Unique behavior of dermal cells from regenerative mammal,the African Spiny Mouse,in response to substrate stiffness

The African Spiny Mouse (Acomys spp.) is a unique outbred mammal capable of full, scar-free skin regeneration. In vivo, we have observed rapid reepithelialization and deposition of normal dermis in Acomys after wounding. Acomys skin also has a lower modulus and lower elastic energy storage than normal lab mice, Mus musculus. To see if the different in vivo mechanical microenvironments retained an effect on dermal cells and contributed to regenerative behavior, we examined isolated keratinocytes in response to physical wounding and fibroblasts in response to varying substrate stiffness. Classic mechanobiology paradigms suggest stiffer substrates will promote myofibroblast activation, but we do not see this in Acomys dermal fibroblasts (DFs). Though Mus DFs increase organization of α-smooth muscle actin (αSMA)-positive stress fibers as substrate stiffness increases, Acomys DFs assemble very few αSMA-positive stress fibers upon changes in substrate stiffness. Acomys DFs generate lower traction forces than Mus DFs on pliable surfaces, and Acomys DFs produce and modify matrix proteins differently than Mus in 2D and 3D culture systems. In contrast to Acomys DFs “relaxed” behavior, we found that freshly isolated Acomys keratinocytes retain the ability to close wounds faster than Mus in an in vitro scratch assay. Taken together, these preliminary observations suggest that Acomys dermal cells retain unique biophysical properties in vitro that may reflect their altered in vivo mechanical microenvironment and may promote scar-free wound healing.     

Decreased expression of Flightless I, a gelsolin family member and developmental regulator, in early-gestation fetal wounds improves healing

Up until late in the third trimester of gestation and through to adulthood, the healing response acts more to regenerate than to repair a wound. The mechanisms underlying this ??scar-free?? healing remain unknown although the actin cytoskeleton has a major role. Flightless I (Flii), an actin-remodelling protein and essential developmental regulator, negatively affects wound repair but its effect on scar-free fetal healing is unknown. Using fetal skin explants from E17 (regenerate) and E19 (repair) rats, the function of Flii in fetal wound repair was determined. Expression of Flii increased between E17 and E19?days of gestation and wounding transiently increased Flii expression in E17 but not E19 wounds. However, both confocal and immunofluorescent analysis showed E17 keratinocytes immediately adjacent to the wounds downregulated Flii. As a nuclear coactivator and inhibitor of proliferation and migration, the absence of Flii in cells at the edge of the wound could be instrumental in allowing these cells to proliferate and migrate into the wound deficit. In contrast, Flii was strongly expressed within the cytoplasm and nucleus of keratinocytes within epidermal cells at the leading edge of E19 wounded fetal skin explants. This increase in Flii expression in E19 wounds could affect the way these cells migrate into the wound space and contribute to impaired wound healing. Neutralising Flii protein improved healing of early- but not late-gestation wounds. Flii did not colocalise with actin cables formed around E17 wounds suggesting an independent mechanism of action distinct from its actin-binding function in scar-free wound repair.     

Skin regeneration in adult axolotls: a blueprint for scar-free healing in vertebrates

While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair.     

Evidence That Mast Cells Are Not Required for Healing of Splinted Cutaneous Excisional Wounds in Mice

Wound healing is a complex biological process involving the interaction of many cell types to replace lost or damaged tissue. Although the biology of wound healing has been extensively investigated, few studies have focused on the role of mast cells. In this study, we investigated the possible role of mast cells in wound healing by analyzing aspects of cutaneous excisional wound healing in three types of genetically mast cell-deficient mice. We found that C57BL/6-Kit^W-sh/W-sh, WBB6F₁-Kit^W/W-v, and Cpa3-Cre; Mcl-1^fl/fl mice re-epithelialized splinted excisional skin wounds at rates very similar to those in the corresponding wild type or control mice. Furthermore, at the time of closure, scars were similar in the genetically mast cell-deficient mice and the corresponding wild type or control mice in both quantity of collagen deposition and maturity of collagen fibers, as evaluated by Masson’s Trichrome and Picro-Sirius red staining. These data indicate that mast cells do not play a significant non-redundant role in these features of the healing of splinted full thickness excisional cutaneous wounds in mice.     

