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2021 Abstracts

D1

Skin Wounding Induces Cognitive And Behavioral Alterations And Modulates Cytokine Expression In The Murine Hippocampus
Daniel Fregoso1, Yasmin Hadian1, Anthony Gallegos1, John Maaga1, Isaiah Kletenik2, Melanie Gareau1, Rivkah Isseroff1
1UC Davis, Sacramento, CA, USA, 2Harvard Medical School, Brigham and Women’s Hospital, Boston, MA, USA
Skin Wounding Induces Cognitive And Behavioral Alterations And Modulates Cytokine Expression In The Murine Hippocampus

Daniel Fregoso1, Yasmin Hadian1, Anthony Gallegos1, John Maaga1, Isaiah Kletenik2, Melanie Gareau1, Rivkah Isseroff1 1UC Davis, Sacramento, CA, USA, 2Harvard Medical School, Brigham and Women’s Hospital, Boston, MA, USA

Background: Patients with chronic wounds often have associated cognitive dysfunction and depression with an as yet unknown mechanism for this association. Methods: To address the possible causality of skin wounding inducing these changes, the behavior and cognitive functions of mice with an excisional skin wound were compared to those of control unwounded animals. Hippocampal tissues were probed for expression of inflammatory mediators and cerebral cortices and sera were analyzed for catecholamines. Results: At six days post wounding, wounded animals exhibited increased depressive and anxiety-like behaviors as well as decreased memory, as elicited by the forced swim, light/dark box and novel object recall tests, respectively (n=8-10, p=<0.05). Wounded animals also had concomitant increased hippocampal expression of TNFa, the pattern recognition receptor NOD2, the glucocorticoid receptors GR/NR3C1 and NR3C2 (n=8 unwounded, 16 wounded p<0.05). Prefrontal cortex serotonin metabolism was increased as evidenced by increase in tryptophan precursor and of the serotonin metabolite 5-HIAA (n=8-10, p=<0.05). The dopamine metabolite, HVA was also elevated in the cortex of wounded animals. In contrast to the central CNS findings, day six post-wounding serum catecholamines did not differ between wounded and unwounded animals. Conclusions: These findings suggest that wounding induces an altered expression of inflammatory mediators and pattern recognition receptor genes in the hippocampus, independent of changes in systemic mediators, which may be responsible for the observed behavioral deficits.

D2

Transcriptome Analysis Identifies Differentially Regulated Genes Contributing To Hematological Response After Radiation Exposure In A Mouse Model
Abdulnaser Alkhalil1, John Clifford2, Stacy Miller3, Ross Campbell4, Aarti Gautam5, Marti Jett5, Rasha Hammamieh5, Lauren Moffatt1, Jeffrey Shupp1
1MedStar Health Research Institute, Washington, DC, USA, 2USAISR, San Antonio, TX, USA, 3The Oak Ridge Institute for Science and Education, Fort Detrick, MD, USA, 4The Geneva Foundation, Washington, DC, USA, 5Walter Reed Army Institute of Research, Silver Spring, MD, USA
Transcriptome Analysis Identifies Differentially Regulated Genes Contributing To Hematological Response After Radiation Exposure In A Mouse Model

Abdulnaser Alkhalil1, John Clifford2, Stacy Miller3, Ross Campbell4, Aarti Gautam5, Marti Jett5, Rasha Hammamieh5, Lauren Moffatt1, Jeffrey Shupp1 1MedStar Health Research Institute, Washington, DC, USA, 2USAISR, San Antonio, TX, USA, 3The Oak Ridge Institute for Science and Education, Fort Detrick, MD, USA, 4The Geneva Foundation, Washington, DC, USA, 5Walter Reed Army Institute of Research, Silver Spring, MD, USA

Background: Risks of radiation exposure in occupational, accidental, or deliberate incidents are on increase. Physiological and biological consequences of ionizing radiation exposures are not well understood but known to be accumulative and lethal at higher doses when repair and restoration mechanisms in affected victims are overwhelmed. Using a genomic approach in a mouse irradiation model we have characterized hematological responses that align with clinical manifestation of radiation exposure at molecular levels. Opposite hematological responses in survivable and lethal IR doses were identified. Methods: Groups of mice n=5 received whole-body x-ray exposures (0, 1, 3, 6, or 20Gy) and skin biopsies were obtained from each animal at times post-irradiation (h2, Days 4, 7, 21, 28). Biopsies were collected from the 20Gy cohort for only days 0, 4, and 7. Total RNA was isolated and microarrays were performed and analyzed using custom R scripts to obtain lists of probe sets differentially expressed. Changes in gene expression at Benjamini-Hochberg FDR adjusted P less than 0.05 and FC greater than 2 were deemed significant. Analyses were performed comparing the different doses of X-ray exposure over all time points. Results: Mice in the 20Gy group were euthanized by d7 and the dose was considered lethal. Animals in 1, 3, and 6Gy groups completed the full experiment to d28. Sammon plot analysis of transcriptomes showed clear separation of samples based on the irradiation levels and time after exposure. Pathways enrichment analysis of the significantly differentially transcribed genes (SDTGs) identified GP6 signaling pathway and the intrinsic prothrombin activation pathway among the top significantly (-log p greater than 1.3 and z-score greater than Abs 2) modulated pathways in lethal and sublethal IR doses. Both pathways were predicted significantly inactivated in lethal doses and activated in at least one time point in the sublethal doses. Dysregulation of collagens, laminin, calmodulin, platelet-derived growth factor like and kallikrein related peptidases underlying the modulations in these pathways. Other genes contributing to heme-binding and movement and activation of leukocytes and blood cells, such as erythroblast membrane associated protein (ERMAP), erythroid differentiation regulator 1 (EERDR1), hemoglobin subunit alpha 2 and beta (HBA1/HBA2, HBB), hemopexin (HPX), heme binding protein 2 (HEBP2), and hemojuvelin BMP co-receptor (HJV) were differentially transcribed after IR exposure. Conclusions: These results introduce the major pathways and genes involved in hematological system responses to IR dose exposures and differentiate between responses to lethal and sublethal doses.

