Smooth muscle cell (SMC) migration and proliferation and extracellular matrix remodeling are essential aspects of the arterial response to injury, vessel development, and atherogenesis. Matrix metalloproteinase (MMP) expression is associated with SMC proliferation and migration after arterial injury. To assess the role of MMPs in SMC proliferation and migration and intimal thickening, we measured the effect of the synthetic MMP inhibitor BB94 (Batimastat) on DNA synthesis and migration of SMCs in vitro as well as the formation of a neointima after balloon injury to the rat carotid artery. BB94 dose-dependently inhibited SMC migration induced by platelet-derived growth factor (PDGF)-BB through a filter coated with a thick basement membrane matrix (Matrigel) layer but did not show any inhibitory effect on SMC migration through a lightly coated filter. At concentrations up to 1 mumol/L, BB94 did not alter DNA synthesis induced by PDGF-AA or PDGF-BB. Treatment with 30 mg BB94.kg-1.d-1 IP for 7 or 14 days after balloon injury to the rat carotid artery decreased the total number of intimal SMC nuclei and suppressed intimal thickening. SMC proliferation (5-bromo-2'-deoxyuridine labeling) was decreased in the media at 2 days, whereas it was increased in the intima at 7 but not 14 days. These results suggest that BB94 inhibits intimal thickening after arterial injury by decreasing SMC migration and proliferation and support the conclusion that MMPs play a significant role in regulating intimal thickening in injured arteries.

Arterial smooth muscle cells (SMCs) are in a quiescent growth state under normal physiological conditions, but they can be stimulated to proliferate and migrate from one tissue compartment to another if the vessel is injured. This response might require a selective and focal increase in tissue degradation, which might be mediated through the increased production of matrix metalloproteinases (MMPs). Blockade of MMP activity might therefore inhibit the SMC response to injury. To test this hypothesis, we developed clones of rat SMCs that overexpress baboon tissue inhibitor of matrix metalloproteinase-I (TIMP-1), using retrovirally mediated gene transfer, and characterized the functional capacity of these cells in vitro and in vivo. SMCs transduced with the TIMP-1 vector (LTSN) grew more slowly and also migrated through a gel matrix in a Boyden chamber assay more slowly than the vector alone (LXSN) cells. The conditioned medium from LTSN cells completely inhibited the platelet-derived growth factor-BB-induced migration of normal SMCs across a matrix-coated filter, while the LXSN cell conditioned medium had no effect. The inhibitor activity in the LTSN conditioned medium could be neutralized with an antibody to TIMP-1. In vivo, local overexpression of TIMP-1 using LTSN cells implanted onto balloon-injured rat carotid artery inhibited intimal hyperplasia. Neutralizing antibodies against TIMP-1 suppressed the effect of LTSN cell seeding on intimal thickening. These data support the conclusion that the process of SMC activation leading to a thickened intima is dependent on MMP activity and that TIMP-1 could be utilized to inhibit this process.

Heparin is a complex glycosaminoglycan that inhibits vascular smooth muscle cell (SMC) growth in vitro and in vivo. To define the mechanism by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the activity of intracellular protein kinase C (PKC). The membrane-associated intracellular PKC activity increased following stimulation of cultured rat SMCs with fetal calf serum and was suppressed by heparin in a time- and dose-dependent manner. Heparin acted through a selective inhibition of the PKC-alpha since preincubation of the cells with a 20-mer phosphorothioate PKC-alpha antisense oligodeoxynucleotide (ODN) eliminated the heparin effect. In vivo, following balloon injury of the rat carotid artery, particulate fraction PKC content increased with a time course and to an extent comparable with the observed changes in vitro. Heparin, administered at the time of injury or shortly thereafter, inhibited the activity of the particulate PKC and suppressed the in situ phosphorylation of an 80-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS), a substrate of PKC. The topical application of the phosphorothioate antisense ODN selectively suppressed the expression of the PKC-alpha isoenzyme in vivo but did not affect injury-induced myointimal proliferation. Topical application of the ODN also eliminated the antiproliferative activity of heparin. These results therefore suggest that heparin might block SMC proliferation by interfering with the PKC pathway through a selective direct inhibition of the PKC-alpha isoenzyme.