The extracellular matrix of lip wounds in fetal, neonatal and adult mice.

Wound healing in the fetus occurs rapidly, by a regenerative process and without an inflammatory response, resulting in complete restitution of normal tissue function. By contrast, in the adult, wounds heal with scar formation, which may impair function and inhibit further growth. The cellular mechanisms underlying these differing forms of wound healing are unknown but the extracellular matrix (ECM), through its effects on cell function, may play a key role. We have studied the ECM in upper lip wounds of adult, neonatal and fetal mice at days 14, 16 and 18 of gestation. The spatial and temporal distribution of collagen types I, III, IV, V and VI, fibronectin, tenascin, laminin, chondroitin and heparan sulphates were examined immunohistochemically. Results from the fetal groups were essentially similar whilst there were distinct differences between fetus, neonate and adult. Fibronectin was present at the surface of the wound in all groups at 1 h post-wounding. Tenascin was also present at the wound surface but the time at which it was first present differed between fetus (1 h), neonate (12 h) and adult (24 h). The time of first appearance paralleled the rate of wound healing which was most rapid in the fetus and slowest in the adult. Tenascin inhibits the cell adhesion effect of fibronectin and during development the appearance of tenascin correlates with the initiation of cell migration. During wound healing the appearance of tenascin preceded cell migration and the rapid closure of fetal wounds may be due to the early appearance of tenascin in the wound. Collagen types I, III, IV, V and VI were present in all three wound groups but the timing and pattern of collagen deposition differed, with restoration of the normal collagen pattern in the fetus and a scar pattern in the adult. This confirms that lack of scarring in fetal wounds is due to the organisation of collagen within the wound and not simply lack of collagen formation. The distribution of chondroitin sulphate differed between normal fetal and adult tissues and between fetal and adult wounds. Its presence in the fetal wound may alter collagen fibril formation. No inflammatory response was seen in the fetal wounds. The differences in the ECM of fetal and adult wounds suggests that it may be possible to alter the adult wound so that it heals by a fetal-like process without scar formation, loss of tissue function or restriction of growth.     

Pirfenidone attenuates the profibrotic contractile phenotype of differentiated human dermal myofibroblasts

Dysregulated wound healing after burn injury frequently results in debilitating hypertrophic scarring and contractures. Myofibroblasts, the main effector cells for dermal fibrosis, develop from normal fibroblasts via transforming growth factor beta 1 (TGF-β1). During wound healing, myofibroblasts produce extracellular matrix (ECM) proteins, modulate ECM stability, and contract the ECM using alpha smooth muscle actin (α-SMA) in contractile stress fibers. The antifibrotic pirfenidone has previously been shown to inhibit the initial differentiation of fibroblasts into myofibroblasts in vitro and act as a prophylactic measure against hypertrophic scar development in a mouse burn model. To test whether pirfenidone affects differentiated myofibroblasts, we investigated the in vitro effects of pirfenidone treatment after three to five days of stimulation with TGF-β1. In assays for morphology, protein and gene expression, and contractility, pirfenidone treatment produced significant effects. Profibrotic gene expression returned to near-normal levels, further α-SMA protein expression was prevented, and cell contraction within a stressed collagen matrix was reduced. These in vitro results promote pirfenidone as a promising antifibrotic agent to treat existing scars and healing wounds by mitigating the effects of differentiated myofibroblasts.     

Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.

