Modeling the effects of transforming growth factor-β on extracellular matrix alignment in dermal wound repair

We present a novel mathematical model for collagen deposition and alignment during dermal wound healing, focusing on the regulatory effects of transforming growth factor-beta (TGFbeta.) Our work extends a previously developed model which considers the interactions between fibroblasts and an extracellular matrix composed of collagen and a fibrin based blood clot, by allowing fibroblasts to orient the collagen matrix, and produce and degrade the extracellular matrix, while the matrix directs the fibroblasts and control their speed. Here we extend the model by allowing a time varying concentration of TGFbeta to alter the properties of the fibroblasts. Thus we are able to simulate experiments which alter the TGFbeta profile. Within this model framework we find that most of the known effects of TGFbeta, i.e., changes in cell motility, cell proliferation and collagen production, are of minor importance to matrix alignment and cannot explain the anti-scarring properties of TGFbeta. However, we find that by changing fibroblast reorientation rates, consistent with experimental evidence, the alignment of the regenerated tissue can be significantly altered. These data provide an explanation for the experimentally observed influence of TGFbeta on scarring.     

Transforming growth factor-β (TGF-β) activity in urine of patients with glomerulonephritis is related to their renal functional and structural changes

Transforming growth factor-β (TGF-β) has been considered the principal cytokine involved in the pathogenesis of renal fibrosis. In the present study, we evaluated TGF-β activity in occasional samples from 22 normal individuals and 29 patients (11 with focal glomerulosclerosis, 11 with membranous nephropathy, five with Berger disease, one with type I membranoproliferative glomerulonephritis and one with postinfectious glomerulonephritis) using a CCL-64 mink lung cell growth inhibition assay.
A significantly increased urinary TGF-β activity (reported in relation to urine creatinine, Ucreat. and median) was observed in patients with glomerulonephritis compared with normal individuals ( P <0.01). The patients with Berger disease [median (Md)=9.96/10 μg Ucreat.], membranous glomerulonephritis (Md=7.23/10 μg Ucreat.) and focal glomerulosclerosis (Md=16.6/10 μg Ucreat.) showed higher urinary TGF-β than normal individuals (Md=1.09/10 μg Ucreat.) ( P <0.01). We found a positive correlation between the TGF-β activity in the urine of these patients and the incidence of segmental glomerulosclerosis ( r =0.45, P <0.05) and their plasma creatinine levels ( r =0.87, P <0.01). A negative correlation was observed between the TGF-β activity in the urine of these patients and their creatinine clearance ( r =−0.75, P <0.01).
Our data suggest that measurement of urinary TGF-β activity could be a useful non-invasive procedure for the evaluation of renal TGF-β production, permitting the assessment of prognosis and the evaluation of therapeutic efficacy in patients with renal disease.     

Transforming growth factor-β (TGF-β) activity in urine of patients with glomerulonephritis is related to their renal functional and structural changes

SUMMARY: Transforming growth factor-β (TGF-β) has been considered the principal cytokine involved in the pathogenesis of renal fibrosis. In the present study, we evaluated TGF-β activity in occasional samples from 22 normal individuals and 29 patients (11 with focal glomerulosclerosis, 11 with membranous nephropathy, five with Berger disease, one with type I membranoproliferative glomerulonephritis and one with postinfectious glomerulonephritis) using a CCL-64 mink lung cell growth inhibition assay.
A significantly increased urinary TGF-β activity (reported in relation to urine creatinine, Ucreat, and median) was observed in patients with glomerulonephritis compared with normal individuals ( P <0.01). the patients with Berger disease [median (Md) = 9.96/10 μg Ucreat.], membranous glomerulonephritis (Md = 7.23/10 μg Ucreat.) and focal glomerulosclerosis (Md = 16.6/10 μg Ucreat.) showed higher urinary TGF-β than normal individuals (Md = 1.09/10 μg Ucreat.) ( P <0.01). We found a positive correlation between the TGF-β activity in the urine of these patients and the incidence of segmental glomerulosclerosis ( r = 0.45, P <0.05) and their plasma creatinine levels ( r = 0.87, P <0.01). A negative correlation was observed between the TGF-β activity in the urine of these patients and their creatinine clearance ( r =−0.75, P <0.01).
Our data suggest that measurement of urinary TGF-β activity could be a useful non-invasive procedure for the evaluation of renal TGF-β production, permitting the assessment of prognosis and the evaluation of therapeutic efficacy in patients with renal disease.     

