Multi-organ single-cell RNA sequencing in mice reveals early hyperglycemia responses that converge on fibroblast dysregulation.

Diabetes causes a range of complications that can affect multiple organs. Hyperglycemia is an important driver of diabetes-associated complications, mediated by biological processes such as dysfunction of endothelial cells, fibrosis, and alterations in leukocyte number and function. Here, we dissected the transcriptional response of key cell types to hyperglycemia across multiple tissues using single-cell RNA sequencing (scRNA-seq) and identified conserved, as well as organ-specific, changes associated with diabetes complications. By studying an early time point of diabetes, we focus on biological processes involved in the initiation of the disease, before the later organ-specific manifestations had supervened. We used a mouse model of type 1 diabetes and performed scRNA-seq on cells isolated from the heart, kidney, liver, and spleen of streptozotocin-treated and control male mice after 8 weeks and assessed differences in cell abundance, gene expression, pathway activation, and cell signaling across organs and within organs. In response to hyperglycemia, endothelial cells, macrophages, and monocytes displayed organ-specific transcriptional responses, whereas fibroblasts showed similar responses across organs, exhibiting altered metabolic gene expression and increased myeloid-like fibroblasts. Furthermore, we found evidence of endothelial dysfunction in the kidney, and of endothelial-to-mesenchymal transition in streptozotocin-treated mouse organs. In summary, our study represents the first single-cell and multi-organ analysis of early dysfunction in type 1 diabetes-associated hyperglycemia, and our large-scale dataset (comprising 67 611 cells) will serve as a starting point, reference atlas, and resource for further investigating the events leading to early diabetic disease.

Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.

AIMS: Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS: Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS: Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis.

BACKGROUND: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS: Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Altered Macrophage Polarization Induces Experimental Pulmonary Hypertension and Is Observed in Patients With Pulmonary Arterial Hypertension.

OBJECTIVE: To determine whether global reduction of CD68 (cluster of differentiation) macrophages impacts the development of experimental pulmonary arterial hypertension (PAH) and whether this reduction affects the balance of pro- and anti-inflammatory macrophages within the lung. Additionally, to determine whether there is evidence of an altered macrophage polarization in patients with PAH. Approach and Results: Macrophage reduction was induced in mice via doxycycline-induced CD68-driven cytotoxic diphtheria toxin A chain expression (macrophage low [MacLow] mice). Chimeric mice were generated using bone marrow transplant. Mice were phenotyped for PAH by echocardiography and closed chest cardiac catheterization. Murine macrophage phenotyping was performed on lungs, bone marrow-derived macrophages, and alveolar macrophages using immunohistochemical and flow cytometry. Monocyte-derived macrophages were isolated from PAH patients and healthy volunteers and polarization capacity assessed morphologically and by flow cytometry. After 6 weeks of macrophage depletion, male but not female MacLow mice developed PAH. Chimeric mice demonstrated a requirement for both MacLow bone marrow and MacLow recipient mice to cause PAH. Immunohistochemical analysis of lung sections demonstrated imbalance in M1/M2 ratio in male MacLow mice only, suggesting that this imbalance may drive the PAH phenotype. M1/M2 imbalance was also seen in male MacLow bone marrow-derived macrophages and PAH patient monocyte-derived macrophages following stimulation with doxycycline and IL (interleukin)-4, respectively. Furthermore, MacLow-derived alveolar macrophages showed characteristic differences in terms of their polarization and expression of diphtheria toxin A chain following stimulation with doxycycline. CONCLUSIONS: These data further highlight a sex imbalance in PAH and further implicate immune cells into this paradigm. Targeting imbalance of macrophage population may offer a future therapeutic option.

A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.

