Differences in Cardiometabolic Proteins in Pregnancy Prioritize Relevant Targets of Preeclampsia.

BACKGROUND: Preeclampsia is a hypertensive disorder of pregnancy characterized by widespread vascular inflammation. It occurs frequently in pregnancy, often without known risk factors, and has high rates of maternal and fetal morbidity and mortality. Identification of biomarkers that predict preeclampsia and its cardiovascular sequelae before clinical onset, or even before pregnancy, is a critical unmet need for the prevention of adverse pregnancy outcomes. METHODS: We explored differences in cardiovascular proteomics (Olink Explore 384) in 256 diverse pregnant persons across 2 centers (26% Hispanic, 21% Black). RESULTS: We identified significant differences in plasma abundance of markers associated with angiogenesis, blood pressure, cell adhesion, inflammation, and metabolism between individuals delivering with preeclampsia and controls, some of which have not been widely described previously and are not represented in the preeclampsia placental transcriptome. While we observed a broadly similar pattern in early (<34 weeks) versus late (≥34 weeks) preeclampsia, several proteins related to hemodynamic stress, hemostasis, and immune response appeared to be more highly dysregulated in early preeclampsia relative to late preeclampsia. CONCLUSIONS: These results demonstrate the value of performing targeted proteomics using a panel of cardiovascular biomarkers to identify biomarkers relevant to preeclampsia pathophysiology and highlight the need for larger multiomic studies to define modifiable pathways of surveillance and intervention upstream to preeclampsia diagnosis.

Capillary blood self-collection for high-throughput proteomics.

In this study, we sought to compare protein concentrations obtained from a high-throughput proteomics platform (Olink) on samples collected using capillary blood self-collection (with the Tasso+ device) versus standard venipuncture (control). Blood collection was performed on 20 volunteers, including one sample obtained via venipuncture and two via capillary blood using the Tasso+ device. Tasso+ samples were stored at 2°C-8°C for 24-hs (Tasso-24) or 48-h (Tasso-48) prior to processing to simulate shipping times from a study participant's home. Proteomics were analyzed using Olink (384 Inflammatory Panel). Tasso+ blood collection was successful in 37/40 attempts. Of 230 proteins included in our analysis, Pearson correlations (r) and mean coefficient of variation (CV) between Tasso-24 or Tasso-48 versus venipuncture were variable. In the Tasso-24 analysis, 34 proteins (14.8%) had both a correlation r > 0.5 and CV < 0.20. In the Tasso-48 analysis, 68 proteins (29.6%) had a correlation r > 0.5 and CV < 0.20. Combining the Tasso-24 and Tasso-48 analyses, 26 (11.3%) proteins met these thresholds. We concluded that protein concentrations from Tasso+ samples processed 24-48 h after collection demonstrated wide technical variability and variable correlation with a venipuncture gold-standard. Use of home capillary blood self-collection for large-scale proteomics should be limited to select proteins with good agreement with venipuncture.

Clonal hematopoiesis of indeterminate potential and outcomes after heart transplantation: A multicenter study.

Cardiac allograft vasculopathy (CAV) is a leading cause of late graft failure and mortality after heart transplantation (HT). Sharing some features with atherosclerosis, CAV results in diffuse narrowing of the epicardial coronaries and microvasculature, with consequent graft ischemia. Recently, clonal hematopoiesis of indeterminate potential (CHIP) has emerged as a risk factor for cardiovascular disease and mortality. We aimed to investigate the relationship between CHIP and posttransplant outcomes, including CAV. We analyzed 479 HT recipients with stored DNA samples at 2 high-volume transplant centers, Vanderbilt University Medical Center and Columbia University Irving Medical Center. We explored the association between the presence of CHIP mutations with CAV and mortality after HT. In this case-control analysis, carriers of CHIP mutations were not at increased risk of CAV or mortality after HT. In a large multicenter genomics study of the heart transplant population, the presence of CHIP mutations was not associated with an increased risk of CAV or posttransplant mortality.

SARS-CoV-2 Initiates Programmed Cell Death in Platelets.

[Figure: see text].

Circulating metabolite profile in young adulthood identifies long-term diabetes susceptibility: the Coronary Artery Risk Development in Young Adults (CARDIA) study.

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/m2) 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/m2), 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<0.0001) and FHS (middle-aged and older adults; HR 1.33 [95% CI 1.14, 1.56], p=0.0004). A metabolite score based on the outcome of diabetes was strongly related to diabetes in CARDIA study participants (fully adjusted HR 3.41 [95% CI 2.85, 4.07], p<0.0001) but not in the older FHS population (HR 1.15 [95% CI 0.99, 1.33], p=0.07). CONCLUSIONS/INTERPRETATION: Selected metabolic abnormalities in young adulthood identify individuals with heightened diabetes risk independent of race, sex and traditional diabetes risk factors. These signatures replicate across the life course.

