De novo synthesis of human dermis in vitro in the absence of a three-dimensional scaffold.

Neonatal human dermal fibroblasts cultured in vitro synthesize an organized and physically substantial three-dimensional extracellular matrix, without the addition of exogenous matrix components or synthetic scaffolds. De novo matrix synthesis proceeds in an orderly manner over a 21-d culture period and beyond. Analysis of the fibroblast phenotype, i.e., matrix synthesis by the fibroblasts, suggests that both serum and serum-free conditions are conducive to the production of a human tissue-engineered "dermal equivalent". We report that given the appropriate permissive environment, the fibroblasts establish and grow a tissue in vitro, which bears striking biochemical and physical resemblance to normal human dermis.

Deep dermal fibroblasts contribute to hypertrophic scarring.

Hypertrophic scar (HTS) following thermal injury is a dermal fibroproliferative disorder that leads to considerable morbidity. The development of HTS involves numerous cell types and cytokines with dermal fibroblasts being a key cell. We have previously reported that the phenotype of fibroblasts isolated from HTS was altered compared to fibroblasts from normal skin. In this study, normal skin was horizontally sectioned into five layers using a dermatome from which fibroblasts were isolated and cultured. Cells from the deeper layers were observed to proliferate at a slow rate, but were morphologically larger. In ELISA and FACS assays, cells from the deeper layers produced more TGF-beta1 and TGF-beta1 producing cells were higher. In quantitative RT-PCR, the cells from the deeper layers had higher CTGF and HSP47 mRNA levels compared to those from superficial layers. In western blot, FACS and collagen gel assays, fibroblasts from the deeper layers produced more alpha-smooth muscle actin (alpha-SMA), had higher alpha-SMA positive cells and contracted collagen gels more. Fibroblasts from the deeper layers were also found to produce more collagen, but less collagenase by mass spectrometry and collagenase assay. Interestingly, cells from the deeper layers also produced more of the proteoglycan, versican, but less decorin. Taken together, these data strongly demonstrate that fibroblasts from the deeper layers of the dermis resemble HTS fibroblasts, suggesting that the deeper layer fibroblasts may be critical in the formation of HTS.

Improved scar in postburn patients following interferon-alpha2b treatment is associated with decreased angiogenesis mediated by vascular endothelial cell growth factor.

Hypertrophic scar (HTS) after thermal injury is a dermal fibroproliferative disorder, which leads to considerable morbidity. Previous clinical studies from our laboratory have suggested that interferon-alpha2b (IFN-alpha2b) improves scar quality as a result of the suppression of fibroblast function. More recently, our work has demonstrated that the improvement of scar in patients with HTS after IFN-alpha2b treatment may be associated with a decreased number of fibrocytes and/or altered fibrocyte function. In this study, we report that the improvement of HTS after IFNalpha-2b treatment may be associated with a decrease in angiogenesis. Using immunohistochemistry, we demonstrate an increase in angiogenesis in HTS compared to normal skin, and also show an increase in the expression of vascular endothelial cell growth factor (VEGF) in HTS. Subsequently, we demonstrate a significant reduction in angiogenesis in HTS tissue from patients after treatment with systemic IFN-alpha2b. By using a [3H] thymidine incorporation assay, we demonstrate that IFN-alpha2b suppresses the proliferation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. In addition, IFN-alpha2b inhibits VEGF-induced proliferation and tube formation by using HUVECs. All these effects may be a result of the blocking of VEGF receptor expression on endothelial cells by IFN-alpha2b. Taken together with previous results, the present study suggests that the improvement of scar quality in HTS patients after IFN-alpha2b treatment may also be associated with decreased angiogenesis in HTS. The current in vitro results may provide some insights into the scar improvement that is seen with systemic IFN-alpha2b treatment.

Increased severity of bleomycin-induced skin fibrosis in mice with leukocyte-specific protein 1 deficiency.

