You J. Shen

Enhanced expression of mRNA for insulin-like growth factor-1 in post-burn hypertrophic scar tissue and its fibrogenic role by dermal fibroblasts.

Hypertrophic scarring (HSc) which frequently develops in patients following severe thermal injury is characterized by accumulation of extracellular matrix (ECM) proteins including type I and type III collagen. In this study, we examined the presence and quantity of IGF-1 mRNA transcripts in post-burn HSc. The results of dot blot experiments showed a 77.5% (100±8.15 vs 177.5±19, p<0.01) increase in expression of IGF-1 IIIRNA in HSc tissue relative to normal dermis obtained from the same patients. A Northern blot analysis confirmed the specificity of the IGF-1 cDNA. This cDNA visualized four different transcripts with apparent sizes of 7.0, 3.9, 1.8 and 1.0 kb, similar to those previously reported. The possible fibrogenic role of IGF-1 was examined by analyzing the effect of this growth factor on the expression of mRNA for the pro α1(I) chain of type I procollagen and the pro α1(III) chain of type III procollagen in dermal fibroblasts. IGF-1 increased the expression of these transcripts as early as 6 h and the effect was maximal at 24 h. Quantitative analysis by densitometry showed a 149 and 166% increase in pro α1(I) and pro α1(III) mRNA after 24 h of IGF-1 treatment, respectively. This effect seems to be specific as the abundance of mRNA for the pro α2(I) chain of type I procollagen or TIMP-II was unchanged. When another 4 strains of dermal fibroblasts were treated with IGF-1, a significant increase (16.94±1.13 vs 10.87±1.79, p<0.01, N=4) in the expression of type I procollagen mRNA was found. This was consistent with a significant increase in collagen production, as measured by hydroxyproline in conditioned medium (2.04±0.3 ng/1000 cells vs 1.35±0.4 ng/1000 cells, p<0.01, N=4). The effects of IGF-1 were temporary, since removal of IGF-1 from media caused a reduction in expression of type I procollagen mRNA to its basal level within 48 h.Enhanced expression of IGF-1 mRNA in post-burn HSc tissues and the potentially fibrogenic effects of this growth factor on dermal fibroblasts, suggest that it could contribute to the accumulation of type I and type III collagen found in many fibroproliferative disorders such as HSc.

Induction of transforming growth factor β1 by insulin‐like growth factor‐1 in dermal fibroblasts

Transforming growth factor β1 (TGF‐β1) belongs to a family of multifunctional modulatory proteins involved in cell growth, differentiation, development, and wound healing. Although the biological activities of TGF‐β1 have been extensively studied, its regulation remains obscure. Here we report the effects of insulin‐like growth factor‐1 (IGF‐1) on the expression of TGF‐β1 by dermal fibroblasts and suggest a possible mechanism. An enzyme‐linked immunosorbent assay (ELISA) specific for TGF‐β revealed a greater than twofold increase (12.3 ± 1.6 vs. 4.8± 0.8 pg/104 cells, n = 7, P < 0.05) in the protein in conditioned medium obtained from IGF‐1‐treated cells compared to that from untreated controls. Similar results were obtained by the mink lung epithelial cell growth inhibition assay. The results of Northern analysis revealed a dose‐dependent increase in TGF‐β1 mRNA in response to IGF‐1 treatment. Using the optimum concentration of IGF‐1 (100 ng/ml), a greater than twofold increase (25.43 ± 5.7 vs. 12.13 ± 4.5, P < 0.05) in TGF‐β1 mRNA was observed. This effect persisted for at least 48 h after IGF‐1 was removed from the culture medium. Nuclear run‐on assay showed that this stimulation was due, at least in part, to an increase in the rate of transcription of the TGF‐β1 gene. Treatment of human dermal fibroblasts with IGF‐1 caused a substantial increase in c‐fos and c‐jun mRNA expression within 30 and 60 min, respectively. In contrast to c‐jun mRNA which was constitutively expressed by dermal fibroblasts, the expression of c‐fos mRNA was transient and only detectable between 15 and 60 min. Greater than 58% of the increase in TGF‐β1 caused by IGF‐1 could be blocked by the addition of anti‐TGF‐β1 neutralizing antibody to the culture medium, suggesting that autoinduction of TGF‐β1 may be involved. An increase in IGF‐1‐induced TGF‐β1 should be important in many different physiological processes such as cellular proliferation, differentiation, and wound healing. These findings also suggest that induction of TGF‐β1 mRNA and protein by IGF‐1 may be a mechanism by which this cytokine is regulated in physiological and/or pathological conditions. J. Cell. Physiol. 174:301–309, 1998.

