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 cellspecific 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. Rupture of coronary atheroma precipitates most acute myocardial infarctions (1). These plaque ruptures occur predictably at sites of thinning of the lesion's fibrous cap, areas frequently associated with accumulations of monocyte-derived macrophages (2-4), an important cellular component of the atherosclerotic plaques (5-8). Under certain conditions, mononuclear phagocytes can produce enzymes of the matrix metalloproteinase (MMP) family that are capable of degrading components of the extracellular matrix (9, 10). Thus, monocyte-derived foam cells may be the sources of matrixdegrading activity that may contribute to lability of the fibrous cap (2). However, the macrophages present in atherosclerotic plaques differ from the mononuclear phagocytes previously studied in relation to MMP production (11). Notably (13) and likely reflect the functions of lesional macrophages more faithfully than foam cells produced in culture. MATERIALS AND METHODSReagents. Cell types were identified using anti-muscle actin monoclonal antibody HHF-35 (Enzo Diagnostics) and anti-rabbit macrophage monoclonal antibody RAM-11 (Dako). Sheep polyclonal antibodies raised against rabbit interstitial collagenase (or MMP-1) (17) and sheep polyclonal antibodies against human stromelysin (or MMP-3) cross-reacting with rabbit MMP-3 were generously provided by Constance Brinckerhoff (Darmouth Medical School, Hanover, NH). Affinity-purified donkey antimouse antibodies conjugated to fluorescein or anti-sheep antibodies conjugated to lissamine rhodamine were from Jackson ImmunoResearch. Phorbol 12-myristate 13-acetate (PMA) and Escherichia coli lipopolysaccharide (LPS) serotype 055:B5 were from Sigma.Lesion Induction. Ten New Zealand male rabbits (2-3 kg, body weight) were divided into two groups for convenience and handled identically during the two experiments. Aortic macrophages were obtained from rabbits treated to produce aortic lesions rich in macrophage-derived foam cells (13, 16) by using a modification of procedures as described (13,18,19). Rabbits consumed a diet supplemented with 0.5% cholesterol a...

Multiple data suggest that the renin-angiotensin system contributes to the pathogenesis of atherosclerosis. The atherogenic effect of the renin-angiotensin system can only in part be explained by the influence of its effector angiotensin II on blood pressure, smooth muscle cell (SMC) growth, or antifibrinolytic activity. Because chronic inflammation of the vessel wall is a hallmark of atherosclerosis, we hypothesized that angiotensin II may elicit inflammatory signals in vascular SMCs. Human vascular SMCs were stimulated with angiotensin. Inflammatory activation was assessed by determination of interleukin-6 (IL-6) release into the culture medium, detection of IL-6 mRNA by RT-PCR, and demonstration of activation of nuclear factor-kappaB in electrophoretic mobility shift assays. Angiotensin II concentration-dependently (1 nmol/L to 1 micromol/L) stimulated IL-6 production by SMCs via activation of the angiotensin II type 1 receptor (demonstrated by the inhibitory action of the receptor antagonist losartan). Angiotensin I increased IL-6 production by SMCs, too. This effect was inhibited by captopril and ramiprilat, suggesting conversion of angiotensin I to angiotensin II by angiotensin-converting enzyme in SMCs. Steady-state mRNA for IL-6 was augmented after stimulation with angiotensin II, suggesting regulation of angiotensin-induced IL-6 release at the pretranslational level. Moreover, the proinflammatory transcription factor nuclear factor-kappaB, which is necessary for transcription of most cytokine genes, was also activated by angiotensin II. Pyrrolidine dithiocarbamate suppressed angiotensin II-induced IL-6 release, a finding compatible with involvement of reactive oxygen species as second messengers in cytokine production mediated by angiotensin. The data demonstrate the ability of angiotensin to elicit an inflammatory response in human vascular SMCs by stimulation of cytokine production and activation of nuclear factor-kappaB. Inflammatory activation of the vessel wall by a dysregulated renin-angiotensin system may contribute to the pathogenesis of atherosclerosis.

No abstract

The potent vasoconstrictor peptide endothelin-1 (ET-1) has been implicated in the pathophysiology of atherosclerosis and its complications. Since inflammation of the vessel wall is a hallmark of atherosclerosis, the purpose of the present study was to investigate the influence of ET-1 on cytokine production in human vascular smooth muscle cells (SMC) as a marker of inflammatory cell activation. ET-1 (100 pM - 1 microM) stimulated interleukin-6 (IL-6) secretion from human vascular SMC in a concentration-dependent manner. The ET-A-receptor antagonist BQ-123 (10 microM), but not the ET-B-receptor antagonist BQ-788, inhibited IL-6 release. ET-1 also transiently increased IL-6 mRNA compatible with regulation of IL-6 release at the pretranslational level. Electrophoretic mobility shift assays demonstrated time- and concentration-dependent activation of the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) in ET-1-stimulated human vascular SMC. A decoy oligodeoxynucleotide bearing the NF-kappaB binding site inhibited ET-1-stimulated IL-6 release to a great extent suggesting that this transcription factor plays a key role for cytokine production elicited by ET-1. Moreover, the antioxidant pyrrolidine dithiocarbamate (10 microM) inhibited ET-1-induced IL-6 release indicating involvement of reactive oxygen species in ET-1 signaling. ET-1-stimulated IL-6 secretion was also suppressed by diphenylene iodonium (40 microM), an inhibitor of flavon-containing enzymes such as NADH/NADPH oxidase. The results demonstrate the ability of ET-1 to induce an inflammatory response in human vascular SMC. These observations may contribute to a better understanding of the role of ET-1 in inflammatory activation of the vessel wall during atherogenesis.