Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine.

Brown adipose tissue (BAT) dissipates energy1,2 and promotes cardiometabolic health3. Loss of BAT during obesity and ageing is a principal hurdle for BAT-centred obesity therapies, but not much is known about BAT apoptosis. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. This apoptotic secretome enhances expression of the thermogenic programme in healthy adipocytes. This effect is mediated by the purine inosine that stimulates energy expenditure in brown adipocytes by the cyclic adenosine monophosphate-protein kinase A signalling pathway. Treatment of mice with inosine increased BAT-dependent energy expenditure and induced 'browning' of white adipose tissue. Mechanistically, the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) regulates inosine levels in BAT: ENT1-deficiency increases extracellular inosine levels and consequently enhances thermogenic adipocyte differentiation. In mice, pharmacological inhibition of ENT1 as well as global and adipose-specific ablation enhanced BAT activity and counteracted diet-induced obesity, respectively. In human brown adipocytes, knockdown or blockade of ENT1 increased extracellular inosine, which enhanced thermogenic capacity. Conversely, high ENT1 levels correlated with lower expression of the thermogenic marker UCP1 in human adipose tissues. Finally, the Ile216Thr loss of function mutation in human ENT1 was associated with significantly lower body mass index and 59% lower odds of obesity for individuals carrying the Thr variant. Our data identify inosine as a metabolite released during apoptosis with a 'replace me' signalling function that regulates thermogenic fat and counteracts obesity.

Novel IL-4/HB-EGF-dependent crosstalk between eosinophils and macrophages controls liver regeneration after ischaemia and reperfusion injury.

OBJECTIVE: Previous studies indicate that eosinophils are recruited into the allograft following orthotopic liver transplantation and protect from ischaemia reperfusion (IR) injury. In the current studies, we aim to explore whether their protective function could outlast during liver repair. DESIGN: Eosinophil-deficient mice and adoptive transfer of bone marrow-derived eosinophils (bmEos) were employed to investigate the effects of eosinophils on tissue repair and regeneration after hepatic IR injury. Aside from exogenous cytokine or neutralising antibody treatments, mechanistic studies made use of a panel of mouse models of eosinophil-specific IL-4/IL-13-deletion, cell-specific IL-4rα-deletion in liver macrophages and hepatocytes and macrophage-specific deletion of heparin-binding epidermal growth factor-like growth factor (hb-egf). RESULT: We observed that eosinophils persisted over a week following hepatic IR injury. Their peak accumulation coincided with that of hepatocyte proliferation. Functional studies showed that eosinophil deficiency was associated with a dramatic delay in liver repair, which was normalised by the adoptive transfer of bmEos. Mechanistic studies demonstrated that eosinophil-derived IL-4, but not IL-13, was critically involved in the reparative function of these cells. The data further revealed a selective role of macrophage-dependent IL-4 signalling in liver regeneration. Eosinophil-derived IL-4 stimulated macrophages to produce HB-EGF. Moreover, macrophage-specific hb-egf deletion impaired hepatocyte regeneration after IR injury. CONCLUSION: Together, these studies uncovered an indispensable role of eosinophils in liver repair after acute injury and identified a novel crosstalk between eosinophils and macrophages through the IL-4/HB-EGF axis.

CD73-generated extracellular adenosine promotes resolution of neutrophil-mediated tissue injury and restrains metaplasia in pancreatitis.

