MetfOrmin BenefIts Lower Extremities with Intermittent Claudication (MOBILE IC): randomized clinical trial protocol.

BACKGROUND: Peripheral artery disease (PAD) affects over 230 million people worldwide and is due to systemic atherosclerosis with etiology linked to chronic inflammation, hypertension, and smoking status. PAD is associated with walking impairment and mobility loss as well as a high prevalence of coronary and cerebrovascular disease. Intermittent claudication (IC) is the classic presenting symptom for PAD, although many patients are asymptomatic or have atypical presentations. Few effective medical therapies are available, while surgical and exercise therapies lack durability. Metformin, the most frequently prescribed oral medication for Type 2 diabetes, has salient anti-inflammatory and promitochondrial properties. We hypothesize that metformin will improve function, retard the progression of PAD, and improve systemic inflammation and mitochondrial function in non-diabetic patients with IC. METHODS: 200 non-diabetic Veterans with IC will be randomized 1:1 to 180-day treatment with metformin extended release (1000 mg/day) or placebo to evaluate the effect of metformin on functional status, PAD progression, cardiovascular disease events, and systemic inflammation. The primary outcome is 180-day maximum walking distance on the 6-min walk test (6MWT). Secondary outcomes include additional assessments of functional status (cardiopulmonary exercise testing, grip strength, Walking Impairment Questionnaires), health related quality of life (SF-36, VascuQoL), macro- and micro-vascular assessment of lower extremity blood flow (ankle brachial indices, pulse volume recording, EndoPAT), cardiovascular events (amputations, interventions, major adverse cardiac events, all-cause mortality), and measures of systemic inflammation. All outcomes will be assessed at baseline, 90 and 180 days of study drug exposure, and 180 days following cessation of study drug. We will evaluate the primary outcome with linear mixed-effects model analysis with covariate adjustment for baseline 6MWT, age, baseline ankle brachial indices, and smoking status following an intention to treat protocol. DISCUSSION: MOBILE IC is uniquely suited to evaluate the use of metformin to improve both systematic inflammatory responses, cellular energetics, and functional outcomes in patients with PAD and IC. TRIAL REGISTRATION: The prospective MOBILE IC trial was publicly registered (NCT05132439) November 24, 2021.

Association of Metformin Use During Hospitalization and Mortality in Critically Ill Adults With Type 2 Diabetes Mellitus and Sepsis.

OBJECTIVES: Whether metformin exposure is associated with improved outcomes in patients with type 2 diabetes mellitus and sepsis. DESIGN: Retrospective cohort study. SETTING: Patients admitted to ICUs in 16 hospitals in Pennsylvania from October 2008 to December 2014. PATIENTS: Adult critical ill patients with type 2 diabetes mellitus and sepsis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We conducted a retrospective cohort study to compare 90-day mortality in diabetic patients with sepsis with and without exposure to metformin during hospitalization. Data were obtained from the electronic health record of a large healthcare system in Pennsylvania from October 2008 to December 2014, on patients admitted to the ICU at any of the 16 hospitals within the system. The primary outcome was mortality at 90 days. The absolute and adjusted odds ratio (OR) with 95% CI were calculated in a propensity score-matched cohort. Among 14,847 patients with type 2 diabetes mellitus and sepsis, 682 patients (4.6%) were exposed to metformin during hospitalization and 14,165 (95.4%) were not. Within a total of 2,691 patients subjected to propensity score-matching at a 1:4 ratio, exposure to metformin (n = 599) was associated with decreased 90-day mortality (71/599, 11.9% vs 475/2,092, 22.7%; OR, 0.46; 95% CI, 0.35-0.60), reduced severe acute kidney injury (50% vs 57%; OR, 0.75; 95% CI, 0.62-0.90), less Major Adverse Kidney Events at 1 year (OR, 0.27; 95% CI, 0.22-0.68), and increased renal recovery (95% vs 86%; OR, 6.43; 95% CI, 3.42-12.1). CONCLUSIONS: Metformin exposure during hospitalization is associated with a decrease in 90-day mortality in patients with type 2 diabetes mellitus and sepsis.