D3

Pseudomonas Aeruginosa Theft Biofilm Requires Host Lipidsof Cutaneous Wound
Nandini Ghosh1, Mithun Sinha1, Dayanjan S. Wijesinghe2, Shomita S. Mathew-Steiner1, Amitava Das1, Kanhaiya Singh1, Mohamed El Masry1, Savita Khanna1, Hiroyuki Inoue3, Katsuhisa Yamazaki3, Manabu Kawada3, Gayle M. Gordillo1, Sashwati Roy1, Chandan K. Sen1
1Indiana University, Indianapolis, IN, USA, 2Virginia Commonwealth University, Richmond, VA, USA, 3Institute of Microbial Chemistry, Microbial Chemistry Research Foundation, Tokyo, Japan
Pseudomonas Aeruginosa Theft Biofilm Requires Host Lipids of Cutaneous Wound

Nandini Ghosh1, Mithun Sinha1, Dayanjan S. Wijesinghe2, Shomita S. Mathew-Steiner1, Amitava Das1, Kanhaiya Singh1, Mohamed El Masry1, Savita Khanna1, Hiroyuki Inoue3, Katsuhisa Yamazaki3, Manabu Kawada3, Gayle M. Gordillo1, Sashwati Roy1, Chandan K. Sen1 1Indiana University, Indianapolis, IN, USA, 2Virginia Commonwealth University, Richmond, VA, USA, 3Institute of Microbial Chemistry, Microbial Chemistry Research Foundation, Tokyo, Japan

Background: PA biofilm causes wound chronicity. Both CDC as well as NIH recognizes biofilm infection as a threat leading to wound chronicity. Chronic wounds on lower extremities often lead to surgical limb amputation. Objective: This work addressing complexities in wound infection, seeks to test the reliance of bacterial pathogen Pseudomonas aeruginosa (PA) on host skin lipids to form biofilm with pathological consequences. Methods: An established pre-clinical porcine chronic wound biofilm model, infected with PA (PAWT) or Pseudomonas ceramidase mutant strains (PADeltaCer) was used. The porcine wounds were followed till day 56 post wounding. Analyses of wound closure (digital planimetry), skin barrier function (TEWL), ceramide levels (targeted lipidomics), wound biofilm infection assay (by scanning electron microscopy, wheat-germ agglutinin, PA quorum sensing genes by quantitative RT-PCR), PPARd, ABCA12 expression (IHC and quantitative RT-PCR) were done. Results: We observed that bacteria drew resource from host lipids to induce PA ceramidase expression by three orders of magnitude (n=5, p<0.05). PA utilized product of host ceramide catabolism to augment transcription of PA ceramidase. Biofilm formation was more robust in PA compared to PADeltaCer (n=6, p<0.05). Downstream products of such metabolism such as sphingosine and sphingosine-1-phosphate were both directly implicated in the induction of ceramidase and inhibition of PPARd, respectively. PA biofilm, in a cermidastin-sensitive manner, also silenced PPARd via induction of miR-106b (n=6, p<0.05). Low PPARd limited ABCA12 expression resulting in disruption of skin lipid homeostasis. Barrier function of the wound-site was thus compromised. Conclusions: This work demonstrates that microbial pathogens must co-opt host skin lipids to generate complex biofilms. Anti-biofilm strategies targeting host lipids at risk of infection could be productive.

D4

Cell Therapy Stimulates Antimicrobial Properties Of Venous Leg Ulcers Via Perforin-2
Vivien Chen, Rivka Stone, Jamie L. Burgess, Natasa Strbo, Irena Pastar, Marjana Tomic-Canic
University of Miami Miller School of Medicine, Miami, FL, USA
Cell Therapy Stimulates Antimicrobial Properties Of Venous Leg Ulcers Via Perforin-2

Vivien Chen, Rivka Stone, Jamie L. Burgess, Natasa Strbo, Irena Pastar, Marjana Tomic-Canic University of Miami Miller School of Medicine, Miami, FL, USA

Background: Unresolved inflammation and persistent infection from both intracellular and extracellular pathogens are major factors that contribute to wound chronicity. The antimicrobial protein Perforin-2 (P-2)/MPEG1 is a highly conserved membrane attack complex pore-forming domain containing protein that plays an indispensable role in the innate immune response against intracellular pathogens. It is downregulated in chronic wounds, which results in increased intracellular bacteria. In this study, we investigated P-2 modulation triggered by treatment of chronic venous leg ulcers (VLU) with bioengineered bilayered living cell construct (BLCC), an FDA-approved therapy demonstrating efficacy in promoting healing of VLUs. Methods: We previously conducted a clinical trial (NCT01327937) (n=30) and comprehensive genomic analyses that showed that application of BLCC shifts nonhealing VLUs to a healing phenotype through promotion of antifibrotic remodeling in the wound bed and inducing acute wound-like inflammatory response in the wound edge. Here we used a similar approach to determine P-2 regulation by BLCC treatment. To further define the broader role of the innate antimicrobial response in chronic wound closure, we also examined expression of antimicrobial peptides (AMPs) in transcriptomic profiles from human acute wounds (n=6), VLUs (n=10), and diabetic foot ulcers (DFUs) (n=8). Results: BLCC treatment upregulates P-2 expression in VLUs to promote an enhanced anti-microbial response as a component of its induction of a healing tissue response. Upon further subgroup analysis, BLCC-triggered P-2 upregulation was significant only in VLUs that subsequently healed and not in their non-healing counterparts. This is the first evidence that a cell-based therapy in chronic wounds triggers antimicrobial effect of the host. Expression levels of AMPs beta defensins, S100 alarmins, RNAses and dermcidin were similarly regulated in acute and chronic wounds. In contrast, P-2 was the only AMP exclusively upregulated in acute wounds, displaying downregulation or no change in non-healing ulcers (DFUs and VLUs, respectively). Conclusions: Taken together, our findings elucidate potential antimicrobial mechanisms of action in BLCC treatment, and support a specific role for P-2 in contributing to the antimicrobial innate immune response necessary for successful wound closure of chronic wounds.

