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Exosome Topical Therapy Delivered In Bioinspired Synthetic Protein Hydrogel Enhances Cutaneous Healing Of Diabetic Wounds [Meeting Abstract]

Troncoso, Juan F. Cortes; Kuhn, Joseph F.; Katyal, Priya; Subhan, Bibi; De La Cruz, Iraines; Meleties, Michael; Montclare, Jin K.; Rabbani, Piul S.
ISSN: 1067-1927
CID: 4893122

Keratinocyte-Macrophage Crosstalk by the Nrf2/Ccl2/EGF Signaling Axis Orchestrates Tissue Repair

Villarreal-Ponce, Alvaro; Tiruneh, Melat Worku; Lee, Jasmine; Guerrero-Juarez, Christian F; Kuhn, Joseph; David, Joshua A; Dammeyer, Kristen; Mc Kell, Renee; Kwong, Jennifer; Rabbani, Piul S; Nie, Qing; Ceradini, Daniel J
Unveiling the molecular mechanisms underlying tissue regeneration provides new opportunities to develop treatments for diabetic ulcers and other chronic skin lesions. Here, we show that Ccl2 secretion by epidermal keratinocytes is directly orchestrated by Nrf2, a prominent transcriptional regulator of tissue regeneration that is activated early after cutaneous injury. Through a unique feedback mechanism, we find that Ccl2 from epidermal keratinocytes not only drives chemotaxis of macrophages into the wound but also triggers macrophage expression of EGF, which in turn activates basal epidermal keratinocyte proliferation. Notably, we find dysfunctional activation of Nrf2 in epidermal keratinocytes of diabetic mice after wounding, which partly explains regenerative impairments associated with diabetes. These findings provide mechanistic insight into the critical relationship between keratinocyte and macrophage signaling during tissue repair, providing the basis for continued investigation of the therapeutic value of Nrf2.
PMID: 33238115
ISSN: 2211-1247
CID: 4680802

Nrf2-activating Therapy Accelerates Wound Healing in a Model of Cutaneous Chronic Venous Insufficiency

Kuhn, Joseph; Sultan, Darren L; Waqas, Bukhtawar; Ellison, Trevor; Kwong, Jennifer; Kim, Camille; Hassan, Absara; Rabbani, Piul S; Ceradini, Daniel J
Chronic venous insufficiency (CVI) stems from venous hypertension, extravasation of blood, and iron-rich skin deposits. The latter is central to ulcer development through generating reactive oxygen species (ROS) that drive persistent local inflammation and the development of lipodermatosclerosis. The ability to study CVI cutaneous inflammation is fundamental to advancing therapies. To address this end, a novel protocol was adapted to investigate cutaneous wound healing in iron-induced inflammation.
PMID: 33299679
ISSN: 2169-7574
CID: 4709092

Noninvasive Monitoring of Allograft Rejection Using a Novel Epidermal Sampling Technique

Rabbani, Piul S; Rifkin, William J; Kadle, Rohini L; Rao, Nakul; Diaz-Siso, J Rodrigo; Abdou, Salma A; Rodriguez, Eduardo D; Ceradini, Daniel J
Despite promising short- and long-term results to date in vascularized composite allotransplantation (VCA), acute rejection remains the most common major complication in recipients. Currently, diagnosis of acute rejection relies on clinical inspection correlated with histopathological analysis. However, disagreement exists regarding the value of full-thickness skin and mucosal biopsies and histopathology remains semiquantitative, subject to sampling bias, and prone to intra- and inter-observer variabilities. Additionally, biopsies may cause infection, scarring, and/or potentially incite rejection through immune activation after injury. Noninvasive methods to diagnose rejection represent a critical unmet need for the emerging field of VCA. Here, we propose a novel technique utilizing skin stripping of the epidermis and subsequent molecular analysis to detect known markers of acute rejection. Using a small animal VCA model, we sought to validate our epidermal sampling technique as a noninvasive diagnostic test for acute rejection.
PMID: 31592385
ISSN: 2169-7574
CID: 4129532

Dysregulation of Nrf2/Keap1 Redox Pathway in Diabetes Affects Multipotency of Stromal Cells

