Intense light-elicited alveolar type 2-specific circadian PER2 protects from bacterial lung injury via BPIFB1.

Circadian amplitude enhancement has the potential to be organ protective but has not been studied in acute lung injury (ALI). Consistent light and dark cycles are crucial for the amplitude regulation of the circadian rhythm protein Period2 (PER2). Housing mice under intense instead of ambient light for 1 wk (light: dark cycle:14h:10h), we demonstrated a robust increase of pulmonary PER2 trough and peak levels, which is consistent with circadian amplitude enhancement. A search for the affected lung cell type suggested alveolar type 2 (ATII) cells as strong candidates for light induction of PER2. A head-to-head comparison of mice with cell-type-specific deletion of Per2 in ATII, endothelial, or myeloid cells uncovered a dramatic phenotype in mice with an ATII-specific deletion of Per2. During Pseudomonas aeruginosa-induced ALI, mice with Per2 deletion in ATII cells showed 0% survival, whereas 85% of control mice survived. Subsequent studies demonstrated that intense light therapy dampened lung inflammation or improved the alveolar barrier function during P. aeruginosa-induced ALI, which was abolished in mice with an ATII-specific deletion of Per2. A genome-wide mRNA array uncovered bactericidal/permeability-increasing fold-containing family B member 1 (BPIFB1) as a downstream target of intense light-elicited ATII-PER2 mediated lung protection. Using the flavonoid and PER2 amplitude enhancer nobiletin, we recapitulated the lung-protective and anti-inflammatory effects of light and BPIFB1, respectively. Together, our studies demonstrate that light-elicited amplitude enhancement of ATII-specific PER2 is a critical control point of inflammatory pathways during bacterial ALI.

Chronic Lactate Exposure Decreases Mitochondrial Function by Inhibition of Fatty Acid Uptake and Cardiolipin Alterations in Neonatal Rat Cardiomyocytes.

Introduction: Lactate is an important signaling molecule with autocrine, paracrine and endocrine properties involved in multiple biological processes including regulation of gene expression and metabolism. Levels of lactate are increased chronically in diseases associated with cardiometabolic disease such as heart failure, type 2 diabetes, and cancer. Using neonatal ventricular myocytes, we tested the hypothesis that chronic lactate exposure could decrease the activity of cardiac mitochondria that could lead to metabolic inflexibility in the heart and other tissues. Methods: Neonatal rat ventricular myocytes (NRVMs) were treated for 48 h with 5, 10, or 20 mM lactate and CPT I and II activities were tested using radiolabelled assays. The molecular species profile of the major mitochondrial phospholipid, cardiolipin, was determined using electrospray ionization mass spectrometry along with reactive oxygen species (ROS) levels measured by Amplex Red and mitochondrial oxygen consumption using the Seahorse analyzer. Results: CPT I activity trended downward (p = 0.07) and CPT II activity significantly decreased with lactate exposure (p < 0.001). Cardiolipin molecular species containing four 18 carbon chains (72 carbons total) increased with lactate exposure, but species of other sizes decreased significantly. Furthermore, ROS production was strongly enhanced with lactate (p < 0.001) and mitochondrial ATP production and maximal respiration were both significantly down regulated with lactate exposure (p < 0.05 and p < 0.01 respectively). Conclusions: Chronic lactate exposure in cardiomyocytes leads to a decrease in fatty acid transport, alterations of cardiolipin remodeling, increases in ROS production and decreases in mitochondrial oxygen consumption that could have implications for both metabolic health and flexibility. The possibility that both intra-, or extracellular lactate levels play roles in cardiometabolic disease, heart failure, and other forms of metabolic inflexibility needs to be assessed in vivo.

Linoleate-Enrichment of Mitochondrial Cardiolipin Molecular Species Is Developmentally Regulated and a Determinant of Metabolic Phenotype.