Regulation of collagen synthesis and messenger RNA levels in normal and hypertrophic scar fibroblasts in vitro by interferon alfa-2b.

Hypertrophic scars, which commonly occur after thermal and traumatic injury of the skin, are a fibroproliferative disorder of the dermal matrix wherein components of the inflammatory process, including the fibrotic growth factor, transforming growth factor‐β, appear to activate dormant fibroblasts leading to cellular proliferation and excessive matrix synthesis. To investigate the potential beneficial role and mechanism of interferon alfa‐2b in controlling excessive collagen production in hypertrophic scar, we measured dose response, time of onset, and duration of action in hypertrophic scar fibroblasts in vitro and compared them with those of site‐matched normal fibroblasts obtained from four patients after thermal injury. Interferon alfa‐2b reduced collagen protein synthesis and type I messenger RNA levels in both hypertrophic scar and normal fibroblasts after treatment, but these changes were apparent only after approximately 72 hours. Significant reductions in collagen synthesis occurred in four pairs of normal and hypertrophic scar fibroblasts (p < 0.05), accompanied by significant reductions in type I (p < 0.05) but not type III procollagen messenger RNA. Hypertrophic scar fibroblasts recovered completely from the effects of interferon alfa‐2b on procollagen type I messenger RNA within 48 hours of cessation of treatment in contrast to normal skin fibroblasts, in which the reduction in type I procollagen messenger RNA by interferon alfa‐2b persisted beyond 72 hours after treatment. These data suggest that interferon alfa‐2b reduces collagen synthesis in both normal and hypertrophic fibroblasts but the hypertrophic fibroblast may remain less sensitive to its effects.

Differential effects of thermal injury on circulating insulin-like growth factor binding proteins in burn patients

The results of this report provide evidence that insulin-like growth factor-1 binding proteins (IGFBPs) in human sera are differentially regulated as a result of severe burn injury. Using the ligand binding technique,125I-IGF-1 visualizes 5 different protein bands corresponding to those previously reported for IGFBP-1 to 4 with apparent sizes of 23–42 kd in serum samples prepared from severely burned patients and healthy individuals. The level of IGFBP-3 was significantly decreased within 3–5 days of injury and remained depressed for up to 20 days post injury. The average level of this binding protein reached its lowest value within 3–5 days of the injury (3.8±1.48% relative to day 0–1 value, n=4, p<0.001). Serum samples from 3 to 4 patients showed no recovery within 20 days post injury and the level of IGFBP-3 remained significantly depressed (p<0.01). In contrast, the levels of IGFBP-2 and IGFBP-4 increased 2 and 3 fold in the same serum samples within 3–5 days of the burn injury, respectively. This increase returns to normal (day 0–1 value) within 7–10 days for IGFBP-2, but the level of IGFBP-4 remained elevated 4 fold relative to the day 0–1 (p<0.01). However, the abundance of IGFBP-1 in these serum samples was not significantly altered by the burn injury. By controlling for protein loading, these apparent alterations of IGFBPs in the sera of burned patients were not due to hemodilution. Similarly, significant reductions in IGFBP-3 were not likely due to IGFBP-3 specific protease activity in the sera of burn patients since incubation of sera from burn patients and normal individuals at 37° C did not alter the pattern of IGFBPs in sera obtained from normal individuals. Of interest, the level of IGF-1 protein in these samples was also markedly reduced following severe burn injury similar to IGFBP-3. The results of this study suggest that a marked reduction of serum IGF-1 seen in burn patients is associated with a significant reduction of IGFBP-3, a major IGF-1 binding protein in human serum.