Transforming growth factor-β receptor-II up-regulation during wound healing in previously irradiated graft beds in vivo

Wound healing disorders may often present in patients with head and neck cancer after surgical interventions, particularly in preirradiated tissue. Inflammatory changes and the expression of cytokines can lead to induction of fibrosis. The isoforms of the transforming growth factor beta (TGFbeta1-3) play a key role for this process. It has been shown that radiation treatment associated fibrosis is induced by TGFbeta1 and TGFbeta2, although the influence of radiation on the expression of the TGFbeta receptor-II (TGFbetaR-II) involved in the signal transduction of TGFbeta remains elusive. The objective of this in vivo study was to analyze the expression profile of TGFbetaR-II in the graft bed and in the transition area between graft and graft bed after surgery with and without prior radiation treatment to compare with the expression profiles of activated TGFbeta1 and latency-associated peptide. A total of 48 Wistar rats (male, weight 300-500 g) were used in the study. Eighteen rats were irradiated in the neck region (3 x 10 Gy) without transplantation. A free myocutaneous gracilis flap was transplanted in 30 rats, of which 16 animals were preirradiated in the neck region (3 x 10 Gy) and 14 animals were not irradiated at all. Tissue samples were taken postoperatively from the transition area between the graft and the graft bed and from the graft bed itself after 3, 7, 14, and 28 days. Tissue samples were taken from the irradiated neck region and the non-irradiated groin region 0, 4, 7, 11, 14, and 28 days after the end of the exposure. The expression of TGFbetaR-II, activated TGFbeta1 and latency-associated peptide was analyzed immunohistochemically both qualitatively and quantitatively (labeling index). The success rate for graft healing was 75% in the previously irradiated group with 30 Gy, and 86% in the non-irradiated group. Following radiation alone a significantly (p = 0.04) increased TGFbetaR-II expression in the neck was revealed 2-4 weeks following irradiation compared to non-irradiated skin. Whereas only minor differences in TGFbetaR-II expression were observed following surgery between the groups with and without prior radiation in the transition area between the graft and the graft bed, the group undergoing prior radiation and subsequent grafting showed significantly increased expression in the bed compared to the non-preirradiated group with a maximum on postoperative day 7 (week 1, p = 0.003; week 2-4, p < 0.001). In irradiated tissues the up-regulation of TGFbetaR-II expression correlated with an increase of activated TGFbeta1 and latency-associated peptide expression compared to non-irradiated tissues. After irradiation, a significantly increased TGFbetaR-II expression was identified in the irradiated graft bed, which may be the reason for delayed reepithelialization and fibrosis. Exogenous blocking or TGFbetaR-II inhibitors could therefore represent a new therapeutic approach for improving wound healing after preoperative radiotherapy.     

Incisional wound healing in transforming growth factor-β1 null mice

Expression of endogenous transforming growth factor-beta1 is reduced in many animal models of impaired wound healing, and addition of exogenous transforming growth factor-beta has been shown to improve healing. To test the hypothesis that endogenous transforming growth factor-beta1 is essential for normal wound repair, we have studied wound healing in mice in which the transforming growth factor-beta1 gene has been deleted by homologous recombination. No perceptible differences were observed in wounds made in 3-10-day-old neonatal transforming growth factor-beta1 null mice compared to wild-type littermates. To preclude interference from maternally transferred transforming growth factor-beta1, cutaneous wounds were also made on the backs of 30-day-old transforming growth factor-beta1 null and littermate control mice treated with rapamycin, which extends their lifetime and suppresses the inflammatory response characteristic of the transforming growth factor-beta1 null mice. Again, no impairment in healing was seen in transforming growth factor-beta1 null mice. Instead these wounds showed an overall reduction in the amount of granulation tissue and an increased rate of epithelialization compared to littermate controls. Our data suggest that release of transforming growth factor-beta1 from degranulating platelets or secretion by infiltrating macrophages and fibroblasts is not critical to initiation or progression of tissue repair and that endogenous transforming growth factor-beta1 may actually function to increase inflammation and retard wound closure.     

