transforming-growth-factor-beta and Skin-Ulcer

The management of systemic sclerosis (SSc) is complex, evolving, and requires a multidisciplinary approach. At diagnosis and throughout the disease course, clinical assessment and monitoring of skin involvement is vital using the modified Rodnan Skin Score, patient-reported outcomes, and new global composite scores (such as the Combined Response Index for Systemic Sclerosis, which also considers lung function). Immunomodulation is the mainstay of skin fibrosis treatment, with mycophenolate mofetil considered first line. Meanwhile vasculopathy-related manifestations (Raynaud's phenomenon, digital ulcers) and calcinosis, require general measures combined with specific pharmacologic (calcium-channel blockers, phosphodiesterase type 5 inhibitors, and prostanoids), nonpharmacologic (digital sympathectomy and botulinum toxin injections), and often multifaceted, management approaches. Patients should be screened at the time of diagnosis specifically for systemic manifestations and then regularly thereafter, with appropriate treatment. Numerous targeted therapeutic options for SSc, including skin fibrosis, are emerging and include B-cell depletion, anti-interleukin 6, Janus kinase, and transforming growth factor β inhibition. This second article in the continuing medical education series discusses these key aspects of SSc assessment and treatment, with particular focus on skin involvement. It is vital that dermatologists play a key role in the multidisciplinary approach to SSc management.

Topics: Adult; Botulinum Toxins; Calcium; Fibrosis; Humans; Janus Kinases; Mycophenolic Acid; Phosphodiesterase 5 Inhibitors; Prostaglandins; Raynaud Disease; Scleroderma, Systemic; Skin Ulcer; Transforming Growth Factor beta

CD271 is common stem cell marker for the epidermis and dermis. We assessed a kinetic movement of epidermal and dermal CD271

Topics: Aging; Animals; Cell Movement; Cell Proliferation; Chronic Disease; Disease Models, Animal; Humans; Mice; Receptors, Nerve Growth Factor; Skin; Skin Physiological Phenomena; Skin Ulcer; Stem Cells; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Wound Healing

Despite the heterogeneity of SSc, almost all patients have skin involvement. As such, skin manifestations are critical in the initial diagnosis of SSc and in the subsequent sub-classification into the different subsets of disease. The two principal subsets are lcSSc and dcSSc. The main difference between these two subsets is the speed of disease progression and the extent and severity of skin and visceral involvement; lcSSc has an insidious onset with skin involvement confined largely to the face and extremities. Whilst vascular manifestations of SSc such as pulmonary arterial hypertension are typically more common in lcSSc, patients in both subsets can develop ischaemic digital ulcers. In dcSSc, disease progression is very rapid, with skin thickening extending beyond the extremities and earlier, more widespread internal organ involvement. DcSSc is generally considered to be the more severe subset of the disease. Skin scores in SSc correlate inversely with survival and are considered a valuable marker of disease severity. Skin involvement is easily detectable and, using the modified Rodnan skin score, the degree of skin fibrosis can be quantified. As well as general management measures, a number of targeted therapies are commonly used for treatment of cutaneous manifestations of SSc. These include the intravenous prostanoid iloprost and the dual endothelin receptor antagonist bosentan, which is approved in Europe for the prevention of new digital ulcers.

Topics: Collagen; Connective Tissue Growth Factor; Diagnosis, Differential; Fibrosis; Humans; Scleroderma, Systemic; Skin; Skin Ulcer; Transforming Growth Factor beta

Topics: Animals; Combined Modality Therapy; Diabetes Complications; Electric Stimulation Therapy; Electroporation; Mice; Mice, Mutant Strains; Skin Ulcer; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

We developed a novel mouse model of human refractory cutaneous ulcers that more faithfully reflects pathology and evaluated the effects of mixed cell sheets comprising peripheral blood mononuclear cells and fibroblasts, which we previously developed for treating refractory cutaneous ulcers. Model development involved sandwiching the skin between two magnets, one of which was implanted under the skin for 7 consecutive days. This magnet-implanted ulcer model produced persistently large amounts of exudate and induced the infiltration of the ulcer with inflammatory cells. The model mice had a thicker epidermis and impaired transforming growth factor-β (TGF-β) signaling followed by SMAD2 down-regulation, which causes epidermal hyperplasia in chronic ulcers. Impaired TGF-β signaling also occurred in the ulcers of critical limb ischemia patients. Mixed cell implantation in this ulcer model reduced TNF-α and IL-6 levels in the tissues surrounding the mixed cell sheet-treated ulcers compared with controls or mice treated with trafermin (FGF2). Seven days after commencing therapy, the epidermis was thinner in mice treated with the mixed cell sheets than in controls. This model may therefore serve as a clinically relevant model of human ulcers, and our mixed cell sheets may effectively relieve chronic inflammation and inhibit refractoriness mechanisms.

Topics: Animals; Disease Models, Animal; Histocytochemistry; Magnets; Mice; Signal Transduction; Skin Ulcer; Smad2 Protein; Transforming Growth Factor beta

Lipoxin A4 (LXA4) is involved in the resolution of inflammation and wound healing; however, it is extremely unstable. Thus, to preserve its biological activities and confer stability, we encapsulated LXA4 in poly-lactic-co-glycolic acid (PLGA) microparticles (LXA4-MS) and assessed its application in treating dorsal rat skin lesions. Ulcers were sealed with fibrin adhesive and treated with either LXA4-MS, unloaded microparticles (Un-MS), soluble LXA4, or PBS/glue (vehicle). All groups were compared at 0, 2, 7, and 14 days post-lesions. Our results revealed that LXA4-MS accelerated wound healing from day 7 and reduced initial ulcer diameters by 80%. Soluble LXA4, Un-MS, or PBS closed wounds by 60%, 45%, and 39%, respectively. LXA4-MS reduced IL-1β and TNF-α, but increased TGF-β, collagen deposition, and the number of blood vessels. Compared to other treatments, LXA4-MS reduced inflammatory cell numbers, myeloperoxidase (MPO) concentration, and metalloproteinase-8 (MMP8) mRNA in scar tissue, indicating decreased neutrophil chemotaxis. In addition, LXA4-MS treatment increased macrophages and IL-4, suggesting a positive impact on wound healing. Finally, we demonstrated that WRW4, a selective LXA4 receptor (ALX) antagonist, reversed healing by 50%, indicating that LXA4 must interact with ALX to induce wound healing. Our results show that LXA4-MS could be used as a pharmaceutical formulation for the treatment of skin ulcers.

