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Human genetic variants are associated with many traits through largely unknown mechanisms. Here, combining approximately 260,000 Japanese study participants, a Japanese-specific genotype reference panel and statistical fine-mapping, we identified 4,423 significant loci across 63 quantitative traits, among which 601 were new, and 9,406 putatively causal variants. New associations included Japanese-specific coding, splicing and noncoding variants, exemplified by a damaging missense variant rs730881101 in TNNT2associated with lower heart function and increased risk for heart failure (P= 1.4 × 10−15and odds ratio = 4.5, 95% confidence interval = 3.1–6.5). Putative causal noncoding variants were supported by state-of-art in silico functional assays and had comparable effect sizes to coding variants. A plausible example of new mechanisms of causal variants is an enrichment of causal variants in 3′ untranslated regions (UTRs), including the Japanese-specific rs13306436 in IL6associated with pro-inflammatory traits and protection against tuberculosis. We experimentally showed that transcripts with rs13306436 are resistant to mRNA degradation by regnase-1, an RNA-binding protein. Our study provides a list of fine-mapped causal variants to be tested for functionality and underscores the importance of sequencing, genotyping and association efforts in diverse populations.

IMPORTANCE: Vasospastic angina (VSA) is vasospasm of the coronary artery and is particularly prevalent in East Asian populations. However, the specific genetic architecture for VSA at genome-wide levels is not fully understood. OBJECTIVE: To identify genetic factors associated with VSA. DESIGN, SETTING, AND PARTICIPANTS: This was a case-control genome-wide association study of VSA. Data from Biobank Japan (BBJ; enrolled patients from 2002-2008 and 2013-2018) were used, and controls without coronary artery disease (CAD) were enrolled. Patients from the BBJ were genotyped using arrays or a set of arrays. Patients recruited between 2002 and 2005 were classified within the first dataset, and those recruited between 2006 and 2008 were classified within the second dataset. To replicate the genome-wide association study in the first and second datasets, VSA cases and control samples from the latest patients in the BBJ recruited between 2013 and 2018 were analyzed in a third dataset. EXPOSURES: Single-nucleotide variants associated with VSA. MAIN OUTCOMES AND MEASURES: Cases with VSA and controls without CAD. RESULTS: A total of 5720 cases (mean [SD] age, 67 [10] years; 3672 male [64.2%]) and 153 864 controls (mean [SD] age, 62 [15] years; 77 362 male [50.3%]) in 3 datasets were included in this study. The variants at the RNF213 locus showed the strongest association with VSA across the 3 datasets (odds ratio [OR], 2.34; 95% CI, 1.99-2.74; P = 4.4 × 10−25). Additionally, rs112735431, an Asian-specific rare deleterious variant (p.Arg4810Lys) experimentally shown to be associated with reduced angiogenesis and a well-known causal risk for Moyamoya disease was the most promising candidate for a causal variant explaining the association. The effect size of rs112735431 on VSA was distinct from that of other CADs. Furthermore, homozygous carriers of rs112735431 showed an association with VSA characterized by a large effect estimate (OR, 18.34; 95% CI, 5.15-65.22; P = 7.0 × 10−6), deviating from the additive model (OR, 4.35; 95% CI, 1.18-16.05; P = .03). Stratified analyses revealed that rs112735431 exhibited a stronger association in males (χ21 = 7.24; P = .007) and a younger age group (OR, 3.06; 95% CI, 2.24-4.19), corresponding to the epidemiologic features of VSA. In the registry, carriers without CAD of the risk allele rs112735431 had a strikingly high mortality rate due to acute myocardial infarction during the follow-up period (hazard ratio, 2.71; 95% CI, 1.57-4.65; P = 3.3 × 10−4). As previously reported, a possible overlap between VSA and Moyamoya disease was not found. CONCLUSIONS AND RELEVANCE: Results of this study suggest that vascular cell dysfunction mediated by variants in the RNF213 locus may promote coronary vasospasm, and the presence of the risk allele could serve as a predictive factor for the prognosis.

IMPORTANCE: The clinical utility of polygenic risk scores (PRS) for coronary artery disease (CAD) has not yet been established. OBJECTIVE: To investigate the ability of a CAD PRS to potentially guide statin initiation in primary prevention after accounting for age and clinical risk. DESIGN, SETTING, AND PARTICIPANTS: This was a longitudinal cohort study with enrollment starting on January 1, 2006, and ending on December 31, 2010, with data updated to mid-2021, using data from the UK Biobank, a long-term population study of UK citizens. A replication analysis was performed in Biobank Japan. The analysis included all patients without a history of CAD and who were not taking lipid-lowering therapy. Data were analyzed from January 1 to June 30, 2022. EXPOSURES: Polygenic risk for CAD was defined as low (bottom 20%), intermediate, and high (top 20%) using a CAD PRS including 241 genome-wide significant single-nucleotide variations (SNVs). The pooled cohort equations were used to estimate 10-year atherosclerotic cardiovascular disease (ASCVD) risk and classify individuals as low (<5%), borderline (5-<7.5%), intermediate (7.5-<20%), or high risk (≥20%). MAIN OUTCOMES AND MEASURES: Myocardial infarction (MI) and ASCVD events (defined as incident clinical CAD [including MI], stroke, or CV death). RESULTS: A total of 330 201 patients (median [IQR] age, 57 [40-74] years; 189 107 female individuals [57%]) were included from the UK Biobank. Over the 10-year follow-up, 4454 individuals had an MI. The CAD PRS was significantly associated with the risk of MI in all age groups but had significantly stronger risk prediction at younger ages (age <50 years: hazard ratio [HR] per 1 SD of PRS, 1.72; 95% CI, 1.56-1.89; age 50-60 years: HR, 1.46; 95% CI, 1.38-1.53; age >60 years: HR, 1.42; 95% CI, 1.37-1.48; P for interaction <.001). In patients younger than 50 years, those with high PRS had a 3- to 4-fold increased associated risk of MI compared with those in the low PRS category. A significant interaction between CAD PRS and age was replicated in Biobank Japan. When CAD PRS testing was added to the clinical ASCVD risk score in individuals younger than 50 years, 591 of 4373 patients (20%) with borderline risk were risk stratified into intermediate risk, warranting initiation of statin therapy and 3198 of 7477 patients (20%) with both borderline or intermediate risk were stratified as low risk, thus not warranting therapy. CONCLUSIONS AND RELEVANCE: Results of this cohort study suggest that the predictive ability of a CAD PRS was greater in younger individuals and can be used to better identify patients with borderline and intermediate clinical risk who should initiate statin therapy.

