Clostridium Collagenase Impact on Zone of Stasis Stabilization and Transition to Healthy Tissue in Burns.

Clostridium collagenase has provided superior clinical results in achieving digestion of immediate and accumulating devitalized collagen tissue. Recent studies suggest that debridement via Clostridium collagenase modulates a cellular response to foster an anti-inflammatory microenvironment milieu, allowing for a more coordinated healing response. In an effort to better understand its role in burn wounds, we evaluated Clostridium collagenase's ability to effectively minimize burn progression using the classic burn comb model in pigs. Following burn injury, wounds were treated with Clostridium collagenase or control vehicle daily and biopsied at various time points. Biopsies were evaluated for factors associated with progressing necrosis as well as inflammatory response associated with treatment. Data presented herein showed that Clostridium collagenase treatment prevented destruction of dermal collagen. Additionally, treatment with collagenase reduced necrosis (HMGB1) and apoptosis (CC3a) early in burn injuries, allowing for increased infiltration of cells and protecting tissue from conversion. Furthermore, early epidermal separation and epidermal loss with a clearly defined basement membrane was observed in the treated wounds. We also show that collagenase treatment provided an early and improved inflammatory response followed by faster resolution in neutrophils. In assessing the inflammatory response, collagenase-treated wounds exhibited significantly greater neutrophil influx at day 1, with macrophage recruitment throughout days 2 and 4. In further evaluation, macrophage polarization to MHC II and vascular network maintenance were significantly increased in collagenase-treated wounds, indicative of a pro-resolving macrophage environment. Taken together, these data validate the impact of clostridial collagenases in the pathophysiology of burn wounds and that they complement patient outcomes in the clinical scenario.

Chronic wound repair and healing in older adults: current status and future research.

The incidence of chronic wounds is increased among older adults, and the impact of chronic wounds on quality of life is particularly profound in this population. It is well established that wound healing slows with age. However, the basic biology underlying chronic wounds and the influence of age-associated changes on wound healing are poorly understood. Most studies have used in vitro approaches and various animal models, but observed changes translate poorly to human healing conditions. The impact of age and accompanying multi-morbidity on the effectiveness of existing and emerging treatment approaches for chronic wounds is also unknown, and older adults tend to be excluded from randomized clinical trials. Poorly defined outcomes and variables, lack of standardization in data collection, and variations in the definition, measurement, and treatment of wounds also hamper clinical studies. The Association of Specialty Professors, in conjunction with the National Institute on Aging and the Wound Healing Society, held a workshop, summarized in this paper, to explore the current state of knowledge and research challenges, engage investigators across disciplines, and identify key research questions to guide future study of age-associated changes in chronic wound healing.

Exon-level expression profiling of ocular tissues.

The normal gene expression profiles of the tissues in the eye are a valuable resource for considering genes likely to be involved with disease processes. We profiled gene expression in ten ocular tissues from human donor eyes using Affymetrix Human Exon 1.0 ST arrays. Ten different tissues were obtained from six different individuals and RNA was pooled. The tissues included: retina, optic nerve head (ONH), optic nerve (ON), ciliary body (CB), trabecular meshwork (TM), sclera, lens, cornea, choroid/retinal pigment epithelium (RPE) and iris. Expression values were compared with publically available Expressed Sequence Tag (EST) and RNA-sequencing resources. Known tissue-specific genes were examined and they demonstrated correspondence of expression with the representative ocular tissues. The estimated gene and exon level abundances are available online at the Ocular Tissue Database.

TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure.

