Carlos Zgheib

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• Zgheib, C. (2022). Please see attached a list of the "BOOK CHAPTERS" that I've co-authored and published.. In Please see attached a list of the "BOOK CHAPTERS" that I've co-authored and published..

Journals/Publications

• Apte, A., Bardill, J. R., Canchis, J., Skopp, S. M., Fauser, T., Lyttle, B., Vaughn, A. E., Seal, S., Jackson, D. M., Liechty, K. W., & Zgheib, C. (2024). Targeting Inflammation and Oxidative Stress to Improve Outcomes in a TNBS Murine Crohn's Colitis Model. Nanomaterials (Basel, Switzerland), 14(10).
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  Inflammation and oxidative stress are implicated in the pathogenesis of Crohn's disease. Cerium oxide nanoparticle (CNP) conjugated to microRNA 146a (miR146a) (CNP-miR146a) is a novel compound with anti-inflammatory and antioxidative properties. We hypothesized that local administration of CNP-miR146a would improve colitis in a 2,4,6-Trinitrobenzenesulfonic acid (TNBS) mouse model for Crohn's disease by decreasing colonic inflammation. Balb/c mice were instilled with TNBS enemas to induce colitis. Two days later, the mice received cellulose gel enema, cellulose gel with CNP-miR146a enema, or no treatment. Control mice received initial enemas of 50% ethanol and PBS enemas on day two. The mice were monitored daily for weight loss and clinical disease activity. The mice were euthanized on days two or five to evaluate their miR146a expression, inflammation on histology, and colonic IL-6 and TNF gene expressions and protein concentrations. CNP-miR146a enema successfully increased colonic miR146a expression at 12 h following delivery. At the end of five days from TNBS instillation, the mice treated with CNP-miR146a demonstrated reduced weight loss, improved inflammation scores on histology, and reduced gene expressions and protein concentrations of IL-6 and TNF. The local delivery of CNP-miR146a in a TNBS mouse model of acute Crohn's colitis dramatically decreased inflammatory signaling, resulting in improved clinical disease.
• Apte, A., Dutta Dey, P., Julakanti, S. R., Midura-Kiela, M., Skopp, S. M., Canchis, J., Fauser, T., Bardill, J., Seal, S., Jackson, D. M., Ghishan, F. K., Kiela, P. R., Zgheib, C., & Liechty, K. W. (2024). Oral Delivery of miR146a Conjugated to Cerium Oxide Nanoparticles Improves an Established T Cell-Mediated Experimental Colitis in Mice. Pharmaceutics, 16(12).
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  Dysregulated inflammation and oxidative stress are strongly implicated in the pathogenesis of inflammatory bowel disease. We have developed a novel therapeutic that targets inflammation and oxidative stress. It is comprised of microRNA-146a (miR146a)-loaded cerium oxide nanoparticles (CNPs) (CNP-miR146a). We hypothesized that oral delivery of CNP-miR146a would reduce colonic inflammation in a mouse model of established, chronic, T cell-mediated colitis. The stability of CNP-miR146a and mucosal delivery was assessed in vitro with simulated gastrointestinal fluid and in vivo after oral gavage by quantitative real-time RT-PCR. The efficacy of orally administered CNP-miR146a was tested in mice with established colitis using the model of adoptive naïve T-cell transfer in recombinant activating gene 2 knockout (Rag2) mice. Measured outcomes included histopathology; CD45 immune cell infiltration; oxidative DNA damage (tissue 8-hydroxy-2'-deoxyguanosine; 8-OHdG); expression of IL-6 and TNF mRNA and protein, and flow cytometry analysis of lamina propria Th1 and Th17 cell populations. miR146a expression remained stable in simulated gastric and intestinal conditions. miR146a expression increased in the intestines of mice six hours following oral gavage of CNP-miR146a. Oral delivery of CNP-miR146a in mice with colitis was associated with reduced inflammation and oxidative stress in the proximal and distal colons as evidenced by histopathology scoring, reduced immune cell infiltration, reduced IL-6 and TNF expression, and decreased populations of CD4TbetIFNg+ Th1, CD4RorgTIL17 Th17, as well as pathogenic double positive IFNgIL17 T cells. : CNP-miR146a represents a novel orally available therapeutic with high potential to advance into clinical trials.
• Apte, A., Liechty, K. W., & Zgheib, C. (2023). Immunomodulatory biomaterials on chemokine signaling in wound healing. Frontiers in pharmacology, 14, 1084948.
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  Normal wound healing occurs through a careful orchestration of cytokine and chemokine signaling in response to injury. Chemokines are a small family of chemotactic cytokines that are secreted by immune cells in response to injury and are primarily responsible for recruiting appropriate immune cell types to injured tissue at the appropriate time. Dysregulation of chemokine signaling is suspected to contribute to delayed wound healing and chronic wounds in diseased states. Various biomaterials are being used in the development of new therapeutics for wound healing and our understanding of their effects on chemokine signaling is limited. It has been shown that modifications to the physiochemical properties of biomaterials can affect the body's immune reaction. Studying these effects on chemokine expression by various tissues and cell type can help us develop novel biomaterial therapies. In this review, we summarize the current research available on both natural and synthetic biomaterials and their effects on chemokine signaling in wound healing. In our investigation, we conclude that our knowledge of chemokines is still limited and that many in fact share both pro-inflammatory and anti-inflammatory properties. The predominance of either a pro-inflammatory or anti-inflammatory profile is mostly likely dependent on timing after injury and exposure to the biomaterial. More research is needed to better understand the interaction and contribution of biomaterials to chemokine activity in wound healing and their immunomodulatory effects.
• Lyttle, B. D., Vaughn, A. E., Bardill, J. R., Apte, A., Gallagher, L. T., Zgheib, C., & Liechty, K. W. (2023). Effects of microRNAs on angiogenesis in diabetic wounds. Frontiers in medicine, 10, 1140979.
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  Diabetes mellitus is a morbid condition affecting a growing number of the world population, and approximately one third of diabetic patients are afflicted with diabetic foot ulcers (DFU), which are chronic non-healing wounds that frequently progress to require amputation. The treatments currently used for DFU focus on reducing pressure on the wound, staving off infection, and maintaining a moist environment, but the impaired wound healing that occurs in diabetes is a constant obstacle that must be faced. Aberrant angiogenesis is a major contributor to poor wound healing in diabetes and surgical intervention is often necessary to establish peripheral blood flow necessary for healing wounds. Over recent years, microRNAs (miRNAs) have been implicated in the dysregulation of angiogenesis in multiple pathologies including diabetes. This review explores the pathways of angiogenesis that become dysregulated in diabetes, focusing on miRNAs that have been identified and the mechanisms by which they affect angiogenesis.
• Peddibhotla, S., Caples, K., Mehta, A., Chen, Q. Y., Hu, J., Idlett-Ali, S., Zhang, L., Zgheib, C., Xu, J., Liechty, K. W., & Malany, S. (2023). Triazolothiadiazine derivative positively modulates CXCR4 signaling and improves diabetic wound healing. Biochemical pharmacology, 216, 115764.
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  Development of specific therapies that target and accelerate diabetic wound repair is an urgent need to alleviate pain and suffering and the huge socioeconomic burden of this debilitating disease. C-X-C Motif Chemokine Ligand 12 (CXCL12) also know an stromal cell-derived factor 1α (SDF-1α) is a chemokine that binds the CXC chemokine receptor type 4 (CXCR4) and activates downstream signaling resulting in recruitment of hematopoietic cells to locations of tissue injury and promotes tissue repair. In diabetes, low expression of CXCL12 correlates with impaired wound healing. Activation of CXCR4 receptor signaling with agonists or positive allosteric modulators (PAMs) provides a potential for small molecule therapeutic discovery and development. We recently reported high throughput screening and identification of the CXCR4 partial agonist UCUF-728, characterization of in vitro activity and reduced wound closure time in diabetic mice at 100 μM as a proof-of-concept study. We report here, the discovery of a second chemical scaffold demonstrating increased agonist potency and represented by thiadiazine derivative, UCUF-965. UCUF-965 is a potent partial agonist of β-arrestin recruitment in CXCR4 receptor overexpressing cell line. Furthermore, UCUF-965 potentiates the CXCL12 maximal response in cAMP signaling pathway, activates CXCL12 stimulated migration in lymphoblast cells and modulates the levels of specific microRNA involved in the complex wound repair process, specifically in mouse fibroblasts. Our results indicate that UCUF-965 acts as a PAM agonist of the CXCR4 receptor. Furthermore, UCUF-965 enhanced angiogenesis markers and reduced wound healing time by 36% at 10.0 μM in diabetic mice models compared to untreated control.
• Vaughn, A. E., Lehmann, T., Sul, C., Wallbank, A. M., Lyttle, B. D., Bardill, J., Burns, N., Apte, A., Nozik, E. S., Smith, B., Vohwinkel, C. U., Zgheib, C., & Liechty, K. W. (2023). CNP-miR146a Decreases Inflammation in Murine Acute Infectious Lung Injury. Pharmaceutics, 15(9).