Atrial fibrillation (AF) is a common cardiac arrhythmia resulting in increased risk of stroke. Despite highly heritable etiology, our understanding of the genetic architecture of AF remains incomplete. Here we performed a genome-wide association study in the Japanese population comprising 9,826 cases among 150,272 individuals and identified East Asian-specific rare variants associated with AF. A cross-ancestry meta-analysis of >1 million individuals, including 77,690 cases, identified 35 new susceptibility loci. Transcriptome-wide association analysis identified IL6Ras a putative causal gene, suggesting the involvement of immune responses. Integrative analysis with ChIP-seq data and functional assessment using human induced pluripotent stem cell-derived cardiomyocytes demonstrated ERRg as having a key role in the transcriptional regulation of AF-associated genes. A polygenic risk score derived from the cross-ancestry meta-analysis predicted increased risks of cardiovascular and stroke mortalities and segregated individuals with cardioembolic stroke in undiagnosed AF patients. Our results provide new biological and clinical insights into AF genetics and suggest their potential for clinical applications.

Mobile genetic elements (MEs) are heritable mutagens that recursively generate structural variants (SVs). ME variants (MEVs) are difficult to genotype and integrate in statistical genetics, obscuring their impact on genome diversification and traits. We developed a tool that accurately genotypes MEVs using short-read whole-genome sequencing (WGS) and applied it to global human populations. We find unexpected population-specific MEV differences, including an Aluinsertion distribution distinguishing Japanese from other populations. Integrating MEVs with expression quantitative trait loci (eQTL) maps shows that MEV classes regulate tissue-specific gene expression by shared mechanisms, including creating or attenuating enhancers and recruiting post-transcriptional regulators, supporting class-wide interpretability. MEVs more often associate with gene expression changes than SNVs, thus plausibly impacting traits. Performing genome-wide association study (GWAS) with MEVs pinpoints potential causes of disease risk, including a LINE-1 insertion associated with keloid and fasciitis. This work implicates MEVs as drivers of human divergence and disease risk.

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR–Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD.

We report a genome-wide association study (GWAS) of coronary artery disease (CAD) incorporating nearly a quarter of a million cases, in which existing studies are integrated with data from cohorts of white, Black and Hispanic individuals from the Million Veteran Program. We document near equivalent heritability of CAD across multiple ancestral groups, identify 95 novel loci, including nine on the X chromosome, detect eight loci of genome-wide significance in Black and Hispanic individuals, and demonstrate that two common haplotypes at the 9p21 locus are responsible for risk stratification in all populations except those of African origin, in which these haplotypes are virtually absent. Moreover, in the largest GWAS for angiographically derived coronary atherosclerosis performed to date, we find 15 loci of genome-wide significance that robustly overlap with established loci for clinical CAD. Phenome-wide association analyses of novel loci and polygenic risk scores (PRSs) augment signals related to insulin resistance, extend pleiotropic associations of these loci to include smoking and family history, and precisely document the markedly reduced transferability of existing PRSs to Black individuals. Downstream integrative analyses reinforce the critical roles of vascular endothelial, fibroblast, and smooth muscle cells in CAD susceptibility, but also point to a shared biology between atherosclerosis and oncogenesis. This study highlights the value of diverse populations in further characterizing the genetic architecture of CAD.

Congenital heart diseases often involve maldevelopment of the evolutionarily recent right heart chamber. To gain insight into right heart structure and function, we fine-tuned deep learning models to recognize the right atrium, right ventricle and pulmonary artery, measuring right heart structures in 40,000 individuals from the UK Biobank with magnetic resonance imaging. Genome-wide association studies identified 130 distinct loci associated with at least one right heart measurement, of which 72 were not associated with left heart structures. Loci were found near genes previously linked with congenital heart disease, including NKX2-5, TBX5/TBX3, WNT9Band GATA4. A genome-wide polygenic predictor of right ventricular ejection fraction was associated with incident dilated cardiomyopathy (hazard ratio, 1.33 per standard deviation; P= 7.1 × 10−13) and remained significant after accounting for a left ventricular polygenic score. Harnessing deep learning to perform large-scale cardiac phenotyping, our results yield insights into the genetic determinants of right heart structure and function.

Background: The association of insomnia treatment with medical costs is not well characterized in Japan, despite the high economic burden of insomnia. Objective: The aim of this study was to investigate the impact of suvorexant, the first dual orexin receptor antagonist, on direct medical costs in insomnia patients. Patients and Methods: This retrospective cohort study, conducted using a large-scale claims database, included Japanese patients with diagnosed insomnia receiving suvorexant who were treatment naïve or treatment switchers (pre-treated with a different hypnotic and switched to suvorexant). Total medical costs were estimated for 1 year before and after suvorexant initiation; p-values were calculated for the difference in costs. Results: Of the 1730 patients included, 1116 were treatment naïve and 614 were treatment switchers. Switching to suvorexant did not change the total treatment cost (US$4693–US$4692; p= 0.9964). Although treatment-naïve patients on average incurred US$3259 after suvorexant initiation, much of the additional cost was attributed to drugs other than hypnotics in the outpatient setting (US$332; p <0.0001). While ~ 10% of the additional medical costs in the outpatient setting were attributable to hypnotics in both groups (treatment naïve: US$106, p< 0.0001; treatment switchers: US$115, p< 0.0001), no difference was observed in the inpatient setting. Conclusion: Suvorexant as an initial insomnia treatment was associated with higher total medical costs, given the additional burden of initiating treatment and monitoring costs associated with a new insomnia diagnosis. However, despite a switch from another hypnotic, suvorexant did not increase the incremental economic burden. The hypnotic cost remained proportionately low, demonstrating that suvorexant initiation did not raise the cost of insomnia treatment.

Current genome-wide association studies do not yet capture sufficient diversity in populations and scope of phenotypes. To expand an atlas of genetic associations in non-European populations, we conducted 220 deep-phenotype genome-wide association studies (diseases, biomarkers and medication usage) in BioBank Japan (n= 179,000), by incorporating past medical history and text-mining of electronic medical records. Meta-analyses with the UK Biobank and FinnGen (ntotal= 628,000) identified ~5,000 new loci, which improved the resolution of the genomic map of human traits. This atlas elucidated the landscape of pleiotropy as represented by the major histocompatibility complex locus, where we conducted HLA fine-mapping. Finally, we performed statistical decomposition of matrices of phenome-wide summary statistics, and identified latent genetic components, which pinpointed responsible variants and biological mechanisms underlying current disease classifications across populations. The decomposed components enabled genetically informed subtyping of similar diseases (for example, allergic diseases). Our study suggests a potential avenue for hypothesis-free re-investigation of human diseases through genetics.

