Role of matrix metalloproteinases and their tissue inhibitors in the regulation of coronary cell migration.

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 (P<0.001). 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, P<0.001), 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 (P<0.001). 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 (P<0.001). 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.

In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2.

The pulmonary tree is exposed to neutrophil-derived serine proteinases and matrix metalloproteinases in inflammatory lung diseases, but the degree to which these enzymes participate in tissue injury remains undefined, as does the therapeutic utility of antiproteinase-based interventions. To address these issues, an in vivo rat model was examined in which the intrapulmonary deposition of immune complexes initiates a neutrophil-mediated acute alveolitis. In vitro studies demonstrated that rat neutrophils can release neutrophil elastase and cathepsin G as well as a neutrophil progelatinase, which was subsequently activated by either chlorinated oxidants or serine proteinases. Based on structural homologies that exist between rat and human neutrophil proteinases, rat neutrophil elastase and cathepsin G activities could be specifically regulated in vitro by recombinant human secretory leukoproteinase inhibitor, and rat neutrophil gelatinase activity proved sensitive to inhibition by recombinant human tissue inhibitor of metalloproteinases 2. When either of the recombinant antiproteinases were instilled intratracheally, in vivo lung damage as assessed by increased permeability or hemorrhage was significantly reduced. Furthermore, the coadministration of the serine and matrix metalloproteinase inhibitors almost completely prevented pulmonary damage while effecting only a modest decrease in neutrophil influx. These data support a critical role for neutrophil-derived proteinases in acute lung damage in vivo and identify recombinant human secretory leukoproteinase and recombinant human tissue inhibitor of metalloproteinases 2 as potentially efficacious interventions in inflammatory disease states.

Proteolytic inactivation of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin by oxidatively activated human neutrophil metalloproteinases.

Human neutrophils use the H2O2-myeloperoxidase-chloride system to generate chlorinated oxidants capable of activating metalloproteinase zymogens that hydrolyze not only native and denatured collagens, but also the serine proteinase inhibitor (serpin) alpha 1-proteinase inhibitor (alpha 1 PI). To identify the metalloenzyme that hydrolyzes and inactivates alpha 1 PI, neutrophil releasates were chromatographed over gelatin-Sepharose and divided into fractions containing either progelatinase or procollagenase. The gelatinase-containing fraction cleaved alpha 1 PI in a manner inhibitable by native type V, but not type I, collagen. Conversely, while the collagenase-containing fraction also cleaved alpha 1 PI, this activity was inhibited by type I, but not type V, collagen. Because type I and V collagens are competitive substrates for collagenase and gelatinase, respectively, each of the metalloproteinase zymogens were purified to apparent homogeneity and examined for alpha 1 PI-hydrolytic activities. Both purified gelatinase and collagenase inactivated alpha 1PI by hydrolyzing the serpin within its active-site loop at the Phe352-Leu353 and Pro357-Met358 bonds, albeit with distinct kinetic properties. Furthermore, purified collagenase, but not gelatinase, cleaved a second serpin, alpha 1-antichymotrypsin, by hydrolyzing the Ala362-Leu363 bond within its active-site loop. These data demonstrate that human neutrophils use chlorinated oxidants to activate collagenolytic metalloproteinases whose substrate specificities can be extended to members of the serpin superfamily.

Interstitial collagenase (matrix metalloproteinase-1) expresses serpinase activity.

Human endothelial cells treated with either interleukin-1 beta, tumor necrosis factor-alpha, or phorbol myristate acetate secreted a metalloproteinase that hydrolyzed and inactivated the two major serine proteinase inhibitors (Serpins) found in plasma, alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin. Surprisingly, the responsible metalloproteinase was identified as human interstitial collagenase (matrix metalloproteinase-1), an enzyme whose only known physiologic substrate has heretofore been believed to be the extracellular matrix molecule, collagen. The metalloproteinase inactivated the Serpins by cleaving peptide bonds at sites unrelated to those hydrolyzed in collagenous macromolecules. NH2-terminal sequence analysis localized the cleavage sites in the Serpins to regions near their respective reactive site centers at three distinct peptide bonds on the amino-terminal side of bulky, hydrophobic residues. Together, these data indicate that matrix metalloproteinase-1 displays an expanded substrate repertoire that supports the existence of a new interface between connective tissue turnover and Serpin function.

