Single-Cell RNA Sequencing Reveals an Immune Landscape of CD4+ T Cells in Coronary Culprit Plaques With Acute Coronary Syndrome in Humans-Brief Report.

BACKGROUND: Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. METHODS: We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45+ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. RESULTS: CD4+ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7+ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)+ regulatory CD4+ T cells. The proportion of central memory CD4+ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4+ T cells into effector CD4+ T cells and that from naive CD4+ T cells into central memory CD4+ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4+ T cells. CONCLUSIONS: For the first time, we revealed single cell-level characteristics of CD4+ T cells in patients with ACS. CD4+ T cells could be therapeutic targets of ACS. REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.

Fat embolism syndrome: A severe case diagnosed early using dual-energy CT and saved with VA-ECMO and inhaled nitric oxide.

Diagnosing FES is difficult and time-consuming, and identify FES as an etiology of right ventricular volume overload for early diagnosis. Because FES is a reversible condition, even severe cases can bse treated if the patient survives the acute phase.

Differential gene regulation by a synthetic vitamin D receptor ligand and active vitamin D in human cells.

Vitamin D (VD) exerts a wide variety of biological functions including calcemic activity. VD nutritional status is closely associated with the onset and development of chronic diseases. To develop a VD analog with the desired VD activity but without calcemic activity, we screened synthetic VDR antagonists. We identified 1α,25-dihydroxyvitamin D3-26-23-lactams (DLAM)-2a-d (DLAM-2s) as nuclear vitamin D receptor (VDR) ligands in a competitive VDR binding assay for 1α,25(OH)2 vitamin D3 (1α,25(OH)2D3), and DLAM-2s showed an antagonistic effect on 1α,25(OH)2 D3-induced cell differentiation in HL60 cells. In a luciferase reporter assay in which human VDR was exogenously expressed in cultured COS-1 cells, DLAM-2s acted as transcriptional antagonists. Consistently, DLAM-2s had an antagonistic effect on the 1α,25(OH)2D3-induced expression of a known VD target gene [Cytochrome P450 24A1 (CYP24A1)], and VDR bound DLAM-2s was recruited to an endogenous VD response element in chromatin in human keratinocytes (HaCaT cells) endogenously expressing VDR. In an ATAC-seq assay, the effects of 1α,25(OH)2 D3 and DLAM-2b on chromatin reorganization were undetectable in HaCaT cells, while the effect of an androgen receptor (AR) antagonist (bicalutamide) was confirmed in prostate cancer cells (LNCaP) expressing endogenous AR. However, whole genome analysis using RNA-seq and ATAC (Assay for Transposase Accessible Chromatin)-seq revealed differential gene expression profiles regulated by DLAM-2b versus 1α,25(OH)2D3. The upregulated and downregulated genes only partially overlapped between cells treated with 1α,25(OH)2D3 and those treated with DLAM-2b. Thus, the present findings illustrate a novel VDR ligand with gene regulatory activity differing from that of 1α,25(OH)2D3.

Advances in the Administration of Vitamin D Analogues to Support Bone Health and Treat Chronic Diseases.

Vitamin D (VD) exerts a wide variety of biological actions in addition to its well-known roles in calcium homeostasis. Nutritional VD deficiency induces rachitic abnormalities in growing children and osteomalacia in adults, and it has been proposed to underlie the onset and development of multiple non-communicable chronic diseases. Therefore, the administration of VD or synthetic VD analogues represents a promising therapeutic strategy; indeed, VD and a VD agonist have shown clinical promise in mitigating osteoporosis and symptoms of insufficient calcium intake. However, even though high doses of VD analogues have shown pre-clinical efficacy against several diseases, including cancers, they have not yet had wide-spread clinical success. This difference may be due to limitation of clinical doses in light of the inherent calcemic action of VD. An approach to overcome this problem involves the development of VD analogues with lower calcemic activity, which could be administered in high doses to attenuate the onset and progress of disease. In a similar strategy, selective estrogen receptor modulators have had success as anti-osteoporosis drugs, and they have shown benefit for other estrogen target organs by serving as partial antagonists or agonists of estrogen receptor α. It is thus conceivable to generate synthetic partial antagonists or agonists for the VD receptor (VDR) that would exert beneficial effects on bone and other VD target organs. In this review, we discuss the molecular basis of the development of such synthetic VDR ligands from the viewpoint of roles of VDR in gene regulation.

The epigenetic function of androgen receptor in prostate cancer progression.

Androgen and androgen deprivation (castration) therapies, including androgen receptor antagonists, are clinically used to treat patients with prostate cancer. However, most hormone-dependent prostate cancer patients progress into a malignant state with loss of hormone-dependency, known as castration (drug)-resistant prostate cancer (CRPC), after prolong androgen-based treatments. Even in the CRPC state with irreversible malignancy, androgen receptor (AR) expression is detectable. An epigenetic transition to CRPC induced by the action of AR-mediated androgen could be speculated in the patients with prostate cancer. Androgen receptors belongs to the nuclear receptor superfamily with 48 members in humans, and acts as a ligand-dependent transcriptional factor, leading to local chromatin reorganization for ligand-dependent gene regulation. In this review, we discussed the transcriptional/epigenetic regulatory functions of AR, with emphasis on the clinical applications of AR ligands, AR protein co-regulators, and AR RNA coregulator (enhancer RNA), especially in chromatin reorganization, in patients with prostate cancer.

