Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis.

Migration of activated regulatory T (Treg) cells to inflamed tissue is crucial for their immune-modulatory function. While metabolic reprogramming during Treg cell differentiation has been extensively studied, the bioenergetics of Treg cell trafficking remains undefined. We have investigated the metabolic demands of migrating Treg cells in vitro and in vivo. We show that glycolysis was instrumental for their migration and was initiated by pro-migratory stimuli via a PI3K-mTORC2-mediated pathway culminating in induction of the enzyme glucokinase (GCK). Subsequently, GCK promoted cytoskeletal rearrangements by associating with actin. Treg cells lacking this pathway were functionally suppressive but failed to migrate to skin allografts and inhibit rejection. Similarly, human carriers of a loss-of-function GCK regulatory protein gene-leading to increased GCK activity-had reduced numbers of circulating Treg cells. These cells displayed enhanced migratory activity but similar suppressive function, while conventional T cells were unaffected. Thus, GCK-dependent glycolysis regulates Treg cell migration.

LDL-Cholesterol-Lowering Therapy.

The causal relation between elevated levels of LDL-C and cardiovascular disease has been largely established by experimental and clinical studies. Thus, the reduction of LDL-C levels is a major target for the prevention of cardiovascular disease. In the last decades, statins have been used as the main therapeutic approach to lower plasma cholesterol levels; however, the presence of residual lipid-related cardiovascular risk despite maximal statin therapy raised the need to develop additional lipid-lowering drugs to be used in combination with or in alternative to statins in patients intolerant to the treatment. Several new drugs have been approved which have mechanisms of action different from statins or impact on different lipoprotein classes.

Recent insights into low-density lipoprotein metabolism and therapy.

PURPOSE OF REVIEW: Elevated levels of low-density lipoprotein cholesterol (LDL-C) are causal to atherosclerosis and, thus, the reduction of LDL-C represents a major objective for the prevention of cardiovascular disease. Aim of this review is to provide an overview on novel strategies to lower LDL-C. RECENT FINDINGS: Although inhibiting liver cholesterol biosynthesis by statins is used as the main therapeutic approach to increase hepatic LDL-receptor expression and lower plasma cholesterol levels, novel insights into lipid and lipoprotein biology have led to the development of additional lipid-lowering therapies that can be used in combination with or as an alternative to statins in patients with statin-intolerance. New approaches include bempedoic acid, proprotein convertase subtilisin/kexin type 9 inhibitors, and angiopoietin-like protein 3 inhibitors. SUMMARY: In the last decade, several novel therapeutic approaches have been tested and some of them have been approved as lipid-lowering agents. Some drugs are already available in clinical practice, whereas others are at late stages of development.

Monoclonal Antibodies in the Management of Familial Hypercholesterolemia: Focus on PCSK9 and ANGPTL3 Inhibitors.

PURPOSE OF REVIEW: Familial hypercholesterolemia (FH) is a monogenic disorder characterized by high plasma levels of low-density lipoprotein cholesterol (LDL-C) since birth and a high risk of premature cardiovascular disease. The genetic defect is carried in only one allele in heterozygous FH (HeFH) or in both in the most severe homozygous FH (HoFH). Current guidelines recommend to reduce substantially LDL-C levels in these high-risk patients, with the need to use association therapy combining agents with different mechanisms of action. As most cases of FH are attributable to mutations in the gene encoding the low-density lipoprotein receptor (LDLR), statins, even in combination with ezetimibe, are less effective in reducing LDL-C plasma levels in FH patients, who require a more intensive approach with additional lipid-lowering agents. Additional targets playing key roles in regulating LDL-C levels are represented by PCSK9 and ANGPTL3. RECENT FINDINGS: Two monoclonal antibodies (mAbs) targeting PCSK9, evolocumab and alirocumab, significantly reduce LDL-C levels in HeFH patients. In patients with HoFH, the efficacy of mAbs to PCSK9 is strictly related to the presence of a residual LDLR activity; thus, patients carrying null mutations do not respond to the therapy with these mAbs, whereas some effects can be appreciated in HoFH bearing defective mutations. Conversely, evinacumab, the mAb targeting ANGPTL3, is highly effective in reducing LDL-C levels even in HoFH patients carrying null LDLR mutations, thanks to its LDLR-independent mechanism of action. Monoclonal antibodies inhibiting PCSK9 have shown a robust effect in FH patients presenting a residual LDLR activity, while ANGPTL3 inhibitors appear to be promising even in patients carrying null LDLR mutations.

