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The mechanisms by which genetic variation affects transcription regulation and phenotypes at the nucleotide level are incompletely understood. Here we use natural genetic variation as an in vivo mutagenesis screen to assess the genome-wide effects of sequence variation on lineage-determining and signal-specific transcription factor binding, epigenomics and transcriptional outcomes in primary macrophages from different mouse strains. We find substantial genetic evidence to support the concept that lineage-determining transcription factors define epigenetic and transcriptomic states by selecting enhancer-like regions in the genome in a collaborative fashion and facilitating binding of signal-dependent factors. This hierarchical model of transcription factor function suggests that limited sets of genomic data for lineage-determining transcription factors and informative histone modifications can be used for the prioritization of disease-associated regulatory variants.

Air pollution is a prominent cause of cardiopulmonary illness, but uncertainties remain regarding the mechanisms mediating those effects as well as individual susceptibility. Macrophages are highly responsive to particles, and we hypothesized that their responses would be dependent on their genetic backgrounds. We conducted a genome-wide analysis of peritoneal macrophages harvested from 24 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). Cells were treated with a DEP methanol extract (DEPe) to elucidate potential pathways that mediate acute responses to air pollution exposures. This analysis showed that 1247 genes were upregulated and 1383 genes were downregulated with DEPe treatment across strains. Pathway analysis identified oxidative stress responses among the most prominent upregulated pathways; indeed, many of the upregulated genes included antioxidants such as Hmox1, Txnrd1, Srxn1, and Gclm, with NRF2 (official gene symbol: Nfe2l2) being the most significant driver. DEPe induced a Mox-like transcriptomic profile, a macrophage subtype typically induced by oxidized phospholipids and likely dependent on NRF2 expression. Analysis of individual strains revealed consistency of overall responses to DEPe and yet differences in the degree of Mox-like polarization across the various strains, indicating DEPe × genetic interactions. These results suggest a role for macrophage polarization in the cardiopulmonary toxicity induced by air pollution., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jesus A. Araujo reports financial support was provided by National Institute of Environmental Health Sciences. May Bhetraratana reports financial support was provided by National Institute of Environmental Health Sciences. Aldons J. Lusis reports financial support was provided by National Heart Lung and Blood Institute. Xia Yang reports financial support was provided by National Heart Lung and Blood Institute. May Bhetraratana reports a relationship with California Air Resources Board that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Age-related macular degeneration (AMD) is a multifactorial retinal disease with a large genetic risk contribution. Reticular pseudodrusen (RPD) is a sub-phenotype of AMD with a high risk of progression to late vision threatening AMD. In a genome-wide association study of 2,165 AMD+/RPD+ and 4,181 AMD+/RPD-compared to 7,660 control participants, both chromosomes 1 ( CFH ) and 10 ( ARMS2/HTRA1 ) major AMD risk loci were reidentified. However association was only detected for the chromosome 10 locus when comparing AMD+/RPD+ to AMD+/RPD-cases. The chromosome 1 locus was notably absent. The chromosome 10 RPD risk region contains a long non-coding RNA (ENSG00000285955/BX842242.1) which colocalizes with genetic markers of retinal thickness. BX842242.1 has a strong retinal eQTL signal, pinpointing the parafoveal photoreceptor outer segment layer. Whole genome sequencing of phenotypically extreme RPD cases identified even stronger enrichment for the chromosome 10 risk genotype.

Rare DRAM2 coding variants cause retinal dystrophy with early macular involvement via unknown mechanisms. We found that DRAM2 is ubiquitously expressed in the human eye and expression changes were observed in eyes with more common maculopathy such as Age-related Macular Degeneration (AMD). To gain insights into pathogenicity of DRAM2-related retinopathy, we used a combination of in vitro and in vivo models. We found that DRAM2 loss in human pluripotent stem cell (hPSC)-derived retinal organoids caused the presence of additional mesenchymal cells. Interestingly, Dram2 loss in mice also caused increased proliferation of cells from the choroid in vitro and exacerbated choroidal neovascular lesions in vivo . Furthermore, we observed that DRAM2 loss in human retinal pigment epithelial (RPE) cells resulted in increased susceptibility to stress-induced cell death in vitro and that Dram2 loss in mice caused age-related photoreceptor degeneration. This highlights the complexity of DRAM2 function, as its loss in choroidal cells provided a proliferative advantage, whereas its loss in post-mitotic cells, such as photoreceptor and RPE cells, increased degeneration susceptibility. Different models such as human pluripotent stem cell-derived systems and mice can be leveraged to study and model human retinal dystrophies; however, cell type and species-specific expression must be taken into account when selecting relevant systems., Competing Interests: At the time of the study, all authors were full time employees of Genentech Inc. (a member of the Roche group) with stock and stock options in Roche., (Copyright © 2023 Jones, Orozco, Qin, Truong, Caplazi, Elstrott, Modrusan, Chaney and Jeanne.)

