A second term for the editors. Status and goals for the review process.
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Cardiology↗
Biomedical subjects
P Libby
Publications and source records attributed to P Libby.
Thrombin potently stimulates cytokine production in human vascular smooth muscle cells but not in mononuclear phagocytes.
Thrombosis frequently occurs during atherogenesis and in response to vascular injury. Accumulating evidence supports a role for inflammation in the same situation. The present study therefore sought links between thrombosis and inflammation by determining whether thrombin, which is present in active form at sites of thrombosis, can elicit inflammatory functions of human monocytes and vascular smooth muscle cells (SMCs), two major constituents of advanced atheroma. Human alpha-thrombin (EC50, approximately equal to 500 pmol/L) potently induced interleukin (IL)-6 release from SMCs. The tethered-ligand thrombin receptor appeared to mediate this effect. Furthermore, alpha-thrombin also rapidly increased levels of mRNA encoding IL-6 and monocyte chemotactic protein-1 (MCP-1) in SMCs. In contrast, only alpha-thrombin concentrations of > or = 100 nmol/L could stimulate release of IL-6 or tumor necrosis factor-alpha (TNF alpha) in peripheral blood monocytes or monocyte-derived macrophages. Lipid loading of macrophages did not augment thrombin responsiveness. Likewise, only alpha-thrombin concentrations of > or = 100 nmol/L increased levels of IL-6, IL-1 beta, MCP-1, or TNF alpha mRNA in monocytes. Differential responses of SMCs and monocytes to thrombin extended to early agonist-mediated increases in [Ca2+]i. SMCs and endothelial cells, but not monocytes, contained abundant mRNA encoding the thrombin receptor and displayed cell surface thrombin receptor expression detected with a novel monoclonal antibody. Thus, the level of thrombin receptors appeared to account for the differential thrombin susceptibility of SMCs and monocytes. These data suggest that SMCs may be more sensitive than monocytes/macrophages to thrombin activation in human atheroma. Cytokines produced by thrombin-activated SMCs may contribute to ongoing inflammation in atheroma complicated by thrombosis or subjected to angioplasty.
Amino Acid Chloromethyl Ketones↗
The direct effect of injectable cyclosporine and its vehicle, cremophor, on endothelial vascular cell adhesion molecule-1 expression. Ricinoleic acid inhibits coronary artery endothelial activation.
As in humans, rabbit coronary artery endothelium basally expresses vascular cell adhesion molecule-1 (VCAM-1). Treatment with parenteral cyclosporine (CsA) to prevent graft rejection in rabbits receiving heterotopic heart transplantation reduced VCAM-1 expression in coronary arteries not only in transplanted, but also in native rabbit hearts. To explore the mechanism of this effect, we co-incubated cultured human saphenous vein endothelial cells for 24 hr with CsA or its vehicle (containing polyoxyethylated castor oil, or Cremophor, and ethanol), at concentrations compatible with those achievable in plasma during administration of parenteral preparations of CsA. Cells were then stimulated with TNF alpha or IL-4 to induce VCAM-1 expression, assessed by a cell-surface enzyme immunoassay. Both CsA and vehicle inhibited IL-4-stimulated VCAM-1 expression in a dose-dependent manner (from [OD mU, mean +/- SEM] 230 +/- 5 to 165 +/- 3 for CsA 50 ng/ml, and to 181 +/- 6 for the corresponding vehicle concentration; P < 0.05 for both comparisons). To investigate whether this vehicle effect also occurs in vivo, we treated 9 New Zealand White rabbits with saline (n = 3), CsA (10 mg/kg/day, n = 3), or vehicle at corresponding doses (n = 3) for 6 weeks. Profiles of coronary arteries (> or = 48 for each group) were semiquantitatively scored (0-5) for VCAM-1 in immunostained heart cross-sections. Administration of both CsA and vehicle significantly reduced VCAM-1 expression compared with saline. Two vehicle components, ethanol and ricinoleic acid, were further evaluated directly on endothelial cells in vitro. While ethanol was ineffective, the monounsaturated fatty acid ricinoleic acid inhibited IL-4-stimulated VCAM-1 expression in a dose-dependent manner (IC50 between 10 and 100 microM). Thus, a fatty acid component of CsA vehicle exerts direct endothelial effects, potentially limiting arterial leukocyte recruitment during parenteral CsA treatment. This observation reveals a novel mechanism for CsA as an inhibitor of leukocyte-endothelial interactions, and furnishes a new potential rationale for the therapeutic action of unsaturated fatty acids in graft coronary disease.