Early Prehospital Tranexamic Acid Following Injury Is Associated With a 30-day Survival Benefit: A Secondary Analysis of a Randomized Clinical Trial.

OBJECTIVE: We sought to characterize the timing of administration of prehospital tranexamic acid (TXA) and associated outcome benefits. BACKGROUND: TXA has been shown to be safe in the prehospital setting post-injury. METHODS: We performed a secondary analysis of a recent prehospital randomized TXA clinical trial in injured patients. Those who received prehospital TXA within 1 hour (EARLY) from time of injury were compared to those who received prehospital TXA beyond 1 hour (DELAYED). We included patients with a shock index of >0.9. Primary outcome was 30-day mortality. Kaplan-Meier and Cox Hazard regression were utilized to characterize mortality relationships. RESULTS: EARLY and DELAYED patients had similar demographics, injury characteristics, and shock severity but DELAYED patients had greater prehospital resuscitation requirements and longer prehospital times. Stratified Kaplan-Meier analysis demonstrated significant separation for EARLY patients (N = 238, log-rank chi-square test, 4.99; P = 0.03) with no separation for DELAYED patients (N = 238, log-rank chi-square test, 0.04; P = 0.83). Stratified Cox Hazard regression verified, after controlling for confounders, that EARLY TXA was associated with a 65% lower independent hazard for 30-day mortality [hazard ratio (HR) 0.35, 95% confidence interval (CI) 0.19-0.65, P = 0.001] with no independent survival benefit found in DELAYED patients (HR 1.00, 95% CI 0.63-1.60, P = 0.999). EARLY TXA patients had lower incidence of multiple organ failure and 6-hour and 24-hour transfusion requirements compared to placebo. CONCLUSIONS: Administration of prehospital TXA within 1 hour from injury in patients at risk of hemorrhage is associated with 30-day survival benefit, lower incidence of multiple organ failure, and lower transfusion requirements.

Prehospital Plasma during Air Medical Transport in Trauma Patients at Risk for Hemorrhagic Shock.

BACKGROUND: After a person has been injured, prehospital administration of plasma in addition to the initiation of standard resuscitation procedures in the prehospital environment may reduce the risk of downstream complications from hemorrhage and shock. Data from large clinical trials are lacking to show either the efficacy or the risks associated with plasma transfusion in the prehospital setting. METHODS: To determine the efficacy and safety of prehospital administration of thawed plasma in injured patients who are at risk for hemorrhagic shock, we conducted a pragmatic, multicenter, cluster-randomized, phase 3 superiority trial that compared the administration of thawed plasma with standard-care resuscitation during air medical transport. The primary outcome was mortality at 30 days. RESULTS: A total of 501 patients were evaluated: 230 patients received plasma (plasma group) and 271 received standard-care resuscitation (standard-care group). Mortality at 30 days was significantly lower in the plasma group than in the standard-care group (23.2% vs. 33.0%; difference, -9.8 percentage points; 95% confidence interval, -18.6 to -1.0%; P=0.03). A similar treatment effect was observed across nine prespecified subgroups (heterogeneity chi-square test, 12.21; P=0.79). Kaplan-Meier curves showed an early separation of the two treatment groups that began 3 hours after randomization and persisted until 30 days after randomization (log-rank chi-square test, 5.70; P=0.02). The median prothrombin-time ratio was lower in the plasma group than in the standard-care group (1.2 [interquartile range, 1.1 to 1.4] vs. 1.3 [interquartile range, 1.1 to 1.6], P<0.001) after the patients' arrival at the trauma center. No significant differences between the two groups were noted with respect to multiorgan failure, acute lung injury-acute respiratory distress syndrome, nosocomial infections, or allergic or transfusion-related reactions. CONCLUSIONS: In injured patients at risk for hemorrhagic shock, the prehospital administration of thawed plasma was safe and resulted in lower 30-day mortality and a lower median prothrombin-time ratio than standard-care resuscitation. (Funded by the U.S. Army Medical Research and Materiel Command; PAMPer ClinicalTrials.gov number, NCT01818427 .).

Activation of AMP-activated protein kinase during sepsis/inflammation improves survival by preserving cellular metabolic fitness.