Background: The interleukin-22 cytokine (IL-22) has demonstrated efficacy in preclinical colitis models with non-immunosuppressive mechanism of action. Efmarodocokin alfa (UTTR1147A) is a fusion protein agonist that links IL-22 to the crystallisable fragment (Fc) of human IgG 4 for improved pharmacokinetic characteristics, but with a mutation to minimise Fc effector functions., Methods: This randomised, phase 1b study evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics of repeat intravenous dosing of efmarodocokin alfa in healthy volunteers (HVs; n=32) and patients with ulcerative colitis (n=24) at 30-90 µg/kg doses given once every 2 weeks or monthly (every 4 weeks) for 12 weeks (6:2 active:placebo per cohort)., Results: The most common adverse events (AEs) were on-target, reversible, dermatological effects (dry skin, erythema and pruritus). Dose-limiting non-serious dermatological AEs (severe dry skin, erythema, exfoliation and discomfort) were seen at 90 μg/kg once every 2 weeks (HVs, n=2; patients, n=1). Pharmacokinetics were generally dose-proportional across the dose levels, but patients demonstrated lower drug exposures relative to HVs at the same dose. IL-22 serum biomarkers and IL-22-responsive genes in colon biopsies were induced with active treatment, and microbiota composition changed consistent with a reversal in baseline dysbiosis. As a phase 1b study, efficacy endpoints were exploratory only. Clinical response was observed in 7/18 active-treated and 1/6 placebo-treated patients; clinical remission was observed in 5/18 active-treated and 0/6 placebo-treated patients., Conclusion: Efmarodocokin alfa had an adequate safety and pharmacokinetic profile in HVs and patients. Biomarker data confirmed IL-22R pathway activation in the colonic epithelium. Results support further investigation of this non-immunosuppressive potential inflammatory bowel disease therapeutic., Trial Registration Number: NCT02749630., Competing Interests: Competing interests: ANL, YW, MK, AD, BB, BH, LDO, JSM, HC and MER are employees and stockholders of Genentech/Roche., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Age-related macular degeneration (AMD) is a leading cause of blindness, affecting 200 million people worldwide. To identify genes that could be targeted for treatment, we created a molecular atlas at different stages of AMD. Our resource is comprised of RNA sequencing (RNA-seq) and DNA methylation microarrays from bulk macular retinal pigment epithelium (RPE)/choroid of clinically phenotyped normal and AMD donor eyes (n = 85), single-nucleus RNA-seq (164,399 cells), and single-nucleus assay for transposase-accessible chromatin (ATAC)-seq (125,822 cells) from the retina, RPE, and choroid of 6 AMD and 7 control donors. We identified 23 genome-wide significant loci differentially methylated in AMD, over 1,000 differentially expressed genes across different disease stages, and an AMD Müller state distinct from normal or gliosis. Chromatin accessibility peaks in genome-wide association study (GWAS) loci revealed putative causal genes for AMD, including HTRA1 and C6orf223 . Our systems biology approach uncovered molecular mechanisms underlying AMD, including regulators of WNT signaling, FRZB and TLE2 , as mechanistic players in disease., Competing Interests: L.D.O., J.H., A.D.S., J.T., S.H., V.T.M., C.C., J.L., A. Sridhar, O.M., J.S.K., M.J.T., B.L.Y., and H.-H.C. are employees and shareholders of Genentech/Roche. I.K.K. is a consultant for Kodiak Sciences and Biophytis and receives research funding from Allergen. M.M.D. has a research grant from Genentech/Roche., (© 2023 The Authors.)

Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are 'omics' technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD., Competing Interests: Declaration of competing interest AIdH is currently an employee of AbbVie; LDO, HHC, HC and LH are employees of Genentech; SK is an employee of Gemini Therapeutics. GSH is a shareholder, consultant, and co-founder of Perceive Biotherapeutics, Inc. and an inventor on patents and patent applications owned by the University of Iowa and the University of Utah. RFM, APV, TS, FG, JLH, JJW, SJT, RA, DS, CCWK, JDB, KAF, BHFW and MBG have no competing interests to declare., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Anti-integrins are therapeutically effective for inflammatory bowel disease, yet the relative contribution of α4β7 and αEβ7 to gut lymphocyte trafficking is not fully elucidated. Here, we evaluate the effect of α4β7 and αEβ7 blockade using a combination of murine models of gut trafficking and longitudinal gene expression analysis in etrolizumab-treated patients with Crohn's disease (CD). Dual blockade of α4β7 and αEβ7 reduces CD8 + T cell accumulation in the gut to a greater extent than blockade of either integrin alone. Anti-αEβ7 reduces epithelial:T cell interactions and promotes egress of activated T cells from the mucosa into lymphatics. Inflammatory gene expression is greater in human intestinal αEβ7 + T cells. Etrolizumab-treated patients with CD display a treatment-specific reduction in inflammatory and cytotoxic intraepithelial lymphocytes (IEL) genes. Concurrent blockade of α4β7 and αEβ7 promotes reduction of cytotoxic IELs and inflammatory T cells in the gut mucosa through a stepwise inhibition of intestinal tissue entry and retention., Competing Interests: All authors, except J.G., A.H., M.D., M.S., and W.A.F., are current or past employees of Genentech, a member of the Roche group, and may hold Roche stock or stock options., (© 2021 The Authors.)

