Hiroyuki Itoh

Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase

PURPOSEnMitogen-activated protein kinase (MAPK) is a ubiquitous signaling protein that has been associated with cellular proliferation; however, its role in cellular migration has not been established. In this study, we investigate the role of MAPK in platelet-derived growth factor (PDGF)-induced migration and proliferation of human vascular smooth muscle cells (SMCs).nnnMETHODSnSMC migration was measured using a microchemotaxis assay (4 hours), and proliferation was assessed using 3H-thymidine uptake and cell counts. PD098059 was used as a specific noncompetitive inhibitor of MAPK activation.nnnRESULTSnCoincubation of SMCs with PD098059 resulted in significant inhibition of PDGF-BB (5 ng/ml)-induced SMC chemotaxis and proliferation. The IC50 for both processes was approximately 10 mumol/L with complete inhibition at 50 mumol/L. Stimulation of SMCs with PDGF produced an early peak in MAPK activity followed by a plateau of activity that persisted for 24 hours. We hypothesized that variations in the temporal activation of MAPK might explain the action of this enzyme on these two disparate cellular events. By adding PD098059 at intervals after stimulation of SMCs with PDGF, we demonstrated an association between MAPK activity within the first 15 minutes and SMC migration, whereas MAPK activity between 1 and 4 hours was associated with SMC proliferation.nnnCONCLUSIONSnMAPK activity is essential for both SMC migration and proliferation, and distinct phases of enzyme activation are required to stimulate these two discrete cellular events. Inhibition of this signaling protein may prove to be a useful method for preventing intimal hyperplasia.

The role of integrins in saphenous vein vascular smooth muscle cell migration.

PURPOSEnSmooth muscle cell (SMC) migration is an essential feature of the intimal hyperplastic process that so frequently limits the patency of vascular reconstructions. The purpose of this investigation was to evaluate the effect of a series of integrins, or cell surface receptors that mediate cellular attachment, on platelet-derived growth factor (PDGF) and extracellular matrix (ECM) protein-induced migration of human SMCs.nnnMETHODSnImmunofluorescence staining was used to search for various integrins and subunits on the surface of SMCs derived from human saphenous vein. Chemotaxis and haptotaxis of SMCs to various matrix proteins and PDGF were assayed using a 48-well microchemotaxis chamber in the presence or absence of antibodies that blocked the function of these integrins.nnnRESULTSnSeveral subunits (beta 1, alpha 2, alpha 5) and one integrin (alpha v beta 3) were identified in saphenous vein SMCs. The beta 1 integrin antibody inhibited chemotaxis to collagen I and IV, laminin, and PDGF. The alpha 2 integrin antibody inhibited collagen I and IV, and laminin-induced chemotaxis. The alpha 5 integrin antibody had no effect on SMC migration. The alpha v beta 3 integrin antibody inhibited chemotaxis to PDGF but not to the ECM proteins.nnnCONCLUSIONSnIntegrins are necessary for SMC migration induced by PDGF and ECM proteins. The integrin or subunits responsible for facilitating migration varies with the stimulant. Agonists designed to inhibit integrin function might be used to suppress SMC migration and suppress the formation of intimal hyperplasia.

Prostacyclin administration suppresses the increase in hepatic levels of COL1A (I) and glyceraldehyde-3-phosphate dehydrogenase mRNAs in the rat treated with carbon tetrachloride

Northern analysis using total RNAs from the component cells of normal rat liver indicated that COL1A(I) mRNA is present in fat-storing cells (Ito cells) and sinusoidal endothelial cells. A fraction for Kupffer cells also contained this mRNA. When CCl4 was given, COL1A(I) mRNA was increased in a factor of 1.5 in the fractions of these component cells. After 48 h of the drug administration, hepatocytes appeared to possess over 60% of liver COL1A(I) mRNA, although in normal hepatocytes its level was below the range detectable by our procedures. Under this injured condition of liver, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level was elevated, while activity of this enzyme was lowered by 50% of the control value. All the changes were obviously suppressed by the simultaneous administration of prostacyclin.