Ten patients with active Crohn's disease who have been managed with parenteral-nutrition therapy were administered a lipid emulsion either with [containing 0.6 g eicosapentaenoic acid (EPA)] or without fish oil for 2 wk. We isolated polymorphonuclear leukocytes (PMNLs) from the patients before and after this treatment and measured the amounts of leukotriene [B.sub.4] (LT[B.sub.4]) and leukotriene [B.sub.5] (LT[B.sub.5]) generated by activated PMNLs by reversed-phase HPLC. The LT[B.sub.5] generation in active Crohn's disease before this treatment was significantly lower than in healthy control subjects. The amount of LT[B.sub.5] and the LT[B.sub.5]-LT[B.sub.4] ratio increased significantly after fish-oil supplementation. The difference with LT[B.sub.4] was not statistically significant. We have shown that daily intravenous administration of 0.6 g EPA influenced the generation of leukotrienes in active Crohn's disease even after short-term treatment. Further investigations are necessary to clarify the correlation between EPA and clinical improvement in Crohn's disease. Am J Clin Nutr 1992;56:938-42.

In a meta-analysis of three GWAS for susceptibility to Kawasaki disease (KD) conducted in Japan, Korea, and Taiwan and follow-up studies with a total of 11,265 subjects (3428 cases and 7837 controls), a significantly associated SNV in the immunoglobulin heavy variablegene (IGHV) cluster in 14q33.32 was identified (rs4774175; OR = 1.20, P= 6.0 × 10−9). Investigation of nonsynonymous SNVs of the IGHVcluster in 9335 Japanese subjects identified the C allele of rs6423677, located in IGHV3-66, as the most significant reproducible association (OR = 1.25, P= 6.8 × 10−10in 3603 cases and 5731 controls). We observed highly skewed allelic usage of IGHV3-66, wherein the rs6423677 A allele was nearly abolished in the transcripts in peripheral blood mononuclear cells of both KD patients and healthy adults. Association of the high-expression allele with KD strongly indicates some active roles of B-cells or endogenous immunoglobulins in the disease pathogenesis. Considering that significant association of SNVs in the IGHVregion with disease susceptibility was previously known only for rheumatic heart disease (RHD), a complication of acute rheumatic fever (ARF), these observations suggest that common B-cell related mechanisms may mediate the symptomology of KD and ARF as well as RHD.

To elucidate the genetics of coronary artery disease (CAD) in the Japanese population, we conducted a large-scale genome-wide association study of 168,228 individuals of Japanese ancestry (25,892 cases and 142,336 controls) with genotype imputation using a newly developed reference panel of Japanese haplotypes including 1,781 CAD cases and 2,636 controls. We detected eight new susceptibility loci and Japanese-specific rare variants contributing to disease severity and increased cardiovascular mortality. We then conducted a trans-ancestry meta-analysis and discovered 35 additional new loci. Using the meta-analysis results, we derived a polygenic risk score (PRS) for CAD, which outperformed those derived from either Japanese or European genome-wide association studies. The PRS prioritized risk factors among various clinical parameters and segregated individuals with increased risk of long-term cardiovascular mortality. Our data improve the clinical characterization of CAD genetics and suggest the utility of trans-ancestry meta-analysis for PRS derivation in non-European populations.

The overwhelming majority of participants in current genetic studies are of European ancestry. To elucidate disease biology in the East Asian population, we conducted a genome-wide association study (GWAS) with 212,453 Japanese individuals across 42 diseases. We detected 320 independent signals in 276 loci for 27 diseases, with 25 novel loci (P< 9.58 × 10−9). East Asian–specific missense variants were identified as candidate causal variants for three novel loci, and we successfully replicated two of them by analyzing independent Japanese cohorts; p.R220W of ATG16L2(associated with coronary artery disease) and p.V326A of POT1(associated with lung cancer). We further investigated enrichment of heritability within 2,868 annotations of genome-wide transcription factor occupancy, and identified 378 significant enrichments across nine diseases (false discovery rate < 0.05) (for example, NKX3-1for prostate cancer). This large-scale GWAS in a Japanese population provides insights into the etiology of complex diseases and highlights the importance of performing GWAS in non-European populations.

Kawasaki disease (KD) is a systemic vasculitis affecting infants and children; it manifests as fever and signs of mucocutaneous inflammation. Intravenous immunoglobulin (IVIG) treatment effectively attenuates the fever and systemic inflammation. However, 10–20% patients are unresponsive to IVIG. To identify genetic variants influencing IVIG non-response in KD, a genome-wide association study (GWAS) and a replication study were performed using a total of 148 IVIG non-responders and 845 IVIG-responders in a Korean population. rs28662 in the sterile alpha motif domain-containing protein 9-like (SAMD9L) locus showed the most significant result in the joint analysis of GWAS and replication samples (odds ratio (OR) = 3.47, P=1.39 × 10−5). The same SNP in the SAMD9Llocus was tested in the Japanese population, and it revealed a more significant association in a meta-analysis with Japanese data (OR = 4.30, P=5.30 × 10−6). These results provide new insights into the mechanism of IVIG response in KD.

Background and Aims: Childhood and adolescent cancer survivors often require a long period of follow-up, carrying health risks even after overcoming cancer, and it is desirable for them to shift departments as they grow physically. In this study, we aimed to survey obstetricians and gynecologists about the actual situation regarding transitional care for patients with pediatric cancer (PPCs) in Japan.Method: We conducted a questionnaire survey on transitional care from January 2021 to March 2022, targeting 579 training facilities registered with the Japanese Society of Obstetrics and Gynecology. This work was supported by MHLW Research for Promotion of Cancer Control Program Grant No. JPMH20EA1004.Results: The total response rate was 58.5%. When asked “if they provide transitional care specifically for PPCs between pediatrics and obstetrics/gynecology”, only 13% responded that they do. Forty percent had received referrals for PPCs. The most common reasons for referral were “irregular menstruation or irregular genital bleeding” and “suspected ovarian dysfunction”. The most common problems with referrals from pediatrics were related to “insufficient explanation” in many cases. In addition, at facilities with no experience in treating PPCs, many respondents commented that they didn’t know how to follow up on the progress of the disease. When asked “about the necessity of obstetrics/gynecology visits for PPCs”, more than half of the facilities with experience in treating PPCs answered that such visits were “necessary”, and only 1% answered that they were “unnecessary”. On the other hand, 37% of the facilities that had no experience in treating PPCs answered that it was “necessary”, and 4% answered that it was “unnecessary”.Conclusion: The results suggest that what needs to be done in the future is to educate healthcare professionals by providing them with knowledge, as well as patient education that leads to patients’ awareness of their own self-management.

Supplemental Digital Content is available in the text.

ORAI1encodes a calcium channel essential in the store-operated calcium entry mechanism. A previous genetic association study identified a rare in-frame insertion variant of ORAI1conferring Kawasaki disease (KD). To deepen our understanding of the involvement of rare variants of ORAI1in KD pathogenesis, we investigated 3812 patients with KD and 2644 healthy individuals for variations in the protein-coding region of ORAI1. By re-sequencing the study participants’ DNA, 27 variants with minor allele frequencies (MAFs) < 0.01 that had not been examined in the previous study were identified. Although no significant association with KD was observed either in single-variant analyses or in a collapsing method analysis of the 27 variants, stratification by MAFs, variant types, and predicted deleteriousness revealed that six rare, deleterious, missense variants (MAF < 0.001, CADD C-score ≥ 20) were exclusively present in KD patients, including three refractory cases (OR = ∞, P= 0.046). The six missense variants include p.Gly98Asp, which has been demonstrated to result in gain of function leading to constitutive Ca2+entry. Conversely, five types of frameshift variants, all identified near the N terminus and assumed to disrupt ORAI1 function, showed an opposite trend of association (OR = 0.35, P= 0.24). These findings support our hypothesis that genetic variations causing the upregulation of the Ca2+/NFAT pathway confer susceptibility to KD. Our findings also provide insights into the usefulness of stratifying the variants based on their MAFs and on the direction of the effects on protein function when conducting association studies using the gene-based collapsing method.

Atrial fibrillation (AF) affects more than 33 million individuals worldwide1and has a complex heritability2. We conducted the largest meta-analysis of genome-wide association studies (GWAS) for AF to date, consisting of more than half a million individuals, including 65,446 with AF. In total, we identified 97 loci significantly associated with AF, including 67 that were novel in a combined-ancestry analysis, and 3 that were novel in a European-specific analysis. We sought to identify AF-associated genes at the GWAS loci by performing RNA-sequencing and expression quantitative trait locus analyses in 101 left atrial samples, the most relevant tissue for AF. We also performed transcriptome-wide analyses that identified 57 AF-associated genes, 42 of which overlap with GWAS loci. The identified loci implicate genes enriched within cardiac developmental, electrophysiological, contractile and structural pathways. These results extend our understanding of the biological pathways underlying AF and may facilitate the development of therapeutics for AF.

Kawasaki disease (KD), a systemic vasculitis of infants and children, manifests as fever and mucocutaneous inflammation. Although its etiology is largely unknown, the epidemiological data suggest that genetic factors are important in KD susceptibility. To identify genetic variants influencing KD susceptibility, we performed a genome-wide association study (GWAS) and replication study using a total of 915 children with KD and 4553 controls in the Korean population. Six single-nucleotide polymorphisms (SNPs) in three loci were associated significantly with KD susceptibility (P<1.0 × 10−5), including the previously reported BLKlocus (rs6993775, odds ratio (OR)=1.52, P=2.52 × 10−11). The other two loci were newly identified: NMNAT2on chromosome 1q25.3 (rs2078087, OR=1.33, P=1.15 × 10−6) and the human leukocyte antigen (HLA) region on chromosome 6p21.3 (HLA-C, HLA-B, MICAand HCP5) (rs9380242, rs9378199, rs9266669 and rs6938467; OR=1.33–1.51, P=8.93 × 10−6to 5.24 × 10−8). Additionally, SNP rs17280682 in NLRP14was associated significantly with KD with a family history (18 cases vs 4553 controls, OR=6.76, P=5.46 × 10−6). These results provide new insights into the pathogenesis and pathophysiology of KD.

Atrial fibrillation is the most common cardiac arrhythmia and leads to stroke. To investigate genetic loci associated with atrial fibrillation in the Japanese population, we performed a genome-wide association study (GWAS) that included 8,180 atrial fibrillation cases and 28,612 controls with follow-up in an additional 3,120 cases and 125,064 controls. We replicated previously reported loci and identified six new loci, near the KCND3, PPFIA4, SLC1A4–CEP68, HAND2, NEBL and SH3PXD2A genes. Five of the six new loci were specifically associated with atrial fibrillation in the Japanese population after comparing our data to those from individuals of European ancestry, suggesting that there might be different genetic factors affecting susceptibility across ancestry groups. Our study discovered variants in the HAND2, KCND3 and NEBL genes, which are relevant to atrial fibrillation susceptibility. The involvement of PPFIA4 and SH3PXD2A in axon guidance also suggested a role in disease pathogenesis. Our findings may contribute to a better understanding of atrial fibrillation susceptibility and pathogenesis.

Background:Inflammation plays a primordial role in atherosclerosis. Perivascular adipose tissue (PVAT) that surrounds the vasculature has been reported to undergo phenotypic changes in response to vascular inflammation. However, regulatory roles of PVAT in vascular inflammation and subsequent pathological remodeling remain unclear.Methods and Results:Endovascular injury was generated by wire insertion into the mice femoral artery. Here, we showed that vascular injury induced the beiging (brown adipose tissue-like phenotype change) of PVAT, which fine-tunes inflammatory response and thus vascular remodeling as a protective mechanism. In a mouse model of endovascular injury, macrophages accumulated in PVAT, causing beiging phenotype change. Inhibition of PVAT beiging by adipose tissue-specific genetic ablation of PRDM16, a key regulator to beiging, exacerbated inflammation and vascular remodeling following injury. Local knockdown of Prdm16in perivascular tissues of wild-type mice using small interfering RNA similarly exacerbated inflammation and vascular remodeling. Conversely, activation of PVAT beiging attenuated inflammation and pathological vascular remodeling. Single-cell RNA sequencing revealed that beige adipocytes abundantly expressed neuregulin 4 (Nrg4) which critically regulated alternative macrophage activation. Furthermore, Nrg4knockdown abolished the PVAT-mediated anti-inflammatory effects on macrophages. Importantly, significant beiging was observed in the diseased aortic PVAT in patients with acute aortic dissection.Conclusions:Endovascular injury induced the beiging of adjacent PVAT with macrophage accumulation, where NRG4 secreted from the beige PVAT facilitated alternative activation of macrophages, leading to the resolution of vascular inflammation. Our study demonstrated the pivotal roles of PVAT in vascular inflammation and remodeling and will open a new avenue for treating atherosclerosis.

