Kidney cell type specific changes in the chromatin and transcriptome landscapes following epithelial Hdac1and Hdac2 knockdown

Recent studies have identified at least 20 different kidney cell types based upon chromatin structure and gene expression. Histone deacetylases (HDACs) are epigenetic transcriptional repressors via deacetylation of histone lysines resulting in inaccessible chromatin. We reported that kidney epithelial HDAC1 and HDAC2 activity is critical for maintaining a healthy kidney and preventing fluid-electrolyte abnormalities. However, to what extent does Hdac1/Hdac2 knockdown affect chromatin structure and subsequent transcript expression in the kidney? To answer this question, we used single nucleus Assay for Transposase-Accessible Chromatin-sequencing (snATAC-seq) and snRNA-seq to profile kidney nuclei from male and female, control and littermate kidney epithelial Hdac1/Hdac2 knockdown mice. Hdac1/Hdac2 knockdown resulted in significant changes in the chromatin structure predominantly within the promoter region of gene loci involved in fluid-electrolyte balance such as the aquaporins, with both increased and decreased accessibility captured. Moreover, Hdac1/Hdac2 knockdown resulted different gene loci being accessible with a corresponding increased transcript number in the kidney, but among all mice only 24-30% of chromatin accessibility agreed with transcript expression (e.g. open chromatin, increased transcript). To conclude, although chromatin structure does affect transcription, ~70% of the differentially expressed genes cannot be explained by changes in chromatin accessibility and HDAC1/HDAC2 had a minimal effect on these global patterns. Yet, the genes that are targets of HDAC1 and HDAC2 are critically important for maintaining kidney function.

Progesterone-dependent and -independent modulation of luminal epithelial transcription to support pregnancy in cattle

In cattle, starting 4-5 days after estrus, pre-implantation embryonic development occurs in the confinement of the uterine lumen. Cells in the endometrial epithelial layer control the molecular traffic to and from the lumen and, thereby determine luminal composition. Starting early post-estrus, endometrial function is regulated by sex-steroids, but the effects of progesterone on luminal cells transcription have not been measured in vivo. First objective was to determine the extent to which progesterone controls transcription in luminal epithelial cells 4 d (D4) after estrus. Second objective was to discover luminal transcripts that predict pregnancy outcomes, when the effect of progesterone is controlled. Endometrial luminal epithelial cells were collected from embryo transfer recipients on D4 using a cytological brush and their transcriptome determined by RNASeq. Pregnancy by embryo transfer was measured on D30 (25 pregnant and 18 non-pregnant). Progesterone concentration on D4 was associated positively (n= 182) and negatively (n= 58) with gene expression. Progesterone-modulated transcription indicated an increase in oxidative phosphorylation, biosynthetic activity and proliferation of epithelial cells. When these effects of progesterone were controlled, different genes affected positively (n= 22) and negatively (n= 292) odds of pregnancy. These set of genes indicated that a receptive uterine environment was characterized by the inhibition of phosphoinositide signaling and innate immune system responses. A panel of 25 genes predicted the pregnancy outcome with sensitivity and specificity ranging from 64-96% and 44-83%, respectively. In conclusion, in the early diestrus, both progesterone-dependent and -independent mechanisms regulate luminal epithelial transcription associated with pregnancy outcomes in cattle.

Unique Circulating MicroRNA Associations with Dysglycemia in People Living with HIV and Alcohol Use

