Gene-regulation modules in nonalcoholic fatty liver disease revealed by single-nucleus ATAC-seq.

We investigated the progression of nonalcoholic fatty liver disease from fatty liver to steatohepatitis using single-nucleus and bulk ATAC-seq on the livers of rats fed a high-fat diet (HFD). Rats fed HFD for 4 wk developed fatty liver, and those fed HFD for 8 wk further progressed to steatohepatitis. We observed an increase in the proportion of inflammatory macrophages, consistent with the pathological progression. Utilizing machine learning, we divided global gene regulation into modules, wherein transcription factors within a module could regulate genes within the same module, reaffirming known regulatory relationships between transcription factors and biological processes. We identified core genes-central to co-expression and protein-protein interaction-for the biological processes discovered. Notably, a large part of the core genes overlapped with genes previously implicated in nonalcoholic fatty liver disease. Single-nucleus ATAC-seq, combined with data-driven statistical analysis, offers insight into in vivo global gene regulation as a combination of modules and assists in identifying core genes of relevant biological processes.

Transcriptomic Response in the Heart and Kidney to Different Types of Antihypertensive Drug Administration.

Certain classes of antihypertensive drug may exert specific, blood pressure (BP)-independent protective effects on end-organ damages such as left ventricular hypertrophy, although the overall evidence has not been definitive in clinical trials. To unravel antihypertensive drug-induced gene expression changes that are potentially related to the amelioration of end-organ damages, we performed in vivo phenotypic evaluation and transcriptomic analysis on the heart and the kidney, with administration of antihypertensive drugs to two inbred strains (ie, hypertensive and normotensive) of rats. We chose 6 antihypertensive classes: enalapril (angiotensin-converting enzyme inhibitor), candesartan (angiotensin receptor blocker), hydrochlorothiazide (diuretics), amlodipine (calcium-channel blocker), carvedilol (vasodilating ß-blocker), and hydralazine. In the tested rat strains, 4 of 6 drugs, including 2 renin-angiotensin system inhibitors, were effective for BP lowering, whereas the remaining 2 drugs were not. Besides BP lowering, there appeared to be some interdrug heterogeneity in phenotypic changes, such as suppressed body weight gain and body weight-adjusted heart weight reduction. For the transcriptomic response, a considerable number of genes showed prominent mRNA expression changes either in a BP-dependent or BP-independent manner with substantial diversity between the target organs. Noticeable changes of mRNA expression were induced particularly by renin-angiotensin system blockade, for example, for genes in the natriuretic peptide system (Nppb and Corin) in the heart and for those in the renin-angiotensin system/kallikrein-kinin system (Ren and rat Klk1 paralogs) and those related to calcium ion binding (Calb1 and Slc8a1) in the kidney. The research resources constructed here will help corroborate occasionally inconclusive evidence in clinical settings.

Integrative genomic analysis of blood pressure and related phenotypes in rats.

Despite remarkable progress made in human genome-wide association studies, there remains a substantial gap between statistical evidence for genetic associations and functional comprehension of the underlying mechanisms governing these associations. As a means of bridging this gap, we performed genomic analysis of blood pressure (BP) and related phenotypes in spontaneously hypertensive rats (SHR) and their substrain, stroke-prone SHR (SHRSP), both of which are unique genetic models of severe hypertension and cardiovascular complications. By integrating whole-genome sequencing, transcriptome profiling, genome-wide linkage scans (maximum n=1415), fine congenic mapping (maximum n=8704), pharmacological intervention and comparative analysis with transcriptome-wide association study (TWAS) datasets, we searched causal genes and causal pathways for the tested traits. The overall results validated the polygenic architecture of elevated BP compared with a non-hypertensive control strain, Wistar Kyoto rats (WKY); e.g. inter-strain BP differences between SHRSP and WKY could be largely explained by an aggregate of BP changes in seven SHRSP-derived consomic strains. We identified 26 potential target genes, including rat homologs of human TWAS loci, for the tested traits. In this study, we re-discovered 18 genes that had previously been determined to contribute to hypertension or cardiovascular phenotypes. Notably, five of these genes belong to the kallikrein-kinin/renin-angiotensin systems (KKS/RAS), in which the most prominent differential expression between hypertensive and non-hypertensive alleles could be detected in rat Klk1 paralogs. In combination with a pharmacological intervention, we provide in vivo experimental evidence supporting the presence of key disease pathways, such as KKS/RAS, in a rat polygenic hypertension model.

