Sarpogrelate inhibits serotonin-induced proliferation of porcine coronary artery smooth muscle cells: implications for long-term graft patency Sushil.

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Sarpogrelate inhibits serotonin-induced proliferation of porcine coronary artery smooth muscle cells: implications for long-term graft patency Sushil K Sharma, PhD, Dario F Del Rizzo, MD, PhD, Peter Zahradka, PhD, Sukhinder K Bhangu, BSc, Jeffrey P Werner, BSc, Hideo Kumamoto, DVM, Nobuakira Takeda, MD, PhD, Naranjan S Dhalla, PhD, MD The Annals of Thoracic Surgery Volume 71, Issue 6, Pages 1856-1864 (June 2001) DOI: 10.1016/S0003-4975(01)02599-1

Fig 1 Effect of sarpogrelate on smooth muscle cell response to mitogenic stimulation. Cell number was measured using a Coulter counter 96 hours after treatment in the absence (open bars) and in the presence (solid bars) of sarpogrelate (1 μmol/L): no mitogen (A), serotonin (5-HT) (1 μmol/L) (B), angiotensin II (1 μmol/L) (C), platelet-derived growth factor (1 μmol/L) (D), and endothelin (1 μmol/L) (E). Data represented the mean ± the standard error of the mean of five determinations. In response to 5-HT, sarpogrelate significantly reduced cell number (p < 0.05). No significant differences were seen in the other groups. The Annals of Thoracic Surgery 2001 71, 1856-1864DOI: (10.1016/S0003-4975(01)02599-1)

Fig 2 Rate of (A) DNA (deoxyribonucleic acid), (B) RNA (ribonucleic acid), and (C) protein synthesis in response to serotonin (5-HT) treatment in the presence of increasing concentrations of sarpogrelate. Synthesis of DNA, RNA, and protein was estimated over a 96-hour period in the presence of increasing log concentrations of sarpogrelate and fixed concentrations of 5-HT (1 μmol/L) using 3H-thymidine, 3H-uridine, and 3H-phenylalanine incorporation (1 μCi/mL) in growth-arrested, synchronized quiescent cells as described in Material and Methods section. (CPM = count per minute.) The Annals of Thoracic Surgery 2001 71, 1856-1864DOI: (10.1016/S0003-4975(01)02599-1)

Fig 3 MTT cell proliferation assay of porcine coronary artery smooth muscle cells (PCASMCs) in presence of log dose of serotonin (5-HT) and sarpogrelate. (A) Concentration curve of sarpogrelate with 1 μmol/L 5-HT. (B) Response of PCASMCs to varying concentrations of 5-HT without (•) or with sarpogrelate (1 μmol/L) (○). Data points are presented as the mean ± the standard error of the mean of samples conducted in triplicate. The Annals of Thoracic Surgery 2001 71, 1856-1864DOI: (10.1016/S0003-4975(01)02599-1)

Fig 4 Flow cytometric analysis of serotonin (5-HT)–stimulated porcine coronary artery smooth muscle cells. Quiescent cells were treated with (B) 5-HT (1 μmol/L) alone or (C) with sarpogrelate (1 μmol/L) for 96 hours and then stained with propidium iodide for FACS analysis as described in Material and Methods section. Peaks a and b represent cells in the G1 and G2 phases, respectively, of the cell cycle. (DNA = deoxyribonucleic acid.) The Annals of Thoracic Surgery 2001 71, 1856-1864DOI: (10.1016/S0003-4975(01)02599-1)

Fig 5 Effect of serotonin (5-HT) and sarpogrelate on intracellular free ionized calcium [Ca2+]i of porcine coronary artery smooth muscle cells. Cells were loaded with Fura-2 for measurements of [Ca2+]i as described in Material and Methods section. (A) Cells were treated with 5-HT (1 μmol/L) ± sarpogrelate, and the change in [Ca2+]i was plotted relative to time. Shown is a typical profile in the presence or absence of sarpogrelate (100 nmol/L). (B) Peak height was measured from the time profiles under the conditions shown and compared with untreated control (set to 100%). The data points correspond to the following: control (no sarpogrelate) (•); 0.1 μmol/L sarpogrelate (○), 1.0 μmol/L sarpogrelate (▾); and 10 μmol/L sarpogrelate (▿). Each data point represents the mean ± the standard error of the mean for six replicates. The Annals of Thoracic Surgery 2001 71, 1856-1864DOI: (10.1016/S0003-4975(01)02599-1)

Fig 6 Effect of serotonin (5-HT) and sarpogrelate on mitogen-activated protein (MAP) kinase activation. Porcine coronary artery smooth muscle cells were harvested for Western blot analysis 10 minutes after treatment. Blots were probed with antibodies specific for dual tyrosine-threonine phosphorylated forms of MAP kinase (p42 and p44). Lane designations are as follows: 1 = untreated; 2 = 5-HT (1 μmol/L); 3 = sarpogrelate (1 μmol/L) and; 4 = 5-HT (1 μmol/L) + sarpogrelate (0.1 μmol/L). The positions of the MAP kinase bands were verified with molecular mass markers run in a parallel lane. The Annals of Thoracic Surgery 2001 71, 1856-1864DOI: (10.1016/S0003-4975(01)02599-1)

Fig 7 Analysis of c-fos and c-jun levels in response to serotonin (5-HT) and sarpogrelate. (A) Total RNA (ribonucleic acid) was extracted from cells left untreated (A) or treated with 1 μmol/L 5-HT (B), 0.1 μmol/L sarpogrelate (C), or 1 μmol/L 5-HT + 0.1 μmol/L sarpogrelate (D). The RNA was amplified in the presence of 32P-dCTP as described in Material and Methods section and subsequently analyzed by agarose gel electrophoresis after staining with ethidium bromide. Lane M shows the molecular mass markers used to verify specific c-fos and c-jun amplification (upper gel). The amplification of GAPDH (lower gel) controls for variation in RNA loading. (B) Bands were excised, and incorporation of 32P-dCTP was measured by scintillation counting. Empty and shaded bars indicate c-fos and c-jun levels of 32P-dCTP incorporation, respectively. Data points are shown as the mean ± the standard error of the mean of five sample replicates. Samples A through D are the same as in 7A. (CPM = count per minute.) Immunoblotting of c-fos and c-jun gene products was used to monitor protein levels. Cells were treated with serotonin (5-HT) ± sarpogrelate (both at 1 μmol/L) and harvested in gel sample buffer. Samples were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the proteins were transferred to poly (vinylidene difluoride) (PVDF) membrane. The membrane was subsequently probed with antibody specific to either the Fos or Jun proteins. Treatment conditions were as follows: untreated control (A), 1 μmol/L 5-HT (B), 0.1 μmol/L sarpogrelate (C), and 1 μmol/L 5-HT + 0.1 μmol/L sarpogrelate (D). The Annals of Thoracic Surgery 2001 71, 1856-1864DOI: (10.1016/S0003-4975(01)02599-1)