Hepatic scavenger receptor class B type 1 knockdown reduces atherosclerosis and enhances the antiatherosclerotic effect of brown fat activation in APOE*3-Leiden.CETP mice

Objective:Brown fat activation attenuates atherosclerosis development by accelerating triglyceride-rich lipoprotein turnover and/or stimulation of reverse cholesterol transport via the SRB1 (scavenger receptor class B type 1). The aim of this study was to investigate the specific role of hepatic SRB1 in the atheroprotective properties of brown fat activation.Approach and Results:APOE*3-Leiden.CETP mice, a well-established model of human-like lipoprotein metabolism and atherosclerosis, were treated with vehicle or adenoassociated virus serotype 8-short hairpin RNA, which decreased hepatic SRB1 protein levels by 40% to 55%. After 2 weeks, mice without or with hepatic SRB1 knockdown were treated with vehicle or the beta 3-adrenergic receptor agonist CL316 243 to activate brown fat for 4 weeks to determine HDL (high-density lipoprotein) catabolism and for 9 weeks to evaluate atherosclerosis. Surprisingly, hepatic SRB1 knockdown additively improved the beneficial effects of beta 3-adrenergic receptor agonism on atherosclerosis development. In fact, hepatic SRB1 knockdown per se not only increased HDL-cholesterol levels but also reduced plasma triglyceride and non-HDL-cholesterol levels, thus explaining the reduction in atherosclerosis development. Mechanistic studies indicated that this is due to increased lipolytic processing and hepatic uptake of VLDL (very low density lipoprotein) by facilitating VLDL-surface transfer to HDL.Conclusions:Hepatic SRB1 knockdown in a mouse model with an intact ApoE (apolipoprotein E)-LDLR (low density lipoprotein receptor) clearance pathway, relevant to human lipoprotein metabolism, reduced atherosclerosis and improved the beneficial effect of brown fat activation on atherosclerosis development, explained by pleiotropic effects of hepatic SRB1 knockdown on lipolytic processing and hepatic uptake of VLDL. Brown fat activation could thus be an effective strategy to treat cardiovascular disease also in subjects with impaired SRB1 function.Functional Genomics of Systemic Disorder

Defective Autophagy in T Cells Impairs the Development of Diet-Induced Hepatic Steatosis and Atherosclerosis

Macroautophagy (or autophagy) is a conserved cellular process in which cytoplasmic cargo is targeted for lysosomal degradation. Autophagy is crucial for the functional integrity of different subsets of T cells in various developmental stages. Since atherosclerosis is an inflammatory disease of the vessel wall which is partly characterized by T cell mediated autoimmunity, we investigated how advanced atherosclerotic lesions develop in mice with T cells that lack autophagy-related protein 7 (Atg7), a protein required for functional autophagy. Mice with a T cell-specific knock-out of Atg7 (Lck-Cre Atg7f/f) had a diminished naïve CD4+ and CD8+ T cell compartment in the spleen and mediastinal lymph node as compared to littermate controls (Atg7f/f). Lck-Cre Atg7f/f and Atg7f/f mice were injected intravenously with rAAV2/8-D377Y-mPCSK9 and fed a Western-type diet to induce atherosclerosis. While Lck-Cre Atg7f/f mice had equal serum Proprotein Convertase Subtilisin/Kexin type 9 levels as compared to Atg7f/f mice, serum cholesterol levels were significantly diminished in Lck-Cre Atg7f/f mice. Histological analysis of the liver revealed less steatosis, and liver gene expression profiling showed decreased expression of genes associated with hepatic steatosis in Lck-Cre Atg7f/f mice as compared to Atg7f/f mice. The level of hepatic CD4+ and CD8+ T cells was greatly diminished but both CD4+ and CD8+ T cells showed a relative increase in their IFNγ and IL-17 production upon Atg7 deficiency. Atg7 deficiency furthermore reduced the hepatic NKT cell population which was decreased to < 0.1% of the lymphocyte population. Interestingly, T cell-specific knock-out of Atg7 decreased the mean atherosclerotic lesion size in the tri-valve area by over 50%. Taken together, T cell-specific deficiency of Atg7 resulted in a decrease in hepatic steatosis and limited inflammatory potency in the (naïve) T cell compartment in peripheral lymphoid tissues, which was associated with a strong reduction in experimental atherosclerosis