Transforming growth factor-β enhances connective tissue repair in perforated rat mesentery but not peritoneal macrophage chemotaxis

The perforated rat mesentery model was used to study the effect of transforming growth factor-beta (TGF-beta) on connective tissue repair and influx of macrophages into the peritoneal cavity during such repair. Sprague-Dawley rats were laparotomized, and mesenteric wounds were made with a scalpel. A daily intraperitoneal injection of 0.5 microg TGF-beta was given for either 2 or 4 days. After 1 to 10 days, the animals received an intravenous injection of tritium-labeled thymidine before decapitation. Macrophages were collected by peritoneal washing, and the number of closed perforations was counted. Peritoneal cells were quantitated and a labeling index was determined by autoradiography. TGF-beta given for either 2 (p < 0.001) or 4 (p < 0.004) days accelerated closure of perforations on days 3 to 7 after injury. Laparotomy as such significantly increased leukocyte influx (p < 0.004), as well as macrophage-labeling index (p < 0.02). However, TGF-beta did not significantly influence either leukocyte influx or macrophage-labeling index. We concluded that TGF-beta significantly enhances connective tissue repair in this perforated rat mesentery model and that TGF-beta-induced stimulation of repair is not caused by an increased influx of macrophages into the peritoneal cavity.     

Transforming growth factor-β1 reduces expansion of open wounds in the fetal rabbit

Open wounds in the fetal rabbit do not heal by contraction and actually expand between 60% and 90% over a period of 5 days. Experiments were carried out to determine whether transforming growth factor-beta1 can reduce expansion of open wounds in the fetal rabbit. This study was based on the concept that transforming growth factor-beta1 causes differentiation of fibroblasts into contractile fibroblasts or "myofibroblasts." To test this hypothesis, pregnant New Zealand White rabbits underwent laparotomy and hysterotomy on day 24 of gestation. A circular full-thickness cutaneous wound was made on the back of each fetus. After wounding, either vehicle alone or vehicle with transforming growth factor-beta1 was applied topically to the wound site, and each fetus was then returned to the uterus. The hysterotomy and laparotomy were closed in standard fashion. On postoperative day 5, fetuses were harvested by repeat Cesarean section. Wound areas were determined from photographs, calculated as percentage of original wound size, and expressed in square millimeters. In addition, a portion of each wound was fixed and processed for histologic and immunohistochemical analysis. At harvest, the control wounds had expanded by an average of 87% of the original area. In marked contrast, the transforming growth factor-beta1-treated wounds had only expanded an average of 16%. Thus, transforming growth factor-beta1 significantly decreased the area of the open fetal wounds compared with control (p < 0.001). By histologic examination, no significant difference was found between the test group and the control group with regards to inflammation, neovascularization, collagen deposition, elastin content, glycosaminoglycan content, or hyaluronic acid content. Most notably, however, there was an increased density of fibroblasts in the transforming growth factor-beta1-treated group. In addition, immunohistochemical staining with an anti-alpha-smooth muscle actin antibody showed the presence of contractile fibroblasts in the wound margins in the transforming growth factor-beta1-treated group but failed to show any positive-staining fibroblasts in the matrices of the control group. These results indicate that open wounds in the fetal rabbit treated in vivo with transforming growth factor-beta1 were significantly smaller than control wounds. This process appears to result from the recruitment and differentiation of normal dermal fibroblasts into contractile fibroblasts containing alpha-smooth muscle actin.     

Spatial and temporal expression of transforming growth factor-β isoforms during ovine excisional and incisional wound repair

