[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to assess the potential differences in antibacterial activity and fluid flow rates of 2 commonly used silver dressings: an alginate-only dressing and an alginate dressing with a nonadherent contact layer.
[Show abstract][Hide abstract] ABSTRACT: Bacterial infection of acute and chronic wounds impedes wound healing significantly. Part of this impediment is the ability of bacterial pathogens to grow in wound dressings. In this study, we examined the effectiveness of a polyurethane foam wound dressings coated with poly diallyl-dimethylammonium chloride (pDADMAC-PU) to inhibit the growth and biofilm development by three main wound pathogens: Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, within the wound dressing. pDADMAC-PU inhibited the growth of all three pathogens. Time-kill curves were conducted both with and without serum to determine the killing kinetic of pDADMAC-PU. pDADMAC-PU killed S. aureus, A. baumannii, and P. aeruginosa. The effect of pDADMAC-PU on biofilm development was analyzed quantitatively and qualitatively. Quantitative analysis, (CFU) assay, revealed that pDADMAC-PU dressing produced more than 8 log reduction in biofilm formation by each pathogen. Visualization of the biofilms by either confocal laser scanning microscopy or scanning electron microscopy confirmed these findings. In addition, it was found that the pDADMAC-PU treated foam totally inhibited migration of bacteria through the foam for all three bacterial strains. These results suggest that pDADMAC-PU is an effective wound dressing that inhibits the growth of wound pathogens both within the wound and in the wound dressing.
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Wound Repair and Regeneration 12/2014; · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvβ6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvβ6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter driven GFP reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvβ6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen inducible Cre-loxP system down-regulated integrin αvβ6 in DDC damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvβ6 by administrating the neutralizing antibody 6.3G9 (10 mg/kg body weight) caused low levels of EpCAM and CK19 mRNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within two weeks after DDC treatment. Associated fibrosis was attenuated as indicated by reduced expression of fibrosis-related genes, smaller areas of α smooth muscle actin staining and low collagen production based on hydroxyproline content and the Sirius red staining. Finally, integrin αvβ6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-β1 activation in vitro. Conclusions: CTGF and integrin αvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-β1. CTGF and integrin αvβ6 are potential therapeutic targets to control DRs and fibrosis in related liver disease. (Hepatology 2014)
[Show abstract][Hide abstract] ABSTRACT: The effects of a triple combination of siRNAs targeting key scarring genes was assessed using an ex vivo organ culture model of excimer ablated rabbit corneas. The central 6 mm diameter region of fresh rabbit globes was ablated to a depth of 155 microns with an excimer laser. Corneas were excised, cultured at the air-liquid interface in defined culture medium supplemented with transforming growth factor beta 1 (TGFB1), and treated with either 1% prednisolone acetate or with 22.5 μM cationic nanoparticles complexed with a triple combination of siRNAs (NP-siRNA) targeting TGFB1, TGFB Receptor (TGFBR2) and connective tissue growth factor (CTGF). Scar formation was measured using image analysis of digital images and levels of smooth muscle actin (SMA) were assessed in ablated region of corneas using qRT-PCR and immunostaining. Ex vivo cultured corneas developed intense haze-like scar in the wounded areas and levels of mRNAs for pro-fibrotic genes were significantly elevated 3 to 8 fold in wounded tissue compared to unablated corneas. Treatment with NP-siRNA or steroid significantly reduced quantitative haze levels by 55% and 68%, respectively, and reduced SMA mRNA and immunohistostaining. This ex vivo corneal culture system reproduced key molecular patterns of corneal scarring and haze formation generated in rabbits. Treatment with NP-siRNAs targeting key scarring genes or an anti-inflammatory steroid reduced corneal haze and SMA mRNA and protein.
Experimental Eye Research 06/2014; · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: This study aimed to elucidate the role of connective tissue growth factor (CTGF) in normal eyes and wounded corneas of mice and rabbits. Conditional knockout mice were utilized to determine the role of CTGF in corneal healing. Methods: CTGF expression was determined using transgenic mice carrying CTGF promoter-driven-eGFP, quantitative RT-PCR, and immunohistochemistry. Mice that carried two floxed CTGF alleles and a Cre/ERT2 transgene under the control of human ubiquitin C (ubc) promoter were utilized to conditionally delete CTGF gene in a tamoxifen-inducible manner. Phototherapeutic keratectomy (PTK) was used to generate an acute corneal wound and corneal re-epithelialization was assessed by fluorescein staining. Results: CTGF expression was found in multiple ocular tissues with relatively high levels in the corneal endothelium, lens subcapsular epithelium, and in the vasculature of the iris and retina. Wounded corneas responded with an immediate up-regulation of CTGF in the epithelium at the wound margin and a sustained CTGF induction during re-epithelialization. At the onset of haze formation, CTGF protein becomes more focused in the basal epithelium. Deletion of the CTGF gene caused a 40% reduction (P<0.01) in the cornea re-epithelialization rate in knockout mice compared with wild type mice. Conclusions: CTGF is expressed in the naïve cornea, lens, iris and retina, and is expressed immediately after epithelial injury. Loss of CTGF impairs efficient re-epithelialization of corneal wounds.