The purpose was to determine the role of AMPK activation in the renal metabolic response to sepsis, the development of sepsis-induced acute kidney injury (AKI) and on survival. In a prospective experimental study, 167 10- to 12-week-old C57BL/6 mice underwent cecal ligation and puncture (CLP) and human proximal tubule epithelial cells (TEC; HK2) were exposed to inflammatory mix (IM), a combination of lipopolysaccharide (LPS) and high mobility group box 1 (HMGB1). Renal/TEC metabolic fitness was assessed by monitoring the expression of drivers of oxidative phosphorylation (OXPHOS), the rates of utilization of OXPHOS/glycolysis in response to metabolic stress, and mitochondrial function by measuring O2 consumption rates (OCR) and the membrane potential (Δψm ). Sepsis/IM resulted in AKI, increased mortality, and in renal AMPK activation 6-24 hours after CLP/IM. Pharmacologic activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or metformin during sepsis improved the survival, while AMPK inhibition with Compound C increased mortality, impaired mitochondrial respiration, decreased OCR, and disrupted TEC metabolic fitness. AMPK-driven protection was associated with increased Sirt 3 expression and restoration of metabolic fitness. Renal AMPK activation in response to sepsis/IM is an adaptive mechanism that protects TEC, organs, and the host by preserving mitochondrial function and metabolic fitness likely through Sirt3 signaling.

Traumatic injury results in prolonged circulation of ultralarge von Willebrand factor and a reduction in ADAMTS13 activity.

BACKGROUND: Increases in plasma von Willebrand Factor (VWF) levels, accompanied by decreases in the metalloprotease ADAMTS13, have been demonstrated soon after traumatic injury while downstream effects remain unclear. STUDY DESIGN AND METHODS: A cohort of 37 injured trauma patients from a randomized control trial investigating the use of prehospital plasma transfusion were analyzed for activity and antigen levels of ADAMTS13 and VWF at 0 and 24 hours after admission. Relevant clinical data were abstracted from the medical records. Trauma patient plasma was analyzed via agarose gel electrophoresis to evaluate the effects of injury on VWF multimer composition compared to healthy controls. RESULTS: von Willebrand factor levels were elevated at presentation (189% [110%-263%] vs. 95% [74%-120%]), persisting through 24 hours (213% [146%-257%] vs. 132% [57%-160%]), compared to healthy controls. Ultralarge VWF (UL-VWF) forms were elevated in trauma patients at both 0 and 24 hours, when compared to pooled normal plasma (10.0% [8.9%-14.3%] and 11.3% [9.1%-21.2%], respectively, vs. 0.6%). Circulating plasma ADAMTS13 activity was decreased at 0 hours (66% [47%-86%] vs. 100% [98%-100%]) and at 24 hours (72.5% [56%-87.3%] vs. 103% [103%-103%]) in trauma patients. ADAMTS13 activity independently predicted the development of coagulopathy and correlated with international normalized ratio, thromboelastography values, injury severity, and blood product transfusion. CONCLUSION: Traumatic injury is associated with acute coagulopathy that is characterized by increased UL-VWF multimers and reduction in ADAMTS13, which correlates with blood loss, transfusion requirement, and injury severity. These findings suggest the potential for future trials targeting ADAMTS13 repletion to enhance clearance of VWF multimers.

Systemic vasoprotection by inhaled carbon monoxide is mediated through prolonged alterations in monocyte/macrophage function.

Carbon monoxide (CO) is anti-inflammatory and protective in models of disease. Its actions in vitro are short-lived but are sustained in vivo. We hypothesize that systemic CO can mediate prolonged phenotype changes in vivo, with a focus on macrophages (Mφs). Mφs isolated from CO treated rats responded to lipopolysaccharide (LPS) with increased IL6, IL10 and iNOS expression but decreased TNF. Conditioned media (CM) collected from peritoneal Mφs isolated from CO treated rats stimulated endothelial cell (EC) proliferation versus CM from Mφs from air treated rats. This effect was mediated by Mφ released VEGF and HMGB1. Inhaled CO reduced LPS induced Mφ M1 inflammatory phenotype for up to 5 days. Mitochondrial oxygen consumption in LPS treated Mφs from CO treated mice was preserved compared to LPS treated Mφs from control mice. Finally, transient reduction of inflammatory cells at the time of inhaled CO treatment eliminated the vasoprotective effect of CO in a rodent carotid injury model. Thus, inhaled CO induces a prolonged mixed phenotype change in Mφs, and potentially other inflammatory cells, that contribute to vasoprotection. These findings demonstrate the ability of inhaled CO to modify Mφs in a sustained manner to mediate its therapeutic actions, supporting the translational potential of inhaled CO.