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  Acute respiratory distress syndrome (ARDS) has approximately 40% in-hospital mortality, and treatment is limited to supportive care. Pneumonia is the underlying etiology in many cases with unrestrained inflammation central to the pathophysiology. We have previously shown that CNP-miR146a, a radical scavenging cerium oxide nanoparticle (CNP) conjugated to the anti-inflammatory microRNA(miR)-146a, reduces bleomycin- and endotoxin-induced acute lung injury (ALI) by decreasing inflammation. We therefore hypothesized that CNP-miR146a would decrease inflammation in murine infectious ALI. Mice were injured with intratracheal (IT) MRSA or saline followed by treatment with IT CNP-miR146a or saline control. Twenty-four hours post-infection, bronchoalveolar lavage fluid (BALF) and whole lungs were analyzed for various markers of inflammation. Compared to controls, MRSA infection significantly increased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1β; < 0.05), BALF proinflammatory cytokines (IL-6, IL-8, TNFα, IL-1β; < 0.01), and inflammatory cell infiltrate ( = 0.03). CNP-miR146a treatment significantly decreased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1β; < 0.05), bronchoalveolar proinflammatory protein leak (IL-6, IL-8, TNFα; < 0.05), and inflammatory infiltrate ( = 0.01). CNP-miR146a decreases inflammation and improves alveolar-capillary barrier integrity in the MRSA-infected lung and has significant promise as a potential therapeutic for ARDS.
• Wallbank, A. M., Vaughn, A. E., Niemiec, S., Bilodeaux, J., Lehmann, T., Knudsen, L., Kolanthai, E., Seal, S., Zgheib, C., Nozik, E., Liechty, K. W., & Smith, B. J. (2023). CNP-miR146a improves outcomes in a two-hit acute- and ventilator-induced lung injury model. Nanomedicine : nanotechnology, biology, and medicine, 50, 102679.
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  Acute respiratory distress syndrome (ARDS) has high mortality (~40 %) and requires the lifesaving intervention of mechanical ventilation. A variety of systemic inflammatory insults can progress to ARDS, and the inflamed and injured lung is susceptible to ventilator-induced lung injury (VILI). Strategies to mitigate the inflammatory response while restoring pulmonary function are limited, thus we sought to determine if treatment with CNP-miR146a, a conjugate of novel free radical scavenging cerium oxide nanoparticles (CNP) to the anti-inflammatory microRNA (miR)-146a, would protect murine lungs from acute lung injury (ALI) induced with intratracheal endotoxin and subsequent VILI. Lung injury severity and treatment efficacy were evaluated via lung mechanical function, relative gene expression of inflammatory biomarkers, and lung morphometry (stereology). CNP-miR146a reduced the severity of ALI and slowed the progression of VILI, evidenced by improvements in inflammatory biomarkers, atelectasis, gas volumes in the parenchymal airspaces, and the stiffness of the pulmonary system.
• Bardill, J. R., Laughter, M. R., Stager, M., Liechty, K. W., Krebs, M. D., & Zgheib, C. (2022). Topical gel-based biomaterials for the treatment of diabetic foot ulcers. Acta biomaterialia, 138, 73-91.
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  Diabetic foot ulcers (DFUs) are a devastating ailment for many diabetic patients with increasing prevalence and morbidity. The complex pathophysiology of DFU wound environments has made finding effective treatments difficult. Standard wound care treatments have limited efficacy in healing these types of chronic wounds. Topical biomaterial gels have been developed to implement novel treatment approaches to improve therapeutic effects and are advantageous due to their ease of application, tunability, and ability to improve therapeutic release characteristics. Here, we provide an updated, comprehensive review of novel topical biomaterial gels developed for treating chronic DFUs. This review will examine preclinical data for topical gel treatments in diabetic animal models and clinical applications, focusing on gels with protein/peptides, drug, cellular, herbal/antioxidant, and nano/microparticle approaches. STATEMENT OF SIGNIFICANCE: By 2050, 1 in 3 Americans will develop diabetes, and up to 34% of diabetic patients will develop a diabetic foot ulcer (DFU) in their lifetime. Current treatments for DFUs include debridement, infection control, maintaining a moist wound environment, and pressure offloading. Despite these interventions, a large number of DFUs fail to heal and are associated with a cost that exceeds $31 billion annually. Topical biomaterials have been developed to help target specific impairments associated with DFU with the goal to improve healing. A summary of these approaches is needed to help better understand the current state of the research. This review summarizes recent research and advances in topical biomaterials treatments for DFUs.
• Dewberry, L. C., Niemiec, S. M., Hilton, S. A., Louiselle, A. E., Singh, S., Sakthivel, T. S., Hu, J., Seal, S., Liechty, K. W., & Zgheib, C. (2022). Cerium oxide nanoparticle conjugation to microRNA-146a mechanism of correction for impaired diabetic wound healing. Nanomedicine : nanotechnology, biology, and medicine, 40, 102483.
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  Diabetic wounds represent a significant healthcare burden and are characterized by impaired wound healing due to increased oxidative stress and persistent inflammation. We have shown that CNP-miR146a synthesized by the conjugation of cerium oxide nanoparticles (CNP) to microRNA (miR)-146a improves diabetic wound healing. CNP are divalent metal oxides that act as free radical scavenger, while miR146a inhibits the pro-inflammatory NFκB pathway, so CNP-miR146a has a synergistic role in modulating both oxidative stress and inflammation. In this study, we define the mechanism(s) by which CNP-miR146a improves diabetic wound healing by examining immunohistochemical and gene expression analysis of markers of inflammation, oxidative stress, fibrosis, and angiogenesis. We have found that intradermal injection of CNP-miR146a increases wound collagen, enhances angiogenesis, and lowers inflammation and oxidative stress, ultimately promoting faster closure of diabetic wounds.
• El Ghzaoui, C., Neal, C. J., Kolanthai, E., Fu, Y., Kumar, U., Hu, J., Zgheib, C., Liechty, K. W., & Seal, S. (2022). Assessing the bio-stability of microRNA-146a conjugated nanoparticles electroanalysis. Nanoscale advances, 5(1), 191-207.
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  The number of diabetics is increasing worldwide and is associated with significant instances of clinical morbidity. Increased amounts of reactive oxygen species (ROS) and proinflammatory cytokines are associated with the pathogenesis of diabetic wounds and result in a significant delay in healing. Our previous studies have shown the ability of a cerium oxide nanoparticle (CNP) formulation conjugated with the anti-inflammatory microRNA miR146a (CNP-miR146a) to enhance the healing of diabetic wounds. The observed therapeutic activity exceeded the combined efficacies of the individual conjugate components (CNPs and miR146a alone), suggesting a synergistic effect. The current study evaluates whether the previously observed enhanced activity arises from increased agent delivery (simple nanocarrier activity) or is specific to the CNP-miR146a formulation (functional, bio-active nanomaterial). Comparison with miR146a conjugated gold (bioactive, metal) and silica (bioinert, oxide) nanoparticles (AuNPs and SiONPs) was performed in the presence of HO, as an analogue to the high levels of ROS present in the diabetic wound environment. Electrochemical studies, materials characterization, and chemical assays showed limited interaction of AuNP-miR146a with HO and instability of SiONP-miR146a over time. In contrast, and in support of our prior results, CNP-miR146a displayed chemical stability and persistent ROS scavenging ability. Furthermore, it was determined that CNPs protect miR146a from oxidative damage under prolonged exposure to HO, whereas AuNPs and SiONPs were shown to be ineffective. Overall, these results reinforce the ability of CNPs to stabilize and protect miRNA while exhibiting robust antioxidant properties, suggesting that therapeutic activity observed in related earlier studies is not limited to a facile nanocarrier function.
• Fu, Y., Kolanthai, E., Neal, C. J., Kumar, U., Zgheib, C., Liechty, K. W., & Seal, S. (2022). Engineered Faceted Cerium Oxide Nanoparticles for Therapeutic miRNA Delivery. Nanomaterials (Basel, Switzerland), 12(24).
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  In general, wound healing is a highly ordered process, with distinct phases of inflammation, proliferation, and remodeling. However, among diabetic patients, the progression through these phases is often impeded by increased level of oxidative stress and persistent inflammation. Our previous studies demonstrated that cerium oxide nanoparticles (CNPs) conjugated with therapeutic microRNA146a (miR146a) could effectively enhance wound healing by targeting the NFκB pathway, reducing oxidative stress and inflammation. In the present study, we consider the potential effects of nanomaterial surface-faceting and morphology on the efficacy of miRNA delivery. Compared with octahedral-CNPs and cubic-CNPs, rod-CNPs exhibited higher loading capacity. In addition, in comparing the influence of particle morphology on wound healing efficacy, several markers for bioactivity were evaluated and ascribed to the combined effects of the gene delivery and reactive oxygen species (ROS) scavenging properties. In the cellular treatment study, rod-CNP-miR146a displayed the greatest miR146a delivery into cells. However, the reduction of IL-6 was only observed in the octahedral-CNP-miR146a, suggesting that the efficacy of the miRNA delivery is a result of the combination of various factors. Overall, our results give enlightenments into the relative delivery efficiency of the CNPs with different morphology enhancing miRNA delivery efficacy.