To elucidate the role for transforming growth factor-beta isoforms (beta(1), -beta(2), and -beta(3)) in wound repair, we used isoform-specific antibodies to detect the spatial and temporal expression of the latent and mature/active transforming growth factor-beta isoforms by immunohistochemical localization through 21 days after excisional and incisional wounding of ovine skin. Although incisional and excisional wounds showed similar patterns of transforming growth factor-beta immunoreactivity, we found a differential temporal and spatial expression of the latent and mature transforming growth factor-beta isoforms throughout wound repair. Specifically, 1 day after wounding, there was a marked increase in transforming growth factor-beta isoforms in the epithelium adjacent to the wound, epidermal appendages, and the cells and matrix of the granulation tissue. At this time, transforming growth factor-beta(3) isoform was the most abundant. Most notably, the epidermis adjacent to the wound was intensely immunoreactive for all transforming growth factor-beta isoforms 1 day after injury. However, the migrating epithelium, derived from both the hair follicles and the wound margins, was completely devoid of immunoreactive transforming growth factor-beta until reepithelialization was complete. Within the inflammatory exudate, there was a distinct band of leukocytes that was immunoreactive for transforming growth factor-beta(2) and -beta(3) 1 day after injury and 1 day later for transforming growth factor-beta(1). Although transforming growth factor-beta(1) and -beta(2), latent transforming growth factor-beta(2), transforming growth factor-beta(3), and latent transforming growth factor-beta(3) immunostaining was present in the numerous fibroblasts and other dermal cells, latent transforming growth factor-beta(1) was only associated with the extracellular matrix. In general, immunoreactivity remained high until day 7 after wounding and slowly subsided over time. However, by day 21, immunostaining had not returned to normal and the original wound was replete with immunoreactive fibroblasts and a dense, immunostained extracellular matrix. Thus, although the dynamic presence of transforming growth factor-beta isoforms exemplifies its positive role in the wound repair process, its persistence together with its known potent effects on matrix accumulation, supports its role in scar formation.     

Abrogation of the fibrotic effect of transforming growth factor-β in dermal wound healing

The growth factor, transforming growth factor-beta1, which under normal circumstances promotes wound healing by stimulating local fibroblasts to produce collagen and other extracellular matrix proteins, has also been implicated as the primary causative agent of fibrosis. Because transforming growth factor-beta1 is capable of stimulating its own production by fibroblasts, its normally beneficial effects may become amplified to the point where excess extracellular matrix accumulation occurs, thereby causing abnormal scarring. Therefore, strategies that block or counter the effects of transforming growth factor-beta1 may be useful in preventing or decreasing fibrosis. One such strategy is the use of glucocorticoid steroids such as dexamethasone, which normally have the opposite effect of transforming growth factor-beta1, namely the impairment of wound healing. When used in conjunction with transforming growth factor-beta1, glucocorticoid steroids may normalize the effect of transforming growth factor-beta1 on collagen synthesis, thereby reducing excessive collagen deposition and fibrosis.     

Tissue expression of transforming growth factor-β1 and transforming growth factor-α during wound healing in human skin explants

In the dynamic and complex process of wound healing, locally produced growth factors are important mediators, although their actual roles have not been fully established. In the present study, the presence of transforming growth factor-beta1 and -alpha during the re-epithelialization of full-thickness wounds was investigated in an in vitro model of wound healing in human skin. The amounts of transforming growth factor-beta1 and -alpha secreted from the wound area were measured with enzyme immunoassays, and immunohistochemistry was used to study the localization of these two growth factors in the healing wound. The wounds were followed until they were completely re-epithelialized. The results showed a continuous increase in secreted transforming growth factor-beta1 throughout the re-epithelialization phase of healing followed by a decrease after its completion. The keratinocytes migrating out from the wound edges showed intense staining for transforming growth factor-beta1 which declined to the level of the surrounding epidermis after the wound was covered by a new epidermis. After the skin was wounded, a decrease both in secreted transforming growth factor-alpha and in immunostaining for this growth factor was apparent. Even though a minor increase in the immunoreactivity for transforming growth factor-alpha occurred after the completion of re-epithelialization, no increase in secreted transforming growth factor-alpha could be detected by enzyme immunoassay. These data suggest that keratinocytes modulate their expression of transforming growth factor-beta1 and -alpha during the wound healing process in human skin and that these changes may be controlled in part by autocrine pathways.     

Transforming growth factor-β levels during second- intention healing are related to the different course of wound contraction in horses and ponies

