Nucleolar dominance and DNA methylation directed by small interfering RNA

In the December 5th issue of Molecular Cell, Preuss et al. (2008) demonstrated a link between small interfering RNA (siRNA)-directed de novo DNA methylation and rDNA silencing in nucleolar dominance

Mitochondrial respiration is reduced in atherosclerosis, promoting necrotic core formation and reducing relative fibrous cap thickness

Objective: Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction, and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques, and whether augmenting mitochondrial respiration affects atherogenesis. Approach and Results: Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells (VSMCs) derived from plaques showed impaired mitochondrial respiration, reduced complex I expression and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E-deficient (ApoE-/-) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species (ROS). To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE-/- mice overexpressing the mitochondrial helicase Twinkle (Tw+/ApoE-/-). Tw+/ApoE-/- mice showed increased mtDNA integrity, copy number, respiratory complex abundance and respiration. Tw+/ApoE-/- mice had decreased necrotic core and increased fibrous cap areas, and Tw+/ApoE-/- bone marrow transplantation also reduced core areas. Twinkle increased VSMC mtDNA integrity and respiration. Twinkle also promoted VSMC proliferation and protected both VSMCs and macrophages from oxidative stress-induced apoptosis. Conclusions: Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decreases necrotic core and increases fibrous cap areas independently of changes in ROS, and may be a promising therapeutic strategy in atherosclerosis.This work was supported by British Heart Foundation (BHF) grants PG/14/69/31032 and RG/13/14/30314, a Wellcome Trust PhD Fellowship to J. Reinhold, the National Institute for Health Research Cambridge Biomedical Research Centre, the BHF Centre for Research Excellence, the Academy of Medical Sciences and by grants to M.P. Murphy from the Medical Research Council UK (MC_U105663142), and by a Wellcome Trust Investigator award (110159/Z/15/Z)

Defective Base Excision Repair of Oxidative DNA Damage in Vascular Smooth Muscle Cells Promotes Atherosclerosis

Background: Atherosclerotic plaques demonstrate extensive accumulation of oxidative DNA damage, predominantly as 8-oxoguanine (8oxoG) lesions. 8oxoG is repaired by base excision repair enzymes; however, the mechanisms regulating 8oxoG accumulation in vascular smooth muscle cells (VSMCs) and its effects on their function and in atherosclerosis are unknown. Methods: We studied levels of 8oxoG and its regulatory enzymes in human atherosclerosis, the mechanisms regulating 8oxoG repair and the base excision repair enzyme 8oxoG DNA glycosylase I (OGG1) in VSMCs in vitro, and the effects of reducing 8oxoG in VSMCs in atherosclerosis in ApoE−/− mice. Results: Human plaque VSMCs showed defective nuclear 8oxoG repair, associated with reduced acetylation of OGG1. OGG1 was a key regulatory enzyme of 8oxoG repair in VSMCs, and its acetylation was crucial to its repair function through regulation of protein stability and expression. p300 and sirtuin 1 were identified as the OGG1 acetyltransferase and deacetylase regulators, respectively, and both proteins interacted with OGG1 and regulated OGG1 acetylation at endogenous levels. However, p300 levels were decreased in human plaque VSMCs and in response to oxidative stress, suggesting that reactive oxygen species–induced regulation of OGG1 acetylation could be caused by reactive oxygen species–induced decrease in p300 expression. We generated mice that express VSMC-restricted OGG1 or an acetylation defective version (SM22α-OGG1 and SM22α-OGG1K-R mice) and crossed them with ApoE−/− mice. We also studied ApoE−/− mice deficient in OGG1 (OGG1−/−). OGG1−/− mice showed increased 8oxoG in vivo and increased atherosclerosis, whereas mice expressing VSMC-specific OGG1 but not the acetylation mutant OGG1K-R showed markedly reduced intracellular 8oxoG and reduced atherosclerosis. VSMC OGG1 reduced telomere 8oxoG accumulation, DNA strand breaks, cell death and senescence after oxidant stress, and activation of proinflammatory pathways. Conclusions: We identify defective 8oxoG base excision repair in human atherosclerotic plaque VSMCs, OGG1 as a major 8oxoG repair enzyme in VSMCs, and p300/sirtuin 1 as major regulators of OGG1 through acetylation/deacetylation. Reducing oxidative damage by rescuing OGG1 activity reduces plaque development, indicating the detrimental effects of 8oxoG on VSMC function

Type-2 innate lymphoid cells control the development of atherosclerosis in mice.