Patient and surrogate attitudes via an interviewer-administered survey on exception from informed consent enrollment in the Prehospital Air Medical Plasma (PAMPer) trial.

OBJECTIVES: With increased focus on early resuscitation methods following injury to improve patient outcomes, studies are employing exception from informed consent (EFIC) enrollment. Few studies have assessed patients' opinions following participation in an EFIC study, and none have been conducted within the realm of traumatic hemorrhage. We surveyed those patients and surrogates previously enrolled in the Prehospital Air Medical Plasma (PAMPer) Trial to clarify their opinions related to consent and emergency research. METHODS: Telephone calls were made between January-June 2019 to all patients who were enrolled under EFIC in the PAMPer study at the Pittsburgh site (169 of the 501 total patients enrolled, May 2014-Oct 2017) and their surrogates. Questions gauging approval of EFIC enrollment were asked before discussion of PAMPer trial outcomes, after disclosure of positive outcomes, and after a hypothetical negative trial outcome was proposed. RESULTS: Of the total 647 telephone calls made, ninety-three interviews, reflecting 70 of 169 patient enrollments, were conducted. This included 13 in which only the patient was interviewed, 23 in which the patient and a surrogate were interviewed, and 34 in which only a surrogate was interviewed. Nearly half (48.4%) of respondents did not recall their personal or family member enrollment in the study. No patients or surrogates recalled hearing about the study through community consultation or being aware of opt out procedures. Patients and surrogates were glad they were enrolled (90.3%), agreed with EFIC use for their personal enrollment (88.17%), and agreed with the general use of EFIC for the PAMPer study (81.7%). Disclosure of the true positive PAMPer study outcome resulted in a significant increase in opinions regarding personal enrollment, EFIC for personal enrollment, and EFIC for general enrollment (all p < 0.001). Disclosure of a hypothetical neutral or negative study outcome resulted in significant decreases in opinions regarding EFIC for personal enrollment (p = 0.003) and EFIC for general enrollment (p < 0.001). CONCLUSIONS: Clinical trial participants with traumatic hemorrhagic shock enrolled with EFIC, and surrogates of such participants, are generally accepting of EFIC. The results of the trial in which EFIC was utilized significantly affected patient and surrogate agreement with personal and general EFIC enrollment.

Blue light reduces organ injury from ischemia and reperfusion.

Evidence suggests that light and circadian rhythms profoundly influence the physiologic capacity with which an organism responds to stress. However, the ramifications of light spectrum on the course of critical illness remain to be determined. Here, we show that acute exposure to bright blue spectrum light reduces organ injury by comparison with bright red spectrum or ambient white fluorescent light in two murine models of sterile insult: warm liver ischemia/reperfusion (I/R) and unilateral renal I/R. Exposure to bright blue light before I/R reduced hepatocellular injury and necrosis and reduced acute kidney injury and necrosis. In both models, blue light reduced neutrophil influx, as evidenced by reduced myeloperoxidase (MPO) within each organ, and reduced the release of high-mobility group box 1 (HMGB1), a neutrophil chemotactant and key mediator in the pathogenesis of I/R injury. The protective mechanism appeared to involve an optic pathway and was mediated, in part, by a sympathetic (ß3 adrenergic) pathway that functioned independent of significant alterations in melatonin or corticosterone concentrations to regulate neutrophil recruitment. These data suggest that modifying the spectrum of light may offer therapeutic utility in sterile forms of cellular injury.

Blue Light Enhances Bacterial Clearance and Reduces Organ Injury During Sepsis.

