EUROPEAN  TISSUE  REPAIR  SOCIETY

5. Chaperones

Efforts to develop strategies to augment cell wound healing involve developing therapeutic molecular templates to speed refolding of denatured proteins as well as strategies to facilitate reassembly of porated cell membranes. There has been some success on both areas. Fershet et al. have reported that a C-terminal fragment of GroEL, which they called a mini-chaperon, retains some of the capability to assist protein folding and does not require ATP for this process. Later, Weissman et al. demonstrated that this mini-chaperon only speeds refolding of proteins that would have spontaneously refolded with additional time. Nonetheless, Tsong has postulated that it may be possible to develop mini-chaperon drugs for treatment of cell injury. In brief, DNA repair is more involved. Several ATP requiring enzymes are involved in repair of DNA. At what extent of protein denaturation is the possibility of renaturation and return to functionality lost is not known.

6. Supporting Co-Therapeutics

Antioxidants
Following cell membrane poration, the production of ROI increases by many orders of magnitude (Gabrielle et al.) leading to additional cellular injury. Antioxidants have been found to be useful following many types of cellular injury. Preliminary data indicate that ascorbate as well as the combination of P188 with ascorbate may provide therapeutic benefits after electrical shock (Abramov et al., 1997, Abra-mov et al., 1998). An important goal is to confirm this response in our in vivo electrical injury model with radio-tracer imaging and surface electromyography.

Figure 3: Schematic drawing to illustrate structural differences between poloxamers (left)
and poloxamines (right). The PEO and PPO chain lenths vary among the members of the
sufactant families.

Co-Enzymes
As described above, loss of cell membrane integrity quickly drains the cell's energy resources as the cell attempts to maintain the normal transmembrane ion gradients using ATP dependent membrane transport enzymes. The critical role of ATP in cell metabolism and tissue respiration is well recognized (Long 1943, Green and Stoner 1950). In an animal ischemic extremity model, nowadays hypothe-tized as free radical mediated cell membrane permeabil-ization injury, the administration of high-energy phosphates, in particular adenine, was effective in reducing ischemic damage (Ablove et al., 1996). Just prior to sealing electroporated cells a temporarily large extracellular concentration of these co-enzymes could perhaps provide transient energy charging of the cell. In addition, it has been shown that extracellular magnesium protects cells against energy-dependent cell damage (Kristensen and Hørder, 1989). Administration of exogenous ATP-MgCl2 has been shown to be beneficial for the survival of experimental animals subjected to hemorrhagic shock (Chaudry et al., 1974, Hirasawa et al., 1983) and varieties of ischemia. Moreover, ATP-MgCl2 has been found to improve mitochondrial function following shock and ischemia (Machiedo et al., 1981).

Recently, our laboratory observed that combination of P188, ATP and ascorbate has been shown to significant enhance the survival of post-mitotic muscle cells permea-bilized by exposure to intense (40 Gy) ionizing radiaton (in press). We are currently examining the effect of post-injury membrane sealing on DNA repair function. With molecular repair of cell membranes and resuscitation of cellular metabolism it appears that acute necrosis following severe membrane injury can be blocked. It remains to be seen whether additional measures are needed to block later cell entry into apoptotic pathways.

Raphael C. Lee