Differentiation of mesenchymal stem cells to support peripheral nerve regeneration in a rat model.

Mesenchymal stem cells (MSCs) support axon regeneration across artificial nerve bridges but their differentiative capacity and ability to promote nerve regeneration remains unclear. In this study, MSCs isolated from bone marrow of Sprague-Dawley rats were characterized by plastic adherence and pluripotency towards mesodermal lineages. Isolated undifferentiated MSCs (uMSCs) were stimulated towards a Schwann cell (SC) phenotype using specific growth factors, and cell marker analysis was performed to verify SC phenotype in vitro. Differentiation resulted in temporally dependent positive immunocytochemical staining for the SC markers, glial fibrillary acidic protein (GFAP), S100, and nerve growth factor receptor (NGFR), with maximal marker expression achieved after 6days of treatment with differentiation media. Quantitative analysis demonstrated that ~50% of differentiated MSCs (dMSCs) have a SC phenotype. Using an indirect co-culture system, we compared the ability of dorsal root ganglion (DRG) cells to extend neurites in indirect contact with uMSCs and dMSCs as compared to SCs. The mean values of the longest length of the DRG neurites were the same for the dMSCs and SCs and significantly higher than the uMSC and DRG mono-culture systems (p < 0.05). In vivo, compared to an empty conduit, dMSC seeded collagen nerve conduits resulted in a greater number of sciatic motoneurons regenerating axons through the conduit into the distal nerve stump. We conclude that bone marrow-derived MSCs differentiate into a SC-phenotype that expresses SC markers transiently and sufficiently to support limited neurite outgrowth in vitro and axonal regeneration equivalent to that of SCs in vitro and in vivo. The nerve autograft remains the most effective conduit for supporting regeneration across nerve gaps.

Hydrofiber dressing with silver for the management of split-thickness donor sites: a randomized evaluation of two protocols of care.

BACKGROUND: This randomized, open-label study evaluated Aquacel Ag Hydrofiber dressing with silver (HDS; ConvaTec, Skillman, NJ, USA) with an adherent or gelled protocol in the management of split-thickness donor sites. METHODS: HDS was the primary dressing in the adherent group (gauze as secondary covering) and gelled group (transparent film as secondary covering). Dressings were changed on study day 1 or 2 and study days 5 (optional), 10 (optional), and 14. The primary outcome was healing (>or=90% re-epithelialization) at study day 14. RESULTS: Seventy subjects were treated (36 adherent, 34 gelled). By study day 14, 77% of donor sites had healed (67% adherent, 88% gelled). Pain scores decreased over time in both treatment groups. Investigators were "very satisfied" or "satisfied" with (adherent, gelled) time required to manage dressing change (89%, 79% of subjects), minimization of donor-site pain (64%, 82%), ease of application (97%, 94%), management of drainage (92%, 82%), ease of removal (77%, 85%), and ability of dressing to remain in place (69%, 76%). Thirty-nine (56%) subjects had adverse events, most commonly non-donor-site infection (11%) and gastrointestinal events (11%). CONCLUSION: In this randomized, open-label study, HDS was well-tolerated, versatile, and effective in the management of split-thickness donor sites.

Healing of burn wounds in transgenic mice overexpressing transforming growth factor-beta 1 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.

Myofibroblasts and apoptosis in human hypertrophic scars: the effect of interferon-alpha2b.

BACKGROUND: Hypertrophic scars (HSc) are a dermal fibroproliferative disorder that leads to considerable morbidity. Preliminary evidence suggests that interferon (IFN) may improve HSc clinically. The aims of this study were (1) to compare the cell density in HSc and in wounds that heal without the development of HSc (normotrophic scars), (2) to examine the presence of myofibroblasts and apoptosis in normotrophic and HSc scars over time, and (3) to determine if the systemic administration of IFN-alpha2b can induce apoptosis. METHODS: Two groups of patients underwent serial tissue biopsies. Six burn patients were studied prospectively by obtaining biopsy specimens from wound granulation tissue, normal skin, post-burn HSc, and normotrophic scars (healed donor sites). A second patient group with HSc was treated with systemic IFN-alpha2b and had biopsy material taken before, during, and after IFN therapy. The tissue was analyzed by immunohistochemical staining for alpha-smooth muscle actin (alpha-SMA) and in situ DNA fragmentation terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay for apoptosis. RESULTS: The total numbers of fibroblasts in HSc were found to be similar to granulation tissue and twice that of normal skin and normotrophic scar. Over time the numbers of cells in HSc tissue decreased toward normal skin levels. There was a significantly higher percentage of fibroblasts staining for alpha-SMA in HSc as compared with normotrophic scar or normal skin obtained from the same patient (P >.05). Serial biopsy specimens of resolving HSc tissue obtained from the patients who received systemic IFN-alpha2b showed a general reduction in total number of fibroblasts and myofibroblasts associated with a significant increase in the percentage of apoptotic cells compared with normal dermis from the same patient. CONCLUSIONS: HSc tissues have greater numbers of fibroblasts and myofibroblasts than normal skin and normotrophic scars. As HSc remodels, the numbers of fibroblasts and myofibroblasts reduces, possibly by the induction of apoptosis. Systemic IFN-alpha2b may contribute to the resolution of HSc in part by the enhanced induction of apoptosis.