OBJECTIVES: The physiology of nearly all mammalian organisms are entrained by light and exhibit circadian rhythm. The data derived from animal studies show that light influences immunity, and these neurophysiologic pathways are maximally entrained by the blue spectrum. Here, we hypothesize that bright blue light reduces acute kidney injury by comparison with either bright red or standard, white fluorescent light in mice subjected to sepsis. To further translational relevance, we performed a pilot clinical trial of blue light therapy in human subjects with appendicitis. DESIGN: Laboratory animal research, pilot human feasibility trial. SETTING: University basic science laboratory and tertiary care hospital. SUBJECTS: Male C57BL/6J mice, adult (> 17 yr) patients with acute appendicitis. INTERVENTIONS: Mice underwent cecal ligation and puncture and were randomly assigned to a 24-hour photoperiod of bright blue, bright red, or ambient white fluorescent light. Subjects with appendicitis were randomized to receive postoperatively standard care or standard care plus high-illuminance blue light. MEASUREMENTS AND MAIN RESULTS: Exposure to bright blue light enhanced bacterial clearance from the peritoneum, reduced bacteremia and systemic inflammation, and attenuated the degree of acute kidney injury. The mechanism involved an elevation in cholinergic tone that augmented tissue expression of the nuclear orphan receptor REV-ERBα and occurred independent of alterations in melatonin or corticosterone concentrations. Clinically, exposure to blue light after appendectomy was feasible and reduced serum interleukin-6 and interleukin-10 concentrations. CONCLUSIONS: Modifying the spectrum of light may offer therapeutic utility in sepsis.

Calcium/calmodulin-dependent protein kinase regulates the PINK1/Parkin and DJ-1 pathways of mitophagy during sepsis.

During sepsis and shock states, mitochondrial dysfunction occurs. Consequently, adaptive mechanisms, such as fission, fusion, and mitophagy, are induced to eliminate damaged portions or entire dysfunctional mitochondria. The regulatory PINK1/Parkin and DJ-1 pathways are strongly induced by mitochondrial depolarization, although a direct link between loss of mitochondrial membrane potential (ΔΨ) and mitophagy has not been identified. Mitochondria also buffer Ca2+, and their buffering capacity is dependent on ΔΨ Here, we characterize a role for calcium/calmodulin-dependent protein kinase (CaMK) I in the regulation of these mechanisms. Loss of ΔΨ with either pharmacologic depolarization or LPS leads to Ca2+-dependent mitochondrial recruitment and activation of CaMKI that precedes the colocalization of PINK1/Parkin and DJ-1. CaMKI is required and serves as both a PINK1 and Parkin kinase. The mechanisms operate in both immune and nonimmune cells and are induced in in vivo models of endotoxemia, sepsis, and hemorrhagic shock. These data support the idea that CaMKI links mitochondrial stress with the PINK1/Parkin and DJ-1 mechanisms of mitophagy.-Zhang, X., Yuan, D., Sun, Q., Xu, L., Lee, E., Lewis, A. J., Zuckerbraun, B. S., Rosengart, M. R. Calcium/calmodulin-dependent protein kinase regulates the PINK1/Parkin and DJ-1 pathways of mitophagy during sepsis.

ADAMTS13: origins, applications, and prospects.

ADAMTS13 is an enzyme that acts by cleaving prothrombotic von Willebrand factor (VWF) multimers from the vasculature in a highly regulated manner. In pathologic states such as thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathies (TMAs), VWF can bind to the endothelium and form large multimers. As the anchored VWF chains grow, they provide a greater surface area to bind circulating platelets (PLTs), generating unique thrombi that characterize TTP. This results in microvasculature thrombosis, obstruction of blood flow, and ultimately end-organ damage. Initial presentations of TTP usually occur in an acute manner, typically developing due to an autoimmune response toward, or less commonly a congenital deficiency of, ADAMTS13. Triggers for TMAs that can be associated with ADAMTS13 deficiency, including TTP, have been linked to events that place a burden on hemostatic regulation, such as major trauma and pregnancy. The treatment plan for cases of suspected TTP consists of emergent therapeutic plasma exchange that is continued on a daily basis until normalization of PLT counts. However, a subset of these patients does not respond favorably to standard therapies. These patients necessitate a better understanding of their diseases for the advancement of future therapeutic options. Given ADAMTS13's key role in the cleavage of VWF and the prevention of PLT-rich thrombi within the microvasculature, future treatments may include anti-VWF therapeutics, recombinant ADAMTS13 infusions, and ADAMTS13 expression via gene therapy.