A Call to Action for New Global Approaches to Cardiovascular Disease Drug Solutions.

While we continue to wrestle with the immense challenge of implementing equitable access to established evidence-based treatments, substantial gaps remain in our pharmacotherapy armament for common forms of cardiovascular disease including coronary and peripheral arterial disease, heart failure, hypertension, and arrhythmia. We need to continue to invest in the development of new approaches for the discovery, rigorous assessment, and implementation of new therapies. Currently, the time and cost to progress from lead compound/product identification to the clinic, and the success rate in getting there reduces the incentive for industry to invest, despite the enormous burden of disease and potential size of market. There are tremendous opportunities with improved phenotyping of patients currently batched together in syndromic "buckets." Use of advanced imaging and molecular markers may allow stratification of patients in a manner more aligned to biological mechanisms that can, in turn, be targeted by specific approaches developed using high-throughput molecular technologies. Unbiased "omic" approaches enhance the possibility of discovering completely new mechanisms in such groups. Furthermore, advances in drug discovery platforms, and models to study efficacy and toxicity more relevant to the human disease, are valuable. Re-imagining the relationships among discovery, translation, evaluation, and implementation will help reverse the trend away from investment in the cardiovascular space, establishing innovative platforms and approaches across the full spectrum of therapeutic development.

The Dynamic Platelet Transcriptome in Obesity and Weight Loss.

OBJECTIVE: Adiposity is associated with oxidative stress, inflammation, and glucose intolerance. Previous data suggest that platelet gene expression is associated with key cardiometabolic phenotypes, including body mass index but stable in healthy individuals over time. However, modulation of gene expression in platelets in response to metabolic shifts (eg, weight reduction) is unknown and may be important to defining mechanism. Approach and Results: Platelet RNA sequencing and aggregation were performed from 21 individuals with massive weight loss (>45 kg) following bariatric surgery. Based on RNA sequencing data, we measured the expression of 67 genes from isolated platelet RNA using high-throughput quantitative reverse transcription quantitative PCR in 1864 FHS (Framingham Heart Study) participants. Many transcripts not previously studied in platelets were differentially expressed with bariatric surgical weight loss, appeared specific to platelets (eg, not differentially expressed in leukocytes), and were enriched for a nonalcoholic fatty liver disease pathway. Platelet aggregation studies did not detect alteration in platelet function after significant weight loss. Linear regression models demonstrated several platelet genes modestly associated with cross-sectional cardiometabolic phenotypes, including body mass index. There were no associations between studied transcripts and incident diabetes or cardiovascular end points. CONCLUSIONS: In summary, while there is no change in platelet aggregation function after significant weight loss, the human platelet experiences a dramatic transcriptional shift that implicates pathways potentially relevant to improved cardiometabolic risk postweight loss (eg, nonalcoholic fatty liver disease). Further studies are needed to determine the mechanistic importance of these observations.

A call to action for new global approaches to cardiovascular disease drug solutions.

Whilst we continue to wrestle with the immense challenge of implementing equitable access to established evidence-based treatments, substantial gaps remain in our pharmacotherapy armament for common forms of cardiovascular disease including coronary and peripheral arterial disease, heart failure, hypertension, and arrhythmia. We need to continue to invest in the development of new approaches for the discovery, rigorous assessment, and implementation of new therapies. Currently, the time and cost to progress from lead compound/product identification to the clinic, and the success rate in getting there reduces the incentive for industry to invest, despite the enormous burden of disease and potential size of market. There are tremendous opportunities with improved phenotyping of patients currently batched together in syndromic 'buckets'. Use of advanced imaging and molecular markers may allow stratification of patients in a manner more aligned to biological mechanisms that can, in turn, be targeted by specific approaches developed using high-throughput molecular technologies. Unbiased 'omic' approaches enhance the possibility of discovering completely new mechanisms in such groups. Furthermore, advances in drug discovery platforms, and models to study efficacy and toxicity more relevant to the human disease, are valuable. Re-imagining the relationships among discovery, translation, evaluation, and implementation will help reverse the trend away from investment in the cardiovascular space, establishing innovative platforms and approaches across the full spectrum of therapeutic development.

Comprehensive Metabolic Phenotyping Refines Cardiovascular Risk in Young Adults.

