CD40 signaling and plaque instability.

Today, multiple lines of evidence support the view of atherosclerosis as a chronic inflammatory disease and implicate components of the immune system in atherogenesis. Recent work has documented overexpression of the potent immune mediator CD40 and its counterpart CD40 ligand (CD40L) in experimental and human atherosclerotic lesions. Notably, interruption of CD40/CD40L interactions not only diminished the formation and progression of mouse atheroma, but also fostered changes in lesion biology and structure, which are associated in humans with "plaque stabilization." In accordance with the hypothesis that CD40 signaling promotes plaque instability, in vitro studies demonstrated that ligation of CD40 on atheroma-associated cell types, namely endothelial cells, smooth muscle cells, and macrophages, mediates functions considered crucial to the process of atherogenesis, such as the expression of cytokines, chemokines, growth factors, matrix metalloproteinases, and procoagulants. The combination of the broad gamut of proatherogenic biological responses triggered by ligation of CD40 on endothelial cells, smooth muscle cells, and macrophages in vitro and the results of in vivo studies of interruption of CD40 signaling suggests a central role for this receptor/ligand dyad during atherogenesis, proposing CD40/CD40L interactions as a novel potential therapeutic target for this prevalent human disease.

Soluble CD40L and cardiovascular risk in women.

BACKGROUND: The immune-signaling dyad CD40/CD40L promotes atherogenesis, and patients with unstable angina have elevated plasma levels of soluble CD40L (sCD40L) and membrane-bound CD40L. It is unknown, however, whether elevations of circulating sCD40L precede the onset of acute cardiovascular symptoms. METHODS AND RESULTS: In a prospective, nested case-control evaluation of healthy middle-aged women, mean concentrations of sCD40L at baseline were significantly higher among 130 participants who subsequently developed myocardial infarction, stroke, or cardiovascular death (cases), compared with 130 age- and smoking-matched women who remained free of cardiovascular disease (controls) during a 4-year follow-up (2.86 ng/mL for cases versus 2.09 ng/mL for controls; P=0.02). Women with concentrations above the 95th percentile of the control distribution (>3.71 ng/mL) had a significantly increased relative risk (RR) of developing future cardiovascular events (RR, 3.3; 95% CI, 1.2 to 8.6; P=0.01) that remained after adjustment for usual cardiovascular risk factors (multivariate RR, 2.8; 95% CI, 0.9 to 8.0; P=0.05). CONCLUSIONS: High plasma concentrations of sCD40L may be associated with increased vascular risk in apparently healthy women.

Expression of neutrophil collagenase (matrix metalloproteinase-8) in human atheroma: a novel collagenolytic pathway suggested by transcriptional profiling.

BACKGROUND: Loss of interstitial collagen, particularly type I collagen, the major load-bearing molecule of atherosclerotic plaques, renders atheroma prone to rupture. Initiation of collagen breakdown requires interstitial collagenases, a matrix metalloproteinase (MMP) subfamily consisting of MMP-1, MMP-8, and MMP-13. Previous work demonstrated the overexpression of MMP-1 and MMP-13 in human atheroma. However, no study has yet evaluated the expression of MMP-8, known as "neutrophil collagenase," the enzyme that preferentially degrades type I collagen, because granulocytes do not localize in plaques. METHODS AND RESULTS: Transcriptional profiling and reverse transcription-polymerase chain reaction analysis revealed inducible expression of MMP-8 transcripts in CD40 ligand-stimulated mononuclear phagocytes. Western blot analysis demonstrated that 3 atheroma-associated cell types, namely, endothelial cells, smooth muscle cells, and mononuclear phagocytes, expressed MMP-8 in vitro upon stimulation with proinflammatory cytokines such as interleukin-1beta, tumor necrosis factor-alpha, or CD40 ligand. MMP-8 protein elaborated from these atheroma-associated cell types migrated as 2 immunoreactive bands, corresponding to the molecular weights of the zymogen and the active molecule. Extracts from atherosclerotic, but not nondiseased arterial tissue, contained similar immunoreactive bands. Moreover, all 3 cell types expressed MMP-8 mRNA and protein in human atheroma in situ. Notably, MMP-8 colocalized with cleaved but not intact type I collagen within the shoulder region of the plaque, a frequent site of rupture. CONCLUSIONS: These data point to MMP-8 as a previously unsuspected participant in collagen breakdown, an important determinant of the vulnerability of human atheroma.