Keratinocyte differentiation inversely regulates the expression of involucrin and transforming growth factor beta1.

Extensive skin loss from a variety of conditions such as severe thermal injury is associated with significant functional morbidity and mortality. In recent years, the healing quality has been improved for patients who suffer burns due in part to the usage of skin replacement mainly prepared from multi-layered sheets of cultured keratinocytes. Although it is known that keratinocytes are a rich source of wound healing promoting factors such as transforming growth factor-beta1 (TGF-beta1), it is not clear whether differentiated keratinocytes in a multi-layer form release this multi-functional growth factor and has any functional influence on dermal fibroblasts. This study examined the hypothesis that keratinocytes in mono- and multi-layer forms express different levels of TGF-beta1. To address this hypothesis, keratinocytes were grown in serum free medium (KSFM) supplemented with bovine pituitary extract (50 microg/ml) and EGF (5 microg/ml). When cells reached confluency, conditioned medium was removed and replaced with 50% KSFM with no additives and 50% DMEM without serum and cells were allowed to form multi-layers and differentiate. The conditioned medium was then collected every 48 h up to 24 days and the level of TGF-beta1 and the efficacy of a keratinocyte released fibroblast mitogenic factor were evaluated by ELISA and (3)H-thymidine incorporation, respectively. Northern analysis was also employed to evaluate the expression of TGF-beta1, involucrin, TIMP-1, and 18 S ribosomal RNA in keratinocytes at different times of the onset of differentiation. The microscopic morphology of keratinocytes at different times of induction of cell differentiation showed detachments (nodules) of many regions of keratinocyte sheet from culture substratum within 1-2 weeks. The numbers and sizes of these nodules were increased as the process of keratinocyte differentiation proceed. The results of TGF-beta1 evaluation revealed that mono-layers of cultured keratinocytes which were round, attached, and proliferating in KSFM + BPE and EGF containing medium released a significantly higher level of TGF-beta1 (196 +/- 58 pg /ml) relative to those grown in multi-layer forms (28 +/- 7.8 pg/ml). A longitudinal experiment was then conducted and the results showed that cells on the onset of differentiation released even greater level of TGF-beta1 (388 +/- 53 pg/ml) relative to those grown in KSFM + BPE and EGF. This finding was consistent with the expression of TGF-beta1 mRNA evaluated in keratinocytes grown in test medium for various duration. In general, the level of TGF-beta1 protein and mRNA gradually reduced to its lowest level within 12 days of growing cells in our test medium. When aliquots of the collected keratinocyte conditioned medium were added to dermal fibroblasts, the level of (3)H-thymidine incorporation increased only in those cells receiving aliquots of conditioned medium containing high levels of TGF-beta1. When involucrin was used as a differentiation marker for keratinocytes at different time points, the highest level of involucrin mRNA expression was found at the later stage of cell differentiation. In conclusion, high involucrin expressing differentiated keratinocytes seem to be quiescent in releasing both TGF-beta1 and a fibroblast mitogenic factor.

Induction of collagenase mRNA expression in dermal fibroblasts by IFN-alpha 2b and determination of the IFN-alpha 2b responsive element on 5'-flanking regions of collagenase promoter.