Fibrosis is a complex process resulting from persistent inflammation following tissue damage. It involves the interaction of numerous cell types, soluble mediators, and extracellular matrix. Recently, a newly identified cell type, the fibrocyte, has been reported to contribute to wound healing and to fibrotic conditions such as hypertrophic scarring. Previously, we established leukocyte-specific protein 1 (LSP1) as a new marker for fibrocytes. In the present study, we examined the biological role of LSP1 in the development of skin fibrosis using bleomycin in an Lsp1(-/-) mice. These animals showed a significant increase in fibrosis, with increased thickness of the skin and collagen content. The skin in Lsp1(-/-) mice injected with bleomycin had higher densities of neutrophils, macrophages, and fibrocytes. In accordance with the increased leukocyte infiltration, the expression levels of macrophage-derived chemokines, transforming growth factor-beta1, and connective tissue growth factor were all upregulated in Lsp1(-/-) mice. These results demonstrate that the absence of LSP1 promotes fibrosis in the skin. The most likely mechanism for this effect seems to be through an increase in leukocyte infiltration, leading to locally elevated synthesis and the release of chemokines and growth factors.

Chimeras of bacterial translation factors Tet(O) and EF-G.

Ribosomal protection proteins (RPPs) confer bacterial resistance to tetracycline by releasing this antibiotic from ribosomes stalled in protein synthesis. RPPs share structural similarity to elongation factor G (EF-G), which promotes ribosomal translocation during normal protein synthesis. We constructed and functionally characterized chimeric proteins of Campylobacter jejuni Tet(O), the best characterized RPP, and Escherichia coli EF-G. A distinctly conserved loop sequence at the tip of domain 4 is required for both factor-specific functions. Domains 3-5: (i) are necessary, but not sufficient, for functional specificity; and (ii) modulate GTP hydrolysis by EF-G, while minimally affecting Tet(O), under substrate turnover conditions.

Improvement in postburn hypertrophic scar after treatment with IFN-alpha2b is associated with decreased fibrocytes.

Hypertrophic scar (HTS) following thermal injury is a dermal fibroproliferative disorder that leads to considerable morbidity. Previous clinical studies from our laboratory have suggested that interferon-alpha2b (IFN-alpha2b) improves scar quality as a result of suppression of fibroblast functions. Fibrocytes, which constitute a unique cell population, have recently been reported to contribute to wound healing and to a variety of fibrotic conditions, including HTS. Therefore, we hypothesize that improvement of scar in HTS patients after IFN-alpha2b treatment may be associated with a decreased number of fibrocytes or altered fibrocyte function. Using flow cytometry, immunofluorescent staining, and confocal microscopy, we demonstrate here that the marker protein leukocyte-specific protein 1 (LSP1) is stably expressed by fibrocytes for at least 2 months, whereas other potential fibrocyte markers, such as CD34 and CD45, gradually disappear. Using dual staining immunohistochemistry for LSP1 and procollagen, we demonstrated a significant reduction in numbers of fibrocytes in HTS tissue from patients after treatment with systemic IFN-alpha2b. IFN-alpha2b was shown to abolish fibrocyte differentiation from peripheral blood mononuclear cells (PBMCs) in vitro in a dose-dependent fashion. In addition, IFN-alpha2b inhibits proliferation of fibrocytes and T lymphocytes and reduces transforming growth factor-beta (TGF-beta)-mediated alpha-smooth muscle actin (alpha-SMA) expression in fibrocytes. Taken together with our previous study in which we showed that fibrocytes could indirectly regulate dermal fibroblasts in burn patients, the present study suggests that the improvement of scar quality in HTS patients after IFN-alpha2b treatment is associated with decreased numbers and activities of fibrocytes.

Accelerated wound healing in leukocyte-specific, protein 1-deficient mouse is associated with increased infiltration of leukocytes and fibrocytes.