Topics: Animals; Cytokines; Interleukin-1beta; Interleukin-6; Lactic Acid; Lipoxins; Male; Neutrophils; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Wistar; Skin Ulcer; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Wound Healing

Impaired wound healing in skin ulcer is one of the major medical issues in the aged society. Wound healing is a complex process orchestrated by a number of humoral factors and cellular components. TGF-β is known to stimulate collagen production in dermal fibroblasts while inhibiting proliferation of epidermal keratinocyte. A screening of small compounds that suppress type I collagen production in fibroblasts has identified HSc025 that antagonizes the TGF-β/Smad signal.. We examined the effects of HSc025 on dermal wound healing and elucidated the underlying mechanisms.. Effects of HSc025 on the wound closure process were evaluated in a murine full-thickness excisional wound healing model. Cell proliferation and migration were estimated using primary cultures of human keratinocytes and fibroblasts. Comprehensive analyses of gene expression profiles were performed using untreated and HSc025-treated fibroblasts.. Oral HSc025 administration suppressed macrophage infiltration and accelerated wound closure as early as at day 2 after the dermal excision. Treatment of cultured keratinocytes with HSc025 counteracted the inhibitory effects of TGF-β on cell proliferation and migration. On the other hand, HSc025 stimulated migration, but not proliferation, of dermal fibroblasts independently of TGF-β. Experiments using an artificial dermis graft revealed that HSc025 stimulated migration of collagen-producing cells into the graft tissue. A cDNA microarray analysis of untreated and HSc025-treated fibroblasts identified pirin as a critical mediator accelerating fibroblast migration.. HSc025 accelerates wound healing by modifying infiltration, proliferation and migration of distinct cellular components, which provides a novel insight into the therapy for intractable skin ulcer.

Topics: Alkadienes; Animals; Carrier Proteins; Cell Movement; Cell Proliferation; Cells, Cultured; Collagen Type I; Dioxygenases; Drug Evaluation, Preclinical; Female; Fibroblasts; Granulation Tissue; Humans; Keratinocytes; Mice; Nuclear Proteins; Skin Ulcer; Transforming Growth Factor beta; Wound Healing

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-β (TGF-β) signaling, and mice lacking components of the TGF-β signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-β pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of β4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-β was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-β. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-β pathway.

Topics: Animals; Cell Adhesion; Cell Movement; Cells, Cultured; Chloride Channels; Cornea; Corneal Injuries; Dose-Response Relationship, Drug; Keratinocytes; Mice; Mice, Knockout; Microscopy, Confocal; Mitochondrial Proteins; Proteins; Signal Transduction; Skin; Skin Ulcer; Time Factors; Transforming Growth Factor beta; Wound Healing

The aim of the present study is to investigate the effect and mechanism of acidic fibroblast growth factor (aFGF) on treating refractory wound of diabetic rats. SD rats were randomly divided into control group, diabetes group, and aFGF group. Ulcer skin tissues of three groups of rats were respectively collected on days 7 and 14 after establishment of ulcer model for biochemical test, pathological section and immunohistochemistry to comprehensively evaluate the treatment effect of aFGF on diabetic ulcer. The results showed that aFGF could significantly increase capillaries and fibroblast amounts of ulcer tissues, enhance the expression of TGF-β and PCNA proliferation proteins, and thus improved diabetic ulcer tissues. The preliminary mechanism that aFGF helps to promote healing of diabetic ulcer is possibly associated with that aFGF stimulated ulcer skins to secrete TGF-β and PCNA proteins and promoted proliferation of capillaries and fibroblasts.

Topics: Animals; Diabetes Mellitus, Experimental; Fibroblast Growth Factor 1; Immunohistochemistry; Male; Proliferating Cell Nuclear Antigen; Random Allocation; Rats; Rats, Sprague-Dawley; Skin Ulcer; Transforming Growth Factor beta; Wound Healing

Radiation therapy (RT) to the head and neck often results in damage to the vocal folds (VF) and surrounding structures. Characterization and treatment of these sequelae is limited, likely due to the lack of experimental models.. Larynges from rats exposed to 2 fractionation schedules (40 Gy total) were analyzed histologically. In vitro, reactive oxygen species (ROS) synthesis, and transcription of select genes associated with ROS, inflammation, and fibrosis were examined in VF fibroblasts after single-dose radiation.. Although radiation-induced histologic alterations are made to VF architecture, 1 fractionation schedule was accompanied by significant morbidity and mortality. In vitro, radiation increased ROS synthesis and inflammatory and profibrotic gene expression.. Our data suggest that hyperfractionated RT is more tolerable. Utilizing this model, RT-induced histologic aberrations are made to the VF mucosa. In addition, a relationship between radiation, ROS, and inflammatory and fibrotic gene expression was observed in vitro.