Pancreatitis is currently the leading cause of gastrointestinal hospitalizations in the US. This condition occurs in response to abdominal injury, gallstones, chronic alcohol consumption or, less frequently, the cause remains idiopathic. CD73 is a cell surface ecto-5'-nucleotidase that generates extracellular adenosine, which can contribute to resolution of inflammation by binding adenosine receptors on infiltrating immune cells. We hypothesized genetic deletion of CD73 would result in more severe pancreatitis due to decreased generation of extracellular adenosine. CD73 knockout (CD73-/- ) and C57BL/6 (wild type, WT) mice were used to evaluate the progression and response of caerulein-induced acute and chronic pancreatitis. In response to caerulein-mediated chronic or acute pancreatitis, WT mice display resolution of pancreatitis at earlier timepoints than CD73-/- mice. Using immunohistochemistry and analysis of single-cell RNA-seq (scRNA-seq) data, we determined CD73 localization in chronic pancreatitis is primarily observed in mucin/ductal cell populations and immune cells. In murine pancreata challenged with caerulein to induce acute pancreatitis, we compared CD73-/- to WT mice and observed a significant infiltration of Ly6G+, MPO+, and Granzyme B+ cells in CD73-/- compared to WT pancreata and we quantified a significant increase in acinar-to-ductal metaplasia demonstrating sustained metaplasia and inflammation in CD73-/- mice. Using neutrophil depletion in CD73-/- mice, we show neutrophil depletion significantly reduces metaplasia defined by CK19+ cells per field and significantly reduces acute pancreatitis. These data identify CD73 enhancers as a potential therapeutic strategy for patients with acute and chronic pancreatitis as adenosine generation and activation of adenosine receptors is critical to resolve persistent inflammation in the pancreas.

Patient-derived iPSCs link elevated mitochondrial respiratory complex I function to osteosarcoma in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies.

Therapeutic targeting of hypoxia inducible factor in acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is characterized by bilateral chest infiltration and acute hypoxic respiratory failure. ARDS carries significant morbidity and mortality despite advancements in medical management, calling for the development of novel therapeutic targets. Hypoxia-inducible factor (HIF) is a heterodimeric protein involved in various essential pathways, including metabolic reprogramming, immune modulation, angiogenesis and cell cycle regulation. HIF is routinely degraded in homeostasis conditions via the prolyl hydroxylase domain/von Hippel-Lindau protein pathway. However, HIF is stabilized in ARDS via various mechanisms (oxygen-dependent and independent) as an endogenous protective pathway and plays multifaceted roles in different cell populations. This review focuses on the functional role of HIF and its target genes during ARDS, as well as how HIF has evolved as a therapeutic target in current medical management.

Adenosine metabolized from extracellular ATP ameliorates organ injury by triggering A2BR signaling.

BACKGROUND: Trauma and a subsequent hemorrhagic shock (T/HS) result in insufficient oxygen delivery to tissues and multiple organ failure. Extracellular adenosine, which is a product of the extracellular degradation of adenosine 5' triphosphate (ATP) by the membrane-embedded enzymes CD39 and CD73, is organ protective, as it participates in signaling pathways, which promote cell survival and suppress inflammation through adenosine receptors including the A2BR. The aim of this study was to evaluate the role of CD39 and CD73 delivering adenosine to A2BRs in regulating the host's response to T/HS. METHODS: T/HS shock was induced by blood withdrawal from the femoral artery in wild-type, global knockout (CD39, CD73, A2BR) and conditional knockout (intestinal epithelial cell-specific deficient VillinCre-A2BRfl/fl) mice. At 3 three hours after resuscitation, blood and tissue samples were collected to analyze organ injury. RESULTS: T/HS upregulated the expression of CD39, CD73, and the A2BR in organs. ATP and adenosine levels increased after T/HS in bronchoalveolar lavage fluid. CD39, CD73, and A2BR mimics/agonists alleviated lung and liver injury. Antagonists or the CD39, CD73, and A2BR knockout (KO) exacerbated lung injury, inflammatory cytokines, and chemokines as well as macrophage and neutrophil infiltration and accumulation in the lung. Agonists reduced the levels of the liver enzymes aspartate transferase and alanine transaminase in the blood, whereas antagonist administration or CD39, CD73, and A2BR KO enhanced enzyme levels. In addition, intestinal epithelial cell-specific deficient VillinCre-A2BRfl/fl mice showed increased intestinal injury compared to their wild-type VillinCre controls. CONCLUSION: In conclusion, the CD39-CD73-A2BR axis protects against T/HS-induced multiple organ failure.

Strategies to Modulate MicroRNA Functions for the Treatment of Cancer or Organ Injury.