Animals↗
Acute rejection accelerates graft coronary disease in transplanted rabbit hearts.
BACKGROUND: The relation between episodes of acute rejection and the development of graft coronary arteriosclerosis remains controversial. We examined the hypothesis that acute rejection episodes accelerate graft coronary arteriosclerosis lesion formation in rabbit allografts. METHODS AND RESULTS: A control group (n = 5) received cyclosporine 5 mg.kg-1.d-1 for 6 weeks after heterotopic heart transplantation. In a rejection group (n = 5), cyclosporine was omitted for 4 days at 1 and 4 weeks after transplantation. We studied cross sections of grafted hearts at 6 weeks and evaluated myocardial rejection grade, incidence, and severity and cell composition of intimal lesions in multiple coronary artery profiles. Episodic withdrawal of cyclosporine augmented myocardial rejection (International Society for Heart and Lung Transplantation grades 0, 0, 0, 0, and 1A in the control group to grades 1A, 1B, 2, 3A, and 3B in the rejection group). Episodes of acute rejection significantly increased the incidence (7.8 +/- 2.7% to 49.7 +/- 1.9%) and severity (from grade 0.10 +/- 0.04 to 0.79 +/- 0.24) of intimal thickening in graft coronary arteries. Most intimal lesions consisted of smooth muscle cells and contained various degrees of T-lymphocyte infiltration but sparse macrophages. CONCLUSIONS: In this experimental model, episodes of acute rejection precipitated by cyclosporine withdrawal accelerated the development of graft vascular lesion formation. Activation of vascular cells and leukocyte recruitment during acute rejection may thus contribute to the pathogenesis of graft arteriosclerosis.
Animals↗
Nitric oxide inhibits macrophage-colony stimulating factor gene transcription in vascular endothelial cells.
Macrophage-colony stimulating factor (M-CSF) contributes to atherogenesis by regulating macrophage-derived foam cells in atherosclerotic lesions. Here we report that nitric oxide (NO) inhibits the expression of M-CSF in human vascular endothelial cells independent of guanylyl cyclase activation. The induction of M-CSF mRNA expression by either oxidized low density lipoprotein (ox-LDL) or tumor necrosis factor-alpha (TNF alpha) was attenuated by NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and 3-morpholinosydnonimine, but not by cGMP analogues, glutathione, or nitrite. Inhibition of endogenous NO production by N-monomethyl-L-arginine (L-NMA) also increased M-CSF expression in control and TNF alpha-stimulated cells. Nuclear run-on assays and transfection studies using M-CSF promoter constructs linked to chloramphenicol acetyltransferase reporter gene indicated that NO repressed M-CSF gene transcription through nuclear factor-kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrated that activation of NF-kappa B by L-NMA, ox-LDL, and TNF alpha was attenuated by GSNO and SNP, but not by glutathione or cGMP analogues. Since the induction of M-CSF expression depends upon NF-kappa B activation, the ability of NO to inhibit NF-kappa B activation and M-CSF expression may contribute to some of NO's antiatherogenic properties.
Base Sequence↗
Induction and stabilization of I kappa B alpha by nitric oxide mediates inhibition of NF-kappa B.
To determine the mechanism(s) by which the endogenous mediator nitric oxide (NO) inhibits the activation of transcription factor NF-kappa B, we stimulated human vascular endothelial cells with tumor necrosis factor-alpha in the presence of two NO donors, sodium nitroprusside and S-nitrosoglutathione. Electrophoretic mobility shift assays demonstrated that both NO donors inhibited NF-kappa B activation by tumor necrosis factor-alpha. This effect was not mediated by guanylyl cyclase activation since the cGMP analogue 8-bromo-cGMP had no similar effect. Inhibition of endogenous constitutive NO production by L-N-monomethylarginine, however, activated NF-kappa B, suggesting tonic inhibition of NF-kappa B under basal conditions. NO had little or no effects on other nuclear binding proteins such as AP-1 and GATA. Immunoprecipitation studies showed that NO stabilized the NF-kappa B inhibitor, I kappa B alpha, by preventing its degradation from NF-kappa B. NO also increased the mRNA expression of I kappa B alpha, but not NF-kappa B subunits, p65 or p50, and transfection experiments with a chloramphenicol acetyltransferase reporter gene linked to the I kappa B alpha promoter suggested transcriptional induction of I kappa B alpha by NO. We propose that the induction and stabilization of I kappa B alpha by NO are important mechanisms by which NO inhibits NF-kappa B and attenuate atherogenesis.