Peroxisome Proliferator-activated Receptor-γ Coactivator 1-α (PGC1α) Protects against Experimental Murine Colitis.

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is the primary regulator of mitochondrial biogenesis and was recently found to be highly expressed within the intestinal epithelium. PGC1α is decreased in the intestinal epithelium of patients with inflammatory bowel disease, but its role in pathogenesis is uncertain. We now hypothesize that PGC1α protects against the development of colitis and helps to maintain the integrity of the intestinal barrier. We selectively deleted PGC1α from the intestinal epithelium of mice by breeding a PGC1α(loxP/loxP) mouse with a villin-cre mouse. Their progeny (PGC1α(ΔIEC) mice) were subjected to 2% dextran sodium sulfate (DSS) colitis for 7 days. The SIRT1 agonist SRT1720 was used to enhance PGC1α activation in wild-type mice during DSS exposure. Mice lacking PGC1α within the intestinal epithelium were more susceptible to DSS colitis than their wild-type littermates. Pharmacologic activation of PGC1α successfully ameliorated disease and restored mitochondrial integrity. These findings suggest that a depletion of PGC1α in the intestinal epithelium contributes to inflammatory changes through a failure of mitochondrial structure and function as well as a breakdown of the intestinal barrier, which leads to increased bacterial translocation. PGC1α induction helps to maintain mitochondrial integrity, enhance intestinal barrier function, and decrease inflammation.

Impact of Volume Change Over Time on Trauma Mortality in the United States.

OBJECTIVE: To evaluate the association of trauma center volume change over time with mortality. BACKGROUND: Regionalization of trauma systems assumes a volume-outcome relationship for severe injury. Whereas this has been shown for cross-sectional volume, it is unclear whether volume changes over time translate into predictable outcome changes. METHODS: Retrospective cohort study of severely injured (injury severity score >15) patients from the National Trauma Databank 2000 to 2012. A center-level standardized mortality ratio (SMR) was constructed (ratio of observed to expected deaths). Expected mortality was obtained from multilevel logistic regression model, adjusting for demographics, mechanism, vital signs, and injury severity. Center-level percent volume change was assessed across early (2000-2006) and late (2007-2012) periods. Longitudinal panel modeling evaluated association between annual SMR change and volume change over preceding years. RESULTS: There were 839,809 patients included from 287 centers. Each 1% increase in volume was associated with 73% increased odds of improving SMR over time [odds ratio (OR) 1.73; 95% confidence interval (CI) 1.03-2.91; P = 0.03]. Each 1% decrease in volume was associated with 2-fold increase in odds of worsening SMR over time (OR 2.14; 95% CI 1.07-4.26, P = 0.03). Significant improvement in the SMR emerged after 3 or more preceding years of increasing volume (SMR change -0.008; 95% CI -0.015, -0.002; P = 0.01). This benefit occurred only in centers that were level I or II verified. CONCLUSIONS: Increasing volume was associated with improving outcomes, whereas decreasing volume was associated with worsening outcomes. High-level trauma center infrastructure seems to facilitate the volume-outcome relationship. The trauma center designation process should consider volume changes in the overall system.

CaMKIV-dependent preservation of mTOR expression is required for autophagy during lipopolysaccharide-induced inflammation and acute kidney injury.

Autophagy, an evolutionarily conserved homeostasis process regulating biomass quantity and quality, plays a critical role in the host response to sepsis. Recent studies show its calcium dependence, but the calcium-sensitive regulatory cascades have not been defined. In this study, we describe a novel mechanism in which calcium/calmodulin-dependent protein kinase IV (CaMKIV), through inhibitory serine phosphorylation of GSK-3ß and inhibition of FBXW7 recruitment, prevents ubiquitin proteosomal degradation of mammalian target of rapamycin (mTOR) and thereby augments autophagy in both the macrophage and the kidney. Under the conditions of sepsis studied, mTOR expression and activity were requisite for autophagy, a paradigm countering the current perspective that prototypically, mTOR inhibition induces autophagy. CaMKIV-mTOR-dependent autophagy was fundamentally important for IL-6 production in vitro and in vivo. Similar mechanisms were operant in the kidney during endotoxemia and served a cytoprotective role in mitigating acute kidney injury. Thus, CaMKIV-mTOR-dependent autophagy is conserved in both immune and nonimmune/parenchymal cells and is fundamental for the respective functional and adaptive responses to septic insult.