LOX-1 (Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1) Deletion Has Protective Effects on Stroke in the Genetic Background of Stroke-Prone Spontaneously Hypertensive Rat.

Background and Purpose- oxLDL (oxidized low-density lipoprotein) has been known for its potential to induce endothelial dysfunction and used as a major serological marker of oxidative stress. Recently, LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a lectin-like receptor for oxLDL, has attracted attention in studies of neuronal apoptosis and stroke. We aim to investigate the impact of LOX-1-deficiency on spontaneous hypertension-related brain damage in the present study. Methods- We generated a LOX-1 deficient strain on the genetic background of stroke-prone spontaneously hypertensive rat (SHRSP), an animal model of severe hypertension and spontaneous stroke. In this new disease model with stroke-proneness, we monitored the occurrence of brain abnormalities with and without salt loading by multiple procedures including T2 weighted magnetic resonance imaging and also explored circulatory miRNAs as diagnostic biomarkers for cerebral ischemic injury by microarray analysis. Results- Both T2 weighted magnetic resonance imaging abnormalities and physiological parameter changes could be detected at significantly delayed timing in LOX-1 knockout rats compared with wild-type SHRSP, in either case of normal rat chow and salt loading (P<0.005 in all instances; n=11-20 for SHRSP and n=13-23 for LOX-1 knockout rats). There were no significant differences in the form of magnetic resonance imaging findings between the strains. A number of miRNAs expressed in the normal rat plasma, including rno-miR-150-5p and rno-miR-320-3p, showed significant changes after spontaneous brain damage in SHRSP, whereas the corresponding changes were modest or almost unnoticeable in LOX-1 knockout rats. There appeared to be the lessening of correlation of postischemic miRNA alterations between the injured brain tissue and plasma in LOX-1 knockout rats. Conclusions- Our data show that deficiency of LOX-1 has a protective effect on spontaneous brain damage in a newly generated LOX-1-deficient strain of SHRSP. Further, our analysis of miRNAs as biomarkers for ischemic brain damage supports a potential involvement of LOX-1 in blood brain barrier disruption after cerebral ischemia. Visual Overview- An online visual overview is available for this article.

Alterations of lipid metabolism, blood pressure and fatty liver in spontaneously hypertensive rats transgenic for human cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) mediates a step in reverse cholesterol transport, which channels cholesterol from peripheral tissues back to the liver. Mice and rats are CETP-deficient species, which assumedly contribute to rodent atherosclerosis resistance. Both pro- and anti-atherogenic effects have been shown in studies of CETP-transgenic rodent models thus far. As the results of pharmacological studies of CETP modification are largely controversial in humans, further knowledge about the impact of CETP on atherogenic phenotypes is required to evaluate its clinical utility for the prevention of cardiovascular and other organ damage associated with metabolic syndrome. Therefore, we newly generated a human CETP-transgenic (Tg[hCETP]) strain on the genetic background of spontaneously hypertensive rats (SHRs), which are characterized by the spontaneous occurrence of hypertension and insulin resistance. This allowed us to assess the in vivo role of CETP on cardiometabolic phenotypes in combination with hypertension. In Tg[hCETP] SHRs fed normal rat chow, systolic blood pressure was markedly elevated by 20-37 mmHg throughout the study period, and the development of fatty liver was accelerated with appreciable changes in the plasma lipid profile (HDL cholesterol reduction and triglyceride elevation). These phenotypic changes are in accordance with the assumption of proatherogenic effects inducible by the overexpression of CETP. However, with plasma LDL cholesterol levels concomitantly reduced, no apparent progression of atherosclerosis was detected in either the aorta or coronary arteries of Tg[hCETP] SHRs fed a high-fat, high-cholesterol diet. Our data provide new insight into the multifaceted regulation of cardiometabolic phenotypes via the modification of CETP.