Topics: Alopecia; Animals; Dehydration; Dose Fractionation, Radiation; Erythema; Fibrosis; Heme Oxygenase-1; Hypertrophy; In Vitro Techniques; Laryngeal Mucosa; Male; Matrix Metalloproteinase 1; Models, Animal; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Skin Ulcer; Transforming Growth Factor beta; Vocal Cords; Weight Loss

The present study describes the production of hyaluronan based porous microparticles by a semi-continuous gas anti-solvent (GAS) precipitation process to be used as a growth factor delivery system for in vivo treatment of ulcers. Operative process conditions, such as pressure, nozzle diameter and HYAFF11 solution concentrations, were adjusted to optimize particle production in terms of morphology and size. Scanning electron microscopy (SEM) and light scattering demonstrated that porous nano-structured particles with a size of 300 and 900 nm had a high specific surface suitable for absorption of growth factors from the aqueous environment within the polymeric matrix. Water acted as a plasticizer, enhancing growth factor absorption. Water contents within the HYAFF11 matrix were analyzed by differential scanning calorimetry (DSC). The absorption process was developed using fluorescence dyes and growth factors. Immunohistochemical analysis confirmed the high efficiency of absorption of growth factor and a mathematical model was generated to quantify and qualify the in vitro kinetics of growth factor release within the polymeric matrix. In vivo experiments were performed with the aim to optimize timed and focal release of PDGF to promote optimal tissue repair and regeneration of full-thickness wounds.

Topics: Absorption; Animals; Biocompatible Materials; Growth Substances; Hyaluronic Acid; Male; Materials Testing; Microscopy, Electron, Scanning; Nanoparticles; Nanotechnology; Platelet-Derived Growth Factor; Rats; Rats, Wistar; Skin Ulcer; Tissue Scaffolds; Transforming Growth Factor beta; Water; Wound Healing

Up to one million people suffer from chronic skin ulcers in the US. Little is known of the mechanisms leading to tissue breakdown, although inadequate circulation and ischemia are common elements in most dermal ulcers. Collagen is the principal source of mechanical strength in most tissues, and its molecular and fibrillar stability is dependent on adequate oxygen supply. In wound repair, localized ischemia leads to fibrogenic responses culminating in elevated collagen synthesis and remodeling. This study examines factors influencing collagen turnover and stabilization before ulceration in "at risk" patients. Severely ischemic but uninjured ischemic skin (IS) was compared with patient- and site-matched non-ischemic skin. Biochemical mechanisms of tissue repair were activated in IS, with increased lactate, transforming growth factor-beta, vascular endothelial growth factor, collagen synthesis and matrix metalloproteinases (MMPs)-1 and 2. The absence of MMP-9 and inflammatory cells confirmed that this upregulation was inappropriate and not in response to injury. Molecular stability of collagen was reduced in IS, and there was increased susceptibility to enzymic degradation. In conclusion, chronic ischemia and long-term hypoxia result in elevated collagen remodeling in an oxygen-poor environment. Unstable collagen molecules are synthesized together with upregulated MMPs, resulting in collagen denaturation, defective angiogenesis, weaker skin, and predisposition to ulceration.

Topics: Aged; Aged, 80 and over; Chronic Disease; Collagen; Disease Susceptibility; Drug Stability; Female; Humans; Intercellular Signaling Peptides and Proteins; Ischemia; Lactates; Male; Middle Aged; Neovascularization, Physiologic; Receptors, Transforming Growth Factor beta; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Skin; Skin Ulcer; Smad Proteins; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Topics: Animals; Immunosuppressive Agents; Rabbits; Skin Ulcer; Tacrolimus; Transforming Growth Factor beta; Wound Healing

During the fall of 2001 and 2002, Atlantic menhaden Brevoortia tyrannus were collected from several creeks in the Pamlico River, North Carolina, to investigate recent fish kills and ulcerative skin lesions. High skin lesion prevalence (>50%) was associated with the Atlantic menhaden kills in fall 2001, whereas there were no fish kills in fall 2002 and skin lesion prevalence was lower (< or =50%). Indicators of tissue damage (histopathological analyses of gills, heart, liver, intestine, and anterior kidney), body condition (liver somatic index), and immune status (transforming growth factor-beta [TGF-beta] messenger RNA [mRNA] production, hematology, plasma chemistry, and splenosomatic index) were compared between Atlantic menhaden with and without ulcerative skin lesions in fall. Atlantic menhaden with ulcerative skin lesions had significantly higher liver somatic indices, neutrophil and monocyte percentages, and splenic mononuclear cell TGF-beta mRNA levels than did fish without lesions. Hematocrit values, plasma protein, and Ca concentrations were significantly lower in fish with ulcerative skin lesions than in those without. The indicators used in this study at multiple levels of biological organization have provided valuable baseline data for understanding the health status of lesioned and nonlesioned Atlantic menhaden in the Pamlico River.

Topics: Actins; Animals; Blood Cell Count; Blood Chemical Analysis; Blood Proteins; Fish Diseases; Fishes; Liver; North Carolina; Prevalence; Rivers; Skin; Skin Diseases; Skin Ulcer; Spleen; Transforming Growth Factor beta

A quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay was developed to measure transforming growth factor-beta (TGF-beta) in Atlantic menhaden (Brevoortia tyrannus), an estuarine-dependent species plagued by ulcerative skin lesions in the estuaries along the eastern United States. Atlantic menhaden were acclimated in a closed system for two weeks prior to initiation of the study. The synthetic glucocorticoid, triamcinolone acetonide (10mg/kg body weight) was administered by intracoelomic injection and its effect on the splenic mononuclear cell TGF-beta mRNA transcription, liver-somatic index, spleno-somatic index, hematology, and plasma chemistry were compared to untreated fish at 48 and 96h post-treatment. Triamcinolone-treated Atlantic menhaden showed suppression of TGF-beta mRNA production, neutrophilia, monocytosis, lymphopenia, and an increase in blood glucose concentrations. The health indices used in this study may help us interpret some of the changes observed during the development of ulcerative skin lesions in wild-caught menhaden.