Fetal wounds pass from scarless repair to healing with scar formation during gestation. This transition depends on both the size of the wound and the gestational age of the fetus. This study defines the transition period in the fetal rat model and provides new insight into scarless collagen wound architecture by using confocal microscopy. A total of 16 pregnant Sprague-Dawley rats were operated on. Open full-thickness wounds, 2 mm in diameter, were created on fetal rats at gestational ages 14.5 days (E14; n = 10), 16.5 days (E16; n = 42), and 18.5 days (E18; n = 42) (term = 21.5 days). Wounds were harvested at 24 (n = 18 per gestational age) and 72 hours (n = 24 per gestational age). Skin at identical gestational ages to wound harvest was used for controls. The wounds were fixed and stained with hematoxylin and eosin, antibody to type I collagen, and Sirius red for confocal microscopic evaluation. No E14 rat fetuses survived to wound harvest. Wounds created on E16 fetal rats healed completely and without scarring. E16 fetal rat hair follicle formation and collagen architecture was similar to that of normal, nonwounded skin. Wounds created on E18 fetal rats demonstrated slower healing; only 50 percent were completely healed at 72 hours compared with 100 percent of the E16 fetal rat wounds at 72 hours. Furthermore, the E18 wounds healed with collagen scar formation and without hair follicle formation. Confocal microscopy demonstrated that the collagen fibers were thin and arranged in a wispy pattern in E16 fetal rat wounds and in nonwounded dermis. E18 fetal rat wounds had thickened collagen fibers with large interfiber distances. Two-millimeter excisional E16 fetal rat wounds heal without scar formation and with regeneration of normal dermal and epidermal appendage architecture. E18 fetal rat wounds heal in a pattern similar to that of adult cutaneous wounds, with scar formation and absence of epidermal appendages. Confocal microscopy more clearly defined the dermal architecture in normal skin, scarless wounds, and scars. These data further define the transition period in the fetal rat wound model, which promises to be an effective system for the study of in vivo scarless wound healing.     

Application of Electron Paramagnetic Resonance (EPR) Oximetry to Monitor Oxygen in Wounds in Diabetic Models

A lack of oxygen is classically described as a major cause of impaired wound healing in diabetic patients. Even if the role of oxygen in the wound healing process is well recognized, measurement of oxygen levels in a wound remains challenging. The purpose of the present study was to assess the value of electron paramagnetic resonance (EPR) oximetry to monitor pO₂ in wounds during the healing process in diabetic mouse models. Kinetics of wound closure were carried out in streptozotocin (STZ)-treated and db/db mice. The pO₂ was followed repeatedly during the healing process by 1 GHz EPR spectroscopy with lithium phthalocyanine (LiPc) crystals used as oxygen sensor in two different wound models: a full-thickness excisional skin wound and a pedicled skin flap. Wound closure kinetics were dramatically slower in 12-week-old db/db compared to control (db/+) mice, whereas kinetics were not statistically different in STZ-treated compared to control mice. At the center of excisional wounds, measurements were highly influenced by atmospheric oxygen early in the healing process. In pedicled flaps, hypoxia was observed early after wounding. While reoxygenation occurred over time in db/+ mice, hypoxia was prolonged in the diabetic db/db model. This observation was consistent with impaired healing and microangiopathies observed using intravital microscopy. In conclusion, EPR oximetry using LiPc crystals as the oxygen sensor is an appropriate technique to follow wound oxygenation in acute and chronic wounds, in normal and diabetic animals. Nevertheless, the technique is limited for measurements in pedicled skin flaps and cannot be applied to excisional wounds in which diffusion of atmospheric oxygen significantly affects the measurements.     

Histological,immunohistochemical, and genomic evaluation of excisional and diabetic wounds treated with human Wharton’s jelly stem cells with and without a nanocarrier

We showed in previous studies that human umbilical cord Wharton’s jelly stem cells (hWJSCs) improved the healing rates of excisional and diabetic wounds in the mouse model. As an extension of those studies, we report here the more detailed quantitative histological, immunohistochemical, and genomic evaluation of biopsies from those excisional and diabetic wounds in an attempt to understand the mechanisms of the enhanced wound healing aided by hWJSCs. Bright-field microscopic observations and ImageJ software analysis on histological sections of the excisional and diabetic wound biopsies collected at different time points showed that the thickness of the epidermis and dermis, and positive picrosirius-red stained areas for collagen, were significantly greater in the presence of hWJSCs compared with controls (P < 0.05). Immunohistochemistry of the diabetic wound biopsies showed increased positive staining for the vascular endothelial marker CD31 and cell proliferation marker Ki67 in the presence of hWJSCs and its conditioned medium (hWJSC-CM). Quantitative real-time polymerase chain reaction showed upregulation of groups of genes involved in extracellular matrix regulation, collagen biosynthesis, angiogenesis, antifibrosis, granulation, and immunomodulation in the presence of hWJSCs. Taken together, the results demonstrated that hWJSCs and hWJSC-CM that contains the paracrine secretions of hWJSCs, enhance the healing of excisional and diabetic wounds via re-epithelialization, collagen deposition, angiogenesis, and immunomodulation. The inclusion of an Aloe vera-polycaprolactone (AV/PCL) nanocarrier did not significantly change the effect of the hWJSCs. However, the topical application of an AV/PCL nanocarrier impregnated with hWJSCs is convenient and less invasive than the administration of hWJSC injections into wounds.     

