Takashi Iwashina

transforming Growth Factor-β in Thermally Injured Patients with Hypertrophic Scars: Effects of Interferon α-2b

Hypertrophic scarring is a common dermal fibroproliferative disorder that leads to poor quality wound healing, prolongs rehabilitation, and increases morbidity following major thermal and other injuries to the deep dermis. Local and systemic transforming growth factor (TGF)-beta has been implicated as a fibrogenic cytokine in the pathogenesis of many fibrotic disorders, whereas interferon (IFN) alpha-2b may improve the pathologic features of dermal fibrosis directly or by antagonizing the effects of TGF-beta and histamine. Nine patients with severe hypertrophic scarring were evaluated for 8 weeks before treatment with subcutaneous recombinant IFN alpha-2b, 2 x 10(6) IU three times per week for 24 weeks. Clinical assessment was performed using standardized photography, a burn scar assessment tool, and serial scar volume measurements. Monthly measurements of serum TGF-beta and plasma Ntau-methylhistamine were made prior to, during, and after IFN alpha-2b therapy and compared with 27 age-matched controls. Serial biopsies of the hypertrophic scars and normal skin were performed for evaluation of mast cell numbers. Significant improvement in scar assessment occurred in 7 of 9 patients, and 3 of 9 demonstrated significant reductions in scar volume with interferon therapy beyond that occurring during the 8-week control period. For the entire group, mean rates of improvement were significantly better during interferon therapy with no recurrence following treatment. Before interferon therapy, serum TGF-beta was significantly higher in the burn patients with hypertrophic scarring than in a control population (123.04 +/- 36.48 vs. 56.85 +/- 8.38 ng/ml, p < 0.05). Within 3 months of IFN alpha-2b therapy, serum TGF-beta levels fell significantly and remained within the normal range during therapy and after interferon therapy was stopped. Plasma Ntau-methylhistamine levels were also significantly elevated in the hypertrophic scar patients as compared with age and sex-matched controls (153.6 +/- 92.07 vs. 48.3 +/- 28.9 pg/ml, p < 0.05), and significant reductions were achieved with interferon therapy and maintained after interferon was discontinued. Paired biopsies of hypertrophic scarring and normal tissue demonstrated increased numbers of mast cells in hypertrophic scars compared with normal uninjured skin from the same patients (2.65 +/- 1.63 vs. 1.04 +/- 0.62 cells/high power field, p < 0.001); however, no significant change in mast cell content of the hypertrophic scars accompanied interferon therapy. Patients with severe hypertrophic scarring demonstrate increased levels of serum TGF-beta and plasma Ntau-methylhistamine following thermal injury. A significant clinical improvement in scar quality and volume occurred during IFN alpha-2b therapy, which was associated with normalization of serum TGF-beta and plasma Ntau-methylhistamine levels. A double-blind, placebo-controlled trial will be required to further assess the usefulness of subcutaneous treatment with IFN alpha-2b for the treatment of hypertrophic scarring.

Healing of Burn Wounds in Transgenic Mice Overexpressing Transforming Growth Factor-β1 in the Epidermis

Transforming growth factor-beta (TGF-beta) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-beta in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-beta on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-beta in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-beta. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-beta 1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-beta 1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO(2) laser. Using this model, we demonstrated activation of latent TGF-beta after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-beta1 gene into keratinocytes markedly increases the release and activation of TGF-beta after burn injury. Elevated local TGF-beta significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls, P < 0.05) and homozygous (25% versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.

Development, characterization, and wound healing of the keratin 14 promoted transforming growth factor‐β1 transgenic mouse

