Tripathi B. Rajavashisth

Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines.

To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF-kappa B. We propose that NOs ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall.

Calcification in atherosclerosis: Bone biology and chronic inflammation at the arterial crossroads

Dystrophic or ectopic mineral deposition occurs in many pathologic conditions, including atherosclerosis. Calcium mineral deposits that frequently accompany atherosclerosis are readily quantifiable radiographically, serve as a surrogate marker for the disease, and predict a higher risk of myocardial infarction and death. Accelerating research interest has been propelled by a clear need to understand how plaque structure, composition, and stability lead to devastating cardiovascular events. In atherosclerotic plaque, accumulating evidence is consistent with the notion that calcification involves the participation of arterial osteoblasts and osteoclasts. Here we summarize current models of intimal arterial plaque calcification and highlight intriguing questions that require further investigation. Because atherosclerosis is a chronic vascular inflammation, we propose that arterial plaque calcification is best conceptualized as a convergence of bone biology with vascular inflammatory pathobiology.

Membrane Type 1 Matrix Metalloproteinase Expression in Human Atherosclerotic Plaques Evidence for Activation by Proinflammatory Mediators

BACKGROUND Matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques, where in their active form, they may contribute to vascular remodeling and plaque disruption. In this study, we tested the hypothesis that membrane type 1 MMP (MT1-MMP), a novel transmembrane MMP that activates pro-MMP-2 (gelatinase A), is expressed in human atherosclerotic plaques and that its expression is regulated by proinflammatory molecules. METHODS AND RESULTS MT1-MMP expression was examined in normal and atherosclerotic human arteries by immunocytochemistry with specific antibodies. MT1-MMP expression in human saphenous vein-derived smooth muscle cells (SMCs) maintained in tissue culture was determined under basal conditions and in response to proinflammatory molecules (interleukin [IL]-1alpha, tumor necrosis factor [TNF]-alpha, and oxidized LDL [ox-LDL]) by use of Northern blot and ribonuclease protection assays for mRNA, Western blot and immunoprecipitation for protein, and gelatin zymography for catalytic activity. Medial SMCs of normal vessel wall expressed MT1-MMP. In atherosclerotic arteries, MT1-MMP expression was noted within the complex atheroma colocalizing with SMCs and macrophages (Mphi). Cultured SMCs constitutively expressed MT1-MMP mRNA and protein, which increased 2- to 4-fold over control in a time-dependent manner within 4 to 8 hours of exposure to IL-1alpha, TNF-alpha, and ox-LDL (thiobarbituric acid-reactive substances, 13.4 nmol/mg LDL protein), whereas native LDL had no effect. Flow cytometry revealed MT1-MMP expression by human monocyte-derived Mphi, which increased 3.8-fold over baseline within 6 hours after exposure to 10 ng/mL TNF-alpha. CONCLUSIONS This study demonstrates that MT1-MMP, an activator of pro-MMP-2, is expressed by SMCs and Mphi in human atherosclerotic plaques. Furthermore, proinflammatory molecules upregulate MT1-MMP expression in vascular SMCs and Mphi. Thus, activation of SMCs and Mphi by proinflammatory molecules may influence extracellular matrix remodeling in atherosclerosis by regulating MT1-MMP expression.

Oxidized Low-Density Lipoprotein Regulates Matrix Metalloproteinase-9 and Its Tissue Inhibitor in Human Monocyte-Derived Macrophages

