Deletion of the PDGFR-beta gene affects key fibroblast functions important for wound healing

This study provides new perspectives of the unique aspects of platelet-derived growth factor beta-receptor (PDGFR-beta) signaling and biological responses through the establishment of a mutant mouse strain in which two loxP sequences were inserted into the introns of PDGFR-beta genome sequences. Isolation of skin fibroblasts from the mutant mice and Cre recombinase transfection in vitro induced PDGFR-beta gene deletion (PDGFR-betaDelta/Delta). The resultant depletion of the PDGFR-beta protein significantly attenuated platelet-derived growth factor (PDGF)-BB-induced cell migration, proliferation, and protection from H2O2-induced apoptosis of the cultured PDGFR-betaDelta/Delta dermal fibroblasts. PDGF-AA and fetal bovine serum were mitogenic and anti-apoptotic but were unable to induce the migration in PDGFR-beta Delta/Delta fibroblasts. Concerning the PDGF signaling, PDGF-BB-induced phosphorylation of Akt, ERK1/2, and JNK, but not p38, decreased in PDGFR-betaDelta/Delta fibroblasts, but PDGF-AA-induced signaling was not altered. Overexpression of the phospholipid phosphatases, SHIP2 and/or PTEN, inhibited PDGF-BB-induced phosphorylation of Akt and ERK1/2 in PDGFR-betaDelta/Delta fibroblasts but did not affect that of JNK and p38. These results indicate that disruption of distinct PDGFR-beta signaling pathways in PDGFR-betaDelta/Delta dermal fibroblasts impaired their proliferation and survival, but completely inhibits migratory response, and that PDGF-BB-induced phosphorylation of Akt and ERK1/2 possibly mediated by PDGFR-alpha is regulated, at least in part, by the lipid phosphatases SHIP2 and/or PTEN. Thus, the PDGFR-beta function on dermal fibroblasts appears to be critical in PDGF-BB action for skin wound healing and is clearly distinctive from that of PDGFR-alpha in the ligand-induced biological responses and the underlying properties of cellular signaling.     

The role of the tetraspanin CD151 in primary keratinocyte and fibroblast functions: implications for wound healing

Previous studies showed that CD151-null mice have a skin wound healing deficit. To gain an understanding of the role of CD151 in re-epithelialisation and dermal contraction, keratinocyte and fibroblast functions were assayed. Primary CD151-null keratinocytes displayed defective migration on Matrigel (a basement membrane equivalent) and laminin-332, the primary adhesion component of basement membranes, but not on collagen-I. Adhesion, spreading and proliferation were also deficient on laminin-332, but not collagen-I. The data suggest that loss of CD151 impairs the function of its primary interaction partners, integrin alpha3beta1- and/or alpha6beta4 which bind to laminin-332. Skin fibroblasts also produce CD151 mRNA. CD151-null fibroblasts migrated significantly faster on collagen I than wild type fibroblasts, confirming that they possess functional collagen receptors. However, no significant decrease in the ability of CD151-null fibroblasts to cause contraction in floating collagen gel assays in response to transforming growth factor beta-1 (TGF-beta1) or platelet derived growth factor (PDGF-BB) was observed, nor was there an effect on fibroblast adhesion or proliferation on collagen-I. The data implicate CD151 as a facilitator of laminin-332-mediated keratinocyte functions that impact on the re-epithelialisation process intrinsic to wound healing and further suggest a potential novel role for CD151 in fibroblast migration.     

JNK signaling pathway required for wound healing in regenerating Drosophila wing imaginal discs

We have examined wound healing during regeneration of Drosophila wing imaginal discs fragments by confocal microscopy and assessed the role of components of the JNK pathway in this process. After cutting, columnar and peripodial epithelia cells at the wound edge start to close the wound through formation and contraction of an actin cable. This is followed by a zipping process through filopodial protrusions from both epithelia knitting the wound edges from proximal to distal areas of the disc. Activation of the JNK pathway is involved in such process. puckered (puc) expression is induced in several rows of cells at the edge of the wound, whereas absence of JNK pathway activity brought about by hemipterous, basket, and Dfos mutants impair wound healing. These defects are accompanied by lowered or loss of expression of puc. In support of a role of puc in wound healing, hep mutant phenotypes are rescued by reducing puc function, whereas overexpression of puc inhibits wound healing. Altogether, these results demonstrate a role for the JNK pathway in imaginal disc wound healing, similar to that reported for other healing processes such as embryonic dorsal closure, thoracic closure, and adult epithelial wound healing in Drosophila. Differences with such processes are also highlighted.     

