Dantzer, Robert

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.

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.

Abstract Background Inflammatory deregulation may be a major factor in cancer related symptoms (Dantzer Nature Rev Clin Oncol 2012). Improved symptoms in myelofibrosis (MF) patients treated with single arm ruxolitinib (JAK1/JAK2 inhibitor) studies has been correlated with normalization of selected cytokines increased pre-therapy (c-reactive protein, IL-1ra, MIP-1b, TNF-a, and IL-6; Verstovsek NEJM 2010). We sought to assess the impact of JAK1/JAK2 inhibitor therapy on cytokine levels and the relationship between cytokine levels and symptoms prior to and during JAK1/JAK2 inhibitor therapy in the phase III placebo controlled COMFORT-I trial (Verstovsek NEJM 2012). Methods Cytokine levels (89 cytokines measured at baseline and at weeks 4 and 24) and MF symptoms (assessed by MFSAF 2.0 – Mesa JCO 2013) were collected during the blinded phase of COMFORT-I. Patients were randomized to ruxolitinib vs. placebo. Plasma was used for the measurement of cytokines using Rules-Based Medicine, Inc. (Austin, TX) Human MAP panel. Associations between the MFSAF total symptom score (TSS) and log2-transformed cytokine data were investigated at baseline using Spearman correlations and linear regression. Mixed models were used to assess cytokine and TSS changes over time within each arm and overall. Logistic regression was used to assess the relationship between baseline cytokines and TSS response (>/=50% reduction from baseline) at week 24, and between week 4 cytokine changes and TSS response (>/=50% reduction from baseline) at week 24 within each arm and overall. Mixed and logistic regression models combining data across arms also included terms for visit, arm, and visit-by-arm interaction. All models also included age, gender, and body mass index (BMI). Given the large number of cytokines being investigated, p<0.001 was considered statistically significant. Cytokine values below the limit of detection were set at the lowest limit of detection and 25 cytokines were excluded from statistical analysis due to having more than 30% of data missing or below the limit of detection. Results Patients: 309 subjects were randomized in COMFORT-I with median age 68 (range 40-91), 46% female, 50% primary myelofibrosis, and 61% high risk. All 309 subjects had cytokines measured at one or more of the three visits included in this analysis, with 308 having cytokine values paired with a TSS score at the same visit. Cytokines at Baseline: At baseline, the highest Spearman correlations with symptomatic burden (as assessed by the TSS) were observed for APOA1 (rho=-.21) and FERRITIN (rho=-.20), followed by INTLK5, MIP1A, MMP3, INTLK2, MGB, INTLK1A, and INTLK7 with correlations between -.15 and -.17 (all p<0.01). After adjusting for age, sex, and BMI, VCAM1 and APOA1 were significantly associated with TSS at baseline. Cytokine and Symptom changes during trial: Changing levels of 5 cytokines were significantly associated with change of TSS over time beyond the change due to visit, arm, visit-by-arm interaction, age, sex and BMI including VCAM1, LEPTIN, TIMP1, B2MICG, and TNFRII. Within the placebo arm, only 5 cytokines significantly changed over time compared to 46 cytokines in the ruxolitinib arm (visit-by-arm interaction was significant for 43 in the overall models). VCAM1, B2MICG, and TNFRII were among the 5 cytokines which changed in the placebo arm, and VCAM1, LEPTIN, TIMP1, B2MICG, and TNFRII were among the 46 cytokines which changed in the ruxolitinib arm. No baseline cytokines and no changes in cytokines at week 4 univariately predicted week 24 TSS response within either arm at the p<0.001 level. Conclusions In the first serial assessment of MF symptoms and plasma cytokine levels in the conduct of a placebo controlled trial we found 5 cytokines in which improved levels correlated with decreased MF symptom burden in ruxolitinib treated patients. Development of a multivariate model for predicting symptom response, correlations with splenic response and impact on survival benefit of therapy is ongoing. Disclosures: No relevant conflicts of interest to declare.