We have previously demonstrated that interferon-alpha2b (IFN-alpha2b) markedly depresses the expression of mRNA for type I procollagen in dermal fibroblasts. In the present study, the effect of various concentrations of IFN-alpha2b on the expression of collagenase mRNA and activity of 5'-flanking regions of collagenase promoter in dermal fibroblasts are presented. The results showed at least a 2-fold increase in the expression of collagenase mRNA in fibroblasts grown at either 70% confluency (40.9 +/- 4.6 vs. 18.5 +/- 1.6, n=4, p<0.05) or 95% confluency (24.7 +/- 6.7 vs. 4.5 +/- 1.6, n=4, p<0.05). The effects of IFN-alpha2b on collagenase mRNA stability and promoter activity were evaluated to determine the mechanism by which IFN-alpha2b increases the expression of collagenase mRNA. IFN-alpha2b-treated and untreated fibroblasts were treated with alpha-amanitin to arrest collagenase mRNA transcription, and total RNA was then harvested at 0, 3, 6, 12, and 24 h. The decay curves of collagenase mRNA as a function of time showed a greater rate of degradation for collagenase mRNA in IFN-alpha2b-treated cells relative to untreated control cells. This difference was more pronounced in cells treated with alpha-amanitin at either 12 or 24 h. To determine the regions of the collagenase promoter that might function as IFN-alpha2b responsive elements, eight different fragments of the collagenase promoter, -518, -300, -171, -161, -127, -91, -74, and -66 to +63 nucleotide (nt), were constructed in a chloramphenicol acetyltransferase (CAT) expression vector. The results of CAT activity of cells transfected with these construct identified three constructs, 171/+63, -161/+63, and -127/+63, as being responsive to IFN-alpha2b treatment in dermal fibroblasts. The CAT activity was increased 279%, 163%, and 261% in -171/+63, -161/+63, and -127/+63-transfected fibroblasts, respectively, in response to IFN-alpha2b treatment relative to untreated control. No significant increase in CAT activity was found in cells transfected with the other constructs of the collagenase promoter. A time response experiment showed a marked increase in CAT activity of cells transfected with either 127/+63 or -171/63 constructs within 6-12 hr of IFN-alpha2b treatment. In conclusion, IFN-alpha2b significantly increases the expression of collagenase mRNA in dermal fibroblasts probably through stimulation of the -127/-91 region of the collagenase promoter. Thus, this region may function as an IFN-alpha2b responsive element on collagenase promoter.

Fatty acid content of plasma lipids and erythrocyte phospholipids are altered following burn injury.

The objective of this study was to examine compositional and quantitative changes in fatty acids of plasma components and red blood cell phospholipids (PL) immediately following and during recovery from burn injury. Subjects (n = 10) with >10% total body surface area burn had blood drawn at specific timepoints (0 to >50 d) following burn injury. Fatty acid composition of red blood cell PL and plasma PL, cholesteryl esters (CE), and triglycerides was determined using gas-liquid chromatography after separating each fraction from extracted lipids by thin-layer chromatography. Total plasma PL and CE in burn patients were lower than in healthy control subjects with reduced 20:4n-6, n-6, and n-3 fatty acids and higher levels of monounsaturated and saturated fatty acids early after burn. CE levels remained half that of healthy control values up to 50 d post-burn. Red blood cell PL had decreased 20:4n-6 content and profiles similar to that of an essential fatty acid deficiency early after burn. These results suggest an impairment in lipoprotein and polyunsaturated fatty acid metabolism in the early post-burn period. Lower levels of 20:4n-6 and n-3 fatty acids in every plasma fraction suggest increased use of these fatty acids for wound healing and immune function following burn injury. Further work is needed to determine the ability of burn patients to utilize essential fatty acids in order to design nutritional intervention that promotes wound healing and immunological functions consistent with recovery in these patients.

Stress-relaxation and contraction of a collagen matrix induces expression of TGF-beta and triggers apoptosis in dermal fibroblasts.

Extracellular matrix serves as a scaffold for cells and can also regulate gene expression and ultimately cell behaviour. In this study, we compared the effects of three forms of type I collagen matrix, which differed only in their mechanical properties, and plastic on the expression of transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinase-1 (collagenase), and type I collagen and on the growth and survival of human dermal fibroblasts. These effects were correlated with alterations in cell morphology and organization of intracellular actin. Cells in detached or stress-relaxed matrices were spherical, lacked stress fibres, and showed increased TGF-beta1 mRNA compared to the cells in anchored collagen matrices or on plastic, which were polygonal or bipolar and formed stress fibres. The levels of TGF-beta measured by bioassay were higher in detached and stress-relaxed collagen matrices, than in anchored collagen matrices. Cells on plastic contained little or no immunoreactive TGF-beta, while most cells in collagen matrices were stained. The levels of collagenase mRNA were significantly higher in all the collagen matrix cultures compared to those on plastic, but there were no statistically significant differences between them. Levels of mRNA for procollagen type I were not significantly affected by culture in the collagen matrices. Apoptotic fibroblasts were detected by the TUNEL assay in detached (5.7%) and to a lesser extent in stress-relaxed (2.2%) matrices, but none were observed in anchored collagen matrices or on plastic. These results show that alterations in the mechanical properties of matrix can induce the expression of TGF-beta and trigger apoptosis in dermal fibroblasts. They further suggest that inability to reorganize this matrix could be responsible for the maintenance of the fibroproliferative phenotype associated with fibroblasts in hypertrophic scarring.