It is suggested that angiotensin II is involved in the pathogenesis of pulmonary hypertension and subsequent right ventricular hypertrophy; therefore, an angiotensin AT1 receptor antagonist could be beneficial for the treatment of this disease. We tested the effect of the new AT1 receptor antagonist olmesartan medoxomil on monocrotaline-induced pulmonary hypertension in rats. At 3 weeks after a single subcutaneous injection of monocrotaline (50 mg/kg), the lung/body weight ratio, the right ventricle/(left ventricle plus septum) weight ratio [RV/(LV+S)], and right ventricular systolic pressure were increased, indicating establishment of pulmonary hypertension and right ventricular hypertrophy. Oral administration of olmesartan medoxomil (2 or 5 mg/kg/day for 3 weeks) restored RV/(LV+S) and right ventricular systolic pressure, and a higher dose (5 mg/kg/day) improved the lung/body weight ratio. Pulmonary arteries isolated from monocrotaline-treated rats exhibited an increase in basal tone in the resting state, indicating that they had intrinsic tone. Three weeks of treatment with olmesartan decreased this intrinsic tone. These data suggest that long-term treatment with olmesartan has beneficial effects on monocrotaline-induced pulmonary hypertension and subsequent right ventricular hypertrophy.

We describe a 54‐year‐old man with hepatitis C virus (HCV) infection‐associated cryoglobulinemia type III. The patient had suffered from cold‐induced urticaria that left purpuric eruptions up to 1 cm in diameter, intermittent migratory joint pain for seven years and mild liver dysfunction for nine years. Hemophilia A was diagnosed when the patient was 26 years old, and he was then given infusions of factor VIII for a short time. In both skin biopsy samples from urticarial and purpuric eruptions, mild inflammatory infiltration by polymorphonuclear leukocytes with nuclear dust, extravasation of erythrocytes and deposition of IgM and C3 in the superficial blood vessels were observed. After antiviral treatment with interferon‐β, the clinical symptoms and the cryoglobulin and HCV‐RNA in the serum disappeared. There has been no recurrence in the subsequent nine years.

When co-expressed with a receptor activity-modifying protein (RAMP) accessory protein, calcitonin receptor-like receptor (CRLR) can function as a calcitonin gene-related peptide receptor (CRLR-RAMP1) or an adrenomedullin (AM) receptor (CRLR-RAMP2/3). Here we report on the structural domain(s) involved in selective AM binding that were examined using various RAMP chimeras and deletion mutants. Co-expression of chimeric RAMPs and CRLR in HEK293 cells revealed that residues 77–101, situated in the extracellular N-terminal domain of human RAMP2 (hRAMP2), were crucial for selective AM-evoked cAMP production. More detailed analysis showed that deletion of hRAMP2 residues 86–92 significantly attenuated high-affinity125I-AM binding and AM-evoked cAMP production despite full cell surface expression of the receptor heterodimer and that deletion of hRAMP3 residues 59–65 had a similar effect. There is little sequence identity between hRAMP3 residues 59–65 and hRAMP2 residues 86–92; moreover, substituting alanine for Trp86(Ala87), Met88, Ile89, Ser90, Arg91, or Pro92of hRAMP2 had no effect on AM-evoked cAMP production. It thus seems unlikely that any one amino acid residue is responsible for determining selective AM binding or that AM binds directly to these peptide segments. Instead these findings suggest that the respective seven-amino acid sequences confer selectivity either by directly contributing to the structure of ligand binding pocket or by allosteric modulation of the conformation of CRLR.

Both endothelin (ET) and adrenomedullin (AM), produced by cardiac myocytes, are thought to be locally-acting hormones in the heart. Recently, calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) have been shown to function together to serve as AM receptors stimulating cAMP production. In the present study, we examined the effects of ET on AM secretion, intracellular cAMP response to AM, and gene expressions of CRLR and RAMPs in cultured cardiac myocytes. Synthetic ET-1 dose-dependently increased AM secretion from the cardiomyocytes. AM increased the intracellular cAMP level in a dose-dependent manner and the cAMP accumulation by AM was significantly amplified by 24 h preincubation with ET-1. 10 nmol/L ET-1 significantly increased the CRLR mRNA level without any effect on RAMP1 mRNA. 1 μmol/L ET-1 significantly reduced the RAMP2 mRNA level, but ET-1 dose-dependently increased the RAMP3 mRNA level in the cardiac myocytes. These findings suggest that ET-1 not only stimulates AM secretion, but also modulates intracellular cAMP responses to AM probably by altering the expressions of CRLR and RAMPs in rat cardiomyocytes.

We compared clinical outcomes in hydrocephalic patients and observed variation in the rate of flow in ventriculoperitoneal shunts with changes in posture in 231 separate examinations of shunt flow in 148 patients. A small cadmium telluride detector was placed over the shunt reservoir, and clearance of radioisotope injected into the reservoir was recorded as a measure of flow. Flow rate tended to increase during head elevation. Four patterns of radioisotope clearance were seen: type I, no flow; type II, adequate flow with moderate opening pressure; type III, adequate flow with low opening pressure; and type IV, excessive flow. This categorisation reflected clinical shunt function. Our method effectively assesses flow rate with the patient in a variety of postures or during movement, yielding useful information for adjustment of shunt valve pressure.

Although seminolipid has long been suspected to play an essential role in spermatogenesis because of its uniquely abundant and temporally regulated expression in the spermatocytes, direct experimental evidence has been lacking. We have tested the hypothesis by examining the testis of the UDP-galactose:ceramide galactosyltransferase-deficient mouse, which is incapable of synthesizing seminolipid. Spermatogenesis in homozygous affected males is arrested at the late pachytene stage and the spermatogenic cells degenerate through the apoptotic process. This stage closely follows the phase of rapid seminolipid synthesis in the wild-type mouse. These observations not only provide the first experimental evidence that seminolipid is indeed essential for normal spermatogenesis but also support the broader concept that cell surface glycolipids are important in cellular differentiation and cell-to-cell interaction.