The growth of neointima and neointimal smooth muscle cells in baboon polytetrafluoroethylene grafts is regulated by blood flow. Because neointimal smooth muscle cells express both platelet-derived growth factor receptor-alpha and -beta (PDGFR-alpha and -beta), we designed this study to test the hypothesis that inhibiting either PDGFR-alpha or PDGFR-beta with a specific mouse/human chimeric antibody will modulate flow-induced neointimal formation. Bilateral aortoiliac grafts and distal femoral arteriovenous fistulae were placed in 17 baboons. After 8 weeks, 1 arteriovenous fistulae was ligated, normalizing flow through the ipsilateral graft while maintaining high flow in the contralateral graft. The experimental groups received a blocking antibody to PDGFR-alpha (Ab-PDGFR-alpha; 10 mg/kg; n=5) or PDGFR-beta (Ab-PDGFR-beta; 10 mg/kg; n=6) by pulsed intravenous administration 30 minutes before ligation and at 4, 8, 15, and 22 days after ligation. Controls received carrier medium alone (n=8). Serum antibody concentrations were followed. Grafts were harvested after 28 days and analyzed by videomorphometry. Serum Ab-PDGFR-alpha concentrations fell rapidly after day 7 to 0, whereas serum Ab-PDGFR-beta concentrations were maintained at the target levels (>50 microg/mL). Compared with controls (3.7+/-0.3), the ratio of the intimal areas (normalized flow/high flow) was significantly reduced in Ab-PDGFR-beta (1.2+/-0.2, P<0.01) but not in Ab-PDGFR-alpha (2.2+/-0.4). Ab-PDGFR-alpha decreased significantly the overall smooth muscle cell nuclear density of the neointima (P<0.01) compared with either the control or Ab-PDGFR-beta treated groups. PDGFR-beta is necessary for flow-induced neointimal formation in prosthetic grafts. Targeting PDGFR-beta may be an effective pharmacological strategy for suppressing graft neointimal development.

Abstract-Elevated levels of plasminogen activator inhibitor type 1 (PAI-1) are found in advanced atherosclerotic plaque compared with normal vessel and may contribute to plaque progression and complications associated with plaque rupture. Increased expression of PAI-1 probably contributes to the thrombotic properties of advanced atherosclerotic plaque by impeding plasmin generation and degradation of fibrin. To test this hypothesis, we have deliberately created synthetic neointimas by seeding onto the denuded luminal surface of rat carotid arteries smooth muscle cells transduced with replication-defective retrovirus encoding rat PAI-1. This cell-based gene transfer method results in stable, long-term, and localized gene expression. PAI-1 overexpression increases mural thrombus accumulation at 4 days but decreases neointimal area by 30% and 25% at 1 week and 2 weeks, respectively. PAI-1 overexpression accelerates reendothelialization of injured arteries compared with control arteries at 1 week, 2 weeks, and 1 month. PAI-1 overexpression does not alter matrix accumulation at 1 week. Increased PAI-1 expression in the rat carotid artery enhances thrombosis and endothelial regeneration while inhibiting intimal thickening. These results suggest that PAI-1 could play a direct role in the development of advanced atherosclerotic plaque and in the repair of the diseased vessel after fibrous cap disruption. Key Words: plasminogen activator inhibitor type 1 Ⅲ carotid arteries Ⅲ thrombosis Ⅲ fibrinolysis Ⅲ endothelium P lasminogen activator inhibitor type 1 (PAI-1) is the primary physiological inhibitor of the plasminogen activator system and thereby blocks the conversion of plasminogen to plasmin. The plasminogen activator (PA) system is composed of urokinase PA (uPA) and tissue PA that proteolytically convert plasminogen to the serine protease plasmin. Components of the PA system are localized to the cell surface by receptors or extracellular matrix binding sites.Increased expression of PAI-1 has been demonstrated in atherosclerotic arteries. 1,2 PAI-1 is elevated in mesenchymalappearing intimal cells at the base of the plaque and in the necrotic core. Its function in the advanced atherosclerotic lesion is not known. PAI-1 may limit the fibrinolytic capacity of the plaque. 3 It also might modulate cellular proliferation or migration in the lesion through changes in matrix composition and growth factor release. The effects of PAI-1 are likely to be exerted locally in view of the fact that a majority of patients with generalized atherosclerosis have normal plasma fibrinolytic profiles. 4,5 The various biological effects of PAI-1 generate a dilemma. 6 On the one hand, local PAI-1 overexpression should enhance fibrin accumulation and thereby contribute to the growth of atherosclerotic lesions. 3,7 On the other hand, increased PAI-1 inhibits smooth muscle cell (SMC) migration and the formation of neointima in injured mouse vessels; this result supports the hypothesis that PAI-1 overexpression retards the growth of atherosclerot...