YIA1

Wnt-active Engrailed-1 Lineage-negative Fibroblasts Mediate Postnatal Skin Regeneration
Shamik Mascharak, Heather E. desJardins-Park, Michael Januszyk, Kellen Chen, Michael F. Davitt, Janos Demeter, Dominic Henn, Michelle Griffin, Clark A. Bonham, Nancie Mooney, Ran Cheng, Peter K. Jackson, Derrick C. Wan, Geoffrey C. Gurtner, Michael T. Longaker
Stanford University School of Medicine, Stanford, CA, USA
Wnt-active Engrailed-1 Lineage-negative Fibroblasts Mediate Postnatal Skin Regeneration

Shamik Mascharak, Heather E. desJardins-Park, Michael Januszyk, Kellen Chen, Michael F. Davitt, Janos Demeter, Dominic Henn, Michelle Griffin, Clark A. Bonham, Nancie Mooney, Ran Cheng, Peter K. Jackson, Derrick C. Wan, Geoffrey C. Gurtner, Michael T. Longaker Stanford University School of Medicine, Stanford, CA, USA

Background: We previously reported that embryonically derived Engrailed-1 (En-1) lineage-positive fibroblasts (EPF) drive dorsal skin scarring. More recently, we showed that En-1-lineage negative fibroblasts (ENFs) activate En-1 expression within the postnatal wound environment, and that blocking this conversion with verteporfin (YAP inhibitor) yields ENF-mediated wound regeneration with full recovery of normal adnexa (hair follicles, glands), matrix ultrastructure, and mechanical strength. This observation presented a unique opportunity to study the molecular events differentiating skin regeneration from scarring. Methods: We compared regenerating (YAP inhibition) and scarring (PBS) excisional wounds in mice at post-operative days (POD) 0 (unwounded), 2, 7, 14, and 30 (n=10 wounds per timepoint). Wounds were compared in a multi-“omic” fashion using single-cell RNA sequencing (scRNA-seq), timsTOF bulk proteomics (a novel mass spectrometry-based technique for quantifying thousands of peptide targets), and a novel image processing algorithm for quantifying 294 extracellular matrix parameters. Using a cell-barcoding and data imputation approach, we correlated these data modalities on a per-mouse basis to infer functional gene-protein-matrix relationships. Results: Pseudotime analysis (Monocle3) of scRNA-seq data revealed that fibroblasts followed two distinct transcriptional trajectories, one characterized by mechanical activation (En-1 lineage-positive, “fibrotic” trajectory) and the other characterized by developmental and regenerative pathways (En-1 lineage-negative; Rspo1, Dkk2/3, Trps1). Similar analyses did not reveal diverging behavior in other cell types (e.g., myeloid or lymphoid cells). Cross-platform data integration confirmed that fibroblasts in the fibrotic trajectory correlated with myofibroblast proteomic signatures (Col1a1/2, Fn1, etc.) and scar ECM. In contrast, fibroblasts in the regenerative trajectory negatively correlated with myofibroblast markers and were instead associated with basket-weave ECM quantitatively indistinguishable from unwounded skin. Our integrated dataset suggested an important role for Wnt pathway proteins in ENF-mediated skin regeneration, so we compared POD 14 scars and regenerated wounds by multiplexed in situ hybridization (RNAScope) for Rspo1 (Wnt agonist), Trps1 (master hair follicle regulator), Ank1 (YAP target gene), and Dpp4 (EPF marker). Quantification of RNA granules across thousands of cells using a custom image analysis pipeline revealed that ENF-mediated healing (low Dpp4) in YAP-inhibited (low Ank1) wounds yielded regeneration of functional hair follicles through Wnt-mediated pathway activation (high Rpos1, Trps1). Conclusions: These data suggest that YAP inhibition unlocks wound regeneration through Wnt-active, En-1 lineage-negative fibroblasts. Furthermore, transcriptional divergence between the default, pro-fibrotic transcriptional trajectory (En-1 lineage-positive fibroblasts) and this regenerative trajectory occurs as early as POD 2, with proteomic and matrix signatures of skin regeneration following on POD 7 and 14.

YIA2

MiR-193b-3p In Diabetic Foot Ulcers: A Master Regulator Of Impaired Wound Healing And A Tumor Protector
Jelena Marjanovic, Horacio A. Ramirez, Ivan Jozic, Rivka Stone, Tongyu C. Wikramanayake, Cheyanne R. Head, Beatriz Abdo Abujamra, Nkemcho Ojeh, Robert S. Kirsner, Hadar Lev-Tov, Irena Pastar, Marjana Tomic-Canic
Wound Healing and Regenerative Medicine Research Program, Dr Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, USA
MiR-193b-3p In Diabetic Foot Ulcers: A Master Regulator Of Impaired Wound Healing And A Tumor Protector

Jelena Marjanovic, Horacio A. Ramirez, Ivan Jozic, Rivka Stone, Tongyu C. Wikramanayake, Cheyanne R. Head, Beatriz Abdo Abujamra, Nkemcho Ojeh, Robert S. Kirsner, Hadar Lev-Tov, Irena Pastar, Marjana Tomic-Canic Wound Healing and Regenerative Medicine Research Program, Dr Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, USA

