Tatiana N. Demidova-Rice

Effect of chitosan acetate bandage on wound healing in infected and noninfected wounds in mice

HemCon® bandage is an engineered chitosan acetate preparation designed as a hemostatic dressing, and is under investigation as a topical antimicrobial dressing. We studied its effects on healing of excisional wounds that were or were not infected with Staphylococcus aureus, in normal mice or mice previously pretreated with cyclophosphamide (CY). CY significantly suppressed wound healing in both the early and later stages, while S. aureus alone significantly stimulated wound healing in the early stages by preventing the initial wound expansion. CY plus S. aureus showed an advantage in early stages by preventing expansion, but a significant slowing of wound healing in later stages. In order to study the conflicting clamping and stimulating effects of chitosan acetate bandage on normal wounds, we removed the bandage from wounds at times after application ranging from 1 hour to 9 days. Three days application gave the earliest wound closure, and all application times gave a faster healing slope after removal compared with control wounds. Chitosan acetate bandage reduced the number of inflammatory cells in the wound at days 2 and 4, and had an overall beneficial effect on wound healing especially during the early period where its antimicrobial effect is most important.

Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy

Background Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy. Methodology/Principal Findings We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection). Conclusions/Significance ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-β1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.

Bioactive peptides derived from vascular endothelial cell extracellular matrices promote microvascular morphogenesis and wound healing in vitro

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.

Human Platelet-Rich Plasma- and Extracellular Matrix-Derived Peptides Promote Impaired Cutaneous Wound Healing In Vivo

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.

Cellular chromophores and signaling in low level light therapy

The use of low levels of visible or near infrared light (LLLT) for reducing pain, inflammation and edema, promoting healing of wounds, deeper tissues and nerves, and preventing tissue damage by reducing cellular apoptosis has been known for almost forty years since the invention of lasers. Originally thought to be a peculiar property of laser light (soft or cold lasers), the subject has now broadened to include photobiomodulation and photobiostimulation using non-coherent light. Despite many reports of positive findings from experiments conducted in vitro, in animal models and in randomized controlled clinical trials, LLLT remains controversial. This likely is due to two main reasons; firstly the biochemical mechanisms underlying the positive effects are incompletely understood, and secondly the complexity of rationally choosing amongst a large number of illumination parameters such as wavelength, fluence, power density, pulse structure and treatment timing has led to the publication of a number of negative studies as well as many positive ones. In recent years major advances have been made in understanding the mechanisms that operate at the cellular and tissue levels during LLLT. Mitochondria are thought to be the main site for the initial effects of light and specifically cytochrome c oxidase that has absorption peaks in the red and near infrared regions of the electromagnetic spectrum matches the action spectra of LLLT effects. The discovery that cells employ nitric oxide (NO) synthesized in the mitochondria by neuronal nitric oxide synthase, to regulate respiration by competitive binding to the oxygen binding of cytochrome c oxidase, now suggests how LLLT can affect cell metabolism. If LLLT photodissociates inhibitory NO from cytochrome c oxidase, this would explain increased ATP production, modulation of reactive oxygen species, reduction and prevention of apoptosis, stimulation of angiogenesis, increase of blood flow and induction of transcription factors. In particular, signaling cascades are initiated via cyclic adenosine monophosphate (cAMP) and nuclear factor kappa B (NF-&kgr;B). These signal transduction pathways in turn lead to increased cell proliferation and migration (particularly by fibroblasts), modulation in levels of cytokines, growth factors and inflammatory mediators, and increases in anti-apoptotic proteins. The results of these biochemical and cellular changes in animals and patients include such benefits as increased healing in chronic wounds, improvements in sports injuries and carpal tunnel syndrome, pain reduction in arthritis and neuropathies, and amelioration of damage after heart attacks, stroke, nerve injury and retinal toxicity.

Identification and Characterization of Novel Matrix-Derived Bioactive Peptides: A Role for Collagenase from Santyl® Ointment in Post-Debridement Wound Healing?

Debridement, the removal of diseased, nonviable tissue, is critical for clinicians to readily assess wound status and prepare the wound bed for advanced therapeutics or downstream active healing. Removing necrotic slough and eschar through surgical or mechanical methods is less specific and may be painful for patients. Enzymatic debridement agents, such as Clostridial collagenase, selectively and painlessly degrade devitalized tissue. In addition to its debriding activities, highly-purified Clostridial collagenase actively promotes healing, and our past studies reveal that extracellular matrices digested with this enzyme yield peptides that activate cellular migratory, proliferative and angiogenic responses to injury in vitro, and promote wound closure in vivo. Intriguingly, while collagenase Santyl® ointment, a sterile preparation containing Clostridial collagenases and other non-specific proteases, is a well-accepted enzymatic debridement agent, its role as an active healing entity has never been established. Based on our previous studies of pure Clostridial collagenase, we now ask whether the mixture of enzymes contained within Santyl® produces matrix-derived peptides that promote cellular injury responses in vitro and stimulate wound closure in vivo. Here, we identify novel collagen fragments, along with collagen-associated peptides derived from thrombospondin-1, multimerin-1, fibronectin, TGFβ-induced protein ig-h3 and tenascin-C, generated from Santyl® collagenase-digested human dermal capillary endothelial and fibroblastic matrices, which increase cell proliferation and angiogenic remodeling in vitro by 50–100% over controls. Using an established model of impaired healing, we further demonstrate a specific dose of collagenase from Santyl® ointment, as well as the newly-identified and chemically-synthesized ECM-derived peptides significantly increase wound re-epithelialization by 60–100% over saline-treated controls. These results not only confirm and extend our earlier studies using purified collagenase- and matrix-derived peptides to stimulate healing in vitro and in vivo, but these Santyl®-generated, matrix-derived peptides may also represent exciting new opportunities for creating advanced wound healing therapies that are enabled by enzymatic debridement and potentially go beyond debridement.

Low-level light therapy for zymosan-induced arthritis in rats

It has been known for many years that low level laser (or light) therapy (LLLT) can ameliorate the pain, swelling and inflammation associated with various forms of arthritis. Light is absorbed by mitochondrial chromophores leading to an increase in ATP, reactive oxygen species and/or cyclic AMP production and consequent gene transcription via activation of transcription factors. However, despite many reports about the positive effects of LLLT in medicine, its use remains controversial. Our laboratory has developed animal models designed to objectively quantify response to LLLT and compare different light delivery regimens. In the arthritis model we inject zymosan into rat knee joints to induce inflammatory arthritis. We have compared illumination regimens consisting of a high and low fluence (3 J/cm2 and 30 J/cm2), delivered at a high and low irradiance (5 mW/cm2 and 50 mW/cm2) using 810-nm laser light daily for 5 days, with the effect of conventional corticosteroid (dexamethasone) therapy. Results indicated that illumination with 810-nm laser is highly effective (almost as good as dexamethasone) at reducing swelling and that longer illumination time was more important in determining effectiveness than either total fluence delivered or irradiance. Experiments carried out using 810-nm LLLT on excisional wound healing in mice also confirmed the importance of longer illumination times. These data will be of value in designing clinical trials of LLLT.