• Lehmann, T., Vaughn, A. E., Seal, S., Liechty, K. W., & Zgheib, C. (2022). Silk Fibroin-Based Therapeutics for Impaired Wound Healing. Pharmaceutics, 14(3).
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  Impaired wound healing can lead to local hypoxia or tissue necrosis and ultimately result in amputation or even death. Various factors can influence the wound healing environment, including bacterial or fungal infections, different disease states, desiccation, edema, and even systemic viral infections such as COVID-19. Silk fibroin, the fibrous structural-protein component in silk, has emerged as a promising treatment for these impaired processes by promoting functional tissue regeneration. Silk fibroin's dynamic properties allow for customizable nanoarchitectures, which can be tailored for effectively treating several wound healing impairments. Different forms of silk fibroin include nanoparticles, biosensors, tissue scaffolds, wound dressings, and novel drug-delivery systems. Silk fibroin can be combined with other biomaterials, such as chitosan or microRNA-bound cerium oxide nanoparticles (CNP), to have a synergistic effect on improving impaired wound healing. This review focuses on the different applications of silk-fibroin-based nanotechnology in improving the wound healing process; here we discuss silk fibroin as a tissue scaffold, topical solution, biosensor, and nanoparticle.
• Louiselle, A. E., Niemiec, S., Azeltine, M., Mundra, L., French, B., Zgheib, C., & Liechty, K. W. (2022). Evaluation of skin care concerns and patient's perception of the effect of NanoSilk Cream on facial skin. Journal of cosmetic dermatology, 21(3), 1075-1085.
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  Skin aging is an inevitable process with one of the key features of aging being dryness or flakiness of the skin. Previous in vivo and in vitro testing has highlighted that a silk-based product may be effective in improving moisture retention in skin.
• Niemiec, S. M., Hilton, S. A., Wallbank, A., Louiselle, A. E., Elajaili, H., Hu, J., Singh, S., Seal, S., Nozik, E., Smith, B., Zgheib, C., & Liechty, K. W. (2022). Lung function improves after delayed treatment with CNP-miR146a following acute lung injury. Nanomedicine : nanotechnology, biology, and medicine, 40, 102498.
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  Acute respiratory distress syndrome (ARDS) is a highly morbid pulmonary disease characterized by hypoxic respiratory failure. Its pathogenesis is characterized by unrestrained oxidative stress and inflammation, with long-term sequelae of pulmonary fibrosis and diminished lung function. Unfortunately, prior therapeutic ARDS trials have failed and therapy is limited to supportive measures. Free radical scavenging cerium oxide nanoparticles (CNP) conjugated to the anti-inflammatory microRNA-146a (miR146a), termed CNP-miR146a, have been shown to prevent acute lung injury in a pre-clinical model. In this study, we evaluated the potential of delayed treatment with CNP-miR146a at three or seven days after injury to rescue the lung from acute injury. We found that intratracheal CNP-miR146a administered three days after injury lowers pulmonary leukocyte infiltration, reduce inflammation and oxidative stress, lower pro-fibrotic gene expression and collagen deposition in the lung, and ultimately improve pulmonary function.
• Stager, M. A., Bardill, J., Raichart, A., Osmond, M., Niemiec, S., Zgheib, C., Seal, S., Liechty, K. W., & Krebs, M. D. (2022). Photopolymerized Zwitterionic Hydrogels with a Sustained Delivery of Cerium Oxide Nanoparticle-miR146a Conjugate Accelerate Diabetic Wound Healing. ACS applied bio materials, 5(3), 1092-1103.
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  In the United States, $87 billion per year is spent on the care of diabetic ulcers alone. Although the pathophysiology of diabetic wound healing is multifaceted, high systemic levels of inflammation and increased reactive oxygen species are often implicated in the wound healing impairment. Zwitterionic materials have been demonstrated to reduce inflammation and increase extracellular matrix deposition in wound beds, and here, we demonstrate a fabrication method for photopolymerized zwitterionic hydrogels that also enables sustained drug delivery over time. A therapeutic molecule of interest that is examined in this work is cerium oxide nanoparticle tagged with microRNA-146a (CNP-miR146a) to combat both oxidative stress and inflammation. The hydrogels are composed of zwitterionic and nonzwitterionic monomers, and the hydrogel formation occurs in the absence of a crosslinker. The hydrogels exhibit a wide range of stiffness and mechanical properties depending on their monomer content. Additionally, these hydrogels exhibit sustained release of nanoparticles and proteins. Finally, when employed in an diabetic mouse wound healing model, the zwitterionic hydrogels alone and laden with the CNP-miR146a conjugate significantly improved the rate of diabetic wound healing. Overall, these materials have excellent potential to be used as a topical treatment for chronic diabetic wounds.
• Xu, J., Hu, J., Idlett-Ali, S., Zhang, L., Caples, K., Peddibhotla, S., Reeves, M., Zgheib, C., Malany, S., & Liechty, K. W. (2022). Discovery of Small Molecule Activators of Chemokine Receptor CXCR4 That Improve Diabetic Wound Healing. International journal of molecular sciences, 23(4).
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  Diabetes produces a chronic inflammatory state that contributes to the development of vascular disease and impaired wound healing. Despite the known individual and societal impacts of diabetic ulcers, there are limited therapies effective at improving healing. Stromal cell-derived factor 1α (SDF-1α) is a CXC chemokine that functions via activation of the CXC chemokine receptor type 4 (CXCR4) receptor to recruit hematopoietic cells to locations of tissue injury and promote tissue repair. The expression of SDF-1α is reduced in diabetic wounds, suggesting a potential contribution to wound healing impairment and presenting the CXCR4 receptor as a target for therapeutic investigations. We developed a high-throughput β-arrestin recruitment assay and conducted structure-activity relationship (SAR) studies to screen compounds for utility as CXCR4 agonists. We identified CXCR4 agonist UCUF-728 from our studies and further validated its activity in vitro in diabetic fibroblasts. UCUF-728 reduced overexpression of miRNA-15b and miRNA-29a, negative regulators of angiogenesis and type I collagen production, respectively, in diabetic fibroblasts. In vivo, UCUF-728 reduced the wound closure time by 36% and increased the evidence of angiogenesis in diabetic mice. Together, this work demonstrates the clinical potential of small molecule CXCR4 agonists as novel therapies for pathologic wound healing in diabetes.
• Hodges, M. M., Zgheib, C., & Liechty, K. W. (2021). A Large Mammalian Model of Myocardial Regeneration After Myocardial Infarction in Fetal Sheep. Advances in wound care, 10(4), 174-190.
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  Ischemic heart disease accounts for over 20% of all deaths worldwide. As the global population faces a rising burden of chronic diseases, such as hypertension, hyperlipidemia, and diabetes, the prevalence of heart failure due to ischemic heart disease is estimated to increase. We sought to develop a model that may more accurately identify therapeutic targets to mitigate the development of heart failure following myocardial infarction (MI). Having utilized fetal large mammalian models of scarless wound healing, we proposed a fetal ovine model of myocardial regeneration after MI. Use of this model has identified critical pathways in the mammalian response to MI, which are differentially activated in the regenerative, fetal mammalian response to MI when compared to the reparative, scar-forming, adult mammalian response to MI. While the foundation of myocardial regeneration research has been built on zebrafish and rodent models, effective therapies derived from these disease models have been lacking; therefore, we sought to develop a more representative ovine model of myocardial regeneration after MI to improve the identification of therapeutic targets designed to mitigate the development of heart failure following MI. To develop therapies aimed at mitigating this rising burden of disease, it is critical that the animal models we utilize closely reflect the physiology and pathology we observe in human disease. We encourage use of this ovine large mammalian model to facilitate identification of therapies designed to mitigate the growing burden of heart failure.
• Louiselle, A. E., Niemiec, S. M., Zgheib, C., & Liechty, K. W. (2021). Macrophage polarization and diabetic wound healing. Translational research : the journal of laboratory and clinical medicine, 236, 109-116.
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  Diabetes mellitus is a costly disease and nearly one-third of these costs are attributed to management of diabetic foot disease including chronic, non-healing, diabetic foot ulcers. Therefore, much effort has been placed into understanding the pathogenesis of diabetic wounds and novel therapeutics. A relatively new area of interest has been macrophage polarization and its role in diabetic wound healing. Diabetic wounds show dysregulated and persistent M1 (pro-inflammatory) macrophage polarization whereas normal wounds will display a transition to M2 (pro-healing) macrophages around day three after wounding. We reviewed factors known to affect macrophage polarization, mostly focused on those that contribute to M2 macrophage polarization, and potential treatments that at least in part target macrophage polarization in the diabetic wound bed. Much of the work has been aimed at reducing hyperglycemia and encouraging pro-inflammatory cytokine neutralization or decreased expression given this has a significant role in producing M1 macrophages. Treatment of diabetic wounds will likely require a multi-modal approach including management of underlying diabetes and control of hyperglycemia, topical therapeutics, and prevention of secondary infection and inflammation.