Cardiolipin (CL), the major mitochondrial phospholipid, regulates the activity of many mitochondrial membrane proteins. CL composition is shifted in heart failure with decreases in linoleate and increases in oleate side chains, but whether cardiolipin composition directly regulates metabolism is unknown. This study defines cardiolipin composition in rat heart and liver at three distinct ages to determine the influence of CL composition on beta-oxidation (ß-OX). CL species, expression of ß-OX and glycolytic genes, and carnitine palmitoyltransferase (CPT) activity were characterized in heart and liver from neonatal, juvenile, and adult rats. Ventricular myocytes were cultured from neonatal, juvenile, and adult rats and cardiolipin composition and CPT activity were measured. Cardiolipin composition in neonatal rat ventricular cardiomyocytes (NRVMs) was experimentally altered and mitochondrial respiration was assessed. Linoleate-enrichment of CL was observed in rat heart, but not liver, with increasing age. ß-OX genes and CPT activity were generally higher in adult heart and glycolytic genes lower, as a function of age, in contrast to liver. Palmitate oxidation increased in NRVMs when CL was enriched with linoleate. Our results indicate (1) CL is developmentally regulated, (2) linoleate-enrichment is associated with increased ß-OX and a more oxidative mitochondrial phenotype, and (3) experimentally induced linoleate-enriched CL in ventricular myocytes promotes a shift from pyruvate metabolism to fatty acid ß-OX.

Circadian Angiopoietin-Like-4 as a Novel Therapy in Cardiovascular Disease.

Angiopoietin-like 4 (ANGPTL4) is critical for regulating plasma lipids, and thus an attractive therapeutic target for cardiovascular diseases. Unfortunately, targeting ANGPTL4 results in a proinflammatory and ultimately lethal phenotype in animals. The serendipitous discovery of cardiac ANGPTL4 as a circadian protein reveals novel mechanistic insight and a solution for this therapeutic dilemma.

Intense light as anticoagulant therapy in humans.

Blood coagulation is central to myocardial ischemia and reperfusion (IR) injury. Studies on the light elicited circadian rhythm protein Period 2 (PER2) using whole body Per2-/- mice found deficient platelet function and reduced clotting which would be expected to protect from myocardial IR-injury. In contrast, intense light induction of PER2 protected from myocardial IR-injury while Per2 deficiency was detrimental. Based on these conflicting data, we sought to evaluate the role of platelet specific PER2 in coagulation and myocardial ischemia and reperfusion injury. We demonstrated that platelets from mice with tissue-specific deletion of Per2 in the megakaryocyte lineage (Per2loxP/loxP-PF4-CRE) significantly clot faster than platelets from control mice. We further found increases in infarct sizes or plasma troponin levels in Per2loxP/loxP-PF4-CRE mice when compared to controls. As intense light increases PER2 protein in human tissues, we also performed translational studies and tested the effects of intense light therapy on coagulation in healthy human subjects. Our human studies revealed that intense light therapy repressed procoagulant pathways in human plasma samples and significantly reduced the clot rate. Based on these results we conclude that intense light elicited PER2 has an inhibitory function on platelet aggregation in mice. Further, we suggest intense light as a novel therapy to prevent or treat clotting in a clinical setting.

Intense Light Pretreatment Improves Hemodynamics, Barrier Function and Inflammation in a Murine Model of Hemorrhagic Shock Lung.

INTRODUCTION: Hemorrhagic shock is a primary injury amongst combat casualties. Hemorrhagic shock can lead to acute lung injury, which has a high mortality rate. Based on studies showing the role of intense light for organ-protection, we sought to evaluate if intense light pretreatment would be protective in a murine model of hemorrhagic shock lung. MATERIALS AND METHODS: After exposure to standard room light or to intense light (10 000 LUX), mice were hemorrhaged for 90 minutes to maintain a mean arterial pressure (MAP) of 30-35 mmHg. Mice were then resuscitated with their blood and a NaCl infusion at a rate of 0.2 ml/h over a 3-hour period. During resuscitation, blood pressure was recorded. At the end of resuscitation, bronchoalveolar lavage was analyzed for alveolar epithelial barrier function and inflammation. To get insight into the relevance of intense light for humans, we performed a proteomics screen for lung injury biomarkers in plasma from healthy volunteers following intense light therapy. RESULTS: We found that intense light pretreated mice had improved hemodynamics and significantly lower albumin, IL-6, and IL-8 levels in their bronchoalveolar lavage than controls. We further discovered that intense light therapy in humans significantly downregulated proinflammatory plasma proteins that are known to cause acute lung injury. CONCLUSIONS: Our data demonstrate that mice exposed to intense light before hemorrhagic shock lung have less lung inflammation and improved alveolar epithelial barrier function. We further show that intense light therapy downregulates lung injury promoting proteins in human plasma. Together, these data suggest intense light as a possible strategy to ameliorate the consequences of a hemorrhagic shock on lung injury.