Topics: Animals; Anti-Inflammatory Agents; Base Sequence; Blood Glucose; Fish Diseases; Fishes; Leukocytes, Mononuclear; Liver; Molecular Sequence Data; Plasma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Ulcer; Spleen; Transforming Growth Factor beta; Triamcinolone Acetonide

A number of studies have implicated transforming growth factor (TGF)-beta1, 2, and 3 (TGF-beta) in wound healing and hypertrophic scarring. We propose that TGF-beta has a temporal effect on these processes. To test this hypothesis, we applied anti-TGF beta1, 2, and 3 monoclonal antibody topically to our dermal ulcer model in the rabbit ear.. Rabbit ear wounds were treated intradermally with anti-TGF-beta1, 2, and 3 antibody at early, middle, and late time points. Treated and untreated control wounds were harvested at various time points and examined histologically to quantify wound healing and scar hypertrophy. Real-time polymerase chain reaction was performed to determine TGF-beta mRNA expression in the treated and control wounds.. The early treatment group demonstrated decreased new epithelium and granulation tissue (p < 0.05 versus controls). Scars harvested on days 28 and 40 displayed no difference in scar hypertrophy. Both the middle and late treatment groups demonstrated a significant decrease in scar hypertrophy (p < 0.05).. Treated wounds from the early treatment group displayed delayed wound healing, with no reduction in scar hypertrophy. Later treatment of wounds with the same antibody, beginning 7 days after wounding, resulted in a reduction in scar hypertrophy. These results support our hypothesis and clearly demonstrate that TGF-beta1, 2, and 3 have differential temporal effects during the wound-healing process, and are important for optimal wound healing in the first week after wounding; beyond 1 week, TGF-beta1, 2, and 3 play a critical role in hypertrophic scar formation.

Topics: Animals; Antibodies, Monoclonal; Cicatrix, Hypertrophic; Ear, External; Female; Immunosuppressive Agents; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Ulcer; Time Factors; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3; Wound Healing

Wound healing is a specific host immune response for restoration of tissue integrity. Experimental studies demonstrated an alteration of growth factors activity due to their reduced synthesis, increased degradation and inactivation. In wound healing platelets play an essential role since they are rich of alpha-granules growth factors (platelet derived growth factor--PDGF; transforming growth factor-beta--TGF-beta; vascular endothelial growth factor--VEGF). Topical use of platelet gel (PG), hemocomponent obtained from mix of activated platelets and cryoprecipitate, gives the exogenous and in situ adding of growth factors (GF). The hemocomponents are of autologous or homologous origin. We performed a technique based on: multicomponent apheretic procedure to obtain plasma rich platelet and cryoprecipitate; manual processing in an open system, in sterile environment, for gel activation. Every step of the gel synthesis was checked by a quality control programme. The therapeutic protocol consists of the once-weekly application of PG. Progressive reduction of the wound size, granulation tissue forming, wound bed detersion, regression and absence of infective processes were considered for evaluating clinical response to hemotherapy. 24 patients were enrolled. They had single or multiple cutaneous ulcers with different ethiopathogenesis. Only 3 patients could perform autologous withdrawal; in the others homologous hemocomponent were used, always considering suitability and traceability criteria for transfusional use of blood. Complete response was observed in 9 patients, 2 were subjected to cutaneous graft, 4 stopped treatment, 9 had partial response and are still receiving the treatment. In each case granulation tissue forming increased following to the first PG applications, while complete re-epithelization was obtained later. Pain was reduced in every treated patient. Topical haemotherapy with PG may be considered as an adjuvant treatment of a multidisciplinary process, useful to enhance therapy of cutaneous ulcers.

Topics: Aged; Aged, 80 and over; Blood Platelets; Blood Proteins; Cryogels; Female; Fibronectins; Gels; Humans; Hydrogels; Male; Middle Aged; Platelet Activation; Platelet-Derived Growth Factor; Skin Ulcer; Thrombin; Transforming Growth Factor beta; Treatment Outcome; Vascular Endothelial Growth Factor A; Wound Healing

Exogenous administration of transforming growth factor-beta (TGF-beta) improves wound healing by affecting cellular and molecular events involved in tissue repair. But mice with a deficiency of a key TGF-beta signaling intermediate, Smad3, paradoxically showed accelerated cutanenous wound healing, suggesting that endogenous Smad3 had inhibitory effect on cutaneous wound healing. Here we investigated the effect of exogenous expression of Smad3 in dermal fibroblasts on cutaneous wound healing. Subcutaneous injection of adenovirus-containing Smad3 complementary DNA (AdCMV-Smad3) targeting mainly dermal fibroblasts accelerated tissue repair following full-thickness dermal round wounds in rabbit ear as judged by the size of granulation tissue area, number of capillaries, and re-epithelialization rate of the wounds. Expressions of alpha-smooth muscle actin (alpha-SMA), vascular endothelial growth factor (VEGF), and fibroblast growth factor receptor were upregulated in the wounded area injected with AdCMV-Smad3. Consistent with the in vivo findings, overexpression of Smad3 induced alpha-SMA, VEGF, and TGF-beta1 expression and augmented chemotactic response in cultured dermal fibroblasts. Therefore, exogenous administration of Smad3 targeting dermal fibroblasts accelerated tissue repair in a rabbit dermal ulcer model by affecting fibroblast responses associated with wound healing. The results suggest that Smad3, when overexpressed in dermal fibroblasts, can promote wound healing.