BACKGROUND: Whereas cardiovascular disease (CVD) metrics define risk in individuals >40 years of age, the earliest lesions of CVD appear well before this age. Despite the role of metabolism in CVD antecedents, studies in younger, biracial populations to define precise metabolic risk phenotypes are lacking. METHODS: We studied 2330 White and Black young adults (mean age, 32 years; 45% Black) in the CARDIA study (Coronary Artery Risk Development in Young Adults) to identify metabolite profiles associated with an adverse CVD phenome (myocardial structure/function, fitness, vascular calcification), mechanisms, and outcomes over 2 decades. Statistical learning methods (elastic nets/principal components analysis) and Cox regression generated parsimonious, metabolite-based risk scores validated in >1800 individuals in the Framingham Heart Study. RESULTS: In the CARDIA study, metabolite profiles quantified in early adulthood were associated with subclinical CVD development over 20 years, specifying known and novel pathways of CVD (eg, transcriptional regulation, brain-derived neurotrophic factor, nitric oxide, renin-angiotensin). We found 2 multiparametric, metabolite-based scores linked independently to vascular and myocardial health, with metabolites included in each score specifying microbial metabolism, hepatic steatosis, oxidative stress, nitric oxide modulation, and collagen metabolism. The metabolite-based vascular scores were lower in men, and myocardial scores were lower in Black participants. Over a nearly 25-year median follow-up in CARDIA, the metabolite-based vascular score (hazard ratio, 0.68 per SD [95% CI, 0.50-0.92]; P=0.01) and myocardial score (hazard ratio, 0.60 per SD [95% CI, 0.45-0.80]; P=0.0005) in the third and fourth decades of life were associated with clinical CVD with a synergistic association with outcome (Pinteraction=0.009). We replicated these findings in 1898 individuals in the Framingham Heart Study over 2 decades, with a similar association with outcome (including interaction), reclassification, and discrimination. In the Framingham Heart Study, the metabolite scores exhibited an age interaction (P=0.0004 for a combined myocardial-vascular score with incident CVD), such that young adults with poorer metabolite-based health scores had highest hazard of future CVD. CONCLUSIONS: Metabolic signatures of myocardial and vascular health in young adulthood specify known/novel pathways of metabolic dysfunction relevant to CVD, associated with outcome in 2 independent cohorts. Efforts to include precision measures of metabolic health in risk stratification to interrupt CVD at its earliest stage are warranted.

A Translational Model for Venous Thromboembolism: MicroRNA Expression in Hibernating Black Bears.

BACKGROUND: Hibernating American black bears have significantly different clotting parameters than their summer active counterparts, affording them protection against venous thromboembolism during prolonged periods of immobility. We sought to evaluate if significant differences exist between the expression of microRNAs in the plasma of hibernating black bears compared with their summer active counterparts, potentially contributing to differences in hemostasis during hibernation. MATERIALS AND METHODS: MicroRNA sequencing was assessed in plasma from 21 American black bears in summer active (n = 11) and hibernating states (n = 10), and microRNA signatures during hibernating and active state were established using both bear and human genome. MicroRNA targets were predicted using messenger RNA (mRNA) transcripts from black bear kidney cells. In vitro studies were performed to confirm the relationship between identified microRNAs and mRNA expression, using artificial microRNA and human liver cells. RESULTS: Using the bear genome, we identified 15 microRNAs differentially expressed in the plasma of hibernating black bears. Of these microRNAs, three were significantly downregulated (miR-141-3p, miR-200a-3p, and miR-200c-3p), were predicted to target SERPINC1, the gene for antithrombin, and demonstrated regulatory control of the gene mRNA expression in cell studies. CONCLUSIONS: Our findings suggest that the hibernating black bears' ability to maintain hemostasis and achieve protection from venous thromboembolism during prolonged periods of immobility may be due to changes in microRNA signatures and possible upregulation of antithrombin expression.

Dynamic Role of trans Regulation of Gene Expression in Relation to Complex Traits.