Rabbani, Piul S; Soares, Marc A; Hameedi, Sophia G; Kadle, Rohini L; Mubasher, Adnan; Kowzun, Maria; Ceradini, Daniel J
The molecular and cellular level reaches of the metabolic dysregulations that characterize diabetes are yet to be fully discovered. As mechanisms underlying management of reactive oxygen species (ROS) gain interest as crucial factors in cell integrity, questions arise about the role of redox cues in regulation and maintenance of bone marrow-derived multipotent stromal cells (BMSCs) that contribute to wound healing, particularly in diabetes. Through comparison of BMSCs from wild type and diabetic mice, with a known redox and metabolic disorder, we found that the cytoprotective Nrf2/Keap1 pathway is dysregulated and functionally insufficient in diabetic BMSCs. Nrf2 is basally active, but in chronic ROS we found irregular inhibition of Nrf2 by Keap1, altered metabolism and limited BMSC multipotency. Forced upregulation of Nrf2-directed transcription, through knockdown of Keap1, restores redox homeostasis. Normalized Nrf2/Keap1 signaling restores multipotent cell properties in diabetic BMSCs through Sox2 expression. These restored BMSCs can resume their role in regenerative tissue repair and promote healing of diabetic wounds. Knowledge of diabetes and hyperglycemia-induced deficits in BMSC regulation, and strategies to reverse them offers translational promise. Our study establishes Nrf2/Keap1 as a cytoprotective pathway, as well as a metabolic rheostat that affects cell maintenance and differentiation switches in BMSCs.
PMID: 30352880
ISSN: 1939-327x
CID: 3384652

Hedgehog stimulates hair follicle neogenesis by creating inductive dermis during murine skin wound healing

Lim, Chae Ho; Sun, Qi; Ratti, Karan; Lee, Soung-Hoon; Zheng, Ying; Takeo, Makoto; Lee, Wendy; Rabbani, Piul; Plikus, Maksim V; Cain, Jason E; Wang, David H; Watkins, D Neil; Millar, Sarah; Taketo, M Mark; Myung, Peggy; Cotsarelis, George; Ito, Mayumi
Mammalian wounds typically heal by fibrotic repair without hair follicle (HF) regeneration. Fibrosis and regeneration are currently considered the opposite end of wound healing. This study sought to determine if scar could be remodeled to promote healing with HF regeneration. Here, we identify that activation of the Sonic hedgehog (Shh) pathway reinstalls a regenerative dermal niche, called dermal papilla, which is required and sufficient for HF neogenesis (HFN). Epidermal Shh overexpression or constitutive Smoothened dermal activation results in extensive HFN in wounds that otherwise end in scarring. While long-term Wnt activation is associated with fibrosis, Shh signal activation in Wnt active cells promotes the dermal papilla fate in scarring wounds. These studies demonstrate that mechanisms of scarring and regeneration are not distant from one another and that wound repair can be redirected to promote regeneration following injury by modifying a key dermal signal.
PMID: 30464171
ISSN: 2041-1723
CID: 3467842

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

Rabbani, Piul S; Abdou, Salma A; Sultan, Darren L; Kwong, Jennifer; Duckworth, April; Ceradini, Daniel J
The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.
PMID: 30507922
ISSN: 1940-087x
CID: 3520222

Dissecting Wnt signaling for melanocyte regulation during wound healing

Sun, Qi; Rabbani, Piul; Takeo, Makoto; Lee, Soung-Hoon; Lim, Chae Ho; Noel, En-Nekema Shandi; Taketo, M Mark; Myung, Peggy; Millar, Sarah; Ito, Mayumi
Abnormal pigmentation is commonly seen in the wound scar. Despite advancements in the research of wound healing, little is known about the repopulation of melanocytes in the healed skin. Previous studies have demonstrated the capacity of melanocyte stem cells (McSCs) in the hair follicle to contribute skin epidermal melanocytes following injury in mice and humans. Here, we focused on the Wnt pathway, known to be a vital regulator of McSCs in efforts to better understand the regulation of follicle-derived epidermal melanocytes during wound healing. We showed that transgenic expression of Wnt inhibitor, Dkk1 in melanocytes reduced epidermal melanocytes in the wound scar. Conversely, forced activation of Wnt signaling by genetically stabilizing β-catenin in melanocytes increases epidermal melanocytes. Furthermore, we reveal that deletion of Wntless, a gene required for Wnt ligand secretion, within epithelial cells, results in failure in activating Wnt signaling in adjacent epidermal melanocytes. These results reveal the essential function of extrinsic Wnt ligands to initiate Wnt signaling in follicle-derived epidermal melanocytes during wound healing. Collectively, our results suggest the potential for Wnt signal regulation to promote melanocyte regeneration and provide a potential molecular window to promote proper melanocyte regeneration following wounding as well as in conditions such as vitiligo.
PMID: 29428355
ISSN: 1523-1747
CID: 2958132

