An angiogenic laminin site and its antagonist bind through the alpha(v)beta3 and alpha5beta1 integrins.

Angiogenesis is important for wound healing, tumor growth, and metastasis. Endothelial cells differentiate into capillary-like structures on a laminin-1-rich matrix (Matrigel). We previously identified 20 angiogenic sites on laminin-1 (alpha1beta1gamma1) by screening 559 overlapping synthetic peptides. C16, the most potent gamma1 chain peptide, blocked laminin-1-mediated adhesion and was the only gamma1 chain peptide to block attachment to both collagen I and fibronectin. This suggested that C16 was acting via a receptor common to these substrates. We demonstrated that C16 is angiogenic in vivo. Affinity chromatography identified the integrins alpha5beta1 and alpha(v)beta3 as surface receptors. Blocking antibodies confirmed the role of these receptors in C16 adhesion. C16 does not contain an RGD sequence and, as expected, an RGD-containing peptide did not block C16 adhesion nor did C16 act via MAP kinase phosphorylation. Furthermore, we identified a C16 scrambled sequence, C16S, which antagonizes the angiogenic activity of bFGF and of C16 by binding to the same receptors. Because the laminin gamma1 chain is ubiquitous in most tissues, C16 is likely an important functional site. Since the biological activity of C16 is blocked by a scrambled peptide, C16S may serve as an anti-angiogenic therapeutic agent.     

Identification of redundant angiogenic sites in laminin alpha1 and gamma1 chains

The degradation of the extracellular matrix is one of the first steps involved in angiogenesis, the formation of new vessels from preexisting ones. Laminin, a large extracellular matrix protein, has many biological activities, including the promotion of angiogenesis. Screening of the laminin-1 chains identified 20 angiogenic peptides, of which, A13 and C16, from the alpha1 and gamma1 chains, respectively, were the most active. We recently identified the receptors for C16 as the integrins alpha5beta1 and alphavbeta3. Here, we show unexpectedly that A13 is a redundant active site to C16 present in the N-terminal globular domain of the alpha1 chain. The peptides are located in homologous sites present in the last globular domains of their respective chains, and their amino acids are 66% conserved, as compared to the inactive homologous site in the beta1 chain, B19 to B20, which is only 18%-23% conserved. Cell attachment studies demonstrated that both A13 and C16 reciprocally inhibited their adhesion activity, whereas the corresponding laminin beta1 chain peptides were inactive. Chorioallantoic membrane assays showed that the in vivo angiogenic activity of A13 is blocked by a C16 antagonist, C16S, which also binds to the same integrin receptors. A13 affinity chromatography and immunoprecipitation analysis showed that the alphavbeta3 and alpha5beta1 integrin receptors bind to this sequence. We have therefore identified redundant activity on two laminin chains. These highly conserved functional sites are likely important mediators of the biological responses of laminins because either one or both of these chains (active sites) are present in almost all laminin isoforms identified to date.     

The basement membrane protein laminin-5 acts as a soluble cell motility factor

One of the basement membrane (BM) proteins, laminin-5 (LN5), is known to support efficient cell adhesion and migration through interaction with integrins on the basal plasma membrane. Here, we show that a soluble form of LN5 induced migration of human epithelial cells and carcinoma cells by interacting with integrins on the apical cell surface. Although both LN5 and laminin-10/11 (LN10/11) promoted cell migration when coated onto a plastic surface as insoluble substrata, only LN5 stimulated cell migration in its soluble form on other substrata such as fibronectin (FN), vitronectin (VN) and collagen. Soluble LN5 interacted with integrins alpha3beta1 and alpha6beta1 on the apical cell surface and stimulated cell migration, while the cell morphology was largely dependent on the underlying substratum. Thus, integrin signals from the apical surface and the basal surface synergistically regulated cytoskeletal organization and cell motility. Soluble and insoluble LN5 induced cell motility by activating signal pathways via protein kinase C (PKC), phosphoinositide 3-OH kinase (PI3-K) and MAP kinase. The PKC dependency was more prominent for soluble LN5 than insoluble LN5, and was absent in the stimulation by insoluble LN10/11. In vitro scratch assays with keratinocytes, self-produced soluble LN5 bound to the apical cell surface of migrating cells at the scratched edges, suggesting that soluble LN5 may contribute to cell migration in pathological conditions such as wound healing and tumor invasion.     

Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.     

Exogenous non-crosslinked collagen enhances granulation tissue formation in dermal excision wounds in guinea pigs

Based on previous observations indicating a role for collagen peptides in eliciting a positive feedback for collagen biosynthesis, this study was initiated to elucidate the effect of non-crosslinked collagen on granulation tissue formation in dermal excision wounds. The wounds were treated with either non-crosslinked or crosslinked native collagen, or left untreated as controls. Granulation tissue was analyzed for collagen type I mRNA, for levels of interstitial collagen and for the number of blood vessels. The results indicated significant increases in procollagen type I mRNA, in interstitial collagen, in the number of blood vessels and in epithelial advance in the non-crosslinked collagen-treated wounds relative to the untreated controls. It is assumed that the presence of non-crosslinked collagen in a healing wound enhances both procollagen type I biosynthesis and the repair process of dermal wounds, due to the more readily released collagen peptides derived from this exogenous collagen dressing.     

Human platelet-rich plasma- and extracellular matrix-derived peptides promote impaired cutaneous wound healing in vivo

Previous work in our laboratory has described several pro-angiogenic short peptides derived from endothelial extracellular matrices degraded by bacterial collagenase. Here we tested whether these peptides could stimulate wound healing in vivo. Our experiments demonstrated that a peptide created as combination of fragments of tenascin X and fibrillin 1 (comb1) applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure. Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury. Results of in vivo experiments where comb1 and UN3 peptides were added together to cranial wounds in cyclophosphamide-treated mice leads to improvement of wound vascularization as shown by an increase of the number of blood vessels present in the wound beds. Application of the peptides markedly promotes cellular responses to injury and essentially restores wound healing dynamics to those of normal, acute wounds in the absence of cyclophosphamide impairment. Our current work is aimed at understanding the mechanisms underlying the stimulatory effects of these peptides as well as identification of the cellular receptors mediating these effects.     

Transforming growth factor-beta (TGF beta) is chemotactic for human monocytes and induces their expression of angiogenic activity

TGF beta stimulates human blood monocyte migration, with peak migratory response occurring consistently at a concentration of 16-100 fg/ml. Checkerboard analysis revealed both chemotactic and chemokinetic components to this response. At higher concentrations (10-100 pg/ml), TGF beta stimulated expression of angiogenic activity by monocytes. While mRNA for TNF alpha was undetectable in resting monocytes, high steady state levels of TNF alpha mRNA were rapidly induced in TGF beta-treated monocytes. TGF beta is secreted by a number of neoplastic cells as well as normal cells such as platelets and lymphocytes. TGF beta may recruit monocytes from the circulation, and subsequently activate them to express angiogenic activities such as TNF alpha, thus playing an important role in wound repair, inflammation and tumor growth.     

Fibroblast growth factor 2-functionalized collagen matrices for skeletal muscle tissue engineering

Fibroblast growth factor 2 (FGF2) protein plays important roles in wound healing and tissue regeneration. Collagen is clinically used for wound care applications. We investigated the potential value of FGF2-functionalized collagen matrices for skeletal muscle tissue engineering. When C2C12 cells were treated with FGF2, cell adhesion increased after 3 and 5 days compared to the control (P < 0.05). Wound healing activity of FGF2 was slightly higher than the control through cell migration. Cell proliferation activity of FGF2-functionalized collagen matrices on C2C12 cells also increased. Taken together, FGF2 stimulated C2C12 myoblast growth by promoting cell adhesion, proliferation and wound healing activity after injury. The potential effect of FGF2-functionalized collagen matrices was also observed. Thus FGF2 stimulates skeletal muscle development and regeneration, thereby leading to potential utility for skeletal muscle tissue engineering.     

