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Synthetic smooth muscle cell phenotype is associated with increased nicotinamide adenine dinucleotide phosphate oxidase activity: Effect on collagen secretion 

Published byPenelope McLaughlin Modified over 5 years ago

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Presentation on theme: "Synthetic smooth muscle cell phenotype is associated with increased nicotinamide adenine dinucleotide phosphate oxidase activity: Effect on collagen secretion "— Presentation transcript:

1 Synthetic smooth muscle cell phenotype is associated with increased nicotinamide adenine dinucleotide phosphate oxidase activity: Effect on collagen secretion  Rajendra Patel, MD, Jeffry D. Cardneau, MD, Scott M. Colles, PhD, Linda M. Graham, MD  Journal of Vascular Surgery  Volume 43, Issue 2, Pages (February 2006) DOI: /j.jvs Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

2 Fig 1 Graft smooth muscle cells (SMCs) secrete more collagen type I than aortic SMCs under basal conditions and in response to LY83583 (LY). A, Graft and aortic SMCs (passage 2) were grown to near confluence in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Cells were made quiescent by placing them in DMEM with 5% FBS for 24 hours. Medium and cells were collected at 1, 2, 3, 6, 9, and 12 hours after the addition of 1 μmol/L LY Collagen type I present in the medium was determined by enzyme-linked immunoabsorbent assay. DNA in the cell layer was quantitated by fluorometric assay. The collagen type I was standardized to μg of DNA. P < .01 for graft SMCs compared with aortic SMCs at all times, P < .01 for graft SMCs + LY compared with graft SMCs at all times after 2 hours, P < .01 for aortic SMCs + LY compared with aortic SMCs at all times after 2 hours. B, Graft and aortic SMCs (passage 2) were grown as above. At 12 hours after the addition of LY83583, collagen type I, and DNA were measured. *P < .01 compared with aortic SMCs at same LY83583 concentration (Student’s t test) #P < .01 for LY83583-treated aortic or graft SMCs compared to untreated aortic or graft SMCs. Representative graphs of five separate experiments are shown in A and B. Values are expressed as the mean ± SD. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

3 Fig 2 Graft smooth muscle cells (SMCs) treated with LY83583 (LY) generate more O2.− than LY83583-treated aortic SMCs. LY83583 (3 μmol/L) and lucigenin (10 μmol/L) were added to aliquots of 200,000 aortic or graft SMCs (passage 3). Luminescence was measured every 2 minutes. The graph depicts mean ± SEM of counts obtained in four separate experiments. P < .01 for all points after t = 0 when comparing either graft SMCs + LY83583 or aortic SMCs + LY83583 with respective controls. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

4 Fig 3 Scavenger of O2.− inhibits collagen type I secretion in smooth muscle cells (SMCs) treated with LY83583 (LY). A, Confluent aortic SMCs were placed in medium containing 5% fetal bovine serum (FBS) for 24 hours. SMCs (200,000) were suspended in phenol-free Roswell Park Memorial Institute medium and incubated with 3 μmol/L LY In parallel wells, 1.5 mmol/L tiron was added for 30 minutes before and during LY83583 treatment. Luminescence was measured as described in Methods and depicted as the fold increase (mean ± SD) in counts per second compared with respective untreated SMCs at t = 0. B, Aortic SMCs (passage 3) were grown to near confluence and placed in media containing 5% FBS for 24 hours. Cells were treated with 1.5 mmol/L tiron for 30 minutes before and during incubation with 1 μmol/L LY83583 as indicated. After 9 hours, medium was collected and the cells were harvested to measure the DNA. Collagen type I was normalized to μg of DNA. C, Graft SMCs were incubated with 3 μmol/L LY83583 with or without 1.5 mmol/L tiron, and O2.− production was measured as described in A. D, Graft SMCs (passage 3) were grown to near confluence and treated in an identical fashion to aortic SMCs described in B. A and C are representative of 3 separate experiments, B and D are representative graphs of 5 separate experiments. In A and C, P < .01 at all time points after t = 0 comparing LY83583-treated SMCs with control SMCs or LY83583-treated SMCs with tiron. In B and D, *P < .01 when compared with control. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

5 Fig 4 Nicotinamide adenine phosphate dinucleotide (NADPH) oxidase activity is higher in graft smooth muscle cells (SMCs) than in aortic SMCs. Graft and aortic SMCs (passage 3) were harvested and suspended in phenol-free Roswell Park Memorial Institute medium. Aliquots of 200,000 graft or aortic SMCs were incubated with 0.1 mmol/L NADPH or solvent control. In parallel wells, SMCs were treated with 2 μmol/L diphenylene iodonium (DPI) for 30 minutes before and during incubation with NADPH. Luminescence was measured as described in Methods, and depicted as fold increase (mean ± SD) in counts per second compared with respective untreated SMCs at t = 0 (n = 3 separate experiments). P < .01 at all times after t = 0 for NADPH-treated graft or aortic SMCs compared with respective controls or NADPH-treated SMCs pretreated with DPI. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

6 Fig 5 Inhibition of nicotinamide adenine (phosphate) dinucleotide (NAD[P]H) oxidase diminishes LY83583 (LY)-induced O2.− and collagen type I production by aortic and graft smooth muscle cells (SMCs). A, Diphenylene iodonium (DPI) (2 μmol/L) and 100 μmol/L hydralazine (H) were added to SMCs (passage 3) for 30 minutes before and during LY83583 (3 μmol/L) treatment. Production of O2.− was measured by lucigenin chemiluminescent assay as described in Methods and depicted as fold increase (mean ± SD) in counts per second compared with respective untreated SMCs at t = 0 (n = 3 separate experiments). P < .01 for all time points when comparing LY83583-treated SMCs with LY83583-treated SMCs pretreated with DPI and hydralazine. B, SMCs (passage 3) were grown to near confluence, made quiescent, then treated with 2 μmol/L DPI and/or 100 μmol/L hydralazine, as indicated, for 30 minutes before and during incubation with 1 μmol/L LY83583 for 9 hours. Conditioned medium was collected and collagen type I measured by enzyme-linked immunoabsorbent assay. SMCs were lysed and DNA measured by fluorometric assay. Collagen type I was standardized to micrograms of DNA. Representative graph of five separate experiments is shown. *P < .01 compared with aortic SMCs under the same conditions, #P < .01 compared with respective LY83583-treated graft or aortic SMCs. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2006 The Society for Vascular Surgery Terms and Conditions

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