Investigation of Functional Genes at Homologous Loci Identified Based on Genome-wide Association Studies of Blood Lipids via High-fat Diet Intervention in Rats using an in vivo Approach.

AIM: It is challenging to identify causal (or target) genes at individual loci detected using genome-wide association studies (GWAS). In order to follow up GWAS loci, we investigated functional genes at homologous loci identified using human lipid GWAS that responded to a high-fat, high-cholesterol diet (HFD) intervention in an animal model. METHODS: The HFD intervention was carried out for four weeks in male rats of the spontaneously hypertensive rat strain. The liver and adipose tissues were subsequently excised for analyses of changes in the gene expression as compared to that observed in rats fed normal rat chow (n=8 per group). From 98 lipid-associated loci reported in previous GWAS, 280 genes with rat orthologs were initially selected as targets for the two-staged analysis involving screening with DNA microarray and validation with quantitative PCR (qPCR). Consequently, genes showing a differential expression due to HFD were examined for changes in the expression induced by atorvastatin, which was independently administered to the rats. RESULTS: Using the HFD intervention in the rats, seven known (Abca1, Abcg5, Abcg8, Lpl, Nr1h3, Pcsk9 and Pltp) and three novel (Madd, Stac3 and Timd4) genes were identified as potential significant targets, with an additional list of 23 suggestive genes. Among these 33 genes, Stac3, Fads1 and six known genes exhibited nominally significant expression changes following treatment with atorvastatin. Six (of 33) genes overlapped with those previously detected in the expression QTL studies. CONCLUSIONS: Our experimental in vivo approach increases the ability to identify target gene(s), when combined with other functional studies, thus improving understanding of the mechanisms by which GWAS variants act.

Deletion of CDKAL1 affects high-fat diet-induced fat accumulation and glucose-stimulated insulin secretion in mice, indicating relevance to diabetes.

BACKGROUND/OBJECTIVE: The CDKAL1 gene is among the best-replicated susceptibility loci for type 2 diabetes, originally identified by genome-wide association studies in humans. To clarify a physiological importance of CDKAL1, we examined effects of a global Cdkal1-null mutation in mice and also evaluated the influence of a CDKAL1 risk allele on body mass index (BMI) in Japanese subjects. METHODS: In Cdkal1-deficient (Cdkal1⁻/⁻) mice, we performed oral glucose tolerance test, insulin tolerance test, and perfusion experiments with and without high-fat feeding. Based on the findings in mice, we tested genetic association of CDKAL1 variants with BMI, as a measure of adiposity, and type 2 diabetes in Japanese. PRINCIPAL FINDINGS: On a standard diet, Cdkal1⁻/⁻ mice were modestly lighter in weight than wild-type littermates without major alterations in glucose metabolism. On a high fat diet, Cdkal1⁻/⁻ mice showed significant reduction in fat accumulation (17% reduction in %intraabdominal fat, P = 0.023 vs. wild-type littermates) with less impaired insulin sensitivity at an early stage. High fat feeding did not potentiate insulin secretion in Cdkal1⁻/⁻ mice (1.0-fold), contrary to the results in wild-type littermates (1.6-fold, P<0.01). Inversely, at a later stage, Cdkal1⁻/⁻ mice showed more prominent impairment of insulin sensitivity and glucose tolerance. mRNA expression analysis indicated that Scd1 might function as a critical mediator of the altered metabolism in Cdkal1⁻/⁻ mice. In accordance with the findings in mice, a nominally significant (P<0.05) association between CDKAL1 rs4712523 and BMI was replicated in 2 Japanese general populations comprising 5,695 and 12,569 samples; the risk allele for type 2 diabetes was also associated with decreased BMI. CONCLUSIONS: Cdkal1 gene deletion is accompanied by modestly impaired insulin secretion and longitudinal fluctuations in insulin sensitivity during high-fat feeding in mice. CDKAL1 may affect such compensatory mechanisms regulating glucose homeostasis through interaction with diet.