Background: Diabetic foot ulcers (DFUs) are marked by characteristic non-migratory, hyperproliferative epidermis resulting from the activation of beta-catenin (Wnt) pathway and overexpression of oncogene c-myc. Despite this hyperproliferative ‘cancer-like’ cellular microenvironment, malignant transformation is a rare event in DFUs. To understand molecular mechanisms that contribute to such unique cellular phenotype, we focused on the role of master-regulators, microRNAs (miRs) in DFUs. Methods: We collected tissue samples from DFUs (n=15), venous leg ulcers (VLUs, n=9), control skin samples (n=15), cutaneous squamous cell carcinomas (cSCC, n=6) and acute wounds (n=3), and performed genomic and qPCR analyses. Network analysis was performed using Ingenuity Pathway analysis. To decipher the role of miR-193-3p in DFUs, we performed gain- and loss-of function experiments. Cell migration was analyzed using scratch assay, re-epithelialization was evaluated using 3D skin organotypic culture and a murine in vivo wound model. Formation of lamellipodia, filopodia and stress fibers were studied using immunocytochemistry. RhoA activity was analyzed using RhoA pull-down assay. Results: We found tumor suppressor miR-193b-3p upregulated specifically in the epidermis of non-healing DFUs, but not in acute wounds, VLUs or cSCC suggesting its unique, dual role. Ectopic overexpression of miR-193b-3p selectively inhibits migration of human keratinocytes and fibroblasts, without affecting proliferation. Moreover, miR-193b-3p showed a dominant negative effect on keratinocyte migration by suppressing wound closure in vitro even in the presence of pro-migratory miR31-5p and miR-15b-5p. We confirmed miR-193b-3p healing-inhibitory effect by overexpressing it in human 3D skin organotypic culture and in a murine in vivo wound model. Further in-depth molecular and cellular analyses revealed that anti-migratory of the miR-193b-3p activity occurs through a disruption of stress fiber formation and suppression of RhoA activity, with no effect on lamellipodia or filopodia formation. To further examine miR-193b-3p mediated suppression of wound epithelialization, we evaluated expression of its target genes in DFUs. We confirmed suppression of downstream targets of miR-193b-3p in DFUs: MAP3K1, TNS1, SOS2, JAK2, LRP6 ARHGEF6 and proto-oncogene, KRAS. We confirmed that KRAS is suppressed upon ectopic expression of miR-193b-3p even in the presence of pro-migratory miRs. Moreover, miR-193b-3p targets involved in malignant transformation were found differentially regulated between DFUs and cSCC, providing a new molecular insight regarding low incidence of tumor in DFUs. Conclusions: We identified miR-193b-3p as important regulator that represents a target for promotion of wound healing and cancer therapeutic.

YIA3

Adipocytes In Dermal Wounds Undergo Conversion To Pro-fibrotic Fibroblasts That Contribute To Scar Formation
Nicholas Guardino, Michelle Griffin, Heather E. desJardins-Park, Megan E. King, Kellen Chen, Khristian E. Bauer-Rowe, Michael T. Longaker
Stanford Medical School, Stanford, CA, USA
Adipocytes In Dermal Wounds Undergo Conversion To Pro-fibrotic Fibroblasts That Contribute To Scar Formation

Nicholas Guardino, Heather E. desJardins-Park, Michelle Griffin, Khristian E. Bauer-Rowe, Megan E. King, Megan E. King, Shamik Mascharak, Michael T. Longaker Stanford Medical School, Stanford, CA, USA

Background: Adipocytes have previously been implicated in skin wound healing in both metabolic and non-metabolic roles. Recent studies have suggested that adipocytes and fibroblasts (cellular mediators of scarring) interconvert in the wound environment. However, the lineage dynamics and molecular properties of wound-resident adipocytes remain undefined. We hypothesized that mature adipocytes directly participate in wound repair via conversion into fibroblasts, and that adipocyte-derived fibroblasts contribute to scarring. Methods: AdipoqCre transgenic driver mice were crossed to R26mTmG reporter mice to generate AdipoqCre;ROSA26mTmG mice, wherein mature adipocytes (Adipoq+) and their progeny express GFP, and other cells express RFP. AdipoqCre;ROSA26mTmG mice underwent splinted dorsal excisional wounding and wounds were harvested at POD7 (mid-healing) and 14 (wounds re-epithelialized) for analysis. Wound sections underwent immunofluorescent staining for fibroblast (e.g., collagen type I/col-I, alpha smooth muscle actin/a-SMA) and adipocyte (e.g., perilipin) cell markers and confocal imaging. For fibroblast subtype analysis, wound cells were stained for fibroblast subtype markers (Sca1, Dlk1, CD26 and analyzed via FACS. To achieve local adipocyte ablation, AdipoqCre;ROSA26mTmG;R26tm1(HBEGF)Awai mice were generated and wounded, and diphtheria toxin (DT; 400 ng/mouse) was injected into the wound base on POD0, 1, and 2. DT- and vehicle control-treated wounds underwent histologic analysis with hematoxylin and eosin (H&E) staining. Results: Using our AdipoqCre;ROSA26mTmG adipocyte lineage-tracing model, we identified significantly greater number of adiponectin lineage-positive cells (GFP+) within wounds at POD 7 and 14 compared to unwounded skin (*P<0.05, n=5). Compared to typical subcutaneous adipocytes, the GFP+ cells that infiltrated the injury site exhibited upregulation of fibrotic/fibroblast markers including col-I, col-III, and a-SMA, and downregulation of adipocyte markers including perilipin, at POD7 and POD 14, indicating conversion of mature (Adipoq-expressing) adipocytes into pro-fibrotic wound fibroblasts. Upon both FACS and immunohistochemical analysis, these adipocyte-derived fibroblasts demonstrated positive expression of fibroblast subpopulation markers including Sca1 and Dlk1, consistent with a hypodermal/lipofibroblast identity. DT-induced ablation of Adipoq lineage-positive cells was confirmed by the absence of GFP+ cells in wounds; these wounds exhibited reduced dermal scarring, with decreased scar thickness and collagen deposition at POD 14 compared to control wounds. Conclusions: Our findings strongly suggest that mature adipocytes in the skin undergo conversion to pro-fibrotic fibroblasts in response to the wound environment. Local adipocyte ablation resulted in reduced scarring, suggesting that adipocyte-derived fibroblasts are important contributors to wound fibrosis. Identification of the specific lineage dynamics and molecular cues driving adipocyte-to-fibroblast transdifferentiation may reveal novel strategies to target skin scarring and fibrosis.

