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Background: Atherosclerosis is an inflammatory vascular disease marked by hyperlipidemia and hematopoietic stem cell expansion. Activin A, a member of the Activin/GDF/TGFβ/BMP (growth/differentiation factor/transforming growth factor beta/bone morphogenetic protein) family is broadly expressed and increases in human atherosclerosis, but its functional effects in vivo in this context remain unclear. Methods: We studied LDLR − /− mice on a Western diet for 12 weeks and used adeno-associated viral vectors with a liver-specific TBG (thyroxine-binding globulin) promoter to express Activin A or GFP (control). Atherosclerotic lesions were analyzed by oil red staining. Blood lipid profiling was performed by high-performance liquid chromatography, and immune cells were evaluated by flow cytometry. Liver RNA-sequencing was performed to explore the underlying mechanisms. Results: Activin A expression decreased in both livers and aortae from LDLR − /− mice fed a Western diet compared with standard laboratory diet. Adenoassociated virus-TBG-Activin A increased Activin A hepatic expression ≈10-fold at 12 weeks; P P Conclusions: Our studies reveal hepatic Activin A expression reduces inflammation, hematopoietic stem cell expansion, liver steatosis, circulating cholesterol, and fat accumulation, which likely all contribute to the observed protection against atherosclerosis. The reduced Activin A observed in LDLR −/− mice on a Western diet seems maladaptive and deleterious for atherogenesis.

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that transfer RNA-derived small RNAs (tDRs) play pivotal roles in stress responses. However, RNA modifications present on tDRs are barriers to accurately quantifying tDRs using traditional small RNA sequencing. Here, AlkB-facilitated methylation sequencing is used to generate a comprehensive landscape of cellular and extracellular tDR abundances in various cell types during different stress responses. Extracellular tDRs are found to have distinct fragmentation signatures from intracellular tDRs and these tDR signatures are better indicators of different stress responses than miRNAs. These distinct extracellular tDR fragmentation patterns and signatures are also observed in plasma from patients on cardiopulmonary bypass. It is additionally demonstrated that angiogenin and RNASE1 are themselves regulated by stressors and contribute to the stress-modulated abundance of sub-populations of cellular and extracellular tDRs. Finally, a sub-population of extracellular tDRs is identified for which AGO2 appears to be required for their expression. Together, these findings provide a detailed profile of stress-responsive tDRs and provide insight about tDR biogenesis and stability in response to cellular stressors.

AIMS/HYPOTHESIS: The aim of this work was to define metabolic correlates and pathways of diabetes pathogenesis in young adults during a subclinical latent phase of diabetes development. METHODS: We studied 2083 young adults of Black and White ethnicity in the prospective observational cohort Coronary Artery Risk Development in Young Adults (CARDIA) study (mean ± SD age 32.1 ± 3.6 years; 43.9% women; 42.7% Black; mean ± SD BMI 25.6 ± 4.9 kg/m(2)) and 1797 Framingham Heart Study (FHS) participants (mean ± SD age 54.7 ± 9.7 years; 52.1% women; mean ± SD BMI 27.4 ± 4.8 kg/m(2)), examining the association of comprehensive metabolite profiles with endophenotypes of diabetes susceptibility (adipose and muscle tissue phenotypes and systemic inflammation). Statistical learning techniques and Cox regression were used to identify metabolite signatures of incident diabetes over a median of nearly two decades of follow-up across both cohorts. RESULTS: We identified known and novel metabolites associated with endophenotypes that delineate the complex pathophysiological architecture of diabetes, spanning mechanisms of muscle insulin resistance, inflammatory lipid signalling and beta cell metabolism (e.g. bioactive lipids, amino acids and microbe- and diet-derived metabolites). Integrating endophenotypes of diabetes susceptibility with the metabolome generated two multi-parametric metabolite scores, one of which (a proinflammatory adiposity score) was associated with incident diabetes across the life course in participants from both the CARDIA study (young adults; HR in a fully adjusted model 2.10 [95% CI 1.72, 2.55], p