[Correlation of DNA-dependent protein kinase catalytic subunit expression to radiosensitivity of non-small cell lung cancer cell lines].

BACKGROUND AND OBJECTIVE: DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role in repairing irradiation-induced DNA double-strand break (DSB), and affects the radiosensitivity of tissue cells. This study was to detect the expression of DNA-PKcs in different non-small cell lung cancer (NSCLC) cell lines and evaluate its correlation to radiosensitivity. METHODS: The content and activity of DNA-PKcs in five NSCLC cell lines A549, H1299, L78, PGCL3 and H460 were measured by Western blot and the DNA-PK activity assay. Cell survival was analyzed using clonogenic formation assay. RESULTS: The radiosensitivities of five NSCLC cell lines were different. The values of survival fraction at 2 Gy (SF2) were 0.74 in A549 cells, 0.25 in H1299 cells, 0.21 in H460 cells, 0.48 in PGCL3 cells, and 0.58 in L78 cells. The protein levels of DNA-PKcs were 3.26+/-0.98 in A549 cells, 0.51+/-0.07 in L78 cells, 0.51+/-0.11 in H1299 cells, 0.86+/-0.23 in H460 cells, and 2.60+/-0.76 in PGCL3 cells. The activity values of DNA-PKcs were 8.30+/-1.03 in A549 cells, 2.45+/-0.52 in H1299 cells, 0.11+/-0.02 in H460 cells, 4.13+/-0.87 in PGCL3 cells, and 0.42+/-0.07 in L78 cells. In adenocarcinoma and large cell carcinoma cell lines, SF2 were correlated to DNA-PKcs content (P<0.05, r=0.95) and activity (P=0.03, r=0.98). CONCLUSION: DNA-PKcs is an important factor to predict the radiosensitivity in adenocarcinoma and large cell lung cancer cell lines.

Candesartan-induced gene expression in five organs of stroke-prone spontaneously hypertensive rats.

To test the functional consequences of blocking the local renin-angiotensin system (RAS), we investigated the effects of an angiotensin II type 1 receptor blocker (ARB), candesartan, on the systemic gene expression profile of five important organs (brain, heart, kidney, liver and adipose tissues) in the stroke-prone spontaneously hypertensive rat (SHRSP), an established model of essential hypertension and cardiovascular disorders, and its normotensive control, the Wistar Kyoto (WKY) rat. Rats were treated with candesartan (5 mg/kg/d) for 4 weeks from 12 to 16 weeks of age. DNA microarray technology was used to identify changes in gene expression. Four weeks of treatment with candesartan significantly lowered systolic blood pressure in male rats of both the SHRSP and the WKY strains (p<0.0005). Candesartan differentially modulated the gene expression profile in an organ-specific manner in male SHRSP; of the five organs tested, gene expression was most prominently altered in the hearts of SHRSP. In contrast, candesartan treatment exerted minimal or no significant effects on the gene expression profile of the corresponding organs of male WKY rats. The inter-strain differences in gene expression changes induced by candesartan were considered to be associated with both blood pressure-dependent and independent mechanisms. These results help to delineate the mechanisms that underlie the organ or tissue protection conferred by ARB at the levels of cellular biology and genomics in the context of the local RAS. Further studies are warranted to investigate not only individual genes of interest but also genetic "networks" that involve differential organ- or tissue-specific gene expression induced by the blockade of RAS in essential hypertension. Tokyo, Japan

Dynamic changes of the renin-angiotensin and associated systems in the rat after pharmacological and dietary interventions in vivo.