Liposome associated interferon-alpha-2b functions as an anti-fibrogenic factor in dermal wounds in the guinea pig.

We have previously reported that interferon-alpha-2b (IFN-alpha-2b) can be encapsulated in liposomes without compromising its anti-fibrogenic effects on dermal fibroblasts in vitro. This study was conducted to determine whether this preparation applied topically to guinea pig wounds can affect their healing. The rationale for this approach is that systemic administration of IFN-alpha-2b by injection for treatment of dermal fibrosis is uncomfortable, requires a large quantity of the cytokine and cannot be easily used in children. Liposomes are potentially useful vehicles for the topical delivery of drugs. Empty sonicated liposome vesicles were mixed with various concentrations of IFN-alpha-2b and then dried and rehydrated. An enzyme-linked immunosorbent assay (ELISA) was used to determine the efficiency of encapsulation and the stability of the preparation under experimental conditions. A total of 36 full thickness skin wounds (6/animal, 3 on each side) were made with an 8 mm disposable punch. Each wound on the right side received cream (100 mg/wound) containing 3000 units of liposome-encapsulated IFN-alpha-2b, while wounds on the left side received cream containing empty liposomes. There was a significant reduction in rate of contraction of wounds treated with IFN-alpha-2b as early as 5 days after wounding. This reduction remained significant up to 10 days. Northern analysis, used to evaluate the expression of mRNAs for type I and type III collagens in response to IFN-alpha-2b showed a marked reduction in abundance of the transcripts for the pro-alpha1(I) chain of type 1 collagen on days 11 and 14 after wounding. Similarly, the level of mRNA for type III procollagen was markedly reduced as early as day 7 and remained depressed up to day 14. These findings were consistent with results obtained for the total collagen content in tissue samples. Cellularity of the IFN-alpha-2b-treated wounds, assessed by vimentin content, was also markedly reduced at day 7 and remained depressed up to day 14. Liposome associated IFN-alpha-2b applied 5 days after completion of epithelialization reduced mRNA for the pro-alpha1(I) chain of type 1 collagen, confirming its transepidermal penetration and effectiveness. The activity of liposome-associated IFN-alpha-2b in vivo supports the concept of the topical use of this anti-fibrogenic agent for treatment of fibroproliferative disorders.

Rating the resolving hypertrophic scar: comparison of the Vancouver Scar Scale and scar volume.

The increased focus of research interests and clinical documentation on outcomes demands that evaluation tools provide reliable and valid data. The Vancouver Scar Scale (VSS) was developed to provide a more objective measurement of burn scars; however, the validity (a test's ability to measure the phenomenon for which it was designed) of the VSS has not been tested. To examine the construct validity of the VSS, we compared it with scar volume, which has established face validity. Burn scars were evaluated monthly for a minimum of 7 months. Three scar volume measurements were performed on each scar. In addition, 3 independent examiners completed the VSS for the same scar. The data generated by these 2 measurements were used to establish the following: (1) the interrater agreement estimated by interclass correlation coefficient, (2) convergence validity, (3) the sensitivity of the assessments to discriminate changes in the scar over time, and (4) the prevalence of related parameters that are not currently being captured by the VSS. In an attempt to address some of the deficiencies of the VSS, we propose several modifications. We anticipate that these changes will increase the reliability and validity of the VSS through an increase in the awareness that training in the use of this scale is required, through improvement in the quality of the subscales, and through the documentation of additional pertinent information.

Hypertrophic scar tissues and fibroblasts produce more transforming growth factor-beta1 mRNA and protein than normal skin and cells.