Identifying causal genetic variants and understanding their mechanisms of effect on traits remains a challenge in genome-wide association studies (GWASs). In particular, how genetic variants (i.e., trans-eQTLs) affect expression of remote genes (i.e., trans-eGenes) remains unknown. We hypothesized that some trans-eQTLs regulate expression of distant genes by altering the expression of nearby genes (cis-eGenes). Using published GWAS datasets with 39,165 single-nucleotide polymorphisms (SNPs) associated with 1,960 traits, we explored whole blood gene expression associations of trait-associated SNPs in 5,257 individuals from the Framingham Heart Study. We identified 2,350 trans-eQTLs (at p < 10-7); more than 80% of them were found to have cis-associated eGenes. Mediation testing suggested that for 35% of trans-eQTL-trans-eGene pairs in different chromosomes and 90% pairs in the same chromosome, the disease-associated SNP may alter expression of the trans-eGene via cis-eGene expression. In addition, we identified 13 trans-eQTL hotspots, affecting from ten to hundreds of genes, suggesting the existence of master genetic regulators. Using causal inference testing, we searched causal variants across eight cardiometabolic traits (BMI, systolic and diastolic blood pressure, LDL cholesterol, HDL cholesterol, total cholesterol, triglycerides, and fasting blood glucose) and identified several cis-eGenes (ALDH2 for systolic and diastolic blood pressure, MCM6 and DARS for total cholesterol, and TRIB1 for triglycerides) that were causal mediators for the corresponding traits, as well as examples of trans-mediators (TAGAP for LDL cholesterol). The finding of extensive evidence of genome-wide mediation effects suggests a critical role of cryptic gene regulation underlying many disease traits.

Circulating Platelets as Mediators of Immunity, Inflammation, and Thrombosis.

Platelets, non-nucleated blood components first described over 130 years ago, are recognized as the primary cell regulating hemostasis and thrombosis. The vascular importance of platelets has been attributed to their essential role in thrombosis, mediating myocardial infarction, stroke, and venous thromboembolism. Increasing knowledge on the platelets' role in the vasculature has led to many advances in understanding not only how platelets interact with the vessel wall but also how they convey changes in the environment to other circulating cells. In addition to their well-described hemostatic function, platelets are active participants in the immune response to microbial organisms and foreign substances. Although incompletely understood, the immune role of platelets is a delicate balance between its pathogenic response and its regulation of thrombotic and hemostatic functions. Platelets mediate complex vascular homeostasis via specific receptors and granule release, RNA transfer, and mitochondrial secretion that subsequently regulates hemostasis and thrombosis, infection, and innate and adaptive immunity.

Proteins Altered by Surgical Weight Loss Highlight Biomarkers of Insulin Resistance in the Community.

Objective- Mechanisms of early and late improvements in cardiovascular risk after bariatric surgery and applicability to larger, at-risk populations remain unclear. We aimed to identify proteins altered after bariatric surgery and their relations to metabolic syndrome and diabetes mellitus. Approach and Results- We identified 19 proteins altered in 32 nonfasting plasma samples from a study of patients undergoing bariatric surgery who were evaluated preoperatively (visit 1) versus both early (visit 2; ≈3 months) and late (visit 3; ≈12 months) postoperative follow-up using predefined protein panels (Olink). Using in silico methods and publicly available gene expression repositories, we found that genes encoding 8 out of 19 proteins had highest expression in liver relative to other assayed tissues, with the top biological and disease processes, including major obesity-related vascular diseases. Of 19 candidate proteins in the surgical cohort, 6 were previously measured in >3000 FHS (Framingham Heart Study) participants (IGFBP [insulin-like growth factor binding protein]-1, IGFBP-2, P-selectin, CD163, LDL (low-density lipoprotein)-receptor, and PAI [plasminogen activator inhibitor]-1). A higher concentration of IGFBP-2 at baseline was associated with a lower risk of incident metabolic syndrome (odds ratio per log-normal unit, 0.45; 95% CI, 0.32-0.64; P=7.7×10-6) and diabetes mellitus (odds ratio, 0.63; 95% CI, 0.49-0.79; P=0.0001) after multivariable adjustment. Conclusions- Using a directed protein quantification platform (Olink), we identified known and novel proteins altered after surgical weight loss, including IGFBP-2. Future efforts in well-defined obesity intervention settings may further define and validate novel targets for the prevention of vascular disease in obesity.

Circulating microRNAs miR-331 and miR-195 differentiate local luminal a from metastatic breast cancer.

BACKGROUND: Breast cancer is the leading cause of cancer related death in women, with metastasis the principle cause of mortality. New non-invasive prognostic markers are needed for the early detection of metastasis, facilitating treatment decision optimisation. MicroRNA (miRNA) are small, non-coding RNAs regulating gene expression and involved in many cellular processes, including metastasis. As biomarkers, circulating miRNAs (in blood) hold great promise for informing diagnosis or monitoring treatment responses. METHODS: Plasma extracted RNA from age matched local Luminal A (n = 4) or metastatic disease (n = 4) were profiled using Next Generation Sequencing. Selected differentially expressed miRNA were validated on a whole blood extracted miRNA cohort [distant metastatic disease (n = 22), local disease (n = 31), healthy controls (n = 21)]. Area Under the Curve (AUC) in Receiver Operating Characteristic (ROC) analyses was performed. RESULTS: Of 4 miRNA targets tested (miR-181a, miR-329, miR-331, miR-195), mir-331 was significantly over-expressed in patients with metastatic disease, compared to patients with local disease (p < 0.001) or healthy controls (p < 0.001). miR-195 was significantly under-expressed in patients with metastatic disease, compared to patients with local disease (p < 0.001) or healthy controls (p = 0.043). In combination, miR-331 and miR-195 produced an AUC of 0.902, distinguishing metastatic from local breast cancer. CONCLUSIONS: We identified and validated two circulating miRNAs differentiating local Luminal A breast cancers from metastatic breast cancers. Further investigation will reveal the molecular role of these miRNAs in metastasis, and determine if they are subtype specific. This work demonstrates the ability of circulating miRNA to identify metastatic disease, and potentially inform diagnosis or treatment effectiveness.

