Abe, Jun-Ichi

Atherosclerotic and high-risk plaques are predominantly localized to vessel wall regions with non-laminar disturbed blood flow (d-flow), leading to the principle that d-flow-, but not laminar flow (l-flow)-driven endothelial cells (EC) dysfunction plays a role in this process. LATS1/2 and YAP are components of the Hippo signaling pathway that are involved in cellular responses to l-flow. The l-flow-mediated inhibition of LATS1/2 activity triggers YAP activation and maintains vascular structure. However, the role of Hippo signaling under d-flow remains unclear. Although both l- and d-flow increased YAP activity, only d-flow decreased LATS1/2 expression. Since the effects of l- and d-flow on EC differ, we studied the role of EC LATS1/2 using EC-specific Lats1 and 2 knock-out (EKO) mice. Within 14 days after inducing Lats1/2 deletion in tamoxifen-inducible Lats1 homo / Lats2 homo EKO mice, all the mice (27/27) died of severe systemic edema, accompanied by increased EC apoptosis and vascular permeability. We generated Lats1 he t / Lats2 homo -EKO mice ( Lats1 het /2 homo -EKO) and induced hypercholesteremia with AAV8-PCSK9 injections and a high-fat diet (HFD). After HFD, we detected plaque erosion-like plaque lesions at the d-flow areas, which revealed strongly fibrin/fibrinogen-positive organized thrombus (OTh) formation without sizeable necrotic cores. In Lats1 het /2 homo -EKO, we found significant increases in 1) EC proliferation, 2) EC apoptosis with senescent phenotype, 3) tissue factor (TF) expression, and 4) inflammation. Therefore, the phenotype observed in Lats1 het /2 homo -EKO represents a unique senescence-associated stemness (SAS) phenotype, which allows EC to escape senescence-induced cell cycle arrest. The EC proliferation induced by depletion of LATS1/2 was YAP-dependent. In contrast, EC senescence induced by CD38-mediated NAD + depletion was YAP-independent. These findings indicate that downregulating LATS1/2 expression-induced SAS leads to OTh via both YAP-dependent and independent mechanisms. OTh plays a key role in human atherosclerosis but is rarely observed in the mouse atherosclerosis model. Lats1 het /2 homo -EKO can provide a valuable platform for studying the mechanism and unique role of EC SAS-induced OTh formation.

Senescence induced by various stresses reprograms cancer cells to acquire senescence-associated (SA) cell growth (SACG), which allows them to escape senescence-induced cell cycle arrest with enhanced growth potential. ERK5 is a dual kinase-transcription factor, containing an N-terminal kinase domain and transactivation domains in the C-terminal half. Recent data have raised questions regarding the functional role of ERK5 kinase inhibitors in cancer and inflammatory diseases. We aimed to investigate how ERK5 reprograms myeloid cells (MC) to the SA secretory phenotype (SASP) and SACG, consequently leading to atherosclerosis (AthS). AthS was inhibited in ERK5 S496A knock-in (KI) mice. Single-cell analysis of p53 and Ki67 with imaging mass cytometry revealed three groups of MCs with different p53 and Ki67 expression patterns in the plaque. One group showed that p53 and Ki67 were co-expressed lineally, suggesting that this group of cells escaped from the growth-suppression effects of p53, which is also known as a senescence marker. The % of ERK5 S496A KI cells in this group was significantly lower than in wild-type (WT). Also, we found an increase of SA β-gal (SAβ-gal) + Ki67 + and 53BP1 + Ki67 + cells in bone narrow-derived macrophage (BMDMs) isolated from hypercholesterolemia (H-chol) WT mice, suggesting H-chol-induced MC SACG. MC SACG were significantly inhibited in ERK5 S496A KI mice, supporting the role of ERK5 S496 phosphorylation on SACG. RNA sequencing analysis identified the ERK5 S496 phosphorylation-dependent AHR induction by H-chol, and AHR depletion significantly inhibited SACG. We also discovered a critical effect of ERK5 S496 phosphorylation on SUMOylation at a novel site of NRF2 (i.e., K518), which inhibited NRF2 transcriptional activity without affecting ERK5 kinase activity and antagonized oxidized LDL (oxLDL)-induced SASP. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) inhibited oxLDL-induced ERK5 S496 phosphorylation, suggesting that ERK5 S496 phosphorylation was involved at least in part of the effects of these inhibitors. We have discovered a novel mechanism in which ERK5 S496 phosphorylation directly inhibits NRF2 activity by upregulating NRF2 K518 SUMOylation and AHR induction, subsequently inducing SACG and AthS.

Background: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. Methods: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow–derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. Results: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow–derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)–induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. Conclusions: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

Background: There is a paucity of data regarding the association between radiation exposure of heart substructures and the incidence of major coronary events (MCEs) in patients with esophageal cancer (ESOC) undergoing chemoradiation therapy. We studied radiation dosimetric determinants of MCE risk and measured their impact on patient prognosis using a cohort of ESOC patients treated at a single institution. Methods: Between March 2005 and October 2015, 355 ESOC patients treated with concurrent chemoradiotherapy were identified from a prospectively maintained and institutional-regulatory-board-approved clinical database. Dose-distribution parameters of the whole heart, the atria, the ventricles, the left main coronary artery, and three main coronary arteries were extracted for analysis. Results: Within a median follow-up time of 67 months, 14 patients experienced MCEs at a median of 16 months. The incidence of MCEs was significantly associated with the left anterior descending coronary artery (LAD) receiving ≥30 Gy (V30Gy) (p = 0.048). Patients receiving LAD V30Gy ≥ 10% of volume experienced a higher incidence of MCEs versus the LAD V30Gy < 10% group (p = 0.044). The relative rate of death increased with the left main coronary artery (LMA) mean dose (Gy) (p = 0.002). Furthermore, a mutual promotion effect of hyperlipidemia and RT on MCEs was observed. Conclusion: Radiation dose to coronary substructures is associated with MCEs and overall survival in patients with ESOC. In this study, the doses to these substructures appeared to be better predictors of toxicity outcomes than mean heart dose (MHD) or whole-heart V30Gy. These findings have implications for reducing coronary events through radiation therapy planning.

The possible association between the membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) and inflammation has been suggested, but the molecular mechanisms underlying this link, especially during atherogenesis, remain unclear. In endothelial cells (ECs) exposed to disturbed flow (d-flow), p90 ribosomal S6 kinase (p90RSK) bound to MAGI1, causing MAGI1-S741 phosphorylation and sentrin/SUMO-specific protease 2 T368 phosphorylation-mediated MAGI1-K931 deSUMOylation. MAGI1-S741 phosphorylation upregulated EC activation via activating Rap1. MAGI1-K931 deSUMOylation induced both nuclear translocation of p90RSK-MAGI1 and ATF-6-MAGI1 complexes, which accelerated EC activation and apoptosis, respectively. Microarray screening revealed key roles for MAGI1 in the endoplasmic reticulum (ER) stress response. In this context, MAGI1 associated with activating transcription factor 6 (ATF-6). MAGI1 expression was upregulated in ECs and macrophages found in atherosclerotic-prone regions of mouse aortas as well as in the colonic epithelia and ECs of patients with inflammatory bowel disease. Further, reduced MAGI1 expression in Magi1 –/+ mice inhibited d-flow–induced atherogenesis. In sum, EC activation and ER stress–mediated apoptosis are regulated in concert by two different types of MAGI1 posttranslational modifications, elucidating attractive drug targets for chronic inflammatory disease, particularly atherosclerosis.