Role of plasma-derived fibrin on keratinocyte and fibroblast wound healing

Fibrin has excellent biocompatibility and biological properties to support tissue regeneration and promote wound healing. However, the role of diluted fibrin in wound healing has yet to be elucidated as it is commonly used in high concentration. This study was aimed to examine the effects of diluted plasma-derived fibrin (PDF) on keratinocyte and fibroblast wound healing in term of cell proliferation, migration, extracellular matrix (ECM) production and soluble factor secretion. Two PDF concentrations, 10 and 20% (v/v) were tested on keratinocytes and fibroblasts indirectly co-cultured in the transwell system. The control group was cultured with 5% FBS. Results showed that PDF reduced the keratinocyte growth rate and fibroblast migration, and increased the fibroblast ECM gene expression whereby significant differences were found between the 20% PDF group and the 5% FBS group. Similar trend was seen for the 10% PDF group but the differences were not significant. Comparison of the soluble factors between the PDF groups demonstrated that the level of growth-related oncogene alpha, interleukin-8 and epithelial neutrophil-activating peptide-78 were significantly higher in the 10% PDF group, whilst interleukin-1 alpha and granulocyte–macrophage colony stimulating factor were significantly more concentrated in the 20% PDF group. Our results suggested that PDF selectively elevated the expression of collagen type 1 and collagen type 3 in fibroblasts but slowed down the migration in concentration-dependent manner. These novel findings provide new insight into the role of PDF in wound healing and may have important implications for the use of fibrin in skin tissue engineering.     

Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor with anti-microbial properties found in mucosal fluids. It is expressed during cutaneous wound healing. Impaired healing states are characterized by excessive proteolysis and often bacterial infection, leading to the hypothesis that SLPI may have a role in this process. We have generated mice null for the gene encoding SLPI (Slpi), which show impaired cutaneous wound healing with increased inflammation and elastase activity. The altered inflammatory profile involves enhanced activation of local TGF-beta in Slpi-null mice. We propose that SLPI is a pivotal endogenous factor necessary for optimal wound healing.     

Opioid activity of peptides and wound healing of the skin

The binding of dalargin, its four analogues and FK-33824, DADLE, met-enkephalin and morphine to peripheral mu- and delta-receptors and to brain receptors has been investigated in comparison with their influence on skin wound healing in rats. It has been shown that only substances with opiate activity, including morphine, stimulated wound healing. No correlation between wound healing effect of peptides and their binding to a definite receptor has been found. Naloxone inhibited wound healing and suppressed opiate peptide-mediated healing process. It is suggested that endogenous opiate peptides are involved in the maintenance of structural homeostasis.     

Effect of fibroblast implants on wound healing of irradiated skin: assay of wound strength and quantitative immunohistology of collagen

The role of dermal fibroblasts in the expression of radiation-induced damage to the skin was studied. Fibroblasts from neonatal mice were cultured, harvested, and injected into full-depth surgical incisions in the dorsal area of mouse skin, which had been previously locally irradiated by 18 Gy X rays. As a control, cells irradiated with a dose of 20 Gy were also injected. The effect of radiation and fibroblast implants on the gain of skin wound strength was assayed. In an additional experiment freshly isolated cells were implanted. Two weeks following wounding the irradiated skin had reached only about a third of the strength of unirradiated skin. A significant increase of wound strength in irradiated skin was observed when 1.5-2 x 10(6) cultured fibroblasts or freshly isolated fibroblasts were injected into the 20-mm-long wound bed. Irradiated cells had significantly less effect. This suggests that implanting isolated syngeneic cells may "rescue" wounds from the effect of prior irradiation. Semiquantitative immunohistology of types I and III collagen was performed in parallel using a video image digitizing system. Levels of both types I and III collagen were altered in the dermis and the wound tissues in irradiated skin, but the implant of cultured fibroblasts did not affect notably the total levels and the disposition of the two collagen isotypes.     