Interferons gamma and alpha‐2b differentially regulate the expression of collagenase and tissue inhibitor of metalloproteinase‐1 messenger RNA in human hypertrophic and normal dermal fibroblasts

We have recently shown that both interferon gamma (γ) and interferon alpha‐2b (α‐2b) markedly depress the expression of messenger RNA for type I procollagen and fibronectin in postburn hypertrophic scar and normal dermal fibroblasts. In this article we examine the effects of these cytokines on the expression of mRNA for collagenase and its natural inhibitor, tissue inhibitor of metalloproteinase‐1. Twelve different fibroblast cell strains, six from postburn hypertrophic scar and six from the normal dermis of the same patients, were established in cell culture. The results of a dose response experiment showed increases in collagenase mRNA up to 4000 U/ml of interferon‐α‐2b, but maximal increases in tissue inhibitor of metalloproteinase mRNA expression and maximal decrease in mRNA for type I procollagen at 2000 U/ml. For subsequent experiments cells were treated with either interferon‐α‐2b (2000 U/ml) or ‐γ (1000 U/ml) for 96 hours. Quantitative analysis showed increases in tissue inhibitor of metalloproteinase‐1 and collagenase mRNA (81% and 54%, respectively) in interferon‐α‐2b‐treated hypertrophic scar fibroblasts. Under the same experimental conditions, interferon‐α‐2b had similar effects on normal dermal fibroblasts; however, interferon‐γ had a differential effect on the expression of mRNA for collagenase and tissue inhibitor of metalloproteinase‐1. Cells treated with interferon‐γ showed increases in tissue inhibitor of metalloproteinase‐1 mRNA (78% in hypertrophic scar and 56% in normal dermal fibroblasts) but decreases (59% and 42%, respectively) in collagenase mRNA. These effects appear to be selective because rehybridization of blots with a complementary DNA for tissue inhibitor of metalloproteinase‐2 mRNA showed no marked alteration in the abundance of this transcript. Significantly greater collagenase activity was found in conditioned medium from interferon‐α‐2b‐treated hypertrophic scar cells compared with that from interferon‐γ‐treated cells. These findings suggest that interferon α‐2b would have some advantages over interferon‐γ for the treatment of dermal fibroproliferative disorders, such as postburn hypertrophic scar.

EXPRESSION AND LOCALIZATION OF INSULIN-LIKE GROWTH FACTOR-1 IN NORMAL AND POST-BURN HYPERTROPHIC SCAR TISSUE IN HUMAN

The migration of epithelial cells from dermal appendages toward the wound surface is essential for re-epithelialization of partial thickness burn injuries. This study provides evidence that these cells in vivo synthesize a mitogenic and fibrogenic factor, insulin-like growth factor-1 (IGF-1), which may promote the development of the post-burn fibroproliferative disorder, hypertrophic scarring (HSc). An evaluation of 7 post-burn hypertrophic scars, 7 normal skin samples obtained from the same patients and 4 mature scars revealed that IGF-1 expressing cells from the disrupted sweat glands tend to reform small sweat glands of 4-10 cells/gland in post-burn HSc. The number of these cells increases with time and the glands become larger in mature scar. Other epithelial cells such as those found in sebaceous glands and basal and suprabasal keratinocytes, also express IGF-1 protein and mRNA as detected by Northern and RT-PCR analysis of RNA obtained from whole skin and separated epidermis and dermis. However, cultured keratinocytes did not express mRNA for IGF-1. Histological comparisons between normal and HSc sections show no mature sebaceous glands in dermal fibrotic tissues but the number of IGF-1 producing cells including infiltrated immune cells was markedly higher in the dermis of hypertrophic scar tissues relative to that of the normal control. In these tissues, but not in normal dermis, IGF-1 protein was found associated with the extracellular matrix. By in situ hybridization, IGF-1 mRNA was localized to both epithelial and infiltrated immune cells. Collectively, these findings suggest that in normal skin, fibroblasts have little or no access to diffusible IGF-1 expressed by epithelial cells of the epidermis, sweat and sebaceous glands; while following dermal injury when these structures are disrupted, IGF-1 may contribute to the development of fibrosis through its fibrogenic and mitogenic functions. Reformation of sweat glands during the later stages of healing may, therefore, limit this accessibility, and lead to scar maturation.

