Browsing by Author "Xuan, Yi"

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    Driving adult tissue repair via re-engagement of a pathway required for fetal healing
    (Elsevier, 2023) Ghatak, Subhadip; Khanna, Savita; Roy, Sashwati; Thirunavukkarasu, Mahesh; Pradeep, Seetur R.; Wulff, Brian C.; El Masry, Mohamed S.; Sharma, Anu; Palakurti, Ravichand; Ghosh, Nandini; Xuan, Yi; Wilgus, Traci A.; Maulik, Nilanjana; Yoder, Mervin C.; Sen, Chandan K.; Surgery, School of Medicine
    Fetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition in vitro and in vivo. Increased NPGPx expression induced increased SOX2 expression and β-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.
  • Thumbnail Image
    Item
    Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing
    (ACS, 2020-09) Zhou, Xiaoju; Brown, Brooke A.; Siegel, Amanda P.; El Masry, Mohamed S.; Zeng, Xuyao; Song, Woran; Das, Amitava; Khandelwal, Puneet; Clark, Andrew; Singh, Kanhaiya; Guda, Poornachander R.; Gorain, Mahadeo; Timsina, Lava; Xuan, Yi; Jacobson, Stephen C.; Novotny, Milos V.; Roy, Sashwati; Agarwal, Mangilal; Lee, Robert J.; Sen, Chandan K.; Clemmer, David E.; Ghatak, Subhadip; Surgery, School of Medicine
    Bidirectional cell–cell communication involving exosome-borne cargo such as miRNA has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte–macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT), and they were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low-base-pair RNA and was selectively engulfed by wound macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFPin vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to the murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to the TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, the TLNPκ/si-hnRNPA2B1 treated group showed a significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to the TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP is recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties postinjury.
  • Thumbnail Image
    Item
    Fabrication and use of silicon hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo
    (Springer Nature, 2021) Xuan, Yi; Ghatak, Subhadip; Clark, Andrew; Li, Zhigang; Khanna, Savita; Pak, Dongmin; Agarwal, Mangilal; Roy, Sashwati; Duda, Peter; Sen, Chandan K.; Surgery, School of Medicine
    Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In <1 s, these silicon hollow-needle arrays can be used to deliver plasmids to a predetermined specific depth in murine skin in response to pulsed nanoporation. Tissue nanotransfection eliminates the need to use viral vectors, minimizing the risk of genomic integration or cell transformation. The TNT chip fabrication process typically takes 5-6 d, and in vivo TNT takes 30 min. This protocol does not require specific expertise beyond a clean room equipped for basic nanofabrication processes.
  • Thumbnail Image
    Item
    Human fetal dermal fibroblast-myeloid cell diversity is characterized by dominance of pro-healing Annexin1-FPR1 signaling
    (Elsevier, 2023-08-02) Srivastava, Rajneesh; Singh, Kanhaiya; Abouhashem, Ahmed S.; Kumar, Manishekhar; Kacar, Sedat; Verma, Sumit S.; Mohanty, Sujit K.; Sinha, Mithun; Ghatak, Subhadip; Xuan, Yi; Sen, Chandan K.; Surgery, School of Medicine
    Fetal skin achieves scarless wound repair. Dermal fibroblasts play a central role in extracellular matrix deposition and scarring outcomes. Both fetal and gingival wound repair share minimal scarring outcomes. We tested the hypothesis that compared to adult skin fibroblasts, human fetal skin fibroblast diversity is unique and partly overlaps with gingival skin fibroblasts. Human fetal skin (FS, n = 3), gingiva (HGG, n = 13), and mature skin (MS, n = 13) were compared at single-cell resolution. Dermal fibroblasts, the most abundant cluster, were examined to establish a connectome with other skin cells. Annexin1-FPR1 signaling pathway was dominant in both FS as well as HGG fibroblasts and related myeloid cells while scanty in MS fibroblasts. Myeloid-specific FPR1-ORF delivered in murine wound edge using tissue nanotransfection (TNT) technology significantly enhanced the quality of healing. Pseudotime analyses identified the co-existence of an HGG fibroblast subset with FPR1high myeloid cells of fetal origin indicating common underlying biological processes.