Role of fibroblast migration in collagen fiber formation during fetal and adult dermal wound healing

Adult dermal wounds, in contrast to fetal wounds, heal with the formation of scar tissue. A crucial factor in determining the degree of scarring is the ratio of types I and III collagen, which regulates the diameter of the combined fibers. We developed a reaction-diffusion model which focuses on the control of collagen synthesis by different isoforms of the polypeptide transforming growth factor-β (TGFβ). We used the model to investigate the current controversy as to whether the fibroblasts migrate into the wound from the surrounding unwounded dermis or from the underlying subcutaneous tissue. Numerical simulations of a spatially independent, temporal model led to a value of the collagen ratio consistent with that of healthy tissue for the fetus, but corresponding to scarring in the adult. We investigated the effect of topical application of TGFβ and show that addition of isoform 3 reduces scar tissue formation, in agreement with the experiment. However, numerical solutions of the reaction-diffusion system do not exhibit this sensitivity to growth factor application. Mathematically, this corresponds to the observation that behind healing wavefront solutions, a particular healed state is always selected independent of transients, even though there is a continuum of possible positive steady states. We explain this phenomenon using a caricature system of equations, which reflects the key qualitative features of the full model but has a much simpler mathematical form. Biologically, our results suggest that the migration into a wound of fibroblasts and TGFβ from the surrounding dermis alone cannot account for the essential features of the healing process, and that fibroblasts entering from the underlying subcutaneous tissue are crucial to the healing process.     

Activation of JNK signaling mediates connective tissue growth factor expression and scar formation in corneal wound healing

Connective Tissue Growth Factor (CTGF) and Transforming growth factor-β1 (TGF-β1) are key growth factors in regulating corneal scarring. Although CTGF was induced by TGF-β1 and mediated many of fibroproliferative effects of TGF-β1, the signaling pathway for CTGF production in corneal scarring remains to be clarified. In the present study, we firstly investigated the effects of c-Jun N-terminal kinase (JNK) on CTGF expression induce by TGF-β1 in Telomerase-immortalized human cornea stroma fibroblasts (THSF). Then, we created penetrating corneal wound model and determined the effect of JNK in the pathogenesis of corneal scarring. TGF-β1 activated MAPK pathways in THSF cells. JNK inhibitor significantly inhibited CTGF, fibronectin and collagen I expression induced by TGF-β1 in THSF. In corneal wound healing, the JNK inhibitor significantly inhibited CTGF expression, markedly improved the architecture of corneal stroma and reduced corneal scar formation, but did not have a measurable impact on corneal wound healing in vivo. Our results indicate that JNK mediates the expression of CTGF and corneal scarring in corneal wound healing, and might be considered as specific targets of drug therapy for corneal scarring.     

Aminoaldehyde dehydrogenase activity during wound healing of mechanically injured pea seedlings

Aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.19) is an enzyme that, in association with amine oxidase, participates in polyamine catabolism. In plants, the enzyme is well characterized in pea seedlings. In this study, we used etiolated and light-grown pea seedlings as model plants to evaluate the possible AMADH role in response to stress caused by mechanical damage. In the beginning, the activity distribution of AMADH, amine oxidase and peroxidase in organs of 7-day-old intact pea seedlings was analyzed. To perform mechanical damage, stems of 10-day-old seedlings were each divided into four segments of equal length. The top (=fourth) segments were then longitudinally cut with a lancet. During healing, the injured segments and their control counterparts were harvested in 1-day intervals and analyzed for activity of the above enzymes, polyamine and 4-aminobutyrate (GABA) concentrations. The injury elicited increases in AMADH, amine oxidase and peroxidase activities in both etiolated and green seedlings, accompanied by parallel increases in putrescine, cadaverine, spermidine and GABA content. Histochemical experiments allowed visualization of increased AMADH activity in cross sections obtained from the injured stem segments. The activity was localized in cortical parenchyma and epidermal cells adjacent to the wound site in spatial correlation with an intensive lignification. In the control seedlings, AMADH activity or lignification in these tissues could not be visualized. Thus, we conclude that, in plants, AMADH may participate in processes of adaptation to stress events caused by mechanical injury, which involve polyamine catabolism, GABA production and lignification.     

