epidermal-growth-factor and nafamostat

The effects of epidermal growth factor (EGF) on Cu, Zn-superoxide dismutase (SOD) in cultured fibroblasts from rat skin exposed to superoxide anions were studied. Cross-linking of [125I]hEGF using disuccinimidyl suberate and immunoblot analysis using anti-EGF receptor antibody to crude plasma membrane fractions of fibroblasts showed that a 170 kDa EGF receptor protein was present on the membrane, as in A431 cells which over express a specific EGF receptor. The cytosolic SOD enzyme activity in fibroblasts exposed to superoxide anions 24 h after treatment with EGF plus nafamostat (NM), a potent protease inhibitor, was increased 1.6-fold compared to control-treated cells. Treatment with either EGF or NM alone, evoked little increase in SOD enzyme activity. The increase in Cu, Zn-SOD protein levels corresponded to the increase in cytosolic SOD enzyme activity in fibroblasts. The Cu, Zn-SOD mRNA level in fibroblasts treated with EGF plus NM at 3 and 6 h was higher than that of the control. Additionally, levels of [125I]hEGF degradation products released into the medium from fibroblasts exposed to superoxide anions were significantly reduced in the presence of NM. These results suggest that the stabilization of EGF by NM in culture is an important factor in the expression of its effects, and that EGF induces Cu, Zn-SOD expression by accelerating transcription of the Cu, Zn-SOD gene in cells, resulting in their protection from the effects of superoxide anion radicals.

Topics: Animals; Benzamidines; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Guanidines; Humans; Rats; RNA, Messenger; Skin; Superoxide Dismutase

When epidermal growth factor (EGF) ointment containing a protease inhibitor, nafamostat (NM), was applied to burn sites in rats, the superoxide dismutase (SOD) enzyme activity and protein content increased 45% and 60%, respectively, at these sites 1 d after the burns compared with the control ointment. Following treatment with EGF plus NM (EGF + NM) ointment, messenger RNA for SOD also increased, to about 1.6 times that of the control at 1 d after the burn, indicating that this ointment stimulates SOD synthesis at burn sites in vivo. In contrast, following treatment with EGF + NM ointment, the content of heat shock protein (HSP 70) in the burned tissue decreased to about 70% of the control value 1 d after the burn. These findings suggest that EGF + NM ointment alleviated tissue damage at burn sites at an early stage, and that this was related to the stimulation of SOD synthesis and reduced HSP 70 levels. We also examined the effects of SOD ointment on wound healing at burn sites. A dose-dependent increase in the dry weight of granulation tissue at wound sites 3 d after the burn was observed following the application of this ointment. These results suggest that SOD may play an important role in wound healing after burns.

Topics: Animals; Benzamidines; Blotting, Northern; Burns; Drug Combinations; Epidermal Growth Factor; Guanidines; Heat-Shock Proteins; Male; Molecular Weight; Ointments; Protease Inhibitors; Proteins; Rats; Rats, Wistar; RNA, Messenger; Skin; Superoxide Dismutase

The systemic effects of epidermal growth factor (EGF) ointment containing nafamostat (NM), gabexate, or gelatin was studied in rats with burns or open wounds. At 1 d after burn, plasma epinephrine, cortisol, and glutamic-oxalacetic transaminase (GOT) levels were elevated, but treatment with EGF plus NM (EGF+NM) ointment significantly suppressed the increase in these levels. Further, there was no loss of body weight in the open wound model following treatment with EGF+NM ointment, while loss of body weight occurred in animals in which EGF ointments without NM were applied. Increases in plasma epinephrine 1 d after open wound formation were also suppressed by the application of EGF+NM ointment. Treatment with EGF ointment containing gabexate (GX) or gelatin (GL) ameliorated changes in body weight that occurred after open wound formation, while loss of body weight in animals with open wounds occurred following the application of ointment base, EGF ointment, GX ointment, or GL ointment. The present study thus indicates that the topical application of EGF ointment containing a protease inhibitor has ameliorative systemic effects.

Topics: Animals; Aspartate Aminotransferases; Benzamidines; Body Weight; Burns; Drug Interactions; Drug Synergism; Epidermal Growth Factor; Epinephrine; Gabexate; Gelatin; Guanidines; Hydrocortisone; Male; Ointments; Protease Inhibitors; Rats; Rats, Wistar; Wounds and Injuries

The healing effect of human epidermal growth factor (hEGF) on second degree burn was studied in rats. No improvement in wound healing was found on topical application of EGF alone to burn sites, but an ointment containing EGF and nafamostat mesilate (NM), a protease inhibitor, accelerated the healing rate of burns. The dry weight of the granulation tissue on the wound site in the group treated with EGF plus NM ointment did not change, although that in other groups decreased. After treatment with EGF ointment containing NM, the content of uronic acid, as an index of acid mucopolysaccharide, at 3 d after burn rapidly increased and had recovered to nearly normal levels at 7 d after burn. However, the uronic acid content in the other groups (control, EGF alone, and NM alone) showed a higher value at 7 d than at 3 d. When compared with the control values significant increases in hydroxyproline, as an index of collagen, in the wound site were observed at 7 d after treatment with EGF ointment containing NM. The degradation of [125I]EGF in burned tissue homogenate decreased significantly in a concentration-dependent manner in the presence of NM. Body weights did not change after treatment with EGF plus NM ointment, although the body weights of other treatment groups decreased after burn, suggesting that EGF ointment containing the protease inhibitor, NM, alleviated the effects of burn shock. These findings indicate that the stabilization of EGF at the wound site is an important factor for the expression of its healing effects.

Topics: Animals; Benzamidines; Burns; Drug Interactions; Drug Stability; Drug Therapy, Combination; Epidermal Growth Factor; Guanidines; Hydroxyproline; Male; Ointments; Rats; Rats, Inbred Strains; Serine Proteinase Inhibitors; Uronic Acids; Wound Healing

The healing effect of human epidermal growth factor (hEGF) on open wounds was studied in rats. No improvement in wound healing was found by topical application of EGF alone to open wound sites. We found an ointment containing EGF and a protease inhibitor, nafamostat mesilate or gabexate mesilate, or gelatin accelerated the healing rate of open wounds. Significant increases in the dry weight of the wound site granulation tissue, uronic acid (as an index of acid mucopolysaccharide) and hydroxyproline (as an index of collagen) were observed by treatment with EGF ointment containing nafamostat compared with the controls. The effects of the protease inhibitor on wound healing were dose dependent. Nafamostat was more efficient than gabexate or gelatin on wound healing. The degradation of 125I-EGF in wound tissue homogenate was significantly decreased in the presence of a protease inhibitor, such as nafamostat or gabexate, or gelatin. These findings indicate that the stabilization of EGF at the wound site is an important factor in permitting the expression of its healing effects and suggest that the ointment containing EGF and a stabilizing agent would be a suitable dosage form for acceleration of wound repair.

Topics: Animals; Benzamidines; Biotransformation; Dose-Response Relationship, Drug; Drug Combinations; Drug Stability; Epidermal Growth Factor; Gabexate; Gelatin; Guanidines; Male; Ointments; Protease Inhibitors; Rats; Rats, Inbred Strains; Wound Healing; Wounds and Injuries