Base Sequence↗
Molecular bases of the acute coronary syndromes.
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Angina, Unstable↗
Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading proteinases.
Monocyte-derived foam cells figure prominently in rupture-prone regions of atherosclerotic plaques. Peripheral blood monocytes in culture can produce certain enzymes that degrade extracellular matrix, known as matrix metalloproteinases (MMPs). Lipid-laden macrophages may thus contribute to weakening of extracellular matrix of rupture-prone atherosclerotic plaques. However, the spectrum and regulation of MMP production by foam cells remain unknown. To investigate this issue, we isolated lipid-laden macrophages from rabbit aortic lesions produced by a combination of hypercholesterolemia and balloon injury. Freshly isolated aortic macrophage foam cells, identified using cell-specific antibodies, contained immunoreactive stromelysin and interstitial collagenase, whereas alveolar macrophages isolated from the lungs of same rabbits did not. Macrophages from both tissue sources released gelatinolytic activity consistent with the 92-kDa gelatinase. In vitro, lipid-laden aortic macrophages, but not alveolar macrophages, synthesized de novo and released immunoprecipitable stromelysin and collagenase, with or without stimulation by phorbol ester or bacterial lipopolysaccharide. These stimuli caused foam cells to release additional gelatinolytic activity that migrated faster than a purified preparation of 92-kDa gelatinase in substrate-containing polyacrylamide gels, indicating activation of the 92-kDa gelatinase or induction of the 72-kDa gelatinase. Our results show that lipid-laden macrophages elaborate MMPs capable of degrading the major constituents of vascular extracellular matrix even without further stimulation. Therefore, these cells may contribute to remodeling of the extracellular matrix during atherogenesis and to the disruption of plaques often responsible for acute clinical manifestations of atherosclerosis.
Animals↗
Cytokines regulate genes involved in atherogenesis.
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Animals↗
Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions.
Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during atherogenesis. The ability of vascular cells to synthesize the components of ECM is well characterized, but less is known about their capacity to degrade ECM and the factors that may regulate this process. We therefore studied the expression of matrix metalloproteinases (MMPs), enzymes that degrade various components of ECM, and of tissue inhibitors of MMPs (TIMPs) by untreated or cytokine-stimulated human smooth muscle cells (SMC). Messenger RNA was studied by Northern blotting, and proteins secreted in culture by SMC were identified by immunoprecipitation. Gelatinolytic and caseinolytic activity of MMPs was detected zymographically. SMC constitutively produced a 72 kDa type IV gelatinase (GL), TIMP-1, and TIMP-2. Upon stimulation with IL1 or TNF alpha, SMC synthesized in addition 92 kDa GL, stromelysin, and interstitial collagenase, MMPs that together can degrade all of the ECM components. IL1 or TNF alpha did not alter the level of TIMP mRNA and protein, suggesting that a net excess of MMP production under these conditions may promote breakdown of the vascular ECM. To test the in vivo relevance of these in vitro findings, we analyzed immunohistochemically normal human arteries and carotid atheromas. Normal tissue and the medial layer underlying lesions stained uniformly for 72 kDa GL and TIMPs 1 and 2. Lesions showed regionally increased MMP expression: the shoulders of atherosclerotic plaques contained stromelysin and 92 kDa GL associated with SMC, and clusters of macrophage-derived foam cells associated with the lipid core stained intensely for all MMPs studied. Endothelial cells covering atheroma or of the plaque microvasculature contained interstitial collagenase. In pathological conditions associated with local release of cytokines in the vessel wall, enhanced regional expression of vascular MMPs may contribute to SMC migration and weakening of matrix that would favor plaque rupture, events associated with the development or complication of the atherosclerotic lesions.
Arteries↗
Activation of smooth muscle and endothelial cells following balloon injury.