YIA4

CRISPR/Cas9 Editing Of Autologous Dendritic Cells To Enhance Angiogenesis And Wound Healing
Dominic Henn1, Dehua Zhao2, Clark A. Bonham1, Kellen Chen1, Autumn H. Greco1, Jagannath Padmanabhan1, Dharshan Sivaraj1, Artem Trotsyuk1, Janos A. Barrera1, Michael Januszyk1, Lei Stanley Qi2, Geoffrey C. Gurtner1
1Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University, Stanford, CA, USA, 2Dept. of Bioengineering, Stanford University, Stanford, CA, USA
CRISPR/Cas9 Editing Of Autologous Dendritic Cells To Enhance Angiogenesis And Wound Healing

Dominic Henn1, Dehua Zhao2, Clark A. Bonham1, Kellen Chen1, Autumn H. Greco1, Jagannath Padmanabhan1, Dharshan Sivaraj1, Artem Trotsyuk1, Janos A. Barrera1, Michael Januszyk1, Lei Stanley Qi2, Geoffrey C. Gurtner1 1Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University, Stanford, CA, USA, 2Dept. of Bioengineering, Stanford University, Stanford, CA, USA

Background: Dendritic cells (DCs) are a heterogeneous cell population which critically regulates the adaptive immune response. Depending on their activation status, DCs can also promote peripheral immune tolerance, thus limiting the activation of the immune system and tissue damage. The N-myc downregulated gene 2 (Ndrg2) is highly expressed in DCs and limits the secretion of vascular endothelial growth factor (VEGF), which is critical for wound healing. Cell based therapy approaches using DCs have been approved by the FDA and clinical trials using DC immunotherapy are being performed against a variety of cancer types. However, the role of DC therapy for wound healing has not yet been investigated. Methods: Hematopoietic progenitor cells were isolated from the bone marrow of mice and differentiated into DCs over a 7-day in vitro culture period. Pharmacologic down-regulation of Ndrg2 was performed by treatment of cultures with 1,25-dihydroxyvitamin D3 (VD3) and the angiogenic potential of the treated cells was evaluated by endothelial cell (EC) tube formation assays. Cytokine secretion of DCs was measured in the conditioned media using Luminex multiplex assays. To permanently knock out Ndrg2 in DCs, a CRISPR/Cas9 gene editing approach was developed, using Cas9/sgRNA-ribonucleoproteins and electroporation. To determine the impact of genetically edited DCs on wound healing, splinted excisional wounds in C57BL6/J (wild-type) mice were treated weekly with pullulan-collagen hydrogels seeded with Ndrg2-knockout (KO) DCs, control DCs which had undergone electroporation only, or blank hydrogels. The transcriptomic impact of Ndrg2 downregulation on DC fate was evaluated by microfluidic single-cell RNA sequencing (scRNA seq) of Ndrg2-KO DCs, VD3-treated DCs and control DCs. Results: Ndrg2 down-regulation lead to a significantly stronger EC tube formation in co-cultures with VD3-treated DCs, and strongly enhanced VEGF secretion compared to untreated DCs in vitro. A CRISPR/Cas9 editing pipeline was developed for KO of Ndrg2 in DCs with a transfection rate and editing efficiency of >90% shown by Sanger Sequencing. Excisional wounds treated with Ndrg2-KO DCs demonstrated significantly accelerated healing compared to control DCs and blank hydrogels. scRNA seq revealed that Ndrg2 downregulation strongly induced Vegfa expression and anti-oxidative transcriptomic signatures. Conclusions: Our data indicate that KO of Ndrg2 in DCs strongly enhances their secretion of VEGF, thus promoting angiogenesis and accelerating wound healing. Given the ready availability of DCs from the human blood through established leukapheresis protocols and easy multiplication in vitro, CRISPR/Cas9 editing of DCs is a promising new approach to induce wound healing and soft-tissue regeneration.