[Show abstract][Hide abstract] ABSTRACT: Negative pressure wound therapy with instillation (NPWTi) is increasingly used as an adjunct therapy for a wide variety of infected wounds. However, the effect of NPWTi on mature biofilm in wounds has not been determined. This study assessed the effects of NPWTi using saline or various antimicrobial solutions on mature Pseudomonas aeruginosa biofilm using an ex vivo porcine skin explant biofilm model. Treatment consisted of six cycles with 10-minute exposure to instillation solution followed by 4 hours of negative pressure at -125 mm Hg over a 24-hour period. NPWTi using saline reduced bacterial levels by 1-log (logarithmic) of 7-log total colony-forming units (CFUs). In contrast, instillation of 1% povidone iodine (2-log), L-solution (3-log), 0·05% chlorhexidine gluconate (3-log), 0·1% polyhexamethylene biguanide (4-log), 0·2% polydiallyldimethylammonium chloride (4-log) and 10% povidone iodine (5-log), all significantly reduced (P < 0·001) total CFUs. Scanning electron micrographs showed disrupted exopolymeric matrix of biofilms and damaged bacterial cells that correlated with CFU levels. Compared with previous studies assessing microbicidal effects of topical antimicrobial dressings on biofilms cultured on porcine skin explants, these ex vivo model data suggest that NPWTi with delivery of active antimicrobial agents enhances the reduction of CFUs by increasing destruction and removal of biofilm bacteria. These results must be confirmed in human studies.
International Wound Journal 12/2013; 10(s1):48-55. · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: Transforming Growth Factor β1 (TGFβ1), TGFβ receptor (TGFβR2) and Connective Tissue Growth Factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin and kidney. We developed an anti-fibrotic treatment targeting these three critical scarring genes using a combination of siRNAs and assessed its effect on downstream scarring genes, Collagen-I (Col-I) and alpha smooth muscle actin (SMA). Methods:Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts (RbCFs) at concentrations from 15 to 90nM. The knock down of target gene proteins were measured by ELISA and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy using Combination Index to predict 'effective' and 'ineffective' triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured using qRT-PCR and levels of SMA protein were assessed by immunohistochemistry. Results:Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5% to 80%). The effective triple siRNA significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and SMA protein (>95%), and significantly reduced cell migration without reducing cell viability. Conclusions:Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knock down of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal haze and scarring in other tissues.
[Show abstract][Hide abstract] ABSTRACT: A bipedicle ischaemic rat skin flap model was used to study the effects of daily topical applications of platelet-derived growth factor (PDGF) on the healing of ischaemic wounds. Levels of tumour necrosis factor-alpha (TNFA), interleukin 1-beta (IL1B) and both the latent and active forms of matrix metalloproteinase 2 (MMP2) and 9 (MMP9) were measured. Full-thickness wounds were made on a total of 72 adult male Sprague-Dawley rats. Each group of 18 rats with normal and ischaemic wounds received either vehicle or 0·01% recombinant PDGF-BB. Additional applications were made on the wounds on a daily basis. Wound areas were measured at 0, 1, 3, 5, 7 9 and 13 days after wounding. Ischaemia caused a delay in wound healing as well as an increase in TNFA, IL1B and both the pro and active forms of MMP2 and MMP9. PDGF accelerated the rate of wound healing in both normal and ischaemic wounds and negated the effect of ischaemia. PDGF reduced the TNFA concentration in both normal and ischaemic wounds, and the rate of wound healing closely resembled the pattern of TNFA protein expression. PDGF also reduced both the magnitude and duration of the increases in IL1B and both the pro and active forms of MMP2 and MMP9 induced by ischaemia.
International Wound Journal 10/2013; · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: The transforming growth factor beta1 (TGFB1) pathway has been linked to fibrosis in sev- eral tissues including skin, liver, kidney and the cor- nea. In this study, a RNA interference-based approach using siRNAs targeting three critical scarring genes, TGFB1, TGFB receptor 2 (TGFBR2) and connective tissue growth factor (CTGF), was tested for effects on reducing alpha smooth muscle actin (SMA) and cor- neal scarring (haze) in excimer laser ablated rabbit corneas. Methods: Levels of TGFB1 and CTGF mRNAs were measured using qRT-PCR in the epithelial and endothelial cell layers of normal and excimer ablated rabbit corneas at 30 minutes, 1 day and 2 days after ablation. Two different scarring models were utilized to assess the effects of the triple siRNA combination on corneal scarring. In the first model, rabbit corneas were unevenly ablated creating a mesh pattern then treated immediately with the triple siRNA combina- tion. After 1 day the ablated areas of corneas were collected and levels of mRNAs for TGFB1, TGFBR2 and CTGF were measured. After 14 days, levels of mRNA for SMA were measured and SMA protein im- munolocalized in frozen sections. In the second model, rabbit corneas were uniformly ablated to a depth of 155 microns followed by three daily doses of the triple combination of siRNA. After 14 days, corneas were photographed and images were analyzed using Image J software to assess corneal scarring. Corneas were also analyzed for levels of SMA mRNA. Results: In both unwounded and wounded corneas, levels of TGFB1 and CTGF mRNA were always significantly higher in endothelial cells than in epithelial cells (10 to 30 fold). Thirty minutes after injury, levels of both TGFB1 and CTGF mRNAs increased approximately 20-fold in both epithelial and endothelial cells, and further in- creased approximately 60-fold in 2 days. In the first therapeutic experiment with a single siRNA dose, two of three rabbits showed substantial reductions of all three target genes after 1 day with a maximum knock down of 80% of TGFb1, 50% reduction of TGFBR2 and 40% reduction of CTGF mRNA levels and reduc- ed SMA mRNA at day 14. In the second therapeutic experiment with multiple doses of siRNA treatment, both rabbits showed a ~22% reduction in scar forma- tion at day 14 as calculated by image analysis. There was also a corresponding 70% and 60% reduction of SMA RNA expression. Conclusion: These results de- monstrate that both TGFB1 and CTGF dramatically increase in rabbit corneal epithelial and endothelial cells after injury. Treatment of excimer ablated rab- bit corneas with a triple combination of siRNAs effec- tively reduced levels of the target genes and SMA, lead- ing to reduced corneal scarring at 14 days, suggesting tha t this triple siRNA combination may be an effective new approach to reducing scarring in cornea and other tissues.
Advances in Bioscience and Biotechnology 10/2013; 4:47-55.
[Show abstract][Hide abstract] ABSTRACT: Purpose: The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes. Methods: Laser ablated mouse corneas were collected at 0, 30 minutes and 2 days. RNA was collected from corneas and analyzed using cell differentiation & development miRNA PCR Arrays (QIAgen). Luciferase assay was used to determine whether miR-133b targeted the 3'UTR of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) in RbCF. qRT-PCR and western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA) and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration. Results: At day 2, 37 out of 86 miRNAs had significant expression fold changes. mir-133b had the greatest fold decreased at -14.33. Pre-miR-133b targeted the 3'UTR of CTGF and caused significant decrease of 38% (p<0.01). TGFβ1 treated RbCF had significant decrease of miR-133b of 49% (p<0.01), whereas, CTGF, SMA, and COL1A1 had significant increase of 20%, 54% and 37% (p<0.01), respectively. RbCF treated with TGFβ1 and pre-miR133b caused significant decrease in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (p<0.01), respectively. Finally, there was significant decrease in migration of mir-133b treated RbCF. Conclusions: Significant changes occur in key miRNAs during early corneal wound healing, suggsting novel miRNA targets to reduce scar formation.
[Show abstract][Hide abstract] ABSTRACT: An ex vivo porcine skin explant biofilm model that preserves key properties of biofilm attached to skin at different levels of maturity (0-3 days) was used to assess the efficacy of commercially available antimicrobial dressings and topical treatments. Assays were also performed on the subpopulation of antibiotic tolerant biofilm generated by 24 hours of pre-treatment with gentamicin (120× minimal inhibitory concentration) prior to agent exposure. Five types of antimicrobial agents (iodine, silver, polyhexamethylene biguanide, honey and ethanol) and four types of moisture dressings (cotton gauze, sodium carboxymethylcellulose fibre, calcium alginate fibre and cadexomer beads) were assessed. Time-release silver gel and cadexomer iodine dressings were the most effective in reducing mature biofilm [between 5 and 7 logarithmic (log) of 7-log total], whereas all other dressing formulations reduced biofilm between 0·3 and 2 log in 24 or 72 hours with a single exposure. Similar results were found after 24-hour exposure to silver release dressings using an in vivo pig burn wound model, demonstrating correlation between the ex vivo and in vivo models. Results of this study indicate that commonly used microbicidal wound dressings vary widely in their ability to kill mature biofilm and the efficacy is influenced by time of exposure, number of applications, moisture level and agent formulation (sustained release).
