Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator inhibitor-1 synthesis in vascular smooth muscle cells

C.F Reilly,R.C Mcfall

doi:10.1016/s0021-9258(18)92836-x

Abstract

Bovine vascular smooth muscle cells (SMC) were examined for production of plasminogen activator inhibitor-1 (PAI-1) which may play a key role in regulating the fibrinolytic system. Growth-arrested SMC released active PAI (101 arbitrary units (AU)/10(6) cells/24 h) and a latent form of PAI (880 AU/10(6) cells/24 h) into the conditioned medium (CM). The levels of PAI were significant since 880 AU of PAI could inhibit approximately 1 microgram of tissue plasminogen activator. The extracellular matrix of SMC also contained PAI activity; however, the level was 17-fold less than that observed in the CM. SMC-PAI was a rapid inhibitor of tissue plasminogen activator (kass greater than 10(7) M-1 S-1) and was identified as a 45-kDa protein immunologically related to endothelial cell PAI-1. PAI-1 comprised 20 and 30%, respectively, of the newly synthesized protein detected in the CM and extracellular matrix of SMC. The SMC growth modulators, platelet-derived growth factor and transforming growth factor-beta, induced PAI-1 activity and protein synthesis by 2- and 3-fold, respectively, in a dose- and time-dependent manner. The increases in PAI-1 activity and protein synthesis were ascribed to elevated levels of PAI-1 mRNA as judged by Northern blot analysis of total RNA prepared from control and platelet-derived growth factor- and transforming growth factor-beta-treated cells. Increases in PAI-1 mRNA levels were evident 1 h after growth factor treatment and were maximal after 4 h. PAI-1 mRNA levels were unaffected by cycloheximide treatment. The results indicate that SMC synthesize and release PAI-1 which could regulate the normal fibrinolytic environment of the arterial wall. During atherosclerosis or after vascular injury increases in platelet-derived or locally produced mitogens may stimulate further PAI-1 synthesis and generate a prothrombotic state.

Highlights

Platelet-derived Growth Factor and Transforming Growth Factor-@ Regulate Plasminogen ActivatorInhibitor-1 Synthesis in Vascular Smooth Muscle Cells*
The increases in PAI-1 activity and protein synthesis were ascribed to elevated levels of PAI-1 mRNA as judged by Northern blot analysis of total RNA preparedfromcontroalndplatelet-derived growthfactor-andtransforminggrowthfactor-& treated cells
Our results indicate that PAI-1is a major biosynthetic product of growth-arrested smooth muscle cells (SMC) and that its production can be up-regulatedfurther by exposure totheplatelet-associated growth factorsplatelet-derived growth factor(PDGF)and fibrin autography

Summary

PAI actiuity levels in the CM and ECM of SMC

Growth-arrestedSMC were placed in 5% PDS-DME f EGF (5 ng/ml), PDGF-BB (10 ng/ml), or TGF-fi (1 ng/ml). To examine SMC productioonf PA1 in more detail, multiple experiments were performed in which the PA1 activity in the CM as well as ECM was quantitated under basal conditions and after growth factor stimulation. Since the PAI-1 that is released by endothelial cells to the CM exists predominantly in the latent sta(t1e7-20), the CM fromSMC was treated with guanidine HCl to convert any latentPA1 to active PAI. PA1 detected in the CM increased 4-8-fold after activation with guanidine HCl,indicatingthatthebulk of the PA1 released intotheCM was in thelatentstate.TGF-Pand. PDGF elicited 2- and %fold increases,respectively,in the amount of active PA1 detected in the CM relative to control. SMC exposed to EGF produced 50% more active PA1 than control cells; there was no detectable increase in latent PA1 produc-

CellTypg BAEC SMSCMSCMSCMC

Findings

CM PDGF