David Hasenstab

Matrix Metalloproteinase-9 Overexpression Enhances Vascular Smooth Muscle Cell Migration and Alters Remodeling in the Injured Rat Carotid Artery

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerosis as well as intimal hyperplasia after vascular injury. We used Fischer rat smooth muscle cells (SMCs) overexpressing MMP-9 to determine the role of MMP-9 in migration and proliferation as well as in vessel remodeling after balloon denudation. Fischer rat SMCs were stably transfected with a cDNA for rat MMP-9 under the control of a tetracycline-regulatable promoter. In this system, MMP-9 was overexpressed in the absence, but not in the presence, of tetracycline. In vitro SMC migration was determined using a collagen invasion assay as well as a Boyden chamber assay. In vivo migration was determined by measuring the invasion into the medial and intimal layers of transduced SMCs seeded on the outside of the artery. Transduced SMCs were also seeded on the luminal surface, and the effect of local MMP-9 overexpression on vascular structure was measured morphometrically at intervals up to 28 days. MMP-9 overexpression enhanced SMC migration in both the collagen invasion assay and Boyden chamber in vitro, increased SMC migration into an arterial matrix in vivo, and altered vessel remodeling by increasing the vessel circumference, thinning the vessel wall and decreasing intimal matrix content. These results demonstrate that MMP-9 enhances vascular SMC migration in vitro and in vivo and alters postinjury vascular remodeling.

Overexpression of Tissue Inhibitor of Matrix Metalloproteinase-1 Inhibits Vascular Smooth Muscle Cell Functions In Vitro and In Vivo

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.

Plasminogen Activator Inhibitor Type 1 and Tissue Inhibitor of Metalloproteinases-2 Increase After Arterial Injury in Rats

Vascular injury induced by angioplasty causes smooth muscle cells to migrate, proliferate, and form a neointima. The neointima is further enlarged by the accumulation of matrix molecules synthesized by smooth muscle cells. Smooth muscle cell migration and matrix accumulation are associated with an increase in the expression of matrix-degrading enzymes and might be regulated by the balance of protease and anti-protease activity. We have studied the inhibitors of two major classes of matrix-degrading enzymes, the plasminogen activators and the matrix metalloproteinases (MMPs) to understand better the regulation of proteolytic activity following balloon catheter injury in the rat carotid artery. At various times after injury, protease inhibitor expression was analyzed by Northern blotting, reverse zymography, immunohistochemistry, and Western blotting. During the first month after injury, we found that the expression of two proteinase inhibitors (plasminogen activator inhibitor type 1 [PAI-1] and tissue inhibitor of metalloproteinases-2 [TIMP-2]) was modulated. PAI-1 mRNA expression reached a maximum 6 hours after injury before tapering off to baseline levels by 3 days. PAI-1 activity, as measured by reverse zymography, followed the same temporal profile. PAI-1, localized by immunohistochemistry, was expressed at low levels in the media of control arteries and was increased after injury primarily in the medial smooth muscle cells. TIMP-2 mRNA levels began to increase 24 hours after injury and reached a maximum at day 7. TIMP-2 activity, measured by reverse zymography, peaked at day 3 after injury. TIMP-2 protein was increased in the intima compared with the media and adventitia at day 7 after injury. The increase of PAI-1 and TIMP-2 after injury supports the hypothesis that changes in the proteolytic balance play an important role in smooth muscle cell migration after arterial injury.

Local Plasminogen Activator Inhibitor Type 1 Overexpression in Rat Carotid Artery Enhances Thrombosis and Endothelial Regeneration While Inhibiting Intimal Thickening

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.

Tissue Factor Overexpression in Rat Arterial Neointima Models Thrombosis and Progression of Advanced Atherosclerosis

BACKGROUND Tissue factor located in the atherosclerotic plaque might cause the clinically significant thrombotic events associated with end-stage disease. It might also affect intimal area by increasing matrix accumulation and stimulating smooth muscle cell (SMC) migration and proliferation. To test this hypothesis, we overexpressed tissue factor in a rat model of the human fibrous plaque. METHODS AND RESULTS A neointima was generated by seeding tissue factor-overexpressing rat SMCs onto the luminal surface of a balloon-injured syngeneic rat carotid artery. The cells attached and expressed tissue factor over the long term. Mural thrombus accumulation was present at 4 and 7 days and increased neointimal SMC numbers and area by 2-fold at 2 and 4 weeks. Tissue factor overexpression accelerated reendothelialization compared with controls at 2 weeks and 1 month. Tissue factor-overexpressing SMCs exhibited increased migration both in vitro and in vivo. The increased migration by tissue factor-overexpressing SMCs in vitro was not dependent on activation of the coagulation cascade and could be blocked by an inhibitor of tissue factor. CONCLUSIONS These results suggest that tissue factor plays a direct role in neointimal development by coagulation-dependent and -independent pathways.