Cancer and organ injury-such as that occurring in the perioperative period, including acute lung injury, myocardial infarction, and acute gut injury-are among the leading causes of death in the United States and impose a significant impact on quality of life. MicroRNAs (miRNAs) have been studied extensively during the last two decades for their role as regulators of gene expression, their translational application as diagnostic markers, and their potential as therapeutic targets for disease treatment. Despite promising preclinical outcomes implicating miRNA targets in disease treatment, only a few miRNAs have reached clinical trials. This likely relates to difficulties in the delivery of miRNA drugs to their targets to achieve efficient inhibition or overexpression. Therefore, understanding how to efficiently deliver miRNAs into diseased tissues and specific cell types in patients is critical. This review summarizes current knowledge on various approaches to deliver therapeutic miRNAs or miRNA inhibitors and highlights current progress in miRNA-based disease therapy that has reached clinical trials. Based on ongoing advances in miRNA delivery, we believe that additional therapeutic approaches to modulate miRNA function will soon enter routine medical treatment of human disease, particularly for cancer or perioperative organ injury. SIGNIFICANCE STATEMENT: MicroRNAs have been studied extensively during the last two decades in cancer and organ injury, including acute lung injury, myocardial infarction, and acute gut injury, for their regulation of gene expression, application as diagnostic markers, and therapeutic potentials. In this review, we specifically emphasize the pros and cons of different delivery approaches to modulate microRNAs, as well as the most recent exciting progress in the field of therapeutic targeting of microRNAs for disease treatment in patients.

Targeting alveolar-specific succinate dehydrogenase A attenuates pulmonary inflammation during acute lung injury.

Acute lung injury (ALI) is an inflammatory lung disease, which manifests itself in patients as acute respiratory distress syndrome (ARDS). Previous studies have implicated alveolar-epithelial succinate in ALI protection. Therefore, we hypothesized that targeting alveolar succinate dehydrogenase SDH A would result in elevated succinate levels and concomitant lung protection. Wild-type (WT) mice or transgenic mice with targeted alveolar-epithelial Sdha or hypoxia-inducible transcription factor Hif1a deletion were exposed to ALI induced by mechanical ventilation. Succinate metabolism was assessed in alveolar-epithelial via mass spectrometry as well as redox measurements and evaluation of lung injury. In WT mice, ALI induced by mechanical ventilation decreased SDHA activity and increased succinate in alveolar-epithelial. In vitro, cell-permeable succinate decreased epithelial inflammation during stretch injury. Mice with inducible alveolar-epithelial Sdha deletion (Sdhaloxp/loxp SPC-CreER mice) revealed reduced lung inflammation, improved alveolar barrier function, and attenuated histologic injury. Consistent with a functional role of succinate to stabilize HIF, Sdhaloxp/loxp SPC-CreER experienced enhanced Hif1a levels during hypoxia or ALI. Conversely, Hif1aloxp/loxp SPC-CreER showed increased inflammation with ALI induced by mechanical ventilation. Finally, wild-type mice treated with intra-tracheal dimethlysuccinate were protected during ALI. These data suggest that targeting alveolar-epithelial SDHA dampens ALI via succinate-mediated stabilization of HIF1A. Translational extensions of our studies implicate succinate treatment in attenuating alveolar inflammation in patients suffering from ARDS.

Hypoxia-inducible factor-dependent induction of myeloid-derived netrin-1 attenuates natural killer cell infiltration during endotoxin-induced lung injury.

Sepsis and sepsis-associated lung inflammation significantly contribute to the morbidity and mortality of critical illness. Here, we examined the hypothesis that neuronal guidance proteins could orchestrate inflammatory events during endotoxin-induced lung injury. Through a targeted array, we identified netrin-1 as the top upregulated neuronal guidance protein in macrophages treated with lipopolysaccharide (LPS). Furthermore, we found that netrin-1 is highly enriched in infiltrating myeloid cells, particularly in macrophages during LPS-induced lung injury. Transcriptional studies implicate hypoxia-inducible factor HIF-1α in the transcriptional induction of netrin-1 during LPS treatment. Subsequently, the deletion of netrin-1 in the myeloid compartment (Ntn1loxp/loxp LysM Cre) resulted in exaggerated mortality and lung inflammation. Surprisingly, further studies revealed enhanced natural killer cells (NK cells) infiltration in Ntn1loxp/loxp LysM Cre mice, and neutralization of NK cell chemoattractant chemokine (C-C motif) ligand 2 (CCL2) reversed the exaggerated lung inflammation. Together, these studies provide functional insight into myeloid cell-derived netrin-1 in controlling lung inflammation through the modulation of CCL2-dependent infiltration of NK cells.