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT–loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72 h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT–loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p < 0.01) and IL-1β (p < 0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p < 0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone.     

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.     

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.     

Enhanced wound healing in animal models by interferon and an interferon inducer.

Healing of wounds is a significant biological process that involves the interactions of different cell types, growth factors, cytokines, and extracellular matrix molecules. Mice and rats were treated in vivo with interferon (IFN) alpha/beta or polyinosinic-polycytidylic acid (Poly I:C), a ds-RNA, a potent inducer of IFN. We observed faster and enhanced closure of wounds as compared to untreated controls on day 7 (wound area measured on Macintosh II CX using NIH image 1.30u program), increased migration of dermal fibroblasts in the wound bed, complete re-epithelialization evidenced by routine histology and scanning electron microscopic procedures, and increased collagen synthesis, which correlates to greater tensile strength. In addition, classical immunofluorescence procedures using frozen sections showed that dermal fibroblasts synthesized much more laminin following Poly I:C treatment, whereas no effect was observed on fibronectin synthesis. These results suggest that Poly I:C and IFN treatment result in a faster restoration of tissue integrity in both full skin punch biopsy and skin incision models.     

The multifunctional role of fibroblasts during wound healing in Hirudo medicinalis (Annelida, Hirudinea)

Extracellular matrix components play a key role during the angiogenic process for a correct development of blood vessels: fibroblasts are the main cell type involved in the regulation of ECM protein production. In this study we characterize H. medicinalis fibroblasts and demonstrate that they take part to the regulation of angiogenesis that occurs during wound healing process. Massive proliferation and phenotypic modification are two distinctive markers of fibroblast activation. These cells, that are usually responsible for collagen production and function as an energy reservoir, are recruited during wound healing to form a collagen scaffold through a direct mechanic action and through secretion of specific proteoglycans. In addition we show that the activity of fibroblasts is modulated by EGF, a growth factor involved in wound healing in vertebrates. The formation of bundles of collagen fibrils by fibroblasts is fundamental for the development and migration of new blood vessels in lesioned areas during wound repair: administration of lovastatin in explanted leeches affects fibroblasts, damages collagen "scaffold" and indirectly causes the reduction of neo-capillary formation.     

Activated protein C mediates a healing phenotype in cultured tenocytes

Tendon injuries cause considerable morbidity in the general adult population. The tenocytes within the tendon have the full capacity to heal the tendon intrinsically. Activated protein C (APC) plays an important role in coagulation and inflammation and more recently has been shown to promote cutaneous wound healing. In this study we examined whether APC can induce a wound healing phenotype in tenocytes. Sheep tenocytes were treated with APC, endothelial protein C receptor (EPCR) blocking antibody (RCR252) and/or EPCR small interfering (si)RNA. Cell proliferation and migration were measured by crystal violet assay and a scratch wounding assay, respectively. The expression of EPCR, matrix metalloproteinase (MMP)-2, type I collagen and MAP kinase activity were detected by real time PCR, zymography, immunofluorescence, immunohistochemistry and Western blotting. APC stimulated proliferation, MMP-2 activity and type I collagen deposition in a dose-dependent manner and promoted migration of cultured tenocytes. APC dose-dependently stimulated phosphorylated (P)-ERK2 and inhibited P-p38. Interestingly, tenocytes expressed EPCR protein, which was up-regulated by APC. When tenocytes were pre-treated with RCR252 or EPCR siRNA the effect of APC on proliferation, MMP-2 and type 1 collagen synthesis and MAP kinases was blocked. APC promotes the growth, MMP-2 activity, type I collagen deposition and migration of tenocytes. Furthermore, EPCR is expressed by tenocytes and mediates the actions of APC, at least partly by signalling through selective MAP kinases. These data implicate APC as a potential healing agent for injured tendons.     

