VSMC Migration Research Articles

Protective Role of Estrogen-induced miRNA-29 Expression in Carbon Tetrachloride-induced Mouse Liver Injury

Previous studies have indicated that female animals are more resistant to carbon tetrachloride (CCl(4))-induced liver fibrosis than male animals, and that estradiol (E(2)) treatment can inhibit CCl(4)-induced animal hepatic fibrosis. The underlying mechanism governing these phenomena, however, has not been fully elucidated. Here we reported the role of estrogen-induced miRNA-29 (miR-29) expression in CCl(4)-induced mouse liver injury. Hepatic miR-29 levels were differentially regulated in female and male mice during CCl(4) treatment. Specifically, the levels of miR-29a and miR-29b expression were significantly decreased in the livers of male, but not female, mice following 4 weeks of CCl(4) treatment. The down-regulation of miR-29a and miR-29b in male mouse livers correlated with the early development of liver fibrosis, as indicated by increased expressions of fibrotic markers in male mice relative to female mice. In addition, E(2) was maintained at a higher level in female mice than in male mice. In contrast to TGF-β1 that decreased miR-29a/b expression in murine hepatoma IAR20 cells and normal hepatocytes, E(2) enhanced the expression of miR-29a/b through suppression of the nuclear factor-κB (NF-κB) signal pathway, which negatively regulates miR-29 expression. Furthermore, both E(2) treatment and intravenous injection of the recombinant adenovirus expressing miR-29a/b markedly increased the miR-29a/b level and attenuated the expression of fibrotic markers in mouse livers during CCl(4) treatment, supporting the protective role of E(2)-induced miR-29 in CCl(4)-induced hepatic injury. In conclusion, our results collectively demonstrate that estrogen can inhibit CCl(4)-induced hepatic injury through the induction of hepatic miR-29.

Cinaciguat prevents neointima formation after arterial injury by decreasing vascular smooth muscle cell migration and proliferation

AimsVascular smooth muscle cell (VSMC) migration, proliferation and remodeling of the extracellular matrix contribute to lumen loss after arterial injury leading to restenosis. Several studies indicated the role of the cyclic guanosine monophosphate signaling in neointimal formation. Cinaciguat, the novel soluble guanylate cyclase activator, currently being in phase IIb clinical trial, has been shown to exert antiplatelet and anti-remodeling effects in animal models of vascular pathology. In this study we investigated the effects of cinaciguat on post-injury arterial stenosis. Methods and ResultsMale Sprague-Dawley rats (n=100) underwent endothelial denudation by wire injury of the right common carotid artery. Cinaciguat (10mg/kg/day orally) were administered to 50 rats (1-, 2-, 3-day and 1-, 3-week treatment time), while 50 rats received placebo. A 3-week treatment resulted in a significantly reduced vascular stenosis (17.53±10.84% in the treatment group vs. 43.25±30.83% in the control wire injury group) and neointima/media area ratio (0.45±0.32 in the treatment group vs. 1.09±0.69 in the control wire injury group). By using quantitative real-time PCR, Western blot and immunohistochemistry, matrix-metallopreoteinase-9 (MMP-9) was found to be upregulated in the control-injured carotids over the whole follow-up, and cinaciguat significantly decreased MMP-9 expression by 3weeks. As assessed by protein immunoblot, injury-induced local decrease of soluble guanylate cyclase β1 subunit could be recovered by cinaciguat. In vitro wound healing assay with VSMCs revealed dose-dependent antimigratory and antiproliferative effects of cinaciguat. Plasma level of cyclic guanosine monophosphate was significantly elevated after 3weeks of treatment. ConclusionOur results show that cinaciguat prevents injury-induced neointimal hyperplasia by decreasing VSMC migration and proliferation through the regulation of MMP-9.

