title: Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway.
abstract: Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN(-/-)) mice were treated in vitro with H(2)O(2) to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN(-/-) cells but was increased to only 20% in WT cells. In contrast, after 1-8 h of treatment with H(2)O(2), the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN(-/-) cells. Electron microscopy of WT cells treated with H(2)O(2) showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H(2)O(2)-treated OPN(-/-) cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN(-/-) and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN(-/-) cells by approximately 30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN(-/-) cells was not altered. Restoration of OPN expression in OPN(-/-) fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H(2)O(2) treatment. Thus H(2)O(2)-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.
authors.affiliation: Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, 124 Edward Street, Rm. 464A, Toronto, Ontario M5G 1G6, Canada. ron.zohar@utoronto.ca
authors.forename: Ron
authors.initials: R
authors.lastname: Zohar
authors.forename: Baoqian
authors.initials: B
authors.lastname: Zhu
authors.forename: Peter
authors.initials: P
authors.lastname: Liu
authors.forename: Jaro
authors.initials: J
authors.lastname: Sodek
authors.forename: C A
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D005347:Fibroblasts / Q000473:pathology* / Q000502:physiology; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D008817:Mice, Mutant Strains; D008928:Mitochondria / Q000502:physiology; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology*; D009206:Myocardium / Q000378:metabolism / Q000473:pathology*; D009336:Necrosis; D053495:Osteopontin; D018384:Oxidative Stress / Q000502:physiology; D012795:Sialoglycoproteins / Q000235:genetics*
chemical_list: D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases