Collagen-Binding Peptidoglycans Inhibit MMP Mediated Collagen Degradation and Reduce Dermal Scarring

Scarring of the skin is a large unmet clinical problem that is of high patient concern and impact. Wound healing is complex and involves numerous pathways that are highly orchestrated, leaving the skin sealed, but with abnormal organization and composition of tissue components, namely collagen and proteoglycans, that are then remodeled over time. To improve healing and reduce or eliminate scarring, more rapid restoration of healthy tissue composition and organization offers a unique approach for development of new therapeutics. A synthetic collagen-binding peptidoglycan has been developed that inhibits matrix metalloproteinase-1 and 13 (MMP-1 and MMP-13) mediated collagen degradation. We investigated the synthetic peptidoglycan in a rat incisional model in which a single dose was delivered in a hyaluronic acid (HA) vehicle at the time of surgery prior to wound closure. The peptidoglycan treatment resulted in a significant reduction in scar tissue at 21 days as measured by histology and visual analysis. Improved collagen architecture of the treated wounds was demonstrated by increased tensile strength and transmission electron microscopy (TEM) analysis of collagen fibril diameters compared to untreated and HA controls. The peptidoglycan's mechanism of action includes masking existing collagen and inhibiting MMP-mediated collagen degradation while modulating collagen organization. The peptidoglycan can be synthesized at low cost with unique design control, and together with demonstrated preclinical efficacy in reducing scarring, warrants further investigation for dermal wound healing

Synthesis and optimization of collagen‐targeting peptide‐glycosaminoglycans for inhibition of platelets following endothelial injury

Many endothelial complications, whether from surgical or pathological origins, can result in the denudation of the endothelial layer and the exposure of collagen. Exposure of collagen results in the activation of platelets, leading to thrombotic and inflammatory cascades that ultimately result in vessel stenosis. We have previously reported the use of peptide-GAG compounds to target exposed collagen following endothelial injury. In this paper we optimize the spacer sequence of our collagen binding peptide to increase its conjugation to GAG backbones and increase the peptide-GAG collagen binding affinity by increasing peptide C-terminal cationic charge. Furthermore, we demonstrate the use of these molecules to inhibit platelet activation through collagen blocking, as well as their localization to exposed vascular collagen following systemic delivery. Altogether, optimization of peptide sequence and linkage chemistry can allow for increased conjugation and function, having implications for glycoconjugate use in other clinical applications

Decorin mimic inhibits vascular smooth muscle proliferation and migration.

Over the past 10 years, the number of percutaneous coronary intervention procedures performed in the United States increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, complicates this procedure. A wide range of anti-restenotic therapeutics have been developed, although many elicit non-specific effects that compromise vessel healing. Drawing inspiration from biologically-relevant molecules, our lab developed a mimic of the natural proteoglycan decorin, termed DS-SILY, which can mask exposed collagen and thereby effectively decrease platelet activation, thus contributing to suppression of vascular intimal hyperplasia. Here, we characterize the effects of DS-SILY on both proliferative and quiescent human SMCs to evaluate the potential impact of DS-SILY-SMC interaction on restenosis, and further characterize in vivo platelet interactions. DS-SILY decreased proliferative SMC proliferation and pro-inflammatory cytokine secretion in vitro in a concentration dependent manner as compared to untreated controls. The addition of DS-SILY to in vitro SMC cultures decreased SMC migration and protein synthesis by 95% and 37%, respectively. Furthermore, DS-SILY decreased platelet activation, as well as reduced neointimal hyperplasia by 60%, in vivo using Ossabaw swine. These results indicate that DS-SILY demonstrates multiple biological activities that may all synergistically contribute to an improved treatment paradigm for balloon angioplasty

Neointimal Hyperplasia.