A Role for the Adenosine ADORA2B Receptor in Midazolam Induced Cognitive Dysfunction.

BACKGROUND: We recently reported a role for the circadian rhythm protein Period 2 (PER2) in midazolam induced cognitive dysfunction. Based on previous studies showing a critical role for the adenosine A2B receptor (ADORA2B) in PER2 regulation, we hypothesized that hippocampal ADORA2B is crucial for cognitive function. METHODS: Midazolam treated C57BL/6J mice were analyzed for Adora2b hippocampal mRNA expression levels, and spontaneous T-maze alternation was determined in Adora2b-/- mice. Using the specific ADORA2B agonist BAY-60-6583 in midazolam treated C57BL/6J mice, we analyzed hippocampal Per2 mRNA expression levels and spontaneous T-maze alternation. Finally, Adora2b-/- mice were assessed for mRNA expression of markers for inflammation or cognitive function in the hippocampus. RESULTS: Midazolam treatment significantly downregulated Adora2b or Per2 mRNA in the hippocampus of C57BL/6J mice, and hippocampal PER2 protein expression or T-maze alternation was significantly reduced in Adora2b-/- mice. ADORA2B agonist BAY-60-6583 restored midazolam mediated reduction in spontaneous alternation in C57BL/6J mice. Analysis of hippocampal Tnf-α or Il-6 mRNA levels in Adora2b-/- mice did not reveal an inflammatory phenotype. However, C-fos, a critical component of hippocampus-dependent learning and memory, was significantly downregulated in the hippocampus of Adora2b-/- mice. CONCLUSION: These results suggest a role of ADORA2B in midazolam induced cognitive dysfunction. Further, our data demonstrate that BAY-60-6583 treatment restores midazolam induced cognitive dysfunction, possibly via increases of Per2. Additional mechanistic studies hint towards C-FOS as another potential underlying mechanism of memory impairment in Adora2b-/- mice. These findings suggest the ADORA2B agonist as a potential therapy in patients with midazolam induced cognitive dysfunction.

RIPK1-mediated induction of mitophagy compromises the viability of extracellular-matrix-detached cells.

For cancer cells to survive during extracellular matrix (ECM) detachment, they must inhibit anoikis and rectify metabolic deficiencies that cause non-apoptotic cell death. Previous studies in ECM-detached cells have linked non-apoptotic cell death to reactive oxygen species (ROS) generation, although the mechanistic underpinnings of this link remain poorly defined. Here, we uncover a role for receptor-interacting protein kinase 1 (RIPK1) in the modulation of ROS and cell viability during ECM detachment. We find that RIPK1 activation during ECM detachment results in mitophagy induction through a mechanism dependent on the mitochondrial phosphatase PGAM5. As a consequence of mitophagy, ECM-detached cells experience diminished NADPH production in the mitochondria, and the subsequent elevation in ROS levels leads to non-apoptotic death. Furthermore, we find that antagonizing RIPK1/PGAM5 enhances tumour formation in vivo. Thus, RIPK1-mediated induction of mitophagy may be an efficacious target for therapeutics aimed at eliminating ECM-detached cancer cells.

The role of multicellular aggregation in the survival of ErbB2-positive breast cancer cells during extracellular matrix detachment.

The metastasis of cancer cells from the site of the primary tumor to distant sites in the body represents the most deadly manifestation of cancer. In order for metastasis to occur, cancer cells need to evade anoikis, which is defined as apoptosis caused by loss of attachment to extracellular matrix (ECM). Signaling from ErbB2 has previously been linked to the evasion of anoikis in breast cancer cells but the precise molecular mechanisms by which ErbB2 blocks anoikis have yet to be unveiled. In this study, we have identified a novel mechanism by which anoikis is inhibited in ErbB2-expressing cells: multicellular aggregation during ECM-detachment. Our data demonstrate that disruption of aggregation in ErbB2-positive cells is sufficient to induce anoikis and that this anoikis inhibition is a result of aggregation-induced stabilization of EGFR and consequent ERK/MAPK survival signaling. Furthermore, these data suggest that ECM-detached ErbB2-expressing cells may be uniquely susceptible to targeted therapy against EGFR and that this sensitivity could be exploited for specific elimination of ECM-detached cancer cells.