Age-related macular degeneration (AMD) is a leading cause of vision loss. To better understand disease pathogenesis and identify causal genes in GWAS loci for AMD risk, we present a comprehensive database of human retina and retinal pigment epithelium (RPE). Our database comprises macular and non-macular RNA sequencing (RNA-seq) profiles from 129 donors, a genome-wide expression quantitative trait loci (eQTL) dataset that includes macula-specific retina and RPE/choroid, and single-nucleus RNA-seq (NucSeq) from human retina and RPE with subtype resolution from more than 100,000 cells. Using NucSeq, we find enriched expression of AMD candidate genes in RPE cells. We identify 15 putative causal genes for AMD on the basis of co-localization of genetic association signals for AMD risk and eye eQTL, including the genes TSPAN10 and TRPM1. These results demonstrate the value of our human eye database for elucidating genetic pathways and potential therapeutic targets for ocular diseases., Competing Interests: Declaration of Interests L.D.O., H.-H.C., C. Cox, K.J.K., R.A., C.E., P.C., S.S.N., Y.-J.C., Z.M., A.D., L.D.G., C. Clarke, T.B., B.Y., M.J., M.J.T., M.v.L.C., and J.A.H. were employees of Genentech at the time these studies were carried out., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Exposure to ambient particulate matter has been shown to promote a variety of disorders, including cardiovascular diseases predominantly of ischemic etiology. However, the mechanisms linking inhaled particulates with systemic vascular effects, resulting in worsened atherosclerosis, are not well defined. We assessed the potential role of macrophages in translating these effects by analyzing gene expression patterns in response to diesel exhaust particles (DEP) at the average cell level, using Affymetrix microarrays in peritoneal macrophages in culture (in vitro), and at the individual cell level, using single-cell RNA sequencing (scRNA-seq) in alveolar macrophages collected from exposed mice (in vivo). Peritoneal macrophages were harvested from C57BL/6J mice and treated with 25 μg/mL of a DEP methanol extract (DEPe). These cells exhibited significant (FDR < 0.05) differential expression of a large number of genes and enrichment in pathways, especially engaged in immune responses and antioxidant defense. DEPe led to marked upregulation of heme oxygenase 1 (Hmox1), the most significantly upregulated gene (FDR = 1.75E-06), and several other antioxidant genes. For the in vivo work, C57BL/6J mice were subjected to oropharyngeal aspiration of 200 μg of whole DEP. The gene expression profiles of the alveolar macrophages harvested from these mice were analyzed at the single-cell level using scRNA-seq, which showed significant dysregulation of a broad number of genes enriched in immune system pathways as well, but with a large heterogeneity in how individual alveolar macrophages responded to DEP exposures. Altogether, DEP pollutants dysregulated immunological pathways in macrophages that may mediate the development of pulmonary and systemic vascular effects., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active β-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human β-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a β-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Folate B 12 -dependent remethylation of homocysteine is important, but less is understood about the importance of the alternative betaine-dependent methylation pathway-catalyzed by betaine-homocysteine methyltransferase (BHMT)-for establishing and maintaining adequate DNA methylation across the genome. We studied C57Bl/6J Bhmt (betaine-homocysteine methyltransferase)-null mice at age 4, 12, 24, and 52 wk ( N = 8) and observed elevation of S -adenosylhomocysteine concentrations and development of preneoplastic foci in the liver (increased placental glutathione S -transferase and cytokeratin 8-18 activity; starting at 12 wk). At 4 wk, we identified 63 differentially methylated CpGs (DMCs; false discovery rate < 5%) proximal to 81 genes (across 14 chromosomes), of which 18 were differentially expressed. Of these DMCs, 52% were located in one 15.5-Mb locus on chromosome 13, which encompassed the Bhmt gene and defined a potentially sensitive region with mostly decreased methylation. Analyzing Hybrid Mouse Diversity Panel data, which consisted of 100 inbred strains of mice, we identified 97 DMCs that were affected by Bhmt genetic variation in the same region, with 7 overlapping those found in Bhmt -null mice ( P < 0.001). At all time points, we found a hypomethylated region mapping to Iqgap2 (IQ motif-containing GTPase activating protein 2) and F2rl2 (proteinase-activated receptor-3), 2 genes that were also silenced and underexpressed, respectively.-Lupu, D. S., Orozco, L. D., Wang, Y., Cullen, J. M., Pellegrini, M., Zeisel, S. H. Altered methylation of specific DNA loci in the liver of Bhmt -null mice results in repression of Iqgap2 and F2rl2 and is associated with development of preneoplastic foci., (© FASEB.)