  • Thumbnail Image
    Item
    Identification of a Biofilm-Inducible Wound Macrophage Subset Characterized by Unique MARCO Expression: Significance in Chronic Wound Infection
    (2023-08) Das Ghatak, Piya; Roy, Sashwati; Gordillo, Gayle M.; Khanna, Savita; Xuan, Yi
    Staphylococcus aureus (SA) biofilm infection, a common occurrence in human chronic wounds, exhibits sustained inflammation resulting in wound chronicity. Current studies claim that SA biofilm transforms host macrophages (m) towards an alternatively activated phenotype that suppresses the inflammatory response. Given a key role of m in wound healing, we investigate the phenotype and function of wound m (wm) in SA biofilm infection. To address the biofilm component of SA pathogenicity, two isogenic mutant strains of wild-type SA (USA300) were used that possess varying degrees of biofilm-forming ability. The strains, USA300::sarA (ΔsarA) and USA300::rexB (ΔrexB), have hypo- and hyper biofilm-forming ability, respectively. Mechanistic studies were performed on peripheral blood monocytes subjected to conditioned media (CM) derived from ΔsarA and ΔrexB biofilm cultures. scRNAseq and bulk RNAseq studies revealed presence of a unique m subtype that displays a distinct gene expression signature when exposed to CM derived from ΔrexB mutant as compared to CM from ΔsarA mutant. We identified macrophage receptors with collagenous structure (MARCO) as one of the highly abundant uniquely expressed biofilm-responsive genes in wm. MARCO, a member of the scavenger receptor, belongs to a larger group of pattern recognition receptors primarily involved in immunosurveillance and plays important roles in host defense and apoptotic cell clearance activities. scRNAseq and immunohistochemistry from human chronic biofilm-infected wounds demonstrated abundance of MARCO expression on wm. Significant upregulation of MARCO was observed in ΔrexB CM treated m in a dose and time-dependent manner as compared to ΔsarA CM treated m. Knockdown of biofilm-induced MARCO in m resulted in dysregulated m functions and inflammatory responses, suggesting its critical significance in biofilm infection. Immunohistochemistry studies in SA biofilm-infected porcine wounds validated the in vitro findings. In summary, this study identifies a SA biofilm inducible m receptor that has a significant role in wound chronicity.
  • Thumbnail Image
    Item
    Modeling the gene delivery process of the needle array-based tissue nanotransfection
    (Springer, 2022) Li, Zhigang; Xuan, Yi; Ghatak, Subhadip; Guda, Poornachander R.; Roy, Sashwati; Sen, Chandan K.; Surgery, School of Medicine
    Hollow needle array-based tissue nanotransfection (TNT) presents an in vivo transfection approach that directly translocate exogeneous genes to target tissues by using electric pulses. In this work, the gene delivery process of TNT was simulated and experimentally validated. We adopted the asymptotic method and cell-array-based model to investigate the electroporation behaviors of cells within the skin structure. The distribution of nonuniform electric field across the skin results in various electroporation behavior for each cell. Cells underneath the hollow microchannels of the needle exhibited the highest total pore numbers compared to others due to the stronger localized electric field. The percentage of electroporated cells within the skin structure, with pore radius over 10 nm, increases from 25% to 82% as the applied voltage increases from 100 to 150 V/mm. Furthermore, the gene delivery behavior across the skin tissue was investigated through the multilayer-stack-based model. The delivery distance increased nonlinearly as the applied voltage and pulse number increased, which mainly depends on the diffusion characteristics and electric conductivity of each layer. It was also found that the skin is required to be exfoliated prior to the TNT procedure to enhance the delivery depth. This work provides the foundation for transition from the study of murine skin to translation use in large animals and human settings.
  • Thumbnail Image
    Item
    Myogenic tissue nanotransfection improves muscle torque recovery following volumetric muscle loss
    (Nature, 2022) Clark, Andrew; Ghatak, Subhadip; Guda, Poornachander Reddy; El Masry, Mohamed S.; Xuan, Yi; Sato, Amy Y.; Bellido, Teresita; Sen, Chandan K.; Surgery, School of Medicine
    This work rests on our non-viral tissue nanotransfection (TNT) platform to deliver MyoD (TNT) to injured tissue in vivo. TNT was performed on skin and successfully induced expression of myogenic factors. TNT was then used as a therapy 7 days following volumetric muscle loss (VML) of rat tibialis anterior and rescued muscle function. TNT is promising as VML intervention.