PURPOSE: Transforming growth factor (TGF)-beta2 levels are elevated in glaucomatous human aqueous humor. TGFbeta is a cytokine that alters extracellular matrix (ECM) metabolism, and excess ECM has been proposed to increase aqueous outflow resistance in the trabecular meshwork (TM) of glaucomatous eyes. This study was undertaken to investigate effects of TGFbeta2 on secretion of fibronectin and the protease inhibitor plasminogen activator inhibitor (PAI)-1 from human TM cell cultures and perfused human ocular anterior segments. METHODS: Total RNA was isolated from pooled human TM cell monolayers and used for a gene microarray expression analysis. Supernatants from treated human TM cells were analyzed by ELISA for fibronectin or PAI-1 content. TGFbeta2 effects on intraocular pressure (IOP) were evaluated in a perfused organ culture model using human anterior segments, and eluates were analyzed for fibronectin and PAI-1 content. RESULTS: Overnight treatment of TM cells with TGFbeta2 upregulated multiple ECM-related genes, such as PAI-1. TGFbeta2 also increased secretion of both fibronectin and PAI-1 from TM cells. TGFbeta2 effects on TM cells were blocked by inhibitors of the TGFbeta type I receptor. In perfused human anterior segments, TGFbeta2 treatment elevated IOP and increased eluate fibronectin and PAI-1 content. CONCLUSIONS: TGFbeta2 effects on IOP may be transduced by TGFbeta type-I receptor-mediated changes in TM secretion of ECM-related factors such as fibronectin and PAI-1. Modulation of TGFbeta2-induced changes in the ECM may provide a novel and viable approach to the management of glaucoma.

Chronic wound repair and healing in older adults: current status and future research.

Older adults are more likely to have chronic wounds than younger people, and the effect of chronic wounds on quality of life is particularly profound in this population. Wound healing slows with age, but the basic biology underlying chronic wounds and the influence of age-associated changes on wound healing are poorly understood. Most studies have used in vitro approaches and various animal models, but observed changes translate poorly to human healing conditions. The effect of age and accompanying multimorbidity on the effectiveness of existing and emerging treatment approaches for chronic wounds is also unknown, and older adults tend to be excluded from randomized clinical trials. Poorly defined outcomes and variables; lack of standardization in data collection; and variations in the definition, measurement, and treatment of wounds also hamper clinical studies. The Association of Specialty Professors, in conjunction with the National Institute on Aging and the Wound Healing Society, held a workshop, summarized in this article, to explore the current state of knowledge and research challenges, engage investigators across disciplines, and identify research questions to guide future study of age-associated changes in chronic wound healing.

Expression of matrix metalloproteinases and their inhibitors in human trabecular meshwork cells.

PURPOSE: Matrix metalloproteinases (MMPs) are involved in trabecular meshwork (TM) extracellular matrix metabolism and have been shown to increase aqueous outflow facility. The purpose of this study was to characterize effects of cytokines, a phorbol ester, and prostanoids on the expression of MMP-1, -2, -3, and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in cultured human TM cells. METHODS: Five human TM cell strains were treated with selected compounds. Levels of proMMPs and TIMPs in cell media were quantified by ELISA. MMP-3 activity was assayed by casein zymography. RESULTS: All human TM cell strains produced detectable basal amounts of proMMPs and TIMPs. 12-O-tetradecanoyl-phorbol-13-acetate was effective in increasing the levels of proMMP-1, -3, and -9 and TIMP-1. Its effect on proMMP-1 was concentration-dependent with an EC(50) of 2 to 3 nM. Interleukin (IL)-1alpha did not affect levels of proMMP-1 and -2 or the TIMPs, but was most efficacious in increasing proMMP-3 production with an EC(50) of 0.5 ng/mL. The IL-1alpha-induced upregulation of proMMP-3 correlated with an increase in MMP-3 activity. Tumor necrosis factor-alpha activated proMMP-3 production in some but not all cell strains. Platelet-derived growth factor-BB was generally ineffective in modulating MMP and TIMP levels. Prostaglandins E(2) and F(2alpha) at 10 micro M did not affect levels of proMMP-1 or -3. CONCLUSIONS: The expression of the different MMPs and TIMPs in human TM cells was independently regulated. Production of MMP-3 was maximally activated by IL-1alpha. The IL-1alpha-stimulated expression of MMP-3 provides a probable mechanism for IL-1alpha-enhanced aqueous outflow.

Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage.

BACKGROUND: Mutations in the gene encoding human myocilin (MYOC) have been shown to cause juvenile- and adult-onset glaucoma. In addition, myocilin has been associated with glucocorticoid-induced ocular hypertension and steroid-induced glaucoma. To better understand the role myocilin plays in steroid-induced glaucoma and open-angle glaucoma, we examined rabbit myocilin for use in the rabbit animal model of steroid-induced glaucoma. RESULTS: We have cloned the rabbit ortholog of human MYOC. Rabbit MYOC consists of three exons and an open reading frame encoding a 490 amino acid, 54,882-Da protein, which is 14 amino acids shorter at the N-terminus than human myocilin but 84% identical overall. Rabbit myocilin migrates as a single electrophoretic band, vs. double-banded human myocilin, by SDS-PAGE/immunoblot analysis. We determined that the differential migration exhibited is due to an N-glycosylation site that is present in human (Asn57), monkey and mouse myocilin but absent in rabbit (Ser43), rat and bovine myocilin. Rabbit myocilin is secreted in vitro in trabecular meshwork cell culture and in vivo in aqueous humor. Secretion of human myocilin is shown to be dependent on the signal peptide and independent of the extra 14 amino acids not found in rabbit myocilin. Many of the amino acids in myocilin that are mutated in glaucoma patients are conserved across species. CONCLUSION: We have cloned the rabbit MYOC cDNA and determined that rabbit myocilin is secreted but not N-linked glycosylated. Knowledge of the rabbit MYOC cDNA sequence will facilitate future studies in the rabbit animal model examining the role of myocilin in steroid-induced glaucoma and the gain-of-function hypothesis in open-angle glaucoma.

Trabecular meshwork bradykinin receptors: mRNA levels, immunohistochemical visualization, signaling processes pharmacology, and linkage to IOP reduction.

PURPOSE: To localize mRNA and protein of bradykinin (BK) receptors, BK precursor polypeptide (kininogen) mRNA, and to study functional biochemical pharmacology of the signal transduction processes mediated by B2-receptors in isolated human trabecular meshwork (h-TM) cells. Intraocular pressure (IOP) lowering effects of 2 kinins were also investigated. METHODS: Previously documented procedures were utilized throughout these studies. RESULTS: Kinninogen mRNA was most abundant in TM, ciliary body (CB), and optic nerve head and appeared elevated in glaucomatous h-TM tissue. High levels of B2-receptor mRNA were found in the sclera, iris, TM, and CB. B2-receptor subtype protein was localized in cells of the monkey and h-TM, and the treatment of isolated h-TM cells with transforming growth factor-ß2 (5 ng/mL) caused significant (P<0.04) downregulation of B2-receptor mRNA. In isolated primary h-TM cells, BK (EC50=0.8±0.2 nM; n=19) and Met-Lys-BK (EC50=6.5±1.5 nM) mobilized intracellular Ca(2+) and induced the release of prostaglandins (PGs) that was blocked by 2 B2-receptor antagonists [HOE-140; (S)-WIN-64338]. The cyclooxygenase inhibitor, bromfenac, abolished BK-induced PGs production. BK concentration dependently increased cell impedance, and it significantly (P<0.05) decreased h-TM cell volume in vitro. Intravitreal (ivt) administration of BK (50 µg), but not a B1-agonist (Sar-[D-Phe(9)]-Des-Arg(9)-BK; also at 50 µg), efficaciously lowered IOP (22.9% to 37% from baseline) of Dutch-Belted rabbits that naturally have high IOPs (27-28 mmHg). CONCLUSIONS: BK activates multiple signal transduction pathways in h-TM cells via B2-receptors that also mediate IOP reduction as observed in rabbits following ivt administration of BK. These ocular hypotensive effects of BK may be physiologically important and suggest a novel therapeutic potential of BK-related B2-agonists.

Identification of PDE6D as a molecular target of anecortave acetate via a methotrexate-anchored yeast three-hybrid screen.