Peer Review Practices for Evaluating Biomedical Research Grants: A Scientific Statement From the American Heart Association.

The biomedical research enterprise depends on the fair and objective peer review of research grants, leading to the distribution of resources through efficient and robust competitive methods. In the United States, federal funding agencies and foundations collectively distribute billions of dollars annually to support biomedical research. For the American Heart Association, a Peer Review Subcommittee is charged with establishing the highest standards for peer review. This scientific statement reviews the current literature on peer review practices, describes the current American Heart Association peer review process and those of other agencies, analyzes the strengths and weaknesses of American Heart Association peer review practices, and recommends best practices for the future.

Challenges and Opportunities in Linking Long Noncoding RNAs to Cardiovascular, Lung, and Blood Diseases.

The new millennium heralds an unanticipated surge of genomic information, most notably an expansive class of long noncoding RNAs (lncRNAs). These transcripts, which now outnumber all protein-coding genes, often exhibit the same characteristics as mRNAs (RNA polymerase II-dependent, 5' methyl-capped, multiexonic, polyadenylated); yet, they do not encode for stable, well-conserved proteins. Elucidating the function of all relevant lncRNAs in heart, vasculature, lung, and blood is essential for generating a complete interactome in these tissues. This is particularly evident because an increasing number of investigators perform RNA-sequencing experiments where, typically, annotated lncRNAs exhibit impressive changes in gene expression. How does one go about evaluating an lncRNA when the sequence of the transcript lends no insight into how it may function within a cell type? Here, we provide a brief overview for the rational study of lncRNAs.

Thrombosis and platelets: an update.

Haemostasis and thrombosis are complex, multifactorial processes. There is an evolving understanding of the mechanisms influencing vascular occlusion and the role of inflammation and immunity. Despite major advances in elucidating the mechanistic pathways mediating platelet function and thrombosis, challenges in the treatment of vascular occlusive diseases persist. Pharmacological advances have greatly affected thrombotic outcomes, but this has led to the unwanted side effect of bleeding. Detailed assessment of the impact of non-thrombotic diseases on haemostasis and thrombosis is necessary to better evaluate thrombotic risk and establish optimal treatment. This review will focus on recent advances in understanding the contribution of evolving risk factors to thrombosis.

Stroke and Circulating Extracellular RNAs.

BACKGROUND AND PURPOSE: There is increasing interest in extracellular RNAs (ex-RNAs), with numerous reports of associations between selected microRNAs (miRNAs) and a variety of cardiovascular disease phenotypes. Previous studies of ex-RNAs in relation to risk for cardiovascular disease have investigated small numbers of patients and assayed only candidate miRNAs. No human studies have investigated links between novel ex-RNAs and stroke. METHODS: We conducted unbiased next-generation sequencing using plasma from 40 participants of the FHS (Framingham Heart Study; Offspring Cohort Exam 8) followed by high-throughput polymerase chain reaction of 471 ex-RNAs. The reverse transcription quantitative polymerase chain reaction included 331 of the most abundant miRNAs, 43 small nucleolar RNAs, and 97 piwi-interacting RNAs in 2763 additional FHS participants and explored the relations of ex-RNAs and prevalent (n=63) and incident (n=51) stroke and coronary heart disease (prevalent=286, incident=69). RESULTS: After adjustment for multiple cardiovascular disease risk factors, 7 ex-RNAs were associated with stroke prevalence or incidence; there were no ex-RNA associated with prevalent or incident coronary heart disease. Statistically significant ex-RNA associations with stroke were specific, with no overlap between prevalent and incident events. CONCLUSIONS: This is the largest study of ex-RNAs in relation to stroke using an unbiased approach in an observational cohort and the first large study to examine human small noncoding RNAs beyond miRNAs. These results demonstrate that when studied in a large observational cohort, extracellular miRNAs are associated with stroke risk.