People living with HIV (PLWH) have increased prevalence of comorbid conditions including insulin resistance and at-risk alcohol use. Circulating microRNAs (miRs) may serve as minimally invasive indicators of pathophysiological states. We aimed to identify whether alcohol modulates circulating miR associations with measures of glucose/insulin dynamics in PLWH. PLWH (N=96; 69.8% male) enrolled in the Alcohol & Metabolic Comorbidities in PLWH: Evidence-Driven Interventions (ALIVE-Ex) study were stratified into negative phosphatidylethanol (PEth<8ng/ml, N=42) and positive PEth (PEth≥8ng/ml, N=54) groups. An oral glucose tolerance test (OGTT) was administered, and total RNA was isolated from fasting plasma to determine absolute miR expression. Circulating miRs were selected based on their role in skeletal muscle (miR-133a, miR-206), pancreatic β-cell (miR-375), liver (miR-20a), and adipose tissue (miR-let-7b, miR-146a, miR-221) function. Correlation and multiple regression analyses between miR expression and adiponectin, 2h glucose, insulin, and C-peptide values were performed adjusting for BMI category, age, sex, and viral load. miR-133a was negatively associated with adiponectin (p=0.002) in the negative PEth group, and miR-20a was positively associated with 2h glucose (p=0.013) in the positive PEth group. Regression analyses combining miRs demonstrated that miR-133a (p<0.001) and miR-221 (p=0.010) together predicted adiponectin in the negative PEth group. miR-20a (p<0.001) and miR-375 (p=0.002) together predicted 2h glucose in the positive PEth group. Our results indicate that associations between miRs and measures of glucose/insulin dynamics differed between PEth groups suggesting that the pathophysiological mechanisms contributing to altered glucose homeostasis in PLWH are potentially modulated by alcohol use.

Single-Cell RNA Sequencing of Mouse Left Ventricle Reveals Cellular Diversity and Intercommunication

Previous studies have revealed the diversity of the whole cardiac cellulome but not refined the left ventricle, which was essential for finding therapeutic targets. Here, we characterized single-cell transcriptional profiles of the mouse left ventricular cellular landscape using single-cell RNA sequencing (10×Genomics). Detailed t-Distributed Stochastic Neighbor Embedding (tSNE) analysis revealed the cell types of left ventricle with gene markers. Left ventricular cellulome contained cardiomyocytes highly expressed Trdn, endothelial cells highly expressed Pcdh17, fibroblast highly expressed Lama2 and macrophages highly expressed Hpgds, also proved by in situ hybridization. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analysis (ListHits>2, p<0.05) were employed with the DAVID database to investigate subtypes of each cell type with the underlying functions of differentially expressed genes (DEGs). Endothelial cells included five subtypes, fibroblasts comprised of seven subtypes and macrophages contained eleven subtypes. The key representative DEGs (p<0.001) were Gja4 and Gja5 in cluster 3 of endothelial cells, Aqp2 and Thbs4 in cluster 2 of fibroblasts, as well as Clec4e and Trem-1 in in cluster 3 of marcophages perhaps involved in the occur of atherosclerosis, heart failure and acute myocardial infarction proved by literature review. We also revealed extensive networks of intercellular communication in left ventricle. We suggested possible therapeutic targets for cardiovascular disease and autocrine and paracrine signaling underpins left ventricular homeostasis. This study provided new insights into the structure and function of the mammalian left ventricular cellulome and offers an important resource that will stimulate studies in cardiovascular research.

The adducin saga: Pleiotropic genomic targets for precision medicine in human hypertension; vascular, renal, and cognitive diseases

Hypertension is a leading risk factor for stroke, heart disease, chronic kidney disease, vascular cognitive impairment, and Alzheimer's disease. Previous genetic studies have nominated hundreds of genes linked to hypertension and renal and cognitive diseases. Some have been advanced as candidate genes by showing that they can alter blood pressure or renal and cerebral vascular function in knockout animals; however, final validation of the causal variants and underlying mechanisms have remained elusive. This review chronicles 40 years of work, from the initial identification of adducin (ADD) as an ACTIN-binding protein suggested to increase blood pressure in Milan hypertensive rats, to the discovery of a mutation in ADD1 as a candidate gene for hypertension in rats that were subsequently linked to hypertension in man. More recently, a recessive K572Q mutation in ADD3 was identified in Fawn-Hooded Hypertensive (FHH) and Milan Normotensive (MNS) rats that develop renal disease, which is absent in resistant strains. ADD3 dimerizes with ADD1 to form functional ADD protein. The mutation in ADD3 disrupts a critical ACTIN-binding site necessary for its interactions with actin and spectrin to regulate the cytoskeleton. Studies using Add3 knockout and transgenic strains, as well as a genetic complementation study in FHH and MNS rats, confirmed that the K572Q mutation in ADD3 plays a causal role in altering the myogenic response and autoregulation of renal and cerebral blood flow, resulting in increased susceptibility to hypertension-induced renal disease and cerebral vascular and cognitive dysfunction.