Jugular venous congestion during off-pump lung transplantation is not associated with an increased risk of postoperative delirium.

INTRODUCTION: Delirium occurs frequently after lung transplantation and is associated with poor clinical outcomes. Significantly prolonged jugular venous congestion (JVC) occurs during off-pump lung transplantation and is thought to impair cerebral perfusion. Our study aimed to test the hypothesis that increased intraoperative JVC is associated with an increased risk of postoperative delirium among lung transplantation recipients. METHODS: This is a retrospective observational cohort study. Adult patients who received off-pump lung transplantation at the Vanderbilt University Medical Center between 2006 and 2016 are included. The magnitude of JVC was calculated by the area under the curve (AUC) of the central venous pressure (CVP) above the threshold of 12 mmHg. Postoperative delirium was assessed by Confusion Assessment Method for the Intensive Care Unit (CAM-ICU) criteria during their ICU stay. Multivariate regression analysis was used to determine the association of intraoperative JVC with postoperative delirium, adjusting for baseline demographics, surgical, and intraoperative characteristics. RESULTS: Thirty-two (23.5%) out of 136 patients developed delirium in the ICU. There was no statistical difference in terms of intraoperative JVC between patients with delirium and those without (4058 ± 6650 vs. 3495 ± 10 151 mmHg min; p = .772). Furthermore, during multivariate regression analysis, JVC was not associated with an increased risk of delirium (odds ratio: 1.03 per 100 mmHg min increase in venous congestion; 95% confidence interval: .31, 3.39; p = .96). CONCLUSIONS: Delirium occurred frequently after off-pump lung transplantation. Although physiologically plausible, the present study did not find an association between increased JVC during off-pump lung transplantation and an increased risk of postoperative delirium.

Diversity and Inclusion in Anesthesiology.

In today's world, departments of anesthesiology and professional organizations are rightfully expected to have racial, ethnic, and gender diversity. Diversity and inclusiveness are considered important contributors to an effective and collaborative work environment by promoting excellence in patient care, education, and research. This has been re-emphasized in the racial reckoning in the summer of 2020, and the ongoing health care disparities manifested by the global coronavirus disease 2019 (COVID-19) pandemic. Moreover, the negative consequences of a lack of diversity and inclusion in health care have been shown to impact recruitment, retention, and the economic well-being of academic departments. In the present article, we review the current state of diversity in anesthesiology departments and professional organizations in the United States. We discuss strategies and important approaches to further enhance diversity to promote an inclusive perioperative work environment.

Incidence and Predictive Factors of Acute Kidney Injury After Off-pump Lung Transplantation.

OBJECTIVES: To determine the incidence and predictive factors of acute kidney injury (AKI) after off-pump lung transplantation. DESIGN: A retrospective cohort study. SETTING: The operating room and intensive care unit. PARTICIPANTS: Adult patients who underwent lung transplant without cardiopulmonary bypass or extracorporeal membrane oxygenator between 2006 and 2016 at the Vanderbilt University Medical Center. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The presence of postoperative AKI was assessed by the Kidney Disease: Improving Global Outcomes criteria in the first seven postoperative days. Multivariate logistic regression analysis was used to determine the independent predictive factors of AKI. One hundred forty-eight patients were included in the final analysis, of whom 63 (42.6%) subsequently developed AKI: 43 (29.0%) stage 1, ten (6.8%) stage 2, and ten (6.8%) stage 3. Patients who had AKI had a longer hospital length of stay (12 days [interquartile range (IQR): 10-17] vs ten days [IQR: 8-12], p < 0.001). For every one-year increase in age, the odds of AKI decreased by 8% (odds ratio [OR] 0.92, 95% confidence interval [CI]: 0.87-0.98, p = 0.008). The odds of having AKI in patients with bilateral lung transplant was lower than patients with unilateral transplant (OR 0.09, 95% CI: 0.01-0.63, p = 0.015). Additionally, a diagnosis of chronic obstructive pulmonary disease increased the odds of AKI by four-fold compared with a diagnosis of idiopathic pulmonary fibrosis (OR 4.73, 95% CI: 1.44-15.56, p = 0.011). CONCLUSIONS: AKI is a common complication after off-pump lung transplantation and is associated with increased hospital length of stay. Younger age, unilateral lung transplant, and diagnosis of chronic obstructive pulmonary disease are independently associated with AKI.

