Cardioprotective properties of OMT-28, a synthetic analog of omega-3 epoxyeicosanoids.

OMT-28 is a metabolically robust small molecule developed to mimic the structure and function of omega-3 epoxyeicosanoids. However, it remained unknown to what extent OMT-28 also shares the cardioprotective and anti-inflammatory properties of its natural counterparts. To address this question, we analyzed the ability of OMT-28 to ameliorate hypoxia/reoxygenation (HR)-injury and lipopolysaccharide (LPS)-induced endotoxemia in cultured cardiomyocytes. Moreover, we investigated the potential of OMT-28 to limit functional damage and inflammasome activation in isolated perfused mouse hearts subjected to ischemia/reperfusion (IR) injury. In the HR model, OMT-28 (1 µM) treatment largely preserved cell viability (about 75 versus 40% with the vehicle) and mitochondrial function as indicated by the maintenance of NAD+/NADH-, ADP/ATP-, and respiratory control ratios. Moreover, OMT-28 blocked the HR-induced production of mitochondrial reactive oxygen species. Pharmacological inhibition experiments suggested that Gαi, PI3K, PPARα, and Sirt1 are essential components of the OMT-28-mediated pro-survival pathway. Counteracting inflammatory injury of cardiomyocytes, OMT-28 (1 µM) reduced LPS-induced increases in TNFα protein (by about 85% versus vehicle) and NF-κB DNA binding (by about 70% versus vehicle). In the ex vivo model, OMT-28 improved post-IR myocardial function recovery to reach about 40% of the baseline value compared to less than 20% with the vehicle. Furthermore, OMT-28 (1 µM) limited IR-induced NLRP3 inflammasome activation similarly to a direct NLRP3 inhibitor (MCC950). Overall, this study demonstrates that OMT-28 possesses potent cardio-protective and anti-inflammatory properties supporting the hypothesis that extending the bioavailability of omega-3 epoxyeicosanoids may improve their prospects as therapeutic agents.

Linoleic acid-derived diol 12,13-DiHOME enhances NLRP3 inflammasome activation in macrophages.

12,13-dihydroxy-9z-octadecenoic acid (12,13-DiHOME) is a linoleic acid diol derived from cytochrome P-450 (CYP) epoxygenase and epoxide hydrolase (EH) metabolism. 12,13-DiHOME is associated with inflammation and mitochondrial damage in the innate immune response, but how 12,13-DiHOME contributes to these effects is unclear. We hypothesized that 12,13-DiHOME enhances macrophage inflammation through effects on NOD-like receptor protein 3 (NLRP3) inflammasome activation. To test this hypothesis, we utilized human monocytic THP1 cells differentiated into macrophage-like cells with phorbol myristate acetate (PMA). 12,13-DiHOME present during lipopolysaccharide (LPS)-priming of THP1 macrophages exacerbated nigericin-induced NLRP3 inflammasome activation. Using high-resolution respirometry, we observed that priming with LPS+12,13-DiHOME altered mitochondrial respiratory function. Mitophagy, measured using mito-Keima, was also modulated by 12,13-DiHOME present during priming. These mitochondrial effects were associated with increased sensitivity to nigericin-induced mitochondrial depolarization and reactive oxygen species production in LPS+12,13-DiHOME-primed macrophages. Nigericin-induced mitochondrial damage and NLRP3 inflammasome activation in LPS+12,13-DiHOME-primed macrophages were ablated by the mitochondrial calcium uniporter (MCU) inhibitor, Ru265. 12,13-DiHOME present during LPS-priming also enhanced nigericin-induced NLRP3 inflammasome activation in primary murine bone marrow-derived macrophages. In summary, these data demonstrate a pro-inflammatory role for 12,13-DiHOME by enhancing NLRP3 inflammasome activation in macrophages.

Cardioprotective Action of a Novel Synthetic 19,20-EDP Analog Is Sirt Dependent.

ABSTRACT: Mounting evidence suggests that cytochrome P450 epoxygenase-derived metabolites of docosahexaenoic acid, called epoxydocosapentaenoic acids (EDPs), limit mitochondrial damage after cardiac injury. In particular, the 19,20-EDP regioisomer has demonstrated potent cardioprotective action. Thus, we investigated our novel synthetic 19,20-EDP analog SA-22 for protection against cardiac ischemia-reperfusion (IR) injury. Isolated C57BL/6J mouse hearts were perfused through Langendorff apparatus for 20 minutes to obtain baseline function, followed by 30 minutes of global ischemia. Hearts were then treated with vehicle, 19,20-EDP, SA-22, or SA-22 with the pan-sirtuin inhibitor nicotinamide or the SIRT3-selective inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) at the start of 40 minutes reperfusion (N = 5-8). We assessed IR injury-induced changes in recovery of myocardial function, using left ventricular developed pressure and systolic and diastolic pressure change. Tissues were assessed for electron transport chain function, SIRT1 and SIRT3, optic atrophy type 1, and caspase-1. We also used H9c2 cells in an in vitro model of hypoxia/reoxygenation injury (N = 3-6). Hearts perfused with SA-22 had significantly improved postischemic left ventricular developed pressure, systolic and diastolic recovery (64% of baseline), compared with vehicle control (15% of baseline). In addition, treatment with SA-22 led to better catalytic function observed in electron transport chain and SIRT enzymes. The protective action of SA-22 resulted in reduced activation of pyroptosis in both hearts and cells after injury. Interestingly, although nicotinamide cotreatment worsened functional outcomes, cell survival, and attenuated sirtuin activity, it failed to completely attenuate SA-22-induced protection against pyroptosis, possibly indicating EDPs exert cytoprotection through pleiotropic mechanisms. In short, these data demonstrate the potential of our novel synthetic 19,20-EDP analog, SA-22, against IR/hypoxia-reoxygenation injury and justify further development of therapeutic agents based on 19,20-EDP.

