Matrix metalloproteinases (MMPs) cooperatively degrade all components of the extracellular matrix (ECM). Remodeling of ECM during skeletal muscle degeneration and regeneration suggests a tight regulation of matrix-degrading activity during muscle regeneration. In this study, we investigated the expression of MMP-2 and MMP-9, in normal muscles and their regulation during regeneration process. We further investigated their secretion by C2C12 myogenic cell line. Two models of muscle degeneration-regeneration were used: (1) normal muscles in which necrosis was experimentally induced by cardiotoxin injection; (2) mdx muscles which exhibit recurrent signs of focal myofiber necrosis followed by successful regeneration. MMPs were studied by zymography; their free activity was quantified using 3H-labeled gelatin substrate and mRNA expression was followed by Northern hybridization. Muscle degeneration-regeneration was analyzed by conventional morphological methods and in situ hybridization was performed on muscle sections to identify the cells expressing these MMPs. Results show that MMP-2, but not MMP-9 expression, is constitutive in normal muscles. Upon injury, the active form of MMP-2 is transiently increased, whereas MMP-9 is induced within 24 h and remains present for several days. Quantitative assays of free gelatinolytic activity show a progressive and steady increase that culminates at 7 days postinjury and slowly returns to normal levels. In adult mdx mice, both pro and active forms of MMP-2 and MMP-9 are expressed. Northern blot results support these findings. Zymography of C2C12-conditioned medium shows that myogenic cells produce MMP-2. By in situ hybridization we localized MMP-9 mRNA in inflammatory cells and putative activated satellite cells in injured muscles. Our data allow the correlation of the differential expression of pro and/or active forms of MMP-2 and MMP-9 with different stages of the degeneration-regeneration process: MMP-9 expression is related to the inflammatory response and probably to the activation of satellite cells, whereas MMP-2 activation is concomitant with the regeneration of new myofibers.

Polymorphonuclear neutrophil (PMN) migration across basement membrane is thought to be dependent on the degradation of membrane constituents. PMN gelatinase B, a metalloproteinase able to degrade type IV collagen, may be involved in this phenomenon. PMN gelatinase B is released in the extracellular medium as a latent proform and then activated, mainly by PMN elastase. We investigated the role of gelatinase B in PMN migration across a Matrigel basement membrane matrix coated onto a filter, in a Boyden chamber. The effects of gelatinase and elastase inhibitors on PMN migration in this system were tested. Chemokinesis of PMN was tested in the same Boyden chamber across a filter free of basement membrane. The agarose method was used to test the same inhibitors for effects on PMN chemotaxis. In both systems, FMLP 10(-7)M was used as a chemoattractant. Addition of 10(-8)M TIMP-1 (the preferential gelatinase B inhibitor) inhibited trans-basement membrane PMN migration by 52 +/- 6% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. Also, (Ala)(2) Pro Val chloromethyl ketone (AAPVCK) 100 micron, a specific elastase inhibitor, inhibited trans-basement membrane PMN migration by 51 +/- 8% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. The AAPVCK-TIMP combination led to a decrease in migration across Matrigel basement membrane (46 +/- 2%, P,0.05)similar to that seen with TIMP alone. AAPVCK was responsible for inhibition of gelatinase B activation, leading to a decrease in activated gelatinase from 14% to 2% of total gelatinase release (P<0.05). All these results strongly suggest that gelatinase B is a major factor of PMN migration across basement membrane and that elastase may contribute to this process by activating pro-gelatinase B.

Pulmonary arterial hypertension (PAH) results from persistent vasoconstriction, smooth muscle growth and extracellular matrix (ECM) remodelling of pulmonary arteries (PAs). Matrix metalloproteinases (MMPs) are matrix-degrading enzymes involved in ECM turnover, and in smooth muscle cell (SMC) and endothelial cell migration and proliferation. MMP expression and activity are increased in experimental PAH. Therefore, this study investigated whether similar changes occur in idiopathic PAH (IPAH; formerly known as primary pulmonary hypertension).Both in situ and in vitro studies were performed on PAs from patients undergoing lung transplantation for IPAH and from patients treated by lobectomy for localised lung cancer, who served as controls.In IPAH, MMP-tissue inhibitor of metalloproteinase (TIMP) imbalance was found in cultured PASMCs, with increased TIMP-1 and decreased MMP-3. MMP-2 activity was markedly elevated as a result of increases in both total MMP-2 and proportion of active MMP-2. In situ zymography and immunolocalisation showed that MMP-2 was associated with SMCs and elastic fibres, and also confirmed the MMP-3-TIMP-1 imbalance.In conclusion, the findings of this study were consistent with a role for the matrix metalloproteinase-tissue inhibitor of metalloproteinase system in pulmonary vascular remodelling in idiopathic pulmonary arterial hypertension. The matrix metalloproteinase-tissue inhibitor of metalloproteinase imbalance may lead to matrix accumulation, and increased matrix metalloproteinase-2 activity may contribute to smooth muscle cell migration and proliferation. Whether these abnormalities are potential therapeutic targets deserves further investigation.

In order to assess inflammatory features related to severe asthma as compared with mild asthma, we investigated the secretion of 92 kDa gelatinase matrix metalloproteinase (MMP-9) in bronchial lavages of six patients undergoing mechanical ventilation (MV) for status asthmaticus (SA) and in six patients with mild asthma. Ten healthy nonventilated patients and four patients under MV without preexisting respiratory disease were also investigated. Patients with SA were characterized by prominent neutrophilic inflammation (82 +/- 4% versus 10% in mild asthma). On the basis of enzymatic and immunological analysis, results showed an acute 10- to 160-fold increase of 92 kDa gelatinase (MMP-9) concentration in epithelial lining fluid (ELF) from patients with SA, together with activated forms (46 and 26 kDa) of stromelysin-1 matrix metalloproteinase (MMP-3) and detectable concentration of free metallogelatinolytic activity (1-5 micrograms gelatin hydrolyzed/48 h/ml ELF). Concomitant elevated level of tissue inhibitor of metalloproteinase-1 (TIMP-1) was shown only in patients with SA, thus counterbalancing, at least partially, excess of activated 92 kDa gelatinase. Acutely enhanced albumin levels were only observed in patients with SA; in addition, 92 kDa gelatinase and albumin levels were significantly and positively correlated (r = 0.96, p < 0.0001), suggesting that 92 kDa gelatinase may account for increased bronchial permeability in patients with SA. Several arguments support that 92 kDa gelatinase during SA originates both from numerous activated chemoattracted neutrophils and from activated bronchial epithelial cells in response to in situ lung injury. The fact that no relevant change in ELF, albumin, MMP-9, MMP-3, TIMP-1, or laminin degradation products was observed during mild asthma, strongly supports that the mechanism of airway inflammation in SA is quite distinct from that observed in mild asthma.