WS5-J-1-03

TRANSFORMING GROWTH FACTOR 13 (TGF-[3) AND ATHEROSCLEROSIS -ACTIVATION OF LATENT TGF-I~ AT THE VASCULAR WALL- Yasufuml Sato, Mayumi Abe, Hiroko Abe, Katsuhiro Tanaka, Chika Iwasaka First Department of Internal Medicine, Oita Medical University, Oita 879-55, Japan

Transforming growth faetor-I~ (TGF-I~) is believed to be involved in the progress of atherosclerosis. Platelets contain large amount of TGF-13 and secrete it during aggregation. Vascular cells including endothelial cells (ECs) and smooth muscle cells (SMCs) produce and secrete TGF-I~. TGF-fl inhibits the migration and proliferation of ECs, while it stimulates or inhibits the proliferation of SMCs depending on its concentration. As TGF-[I is always secreted in a latent form, post-secretional activation is required to express its activity. It is found that latent TGF-I~ (LTGF-[3) is activated during co-culture of ECs and SMCs, and plasminogen activator (PA), plasminogen, and plasmin located on the surface of ECs is required for the activation. Moreover, recent observations suggested that the targeting of LTGF-ll to the cellular surface is requisite for its activation. To further clarify the mechanism of activation of LTGF-I~ during co-culture, the cellular targeting of LTGF-13 was examined. We detected the specific binding of large LTGF-fll to porcine aortic SMCs (PASMCs) but not to porcine aortic ECs (PAECs). A monolonal antibody, KM704, against the latency associated peptide (LAP) of large LTGF-I~I complex, which blocked the binding of large LTGF-fil to PASMCs, inhibited the activation of LTGF-I3 during co-culture. These results indicate that the targeting of LTGF-I~ to SMCs is required for the activation of LTGF-ti during co-culture, and the targeting of LTGF-~ is mediated through LAP. Thus, the activation of LTGF-I~ requires two components: the plasminogen aetivator~plasmin activity expressed on EC and the targeting of LTGF-I~ to SMC. Since SMCs are not always in contact or in close proximity with ECs in the vascular wall, a mechanism for the activation of LTGF-I3 in homotypie SMCs needs to be further elucidated. Low-dose (10-100 pg/rnl) recombinant human TGF-131 stimulated proliferation of PASMCs, while high-dose (1,000 pg/ml) TGF-I~I inhibited it. Human urokinase-type plasminogen activator (u-PA) enhanced the proliferation of PASMCs, and the stimulatory effect of u-PA on PASMCs was comparable with that of low dose TGF-I31. The effect of u-PA on the proliferation of PASMCs was blocked by co-administration of either neutralizing anti TGF-I3 antibody or 02 plasmin inhibitor, suggesting that exogenous u-PA generates active TGF-~ via plasmin activity. When PASMCs were pre-incubated with u-PA for 2 h at room temperature and then washed, the stimulatory effect of u-PA on the proliferation of PASMCs persisted. Thus, cell-associated u-PA is involve in this reaction. A binding experiment using human u-PA indicated that PASMCs expressed specific receptors for u-PA. Finally, the stimulatory effect of u-PA on the proliferation of PASMCs was blocked by KM704, the mAb against LAP which inhibits targeting of latent TGF-13 to PASMCs. These results indicate that the simultaneous targeting of u-PA and LTGF-I~ generates active TGF-I~ in homotypic SMC and stimulates the proliferation of SMCs.

WS5-J-1-04

IN VIVO EFFECT OF TRANSFORMING GROWTH FACTOR-B1 (TGF-81) ON THE INTIMAL THICKENING OF RABBIT ARTERIES INJURED WITH A BALLOON CATHETER ACCOMPANIED BY TGF-B RECEPTOR EXPRESSION T. Kanzaki, N. Morisaki, K. Takaheshi, S, Yoshida 1 , Y. Saito 2, H. Oohashi 3 1 ) Seo:~d Dept. of Internal Medicine, School of Medicine, Chiba University, Chiba, Japan 2) Dept. of Laboratory Medicine, Yamegata University School of Medicine, Yamagata, Japan 3) Kirin Brewery Co., Gunma, Japan

Transforming growth factor-8 (TGF-f~) is a multifunctional protein that has been implicated in ~e regulations of a variety of physiological and developmental processes. The migration and proliferation of arterial smooth muscle cells (SMC) of bhe medial layer into the intima are important in arterial intimal thickening of atherosclerosis. In vitro system, TGF-81 is a bifunclJonal regulator of migration and prolifetation of SMC, dependent on ~le species, cell phenotype, growth conditions and interactions with other growth factors. Moreover, TGF-81 is a potent stimulator of lhe synthesis of extracellular matrix. It is already known that TGF-B exerts its effects by binding to type I and II receptors of cell surface. To clarify lhe in vivo effect of TGF-B1 on atherosclerosis, the intimal thickening and TGF-B receptor expression of arteries were studied in a model system in which arterial intimal thickening was induced by injury of rabbit arteries with a balloon catheter. Balloon catheter injury (BCI) was carried out in ~e carotid arteries of male rabbits. Animals then received intravenous injections of 10 IJg/kg of TGF-81 and 10 rng/kg of aspirin once a day for 14 days (TGF-i3 group) or 10 mg&g of aspirin only (control group). In TGF-8 and control groups, ~e areas of the intimal layer of carotid arteries with BCI were 0.418 (range, 0263-0.471; n=7) and 0251 (range, 0.03-0.322; n=5) mm 2, respectively, which were significantly different (p<0.05); the areas of '~e medial layers were 0.782 (range, 0.600-1.137; n=7) and 0.731 (range, 0.667-0.890; n=5) mm 2, respectively, which were not significantly different, ard so the intimal/medial ratios of thickening [46.0% (range, 332-67.9%; n=7) in the TGF-B group and 292% (range, 4.5-39.7%; n=5) in the control group] were significantly different. Intimal cell numbers in lhe TGF-B and control groups were not significantly different, but the matrix volume of the arterial intimal layer in lhe TGF-8 was significantly more than that of the control group [72.1% (range, 63.9-77.7%; n=7) vs. 62.0% (range, 48.1-65.5%; n=5); p<0.05], indicating that TGF-81 injection resulted in increased production of extracellular matrix per SMC. Moreover, mRNA of lhe TGF-B type I and II receptors was detected in carotid arteries 7 and 14 days after BCI. These results indicate that TGF-81 influences the process of intimal thickening induced by BCI directly through a receptor- mediated mechanism in vivo, suggesting the significance of TGF-81 in relation to ~e development of atherosclerosis.

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