PINK1-dependent and Parkin-independent mitophagy is involved in reprogramming of glycometabolism in pancreatic cancer cells.

Cancer cells rely on glycolysis to generate ATP for survival. However, inhibiting glycolysis is insufficient for the eradication of cancer cells because glycolysis-suppressed cells undergo metabolic reprogramming toward mitochondrial oxidative phosphorylation. We previously described that upon glycolytic suppression in pancreatic cancer cells, intracellular glycometabolism is shifted toward mitochondrial oxidative phosphorylation in an autophagy-dependent manner for cellular survival. Here, we hypothesized that mitophagy, which selectively degrades mitochondria via autophagy, is involved in mitochondrial activation under metabolic reprogramming. We revealed that glycolytic suppression notably increased mitochondrial membrane potential and mitophagy in a pancreatic cancer cell model (PANC-1). PTEN-induced kinase 1 (PINK1), a ubiquitin kinase that regulates mitophagy in healthy cells, regulated mitochondrial activation through mitophagy by glycolytic suppression. However, Parkin, a ubiquitin ligase regulated by PINK1 in healthy cells to induce mitophagy, was not involved in the PINK1-dependent mitophagy of the cancer glycometabolism. These results imply that cancer cells and healthy cells have different regulatory pieces of machinery for mitophagy, and inhibition of cancer-specific mechanisms may be a potential strategy for cancer therapy targeting metabolic reprogramming.

Potential role of lipophagy impairment for anticancer effects of glycolysis-suppressed pancreatic ductal adenocarcinoma cells.

Although increased aerobic glycolysis is common in various cancers, pancreatic ductal adenocarcinoma (PDAC) cells can survive a state of glycolysis suppression. We aimed to identify potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anticancer metabolic compounds, we identified SP-2509, an inhibitor of lysine-specific histone demethylase 1A (LSD1), which dramatically decreased the growth of PDAC PANC-1 cells and showed an anti-tumoral effect in tumor-bearing mice. The growth of glycolysis-suppressed PANC-1 cells was also inhibited by another LSD1 inhibitor, OG-L002. Similarly, the other two PDAC cells (PK-1 and KLM-1) with suppressed glycolysis exhibited anticancer effects against SP-2509. However, the anticancer effects on PDAC cells were unrelated to LSD1. To investigate how PDAC cells survive in a glycolysis-suppressed condition, we conducted proteomic analyses. These results combined with our previous findings suggested that glucose-starvation causes PDAC cells to enhance mitochondrial oxidative phosphorylation. In particular, mitochondrial fatty acid metabolism was identified as a key factor contributing to the survival of PDAC cells under glycolysis suppression. We further demonstrated that SP-2509 and OG-L002 disturbed fatty acid metabolism and induced lipid droplet accumulation through the impairment of lipophagy, but not bulk autophagy. These findings indicate a significant potential association of lipophagy and anticancer effects in glycolysis-suppressed PDAC cells, offering ideas for new therapeutic strategies for PDAC by dual inhibition of glycolysis and fatty acids metabolism.

Loss of ADAM15 Exacerbates Transition to Decompensated Myocardial Hypertrophy and Dilation Through Activation of the Calcineurin Pathway.

BACKGROUND: Myocardial hypertrophy and dilation are key features of cardiomyopathies and involve several cellular and molecular events. ADAMs (a disintegrin and metalloproteinases) are membrane-bound proteinases with diverse functions whose role in heart disease remains underexplored. ADAM15 is expressed in the heart and is downregulated in the failing human heart. We investigated the role ADAM15 in pressure overload cardiomyopathy. METHODS: We assessed ADAM15 levels in myocardial specimens from patients. Its direct role in pressure overload was investigated by subjecting wildtype and Adam15-deficient mice to transverse aortic constriction (TAC). RESULTS: ADAM15 levels did not change in patients with concentric hypertrophy, but markedly decreased in eccentric hypertrophy and heart failure. Loss of ADAM15 alone did not cause cardiomyopathy in mice (1 year old). After TAC, Adam15-/- mice exhibited worsened eccentric hypertrophy and dilation with greater increase in hypertrophy markers (pJNK, pERK1/2; Nppb, Nppa, Myh7, Acta1) compared with wildtype-TAC. Expression of integrin-α7 (but not integrin ß1) increased significantly more in Adam15-/--TAC hearts, while the interaction of these integrins with basement membrane (laminin), decreased consistent with worsened left ventricle dilation. In vitro, ADAM15 knockdown increased cardiomyocyte hypertrophy in response to mechanical stretch. Adam15-/--TAC hearts exhibited increased calcineurin activity and de-phosphorylation of nuclear factor of activated T cells. Calcineurin inhibition (cyclosporin-A) blocked the excess hypertrophy and dilation in Adam15-/--TAC mice. Proteome profiling demonstrated the increased abundance of the key proteins linked to worsened DCM in Adam15-/--TAC. CONCLUSION: This is the first report demonstrating that ADAM15 can suppress hypertrophy through regulating the integrin-laminin interaction and the calcineurin pathway.