• Niemiec, S. M., Hilton, S. A., Wallbank, A., Azeltine, M., Louiselle, A. E., Elajaili, H., Allawzi, A., Xu, J., Mattson, C., Dewberry, L. C., Hu, J., Singh, S., Sakthivel, T. S., Sea, S., Nozik-Grayck, E., Smith, B., Zgheib, C., & Liechty, K. W. (2021). Cerium oxide nanoparticle delivery of microRNA-146a for local treatment of acute lung injury. Nanomedicine : nanotechnology, biology, and medicine, 34, 102388.
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  Acute respiratory distress syndrome (ARDS) is a devastating pulmonary disease with significant in-hospital mortality and is the leading cause of death in COVID-19 patients. Excessive leukocyte recruitment, unregulated inflammation, and resultant fibrosis contribute to poor ARDS outcomes. Nanoparticle technology with cerium oxide nanoparticles (CNP) offers a mechanism by which unstable therapeutics such as the anti-inflammatory microRNA-146a can be locally delivered to the injured lung without systemic uptake. In this study, we evaluated the potential of the radical scavenging CNP conjugated to microRNA-146a (termed CNP-miR146a) in preventing acute lung injury (ALI) following exposure to bleomycin. We have found that intratracheal delivery of CNP-miR146a increases pulmonary levels of miR146a without systemic increases, and prevents ALI by altering leukocyte recruitment, reducing inflammation and oxidative stress, and decreasing collagen deposition, ultimately improving pulmonary biomechanics.
• Azeltine, M. W., Chavez, E. J., Nemec, K. M., Bednarek, J. M., Asokan, R., Balasubramaniyan, N., Zgheib, C., Mack, C. L., & Roach, J. P. (2020). Inflammation Drives MicroRNAs to Limit Hepatocyte Bile Acid Transport in Murine Biliary Atresia. The Journal of surgical research, 256, 663-672.
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  Biliary atresia (BA) is an inflammatory pediatric cholangiopathy with only surgical means for treatment. Many contributors to bile acid synthesis and transport have previously been reported to be downregulated in patients with BA; yet, the driving factors of the abnormal bile acid synthesis and transport in regard to BA have not been previously studied.
• Hu, J., Zhang, L., Liechty, C., Zgheib, C., Hodges, M. M., Liechty, K. W., & Xu, J. (2020). Long Noncoding RNA GAS5 Regulates Macrophage Polarization and Diabetic Wound Healing. The Journal of investigative dermatology, 140(8), 1629-1638.
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  A central feature of diabetic (Db) wounds is the persistence of chronic inflammation, which is partly due to the prolonged presence of proinflammatory (M1) macrophages. Using in vivo and in vitro analyses, we have tested the hypothesis that long noncoding RNA GAS5 is dysregulated in Db wounds. We have assessed the contribution of GAS5 to the M1 macrophage phenotype, as well as the functional consequences of knocking down its expression. We found that expression of GAS5 is increased significantly in Db wounds and in cells isolated from Db wounds. Hyperglycemia induced GAS5 expression in macrophages in vitro. Overexpression of GAS5 in vitro promoted macrophage polarization toward an M1 phenotype by upregulating signal transducer and activator of transcription 1. Of most significance in our judgment, GAS5 loss-of-function enhanced Db wound healing. These data indicate that the relative level of long noncoding RNA GAS5 in wounds plays a key role in the wound healing response. Reductions in the levels of GAS5 in wounds appeared to enhance healing by promoting transition of M1 macrophages to M2 macrophages. Thus, our results suggest that targeting long noncoding RNA GAS5 may provide a therapeutic intervention for correcting impaired Db wound healing.
• Niemiec, S. M., Louiselle, A. E., Hilton, S. A., Dewberry, L. C., Zhang, L., Azeltine, M., Xu, J., Singh, S., Sakthivel, T. S., Seal, S., Liechty, K. W., & Zgheib, C. (2020). Nanosilk Increases the Strength of Diabetic Skin and Delivers CNP-miR146a to Improve Wound Healing. Frontiers in immunology, 11, 590285.
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  Diabetes mellitus is a metabolic disorder associated with properties and an increased risk of chronic wounds due to sustained pro-inflammatory response. We have previously of radical scavenging cerium oxide nanoparticles (CNP) conjugated to the anti-inflammatory microRNA (miR)-146a, termed CNP-miR146a, improves diabetic wound healing by synergistically lowering oxidative stress and inflammation, and we sought to evaluate this treatment in a topical application. Silk fibroin is a biocompatible polymer that can be fabricated into nanostructures, termed nanosilk. Nanosilk is characterized by a high strength-to-density ratio and an ability to exhibit strain hardening. We therefore hypothesized that nanosilk would strengthen the biomechanical properties of diabetic skin and that nanosilk solution could effectively deliver CNP-miR146a to improve diabetic wound healing. The ability of nanosilk to deliver CNP-miR146a to murine diabetic wounds and improve healing was assessed by the rate of wound closure and inflammatory gene expression, as well as histologic analysis. The effect of nanosilk on the properties of human diabetic skin was evaluated by testing the biomechanical properties following topical application of a 7% nanosilk solution. Diabetic murine wounds treated with topical nanosilk and CNP-miR146a healed by day 14.5 compared to day 16.8 in controls (p = 0.0321). Wounds treated with CNP-miR146a had higher collagen levels than controls (p = 0.0126) with higher pro-fibrotic gene expression of TGFβ-1 (p = 0.0092), Col3α1 (p = 0.0369), and Col1α2 (p = 0.0454). Treatment with CNP-miR146a lowered pro-inflammatory gene expression of IL-6 (p = 0.0488) and IL-8 (p = 0.0009). Treatment of human diabetic skin with 7% nanosilk solution resulted in significant improvement in maximum load and modulus (p < 0.05). Nanosilk solution is able to strengthen the biomechanical properties of diabetic skin and can successfully deliver CNP-miR146a to improve diabetic wound healing through inhibition of pro-inflammatory gene signaling and promotion of pro-fibrotic processes.
• Niemiec, S. M., Louiselle, A. E., Liechty, K. W., & Zgheib, C. (2020). Role of microRNAs in Pressure Ulcer Immune Response, Pathogenesis, and Treatment. International journal of molecular sciences, 22(1).
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  Pressure ulcers are preventable, yet highly prevalent, chronic wounds that have significant patient morbidity and high healthcare costs. Like other chronic wounds, they are characterized by impaired wound healing due to dysregulated immune processes. This review will highlight key biochemical pathways in the pathogenesis of pressure injury and how this signaling leads to impaired wound healing. This review is the first to comprehensively describe the current literature on microRNA (miRNA, miR) regulation of pressure ulcer pathophysiology.
• Sener, G., Hilton, S. A., Osmond, M. J., Zgheib, C., Newsom, J. P., Dewberry, L., Singh, S., Sakthivel, T. S., Seal, S., Liechty, K. W., & Krebs, M. D. (2020). Injectable, self-healable zwitterionic cryogels with sustained microRNA - cerium oxide nanoparticle release promote accelerated wound healing. Acta biomaterialia, 101, 262-272.
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  Diabetics are prone to chronic wounds that have slower healing, and methods of accelerating the wound closure and to ensure protection from infections are critically needed. MicroRNA-146a gets dysregulated in diabetic wounds and injection of this microRNA combined with reactive oxygen species-scavenging cerium oxide nanoparticles (CNPs) can reduce inflammation and improve wound healing; however, a better delivery method than intradermal injections is needed. Here we demonstrate a biomaterial system of zwitterionic cryogels (gels formed below freezing temperatures) laden with CNP-miR146a that are topically applicable, injectable, self-healable, and provide sustained release of the therapeutic molecules. These cryogels are comprised of CBMA or SBMA and HEMA, and do not contain chemical crosslinkers. Properties of the gels can be manipulated by changing monomer type and ratio. These materials have demonstrated efficacy and viability in vivo with a diabetic mouse wound healing model. Overall, these materials have a high potential for application in wound treatments due to their ease of production, antifouling characteristics, durability, topical application, and sustained release mechanics. STATEMENT OF SIGNIFICANCE: This work presents the development of zwitterionic cryogels with unique physical properties including injectability and self-healing, that also offer highly sustained release of nanoparticles over time to improve wound healing in a diabetic mouse model. The nanoparticles are made of cerium oxide, which is known to scavenge reactive oxygen species and reduce oxidative stress, and these particles have been further tagged with a microRNA146a that has been shown to reduce inflammation. Zwitterionic materials are known for their superior antifouling properties and good biocompatibility and ability to incorporate bioactive factors. Given these properties, the use of these materials as wound healing dressings would be exciting, yet to date it has been difficult to prolong the release of bioactive factors from them due to their hydrophilicity. Previously we developed zwitterionic cyrogels with very sustained protein release over time, but those materials were quite brittle and difficult to handle. Here, we demonstrate for the first time that by removing the crosslinker molecule from our reaction and polymerizing gels under cryo-conditions, we are able to form zwitterionic cryogels that are injectable, self-healing, and with sustained release profiles. The sustained release of miRNA146a-tagged cerium oxide nanoparticles from these gels is demonstrated to speed up diabetic wound healing time and significantly reduce inflammation.
