Despite improvements in awareness and treatment of type II diabetes mellitus (TIIDM), this disease remains a major source of morbidity and mortality worldwide, and prevalence continues to rise. ...Oxidative damage caused by free radicals has long been known to contribute to the pathogenesis and progression of TIIDM and its complications. Only recently, however, has the role of the Nrf2/Keap1/ARE master antioxidant pathway in diabetic dysfunction begun to be elucidated. There is accumulating evidence that this pathway is implicated in diabetic damage to the pancreas, heart, and skin, among other cell types and tissues. Animal studies and clinical trials have shown promising results suggesting that activation of this pathway can delay or reverse some of these impairments in TIIDM. In this review, we outline the role of oxidative damage and the Nrf2/Keap1/ARE pathway in TIIDM, focusing on current and future efforts to utilize this relationship as a therapeutic target for prevention, prognosis, and treatment of TIID.
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 the 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 nuclear factor erythroid-related factor 2 (Nrf2)/kelch-like erythroid cell-derived protein 1 (Keap1) pathway is dysregulated and functionally insufficient in diabetic BMSCs (dBMSCs). 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 dBMSCs 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.
Hair follicle neogenesis (HFN) occurs after large skin excisions in mice, serving as a rare regenerative model in mammalian wound healing. Wound healing typically results in fibrosis in mice and ...humans. We previously showed that small skin excisions in mice result in scarring devoid of HFN, displaying features of nonregenerative healing, and hedgehog (Hh) activation in the dermis of such wounds can induce HFN. In this study, we sought to verify the role of dermal Wnt/β-catenin signaling in HFN because this pathway is essential for hair follicle development but is also paradoxically well-characterized in fibrosis of adult wounds. By deletion of β-catenin in large wound myofibroblasts, we show that Wnt/β-catenin signaling is required for endogenous mechanisms of HFN. By utilizing a combined mouse model that simultaneously induces deletion of β-catenin and constitutive activation of Smoothened in myofibroblasts, we also found that β-catenin is required for Hh-driven dermal papilla formation. Transcriptome analysis confirms that Wnt/β-catenin and Hh pathways are activated in dermal papilla cells. Our results indicate that Wnt-active fibrotic status may also create a permissive state for the regenerative function of Hh, suggesting that activation of both Wnt and Hh pathways in skin wound fibroblasts must be ensured in future strategies to promote HFN.
Cutaneous wounds in patients with diabetes exhibit impaired healing due to physiological impediments and conventional care options are severely limited. Multipotent stromal cells (MSCs) have been ...touted as a powerful new therapy for diabetic tissue repair owing to their trophic activity and low immunogenicity. However, variations in sources and access are limiting factors for broader adaptation and study of MSC-based therapies. Amniotic fluid presents a relatively unexplored source of MSCs and one with wide availability. Here, we investigate the potential of amniotic fluid-derived multipotent stromal cells (AFMSCs) to restore molecular integrity to diabetic wounds, amend pathology and promote wound healing.
We obtained third trimester amniotic fluid from term cesarean delivery and isolated and expanded MSCs in vitro. We then generated 10 mm wounds in Lepr
diabetic mouse skin, and splinted them open to allow for humanized wound modeling. Immediately after wounding, we applied AFMSCs topically to the sites of injuries on diabetic mice, while media application only, defined as vehicle, served as controls. Post-treatment, we compared healing time and molecular and cellular events of AFMSC-treated, vehicle-treated, untreated diabetic, and non-diabetic wounds. A priori statistical analyses measures determined significance of the data.
Average time to wound closure was approximately 19 days in AFMSC-treated diabetic wounds. This was significantly lower than the vehicle-treated diabetic wounds, which required on average 27.5 days to heal (p < 0.01), and most similar to time of closure in wild type untreated wounds (an average of around 18 days). In addition, AFMSC treatment induced changes in the profiles of macrophage polarizing cytokines, resulting in a change in macrophage composition in the diabetic wound bed. We found no evidence of AFMSC engraftment or biotherapy induced immune response.
Treatment of diabetic wounds using amniotic fluid-derived MSCs encourages cutaneous tissue repair through affecting inflammatory cell behavior in the wound site. Since vehicle-treated diabetic wounds did not demonstrate accelerated healing, we determined that AFMSCs were therapeutic through their paracrine activities. Future studies should be aimed towards validating our observations through further examination of the paracrine potential of AFMSCs. In addition, investigations concerning safety and efficacy of this therapy in clinical trials should be pursued.