Wound healing in horses is often complicated by wound infection, exuberant granulation tissue, and hypertrophic scars, especially when wounds are located on the limbs. Wound healing in ponies is less problematic, characterized by a greater degree of wound contraction and a more intense initial inflammatory response. Because both processes are influenced by transforming growth factor-beta (TGF-beta), it was hypothesized that the better wound healing in ponies was associated with different TGF-beta profiles. A series of small wounds was created on the distal limbs and hindquarters of ponies and horses. Tissue samples were harvested on alternate days until day 13 postwounding, and levels of total and active TGF-beta were determined. Levels of TGF-beta were significantly higher in pony wounds than in those of horses. The TGF-beta profile differed between limb and body wounds, with levels in body wounds decreasing at the end of the experiment and persisting in limb wounds. In ponies, the higher TGF-beta levels can, to a large extent, explain the more intense inflammatory response and may explain the greater degree of wound contraction. Apparently adequate levels in the limbs fail to result in greater wound contraction, probably because of a stronger fixation of the skin. The persistence of elevated levels of TGF-beta may result in the production of exuberant granulation tissue. Further research on the temporal patterns of the different TGF-beta isoforms seems indicated, because manipulation of TGF-beta levels appears to be a promising option for intervention in problematic wound healing in horses.     

Transforming growth factor-β and its effect on reepithelialization of partial-thickness ear wounds in transgenic mice

Transforming growth factor-beta (TGF-beta) is known to affect nearly every aspect of wound repair. Many of the effects have been extensively investigated; however, the primary effect of endogenously derived TGF-beta on wound reepithelialization is still not completely understood. To examine this, two types of wounds were made on a transgenic mouse over-expressing TGF-beta1. Full-thickness back wounds were made to compare the wound healing process in the presence of compensatory healing mechanisms. Superficial partial-thickness ear wounds involving only the epidermis were made to determine the effect of TGF-beta on reepithelialization. In the partial-thickness ear wounds, at later time points, the transgenic group had smaller epithelial gaps than the wild-type mice. A greater number of actively proliferating cells, as determined by bromodeoxyuridine incorporation, was also found in the transgenic mice at post-injury day 8. These results show that TGF-beta1 stimulates the rate of reepithelialization at later time points in partial-thickness wounds. However, in the full-thickness back wounds, the transgenic animals exhibited a slower reepithelialization rate at all time points and the number of bromodeoxyuridine-positive cells was fewer. Our findings would suggest that the overexpression of TGF-beta1 speeds the rate of wound closure in partial-thickness wounds by promoting keratinocyte migration. In full-thickness wounds, however, the overexpression of TGF-beta1 slows the rate of wound reepithelialization.     

Increase in wound breaking strength in rats in the presence of positively charged dextran beads correlates with an increase in endogenous transforming growth factor-β1 and its receptor TGF-βRI in close proximity to the wound

We have previously shown that positively charged beads (DEAE A25) increase wound breaking strength in linear incisions in rats and nonhuman primates at days 10-14 post-wounding. The increased wound strength may result in part from a stimulation of cells adjacent to the DEAE A25 beads to produce growth factors important for wound healing. In this report, we investigate this hypothesis by comparing the relative expression levels of transforming growth factor-beta1 and its receptor transforming growth factor-beta receptor type I in DEAE A25-treated and contralateral untreated rat linear incisions. DEAE A25-treated incisions were stronger than untreated control wounds at 3 days post-wounding, and the difference in breaking strength reached statistical significance at days 5, 7 and 10. Immunohistochemical analysis revealed a significant increase in transforming growth factor-beta1 and transforming growth factor-beta receptor type I expression in DEAE A25-treated incisions, up to 7 days post-wounding, as compared to untreated control wounds. FACS analysis revealed that macrophage cell lines exposed to DEAE A25 in vitro upregulate transforming growth factor-beta1 and transforming growth factor-beta receptor type I expression by 2-3 fold. Therefore, the increase in expression of transforming growth factor-beta1 and transforming growth factor-beta receptor type I in DEAE A25-treated incisions may be due to an increase in the concentration of macrophages adjacent to DEAE A25 beads, as well as the stimulation of individual macrophages to produce greater amounts of transforming growth factor-beta1 and transforming growth factor-beta receptor type I. This study also supports the significance of transforming growth factor-beta1 in wound healing.     