International Wound Journal 09/2013; · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial-thickness wound in 12-mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a ΔlasI quorum-sensing mutant of P. aeruginosa developed biofilm as antibiotic-tolerant as wild-type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms.
Wound Repair and Regeneration 08/2013; · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the topographical location and time course of development of corneal haze in a phototherapeutic keratectomy model using slit lamp examination, macrophotography, quantitative image analysis, and immunofluorescence staining of corneal sections.
Rabbit corneas were ablated with an excimer laser and were observed and graded for haze via slit lamp, imaged and graded by macrophotography. Corneal sections were stained for α-smooth muscle actin (α-SMA) and tenascin-C (TNC). The distribution of haze imaged in the macrophotographs and density of α-SMA and TNC staining were compared. A daily image time course of haze formation was generated using macrophotography.
The first signs of corneal haze were apparent shortly after re-epithelialization. The haze was distributed as a ring at the wound margin in all cases, while nearly all corneas also had some central islands of haze initiation. With time, the haze spread within the ablated zone and intensified. The pattern of immunofluorescent staining for α-SMA and TNC at the wound margin mirrored the haze distribution, spread, and intensification with time.
The initiation and spread of sub-epithelial haze begins shortly after re-epithelialization. The haze then spreads from the loci of initiation and becomes more dense with time; maturing as early as 14 days after wounding. The improved temporal and spatial resolution provided by these data improve the current model of generation of light scattering haze formation in wounded corneas, which will improve design of studies aimed at maintaining corneal clarity following acute injury or surgery.
[Show abstract][Hide abstract] ABSTRACT: Chronic wounds are a signi�cant health problem in the United States, with annual associated costs exceeding $20 billion annually. Traditional wound care consists of surgical debridement, manual irrigation, moisture retentive dressings, and topical and/or systemic antimicrobial therapy. However, despite progress in the science of wound healing, the prevalence and incidence of chronic wounds and their complications are escalating. e presence � complexity of bacterial bio�lms in chronic wounds has recently been recognized as a key aspect of non-healing wounds. �acterial bio�lms are sessile colonies of polymicrobial organisms (bacteria, fungus, etc.) enclosed within a self-produced exopolymeric matrix that provides high levels of tolerance to host defenses, antibiotics and antiseptics. us, there is a need for alternative therapies to reduce bio�lms in chronic wounds. In this report, we present initial �ndings from in vitro experiments which show that larval debridement therapy with disinfected blow �y larvae (Phaenicia sericata) reduced total CFUs (�-logs) of planktonic and mature bio�lms of Pseudomonas aeruginosa or Staphylococcus aureus grown on dermal pig skin explants by 5-logs a�er 24 hours of exposure, and eliminated bio�lms (no measurable CFUs) a�er 48 hours of exposure.
[Show abstract][Hide abstract] ABSTRACT: The process of wound healing includes the regulated destruction of proteins via enzymes called proteinases. However, when the proteolytic process becomes excessive, pro-healing factors are destroyed and the wound healing process stalls. Matrix metalloproteinases (MMPs) are one key class of proteinases that have been observed to be elevated in many cases of failed wound healing.
Two key advances have been made in recent years. First is that, until recently, MMPs were only implicated in impaired healing of chronic wounds. Measurements of MMPs in wound fluids and serum from individuals with acute traumatic wounds have revealed that elevated MMPs are predictive of both impaired healing and of dehiscence of surgically closed wounds. The second advance is in the development of at least three clinically viable methods for measuring MMPs at the point of care.
At present there is no objective method of determining proteinase levels within a wound. Since elevated MMPs have now been shown to be predictive of dehiscence in surgically closed acute wounds, a new clinical utility for measuring MMPs has been established. With the advent of several new technologies to measure MMPs, the translation of this valuable molecular knowledge into improved therapeutic regimens is nearly complete.
The clinical utility of measuring MMPs continues to expand and be further validated with each new investigation. The tools that will enable clinicians to leverage this valuable information are nearing maturity and integration into the clinic.