Type-2 innate lymphoid cells (ILC2) are a prominent source of type II cytokines and are found constitutively at mucosal surfaces and in visceral adipose tissue. Despite their role in limiting obesity, how ILC2s respond to high fat feeding is poorly understood, and their direct influence on the development of atherosclerosis has not been explored. Here, we show that ILC2 are present in para-aortic adipose tissue and lymph nodes and display an inflammatory-like phenotype atypical of adipose resident ILC2. High fat feeding alters both the number of ILC2 and their type II cytokine production. Selective genetic ablation of ILC2 in Ldlr-/- mice accelerates the development of atherosclerosis, which is prevented by reconstitution with wild type but not Il5-/- or Il13-/- ILC2. We conclude that ILC2 represent a major innate cell source of IL-5 and IL-13 required for mounting atheroprotective immunity, which can be altered by high fat diet

MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

Excessive activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including cardiovascular and Alzheimer's disease. Here we show that microtubule-affinity regulating kinase 4 (MARK4) binds to NLRP3 and drives it to the microtubule-organizing centre, enabling the formation of one large inflammasome speck complex within a single cell. MARK4 knockdown or knockout, or disruption of MARK4-NLRP3 interaction, impairs NLRP3 spatial arrangement and limits inflammasome activation. Our results demonstrate how an evolutionarily conserved protein involved in the regulation of microtubule dynamics orchestrates NLRP3 inflammasome activation by controlling its transport to optimal activation sites, and identify a targetable function for MARK4 in the control of innate immunity.The work was supported by NNSFC grant (81370620 to G.M.), BHF grant (CH/10/001/27642 to Z.M.), ERC grant (2891164 to Z.M.), and BHF fellowship grant (FS/14/28/30713 to X.L.)

Recurrent Recruitment Manoeuvres Improve Lung Mechanics and Minimize Lung Injury during Mechanical Ventilation of Healthy Mice

INTRODUCTION: Mechanical ventilation (MV) of mice is increasingly required in experimental studies, but the conditions that allow stable ventilation of mice over several hours have not yet been fully defined. In addition, most previous studies documented vital parameters and lung mechanics only incompletely. The aim of the present study was to establish experimental conditions that keep these parameters within their physiological range over a period of 6 h. For this purpose, we also examined the effects of frequent short recruitment manoeuvres (RM) in healthy mice. METHODS: Mice were ventilated at low tidal volume V(T) = 8 mL/kg or high tidal volume V(T) = 16 mL/kg and a positive end-expiratory pressure (PEEP) of 2 or 6 cm H(2)O. RM were performed every 5 min, 60 min or not at all. Lung mechanics were followed by the forced oscillation technique. Blood pressure (BP), electrocardiogram (ECG), heart frequency (HF), oxygen saturation and body temperature were monitored. Blood gases, neutrophil-recruitment, microvascular permeability and pro-inflammatory cytokines in bronchoalveolar lavage (BAL) and blood serum as well as histopathology of the lung were examined. RESULTS: MV with repetitive RM every 5 min resulted in stable respiratory mechanics. Ventilation without RM worsened lung mechanics due to alveolar collapse, leading to impaired gas exchange. HF and BP were affected by anaesthesia, but not by ventilation. Microvascular permeability was highest in atelectatic lungs, whereas neutrophil-recruitment and structural changes were strongest in lungs ventilated with high tidal volume. The cytokines IL-6 and KC, but neither TNF nor IP-10, were elevated in the BAL and serum of all ventilated mice and were reduced by recurrent RM. Lung mechanics, oxygenation and pulmonary inflammation were improved by increased PEEP. CONCLUSIONS: Recurrent RM maintain lung mechanics in their physiological range during low tidal volume ventilation of healthy mice by preventing atelectasis and reduce the development of pulmonary inflammation

Response of bone turnover markers to raloxifene treatment in postmenopausal women with osteopenia.