Src Regulates Sphingosine-1-Phosphate Mediated Smooth Muscle Cell Migration

Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid released from activated platelets at sites of arterial injury that stimulates migration of smooth muscle cells (SMC). The kinase src is a significant focal point in transmembrane signaling. This study examines the role of src during smooth muscle cell migration in response to S-1-P. Human coronary arterial SMCs were cultured in vitro. Boyden microchemotaxis assays of migration were performed in response to S-1-P in the presence and absence the src inhibitor (PP2, 10 μM) and a dominant negative src construct (DNsrc). siRNA to S-1-P receptors was used to down-regulate the S-1-P receptors. Western blotting was performed for src and MAPK phosphorylation. Inhibition of src with PP2 but not PP3 partially blocked S-1-P-mediated cell migration. S-1-P induced time-dependent activation of src, which was inhibited by PP2 and adenoviral DNsrc. PP3 or an empty vector had no effect. Activation of src by S-1-P was inhibited by siRNA to S-1-PR1 and S-1-PR3 but not by S-1-PR2. When the VSMC were transfected with adenovirus containing βARK(CT), an inhibitor to Gβγ, src activation was significantly attenuated. Src inhibition with PP2 reduced p38(MAPK) and JNK activation but did not alter ERK1/2 activation. S-1-P mediated VSMC migration is modulated by a G-protein-coupled src pathway partially through src-mediated p38(MAPK) and JNK signaling and requires S-1-PR1 and S-1-PR3 receptors.

Asymmetric dimethylarginine confers the communication between endothelial and smooth muscle cells and leads to VSMC migration through p38 and ERK1/2 signaling cascade

Communication between endothelial and smooth muscle cells (SMCs) contributes to atherosclerosis induced by atherogenic factors, such as oxide LDL. Asymmetric dimethylarginine (ADMA), a newly found cardiovascular risk factor, accumulates in the culture medium of oxide LDL (oxLDL)-treated endothelial cells and positively correlates with atherosclerosis. This study demonstrates that ADMA mediates the communication between endothelial cells and SMCs induced by oxLDL leading to SMC migration. In addition, the present study suggests exogenous ADMA directly induces SMC migration via p38 and ERK1/2 MAPK signaling transduction way. Investigations to identify the factors regulating VSMC migration may provide novel insights into atherosclerosis and its complications.

MiR-143 and miR-145

Vascular smooth muscle cells (VSMCs) are able to perform both contractile and synthetic functions, which are associated with changes in morphology, proliferation, and migration rates and are characterized by the specific expression of different marker proteins. Under normal physiological conditions, VSMC rarely proliferate in adult tissues, but undergo major phenotypic changes from the contractile to the synthetic in response to environmental cues, a phenomenon known as switching, or phenotypic modulation.1,2 Phenotypic switching is accompanied by production of abundant cytokines, extracellular matrix, and an increased rate of proliferation and migration. Therefore, the transition of VSMCs from a differentiated phenotype to a dedifferentiated state plays a critical role in the pathogenesis of cardiovascular diseases such as hypertension, vascular injury, and arteriosclerosis.2,3 However, the molecular mechanisms involved in phenotypic switching remain elusive. The last decade has witnessed an exciting discovery that led to a revolution in our understanding of the extensive regulatory gene expression networks modulated by small, untranslated RNAs, microRNAs (miRNAs).4 miRNAs comprise a novel class of endogenous, small RNAs of ≈20 to 25 nucleotides. Although the mature miRNA is very small, it is derived from a transcriptional product of a few hundred to a few thousand nucleotides. This process of maturation is known as miRNA biogenesis, extensively reviewed by Kim.5 Biogenesis of miR-143 and miR-145 is pictorially presented in Figure 1. Functionally, miRNAs are noncoding RNAs that negatively regulate gene expression. In the current, generally accepted model, they act mostly by inducing an inhibition of the translation of their target mRNAs, and, in a minority of cases, via their degradation.6,7 Very recently, however, Bartel's team challenged this view by showing that, in a vast majority of cases, mammalian microRNAs act by destabilizing their target mRNAs and decreasing their levels. …

