordiner and R.M. Aspden
Department of Medicine (Rheumatology), Thomas Jefferson University, Philadelphia, PA 19107, USA Angiotensin (Ang) II exerts direct action on the heart and is a stimulus for cardiac growth which may involve myocyte hypertrophy as well as proliferation of nonmyocytes. Clinical studies show that angiotensin converting enzyme (ACE) inhibitors are useful in the treatment of heart failure, myocardial ischemia and hypertension. In experimental hypertension ACE inhibitors lower high blood pressure, and reduce myocardial hypertrophy. The aim of this study was to determine if Ang II has any role in myocardial fibrosis associated with cardiac hypertrophy. Confluent rat myocardial fibroblasts in culture were treated with Ang II for 24 h and incubated with [14C]proline in the presence of 0.2% FCS. Total 14Cincorporation and collagen synthesis was determined by collagenase digestion assay. 14C-labeled proteins were further characterized by SDS gel electrophoresis. Total RNA was isolated from these cultures and used for Northern-blot hybridization using a specific cDNA for 0tl(I) procollagen. Biosynthetic labeling showed that proline incorporation and 14C-collagen synthesis were higher by 40% in fibroblasts treated with Ang II. The increment in collagen synthesis by Ang II was confirmed by SDS gel electrophoresis. The steady-state mRNA for 0tl(I) procollagen showed a 1.8-fold increase in &l(I) procollagen mRNA in cells treated with Ang II. The data demonstrate that Ang II stimulates collagen synthesis causing increased expression of the gene for 0~1(I) procollagen and may contribute to myocardial fibrosis in the hypertrophic heart.
Departmentof Orthopaedics, University of Aberdeen, AberdeenAB9 2ZD, Scotland; Department of Pathology, University of Manchester,ManchesterM13 9PT, UK; and Max-Planck Society, Clinical Research Units for Rheumatology, 8520 Erlangen, Germany It is well established that mechanical load has a regulatory effect on the metabolism of articular cartilage chondrocytes. Loads applied at walking frequencies, -1 Hz, are stimulatory while low frequency and static loads are inhibitory. However, the mechanism is unknown. This study investigates alterations in the expression of mRNA for the major matrix components in response to mechanical loads and whether an altered expression of cytokines could be at least partly responsible as an autocrine mechanism. Full depth samples of human articular cartilage are maintained in culture and subjected to cyclic loads of known frequency and magnitude for a predetermined period of up to 4 h. In-situ hybridisation is being used for two purposes; firstly to localise and quantify the major matrix components and secondly to investigate the expression of cytokines where load induced changes can first be detected. The matrix components being studied at present are type II collagen and aggrecan core protein as these form the principal structural components of the tissue, but we intend also to probe for other collagen types. The cytokines being studied are PDGF, IGF-1 and IL-1 as the first two have previously been shown to be stimulatory of proteoglycan production while IL-1 is known to induce cartilage breakdown in vitro. Preliminary results suggest that ISH can be used to detect and localise expression of the major tissue components but that it may not be sensitive enough to detect the small quantities of more minor constituents.
Fifth International Conference on the Molecular Biology and Pathology of Matrix
Angiotensin II Stimulates Collagen Synthesis and Expression of Collagen Type I Gene in Adult Rat Cardiac Fibroblasts
Locahzation and Characterization of the Response of Chondrocytes to Mechanical Load Using In-Situ Hybridization
T. Liu, J. Kyle, S. A. Jimenez and R. I. Bashey
A.K. Macfarland, S. Ewen, J.A. Hoyland, T. Aigner, G. Keenan, D. Cordiner and R.M. Aspden
Department of Medicine (Rheumatology), Thomas Jefferson University, Philadelphia, PA 19107, USA Angiotensin (Ang) II exerts direct action on the heart and is a stimulus for cardiac growth which may involve myocyte hypertrophy as well as proliferation of nonmyocytes. Clinical studies show that angiotensin converting enzyme (ACE) inhibitors are useful in the treatment of heart failure, myocardial ischemia and hypertension. In experimental hypertension ACE inhibitors lower high blood pressure, and reduce myocardial hypertrophy. The aim of this study was to determine if Ang II has any role in myocardial fibrosis associated with cardiac hypertrophy. Confluent rat myocardial fibroblasts in culture were treated with Ang II for 24 h and incubated with [14C]proline in the presence of 0.2% FCS. Total 14Cincorporation and collagen synthesis was determined by collagenase digestion assay. 14C-labeled proteins were further characterized by SDS gel electrophoresis. Total RNA was isolated from these cultures and used for Northern-blot hybridization using a specific cDNA for 0tl(I) procollagen. Biosynthetic labeling showed that proline incorporation and 14C-collagen synthesis were higher by 40% in fibroblasts treated with Ang II. The increment in collagen synthesis by Ang II was confirmed by SDS gel electrophoresis. The steady-state mRNA for 0tl(I) procollagen showed a 1.8-fold increase in &l(I) procollagen mRNA in cells treated with Ang II. The data demonstrate that Ang II stimulates collagen synthesis causing increased expression of the gene for 0~1(I) procollagen and may contribute to myocardial fibrosis in the hypertrophic heart.
Departmentof Orthopaedics, University of Aberdeen, AberdeenAB9 2ZD, Scotland; Department of Pathology, University of Manchester,ManchesterM13 9PT, UK; and Max-Planck Society, Clinical Research Units for Rheumatology, 8520 Erlangen, Germany It is well established that mechanical load has a regulatory effect on the metabolism of articular cartilage chondrocytes. Loads applied at walking frequencies, -1 Hz, are stimulatory while low frequency and static loads are inhibitory. However, the mechanism is unknown. This study investigates alterations in the expression of mRNA for the major matrix components in response to mechanical loads and whether an altered expression of cytokines could be at least partly responsible as an autocrine mechanism. Full depth samples of human articular cartilage are maintained in culture and subjected to cyclic loads of known frequency and magnitude for a predetermined period of up to 4 h. In-situ hybridisation is being used for two purposes; firstly to localise and quantify the major matrix components and secondly to investigate the expression of cytokines where load induced changes can first be detected. The matrix components being studied at present are type II collagen and aggrecan core protein as these form the principal structural components of the tissue, but we intend also to probe for other collagen types. The cytokines being studied are PDGF, IGF-1 and IL-1 as the first two have previously been shown to be stimulatory of proteoglycan production while IL-1 is known to induce cartilage breakdown in vitro. Preliminary results suggest that ISH can be used to detect and localise expression of the major tissue components but that it may not be sensitive enough to detect the small quantities of more minor constituents.