Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory and immunomodulatory cytokine implicated in inflammatory conditions such as rheumatoid arthritis, Crohn's disease, multiple sclerosis and the cachexia associated with cancer or human immunodeficiency virus infection. TNF-alpha is initially expressed as a 233-amino-acid membrane-anchored precursor which is proteolytically processed to yield the mature, 157-amino-acid cytokine. The processing enzyme(s) which cleave TNF-alpha are unknown. Here we show that the release of mature TNF-alpha from leukocytes cultured in vitro is specifically prevented by synthetic hydroxamic acid-based metalloproteinase inhibitors, which also prevent the release of TNF-alpha into the circulation of endotoxin challenged rats. A recombinant, truncated TNF-alpha precursor is cleaved to biologically active, mature TNF-alpha by several matrix metalloproteinase enzymes. These results indicate that processing of the TNF-alpha precursor is dependent on at least one matrix metalloproteinase-like enzyme, inhibition of which represents a novel therapeutic mechanism for interfering with TNF-alpha production.

Tumor necrosis factor-alpha (TNF-alpha) is released from a cell membrane-anchored precursor by proteolytic cleavage. We have shown that broad spectrum synthetic inhibitors of matrix metalloproteinases (MMPs) prevent the processing of the TNF precursor but do not inhibit the release of other cytokines. Purified MMPs, stromelysin, matrilysin, collagenase, and the gelatinases can all cleave a recombinant pro-TNF substrate to yield mature TNF. MMP inhibitors prevent the rise in blood levels of TNF after endotoxin administration in rats and are effective in animal models of inflammatory disease such as adjuvant arthritis. Drugs that inhibit MMP action and TNF release show great promise for the treatment of autoimmune inflammatory diseases.

Abstract-Reactive oxygen species (ROS), produced by cellular constituents of the arterial wall, provide a signaling mechanism involved in vascular remodeling. Because adventitial fibroblasts are actively involved in coronary remodeling, we examined whether the changes in the redox state affect their phenotypic characteristics. To this end, superoxide anion production and NAD(P)H oxidase activity were measured in porcine coronary arteries in vivo, and the effect of ROS generation on adventitial fibroblast proliferation was examined in vitro. Superoxide production (SOD-and Tiron-inhibitable nitro blue tetrazolium [NBT] reduction) increased significantly within 24 hours after balloon-induced injury, with the product of NBT reduction present predominantly in adventitial fibroblasts. These changes were NAD(P)H oxidase-dependent, because diphenyleneiodonium (DPI) abolished superoxide generation (PϽ0.001). Furthermore, the injury-induced superoxide production was associated with augmented NAD(P)H oxidase activity and upregulation of p47 phox and p67 phox in adventitial fibroblasts (immunohistochemistry). Serum stimulation of isolated adventitial fibroblasts produced time-dependent increases in ROS production (peak 3 to 6 hours). The inhibition of ROS generation with NAD(P)H oxidase inhibitor (DPI) or the removal of ROS with antioxidants (Tiron, catalase) abrogated proliferation of adventitial fibroblasts. These results indicate that vascular NAD(P)H oxidase plays a central role in the upregulation of oxidative stress after coronary injury, providing pivotal growth signals for coronary fibroblasts.

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Coronary SMCs appeared to maintain highly differentiated phenotype in response to stimulation, whereas coronary adventitial fibroblasts demonstrated several characteristics that are essential during vascular repair. Coronary SMCs, however, were distinct from noncoronary medial cells, which displayed greater phenotypic heterogeneity and versatility in culture. We postulate that the mechanism of vascular repair may differ among vascular beds, pointing to the importance of coronary artery-specific investigations in vascular biology.

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Abstract-The migration of vascular cells is regulated by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Because the activation of adventitial fibroblasts has been implicated in coronary repair, we have examined regional differences in cell outgrowth and the synthesis of MMPs/TIMPs in different layers of porcine coronary arteries. Coronary medial explants demonstrated significantly slower cell outgrowth than coronary adventitia in culture (PϽ0.001). These observations were paralleled by the predominant expression of TIMP-1 and -2 in the media (14-fold and 37-fold higher than in adventitia, respectively, PϽ0.001), whereas higher gelatinolytic activities (MMP-2 and -9) were released from adventitial explants. Smooth muscle cell outgrowth from the media was regulated by endogenous TIMPs, since TIMP inhibition (recombinant MMP-2 or neutralizing anti-TIMP antibodies) facilitated cell outgrowth (PϽ0.001). In contrast, the addition of recombinant TIMP-1 or -2 decreased adventitial cell outgrowth. In the coculture experiments, the presence of coronary media retarded adventitial cell outgrowth, whereas medial damage abrogated these effects, allowing for fibroblast migration (PϽ0.001).In conclusion, this study demonstrated differential migratory properties and distinct MMP/TIMP synthesis by coronary fibroblasts and smooth muscle cells. Endogenous TIMPs in the media may play an important role in maintaining coronary arterial wall homeostasis, whereas high levels of matrix-degrading activities confer the "invasive" characteristics of adventitial fibroblasts. Key Words: migration Ⅲ matrix metalloproteinases Ⅲ tissue inhibitor of matrix metalloproteinase Ⅲ fibroblasts Ⅲ smooth muscle cells A balance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) contributes to tissue remodeling under physiological and pathological conditions. 1-4 Migration of resident vascular cells, a key event in the development of many vascular diseases, 5 is associated with enzymatic degradation of the intricate network of extracellular matrix (ECM) proteins. The upregulation of MMPs and serine proteases coincides with the formation of a hypercellular neointima after arterial injury. 6 -9 Previous studies have emphasized the expression of proteolytic enzymes by smooth muscle (SM) cells, which is consistent with the migration of medial cells during remodeling of noncoronary vascular beds. 10 -14 Recent observations regarding coronary response to injury, however, have pointed to a number of cellular mechanisms not included in the classic paradigm of vascular repair. 15 They involved the phenotypic versatility of adventitial fibroblasts that contribute to coronary repair, 16,17 similar to nonvascular fibroblasts during wound healing. 18,19 Translocation of coronary adventitial fibroblasts/myofibroblasts to the neointima contrasted with a limited response of medial SM cells in a porcine model of severe coronary injury. 20,21 These observations have prompted a question regarding the mechanisms regulating distincti...

Almost every pediatric resident was involved in attempting to resuscitate a child suffering a CPA, yet many were inadequately trained to respond. Formal mechanisms are needed to guarantee adequate resuscitation training for pediatric residents, especially regarding participation in basic life support and hands on defibrillator training.