To address the multiplicity of the renin-angiotensin system (RAS) with particular interest in its local, synergistic regulation, we investigate dynamic changes of the RAS and associated systems in response to external stimuli in the rat. We tested influences of the RAS blockade (candesartan and enalapril), diuretics (hydrochlorothiazide), high lipid diet, and salt loading on tissue mRNA level of 12 principal genes. Under the hemodynamic conditions appropriately predetermined, we quantitatively evaluated mRNA level changes with and without each intervention in five organs-the brain, heart, kidney, liver, and adipose tissues-of male rats (n = 5 each). A total of 250 tissues were examined by real-time PCR. Significant changes in mRNA level (P < 0.05) were found in a drug-, diet- and tissue-specific manner. For instance, 29% of genes (14 out of 48 tissues showing detectable mRNA levels) were differentially regulated by candesartan and enalapril, although both drugs reduced blood pressure to similar extents. When the overall interactions among 12 genes were compared between interventions, the RAS and associated systems appeared to change in the opposite direction between candesartan and high lipid diet in the adipose tissue and between candesartan and salt loading in the heart. Enalapril, however, induced unique patterns of perturbation in the local RAS under the corresponding conditions. Thus, this study provides a fundamental picture of gene expression profile in vivo in the RAS and associated systems. In particular, our data highlight differential regulation between candesartan and enalapril, which may reflect the individual pharmacological properties regarding clinical implications.

Systemic evaluation of gene expression changes in major target organs induced by atorvastatin.

Statins have been reported to protect against end-organ damage in essential hypertension; however, detailed mechanisms underlying organ-protective actions of statins remain unclear. Statins can exert pleiotropic effects aside from lowering cholesterol and blood pressure levels through several different pathways, which may lead to distinct patterns of changes in gene expression in vital end-organs. The aim of the present study was to systemically evaluate gene expression changes in three major end-organs (the brain, heart and kidney) induced by atorvastatin at a dose that altered neither blood pressure nor plasma total cholesterol levels. The stroke-prone spontaneously hypertensive (SHRSP) rats, an established model of hypertension and end-organ damage, was treated with atorvastatin (15 mg/kg/day) for 4 weeks from 12 to 16 weeks of age. DNA microarray technology was used to identify gene expression changes in three end-organs. In the current experimental setting, 4 weeks of atorvastatin treatment lowered plasma levels of non-esterified fatty acid significantly (P=0.0012) and triglyceride modestly (P=0.07) without altering blood pressure and plasma total cholesterol levels in male SHRSP rats. The level of expression of a number of genes was changed in an organ-specific manner after 4 weeks of drug administration to SHRSP rats. Among the end-organs studied, the most prominent alteration in gene expression was observed in the heart. The identical treatment protocol was applied to age-matched normotensive control rats, Wistar Kyoto rats, and this also caused a number of genes to be differentially expressed in an organ-specific manner. These results provide new insights into the mechanisms underlying the potential efficacy of statins in protecting against end-organ damage in essential hypertension and thus lay the foundation for future studies.

Quantum dot as a drug tracer in vivo.

Quantum dots (QDs) have been applied to a wide range of biological studies by taking advantage of their fluorescence properties. There is almost no method to trace small molecules including medicine. Here, we used QDs for fluorescent tracers for medicine and analyzed their kinetics and dynamics. We conjugated QDs with captopril, anti-hypertensive medicine, by an exchange reaction while retaining the medicinal properties. We investigated the medicinal effect of QD-conjugated captopril (QD-cap) in vitro and in vivo. We also evaluated the concentration and the distribution of the QD-cap in the blood and the organs with their fluorescence. We demonstrate that the QD-cap inhibits the activity of ACE in vitro. The QD-cap reduced the blood pressure of hypertensive model rats. The concentration of the QD-cap in the blood was measured by using the standard curve of the fluorescence intensity. The blood concentration of the QD-cap decrease exponentially and QD-cap has approximately the same half-life as that of captopril. In addition, the fluorescence of the QDs revealed that QD-cap accumulates in the liver, lungs, and spleen. We succeeded in analyzing the dynamics and kinetics of small molecules using fluorescence of QDs.

Identification of quantitative trait loci for cardiac hypertrophy in two different strains of the spontaneously hypertensive rat.