YIA5

Energy Metabolism Governs The Degree Of Fibrosis In Dermal Wound Healing
Umang M. Parikh1, Hima V. Vangapandu1, Harrison Strang1, Aditya Kaul1, Hui Li1, Cristian Coarfa1, Swathi Balaji2
1Baylor College of Medicine, Houston, TX, USA, 2Surgery, Baylor College of Medicine, Houston, TX, USA
Energy Metabolism Governs The Degree Of Fibrosis In Dermal Wound Healin

Umang M. Parikh1, Hima V. Vangapandu1, Harrison Strang1, Aditya Kaul1, Hui Li1, Cristian Coarfa1, Swathi Balaji2 1Baylor College of Medicine, Houston, TX, USA, 2Surgery, Baylor College of Medicine, Houston, TX, USA

Background: Similar dermal injuries in different patients can result in varying degrees of fibrosis during wound healing. While the reasons for this skin fibrosis/scarring heterogeneity are unknown, responses to injury-induced oxidative stress involve energy metabolism through both oxidative phosphorylation (OXPHOS) and glycolysis to generate ATP and fuel tissue repair. Aerobic glycolysis is emerging as a mechanism that underlie stressed cells during fibrosis. Since energy-intensive proliferation and ECM production by fibroblasts relies on increased lactate production, indicating a shift to aerobic glycolysis, we hypothesize differences in intrinsic fibroblast bioenergetic metabolism underlie differential scarring. Methods: We created a biorepository of paired normal-skin and c-section scar tissue and fibroblasts from abdominoplasty samples derived from patients with clinically stratified low (LS) and high scar (HS) phenotypes using Vancouver Scar Scale <3 vs.>6 respectively. Oxidative phosphorylation (OCR), glycolysis (ECAR), ATP-production at resting and stress/hypoxia (seahorse-assays), mitochondrial membrane potential (DeltaPsim; JC-1) and mitoROS (MitoSOX) were measured. LS/HS fibroblasts (10^6 cells/wound) were transferred to 6mm full thickness-stented wounds on SCID mice (female, 8-10wk). Wounds were harvested at d7, 28, and wound closure (H&E), a-SMA and UPC2-3 (RT-PCR), inflammatory profile (Luminex; IHC), collagen expression (trichrome) were analyzed. n=3-4 independent cells/group; p-values were calculated by ANOVA. Results: Both normal-skin and scar fibroblasts from high-scarrers had higher basal OCR and ECAR than low-scarrers (p<0.01), suggesting more bioenergetic metabolism in high-scarrers. We observed no difference in the %ATP production between high-scarrer and low-scarrer normal-skin fibroblasts. High-scarrer fibroblasts responded to stress (FCCP/Oligomycin treatments) with a significant increase in spare respiratory and glycolytic reserve capacity than low-scarrers (p<0.01). Under hypoxia, OCR decreased in both high-scarrer and low-scarrer fibroblasts, but only high-scarrers showed a significant increase in glycolysis. High-scarrer normal-skin fibroblasts had more depolarized mitochondria and mitochondrial-ROS than low-scarrers (p<0.01). Proteome-profiler assay showed Hsp-27 phosphorylation (p-Hsp-27Ser82) was significantly lower (p<0.001) in high-scarrer fibroblasts and correlated with the scarring in skin tissue. HSP27/p38 inhibitor (SB203580) significantly reduced low-scarrer fibroblast migration to match high-scarrer fibroblast phenotype. In vivo, murine wounds with high-scarrer fibroblasts showed expedited wound closure at d7 with increase in a-SMA (25-fold) and UCP2 (18-fold), and pronounced collagen staining at d28. D7 wounds demonstrated reduced IL-10, IL-17, MIP-1a/b, and G-CSF in high-scarrer fibroblast-injected wounds. Conclusions: Fibroblasts of different scarring phenotypes display characteristic bioenergetic metabolism profiles and produces distinct scarring in murine wounds. Our data suggests that a shift to aerobic glycolysis (Warburg effect) is associated with increased fibrosis (high scar). Testing the role of Hsp-27 as a key regulator that responds to metabolic alterations to drive the heterogeneity of human scarring is significant because regulators of p-Hsp-27 are readily available.

YIA6

Exosome Topical Therapy Delivered In Bioinspired Synthetic Protein Hydrogel Enhances Cutaneous Healing Of Diabetic Wounds
Juan F. Cortes Troncoso1, Joseph F. Kuhn1, Priya Katyal2, Bibi Subhan1, Iraines De La Cruz1, Michael Meleties2, Jin K. Montclare2, Piul S. Rabbani1
1New York University School of Medicine, New York, NY, USA, 2NYU Tandon School of Engineering, New York, NY, USA
Exosome Topical Therapy Delivered In Bioinspired Synthetic Protein Hydrogel Enhances Cutaneous Healing Of Diabetic Wounds

Juan F. Cortes Troncoso1, Joseph F. Kuhn1, Priya Katyal2, Bibi Subhan1, Iraines De La Cruz1, Michael Meleties2, Jin K. Montclare2, Piul S. Rabbani1 1New York University School of Medicine, New York, NY, USA, 2NYU Tandon School of Engineering, New York, NY, USA

Background: We have used human bone marrow multipotent stromal cells (MSCs) and their secreted nanoscale exosomes (Exo) to successfully promote wound closure in diabetic animal models of delayed healing. However, safe and easy delivery platforms that maintain exosome efficacy are necessary to translate these therapies to the bedside. Here, we describe the development of Exo-Q, a thermoresponsive soft protein matrix loaded with MSC-derived exosomes that accelerates wound closure in a model of Type II diabetes. Methods: We isolated exosome preps by differential ultracentrifugation of conditioned media from human bone marrow-derived MSCs, prior to detailed characterization. We synthesized Q, an engineered variant of the coiled-coil domain of cartilage oligomeric matrix protein, and incorporated 3×10^9 exosomes during gelation to yield Exo-Q hydrogels. We analyzed Exo-Q using transmission electron microscopy (TEM) and performed rheology. We used excisional stented wounds on 12-16 weeks old LepRdb/db mice and either topically pipetted on exosome preps or applied Exo-Q hydrogels. We observed the wounds for closure and collected intermediate time point tissues for biomolecular analysis and histology. Results: Q self-assembles into a fibrous matrix at low temperatures and exhibits an upper critical solution temperature phase behavior. Exo-Q shows entanglement of protein fibers uniformly interspersed with exosomes in TEM and increased the storage modulus of Q, indicating increased elasticity of a pliable hydrogel. Exo-Q solubilizes at skin wound temperature (~31C) for sustained delivery of exosomes into wounds, without invasive or painful routes. Pipetted single doses 1×10^9 or 3×10^9 exosomes at post-operative day 1 (POD1) demonstrated non-significant changes in closure time (30.25+/-1.5 and 27+/-2 days, respectively, n=4) vs PBS/sham-treated diabetic wounds. Exo-Q application at POD1 decreased time to closure of diabetic wounds to 17+/-1.4 days compared to 28+/-1.5 days for Q vehicle alone (p<0.01, n=3), and correspondingly also reduced the wound burden relative to that with Q alone. Exo-Q administration also generated extensive CD31+ neovascularization in large areas of granulation tissue in the Leprdb/db diabetic wound bed by POD10. Exo-Q does not interfere with wound healing progression. The single Exo-Q dose also resulted in upregulated gene expression of angiogenic and wound healing associated factors VEGF, SDF1 and PDGF in diabetic wound beds, compared to Q vehicle-only treated wounds (all p<0.05, n>3). Conclusions: Exosome delivery in our novel protein construct is an efficacious and translatable therapy that may reverse pathologic healing of diabetic wounds. Future iterations could incorporate drugs into Q, with exosomes, for a compound therapeutic hydrogel.