BACKGROUND Macrophages in human atherosclerotic plaques produce a family of matrix metalloproteinases (MMPs), which may influence vascular remodeling and plaque disruption. Because oxidized LDL (ox-LDL) is implicated in many proatherogenic events, we hypothesized that ox-LDL would regulate expression of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in monocyte-derived macrophages. MWRHOSA AND RESULTS: Mononuclear cells were isolated from normal human subjects with Ficoll-Paque density gradient centrifugation, and adherent cells were allowed to differentiate into macrophages during 7 days of culture in plastic dishes. On day 7, by use of serum-free medium, the macrophages were incubated with various concentrations of native LDL (n-LDL) and copper-oxidized LDL. Exposure to ox-LDL (10 to 50 microg/mL) increased MMP-9 mRNA expression as analyzed by Northern blot, protein expression as measured by ELISA and Western blot, and gelatinolytic activity as determined by zymography. The increase in MMP-9 expression was associated with increased nuclear binding of transcription factor NF-kappaB and AP-1 complex on electromobility shift assay. In contrast, ox-LDL (10 to 50 microg/mL) decreased TIMP-1 expression. Ox-LDL-induced increase in MMP-9 expression was abrogated by HDL (100 microg/mL). n-LDL had no significant effect on MMP-9 or TIMP-1 expression. CONCLUSIONS These data demonstrate that unlike n-LDL, ox-LDL upregulates MMP-9 expression while reducing TIMP-1 expression in monocyte-derived macrophages. Furthermore, HDL abrogates ox-LDL-induced MMP-9 expression. Thus, ox-LDL may contribute to macrophage-mediated matrix breakdown in the atherosclerotic plaques, thereby predisposing them to plaque disruption and/or vascular remodeling.

Heterozygous osteopetrotic (op) mutation reduces atherosclerosis in LDL receptor- deficient mice.

Previous studies of osteopetrotic (op) mice lacking macrophage colony-stimulating factor (M-CSF) have revealed an inhibition of atherosclerosis development in the apolipoprotein E (apo E)-deficient model and in a diet-induced model. Using LDL receptor-deficient mice, we now show that atheroma development depends on M-CSF concentration, as not only did homozygous osteopetrotic (op/op) mice have dramatically reduced lesions (approximately 0.3% of control lesion size) but heterozygous (op/+) mice had lesions < 1% of controls. Mice heterozygous for the op mutation (op/+) had plasma levels of M-CSF about half those in controls (+/+). The finding that an approximately 2-fold reduction in M-CSF expression reduced lesion size approximately 100-fold suggests the requirement for a threshold level of M-CSF. The effect of M-CSF on atherosclerosis did not appear to be mediated either by changes in plasma lipoprotein levels or alterations in the number of circulating monocytes, since both op/op and op/+ mice exhibited higher levels of atherogenic lipoprotein particles and (op/+) mice showed a near normal number of circulating monocytes. LDL receptor-null littermates of genotypes from op/op, op/+, to +/+ showed monocyte differentials of approximately 4.5, 8, and 10%, respectively. Taken together, these results suggest that the effects of M-CSF on atherogenesis may not be mediated by expression of M-CSF systemically or by modulation of the number of circulating monocytes. These studies support the conclusion that M-CSF participates critically in fatty streak formation and progression to a complex fibrous lesion.

Inflammatory Cytokines and Oxidized Low Density Lipoproteins Increase Endothelial Cell Expression of Membrane Type 1-Matrix Metalloproteinase

We investigated whether inflammatory cytokines or oxidized low density lipoproteins (Ox-LDL) present in human atheroma modulate extracellular matrix degradation by inducing membrane type 1-matrix metalloproteinase (MT1-MMP) expression. Cultured human endothelial cells (EC) constitutively expressed MT1-MMP mRNA and protein with enzymatic activity. Tumor necrosis factor-α (TNF-α), interleukin-1α, or interleukin-1β caused a time-dependent increase in the steady-state MT1-MMP mRNA levels within 4 h of exposure, peaking about 4-fold by 6 h, and remaining elevated for 12 h. Increased MT1-MMP mRNA correlated with a 2.5-fold increase in MT1-MMP protein in EC membranes. Ox-LDL also increased MT1-MMP mRNA levels that varied with the duration of exposure and degree of LDL oxidation. The increase in MT1-MMP mRNA occurred within 6 h of exposure to Ox-LDL and peaked over 3-fold by 6 h. Ox-LDL, but not native LDL, increased MT1-MMP protein by 2-fold in EC membranes. A combination of TNF-α and Ox-LDL was additive in increasing MT1-MMP expression. Nuclear run-on assays showed that TNF-α or Ox-LDL augmented steady-state mRNA levels by increased transcription of the MT1-MMP gene. These findings indicate that activation of EC by inflammatory cytokines and/or Ox-LDL increase MT1-MMP expression. Since MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokines or Ox-LDL may influence extracellular matrix remodeling.