[Show abstract][Hide abstract] ABSTRACT: Type 2 diabetes mellitus is a metabolic noncommunicable disease with an expanding pandemic magnitude. Diabetes predisposes to lower extremities ulceration and impairs the healing process leading to wound chronification. Diabetes also dismantles innate immunity favoring wound infection. Amputation is therefore acknowledged as one of the disease's complications. Hyperglycemia is the proximal detonator of systemic and local toxic effectors including proinflammation, acute-phase proteins elevation, and spillover of reactive oxygen and nitrogen species. Insulin axis deficiency weakens wounds' anabolism and predisposes to inflammation. The systemic accumulation of advanced glycation end-products irreversibly impairs the entire physiology from cells-to-organs. These factors in concert hamper fibroblasts and endothelial cells proliferation, migration, homing, secretion, and organization of a productive granulation tissue. Diabetic wound bed may turn chronically inflammed, procatabolic, and an additional source of circulating pro-inflammatory cytokines, establishing a self-perpetuating loop. Diabetic fibroblasts and endothelial cells may bear mitochondrial damages becoming prone to apoptosis, which impairs granulation tissue cellularity and perfusion. Endothelial progenitor cells recruitment and tubulogenesis are also impaired. Failure of wound reepithelialization remains a clinical challenge while it appears to be biologically multifactorial. Ulcer prevention by primary care surveillance, education, and attention programs is of outmost importance to reduce worldwide amputation figures.
BioMed research international. 01/2013; 2013:256043.
[Show abstract][Hide abstract] ABSTRACT: Corneal opacification (i.e., haze) following a non-denaturing acute injury to the cornea is a process which takes about 5 days to manifest itself, indicating that it is the consequence of cellular and molecular biological processes. In order to obtain a better understanding of the haze development process, and to test candidate anti-haze therapies, we use a corneal scarring model whereby we create an excimer laser wound in the center of rabbit corneas. The primary data generated by this model are (1) changes in corneal thickness with time; (2) wound closure rates, or re-epithelialization; (3) changes in the location and density of corneal sub-epithelial haze; and (4) molecular and histological changes leading up to, during, and following the formation of haze.While the use of excimer lasers to generate consistent wounds in rabbit corneas is not a novel protocol for the study of corneal haze, the photographic technique presented here for the more objective recording and quantification of corneal haze is. At present, a qualitative, semiquantitative, grading system is employed whereby the amount of iris detail discernible through the scar is assigned a value between 0 and 4. Such a system makes direct comparisons amongst reported anti-haze trials nearly impossible. Furthermore, the additional "geographic" detail provided by the image provides a new layer of information about the formation of haze and the ability to troubleshoot dosing regimens. Altogether, with the information present herein, we believe that the study of corneal haze formation and the ability to compare and contrast candidate therapies are both greatly improved.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: To investigate signal transduction pathways for connective tissue growth factor (CTGF) in human corneal fibroblasts(HCF). METHODS: Expression of 75 kinases in cultures of HCF serum-starved were investigated using protein kinase screens (Kinetworks™), and changes in levels of phosphorylation of 31 different phosphoproteins were determined at 0, 5 and 15 minutes after treatment with CTGF. Levels of phosphorylation of three signal transducing phosphoproteins (ERK1, ERK2 [MAPKs], and STAT3) were measured at nine time points after exposure to CTGF using Western immunoblots. Inhibition of Ras, MEK1/2 (MAPKK), and ERK1/2, on CTGF-stimulated fibroblast proliferation and collagen gel contraction was assessed using selective inhibitors farnesylthiosalicylic acid, PD-98059, and SB203580, respectively. RESULTS: Thirty two of the 75 kinases (43%) evaluated by the kinase screen were detected in extracts of quiescent HCF, suggesting these kinases are available to respond acutely to CTGF exposure. Addition of CTGF increased levels of phosphorylation of 5 phosphoproteins (ERK1&2, MEK1/2 [MAPKK], STAT3, and SAPK/JNK), and decreased levels of phosphorylation of 14 phosphoproteins (including protein kinases B and C) after 5 and 15 minutes. Further analysis of ERK1&2 and STAT3 phosphorylation showed rapid increases within 1 minute of CTGF exposure that peaked between 5 and 10 minutes then returned to pretreatment levels by 30 minutes. Treatment of HCF with selective inhibitors of Ras, MEK 1/2, and ERK1/2 individually blocked both CTGF induced cell proliferation, and collagen gel contraction. CONCLUSIONS: Results from protein kinase screens and selective kinase inhibitors demonstrate Ras/MEK/ERK/STAT3 pathway is required for CTGF signaling in human corneal fibroblasts.