Xanthine Oxidoreductase Function Contributes to Normal Wound Healing.

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H2O2, 0.15%) or allopurinol (30 µg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H2O2 and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H2O2 restored wound healing in tungsten-fed mice. In vitro, nitrite and H2O2 both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.

Benzyl alcohol attenuates acetaminophen-induced acute liver injury in a Toll-like receptor-4-dependent pattern in mice.

UNLABELLED: Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in industrialized countries. Understanding the mechanisms of APAP-induced liver injury as well as other forms of sterile liver injury is critical to improve the care of patients. Recent studies demonstrate that danger signaling and inflammasome activation play a role in APAP-induced injury. The aim of these investigations was to test the hypothesis that benzyl alcohol (BA) is a therapeutic agent that protects against APAP-induced liver injury by modulation of danger signaling. APAP-induced liver injury was dependent, in part, on Toll-like receptor (TLR)9 and receptor for advanced glycation endproducts (RAGE) signaling. BA limited liver injury over a dose range of 135-540 µg/g body weight or when delivered as a pre-, concurrent, or post-APAP therapeutic. Furthermore, BA abrogated APAP-induced cytokines and chemokines as well as high-mobility group box 1 release. Moreover, BA prevented APAP-induced inflammasome signaling as determined by interleukin (IL)-1ß, IL-18, and caspase-1 cleavage in liver tissues. Interestingly, the protective effects of BA on limiting liver injury and inflammasome activation were dependent on TLR4 signaling, but not TLR2 or CD14. Cell-type-specific knockouts of TLR4 were utilized to further determine the protective mechanisms of BA. These studies found that TLR4 expression specifically in myeloid cells (LyzCre-tlr4-/-) were necessary for the protective effects of BA. CONCLUSION: BA protects against APAP-induced acute liver injury and reduced inflammasome activation in a TLR4-dependent manner. BA may prove to be a useful adjunct in the treatment of APAP and other forms of sterile liver injury.

Inhaled, nebulized sodium nitrite protects in murine and porcine experimental models of hemorrhagic shock and resuscitation by limiting mitochondrial injury.

OBJECTIVE: The cellular injury that occurs in the setting of hemorrhagic shock and resuscitation (HS/R) affects all tissue types and can drive altered inflammatory responses. Resuscitative adjuncts hold the promise of decreasing such injury. Here we test the hypothesis that sodium nitrite (NaNO2), delivered as a nebulized solution via an inhalational route, protects against injury and inflammation from HS/R. METHODS: Mice underwent HS/R to a mean arterial pressure (MAP) of 20 or 25 mmHg. Mice were resuscitated with Lactated Ringers after 90-120 min of hypotension. Mice were randomized to receive nebulized NaNO2 via a flow through chamber (30 mg in 5 mL PBS). Pigs (30-35 kg) were anesthetized and bled to a MAP of 30-40 mmHg for 90 min, randomized to receive NaNO2 (11 mg in 2.5 mL PBS) nebulized into the ventilator circuit starting 60 min into the hypotensive period, followed by initial resuscitation with Hextend. Pigs had ongoing resuscitation and support for up to four hours. Hemodynamic data were collected continuously. RESULTS: NaNO2 limited organ injury and inflammation in murine hemorrhagic shock. A nitrate/nitrite depleted diet exacerbated organ injury, as well as mortality, and inhaled NaNO2 significantly reversed this effect. Furthermore, NaNO2 limited mitochondrial oxidant injury. In porcine HS/R, NaNO2 had no significant influence on shock induced hemodynamics. NaNO2 limited hypoxia/reoxia or HS/R-induced mitochondrial injury and promoted mitochondrial fusion. CONCLUSION: NaNO2 may be a useful adjunct to shock resuscitation based on its limitation of mitochondrial injury.