Tissue factor pathway inhibitor-2 is a novel inhibitor of matrix metalloproteinases with implications for atherosclerosis.

Degradation of ECM, particularly interstitial collagen, promotes plaque instability, rendering atheroma prone to rupture. Previous studies implicated matrix metalloproteinases (MMPs) in these processes, suggesting that dysregulated MMP activity, probably due to imbalance with endogenous inhibitors, promotes complications of atherosclerosis. We report here that the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) can function as an MMP inhibitor. TFPI-2 diminished the ability of the interstitial collagenases MMP-1 and MMP-13 to degrade triple-helical collagen, the primary load-bearing molecule of the ECM within human atheroma. In addition, TFPI-2 also reduced the activity of the gelatinases MMP-2 and MMP-9. In contrast to the "classical" tissue inhibitors of MMPs (TIMPs), TFPI-2 expression in situ correlated inversely with MMP levels in human atheroma. TFPI-2 colocalized primarily with smooth muscle cells in the normal media as well as the plaque's fibrous cap. Conversely, the macrophage-enriched shoulder region, the prototypical site of matrix degradation and plaque rupture, stained only weakly for TFPI-2 but intensely for gelatinases and interstitial collagenases. Evidently, human mononuclear phagocytes, an abundant source of MMPs within human atheroma, lost their ability to express this inhibitor during differentiation in vitro. These findings establish a new, anti-inflammatory function of TFPI-2 of potential pathophysiological significance for human diseases, including atherosclerosis.

The CD40/CD154 receptor/ligand dyad.

Until recently, the expression and primary function of the cell surface receptor CD40 and its ligand CD154 were considered restricted to B and T lymphocytes, and their interactions required for the thymus-dependent humoral response. However, current work from several groups challenges this view of the CD40/CD154 dyad as a mere mediator of lymphocyte communication. A variety of non-lymphocytic cell types express both receptor and ligand, including hematopoetic and non-hematopoetic cells, such as monocytes, basophils, eosinophils, dendritic cells, fibroblasts, smooth muscle, and endothelial cells. Accordingly, ligation of CD40 mediates a broad variety of immune and inflammatory responses, such as the expression of adhesion molecules, cytokines, matrix-degrading enzymes, prothrombotic activities, and apoptotic mediators. Consequently, CD40 signaling has been associated with pathogenic processes of chronic inflammatory diseases, such as autoimmune diseases, neurodegenerative disorders, graft-versus-host disease, cancer, and atherosclerosis. This review focuses on the synthesis and structure of CD40 and outlines CD154/CD40 signaling pathways, and emphasizes the previously unexpected importance of the CD40/CD154 receptor/ligand dyad in a spectrum of immunoregulatory processes and prevalent human diseases.

PPARalpha activators inhibit tissue factor expression and activity in human monocytes.

BACKGROUND: Tissue factor (TF), expressed on the surface of monocytes and macrophages in human atherosclerotic lesions, acts as the major procoagulant initiating thrombus formation in acute coronary syndromes. Peroxisome proliferator-activated receptor-alpha (PPARalpha), a nuclear receptor family member, regulates gene expression in response to certain fatty acids and fibric acid derivatives. Given that some of these substances reduce TF activity in patients, we tested whether PPARalpha activators limit TF responses in human monocytic cells. METHODS AND RESULTS: Pretreatment of freshly isolated human monocytes or monocyte-derived macrophages with PPARalpha activators WY14643 and eicosatetraynoic acid (ETYA) led to reduced lipopolysaccharide (LPS)-induced TF activity in a concentration-dependent manner (maximal reduction to 43+/-8% with 250 micromol/L WY14643 [P:<0.05, n=5] and to 42+/-12% with 30 micromol/L ETYA [P:>0.05, n=3]). Two different PPARgamma activators (15-deoxy(_Delta12,14)-prostaglandin J(2) and BRL49653) lacked similar effects. WY14643 also decreased tumor necrosis factor-alpha protein expression in supernatants of LPS-stimulated human monocytes. Pretreatment of monocytes with WY14643 inhibited LPS-induced TF protein and mRNA expression without altering mRNA half-life. Transient transfection assays of a human TF promoter construct in THP-1 cells revealed WY14643 inhibition of LPS-induced promoter activity, which appeared to be mediated through the inhibition of nuclear factor-kappaB but not to be due to reduced nuclear factor-kappaB binding. CONCLUSIONS: PPARalpha activators can reduce TF expression and activity in human monocytes/macrophages and thus potentially reduce the thrombogenicity of atherosclerotic lesions. These data provide new insight into how PPARalpha-activating fibric acid derivatives and certain fatty acids might influence atherothrombosis in patients with vascular disease.