Transforming growth factor-beta1 is a well-known fibrogenic cytokine produced by many types of cells including dermal fibroblasts. To investigate whether this fibrogenic cytokine is involved in development of hypertrophic scar, transforming growth factor-beta1 gene expression was evaluated in small skin samples. Because a sufficient quantity of normal skin from patients with hypertrophic scar is not readily available, a reverse transcription-polymerase chain reaction technique was used. Quantitation of gene expression by reverse transcription-polymerase chain reaction is difficult partly due to the lack of suitable complementary RNA standards. We have established a convenient, reliable procedure to construct an internal standard for transforming growth factor-beta1 starting with a gene specific polymerase chain reaction product. After digestion of the polymerase chain reaction product with endonuclease, a small piece of cDNA from human procollagen alpha1(I) cDNA with compatible ends was inserted into the polymerase chain reaction-DNA fragment. The recombinant cDNA was re-amplified by polymerase chain reaction and subcloned into a plasmid containing bacteriophage T7 and T3 promoters. Complementary RNA was prepared from the recombinant plasmid and amplified by reverse transcription-polymerase chain reaction together with the tissue or cellular RNA. After amplification, the products were electrophoresed in an agarose gel containing ethidium bromide. The bands for internal standard and transforming growth factor-beta1 mRNA were scanned, digitized, and plotted against the amount of internal standard complementary RNA added in the reverse transcription-polymerase chain reaction. The number of mRNA molecules/cell was calculated. We examined the transforming growth factor-beta1 mRNA in hypertrophic scar tissue and in normal skin and found that hypertrophic scar tissues expressed five-fold more transforming growth factor-beta1 mRNA than normal skin per unit of wet weight. We used this procedure to quantitate transforming growth factor-beta1 mRNA expression in 5 pairs of fibroblast cultures derived from hypertrophic scar and normal skin. The results showed that hypertrophic scar fibroblast cultures contain significantly more molecules of mRNA for transforming growth factor-beta1 than normal cells (116 +/- 6 vs. 97 +/- 7, p = 0.017, n = 5). These results were supported by Northern analysis for transforming growth factor-beta1 mRNA in the cells and enzyme-linked immunosorbent assay for TGF-beta1 protein in fibroblast-conditioned medium. In conclusion, hypertrophic scar tissue and fibroblasts produce more mRNA and protein for transforming growth factor-beta1, which may be important in hypertrophic scar formation. The construction of the gene specific internal standard for reverse transcription-polymerase chain reaction is a simple and reliable procedure useful to quantitate gene expression in a small amount of tissue or number of cells.

Management of the acutely burned upper extremity.

In summary, thermal injury is a frequent from of upper extremity trauma that often affects young working-class males. Its management must be designed with preserving life as a priority, recognizing the additional risk factor of inhalation injury as well as the traditional predictors of mortality--age and proportion of the TBSA burned. The aim of care in dorsal hand burns is to restore wound coverage and movement within 14 days of injury. Determining the depth of thermal injury to the upper extremity is notoriously difficult and requires serial assessment over time to most accurately predict the time of healing, still considered the standard measure of burn depth despite newly developing technical assessment tools. Tangential excision and split-thickness autograft coverage are predictably uniformly successful for deep dermal burns in restoring function in both the short and long term; however, burns exposing underlying tendons, bones, or joints require individualized reconstructive procedures, including local and distant flaps, free-tissue transfers, or combinations of these procedures. Early TAM is the most successful modality for recovery of function and to minimize any one of many potential complications associated most commonly with deep wounds and prolonged periods of healing.

Molecular and cellular aspects of fibrosis following thermal injury.

The pathogenesis of hypertrophic scars following thermal injury remains a complex and incompletely understood process but recent investigations into the composition of the tissue itself, the activities of the scar fibroblasts, and the effects of various cytokines and growth factors, have all contributed to the emergence of an increasingly clear picture. Although it may be considered just one example of a broad range of fibroproliferative disorders that afflict many different organs, often in response to diverse environmental insults, the nature of the burn injury and the special properties of skin probably play important roles in promoting the development of this especially troublesome variety of excessive connective tissue. This knowledge has provided the rationale for a number of experimental therapies that, individually or in some combination, may augment or one day supplant the more commonly employed surgical or physical treatments.

Control of wound contraction. Basic and clinical features.

