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UN 1,2,3 / Thrombin Precursor & Comb 1 |
Extracellular Matrix and Platelet-Rich Plasma Derived Peptides Promoting Wound Healing |
Comb 1, a peptide created as combination of fragments of tenascin X and fibrillin 1, applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure.
UN3, a novel peptide created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury. Results of in vivo experiments where comb1 and UN3 peptides were added together to cranial wounds in cyclophosphamide-treated mice leads to improvement of wound vascularization as shown by an increase of the number of blood vessels present in the wound beds.
Application of the peptides markedly promotes cellular responses to injury and essentially restores wound healing dynamics to those of normal, acute wounds in the absence of cyclophosphamide impairment.
Chrysalin, or TP508, is a 23-amino acid synthetic peptide representing a receptor-binding domain of the human thrombin molecule, a naturally occurring molecule in the body responsible for both blood clotting and initiating many of the cellular events responsible for tissue repair.
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Demidova-Rice TN, et al., PLoS One. 2012;7(2):e32146. Epub 2012 Feb 23.
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Previous work in our laboratory has described several pro-angiogenic short peptides derived from endothelial extracellular matrices degraded by bacterial collagenase. Here we tested whether these peptides could stimulate wound healing in vivo. Our experiments demonstrated that a peptide created as combination of fragments of tenascin X and fibrillin 1 (comb1) applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure. Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury. Results of in vivo experiments where comb1 and UN3 peptides were added together to cranial wounds in cyclophosphamide-treated mice leads to improvement of wound vascularization as shown by an increase of the number of blood vessels present in the wound beds. Application of the peptides markedly promotes cellular responses to injury and essentially restores wound healing dynamics to those of normal, acute wounds in the absence of cyclophosphamide impairment. Our current work is aimed at understanding the mechanisms underlying the stimulatory effects of these peptides as well as identification of the cellular receptors mediating these effects.
Demidova-Rice TN, et al., PLoS One. 2012;7(2):e32146. Epub 2012 Feb 23.
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Demidova-Rice TN, et al., Wound Repair Regen. 2011 Jan-Feb;19(1):59-70. doi: 10.1111/j.1524-475X.2010.00642.x. Epub 2010 Dec 6.
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Studies in our laboratory indicate that collagenase from Clostridium histolyticum promotes endothelial cell and keratinocyte responses to injury in vitro and wound healing in vivo. We postulate that matrix degradation by Clostridial collagenase creates bioactive fragments that can stimulate cellular responses to injury and angiogenesis. To test this hypothesis, we performed limited digestion of defined capillary-endothelial-derived extracellular matrices using purified human or bacterial collagenases. Immunoprecipitation with antibodies recognizing collagens I, II, III, IV, and V, followed by mass spectrometry reveals the presence of unique fragments in bacterial, but not human-enzyme-digested matrix. Results show that there are several bioactive peptides liberated from Clostridial collagenase-treated matrices, which facilitate endothelial responses to injury, and accelerate microvascular remodeling in vitro. Fragments of collagen IV, fibrillin-1, tenascin X, and a novel peptide created by combining specific amino acids contained within fibrillin 1 and tenascin X each have profound proangiogenic properties. The peptides used at 10-100 nM increase rates of microvascular endothelial cell proliferation by up to 47% and in vitro angiogenesis by 200% when compared with serum-stimulated controls. Current studies are aimed at revealing the molecular mechanisms regulating peptide-induced wound healing while extending these in vitro observations using animal modeling.
Demidova-Rice TN, et al., Wound Repair Regen. 2011 Jan-Feb;19(1):59-70. doi: 10.1111/j.1524-475X.2010.00642.x. Epub 2010 Dec 6.
Thrombin and thrombin peptides play a role in initiating tissue repair. The potential safety and efficacy of TP508 (Chrysalin) treatment of diabetic foot ulcers was evaluated in a 60-subject, prospective, randomized, double-blind, placebo-controlled phase I/II clinical trial. Chrysalin in saline or saline alone was applied topically, twice weekly, to diabetic ulcers with standardized care and offloading. A dose-dependent effect was seen in the per-protocol population where 1 and 10 mug Chrysalin treatment resulted in 45 and 72% more subjects with complete healing than placebo treatment. Chrysalin treatment of foot ulcers more than doubled the incidence of complete healing (p<0.05), increased mean closure rate approximately 80% (p<0.05), and decreased the median time to 100% closure by approximately 40% (p<0.05). Chrysalin treatment of heel ulcers within this population resulted in mean closure rates 165% higher than placebos (p<0.02) and complete healing in 86% (6/7) of ulcers compared with 0% (0/5) of placebo ulcers (p<0.03). Local wound reactions and adverse events (AEs) were equal between groups with no reported drug-related changes in laboratory tests or serious AEs. These results indicate the potential safety and efficacy of Chrysalin for treatment of diabetic foot ulcers
Fife C. et al., Wound Repair Regen. 15:23-34(2007)
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A¨C In vitro angiogenesis assay. Human capillary endothelial cells were plated on growth factor reduced Matrigel. The media was supplemented with either DMEM supplemented with 1% BCS or 250 nM of UN1, UN2 or UN3 peptides. Cells that have received DMEM/1% in the presence of 10 ng/ml VEGF served as positive control. Total tube length was measured at 4 h post-plating. Relative tube length compared to control is shown.
B – In vitro epithelial wound healing assay. NHEK cells were plated and injured as described in Material ad methods. The following experimental conditions were used: basal keratinocyte growth media (control), 10 ng/ml HB-EGF (positive control), 0.5 nM UN1, UN2 or UN3 peptides. Relative wound closure is shown.
Tatiana N. Demidova-Rice et al. PLoS One. 2012; 7(2): e32146. Published online 2012 February 23. doi: 10.1371/journal.pone.0032146 |
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A – In vitro angiogenesis assay. Bovine capillary endothelial cells were plated on the surface of growth factor reduced Matrigel in the presence or absence of 100 nM comb1 or 250 nM UN3, or the two peptides combined. DMEM supplemented with 1% BCS was used as control, cells that have received DMEM/1% supplemented with 10 ng/ml bFGF or VEGF served as positive control. Total tube length was measured at 7 h post-plating. Relative tube length compared to control is shown.
B – In vitro epithelial proliferation assay. Hacat cells were plated. The peptides were added at 100 or 250 nM (comb1 and UN3 respectively). Cell counting was performed at 5 days post-plating. Relative cells numbers as compared to control are shown
Tatiana N. Demidova-Rice et al. PLoS One. 2012; 7(2): e32146. Published online 2012 February 23. doi: 10.1371/journal.pone.0032146 |
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