Topics: Actins; Adenoviridae; Animals; Dermis; Disease Models, Animal; DNA-Binding Proteins; Female; Fibroblasts; Gene Expression; Genetic Therapy; In Vitro Techniques; Mice; Rabbits; Skin Ulcer; Smad3 Protein; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Wound Healing

Electrical stimulation (ES) is a therapeutic treatment for wound healing. Electroporation, a type of ES, is a well-established method for gene delivery. We hypothesize that proper conditions can be found with which both electrical and gene therapies can be additively applied to treat diabetic wound healing. For the studies of transforming growth factor-beta1 (TGF-beta1) local expression and therapeutic effects, full thickness excisional wound model of db/db mice was used, we measured TGF-beta1 cytokine level at 24 h postwounding and examined wounds histologically. Furthermore, wound closure was evaluated by wound-area measurements at each day for 14 d. We found that syringe electrodes are more effective than the conventional caliper electrodes. Furthermore, diabetic skin was more sensitive to the electroporative damage than the normal skin. The optimal condition for diabetic skin was six pulses of 100 V per cm for 20 ms. Under such condition, the healing rate of electrically treated wound was significantly accelerated. Furthermore, when TGF-beta1 gene was delivered by electric pulses, the healing rate was further enhanced. Five to seven days postapplication of intradermal injection of plasmid TGF-beta1 followed by electroporation, the wound bed showed an increased reepithelialization rate, collagen synthesis, and angiogenesis. The data indicates that indeed the electric effect and gene effect work synergistic in the genetically diabetic model.

Topics: Animals; Collagen; Combined Modality Therapy; Diabetes Complications; Electric Stimulation Therapy; Electroporation; Female; Genetic Therapy; Humans; Keratinocytes; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neovascularization, Physiologic; Plasmids; Skin Ulcer; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

Type VII collagen is the major component of anchoring fibrils within the cutaneous basement membrane zone. The large amino-terminal noncollagenous domain of type VII collagen interacts with various extracellular matrix proteins and contributes to the dermal-epidermal attachment. The purpose of this study was to detect alternative splicing of COL7A1 transcript encoding the noncollagenous 1 domain. The alternative splicing in this region may affect interactions of the noncollagenous 1 domain with extracellular matrix proteins and also dermal-epidermal adhesion. Thus we examined expression of the alternative splicing in situations relating to wound healing and skin remodeling that required dermal-epidermal binding and detachment. Amplification of overlapping cDNA from keratinocytes using reverse transcription-polymerase chain reaction identified alternative splicing, which was generated by a different exon 18 acceptor site 27 bp upstream from the common acceptor site. Expression of this alternatively spliced transcript differed among several cell types. The nine amino acid residues GPLTLPLSP from the 27 bp nucleotides were inserted into the linker of fibronectin type III domains. This insertion was suggested to contribute to flexibility of the linker of fibronectin type III domains and may affect the interactions between the noncollagenous 1 domain and extracellular matrix proteins. Treatment with transforming growth factor-beta 1, which is known to promote wound healing and skin remodeling, enhanced the expression of this 27 bp transcript. Furthermore, keratinocyte biopsies from the wound edge of patients with epithelizing skin ulcers showed a significant increase in the 27 bp transcript expression compared with normal keratinocytes from steady-state body sites. These results suggest that amino acid variation of this alternative splicing may have some role in dermal-epidermal adhesion, wound healing, and skin remodeling. To the best of our knowledge, this is the first evidence of alternative splice insertion of a small peptide into the linker region of the fibronectin type III domains, a common motif within modular proteins.

Topics: Alternative Splicing; Amino Acid Sequence; Base Sequence; Cells; Cells, Cultured; Collagen Type VII; DNA Transposable Elements; Fibronectins; Humans; Keratinocytes; Molecular Sequence Data; Protein Structure, Tertiary; RNA, Messenger; Skin Ulcer; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

To characterize the impact of increased production of TGF-beta in a xenograft model of human breast cancer, TGF-beta-responsive MDA-231 cells were genetically modified by stable transfection so as to increase their production of active TGF-beta1. Compared with control cells, cells that produced increased amounts of TGF-beta proliferated in vitro more slowly. In vivo, however, tumors derived from these cells exhibited increased proliferation and grew at an accelerated pace. To evaluate the role of autocrine TGF-beta signaling, cells were also transfected with a dominant-negative truncated type II TGF-beta receptor (TbetaRII). Disruption of autocrine TGF-beta signaling in the TGF-beta-overexpressing cells reduced their in vivo growth rate. Co-inoculation of Matrigel with the TGF-beta-overexpressing cells expressing the truncated TbetaRII compensated for their diminished in vivo growth capacity, compared with the TGF-beta-overexpressing cells with an intact autocrine loop. Tissue invasion by the tumor was a distinctive feature of the TGF-beta-overexpressing cells, whether or not the autocrine loop was intact. Furthermore, tumors derived from TGF-beta-overexpressing cells, irrespective of the status of the autocrine TGF-beta-signaling pathway, had a higher incidence of lung metastasis. Consistent with the suggestion that TGF-beta's enhancement of invasion and metastasis is paracrine-based, we observed no significant differences among the cell clones in an in vitro invasion assay. Thus, in this experimental model system in vitro assays of cell proliferation and invasion do not accurately reflect in vivo observations, perhaps due to autocrine and paracrine effects of TGF-beta that influence the important in vivo-based phenomena of tumor growth, invasion, and metastasis.

Topics: Animals; Autocrine Communication; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Division; Collagen; Culture Media, Conditioned; Drug Combinations; Female; Gene Expression Regulation, Neoplastic; Genes, Dominant; Hemorrhage; Humans; Laminin; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Paracrine Communication; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proteoglycans; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; Sequence Deletion; Skin Ulcer; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Decorin belongs to a family of small leucine-rich dermatan sulfate proteoglycans that are involved in the control of matrix organization and cell growth. Here, we described a patient whose skin glycosaminoglycans showed extremely decreased amount of dermatan sulfate compared with a normal control skin. This patient presented clinical features of Ehlers-Danlos syndrome with a chronic skin ulcer. Western blotting revealed that the deficiency of dermatan sulfate was due to the defect of decorin core protein. Beta-xyloside, an initiator of dermatan sulfate glycosaminoglycan chain elongation, enhanced the synthesis of dermatan sulfate in the fibroblasts of the patient to a similar extent to that of control. This result indicated that the enzymes for the elogation of dermatan sulfate side chains were normal. Northern blotting demonstrated remarkable reduction of decorin mRNA level, while biglycan mRNA level was concomitantly increased and procollagen alpha1(I) mRNA level was normal. cDNA and exons sequencing analysis showed there was no mutation in decorin gene of the patient. IL-1beta stimulated decorin expression to about 140% in control fibroblasts while about 110% in patient fibroblasts. On the other hand, TGF-beta1 resulted in 40% reductions of decorin expression in both control and patient fibroblasts. These data suggested that reduced decorin expression of fibroblasts from the patient of Ehlers-Danlos syndrome may be due to abnormalities in the regulatory regions, which is responsible for the IL-1beta stimulation.