Although a substantial amount of molecular and cellular data have been generated in an effort to understand the process of wound contraction and scar contracture formation, questions remain. What seems apparent is that the myofibroblast is not the only cell that generates contractile forces within wounds, but it does appear to be intrinsically linked to the development of hypertrophic scars. The supposition that the formation of scar contractures is solely the result of a continuation of wound contraction is an oversimplification. Figure 4 provides a model of the possible evolution of contractile forces during the wound healing process and their role in the development of scar contractures. Migration of fibroblasts into and through the extracellular matrix during the initial phase of wound healing, prior to the expression of alpha-SMA, appears to be a fundamental component of wound contraction. During this migration, the pulling of collagen fibrils into a streamlined pattern in their wake, and the associated production of collagenase, may facilitate a more normal arrangement of collagen. Once the wound has been repopulated and the chemotactic gradient that was established by inflammatory cells is decreased, fibroblast migration will cease. It is at this point that myofibroblasts appear and play a key role in the production of hypertrophic scars, given that their prolonged presence and over-representation are hallmarks of this pathology. One of the pivotal differences between wounds that proceed to normal scar compared with those that develop hypertrophic scars and scar contractures may be a lack (or late induction) of myofibroblast apoptotic cell death. The combined contribution of fibroblasts and myofibroblasts to abnormal extracellular matrix protein production results in an excessive and rigid scar. The isometric application of contractile forces by myofibroblasts probably contributes to the formation of the whorls, nodules, and scar contractures characteristic of hypertrophic scars. Because the prolonged presence of myofibroblasts, producing an imbalance in extracellular matrix proteins and proteases, probably exacerbates hypertrophic scars and wound contraction, accelerating the rate of apoptotic cell death to reduce the cell number to that seen in normal scar may be a useful strategy for providing effective and efficient treatment of scar contracture.

Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivo.

Hypertrophic scarring (HSc) following burn injury is a common, disfiguring, and functionally limiting form of dermal fibrosis, compromising recovery. Previously, elevated levels of transforming growth factor-beta1 (TGF-beta1), a fibrogenic cytokine, were found in wounds and serum of severely injured patients, antagonized in part by treatment with systemic interferon-alpha2b (IFN-alpha2b) both in vitro and in vivo. It is hypothesized that in wound healing after injury, platelets are an initial source of TGF-beta, but wound fibroblasts may be capable, after activation, of autoamplification of the initial response to injury by increasing TGF-beta mRNA and protein that may subsequently be responsive to IFN therapy with IFN-alpha or IFN-gamma or both. Using three pairs of site-matched HSc and normal fibroblasts from the same individuals, nonconfluent and near confluent fibroblasts were treated with TGF-beta, and cell proliferation and collagen production were assayed using cell counting and 18O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry (GC-MS). HSc and normal fibroblasts were assayed for the production of TGF-beta protein secretion using ELISA for TGF-beta1, TGF-beta2, and TGF-beta3 after acidification of medium samples from 96-h cultures. HSc and normal fibroblasts were treated with IFN-alpha2b or IFN-gamma or both for 96 h. Quantitative RT-PCR and Northern analysis were performed using newly synthesized internal standards for human TGF-beta1. TGF-beta stimulates both HSc and normal fibroblast proliferation. Collagen synthesis is greater in HSc than in normal fibroblasts and is maximally stimulated at 75 pM TGF-beta. TGF-beta stimulated collagen metabolism is antagonized by IFN-alpha or IFN-gamma or both in an additive fashion. HSc and normal fibroblasts not only possess the mRNA for TGF-beta1 but also secrete mature TGF-beta protein. Treatment of HSc and normal fibroblasts with IFN-alpha2b or IFN-gamma antagonizes TGF-beta protein production, and additive effects occur. RT-PCR demonstrates that after IFN treatment, downregulation of TGF-beta1 mRNA accounts in part for the reduction in protein secretion in HSc fibroblasts. Elevations of systemic TGF-beta may be due to wound fibroblasts. TGF-beta synthesis and antagonism of fibroblast TGF-beta protein secretion occurs with either IFN-alpha or IFN-gamma, in part by downregulation of TGF-beta1 mRNA levels.

Mannose-6-phosphate/IGF-II receptors mediate the effects of IGF-1-induced latent transforming growth factor beta 1 on expression of type I collagen and collagenase in dermal fibroblasts.