Cardiac hypertrophy and left ventricular hypertrophy are known to be substantially controlled by genetic factors. As an experimental model, we undertook genome-wide screens for cardiac mass in F2 populations bred from the stroke-prone spontaneously hypertensive rats (SHRSP) and normal spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) of a Japanese colony. Two F2 cohorts were independently produced: F2(SHRSP x WKY) (110 male and 110 female rats) and F2(SHR x WKY) (151 male rats). The ratio of heart weight to body weight (Hw/Bw) was evaluated at 12 months of age in F2(SHRSP x WKY) after salt-loading for 7 months, and at around 15 weeks of age in F2(SHR x WKY) who had been fed a normal rat chow diet. Subsequent to an initial screen with 251 markers in F2(SHRSP x WKY) male progeny, 170 and 161 markers were selected and characterized in F2(SHRSP x WKY) female progeny and F2(SHR x WKY) male progeny, respectively. Markers from four chromosomal regions showed suggestive or significant linkage to Hw/Bw. The strongest and the most consistent linkage was found in the vicinity of D3Mgh16 on rat chromosome (RNO) 3 (a maximal log of the odds score reached 4.0 to 6.6 across the F2 populations studied). In the other three regions on RNO6, RNO10 and RNO13, the degree of linkage was more prominent in either males or females. These data provide solid evidence for a "principal" RNO3 quantitative trait loci regulating Hw/Bw in SHRSP and SHR, and also suggest the possible presence of sexual dimorphism in regard to genetic susceptibility for cardiac hypertrophy.

Evaluation of insulin resistance linkage to rat chromosome 4 in SHR of a Japanese colony.

The spontaneously hypertensive rat (SHR) is a model of human insulin resistance syndrome. Quantitative trait loci for cellular defects in glucose and fatty acid metabolism have been mapped to an overlapping region of rat chromosome (RNO) RNO4 in SHR of the National Institute of Health colony, where a deletion in the Cd36 gene has been implicated as the causative mutation of insulin resistance. The present study has examined the potential presence of RNO4 linkage to a series of metabolic phenotypes in F(2) progeny derived from SHR of a Japanese colony (SHR/Izm) without the Cd36 mutation. Our data demonstrate that 'major' insulin resistance gene(s) are unlikely to exist on RNO4 in SHR/Izm and in vitro phenotypes measured in isolated adipocytes do not cosegregate in the F(2) population studied. Thus, it seems to be difficult to explain the underlying genetic mechanisms of insulin resistance by a single major gene on RNO4.

Isolation of a chromosome 1 region affecting blood pressure and vascular disease traits in the stroke-prone rat model.

Recently, a genome-wide screen has shown a major quantitative trait locus (QTL) for a stroke-associated phenotype on rat chromosome 1 (RNO1) independent of QTL for blood pressure (BP) in the stroke-prone spontaneously hypertensive rat (SHRSP) of a Heidelberg colony. However, it remains to be elucidated whether these observations reflect the existence of different genes predisposing to each of the disorders. To address this issue, we performed comprehensive approaches in a Japanese colony, Izm, as follows. First, we undertook genome-wide searches in F1(SHRSP/IzmxWKY/Izm)xSHRSP/Izm back-cross (n=63) to pursue a causal relation between hypertension and stroke. Although the strongest linkage to BP (LOD score of 3.4) was identified on RNO1, its relevance to stroke was not supported in the F1 back-cross studied. Second, we also investigated linkage to BP in F2 progeny (n=175) involving the stroke-resistant (or normal) spontaneously hypertensive rat (SHR). In F2 studies of SHR/Izm, this locus did not appear to constitute a principal BP QTL. Third, we constructed congenic animals with detailed phenotype characterization. Transfer of a chromosomal fragment between markers Klk1 and D1Rat116 from WKY/Izm onto the SHRSP/Izm background lowered systolic BP by 20 to 80 mm Hg, prevented development of apparent stroke, and exaggerated impaired glucose tolerance. In conclusion, we have successfully isolated an RNO1 region affecting BP, stroke, and glucose tolerance in SHRSP/Izm-derived congenic rats. The size of the introgressed region is large, but our novel congenic strain should help delineate complex, genetic impairments underlying BP and associated vascular disease phenotypes.