Trypsin,Tryptase, and Thrombin Polarize Macrophages towards a Pro-Fibrotic M2a Phenotype

For both wound healing and the formation of a fibrotic lesion, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes and pro-fibrotic M2a macrophages, which together with fibroblasts form scar tissue. Monocytes can also differentiate into classically activated M1 macrophages and alternatively activated M2 macrophages. The proteases thrombin, which is activated during blood clotting, and tryptase, which is released by activated mast cells, potentiate fibroblast proliferation and fibrocyte differentiation, but their effect on macrophages is unknown. Here we report that thrombin, tryptase, and the protease trypsin bias human macrophage differentiation towards a pro-fibrotic M2a phenotype expressing high levels of galectin-3 from unpolarized monocytes, or from M1 and M2 macrophages, and that these effects appear to operate through protease-activated receptors. These results suggest that proteases can initiate scar tissue formation by affecting fibroblasts, fibrocytes, and macrophages.     

Trypsin Potentiates Human Fibrocyte Differentiation

Trypsin-containing topical treatments can be used to speed wound healing, although the mechanism of action is unknown. To help form granulation tissue and heal wounds, monocytes leave the circulation, enter the wound tissue, and differentiate into fibroblast-like cells called fibrocytes. We find that 20 to 200 ng/ml trypsin (concentrations similar to those used in wound dressings) potentiates the differentiation of human monocytes to fibrocytes in cell culture. Adding trypsin inhibitors increases the amount of trypsin needed to potentiate fibrocyte differentiation, suggesting that the potentiating effect is dependent on trypsin proteolytic activity. Proteases with other site specificities such as pepsin, endoprotease GluC, and chymotrypsin do not potentiate fibrocyte differentiation. This potentiation requires the presence of albumin in the culture medium, and tryptic fragments of human or bovine albumin also potentiate fibrocyte differentiation. These results suggest that topical trypsin speeds wound healing by generating tryptic fragments of albumin, which in turn potentiate fibrocyte differentiation.     

Appearance of tenascin in healing skin of the mouse: possible involvement in seaming of wounded tissues

Distribution of the extracellular matrix glycoprotein tenascin during wound healing in mouse skin was studied immunohistochemically. Within 24 hours after wounding, and preceding the formation of granulation tissue, tenascin appeared in the basement membranes beneath epidermis and hair follicles adjacent to the wound edges and in the wounded edges of cutaneous muscle layer. Granulation tissue began to form in the wound space at about 1-2 days and was immediately covered by epidermis. Tenascin first appeared in the periphery of the granulation tissue beneath healing epidermis and around the wounded edges of cutaneous muscle layer. Then the tenascin-positive area extended into the inner region of granulation tissue. At about 5-7 days, all of the granulation tissue was intensely stained with anti-tenascin serum. Tenascin immunoreactivity decreased as granulation tissue was replaced with reconstructed dermal tissue at 7-14 days. In most cases, tenascin staining persisted longest in the dermis beneath the healing epidermis and at the juncture of healing edges of cutaneous muscle layer. It disappeared at about 10-14 days after wounding. These findings suggest that tenascin may play an important role in the seaming of wounded tissues.     

Differential stimulation of collagenase and chemotactic activity in fibroblasts derived from rat wound repair tissue and human skin by growth factors

Epidermal growth factor and cartilage-derived basic fibroblast growth factor (EGF and CD-bFGF) are mitogens shown to increase the rate of wound repair in animal models. In addition to being a mitogen for granulation tissue, CD-bFGF stimulates the recruitment of cells to the wound site. CD-bFGF and a closely-related chondrosarcoma-derived fibroblast growth factor stimulated chemotaxis of granulation tissue cells in vitro, each factor having a maximum activity at a concentration of 55 pM. Epidermal growth factor was also a potent chemoattractant for rat granulation tissue fibroblasts; however, maximum activity was obtained at 1.7 nM. Cells from all stages of wound repair were chemotactically responsive to these factors, but there was some attenuation of the response to bFGF in cells derived from fully-organized day 28 granulation tissue. Collagenase-catalyzed restructuring of collagen, an additional significant feature of wound repair, is probably critical to cell movement in an extracellular matrix. Cells derived from organizing (6-day old) sponge granulation tissue secreted latent collagenase constitutively in vitro. In the presence of serum, the production of collagenase was stimulated three-four fold by 1.8 nM bFGF derived either from cartilage or chondrosarcoma. When serum was present, as at a wound site, collagenase production was not enhanced by the addition of EGF. Cells from fully organized, day 21 sponge granulation tissue did not secrete latent collagenase constitutively and could not be stimulated to do so by the addition of EGF, bFGF, or phorbol ester. Human skin fibroblast collagenase production was also stimulated by bFGF and was refractory to EGF. While both classes of growth factor have the ability to promote wound healing, the varying responses they elicit in cell populations from the wound site emphasize the different pathways of cellular activation.     

Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors

Wound contraction is one function of granulation tissue which is critical to repair. This study compares the ability of fibroblast-like cells derived from granulation tissue of various ages to contract a tissue equivalent, or a collagen gel, and examines the influence of growth factors implicated in wound repair on collagen gel contraction by these different cell populations. Cells from older granulation tissue (21 and 28 days) have an enhanced ability to contract a tissue equivalent when compared to cells from younger granulation tissue (7 and 14 days) or normal rat skin fibroblasts. Transforming growth factor-beta 1 (TGF-beta 1) enhanced contractility most in those cells which had a greater basal contractile ability. While basic fibroblast growth factor (bFGF) alone had moderately stimulatory effects at low doses (0.1-1.0 ng/ml), higher doses (greater than or equal to 10 ng/ml) inhibited basal contraction. Pretreatment with bFGF followed by exposure to TGF-beta 1, with or without the continued presence of bFGF, delayed gel contraction by cells from skin and early granulation tissue, but bFGF enhanced TGF-beta 1 activity in highly contractile cells. Transforming growth factor-alpha moderately enhanced contraction by cells from older granulation tissue. While both TGF-beta 1 and bFGF enhanced wound repair, their differential effects on the fibroblast-like cell derived from granulation tissue of different ages suggest that phenotypic differences exist between these cell populations. In addition, our results predict significant interactions between polypeptide cytokines at the site of repair.     

Ascorbic acid and hydroxyproline levels in the serum and granulation tissue of rats with aseptic and infected wounds

Hydroxyproline and ascorbic acid were measured in wound tissues and ascorbic acid was determined in the blood sera of 230 mature male Wistar rats with aseptic and infected surface wounds on days 1-10 (daily), 12, and 15. The principal morphologic characteristics of the granulation tissue were assessed by semiquantitative methods. An infected wound is characterized by increased hydroxyproline levels in the granulation tissue and elevated ascorbic acid concentration in the blood serum. The granulation tissue ascorbic acid level augments during the first nine days in case of an aseptic wound and during twelve days in case of an infected wound. Morphologic and biochemical correlations are indicative of a relationship between the ascorbic acid level on the one hand and granulation tissue vascularization and fibroblast proliferation on the other.     

Topical application of aminopeptidase N-neutralizing antibody accelerates wound closure

Upon release from keratinocytes, 14-3-3 sigma (also known as stratifin) acts on the dermal fibroblast and modulates its production of extracellular matrix proteins. Subsequent to the recent identification as a receptor responsible for stratifin-mediated matrix turnover in dermal fibroblasts, aminopeptidase N has been implicated in the regulation of epidermal?Cdermal communication and expression of key matrix proteases and adhesion molecules. In light of the growing importance of aminopeptidase N in modulation of the fibroblast phenotype, the present study evaluates the potential of targeting the ectoenzyme in cutaneous repair, and demonstrates that neutralization of aminopeptidase N led to acceleration of wound closure. This was attributed to at least in part an increase of collagen deposition and fibroblast contractility in the granulation tissue. These findings confirmed the important role of aminopeptidase N in post-injury tissue remodeling and wound contraction.     

Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.

BACKGROUND: The host response to tissue injury requires a complex interplay of diverse cellular, humoral, and connective tissue elements. Fibroblasts participate in this process by proliferating within injured sites and contributing to scar formation and the longterm remodeling of damaged tissue. Fibroblasts present in areas of tissue injury generally have been regarded to arise by recruitment from surrounding connective tissue; however this may not be the only source of these cells. MATERIALS AND METHODS: Long-term culture of adherent, human, and murine leukocyte subpopulations was combined with a variety of immunofluorescence and functional analyses to identify a blood-borne cell type with fibroblast-like properties. RESULTS: We describe for the first time a population of circulating cells with fibroblast properties that specifically enter sites of tissue injury. This novel cell type, termed a "fibrocyte," was characterized by its distinctive phenotype (collagen+/vimentin+/CD34+), by its rapid entry from blood into subcutaneously implanted wound chambers, and by its presence in connective tissue scars. CONCLUSIONS: Blood-borne fibrocytes contribute to scar formation and may play an important role both in normal wound repair and in pathological fibrotic responses.     