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Animals↗
Human vascular smooth muscle cells poorly co-stimulate and actively inhibit allogeneic CD4+ T cell proliferation in vitro.
We compared immunologic functions of human vascular smooth muscle cells (VSMC) with those of endothelial cells (EC) cultured from saphenous vein. Both cell types can express comparable levels of MHC class II molecules. However, class II-positive VSMCs, unlike ECs, do not stimulate resting CD4+ T cell proliferation. Limiting dilution analyses revealed IL-2-producing cells alloreactive to class II-positive ECs but not VSMCs. Class II molecules on VSMCs are functional, inducing CD25 expression on resting CD4+ T cells and stimulating proliferation of CD4+ T cells that have been pre-activated by ECs. VSMC expression of the co-stimulator molecules CD44, CD54, CD58, and CD59 is comparable to EC expression, and neither VCAM-1 nor B7 are expressed on either cell. However, VSMCs are less efficient than ECs at co-stimulating IL-2 production by PHA-stimulated PBL. VSMCs but not ECs cultured across a Transwell inhibit CD4+ T cell proliferation to allogeneic ECs and, to a lesser extent, IL-2 production in the same assay. Inhibition of proliferation cannot be transferred by VSMC-conditioned media, nor reversed by inhibitors of prostaglandin synthesis, TGF-beta 1, or nitric oxide synthesis. CD4+ T cells cocultured with class II-positive VSMCs proliferate to a subsequent challenge with ECs from the same donor as well as freshly isolated T cells. We conclude that VSMCs express functional MHC class II molecules and stimulate pre-activated T cells. However, VSMCs lack adequate costimulators to fully stimulate resting T cells, and VSMCs inhibit T cell proliferation.
Antigen-Presenting Cells↗
Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue.
Many physiological and pathological processes involve tissue remodeling due in part to degradation of extracellular matrix. Several factors limit current approaches used for detection of matrix-degrading enzymes in tissues. Matrix metalloproteinases (MMPs), enzymes specialized in catabolism of extracellular matrix constituents, require processing from inactive zymogen precursors to gain enzymatic function. Presently available antibodies do not distinguish between precursor and proteolytically processed forms of MMPs. Also, ubiquitous tissue inhibitors of metalloproteinases (TIMPs) could prevent matrix degradation by MMPs even if the enzymes were in an active form. For these reasons immunocytochemistry does not provide information regarding the functional state of these enzymes. Biochemical studies of tissue extracts preclude localization and entail the possibility of artifactual activation of the enzymes consequent to tissue disruption. To obviate these problems, we have adapted substrate zymography to frozen tissue sections to assess net proteolytic activity in situ. We report here the details and the validation of this methodology. Initial experiments defined casein fluorescently labeled with resorufin as a useful substrate for detecting stromelysin, and fluoresceinated gelatin or autoradiographic emulsion as suitable for detecting gelatinolytic activity by this approach. Either TIMP-1 or the Zn chelator 1,10-phenanthroline reduced the zymographic activity in cryosections of atheroma from humans or rabbits. Inhibitors of serine proteases did not reduce the extent of substrate lysis substantially. In situ zymography preserves the fine morphological details of the tissue and can complement the study of enzyme expression by other microscopic techniques, such as immunocytochemistry. This approach may prove generally applicable for the detection of protease activity in tissue sections permitting exploration of the roles of these enzymes in pathobiology.
Animals↗
Cytokines regulate vascular functions related to stability of the atherosclerotic plaque.