CD44-dependent inflammation,fibrogenesis, and collagenolysis regulates extracellular matrix remodeling and tensile strength during cutaneous wound healing

Cutaneous wound healing consists of three main phases: inflammation, re-epithelialization, and tissue remodeling. During normal wound healing, these processes are tightly regulated to allow restoration of skin function and biomechanics. In many instances, healing leads to an excess accumulation of fibrillar collagen (the principal protein found in the extracellular matrix - ECM), and the formation of scar tissue, which has compromised biomechanics, tested using ramp to failure tests, compared to normal skin (Corr and Hart, 2013 [1]). Alterations in collagen accumulation and architecture have been attributed to the reduced tensile strength found in scar tissue (Brenda et al., 1999; Eleswarapu et al., 2011). Defining mechanisms that govern cellular functionality and ECM remodeling are vital to understanding normal versus pathological healing and developing approaches to prevent scarring. CD44 is a cell surface adhesion receptor expressed on nearly all cell types present in dermis. Although CD44 has been implicated in an array of inflammatory and fibrotic processes such as leukocyte recruitment, T-cell extravasation, and hyaluronic acid (the principal glycosaminoglycan found in the ECM) metabolism, the role of CD44 in cutaneous wound healing and scarring remains unknown. We demonstrate that in an excisional biopsy punch wound healing model, CD44-null mice have increased inflammatory and reduced fibrogenic responses during early phases of wound healing. At wound closure, CD44-null mice exhibit reduced collagen degradation leading to increased accumulation of fibrillar collagen, which persists after wound closure leading to reduced tensile strength resulting in a more severe scarring phenotype compared to WT mice. These data indicate that CD44 plays a previously unknown role in fibrillar collagen accumulation and wound healing during the injury response.     

Matrix metalloproteinase 9 (MMP-9) is upregulated during scarless wound healing in athymic nude mice

Cutaneous wound healing is associated with migratory and remodeling events that require the action of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Differences in their expressions were observed during scar-forming and scar-free skin wound healing. We previously found that athymic nude mice are exceptional among mature mammals in their ability to heal injured skin scarlessly. The present study was undertaken to determine whether the modulation of MMP-2 and MMP-9 expression during scarless healing in nude mice was different from scar-forming animals. Full thickness skin wounds were made into the back of nude, wild-type controls (C57BL/6J), immunodeficient SCID and Rag, thymectomized neonates and adults, and cyclosporin A treated mice. Post-injured skin tissues were harvested at Day 7 and 24 after injury. Quantitative RT-PCR, Western blot, gelatin zymography and immunohistochemical assays were performed. Our results show that MMP-2 protein was high but similarly expressed in all post-injured animals on Day 7 after injury. Late phase (Day 24) of wound repair was characterized by a decrease in mRNA and protein expression and a decrease in gelatinolytic activity of MMP-2 in all post-injured samples. On the contrary, high (p < 0.001) levels of mRNA expression, prominent pro-and active forms of MMP-9 and cells immunopositive for MMP-9 were present exclusively in the post-injured tissues from nude mice on Day 24 after wounding. This data suggest that MMP-9 expression in the remodeling phase of wound healing in nude mice could be a major component of their ability for scar-free healing.     

Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis

The biological roles of hyaluronan (HA) fragments in angiogenesis acceleration have been investigated recently. Studies have confirmed that oligosaccharides of HA (o-HA) are capable of stimulating neovascularization in vitro and promoting blood flow or angiogenesis in animal models. However, few laboratories have studied the function of o-HA as an exogenous treatment in injured tissue repair in vivo. It is thought that o-HA may lose its activities when used topically in vivo due to its small size, which may be absorbed quickly by the surrounding tissues. In this study, we prepared a special slow-releasing gel that contains a mixture of defined size of o-HA and studied the healing effects of o-HA by topical application to an acute wound model. We report that o-HA complex promotes the repair of tissue injury of a murine excisional dermal wound. The therapy by o-HA was compared with high molecular weight HA (HMW-HA) and the known angiogenesis stimulator, VEGF. At days 6 to 8 after treatment, significant differences were seen in wound closure rates between o-HA and control or HMW-HA groups, in which o-HA showed an increased wound recovery. Histological analysis revealed that increased neo-blood and lymph vessels were formed in wounded tissues treated by o-HA. In addition, treatments of wounds with o-HA resulted in more granulation production, collagen deposition, and fibroblast proliferation. Analysis of gene expression by real-time RT-PCR demonstrated a significant up-regulation of some cytokines or adhesion molecules in o-HA-treated wounds, which corresponds with the increased granulation tissue in these wounds. Our findings suggested that o-HA therapy may be useful in acute wound repair.     

Mesenchymal stromal cells enhance wound healing by ameliorating impaired metabolism in diabetic mice

Background aimsMesenchymal stromal cells (MSCs) have been documented to improve delayed wound healing in diabetes, but the underlying mechanism remains obscure. We aimed to investigate whether the therapeutic effects on wounds was associated with metabolic alterations by paracrine action of MSCs.MethodsMSCs from mice with high-fat diet/streptozotocin–induced diabetes or wild-type C57BL/6 mice were evaluated for their paracrine potential in vitro using enzyme-linked immunosorbent assay and immunohistochemical staining assay. MSCs were then evaluated for their therapeutic potential in vivo using an excisional cutaneous wound model in mice with diabetes. Metabolic alterations and glucose transporter four (GLUT4) as well as PI3K/Akt signaling pathway expression after wounding were also examined.ResultsMSCs from normal mice expressed even more insulin-like growth factor-1 (IGF-1) than mice with diabetes, suggesting putative paracrine action. Furthermore, compared with IGF-1 knockdown MSCs, normal MSCs markedly accelerated wound healing, as revealed by higher wound closure rate and better healing quality at 21 days post-wound. By contrast, MSCs administration increased the level of insulin as well as GLUT4 and PI3K/Akt signaling pathway expression but repressed the biochemical indexes of glucose and lipid, resulting in obvious metabolic improvement.ConclusionsThese findings suggest that IGF-1 is an important paracrine factor that mediates the therapeutic effects of MSCs on wound healing in diabetes, and the benefits of MSCs may be associated with metabolism improvements, which would provide a new target for treatment.     

Discoidin domain receptors and their ligand, collagen, are temporally regulated in fetal rat fibroblasts in vitro

The biochemical regulation of collagen deposition during adult cutaneous wound repair is poorly understood. Likewise, how collagen is perceived and modulated in fetal scarless healing remains unknown. Recently, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. In light of these findings, it was speculated that the production of collagen receptors DDR1 and DDR2 by fetal fibroblasts may be temporally regulated to correlate with the ontogeny of embryonic scar formation. More specifically, because DDRs directly bind collagen and transmit the signals intracellularly, it was hypothesized that they may play an important role in fetal scarless healing by ultimately regulating and modulating collagen production and organization. As part of a fundamental assessment to elucidate the role of DDRs in scarless fetal wound repair, the endogenous expression of DDR1, DDR2, collagen I, and total collagen, as a function of fetal Sprague-Dawley rat skin fibroblasts of different gestational ages, representing scar-free (E16.5) periods was determined. Using explanted dermal fibroblasts of gestational days E13.5, E16.5, E18.5, and E21.5 (term gestation = 21.5 days) fetuses (n = 92), [3H]proline incorporation assay and Northern and Western blotting analysis were performed to compare the expressions of these molecules with scar-free and scar-forming stages of embryonic development. These results revealed a pattern of increasing collagen production with increasing gestational ages, whereas DDR1 expression decreased with increasing gestational age. This observation suggests that elevated levels of DDR1 may play an important role in scarless tissue regeneration by early gestation fetal fibroblasts. In contrast, DDR2 was expressed by fetal rat fibroblasts at a similar level throughout gestation. These data demonstrate for the first time the temporal expression of collagen and DDR tyrosine kinases in fetal rat fibroblasts as a function of gestational ages. Overall, these data suggest that differential temporal expression of the above-mentioned molecules during fetal skin development may play an important role in the ontogeny of scar formation. Future studies will involve the characterization of the biomolecular functions of these receptor kinases during fetal wound repair.     

