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Topical Ozone Accelerates Diabetic Wound Healing by Promoting Re-Epithelialization through the Activation of IGF1R-EGFR Signaling
Summary: Ozonated oil increases the healing of chronic diabetic wounds, but the underlying mechanisms remain unclear. We investigated the effect of topical ozonated oil on wound healing in mice with diabetes with diet-induced obesity and further elucidated the role of EGFR and IGF1R signaling in diabetic wound healing. We found that topical ozonated oil accelerated wound healing; increased phosphorylation of IGF1R, EGFR, and VEGFR; and improved vascularization at the wound leading edge in mice with diabetes with diet-induced obesity. Exposure of normal epidermal keratinocytes to ozonated medium (20 μM for 2 hours daily) increased cell proliferation and migration distance by increasing phosphorylation of IGF1R and EGFR and downstream phosphoinositide 3-kinase, protein kinase B, and extracellular signal-regulated kinase. These findings shed light on the mechanism for topical ozone action in chronic wounds and support its potential therapeutic application.
Cutaneous innervation in impaired diabetic wound healing
Summary: Type 2 diabetes is associated with several potential comorbidities, among them impaired wound healing, chronic ulcerations, and the requirement for lower extremity amputation. Disease-associated abnormal cellular responses, infection, immunological and microvascular dysfunction, and peripheral neuropathy are implicated in the pathogenesis of the wound healing impairment and the diabetic foot ulcer. The skin houses a dense network of sensory nerve afferents and nerve-derived modulators, which communicate with epidermal keratinocytes and dermal fibroblasts bidirectionally to effect normal wound healing after trauma. However, the mechanisms through which cutaneous innervation modulates wound healing are poorly understood, especially in humans. Better understanding of these mechanisms may provide the basis for targeted treatments for chronic diabetic wounds. This review provides an overview of wound healing pathophysiology with a focus on neural involvement in normal and diabetic wound healing, as well as future therapeutic perspectives to address the unmet needs of diabetic patients with chronic wounds.
Epidermal SR-A Complexes Are Lipid Raft Based and Promote Nucleic Acid Nanoparticle Uptake
Qian Song, Xiao-Qi Wang, Thomas R Holmes, Michael Bonkowski, Eric W Roth, Adam Ponedal, Chad Mirkin, Amy S Paller
Summary: Scavenger receptors clear pathogens, transport lipid, and mediate polyanionic ligand uptake in macrophages, but their expression and role in the skin are poorly understood. Although the epidermal barrier typically excludes nucleic acid entry, topically applied, spherically arranged oligonucleotide nanoconjugates (spherical nucleic acids [SNAs]) penetrate mouse skin, three-dimensional (3D) skin equivalents, and human skin. We explored the mechanism of SNA uptake in normal human epidermal keratinocytes and 3D skin equivalents. Normal human epidermal keratinocytes and 3D raft treatment with SR-A inhibitors reduced SNA uptake by >80%. The human epidermis expresses SR-As SCARA3 and, to a lesser extent, MARCO. Simultaneous lentiviral knockdown of SCARA3 and MARCO reduced SNA uptake in normal human epidermal keratinocytes and 3D rafts after topical application, affirming a role for SR-As in SNA uptake and 3D raft penetration. Incubation of normal human epidermal keratinocytes at 4oC or with sodium azide prevented SNA uptake, suggesting active endocytosis. Endocytosis inhibitors, immunofluorescence, immunoprecipitation, and knockdown studies localized functional SR-As to FLOT-1-containing lipid rafts throughout the epidermis and CAV-1-containing rafts only in the upper epidermis. These studies suggest a central role for SR-A complexes in epidermal lipid rafts in mediating the uptake of nucleic acid‒laden nanoparticles.
Gene Regulation Using Spherical Nucleic Acids to Treat Skin Disorders
Summary: Spherical nucleic acids (SNAs) are nanostructures consisting of nucleic acids in a spherical configuration, often around a nanoparticle core. SNAs are advantageous as gene-regulating agents compared to conventional gene therapy owing to their low toxicity, enhanced stability, uptake by virtually any cell, and ability to penetrate the epidermal barrier. In this review we: (i) describe the production, structure and properties of SNAs; (ii) detail the mechanism of SNA uptake in keratinocytes, regulated by scavenger receptors; and (iii) report how SNAs have been topically applied and intralesionally injected for skin disorders. Specialized SNAs called nanoflares can be topically applied for gene-based diagnosis (scar vs. normal tissue). Topical SNAs directed against TNFα and interleukin-17A receptor reversed psoriasis-like disease in mouse models and have been tested in Phase 1 human trials. Furthermore, SNAs targeting ganglioside GM3 synthase accelerate wound healing in diabetic mouse models. Most recently, SNAs targeting toll-like receptor 9 are being used in Phase 2 human trials via intratumoral injection to induce immune responses in Merkel cell and cutaneous squamous cell carcinoma. Overall, SNAs are a valuable tool in bench-top and clinical research, and their advantageous properties, including penetration into the epidermis after topical delivery, provide new opportunities for targeted therapies.
