OBJECTIVE: The function of angiotensin converting enzyme (ACE) at cell sites of high collagen turnover, such as heart valves, is uncertain. The aim of this study was to assess ACE and kininase-II-like activities and collagen turnover in cultured valvular interstitial cells of the adult rat heart. METHODS: The valvular interstitial cell phenotype was determined by immunolabelling (rhodamine phalloidin, desmin, and Griffonia simplicifolia lectin), and the presence of ACE mRNA and protein was confirmed by reverse transcriptase-polymerase chain reaction analysis, ACE monoclonal antibody and in vitro autoradiography, respectively. ACE and kininase-II-like activities in valvular interstitial cells were analysed by high performance liquid chromatography. Angiotensin II (AT1) and bradykinin receptors in valvular interstitial cell membranes were examined by western immunoblotting and binding assay. Type I collagen and collagenase in valvular interstitial cell culture media were determined by ELISA and zymography, respectively. Type I collagen mRNA expression in cultured valvular interstitial cells was determined by northern blot analysis and in situ hybridisation. RESULTS: In intact valvular interstitial cells or their cell membrane we found: (1) actin microfilaments, but not desmin or lectin labelling; (2) ACE mRNA expression and binding activity; (3) conversion of angiotensin I to angiotensin II, which was completely inhibited by 50 microM lisinopril, while kinase-II-like activity exceeded ACE activity and was not inhibited by lisinopril; (4) AT1 and bradykinin receptors in valvular interstitial cell membrane preparations; (5) type I collagen mRNA expression and collagenase activity; and (6) angiotensin II induced increase in type I collagen synthesis and mRNA expression. CONCLUSIONS: Cultured valvular interstitial cells represent a nonendothelial, non-smooth-muscle cell type that expresses mRNA for ACE and type I collagen. ACE and kininase-II-like activities in valvular interstitial cells may be involved in the regulation of peptides that influence collagen turnover. Angiotensin II stimulates type I collagen synthesis and mRNA expression in these cells.
OBJECTIVE: The function of angiotensin converting enzyme (ACE) at cell sites of high collagen turnover, such as heart valves, is uncertain. The aim of this study was to assess ACE and kininase-II-like activities and collagen turnover in cultured valvular interstitial cells of the adult rat heart. METHODS: The valvular interstitial cell phenotype was determined by immunolabelling (rhodamine phalloidin, desmin, and Griffonia simplicifolia lectin), and the presence of ACE mRNA and protein was confirmed by reverse transcriptase-polymerase chain reaction analysis, ACE monoclonal antibody and in vitro autoradiography, respectively. ACE and kininase-II-like activities in valvular interstitial cells were analysed by high performance liquid chromatography. Angiotensin II (AT1) and bradykinin receptors in valvular interstitial cell membranes were examined by western immunoblotting and binding assay. Type I collagen and collagenase in valvular interstitial cell culture media were determined by ELISA and zymography, respectively. Type I collagen mRNA expression in cultured valvular interstitial cells was determined by northern blot analysis and in situ hybridisation. RESULTS: In intact valvular interstitial cells or their cell membrane we found: (1) actin microfilaments, but not desmin or lectin labelling; (2) ACE mRNA expression and binding activity; (3) conversion of angiotensin I to angiotensin II, which was completely inhibited by 50 microM lisinopril, while kinase-II-like activity exceeded ACE activity and was not inhibited by lisinopril; (4) AT1 and bradykinin receptors in valvular interstitial cell membrane preparations; (5) type I collagen mRNA expression and collagenase activity; and (6) angiotensin II induced increase in type I collagen synthesis and mRNA expression. CONCLUSIONS: Cultured valvular interstitial cells represent a nonendothelial, non-smooth-muscle cell type that expresses mRNA for ACE and type I collagen. ACE and kininase-II-like activities in valvular interstitial cells may be involved in the regulation of peptides that influence collagen turnover. Angiotensin II stimulates type I collagen synthesis and mRNA expression in these cells.
Authors: Cui Lin; Dongxing Zhu; Greg Markby; Brendan M Corcoran; Colin Farquharson; Vicky E Macrae Journal: J Vis Exp Date: 2017-11-20 Impact factor: 1.355