A role for endogenous glucocorticoids in wound repair

Exogenous glucocorticoids are known to inhibit wound repair, but the roles and mechanisms of action of endogenous glucocorticoids during the healing process are as yet unknown. Therefore, we wounded mice expressing a DNA-binding-defective mutant version of the glucocorticoid receptor (GR^dim mice) and also analysed fibroblasts from these animals in vitro. We found a remarkably enlarged granulation tissue with a high fibroblast density in GR^dim mice. This difference is likely to result from an increased migratory and proliferative capacity of GR^dim fibroblasts and from elevated expression levels of soluble factors involved in granulation tissue formation in wounds of GR^dim mice. In spite of the larger granulation tissue seen in early wounds, late wounds appeared normal, most likely due to an enhanced ability of GR^dim fibroblasts to contract collagen. These results demonstrate an as yet unidentified role of endogenous glucocorticoids in the regulation of wound repair.     

Electron-radioautographic study of the effect of potassium orotate on RNA and protein synthesis in fibroblasts during experimental wound healing

Electron microscopic radioautography with the use of the double-labeling technique was applied to study potassium orotate influence on RNA and protein synthesis in fibroblasts of intact mouse skin and in granulation tissue on days 3 and 7 of the healing of a musculocutaneous wound. Experimental animals received potassium orotate per os in a daily dose of 0.2 g/kg immediately after operation. Intact animals received the drug for 3 days. According to the assay data potassium orotate did not potentiate the protein-synthesizing activity of an individual cell but apparently activated the proliferation of wound fibroblasts.     

Transgenic mice reveal novel activities of growth hormone in wound repair, angiogenesis, and myofibroblast differentiation

An increasing number of patients are being treated with growth hormone (GH) for the enhancement of body growth but also as an anti-aging strategy. However, the side effects of GH have been poorly defined. In this study we determined the effect of GH on wound repair and its mechanisms of action at the wound site. For this purpose, we performed wound healing studies in transgenic mice overexpressing GH. Full thickness incisional and excisional wounds of transgenic animals developed extensive, highly vascularized granulation tissue. However, wound bursting strength was not increased. Wound closure was strongly delayed as a result of enhanced granulation tissue formation and impaired wound contraction. The latter effect is most likely due to a significantly reduced number of myofibroblasts at the wound site. By using in vitro studies with stressed collagen lattices, we identified GH as an inhibitor of transforming growth factor beta-induced myofibroblast differentiation, resulting in a reduction in fibroblast contractile activity. These results revealed novel roles of GH in angiogenesis and myofibroblast differentiation, which are most likely not mediated via insulin-like growth factors at the wound site. Furthermore, our data suggested that systemic GH treatment is detrimental for wound healing in healthy individuals.     

Quantitative analysis of collagen gel contractile forces generated by dermal fibroblasts and the relationship to cell morphology

The force generated in granulation tissue during wound contraction is thought to be cell mediated; however, it is unclear whether contractile forces are generated by fibroblast locomotion or contraction of myofibroblasts. To help clarify this question the force of this contraction can now be determined accurately in a human dermal fibroblast collagen lattice system using a novel instrument known as a Culture Force Monitor. Three distinct phases of contraction of such collagen gels could be identified over the first 24 hours. Most of the force generated by human dermal fibroblasts was produced during the first stage in parallel with cell attachment and associated changes in cell shape, and the appearance of cell processes. During this initial 24 hours no evidence could be found for the presence of myofibroblasts, but stereoscopic and electron microscopic analysis at a range of time points indicated that migratory fibroblasts were present in the system. Comparison of the contraction profiles of cells extracted from other tissues (tendon and articular cartilage), and extracted by different means from the same tissue specimen, indicated that different populations of fibroblasts can be distinguished on the basis of their pattern of contractions. It would seem that most of the force generated in this model is a result of fibroblast attachment and movement within the collagen lattice. Furthermore, different groups of fibroblasts, even within the same tissue, may vary in their contraction (hence locomotory) activity. © 1996 Wiley-Liss, Inc.     