• Singh, S., Cortes, G., Kumar, U., Sakthivel, T. S., Niemiec, S. M., Louiselle, A. E., Azeltine-Bannerman, M., Zgheib, C., Liechty, K. W., & Seal, S. (2020). Silk fibroin nanofibrous mats for visible sensing of oxidative stress in cutaneous wounds. Biomaterials science, 8(21), 5900-5910.
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  Wound healing is of major clinical concern and is constantly being explored for early restoration and enhanced recovery. While the etiology of the wound healing is multifactorial, high inflammation and increased oxidative stress which results in chronic inflammation, endothelial dysfunction and collagen degradation, delay the overall healing process. Thus, visual sensing of the oxidative stress would be highly informative in the successful implementation of wound healing therapies based on specific requirements. In this study, electrospinning was used to fabricate silk fibroin nanofibrous mats infused with Amplex red capable of detecting hydrogen peroxide, a reactive oxygen molecule. These mats produced a visible change in color with the limit of detection at 1 μM HO concentration. In vivo studies carried out in diabetic mice with impaired wounds also displayed a visible change in color of the mats infused with Amplex red within 24 hours. These electrospun silk fibroin nanofibrous Amplex infused mats has the potential to enable a futuristic platform where decisions can be made for enhanced wound healing therapy.
• Hilton, S. A., Dewberry, L. C., Hodges, M. M., Hu, J., Xu, J., Liechty, K. W., & Zgheib, C. (2019). Mesenchymal stromal cells contract collagen more efficiently than dermal fibroblasts: Implications for cytotherapy. PloS one, 14(7), e0218536.
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  Stem cell therapy is the next generation a well-established technique. Cell therapy with mesenchymal stem cells (MSC) has been demonstrated to enhance wound healing in diabetic mice, at least partly due to improved growth factor production. However, it is unclear whether MSC can biomechanically affect wound closure. Utilizing the well-established cell-populated collagen gel contraction model we investigated the interactions between MSC and the extracellular matrix.
• Hodges, M. M., Zgheib, C., Xu, J., Hu, J., Dewberry, L. C., Hilton, S. A., Allukian, M. W., Gorman, J. H., Gorman, R. C., & Liechty, K. W. (2019). Differential Expression of Transforming Growth Factor-β1 Is Associated With Fetal Regeneration After Myocardial Infarction. The Annals of thoracic surgery, 108(1), 59-66.
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  Global extracellular matrix (ECM)-related gene expression is decreased after myocardial infarction (MI) in fetal sheep when compared with adult sheep. Transforming growth factor (TGF)-β1 is a key regulator of ECM; therefore we hypothesize that TGF-β1 is differentially expressed in adult and fetal infarcts after MI.
• Zgheib, C., Hilton, S. A., Dewberry, L. C., Hodges, M. M., Ghatak, S., Xu, J., Singh, S., Roy, S., Sen, C. K., Seal, S., & Liechty, K. W. (2019). Use of Cerium Oxide Nanoparticles Conjugated with MicroRNA-146a to Correct the Diabetic Wound Healing Impairment. Journal of the American College of Surgeons, 228(1), 107-115.
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  Diabetic wounds have become one of the most challenging public health issues of the 21st century, yet there is no effective treatment available. We have previously shown that the diabetic wound healing impairment is associated with increased inflammation and decreased expression of the regulatory microRNA miR-146a. We have conjugated miR-146a to cerium oxide nanoparticles (CNP-miR146a) to target reactive oxygen species (ROS) and inflammation. This study aimed to evaluate the consequences of CNP-miR146a treatment of diabetic wounds.
• Kurdi, M., Zgheib, C., & Booz, G. W. (2018). Recent Developments on the Crosstalk Between STAT3 and Inflammation in Heart Function and Disease. Frontiers in immunology, 9, 3029.
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  The transcription factor STAT3 has a protective function in the heart. Until recently, the role of STAT3 in hypertension-induced cardiac hypertrophy was unsettled. Earlier studies revealed that global reduction of STAT3 activity reduced cardiac hypertrophy with hypertension, but caused a disruption of myofilaments and increased contractile dysfunction. However, newer studies with cardiomyocyte-specific deletion of STAT3 indicate that STAT3 does not cause cardiac hypertrophy with increased blood pressure. Rather, cardiac STAT3 is important for maintaining metabolic homeostasis, and loss of STAT3 in cardiomyocytes makes the heart more susceptible to chronic pathological insult, for example by disrupting glucose metabolism and protective signaling networks via the upregulation of certain microRNAs. This scenario has implications for understanding peripartum cardiomyopathy as well. In viral myocarditis, STAT3 opposes the initiation of the dilated phenotype by maintaining membrane integrity via the expression of dystrophin. STAT3 signaling was also found to attenuate myocarditis by polarizing macrophages to a less inflammatory phenotype. On the other hand, STAT3 contributes to immune-mediated myocarditis due to IL-6-induced complement component C3 production in the liver, as well as the differentiation of Th17 cells, which play a role in initiation and development of myocarditis. Besides canonical signaling pathways, unphosphorylated STAT3 (U-STAT3) and redox-activated STAT3 have been shown to couple to transcription in the heart. In addition, tissue signaling cytokines such as IL-22 and IL-17 have been proposed to have actions on the heart that involve STAT3, but are not fully defined. Understanding the novel and often protective aspects of STAT3 in the myocardium could lead to new therapeutic approaches to treat heart disease.
• Xu, J., Zgheib, C., Hodges, M. M., Caskey, R. C., Hu, J., & Liechty, K. W. (2017). Mesenchymal stem cells correct impaired diabetic wound healing by decreasing ECM proteolysis. Physiological genomics, 49(10), 541-548.
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  Impaired diabetic wound healing is associated with a dermal extracellular matrix protein profile favoring proteolysis; within the healing diabetic wound, this is represented by an increase in activated matrix metalloproteinase (MMPs). Treatment of diabetic wounds with mesenchymal stem cells (MSCs) has been shown to improve wound healing; however, there has not yet been an assessment of their ability to correct dysregulation of MMPs in diabetic wounds. Furthermore, there has been no prior assessment of the role of microRNA29b (miR-29b), an inhibitory regulatory molecule that targets MMP-9 mRNA. Using in vitro models of fibroblast coculture with MSCs and in vivo murine wound healing models, we tested the hypothesis that MSCs correct dysregulation of MMPs in a microRNA-29b-dependent mechanism. In this study, we first demonstrated that collagen I and III protein content is significantly reduced in diabetic wounds, and treatment with MSCs significantly improves collagen I content in both nondiabetic and diabetic wounds. We then found that MMP-9 gene expression and protein content were significantly upregulated in diabetic wounds, indicating elevated proteolysis. Treatment with MSCs resulted in a decrease in MMP-9 gene expression and protein content level in diabetic wounds 3 and 7 days after wounding. Zymographic analysis indicated that MSC treatment also decreased the amount of activated MMP-9 present in diabetic wounds. Furthermore, miR-29b expression was inversely associated with MMP-9 gene expression; miR-29b expression was decreased in diabetic wounds and diabetic fibroblast. Following treatment of diabetic wounds with MSCs, as well as in diabetic fibroblasts cocultured with MSCs, miR-29b was significantly increased. These findings suggest a potential mechanism through which MSCs enhance diabetic wound healing by improving collagen I content in diabetic wounds through decreasing MMP-9 expression and increasing miR-29b expression.
• Zgheib, C., Hodges, M. M., Allukian, M. W., Xu, J., Spiller, K. L., Gorman, J. H., Gorman, R. C., & Liechty, K. W. (2017). Cardiac Progenitor Cell Recruitment Drives Fetal Cardiac Regeneration by Enhanced Angiogenesis. The Annals of thoracic surgery, 104(6), 1968-1975.
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  In contrast to adults, the fetal response to myocardial infarction (MI) is regenerative, requiring the recruitment of cardiac progenitor cells to replace infarcted myocardium. Macrophage contribution to tissue repair depends on their phenotype: M1 are proinflammatory and initiate angiogenesis; M2a are profibrotic and contribute to blood vessels maturation; and M2c are proremodeling and proangiogenesis. The goal of the present study was to expand on this work by examining cardiac progenitor cells recruitment, and the role of macrophages in promoting angiogenesis and cardiac regeneration in the fetal heart after MI.
• Zgheib, C., Hodges, M. M., Hu, J., Liechty, K. W., & Xu, J. (2017). Long non-coding RNA Lethe regulates hyperglycemia-induced reactive oxygen species production in macrophages. PloS one, 12(5), e0177453.