Phosphatase activity of blood leukocytes and wound exudate during the experimental healing of an aseptic wound

Cytochemical investigations of plain aseptic wounds simulated in 110 Wistar rats revealed a clear-cut dependence between the variations in the activity of alkaline and acid phosphatases in neutrophilic leukocytes, monocytes and lymphocytes of the blood and wound exudate and the stage of the healing process. Elevated activity of the blood alkaline phosphatase correlated with the term of inflammation phase.     

Histatin 1 enhanced the speed and quality of wound healing through regulating the behaviour of fibroblast

ObjectivesHistatin 1(Hst 1) has been proved to promote wound healing. However, there was no specific study on the regulation made by Hst 1 of fibroblasts in the process of wound healing. This research comprehensively studied the regulation of Hst 1 on the function of fibroblasts in the process of wound healing and preliminary mechanism about it.Materials and methodsThe full‐thickness skin wound model was made on the back of C57/BL6 mice. The wound healing, collagen deposition and fibroblast distribution were detected on days 3, 5 and 7 after injury. Fibroblast was cultured in vitro and stimulated with Hst 1, and then, their biological characteristics and functions were detected.ResultsHistatin 1 can effectively promote wound healing, improve collagen deposition during and after healing and increase the number and function of fibroblasts. After healing, the mechanical properties of the skin also improved. In vitro, the migration ability of fibroblasts stimulated by Hst 1 was significantly improved, and the fibroblasts transformed more into myofibroblasts, which improved the function of contraction and collagen secretion. In fibroblasts, mTOR signalling pathway can be activated by Hst 1.ConclusionsHistatin 1 can accelerate wound healing and improve the mechanical properties of healed skin by promoting the function of fibroblasts. The intermolecular mechanisms need to be further studied, and this study provides a direction about mTOR signalling pathway.     

Disruption of interdomain interactions in the glutamate binding pocket affects differentially agonist affinity and efficacy of N-methyl-D-aspartate receptor activation

In ionotropic glutamate receptors, agonist binding occurs in a conserved clam shell-like domain composed of the two lobes D1 and D2. Docking of glutamate into the binding cleft promotes rotation in the hinge region of the two lobes, resulting in closure of the binding pocket, which is thought to represent a prerequisite for channel gating. Here, we disrupted D1D2 interlobe interactions in the NR2A subunit of N-methyl-d-aspartate (NMDA) receptors through systematic mutation of individual residues and studied the influence on the activation kinetics of currents from NR1/NR2 NMDA receptors heterologously expressed in HEK cells. We show that the mutations affect differentially glutamate binding and channel gating, depending on their location within the binding domain, mainly by altering k(off) and k(cl), respectively. Whereas impaired stability of glutamate in its binding site is the only effect of mutations on one side of the ligand binding pocket, close to the hinge region, alterations in gating are the predominant consequence of mutations on the opposite side, at the entrance of the binding pocket. A mutation increasing D1D2 interaction at the entrance of the pocket resulted in an NMDA receptor with an increased open probability as demonstrated by single channel and whole cell kinetic analysis. Thus, the results indicate that agonist-induced binding domain closure is itself a complex process, certain aspects of which are coupled either to binding or to gating. Specifically, we propose that late steps of domain closure, in kinetic terms, represent part of channel gating.     

An activity of Notch regulates JNK signalling and affects dorsal closure in Drosophila.