Omega n-3 Supplementation: Exploring the Cardiovascular Benefits Beyond Lipoprotein Reduction.

PURPOSE OF REVIEW: Hypertriglyceridaemia is a highly prevalent disorder worldwide. Genetic and Mendelian randomization studies have suggested that triglyceride (TG)-rich lipoproteins are causal risk factors for coronary heart disease and contribute to the residual cardiovascular risk observed in patients optimally treated with statins. However, clinical trials failed to show cardiovascular benefits of TG-lowering; in this context, trials with omega-3 fatty acids provided contrasting results. Few trials have tested the supplementation of EPA alone rather than the combination of EPA + DHA. The JELIS study showed that EPA 1.8 g/day significantly reduced CV events in hypercholesterolaemic patients given statins, an effect that was independent on lipid reduction. RECENT FINDINGS: The REDUCE-IT trial showed that high-dose (4 g/day) EPA significantly reduces the incidence of major cardiovascular events compared with placebo in patients with elevated TG levels. The clinical benefit was higher than expected by the reduction of TG-rich lipoprotein levels. Recent data support the efficacy of high-dose EPA supplementation on a background of optimal LDL-C-lowering therapy as a key approach to achieve a further and significant reduction of CV events in very-high CV risk patients with persistent hypertriglyceridaemia. The effect on lipids does not appear to fully explain the CV benefit, and additional mechanisms of action of EPA likely contribute to the cardiovascular protection, including the reduction of inflammation and platelet aggregation. Current guidelines recommend using high-dose EPA in combination with a statin in high/very-high CV risk patients with mild-to-moderate elevation of plasma TG to reduce the residual CV risk.

New Pharmacological Approaches to Target PCSK9.

PURPOSE OF REVIEW: Proprotein convertase subtilisin kexin 9 (PCSK9) plays a crucial role in regulating circulating levels of LDL-C as a consequence of its ability to inhibit LDL receptor recycling in the liver. Loss of function variants in the PCSK9 gene result in low LDL-C levels and associate with reduced cardiovascular risk, whereas gain of-function variants associate with hypercholesterolemia and increased risk of early cardiovascular events. Thus, PCSK9 inhibition has been established as an additional approach for the treatment of hypercholesterolemia. The aim of this review is to provide a brief overview of current strategies targeting PCSK9 and discuss clinical results of the emerging approaches. RECENT FINDINGS: Two monoclonal antibodies targeting circulating PCSK9 (evolocumab and alirocumab) have been approved for the treatment of hypercholesterolemia and cardiovascular disease. Later, a gene silencing approach (inclisiran), which inhibits hepatic PCSK9 synthesis, was shown to be as effective as monoclonal antibodies but with a twice a year injection and is currently under evaluation for approval. Due to the elevated costs of such therapies, several other approaches have been explored, including peptide-based anti PCSK9 vaccination, and small oral PCSK9 inhibitors, which are still in preclinical phase. In the coming years, we will assist to a progressive introduction of novel anti-PCSK9 approaches in the clinical practice for the treatment of patients with hypercholesterolemia as well as patients at high cardiovascular risk.

Cholesterol membrane content has a ubiquitous evolutionary function in immune cell activation: the role of HDL.

PURPOSE OF REVIEW: Cellular cholesterol content influences the structure and function of lipid rafts, plasma membrane microdomains essential for cell signaling and activation. HDL modulate cellular cholesterol efflux, thus limiting cholesterol accumulation and controlling immune cell activation. Aim of this review is to discuss the link between HDL and cellular cholesterol metabolism in immune cells and the therapeutic potential of targeting cholesterol removal from cell membranes. RECENT FINDINGS: The inverse relationship between HDL-cholesterol (HDL-C) levels and the risk of cardiovascular disease has been recently challenged by observations linking elevated levels of HDL-C with increased risk of all-cause mortality, infections and autoimmune diseases, paralleled by the failure of clinical trials with HDL-C-raising therapies. These findings suggest that improving HDL function might be more important than merely raising HDL-C levels. New approaches aimed at increasing the ability of HDL to remove cellular cholesterol have been assessed for their effect on immune cells, and the results have suggested that this could be a new effective approach. SUMMARY: Cholesterol removal from plasma membrane by different means affects the activity of immune cells, suggesting that approaches aimed at increasing the ability of HDL to mobilize cholesterol from cells would represent the next step in HDL biology.