Wound healing is a complex process involving the integrated actions of numerous cell types, soluble mediators, and ECM. Recently, a newly identified cell type, the fibrocyte, has been reported to contribute to wound healing and fibrotic conditions such as hypertrophic scarring. We previously established leukocyte-specific protein 1 (LSP1) as a marker for fibrocytes. LSP1 is an F-actin binding protein and substrate of p38 mitogen-activated protein kinase and protein kinase C, and has been reported to be important in leukocyte chemotaxis. We examine the biological roles of LSP1 in skin wound healing using Lsp1(-/-) null mice. These animals showed accelerated healing of full-thickness skin wounds, with increased re-epithelialization rates, collagen synthesis, and angiogenesis. Healing wounds in Lsp1(-/-) mice had higher densities of neutrophiles, macrophages, and fibrocytes. Along with increased leukocyte infiltration, levels of macrophage-derived chemokine expression, TGF-beta1, and VEGF were all up-regulated. These results demonstrate that the absence of LSP1 promotes healing of skin wounds. The primary mechanism seems to be an increase in leukocyte infiltration, leading to locally elevated synthesis and release of chemokines and growth factors. Further analysis of Lsp1(-/-) mice may suggest ways to improve wound healing and/or treat fibrotic conditions of skin and other tissue.

Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis.

Although chronic wounds are common, treatment for these disabling conditions remains limited and largely ineffective. In this study, we examined the benefit of bone marrow-derived mesenchymal stem cells (BM-MSCs) in wound healing. Using an excisional wound splinting model, we showed that injection around the wound and application to the wound bed of green fluorescence protein (GFP)(+) allogeneic BM-MSCs significantly enhanced wound healing in normal and diabetic mice compared with that of allogeneic neonatal dermal fibroblasts or vehicle control medium. Fluorescence-activated cell sorting analysis of cells derived from the wound for GFP-expressing BM-MSCs indicated engraftments of 27% at 7 days, 7.6% at 14 days, and 2.5% at 28 days of total BM-MSCs administered. BM-MSC-treated wounds exhibited significantly accelerated wound closure, with increased re-epithelialization, cellularity, and angiogenesis. Notably, BM-MSCs, but not CD34(+) bone marrow cells in the wound, expressed the keratinocyte-specific protein keratin and formed glandular structures, suggesting a direct contribution of BM-MSCs to cutaneous regeneration. Moreover, BM-MSC-conditioned medium promoted endothelial cell tube formation. Real-time polymerase chain reaction and Western blot analysis revealed high levels of vascular endothelial growth factor and angiopoietin-1 in BM-MSCs and significantly greater amounts of the proteins in BM-MSC-treated wounds. Thus, our data suggest that BM-MSCs promote wound healing through differentiation and release of proangiogenic factors. Disclosure of potential conflicts of interest is found at the end of this article.

Increased TGF-beta-producing CD4+ T lymphocytes in postburn patients and their potential interaction with dermal fibroblasts in hypertrophic scarring.

The development of hypertrophic scar involves a complex interplay between cells and cytokines. Although the mechanism underlying its pathogenesis is not well understood, a polarized T-helper type 2 immune response has been reported, indicating a role for CD4+ T lymphocytes in hypertrophic scarring. Here, we report an increased frequency of CD4+/transforming growth factor-beta (TGF-beta)-producing T cells in the peripheral blood and hypertrophic scar tissue of burn patients. These cells may play an indirect regulatory role in hypertrophic scar by affecting the functions of dermal fibroblasts. Our results show an increase in cell proliferation and collagen synthesis by dermal fibroblasts treated with medium derived from burn patient CD4+ T lymphocytes but not from the CD4+ T cells of normal subjects. Using confocal microscopy and immunoblotting, we found the level of alpha-smooth muscle actin to be elevated in these treated dermal fibroblasts, which also showed an enhanced ability to contract collagen lattices. TGF-beta levels in medium conditioned by the culture of CD4+ T lymphocytes from burn patients were significantly higher than in the conditioned medium from CD4+ T lymphocytes of normal subjects. In addition, the application of a TGF-beta-neutralizing antibody significantly reduced the effect of burn patient CD4+ T lymphocyte medium on dermal fibroblast proliferation and collagen lattice contraction. Our study suggests that CD4+/TGF-beta-producing T lymphocytes may play an important role in postburn hypertrophic scarring.