We have previously shown that insulin-like growth factor-1 (IGF-1) induces the expression of latent transforming growth factor beta 1 (LTGF-beta 1) through activation of c-fos and c-jun oncogenes. In this study we investigated whether IGF-1 induced latent TGF-beta 1 has autocrine effects on dermal fibroblasts and described a possible mechanism. Human dermal fibroblasts were treated with either vehicle, IGF-1 alone, or IGF-1 with either anti-TGF-beta 1 neutralizing antibody or mannose-6-phosphate (M6P) and levels of mRNAs for TGF-beta 1, collagenase and the pro alpha 1(I) chain of type I collagen were then evaluated by Northern analysis. Conditioned medium was also collected from treated and untreated cells and assayed for TGF-beta 1 protein by enzyme-linked immunosorbent assay. The results of the Northern analysis revealed a differential effect on the expression of the pro alpha 1(I) chain of type I collagen and collagenase in dermal fibroblasts: mRNA for the former being significantly increased in response to IGF-1 treatment while that for collagenase was markedly suppressed. These effects of IGF-1 were blocked to a significant extent by TGF-beta 1 neutralizing antibody at a concentration of 0.5-2.0 micrograms/ml. As the TGF-beta 1 induced by IGF-1 is inactive in the traditionally used mink lung epithelial cell growth inhibition assay, we explored the possible role of IGF-II/M6P receptors in facilitating these autocrine effects. The results showed that the greater than two-fold increase (201.9 +/- 38 vs 81.8 +/- 13, p < 0.05) in mRNA for the pro alpha 1(I) chain of type I collagen induced by IGF-1 was at least 60% inhibited by M6P in a time-dependent fashion. A direct correlation between the expression of TGF-beta 1 and the pro alpha 1(I) chain of type I collagen was found in response to either IGF-1 alone or IGF-1 with M6P. Treatment of cell cultures with TGF-beta 1 neutralizing antibody mimicked the effect of M6P. In contrast to the effects on expression of type I collagen, the level of collagenase mRNA was markedly reduced by IGF-1 alone and was restored by the administration of M6P. The levels of TGF-beta 1 in conditioned medium from treated and untreated cells showed a similar pattern to that of the mRNA detected by Northern analysis. These findings suggest that IGF-1 induces latent TGF-beta 1 and that the matrix-modulating autocrine effects of LTGF-beta 1 on dermal fibroblasts are facilitated by M6P/IGF-II receptors on these cells.

Delayed appearance of decorin in healing burn scars.

AIMS: We have previously shown that hypertrophic scar tissue from burn patients contains abnormally high amounts of the proteoglycans versican and biglycan and reduced amounts of decorin, in comparison with normal dermis or mature scar. The lack of decorin may account for the poor organization of collagen fibrils in the nodular areas of these scars. Decorin has also been reported to neutralize the fibrogenic growth factor TGF-beta1. This study was conducted to monitor the time-course of expression of decorin in healing burn wounds by in-situ hybridization to determine whether its absence from hypertrophic scars could result from reduced synthesis. METHODS AND RESULTS: Scar tissue from 19 patients and normal dermis from six patients, was fixed in paraformaldehyde, embedded in paraffin and sectioned. Digoxigenin-labelled cRNA probes were prepared from a plasmid containing a 622-bp insert of human decorin cDNA and used for in-situ hybridization. Total numbers of connective tissue cells and cells positive for decorin mRNA were counted in 10 random fields in the upper (papillary), middle and lower (reticular) one-thirds of the dermis. In all regions the number and percentages of cells with decorin mRNA were low during the first 12 months after injury (eight samples), much higher between 12 and 36 months (seven samples) and low and similar to those in normal skin after 36 months (five samples). The differences between intermediate and early or late stage samples were statistically significant (one-way ANOVA). Immunohistochemistry showed little staining for decorin in early stage samples and much stronger staining in mid-stage. Late stage tissue showed intense staining for decorin, almost comparable to that in normal dermis. CONCLUSION: Expression of decorin in burn wounds is suppressed for about 12 months and then increases at a time when resolution of hypertrophic scarring is generally considered to occur.

Activation of latent transforming growth factor-beta1 is induced by mannose 6-phosphate/insulin-like growth factor-II receptor.