Introduction: The change in bone turnover markers (BTM) in response to osteoporosis therapy can be assessed by a decrease beyond the least significant change (LSC) or below the mean of the reference interval (RI). We compared the performance of these two approaches in women treated with raloxifene. Methods: Fifty postmenopausal osteopenic women, (age 51-72y) were randomised to raloxifene or no treatment for 2 years. Blood samples were collected for the measurement of BTM. The LSC for each marker was calculated from the untreated women and the RI obtained from healthy premenopausal women (age 35-40y). Bone mineral density (BMD) was measured at the spine and hip. Results: There was a decrease in BTM in response to raloxifene treatment; percentage change at 12 weeks, CTX -39% (95% CI -48 to -28) and PINP -32% (95% CI -40 to -23) P<0.001. The proportion of women classified as responding to treatment using LSC at 12 weeks was: CTX 38%, PINP 52%, at 48 weeks CTX 60%, PINP 65%. For the RI approach; at 12 weeks CTX and PINP 38%, at 48 weeks CTX 40%, PINP 45%. There was a significant difference in the change in spine BMD in the raloxifene treated group compared to the no-treatment group at week 48; difference 0.031 g/cm2, (95% CI 0.016 to 0.046, P<0.001). Conclusions: The two approaches identified women that reached the target for treatment using BTM. Both LSC and RI criteria appear useful in identifying treatment response but the two approaches do not fully overlap and may be complementary

Effectiveness of Teriparatide in Women Over 75 Years of Age with Severe Osteoporosis: 36-Month Results from the European Forsteo Observational Study (EFOS)

This predefined analysis of the European Forsteo Observational Study (EFOS) aimed to describe clinical fracture incidence, back pain, and health-related quality of life (HRQoL) during 18 months of teriparatide treatment and 18 months post-teriparatide in the subgroup of 589 postmenopausal women with osteoporosis aged ≥75 years. Data on clinical fractures, back pain (visual analogue scale, VAS), and HRQoL (EQ-5D) were collected over 36 months. Fracture data were summarized in 6-month intervals and analyzed using logistic regression with repeated measures. A repeated-measures model analyzed changes from baseline in back pain VAS and EQ-VAS. During the 36-month observation period, 87 (14.8 %) women aged ≥75 years sustained a total of 111 new fractures: 37 (33.3 %) vertebral fractures and 74 (66.7 %) nonvertebral fractures. Adjusted odds of fracture was decreased by 80 % in the 30 to <36–month interval compared with the first 6-month interval (P < 0.009). Although the older subgroup had higher back pain scores and poorer HRQoL at baseline than the younger subgroup, both age groups showed significant reductions in back pain and improvements in HRQoL postbaseline. In conclusion, women aged ≥75 years with severe postmenopausal osteoporosis treated with teriparatide in normal clinical practice showed a reduced clinical fracture incidence by 30 months compared with baseline. An improvement in HRQoL and, possibly, an early and significant reduction in back pain were also observed, which lasted for at least 18 months after teriparatide discontinuation when patients were taking other osteoporosis medication. The results should be interpreted in the context of an uncontrolled observational study

Marginal zone B cells control the response of follicular helper T cells to a high-cholesterol diet

Splenic marginal zone B (MZB) cells, positioned at the interface between circulating blood and lymphoid tissue, detect and respond to blood-borne antigens. Here we show that MZB cells in mice activate a homeostatic program in response to a high-cholesterol diet (HCD) and regulate both the differentiation and accumulation of T follicular helper (TFH) cells. Feeding mice an HCD resulted in upregulated MZB cell surface expression of the immunoregulatory ligand PDL1 in an ATF3-dependent manner and increased the interaction between MZB cells and pre-TFH cells, leading to PDL1-mediated suppression of TFH cell motility, alteration of TFH cell differentiation, reduced TFH abundance and suppression of the proatherogenic TFH response. Our findings reveal a previously unsuspected role for MZB cells in controlling the TFH–germinal center response to a cholesterol-rich diet and uncover a PDL1-dependent mechanism through which MZB cells use their innate immune properties to limit an exaggerated adaptive immune response.This work was supported by BHF grant no. PG/15/76/31756, BHF grant no. PG/13/73/30466, ERC grant no. 2891164 and EC FP7 VIA grant no. HEALTH-F4- 2013-603131 to Z.M. and by SAF2013-45543-R from the Spanish Ministry of Economy and Competitiveness (MINECO) to J.L.d.l.P. M.N. was first supported by a Sara Borrell grant (CD09/00452) from the Instituto Nacional de Salud Carlos III (Spain) and then by a 2-year BHF Project Grant. M.N. has also received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. 608765. The Wellcome Trust supported the Cambridge Mouse Biochemistry Laboratory