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  Type 2 diabetes mellitus is a complex, systemic metabolic disease characterized by insulin resistance and resulting hyperglycemia, which is associated with impaired wound healing. The clinical complications associated with hyperglycemia are attributed, in part, to the increased production of reactive oxygen species (ROS). Recent studies revealed that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological processes. Specifically, lncRNA Lethe has been described as exhibiting an anti-inflammatory effect by binding to the p65 subunit of NFκB and blocking its binding to DNA and the subsequent activation of downstream genes. We therefore hypothesize that dysregulation of Lethe's expression plays a role in hyperglycemia-induced ROS production. To test our hypothesis, we treated RAW264.7 macrophages with low glucose (5 mM) or high glucose (25 mM) for 24h. High glucose conditions significantly induced ROS production and NOX2 gene expression in RAW cells, while significantly decreasing Lethe gene expression. Overexpression of Lethe in RAW cells eliminated the increased ROS production induced by high glucose conditions, while also attenuating the upregulation of NOX2 expression. Similar results was found also in non-diabetic and diabetic primary macrophage, bone marrow derived macrophage (BMM). Furthermore, overexpression of Lethe in RAW cells treated with high glucose significantly reduced the translocation of p65-NFkB to the nucleus, which resulted in decreased NOX2 expression and ROS production. Interestingly, these findings are consistent with the decreased Lethe gene expression and increased NOX2 gene expression observed in a mouse model of diabetic wound healing. These findings provide the first evidence that lncRNA Lethe is involved in the regulation of ROS production in macrophages through modulation of NOX2 gene expression via NFκB signaling. Moreover, this is the first report to describe a role of lncRNAs, in particular Lethe, in impaired diabetic wound healing. Further studies are warranted to determine if correction of Lethe expression in diabetic wounds could improve healing.
• Zgheib, C., & Liechty, K. W. (2016). Shedding light on miR-26a: Another key regulator of angiogenesis in diabetic wound healing. Journal of molecular and cellular cardiology, 92, 203-5.
• Zgheib, C., Hodges, M., Hu, J., Beason, D. P., Soslowsky, L. J., Liechty, K. W., & Xu, J. (2016). Mechanisms of mesenchymal stem cell correction of the impaired biomechanical properties of diabetic skin: The role of miR-29a. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 24(2), 237-46.
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  Diabetic skin has impaired wound healing properties following injury. We have further shown that diabetic skin has weakened biomechanical properties at baseline. We hypothesize that the biomechanical properties of diabetic skin decline during the progression of the diabetic phenotype, and that this decline is due to the dysregulation of miR-29a, resulting in decreased collagen content. We further hypothesize that treatment with mesenchymal stem cells (MSCs) may improve diabetic wound healing by correction of the dysregulated miR-29a expression. We analyzed the biomechanical properties, collagen gene expression, collagen protein production, and miR-29a levels in skin harvested from 6 to 18 week old mice during the development of the diabetic phenotype. We also examined the correction of these impairments by both MSC treatment and the inhibition of miR-29a. Diabetic skin demonstrated a progressive impairment of biomechanical properties, decreased collagen content, and increased miR-29a levels during the development of the diabetic phenotype. MSC treatment decreased miR-29a levels, increased collagen content, and corrected the impaired biomechanical properties of diabetic skin. Additionally, direct inhibition of miR-29a also increased collagen content in diabetic skin. This decline in the biomechanical properties of diabetic skin during the progression of diabetes may increase the susceptibility of diabetic skin to injury and miR-29a appears to play a key role in this process.
• Gergi, R., Osta, N., Bourbouze, G., Zgheib, C., Arbab-Chirani, R., & Naaman, A. (2015). Effects of three nickel titanium instrument systems on root canal geometry assessed by micro-computed tomography. International endodontic journal, 48(2), 162-70.
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  To compare and evaluate the shaping ability of several nickel titanium instrument systems with different motions: two reciprocating single-file systems (Reciproc and WaveOne) and one continuous rotation/reciprocation full-sequence system [Twisted File (TF) Adaptive] using micro-computed tomography.
• Zgheib, C., Xu, J., Mallette, A. C., Caskey, R. C., Zhang, L., Hu, J., & Liechty, K. W. (2015). SCF increases in utero-labeled stem cells migration and improves wound healing. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 23(4), 583-90.
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  Diabetic skin wounds lack the ability to heal properly and constitute a major and significant complication of diabetes. Nontraumatic lower extremity amputations are the number one complication of diabetic skin wounds. The complexity of their pathophysiology requires an intervention at many levels to enhance healing and wound closure. Stem cells are a promising treatment for diabetic skin wounds as they have the ability to correct abnormal healing. Stem cell factor (SCF), a chemokine expressed in the skin, can induce stem cells migration, however the role of SCF in diabetic skin wound healing is still unknown. We hypothesize that SCF would correct the impairment and promote the healing of diabetic skin wounds. Our results show that SCF improved wound closure in diabetic mice and increased HIF-1α and vascular endothelial growth factor (VEGF) expression levels in these wounds. SCF treatment also enhanced the migration of red fluorescent protein (RFP)-labeled skin stem cells via in utero intra-amniotic injection of lenti-RFP at E8. Interestingly these RFP+ cells are present in the epidermis, stain negative for K15, and appear to be distinct from the already known hair follicle stem cells. These results demonstrate that SCF improves diabetic wound healing in part by increasing the recruitment of a unique stem cell population present in the skin.
• Caskey, R. C., Zgheib, C., Morris, M., Allukian, M., Dorsett-Martin, W., Xu, J., Wu, W., & Liechty, K. W. (2014). Dysregulation of collagen production in diabetes following recurrent skin injury: contribution to the development of a chronic wound. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 22(4), 515-20.
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  Recurrent injury has been implicated in the development of chronic diabetic wounds. We have developed a chronic diabetic wound model based upon recurrent injury in diabetic mice. We hypothesized that dysregulation of collagen production at both the mRNA and microRNA levels contributes to the development of chronic diabetic wounds. To test this, both diabetic and nondiabetic mice were made to undergo recurrent injury. Real-time PCR for TGF-β1, SMAD-3, Col1α1, Col3α1, microRNA-25, and microRNA-29a and Western blot for collagen I and III were performed 7 days following each injury. Diabetic wounds displayed decreased collagen at all time points. This was associated with dysregulated collagen production at both the gene and microRNA levels at all time points. Following the final injury, however, diabetic collagen production significantly improved. This appeared to be due to a substantial decrease in both microRNAs as well as an increase in the expression of collagen pathway genes. That dysregulated collagen production progressed throughout the course of wounding suggests that this is one factor contributing to the development of chronic diabetic wounds. Future studies using this model will allow for the determination of other factors that may also contribute to the development and/or persistence of chronic diabetic wounds.
• Morris, M. W., Allukian, M., Herdrich, B. J., Caskey, R. C., Zgheib, C., Xu, J., Dorsett-Martin, W., Mitchell, M. E., & Liechty, K. W. (2014). Modulation of the inflammatory response by increasing fetal wound size or interleukin-10 overexpression determines wound phenotype and scar formation. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 22(3), 406-14.
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  Wound size impacts the threshold between scarless regeneration and reparative healing in the fetus with increased inflammation showed in fetal scar formation. We hypothesized that increased fetal wound size increases pro-inflammatory and fibrotic genes with resultant inflammation and fibroplasia and that transition to scar formation could be reversed by overexpression of interleukin-10 (IL-10). To test this hypothesis, 2-mm and 8-mm dermal wounds were created in mid-gestation fetal sheep. A subset of 8-mm wounds were injected with a lentiviral vector containing the IL-10 transgene (n = 4) or vehicle (n = 4). Wounds were harvested at 3 or 30 days for histology, immunohistochemistry, analysis of gene expression by microarray, and validation with real-time polymerase chain reaction. In contrast to the scarless 2-mm wounds, 8-mm wounds showed scar formation with a differential gene expression profile, increased inflammatory cytokines, decreased CD45+ cells, and subsequent inflammation. Lentiviral-mediated overexpression of the IL-10 gene resulted in conversion to a regenerative phenotype with decreased inflammatory cytokines and regeneration of dermal architecture. In conclusion, increased fetal wounds size leads to a unique gene expression profile that promotes inflammation and leads to scar formation and furthermore, these results show the significance of attenuated inflammation and IL-10 in the transition from fibroplasia to fetal regenerative healing.
• Xu, J., Zgheib, C., Hu, J., Wu, W., Zhang, L., & Liechty, K. W. (2014). The role of microRNA-15b in the impaired angiogenesis in diabetic wounds. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 22(5), 671-7.
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  The impairment in diabetic wound healing represents a significant clinical problem. Decreased angiogenesis is thought to play a central role in the pathogenesis of this impairment. We have previously shown that treatment of diabetic murine wounds with mesenchymal stem cells can improve healing, but the mechanisms are not completely defined. MicroRNA-15b (miR-15b) has been implicated in the regulation of the angiogenic response. We hypothesized that abnormal miR-15b expression may contribute to the impaired angiogenesis observed in impaired diabetic wound healing. To test this hypothesis, we examined the expression of miR-15b and its target genes in diabetic and nondiabetic mice before and after injury. MiR-15b expression was significantly up-regulated in diabetic mouse wounds during the wound healing response. Increased miR-15b levels also closely correlated with decreased gene expression of its proangiogenic target genes. Furthermore, the correction of the diabetic wound healing impairment with mesenchymal stem cell treatment was associated with a significant decrease in miR-15b expression level and increased gene expression of its proangiogenic target genes. These results provide the first evidence that increased expression of miR-15b in diabetic wounds in response to injury may, in part, be responsible for the abnormal angiogenic response seen in diabetic wounds and may contribute to the observed wound healing impairment.