Proteolytic inactivation of alpha-1-proteinase inhibitor by a neutrophil metalloproteinase.

Human neutrophils triggered with phorbol myristate acetate or opsonized zymosan particles released a metalloproteinase (MP) capable of cleaving and inactivating alpha-1-proteinase inhibitor (alpha-1-PI). Sequence analysis of the amino acids in proteolyzed, native alpha-1-PI revealed a unique single cleavage site between Phe-352 and Leu-353. An analysis of the process regulating the enzyme's activity revealed that the neutrophil MP was released from cells in a latent form whose activation was tightly linked to the generation of hypochlorous acid. These results indicate that human neutrophils use chlorinated oxidants to activate a latent MP that is capable of proteolytically inactivating alpha-1-PI by cleaving the antiproteinase at a unique point in its inhibitory site region.

Oxygen diffusion in the trout retina.

Oxygen tensions were measured in vivo within the different layers of the rainbow trout retina. Oxygen microelectrodes were advanced in 10 micron increments through the retinas and the PO2 measured at each location. Mean retinal PO2 ranged from 124 mmHg at the retinal-vitreal interface to 381 mmHg at the choriocapillaris. These data reaffirm that the cellular layers of the normal trout retina are continually exposed to supra-arterial oxygen tensions that are known to cause toxicity in other species. The intraretinal PO2 gradient was mathematically characterized and results indicate that the in vivo oxygen profile can be described by a two-component exponential function. In order to gain a better understanding of oxygen delivery to the retina, the data were also subjected to an analysis based upon the classical equation for planar diffusion. The trout retina is an excellent model for studying oxygen diffusion in vivo since this tissue is supplied with oxygen from a single source, thereby simplifying the mathematical analysis. Calculations yielded values of 1.86 X 10(-5) and 0.58 X 10(-5) ml O2 min-1 cm-1 atm-1 (at 9 degrees C) for the Krogh permeation coefficient (DS) for the photoreceptor region and the remaining neural retina, respectively.

Superoxide dismutase provides protection against the hyperoxia in the retina of the rainbow trout (Salmo gairdneri).

A modified hydroxylamine oxidation assay method coupled with a parallel line analysis to detect endogenous interfering substances, was used to determine superoxide dismutase (SOD). Retinal SOD activities in trout, frog and rat were compared. SOD activity in liver and brain tissues was also determined. Based on estimates of superoxide anion production there appears to be excess SOD protection in the rainbow trout retina, which could, at least in part, account for its resistance to oxygen toxicity.

Quantification of hyperbaric oxygen-induced toxicity utilizing a malarial system.

This study was undertaken in recognition of the need to develop quantitative systems to evaluate the toxicity associated with hyperbaric oxygen (HBO) exposure. Malaria-infected (P. berghei berghei) mice were briefly exposed to 100% oxygen at 3 ATA on day 10 of infection. At 25, 48, and 72 h thereafter, the levels of circulating erythrcytes and percent parasitized RBC were monitored and compared to those of infected non-exposed controls. The total erythrocyte counts of the infected HBO-exposed and non-exposed mice did not differ significantly. In contrast, percent parasitized cells in the oxygen-exposed mice were lowered to 55-60% control values at 24, 48, and 72 h. The mechanism of this difference needs further study, but we believe that P. berghei-infected erythrocytes are preferentially hemolyzed as a consequence of HBO exposure. this mode system is useful in the study of HBO-induced toxicity because of its high degree of selectivity and sensitivity and its amenability to strict quantification over a period of at least several days.

Stereotactic lesions in primary epilepsy of the limbic system.

Chronic depth electrodes have proven useful in diagnosing primary epilepsy of the limbic system. Five patients had small lesions in the amygdala and hippocampus. There was a 50% reduction of the seizures frequency. No complication were observed and patients were ambulant the day following surgery. It is felt that stereotactic lesions larger than produced by the leucotome used should be performed provided we have proofs that the epileptogenic focus is in a restricted area. Stereotactic lesions may also have a role in interrupting pathways clearly proven as participating in the epileptic discharge. Only under these conditions, will it be possible to say that stereotactic lesions are effective in specific type of epilepsy.