We have characterized matrix metalloproteinase expression in the rat carotid artery after two forms of arterial injury, balloon catheter denudation and nylon filament denudation. Gelatinolytic enzymes with molecular masses of 70 and 62 kD were produced constitutively in the rat carotid. Production of an 88-kD gelatinase was induced after balloon catheter injury, and proteinase production continued during the period of migration of smooth muscle cells from the media to the intima, from 6 hours to 6 days after balloon catheter injury. In addition, a marked increase in 62-kD gelatinolytic activity was observed between 4 and 14 days after arterial injury. Gelatinase activities (88 and 62 kD) were also increased after nylon filament denudation but were markedly less after this injury than after balloon catheter injury. These results suggested a correlation between gelatinase activity and smooth muscle cell migration after arterial injury. Administration of a metalloproteinase inhibitor after balloon catheter injury resulted in a 97% reduction in the number of smooth muscle cells migrating into the intima. Therefore, we hypothesize that gelatinase expression directly facilitates smooth muscle cell migration within the media and into the intima. These results suggest that gelatinases are involved in the vascular smooth muscle cell activation and neointimal formation that characterize arterial tissue remodeling after injury.

The endothelial cell (EC) specific tyrosine kinase receptor, Tie2, interacts with at least two ligands, angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 stimulates Tie2 receptor autophosphorylation, while Ang2 has been reported to inhibit Ang1-induced Tie2 receptor autophosphorylation. We studied the effects of Ang1 and Ang2 in an in vitro model of angiogenesis. Human ECs (HUVEC), cultured on 3-D fibrin matrices, were treated with conditioned media (CM) from stably transfected cells expressing human Ang1 or Ang2, or with purified recombinant proteins. EC tube formation was measured as a differentiation index (DI), calculated as the ratio of total tube length over residual of EC monolayer. CM from Ang1 overexpressing A10 SMC or HEK293T cells induced profound HUVEC differentiation, resulting in the formation of extensive capillary-like tubes within 48 h (DI: 24.58+/-5.91 and 19.13+/-7.86, respectively) vs. control (DI: 2.73+/-1.68 and 2.15+/-1.45, respectively, both P<0.001). Interestingly, CM from two independent cell lines overexpressing Ang2 also produced a significant increase in EC differentiation (DI: 9.22+/-3.00 and 9.72+/-4.84, both P<0.005 vs. control) although the degree of angiogenesis was significantly less then that seen with Ang1. Addition of Ang1* (a genetically engineered variant of naturally occurring Ang1) or Ang2 also resulted in dose dependent increases in DI, which were blocked by an excess of soluble Tie2 receptor (20 microg/ml). Both Ang1* and Ang2 induced modest increases in [3H]thymidine incorporation into HUVECs (20 and 26%, respectively), which were inhibited by excess soluble Tie2. Although Ang2 was unable to induce significant Tie2 receptor phosphorylation during a 5-min exposure, a 24-h pretreatment with Ang2, followed by brief re-exposure, produced Tie2 phosphorylation in HUVEC comparable to that produced by Ang1*. These results demonstrate for the first time that Ang2 may have a direct role in stimulating Tie2 receptor signaling and inducing in vitro angiogenesis. Our findings suggest that the physiological role of Ang2 is more complex than previously recognized: acting alternately to promote or blunt Tie2 receptor signaling in endothelial cells, depending on local conditions.

Smooth muscle cell (SMC) migration and replication are important for neointimal formation after arterial injury. Migration of SMCs requires degradation of basement membrane and extracellular matrix surrounding the cell, and our previous work has shown a correlation between expression of two matrix-degrading metalloproteinases (MMPs), MMP-2 and MMP-9, and smooth muscle migration into the intima in the balloon catheter-injured rat carotid artery. In the present study, an MMP inhibitor, GM 6001, was administered to rats for various times after balloon injury of the carotid artery. Inhibition of MMP activity resulted in a 97% decrease in the number of SMCs that migrated into the intima by 4 days after injury, and lesions growth was retarded by continuous treatment with GM 6001-treated rats was 0.035 +/- 0.008 mm2 compared with 0.095 +/- mm2 in the control group. Neither intimal nor medical SMC replication rates were decreased by GM 6001 treatment, supporting our hypothesis that the decrease in lesion size was due to inhibition of MMP-mediated migration and not inhibition of replication. By 14 days after injury, however, intimal area and SMC number were the same control and inhibitor-treated rats. An increased rate of SMC replication in the GM 6001 rats (replication rates at 10 days were 56.7 +/- 10.0% in the GM 6001 group and 16.97 +/- 1.73% in the control group) contributed to "catch-up" growth of the neointima. Thus, it appears that inhibiting SMC migration with MMP inhibitors is not sufficient to inhibit lesion growth, and lesion size eventually catches up to control via increased SMC replication.

IntroductionAtherosclerosis and restenosis are characterized by the development of a thickened neointimal layer in the blood vessel wall. Smooth muscle cells (SMCs) are activated after arterial injury and contribute to neointimal lesion development through proliferation, migration, and ECM synthesis. Recent research suggests that the ECM is not simply an inert scaffold, but instead there are dynamic interactions between cells and matrix that contribute to SMC responses. After arterial injury, SMCs synthesize the fibrillar type I and III collagens (1, 2) and the short-chain type VIII collagen (3-6). Recently, the discoidin domain receptor tyrosine kinases (DDRs) were shown to function as collagen receptors and to increase matrix metalloproteinase (MMP) production in a fibrosarcoma cell line (7,8). The MMPs (including MMP-1, MMP-2, MMP-3, MMP-9, and MT1-MMP) are upregulated after injury and facilitate SMC migration in the vessel wall (9-12). Given the role of the DDRs in mediating interactions with collagen and stimulating MMP synthesis, we posit here that the DDRs are important mediators of the SMC response to injury.The DDRs are distinguished by an extracellular domain of 160 amino acids that is homologous to the Dictyostelium discoideum protein discoidin-I (13). There are two distinct gene products, DDR1 and DDR2, and DDR1 appears in three alternative splice variants, 1a, 1b, and 1c (13). DDR1 is widely expressed during embryonic development and in adult tissues, particularly in the epithelium of skin, kidney, gut, and brain, and the splice variant DDR1b increases considerably during postnatal development. DDR2 is restricted to skeletal muscle, heart, and connective tissues (13). Strikingly high levels of DDR1 and 2 are seen in fastgrowing invasive mammary, ovarian, and lung tumors (14), in keeping with the increased proliferative rates and MMP production in these tumors. To date, one abstract has reported DDR1 and DDR2 expression in atherosclerotic lesions of nonhuman primates fed a high-cholesterol diet (15); however, nothing is known about the function of DDRs in the vascular system. In the current study we have examined DDR1, the most widely expressed DDR in adult mammals.The arterial collagens that are upregulated after injury can be classified broadly into two categories based on their structure; fibril-forming (type I and III) and short-chain (type VIII) collagens. Tissue-culture studies show that type I collagen affects SMC growth and migration (16)(17)(18). Type VIII collagen is a shortchain collagen expressed during active remodeling in angiogenesis (19), embryonic development of the heart (20), and glomerulonephritis (21). We and others have shown that it is dramatically upregulated following experimental arterial injury (3-5) and in human atherosclerotic plaques (6), and we have shown The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair Collagens act as important signaling molecules regulating vascular smooth muscle cell responses during arterial wound repair. Discoidin domain...