NADPH oxidase upregulates intermediate‐conductance Ca2+ ‐ activated K+ channels (KCa3.1) in coronary smooth muscle

Oxidative stress is known to play a role in cardiovascular disease including atherosclerosis. While several ROS may be involved, NADPH oxidase appears to play a key role. Studies have shown VSMC phenotypic modulation is critical to the development of atherosclerotic plaques. KCa3.1 is one mechanism by which increased VSMC proliferation and migration occurs. Our objective was to determine if NADPH oxidase plays a role in KCa3.1 upregulation. In coronary SMCs, bFGF increased superoxide (O2.‐) production; increased KCa3.1 mRNA levels ~2.5 fold, protein expression ~2.5 fold, and channel activity ~4.0 fold; and increased AP‐1 activity ~2.0 fold. Apo inhibited the bFGF increase in O2.‐, KCa3.1 message and activity, and AP‐1 activity. Coronary smooth muscle expressed all four cardiovascular Nox isoforms (Nox1,2,4, and 5) with Nox4 being the predominant isoform. bFGF decreased Nox1,2, and 4 message, while Apo increased message of all four isoforms. Nox2 and Nox4 siRNA did not affect KCa3.1 message response to bFGF or Apo. Our findings provide novel evidence that NADPH oxidase contributes to VSMC phenotypic modulation through AP‐1 transcriptional upregulation of KCa3.1.

PS218. Effect of Remote Ischemic Preconditioning in Liver Ischemia—Reperfusion Injury Produced by Supraceliac Aortic Cross-clamping in a Swine Model of Open Repair of Thoracoabdominal Aortic Aneurysm

by 34 2% (p 0.01; n 4-8), and Est-stimulated proliferation by 9 3% (p 0.05; n 3), indicating they occur in an ERK1/2 dependent manner. Conclusions: Estrogen stimulates the cellular processes of IH development via ERK1/2 dependent signaling. Inhibition of ERK1/2 results in downregulation of Est-mediated MMP activity and Est-induced VS MC migration and proliferation. Future studies will include targeted ERK1/2 silencing to assay a possible mechanism for effective IH inhibition. Understanding the signaling mechanisms involved in hormone-mediated IH development could provide a basis for therapeutic strategies of inhibition.

Role of Phosphodiesterase 1A in Cold‐induced Hypertension and Cardiac Hypertrophy

The calcium/calmodulin‐dependent phosphodiesterase 1A (PDE1A) hydrolyzes the intracellular second messengers cAMP and cGMP. Cold exposure to (6 hr/day) results in hypertension and cardiac hypertrophy. PDE1A expression was up‐regulated in resistance arteries, kidneys and hearts of cold‐exposed rats. We hypothesized that RNAi inhibition of PDE1A would attenuate cold‐induced hypertension (CIH) and cardiac hypertrophy (CICH).We constructed AAV carrying PDE1AshRNA, ControlshRNA, and LacZ driven by a CMV promoter. Four groups received an intravenous injection of AAV.PDE1A‐shRNA, AAV.Control‐shRNA, AAV.LacZ, and PBS, respectively, before exposure to cold (5°C), while 1 group received PBS and was kept at room temperature as a control. BP was increased significantly in all cold‐exposed groups except the group treated with PDE1AshRNA, indicating that PDE1AshRNA prevented CIH. PDE1AshRNA enhanced hypothesive responses to acetylcholine in cold‐exposed rats, indicating improved endothelial dysfunction. The in vivo left ventricle wall thickness was also decreased significantly in the cold‐exposed rats treated with PDE1AshRNA. PDE1A‐shRNA attenuated the cold‐induced increases in heart weight, cardiomyocyte sizes, and vascular wall thickness. PDE1AshRNA increased intracellular cGMP and cAMP levels and decreased VSMC cell proliferation and migration. Thus, PDE1A plays a critical role in the pathogenesis of CIH and CICH.