Strategies for the use of nonstatin therapies.

PURPOSE OF REVIEW: Dyslipidaemias are a major risk factor for cardiovascular disease (CVD); in particular, high levels of low-density lipoprotein cholesterol (LDL-C) have been associated to a higher cardiovascular risk. Reducing LDL-C levels decreases the risk of coronary heart disease (CHD), and the greater the LDL-C reduction, the greater the decrease in cardiovascular risk. Although statins represent the first line lipid-lowering therapy, many patients do not reach the recommended goals or exhibit adverse side effects leading to therapy discontinuation; in addition, a significant percentage of statin-treated patients continue to experience cardiovascular events even in the presence of well controlled LDL-C levels, because of alterations in other lipid/lipoprotein classes, including triglycerides and high-density lipoprotein cholesterol. RECENT FINDINGS: These conditions require further therapeutic interventions to achieve the recommended lipid goals. Several drugs have been developed to address these needs. Recent studies have shown that the association of ezetimibe with rosuvastatin or atorvastatin results in a better hypolipidaemic effect; in addition to this, PCSK9 inhibitors significantly reduce LDL-C levels and cardiovascular events. SUMMARY: For patients who are intolerant to statins or not able to reach the recommended LDL-C levels, despite maximal tolerated dose of statin, or exhibiting additional lipid alterations, several drugs are available that can be used either in monotherapy or on top of the maximally tolerated dose of statins.

Effect of treatment with pravastatin or ezetimibe on endothelial function in patients with moderate hypercholesterolemia.

BACKGROUND/AIM: Statin treatment improves endothelial function. It is matter of debate, however, if this effect of statins is due to their action on low-density lipoprotein cholesterol (LDL-C) or to other non-lipidic (pleiotropic) effects. The aim of this study was to evaluate whether the effect of pravastatin on endothelial function is mediated by pleiotropic effects. We therefore compared the effect of pravastatin and ezetimibe, a cholesterol absorption inhibitor, at doses yielding similar reductions in LDL-C and examined the effect of the two treatments on flow-mediated dilation (FMD) in hypercholesterolemic subjects. METHODS: A total of 33 moderately hypercholesterolemic patients were randomized into three treatment groups to receive ezetimibe 10 mg/day (n = 10), pravastatin 10 mg/day (n = 13) or no treatment (control, n = 10) for 6 weeks. To assess endothelial function, we determined FMD of the brachial artery non-invasively by high-resolution ultrasound before and after treatment. RESULTS: Ezetimibe and pravastatin treatment reduced LDL-C (mean ± standard error) to a similar extent (-20.6 ± 4.1 vs. -24.1 ± 4.0 %, respectively; P = 0.4771), while no decrease was observed in the control group. FMD increased significantly after treatment with ezetimibe (from 11.4 ± 5.7 to 16.8 ± 3.6 %; P = 0.022) and with pravastatin (from 13.7 ± 4.9 to 17.5 ± 4.4 %; P = 0.0466), but not in the control group. There were no differences in the endothelial function changes between the two treatment groups. CONCLUSIONS: In this study, two treatments that lower cholesterol via different mechanisms improved endothelial function to a similar extent, suggesting that the observed effect can be explained by the reduction of cholesterol levels.

Gonadal sex vs genetic sex in experimental atherosclerosis.

Epidemiological data and interventional studies with hormone replacement therapy suggest that women, at least until menopause, are at decreased cardiovascular risk compared to men. Still the molecular mechanisms beyond this difference are debated and the investigation in experimental models of atherosclerosis has been pivotal to prove that the activation of the estrogen receptor is atheroprotective, despite not enough to explain the differences reported in cardiovascular disease between male and female. This casts also for investigating the importance of the sex chromosome complement (genetic sex) beyond the contribution of sex hormones (gonadal sex) on atherosclerosis. Aim of this review is to present the dualism between gonadal sex and genetic sex with a focus on the data available from experimental models. The molecular mechanisms driving changes in lipid metabolism, immuno-inflammatory reactivity and vascular response in males and females that affect atherosclerosis progression will be discussed.