Acute Coronary Syndrome Due to Intraplaque Hemorrhage in a Post-gastrectomy Patient with a Latent Severe Glycemic Disorder.

Glycemic disorders involving large glucose fluctuations and recurrent hypoglycemia may lead to adverse cardiovascular events, including acute coronary syndrome (ACS). Flash glucose monitoring (FGM) has reportedly been useful for detecting latent glycemic disorders. However, only a few studies have so far reported latent glycemic disorders in coronary artery disease. Thus, we herein present a unique case of ACS due to intraplaque hemorrhage in a post-gastrectomy patient who had no apparent coronary risk, except for a latent severe glycemic disorder detected via FGM. This masked etiology should be considered in ACS patients who have no apparent cardiovascular risks in order to improve their cardiovascular outcomes.

Pacemaker implantation for persistent sinus node dysfunction in a patient with COVID-19.

A 46-year-old woman was admitted with coronavirus disease-2019 infection. Symptomatic sinus bradycardia occurred, followed by congestive heart failure. Therapeutics such as isoproterenol, theophylline, and cilostazol could not safely improve her symptoms. She underwent pacemaker implantation 53 days after admission. Atrial pacing remained was at 60% after 6 months.

Identification of cell cycle-associated and -unassociated regulators for expression of a hepatocellular carcinoma oncogene cyclin-dependent kinase inhibitor 3.

Human cyclin-dependent kinase inhibitor 3 (CDKN3) is a known oncogene in hepatocellular carcinoma (HCC) and its expression is promoted during tumor development. CDKN3 serves as a cell cycle regulator and its dysregulation is considered to be a causal factor for tumor progression. However, the molecular basis of the regulation of CDKN3 expression remains largely elusive. Using in silico approach, we identified CDKN3SE, a super enhancer (SE), and enhancer RNA (eRNA) candidates transcribed from this SE. Among the eRNA candidates, the expression of CDKN3eRNA was detected in the human HCC model cell line HepG2, and was found to facilitate the expression of CDKN3 without affecting the cell proliferation rate. In silico screening revealed two DNA-binding transcription factors, upstream stimulatory factor (USF) 1 and 2, involved in the regulation of CDKN3eRNA expression on CDKN3SE. A knock-down of USF1/USF2 expression in the HepG2 cells did not affect CDKN3eRNA expression, while the expression of CDKN3 was down-regulated. In a USF2 dominant negative HepG2 cell line generated by genome editing, a drastically altered cell shape and lowered cell proliferation rate were found; however, the expression of CDKN3eRNA appeared unaffected. Thus, the present study illustrated two regulators for CDKN3 expression: USF2, as a cell cycle-associated protein regulator, and CDKN3eRNA, as a cell cycle-unassociated RNA regulator.

A long non-coding RNA as a direct vitamin D target transcribed from the antisense strand of the human HSD17B2 locus.

Vitamin D (VD) exerts a wide variety of actions via gene regulation mediated by the nuclear vitamin D receptor (VDR) under physiological and pathological settings. However, the known target genes of VDR appear unlikely to account for all VD actions. We used in silico and transcriptomic approaches in human cell lines to search for non-coding RNAs transcriptionally regulated by VD directly. Four long non-coding RNAs (lncRNAs), but no microRNAs (miRNAs), were found, supported by the presence of consensus VDR-binding motifs in the coding regions. One of these lncRNAs (AS-HSD17ß2) is transcribed from the antisense strand of the HSD17ß2 locus, which is also a direct VD target. AS-HSD17ß2 attenuated HSD17ß2 expression. Thus, AS-HSD17ß2 represents a direct lncRNA target of VD.

Antagonistic action of a synthetic androgen ligand mediated by chromatin remodeling in a human prostate cancer cell line.

The clinical use of androgen receptor (AR) antagonists has been successful in treating prostate cancer patients, inducing remission of androgen-dependent tumors. However, a couple of years after treatment, prostate tumors transition into an androgen-independent state with altered gene expression profiles, but the molecular basis is not understood. Since the AR antagonists trigger this transition, we assessed whether AR antagonists induce chromatin reorganization in an androgen-dependent prostate cancer cell line (LNCaP). Treatment of LNCaP cells with two clinically used AR antagonists (bicalutamide [Bic] and enzalutamide [Enz]) expectedly resulted in antagonistic effects on cell proliferation, AR transactivation, and dihydrotestosterone (DHT)-induced expression of AR target genes. Thus, the antagonists expectedly acted to antagonize the transactivation function of AR activated by androgen binding. By ChIP-qPCR assay, AR bound to Bic, but not Enz, was recruited to an endogenous consensus AR-binding site within the kallikrein-related peptidase 3 gene promoter after treatment with Bic, similar to the effect of DHT. By ATAC-seq analysis of the cells after long-term treatment for 5 days, Bic and dihydrotestosterone DHT induced different chromatin reorganization patterns and gene expression profiles, suggesting that Bic exhibited a distinct action from that by DHT. Thus, these results suggest that the action of a known AR antagonist is mediated by chromatin reorganization in a prostate cancer cell line.

