in-vivo-imaging-of-reactive-oxygen-species-in-a-murine-wound-model

<em>In Vivo</em> Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

New York University School of Medicine

The trafficking of circulating stem and progenitor cells to areas of tissue damage is poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) mediates homing of stem cells to bone marrow by binding to CXCR4 on circulating cells. SDF-1 and CXCR4 are expressed in complementary patterns during embryonic organogenesis and guide primordial stem cells to sites of rapid vascular expansion. However, the regulation of SDF-1 and its physiological role in peripheral tissue repair remain incompletely understood. Here we show that SDF-1 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of SDF-1 in ischemic tissue in direct proportion to reduced oxygen tension. HIF-1-induced SDF-1 expression increases the adhesion, migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue. Blockade of SDF-1 in ischemic tissue or CXCR4 on circulating cells prevents progenitor cell recruitment to sites of injury. Discrete regions of hypoxia in the bone marrow compartment also show increased SDF-1 expression and progenitor cell tropism. These data show that the recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by hypoxic gradients via HIF-1-induced expression of SDF-1.

Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.

The identification of bone marrow-derived endothelial progenitor cells has altered our understanding of new blood vessel growth and tissue regeneration. Previously, new blood vessel growth in the adult was thought to only occur through angiogenesis, the sprouting of new vessels from existing structures. However, it has become clear that circulating bone marrow-derived cells can form new blood vessels through a process of postnatal vasculogenesis, with endothelial progenitor cells selectively recruited to injured or ischemic tissue. How this process occurs has remained unclear. One common element in the different environments where vasculogenesis is believed to occur is the presence of a hypoxic stimulus. We have identified the chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 as critical mediators for the ischemia-specific recruitment of circulating progenitor cells. We have found that the endothelial expression of SDF-1 acts as a signal indicating the presence of tissue ischemia, and that its expression is directly regulated by hypoxia-inducible factor-1. Stromal cell-derived factor 1 is the only chemokine family member known to be regulated in this manner. Later events, including proliferation, patterning, and assembly of recruited progenitors into functional blood vessels, are also influenced by tissue oxygen tension and hypoxia. Interestingly, both SDF-1 and hypoxia are present in the bone marrow niche, suggesting that hypoxia may be a fundamental requirement for progenitor cell trafficking and function. As such, ischemic tissue may represent a conditional stem cell niche, with recruitment and retention of circulating progenitors regulated by hypoxia through differential expression of SDF-1.

Homing to hypoxia: HIF-1 as a mediator of progenitor cell recruitment to injured tissue.

Vascular complications in diabetes are a significant source of human morbidity and mortality, affecting multiple organ systems and persisting despite tight glucose control. Many of these complications can be linked to impairments in vasculogenesis, the process by which circulating and bone marrow-derived endothelial progenitor cells (EPCs) contribute to new vessel formation. Recent evidence suggests that hyperglycemia alone, through the mitochondrial overproduction of reactive oxygen species (ROS), can induce changes in gene expression and cellular behavior in diabetes. In this review, we examine how hyperglycemia-induced overproduction of ROS could explain EPC impairments observed in diabetes. Experimentally, impairments in EPC function prevent new blood vessel growth and are potentially reversible by manipulations to decrease ROS. Novel strategies aimed at reducing hyperglycemia-induced ROS may be a useful adjuvant to antihyperglycemic therapies in the restoration of vasculogenesis and the prevention of diabetic complications.

Hyperglycemia-induced reactive oxygen species and impaired endothelial progenitor cell function.

Treatment of metachronous lower extremity defects with delay and splitting of a previously advanced reverse sural artery flap.

Diabetic patients exhibit dysfunction of the normal wound healing process, leading to local ischemia by vascular occlusive disease as well as sustained increases in the proinflammatory cytokines and overproduction of reactive oxygen species (ROS). Of the many sources of ROS, the enzyme xanthine oxidase (XO) has been linked to overproduction of ROS in diabetic environment, and studies have shown that treatment with XO inhibitors decreases XO overactivity and XO-generated ROS. This study evaluates the role of XO in the diabetic wound and the impact of specifically inhibiting its activity on wound healing. Treatment of diabetic wounds with siXDH (xanthine dehydrogenase siRNA) decreased XDH mRNA expression by 51.6%, XO activity by 35.9%, ROS levels by 78.1%, pathologic wound burden by 31.5%, and accelerated wound healing by 7 days (23.3%). Polymerase chain reaction analysis showed that increased XO activity in wild-type wound may be due to XDH to XO conversion and/or XO phosphorylation, but not to gene transcription, whereas increased XO activity in diabetic wounds may also be from gene transcription. These results suggest that XO may be responsible for large proportion of elevated oxidative stress in the diabetic wound environment and that normalizing the metabolic activity of XO using targeted delivery of siXDH may decrease overproduction of ROS and accelerate wound healing in diabetic patients. 

Normalizing dysfunctional purine metabolism accelerates diabetic wound healing.

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.

Restoration of Nrf2 Signaling Normalizes the Regenerative Niche.

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 siKeap1 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.

Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing.

Intraoperative instrument recounts are performed to avoid retained foreign surgical items. These additional counts, however, beget risks of their own, including prolonged operative times, exposure to radiation, and increased cost. Our study aimed to identify factors that increase the likelihood of instrument recounts during plastic surgery procedures, and use our findings to guide potential solutions for preventing unnecessary recounts across all surgical fields.

How many people work in your operating room? An assessment of factors associated with instrument recounts within plastic surgery.

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 Nrf2/Keap1/ARE Pathway and Oxidative Stress as a Therapeutic Target in Type II Diabetes Mellitus.

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.

Targeted Nrf2 activation therapy with RTA 408 enhances regenerative capacity of diabetic wounds.

Widespread adoption of bariatric surgery in the treatment of obesity has led to greater numbers of patients seeking panniculectomy, including aged patients, who represent a rapidly growing proportion of the U.S population. Although the quality of life and functional benefits of abdominal panniculectomy have been established, its safety in patients 65 years and older has not been evaluated.

Advanced Age Is a Risk Factor for Complications Following Abdominal Panniculectomy.

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 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 Nrf2/Keap1 pathway is dysregulated and functionally insufficient in diabetic BMSCs. 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 diabetic BMSCs 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.

Daniel J. Ceradini

Hansjörg Wyss Department of Plastic Surgery

New York University School of Medicine

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

Daniel J. Ceradini

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Hansjörg Wyss Department of Plastic Surgery

New York University School of Medicine

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

Hansjörg Wyss Department of Plastic Surgery