Dendritic cell immunoreceptor 2 (DCIR2) deficiency decreases hepatic conventional dendritic cell content but not the progression of diet-induced obesity.

AIMS: Inflammatory pathways and immune system dysregulation participate in the onset and progression of cardiometabolic diseases. The dendritic cell immunoreceptor 2 (DCIR2) is a C-type lectin receptor mainly expressed by conventional type 2 dendritic cells, involved in antigen recognition and in the modulation of T cell response. Here, we investigated the effect of DCIR2 deficiency during the development of obesity. METHODS: DCIR2 KO mice and the WT counterpart were fed with high-fat diet (HFD) for 20 weeks. Weight gain, glucose and insulin tolerance were assessed, parallel to immune cell subset profiling and histological analysis. RESULTS: After HFD feeding, DCIR2 KO mice presented altered conventional dendritic cell distribution within the liver without affecting markers of hepatic inflammation. These observations were liver restricted, since immune profile of metabolic and lymphoid organs-namely adipose tissue, spleen and mesenteric lymph nodes-did not show differences between the two groups. This reflected in a similar metabolic profile of DCIR2 KO compared to WT mice, characterized by comparable body weight gain as well as adipose tissues, spleen, Peyer's patches and mesenteric lymph nodes weight at sacrifice. Also, insulin response was similar in both groups. CONCLUSION: Our data show that DCIR2 has a redundant role in the progression of diet-induced obesity and inflammation.

Hepatic SREBP signaling requires SPRING to govern systemic lipid metabolism in mice and humans.

The sterol regulatory element binding proteins (SREBPs) are transcription factors that govern cholesterol and fatty acid metabolism. We recently identified SPRING as a post-transcriptional regulator of SREBP activation. Constitutive or inducible global ablation of Spring in mice is not tolerated, and we therefore develop liver-specific Spring knockout mice (LKO). Transcriptomics and proteomics analysis reveal attenuated SREBP signaling in livers and hepatocytes of LKO mice. Total plasma cholesterol is reduced in male and female LKO mice in both the low-density lipoprotein and high-density lipoprotein fractions, while triglycerides are unaffected. Loss of Spring decreases hepatic cholesterol and triglyceride content due to diminished biosynthesis, which coincides with reduced very-low-density lipoprotein secretion. Accordingly, LKO mice are protected from fructose diet-induced hepatosteatosis. In humans, we find common genetic SPRING variants that associate with circulating high-density lipoprotein cholesterol and ApoA1 levels. This study positions SPRING as a core component of hepatic SREBP signaling and systemic lipid metabolism in mice and humans.

HDLs, immunity, and atherosclerosis.

PURPOSE OF REVIEW: HDLs possess several physiological activities that may explain their antiatherosclerotic properties. Among them, the most relevant is the ability of HDL to promote the efflux of excess cholesterol from peripheral tissues to the liver for excretion. RECENT FINDINGS: The ability of HDL to promote cholesterol efflux results also in the modulation of a series of responses in the immune cells involved in atherosclerosis, including monocyte-macrophages, B and T lymphocytes. HDL also acts as a reservoir for a number of biologically active substances that may impact the immune system, and as the HDL composition varies to a large extent during inflammation. SUMMARY: The understanding of how these interactions take place and how biologically active substances can be delivered to relevant targets during atherogenesis is of great interest and may provide a better understanding for the role of HDL in atherogenesis.

Predictive value of HDL function in patients with coronary artery disease: relationship with coronary plaque characteristics and clinical events.