Flotillin and AP2A1/2 Promote IGF-1 Receptor Association with Clathrin and Internalization in Primary Human Keratinocytes
Duncan Hieu M Dam, Sophia A Jelsma, Jeong Min Yu, Haoming Liu, Betty Kong, Amy S Paller
Summary: Considering that IGF-1 receptor (IGF1R) signaling promotes keratinocyte proliferation, migration, and survival. However, the mechanism of IGF1R endocytosis in normal keratinocytes remains unclear. In the study we found that keratinocytes express Flot-1 that is more responsive to low IGF-1 concentrations, whereas the AP2A1/2 pathway predominates at higher IGF-1 concentrations. Selective association of IGF1R–Flot-1–clathrin with Rab4, but IGF1R–AP2A1/2–clathrin with Rab11, implicates Flot-1 as the adaptor for faster recycling and AP2A1/2 as the adaptor for slower IGF1R recycling. These dual pathways, particularly flotillin-dependent, clathrin-mediated endocytosis, provide a new avenue for drug targeting in disorders with aberrant regulation of IGF1R signaling.
Gangliosides in Diabetic Wound Healing
Duncan Hieu M Dam, Amy S Paller
Summary: This review outlines the organized series of complicated biological and molecular phenomena is required for normal skin wound healing. These processes depend on normal cellular responses to cytokines, growth factors, and other mediators, such as clotting factors, prostaglandins, free radicals, and nitric oxide. In diabetic ulcers, impaired responses to these molecules lead to abnormalities in vascularization, innervation, matrix reconstruction, and reepithelialization of wounds. keratinocyte migration and proliferation on an extracellular matrix is critical in reepithelialization, but the response to growth factors is blunted in diabetes, including the insulin/IGF-1signaling axis. Ganglioside GM3, a sialylated epidermal glycosphingolipid, has been identified as a key mediator of the inhibition of insulin/IGF-1 signaling in response to factors, such as tumor necrosis factor-alpha (TNF-α) and hyperglycemia. Decreased expression of GM3 and the enzyme required for its synthesis, GM3 synthase (GM3S), leads to increased insulin/IGF-1 receptor signaling and accelerated keratinocyte migration, even in the presence of high glucose levels. GM3 depletion in GM3S knockout diabetic mice and diet-induced diabetic mice treated topically with nanoconstruct-mediated GM3S-targeting gene regulation also accelerates wound healing. These recent observations, coupled with evidence that GM3 depletion reverses distal innervation abnormalities in diabetic mice, suggest that GM3-depleting strategies are a promising new approach for human diabetic wounds.
Ganglioside GM3 Mediates Glucose-Induced Suppression of IGF-1 Receptor-Rac1 Activation to Inhibit Keratinocyte Motility
Duncan Hieu M Dam, Xiao-Qi Wang, Sarah Sheu, Mahima Vijay, Desmond Shipp, Luke Miller, Amy S Paller
Summary: Activation of insulin-like growth factor-1 (IGF-1) receptor (IGF1R) signaling induces keratinocyte migration, but little is known about its regulation, including in diabetic wounds. GM3, a lipid raft ganglioside synthesized by GM3 synthase (GM3S), regulates receptor signaling. In diabetic mice, knockout or topically applied nanoconstruct-mediated knockdown of GM3S promotes wound edge IGF1R phosphorylation and re-epithelialization. Through modulating GM3 expression, we explored the role of GM3 in regulating human keratinocyte IGF1R signaling. Increases in GM3 and GM3S expression, including by exposure to high glucose, inhibit keratinocyte migration and IGF-1-induced chemotaxis in association with inhibition of IGF1R phosphorylation, suppression of Rac1 signaling, and activation of RhoA signaling. In contrast, GM3 depletion accelerates cell migration; increases cell velocity, displacement, and persistence; and activates IGF1R-Rac1 signaling. These data implicate GM3 in mediating glucose-induced suppression of IGF1R-Rac1 signaling. Furthermore, our findings provide evidence of a pivotal role for GM3-induced insulin resistance in impairing keratinocyte migration and reinforce the previously published studies in diabetic mice supporting GM3-depleting strategies as an approach for accelerating the healing of human diabetic wounds.