Background: Complex diseases are characterized by multiple subtle perturbations to biological processes. New omics platforms can detect these perturbations, but translating the diverse molecular and statistical information into testable mechanistic hypotheses is challenging. Therefore, we set out to create a public tool that integrates these data across multiple datasets, platforms, study designs and species in order to detect the most promising targets for further mechanistic studies., Results: We developed Mergeomics, a computational pipeline consisting of independent modules that 1) leverage multi-omics association data to identify biological processes that are perturbed in disease, and 2) overlay the disease-associated processes onto molecular interaction networks to pinpoint hubs as potential key regulators. Unlike existing tools that are mostly dedicated to specific data type or settings, the Mergeomics pipeline accepts and integrates datasets across platforms, data types and species. We optimized and evaluated the performance of Mergeomics using simulation and multiple independent datasets, and benchmarked the results against alternative methods. We also demonstrate the versatility of Mergeomics in two case studies that include genome-wide, epigenome-wide and transcriptome-wide datasets from human and mouse studies of total cholesterol and fasting glucose. In both cases, the Mergeomics pipeline provided statistical and contextual evidence to prioritize further investigations in the wet lab. The software implementation of Mergeomics is freely available as a Bioconductor R package., Conclusion: Mergeomics is a flexible and robust computational pipeline for multidimensional data integration. It outperforms existing tools, and is easily applicable to datasets from different studies, species and omics data types for the study of complex traits.

Rationale: Only a small portion of the known heritability of cardiovascular diseases, such as heart failure, can be explained based on single-gene mutations. Chromatin structure and regulation provide a substrate through which genetic differences in noncoding regions may affect cellular function and response to disease, but the mechanisms are unknown., Objective: We conducted genome-wide measurements of DNA methylation in different strains of mice that are susceptible and resistant to isoproterenol-induced dysfunction to test the hypothesis that this epigenetic mark may play a causal role in the development of heart failure., Methods and Results: BALB/cJ and BUB/BnJ mice, determined to be susceptible and resistant to isoproterenol-induced heart failure, respectively, were administered the drug for 3 weeks via osmotic minipump. Reduced representational bisulfite sequencing was then used to compare the differences between the cardiac DNA methylomes in the basal state between strains and then after isoproterenol treatment. Single-base resolution DNA methylation measurements were obtained and revealed a bimodal distribution of methylation in the heart, enriched in lone intergenic CpGs and depleted from CpG islands around genes. Isoproterenol induced global decreases in methylation in both strains; however, the basal methylation pattern between strains shows striking differences that may be predictive of disease progression before environmental stress. The global correlation between promoter methylation and gene expression (as measured by microarray) was modest and revealed itself only with focused analyses of transcription start site and gene body regions (in contrast to when gene methylation was examined in toto). Modules of comethylated genes displayed correlation with other protein-based epigenetic marks, supporting the hypothesis that chromatin modifications act in a combinatorial manner to specify transcriptional phenotypes in the heart., Conclusions: This study provides the first single-base resolution map of the mammalian cardiac DNA methylome and the first case-control analysis of the changes in DNA methylation with heart failure. The findings demonstrate marked genetic differences in DNA methylation that are associated with disease progression., (© 2016 American Heart Association, Inc.)

Background: Hepatic angiosarcoma is a rare vascular malignancy that accounts for 2% of all hepatic primary tumours. The diagnosis is difficult, especially if the patient does not have history of exposure to carcinogens, which are considered as risk factors. The diagnosis is made by histopathology, but in a considerable percentage it can only be accomplished by autopsy. The performing of fine needle aspiration biopsy can lead to bleeding, with limitations in its use., Clinical Case: A 41 year-old male, with no history of exposure to carcinogens, who developed abdominal pain secondary to a haemoperitoneum due to tumour rupture, was diagnosed by imaging methods with a giant cavernous hepatic haemangioma. He was initially treated with embolisation, and later with a liver transplant. After six months he developed haemoptysis secondary to lung metastasis. The autopsy reported metastatic hepatic angiosarcoma., Discussion: This condition has been related to carcinogen exposure, with malignant transformation from a benign vascular neoplasia being proposed as a hypothesis. The differential diagnosis can be achieved with imaging studies such as CT scan, and the definitive diagnosis is made by histopathology with immunohistochemistry tests, with 35%-100% being made in the autopsy., Conclusion: Hepatic angiosarcoma is a malignant vascular neoplasia, the potential curative option is surgery with tumour free margins. Liver transplantation remains controversial because of its poor prognosis in the short term., (Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.)