Atrial fibrillation (AF) is the most common cardiac arrhythmia associated with increased risks of morbidity and mortality. We performed a large-scale genome-wide association study (GWAS) of 150,272 individuals of Japanese ancestry (9,826 cases and 140,446 controls) and identified five new susceptibility loci including Japanese-specific rare variants associated with the pathogenesis of cardiomyopathy. We then conducted a trans-ancestry meta-analysis of more than 1 million individuals including 77,690 cases and discovered 35 novel loci in total. Using the GWAS result, epigenetic-feature and gene-set analyses revealed the transcriptional landscape of AF, elucidating the underlying biological pathways. Additionally, we created polygenic risk scores (PRS) for AF, where PRS derived from trans-ancestry GWAS surpassed those derived from a single population. The PRS was associated with increased risk of long-term cardiovascular and stroke mortalities and segregated individuals with cardioembolic stroke in undiagnosed AF patients, suggesting that it may play an important role in early detection of subclinical AF or the pathophysiology underlying thromboembolism. Our results provide novel biological and clinical insights into AF genetics and broaden its clinical applications.

Abstract:  We investigated the role of 20 kDa myosin light chain (MLC20) phosphorylation in contractions following protein kinase C (PKC) activation by 12-deoxyphorbol-13-isobutyrate (DPB) in rabbit aortae. DPB induced a sustained contraction and phosphorylation of MLC20 independent of a change in cytosolic Ca2+ ([Ca2+]i). Phosphorylation on Ser19 of MLC20, which is a target site of MLC kinase (MLCK), was 9.2 ± 5.1% and 22.3 ± 4.9% of the phosphorylation caused by KCl, at 5 and 30 min of application of DPB, respectively. When KCl-precontracted muscles were rinsed with Ca2+-free, EGTA solution, [Ca2+]i rapidly declined, MLC20 was dephosphorylated and the tension decreased. If DPB was present in the Ca2+-free solution, the relaxation and the dephosphorylation of either total MLC20 or Ser19 were inhibited. The phospholipase A2 inhibitor ONO-RS-082 partially antagonized the effects of DPB on the tension and the MLC20 dephosphorylation. In Ca2+-free solution, DPB induced a contraction smaller than that in normal solution without an increase in MLC20 phosphorylation, and the contraction was also sensitive to ONO-RS-082. These results suggest that a part of MLC20 phosphorylation following PKC activation is due to inhibition of MLC20 phosphatase and the phosphorylation is responsible for the contraction. Furthermore, a mechanism independent of [Ca2+]i and phosphorylation may play a significant role in the PKC-dependent contraction. The involvement arachidonic acid is suggested, not only in the inhibition of dephosphorylation but also in the Ca2+-independent regulation of contractile proteins.

Semotiadil fumarate (SD–3211), a Ca2+channel blocker of benzothiazine derivative and its (S)-(-)–enantiomer (SD–3212), inhibited K+— and norepinephrine (NE)-induced contractions in isolated rat aortas. Inhibition of NE contraction induced by both drugs was greater than that induced by diltiazem or bepridil, whereas inhibition of K+—contraction was similar to that induced by diltiazem or bepridil. Semotiadil and SD–3212 (10 μM) inhibited the increase in cytosolic Ca2+([Ca2+Ji) induced by 65.4 mMK+in fura-2-loaded preparations as well as diltiazem and bepridil (10 μM). On the other hand, semotiadil and SD–3212 (10 μM) inhibited only the early phase of increase in [Ca2+]iinduced by 1 μMNE. After 5 min, no significant effect on [Ca2+]iwas observed with these compounds despite the significant decrease in the contraction. In contrast to these compounds, diltiazem and bepridil 10 μMaffected neither the increase in [Ca2+]inor the contraction induced by NE. Semotiadil and SD–3212 inhibited the transient contraction induced by 1 μMNE in the absence of external Ca2+. Both compounds partially but significantly inhibited the NE-induced contraction in nifedipine-treated muscles. These results suggest that semotiadil and SD–3212 inhibit contractions of vascular smooth muscle (VSM) not only through blockade of voltage-dependent Ca2+channels but also through other mechanisms, such as inhibition of Ca2+release from Ca2+stores or decrease in sensitivity of the contractile elements to Ca2+.

Abstract: The sporangiophores (spphs) of the fungusPhycomyces blakesleeanus bend upward in a negative gravitropic response when placed in a horizontal position in the dark. The spphs of a hypergravitropic mutant showed higher bending rate and shorter latency period than those of the wild type. In both strains, spphs of smaller diameter had higher bending rates. No significant differences were found between the wild type and the mutant and between the thin spphs and the spphs of standard diameter in respect to their elongation rates. Phototropic rate was also the same between the wild type and the mutant. Parameters influencing the gravitropic response such as diameter of the spph, absolute elongation rate, and ratio of differential growth between the upper and the lower sides of the extension zone of spph were investigated to elucidate the kinetics of bending in the mutant. The results demonstrate that the rapid gravitropic response in the mutant is due to its higher (about 5–6 times) differential-growth rate compared with the wild type.

Vasoconstrictor responses to endothelin-1 (ET) were compared between endothelium-denuded strips of cerebral, coronary and mesenteric arteries of the dog. Contractile responses to lower concentrations (below 3×10−10M) of ET were significantly greater in the cerebral and coronary arteries than in the mesenteric artery. The cerebral and coronary arteries, but not the mesenteric artery, relaxed significantly from the resting level when placed in a 0-Ca solution. Readdition of Ca2+to the cerebral and coronary arteries placed in the 0-Ca solution caused a biphasic contraction which was susceptible to inhibition by nifedipine. When ET below 10−10M was introduced before the Ca2+contraction, this peptide produced no detectable contraction, but augmented the Ca2+contraction. The augmented Ca2+contractions were abolished by 10−7M nifedipine. These effects of ET were not observed in the mesenteric artery. The contractile responses of th e mesenteric artery to the ET determined in the presence of elevated extracellular K+concentrations were comparable to the responses of the cerebral artery to this peptide determined in the presence of normal K+concentrations. These results indicate that the enhanced responses to ET in the cerebral and coronary arteries were dependent on the Ca2+influx through voltage-dependent Ca2+channels and suggest that these channels are in an activated state when these arteries are in a resting state.