Introduction: CITED4 (CBP/p300-interacting transactivators with E [glutamic acid]/D [aspartic acid]-rich-carboxylterminal domain 4) is a transcriptional regulator which limits pathologic remodeling after ischemic and pressure overload models of cardiac stress. It is also upregulated in the heart with exercise, an effective intervention which induces physiological hypertrophy and cardiomyogenesis in adult mice. We hypothesized that CITED4 is required for exercise-induced cardiac growth and cardiomyogenesis. Methods and Results: Eight- to ten-week-old mice with cardiomyocyte-specific CITED4 knockout (C4KO) and littermate controls underwent 8 weeks of voluntary wheel running in comparison to sedentary controls. 5-Ethynyl-2′-deoxyuridine (EdU) was injected subcutaneously every two days for the first two weeks. There were no significant differences in cardiac size and function at baseline between C4KO and control mice. In control mice, running increased heart weight to tibial length ratio (HW/TL, 6.26±0.25 vs 7.37±0.75, p=0.01, n=5-9 per group), heart weight to body weight ratio (HW/BW, 4.08±0.40 vs 4.93±0.48, p=0.01), and cardiomyocyte (CM) size without affecting fractional shortening (FS), consistent with physiologic remodeling. There was also a nonsignificant trend to increased EdU+ CMs in the control exercised mice (2.96±2.34% vs 5.23±6.28%, p=0.44), suggestive of previously documented exercise-induced cardiomyogenesis. In contrast, running did not change HW/TL and CM size in C4KO mice (HW/TL, 6.44±0.48 vs 6.66±0.29, p=0.22, n= 0-13 per group) but reduced %FS (58.78±3.27 vs 53.67±4.59, p=0.03) and increased chamber size (LVIDs, 1.32±0.13 vs 1.50±0.17, p=0.032), suggesting maladaptive cardiac remodeling with exercise, confirming prior findings observed in a more extreme swim-exercise model. Rather than the expected increase, hearts from exercised C4KO mice had fewer EdU+ CMs compared to sedentary C4KO mice (3.76±2.60% vs 2.11±0.76%, p=0.05) and reduced expression of cell cycle genes Cdkn1b, Cyclin B1, Cdkn2a, and Ki-67. Conclusions: CITED4 is required for exercise-induced cardiomyogenesis, CM hypertrophy, and adaptive remodeling. CITED4 deficiency leads to maladaptive cardiac remodeling with physiologic stress.

Introduction: Peripartum cardiomyopathy (PPCM) is a rare form of pregnancy-related heart failure associated with preeclampsia. Our understanding of their shared pathophysiology is limited as are treatment options. Hypothesis: We hypothesized that plasma proteomic profiling would identify deleterious circulating proteins and provide insights into shared mechanisms driving preeclampsia and PPCM. Methods: We performed serum proteomic case-control studies in women with preeclampsia or PPCM to identify biological processes common to both conditions. The top candidate was validated in independent preeclampsia (n=58) and PPCM (n=114) cohorts. Preeclamptic placenta was examined as a potential source of circulating proteins. Therapeutic targeting the top candidate was examined in an animal model of PPCM. Results: Paradoxically, we found the senescence-associated secretory phenotype (SASP), a marker of biological aging, to be the most upregulated process in relatively young women with preeclampsia (normalized enrichment score [NES]=3.3;p adj =4.0x10 -6 ) or PPCM (NES=3.2;p adj =1.9x10 -5 ). 28 circulating proteins contributed to this common signal, and were strongly enriched in preeclamptic placenta, which expressed markers of increased senescence. The TGFb family member, Activin-A, was the most highly upregulated SASP gene in preeclamptic placentas (5-fold;p Conclusions: These data implicate placental senescence in the increased deleterious circulating proteins seen in preeclampsia and PPCM. We identify multiple new circulating factors associated with these diseases and demonstrate that targeting SASP proteins, such as Activin-A, can mitigate PPCM in mice.