Increased Expression of Membrane Type 3-Matrix Metalloproteinase in Human Atherosclerotic Plaque Role of Activated Macrophages and Inflammatory Cytokines

Background—Matrix metalloproteinases (MMPs) are thought to play a prominent role in atherogenesis and destabilization of plaque. Pericellularly localized membrane-type (MT)-MMPs activate secreted MMPs. We investigated the hypothesis that MT3-MMP is expressed in human atherosclerotic plaques and is regulated by locally produced inflammatory cytokines and oxidized low-density lipoprotein (Ox-LDL). Methods and Results—Expression and cellular localization of MT3-MMP in normal and atherosclerotic human coronary arteries were examined using specific antibodies. Abundant MT3-MMP expression was noted in medial smooth muscle cells (SMCs) of normal arteries. In atherosclerotic arteries, MT3-MMP expression was observed within complex plaques and colocalized with SMCs and macrophages (M&phgr;). Cultured human monocyte-derived M&phgr; constitutively expressed MT3-MMP mRNA and proteolytically active protein, as demonstrated by mRNA analyses, immunoblotting, and gelatin zymography, respectively. Ox-LDL, tumor necrosis factor-&agr;, or macrophage colony-stimulating factor caused dose- and time-dependent increases in steady-state levels of MT3-MMP mRNA in cultured M&phgr;. This correlated with a 2- to 4-fold increase in levels of MT3-MMP immunoreactive protein and enzymatic activity in M&phgr; membranes. Confocal microscopy and flow cytometry confirmed induction and spatial distribution of MT3-MMP protein from intracellular domains to the M&phgr; plasma membrane by Ox-LDL, tumor necrosis factor-&agr;, or macrophage colony-stimulating factor. Conclusions—MT3-MMP is expressed by SMCs and M&phgr; in human atherosclerotic plaques. Proinflammatory molecules cause a progressive increase in the expression of MT3-MMP in cultured M&phgr;. Our results suggest a mechanism by which inflammatory molecules could promote M&phgr;-mediated degradation of extracellular matrix and thereby contribute to plaque destabilization.

Rationale for the role of osteoclast-like cells in arterial calcification

Atherosclerotic arteries frequently become calcified, and these calcium deposits are associated with a high risk of adverse clinical events. Descriptive studies suggest calcification is an organized and regulated process with many similarities to osteogenesis, yet the mechanism and its relationship to atherosclerosis remain largely unknown. In bone development and homeostasis, mineral deposition by osteoblasts and mineral resorption by osteoclasts are delicately balanced such that there is no overall gain or loss in bone mass. We hypothesize that there exists in arteries a mechanism that similarly balances mineral deposition with resorption. We propose that the cellular mediators of arterial mineral resorption are osteoclast‐like cells (OLCs) derived from hematopoietic precursors of the mononuclear phagocytic lineage. In arterial microenvironments, mononuclear precursors are induced to differentiate toward OLCs by macrophage‐colony stimulating factor and receptor activator of NF‐κB ligand, both of which are necessary and sufficient for osteoclastogenesis and mineral resorption in bone. OLCs may participate in normal mineral homeostasis within the arterial wall or, alternatively, may be recruited to specific sites within developing plaque. Net calcium deposition occurs as a result of focal perturbation of the balance between the activity of osteoblast‐like cells and OLCs. Our proposed mechanism thus views arterial mineral deposition not so much as an active pathological process, but as a localized failure of protective mechanisms that actively oppose mineral deposition within the disordered metabolic milieu of developing atherosclerotic plaque.—Doherty, T. M., Uzui, H., Fitzpatrick, L. A., Tripathi, P. V., Dunstan, C. R., Asotra, K., Rajavashisth, T. B. Rationale for the role of osteoclast‐like cells in arterial calcification. FASEB J. 16, 577–582 (2002)