Utility of coronary orbital atherectomy with guide-extension system for distally located undilatable in-stent restenosis: A case report.

Orbital atherectomy (OA) may be effective in managing undilatable in-stent restenosis (ISR) despite off-label indications. We demonstrated that optical frequency domain imaging (OFDI)-guided OA, with a guide-extension system was effective even in distally located, undilatable ISR. However, OFDI revealed that inter-struts calcified neoatherosclerosis remained a challenging issue.

Non-surgical management of an acute decompensated heart failure patient with severe aortic stenosis and concomitant left ventricular outflow tract obstruction.

Acute decompensated heart failure (ADHF) due to severe aortic stenosis (AS) and concomitant left ventricular outflow tract (LVOT) obstruction is a serious condition. Treatment with medication alone is sometimes difficult, and the efficacy of further interventional strategies has not been fully elucidated. In patients with high surgical risks, combination therapy using transcatheter aortic valve replacement (TAVR) and percutaneous alcohol septal ablation (ASA) is used as a non-surgical intervention. However, this treatment cannot be performed under unstable hemodynamic conditions. This report highlights the utility and efficacy of emergent balloon aortic valvuloplasty (BAV) in controlling heart failure and its potential to serve as a bridge to curative therapy, even in older patients with ADHF due to severe AS with concomitant LVOT obstruction. Furthermore, combination therapy with TAVR and percutaneous ASA could be safely performed after controlling for ADHF using BAV. Non-surgical management is a more feasible treatment option in older patients with ADHF who are at higher risk of complications during surgical intervention. .

Skeletal and gene-regulatory functions of nuclear sex steroid hormone receptors.

The wide variety of sex hormone actions underlie bone growth and health, and their actions mediate gene regulation by the cognate nuclear receptors. Nuclear androgen and estrogen receptors (AR, and ERα/ERß) are hormone-dependent and DNA binding- transcription regulatory factors, and gene regulation by sex hormones often accompany with chromatin remodeling under aid of a number of co-regulators. As sex hormone biosynthesis is under highly regulated systemic and local regulations, the skeletal actions of sex hormones could be inferred from only the phenotypic abnormalities in skeleton in mouse genetic models deficient of nuclear receptors selectively in specific types of bone cells as well as at specific cell differentiation stages. Anabolic androgen actions and anti-bone resorptive estrogen actions are discussed here from the phenotypic abnormalities in such model mice. Though rapid gene regulation by sex hormones may not require chromatin reorganization, dynamic chromatin reconfiguration looks to facilitate profound and long-term hormonal actions. In this review, we focus the recent findings in gene regulation at a chromatin level, particularly of the function of enhancer RNAs transcribed from strong enhancers, and in the role of liquid-liquid phase separation state in transcription initiation through chromatin reconfiguration.

Profiling of Androgen-Dependent Enhancer RNAs Expression in Human Prostate Tumors: Search for Malignancy Transition Markers.

INTRODUCTION: Although the ability of androgens to promote prostate cancer development has been known for decades, the molecular mechanisms of androgen receptor (AR) signaling in the tumorigenesis remain unclear. Enhancer RNAs (eRNAs) transcribed from strong enhancers, or super-enhancers (SEs), have recently emerged as a novel class of regulatory non-coding RNAs (ncRNAs) that facilitate transcription, including that of androgen target genes, through chromatin looping to position enhancers proximate to the promoters. The aim of this study was to assess androgen-dependent transcription in prostate tumors of eRNAs (designated as KLK3eRNAs) from the SE of the KLK3 gene encoding the prostate-specific antigen (PSA) protein, a clinical marker of prostate carcinogenesis. MATERIALS AND METHODS: The androgen-induced KLK3eRNAs were identified in the LNCaP human prostate cancer cell line. The expressions of these KLK3eRNAs together with KLK3 and AR mRNA transcripts were assessed by qRT-PCR in prostate tumor samples from five prostate cancer patients. RESULTS: Androgen-induced KLK3eRNAs have been identified in the LNCaP cells, and their expression was further analyzed in tumors of prostate cancer patients. Transcripts of the tested KLK3eRNAs have been detected in all clinical samples, but their expression patterns differed between individual tumor specimens. We found a statistically significant correlation between the levels of the KLK3 and AR mRNAs with those of the previously reported KLK3eRNAs, while such correlation was not observed for novel KLK3eRNAs described in our recent report. CONCLUSION: Presented data suggest that prostate tumor development may associate with epigenetic reorganization in the KLK3 genomic regulatory elements reflected by changes of the KLK3eRNA expression. Our findings support a potential of eRNAs profiling to be used as diagnostic marker.