AIM:Sepsis-induced myocardial injury (SIMI) may be associated with insufficient mitophagy in cardiomyocytes, but the exact mechanism involved remains unknown. Sirtuin 3 (Sirt3) is mainly found in the mitochondrial matrix and is involved in repairing mitochondrial function through means such as the activation of autophagy. Previously, we demonstrated that the annexin-A1 small peptide (ANXA1sp) can promote Sirt3 expression in mitochondria. In this study, we hypothesized that the activation of Sirt3 by ANXA1sp induces mitophagy, thereby providing a protective effect against SIMI in mice. METHODS:A mouse model of SIMI was established via cecal ligation and puncture. Intraperitoneal injections of ANXA1sp, 3TYP, and 3MA were administered prior to modeling. After successful modeling, IL-6, TNF-α, CK-MB, and CTn-I levels were measured; cardiac function was assessed using echocardiography; myocardial mitochondrial membrane potential, ROS, and ATP production were determined; myocardial mitochondrial ultrastructure was observed using transmission electron microscopy; and the expression levels of Sirt3 and autophagy-related proteins were detected using western blotting. RESULTS:ANXA1sp significantly reduced serum IL-6, TNF-α, CK-MB, and CTn-I levels; decreased myocardial ROS production; increased mitochondrial membrane potential and ATP synthesis; and improved myocardial mitochondrial ultrastructure in septic mice. Furthermore, ANXA1sp promoted Sirt3 expression and activated the AMPK-mTOR pathway to induce myocardial mitophagy. These protective effects of ANXA1sp were reversed upon treatment with the Sirt3 blocker, 3-TYP. CONCLUSION:ANXA1sp can reverse SIMI, and the underlying mechanism may be related to the activation of the AMPK-mTOR pathway following upregulation of Sirt3 by ANXA1sp, which, in turn, induces autophagy.

Sepsis-associated encephalopathy (SAE) is characterized by high incidence and mortality rates, with limited treatment options available. The underlying mechanisms and pathogenesis of SAE remain unclear. Annexin A1 (ANXA1), a membrane-associated protein, is involved in various in vivo pathophysiological processes. This study aimed to explore the neuroprotective effects and mechanisms of a novel bioactive ANXA1 tripeptide (ANXA1sp) in SAE. Forty Sprague-Dawley rats were randomly divided into four groups (n = 10 each): control, SAE (intraperitoneal injection of lipopolysaccharide), vehicle (SAE + normal saline), and ANXA1sp (SAE + ANXA1sp) groups. Changes in serum inflammatory factors (interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α]), hippocampal reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and adenosine triphosphate (ATP) levels were measured. The Morris water maze and Y maze tests were used to assess learning and memory capabilities in the rats. Further, changes in peroxisome proliferator-activated receptor-gamma (PPAR-γ) and apoptosis-related protein expression were detected using western blot. The IL-6, TNF-α, and ROS levels were significantly increased in the SAE group compared with the levels in the control group. Intraperitoneal administration of ANXA1sp led to a significant decrease in the IL-6, TNF-α, and ROS levels (p < 0.05). Compared with the SAE group, the ANXA1sp group exhibited reduced escape latency on day 5, a significant increase in the number of platform crossings and the percent spontaneous alternation, and significantly higher hippocampal MMP and ATP levels (p < 0.05). Meanwhile, the expression level of PPAR-γ protein in the ANXA1sp group was significantly increased compared with that in the other groups (p < 0.05). The expressions of apoptosis-related proteins (nuclear factor-kappa B [NF-κB], Bax, and Caspase-3) in the SAE and vehicle groups were significantly increased, with a noticeable decrease in Bcl-2 expression, compared with that noted in the control group. Moreover, the expressions of NF-κB, Bax, and Caspase-3 were significantly decreased in the ANXA1sp group, and the expression of Bcl-2 was markedly increased (p < 0.05). ANXA1sp can effectively reverse cognitive impairment in rats with SAE. The neuroprotective effect of ANXA1sp may be attributed to the activation of the PPAR-γ pathway, resulting in reduced neuroinflammatory response and inhibition of apoptosis.