Inhibition by the soluble syndecan-1 ectodomains delays wound repair in mice overexpressing syndecan-1

Wound repair is a tightly regulated process stimulated by proteases, growth factors, and chemokines, which are modulated by heparan sulfate. To characterize further the role of the heparan sulfate proteoglycan syndecan-1 in wound repair, we generated mice overexpressing syndecan-1 (Snd/Snd) and studied dermal wound repair. Wound closure, reepithelialization, granulation tissue formation, and remodeling were delayed in Snd/Snd mice. Soluble syndecan-1 was increased, and shedding was prolonged in wounds from Snd/Snd mice. Excess syndecan-1 increased the elastolytic activity of wound fluids. Additionally, cells in the granulation tissue and keratinocytes at wound edges showed markedly reduced proliferation rates in Snd/Snd mice. Skin grafting experiments between Snd/Snd and control mice indicated that the slower growth rate was mainly due to a soluble factor in the Snd/Snd mouse skin. Syndecan-1 immunodepletion and further degradation experiments identified syndecan-1 ectodomain as a dominant negative inhibitor of cell proliferation. These studies indicate that shed syndecan-1 ectodomain may enhance proteolytic activity and inhibit cell proliferation during wound repair.     

Spectroscopic and histological evaluation of wound healing progression following Low Level Laser Therapy (LLLT)

The present study focuses on the evaluation of the effect of He‐Ne laser on tissue regeneration by monitoring collagen synthesis in wound granulation tissues in Swiss albino mice using analysis of laser induced fluorescence (LIF) and light microscopy techniques. The spectral analyses of the wound granulation tissues have indicated a dose dependent increase in collagen levels during the post‐wounding days. The histological examinations on the other hand have also shown a significant increase in collagen deposition along with the reduced edema, leukocytes, increased granulation tissue, and fibroblast number in the optimal laser dose treated group compared to the non‐illuminated controls. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

创伤合并海水浸泡后愈合过程的病理学观察

目的观察创伤合并海水浸泡后对伤口愈合时间的影响及愈合过程中的病理学改变。方法以大鼠为实验动物,建立背部双侧圆形创伤模型,创后随机分为对照组和实验组,每组50只,实验组创后置海水中浸泡30min。观察2组创面的愈合生长状况。观察伤口局部愈合过程中的病理学改变,应用免疫组化方法观察愈合过程中伤口修复细胞增殖指数及微血管密度变化。结果对照组平均愈合时间为（12.0±1.0）d,而实验组的平均愈合时间为（14.3±0.8）d;两组在肉芽组织形成时间上以及修复细胞增殖能力上存在差异。结论创伤合并海水浸泡可导致伤口愈合时间延迟,加重创伤局部的炎症反应、延缓肉芽组织形成时间。     

Epidermal growth factor increases collagen production in granulation tissue by stimulation of fibroblast proliferation and not by activation of procollagen genes.

The effects of epidermal growth factor (EGF) on granulation-tissue formation and collagen-gene expression were studied in experimental sponge-induced granulomas in rats. After daily administration of 5 micrograms of EGF into the sponge, total RNA was extracted from the ingrown granulation tissue at days 4 and 7 and analysed by Northern hybridization for the contents of mRNAs for types I and III procollagens. EGF treatment increased procollagen mRNA, particularly at day 4. To determine whether this elevation was due to increased proliferation of collagen-producing fibroblasts or to activation of collagen-gene expression in these cells, fibroblast cultures were started from granulation tissue and treated with EGF. These experiments confirmed that EGF is a potent mitogen for granuloma fibroblasts in a dose-dependent manner. The effect of EGF treatment on radioactive hydroxyproline production in cultured cells was inhibitory. The decreased rate of collagen synthesis was also indicated by decreased amounts of procollagen mRNAs. The results suggest that the stimulation of wound healing and collagen production by EGF is due to increased fibroblast proliferation, and not to increased expression of type I and III procollagen genes.     

Autoradiographic study of the rate of collagen synthesis under conditions of stimulation of the wound process]

A study was made of the rate of the tropocollagen synthesis by the granulation tissue fibroblasts and of its passage into the intercellular space in control animals and under conditions of stimulation of the wound process by potassium orotate, one of the pyrimidine series derivatives. It appeared that the process of tropocollagen synthesis became accelerated under the effect of the stimulant; collagen fiber precursor appeared in the intercellular space earlier than in control and became included into the fibrous structures of the granulation tissue, this correlating with the intensification of the RNA synthesis in the fibroblast nuclei and an accelerated passage of the newly-synthesized RNA from the nucleus into the cell cytoplasm under analogous conditions. There was noted no sharp excess of collagen in the granulation tissue of animals given potassium orotate.     