KIS protects against adverse vascular remodeling by opposing stathmin-mediated VSMC migration in mice

Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS-/- mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS-/- VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

The expression of PKA and VASP within migrating VSMCs in the rat model of type 2 diabetic macroangiopathy

Vasodilator‐stimulated phosphoprotein (VASP), an important substrate of PKA, plays a critical role in the regulation of actin‐based cell motility in vitro. But the role of VASP in cell migration in vivo is not entirely clear. In present study, the relationship between VSMC migration and VASP expression in vivo were investigated. Eight‐week‐old male SD rats were fed with normal diet (control group, n=10) and fed with high fat diet and intraperitoneally injected with STZ (25mg/kg) to induce a model of type 2 diabetes (DM group, n=12). After feeding for 8 weeks, DM group showed significantly increased levels of the plasma glucose, HbAlc and blood cholesterin. Insulin sensitivity was reduced, identified with a glucose test using HOMA‐IR index. Morphological changes of aorta macroangiopathy in DM group were observed by HE staining and transmission electron microscope. The thickness of media was increased and the VSMCs in the media proliferated, the elastic lamella within the intima was destroyed and VSMCs in the media migrated to the intima. Immunohistochemistry showed that expression of PKA and phosphorylated VASP were distributed in the cytoplasm of VSMCs. Compared with diffused distribution within media in control group, the number of VSMCs expressed PKA was increased and mainly distributed in the region near the intima in DM group. Similarly, the distribution of VSMCs expressed VASP was consistent with that of PKA. Our study indicates that the rat model of type 2 diabetic macroangiopathy was successfully established, in which the expression of PKA and phosphorylated VASP were correlated with the migration of VSMCs. (The research is supported by national natural science foundation of China granted NO.30470680)

Neointimal formation is exacerbated following vascular injury in p22phox overexpressing transgenic mice

These studies tested the hypothesis that transgenic mice overexpressing the p22phox subunit of the NAD(P)H oxidase selectively in smooth muscle (Tgp22smc) would exhibit exacerbated neointimal formation following mechanically induced transluminal vascular injury compared to wild‐type control mice. Tgp22smc mice have a two‐fold increase in p22phox expression and H2O2 production; thus providing a model to examine the effects of elevated vascular ROS production on VSMC function in vivo. To induce vascular injury, a 0.36 mm steel catheter was guided 4–5 mm into the left common carotid via the external carotid. Following one minute the catheter was removed and reperfusion verified. The degree of neointimal formation was assessed by morphological analysis of wall thickness (WT) and cross‐sectional wall area (CSWA) in male Tgp22smc mice and littermate controls 14 days following injury. WT following injury indicated significantly greater neoinitimal formation in Tgp22smc mice (54.3 ± 7.3 vs. 43.7 ± 4.1 μm), while non‐injured contralateral carotids were similar between groups (28.5 ± 3.3 vs. 33.8 ± 2.8 μm). Moreover, the increase in carotid artery CSWA following injury was approximately four‐fold greater in Tgp22smc mice compared to control animals. Findings from these in vivo studies strongly support in vitro data which has implicated ROS as a crucial mediator of both VSMC proliferation and migration. AHA 03300119N