Toll-like Receptor 3 Is a Therapeutic Target for Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by vascular cell proliferation and endothelial cell apoptosis. TLR3 (Toll-like receptor 3) is a receptor for double-stranded RNA and has been recently implicated in vascular protection. OBJECTIVES: To study the expression and role of TLR3 in PAH and to determine whether a TLR3 agonist reduces pulmonary hypertension in preclinical models. METHODS: Lung tissue and endothelial cells from patients with PAH were investigated by polymerase chain reaction, immunofluorescence, and apoptosis assays. TLR3-/- and TLR3+/+ mice were exposed to chronic hypoxia and SU5416. Chronic hypoxia or chronic hypoxia/SU5416 rats were treated with the TLR3 agonist polyinosinic/polycytidylic acid (Poly[I:C]). MEASUREMENTS AND MAIN RESULTS: TLR3 expression was reduced in PAH patient lung tissue and endothelial cells, and TLR3-/- mice exhibited more severe pulmonary hypertension following exposure to chronic hypoxia/SU5416. TLR3 knockdown promoted double-stranded RNA signaling via other intracellular RNA receptors in endothelial cells. This was associated with greater susceptibility to apoptosis, a known driver of pulmonary vascular remodeling. Poly(I:C) increased TLR3 expression via IL-10 in rat endothelial cells. In vivo, high-dose Poly(I:C) reduced pulmonary hypertension in both rat models in proof-of-principle experiments. In addition, Poly(I:C) also reduced right ventricular failure in established pulmonary hypertension. CONCLUSIONS: Our work identifies a novel role for TLR3 in PAH based on the findings that reduced expression of TLR3 contributes to endothelial apoptosis and pulmonary vascular remodeling.

Divergent Roles for TRAIL in Lung Diseases.

The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a widely expressed cytokine that can bind five different receptors. TRAIL has been of particular interest for its proposed ability to selectively induce apoptosis in tumour cells. However, it has also been found to regulate a wide variety of non-canonical cellular effects including survival, migration and proliferation via kinase signalling pathways. Lung diseases represent a wide range of conditions affecting multiple tissues. TRAIL has been implicated in several biological processes underlying lung diseases, including angiogenesis, inflammation, and immune regulation. For example, TRAIL is detrimental in pulmonary arterial hypertension-it is upregulated in patient serum and lungs, and drives the underlying proliferative pulmonary vascular remodelling in rodent models. However, TRAIL protects against pulmonary fibrosis in mice models-by inducing apoptosis of neutrophils-and reduced serum TRAIL is found in patients. Conversely, in the airways TRAIL positively regulates inflammation and immune response. In COPD patients and asthmatic patients challenged with antigen, TRAIL and its death receptors are upregulated in serum and airways. Furthermore, TRAIL-deleted mouse models have reduced airway inflammation and remodelling. In the context of respiratory infections, TRAIL assists in immune response, e.g., via T-cell toxicity in influenza infection, and neutrophil killing in S. pneumoniae infection. In this mini-review, we examine the functions of TRAIL and highlight the diverse roles TRAIL has in diseases affecting the lung. Disentangling the facets of TRAIL signalling in lung diseases could help in understanding their pathogenic processes and targeting novel treatments.

Eplerenone attenuates pathological pulmonary vascular rather than right ventricular remodeling in pulmonary arterial hypertension.

BACKGROUND: Aldosterone is a mineralocorticoid hormone critically involved in arterial blood pressure regulation. Although pharmacological aldosterone antagonism reduces mortality and morbidity among patients with severe left-sided heart failure, the contribution of aldosterone to the pathobiology of pulmonary arterial hypertension (PAH) and right ventricular (RV) heart failure is not fully understood. METHODS: The effects of Eplerenone (0.1% Inspra® mixed in chow) on pulmonary vascular and RV remodeling were evaluated in mice with pulmonary hypertension (PH) caused by Sugen5416 injection with concomitant chronic hypoxia (SuHx) and in a second animal model with established RV dysfunction independent from lung remodeling through surgical pulmonary artery banding. RESULTS: Preventive Eplerenone administration attenuated the development of PH and pathological remodeling of pulmonary arterioles. Therapeutic aldosterone antagonism - starting when RV dysfunction was established - normalized mineralocorticoid receptor gene expression in the right ventricle without direct effects on either RV structure (Cardiomyocyte hypertrophy, Fibrosis) or function (assessed by non-invasive echocardiography along with intra-cardiac pressure volume measurements), but significantly lowered systemic blood pressure. CONCLUSIONS: Our data indicate that aldosterone antagonism with Eplerenone attenuates pulmonary vascular rather than RV remodeling in PAH.

Selective improvement of pulmonary arterial hypertension with a dual ETA/ETB receptors antagonist in the apolipoprotein E-/- model of PAH and atherosclerosis.