Ligation of CD40 onvascular smooth muscle cells mediates loss of interstitial collagen via matrix metalloproteinase activity.

Interstitial collagen constitutes the predominant structural component of the fibrous cap of atherosclerotic plaque. The balance between synthesis and degradation of this extracellular matrix protein probably determines plaque stability and hence the tendency for plaque rupture. The CD40/CD40L signaling dyad has been implicated as an important regulatory pathway of collagen-degrading activity in atherosclerosis via the induction of matrix metalloproteinases (MMPs). However, the role of CD40 signaling in the synthesis of interstitial collagen and thus in the overall rate of collagen turnover has remained unknown. We demonstrate here that CD40 ligation on cultured human vascular smooth muscle cells (SMCs) diminishes the detectable content of de novo synthesized interstitial procollagens. Notably, the loss of collagen is not accompanied by a reduction in collagen transcript expression but can be prevented by MMP inhibition. These data demonstrate that CD40 signaling in human vascular SMC shifts interstitial collagen turnover towards the loss of this extracellular matrix protein by accelerating its degradation without concomitantly diminishing its synthesis. Thus, CD40/CD40L interactions might play a key role in rendering atheromatous lesions prone to rupture.

Host CD40 ligand deficiency induces long-term allograft survival and donor-specific tolerance in mouse cardiac transplantation but does not prevent graft arteriosclerosis.

Although interruption of CD40-CD40L interactions via their respective mAbs yields prolonged allograft survival, the relative importance of CD40 or CD40L on donor or host cells remains unknown. Moreover, it is uncertain whether any allospecific tolerance occurring with CD40-CD40L blockade will also prevent allograft arteriopathy, the major long-term limitation to transplantation. Therefore, we performed cardiac transplantations using CD40L-deficient (CD40L-/-) mice to investigate the mechanisms underlying prolonged allograft survival. Without immunosuppression, wild-type (WT) hosts rejected allo-mismatched WT or CD40L-/- heart allografts within 2 wk. Conversely, allografts in CD40L-/- hosts beat vigorously for 12 wk. Anti-CD40 treatment did not induce graft failure in CD40L-/- recipients. Although graft-infiltrating cells were reduced approximately 50% in CD40L-/- hosts, the relative percentages of macrophages and T cell subsets were comparable to WT. IFN-gamma, TNF-alpha, and IL-10 were diminished commensurate with the reduced cellular infiltrate; IL-4 was not detected. CD40L-/- recipients did not develop IgG alloantibodies and showed diminished B7 and CD28 expression on subsets of graft-infiltrating cells. CD40L-/- transplant recipients developed allospecific tolerance to the donor haplotype; second set donor skin grafts engrafted well, whereas third-party skin grafts were vigorously rejected. By MLR, splenocytes from CD40L-/- allograft recipients also demonstrated allo-specific hyporesponsiveness. Nevertheless, allografts in CD40L-/- hosts developed significant graft arteriosclerosis by 8-12 wk posttransplant. Therefore, we propose that early alloresponses, without CD40-CD40L costimulation, induce allospecific tolerance but may trigger allo-independent mechanisms that ultimately result in graft vasculopathy.

CD154 (CD40 ligand).

CD40 ligand, a type II transmembrane protein recently renamed CD154, was originally considered restricted to activated T lymphocytes, functioning as a mediator of T cell-dependent B cell activation, proliferation, and differentiation. However, the spectrum of CD154 expression and function has broadened considerably during recent years, establishing new roles as a central mediator of immunity and inflammation for this member of the tumor necrosis factor (TNF) gene superfamily. The emerging picture indicates that ligation of the receptor CD40 via CD154, most potently in its trimeric form, functions in two ways. CD154 modulates physiologic processes, such as T cell-mediated effector functions and general immune responses required for appropriate host defense, but also triggers the expression of pro-inflammatory mediators, such as cytokines, adhesion molecules, and matrix degrading activities, all of which are associated with the pathogenesis of chronic inflammatory diseases, e.g., autoimmune disorders, arthritis, atherosclerosis, and cancer. Accordingly, CD40/CD154 interactions have advanced as a potential therapeutic target for these diseases, whereby two opposing strategies, interruption as well as enhancement of CD40 signaling, are explored for beneficial outcomes.

Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice.

Interruption of inflammatory pathways may provide a novel approach to the therapy of atherosclerosis. Recently, we and others have implicated the immune mediator dyad CD40/CD40L (CD40 ligand), which is expressed on endothelial and smooth muscle cells, macrophages, and T lymphocytes within human atherosclerotic lesions, in aspects of atherogenesis and the acute coronary syndromes, including regulation of matrix metalloproteinases, procoagulant activity, cytokines, etc. In vivo, interruption of CD40 signaling reduced the initiation and early phases of atheroma formation in hypercholesterolemic mice. However, whether interruption of CD40 signaling can retard the progression or even regress established lesions remains unknown. We report here that anti-CD40L antibody treatment of randomly assigned low-density lipoprotein receptor-deficient mice during the second half of a 26-week regimen of high-cholesterol diet did not regress, but did significantly reduce further evolution of established atherosclerotic lesions within the aortic arch and particularly the thoracic and abdominal aorta, as compared with control treatment (application of rat-IgG or saline; 13 weeks, continued high-cholesterol diet). In addition to limiting lesion progression, anti-CD40L treatment changed the composition of atheroma in manners thought to favor plaque stability, e.g., reduced relative content of macrophages and lipid, as well as increased relative content of smooth muscle cells and collagen. These data implicate CD40/CD40L as crucial mediators not only in the initial events of atherogenesis but also during the evolution of established atheroma. This study lends further support to the importance of this specific inflammatory signaling pathway in atherosclerosis and its complications.

The serpin proteinase inhibitor 9 is an endogenous inhibitor of interleukin 1beta-converting enzyme (caspase-1) activity in human vascular smooth muscle cells.

Interleukin-1beta-converting enzyme (ICE, caspase-1) regulates key steps in inflammation and immunity, by activating the proinflammatory cytokines interleukin (IL-)1beta and IL-18, or mediating apoptotic processes. We recently provided evidence for the regulation of caspase-1 activity via an endogenous inhibitor expressed by human vascular smooth muscle cells (SMCs) (Schönbeck, U., M. Herzberg, A. Petersen, C. Wohlenberg, J. Gerdes, H.-D. Flad, and H. Loppnow. 1997. J. Exp. Med. 185:1287-1294). However, the molecular identity of this endogenous inhibitor remained undefined. We report here that the serine proteinase inhibitor (serpin) PI-9 accounts for the endogenous caspase-1 inhibitory activity in human SMCs and prevents processing of the enzyme's natural substrates, IL-1beta and IL-18 precursor. Treatment of SMC lysates with anti-PI-9 antibody abrogated the caspase-1 inhibitory activity and coprecipitated the enzyme, demonstrating protein-protein interaction. Furthermore, PI-9 antisense oligonucleotides coordinately reduced PI-9 expression and promoted IL-1beta release. Since SMCs comprise the majority of cells in the vascular wall, and because IL-1 is implicated in atherogenesis, we tested the biological validity of our in vitro findings within human atheroma in situ. The unaffected arterial wall contains abundant and homogeneously distributed PI-9. In human atherosclerotic lesions, however, PI-9 expression correlated inversely with immunoreactive IL-1beta, supporting a potential role of the endogenous caspase-1 inhibitor in this chronic inflammatory disease. Thus, our results provide new insights into the regulation of this enzyme involved in immune and inflammatory processes of chronic inflammatory diseases, and point to an endogenous antiinflammatory action of PI-9, dysregulated in a prevalent human disease.

CD40 ligation induces tissue factor expression in human vascular smooth muscle cells.

Tissue factor (TF) instigates the extrinsic pathway of blood coagulation. Plaque disruption and exposure of circulating factor VII/VIIa to subendothelial procoagulants such as TF leads to intravascular thrombosis, a frequent cause of acute atherosclerotic events. Although the expression of TF in the intima of human atherosclerotic lesions is well established, little is known about the mechanisms of TF regulation in vascular smooth muscle cells (SMC). We demonstrate here that TF colocalizes with the receptor CD40 on lesional SMC within atherosclerotic lesions in situ. In cultured vascular SMC, ligation of CD40 with native CD40 ligand (CD40L) derived from activated T lymphocytes or recombinant human CD40L (rCD40L) induced the transient expression of TF on the cell surface (as determined by FACS analysis) in a concentration- and time-dependent manner and enhanced total cell-associated TF (as determined by ELISA). CD40L-induced TF on vascular SMC is functional and activates coagulation. In accordance with the increased TF activity, stimulation of vascular SMC with rCD40L did not affect either protein expression or activity of tissue factor pathway inhibitors. In summary, these findings demonstrate the potential of the CD40/CD40L signaling pathway to augment the procoagulant activity in human vascular SMC. Because TF and CD40 colocalize on lesional SMC in human atheroma, CD40/CD40L signaling may contribute to the TF expression and hence to increased thrombogenicity of plaques during the inflammatory responses of atherogenesis and arterial injury.

Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions.

Cyclooxygenase-1 (Cox-1) and Cox-2 convert arachidonic acid to prostaglandin H(2), the precursor of other prostaglandins and thromboxanes, eicosanoids important in vascular pathophysiology. However, knowledge of the expression of cyclooxygenases within atherosclerotic lesions is scant. This study tested the hypothesis that human atheroma and nonatherosclerotic arteries express the two Cox isoforms differentially. Cox-1 mRNA and protein localized on endothelial and medial smooth muscle cells of normal arteries (n = 5), whereas Cox-2 expression was not detectable. In contrast, atheromatous (n = 7) lesions contained both Cox-1 and Cox-2, colocalizing mainly with macrophages of the shoulder region and lipid core periphery, whereas smooth muscle cells showed lower levels, as demonstrated by immunohistochemical and in situ hybridization analysis. Furthermore, microvascular endothelium in plaques showed notable staining for both isoforms. In accord with immunohistochemical studies, Western blot analysis of protein extracts from normal arteries revealed constitutive Cox-1, but not Cox-2, expression. Extracts of atheromatous lesions, however, contained both Cox-1 and Cox-2 protein, detected as two immunoreactive proteins of approximately 70 and 50 kd. Macrophages expressed the short form of Cox-1/-2 constitutively after several days of in vitro culture, rather than the 70-kd protein. These results shed new light on the inflammatory pathways that operate in human atheroma. In particular, the expression of Cox-2 in atheromatous, but not in unaffected, arteries has therapeutic implications, given the advent of selective Cox-2 inhibitors.

Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques.

BACKGROUND: Several recent studies attempted to classify plaques as those prone to cause clinical manifestations (vulnerable, atheromatous plaques) or those less frequently associated with acute thrombotic complication (stable, fibrous plaques). Defining the cellular and molecular mechanisms that underlie these morphological features remains a challenge. Because interstitial forms of collagen determine the biomechanical strength of the atherosclerotic lesion, this study investigated expression of the collagen-degrading matrix metalloproteinase (MMP) interstitial collagenase-3 (MMP-13) and the previously studied MMP-1 in human atheroma and used a novel technique to test the hypothesis that collagenolysis in atheromatous lesions exceeds that in fibrous human atherosclerotic lesions. METHODS AND RESULTS: Human carotid atherosclerotic plaques, similar in size, were separated by conventional morphological characteristics into fibrous (n=10) and atheromatous (n=10) lesions. Immunohistochemical and Western blot analysis demonstrated increased levels of MMP-1 and MMP-13 in atheromatous versus fibrous plaques. In addition, collagenase-cleaved type I collagen, demonstrated by a novel cleavage-specific antibody, colocalized with MMP-1- and MMP-13-positive macrophages. Macrophages, rather than endothelial or smooth muscle cells, expressed MMP-13 and MMP-1 on stimulation in vitro. Furthermore, Western blot analysis demonstrated loss of interstitial collagen type I and increased collagenolysis in atheromatous versus fibrous lesions. Finally, atheromatous plaques contained higher levels of proinflammatory cytokines, activators of MMPs. CONCLUSIONS: This report demonstrates that atheromatous rather than fibrous plaques might be prone to rupture due to increased collagenolysis associated with macrophages, probably mediated by the interstitial collagenases MMP-1 and MMP-13.

Expression of stromelysin-3 in atherosclerotic lesions: regulation via CD40-CD40 ligand signaling in vitro and in vivo.