YIA7

Interleukin-10 Producing T-Lymphocytes Mitigate Dermal Fibrosis
Walker D. Short, Xinyi Wang, Hui Li, Ling Yu, Aditya Kaul, Swathi Balaji, Sundeep G. Keswani
Baylor College of Medicine, Houston, TX, USA
Interleukin-10 Producing T-Lymphocytes Mitigate Dermal Fibrosis

Walker D. Short, Xinyi Wang, Hui Li, Ling Yu, Aditya Kaul, Swathi Balaji, Sundeep G. Keswani Baylor College of Medicine, Houston, TX, USA

Background: CD4+ T-lymphocytes are important regulators of the immune system, which we have shown to be essential in regulating dermal fibrosis. Specific subsets of CD4+ cells, Regulatory T-lymphocyte (Treg) and Type-1 regulatory T-lymphocyte (Tr1), produce IL-10, an anti-inflammatory cytokine that we have previously shown to reduce dermal fibrosis. However, the role of Treg and Tr1 in scarring is unclear. We therefore hypothesize that IL-10 producing CD4+ T-lymphocyte subsets attenuate dermal wound fibrosis via mediation of inflammation and altering dermal fibroblast extracellular matrix (ECM) production. Methods: C57BL/6J murine (male♀ 6-10wk) splenocytes were enriched for CD4+ cells and sorted into IL-10 producing subsets (Treg; Tr1). In vitro, dermal fibroblasts were co-cultured with Treg or Tr1, and fibrotic (Col-1A, a-SMA), ECM remodeling (Hyaluronan synthases (HAS) 1-3) and inflammatory (IL-13Ra2) markers were analyzed at 24h (qRT-PCR). In vivo, we performed 6mm bilateral dorsal full-thickness wounds on SCID mice (female, 8-10wk), followed by adoptive transfer of 10^6 CD4+, Treg, or Tr1 cells. Wounds and spleens were harvested at days 7, 28. Graft uptake, wound closure (H&E), myofibroblasts (a-SMA), inflammatory profile (Luminex; IHC), fibrosis (trichrome), and cellular interactions with imaging mass cytometry (IMC) were analyzed. Results: Treg (1,918-pg/ml) and Tr1 (1,164-pg/ml) showed elevated IL-10 production by ELISA. In the co-culture experiments, Col-1A level of Tr1 and Treg treated fibroblasts was reduced by 52.4% and 43.5%, and a-SMA was reduced by 25.2% and 44.7%, respectively as compared to untreated fibroblasts. HAS2, a producer of high molecular weight hyaluronan implicated in regeneration, increased 3.11-fold and 2.95-fold; and IL-13Ra2 increased 3.66-fold and 1.94-fold with Tr1 and Treg respectively by qPCR. In vivo, amongst all, Tr1 treated mice showed expedited wound closure at d7 and significantly greater aSMA (IHC), though both CD4+ and Tr1 treatments resulted in significantly reduced collagen content at d28 (trichrome). Wounds of mice treated with CD4+, Tr1, and Treg showed reduced F4/80 compared to untreated wounds (IHC). D7 wounds demonstrated decreased IL-6, MCP-1, and GM-CSF in the mice treated with Tr1 and Treg (Luminex). IMC performed on d7 wounds allowed simultaneous visualization of IL-10, CD4+ cells, F4/80 and CD206 positive macrophages providing preliminary insight into their juxtacrine signaling mechanisms. Conclusions: IL-10 producing CD4+ subsets regulate fibroblast ECM deposition and inflammatory cytokine balance to attenuate fibrosis. Promoting Tr1 recruitment and IL-10 production in wounds may be a therapeutic target to promote regenerative wound healing. Our use of IMC demonstrates the novel utility of this advanced technology, with the capacity to measure up to 40 targets in a single histological section, for uncovering the spatiotemporal interactions of immune cells and fibroblasts in dermal wounds.