Tim-1 mucin domain-mutant mice display exacerbated atherosclerosis

Increasing evidence has shown that immune checkpoint molecules of the T-cell immunoglobulin and mucin domain (Tim) family are associated with diverse physiologic and pathologic processes. Previous studies of the role of Tim-1 in atherosclerosis using anti-Tim-1 antibodies have yielded contradictory results. We thus aimed to investigate atherosclerosis development in Tim-1 deficient mice.Mice with a specific loss of the Tim-1 mucin-domain (Tim-1Δmucin) and C57BL/6 (WT) mice received a single injection of a recombinant adeno-associated virus encoding murine Pcsk9 (rAAV2/8-D377Y-mPcsk9) and were fed a Western type diet for 13 weeks to introduce atherosclerosis.Tim-1Δmucin mice developed significantly larger lesions in the aortic root compared to WT mice, with significantly more macrophages and a trend towards a larger necrotic core. Furthermore, Tim-1Δmucin mice showed a significant loss of IL-10+ B cells and regulatory B cell subsets and increased pro-atherogenic splenic follicular B cells compared to WT mice. Moreover, Tim-1Δmucin mice displayed a dramatic reduction in Th2-associated immune response compared to controls but we did not observe any changes in humoral immunity.In summary, Tim-1Δmucin mice displayed a profound impairment in IL-10+ B cells and an imbalance in the Th1/Th2 ratio, which associated with exacerbated atherosclerosis.Biopharmaceutic

Diet-induced dyslipidemia induces metabolic and migratory adaptations in regulatory T cells

A hallmark of advanced atherosclerosis is inadequate immunosuppression by regulatory T(Treg) cells inside atherosclerotic lesions. Dyslipidemia has been suggested to alter Treg cellmigration by affecting the expression of specific membrane proteins, thereby decreasing Treg cellmigration towards atherosclerotic lesions. Besides membrane proteins, cellular metabolism has beenshown to be a crucial factor in Treg cell migration. We aimed to determine whether dyslipidemiacontributes to altered migration of Treg cells, in part, by affecting cellular metabolism.Dyslipidemia was induced by feeding Ldlr-/- mice a Western-type diet for 16-20weeks and intrinsic changes in Treg cells affecting their migration and metabolism were examined.Dyslipidemia was associated with altered mTORC2 signaling in Treg cells, decreased expression ofmembrane proteins involved in migration, including CD62L, CCR7 and S1Pr1, and decreased Tregcell migration towards lymph nodes. Furthermore, we discovered that diet-induced dyslipidemiainhibited mTORC1 signaling, induced PPARδ activation and increased fatty acid (FA) oxidation inTreg cells. Moreover, mass-spectrometry analysis of serum from Ldlr-/- mice with normolipidemia ordyslipidemia showed increases in multiple PPARδ ligands during dyslipidemia. Treatment with asynthetic PPARδ agonist increased the migratory capacity of Treg cells in vitro and in vivo in an FAoxidation dependent manner. Furthermore, diet-induced dyslipidemia actually enhanced Treg cellmigration into the inflamed peritoneum and into atherosclerotic lesions in vitro.Altogether, our findings implicate that dyslipidemia does not contribute toatherosclerosis by impairing Treg cell migration as dyslipidemia associated with an effector-likemigratory phenotype in Treg cells.Analytical BioScience

IFNγ-stimulated B cells inhibit T follicular helper cells and protect against atherosclerosis

B and T cells are interconnected in the T follicular helper-germinal center B cell (TFH-GC B cell) axis, which is hyperactive during atherosclerosis development and loss of control along this axis results in exacerbated atherosclerosis. Inhibition of the TFH-GC B cell axis can be achieved by providing negative co-stimulation to TFH cells through the PD-1/PD-L1 pathway. Therefore, we investigated a novel therapeutic strategy using PD-L1-expressing B cells to inhibit atherosclerosis. We found that IFNγ-stimulated B cells significantly enhanced PD-L1 expression and limited TFH cell development. To determine whether IFNγ-B cells can reduce collar-induced atherosclerosis, apoE -/- mice fed a Western-type diet were treated with PBS, B cells or IFNγ-B cells for a total of 5 weeks following collar placement. IFNγ-B cells significantly increased PD-L1hi GC B cells and reduced plasmablasts. Interestingly, IFNγ-B cells-treated mice show increased atheroprotective Tregs and T cell-derived IL-10. In line with these findings, we observed a significant reduction in total lesion volume in carotid arteries of IFNγ-B cells-treated mice compared to PBS-treated mice and a similar trend was observed compared to B cell-treated mice. In conclusion, our data show that IFNγ-stimulated B cells strongly upregulate PD-L1, inhibit TFH cell responses and protect against atherosclerosis.Biopharmaceutic

CD8+ T-cells contribute to lesion stabilization in advanced atherosclerosis by limiting macrophage content and CD4+ T-cell responses

Human endarterectomy samples analyzed by flow cytometry showed a negative correlation between the percentage of CD8+ T-cells and macrophages, suggesting a possible protective role for these cells in lesion development. To further test this hypothesis, LDLr-/- mice were fed a Western-type diet (WTD) for 10 weeks to induce atherosclerosis, after which they received CD8α-depleting or isotype control antibody for six weeks. Depletion of CD8+ T-cells in advanced atherosclerosis resulted in less stable lesions, with significantly reduced collagen content in the trivalve area, increased macrophage content and increased necrotic core area compared to controls. Mechanistically, we observed that CD8 depletion specifically increased the fraction of Th1 CD4+ T-cells in the lesions. Treatment of WTD-fed LDLr-/- mice with a FasL-neutralizing antibody resulted in similar changes in macrophages and CD4+ T-cell skewing as CD8+ T-cell depletion. T lymphocytes play an important role in atherosclerosis development, but the role of the CD8+ T-cell remains debated, especially in the clinically relevant advanced stages of atherosclerosis development. Here, we set out to determine the role of CD8+ T-cells in advanced atherosclerosis. These findings demonstrate for the first time a local, protective role for CD8+ T-cells in advanced atherosclerosis, through limiting accumulation of Th1 cells and macrophages, identifying a novel regulatory mechanism for these cells in atherosclerosis. Methods and Results Aims ConclusionDrug Delivery Technolog