BACKGROUND: The Drosophila Notch protein is a receptor that controls cell fate during embryonic development, particularly in lateral inhibition, a process that acts on groups of cells that share a particular developmental potential to restrict the number of cells that will adopt that cell fate. The process of lateral inhibition is implemented by the nuclear protein Suppressor of Hairless (Su(H)) and is triggered by the ligand Delta. Recent results have shown that the interaction between Delta and Notch triggers the cleavage of the intracellular domain of Notch which then translocates to the nucleus and binds to Su(H). RESULTS: We find that Notch plays a role in the patterning of the dorsal epidermis of the Drosophila embryo and that this function of Notch is independent of Su(H), requires Notch at the plasma membrane and targets the c-Jun N-terminal kinase (JNK) signalling pathway. Notch mutants show high levels of JNK activity and can rescue the effects of lowered JNK signalling resulting from mutations in the hemipterous and basket genes. Two regions of the intracellular domain of Notch are involved: the Cdc10/ankyrin repeats, which downregulate signalling through the JNK pathway, and a region carboxy-terminal to these repeats, which regulates this negative function. CONCLUSIONS: Our results reveal a novel signalling activity of Notch that does not require its cleavage and acts by modulating signalling through the JNK pathway. In the Drosophila embryo, this activity plays an important role in the morphogenetic movements that drive dorsal closure.     

Pharmacological manipulation of the dopaminergic system affects wheel-running activity in differentially active mice

The genetic factors involved in the regulation of physical activity are not well understood. The dopamine system has been implicated in the control of voluntary locomotion and wheel running (WR) in mice and is thus a likely candidate as a genetic/biological system important to the regulation of physical activity. This study evaluated the effects of four different dopaminergic acting drugs on WR in differentially active inbred strains of mice. High active C57L/J (n=7, 3 controls, 4 experimental) and low active C3H/HeJ (n=8, 3 controls, 5 experimental) were analyzed for baseline wheel-running indices of distance (km/day), duration (mins/day), and speed (m/min) for 21 days. Experimental mice received increasing doses over four days of each of the following drugs: SKF 81297 (D1 agonist), SCH 23390 (D1 antagonist), GBR 12783 (DAT inhibitor), and AMPT (tyrosine hydroxylase inhibitor). Each drug dose response treatment was separated by three days of recovery (no drug injections). WR indices were monitored during drug treatments and during drug wash-out phases. SKF 81297 significantly reduced (p=0.0004) WR in the C57L/J mice, but did not affect WR in the C3H/HeJ mice. GBR 12783 significantly increased (p=0.0005) WR in C3H/HeJ mice, but did not affect WR in C57L/J mice. Only duration (not overall WR) was significantly reduced in C57L/J mice in response to SCH 23390 (p=0.003) and AMPT (p=0.043). SCH 23390 (p=0.44) and AMPT (p=0.98) did not significantly affect WR in C3H/HeJ mice. These results suggest that genetic differences in dopamine signaling may play a role in the WR response to dopaminergic-acting drugs in inbred strains of mice. The high activity in the C57L/J strain appears most responsive to D1-like receptor acting drugs, while in the C3H/HeJ strain, dopamine re-uptake appears to have an influence on activity level.     

Curcumin nanofibers for the purpose of wound healing

Poor wound healing is a highly prevalent clinical problem with, as yet, no entirely satisfactory solution. A new technique, termed electrospinning, may provide a solution to improve wound healing. Due to their large surface area to volume ratio and porosity, the nanofibers created by electrospinning are able to deliver sustained drug release and oxygen to the wound. Using different types of polymers with varying properties helps strengthening nanofiber and exudates absorption. The nanofibers appear to have an ideal structure applicable for wound healing and, in combination with curcumin, can blend the anti-inflammatory and antioxidant properties of curcumin into a highly effective wound dressing. The use of suitable curcumin solvents and the slow release of curcumin from the nanofiber help in overcoming the known limitations of curcumin, specifically its low stability and limited bioavailability. Here, we review the studies which have been done on synthesized nanofibers containing curcumin, produced by the electrospinning technique, for the purpose of wound healing.