Transforming growth factor‐β1 is a fibrogenic cytokine that is important in the development of fibroproliferative disorders of the skin after injury. To investigate the role of transforming growth factor‐β1 produced by keratinocytes during wound healing, a plasmid with the human transforming growth factor‐β1 gene coupled with the keratin 14 promoter (pG3Z: K14‐TGF‐β1) was constructed. The construct was tested successfully in vitro before being used to generate transgenic animals, which were subsequently bred into homozygous and heterozygous lines. Genotype screening of founders and progeny was performed by Southern blotting and targeting of the transgene to the epidermis by the keratin 14 promoter was shown by reverse transcription polymerase chain reaction. The major phenotypic change observed in the transgenic animals was “scruffiness” of the fur attributed to transgene expression in the skin, seen primarily in the homozygous line. A significant reduction in the rate of reepithelialization of full‐thickness excisional wounds of dorsal skin was seen in homozygous animals compared with normal litter‐mate controls at day 7 (p < 0.05, Fishers Exact test) and day 9 (p < 0.01) postwounding. Wounds in heterozygous animals also healed more slowly at day 9 (p < 0.01). Northern analysis of mRNA extracted from the wounds showed increased human transforming growth factor‐β1 message levels in homozygous and heterozygous animals, maximal at day 5. Significant increases in transforming growth factor‐β1 activity in healing wounds measured using the plasminogen activator inhibitor‐1/luciferase assay were found in the transgenic strains at day 9 postinjury as compared with the normal litter‐mate control mice (p < 0.001, ANOVA). Type I procollagen mRNA expression was higher in the homozygous and heterozygous animals, with the highest levels reached at day 9. By day 5 postwounding, biopsies of both homozygous and heterozygous tissues were significantly higher in collagen as compared with wounds in control animals (p < 0.05, ANOVA). Based on these data, the K14‐TGF‐β1 transgenic mouse shows that excessive latent transforming growth factor‐β1 produced in the epidermal layer of the skin delays reepithelialization in excisional wounds but subsequently the cells of the epidermis stimulate dermal fibroblasts leading to fibrosis through a paracrine mechanism. (WOUND REP REG 2002;10:)

Immune cell proliferation is suppressed by the interferon‐γ‐induced indoleamine 2,3‐dioxygenase expression of fibroblasts populated in collagen gel (FPCG)

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan‐catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we report the possible use of this enzyme to suppress proliferation of immune cells cocultured with IDO‐expressing fibroblasts of an allogenic skin substitute. Fetal skin fibroblasts embedded within bovine collagen were treated with cytokine interferon‐γ (IFN‐γ) to induce expression of IDO mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by measurement of kynurenine and tryptophan levels in the IFN‐γ untreated and treated fibroblasts. The results of Northern analysis showed a dose‐dependent increase in expression of IDO mRNA in response to various concentrations of IFN‐γ used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the IFN‐γ treated fibroblasts, compared to those of controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA was gradually reduced to an undetectable level within 32 h of IFN‐γ removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN‐γ on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation of immune cells, IDO‐expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of 3H‐thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO‐expressing fibroblasts, compared to those cocultured with non‐IDO‐expressing fibroblasts (P < 0.001). Furthermore, addition of IDO‐inhibitor (1‐methyl‐d‐tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose‐dependant fashion. To test the viability of immune cells cocultured with IDO‐expressing fibroblasts, FACS analysis of the PI stained PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO‐expressing fibroblasts are also the same as those of control cells. In conclusion, IDO‐expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO‐induced tryptophan‐deficient culture environment. J. Cell. Biochem. 90: 206–217, 2003.

Temperature-sensitive polymer-conjugated IFN-γ induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG)

Indoleamine 2,3‐dioxygenase (IDO) is an intracellular tryptophan‐catabolizing enzyme possessing various immunosuppressive properties. Here, we report the use of this enzyme to suppress the proliferation of peripheral blood mononuclear cells (PBMC) co‐cultured with IDO‐expressing fibroblasts of an allogeneic skin substitute in vitro. Fetal foreskin fibroblasts populated within collagen gel (FPCG) were treated with interferon‐gamma (IFN‐γ) conjugated with a temperature‐sensitive polymer to induce the expression of IDO mRNA and protein. SDS–PAGE showed successful conjugation of IFN‐γ with the temperature‐sensitive polymer. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by the measurement of kynurenine levels. The results of Northern blot analysis showed an induction of IDO mRNA expression when treated with polymer‐conjugated IFN‐γ. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN‐γ‐treated fibroblasts than in controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA in FPCG treated with polymer‐conjugated IFN‐γ was significantly longer than in those treated with free (non‐conjugated) IFN‐γ (P < 0.001). IFN‐γ radiolabeling showed a prolonged retention of IFN‐γ within collagen gel in its polymer‐conjugated form, compared to its free form. Presence of IDO protein in FPCG was demonstrated by Western analysis even 16 days after removal of the conditioned medium (containing released IFN‐γ). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO‐expressing FPCG treated with polymer‐conjugated IFN‐γ were co‐cultured with PBMC for a period of 5 days. The results showed a significant reduction in proliferation of PBMC co‐cultured with IFN‐γ‐treated IDO‐expressing fibroblasts, compared to those co‐cultured with non‐IDO‐expressing fibroblasts (P < 0.001). The addition of an IDO inhibitor (1‐methyl‐D‐tryptophan) reversed the suppressive effects of IDO on PBMC proliferation. In conclusion, IDO expression in FPCG suppresses the proliferation of immune cells in vitro. The use of a temperature‐sensitive polymer further prolongs the effect of IFN‐γ on the expression of IDO. Therefore, modulating IDO levels in situ might be an alternative for prolonging the survival of skin allografts. J. Cell. Physiol. 201: 146–154, 2004.