Heritable epigenetic factors can contribute to complex disease etiology. Here we examine the contribution of DNA methylation to complex traits that are precursors to heart disease, diabetes, and osteoporosis. We profiled DNA methylation in the liver using bisulfite sequencing in 90 mouse inbred strains, genome-wide expression levels, proteomics, metabolomics, and 68 clinical traits and performed epigenome-wide association studies (EWAS). We found associations with numerous clinical traits including bone density, insulin resistance, expression, and protein and metabolite levels. A large proportion of associations were unique to EWAS and were not identified using GWAS. Methylation levels were regulated by genetics largely in cis, but we also found evidence of trans regulation, and we demonstrate that genetic variation in the methionine synthase reductase gene Mtrr affects methylation of hundreds of CpGs throughout the genome. Our results indicate that natural variation in methylation levels contributes to the etiology of complex clinical traits., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: DNA methylation is a contributing factor to both rare and common human diseases, and plays a major role in development and gene silencing. While the variation of DNA methylation among individuals has been partially characterized, the degree to which methylation patterns are preserved across generations is still poorly understood. To determine the extent of methylation differences between two generations of mice we examined DNA methylation patterns in the livers of eight parental and F1 mice from C57BL/6J and DBA/2J mouse strains using bisulfite sequencing., Results: We find a large proportion of reproducible methylation differences between C57BL/6J and DBA/2J chromosomes in CpGs, which are highly heritable between parent and F1 mice. We also find sex differences in methylation levels in 396 genes, and 11% of these are differentially expressed between females and males. Using a recently developed approach to identify allelically methylated regions independently of genotypic differences, we identify 112 novel putative imprinted genes and microRNAs, and validate imprinting at the RNA level in 10 of these genes., Conclusions: The majority of DNA methylation differences among individuals are associated with genetic differences, and a much smaller proportion of these epigenetic differences are due to sex, imprinting or stochastic intergenerational effects. Epigenetic differences can be a determining factor in heritable traits and should be considered in association studies for molecular and clinical traits, as we observed that methylation differences in the mouse model are highly heritable and can have functional consequences on molecular traits such as gene expression.

Maternal nutrient restriction causes the development of adult onset chronic diseases in the intrauterine growth restricted (IUGR) fetus. Investigations in mice have shown that either protein or calorie restriction during pregnancy leads to glucose intolerance, increased fat mass, and hypercholesterolemia in adult male offspring. Some of these phenotypes are shown to persist in successive generations. The molecular mechanisms underlying IUGR remain unclear. The placenta is a critical organ for mediating changes in the environment and the development of embryos. To shed light on molecular mechanisms that might affect placental responses to differing environments we examined placentas from mice that had been exposed to different diets. We measured gene expression and whole genome DNA methylation in both male and female placentas of mice exposed to either caloric restriction or ad libitum diets. We observed several differentially expressed pathways associated with IUGR phenotypes and, most importantly, a significant decrease in the overall methylation between these groups as well as sex-specific effects that are more pronounced in males. In addition, a set of significantly differentially methylated genes that are enriched for known imprinted genes were identified, suggesting that imprinted loci may be particularly susceptible to diet effects. Lastly, we identified several differentially methylated microRNAs that target genes associated with immunological, metabolic, gastrointestinal, cardiovascular, and neurological chronic diseases, as well as genes responsible for transplacental nutrient transfer and fetal development.

Summary: Preliminary experience using a balloon assisted technique (BAT) for embolization of arteriovenous malformations (AVM) is reported. Two patients with large pericallosal AVMs were successfully embolized with Onyx under Scepter C balloon catheter flow arrest., Clinical Presentation: One patient presented with a large intraventricular hemorrhage and hydrocephalus. The second patient presented with a long history of seizures and a small intracerebral hemorrhage. Both patients demonstrated extensive interhemispheric AVMs with multiple arterial feeders, predominantly from the pericallosal arteries., Intervention: A Marathon microcatheter was navigated into the target arterial feeders and a Scepter C occlusion balloon catheter was inflated immediately proximal. Under flow arrest, Onyx was injected via the microcatheter with excellent nidal penetration. In both cases, there was complete angiographic obliteration of the treated component of the AVM., Conclusions: Onyx embolization under balloon catheter flow arrest allows for greater nidal penetration of embolic material and improved reflux control. The technique is limited by the current deliverability of balloon catheters and the potential risk for earlier embolization of dangerous anastomosis.