Topics: Asian People; Chronic Disease; Decorin; Ehlers-Danlos Syndrome; Extracellular Matrix Proteins; Female; Fibroblasts; Gene Expression Regulation; Genetic Variation; Glycosaminoglycans; Humans; Interleukin-1; Japan; Mastectomy; Middle Aged; Postoperative Complications; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; Skin Ulcer; Transcription, Genetic; Transforming Growth Factor beta

To localize the distribution of basic fibroblast growth factor (bFGF) and transforming growth factor-beta(TGF-beta) in tissues from dermal chronic ulcer and hypertrophic scar and to explore their effects on tissue repair.. Twenty-one cases were detected to localize the distribution of bFGF and TGF-beta, among them, there were 8 cases with dermal chronic ulcers, 8 cases with hypertrophic scars, and 5 cases of normal skin.. Positive signal of bFGF and TGF-beta could be found in normal skin, mainly in the keratinocytes. In dermal chronic ulcers, positive signal of bFGF and TGF-beta could be found in granulation tissues. bFGF was localized mainly in fibroblasts cells and endothelial cells and TGF-beta mainly in inflammatory cells. In hypertrophic scar, the localization and signal density of bFGF was similar with those in granulation tissues, but the staining of TGF-beta was negative.. The different distribution of bFGF and TGF-beta in dermal chronic ulcer and hypertrophic scar may be the reason of different results of tissue repair. The pathogenesis of wound healing delay in a condition of high concentration of growth factors may come from the binding disorder of growth factors and their receptors. bFGF may be involved in all process of formation of hypertrophic scar, but TGF-beta may only play roles in the early stage.

Topics: Adult; Burns; Chronic Disease; Cicatrix, Hypertrophic; Female; Fibroblast Growth Factor 2; Humans; Male; Skin Ulcer; Transforming Growth Factor beta; Wound Healing

Stromelysin-2 is a matrix metalloproteinase that degrades in vitro several protein components relevant to wound repair such as collagens III and IV, gelatin, nidogen, laminin-1, proteoglycans, and elastin. Furthermore, it can activate other matrix metalloproteinases, such as collagenase-1 (matrix metalloproteinase-1) and collagenase-2 (matrix metalloproteinase-8), as well as 92 kDa gelatinase. The aim of this study was to determine in a large variety of wounds (normally healing dermal and mucosal wounds, suction blisters, ex vivo cultures, diabetic, decubitus, rheumatic, and venous ulcers) and keratinocyte cultures, which factors contribute to stromelysin-2 expression and how it is induced in relation to other matrix metalloproteinases. Our results show that stromelysin-2 mRNA and protein are upregulated later (at 3 d) than matrix metalloproteinase-1 in normally healing wounds and ex vivo explants, in which stromelysin-2 is invariably expressed by keratinocytes migrating over dermal matrix. The number of keratinocytes expressing stromelysin-2 was greatest in chronic inflamed diabetic and venous ulcers compared with rheumatoid and decubitus ulcers, six of which had no signal. In keratinocyte cultures, tumor necrosis factor-alpha, epidermal growth factor, and transforming growth factor-beta1 induced stromelysin-2 expression as measured by quantitative reverse transcriptase-polymerase chain reaction, whereas different matrices did not upregulate the mRNA. Immunostaining demonstrated stromal transforming growth factor-beta1 in contact with the stromelysin-2-positive keratinocytes. Our results suggest that stromelysin-2 expression is important for the normal repair process and is upregulated by cytokines rather than cell-matrix interactions. Stromelysin-2 is most likely to participate in the remodeling of the newly formed basement membrane, and is not overexpressed in retarded wound healing.

Topics: Cell Adhesion Molecules; Cell Communication; Cell Movement; Cytokines; Epidermal Growth Factor; Epithelial Cells; Kalinin; Keratinocytes; Matrix Metalloproteinase 10; Metalloendopeptidases; Neutrophils; RNA, Messenger; Skin Ulcer; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Up-Regulation; Wound Healing

Clinical trials of exogenous growth factors in treating chronic wounds have been less successful than expected. One possible explanation is that most studies used animal models of acute wounds in young animals, whereas most chronic wounds occur in elderly patients with tissue ischemia. We described an animal model of age- and ischemia-impaired wound healing and analyzed the wound-healing response as well as the transforming growth factor (TGF)-beta1 effect in this model. Rabbits of increasing ages were made ischemic in the ear where dermal ulcers were created. Histological analysis showed that epithelium ingrowth and granulation tissue deposition were significantly impaired with increased age under ischemia. TGF-beta1 stimulated wound repair under both ischemic and non-ischemic conditions in young animals, although it showed no statistical difference in aged animals. Procollagen mRNA expression decreased under ischemic conditions and with aging. Neither TGF-beta1 nor procollagen alpha1(I) mRNA expression increased in response to TGF-beta1 treatment under ischemia in aged animals. Therefore, the wound-healing process is impaired additively by aging and ischemia. The lack of a wound-healing response to TGF-beta1 in aged ischemic wounds may play a role in the chronic wounds.