One promising goal for utilizing the molecular information circulating in biofluids is the discovery of clinically useful biomarkers. Extracellular RNAs (exRNAs) are one of the most diverse classes of molecular cargo, easily assayed by sequencing and with expressions that rapidly change in response to subject status. Despite diverse exRNA cargo, most evaluations from biofluids have focused on small RNA sequencing and analysis, specifically on microRNAs (miRNAs). Another goal of characterizing circulating molecular information, is to correlate expression to injuries associated with specific tissues of origin. Biomarker candidates are often described as being specific, enriched in a particular tissue or associated with a disease process. Likewise, miRNA data is often reported to be specific, enriched for a tissue, without rigorous testing to support the claim. Here we provide a tissue atlas of small RNAs from 30 different tissues and three different blood cell types. We analyzed the tissues for enrichment of small RNA sequences and assessed their expression in biofluids: plasma, cerebrospinal fluid, urine, and saliva. We employed published data sets representing physiological (resting vs. acute exercise) and pathologic states (early- vs. late-stage liver fibrosis, and differential subtypes of stroke) to determine differential tissue-enriched small RNAs. We also developed an online tool that provides information about exRNA sequences found in different biofluids and tissues. The data can be used to better understand the various types of small RNA sequences in different tissues as well as their potential release into biofluids, which should help in the validation or design of biomarker studies.

BackgroundPostoperative delirium (POD) is an acute altered mental state commonly encountered after cardiac surgery. The pathophysiological mechanisms underlying POD remain unclear. We aimed to identify circulating proteins significantly altered after major cardiac surgery with cardiopulmonary bypass (CPB). We also aimed to enable inferences on associations with POD.MethodsSerum and whole blood samples were collected before CPB (n = 16 patients; n = 8 with POD) and again from the same patients on postoperative day 1. All patients were clinically evaluated for POD on postoperative days 1–3. An aptamer-based proteomics platform (SOMAscan) was used to quantify serum protein abundance in patients with POD compared with non-POD controls. We also performed a lipopolysaccharide (LPS)-based in vitro functional analysis (TruCulture) on whole blood samples from patients with POD and non-POD controls to approximate surgical stress. Cytokine levels were determined using a Luminex immunoassay.ResultsCardiac surgery with CPB resulted in a significant (padj < 0.01) change in 48.8% (637 out of 1,305) of proteins detected by SOMAscan. Gene set enrichment showed that the most impacted biological processes involved myeloid cell activation. Specifically, activation and degranulation of neutrophils were the top five highest-scoring processes. Pathway analyses with the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that metabolic enzymes, particularly those of glycolysis, were elevated in serum concentration after surgery. Several proteins were significantly increased postoperatively in patients diagnosed with POD relative to the non-POD controls, with interleukin-6 (IL-6) showing the greatest fold-change. LPS stimulation of whole blood samples confirmed these findings. Linear regression analysis showed a highly significant correlation between Confusion Assessment Method (CAM) scores and CPB-mediated changes in cGMP-inhibited 3′,5′-cyclic phosphodiesterase A (PDE3A).ConclusionsCardiac surgery with CPB resulted in inflammasome changes accompanied by unexpected increases in metabolic pathways. In exploratory analyses, we found that POD was associated with changes in the expression level of various proteins, most notably IL-6 and PDE3A. This study and ongoing protein biomarker studies will likely help quantify risk or confirm the diagnosis for POD and increase understanding of its pathophysiological mechanisms.

Summary: Dynamic profiling of changes in gene expression in response to stressors in specific microenvironments without requiring cellular destruction remains challenging. Current methodologies that seek to interrogate gene expression at a molecular level require sampling of cellular transcriptome and therefore lysis of the cell, preventing serial analysis of cellular transcriptome. To address this area of unmet need, we have recently developed a technology allowing transcriptomic analysis over time without cellular destruction. Our method, TRACE-seq (TRanscriptomic Analysis Captured in Extracellular vesicles using sequencing), is characterized by a cell-type specific transgene expression. It provides data on the transcriptome inside extracellular vesicles that provides an accurate representation of stress-responsive cellular transcriptomic changes. Thus, the transcriptome of cells expressing TRACE can be followed over time without destroying the source cell, which is a powerful tool for many fields of fundamental and translational biology research.

© 2019 Elsevier Inc. The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data. The Extracellular RNA Communication Consortium (ERCC) presents progress toward understanding the biology of extracellular RNAs and their use as biomarkers and therapeutics.

More than a year ago (Eos 85(49), 523, 2004), the Eos Forum column featured an article by Vice Admiral Conrad Lautenbacher, administrator for the U.S. National Oceanic and Atmospheric Administration, and Charles Groat, who at that time was Director of the U.S. Geological Survey, concerning the status of the Earth sciences in the public school curriculum. A very small percentage of high school students take an Earth science course, and that number is dropping every day As a retired high school Earth science teacher and science department chair, I often found myself defending our Earth science requirement to some parents and school committee members.