Idiopathic pulmonary arterial hypertension (IPAH) is increasingly diagnosed in elderly patients who also have an increased risk of co-morbid atherosclerosis. Apolipoprotein E-deficient (ApoE-/-) mice develop atherosclerosis with severe PAH when fed a high-fat diet (HFD) and have increased levels of endothelin (ET)-1. ET-1 receptor antagonists (ERAs) are used for the treatment of PAH but less is known about whether ERAs are beneficial in atherosclerosis. We therefore examined whether treatment of HFD-ApoE-/- mice with macitentan, a dual ETA/ETB receptor antagonist, would have any effect on both atherosclerosis and PAH. ApoE-/- mice were fed chow or HFD for eight weeks. After four weeks of HFD, mice were randomized to a four-week treatment of macitentan by food (30 mg/kg/day dual ETA/ETB antagonist), or placebo groups. Echocardiography and closed-chest right heart catheterization were used to determine PAH phenotype and serum samples were collected for cytokine analysis. Thoracic aortas were harvested to assess vascular reactivity using wire myography, and histological analyses were performed on the brachiocephalic artery and aortic root to assess atherosclerotic burden. Macitentan treatment of HFD-fed ApoE-/- mice was associated with a beneficial effect on the PAH phenotype and led to an increase in endothelial-dependent relaxation in thoracic aortae. Macitentan treatment was also associated with a significant reduction in interleukin 6 (IL-6) concentration but there was no significant effect on atherosclerotic burden. Dual blockade of ETA/ETB receptors improves endothelial function and improves experimental PAH but had no significant effect on atherosclerosis.

CDKN2B expression in adipose tissue of familial combined hyperlipidemia patients.

The purpose of this study was to determine the core biological processes perturbed in the subcutaneous adipose tissue of familial combined hyperlipidemia (FCHL) patients. Annotation of FCHL and control microarray datasets revealed a distinctive FCHL transcriptome, characterized by gene expression changes regulating five overlapping systems: the cytoskeleton, cell adhesion and extracellular matrix; vesicular trafficking; lipid homeostasis; and cell cycle and apoptosis. Expression values for the cell-cycle inhibitor CDKN2B were increased, replicating data from an independent FCHL cohort. In 3T3-L1 cells, CDKN2B knockdown induced C/EBPα expression and lipid accumulation. The minor allele at SNP site rs1063192 (C) was predicted to create a perfect seed for the human miRNA-323b-5p. A miR-323b-5p mimic significantly reduced endogenous CDKN2B protein levels and the activity of a CDKN2B 3'UTR luciferase reporter carrying the rs1063192 C allele. Although the allele displayed suggestive evidence of association with reduced CDKN2B mRNA in the MuTHER adipose tissue dataset, family studies suggest the association between increased CDKN2B expression and FCHL-lipid abnormalities is driven by factors external to this gene locus. In conclusion, from a comparative annotation analysis of two separate FCHL adipose tissue transcriptomes and a subsequent focus on CDKN2B, we propose that dysfunctional adipogenesis forms an integral part of FCHL pathogenesis.

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

Field strength and diffusion encoding technique affect the apparent diffusion coefficient measurements in diffusion-weighted imaging of the abdomen.

OBJECTIVES: The purpose of this study is to determine what effects a variety of diffusion encoding techniques at 1.5 T and 3 T have on measured abdominal apparent diffusion coefficient (ADC) values obtained in a healthy population. MATERIALS AND METHODS: Sixteen healthy male volunteers were enrolled in this prospective Institutional Review Board-approved study following written informed consent. Imaging was performed on a 1.5 T and a 3 T magnetic resonance system (Siemens, Erlangen) with several abdominal axial diffusion weighted imaging (DWI) acquisitions: an orthogonal diffusion encoding with b-values of 0/400 seconds/mm, and a series of four 3-scan trace weighted acquisitions with b-values of 0/50, 0/400, 0/800, 0/50/400/800 seconds/mm, respectively. The mean ADC values were calculated for 3 regions of interest (ROI) in 5 locations (right hepatic lobe, spleen, pancreatic head, body, and tail). The ADC data were analyzed using a repeated-measures analysis of variance. RESULTS: There was a significant difference between measured ADC values at 1.5 T and 3 T for liver (P < 0.001), but not for pancreas (P = 0.427) or spleen (P = 0.167). There was no significant difference (P > 0.999) in the measured ADC values between the orthogonal encodings and the 3-scan trace weighted encoding with the same b-value. There were significant differences (P < 0.001) between all 4 weighting schemes for the 3-scan trace with the measured ADC decreasing with increasing b-value. CONCLUSION: Measured abdominal ADC values depend on the exact selection of b-value used for encoding for liver, pancreas, and spleen. In addition, the measured ADC values depend on the field strength of the scanner for liver.