Topics: Aging; Animals; Ear; Ischemia; Male; Oxygen; Procollagen; Rabbits; RNA, Messenger; Signal Transduction; Skin Ulcer; Transforming Growth Factor beta; Wound Healing

Transforming growth factor (TGF) beta 3 is a new isoform of the TGF beta superfamily and is presumed to play an important role in wound repair and scarring.. To examine the effects of TGF beta 3 on wound healing and on reducing scarring.. Dermal ulcers were created on the ears of 75 anesthetized young female rabbits. Either TGF beta 3 or vehicle was applied topically to the wounds. Wounds were bisected and analyzed histologically at postwounding day 7. A second group of wounds was treated with topical TGF beta 3 and TGF beta 2 or vehicle at days 0 and 3 and harvested at days 21 through 42 as an excessive scarring model. The third group of wounds was treated with TGF beta 1, TGF beta 2, and TGF beta 3 and vehicle. The granulation tissue was harvested at day 7, and cellular RNA was extracted for performing competitive reverse-transcription polymerase chain reaction.. The amount of new epithelium and granulation tissue was measured in TGF beta 3- and vehicle-treated wounds. The hypertrophic index was calculated for scarring wounds treated with TGF beta 2 and TGF beta 3 or vehicle. Levels of TGF beta 1 messenger RNA were measured in those wounds that were treated with TGF beta 1, TGF beta 2, and TGF beta 3 and in their controls.. The use of TGF beta 3 (0.3-0.75 microgram per wound) increased granulation tissue formation by more than 100% (P < .005). Epithelialization showed a biphase, either increasing 30% (P < .04) or decreasing 25% (P < .001) dependent on dose. No significant difference in the hypertrophic index was noted in TGF beta 3-treated wounds compared with controls. Levels of TGF beta 1 messenger RNA increased (7.1- to 14.9-fold) in those wounds treated with TGF beta s compared with controls at day 7.. Exogenous TGF beta 3 displays substantial vulnerary properties in wound healing and may be useful in treating nonhealing wounds. However, the observation that TGF beta 3 can reduce scarring was not confirmed in this study, and the messenger RNA level in response to TGF beta 3 suggests that it behaves similarly to TGF beta 1.

Topics: Animals; Cicatrix; Cicatrix, Hypertrophic; DNA Probes; Female; Humans; Polymerase Chain Reaction; Rabbits; RNA-Directed DNA Polymerase; Skin Ulcer; Transforming Growth Factor beta; Wound Healing

Macrophage colony-stimulating factor (M-CSF) is produced by many cell types involved in wound repair, yet it acts specifically on monocytes and macrophages. The monocyte-derived cell is thought to be important in wound healing, but the importance of the role of tissue macrophages in wound healing has not been well defined. Dermal ulcers were created in normal and ischemic ears of young rabbits. Either rhM-CSF (17 microg/wound) or buffer was applied to each wound. Wounds were bisected and analyzed histologically at Days 7 and 10 postwounding. The amounts of epithelial growth and granulation tissue deposition were measured in all wounds. The level of increase of TGF-beta1 mRNA level in M-CSF-treated wounds was examined using competitive RT-PCR. M-CSF increased new granulation tissue formation by 37% (N = 21, P < 0.01) and 50% (P < 0.01) after single and multiple treatments, respectively, in nonischemic wounds. TGF-beta1 mRNA levels in rhM-CSF-treated wounds increased 5.01-fold (N = 8) over vehicle-treated wounds under nonischemic conditions. In contrast, no effect could be detected in ischemic wounds treated with rhM-CSF, and these wounds only showed a 1.66-fold increase in TGF-beta1 mRNA levels when compared to ischemic wounds treated with vehicle alone. GAPDH, a housekeeping gene, showed no change. As mesenchymal cells lack receptors for M-CSF, the improved healing of wounds treated with topical rhM-CSF must reflect a generalized enhancement of activation and function of tissue macrophages, as demonstrated by upregulation of TGF-beta. The lack of effect under ischemic conditions suggests that either macrophage activity and/or response to M-CSF is adversely affected under those conditions; this may suggest the pathogenesis of impaired wound healing at the cellular level.

Topics: Animals; Base Sequence; DNA Primers; Dose-Response Relationship, Drug; Female; Glyceraldehyde-3-Phosphate Dehydrogenases; Ischemia; Macrophage Colony-Stimulating Factor; Macrophages; Monocytes; Polymerase Chain Reaction; Rabbits; Recombinant Proteins; RNA, Messenger; Skin; Skin Ulcer; Transforming Growth Factor beta; Up-Regulation; Wound Healing

To test the influence of hyperbaric oxygen (HBO), platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-beta 1 (TGF-beta 1) on the deficit in wound healing produced by ischemia in a noncontractive dermal ulcer standardized model in the rabbit ear.. Dermal ulcers were created in the ischemic ears of 42 anesthetized young female New Zealand white rabbits. The controls were ulcers created in nonischemic ears of eight anesthetized young female New Zealand white rabbits. Either PDGF-BB (5 micrograms), TGF-beta 1 (1 microgram), or buffer alone was applied to each wound, which was then covered. Some groups were treated with HBO on days 0 through 4. Wounds were harvested on day 7 and were evaluated histologically.. The amount of epithelial regrowth and granulation tissue production were measured. The wounds were evaluated for glycosaminoglycan and collagen content. Angiogenesis was measured.. Hyperbaric oxygen alone, in the ischemic model, increased the production of new granulation tissue by approximately 100% at 7 days without significantly affecting new epithelial growth (P = .03). In contrast, PDGF-BB and TGF-beta 1 each increased the new granulation tissue volume by greater than 200% in 7 days (P = .0001) and also had a statistically significant effect on new epithelial growth. However, the addition of growth factors to HBO treatment produced a synergistic total reversal of the wound-healing deficit produced by ischemia (P = .0001).. Both PDGF-BB and TGF-beta 1 alone are more effective than HBO treatment by itself in accelerating the impaired wound healing produced by ischemia. However, the combination of HBO with either of the growth factors has a synergistic effect that totally reverses the deficit produced by ischemia.

