pepstatin and Wounds-and-Injuries

In response to severe tissue trauma several danger sensing and signalling cascades are activated, including the complement and the apoptosis systems. In polytrauma patients, both the early activation of the complement cascade with an excessive generation of the potent anaphylatoxin C5a and the induction of apoptosis have been shown to modulate the post-traumatic immune response. However, little is known about a direct interaction between the complement and apoptosis systems after severe tissue trauma. Therefore the focus of the present study was to elucidate the interplay between the central complement component C5 and the pro-apoptotic aspartic protease cathepsin D. In vivo, the cathepsin D plasma concentration of multiple injured patients was markedly increased when compared to healthy volunteers. In vitro incubation of C5 with cathepsin D resulted in a concentration- and time-dependent generation of C5a, which was inhibited by the aspartate protease inhibitor pepstatin A. Immunoblotting and sequencing analysis indicated that the C5 cleavage product represents the native form of human C5a, also exhibiting chemotactic activity for human neutrophils. In conclusion, these data show for the first time that cathepsin D is increased in plasma early after severe tissue injury. Furthermore, the results provide in vitro evidence of cleavage of C5 by an aspartic protease with subsequent generation of functional C5a, which represents a new path of complement activation.

Topics: Biocatalysis; Blotting, Western; Cathepsin D; Chemotaxis, Leukocyte; Complement Activation; Complement C5; Complement C5a; Enzyme-Linked Immunosorbent Assay; Humans; Neutrophils; Pepstatins; Protease Inhibitors; Time Factors; Wounds and Injuries

Previous studies established that the populations of neurons that frequently degenerate in Alzheimer's disease exhibit robust up-regulation of the lysosomal system. In this study, we investigated alterations of the lysosomal system during different forms of experimental injury in rat hippocampal neurons in culture, utilizing a combination of immunocytochemical and biochemical methods. Using triple-label immnocytochemistry for activated caspase-3, fragmentation of DNA and the microtubule-associated protein-2, we characterized treatment paradigms as models of the apoptotic (staurosporine, camptothecin), the oncotic (high-dose menadione, glutamate), and the mixed apoptotic and oncotic (low-dose menadione) pathways of neuronal death. Slowly developing apoptotic or slowly developing mixed apoptotic and oncotic forms of neuronal injury were associated with substantial increases in the number and size of cathepsin D-positive vesicles (late endosomes and mature lysosomes) as determined by immunocytochemistry, and elevated levels of cathepsin D by western blotting. In agreement with our previous findings in Alzheimer's disease, where lysosomal system activation was not restricted to overtly degenerating neurons, up-regulation of this system was also detected quite early during the course of experimental neuronal injury, preceding the development of dystrophic neurites, nuclear segmentation or fragmentation of DNA. These findings implicate lysosomal system activation, both in Alzheimer's disease and in experimental models of neuronal injury, as an important event associated with early stages of neurodegeneration.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Astrocytes; Blotting, Western; Cathepsin D; Female; Hippocampus; Humans; Lysosomes; Male; Middle Aged; Neurons; Pepstatins; Up-Regulation; Wounds and Injuries