BACKGROUND: HDL is endowed with several metabolic, vascular, and immunoinflammatory protective functions. Among them, a key property is to promote reverse cholesterol transport from cells back to the liver. The aim of this study was to estimate the association of scavenger receptor class B type I (SR-BI)- and ATP binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux (the two major routes for cholesterol efflux to HDL) with the presence, extent, and severity of coronary artery disease (CAD), vascular wall remodelling processes, coronary plaque characteristics, and the incidence of myocardial infarction in the different subgroups of patients from the CAPIRE study. METHODS: Patients (n = 525) from the CAPIRE study were divided into two groups: low-risk factors (RF), with 0-1 RF (n = 263), and multiple-RF, with ≥2 RFs; within each group, subjects were classified as no-CAD or CAD based on the segment involvement score (SIS) evaluated by coronary computed tomography angiography (SIS = 0 and SIS > 5, respectively). SR-BI- and ABCA1-mediated cholesterol efflux were measured using the plasma of all patients. RESULTS: SR-BI-mediated cholesterol efflux was significantly reduced in patients with CAD in both the low-RF and multiple-RF groups, whereas ABCA1-mediated cholesterol efflux was similar among all groups. In CAD patients, multivariable analysis showed that SR-BI-mediated cholesterol efflux <25th percentile predicted cardiovascular outcome (odds ratio 4.1; 95% CI: 1.3-13.7; p = .019), whereas ABCA-1-mediated cholesterol efflux and HDL-C levels significantly did not. Despite this finding, reduced SR-BI-mediated cholesterol efflux was not associated with changes in high-risk plaque features or changes in the prevalence of elevated total, non-calcified, and low-attenuation plaque volume. CONCLUSION: SR-BI-mediated cholesterol efflux capacity is lower in patients with diffuse coronary atherosclerosis. In addition, a lower SR-BI-mediated cholesterol efflux capacity is associated with the worst clinical outcomes in patients with CAD, independently of atherosclerotic plaque features. Key MessagesIncreased cholesterol efflux capacity, an estimate of HDL function, is associated with a reduced CVD risk, regardless of HDL-C levels.HDL-C levels are significantly lower in patients with CAD.Lower SR-BI-mediated cholesterol efflux capacity is observed in patients with diffuse coronary atherosclerosis and is associated with the worst clinical outcomes in patients with CAD, independently of atherosclerotic plaque features.

Genetically determined hypercholesterolaemia results into premature leucocyte telomere length shortening and reduced haematopoietic precursors.

AIMS: Leucocyte telomere length (LTL) shortening is a marker of cellular senescence and associates with increased risk of cardiovascular disease (CVD). A number of cardiovascular risk factors affect LTL, but the correlation between elevated LDL cholesterol (LDL-C) and shorter LTL is debated: in small cohorts including subjects with a clinical diagnosis of familial hypercholesterolaemia (FH). We assessed the relationship between LDL-C and LTL in subjects with genetic familial hypercholesterolaemia (HeFH) compared to those with clinically diagnosed, but not genetically confirmed FH (CD-FH), and normocholesterolaemic subjects. METHODS AND RESULTS: LTL was measured in mononuclear cells-derived genomic DNA from 206 hypercholesterolaemic subjects (135 HeFH and 71 CD-FH) and 272 controls. HeFH presented shorter LTL vs. controls (1.27 ± 0.07 vs. 1.59 ± 0.04, P = 0.045). In particular, we found shorter LTL in young HeFH as compared to young controls (<35 y) (1.34 ± 0.08 vs. 1.64 ± 0.08, P = 0.019); moreover, LTL was shorter in statin-naïve HeFH subjects as compared to controls (1.23 ± 0.08 vs. 1.58 ± 0.04, P = 0.001). HeFH subjects presented shorter LTL compared to LDL-C matched CD-FH (1.33 ± 0.05 vs. 1.55 ± 0.08, P = 0.029). Shorter LTL was confirmed in leucocytes of LDLR-KO vs. wild-type mice and associated with lower abundance of long-term haematopoietic stem and progenitor cells (LT-HSPCs) in the bone marrow. Accordingly, HeFH subjects presented lower circulating haematopoietic precursors (CD34 + CD45dim cells) vs. CD-FH and controls. CONCLUSIONS: We found (i) shorter LTL in genetically determined hypercholesterolaemia, (ii) lower circulating haematopoietic precursors in HeFH subjects, and reduced bone marrow resident LT-HSPCs in LDLR-KO mice. We support early cellular senescence and haematopoietic alterations in subjects with FH.

HDL in Immune-Inflammatory Responses: Implications beyond Cardiovascular Diseases.