Preventing restenosis after angioplasty: a multistage approach

Arterial reconstruction procedures, including balloon angioplasty, stenting and coronary artery bypass, are used to restore blood flow in atherosclerotic arteries. Restenosis of these arteries has remained a major limitation of the application of these procedures, especially in the case of balloon angioplasty. Post-angioplasty restenosis results from two major processes: neointimal formation and constrictive remodelling. Neointimal formation is initiated by arterial injury with a resultant loss of contractile phenotype in tunica media, leading to VSMC [vascular SM (smooth muscle) cell] migration from the tunica media to the intima. Migrated VSMCs contribute to the intimal thickening by the excessive synthesis of ECM (extracellular matrix) and proliferation. However, increased neointimal mass is not solely responsible for luminal narrowing. Inward constrictive remodelling is also considered as a major cause of delayed failure of angioplasty. At later stages after angioplasty, the increase in contractile forces leads to lumen narrowing. Recent studies show that SM contractile proteins are re-expressed in the neointima, concomitant with late lumen loss. Therefore one important question is whether the restoration of contractile phenotype, which can suppress VSMC migration, is favourable or detrimental. In this review, the importance of viewing restenosis as a multistage process is discussed. Different stages of restenosis occur in a sequential manner and are related to each other, but in each stage a different strategy should be taken into consideration to reduce restenosis. Defining the role of each process not only reshapes the current concept, but also helps us to target restenosis with more efficacy.

Gremlin promotes vascular smooth muscle cell proliferation and migration

Recent reports highlight the importance of BMP in the vasculature. We investigated the expression pattern and role of the BMP antagonist gremlin in VSMC. We detected gremlin mRNA constitutive expression in adult and embryonic rat aortic VSMC, and in rat carotids. In vitro analysis demonstrated that angiotensin II, TGF-β1 and PDGF induced significant changes in gremlin mRNA expression. Gremlin stable overexpression in A7r5 cells blocked BMP signaling. BMP-induced reduction in VSMC DNA synthesis was markedly inhibited by gremlin overexpression. In fact, gremlin overexpression increased DNA synthesis and cell counts, and accelerated cell cycle progression of VSMC, through mechanisms that include p27kip1 down-regulation. Gremlin also led to marked increments in VSMC migration. In addition, gremlin gene silencing promoted a significant blockade on cell proliferation and migration. In vivo studies disclosed increased gremlin protein expression in the neointima of balloon-injured carotid arteries. In summary, the BMP antagonist gremlin is constitutively expressed in the normal vasculature. Gremlin induces VSMC proliferation and migration and is significantly regulated by growth factors and injury. We postulate that gremlin plays a part in the development of pathological phenotypic changes of adult VSMC.

59: S1P induced VSMC migration via activation of NAD(P)H oxidase requires GΑ12/13 mediated phospholipase C activation

Objective: To examine the role of the phospholipase C signaling during NAD(P)H oxidase activation in response to sphingosine-1-phosphate (S-1-P). Background: S-1-P is a bioactive sphingolipid that stimulates migration of smooth muscle cells (VSMC) through Gαi, Gαq and Gα12/13 coupled receptors and has been shown to activate NAD(P)H oxidase. The role of phospholipase C (PLC) in NAD(P)H oxidase regulation in response to S-1-P is poorly understood. Methods: Rat arterial VSMCs were cultured in vitro.

20-Hydroxyeicosatetraenoic Acid (20-HETE) Stimulates Migration of Vascular Smooth Muscle Cells

Aim: We tested the hypothesis that 20-HETE production contributes to platelet derived growth factor (PDGF)-BB stimulated migration of VSMC in a cell culture model. Methods: Studies were performed with A10 cells which are a rat vascular smooth muscle derived cell line. Migration was determined using a Boyden chamber chemotactic assay. Results: Pre-treatment of cells with two doses of 20-HETE (100 and 500 nM) significantly increased PDGF-BB stimulated VSMC migration by 34-58% of control; whereas, prior incubation of cells with inhibitors of 20-HETE production, 17-ODYA (1-25 M) or HET0016 (100 nM), significantly decreased PDGF-BB stimulated migration by 40-90%. 20-HETE mediated increase in PDGF-BB migration was completely prevented by the 20-HETE antagonist, WIT-002. In order to determine what second messenger pathways are involved in the 20-HETE mediated stimulation of VSM migration, experiments were performed with specific inhibitors of tyrosine kinase (tyrphostin 25, 10 µM), mitogen-activated extracellular signal-regulated kinase (MEK, PD98059, 20 µM and U0126, 10 µM), protein kinase C (Myr-PKC, 50 µM), and phosphoinositide 3-kinases (PI3Ks) (wortmannin, 50 nM). Blockade of MEK and PI3K all abolished the increase in 20-HETE mediated migration. Conclusion: 20-HETE stimulates PDGF-mediated VSM migration acting through pathways that involve MEK and PI3K.