This study was conducted to further explore the mechanism of transforming growth factor beta1 (TGF-beta1) activation, which plays a critical role in many physiological and pathological conditions. We have previously shown that the large (270 kDa), but not small (40 kDa), mannose 6-phosphate receptors facilitate the cellular response to latent TGF-beta1 released from genetically modified cells. In this study, we explored the role of cell membrane associated transglutaminase and plasmin in mannose 6-phosphate receptor induced latent TGF-beta activation using MS and MS-9 cells bearing either no receptors or the 270 kDa mannose 6-phosphate/insulin-like growth factor II receptors, respectively. As a source of latent TGF-beta1, PA317 cells were transfected with either pLin-TGF-beta1 vector or pLin retroviral vector with no TGF-beta1 insert using calcium phosphate precipitation. The latency and bioactivity of TGF-beta1 in conditioned medium derived from transfected PA317 cells were evaluated by enzyme-linked immunosorbent assay and mink lung epithelial cell growth inhibition assay, respectively. The level of latent TGF-beta1 was 13-fold higher (20.1 +/- 0.4 vs. 1.5 +/- 0.03 ng/ml) in conditioned medium from pLin-TGF-beta1 transfected cells than that of control. The latency and bioactivity of TGF-beta1 released from pLin-TGF-beta1 transfected cells were confirmed by evaluation of 3H-thymidine incorporation in Mv1Lu epithelial cells treated with non- and heat-activated 10% conditioned medium. The results showed a significantly lower 3H-thymidine incorporation in Mv1Lu epithelial cells treated with heat-activated PA317 conditioned medium (4% of control) relative to those treated with either control or nonheated conditioned medium. This inhibition was abrogated by addition of 40 microg/ml of TGF-beta1 neutralizing antibody. The level of 3H-thymidine incorporation was then evaluated in MS-9 cells receiving Dulbecco's modified Eagle medium containing either 0% 10%, 30% or 50% volumes of nonactivated PA317 conditioned medium for 24 hours. The results showed a markedly lower proliferation in response to 30% and 50% conditioned medium used in MS-9 cells. Under similar experimental conditions, addition of only mannose 6-phosphate, but not fructose 6-phosphate or mannose 1-phosphate, at 1 mM concentration restored the MS-9 cell proliferative response to latent TGF-beta1. The inhibitory effects of latent TGF-beta1 on MS-9 cell proliferation were restored by addition of either TGF-beta1 neutralizing antibody or cystamine, a transglutaminase inhibitor. In contrast, addition of aprotinin, a plasmin inhibitor, had a marginal influence on inhibitory effects of latent TGF-beta1 on MS-9 cell proliferation. Interestingly, a mixture of latent TGF-beta1 + MS-9 cell membranes, but not MS cell membranes, also inhibited the mink lung epithelial cell proliferation (34% of control). These findings indicate that mannose 6-phosphate/insulin-like growth factor II receptors are involved in latent TGF-beta activation and that is at least partly dependent on cell membrane associated transglutaminase, but not on plasmin.

Cellular response to latent TGF-beta1 is facilitated by insulin-like growth factor-II/mannose-6-phosphate receptors on MS-9 cells.

This study was conducted to explore the mechanism of activation of TGF-beta1 which is critical to its role in many physiological and pathological conditions. We have previously demonstrated that latent TGF-beta1 modulates ECM through interaction with IGF-II/M6P receptors on dermal fibroblasts. In this report, we provide evidence that large (270 kDa) but not small (46 kDa) M6P receptors facilitate the cellular response to LTGF-beta1 released from genetically modified cells. As a source of LTGF-beta1, PA317 cells were transfected with either pLin-TGF-beta1 vector or pLin vector with no TGF-beta1 insert using calcium phosphate precipitation. Conditioned medium from transfected cells was removed after 3 days and used to evaluate the latency and bioactivity of TGF-beta1 using ELISA and mink lung epithelial cell growth inhibition assay, respectively. The level of TGF-beta1 was 20-fold greater (2142 +/- 369 vs 102 +/- 23 pg/ml) in conditioned medium derived from pLin-TGF-beta1-transfected cells than in that of controls. Various volumes of this conditioned medium were then used to treat MS-9, SR-2, and MS cells bearing the large, small, and no IGF-II/M6P receptors, respectively, for 24 h. [(3)H]Thymidine incorporation, used as an index for cell proliferation, showed a markedly lower level of proliferation in MS-9 cells in response to a given concentration of LTGF-beta1 than was seen in SR-2 and MS cells. Interestingly, under similar experimental conditions, either addition of M6P at 1 mM concentration or anti-TGF-beta1 antibody abrogated the MS-9 cell proliferative response to LTGF-beta1. In contrast, the inhibitory response of these three cell strains to heat-activated conditioned medium was the same. As another measure of LTGF-beta1-induced cellular response, the expression of mRNA for pro alpha1(I) of type I collagen was also evaluated. A marked increase in expression of this transcript in MS-9 cells in response to LTGF-beta1 was observed. To further examine the possible correlation between the large IGF-II/M6P receptors and cellular responses to LTGF-beta1, expression of IGF-II/M6P receptors at the protein and mRNA levels were evaluated by ligand binding and RT-PCR, respectively. Using (125)I-IGF-II as a ligand, the number of specific IGF-II/M6P receptors was found to be threefold greater on MS-9 than on SR-2 and MS cells. This finding was consistent with the level of IGF-II/M6P receptor mRNA detected by RT-PCR in MS-9 cells. In conclusion, the result of this study shows a direct link between large but not small IGF-II/M6P receptors on MS-9 cells and their response to LTGF-beta1.