Site-specific production of TGF-β in oral mucosal and cutaneous wounds

Wound healing in the oral mucosa is clinically distinguished by rapid healing and lack of scar formation compared with dermal wounds. Mechanisms of favorable mucosal healing are yet to be elucidated. Utilizing a murine model of equivalent-size mucosal and skin wounds, we verified the rapid reepithelializaton and reduction in scarring of oral wounds reported in humans. Collagen fibrillar structure in oral wounds rapidly approached the size of normal collagen fibrils, while the collagen ultrastructure in skin remained immature through the later phases of healing. To determine whether the transforming growth factor-β (TGF-β) contributes to the lack of scar formation in oral mucosa, we compared the expression and production in oral and skin wounds. The RNase protection assay demonstrated significantly lower levels of TGF-β1 expression in oral wounds compared with dermal wounds, and no changes were observed in the expression levels of TGF-β2 or TGF-β3. ELISA analysis confirmed that oral wounds contained lower levels of TGF-β1 levels compared with dermal wounds, along with a significant increase in the ratio of TGF-β3 to -β1. These findings showed reduced scarring in oral wounds at the ultrastructural level, and provide evidence that site-specific differences in TGF-β production contributes to the superior healing of oral wounds.     

Activation of latent TGF-β1 by low-power laser in vitro correlates with increased TGF-β1 levels in laser-enhanced oral wound healing

The term Laser "Photobiomodulation" was coined to encompass the pleiotropic effects of low-power lasers on biological processes. The purpose of this study was to investigate whether transforming growth factor (TGF)-beta had a role in mediating the biological effects of low-power far-infrared laser irradiation. We assayed for in vitro activation using various biological forms of cell-secreted, recombinant, and serum latent TGF-beta using the p3TP reporter and enzyme-linked immunosorbent assays. We demonstrate here that low-power lasers are capable of activating latent TGF-beta1 and -beta3 in vitro and, further, that it is capable of "priming" these complexes, making them more amenable to physiological activation present in the healing milieu. Using an in vivo oral tooth extraction-healing model, we observed an increased TGF-beta1, but not beta3, expression by immunohistochemistry immediately following laser irradiation while TGF-beta3 expression was increased after 14 days, concomitant with an increased inflammatory infiltrate. All comparisons were performed between laser-irradiated wounds and nonirradiated wounds in each subject essentially using them as their own control (paired T-test p<0.05). Low-power laser irradiation is capable of activating the latent TGF-beta1 complex in vitro and its expression pattern in vivo suggests that TGF-beta play a central role in mediating the accelerated healing response.     

Transforming growth factor-β: crossroad of glucocorticoid and bleomycin regulation of collagen synthesis in lung fibroblasts

Fibrosis is a consequence of injury which is characterized by accumulation of excess collagen and other extracellular matrix components, resulting in the destruction of normal tissue architecture and function. Transforming growth factor-beta, a potent wound healing agent, has also been shown to be an agent that can produce fibrosis because it is a potent stimulator of collagen synthesis. Both glucocorticoids and bleomycin have recently been shown to affect collagen synthesis in opposite directions, by utilizing a common pathway of involving transforming growth factor-beta activator protein binding to the transforming growth factor-beta element. This article presents a mechanistic overview of collagen synthesis regulation by glucocorticoids and bleomycin through the transforming growth factor-beta pathway.     

Mechanisms of TGF-β action in connective tissue repair of rat mesenteric wounds

We have recently reported that transforming growth factor-beta stimulates genuine connective tissue repair in the perforated rat mesentery and that this stimulation is not caused by increased macrophage chemotaxis. To further characterize the effect of transforming growth factor-beta(1) on the enhanced rate of wound closure, we performed a series of morphometric analyses with determination of mitotic index, fibroblast labeling index, cellular density, neovascularization, and scar tissue formation. Actin expression close to the wound margin was also evaluated morphologically. Fibroblast cell proliferation was not stimulated by transforming growth factor-beta(1) in either wounded or unwounded tissue. Transforming growth factor-beta(1) did, however, significantly increase the formation of healing tissue postoperative days 5 to 10 (p < 0.05) and angiogenesis was significantly stimulated by transforming growth factor-beta(1) postoperative days 7 and 10 (p < 0.005). The mean cellular density was significantly increased in unperforated, transforming growth factor-beta(1)-treated membranes from days 3 to 10, and increased expression of actin with time was observed close to the wound margin. Transforming growth factor-beta(1) was thus shown to be a potent stimulator of angiogenesis and healing tissue formation in connective tissue repair, but this stimulation mainly occurred after closure of perforations. The increased cellular density in the absence of stimulated proliferation and increased actin expression in wound cells indicate that contraction may be an important mechanism of connective tissue repair in the perforated rat mesentery.