Expression of fibronectin messenger RNA in hypertrophic and normal dermal tissues and in vitro regulation by interferon alfa-2b.

After severe thermal injury, hypertrophic scarring which is associated with accumulation of extracellular matrix proteins including fibronectin, frequently develops. We have recently demonstrated that interferon alfa‐2b significantly reduces the level of type 1 procollagen messenger RNA expressed by both hypertrophic and normal dermal fibroblasts. In this report, we provide evidence that this cytokine also significantly decreases the expression of fibronectin messenger RNA in human hypertrophic scar and normal dermal fibroblasts. Four dermal fibroblast cell strains were established in cell culture from four human postburn hypertrophic scar tissues with the use of normal dermal fibroblasts from the same patients as controls. These cells were then treated with 2000 U/ml interferon alfa‐2b in culture medium at various times. The results of Northern analysis of interferon‐treated dermal fibroblasts indicate that this cytokine reduced the expression of fibronectin messenger RNA as early as 12 hours after treatment and reached its lowest level (24% relative to untreated fibroblasts) after 96 hours. When the expression of fibronectin messenger RNA was quantified by densitometry for each individual paired cell strain, a differential response to interferon treatment was found among cell strains. The level of fibronectin messenger RNA expression decreased from 17.2% to 69% in hypertrophic scar fibroblasts and 47% to 83.7% in normal fibroblasts relative to that of untreated control values. Although this decrease was less pronounced in normal fibroblasts than in hypertrophic scar fibroblasts, this reduction was significant in both interferon alfa‐2b treated hypertrophic scar fibroblasts (6.39 ± 0.71 versus 2.88 ± 0.9, n = 4, p < 0.05) and normal cells compared with untreated controls (5.47 ± 0.89 versus 3.64 ± 0.99, n = 4, p < 0.05) as assessed with Students paired t test. Rehybridization of the RNA blot prepared from interferon alfa‐2b treated and untreated hypertrophic scar fibroblasts with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 gelatinase inhibitor showed no significant changes in abundance of this transcript. This result suggests that this cytokine selectively suppresses the expression of fibronectin messenger RNA and that this reduction is not due to RNA loading. A dot blot analysis of total RNA extracted from these tissues was carried out to compare the expression of fibronectin messenger RNA between human hypertrophic scar tissues and normal dermis obtained from the same patients. The blot was initially hybridized with fibronectin complementary DNA and subsequently with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 to correct for RNA loading. When the ratio of fibronectin to tissue inhibitor or metalloproteinase type 2 messenger RNA expression for each hypertrophic scar tissue was compared with its normal control, this ratio was fourfold higher in human hypertrophic scar tissues relative to normal controls. In contrast, the expression of this message in cultured hypertrophic scar fibroblasts was not significantly different from that in normal fibroblasts. The results of this study suggest that hypertrophic scarring developing after thermal injury is associated with an overexpression of fibronectin messenger RNA, and interferon alfa‐2b may be of therapeutic value to down‐regulate the expression of this transcript.