Changes in adipose tissue microRNA expression across the menstrual cycle in regularly menstruating females: a pilot study

Cyclical changes in hormone profiles across the menstrual cycle are associated with alterations in metabolic control. MicroRNAs (miRNA) contribute to regulating metabolic control, including adipose tissue metabolism. How fluctuations in hormonal profiles across the menstrual cycle affect adipose tissue miRNA expression remain unknown. Eleven healthy, regularly menstruating females underwent four sampling visits across their menstrual cycle. Subcutaneous abdominal adipose tissue and venous blood samples were collected each at sampling visit. Luteinizing hormone (LH) tests, calendar counting, and serum hormone concentrations were used to determine menstrual cycle phases: early-follicular (EF); late-follicular (LF); post-ovulatory (PO) and mid-luteal (ML). Serum follicle-stimulating hormone, LH, estrogen, progesterone and testosterone were determined using multiplex magnetic bead panels and enzyme-linked immunosorbent assays. Global adipose tissue miRNA expression levels were determined via microarray in a subset of participants (N=8) and 17 candidate miRNAs validated by RT-qPCR in the whole cohort (N=11). Global analysis of adipose tissue miRNA expression identified 33 miRNAs significantly altered across the menstrual cycle; however, no significant differences remained after correcting for multiple testing (p>0.05). RT-qPCR analysis of candidate miRNAs revealed miR-497-5p expression was significantly altered across the menstrual cycle (np2=0.18, p=0.03); however, post-hoc tests did not reveal any significant differences between menstrual cycle phases (p> 0.05). miR-30c-5p associated with testosterone concentration (R2=0.13, p=0.033). These pilot data indicate differences in adipose tissue miRNAs in healthy women across the menstrual cycle and a weak association with ovarian hormones. Further research in larger sample sizes is required to confirm regulation of miRNA expression across the menstrual cycle.

Dysregulation of miR-21-associated miRNA regulatory networks by chronic ethanol consumption impairs liver regeneration

Impaired liver regeneration has been considered as a hallmark of progression of alcohol-associated liver disease. Our previous studies demonstrated that in vivo inhibition of the microRNA (miRNA) miR21 can restore regenerative capacity of the liver in chronic ethanol-fed animals. The present study focuses on the role of microRNA regulatory networks that are likely to mediate the miR-21 action. Rats were chronically fed an ethanol-enriched diet along with pair-fed control animals and treated with AM21 (anti-miR-21), a locked nucleic acid antisense to miR-21. Partial hepatectomy (PHx) was performed and miRNA expression profiling over the course of liver regeneration was assessed. Our results showed dynamic expression changes in several miRNAs post PHx, notably with altered miRNA expression profiles between ethanol versus control groups. We found that in vivo inhibition of miR-21 led to correlated differential expression of miR-340-5p, and anti-correlated expression of miR-365, let-7a, miR-1224 and miR-146a across all sample groups post PHx. Gene set enrichment analysis identified a miRNA signature significantly associated with hepatic stellate cell activation within whole-liver tissue data. We hypothesized that at least part of the PHx-induced miRNA network changes responsive to miR-21 inhibition is localized to hepatic stellate cells. We validated this hypothesis using AM21 and TGF-β treatments in LX-2 human hepatic stellate cells in culture, and measured expression levels of select miRNAs by quantitative RT-PCR. Based on the in vivo and in vitro results, we propose a hepatic stellate cell miRNA regulatory network as contributing to the restoration of liver regenerative capacity by miR-21 inhibition.

Systems Genetics Analysis Defines Importance Of TMEM43/LUMA For Cardiac And Metabolic Related Pathways