20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to the migration and proliferation of vascular smooth muscle cells (VSMC) in vitro, but there are few studies that address its effects on vascular remodeling in vivo. The present study determined whether inhibition of 20-HETE production attenuates intimal hyperplasia (IH) and vascular remodeling after balloon injury (BI). Sprague Dawley rats underwent BI of the common carotid artery and were treated with vehicle, 1-aminobenzotriazole (ABT, 50 mg/kg i.p. once daily), or HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (2 mg/kg s.c. twice daily) for 14 days. Fourteen days after BI and treatment, the animals underwent carotid angiography, and the arteries were harvested for morphometric, enzymatic and immunohistochemical analysis. There was a 96% reduction of angiographic stenosis in the rats treated with 1-ABT. There was a 61 and 66% reduction of the intima/media area ratios in the 1-ABT and HET0016 treated rats compared with the vehicle-treated group. 20-HETE levels were elevated in BI carotid arteries, and the levels were markedly suppressed in the groups treated with 1-ABT and HET0016 (P < 0.001). Immunostaining revealed that the expression of CYP4A enzyme was markedly increased in the neointima of BI arteries, and it colocalized with the expression of smooth muscle-specific actin, indicating increased proliferation of VSMC. An increase in the expression of CYP4A and the production of 20-HETE contributes to neointimal growth in BI rat carotid arteries. Systemic administration 1-ABT or HET0016 prevents the increase in 20-HETE levels and attenuates VSMC migration and proliferation, resulting in a marked reduction in IH and vascular remodeling after endothelial injury.

The Fogarty catheter represents a major advancement for the effective removal of distal thrombi during vascular surgery, including carotid endarterectomy (CEA). One complication related to its use is injury to the cavernous carotid artery with development of a carotid cavernous fistula (CCF). Including a recent case at our institution, 21 patients with a Fogarty-related CCF have been reported since 1967. We performed a detailed review of all Fogarty-related CCFs during the treatment of carotid occlusive disease. We suggest a management algorithm for post-CEA acute carotid occlusion.

Background: Network construction and analysis algorithms provide scientists with the ability to sift through high-throughput biological outputs, such as transcription microarrays, for small groups of genes (modules) that are relevant for further research. Most of these algorithms ignore the important role of non-linear interactions in the data, and the ability for genes to operate in multiple functional groups at once, despite clear evidence for both of these phenomena in observed biological systems., Results: We have created a novel co-expression network analysis algorithm that incorporates both of these principles by combining the information-theoretic association measure of the maximal information coefficient (MIC) with an Interaction Component Model. We evaluate the performance of this approach on two datasets collected from a large panel of mice, one from macrophages and the other from liver by comparing the two measures based on a measure of module entropy, Gene Ontology (GO) enrichment, and scale-free topology (SFT) fit. Our algorithm outperforms a widely used co-expression analysis method, weighted gene co-expression network analysis (WGCNA), in the macrophage data, while returning comparable results in the liver dataset when using these criteria. We demonstrate that the macrophage data has more non-linear interactions than the liver dataset, which may explain the increased performance of our method, termed Maximal Information Component Analysis (MICA) in that case., Conclusions: In making our network algorithm more accurately reflect known biological principles, we are able to generate modules with improved relevance, particularly in networks with confounding factors such as gene by environment interactions.

Many common diseases have an important inflammatory component mediated in part by macrophages. Here we used a systems genetics strategy to examine the role of common genetic variation in macrophage responses to inflammatory stimuli. We examined genome-wide transcript levels in macrophages from 92 strains of the Hybrid Mouse Diversity Panel. We exposed macrophages to control media, bacterial lipopolysaccharide (LPS), or oxidized phospholipids. We performed association mapping under each condition and identified several thousand expression quantitative trait loci (eQTL), gene-by-environment interactions, and eQTL "hot spots" that specifically control LPS responses. We used siRNA knockdown of candidate genes to validate an eQTL hot spot in chromosome 8 and identified the gene 2310061C15Rik as a regulator of inflammatory responses in macrophages. We have created a public database where the data presented here can be used as a resource for understanding many common inflammatory traits that are modeled in the mouse and for the dissection of regulatory relationships between genes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We have developed an association-based approach using classical inbred strains of mice in which we correct for population structure, which is very extensive in mice, using an efficient mixed-model algorithm. Our approach includes inbred parental strains as well as recombinant inbred strains in order to capture loci with effect sizes typical of complex traits in mice (in the range of 5% of total trait variance). Over the last few years, we have typed the hybrid mouse diversity panel (HMDP) strains for a variety of clinical traits as well as intermediate phenotypes and have shown that the HMDP has sufficient power to map genes for highly complex traits with resolution that is in most cases less than a megabase. In this essay, we review our experience with the HMDP, describe various ongoing projects, and discuss how the HMDP may fit into the larger picture of common diseases and different approaches.