Glaucoma and age-related macular degeneration are ocular diseases targeted clinically by anecortave acetate (AA). AA and its deacetylated metabolite, anecortave desacetate (AdesA), are intraocular pressure (IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid activity but with an unknown mechanism of action. We used a methotrexate-anchored yeast three-hybrid (Y3H) technology to search for binding targets for AA in human trabecular meshwork (TM) cells, the target cell type that controls IOP, a major risk factor in glaucoma. Y3H hits were filtered by competitive Y3H screens and coimmunoprecipitation experiments and verified by surface plasmon resonance analysis to yield a single target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding protein with additional function outside the PDE6 phototransduction system. Overexpression of PDE6D in mouse eyes caused elevated IOP, and this elevation was reversed by topical ocular application of either AA or AdesA. The identification of PDE6D as the molecular binding partner of AA provides insight into the role of this drug candidate in treating glaucoma.

Adenoviral gene transfer of active human transforming growth factor-{beta}2 elevates intraocular pressure and reduces outflow facility in rodent eyes.

Purpose. Glaucoma is a leading cause worldwide of blindness and visual impairment. Transforming growth factor-beta2 (TGFbeta2) has been implicated in the pathogenesis of primary open-angle glaucoma (POAG) based on elevated levels in glaucomatous aqueous humor and its ability to induce extracellular matrix (ECM) remodeling in the trabecular meshwork (TM). The goal of this study was to generate a rodent model of POAG using viral gene transfer of human TGFbeta2. Methods. Latent (hTGFbeta2(WT)) or active (C226S, C228S; hTGFbeta2(226/228)) TGFbeta2-encoding cDNA was cloned into the pac.Ad5.CMV.K-N.pA shuttle vector for generation of replication-deficient adenovirus. Empty adenovirus (Ad5.CMV.K-N.pA) was used as a control. Adenoviral expression of active and total TGFbeta2 was assayed in vitro by the transduction of Chinese hamster ovary and trabecular meshwork cells. BALB/cJ mice or Wistar rats were injected either intracamerally or intravitreally with the adenovectors and assessed for changes in intraocular pressure (IOP) using the rebound tonometer. At peak IOP, aqueous outflow facility and total TGFbeta2 levels in aqueous humor were measured. Mouse eye morphology was assessed by hematoxylin and eosin staining. Results. Adenoviral gene transfer of hTGFbeta2(226/228), but not hTGFbeta2(WT), to the rodent eye elevated IOP in rat (43%, P < 0.001) and mouse (110%, P < 0.001) and reduced aqueous humor outflow facility in the mouse. The TGFbeta2-induced ocular hypertension correlated with anterior segment TGFbeta2 expression levels (P < 0.0001). Conclusions. The adenoviral TGFbeta2 rodent model displays the glaucoma risk factors of elevated IOP and decreased aqueous outflow facility and may potentially serve as a model for studying glaucoma.

Glaucoma-causing myocilin mutants require the Peroxisomal targeting signal-1 receptor (PTS1R) to elevate intraocular pressure.

Glaucoma is a leading cause of worldwide irreversible visual impairment and blindness and is a clinically and genetically heterogenous group of optic neuropathies. Specific mutations in the myocilin (MYOC) gene cause primary open angle glaucoma (POAG) with varying age-of-onset and degree of severity. We show a mutation-dependent, gain-of-function association between human myocilin and the peroxisomal targeting signal type 1 receptor (PTS1R). There was correlation between the glaucoma phenotype and the specific MYOC mutations, with the more severe early-onset POAG mutations having a higher degree of association with PTS1R. Expression of human myocilin glaucomatous mutations in mouse eyes causes elevated intraocular pressure, which is a major phenotype of MYOC glaucoma. This is the first demonstration of a disease resulting from mutation-induced exposure of a cryptic signaling site that causes mislocalization of mutant protein to peroxisomes and the first disease-gene-based animal model of human POAG.