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Insulin-like growth factor-I (IGF-I) and keratinocyte growth factor (KGF) cDNA gene transfer individually improves dermal and epidermal regeneration. The aim of the present study was to determine whether the combination of IGF-I plus KGF cDNA further improves wound healing and by which mechanisms these changes occur. Rats received an acute wound and were divided into four groups to receive weekly subcutaneous injections of liposomes plus Lac Z cDNA, liposomes plus IGF-I cDNA, liposomes plus KGF cDNA, or liposomes plus IGF-I/KGF cDNA. Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine IGF-I, KGF, platelet-derived growth factor, fibroblast growth factor (FGF), transforming growth factor-beta and vascular endothelial growth factor (VEGF) expression and different types of collagen (I, III and IV). IGF-I, KGF and their combination cDNA treatment significantly (P<0.05) accelerated re-epithelization, increased IGF-I, KGF, FGF, VEGF and collagen type IV expression, while it had no effect on collagen type I and III expression. The combination of IGF-I plus KGF cDNA increased (P<0.05) neovascularization and VEGF expression when compared to IGF-I cDNA, KGF cDNA groups and controls. In conclusion, exogenous administration of liposomal IGF-I plus KGF cDNA enhanced dermal and epidermal regeneration which is due to increased neovascularization.

After a severe trauma, such as a cutaneous thermal injury, an increase in hepatocyte apoptosis has been associated with hepatocyte damage and impairment in hepatic function. Insulinlike growth factor-I (IGF-I) exerts antiapoptotic effects in several organs, thus improving organ homeostasis. The purpose of the present study was to determine whether IGF-I in combination with its principle binding protein-3 (BP-3) attenuates liver damage after a burn and whether this attenuation is through signals of the apoptotic-proliferative axis of hepatocytes. Sprague-Dawley rats (56 males) received a 60% total body surface area third-degree scald burn and were randomly divided to receive either rhlGF-I/BP3 (10 mg/kg/day s.c.) or saline (control). Serum aspartate transaminase (AST) and nitric oxide (NO), and hepatocyte proliferation and apoptosis, were measured on postburn days 1, 2, 5, and 7. Hepatic interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) mRNA and hepatic nuclear-factor kappa B (NF-kappa B) were determined at 1 and 2 days postburn. IGF-I/BP-3 decreased serum AST and increased serum NO at 1, 2, and 5 days after burn when compared with controls (P < 0.05). IGF-I/BP-3 increased hepatocyte proliferation on the first day after burn and decreased hepatocyte apoptosis at day 7 postburn when compared with controls (P < 0.05). IGF-I/BP-3 decreased hepatic IL-1 beta and TNF-alpha mRNA 1 day after burn (P < 0.05). IGF-I/BP-3 further increased hepatic NF-kappa B concentration 1 and 2 days postburn when compared with controls (P < 0.05). Recombinant hIGF-I in combination with its principle binding protein conserves hepatic homeostasis, which is associated with a transient increase in hepatocyte proliferation and decrease in hepatocyte apoptosis possibly through NO and hepatic NF-kappa B.

Objective: To modulate the hepatic acute phase response after a thermal injury by the administration of insulin-like growth factor I (IGF-I) in combination with its principal binding protein 3 (IGFBP-3)., Summary Background Data: The hepatic acute phase response is a cascade of events initiated to restore homeostasis after trauma; however, a prolonged response contributes to multiorgan failure, hypermetabolism, complications, and death. Although IGF-1 has been shown to improve cell recovery and play a major role in liver regeneration, its effect on the hepatic acute phase response is not known., Methods: Sprague-Dawley rats (56 males) received a 60% total body surface area third-degree scald burn and were randomly divided to receive either rhIGF-I/BP-3 (10 mg/kg/day given subcutaneously) or saline (control). Rats were killed on postburn days 1, 2, 5, and 7 and serum glucose, electrolytes, acute phase reactant proteins, tumor necrosis factor alpha, interleukin 1 beta, interleukin 6, and rat and human serum IGF-I and IGFBP-3 were measured. Hepatic protein concentrations, hepatocyte proliferation, and hepatocyte apoptosis were determined., Results: No hypoglycemia or electrolyte imbalance could be shown in rats receiving the growth factor complex compared with saline. rhIGF-I/BP-3 increased serum protein on postburn days 2 and 7, albumin on days 5 and 7, and transferrin on days 1, 5, and 7, and decreased haptoglobin and alpha1-acid glycoprotein on postburn days 5 and 7 compared with controls. IGF-I/ BP-3 had no effect on type II acute phase proteins. Rats receiving IGF-I/BP-3 had lower serum levels of interleukin 1 beta and tumor necrosis factor alpha on the first day after burn compared with controls, whereas serum levels of interleukin 6 did not change. rhIGF-I/BP-3 significantly increased total liver protein content on postburn days 1, 2, 5, and 7 compared with controls. IGF-I/BP-3 increased hepatocyte proliferation and decreased hepatocyte apoptosis versus controls., Conclusion: In combination with its principal binding protein, rhIGF-I decreases the proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha, followed by a decrease in type I acute phase proteins. IGF-I/BP-3 had no effect on interleukin 6 and type II acute phase proteins. Decreases in acute phase protein and proinflammatory cytokine synthesis were associated with increases in constitutive hepatic proteins, total liver protein content, and hepatocyte proliferation. IGF-I/BP-3 attenuates the hypermetabolic response after thermal injury and may improve the clinical outcome.