MMP-12 is important for VSMC proliferation and migration: role of B-catenin signalling

MMP-12 is important for VSMC proliferation and migration: role of B-catenin signalling

Dominant negative insulin-like growth factor-1 receptor inhibits neointimal formation through suppression of vascular smooth muscle cell migration and proliferation, and induction of apoptosis

Blocking of the IGF-1 signaling pathway targeting the IGF-1 receptor (IGF-1R) provides a potential treatment strategy for restenosis. In this study, we have examined the effects of a dominant negative IGF-1R (IGF-1Rt) on primary rat VSMCs in vitro and on injured rat carotid artery in vivo. Ad/IGF-1Rt infection inhibited VSMC migration and proliferation, and it also induced apoptosis by inhibiting phosphorylation of Akt and phosphorylation of ERK1/2. Consistent with the anti-proliferative and apoptotic effects in vitro, the Ad/IGF-1Rt infection markedly reduced neointimal formation in carotid injury model. Ad/IGF-1Rt treated carotid arteries exhibited a suppressed proliferation index, PCNA expression, and also were stained positive for TUNEL assay. These results indicate that a dominant negative IGF-1R has the potential to reduce neointimal formation of injured rats’ carotid arteries. The delivery of dominant negative IGF-1R by adenoviral or other vectors may provide a useful strategy for inhibiting restenosis after angioplasty.

Effects of alpinetin on rat vascular smooth muscle cells

The object of this work was to study the effects of alpinetin on cultured rat aortic smooth muscle cells. It was observed that H2O2 (100 μmol L-1) induced increase of LDH (lactate dehydrogenase) leakage in the medium of VSMC by 7.4% (p<0.01) and 10-7 mol L-1 alpinetin significantly decreased LDH leakage induced by H2O2 (p<0.01). Alpinetin had the effects of inhibiting VSMC proliferation in a dose-dependent manner under the condition of serum stimulation for 24 and 48 h, but with serum stimulation for 72 h adverse effects on VSMC proliferation appeared. 10-7 and 10-8 mol L-1 alpinetin had significantly inhibitory effects on VSMC migration by 67.9% (p<0.001) and 34.1% (p<0.01) respectively. It was also found that alpinetin (10-7–10-9 mol L-1) could significantly inhibit the production of NO in cultured VSMC induced by TNFα (200 U ml-1). At 10-7, 10-8 and 10-9 mol L-1 the modulation of NO was by 22.6% (p<0.001), 20.6% (p<0.01) and 13.9% (p<0.05), respectively. In summary, the data show that alpinetin has, to some extent, protective effects on VSMC.

QUAKING REGULATION OF VISCERAL ENDODERM FUNCTION IS REQUIRED FOR VASCULAR REMODELING AND RETINOIC ACID-MEDIATED CONTROL OF ENDOTHELIAL CELL PROLIFERATION

markedly reduced following over-expression of ILK-KD compared to control infected cells (5.1 + 0.8 fold) and similar findings were observed endothelial cells. VCAM-1 activates endothelial cell NADPH oxidase in minutes and stimulates lymphocyte matrix metalloproteinase (MMP) activafter 72 hr (52.0 + 6.8%, n = 3). Furthermore, expression of proliferating cell nuclear antigen levels was reduced to 35.2 + 1.7% of RAd LacZ control levels, after 24 hr. Wounding assays illustrated VSMC migration was