Short- and midterm reproducibility of apparent diffusion coefficient measurements at 3.0-T diffusion-weighted imaging of the abdomen.

PURPOSE: To test the hypothesis that there is no significant variability in apparent diffusion coefficients (ADCs) at assessment of the short- and midterm reproducibility of ADC measurements in a healthy population. MATERIALS AND METHODS: Twenty healthy male volunteers were enrolled in this prospective institutional review board-approved study after they provided written informed consent. A 3.0-T magnetic resonance (MR) system was used to perform five axial diffusion-weighted (DW) abdominal acquisitions (session 1). A mean of 147 days +/- 20 (standard deviation) later, 16 of the 20 volunteers were imaged again with use of the same protocol and MR system (session 2). The mean ADCs for three regions of interest (ROIs) in five anatomic locations (right hepatic lobe, spleen, and head, body, and tail of pancreas) were calculated. The coefficient of variation (CV, equal to standard deviation divided by mean) was calculated for each subject, session, and anatomic location. The ADC and CV data were then analyzed by using repeated-measures analysis of variance. RESULTS: There were significant differences (P < .001) in mean ADCs among the five anatomic locations. There were no significant differences in ADCs among the various repeated sequence acquisitions or the three ROIs. There were no significant differences in ADCs between imaging sessions 1 and 2. ADCs were fairly stable over the midterm within a given individual. Finally, there were no significant differences in resulting CVs between the imaging sessions or anatomic locations. The mean CV for ADC measurement reproducibility was 14% (95% confidence interval: 13%, 15%). CONCLUSION: The mean CV for short- and midterm ADC reproducibility was 14% at abdominal DW imaging. Treatment effects of less than approximately 27% (change in ADC divided by pretreatment ADC) will not be clinically detectable with confidence with one acquisition in a single individual.

In vivo silencing of alpha-synuclein using naked siRNA.

BACKGROUND: Overexpression of alpha-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinson's disease (PD), possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD. RESULTS: We have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion. CONCLUSION: We have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for alpha-synucleinopathies resulting from SNCA overexpression.

Genomic investigation of alpha-synuclein multiplication and parkinsonism.

OBJECTIVE: Copy number variation is a common polymorphic phenomenon within the human genome. Although the majority of these events are non-deleterious they can also be highly pathogenic. Herein we characterize five families with parkinsonism that have been identified to harbor multiplication of the chromosomal 4q21 locus containing the alpha-synuclein gene (SNCA). METHODS: A methodological approach using fluorescent in situ hybridization and Affymetrix (Santa Clara, CA) 250K SNP microarrays was used to characterize the multiplication in each family and to identify the genes encoded within the region. The telomeric and centromeric breakpoints of each family were further narrowed using semiquantitative polymerase chain reaction with microsatellite markers and then screened for transposable repeat elements. RESULTS: The severity of clinical presentation is correlated with SNCA dosage and does not appear to be overtly affected by the presence of other genes in the multiplicated region. With the exception of the Lister kindred, in each family the multiplication event appears de novo. The type and position of Alu/LINE repeats are also different at each breakpoint. Microsatellite analysis demonstrates two genomic mechanisms are responsible for chromosome 4q21 multiplications, including both SNCA duplication and recombination. INTERPRETATION: SNCA dosage is responsible for parkinsonism, autonomic dysfunction, and dementia observed within each family. We hypothesize dysregulated expression of wild-type alpha-synuclein results in parkinsonism and may explain the recent association of common SNCA variants in sporadic Parkinson's disease. SNCA genomic duplication results from intraallelic (segmental duplication) or interallelic recombination with unequal crossing over, whereas both mechanisms appear to be required for genomic SNCA triplication.