Hypoxia-inducible factor-dependent induction of netrin-1 dampens inflammation caused by hypoxia.

The neuronal guidance molecule netrin-1 is linked to the coordination of inflammatory responses. Given that mucosal surfaces are particularly prone to hypoxia-elicited inflammation, we sought to determine the function of netrin-1 in hypoxia-induced inflammation. We detected hypoxia-inducible factor 1alpha (HIF-1alpha)-dependent induction of expression of the gene encoding netrin-1 (Ntn1) in hypoxic epithelia. Neutrophil transepithelial migration studies showed that by engaging A2B adenosine receptor (A2BAR) on neutrophils, netrin-1 attenuated neutrophil transmigration. Exogenous netrin-1 suppressed hypoxia-elicited inflammation in wild-type but not in A2BAR-deficient mice, and inflammatory hypoxia was enhanced in Ntn1(+/-) mice relative to that in Ntn1(+/+) mice. Our studies demonstrate that HIF-1alpha-dependent induction of netrin-1 attenuates hypoxia-elicited inflammation at mucosal surfaces.

Hypoxia-Inducible Factor-2α Reprograms Liver Macrophages to Protect Against Acute Liver Injury Through the Production of Interleukin-6.

BACKGROUND AND AIMS: Acetaminophen (APAP) overdose represents the most frequent cause of acute liver failure, resulting in death or liver transplantation in more than one third of patients in the United States. The effectiveness of the only antidote, N-acetylcysteine, declines rapidly after APAP ingestion, long before patients are admitted to the clinic with symptoms of severe liver injury. The direct hepatotoxicity of APAP triggers a cascade of innate immune responses that may exacerbate or limit the progression of tissue damage. A better understanding of this complex mechanism will help uncover targets for therapeutic interventions. APPROACH AND RESULTS: We observed that APAP challenge caused stabilization of hypoxia-inducible factors (HIFs) in the liver and hepatic macrophages (MΦs), particularly HIF-2α. Genetic deletion of the HIF-2α gene in myeloid cells (HIF-2αmye/- ) markedly exacerbated APAP-induced liver injury (AILI) without affecting APAP bioactivation and detoxification. In contrast, hepatic and serum levels of the hepatoprotective cytokine interleukin 6 (IL-6), its downstream signal transducer and transcription factor 3 activation in hepatocytes, as well as hepatic MΦ IL-6 expression were markedly reduced in HIF-2αmye/- mice compared to wild-type mice post-APAP challenge. In vitro experiments revealed that hypoxia induced IL-6 production in hepatic MΦs and that such induction was abolished in HIF-2α-deleted hepatic MΦs. Restoration of IL-6 by administration of exogenous IL-6 ameliorated AILI in HIF-2αmye/- mice. Finally, IL-6-mediated hepatoprotection against AILI was abolished in hepatocyte-specific IL-6 receptor knockout mice. CONCLUSIONS: The data demonstrate that APAP treatment leads to HIF-2α stabilization in hepatic MΦs and that HIF-2α subsequently reprograms hepatic MΦs to produce the hepatoprotective cytokine IL-6, thereby ameliorating AILI.

Neuronal modulation of hepatic lipid accumulation induced by bingelike drinking.

Excessive alcohol consumption, including binge drinking, is a common cause of fatty liver disease. Binge drinking rapidly induces hepatic steatosis, an early step in the pathogenesis of chronic liver injury. Despite its prevalence, the process by which excessive alcohol consumption promotes hepatic lipid accumulation remains unclear. Alcohol exerts potent effects on the brain, including hypothalamic neurons crucial for metabolic regulation. However, whether or not the brain plays a role in alcohol-induced hepatic steatosis is unknown. In the brain, alcohol increases extracellular levels of adenosine, a potent neuromodulator, and previous work implicates adenosine signaling as being important for the development of alcoholic fatty liver disease. Acute alcohol exposure also increases both the activity of agouti-related protein (AgRP)-expressing neurons and AgRP immunoreactivity. Here, we show that adenosine receptor A2B signaling in the brain modulates the extent of alcohol-induced fatty liver in mice and that both the AgRP neuropeptide and the sympathetic nervous system are indispensable for hepatic steatosis induced by bingelike alcohol consumption. Together, these results indicate that the brain plays an integral role in alcohol-induced hepatic lipid accumulation and that central adenosine signaling, hypothalamic AgRP, and the sympathetic nervous system are crucial mediators of this process.