Primary versus early secondary referral to a specialized neurotrauma center in patients with moderate/severe traumatic brain injury: a CENTER TBI study.

BackgroundPrehospital care for patients with traumatic brain injury (TBI) varies with some emergency medical systems recommending direct transport of patients with moderate to severe TBI to hospitals with specialist neurotrauma care (SNCs). The aim of this study is to assess variation in levels of early secondary referral within European SNCs and to compare the outcomes of directly admitted and secondarily transferred patients.MethodsPatients with moderate and severe TBI (Glasgow Coma Scale ResultsA total of 1347 moderate/severe TBI patients from 53 SNCs in 18 European countries were included. Of these 1347 patients, 195 (14.5%) were admitted after early secondary referral. Secondarily referred moderate/severe TBI patients presented more often with a CT abnormality: mass lesion (52% vs. 34%), midline shift (54% vs. 36%) and acute subdural hematoma (77% vs. 65%). After adjusting for case-mix, there was a large European variation in early secondary referral, with a median OR of 1.69 between countries. Early secondary referral was not associated with functional outcome (adjusted OR 1.07, 95% CI 0.78-1.69), nor with survival at discharge (1.05, 0.58-1.90).ConclusionsAcross Europe, substantial practice variation exists in the proportion of secondarily referred TBI patients at SNCs that is not explained by case mix. Within SNCs early secondary referral does not seem to impact functional outcome and survival after stabilisation in a non-specialised hospital. Future research should identify which patients with TBI truly benefit from direct transportation

Predictors of Access to Rehabilitation in the Year Following Traumatic Brain Injury : A European Prospective and Multicenter Study

Background Although rehabilitation is beneficial for individuals with traumatic brain injury (TBI), a significant proportion of them do not receive adequate rehabilitation after acute care. Objective Therefore, the goal of this prospective and multicenter study was to investigate predictors of access to rehabilitation in the year following injury in patients with TBI. Methods Data from a large European study (CENTER-TBI), including TBIs of all severities between December 2014 and December 2017 were used (N = 4498 patients). Participants were dichotomized into those who had and those who did not have access to rehabilitation in the year following TBI. Potential predictors included sociodemographic factors, psychoactive substance use, preinjury medical history, injury-related factors, and factors related to medical care, complications, and discharge. Results In the year following traumatic injury, 31.4% of patients received rehabilitation services. Access to rehabilitation was positively and significantly predicted by female sex (odds ratio [OR] = 1.50), increased number of years of education completed (OR = 1.05), living in Northern (OR = 1.62; reference: Western Europe) or Southern Europe (OR = 1.74), lower prehospital Glasgow Coma Scale score (OR = 1.03), higher Injury Severity Score (OR = 1.01), intracranial (OR = 1.33) and extracranial (OR = 1.99) surgery, and extracranial complication (OR = 1.75). On contrast, significant negative predictors were lack of preinjury employment (OR = 0.80), living in Central and Eastern Europe (OR = 0.42), and admission to hospital ward (OR = 0.47; reference: admission to intensive care unit) or direct discharge from emergency room (OR = 0.24). Conclusions Based on these findings, there is an urgent need to implement national and international guidelines and strategies for access to rehabilitation after TBI.Peer reviewe

Brain death and postmortem organ donation: Report of a questionnaire from the CENTER-TBI study

Background: We aimed to investigate the extent of the agreement on practices around brain death and postmortem organ donation. Methods: Investigators from 67 Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study centers completed several questionnaires (response rate: 99%). Results: Regarding practices around brain death, we found agreement on the clinical evaluation (prerequisites and neurological assessment) for brain death determination (BDD) in 100% of the centers. However, ancillary tests were required for BDD in 64% of the centers. BDD for nondonor patients was deemed mandatory in 18% of the centers before withdrawing life-sustaining measures (LSM). Also, practices around postmortem organ donation varied. Organ donation after circulatory arrest was forbidden in 45% of the centers. When withdrawal of LSM was contemplated, in 67% of centers the patients with a ventricular drain in situ had this removed, either sometimes or all of the time. Conclusions: This study showed both agreement and some regional differences regarding practices around brain death and postmortem organ donation. We hope our results help quantify and understand potential differences, and provide impetus for current dialogs toward further harmonization of practices around brain death and postmortem organ donation