Supplementary Figure 3 from Annexin 1 Induced by Anti-Inflammatory Drugs Binds to NF-κB and Inhibits Its Activation: Anticancer Effects In vitro and In vivo

Neuroinflammation is a growing hallmark of perioperative neurocognitive disorders (PNDs), including delirium and longer-lasting cognitive deficits. We have developed a clinically relevant orthopedic mouse model to study the impact of a common surgical procedure on the vulnerable brain. The mechanism underlying PNDs remains unknown. Here we evaluated the impact of surgical trauma on the NLRP3 inflammasome signaling, including the expression of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and IL-1β in the hippocampus of C57BL6/J male mice, adult (3-months) and aged (>18-months). Surgery triggered ASC specks formation in CA1 hippocampal microglia, but without inducing significant morphological changes in NLRP3 and ASC knockout mice. Since no therapies are currently available to treat PNDs, we assessed the neuroprotective effects of a biomimetic peptide derived from the endogenous inflammation-ending molecule, Annexin-A1 (ANXA1). We found that this peptide (ANXA1sp) inhibited postoperative NLRP3 inflammasome activation and prevented microglial activation in the hippocampus, reducing PND-like memory deficits. Together our results reveal a previously under-recognized role of hippocampal ANXA1 and NLRP3 inflammasome dysregulation in triggering postoperative neuroinflammation, offering a new target for advancing treatment of PNDs through the resolution of inflammation.

Background: Acute kidney injury (AKI) is one of the most common organ failures following surgery. We have developed a tripeptide mimetic (ANXA1sp) of the parent annexin A1 molecule that shows promise as an organ protectant limiting cellular stress; however, its potential as a kidney protective agent remains unexplored, and its mechanism of action is poorly understood. Our hypothesis was that ANXA1sp would limit kidney injury following surgical ischemic kidney injury. Methods: In a blinded fashion, wildtype mice were assigned to receive vehicle control or ANXA1sp one hour prior to and one hour after kidney vascular clamping. Our primary outcomes were markers of kidney injury and function as measured by serum creatinine and histologic injury scoring of kidney tissue sections. Immunofluorescence microscopy, real-time PCR, and Western blot were used to assess cell death, oxidative stress, and mitochondrial biomarkers. An in vitro model of oxygen-glucose deprivation in immortalized kidney tubule cells was used. Results: ANXA1sp given prior to and after ischemic kidney injury abrogated ischemic kidney injury. ANXA1sp limited cell death both in vivo and in vitro and abrogated oxidative stress following ischemia. ANXA1sp significantly increased the expression of markers associated with protective mitophagy and limited the expression of markers associated with detrimental mitochondrial fission. ANXA1sp upregulated the expression of the mitochondrial protectant sirtuin-3 (SIRT3) in the mitochondria of kidney tubular cells. Silencing of SIRT3 reversed ANXA1sp-mediated protection against hypoxic cell death. Conclusions: ANXA1sp limits kidney injury, upregulates SIRT3, and preserves mitochondrial integrity following ischemic kidney injury. ANXA1sp holds considerable promise as a perioperative kidney protectant prior to ischemia inducing surgery and kidney transplantation.

Neuroinflammation initiated by damage-associated molecular patterns, including high mobility group box 1 protein (HMGB1), has been implicated in adverse neurological outcomes following lethal hemorrhagic shock and polytrauma. Emergency preservation and resuscitation (EPR) is a novel method of resuscitation for victims of exsanguinating cardiac arrest, shown in preclinical studies to improve survival with acceptable neurological recovery. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has emerged as a key regulator of metabolic and energy stress response pathways in the brain and a pharmacological target to induce a neuronal pro-survival phenotype. This study aims to examine whether systemic administration of an Annexin-A1 bioactive peptide (ANXA1sp) could resolve neuroinflammation and induce sirtuin-3 regulated cytoprotective pathways in a novel rat model of exsanguinating cardiac arrest and EPR. Adult male rats underwent hemorrhagic shock and ventricular fibrillation, induction of profound hypothermia, followed by resuscitation and rewarming using cardiopulmonary bypass (EPR). Animals randomly received ANXA1sp (3 mg/kg, in divided doses) or vehicle. Neuroinflammation (HMGB1, TNFα, IL-6, and IL-10 levels), cerebral cell death (TUNEL, caspase-3, pro and antiapoptotic protein levels), and neurologic scores were assessed to evaluate the inflammation resolving effects of ANXA1sp following EPR. Furthermore, western blot analysis and immunohistochemistry were used to interrogate the mechanisms involved. Compared to vehicle controls, ANXA1sp effectively reduced expression of cerebral HMGB1, IL-6, and TNFα and increased IL-10 expression, which were associated with improved neurological scores. ANXA1sp reversed EPR-induced increases in expression of proapoptotic protein Bax and reduction in antiapoptotic protein Bcl-2, with a corresponding decrease in cerebral levels of cleaved caspase-3. Furthermore, ANXA1sp induced autophagic flux (increased LC3II and reduced p62 expression) in the brain. Mechanistically, these findings were accompanied by upregulation of the mitochondrial protein deacetylase Sirtuin-3, and its downstream targets FOXO3a and MnSOD in ANXA1sp-treated animals. Our data provide new evidence that engaging pro-resolving pharmacological strategies such as Annexin-A1 biomimetic peptides can effectively attenuate neuroinflammation and enhance the neuroprotective effects of EPR after exsanguinating cardiac arrest.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