YIA8

Novel Antibiotic-Free Synthetic Wound Matrix Prevents Wound Infection And Promotes Healing
Ana Tellechea, Kushee-Nidhi Kumar, Ravi K. Mekala, Yewoo Lee, Seyedeh Zahra M. Madani, Hung-Yi Liu, Manav Mehta
Gel4Med Inc, Lowel, MA, USA
Novel Antibiotic-Free Synthetic Wound Matrix Prevents Wound Infection And Promotes Healing

Ana Tellechea, Kushee-Nidhi Kumar, Ravi K. Mekala, Yewoo Lee, Seyedeh Zahra M. Madani, Hung-Yi Liu, Manav Mehta Gel4Med Inc, Lowell, MA, USA

Background: Chronic wounds affect 6-8 million patients in the US, resulting in >$25 billion annual healthcare expenses, mostly due to antimicrobial resistance and infection recurrence. We developed a novel antibiotic-free Synthetic Wound Matrix (SWM) and evaluated its antimicrobial activity and wound healing potential. Methods: SWM was manufactured as sterile self-assembly peptide hydrogels (0.75 and 1.5 w/v%). Antimicrobial effectiveness was tested in vitro using standard assays. Antibiofilm activity was evaluated in vitro and ex vivo (porcine skin model). Antibacterial activity was confirmed in CD-1 mice. Briefly, 5 mm diameter full-thickness wounds were inoculated with MRSA (10^4-10^6 CFU/wound), treated with SWM, collagen, silver gel, or left untreated, and tissue was processed to quantify bioburden at 6, 24, and 72 hours. A swine model was used to evaluate healing potential. 2 cm full-thickness wounds were created in the dorsal skin, treated with (i) PBS, (ii) SWM 0.75%, (iii) SWM 1.5%, (iv) collagen, or (v) silver gel, and monitored up to 14 days. Wound closure and re-epithelialization were analyzed using Tissue Analytics platform and histopathology. Results: In vitro, SWM eliminated >5 log10 CFU of several clinically relevant Gram-positive (Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus haemolyticus, MRSA) and Gram-negative (Pseudomonas aeruginosa (PaO1), ESBL Escherichia coli, ESBL Klebsiella pneumoniae, Acinetobacter baumannii) bacteria by 24 hours. In a timekill study, SWM eradicated 6 log10 CFU of MRSA and PaO1 within 20 minutes. Moreover, SWM cleared 6 log10 CFU of two model fungal pathogens, Candida albicans and Aspergillus fumigatus. Notably, when tested against established PaO1 and/or MRSA biofilms, SWM’s effectiveness was comparable to bleach and superior to multiple silver-containing gels. Activity against PaO1 biofilm was confirmed in ex vivo pig skin after 3 days of treatment. In CD-1 mouse wounds inoculated with MRSA (10^4 CFU/wound), SWM reduced wound bioburden compared to all groups (n>=8, p<0.0001 vs. untreated, p<0.0001 vs. collagen, p<0.01 vs. silver gel) at 24 hours. SWM's wound bioburden reduction was sustained after 72 hours, as well as at higher doses of bacterial inoculum (10^6 CFU/wound). From all treatment groups, SWM-treated swine wounds showed the greatest extent of closure (92% and 94% average wound closure for 0.75% and 1.5%, respectively, n=5), and resulted in healthy granulation tissue with no signs of necrotic or sloughy tissue. Importantly, SWM was the only treatment achieving complete wound re-epithelialization within 14 days. Conclusions: SWM shows broad-spectrum antibacterial and antifungal activity including against multidrug-resistant organisms, as well as biocompatibility and wound healing potential.

YIA9

ETRS WINNER No-GC in Pericytes as Modulator of Angiogenesis in Skin Fibrosis and How to Generate a Human IPSC Derived 3D in Vitro Test system
Amelie Reigl1, Florian Groeber-Becker2, Andreas Friebe1, Dieter Groneberg1
1Institute of Physiology, Julius-Maximilians University Würzburg, Germany 2Institute of Tissue Engineering and Regenerative Medicine, Würzburg, Germany
No-GC in Pericytes as Modulator of Angiogenesis in Skin Fibrosis and How to Generate a Human IPSC Derived 3D in Vitro Test system

Amelie Reigl, Florian Groeber-Becker2, Andreas Friebe1, Dieter Groneberg1 1Institute of Physiology, Julius-Maximilians University Würzburg, Germany, 2Institute of Tissue Engineering and Regenerative Medicine, Würzburg, Germany

Background: Angiogenesis is a crucial phase in skin fibrosis and wound healing. There is evidence that nitric oxide (NO) plays an important role in angiogenesis during skin fibrosis. NO-sensitive guanylyl cyclase (NO-GC) is the main target for NO. In skin, the exact cell type expressing NO-GC as well its role in angiogenesis has yet to be identified. For this reason, a suitable murine model as well as a human in vitro test system are necessary. Methods: To investigate the function of NO-GC in skin, we used the bleomycin model of skin fibrosis combined with reporter mice. These were used to lineage trace NO-GC+ cells within the course of the fibrotic reaction. In addition, we used human in vitro 3D full-thickness skin equivalents (FTSE) and iPSC-derived skin organoids to optimize the in vitro test system. Results: We found NO-GC expression in skin pericytes indicated by colocalisation with the pericyte marker PDGFRb and closely associated to CD31+ endothelial cells in the skin. Tamoxifen-induced expression of the reporter dye tdTomato under the control of SMMHC (smooth muscle myosin heavy chain) promotor was found to colocalize with NO-GC+ pericytes. They were mainly located within a collagen IV structure. Collagen (col III and IV) was increased and a higher density of fibroblasts could be shown. Interestingly, the tdTomato labeled pericytes remain in the collagen IV matrix. On the other side, we pharmacological modulate NO-GC+ cells in the human 3D FTSE during in vitro wound healing and optimized FTSE by adding iPSC derived cells to study angiogenesis. Conclusion: We assume that NOGC+ pericytes constitute an interesting therapeutic target to treat skin fibrosis. In the next step, we want to identify the role of NO-GC using additional promotor-specific lineage tracing of NOGC+ cells and knockdown of NO-GC in the mouse model. The lack of in vitro vascularization is crucial for further research; therefore, we add a vascular network using iPSC to optimize the 3D in vitro test systems.