Background and Importance: Prominent intercavernous sinuses may result in vigorous bleeding during transsphenoidal resection of pituitary microadenomas and lead to incomplete or aborted tumor resection. We report the use of coil embolization of the intercavernous sinuses to prevent uncontrollable bleeding before transsphenoidal surgery is reattempted., Clinical Presentation: A 40-year-old man with Cushing disease underwent an attempt for transsphenoidal resection of an adrenocorticotrophic hormone--producing pituitary microadenoma. This approach was aborted secondary to profuse intercavernous sinus bleeding. The patient underwent endovascular coil embolization of the anterior intercavernous sinuses with complete obliteration. Six weeks later, he underwent successful transsphenoidal resection of the microadenoma., Conclusion: To the best of our knowledge, this is the first report of successful coil embolization of the intercavernous sinuses to prevent uncontrolled bleeding before transsphenoidal resection of pituitary microadenomas.

The small diameter of the carotid artery is not compatible with the evaluation of clinically available endovascular devices in the carotid balloon-injury (BI) model. We developed an endovascular BI model in the rat descending aorta, whose size is compatible with available endovascular instruments. We also tested the hypothesis that neointima formation is enhanced in the aorta of obese Zucker rats (OZR) compared with lean Zucker rats (LZR). Left external carotid arteriotomies and BI of the thoracic and abdominal aorta were performed by using a balloon catheter. Aortograms and aortic pathology were examined at 2, 4, and 10 wk after BI. At 10 wk after BI, the abdominal aorta in OZR had narrowed 8.3% ± 1.1% relative to baseline compared with an expansion of 2.4% ± 2.2% in LZR. Simultaneously, the thoracic aorta had expanded 9.5% ± 4.3% in LZR compared with stenosis of 2.8% ± 1.6% in OZR. Calculation of the intimal:medial thickness ratio revealed significantly increased neointimal formation in the OZR descending aorta compared with that in LNR. In conclusion, this minimally invasive BI model involving the rat descending aorta is compatible with available endovascular instruments. The descending aorta of OZR demonstrates enhanced neointimal formation and constrictive vascular remodeling after BI.

Rationale: The rat carotid balloon-injury (BI) model is a widely used model of intimal hyperplasia (IH) and vascular remodeling. A variable degree of IH after BI has been previously reported, and we have encountered technical challenges and suboptimal results with the original method., Objective: To evaluate the original rat carotid artery BI method with the use of micro-angiography. We tested the hypothesis that in order to obtain an optimal arterial response, BI should be limited to the common carotid artery with preservation of blood flow., Methods and Results: The left common carotid artery (CCA) was injured by one of three different methods. Carotid angiograms and pathology were examined 14 days after BI. A 2F Fogarty balloon catheter inflated to 2 atm inside the aortic arch would not slide back into the common carotid artery until deflation to 0.5 to 0.7 atm. Four out of five (80%) vessels injured with this method developed excessive inflammation without discernible IH. Six out of nine (66%) arteries that underwent BI limited to the CCA at 2 atm developed the largest angiographic stenosis (p=0.003) and IH (0.20±0.03 mm(2), p=0.028). Ten out of eleven (91%) arteries injured with a variable pressure of 1.5 to 2.2 atm, based on the operator's feedback, developed considerable IH (0.12±0.02 mm(2)). All injured carotid arteries with preserved blood flow on angiography developed IH with intact histological boundaries., Conclusions: Optimal IH with preservation of histological boundaries is achieved by graded BI limited to the CCA that preserves carotid blood flow., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Exophytic ependymomas of the spinal cord are very rare outside the filum or conus region. We present a patient with a thoracic spinal cord intradural extramedullary and intramedullary, World Health Organization grade II ependymoma. Gross total resection of the extramedullary component with subtotal resection of the intramedullary tumor was achieved, since there was no clear distinction between cord and tumor. The patient received postoperative external beam radiotherapy for residual tumor, and at a 2-year follow-up he is ambulatory without evidence of tumor recurrence., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This is the first report to our knowledge of the successful treatment of an asymptomatic mycotic aneurysm associated with Balamuthia mandrillaris encephalitis. A 27-year-old male with end-stage renal disease presented with generalized seizures following renal transplantation. MRI demonstrated multiple brain masses and an aneurysm of the cavernous and supraclinoid carotid artery. Autopsy of the donor's brain revealed Balamuthia encephalitis. The patient was placed on an anti-amebic regimen, his condition improved, and 126 days after the kidney transplant, MRI brain showed resolution of the aneurysm and improvement of the enhancing lesions. Balamuthia mandrillaris has been shown to cause a granulomatous encephalitis, with prominent vasculitis. This is the first report to demonstrate the risk of aneurysm formation associated with this infection. Prolonged anti-amebic treatment resulted in resolution of the aneurysm without clinical evidence of subarachnoid hemorrhage., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: To test the hypothesis that NF-E2-related factor 2 (Nrf2) expression plays an antiatherogenic role by its vascular antioxidant and anti-inflammatory properties., Methods and Results: Nrf2 is an important transcription factor that regulates the expression of phase 2 detoxifying enzymes and antioxidant genes. Its expression in vascular cells appears to be an important factor in the protection against vascular oxidative stress and inflammation. We developed Nrf2 heterozygous (HET) and homozygous knockout (KO) mice on an apolipoprotein (apo) E-null background by sequential breeding, resulting in Nrf2(-/-), apoE(-/-) (KO), Nrf2(-/+), apoE(-/-) (HET) and Nrf2(+/+), and apoE(-/-) wild-type littermates. KO mice exhibited decreased levels of antioxidant genes with evidence of increased reactive oxygen species generation compared with wild-type controls. Surprisingly, KO males exhibited 47% and 53% reductions in the degree of aortic atherosclerosis compared with HET or wild-type littermates, respectively. Decreased atherosclerosis in KO mice correlated with lower plasma total cholesterol in a sex-dependent manner. KO mice also had a decreased hepatic cholesterol content and a lower expression of lipogenic genes, suggesting that hepatic lipogenesis could be reduced. In addition, KO mice exhibited atherosclerotic plaques characterized by a lesser macrophage component and decreased foam cell formation in an in vitro lipid-loading assay. This was associated with a lower rate of cholesterol influx, mediated in part by decreased expression of the scavenger receptor CD36., Conclusions: Nrf2 expression unexpectedly promotes atherosclerotic lesion formation in a sex-dependent manner, most likely by a combination of systemic metabolic and local vascular effects.