Determination of plasma Nτ-methylhistamine in vivo by isotope dilution using benchtop gas chromatography–mass spectrometry

A practical sensitive and specific method for determination of the stable metabolite of histamine, Ntau-methylhistamine, in human plasma using benchtop gas chromatography-stable isotope dilution mass spectrometry has been developed. Ntau-Methylhistamine, a principal metabolite of histamine in humans, was extracted and purified from human plasma using a two-step procedure with Sep-Pak silica cartridges. Quantitation of Ntau-methylhistamine was made possible by the synthesis of Ntau-[2H3]methylhistamine used as an internal standard. Derivatization with pentafluoropropionyl anhydride of extracts of human plasma yielded the bis-pentafluoropropionyl derivative of Ntau-methylhistamine for measurement using selected ion monitoring of the m/z 417/420 ion pair after electron impact on a benchtop gas chromatography-mass spectrometry (GC-MS). By improvements in the plasma extraction technique, inclusion of a synthetic internal standard and the development of a sensitive and stable derivative of the histamine metabolite, Ntau-methylhistamine was found to be significantly elevated in the plasma of patients with the dermal fibroproliferative disorder, hypertrophic scarring as compared to age-matched normal volunteers (98.5+/-29.5 pg/ml, n=9, versus 43.3+/-16.5 pg/ml, n=8, p<0.05). As such, this method affords a sensitive, specific and practical approach to measurement of histamine metabolites in plasma and other biological fluids.

The JCR:LA-cp rat: A novel model for impaired wound healing

JCR:LA‐cp/cp obese rats and their lean controls were evaluated as a type 2 diabetic wound healing model and the healing quality was characterized. This model of insulin resistance has been used extensively to study atherosclerosis but has not previously been used to study wound healing. Six circular excisional wounds were made on the dorsum of each rat and followed to day 21.Tracings of the wounds were made and used to assess the rate of wound closure. Planimetry showed a significantly diminished contraction of wounds in obese rats, but no significant difference in reepithelialization was observed. Collagen content was determined from the hydroxyproline content in wounded and unwounded skin. There were significantly lower levels of hydroxyproline in the wounds of obese compared to lean animals at day 21. Histology showed adipose tissue in place of dermal tissue in the JCR:LA‐cp/cp rat in both unwounded tissue and in the wound at day 21. Active transforming growth factor‐β1 (TGF‐β1) was measured in the serum using the plasminogen activator inhibitor‐1/luciferase assay and serum total TGF‐β was measured using an enzyme‐linked immunosorbent assay. Active TGF‐β was significantly higher in the serum of obese animals compared with lean animals, while total TGF‐β1 was not significantly different between the groups. Both active and total TGF‐β was measured in tissue sections using the plasminogen activator inhibitor‐1/luciferase assay. There was no significant difference in active TGF‐β between genotypes, while obese rats had significantly higher levels of total TGF‐β at day 21. These results indicate a deficiency in wound healing in obese animals characterized by decreased wound contraction, decreased collagen production, and changes in histology. The JCR:LA‐cp rat develops insulin resistance, atherosclerosis and early type 2 diabetes and may be a good model for impairment of wound healing in humans with metabolic syndrome.

Systemic depletion of macrophages in the subacute phase of wound healing reduces hypertrophic scar formation.