• Zgheib, C., Allukian, M. W., Xu, J., Morris, M. W., Caskey, R. C., Herdrich, B. J., Hu, J., Gorman, J. H., Gorman, R. C., & Liechty, K. W. (2014). Mammalian fetal cardiac regeneration after myocardial infarction is associated with differential gene expression compared with the adult. The Annals of thoracic surgery, 97(5), 1643-50.
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  In adults, myocardial infarction (MI) results in a brisk inflammatory response, myocardium loss, and scar formation. We have recently reported the first mammalian large-animal model of cardiac regeneration after MI in fetal sheep. We hypothesize that the ability of the fetus to regenerate functional myocardium after MI is owing to differential gene expression regulating the response to MI in the fetus compared with the adult.
• Werner, T., Dombrowski, S. M., Zgheib, C., Zouein, F. A., Keen, H. L., Kurdi, M., & Booz, G. W. (2013). Elucidating functional context within microarray data by integrated transcription factor-focused gene-interaction and regulatory network analysis. European cytokine network, 24(2), 75-90.
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  Microarrays do not yield direct evidence for functional connections between genes. However, transcription factors (TFs) and their binding sites (TFBSs) in promoters are important for inducing and coordinating changes in RNA levels, and thus represent the first layer of functional interaction. Similar to genes, TFs act only in context, which is why a TF/TFBS-based promoter analysis of genes needs to be done in the form of gene(TF)-gene networks, not individual TFs or TFBSs. In addition, integration of the literature and various databases (e.g. GO, MeSH, etc) allows the adding of genes relevant for the functional context of the data even if they were initially missed by the microarray as their RNA levels did not change significantly. Here, we outline a TF-TFBSs network-based strategy to assess the involvement of transcription factors in agonist signaling and demonstrate its utility in deciphering the response of human microvascular endothelial cells (HMEC-1) to leukemia inhibitory factor (LIF). Our strategy identified a central core of eight TFs, of which only STAT3 had previously been definitively linked to LIF in endothelial cells. We also found potential molecular mechanisms of gene regulation in HMEC-1 upon stimulation with LIF that allow for the prediction of changes of genes not used in the analysis. Our approach, which is readily applicable to a wide variety of expression microarray and next generation sequencing RNA-seq results, illustrates the power of a TF-gene networking approach for elucidation of the underlying biology.
• Zouein, F. A., Zgheib, C., Hamza, S., Fuseler, J. W., Hall, J. E., Soljancic, A., Lopez-Ruiz, A., Kurdi, M., & Booz, G. W. (2013). Role of STAT3 in angiotensin II-induced hypertension and cardiac remodeling revealed by mice lacking STAT3 serine 727 phosphorylation. Hypertension research : official journal of the Japanese Society of Hypertension, 36(6), 496-503.
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  STAT3 is involved in protection of the heart provided by ischemic preconditioning. However, the role of this transcription factor in the heart in chronic stresses such as hypertension has not been defined. We assessed whether STAT3 is important in hypertension-induced cardiac remodeling using mice with reduced STAT3 activity due to a S727A mutation (SA/SA). Wild type (WT) and SA/SA mice received angiotensin (ANG) II or saline for 17 days. ANG II increased mean arterial and systolic pressure in SA/SA and WT mice, but cardiac levels of cytokines associated with heart failure were increased less in SA/SA mice. Unlike WT mice, hearts of SA/SA mice showed signs of developing systolic dysfunction as evidenced by reduction in ejection fraction and fractional shortening. In the left ventricle of both WT and SA/SA mice, ANG II induced fibrosis. However, fibrosis in SA/SA mice appeared more extensive and was associated with loss of myocytes. Cardiac hypertrophy as indexed by heart to body weight ratio and left ventricular anterior wall dimension during diastole was greater in WT mice. In WT+ANG II mice there was an increase in the mass of individual myofibrils. In contrast, cardiac myocytes of SA/SA+ANG II mice showed a loss in myofibrils and myofibrillar mass density was decreased during ANG II infusion. Our findings reveal that STAT3 transcriptional activity is important for normal cardiac myocyte myofibril morphology. Loss of STAT3 may impair cardiac function in the hypertensive heart due to defective myofibrillar structure and remodeling that may lead to heart failure.
• Alturkmani, H. J., Zgheib, C., Zouein, F. A., Alshaaer, N. E., Kurdi, M., & Booz, G. W. (2012). Selenate enhances STAT3 transcriptional activity in endothelial cells: differential actions of selenate and selenite on LIF cytokine signaling and cell viability. Journal of inorganic biochemistry, 109, 9-15.
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  Sodium selenate may have utility in treating Alzheimer's disease and diabetes; however, its impact on the associated proinflammatory cytokine signaling of endothelial cells has not been investigated. We report that treatment of human microvascular endothelial cells with sodium selenate at a pharmacological dose (100 μM) enhanced tyrosine phosphorylation of nuclear STAT3 on Y705 in response to IL-6-type cytokine, leukemia inhibitory factor (LIF), indicative of enhanced STAT3 activity. Accordingly, STAT3 nuclear binding to DNA was increased, as well as LIF-induced gene expression of chemokine (C-C motif) ligand 2 (CCL2). CCL2 plays a key role in inflammatory processes associated with neuronal degenerative and vascular diseases. The enhancing action of selenate on LIF-induced STAT3 Y705 phosphorylation was replicated by vanadate and a specific inhibitor of protein tyrosine phosphatase, non-receptor type 1 (PTP1B). Moreover, we observed that selenite, the cellular reduction bioproduct of selenate but not selenate itself, inhibited enzymatic activity of human recombinant PTP1B. Our findings support the conclusion that in human microvascular endothelial cells selenate has a vanadate-like effect in inhibiting PTP1B and enhancing proinflammatory STAT3 activation. These findings raise the possibility that beneficial actions of supranutritional levels of selenate for treating Alzheimer's and diabetes may be offset by a proinflammatory action on endothelial cells.
• Zgheib, C., Kurdi, M., Zouein, F. A., Gunter, B. W., Stanley, B. A., Zgheib, J., Romero, D. G., King, S. B., Paolocci, N., & Booz, G. W. (2012). Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive. PloS one, 7(8), e43313.
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  We previously showed that oxidative stress inhibits leukemia inhibitory factor (LIF) signaling by targeting JAK1, and the catalytic domains of JAK 1 and 2 have a cysteine-based redox switch. Thus, we postulated that the NO sibling and thiophylic compound, nitroxyl (HNO), would inhibit LIF-induced JAK-STAT3 activation. Pretreatment of human microvascular endothelial cells (HMEC-1) or neonatal rat cardiomyocytes with the HNO donors Angeli's salt or nitrosocyclohexyl acetate (NCA) inhibited LIF-induced STAT3 activation. NCA pretreatment also blocked the induction of downstream inflammatory genes (e.g. intercellular adhesion molecule 1, CCAAT/enhancer binding protein delta). The related 1-nitrosocyclohexyl pivalate (NCP; not a nitroxyl donor) was equally effective in inhibiting STAT3 activation, suggesting that these compounds act as thiolate targeting electrophiles. The JAK1 redox switch is likely not a target of acyloxy nitroso compounds, as NCA had no effect on JAK1 catalytic activity and only modestly affected JAK1-induced phosphorylation of the LIF receptor. However, pretreatment of recombinant human STAT3 with NCA or NCP reduced labeling of free sulfhydryl residues. We show that NCP in the presence of diamide enhanced STAT3 glutathionylation and dimerization in adult mouse cardiac myocytes and altered STAT3 under non-reducing conditions. Finally, we show that monomeric STAT3 levels are decreased in the Gαq model of heart failure in a redox-sensitive manner. Altogether, our evidence indicates that STAT3 has redox-sensitive cysteines that regulate its activation and are targeted by HNO donors and acyloxy nitroso compounds. These findings raise the possibility of new therapeutic strategies to target STAT3 signaling via a redox-dependent manner, particularly in the context of cardiac and non-cardiac diseases with prominent pro-inflammatory signaling.
• Zgheib, C., Zouein, F. A., Chidiac, R., Kurdi, M., & Booz, G. W. (2012). Calyculin A reveals serine/threonine phosphatase protein phosphatase 1 as a regulatory nodal point in canonical signal transducer and activator of transcription 3 signaling of human microvascular endothelial cells. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 32(2), 87-94.