Regulation of Myogenic Differentiation by Androgens: Cross Talk between Androgen Receptor/ β-Catenin and Follistatin/Transforming Growth Factor-β Signaling Pathways

Androgens are important regulators of body composition and promote myogenic differentiation and inhibit adipogenesis of mesenchymal, multipotent cells. Here, we investigated the mechanisms by which androgens induce myogenic differentiation of mesenchymal multipotent cells. Incubation of mesenchymal multipotent C3H 10T1/2 cells with testosterone and dihydrotestosterone promoted nuclear translocation of androgen receptor (AR)/beta-catenin complex and physical interaction of AR, beta-catenin, and T-cell factor-4 (TCF-4). Inhibition of beta-catenin by small inhibitory RNAs significantly decreased testosterone-induced stimulation of myogenic differentiation. Overexpression of TCF-4, a molecule downstream of beta-catenin in Wnt signaling cascade, in C3H 10T1/2 cells significantly up-regulated expression of myoD and myosin heavy chain II proteins and of follistatin (Fst), which binds and antagonizes native ligands of the TGF-beta/Smad pathway. Gene array analysis of C3H 10T1/2 cells treated with testosterone revealed that testosterone up-regulated the expression of Fst and modified the expression of several signaling molecules involved in the TGF-beta/Smad pathway, including Smad7. Lowering of testosterone levels in mice by orchidectomy led to a significant decrease in Fst and Smad7 expression; conversely, testosterone supplementation in castrated mice up-regulated Fst and Smad7 mRNA expression in androgen-responsive levator ani muscle. Testosterone-induced up-regulation of MyoD and myosin heavy chain II proteins in C3H 10T1/2 cells was abolished in cells simultaneously treated with anti-Fst antibody, suggesting an essential role of Fst during testosterone regulation of myogenic differentiation. In conclusion, our data suggest the involvement of AR, beta-catenin, and TCF-4 pathway during androgen action to activate a number of Wnt target genes, including Fst, and cross communication with the Smad signaling pathway.

Polymeric-based perivascular delivery of a nitric oxide donor inhibits intimal thickening after balloon denudation arterial injury: role of nuclear factor-kappaB

OBJECTIVES To examine the effect of a polymeric-based periadventitial delivery of a nitric oxide (NO)-releasing diazeniumdiolate, spermine/NO (SPER/NO), on balloon injury-induced neointimal hyperplasia in rat ileofemoral arteries. BACKGROUND Reduced local bioavailability and adverse side effects limit systemic administration of NO to modulate vascular response to injury. METHODS A polylactic-polyglycolic acid polymeric matrix containing 2.5% SPER/NO (w/w) was applied around the injured arteries. Quantitative histomorphometry was performed at day 14, proliferating cell nuclear antigen (PCNA) immunohistochemistry at day 3 to assess effects on smooth muscle proliferation and electrophoretic mobility shift assay to evaluate effects on transcription factor, nuclear factor-kappaB (NF-kappaB). RESULTS Treatment with SPER/NO reduced the intimal area (0.011 +/- 0.009 vs. 0.035 +/- 0.006 mm2 control, p < 0.01) and the intima to media ratio (0.089 +/- 0.062 vs. 0.330 +/- 0.057 control, p < 0.005). Spermine/nitric oxide produced a profound inhibition of PCNA-positive cells (>75%, p < 0.005) and significantly suppressed the injury-induced activation of NF-kappaB. Vascular cyclic guanosine monophosphate (cGMP) levels were elevated after treatment with the SPER/NO (0.28 +/- 0.03 vs. 0.17 +/- 0.02 pmol/mg tissue control, p < 0.01). The inhibitory effects on neointimal proliferation were localized to the site of application of SPER/NO and were not associated with any changes in platelet aggregation or bleeding time. Neither SPER nor polymer alone had any significant effects on any of the variables examined. CONCLUSIONS Polymeric-based perivascular delivery of a NO donor produces a marked localized inhibition of neointimal proliferation in balloon-injured arteries. This phenomenon is associated with suppression of NF-kappaB activation and elevation of the vascular cGMP at the site of injury.