Hypertrophic scars are caused by trauma or burn injuries to the deep dermis and can cause cosmetic disfigurement and psychological issues. Studies suggest that M2‐like macrophages are pro‐fibrotic and contribute to hypertrophic scar formation. A previous study from our lab showed that M2 macrophages were present in developing hypertrophic scar tissues in vivo at 3–4 weeks after wounding. In this study, the effect of systemic macrophage depletion on scar formation was explored at subacute phase of wound healing. Thirty‐six athymic nude mice that received human skin transplants were randomly divided into macrophage depletion group and control group. The former received intraperitoneal injections of clodronate liposomes while the controls received sterile saline injections on day 7, 10, and 13 postgrafting. Wound area, scar thickness, collagen abundance and collagen bundle structure, mast cell infiltration, myofibroblast formation, M1, and M2 macrophages together with gene expression of M1 and M2 related factors in the grafted skin were investigated at 2, 4, and 8 weeks postgrafting. The transplanted human skin from the control group developed contracted, elevated, and thickened scars while the grafted skin from the depletion group healed with significant less contraction and elevation. Significant reductions in myofibroblast number, collagen synthesis, and hypertrophic fiber morphology as well as mast cell infiltration were observed in the depletion group compared to the control group. Macrophage depletion significantly reduced M1 and M2 macrophage number in the depletion group 2 weeks postgrafting as compared to the control group. These findings suggest that systemic macrophage depletion in subacute phase of wound healing reduces scar formation, which provides evidence for the pro‐fibrotic role of macrophages in fibrosis of human skin as well as insight into the potential benefits of specifically depleting M2 macrophages in vivo.

The therapeutic potential of a C‐X‐C chemokine receptor type 4 (CXCR‐4) antagonist on hypertrophic scarring in vivo

Effective prevention and treatment of hypertrophic scars (HTSs), a dermal form of fibrosis that frequently occurs following thermal injury to deep dermis, are unsolved significant clinical problems. Previously, we have found that stromal cell‐derived factor 1/CXCR4 signaling is up‐regulated during wound healing in burn patients and HTS tissue after thermal injury. We hypothesize that blood‐borne mononuclear cells are recruited into wound sites after burn injury through the chemokine pathway of stromal cell‐derived factor 1 and its receptor CXCR4. Deep dermal injuries to the skin are often accompanied by prolonged inflammation, which leads to chemotaxis of mononuclear cells into the wounds by chemokine signaling where fibroblast activation occurs and ultimately HTS are formed. Blocking mononuclear cell recruitment and fibroblast activation, CXCR4 antagonism is expected to reduce or minimize scar formation. In this study, the inhibitory effect of CXCR4 antagonist CTCE‐9908 on dermal fibrosis was determined in vivo using a human HTS‐like nude mouse model, in which split‐thickness human skin is transplanted into full‐thickness dorsal excisional wounds in athymic mice, where these wounds subsequently develop fibrotic scars that resemble human HTS as previously described. CTCE‐9908 significantly attenuated scar formation and contraction, reduced the accumulation of macrophages and myofibroblasts, enhanced the remodeling of collagen fibers, and down‐regulated the gene and protein expression of fibrotic growth factors in the human skin tissues. These findings support the potential therapeutic value of CXCR4 antagonist in dermal fibrosis and possibly other fibroproliferative disorders.

Impaired cutaneous wound healing in transforming growth factor‐β inducible early gene1 knockout mice

Transforming growth factor‐β inducible early gene (TIEG) is induced by transforming growth factor‐β (TGF‐β) and acts as the primary response gene in the TGF‐β/Smad pathway. TGF‐β is a multifunctional growth factor that affects dermal wound healing; however, the mechanism of how TGF‐β affects wound healing is still not well understood because of the complexity of its function and signaling pathways. We hypothesize that TIEG may play a role in dermal wound healing, with involvement in wound closure, contraction, and reepithelialization. In this study, we have shown that TIEG1 knockout (TIEG1–/–) mice have a delay in wound closure related to an impairment in wound contraction, granulation tissue formation, collagen synthesis, and reepithelialization. We also found that Smad7 was increased in the wounds and appeared to play a role in this wound healing model in TIEG1–/– mice.