Abstract Therapeutics utilizing siRNA are currently limited by the availability of safe and effective delivery systems. Cutaneous diseases, specifically ones with significant genetic components are ...ideal candidates for topical siRNA based therapy but the anatomical structure of skin presents a considerable hurdle. Here, we optimized a novel liposome and protein hybrid nanoparticle delivery system for the topical treatment of diabetic wounds with severe oxidative stress. We utilized a cationic lipid nanoparticle (CLN) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the edge activator sodium cholate (NaChol), in a 6:1 ratio of DOTAP:NaChol (DNC). Addition of a cationic engineered supercharged coiled-coil protein (CSP) in a 10:1:1 ratio of DNC:CSP:siRNA produced a stable lipoproteoplex (LPP) nanoparticle, with optimal siRNA complexation, minimal cytotoxicity, and increased transfection efficacy. In a humanized murine diabetic wound healing model, our optimized LPP formulation successfully delivered siRNA targeted against Keap1 , key repressor of Nrf2 which is a central regulator of redox mechanisms. Application of LPP complexing si Keap1 restored Nrf2 antioxidant function, accelerated diabetic tissue regeneration, and augmented reduction-oxidation homeostasis in the wound environment. Our topical LPP delivery system can readily be translated into clinical use for the treatment of diabetic wounds and can be extended to other cutaneous diseases with genetic components.
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.
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•Epidermal Nrf2 initiates a regenerative response through Ccl2 regulation•Ccl2 release by basal stem/progenitor keratinocytes prompts macrophage trafficking•Ccl2 regulates EGF production in macrophages trafficked to the injury site•EGF from macrophages stimulates basal keratinocyte proliferation
Villarreal-Ponce et al. show that diabetic wounds fail to activate Nrf2 in keratinocytes, which functions in promoting an early regenerative response in basal keratinocytes by mediating macrophage trafficking through release of Ccl2. Keratinocyte-derived Ccl2 promotes macrophage production of EGF to induce keratinocyte proliferation to promote wound repair.
Though unmitigated oxidative stress in diabetic chronic non-healing wounds poses a major therapeutic challenge, currently, there are no effective pharmacological agents. We targeted the ...cytoprotective Nrf2/Keap1 pathway, which is dysfunctional in diabetic skin and the regenerative environment in the diabetic wound. We assessed the efficacy of a potent Nrf2-activator, RTA 408, a semi-synthetic oleanane triterpenoid, on accelerating diabetic wound healing.
Using Leprdb/dbmice, we made 10 mm-diameter excisional humanized wounds in dorsal skin. We administered RTA 408 formulations daily, and used ANOVA for comparison of time to closure, in vivo real-time ROS, histology, molecular changes.
We found that RTA 408, specifically a 0.1% formulation, significantly reduced wound healing time and increased wound closure rate. While either systemic or topical administration of RTA 408 is effective, wound closure time with the latter was far superior. RTA 408-treated diabetic wounds upregulated Nrf2 and downstream antioxidant genes, and exhibited well-vascularized granulation tissue that aided in re-epithelialization. Reintroduction of redox mechanisms via RTA 408-induced Nrf2 resulted in reduction of the oxidative status of wounds, to coordinate successful wound closure.
This preclinical study shows that promoting Nrf2-mediated antioxidant activity in the localized regenerative milieu of a diabetic wound markedly improves the molecular and cellular composition of diabetic wound beds. RTA 408 treats and corrects the irregularity in redox balance mechanisms involving Nrf2, in an avenue not explored previously for treatment of diabetic wounds and tissue regeneration. Our study supports development of RTA 408 as a therapeutic modality for chronic diabetic wounds.