<p>Representative histology sections of (A, B) arteries with stents and (C, D) arteries without stents treated with (A, C) saline or (B, D) 10 μM DS-SILY₂₀. Arrowheads indicate location of some stent struts; internal elastic lamina (dotted line) and lumenal border (solid line) are identified, indicating the boundaries of the neointima formed following injury. (E) Neointimal hyperplasia was quantified by measuring the distance from a stent strut or the elastic lamina to the vessel lumen in arteries with or without stents, respectively. A significant reduction in neointimal hyperplasia with DS-SILY₂₀ treatment was observed, compared with saline sham controls, for both stented and non-stented artery segments. Six measurements were taken for each artery. Analysis with stents: sham (n=4), DS-SILY₂₀ (n=3); without stents: sham (n=8), DS-SILY₂₀ (n=5). Scale bar = 1 mm.* represents significance from sham-treated vessels.</p

Decorin Mimic Inhibits Vascular Smooth Muscle Proliferation and Migration

<div><p>Over the past 10 years, the number of percutaneous coronary intervention procedures performed in the United States increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, complicates this procedure. A wide range of anti-restenotic therapeutics have been developed, although many elicit non-specific effects that compromise vessel healing. Drawing inspiration from biologically-relevant molecules, our lab developed a mimic of the natural proteoglycan decorin, termed DS-SILY, which can mask exposed collagen and thereby effectively decrease platelet activation, thus contributing to suppression of vascular intimal hyperplasia. Here, we characterize the effects of DS-SILY on both proliferative and quiescent human SMCs to evaluate the potential impact of DS-SILY-SMC interaction on restenosis, and further characterize <i>in vivo</i> platelet interactions. DS-SILY decreased proliferative SMC proliferation and pro-inflammatory cytokine secretion <i>in vitro</i> in a concentration dependent manner as compared to untreated controls. The addition of DS-SILY to <i>in vitro</i> SMC cultures decreased SMC migration and protein synthesis by 95% and 37%, respectively. Furthermore, DS-SILY decreased platelet activation, as well as reduced neointimal hyperplasia by 60%, <i>in vivo</i> using Ossabaw swine. These results indicate that DS-SILY demonstrates multiple biological activities that may all synergistically contribute to an improved treatment paradigm for balloon angioplasty.</p> </div

Cytokine Production.

<p>Cytokine produced (A, C, E, G) proliferative and (B, D, F, H) quiescent SMCs in response to DS-SILY₂₀. The amount of (A, B) IFN-γ, (C, D) IL-1β, (E, F) IL-6, and (G, H) TNF-α produced by cultured SMCs was measured 24 hrs post-treatment. For proliferative SMC cultures, a general trend was observed such that as the concentration of DS-SILY₂₀ increased, cytokine production decreased. Significant reductions in IFN-γand TNF-α were also observed in quiescent SMC cultures with the addition of DS-SILY₂₀. * represents significance from control non-treated cells. (Nγ6).</p

Platelet Deposition and Activation.

<p>SEM images of acute platelet response on artery wall in vessels treated with (A, C) DS-SILY₂₀ and (B, D) sham saline control. Saline-treated arteries exhibited significant platelet coverage with numerous projections extending from activated platelets; platelets were scarcely visible on denuded arteries treated with DS-SILY₂₀. Arrowheads indicate areas of platelet coverage. Magnification = 3,500x (A, B); 35,000x (C, D). Scale bar = 20 μm (A, B); 2 μm (C, D).</p

Protein Synthesis.

<p>Protein expression of (A) proliferative and (B) quiescent SMCs in response to treatment with DS-SILY₂₀. A significant decrease in protein expression was observed in proliferative SMC cultures treated with 10 μM DS-SILY₂₀, while no changes in protein synthesis was exhibited in proliferative SMC cultures treated with lower concentrations of DS-SILY₂₀ or quiescent cultures with the addition of any concentration DS-SILY₂₀. * represents significance from control non-treated cells. (N>6).</p

Proliferation and Migration.

<p>Proliferation (A, B) and migration (C, D) of proliferative (A, C) and quiescent (B, D) SMCs in response to treatment with DS-SILY₂₀. Proliferation and migration of proliferative SMCs significantly decreased with increased concentrations of DS-SILY₂₀. Proliferation in quiescent SMC cultures was not altered with addition of DS-SILY₂₀ at any concentration; however, quiescent SMC migration decreased with the addition of 1 and 10 μM DS-SILY₂₀. * represents significance from control non-treated cells. (N>6).</p