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 (Exo κ-GFP ) using tissue nanotransfection and were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exo κ-GFP were also characterized. Unlike skin exosome, wound-edge Exo κ-GFP demonstrated characteristic N-glycan ions with abundance of low base pair RNA and were selectively engulfed by woundmacrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exo κ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exo κ-GFP in vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNP κ ) were designed with 94.3% encapsulation *

BackgroundIn multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), inflammation is perpetuated by both infiltrating leukocytes and astrocytes. Recent work implicated SUR1-TRPM4 channels, expressed mostly by astrocytes, in murine EAE. We tested the hypothesis that pharmacological inhibition of SUR1 during the chronic phase of EAE would be beneficial.MethodsEAE was induced in mice using myelin oligodendrocyte glycoprotein (MOG) 35–55. Glibenclamide (10 μg/day) was administered beginning 12 or 24 days later. The effects of treatment were determined by clinical scoring and tissue examination. Drug within EAE lesions was identified using bodipy-glibenclamide. The role of SUR1-TRPM4 in primary astrocytes was characterized using patch clamp and qPCR. Demyelinating lesions from MS patients were studied by immunolabeling and immunoFRET.ResultsAdministering glibenclamide beginning 24 days after MOG35–55 immunization, well after clinical symptoms had plateaued, improved clinical scores, reduced myelin loss, inflammation (CD45, CD20, CD3, p65), and reactive astrocytosis, improved macrophage phenotype (CD163), and decreased expression of tumor necrosis factor (TNF), B-cell activating factor (BAFF), chemokine (C-C motif) ligand 2 (CCL2) and nitric oxide synthase 2 (NOS2) in lumbar spinal cord white matter. Glibenclamide accumulated within EAE lesions, and had no effect on leukocyte sequestration. In primary astrocyte cultures, activation by TNF plus IFNγ induced de novo expression of SUR1-TRPM4 channels and upregulated Tnf, Baff, Ccl2, and Nos2 mRNA, with glibenclamide blockade of SUR1-TRPM4 reducing these mRNA increases. In demyelinating lesions from MS patients, astrocytes co-expressed SUR1-TRPM4 and BAFF, CCL2, and NOS2.ConclusionsSUR1-TRPM4 may be a druggable target for disease modification in MS.

Therapeutic VEGF replenishment has met with limited success for the management of critical limb threatening ischemia (CLTI). To improve outcomes of VEGF therapy we applied single-cell RNA sequencing technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic vs non-diabetic skin, phospholipase-C-Gamma-2 (PLCγ2) was down-regulated. The significance of PLCγ2 in VEGF mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In HMECs, VEGF enhanced mitochondrial function as indicated by elevation in oxygen consumption rate and extracellular acidification rate. VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type-1 (streptozotocin-injected) and type-2 (db/db) diabetic ischemic tissue. Non-viral topical tissue nanotransfection (TNT) delivery of CDH5 promoter driven PLCγ2-ORF promoted the rescue of hind-limb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with pro-angiogenic gene therapies. Taken together our study demonstrates that TNT mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.

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.