Chronic hyperglycemia impairs intracellular redox homeostasis and contributes to impaired diabetic tissue regeneration. The Keap1/Nrf2 pathway is a critical regulator of the endogenous antioxidant ...response system, and its dysfunction has been implicated in numerous pathologies. Here we characterize the effect of chronic hyperglycemia on Nrf2 signaling within a diabetic cutaneous regeneration model. We characterized the effects of chronic hyperglycemia on the Keap1/Nrf2 pathway within models of diabetic cutaneous wound regeneration. We assessed reactive oxygen species (ROS) production and antioxidant gene expression following alterations in the Nrf2 suppressor Keap1 and the subsequent changes in Nrf2 signaling. We also developed a topical small interfering RNA (siRNA)-based therapy to restore redox homeostasis within diabetic wounds. Western blotting demonstrated that chronic hyperglycemia-associated oxidative stress inhibits nuclear translocation of Nrf2 and impairs activation of antioxidant genes, thus contributing to ROS accumulation. Keap1 inhibition increased Nrf2 nuclear translocation, increased antioxidant gene expression, and reduced ROS production to normoglycemic levels, both in vitro and in vivo. Topical siKeap1 therapy resulted in improved regenerative capacity of diabetic wounds and accelerated closure. We report that chronic hyperglycemia weakens the endogenous antioxidant response, and the consequences of this defect are manifested by intracellular redox dysregulation, which can be restored by Keap1 inhibition. Targeted siRNA-based therapy represents a novel, efficacious strategy to reestablish redox homeostasis and accelerate diabetic cutaneous tissue regeneration.
Exosomes, nanosized extracellular vesicles, may be key to translating multipotent stromal cell (MSC) therapy to the bedside. We previously found that nuclear factor erythroid 2-related factor 2 ...(Nrf2) regulates MSC promotion of tissue repair in diabetes. Here, we explore a novel role of Nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects MSCs have on wound healing in mice with type 2 diabetes. Exosomes were harvested by differential ultracentrifugation of conditioned human bone marrow derived MSC media. For Nrf2-active exosomes, MSCs were incubated with potent Nrf2 activator, CDDO-Im. MSC characterization demonstrated robust tri-lineage differentiation, >95% expression of positive markers (CD44/CD73/CD90/CD105/CD106), and <5% expression of negative markers (CD45/CD31/CD14/CD19/HLA-DR). Immunoblotting of exosome isolates demonstrates enrichment for CD81, CD9 and TSG101. Nanoparticle tracking analysis demonstrates that Nrf2-active MSCs increase exosome secretion by 54%, compared to Nrf2-baseline MSCs (p<0.05). Exosomes were injected into the wound margin of full-thickness wounds on Leprdb/db (db/db) mice. Both Nrf2-baseline and Nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p<0.05). This reduction eliminated the delay in closure time compared to wounds of C57/B6 mice. Nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated C57/B6 wounds. At day 10, exosome treated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of CD31+ vessels compared to vehicle-treated wounds. We conclude that activating Nrf2 in MSCs increases isolation efficiency by amplifying exosome yield and content. MSC exosome based therapies have potential for rapid translation to improve clinical wound healing outcomes.
Disclosure
J. Kuhn: None. A. Hassan: None. S. Sharma: None. J. Kwong: None. M. Rahman: None. S. Adam: None. J. Lee: None. A.P. Villarreal Ponce: None. P.S. Rabbani: None.
Funding
Plastic Surgery Foundation; Wound Healing Society; New York University Clinical and Translational Science Institute
The current standard of care for an alveolar cleft defect is an autogenous bone graft, typically from the iliac crest. Given the limitations of alveolar bone graft surgery, such as limited supply, ...donor site morbidity, graft failure, and need for secondary surgery, there has been growing interest in regenerative medicine strategies to supplement and replace traditional alveolar bone grafts. Though there have been preliminary clinical studies investigating bone tissue engineering methods in human subjects, lack of consistent results as well as limitations in study design make it difficult to determine the efficacy of these interventions. As the field of bone tissue engineering is rapidly advancing, reconstructive surgeons should be aware of the preclinical studies informing these regenerative strategies. We review preclinical studies investigating bone tissue engineering strategies in large animal maxillary or mandibular defects and provide an overview of scaffolds, stem cells, and osteogenic agents applicable to tissue engineering of the alveolar cleft. An electronic search conducted in the PubMed database up to December 2021 resulted in 35 studies for inclusion in our review. Most studies showed increased bone growth with a tissue engineering construct compared to negative control. However, heterogeneity in the length of follow up, method of bone growth analysis, and inconsistent use of positive control groups make comparisons across studies difficult. Future studies should incorporate a pediatric study model specific to alveolar cleft with long-term follow up to fully characterize volumetric defect filling, cellular ingrowth, bone strength, tooth movement, and implant support.
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