Targeting the IL-17 Receptor Using Liposomal Spherical Nucleic Acids as Topical Therapy for Psoriasis
Haoming Liu, Richard S Kang, Katherine Bagnowski, Jeong Min Yu, Sara Radecki, Weston L Daniel, Bart R Anderson, Subbarao Nallagatla, Andrew Schook, Rishika Agarwal, David A Giljohann, Amy S Paller
Summary: The activation of T helper 17 signaling plays a critical role in psoriasis pathogenesis, and systemically-administered IL-17 inhibitors are highly effective therapy for moderate-to-severe disease. We generated topically-delivered gene-regulating nanoconstructs, comprised of spherically-arrayed antisense DNA (liposomal spherical nucleic acids [L-SNAs]), which are able to penetrate human skin to knock down cutaneous gene targets. Topically-applied L-SNAs targeting the gene encoding the mouse IL-17A receptor (Il17ra) reversed the development of psoriasis clinically, histologically, and transcriptionally in imiquimod-treated psoriasis-like mouse skin. Il17ra L-SNAs reduced the modified PASI by 74% versus controls and decreased epidermal thickness by 56%. Il17ra L-SNA reduced Il17ra protein expression by 75% and significantly decreased the mRNA expression of psoriasis markers, including Defb4, Il17c, S100a7, Pi3, Krt16, and Tnfa versus scrambled spherical nucleic acid (Scr SNA) controls. A human IL17RA L-SNA penetrates 3-dimensional cultures and normal human explants to knock down IL17RA mRNA by 63% and 66%, respectively. After topical application to psoriatic 3-dimensional rafts, anti-human IL17RA L-SNAs reduced the expression of IL17RA (by 72%) and the IL-17-induced genes IL17C (by 85%), DEFB4 (by 83%), TNFA (by 77%), and PI3 (by 65%) versus scrambled L-SNA and vehicle controls (all P < 0.001). Taken together, these data suggest that targeted suppression of IL17RA is a promising new topical treatment strategy for psoriasis.
Topically Delivered Tumor Necrosis Factor-α–Targeted Gene Regulation for Psoriasis
Katherine T.Lewandowski, Rebecca Thiede, Nicholas Guido, Weston L.Daniel, RichardKang, Mara-Isel Guerrero-Zayas, Mark A.Seeger, Xiao-QiWang, David A.Giljohann, Amy S.Paller
Summary: We have generated a TNF-α–suppressing antisense spherical nucleic acid (SNA), a promising construct to emerge from the field of nanotechnology (Banga et al., 2014, Cutler et al., 2012, Giljohann et al., 2009, Zheng et al., 2012). These 3-dimensional (3D) arrangements of densely packed and radially oriented oligonucleotides (see Supplementary Figure S1 online) impart properties distinct from linear nucleic acids, especially skin penetration capability without physical or chemical skin disruptors and increased cellular uptake. SNAs use scavenger receptors to enter cells, whereas other oligonucleotide delivery systems (e.g., cationic lipids or polymers) often disrupt anionic cell membranes for delivery (Choi et al., 2013). Liposomal-cored SNAs (L-SNAs) are physiologically compatible but share characteristics of the early-generation trackable gold-cored SNAs (Banga et al., 2014, Randeria et al., 2015, Zheng et al., 2012). Using both a human 3D cytokine-induced raft model and the mouse imiquimod (IMQ)-generated psoriasis-like model, we found that TNF-targeting L-SNAs prevent the phenotype clinically, histologically, and transcriptionally, suggesting a topical treatment paradigm for psoriasis.
siRNA-based spherical nucleic acids reverse impaired wound healing in diabetic mice by ganglioside GM3 synthase knockdown
Pratik S Randeria, Mark A Seeger, Xiao-Qi Wang, Heather Wilson, Desmond Shipp, Chad A Mirkin, Amy S Paller
Summary: Spherical nucleic acid (SNA) gold nanoparticle conjugates (13-nm-diameter gold cores functionalized with densely packed and highly oriented nucleic acids) dispersed in Aquaphor have been shown to penetrate the epidermal barrier of both intact mouse and human skin, enter keratinocytes, and efficiently down-regulate gene targets. ganglioside-monosialic acid 3 synthase (GM3S) is a known target that is overexpressed in diabetic mice and responsible for causing insulin resistance and impeding wound healing. GM3S SNAs increase keratinocyte migration and proliferation as well as insulin and insulin-like growth factor-1 (IGF1) receptor activation under both normo- and hyperglycemic conditions. The topical application of GM3S SNAs (50 nM) to splinted 6-mm-diameter full-thickness wounds in diet-induced obese diabetic mice decreases local GM3S expression by >80% at the wound edge through an siRNA pathway and fully heals wounds clinically and histologically within 12 d, whereas control-treated wounds are only 50% closed. Granulation tissue area, vascularity, and IGF1 and EGF receptor phosphorylation are increased in GM3S SNA-treated wounds. These data capitalize on the unique ability of SNAs to naturally penetrate the skin and enter keratinocytes without the need for transfection agents. Moreover, the data further validate GM3 as a mediator of the delayed wound healing in type 2 diabetes and support regional GM3 depletion as a promising therapeutic direction.