Copy number variants (CNVs) are genomic segments which are duplicated or deleted among different individuals. CNVs have been implicated in both Mendelian and complex traits, including immune and behavioral disorders, but the study of the mechanisms by which CNVs influence gene expression and clinical phenotypes in humans is complicated by the limited access to tissues and by population heterogeneity. We now report studies of the effect of 19 CNVs on gene expression and metabolic traits in a mouse intercross between strains C57BL/6J and C3H/HeJ. We found that 83% of genes predicted to occur within CNVs were differentially expressed. The expression of most CNV genes was correlated with copy number, but we also observed evidence that gene expression was altered in genes flanking CNVs, suggesting that CNVs may contain regulatory elements for these genes. Several CNVs mapped to hotspots, genomic regions influencing expression of tens or hundreds of genes. Several metabolic traits including cholesterol, triglycerides, glucose and body weight mapped to three CNVs in the genome, in mouse chromosomes 1, 4 and 17. Predicted CNV genes, such as Itlna, Defcr-1, Trim12 and Trim34 were highly correlated with these traits. Our results suggest that CNVs have a significant impact on gene expression and that CNVs may be playing a role in the mechanisms underlying metabolic traits in mice.

Heme oxygenase (HO-1) is the rate-limiting enzyme in the catabolism of heme, which leads to the generation of biliverdin, iron, and carbon monoxide. It has been shown to have important antioxidant and antiinflammatory properties that result in a vascular antiatherogenic effect. To determine whether HO-1 expression in macrophages constitutes a significant component of the protective role in atherosclerosis, we evaluated the effect of decreased or absent HO-1 expression in peritoneal macrophages on oxidative stress and inflammation in vitro, and the effect of complete deficiency of HO-1 expression in macrophages in atherosclerotic lesion formation in vivo. We found that compared with HO-1(+/+) controls, peritoneal macrophages from HO-1(-/-) and HO-1(+/-) mice exhibited (1) increased reactive oxygen species (ROS) generation, (2) increased proinflammatory cytokines such as monocyte chemotactic protein 1 (MCP-1) and interleukin 6 (IL-6), and (3) increased foam cell formation when treated with oxLDL, attributable in part to increased expression of scavenger receptor A (SR-A). Bone marrow transplantation experiments performed in lethally irradiated LDL-R null female mice, reconstituted with bone marrow from HO-1(-/-) versus HO-1(+/+) mice, revealed that HO-1(-/-) reconstituted animals exhibited atherosclerotic lesions with a greater macrophage content as evaluated by immunohistochemistry and planimetric assessment. We conclude that HO-1 expression in macrophages constitutes an important component of the antiatherogenic effect by increasing antioxidant protection and decreasing the inflammatory component of atherosclerotic lesions.