Markers of Accelerated Skeletal Muscle Regenerative Response in Murphy Roths Large Mice: Characteristics of Muscle Progenitor Cells and Circulating Factors.

The "super-healing" Murphy Roths Large (MRL/MpJ) mouse possesses a superior regenerative capacity for repair of many tissues, which makes it an excellent animal model for studying molecular and cellular mechanisms during tissue regeneration. As the role of muscle progenitor cells (MPCs) in muscle-healing capacity of MRL/MpJ mice has not been previously studied, we investigated the muscle regenerative capacity of MRL/MpJ mice following muscle injury, and the results were compared to results from C57BL/6J (B6) age-matched control mice. Our results show that muscle healing upon cardiotoxin injury was accelerated in MRL/MpJ mice and characterized by reduced necrotic muscle area, reduced macrophage infiltration, and more regenerated myofibers (embryonic myosin heavy chain+/centronucleated fibers) at 3, 5, and 12 days postinjury, when compared to B6 age-matched control mice. These observations were associated with enhanced function of MPCs, including improved cell proliferation, differentiation, and resistance to stress, as well as increased muscle regenerative potential when compared to B6 MPCs. Mass spectrometry of serum proteins revealed higher levels of circulating antioxidants in MRL/MpJ mice when compared to B6 mice. Indeed, we found relatively higher gene expression of superoxide dismutase 1 (Sod1) and catalase (Cat) in MRL/MpJ MPCs. Depletion of Sod1 or Cat by small interfering RNA impaired myogenic potential of MRL/MpJ MPCs, indicating a role for these antioxidants in muscle repair. Taken together, these findings provide evidence that improved function of MPCs and higher levels of circulating antioxidants play important roles in accelerating muscle-healing capacity of MRL/MpJ mice. Stem Cells 2019;37:357-367.

Characterization of articular cartilage homeostasis and the mechanism of superior cartilage regeneration of MRL/MpJ mice.

This study investigated articular cartilage (AC) homeostasis and different signaling pathways involved in the superior cartilage regeneration of Murphy Roths large (MRL/MpJ) mice previously reported. We collected uninjured and destabilized medial meniscus (DMM)-injured knees from 8-wk-old C57BL/6J and MRL/MpJ mice. We used micro-computed tomography (microCT), histology, and immunohistochemistry to evaluate AC homeostasis and repair. We used the ear punch model to investigate the role of angiogenesis and inflammation in the superior healing of MRL/MpJ mice. We found fewer ß-catenin and more pSMAD5 positive cells in the uninjured AC of MRL/MpJ mice than that from C57BL/6J mice. MRL/MpJ mice exhibited better AC repair in DMM-induced OA, as indicated by microCT results, Alcian blue, and Safranin O staining. Mechanistically, fewer ß-catenin, pSMAD2-, pSMAD3-, a disintegrin and metalloproteinase with thrombospondin motifs 4-, matrix metalloproteinase (MMP) 9-, and MMP13-positive cells and more proliferating cell nuclear antigen- and pSMAD5-positive cells were found in the DMM-injured AC in MRL/MpJ mice than in normal mice. The accelerated ear wound healing of MRL/MpJ mice correlated with enhanced angiogenesis and macrophage polarization toward the M2a phenotype through elevated IL-10 and IL-4 expressing cells. Collectively, our study revealed that down-regulation of pSMAD2/3, ß-catenin, and MMPs and up-regulation of pSMAD5 and M2a macrophage polarization contribute to the enhanced cartilage repair observed in MRL/MpJ mice.-Deng, Z., Gao, X., Sun, X., Amra, S., Lu, A., Cui, Y., Eltzschig, H. K., Lei, G., Huard, J. Characterization of articular cartilage homeostasis and the mechanism of superior cartilage regeneration of MRL/MpJ mice.