Ex vivo allotransplantation engineering: Delivery of mesenchymal stem cells prolongs rejection-free allograft survival

Soares, Marc A; Massie, Jonathan P; Rifkin, William J; Rao, Nakul; Duckworth, April M; Park, Chin; Kadle, Rohini L; David, Joshua A; Rabbani, Piul S; Ceradini, Daniel J
Current pharmacologic regimens in transplantation prevent allograft rejection through systemic recipient immunosuppression but are associated with severe morbidity and mortality. The ultimate goal of transplantation is the prevention of allograft rejection while maintaining recipient immunocompetence. We hypothesized that allografts could be engineered ex vivo (after allotransplant procurement but before transplantation) by using mesenchymal stem cell-based therapy to generate localized immunomodulation without affecting systemic recipient immunocompetence. To this end, we evaluated the therapeutic efficacy of bone marrow-derived mesenchymal stem cells in vitro and activated them toward an immunomodulatory fate by priming in inflammatory or hypoxic microenvironments. Using an established rat hindlimb model for allotransplantation, we were able to significantly prolong rejection-free allograft survival with a single perioperative ex vivo infusion of bone marrow-derived mesenchymal stem cells through the allograft vasculature, in the absence of long-term pharmacologic immunosuppression. Critically, transplanted rats rejected a second, nonengineered skin graft from the same donor species to the contralateral limb at a later date, demonstrating that recipient systemic immunocompetence remained intact. This study represents a novel approach in transplant immunology and highlights the significant therapeutic opportunity of the ex vivo period in transplant engineering.
PMID: 29359512
ISSN: 1600-6143
CID: 2979132

Ex Vivo Major Histocompatibility Complex I Knockdown Prolongs Rejection-free Allograft Survival

Chang, Jessica B; Rifkin, William J; Soares, Marc A; Duckworth, April; Rao, Nakul; Low, Yee Cheng; Massie, Jonathan P; Rabbani, Piul S; Saadeh, Pierre B; Ceradini, Daniel J
Background/UNASSIGNED:Widespread application of vascularized composite allotransplantation (VCA) is currently limited by the required lifelong systemic immunosuppression and its associated morbidity and mortality. This study evaluated the efficacy of ex vivo (after procurement but before transplantation) engineering of allografts using small interfering RNA to knockdown major histocompatibility complex I (MHC-I) and prolong rejection-free survival. Methods/UNASSIGNED:Endothelial cells (ECs) were transfected with small interfering RNA targeted against MHC-I (siMHC-I) for all in vitro experiments. MHC-I surface expression and knockdown duration were evaluated using quantitative polymerase chain reaction (qPCR) and flow cytometry. After stimulating Lewis recipient cytotoxic lymphocytes (CTL) with allogeneic controls or siMHC-I-silenced ECs, lymphocyte proliferation, CTL-mediated and natural killer-mediated EC lysis were measured. Using an established VCA rat model, allografts were perfused ex vivo with siMHC-I before transplantation. Allografts were analyzed for MHC-I expression and clinical/histologic evidence of rejection. Results/UNASSIGNED:< 0.05). Conclusions/UNASSIGNED:Ex vivo siMHC-I engineering can effectively modify allografts and significantly prolong rejection-free allograft survival. This novel approach may help reduce future systemic immunosuppression requirements in VCA recipients.
PMID: 30276052
ISSN: 2169-7574
CID: 3327792