Peripheral blood fibrocytes: differentiation pathway and migration to wound sites

Fibrocytes are a distinct population of blood-borne cells that display a unique cell surface phenotype (collagen I+/CD11b+/CD13+/CD34+/CD45RO+/MHC class II+/CD86+) and exhibit potent immunostimulatory activities. Circulating fibrocytes rapidly enter sites of tissue injury, suggesting an important role for these cells in wound repair. However, the regulatory processes that govern the differentiation of blood-borne fibrocytes and the mechanisms that underlie the migration of these cells to wound sites are currently not known. We report herein that ex vivo cultured fibrocytes can differentiate from a CD14+-enriched mononuclear cell population and that this process requires contact with T cells. Furthermore, we demonstrate that TGF-beta1 (1-10 ng/ml), an important fibrogenic and growth-regulating cytokine involved in wound healing, increases the differentiation and functional activity of cultured fibrocytes. Because fibrocytes home to sites of tissue injury, we examined the role of chemokine/chemokine receptor interactions in fibrocyte trafficking. We show that secondary lymphoid chemokine, a ligand of the CCR7 chemokine receptor, acts as a potent stimulus for fibrocyte chemotaxis in vitro and for the homing of injected fibrocytes to sites of cutaneous tissue injury in vivo. Finally, we demonstrate that differentiated, cultured fibrocytes express alpha smooth muscle actin and contract collagen gels in vitro, two characteristic features of wound-healing myofibroblasts. These data provide important insight into the control of fibrocyte differentiation and trafficking during tissue repair and significantly expand their potential role during wound healing.     

Vanadate ingestion increases the gain in wound breaking strength and leads to better organized collagen fibers in rats during healing

Repair of incision wounds closed by suturing is evaluated by the progressive gain in wound breaking strength. Previously the closure of open wounds in rats ingesting vanadate, an inhibitor of tyrosine phosphate phosphatases, was shown to occur with deposition of more uniformly organized collagen fiber bundles. The hypothesis of this study was that deposition of more uniformly organized collagen fibers would enhance the gain in wound breaking strength of incisional wounds. Six adult rats received vanadate-supplemented saline drinking water for 1 week before placement of two 6-cm, parallel, suture-closed wounds on their backs. Six control rats received identical wounds and were given saline drinking water. The drinking water regimen was continued for 1 week after wounding, and then wound strength was tested with a tensiometer and tissue samples were obtained for histologic evaluation. Wound breaking strength doubled in vanadate-treated rats compared with controls. Bright-field and polarized light microscopy showed that the connective tissue matrix of granulation tissue from control rats was oriented perpendicular to the surface of the skin. In contrast, the connective tissue matrix of granulation tissue from vanadate-treated rats was oriented parallel to the skin surface. The gap in granulation tissue between the edges of the wounds in the vanadate-treated rats was greater than that in controls. Electron microscopy showed that wounds in the vanadate-treated contained uniform collagen fibers that were 20 percent greater in diameter and more evenly spaced than they were in controls. It is proposed that these changes in the organization of collagen fibers within incisional wounds were responsible for the increased wound breaking strength observed in rats ingesting vanadate.     

Matrix metalloproteinase inhibitor GM 6001 attenuates keratinocyte migration, contraction and myofibroblast formation in skin wounds

In this study, we examined the impact of matrix metalloproteinases (MMP) on epithelialization, granulation tissue development, wound contraction, and alpha-smooth muscle actin (ASMA) expression during cutaneous wound repair through systemic administration of the synthetic broad-spectrum MMP inhibitor GM 6001 (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide). Four full-thickness excisional wounds (50 mm2) on the back of 22 young female Sprague-Dawley rats, 12 treated with GM 6001 100 mg/kg and 10 with vehicle, were allowed to heal by secondary intention. GM 6001-treated wounds were minimally resurfaced with neoepithelium, despite unaltered keratinocyte proliferation in wound edges, whereas control wounds were completely covered with 3-7 cell layers of parakeratinized epithelium on post-wounding day 7. Hydroxyproline concentration, a marker of collagen, and cell proliferation in granulation tissue did not differ significantly between GM 6001-treated and control groups. Impaired wound contraction (P < 0.01) was associated with a dramatic reduction of ASMA-positive myofibroblasts in granulation tissue of GM 6001 wounds. This was not due to GM6001 blocking transforming growth factor-beta1 (TGF-beta1)-induced myofibroblast differentiation since GM 6001 did not inhibit TGF-beta1-induced ASMA expression and force generation in cultured rat dermal fibroblasts. The profound impairment of skin repair by the nonselective MMP inhibitor GM 6001 suggests that keratinocyte resurfacing, wound contraction, and granulation tissue organization are highly MMP-dependent processes.