• The plantar hyperkeratotic skin lesion in a case of Richner-Hanhart's syndrome was investigated using ultrastructural examination. Light microscopic examination showed remarkable hyperkeratosis and some aberrant keratinocytes with multiple nuclei. On ultrastructural examination, some abnormal structures were seen in the affected keratinocytes: aggregations of tonofilaments and intracytoplasmic inclusions. The inclusions were needle shaped and were considered to be "crystal ghosts," presumably of tyrosine. The formation of tyrosine crystalline inclusions seems to be an important factor in the pathogenesis of the cutaneous lesions in Richner-Hanhart's syndrome.(Arch Dermatol. 1990;126:1342-1346)

• Long-term use of a nylon brush for back scratching by a 53-year-old white woman was associated with the development of typical macular amyloidosis. EKH4 monoclonal antikeratin antibody, which recognizes 50-kd neutral and acidic keratin species, labeled this amyloid. Confirmation of amyloid substance in the lesion included positive staining with Dylon and thioflavin T; immunohistochemical reactions with monoclonal and polyclonal antibodies against elastic fiber microfibrils (NKH1 and anti-P component), immunoglobulins (IgG, IgM, and IgA), and complement (C3); and electron microscopic identification of 6- to 10-nm straight filaments. Type IV collagen staining demonstrated a breakage and/or thickening of the dermoepidermal basement membrane above the amyloid deposition in the papillary dermis. Electron microscopic findings confirmed this phenomenon.(Arch Dermatol 1987;123:633-637)

BALB/c mice were immunized with human hair fibrous proteins. Then their spleen cells were fused with Sp2/0‐Agl4 mouse myeloma cells. Antibody‐producing hybridomas were selected by ELISA method and cloned by limiting dilution. A monoclonal antibody was chosen and designated as HKN‐2. Immunohistochemically, on frozen sections of normal human skin, HKN‐2 was found to recognize the suprabasal cells of epidermis and hair follicle, the cells in the keratogenous zone of hair shaft, the sebaceous cells and the ductal and myoepithelial cells of sweat glands. The basal cells of the epidermis and lower hair follicle, hair matrix cells and secretory cells of sweat glands did not react with HKN‐2. An immunoelectron microscopic method showed that the positive reaction to HKN‐2 was located on the tonofilaments in the cytoplasm. It was concluded that there might be a common antigenic determinant between hair and other skin epithelial tissues. A complexity in keratin manifestation in each epithelial tissue of the skin was suspected.

We examined whether Ca2+channel function in the resting state alters the resting tone and Ca2+-activated K+(KCa) channel function in dog basilar artery: data were compared with findings in the mesenteric artery. Isolated dog basilar artery maintained a myogenic tone; that is, the resting tone decreased when either the Krebs solution was replaced with a Ca2+-free solution or nifedipine was added. The basal 45Ca influx in the resting state of the basilar artery was significantly increased compared with that in the mesenteric artery, and this increase in the basilar artery was reduced by nifedipine. The addition of charybdotoxin (ChTX), a blocker of large-conductance KCachannels, to the resting strips caused a concentration-dependent contraction in the basilar artery but not in the mesenteric artery. The ChTX-induced contraction in the basilar artery was abolished by nifedipine. In resting strips preloaded with 86Rb, the basal 86Rb efflux rate constant was significantly greater in the basilar artery than in the mesenteric artery. The addition of nifedipine to the resting strips decreased the basal 86Rb efflux rate constant only in the basilar artery. These results suggest that the transmembrane Ca2+influx via L-type voltage-dependent Ca2+channels was significantly increased in the resting state of the basilar artery and that the myogenic tone was therefore maintained and the ChTX-sensitive KCachannels were highly activated.

The sporangiophores (spphs) of the fungus Phycomyces blakesleeanusbend upward in a negative gravitropic response when placed in a horizontal position in the dark. The spphs of a hypergravitropic mutant showed higher bending rate and shorter latency period than those of the wild type. In both strains, spphs of smaller diameter had higher bending rates. No significant differences were found between the wild type and the mutant and between the thin spphs and the spphs of standard diameter in respect to their elongation rates. Phototropic rate was also the same between the wild type and the mutant. Parameters influencing the gravitropic response such as diameter of the spph, absolute elongation rate, and ratio of differential growth between the upper and the lower sides of the extension zone of spph were investigated to elucidate the kinetics of bending in the mutant. The results demonstrate that the rapid gravitropic response in the mutant is due to its higher (about 5-6 times) differential-growth rate compared with the wild type.

Vasoconstrictor actions of endothelin-1 (ET) were compared between endothelium-removed strips of cerebral (basilar, posterior cerebral, and middle cerebral) and peripheral (coronary and mesenteric) arteries of the dog. ET produced a concentration-dependent contraction in these arteries. A threshold concentration and EC50value for ET were significantly lower in the basilar, posterior cerebral, middle cerebral, and coronary arteries than in the mesenteric artery. In the basilar artery, nifedipine caused a rightward displacement of the concentration-response curve for ET with a significant reduction in the maximum response to ET. On the other hand, nifedipine showed a typical noncompetitive antagonism against ET in the mesenteric artery. Contractile responses of the mesenteric artery to ET determined under an elevation of extracellular Kconcentration were comparable to the responses of the basilar artery to this peptide determined under normal Kconcentrations. The cerebral and coronary arteries, but not the mesenteric artery, relaxed significantly from the resting level when placed in a Ca2-free solution containing 0.1 mM EGTA (0-Ca solution). The readdition of Ca2to the cerebral and coronary arteries soaked in the 0-Ca solution caused a biphasic contraction that was susceptible to inhibition by nifedipine. When ET in concentrations below 10-9Mwas introduced before the Ca2-induced contraction, this peptide produced no detectable contraction, but potentiated the Ca2-induced contraction. The extent of potentiation induced by ET was much greater in the cerebral and coronary arteries than in the mesenteric artery. Even in the 0-Ca solution, higher concentrations of ET (1 × 10 -8and 3 × 10 -8M) produced a contraction that was weaker in the basilar artery than in the mesenteric artery. These results indicate that the cerebral and coronary arteries exhibited more potent contractions in response to lower concentrations (below 10 -9M) of ET than the mesenteric artery. A likely possibility for these enhanced responses to ET in the cerebral and coronary arteries appears to be that the voltage-dependent Ca2 channels in these arteries are more activated in the resting state than those in the mesenteric artery.

The clinical and histopathological findings in a 25‐year‐old Japanese male patient who suffered from chronic graft‐versus‐host‐disease (GVHD) with follicular involvement are described. The patient had been diagnosed as aplastic anemia and underwent an allogeneic bone marrow transplant (BMT). In the eighth month thereafter, pruritic follicular red papules developed over his trunk and extremities. A biopsy specimen revealed histological exocytosis of lymphocytes into the hair follicles and basal‐cell vacuolization of the follicular epithelium. Analysis of T‐lymphocyte subpopulations in the dermis revealed a predominance of CD4 positive cells. To our knowledge, several cases of acute follicular GVHD have been reported (1, 2); however, the occurrence of chronic GVHD with follicular involvement (chronic follicular GVHD) has not been clearly documented.