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  Vascular inflammation is initiated by stimuli acting on endothelial cells. A clinical feature of vascular inflammation is increased circulating interleukin 6 (IL-6) type cytokines such as leukemia inhibitory factor (LIF), but their role in vascular inflammation is not fully defined. IL-6 type cytokines activate transcription factor signal transducer and activator of transcription 3 (STAT3), which has a key role in inflammation and the innate immune response. Canonical STAT3 gene induction is due to phosphorylation of (1) Y705, leading to STAT3 dimerization and DNA binding and (2) S727, enhancing homodimerization and DNA binding by recruiting p300/CBP. We asked whether enhancing S727 STAT3 phosphorylation using the protein phosphatase 1 (PP1) inhibitor, calyculin A, would enhance LIF-induced gene expression in human microvascular endothelial cells (HMEC-1). Cotreatment with calyculin A and LIF markedly increased STAT3 S727 phosphorylation, without affecting the increase in the nuclear fraction of STAT3 phosphorylated on Y705. PP2A inhibitors, okadaic acid and fostriecin, did not enhance STAT3 S727 phosphorylation. Surprisingly, calyculin A eliminated LIF-induced gene expression: (1) calyculin A reduced binding of nuclear extracts to a STAT3 consensus site, thereby reducing the overall level of binding observed with LIF; and (2) calyculin A caused p300/CBP phosphorylation, thus resulting in reduced acetylation activity and degradation. Together, these findings reveal a pivotal role of a protein serine/threonine phosphatases that is likely PP1 in HMEC in controlling STAT3 transcriptional activity.
• Zgheib, C., Zouein, F. A., Kurdi, M., & Booz, G. W. (2012). Chronic treatment of mice with leukemia inhibitory factor does not cause adverse cardiac remodeling but improves heart function. European cytokine network, 23(4), 191-7.
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  Recent evidence suggests that the IL-6 family cytokine, leukemia inhibitory factor (LIF) is produced by cardiac cells under stress conditions including myocardial infarction and heart failure. Additionally, short-term delivery of LIF has been shown to have preconditioning effects on the heart and to limit infarct size. However, cell culture studies have suggested that LIF may exert harmful effects on cardiac myocytes, including pathological hypertrophy and contractile dysfunction. Long-term effects of LIF on the heart in vivo have not been reported and were the focus of this study. Adult male mice were injected daily with LIF (2 μg/30 g) or saline for 10 days. LIF treatment caused an approximate 11% loss in body weight. Cardiac function as assessed by echocardiography was improved in LIF-treated mice. Ejection fraction and fractional shortening were increased by 21% and 32%, respectively. No cardiac hypertrophy was seen on histology in LIF-treated mice,, there was no change in the heart-to-tibia length ratio, and no cardiac fibrosis was observed. STAT3 was markedly activated by LIF in the left ventricle. Different effects of LIF were seen in protein levels of genes associated with STAT3 in the left ventricle: levels of SOD2 and Bcl-xL were unchanged, but levels of total STAT3 and MCP-1 were increased. There was a trend towards increased expression of miR-17, miR-21, and miR-199 in the left ventricle of LIF-treated mice, but these changes were not statistically significant. In conclusion, effects of chronic LIF treatment on the heart, although modest, were positive for systolic function: adverse cardiac remodeling was not observed. Our findings thus lend further support to recent proposals that LIF may have therapeutic utility in preventing injury to or repairing the myocardium.
• Zgheib, C., Zouein, F. A., Kurdi, M., & Booz, G. W. (2012). Differential STAT3 signaling in the heart: Impact of concurrent signals and oxidative stress. JAK-STAT, 1(2), 101-10.
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  Multiple lines of evidence suggest that the transcription factor STAT3 is linked to a protective and reparative response in the heart. Thus, increasing duration or intensity of STAT3 activation ought to minimize damage and improve heart function under conditions of stress. Two recent studies using genetic mouse models, however, report findings that appear to refute this proposition. Unfortunately, studies often approach the question of the role of STAT3 in the heart from the perspective that all STAT3 signaling is equivalent, particularly when it comes to signaling by IL-6 type cytokines, which share the gp130 signaling protein. Moreover, STAT3 activation is typically equated with phosphorylation of a critical tyrosine residue. Yet, STAT3 transcriptional behavior is subject to modulation by serine phosphorylation, acetylation, and redox status of the cell. Unphosphorylated STAT3 is implicated in gene induction as well. Thus, how STAT3 is activated and also what other signaling events are occurring at the same time is likely to impact on the outcome ultimately linked to STAT3. Notably STAT3 may serve as a scaffold protein allowing it to interact with other singling pathways. In this context, canonical gp130 cytokine signaling may function to integrate STAT3 signaling with a protective PI3K/AKT signaling network via mutual involvement of JAK tyrosine kinases. Differences in the extent of integration may occur between those cytokines that signal through gp130 homodimers and those through heterodimers of gp130 with a receptor α chain. Signal integration may have importance not only for deciding the particular gene profile linked to STAT3, but for the newly described mitochondrial stabilization role of STAT3 as well. In addition, disruption of integrated gp130-related STAT3 signaling may occur under conditions of oxidative stress, which negatively impacts on JAK catalytic activity. For these reasons, understanding the importance of STAT3 signaling to heart function requires a greater appreciation of the plasticity of this transcription factor in the context in which it is investigated.
• Zouein, F. A., Zgheib, C., Liechty, K. W., & Booz, G. W. (2012). Post-infarct biomaterials, left ventricular remodeling, and heart failure: is good good enough?. Congestive heart failure (Greenwich, Conn.), 18(5), 284-90.
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  Infarct expansion and extension of the border zone play a key role in the progression of heart failure after myocardial infarction. Increased wall stress, along with complex cellular and extracellular changes in the surviving myocardium, underlie these events and contributes to the adverse cardiac remodeling that drives ventricular dilation and progression of heart failure. Recently, there has been much interest in the development of biopolymers that can be injected into the infarcted myocardium in order to increase its stiffness and thus reduce mechanical stress on the surrounding myocardium. Here we discuss the findings of recent animal studies that have noted improvements in contractile function or cardiac remodeling using either natural or synthetic biomaterials, as well as several that did not. Besides offering physical support to the injured myocardium, injectable biomaterials could also serve the purpose of fostering cardiac repair by functioning as a protective scaffold for stem cell or drug delivery.
• Kurdi, M., Chidiac, R., Hoemann, C., Zouein, F., Zgheib, C., & Booz, G. W. (2010). Hydrogels as a platform for stem cell delivery to the heart. Congestive heart failure (Greenwich, Conn.), 16(3), 132-5.
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  Stem cell therapy offers great promise to repair the injured or failing heart. The outcomes of clinical trials to date, however, have shown that the actual benefit realized falls far short of the promise. A number of factors may explain why that is the case, but poor stem cell retention and engraftment in the hostile environment of the injured heart would seem to be a major factor. Improving stem cell retention and longevity once delivered would seem a logical means to enhance their reparative function. One way to accomplish this goal may be injectable hydrogels, which would serve to fix stem cells in place while providing a sheltering environment. Hydrogels also provide a means to allow for the paracrine factors produced by encapsulated stem cells to diffuse into the injured myocardium. Alternatively, hydrogels themselves can be used for the sustained delivery of reparative factors. Here the authors discuss chitosan-based hydrogels.

Presentations

• Zgheib, C. (2023, April). Inhibition of Pressure Ulcers Development with Nanosilk. Corius Innovation LectureWound Healing Society, Wound Healing Foundation.
• Zgheib, C. (2023, August). Nanosilk for the prevention of Pressure Injury. Surgery Grand RoundsUniversity of Arizona.
• Zgheib, C. (2023, March). The Laboratory of Fetal and Regenerative Biology Research Overview. University of Arizona, Dept of Biomedical Engineering.
• Zgheib, C. (2023, October). Nanosilk, a novel preventative strategy for pressure ulcers.. European Tissue Repair Society 2023. Coimbra, Portugal: European Tissue Repair Society.
• Zgheib, C. (2023). Please see attached a list of the "Invited Presentations".. Please see attached.

Reviews

• Zgheib, C., Xu, J., & Liechty, K. W. (2014. Targeting Inflammatory Cytokines and Extracellular Matrix Composition to Promote Wound Regeneration(pp 344-355).
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  Delayed wound healing is one of the most challenging complications of several diseases, including diabetes. There is a vast interest in finding efficient treatments that promote scarless wound healing. The ability of the fetus to regenerate skin wounds after injury has generated much interest in the fetus as a model of regeneration. In this review, we evaluate the role and differential regulation of inflammation, extracellular matrix (ECM) composition, and mechanical stress in determining wound phenotype after injury. Comparisons between postnatal and fetal wounds have revealed many differences in the healing process. Fetal skin wound healing is characterized by a reduced inflammatory response, an ECM rich in type III collagen and high-molecular-weight hyaluronic acid (HMW-HA), and minimal mechanical stress. In contrast, adult wounds have a sustained inflammatory response, an ECM with increased type I collagen, and low-molecular-weight (LMW-HA) and are subject to significant mechanical load. The differential regulation of these processes in the fetus compared with the adult plays a critical role in promoting regeneration in the fetus while resulting in scar formation in the adult. Understanding the significance of inflammation and biomechanical forces in wound healing may help in designing therapeutic strategies for the management of chronic nonhealing wounds.