An improved procedure for expression and purification of ribosomal protection protein Tet(O) for high-resolution structural studies.

Tetracycline (Tc) is a broad spectrum antibiotic that binds to the A site of the bacterial ribosome inhibiting delivery of aminoacyl-tRNA to the A site for productive protein biosynthesis. Tet(O) is in a class of the ribosomal protection proteins (RPPs) found in many pathogenic bacteria, that dislodges Tc from the A site of 70S ribosome to restore polypeptide elongation and confer Tc resistance to the bacteria. Considerable difficulty has been encountered in overexpressing and purifying Tet(O) from various Escherichia coli strains using lambdaPI, tac or T7 promoters. Here we report molecular cloning, overexpression of His-tagged Tet(O) in E. coli, an improved purification procedure and initial biochemical and biophysical characterization of His-tagged Tet(O).

Fibrocytes from burn patients regulate the activities of fibroblasts.

Wound healing requires an elaborate interplay between numerous cell types that orchestrate a series of regulated and overlapping events. Fibrocytes are a unique leukocyte subpopulation implicated in this process. One role proposed for these cells in wound healing is to synthesize extracellular matrix. Interestingly, using mass spectrometry to quantify hydroxyproline, we discovered that the capacity of fibrocytes from normal subjects or from burn patients to produce collagen is much less than that of dermal fibroblasts. Therefore, we investigated whether fibrocytes could play an indirect, regulatory, role in the healing of burn wounds by affecting the functions of dermal fibroblasts. Dermal fibroblasts treated with medium conditioned by burn patient fibrocytes, but not by those derived from normal subjects, showed an increase in cell proliferation and migration. Using confocal microscopy, flow cytometry, and immunoblotting, we found the level of alpha-smooth muscle actin (alpha-SMA) expression to be increased in these treated dermal fibroblasts, which also showed an enhanced ability to contract collagen lattices. To determine whether these effects could be attributed to transforming growth factor beta (TGF-beta1) or to connective tissue growth factor (CTGF), we measured total TGF-beta1 levels in the conditioned medium by an enzyme-linked immunosorbtion assay and assessed levels of CTGF mRNA and protein in fibroblasts and fibrocytes by reverse transcription-polymerase chain reaction and Western blotting. The results showed significantly higher levels of TGF-beta1 and CTGF produced by burn patient fibrocytes. In addition, the application of a TGF-beta1 neutralizing antibody significantly reduced the effect of burn patient fibrocyte medium on dermal fibroblast proliferation, migration, and collagen lattice contraction. Our results suggest that in healing burn wounds, fibrocytes could regulate the activities of local fibroblasts.

Bone marrow-derived stem cells in wound healing: a review.

Optimum healing of a cutaneous wound requires a well-orchestrated integration of the complex biological and molecular events of cell migration and proliferation, and of extracellular matrix deposition and remodeling. Several studies in recent years suggest that bone marrow derived stem cells such as mesenchymal stem cells, progenitor cells such as endothelial progenitor cells and fibrocytes may be involved in these processes, contributing to skin cells or releasing regulatory cytokines. Stem/progenitor cells may be mobilized to leave the bone marrow, home to injured tissues and participate in the repair and regeneration. Direct injection of bone marrow derived mesenchymal stem cells or endothelial progenitor cells into injured tissues shows improved repair through mechanisms of differentiation and/or release of paracrine factors. Enhanced understanding of these cells may help develop novel therapies for difficult cutaneous conditions such as non-healing chronic wounds and hypertrophic scarring as well as engineering cutaneous substitutes.

Cellular and molecular pathology of HTS: basis for treatment.

