Development and application of anti-fibrogenic dressings

It is well established that more than 15 million patients develop pathological scarring each year following elective operations, surgical procedures, deep trauma, thermal and electrical injuries. These scars, that cause major functional, cosmetic, psychological, and social consequences for the patients, impose a significant financial burden on health care systems. The current treatment modalities for these pathological conditions vary from topical application and intralesional injection of anti-scarring agents to surgical revisions and radiotherapy. The limited efficacy of these therapeutics for prevention of scar formation raised a great need for innovation within the wound care industry. Recently Kynurenine (Kyn), a tryptophan metabolite, has been identified as a potent anti-fibrotic agent. Kyn prevents scar formation by enhancing the expression of ECM degrading enzymes, matrix metalloproteinases (MMPs), and suppressing the expression of collagen. Although daily topical application of Kyn-cream improved the wound healing outcome in animal models, this method of drug delivery is not clinically practical in situations where dressings need to be kept on for 3-5 days. In this dissertation, it is hypothesized that topical application of a slow and controlled releasing Kyn or its metabolites from nanofiber dressing at the wound site improves and/or prevents dermal fibrosis by modulating the expression of the key ECM components involved in dermal fibrotic conditions. To test this hypothesis, three specific objectives were employed: (1) Evaluating and comparing the anti-fibrotic effects of Kynurenic acid (KynA) and Kyn, (2) Developing, characterizing and optimizing the nanofibrous dressings as a slow releasing drug delivery system for Kyn and KynA, (3) Examining the functionality of the developed anti-fibrotic dressings in open wounds in animal models. The findings of these specific objectives of this work demonstrated that topical application of the developed polymeric dressings, which slowly release Kyn/KynA over the course of 4 days, effectively reduces dermal fibrosis by modulating the key ECM components such as MMPs, collagen and fibronectin. The findings of this study support our hypothesis that development of an anti-fibrogenic dressing is feasible and as such its application would overcome the difficulties associated with development of hypertrophic scarring frequently seen in millions of patients worldwide.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Effect of kynurenines on fibroblast and keratinocyte migration.

<p><b>A & B</b>: Images of human fibroblasts and keratinocytes taken immediately, 12 and 24 h after addition of KynA or Kyn (100 μg/ml) in an <i>in vitro</i> wound scratch assay, respectively. <b>C</b>: Reduction of fibroblast migration in the presence of KynA after 12 and 24 hours of treatment. <b>D</b>: Enhancement of keratinocyte migration in the presence of KynA and Kyn. Cell migration was evaluated by manually counting the total number of cells migrated from the edges of the wound into the denuded area (* P-value<0.05 and ** P-value<0.01, n = 4).</p

Effect of kynurenines on fibroblast and keratinocyte proliferation rate and viability.

<p><b>A & B</b>: To determine the effect of different kynurenines on dermal cell proliferation rate, fibroblast and keratinocytes were treated with KynA or Kyn (100 μg/ml). Cells were harvested and total cell number was counted after 36, 72 and 108 hours of incubation (* P-value<0.05 and ** P-value<0.01, n = 6). <b>C</b>: Determination of cellular viability by FACs analysis using live/dead, viability/cytotoxicity assay kit. Fibroblasts and keratinocytes were either cultured in DMEM+2% FBS or DMEM+2% FBS supplemented with increasing concentrations of KynA or Kyn (50 and 150 μg/ml). The viability of cells was determined by FACS analysis following 3 days of incubation.</p

Anti-Scarring Properties of Different Tryptophan Derivatives

<div><p>Hypertrophic scars are associated with prolonged extracellular matrix (ECM) production, aberrant ECM degradation and high tissue cellularity. Routinely used antifibrotic strategies aim to reduce ECM deposition and enhance matrix remodeling. Our previous study investigating the antifibrotic effects of indoleamine2, 3 dioxygenase (IDO) led to the identification of kynurenine (Kyn) as an antiscarring agent. A topical antifibrogenic therapy using Kyn is very attractive; however, it is well established that Kyn passes the blood brain barrier (BBB) which causes complications including excitatory neuronal death. Here we investigated the antiscarring properties of kynurenic acid (KynA), a downstream end product of Kyn that is unlikely to pass the BBB, as an effective and safe replacement for Kyn. Our results indicated that while not having any adverse effect on dermal cell viability, KynA significantly increases the expression of matrix metalloproteinases (MMP1 and MMP3) and suppresses the production of type-I collagen and fibronectin by fibroblasts. Topical application of cream containing KynA in fibrotic rabbit ear significantly decreased scar elevation index (1.13±0.13 vs. 1.61±0.12) and tissue cellularity (221.38±21.7 vs. 314.56±8.66 cells/hpf) in KynA treated wounds compared to controls. KynA treated wounds exhibited lower levels of collagen deposition which is accompanied with a significant decrease in type-I collagen and fibronectin expression, as well as an increase in MMP1 expression compared to untreated wounds or wounds treated with cream only. The results of this study provided evidence for the first time that KynA is promising candidate antifibrogenic agent to improve healing outcome in patients at risk of hypertrophic scarring.</p></div