Objective: To determine the effect of insulinlike growth factor I (IGF-I) in combination with its principal binding protein (IGFBP-3) on the hepatic acute phase response in severely burned children., Summary Background Data: The hepatic acute phase response is a cascade of events initiated to restore homeostasis after trauma. A prolonged response, however, may contribute to multiple organ failure, hypermetabolism, complications, and death., Methods: Twenty-two children with a mean total body surface area (TBSA) burn of 57 +/- 3% were given a continuous infusion of 1 to 4 mg/kg/day IGF-I/BP-3 for 5 days after wound excision and grafting. Eight children with a TBSA burn of 54 +/- 4% were given saline as controls. Before and 5 days after excision and grafting, blood samples were taken for serum hepatic constitutive protein, acute phase protein, and proinflammatory cytokine analysis., Results: Serum IGF-I levels in burned children given the IGF-I/BP-3 complex increased from 113 +/- 15 to 458 +/- 40 ng/mL and IGFBP-3 levels increased from 1.8 +/- 0.2 to 3.1 +/- 0.3 ng/mL. Levels of serum constitutive hepatic proteins (prealbumin, retinol-binding protein, and transferrin) increased with IGF-I/BP-3, whereas levels of type I acute phase proteins (C-reactive protein, alpha1-acid glycoprotein, and complement C-3) decreased when compared with controls. The complex had no effect on type II acute phase proteins. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) levels decreased with IGF-I/BP-3 compared with controls, with no effect on interleukin-6., Conclusion: Severely burned children receiving IGF-I/BP-3 showed a decrease in IL-1beta and TNF-alpha followed by a decrease in type I acute phase proteins that was associated with a concomitant increase in constitutive hepatic proteins. Attenuating the proinflammatory acute phase with IGF-1/BP-3 response may prevent multiple organ failure and improve clinical outcomes after thermal injury without any detectable adverse side effects.

Gene therapy using cationic liposomes containing cDNA is a relatively new approach with great potential; however, little is known about the mechanisms of dermal gene transfer, its biodistribution, systemic transfection, and cellular uptake. This study identifies mechanisms, transfection rates, and biodistribution of liposomal gene transfers in the skin of thermally injured rats using cDNA gene constructs coding for insulin-like growth factor-I (IGF-I) and Lac Z. Male Sprague-Dawley rats (350 to 375 g) were given a 60% total body surface area full-thickness scald burn that was followed by weekly subcutaneous injections of normal saline (control, n = 10), liposomes plus 0.2 microg Lac Z cDNA construct driven by a cytomegalovirus (CMV) promoter (vehicle, n = 10), or liposomes containing 2.2 microg cDNA coding for IGF-I plus 0.2 microg Lac Z cDNA construct driven by a CMV promoter (IGF-I cDNA, n = 10). Gene transfection was determined by histochemical and luminescent beta-galactosidase assays of blood, skin, liver, spleen, and kidney. Transcription of IGF-I cDNA to IGF-I mRNA was determined in skin cells by Northern blot analyses. Levels of IGF-I protein in blood, skin, liver, spleen, and kidney were measured by radioimmunoassay. The biological activity of the translated IGF-I was evaluated by the mitogenic activity in dermal cells and the rate of re-epithelization. Gene transfection was observed only in skin cells. The expression of IGF-I mRNA increased in skin cells of burned rats receiving liposomes containing the IGF-I cDNA construct compared with liposomes without the construct or normal saline. IGF-I protein levels in the skin of rats receiving the IGF-I cDNA was 176 +/- 4 ng/ml compared with 105 +/- 6 ng/ml for liposomes alone or 90 +/-3 ng/ml for saline (p < 0.05). The translated IGF-I protein was found biologically active in the skin by increasing skin cell proliferation and accelerating re-epithelization 33 days after thermal injury (p < 0.05). No systemic transfection could be detected. Skin cells transfected with liposomes encapsulating the IGF-I cDNA constructs increased the expression of IGF-I mRNA transcript and the expression of a biologically active IGF-I protein. Liposomes containing the cDNA coding for IGF-I present an effective approach to gene therapy in the skin.