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.     

SPARC-null mice exhibit accelerated cutaneous wound closure.

Expression of SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40), an extracellular matrix (ECM) associated protein, is coincident with matrix remodeling. To further identify the functions of SPARC in vivo, we have made excisional wounds on the dorsa of SPARC-null and wild-type mice and monitored closure over time. A significant decrease in the size of the SPARC-null wounds, in comparison to that of wild-type, was observed at Day 4 and was maximal at Day 7. Although substantial differences in the percentage of proliferating cells were not apparent in SPARC-null relative to wild-type wounds, primary cultures of SPARC-null dermal fibroblasts displayed accelerated migration, relative to wild-type fibroblasts, in wound assays in vitro. Although the expression of collagen I mRNA in wounds, as measured by in situ hybridization (ISH), was not significantly different in SPARC-null vs wild-type mice, the collagen content of unwounded skin appeared to be substantially lower in the SPARC-null animals. By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of wild-type skin. Moreover, we found an inverse correlation between the efficiency of collagen gel contraction by dermal fibroblasts and the concentration of collagen within the gel itself. We propose that the accelerated wound closure seen in SPARC-null dermis results from its decreased collagen content, a condition contributing to enhanced contractibility.     

Lentiviral-mediated over-expression of hyaluronan synthase-1 (HAS-1) decreases the cellular inflammatory response and results in regenerative wound repair

Fetal wounds have been found to have increased levels of high-molecular-weight hyaluronan (HMW-HA) compared with those of adults. The primary enzyme responsible for producing HMW-HA is hyaluronic acid synthase-1 (HAS-1). We hypothesized that over-expression of HAS-1 in adult dermal wounds would decrease inflammation and promote regenerative healing. To test this hypothesis, the flanks of adult C57Bl/6 mice were treated with a lentiviral construct containing either HAS-1-GFP or GFP transgenes. After 48 h, a 4-mm excisional wound was made at the site of treatment. Wounds were harvested at days 3, 7, or 28 after wounding. Wound phenotype was assessed by histology to examine tissue architecture and immunohistochemistry for CD45. At 7 and 28 days, lenti-HAS-1-treated wounds demonstrated the restoration of the normal dermal elements and organized collagen fiber orientation. In contrast, the lenti-GFP-treated wounds lacked normal dermal architecture and demonstrated a disorganized collagen scar. At 3 and 7 days, wounds treated with lenti-HAS-1 exhibited a significant decrease in the number of inflammatory cells when compared with wounds treated with lenti-GFP. Thus, HAS-1 over-expression promotes dermal regeneration, in part by decreasing the inflammatory response and by recapitulation of fetal extracellular matrix HMW-HA content.     

Endoglin Haploinsufficiency Promotes Fibroblast Accumulation during Wound Healing through Akt Activation

Accurate regulation of dermal fibroblast function plays a crucial role in wound healing. Many fibrotic diseases are characterized by a failure to conclude normal tissue repair and the persistence of fibroblasts inside lesions. In the present study we demonstrate that endoglin haploinsufficiency promotes fibroblast accumulation during wound healing. Moreover, scars from endoglin-heterozygous (Eng^+/−) mice show persisting fibroblasts 12 days after wounding, which could lead to a fibrotic scar. Endoglin haploinsufficiency results in increased proliferation and migration of primary cultured murine dermal fibroblasts (MDFs). Moreover, Eng^+/− MDF have diminished responses to apoptotic signals compared with control cells. Altogether, these modifications could explain the augmented presence of fibroblasts in Eng^+/− mice wounds. We demonstrate that endoglin expression regulates Akt phosphorylation and that PI3K inhibition abolishes the differences in proliferation between endoglin haploinsufficient and control cells. Finally, persistent fibroblasts in Eng^+/− mice wound co-localize with a greater degree of Akt phosphorylation. Thus, endoglin haploinsufficiency seems to promote fibroblast accumulation during wound healing through the activation of the PI3K/Akt pathway. These studies open new non-Smad signaling pathway for endoglin regulating fibroblast cell function during wound healing, as new therapeutic opportunities for the treatment of fibrotic wounds.     

Role of platelet-derived growth factor in wound healing

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.