Stromelysin-3 is an unusual matrix metalloproteinase, being released in the active rather than zymogen form and having a distinct substrate specificity, targeting serine proteinase inhibitors (serpins), which regulate cellular functions involved in atherosclerosis. We report here that human atherosclerotic plaques (n = 7) express stromelysin-3 in situ, whereas fatty streaks (n = 5) and normal arterial specimens (n = 5) contain little or no stromelysin-3. Stromelysin-3 mRNA and protein colocalized with endothelial cells, smooth muscle cells, and macrophages within the lesion. In vitro, usual inducers of matrix metalloproteinases such as interleukin-1, interferon-gamma, or tumor necrosis factor alpha did not augment stromelysin-3 in vascular wall cells. However, T cell-derived as well as recombinant CD40 ligand (CD40L, CD154), an inflammatory mediator recently localized in atheroma, induced de novo synthesis of stromelysin-3. In addition, stromelysin-3 mRNA and protein colocalized with CD40L and CD40 within atheroma. In accordance with the in situ and in vitro data obtained with human material, interruption of the CD40-CD40L signaling pathway in low density lipoprotein receptor-deficient hyperlipidemic mice substantially decreased expression of the enzyme within atherosclerotic plaques. These observations establish the expression of the unusual matrix metalloproteinase stromelysin-3 in human atherosclerotic lesions and implicate CD40-CD40L signaling in its regulation, thus providing a possible new pathway that triggers complications within atherosclerotic lesions.

T lymphocytes induce endothelial cell matrix metalloproteinase expression by a CD40L-dependent mechanism: implications for tubule formation.

Neovascularization frequently accompanies chronic immune responses characterized by T cell infiltration and activation. Angiogenesis requires endothelial cells (ECs) to penetrate extracellular matrix, a process that involves matrix metalloproteinases (MMPs). We report here that activated human T cells mediate contact-dependent expression of MMPs in ECs through CD40/CD40 ligand signaling. Ligation of CD40 on ECs induced de novo expression of gelatinase B (MMP-9), increased interstitial collagenase (MMP-1) and stromelysin (MMP-3), and activated gelatinase A (MMP-2). Recombinant human CD40L induced expression of MMPs by human vascular ECs to a greater extent than did maximally effective concentrations of interleukin-1beta or tumor necrosis factor-alpha. Moreover, activation of human vascular ECs through CD40 induced tube formation in a three-dimensional fibrin matrix gel assay, an effect antagonized by a MMP inhibitor. These results demonstrated that activation of ECs by interaction with T cells induced synthesis and release of MMPs and promoted an angiogenic function of ECs via CD40L-CD40 signaling. As vascular cells at the sites of chronic inflammation, such as atherosclerotic plaques, express CD40 and its ligand, our findings suggest that ligation of CD40 on ECs can mediate aspects of vascular remodeling and neovessel formation during atherogenesis and other chronic immune reactions.

Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells.

Migration of vascular smooth muscle cells (VSMCs) plays an important role in atherogenesis and restenosis after arterial interventions. The expression of matrix metalloproteinases (MMPs), particularly MMP-9, contributes to VSMC migration. This process requires degradation of basal laminae and other components of the arterial extracellular matrix. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor family, regulate gene expression after activation by various ligands. Recent studies have suggested opposing effects of PPAR gamma (PPARgamma) activation on atherogenesis. The present study tested the hypotheses that human VSMCs express PPAR alpha (PPARalpha) and PPARgamma and that PPAR agonists in VSMCs modulate MMP-9 expression and activity, as well as VSMC migration. Human VSMCs expressed PPARalpha and PPARgamma mRNA and protein. Treatment of VSMCs with the PPARgamma ligands troglitazone and the naturally occurring 15-deoxy-Delta12, 14-prostaglandin J2 (15d-PGJ2) decreased phorbol 12-myristate 13-acetate-induced MMP-9 mRNA and protein levels, as well as MMP-9 gelatinolytic activity in the supernatants in a concentration-dependent manner. Six different PPARalpha activators lacked such effects. Addition of prostaglandin F2alpha, known to limit PPARgamma activity, diminished the MMP-9 inhibition seen with either troglitazone or 15d-PGJ2, further implicating PPARgamma in these effects. Finally, troglitazone and 15d-PGJ2 inhibited the platelet-derived growth factor-BB-induced migration of VSMCs in vitro in a concentration-dependent manner. PPARgamma activation may regulate VSMC migration and expression and activity of MMP-9. Thus, PPARgamma activation in VSMCs, via the antidiabetic agent troglitazone or naturally occurring ligands, may act to counterbalance other potentially proatherosclerotic PPARgamma effects.