Background: The goal of this study was to investigate the effects of pulmonary static inflation with 50% xenon on postoperative oxygen impairment during cardiopulmonary bypass (CPB) for Stanford type A acute aortic dissection (AAD).Methods: This prospective single-center nonrandomized controlled clinical trial included 100 adult patients undergoing surgery for Stanford type A AAD at an academic hospital in China. Fifty subjects underwent pulmonary static inflation with 50% oxygen from January 2013 to January 2014, and 50 underwent inflation with 50% xenon from January 2014 to December 2014. During CPB, the lungs were inflated with either 50% xenon (xenon group) or 50% oxygen (control group) to maintain an airway pressure of 5cm H2O. The primary outcome was oxygenation index (OI) value after intubation, and 10minutes and 6hours after the operation. The second outcome was cytokine and reactive oxygen species levels after intubation and 10minutes, 6hours, and 24hours after the operation.Results: Patients treated with xenon had lower OI levels compared to the control group before surgery (P= 0.002); however, there was no difference in postoperative values between the 2 groups. Following surgery, mean maximal OI values decreased by 18.8% and 33.8%, respectively, in the xenon and control groups. After surgery, the levels of interleukin-6 (IL-6), tumor necrosis factor alpha, and thromboxane B-2 decreased by 23.5%, 9.1%, and 30.2%, respectively, in the xenon group, but increased by 10.8%, 26.2%, and 26.4%, respectively, in the control group. Moreover, IL-10 levels increased by 28% in the xenon group and decreased by 7.5% in the control group. There were significant time and treatment-time interaction effects on methane dicarboxylic aldehyde (P= 0.000 and P= 0.050, respectively) and myeloperoxidase (P= 0.000 and P= 0.001 in xenon and control groups, respectively). There was no difference in hospital mortality and 1-year survival rate between the 2 groups.Conclusion: Pulmonary static inflationwith 50% xenon duringCPBcould attenuate OI decreases at the end of surgery for Stanford type A AAD. Thus, xenon may function by triggering anti-inflammatory responses and suppressing pro-inflammatory and oxidative effects.

Resolution agonists, including lipid mediators and peptides such as annexin A1 (ANXA1), are providing novel approaches to treat inflammatory conditions. Surgical trauma exerts a significant burden on the immune system that can affect and impair multiple organs. Perioperative cerebral injury after cardiac surgery is associated with significant adverse neurological outcomes such as delirium and postoperative cognitive dysfunction. Using a clinically relevant rat model of cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA), we tested the pro-resolving effects of a novel bioactive ANXA1 tripeptide (ANXA1sp) on neuroinflammation and cognition. Male rats underwent 2 h CPB with 1 h DHCA at 18 degrees C, and received vehicle or ANXA1sp followed by timed reperfusion up to postoperative day 7. Immortalized murine microglial cell line BV2 were treated with vehicle or ANXA1sp and subjected to 2 h oxygen-glucose deprivation followed by timed reoxygenation. Microglial activation, cell death, neuroinflammation, and NF-kappa B activation were assessed in tissue samples and cell cultures. Rats exposed to CPB and DHCA had evident neuroinflammation in various brain areas. However, in ANXA1sp-treated rats, microglial activation and cell death (apoptosis and necrosis) were reduced at 24 h and 7 days after surgery. This was associated with a reduction in key pro-inflammatory cytokines due to inhibition of NF-kappa B activation in the brain and systemically. Treated rats also had improved neurologic scores and shorter latency in the Morris water maze. In BV2 cells treated with ANXA1sp, similar protective effects were observed including decreased pro-inflammatory cytokines and cell death. Notably, we also found increased expression of ANXA1, which binds to NF-kappa B p65 and thereby inhibits its transcriptional activity. Our findings provide evidence that treatment with a novel pro-resolving ANXA1 tripeptide is neuroprotective after cardiac surgery in rats by attenuating neuroinflammation and may prevent postoperative neurologic complications.