Hypothesis: Multiple subcutaneous injections of cholesterol-containing cationic liposomes encapsulating the complementary DNA (cDNA) gene for insulinlike growth factor I (IGF-I) increase the rate of transfected skin cells and result in increased IGF-I protein levels in the skin with subsequent improvement in wound healing when compared with a single injection., Setting: Laboratory., Intervention: Twenty-four adult male Sprague-Dawley rats (350-375 g) received a full-thickness scald burn on 60% of their body surface. These rats were randomly divided to receive either 1 injection of liposomes containing 2.2 microg-cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct, or 2 injections of liposomes containing 2.2 microg cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct., Main Outcome Measures: Transfection rates and IGF-I protein levels in the skin and physiological responses to the IGF-I gene therapy, evaluated from changes in body weight, protein content in serum and liver, and the rate of burn wound healing., Results: There was a significant decrease in transfection rate and IGF-I protein expression distal from the injection site in animals receiving 1 injection, as compared with a consistent increase in rats receiving multiple injections. Multiple injections improved the response to thermal trauma by increasing the extent of the healed burn wound 33 days after thermal injury (single injection, 31% +/- 1% vs multiple injections, 38% +/- 2%), total serum protein (single injection, 52 +/- 0.5 g/L vs multiple injections, 55 +/- 0.6 g/L), and total liver protein (single injection, 82.0 +/- 0.3 mg/mL vs multiple injections, 91.0 +/- 3.8 mg/mL), P<.05., Conclusions: Gene transfer rates can be increased by multiple injections of liposomes encapsulating IGF-I cDNA constructs. Increased transfer results in greater IGF-I protein skin concentrations, accelerated wound healing, and increased serum and liver protein concentrations. The clinical relevance of these findings is that liposomal gene constructs should be applied in well-defined distances to improve gene transfer in the skin, and thus clinical outcome after thermal injury.

Background: Recombinant human growth hormone (rhGH) has been shown to modulate the hypermetabolic response and the hepatic acute-phase response after thermal injury. In vitro studies, however, demonstrated that rhGH activates insulin-like growth factor-I (IGF-I) gene transcription and production, suggesting that rhGH may exert some of its effects indirectly through IGF-I stimulation. The purpose of this study was to determine the effects of rhGH on serum and hepatic IGF-I in thermally injured rats., Methods: Sprague-Dawley rats (56 males) receiving a 60% TBSA third-degree scald burn were randomly divided to receive either rhGH (2.5 mg/kg/day im) or saline (control). Rats were sacrificed on postburn days 1, 2, 5, and 7 and serum IGF-I, hepatic IGF-I mRNA, and IGF-I protein concentration were measured. The physiologic response to changes in IGF-I levels was evaluated by measuring hepatocyte proliferation, total liver protein concentration, and muscle dry/wet weights., Results: Serum IGF-I was increased from postburn day 1 through day 7 in rats receiving rhGH compared to controls (P < 0.05). Hepatic IGF-I mRNA and IGF-I protein expression were increased from day 1 to 7 after burn in animals receiving rhGH when compared to controls (P < 0.05). Recombinant hGH increased hepatocyte proliferation at 5 days and total liver protein concentration at 5 and 7 days postburn compared to controls (P < 0.05). Muscle dry/wet weights increased in rats receiving rhGH at 7 days after burn compared to controls (P < 0.05)., Summary: Liver and serum IGF-I levels decreased after a thermal injury. Recombinant hGH attenuated this decrease by stimulating hepatic IGF-I expression. Increases in IGF-I were associated with increases in hepatocyte proliferation and protein concentration in liver and muscle., Conclusion: We suggest that rhGH modulates the hypermetabolic response through IGF-I stimulation in the hepatic parenchyma., (Copyright 1999 Academic Press.)

Exogenous insulin-like growth factor-I (IGF-I) is known to improve the pathophysiology of a thermal injury, however, deleterious side-effects have limited its utility. Cholesterol-containing cationic liposomes that encapsulate complementary DNA (cDNA) are nonviral carriers used for in vivo gene transfection. We propose that liposome IGF-I gene transfer will accelerate wound healing in burned rats and attenuate deleterious side-effects associated with high levels of IGF-I. To test this hypothesis IGF-I gene constructs, encapsulated in liposomes, were studied for their efficacy in modulating the thermal injury response. Thirty adult male Sprague-Dawley rats were given a 60% TBSA scald burn and randomly divided into three groups to receive weekly subcutaneous injections of liposomes plus the lacZ gene coding for beta-galactosidase, liposomes plus cDNA for IGF-I and beta-galactosidase or liposomes plus the rhIGF-I protein. Body weights and wound healing were measured. Muscle and liver dry/wet weights and IGF-I concentrations in serum, skin and liver were measured by radioimmunoassay. Transfection was confirmed by histochemical staining for beta-galactosidase. Rats receiving the IGF-I cDNA constructs exhibited the most rapid wound re-epithelialization and greatest increase in body weight and gastrocnemius muscle protein content (P < 0.05). Local IGF-I protein concentrations in the skin were higher when compared to liposomes containing only the lacZ gene (P < 0.05) Transfection was apparent in the cytoplasm of myofibroblasts, endothelial cells and macrophages of the granulation tissue. Liposomes containing the IGF-I gene constructs proved effective in preventing muscle protein wasting and preserving total body weight after a severe thermal injury.