title: Calphostin C as a rapid and strong inducer of apoptosis in human coronary artery smooth muscle cells.
abstract: Vascular smooth muscle cells (VSMCs) play a major role in the development of atherosclerotic and restenotic lesions. The apoptotic process has been implicated in the development of this pathology. In this study, we characterized the induction of apoptosis by calphostin C (CC), a protein kinase C (PKC) inhibitor, in primary human coronary artery smooth muscle cells in the presence and absence of insulin-like growth factor-I (IGF-I). Additionally, we investigated the signal transduction pathways important for IGF-I mediated protection. Calphostin C induced apoptosis, as measured by terminal deoxy-UTP nick-end labeling (TUNEL), in a time- and dose-dependent manner, approaching 20% within 6 h of 50 nM calphostin C treatment. The amount of apoptosis increased to 44.58+/-8.08%, 47.54+/-1.66% and 78.1+/-11.9% after 8, 10 and 12 h of treatment, respectively (p<0.01 vs. control). IGF-I offered significant protection (p<0.05) at 8 and 10 h of treatment (60.6% and 52.5% protection, respectively). DNA ELISA confirmed the apoptotic effect of calphostin C and the protective effect of IGF-I. After 6 h of calphostin C treatment, DNA ELISA revealed 11.20+/-1.53 fold greater apoptosis as compared to baseline values. IGF-I treatment offered a level of protection of 46.6% as measured by DNA ELISA (p=0.06). Apoptosis was further qualitatively confirmed by time-lapse video microscopy and scanning electron microscopy. Interestingly, inhibitors of phosphatidylinositol-3-kinase (PI-3-K), p38 and extracellular regulated kinase (ERK) activation significantly (p<0.05 vs. calphostin C only treatment) increased apoptosis when used in conjunction with calphostin C. Inhibitors of phospatidylinositol-3-kinase and ERK activation reversed IGF-I protection. However, the p38 inhibitor SB203580 failed to reverse IGF-I protection. This study characterized an apoptotic system for human coronary artery smooth muscle cells offering a rapid and strong induction of programmed cell death (PCD) that remains responsive to the survival effects of IGF-I. Studies utilizing this system may prove useful in understanding the apoptotic response of VSMCs in the arterial wall.
authors.affiliation: Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178, USA.
authors.forename: Kristopher D
authors.initials: KD
authors.lastname: Krueger
authors.forename: William J
authors.initials: WJ
authors.lastname: Hunter
authors.forename: Michael G
authors.initials: MG
authors.lastname: DelCore
authors.forename: Devendra K
authors.initials: DK
authors.lastname: Agrawal
mesh_terms: D000730:Androstadienes / Q000009:adverse effects; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D002867:Chromones / Q000009:adverse effects; D003331:Coronary Vessels / Q000187:drug effects* / Q000473:pathology / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D004797:Enzyme-Linked Immunosorbent Assay; D005419:Flavonoids / Q000009:adverse effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020287:In Situ Nick-End Labeling / Q000379:methods; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000627:therapeutic use; D008855:Microscopy, Electron, Scanning / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D020928:Mitogen-Activated Protein Kinases / Q000009:adverse effects / Q000037:antagonists & inhibitors; D009025:Morpholines / Q000009:adverse effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000473:pathology / Q000648:ultrastructure; D009281:Naphthalenes / Q000009:adverse effects* / Q000037:antagonists & inhibitors; D019869:Phosphatidylinositol 3-Kinases / Q000009:adverse effects; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D000730:Androstadienes; D002867:Chromones; D005419:Flavonoids; D007093:Imidazoles; D007334:Insulin-Like Growth Factor I; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D009281:Naphthalenes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011725:Pyridines; D019311:Staurosporine; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C058819:calphostin C; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one