Introduction: We reported cardioprotective efficacy of a novel Annexin-A1 peptide mimetic (Ac10-12, ANXA1sp) in multiple experimental models of cold myocardial ischemia-reperfusion (IR), across spe...

BACKGROUND:Hibernation is an adaptation to extreme environments known to provide organ protection against ischemia-reperfusion (I/R) injury. An unbiased systems approach was utilized to investigate hibernation-induced changes that are characteristic of the hibernator cardioprotective phenotype, by comparing the myocardial proteome of winter hibernating arctic ground squirrels (AGS), summer active AGS, and rats subjected to I/R, and further correlating with targeted metabolic changes. METHODS:In a well-defined rodent model of I/R by deep hypothermic circulatory arrest followed by 3 or 24 h of reperfusion or sham, myocardial protein abundance in AGS (hibernating summer active) and rats (n = 4 to 5/group) was quantified by label-free proteomics (n = 4 to 5/group) and correlated with metabolic changes. RESULTS:Compared to rats, hibernating AGS displayed markedly reduced plasma levels of troponin I, myocardial apoptosis, and left ventricular contractile dysfunction. Of the 1,320 rat and 1,478 AGS proteins identified, 545 were differentially expressed between hibernating AGS and rat hearts (47% up-regulated and 53% down-regulated). Gene ontology analysis revealed down-regulation in hibernating AGS hearts of most proteins involved in mitochondrial energy transduction, including electron transport chain complexes, acetyl CoA biosynthesis, Krebs cycle, glycolysis, and ketogenesis. Conversely, fatty acid oxidation enzymes and sirtuin-3 were up-regulated in hibernating AGS, with preserved peroxisome proliferator-activated receptor-α activity and reduced tissue levels of acylcarnitines and ceramides after I/R. CONCLUSIONS:Natural cardioprotective adaptations in hibernators involve extensive metabolic remodeling, featuring increased expression of fatty acid metabolic proteins and reduced levels of toxic lipid metabolites. Robust up-regulation of sirtuin-3 suggests that posttranslational modifications may underlie organ protection in hibernating mammals.

Introduction: Neuroinflammation triggered by sterile surgical trauma, cardiopulmonary bypass and ischemia/reperfusion (I/R) is a major factor in cerebral injury and cognitive decline after cardiac ...

INTRODUCTION:Pulmonary arterial hypertension (PAH) is a common and fatal complication of congenital heart disease (CHD). PAH-CHD increases the risk for postoperative complications. Recent evidence suggests that perioperative goal-directed hemodynamic optimization therapy (GDHOT) significantly improves outcomes in surgery patients. Standard GDHOT is based on major solution volume, vasodilators and inotropic therapy, while novel GDHOT is based on major vasopressor and inotropic therapy. Therefore, we tested whether standard or novel GDHOT improves surgical outcomes in PAH-CHD patients.METHODS:Forty PAH-CHD patients with a ventricular septal defect (VSD) and mean pulmonary arterial pressure (mPAP) >50 mmHg, who were scheduled for corrective surgery, were randomly assigned to 2 groups: SG (study group, n = 20) and CG (control group, n = 20). SG patients received perioperative hemodynamic therapy guided by novel GDHOT, while CG patients received standard GDHOT. Outcome data were recorded up to 28 days postoperatively. Ventilator time, length of ICU stay, and mortality were the primary endpoints.RESULTS:There were no significant differences in preoperative data, surgical procedure, and hospital mortality rates between the 2 groups. Time of mechanical ventilation and length of ICU stay were significantly shorter in SG patients compared to CG patients (P < .05, n = 20). Patients in SG showed a significantly increased systemic vascular resistance index and decreased cardiac index, but no change in pulmonary vascular resistance index at 12 and 24 hours after surgery compared to the controls (P < .05). Patients in SG had significantly decreased PAP, pulmonary arterial pressure/systemic arterial pressure (Pp/Ps), and RVSWI (right ventricular stroke work index) at 12 and 24 hours after surgery (P < .05, respectively). Patients in SG also showed significantly decreased central venous pressure at 4, 12, and 24 hours after surgery compared to those treated with standard protocol (P < .05).CONCLUSION:Our study provides clinical evidence that perioperative goal-directed hemodynamic optimization therapy based on major vasopressor is associated with reduced duration of postoperative respiratory support, and length of ICU stay in PAH-CHD patients undergoing elective surgery. These outcomes, then, may be linked to improved hemodynamics and preservation of right ventricular dynamic function.