Acute ethanol exposure impairs angiogenesis and the proliferative phase of wound healing

Acute ethanol exposure represents an increased risk factor for morbidity and mortality associated with surgical or traumatic injury. Despite clinical observations suggesting that ethanol exposure before injury alters tissue repair processes, little direct evidence about the mechanism by which ethanol affects the wound healing process is available. In this study, excisional wounds from female BALB/c mice with or without circulating ethanol levels of 100 mg/dl were used to assess wound closure, angiogenesis, and collagen content. Ethanol exposure resulted in a significant but transient delay in wound closure at day 2 postwounding (28 +/- 4% vs. 17 +/- 1%). In addition, total collagen content was significantly reduced by up to 37% in wounds from ethanol-treated mice compared with controls. The most significant effect of ethanol exposure on wounds was on vascularity because angiogenesis was reduced by up to 61% in wounds from ethanol-treated mice. The reduction in vessel density occurred despite near-normal levels of proangiogenic factors VEGF and FGF-2, suggesting a direct effect of ethanol exposure on endothelial cell function. Further evidence for a direct effect was observed in an in vitro angiogenesis assay because the exposure of endothelial cells to ethanol reduced angiogenic responsiveness to just 8.33% of control in a cord-forming assay. These studies provide novel information regarding the effect of a single dose of ethanol on multiple parameters of the wound healing process in vivo and suggest a potential mechanism by which ethanol impairs healing after traumatic injury.     

The activity of soluble VCAM-1 in angiogenesis stimulated by IL-4 and IL-13

IL-13 is a multifunctional lymphokine sharing a number of biological properties with IL-4. We previously observed that IL-4 shows angiogenic activities in vitro as well as in vivo. In this study we examined the effect of IL-13 on angiogenesis in vitro and in vivo and also the underlying mechanisms. Human IL-13 significantly stimulated the formation of tube-like structures in collagen gels by human microvascular endothelial cells and bovine aortic endothelial cells by about 3-fold over the controls in the absence of the cytokines. Administration of murine IL-13 led to neovascularization when implanted in the rat cornea. Coadministration of neutralizing mAb to the IL-4R inhibited both tubular morphogenesis in vitro and activation of STAT6 induced by IL-4 or IL-13. Both IL-4 and IL-13 markedly increased mRNA levels of VCAM-1 in vascular endothelial cells, and the production of the soluble form of VCAM-1 was also stimulated in response to IL-4 or IL-13. Administration of anti-VCAM-1 Ab in vitro blocked tubular morphogenesis induced by IL-4 and IL-13. Angiogenesis induced in vivo in rat cornea by IL-4 and IL-13 was also inhibited by Ab against the rat alpha4 integrin subunit. These findings suggest that angiogenesis dependent on IL-4 and IL-13 is mainly mediated through a soluble VCAM-1/alpha4 integrin pathway.     

Biological effects of collagen I and IV peptides

Various biological events, such as cell differentiation, cell migration or gene expression, are controlled by cell-cell interactions or by cytokines, as well as by interactions between cells and extracellular matrix. The regulation of these events involves a directed and limited proteolysis of matrix macromolecules, that induces the release of proteic domains and peptides exhibiting biological activities. In this review, we summarise several data from our laboratory showing that peptides from type I and type IV collagens play an important role in the control of inflammation and tumor progression. Type I collagen peptides stimulate respiratory burst, granule exocytosis and cytokine secretion by human leukocytes (polymorphonuclear neutrophils or monocytes) for the detersion of inflammatory sites and then for the chemoattraction of various cell types needed for wound healing. A peptide of the NC1 domain of the alpha 3(IV) collagen chain prevents leukocyte activation. In addition, this peptide is also capable of limiting tumor progression by downregulating in vitro and in vivo invasive properties of melanoma cells.