Adenosine monophosphate-activated protein kinase activation protects against sepsis-induced organ injury and inflammation.

BACKGROUND: Mortality in sepsis is most often attributed to the development of multiple organ failure. In sepsis, inflammation-mediated endothelial activation, defined as a proinflammatory and procoagulant state of the endothelial cells, has been associated with severity of disease. Thus, the objective of this study was to test the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation limits inflammation and endothelium activation to protect against organ injury in sepsis. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is an adenosine monophosphate analog, has been used to upregulate activity of AMPK. Compound C is a cell-permeable pyrrazolopyrimidine compound that inhibits AMPK activity. METHODS: Wild-type mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice were randomized to vehicle, AICAR, or compound C. Mouse kidney endothelial cells were used for in vitro experiments. Renal and liver function were determined by serum cystatin C, blood urea nitrogen (BUN), creatinine, and alanine aminotransferase. Serum cytokines were measured by enzyme-linked immunosorbent assay. Microvascular injury was determined using Evans blue dye and electron microscopy. Immunohistochemistry was used to measure protein levels of phospho-AMPK (p-AMPK), microtubule-associated protein 1A/1B-light chain 3 (LC3), and intracellular adhesion molecule. LC3 levels were used as a measure of autophagosome formation. RESULTS: AICAR decreased liver and kidney injury induced by CLP and minimized cytokine elevation in vivo and in vitro. CLP increased renal and hepatic phosphorylation of AMPK and autophagic signaling as determined by LC3. Inhibition of AMPK with compound C prevented CLP-induced autophagy and exacerbated tissue injury. Additionally, CLP led to endothelial injury as determined by electron microscopy and Evans blue dye extravasation, and AICAR limited this injury. Furthermore, AICAR limited CLP and lipopolysaccharide (LPS)-induced upregulation of intracellular adhesion molecule in vivo and in vitro and decreased LPS-induced neutrophil adhesion in vitro. CONCLUSIONS: In this model, activation of AMPK was protective, and AICAR minimized organ injury by decreasing inflammatory cytokines and endothelial activation. These data suggest that AMPK signaling influences sepsis or LPS-induced endothelial activation and organ injury.

CaMKIα regulates AMP kinase-dependent, TORC-1-independent autophagy during lipopolysaccharide-induced acute lung neutrophilic inflammation.

Autophagy is an evolutionarily conserved cytoplasmic process regulated by the energy rheostats mammalian target of rapamycin and AMP kinase (AMPK) that recycles damaged or unused proteins and organelles. It has been described as an important effector arm of immune cells. We have shown that the cytoplasmically oriented calcium/calmodulin-dependent protein kinase (CaMK)Iα regulates the inflammatory phenotype of the macrophage (M). In this study, we hypothesize that CaMKIα mediates M autophagy. LPS induced autophagy in RAW 264.7 cells and murine peritoneal M that was attenuated with biochemical CaMK inhibition or CaMKIα small interfering RNA (siRNA). Inhibition of CaMKIα reduced LPS-induced p-Thr(172)AMPK and target of rapamycin complex-1 activity, and expression of a constitutively active CaMKIα but not a kinase-deficient mutant induced p-Thr(172)AMPK and autophagy that was attenuated by the AMPK inhibitor compound C. Coimmunoprecipitation and in vitro kinase assays demonstrated that CaMKIα activates AMPK, thereby inducing ATG7, which also localizes to this CaMKIα/AMPK complex. During LPS-induced lung inflammation, C57BL/6 mice receiving CaMKIα(siRNA) displayed reduced lung and bronchoalveolar immune cell autophagy that correlated with reduced neutrophil recruitment, myeloperoxidase activity, and air space cytokine concentration. Independently inhibiting autophagy, using siRNA targeting the PI3K VPS34, yielded similar reductions in lung autophagy and neutrophil recruitment. Thus, a novel CaMKIα/AMPK pathway is rapidly activated in M exposed to LPS and regulates an early autophagic response, independent of target of rapamycin complex-1 inhibition. These mechanisms appear to be operant in vivo in orchestrating LPS-induced lung neutrophil recruitment and inflammation.