Metalloproteinase Blockade by Local Overexpression of TIMP-1 Increases Elastin Accumulation in Rat Carotid Artery Intima

We have recently demonstrated that the blockade of matrix metalloproteinases by local overexpression of the intrinsic inhibitor tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) reduces intimal hyperplasia. We now report a major change in the elastin content of the intima of rat carotid arteries seeded with TIMP-1-overexpressing smooth muscle cells. To understand the mechanism responsible for elastin accumulation, synthesis and degradation of elastin in TIMP-1 and control cell-seeded rats were measured. There were no differences in elastin mRNA or elastin synthesis, as documented by 14[C]proline incorporation between TIMP-1 and control cell-seeded arteries. In contrast, there was an increase in cross-linked elastin in the TIMP-1 group. In addition, in TIMP-1 and control rats, an elastase activity of approximately 28 kD was detected by elastin zymography and was decreased in TIMP-1 cell-seeded vessels. The 28 kD elastolytic activity was inhibited by exogenously added TIMP-1 and EDTA but not by PMSF, suggesting that it was a metalloelastase. Therefore, we have demonstrated that a shift of the proteolytic balance toward protease inhibition by TIMP-1 overexpression does not change elastin synthesis but rather changes posttranslational processing, resulting in increased elastin accumulation.

Membrane-type matrix metalloproteinase-1 and -3 activity in primate smooth muscle cells

Membrane‐type matrix metalloproteinases‐1 and ‐3 (MT1‐ and MT3‐MMPs) are expressed by activated smooth muscle cells (SMCs) both in vitro and in vivo (19). To define their functions in SMCs, we transduced MT1‐ and MT3‐MMP cDNAs into baboon SMCs by using adenoviral vectors. Overexpression of MT1‐MMP increased the conversion of proMMP‐2 to the intermediate and active forms. In contrast, in MT3‐MMP‐overexpressing cells, MMP‐2 was activated partially. Immunoblot analyses revealed that MT1‐MMP protein was present in the SMCs and accumulated in the presence of the synthetic MMP inhibitor, BB94, or tissue inhibitor of metalloproteinase‐2 (TIMP‐2). However, MT3‐MMP protein was detectable only when BB94, but not TIMP‐2, was present. Zymographic analyses showed that MT3‐MMP had much stronger casein‐ and gelatin‐degrading activities than did MT1‐MMP. Furthermore, when MT3‐MMP and MT1‐MMP were coexpressed, MT1‐MMP degradation was enhanced; this result supports the possibility that MT3‐MMP can degrade MT1‐MMP. SMCs overexpressing either MT1‐ or MT3‐MMP exhibited altered morphology, without changing their proliferation. This alteration was prevented by BB94 addition. The cells, which underwent this change, showed reduced adhesion to both collagen and fibronectin and increased migration in a Boyden chamber. The present study demonstrates that MT1‐ and MT3‐MMPs have different enzymatic activities but may nevertheless affect SMC function in the same way.

Cloning and characterization of a cDNA encoding the baboon tissue inhibitor of matrix metalloproteinase-1 (TIMP-1)

A baboon aortic smooth muscle cell (SMC) cDNA library was screened for the presence of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) by polymerase chain reaction (PCR); oligodeoxyribonucleotide primers corresponding to the coding frame of the known human TIMP-1 gene were used as primers. Sequencing of the PCR-amplified baboon cDNA demonstrated only eight single-nucleotide (nt) mismatches, when compared with the coding frame of human TIMP-1. The authenticity of the PCR-amplified TIMP-1 cDNA was further confirmed by clonal screening of the library with the PCR probe and sequencing of positive clones. On Northern blots from cultured baboon SMC, the baboon cDNA hybridized to a TIMP-1-specific mRNA of 800 bp. Phorbol ester (PMA) treatment of cultured baboon SMC produced a 2.5-fold increase in TIMP-1 transcript. TIMP-1 transcripts were also demonstrated in cultures of endothelial cells and fibroblasts obtained from baboon arteries. Immunohistochemical analysis demonstrated that TIMP-1 protein is localized to the adventitial layer of baboon artery. We conclude that TIMP-1 is a conserved molecule across species and localized to the tunica adventitia of baboon vessels.

Control of smooth muscle cell function by membrane-type matrix metalloproteinases.

Abstract: Vascular smooth muscle cells (SMCs) express membrane‐type matrix metalloproteinases‐1 and ‐3 (MT1‐ and MT3‐MMPs). Expression is induced by PDGF in culture or by balloon injury in rat carotid arteries. In this study, we tried to define their functions in SMCs by transducing MT1‐ and MT3‐MMP cDNAs into baboon‐cultured SMCs, using adenoviral vectors. Overexpression of MT1‐MMP increased the conversion of proMMP‐2 to the activated form. In contrast, in MT3‐MMP overexpressing cells, MMP‐2 activation was partial. However, both MT1‐ and MT3‐MMP overexpression elicited morphological alterations (cell rounding), which was prevented by BB94 addition. The cells, which unverwent this change, showed reduced adhesion to matrices and increased migration in a Boyden chamber.