The cytokines are multipotent mediators of inflammation and immunity that can affect key functions of vascular wall cells. Growing evidence suggests that cytokines participate as autocrine or paracrine mediators in atherogenesis, as cells in lesions can both produce and respond to these mediators. The functions of vascular wall cells regulated by cytokines may influence lesion initiation, progression, or complication. For example, cytokines can regulate the expression of adhesion molecules crucial to the recruitment of leukocytes to lesions, including vascular cell adhesion molecule-1 (VCAM-1). Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) can regulate the production of monocyte chemoattractant protein-1 (MCP-1), a potential signal for directed migration of monocytes into the intima. Cytokines can also regulate genes that encode other growth factors and cytokines themselves. TNF-alpha can induce IL-1 mRNA in human endothelial (EC) and smooth-muscle cells (SMC). IL-1 and TNF-alpha can augment the production by vascular cells of macrophage-colony stimulating factor (M-CSF), which may promote growth and activation of mononuclear phagocytes. Cytokines can exert both pro-and antiatherogenic actions. Activated T cells in human atheroma may secrete the lymphokine IFN-gamma, an inhibitor of SMC proliferation. Cytokines influence vasomotor tone in arteries, e.g., by inducing a form of nitric oxide synthase, the enzyme that synthesizes the vasodilatory nitric oxide radical. The cytokines also modulate endothelial functions that govern the formation and stability of blood thrombi. Finally, in the late stages of the disease, matrix metalloproteinases derived from macrophages or smooth-muscle cells themselves may contribute to weakening of the fibrous cap in the vulnerable shoulder area, promoting plaque rupture and occlusive thrombosis, culminating in the dramatic clinical manifestations of atherosclerosis, including myocardial infarction and stroke. Thus, cytokines can influence multiple aspects of atherogenesis and provide new and interesting targets for therapeutic intervention.
Animals↗
Human vascular smooth muscle cell-monocyte interactions and metalloproteinase secretion in culture.
Degradation of the atherosclerotic plaque extracellular matrix could destabilize the lesion, rendering it more prone to rupture. Both macrophages and vascular smooth muscle cells (SMCs) are potential sources of matrix metalloproteinases (MMPs), secreted enzymes that can digest vascular matrix. We explored interactions between human vascular SMCs and human monocytes that result in the secretion of interstitial collagenase (MMP-1) and stromelysin (MMP-3). Monocytes alone or those treated with SMC-conditioned media did not secrete these metalloproteinases as detectable by Western blot analysis. SMCs increased secretion of both MMP-1 and MMP-3 greater than 20-fold when cocultured with monocytes or when treated with monocyte-conditioned media. Addition of macrophage colony stimulating factor (< or = 1000 U/mL) to cocultures of monocytes and SMCs did not affect metalloproteinase secretion. Recombinant interleukin (IL)-1 receptor antagonist inhibited MMP-1 and MMP-3 induction in SMC cultures treated with monocyte-conditioned media (94% and 96% reduction, respectively), while a neutralizing antibody to tumor necrosis factor-alpha had no significant effect on metalloproteinase secretion. In contrast to the induction by monocyte-conditioned media of MMP-1 and MMP-3 secretion by SMCs, monocyte-conditioned media did not increase secretion of 72-kD gelatinase (MMP-2). Thus, monocytes induce MMP-1 and MMP-3 secretion by vascular SMCs through an IL-1-dependent mechanism. This response of SMCs to a defined macrophage product may contribute to plaque destabilization by mononuclear phagocytes in the lesion.
Cells, Cultured↗
Regulation of smooth muscle cell scavenger receptor expression in vivo by atherogenic diets and in vitro by cytokines.
Scavenger receptor (ScR)-mediated uptake of modified lipoproteins may contribute to the transformation of smooth muscle cells into lipid-laden foam cells during atherogenesis. This study examined the in vivo expression of ScRs in aortas, with or without balloon injury, taken from hypercholesterolemic or normocholesterolemic rabbits. Numerous intimal cells in the rabbit aortic lesions expressed ScRs as detected by immunocytochemical staining with a goat anti-rabbit ScR antibody. Single immunostaining for cell identification markers in serial sections, as well as double staining, confirmed the expression of ScRs by both intimal smooth muscle cells and macrophages. To explore potential inducers of ScR expression by smooth muscle cells in vivo, we studied the regulation of ScR expression in vitro by cytokines known to be present in atherosclerotic lesions. Tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) increased ScR mRNA levels, protein expression, and AcLDL degradative activity in cultured rabbit aortic smooth muscle cells. The induction of ScR expression in intimal smooth muscle cells in vivo could be a useful marker of smooth muscle cell activation during atherogenesis and may contribute to foam cell formation by this cell type following balloon injury and/or hypercholesterolemia. Cytokines, such as TNF-alpha or IFN-gamma, may stimulate some of the phenotypic changes that characterize the alteration in gene expression of intimal smooth muscle cells in rabbit atherosclerotic lesions.
Animals↗
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 NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall.
Base Sequence↗