An 83‐year‐old woman had developed an asymptomatic, yellowish, dome‐shaped skin tumor on her scalp which had enlarged for a period of 60 years to 10 times 10 times 10 mm in size. Histopathologically, the tumor consisted of undifferentiated basaloid cells, differentiated sebaceous cells and transitional cells. Although the basaloid cells resembled those of basal cell epithelioma (BCE), the tumor was distinct from BCE in the following points; existence of cystic spaces, no peripheral palisading, no proliferation of connective tissue stroma, and no tendency toward local invasion. Immunohistochemical studies using antikeratin monoclonal antibodies revealed that the tumor contained both the keratin types of BCE and of sebaceous glands. Electron microscopically, the tumor cells contained lipid droplets and keratohyaline granules in their cytoplasm. It is suggested that “sebaceous epithelioma” is a benign skin tumor which may be distinguished cytologically from BCE.

During hair follicle differentiation, several families of keratin proteins are synthesized sequentially. In the present study, cDNA clones encoding six members of mouse high-glycine/tyrosine protein were isolated by screening a cDNA library prepared from the mouse skin of an anagen phase with a differential hybridization technique. On the basis of their nucleotide and deduced amino acid sequences, they were found to encode two members of high-glycine/tyrosine protein type I and four of type II. Interestingly, one of the four type II proteins had been encoded by two distinct cDNAs. Among the cDNA clones isolated were included the ones encoding a new member of type I and II protein, respectively, which possessed an entire open reading frame. Novel type II protein, termed type II.4, with a molecular mass of 15,130 Da was revealed to have significant direct repeats and a cysteine residue at the carboxyl terminus, which indicates that this protein has characteristics intermediate between high-glycine/tyrosine proteins and cysteine-rich proteins. In addition, the new member of type I protein has some features common with type II protein. We propose to term this protein type Iα until it is further characterized. Northern blot analysis demonstrated that gene expression of mouse high-glycine/tyrosine proteins followed the hair cycle growth fundamentally and reached its peak at day 9 in the first hair cycle, while two peaks of their expression were observed at day 33 and day 39 in the second cycle. Their transcripts were expressed in the cortical cells of hair follicles but not in the cells of the outer root sheath, inner root sheath, or medulla. Moreover, their gene expression commenced at different levels in cortical cells. The novel findings that each gene is activated transcriptionally with a distinct expression pattern spatially and temporally suggest that there is a remarkable difference in the distribution of these proteins in hair.

Human hair follicles were isolated from the scalp by dispase and collagenase treatment and dispersed into a cell suspension by trypsin. These cells proliferated well and could be subcultured 7 to 8 times. The medium used was MCDB 153 HAA medium further supplemented with some amino acids, hydrocortisone, insulin, EGF, and bovine brain extract. The concentration of Ca++was adjusted to 0.1 mM. Immunohistochemically, these cells were proved to possess keratins specific to hair forming cells.

TO THE EDITOR.— The association of lithium and psoriasis, acne and acneiform eruptions, and folliculitis is relatively well known. There have been other rare and isolated reports of the association of lithium with alopecia, exfoliative dermatitis, maculopapular rash, ulcer, stomatitis, ichthyosis, xerosis, eczema, and pruritus, and a single case of dermatitis herpetiformis.1 The last case was reported by Posey in 19722; in his short report he described an adult woman who had been taking lithium carbonate for a manicdepressive illness and in whom an intensely pruritic papular dermatitis developed that appeared on the elbows and resembled dermatitis herpetiformis clinically. This diagnosis was not histologically confirmed. Sulfoxone sodium therapy was started with cessation of symptoms, but lithium therapy could not be reinstituted without new lesion formation. REPORT OF A CASE.— A 26-year-old man presented with a six-month history of blisters involving his arms and hands, left lower leg and

Background:Perivascular adipose tissue (PVAT) possibly plays a pivotal role in vascular remodeling through its direct local action on the vessels. Some specific conditions such as cold exposure provoke a brown adipose tissue-like phenotype in white adipose tissues, i.e. browning. We here investigated the pathophysiological role of browning of PVAT in the development of vascular remodeling after endovascular injury.Methods and Results:Endovascular injury was generated by wire insertion into the femoral artery of C57BL/6 mice. Transcriptome analysis revealed robust upregulation of brown adipose markers such as Ucp1, Elovl3, Cox8band Cideain injured arteries. The upregulation of browning markers in PVAT after vascular injury were confirmed by quantitative real-time PCR, western blot and immunohistochemical staining. Of note, local perivascular introduction of small interfering RNA targeting Pgc1a, a regulator of browning, exacerbated neointima formation after injury. Meanwhile, a ?3 receptor agonist CL316243 that was administrated locally to PVAT accelerated PVAT browning and further significantly inhibited neointima formation after vascular injury. Interestingly, vascular injury promoted F4/80+ macrophage accumulation and inflammatory cytokines production in PVAT. In vitro study showed that conditioned medium derived from stromal vascular fraction (SVF) of PVAT significantly inhibited expression levels of inflammatory cytokines in a macrophage cell line, RAW 264.7. On the other hand, conditioned medium derived from RAW 264.7 cells upregulated expressions of browning marker genes in PVAT SVF. Furthermore, in vivo depletion of macrophage with clodronate-containing liposomes markedly inhibited upregulation of browning markers in PVAT after injury.Conclusions:We observed the browning phenomenon in PVAT after endovascular injury. The PVAT browning is triggered by macrophage activation in PVAT, and then inhibits excessive inflammation through promoting transformation of activated macrophage to an alternative activated subtype, which leads to prevention of pathological vascular remodeling. These findings may provide novel insights into pathogenesis of atherosclerosis.

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Identification of sequence variants that create or eliminate splice sites has proven to be a significant challenge and represents one of many roadblocks in the clinical interpretation of rare genetic variation. Current methods of identifying splice‐altering sequence variants are limited by an imperfect understanding of splice signals and the need for cumbersome functional assays. We have recently developed a computational tool that prioritizes putative splice‐altering sequence variants and a moderate‐throughput minigene assay that confirms the variants that alter splicing. This bioinformatic strategy represents a substantial increase in accuracy and efficiency with respect to historical in vitro splicing assays. In this article, we give detailed instructions on how to organize, run, and interpret various features of this procedure. We expect that splice‐altering variants revealed through this protocol can be reliably carried forward for further clinical and biological analyses. © 2018 by John Wiley & Sons, Inc.