High density lipoproteins (HDL) are heterogeneous particles composed by a vast array of proteins and lipids, mostly recognized for their cardiovascular (CV) protective effects. However, evidences from basic to clinical research have contributed to depict a role of HDL in the modulation of immune-inflammatory response thus paving the road to investigate their involvement in other diseases beyond those related to the CV system. HDL-C levels and HDL composition are indeed altered in patients with autoimmune diseases and usually associated to disease severity. At molecular levels, HDL have been shown to modulate the anti-inflammatory potential of endothelial cells and, by controlling the amount of cellular cholesterol, to interfere with the signaling through plasma membrane lipid rafts in immune cells. These findings, coupled to observations acquired from subjects carrying mutations in genes related to HDL system, have helped to elucidate the contribution of HDL beyond cholesterol efflux thus posing HDL-based therapies as a compelling interventional approach to limit the inflammatory burden of immune-inflammatory diseases.

Global epidemiology of dyslipidaemias.

Dyslipidaemias are alterations to the plasma lipid profile that are often associated with clinical conditions. Dyslipidaemias, particularly elevated plasma LDL-cholesterol levels, are major risk factors for cardiovascular disease, but some forms, such as hypertriglyceridaemia, are associated with severe diseases in other organ systems, including non-alcoholic fatty liver disease and acute pancreatitis. Dyslipidaemias can be genetically determined (primary or familial dyslipidaemias) or secondary to other conditions (such as diabetes mellitus, obesity or an unhealthy lifestyle), the latter being more common. Hypercholesterolaemia is the most common form of dyslipidaemia and is associated with an increased risk of cardiovascular disease, with elevated plasma LDL-cholesterol levels being the 15th leading risk factor for death in 1990, rising to 11th in 2007 and 8th in 2019. The global burden of dyslipidaemias has increased over the past 30 years. Furthermore, the combination of high triglyceride levels and low HDL-cholesterol levels (together with the presence of small, dense LDL particles), referred to as atherogenic dyslipidaemia, is highly prevalent in patients with diabetes or metabolic syndrome and increases their risk of cardiovascular disease. Given the increasing prevalence of diabetes worldwide, treating lipid abnormalities in these patients might reduce their risk of cardiovascular disease.

Endothelial function in cardiovascular medicine: a consensus paper of the European Society of Cardiology Working Groups on Atherosclerosis and Vascular Biology, Aorta and Peripheral Vascular Diseases, Coronary Pathophysiology and Microcirculation, and Thrombosis.

Endothelial cells (ECs) are sentinels of cardiovascular health. Their function is reduced by the presence of cardiovascular risk factors, and is regained once pathological stimuli are removed. In this European Society for Cardiology Position Paper, we describe endothelial dysfunction as a spectrum of phenotypic states and advocate further studies to determine the role of EC subtypes in cardiovascular disease. We conclude that there is no single ideal method for measurement of endothelial function. Techniques to measure coronary epicardial and micro-vascular function are well established but they are invasive, time-consuming, and expensive. Flow-mediated dilatation (FMD) of the brachial arteries provides a non-invasive alternative but is technically challenging and requires extensive training and standardization. We, therefore, propose that a consensus methodology for FMD is universally adopted to minimize technical variation between studies, and that reference FMD values are established for different populations of healthy individuals and patient groups. Newer techniques to measure endothelial function that are relatively easy to perform, such as finger plethysmography and the retinal flicker test, have the potential for increased clinical use provided a consensus is achieved on the measurement protocol used. We recommend further clinical studies to establish reference values for these techniques and to assess their ability to improve cardiovascular risk stratification. We advocate future studies to determine whether integration of endothelial function measurements with patient-specific epigenetic data and other biomarkers can enhance the stratification of patients for differential diagnosis, disease progression, and responses to therapy.

Individual progression of carotid intima media thickness as a surrogate for vascular risk (PROG-IMT): Rationale and design of a meta-analysis project.

Carotid intima media thickness (IMT) progression is increasingly used as a surrogate for vascular risk. This use is supported by data from a few clinical trials investigating statins, but established criteria of surrogacy are only partially fulfilled. To provide a valid basis for the use of IMT progression as a study end point, we are performing a 3-step meta-analysis project based on individual participant data. Objectives of the 3 successive stages are to investigate (1) whether IMT progression prospectively predicts myocardial infarction, stroke, or death in population-based samples; (2) whether it does so in prevalent disease cohorts; and (3) whether interventions affecting IMT progression predict a therapeutic effect on clinical end points. Recruitment strategies, inclusion criteria, and estimates of the expected numbers of eligible studies are presented along with a detailed analysis plan.