Hypertrophic scar and keloids are fibroproliferative disorders of the skin which occur often unpredictably, following trauma and inflammation that compromise cosmesis and function and commonly recur following surgical attempts for improvement. Despite decades of research in these fibrotic conditions, current non-surgical methods of treatment are slow, inconvenient and often only partially effective. Fibroblasts from these conditions are activated to produce extracellular matrix proteins such as collagen I and III, proteoglycans such as versican and biglycan and growth factors, including transforming growth factor-beta and insulin like growth factor I. However, more consistently these cells produce less remodeling enzymes including collagenase and other matrix metalloproteinases, as well as the small proteoglycan decorin which is important for normal collagen fibrillogenesis. Recently, the systemic response to injury appears to influence the local healing process whereby increases in Th2 and possibly Th3 cytokines such as IL-2, IL-4 and IL-10 and TGF-beta are present in the circulating lymphocytes in these fibrotic conditions. Finally, unique bone marrow derived cells including mesenchymal and endothelial stem cells as well as fibrocytes appear to traffic into healing wounds and influence the healing tissue. On this background, clinicians are faced with patients who require treatment and the pathophysiologic basis as currently understood is reviewed for a number of emerging modalities.

Novel mutations in the small leucine-rich repeat protein/proteoglycan (SLRP) genes in high myopia.

The importance of the genetic component in high myopia has been well established in population and family studies, but only a few candidate genes have been explored to date. The extracellular matrix small leucine-rich repeat proteins/proteoglycans (SLRPs) regulate collagen fibril diameter and spacing. Given their role in extracellular matrix assembly and expression in the eye, they are likely to regulate its shape and size. Analysis of 85 English and 40 Finnish subjects with high myopia (refractive error of -6 diopters [D] or greater) resulted in 23 sequence variations in four SLRP genes, LUM, FMOD, PRELP, and OPTC. We observed higher number of variations in OPTC in English patients than in controls (p=0.042), and a possibly protective variation in LUM (c.893-105G>A) with p-value of 0.0043. Two intronic variations, six nonsynonymous and one synonymous amino acid changes, were not found in any of the nonmyopic controls. Five changes were detected in opticin, Thr177Arg, Arg229His, Arg325Trp, Gly329Ser, and Arg330His, and all but one (Arg229His) were shown to cosegregate with high myopia in families with incomplete penetrance. A homology model for opticin revealed that Arg229His and Arg325Trp are likely to disrupt the protein structure, and PolyPhen analysis suggested that Thr177Arg, Arg325Trp, and Gly329Ser changes may be damaging. A Leu199Pro change in lumican and Gly147Asp and Arg324Thr variations in fibromodulin are located in the highly conserved leucine-rich repeat (LRR) domains. This study provides new insight into the genetics of high myopia, suggesting that sequence variations in the SLRP genes expressed in the eye may be among the genetic risk factors underlying the pathogenesis of high myopia.

Structural correlations in the family of small leucine-rich repeat proteins and proteoglycans.

The family of small leucine-rich repeat proteins and proteoglycans (SLRPs) contains several extracellular matrix molecules that are structurally related by a protein core composed of leucine-rich repeats (LRRs) flanked by two conserved cysteine-rich regions. The small proteoglycan decorin is the archetypal SLRP. Decorin is present in a variety of connective tissues, typically "decorating" collagen fibrils, and is involved in important biological functions, including the regulation of the assembly of fibrillar collagens and modulation of cell adhesion. Several SLRPs are known to regulate collagen fibrillogenesis and there is evidence that they may share other biological functions. We have recently determined the crystal structure of the protein core of decorin, the first such determination of a member of the SLRP family. This structure has highlighted several correlations: (1) SLRPs have similar internal repeat structures; (2) SLRP molecules are far less curved than an early model of decorin based on the three-dimensional structure of ribonuclease inhibitor; (3) the N-terminal and C-terminal cysteine-rich regions are conserved capping motifs. Furthermore, the structure shows that decorin dimerizes through the concave surface of its LRR domain, which has been implicated previously in its interaction with collagen. We have established that both decorin and opticin, another SLRP, form stable dimers in solution. Conservation of residues involved in decorin dimerization suggests that the mode of dimerization for other SLRPs will be similar. Taken together these results suggest the need for reevaluation of currently accepted models of SLRP interaction with their ligands.

Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM.

TraM is essential for F plasmid-mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. Here we report the 1.40 A crystal structure of the TraM core tetramer (TraM58-127). TraM58-127 is a compact eight-helical bundle, in which the N-terminal helices from each protomer interact to form a central, parallel four-stranded coiled-coil, whereas each C-terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen-bonded arrangement within the central four-helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.

Polarized Th2 cytokine production in patients with hypertrophic scar following thermal injury.

Following thermal injury, hypertrophic scar (HSc) is a frequent and severe form of fibrosis of the skin, which limits movement and compromises the cosmetic appearance and function of the skin. Prolonged pruritus and dysesthesia are also common problems in the previously injured, fibrotic tissues, as current understanding of the pathogenesis is limited, and few effective therapies exist, as with other fibroproliferative disorders (FPD). To investigate the role of T cells and their cytokines in the development of HSc, intracellular cytokine synthesis of circulating T cells was measured serially in burn patients using flow cytometry from the time of injury to over a 1-year period during which many patients developed HSc. Within 1 month of injury, low interferon-gamma (IFN-gamma)-positive T cells (Th1) were found in association with low interleukin-12 (IL-12) and absent IFN-gamma cytokine levels in the serum. IL-4-positive Th 2 cells, however, were significantly increased compared with normal controls by 2 months postinjury. In burn patients with HSc, serum IL-10 and transforming growth factor-beta (TGF-beta) levels were also significantly increased early after burn injury in patients who later developed HSc compared with normal volunteers and with a subset of burn patients who did not develop HSc, before returning to normal levels after 6 months. Activated peripheral blood mononuclear cells (PBMC) demonstrated that mRNA for IFN-gamma was present only in normal volunteers or patients without HSc but was undetectable in HSc patients. IL-4 mRNA levels were increased in the PBMCs of burn patients with HSc. In HSc tissues, IL-4 mRNA was increased, whereas, IFN-gamma mRNA was reduced compared with normal skin and mature scar. Increased CD3(+) and CD4(+) cells were present in HSc tissues compared with normal skin and were coexpressed with the fibrogenic cytokine TGF-beta. These longitudinal studies in human patients with HSc suggest that fibrosis in the skin is associated with a polarized Th2 systemic response to injury that leads to increased T cells and their Th2 fibrogenic cytokines in tissues and the development of fibrosis and HSc.

Crystal structure of the biglycan dimer and evidence that dimerization is essential for folding and stability of class I small leucine-rich repeat proteoglycans.

Biglycan and decorin are two closely related proteoglycans whose protein cores contain leucine-rich repeats flanked by disulfides. We have previously shown that decorin is dimeric both in solution and in crystal structures. In this study we determined whether biglycan dimerizes and investigated the role of dimerization in the folding and stability of these proteoglycans. We used light scattering to show that biglycan is dimeric in solution and solved the crystal structure of the glycoprotein core of biglycan at 3.40-angstroms resolution. This structure reveals that biglycan dimerizes in the same way as decorin, i.e. by apposition of the concave inner surfaces of the leucine-rich repeat domains. We demonstrate that low concentrations of guanidinium chloride denature biglycan and decorin but that the denaturation is completely reversible following removal of the guanidinium chloride, as assessed by circular dichroism spectroscopy. Furthermore, the rate of refolding is dependent on protein concentration, demonstrating that it is not a unimolecular process. Upon heating, decorin shows a single structural transition at a T(m) of 45-46 degrees C but refolds completely upon cooling to 25 degrees C. This property of decorin enabled us to show both by calorimetry and light scattering that dimer to monomer transition coincided with unfolding and monomer to dimer transition coincided with refolding; thus these processes are inextricably linked. We further conclude that folded monomeric biglycan or decorin cannot exist in solution. This implies novel interrelated functions for the parallel beta sheet faces of these leucine-rich repeat proteoglycans, including dimerization and stabilization of protein folding.