Inhibitory effects of fluvastatin on the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

172A

POSTERS: Cell Biology/Growth Factors

the intracellular Ca2⫹ concentration in Fura2-labeled VSMCs and the incorporation of BrdU, respectively. Expression of c-myc, c-fos and c-jun proto-oncogenes was evaluated by semi-quantitative RT-PCR. The activation of p42/p44 mitogen-activated protein kinases (ERK 1/2) was evaluated by Western blots using an antibody against the activated form of ERK 1/2, i.e. phospho-ERK 1/2. Results demonstrated that amlodipine inhibited thrombin-induced Ca2⫹ mobilization and its associated capacitative entry. These effects could not be observed with isradipine, verapamil or diltiazem. Amlodipine, like isradipine or nifedipine, inhibited bFGF-triggered voltage-dependent Ca2⫹ influx and the resulting ERK 1/2 activation as well as thrombin- and bFGF-induced c-myc, c-fos and c-jun expression and DNA synthesis. The L-type Ca2⫹ channel agonist Bay K 8644 could antagonize the inhibitory effect of nifedipine but not of amlodipine on DNA synthesis. Kinetic experiments indicated that the effect of amlodipine on Ca2⫹ mobilization clearly dissociated from its effect on L-type Ca2⫹ channels. All the effects of amlodipine were dose-dependent and could be observed with concentrations as low as 1-10 nM, i.e. concentrations that correspond to the peak plasma level of treated subjects. These data indicated that amlodipine exhibits specific properties, additional to its CCB activity, that affect Ca2⫹ movements. Our results strongly suggest that it is by interfering with two branches of growth factor mitogenic pathways, namely Ca2⫹ mobilization and ERK 1/2 activation, that amlodipine exerts its inhibitory activity on VSMC growth/proliferation. Amlodipine has also been reported to possess potent antioxidant and NO release activator properties. Along with these characteristics, the various properties of amlodipine reported in our investigations are likely to participate in amlodipine anti-atherogenic properties. Key Words: calcium movements, MAP Kinases, cell proliferation

P-422 INHIBITORY EFFECTS OF FLUVASTATIN ON THE PROLIFERATION OF VASCULAR SMOOTH MUSCLE CELLS FROM SPONTANEOUSLY HYPERTENSIVE RATS Xie Liangdi, Lin Zhihong, Wu Kegui, Sun Ming, Zhou Hongyan. 1 Hypertension Division, The First Affiliated Hospital of Fujian Medical College, Fuzhou, China,2Department of Cardiology, Xiangya Hospital, Hunan Medical College, China This study was to investigate the effects of fluvastatin on the proliferation of vascular smooth muscle cells derived from spontaneously hyperten-

AJH–April 2001–VOL. 14, NO. 4, PART 2

sive rats (SHR). The aorta smooth muscle cells (ASMC) derived from SHR were cultured. Proliferation of cultured ASMC were determined by direct cell number counting and 3H-TdR incorporation. Data expressed as mean⫾SD of 5 determinations performed in triplicate. It was showed that the increased cell number and 3H-TdR incorporation induced by 10-6M AngII and 10ng ●mL-1PDGF was significantly inhibited by fluvastatin in a concentration-dependent manner from 10-7 to 10-5M. 10-5M fluvastatin produced a 73% and 64% inhibition of cell number increase, 75% and 81% inhibition of 3H-TdR incorporation induced by 10-6M AngII and 10ng ●mL-1PDGF (P⬍0.01 respectively). This inhibitory effect was nearly completely prevented by 10-3M mevalonate acid. It is concluded that mevalonate acid pathway may play an important role in the proliferation of ASMCs. Key Words: Fluvastatin, Mevalonate acid, Vascular smooth muscle cell

P-423 EFFECTS OF TGF—␤1 AND PDGF ON COLLAGEN SYNTHESIS OF CARDIAC FIBROBLASTS FROM SPONTANEOUSLY HYPERTENSIVE RATS Wu Kegui, Xie Liangdi, Chen Shuilong. 1Hypertension Division, The First Affiliated Hospital of Fujian Medical College, Fuzhou, China This study was to investigate the effects of transforming growth factor ␤1(TGF—␤1) and platelets derived growth factor (PDGF) on the collagen synthesis in cultured rat cardiac fibroblasts (CFb) derived from SHR (CFb SHR) and WKY (CFbWKY). CFb derived from 12-week-old SHR and WKY were cultured by outgrowth of tissue block. Cell proliferation of CFb was measured by direct cell counting. Collagen synthesis was determined by 3H-proline incorporation. Data expressed as mean⫾SD of (n) determinations performed in triplicate. It was showed that proliferation of CFb was significantly promoted by PDGF in a dose dependent manner from a concentration range of 3-30ng/ml. Similarly, 1 to 10 ng/ml TGF-␤1 stimulated a markedly dose dependent increase in cell number. TGF-␤1 at concentration of 10 ng/ml provoked a 152% and 143% increase in cell number of CFbSHR and CFbWKY respectively in the medium containing 0.4% FCS. 3H-proline incorporation of CFb was promoted by 1-10 ng/ml TGF-␤1 and 3-30ng/ml in a concentration dependent manner in both CFb SHR and CFbWKY. It is concluded that cell proliferation and collagen synthesis of CFb was promoted by TGF—␤1 and PDGF. Key Words: Cardiac Fibroblasts, TGF, PDGF