Background: Broad cellular functions and diseases including muscular dystrophy, arrhythmogenic right ventricular cardiomyopathy (ARVC5) and cancer are associated with transmembrane protein43 (TMEM43/LUMA). Objective: The study aimed to investigate biological roles of TMEM43 through genetic regulation, gene pathways and gene networks, candidate interacting genes and up- or down-stream regulators. Methods: Cardiac transcriptomes from 40 strains of recombinant inbred BXD mice and two parental strains representing murine genetic reference population (GRP) was applied for genetic correlation, functional enrichment and co-expression network analysis using systems genetics approach. The results were validated in a newly created knock-in Tmem43-S358L mutation mouse model (Tmem43S358L) that displayed signs of cardiac dysfunction, resembling ARVC5 phenotype seen in humans. Results: We found high Tmem43 levels among BXDs with broad variability in expression. Expression of Tmem43 highly negatively correlated with heart mass and heart rate among BXDs, while levels of Tmem43 highly positively correlated with plasma high density lipoproteins (HDL). Through finding differentially expressed genes (DEGs) between Tmem43S358L mutant and wild type (Tmem43WT) lines, 18 pathways (out of 42 found in BXDs GRP) that are involved in ARVC, Hypertrophic cardiomyopathy, Dilated cardiomyopathy, Non-alcoholic fatty liver disease, Alzheimer disease, Parkinson disease and Huntington disease were verified. We further constructed Tmem43-mediated gene network, in which Ctnna1, Adcy6, Gnas, Ndufs6 and Uqcrc2 were significantly altered in Tmem43S358L mice vs Tmem43WT controls. Conclusions: Our study defined the importance of Tmem43 for cardiac and metabolism related pathways, suggesting that cardiovascular disease-relevant risk factors may also increase risk of metabolic and neurodegenerative diseases via TMEM43-mediated pathways.

RNO3 QTL Regulates Vascular Structure and Arterial Stiffness in the Spontaneously Hypertensive Rat

Increased arterial stiffness is an independent risk factor for hypertension, stroke, and cardiovascular morbidity. Thus, understanding the factors contributing to vascular stiffness is of critical importance. Here, we utilized a rat model containing a known quantitative trait loci (QTL) on chromosome 3 (RNO3) for vasoreactivity to assess potential genetic elements contributing to blood pressure, arterial stiffness and their downstream effects on cardiac structure and function. While no differences were found in blood pressure at any time point between parental Spontaneously Hypertensive Rats (SHR) and congenic SHR.BN3 rats, the SHR showed a significant increase in arterial stiffness measured by pulse wave velocity. The degree of arterial stiffness increased with age in the SHR and was associated with compensatory cardiac changes at 16 weeks of age, and decompensatory changes at 32 weeks, with no change in cardiac structure or function in the SHR.BN3 hearts at these time points. To evaluate the arterial wall structure, we utilized multiphoton microscopy to quantify cells and collagen content within the adventitia and media of SHR and SHR.BN3 arteries. No difference in cell numbers or proliferation rates were found, although phenotypic diversity was characterized in vascular smooth muscle cells. Herein, significant anatomical and physiological differences related to arterial structure and cardiovascular tone including, collagen, PWV, LV geometry and function, and VSMC contractile apparatus proteins were associated with the RNO3 QTL, thus providing a novel platform for studying arterial stiffness. Future studies delimiting the RNO3 QTL could aid in identifying genetic elements responsible for arterial structure and function.

Reconstitution of the host holobiont in germ-free born male rats acutely increases bone growth and affects marrow cellular content

Integration of microbiota in a host begins at birth and progresses during adolescence, forming a multidirectional system of physiologic interactions. Here, we present an instantaneous effect of natural, bacterial gut colonization on the acceleration of longitudinal and radial bone growth in germ-free born, 7-week-old male rats. Changes in bone mass and structure were analyzed after 10 days following the onset of colonization through cohousing with conventional rats and revealed unprecedented acceleration of bone accrual in cortical and trabecular compartments, increased bone tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. In addition, the number of small in size adipocytes increased, while the number of megakaryocytes decreased, in the bone marrow of conventionalized germ-free rats indicating that not only bone mass but also bone marrow environment is under control of gut microbiota signaling. The changes in bone status paralleled with a positive shift in microbiota composition toward short chain fatty acids (SCFA)-producing microbes and a considerable increase in cecal SCFA concentrations, specifically butyrate. Further, reconstitution of the host holobiont increased hepatic expression of IGF-1 and its circulating levels. Elevated serum levels of 25-hydroxy vitamin D and alkaline phosphatase pointed toward an active process of bone formation. The acute stimulatory effect on bone growth occurred independently of body mass increase. Overall, the presented model of conventionalized germ-free rats could be used to study microbiota-based therapeutics for combatting dysbiosis-related bone disorders.