Introduction: Acute kidney injury (AKI) is a prevalent and prognostically important complication of cardiac surgery. The complex multifactorial pathogenesis and lack of appropriate animal models to recapitulate the clinical insults leading to CSA-AKI have been implicated in the failure of multiple pharmacologic renoprotective strategies. We have reported anti-inflammatory and renoprotective effects of a novel Annexin-A1 (ANXA1) tripeptide (Ac-QAW) in a rodent model of experimental cardiac surgery. Here, we tested the hypothesis that Ac-QAW attenuates CSA-AKI by upregulating Sirtuin6 and Forkhead box protein O3 (SIRT6/FoxO3), key players in stress resistance, cell survival, and life span. Methods: Male Sprague-Dawley rats underwent 75 min of cardiopulmonary bypass (CPB) with 45 min of cardioplegic arrest (CA). Animals were treated (iv) with 1 mg/kg Ac-QAW (n = 6), the commercially available ANXA1 peptide Ac2-26 (as a positive control; n = 5), or vehicle (n = 6) at 1 h before CPB, during CA, and 1 h after CPB. At 24 h post-reperfusion, renal levels of activated caspase-3, ANXA1, SIRT6, and FoxO3 were determined by Western blot; renal and plasma levels of myeloperoxidase (MPO) were determined by ELISA. Results: At 24 hours post-reperfusion following CPB/CA, rats treated with Ac-QAW showed a) reduced renal caspase-3 activity (P < 0.05); b) decreased MPO in both blood and kidney; and c) increased renal levels of ANXA1 (P < 0.05), SIRT6, and FoxO3 (P < 0.01) (Figure). Conclusions: Using a clinically relevant animal model, we provide preliminary translatable evidence that administration of Ac-QAW attenuates CSA-AKI. This may result from action by Ac-QAW to 1) reduce inflammation by increasing inflammation-resolving molecule ANXA1; 2) inhibit neutrophil transmigration; and 3) promote pro-survival mechanisms by increasing SIRT6/FoxO3 expression. More Ac-QAW studies are needed to define its exact mechanism of action and its impact on long-term functional outcomes.

Objective: Activation of presynaptic voltage-gated calcium channels and release of glutamate play a central role in neuronal necrosis after cardiopulmonary bypass with deep hypothermic circulatory arrest. Pregabalin binds to the alpha 2-delta subunit of voltage-gated calcium channels resulting in reduced glutamate release. The aim of this study is to evaluate the effect of pregabalin on cerebral outcome after cardiopulmonary bypass with deep hypothermic circulatory arrest through an established rat model allowing long-term survival.Methods: Male Sprague-Dawley rats were randomized to receive intraperitoneal injection of 30 mg/kg of pregabalin or an equal amount of normal saline 1 hour before cardiopulmonary bypass (n = 10, each). Rats were cooled to a pericranial temperature of 18 degrees C and underwent deep hypothermic circulatory arrest for 60 minutes. Neurologic performance was assessed at postoperative days 3, 7, and 12. Cognitive performance (Morris water maze) was assessed daily from postoperative day 3 to 12 when histologic assessment was performed.Results: Neurologic scores were significantly better in the pregabalin group than in the control group at all time points of measurements. Morris water maze latencies were not statistically different between the groups. The percentage of necrotic neurons in the cerebral cortex was significantly less in the pregabalin group compared with the control group (8.6% [interquartile range, 5.0-8.9] vs 13.6% [interquartile range, 6.91-8.6], P = .045), whereas no difference was observed in the hippocampus.Conclusions: Preemptive treatment with pregabalin conveyed a beneficial influence on functional and histologic cerebral outcome in rats undergoing 60 minutes of deep hypothermic circulatory arrest after cardiopulmonary bypass without any noticeable side effects.