Topics: Animals; Becaplermin; Combined Modality Therapy; Ear; Female; Hyperbaric Oxygenation; Ischemia; Oxygen; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rabbits; Recombinant Proteins; Skin Ulcer; Transforming Growth Factor beta; Treatment Outcome

Recombinant platelet-derived growth factor (BB homodimer, rPDGF-BB), transforming growth factor beta 1 (rTGF-beta 1), and basic fibroblast growth factor (rbFGF) can accelerate healing of soft tissues. However, little information is available characterizing the components of wound matrix induced by these growth factors and the molecular mechanisms underlying accelerated repair and wound maturation. In this study, the composition, quantity, and rate of extracellular matrix deposition within growth factor-treated lapine ear excisional wounds were analyzed at different stages of healing using specific histochemical and immunohistochemical stains, coupled with image analysis techniques. Single application of optimal concentrations of each growth factor accelerated normal healing by 30% (P less than 0.0003); rPDGF-BB markedly augmented early glycosaminoglycan (GAG) and fibronectin deposition, but induced significantly greater levels of collagen later in the repair process, compared with untreated wounds rTGF-beta 1 treatment led to rapidly enhanced collagen synthesis and maturation, without increased GAG deposition. In contrast, rbFGF treatment induced a predominantly angiogenic response in wounds, with a marked increase in endothelia and neovessels (P less than 0.0001), and increased wound collagenolytic activity (P less than 0.03). rbFGF-treated wounds did not evolve into collagen-containing scars and continued to accumulate only provisional matrix well past wound closure. These results provide new evidence that growth factors influence wound repair via different mechanisms: 1) rPDGF-BB accelerates deposition of provisional wound matrix; 2) rTGF-beta 1 accelerates deposition and maturation of collagen; and 3) rbFGF induces a profound monocellular angiogenic response which may lead to a marked delay in wound maturation, and the possible loss of the normal signal(s) required to stop repair. These results suggest that specific growth factors may selectively regulate components of the repair response by differing mechanisms, offering the potential for targeted therapeutic intervention.

Topics: Animals; Collagen; Extracellular Matrix; Fibroblast Growth Factor 2; Fibronectins; Glycosaminoglycans; Granulation Tissue; Growth Substances; Kinetics; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Rabbits; Skin; Skin Ulcer; Transforming Growth Factor beta; Wound Healing

The effects of recombinant human transforming growth factor beta 1 (rhTGF-beta 1) on wound healing were examined in a rabbit ear ulcer model in which rhTGF-beta 1 was applied to full-thickness biopsy ulcers on the ears. The influence of perichondrium on healing was studied by comparing ulcers with and without perichondrium on 1) formation of total healing wound area (HWA, the newly formed connective and granulation tissues within the ulcer) over time and 2) the amount of collagen synthesized by the wound tissue at day 5. The HWA of ulcers with intact perichondrium increased sharply with time and reached a plateau at day 7, whereas a slower healing occurred in the perichondrium-free model where maximal HWA appeared at day 14. Topical application of 100 ng of rhTGF-beta 1 per wound accelerated healing by increasing HWA in both models. The enhancement of healing by rhTGF-beta 1 was associated with increased collagen synthesis. The percent collagen synthesis in the rhTGF-beta 1 was doubled in the perichondrium-intact ulcers and increased 40% in the perichondrium-free ulcers. DNA synthesis in the perichondrium-intact ulcers was not altered by rhTGF-beta 1 when measured at day 5 by in vitro labeling with [3H]thymidine ([3H]TdR). Autoradiography indicated that the primary cells labeled in the wound tissue were epithelial cells and rhTGF-beta 1 enhanced the migration of these cells from the wound margin towards the center. To evaluate the effects of rhTGF-beta 1 on fibroblasts derived from the granulation tissue of the wound, cells were treated with increasing concentrations of rhTGF-beta 1 and DNA and collagen synthesis were determined. rhTGF-beta 1 elicited a biphasic change in percent collagen synthesis with a maximal increase of 50% at 20 pM followed by a decline. A twofold increase in [3H]TdR incorporation that plateaued at 1 nM was also observed. Our results indicate that the cellular responses to rhTGF-beta 1 differ in vivo and in vitro. The perichondrium-intact ulcers contain more wound tissue and have larger responses to rhTGF-beta 1 stimulation, which allows better examination of biochemical and cellular events. The in vivo mechanisms are multi-factorial, which may involve cell migration and recruitment as results of numerous cell/cell and cell/matrix interactions.

Topics: Animals; DNA; Ear Diseases; Fibroblasts; Models, Biological; Protein Biosynthesis; Rabbits; Recombinant Proteins; Skin Ulcer; Stimulation, Chemical; Transforming Growth Factor beta; Wound Healing

A new animal model to study secondary intention wound healing and the effects of topically applied rhTGF-beta 1 was developed. A time course study was performed of full thickness 6 mm punch wounds placed on the backs of anesthetized pigs and treated once with either 3% methylcellulose or rhTGF-beta 1 in 3% methylcellulose or left untreated. Wounds receiving rhTGF-beta 1 had enhanced tensile strength at days 4 and 7 compared to controls. Studies of the response on days 4 and 7 to graded doses of rhTGF-beta 1 showed that a dose of 250 or 2500 ng rhTGF-beta 1 gave a similar enhanced wound strength, while 25 ng rhTGF-beta 1 had no effect. Blood flow to treated granulating wounds as measured by 141Ce microspheres indicate an increase in flow in wounds treated with 250, 500 or 2500 ng rhTGF-beta 1 compared to controls. These results indicate a possible use for rhTGF-beta 1 in enhancing wound healing clinically.

Topics: Animals; Disease Models, Animal; Granuloma; Humans; Male; Recombinant Proteins; Skin; Skin Ulcer; Swine; Tensile Strength; Time Factors; Transforming Growth Factor beta; Wound Healing