Effect of Kyn and KynA topical application on collagen deposition, tissue cellularity and ECM expression.

<p>A: Evaluation of collagen deposition in tissue samples using Masson's Trichrome staining at day 35 post-wounding. In this staining collagen fibers are stained blue, keratin and muscle fibers are stained red, and cell cytoplasm and nuclei are stained light pink and dark brown, respectively. B: Quantification and statistical analysis of tissue cellularity. Q-PCR analysis of relative MMP1 (C), type-I collagen (D) and fibronectin (E) mRNA expression in tissue samples (* P-value<0.05 and ** P-value<0.01, n = 4).</p

Inhibition of type-I collagen and fibronectin expression in dermal fibroblasts by kynurenines.

<p>Type-I Collagen and fibronectin expression at the mRNA and protein level in cultured fibroblasts treated with increasing concentrations (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn. <b>A & B</b>: Relative type-I collagen and fibronectin mRNA expression in treated fibroblasts, respectively. GAPDH was used as the reference gene. <b>C</b>: Evaluation of type-I collagen and fibronectin expression at the protein level using Western blotting. <b>D & E</b>: The Mean±SEM ratio of type-I collagen and fibronectin density to β-actin at the protein level, respectively. β-actin was used as protein loading control.</p

Stimulatory effect of kynurenines on MMP1 expression.

<p><b>A</b>: Dermal fibroblasts were treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn. Following 24 hours of treatment cells were collected, and MMP1 expression was determined by Q-PCR after RNA extraction and cDNA synthesis. <b>B</b>: Evaluation of MMP1 expression at the protein level by Western blotting after 48 hours of treatment. <b>C</b>: The Mean±SEM ratio of MMP1 to β-actin density at the protein level. β-actin and GAPDH were used as loading controls for western blotting and Q-PCR, respectively.</p

Stimulatory effect of Kynurenines on MMP activity and Kynurenines lasting effect on MMP1 expression.

<p><b>A</b>: The effect of kynurenines of MMP activity. To determine MMP activity in the fibroblasts conditioned medium, cells were treated with 100 μg/ml of KynA, Kyn, L-Kyn or D-Kyn for 48 hours and MMP activity was evaluated using SensoLyte Plus 520 generic MMP Assay Kit (** P-value<0.001, n = 4). <b>B</b>: Kynurenines lasting effect on the MMP1 expression. To determine the lasting effect of kynurenines on MMP1 expression, fibroblasts were treated with KynA or Kyn (100 μg/ml) for 48 hours. The medium was replaced and cells were harvested immediately, 12, 24, and 48 hours after treatment removal. The MMP1 expression in dermal fibroblasts was evaluated using western blotting. <b>C</b>: MMP1/β-actin expression ratio was calculated in treated fibroblasts. Data is mean±SEM of 4 independent experiments (* P-value<0.05 and ** P-value<0.01, n = 4).</p

Stimulatory effect of kynurenines on MMP3 secretion by fibroblasts.

<p><b>A</b>: Evaluation of MMP3 mRNA expression in fibroblasts treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn following 24 hours of treatment. GAPDH was used as loading control for Q-PCR. <b>B</b>: Evaluation of MMP3 presence in the fibroblast conditioned medium using Western blotting after 48 hours of treatment. <b>C</b>: The Mean±SEM ratio of MMP3 density at the protein level.</p

Immuno-Regulatory Function of Indoleamine 2,3 Dioxygenase through Modulation of Innate Immune Responses

<div><p>Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO–expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11±1.47 vs. 70.5±7.57 cells/HPF), T-cells (8.75±1.03 vs. 75.75±5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.</p></div