Comprehensive Phenotyping Research Articles

Comprehensive phenotyping of endothelial cells using flow cytometry 2: Human.

In vascular research, clinical samples and samples from animal models are often used together to foster translation of preclinical findings to humans. General concepts of endothelia and murine-specific endothelial phenotypes were discussed in part 1 of this two part series. Here, in part 2, we present a comprehensive overview of human-specific endothelial phenotypes. Pan-endothelial cell markers, organ specific endothelial antigens, and flow cytometric immunophenotyping of blood-borne endothelial cells are reviewed.

Comprehensive Phenotyping of Human Dendritic Cells and Monocytes.

Professional antigen-presenting cells (APCs), which include dendritic cells (DCs) and monocytes are essential for inducing and steering adaptive T-cell responses. Recent technological developments in single-cell analysis have significantly advanced our understanding of APC subset heterogeneity. To accurately resolve this functional diversity and to account for tissue-specific adaptation, novel phenotyping markers have been described more recently. While some of these largely overlap with traditionally used markers, more fine-grained phenotyping might be essential during inflammatory settings, where the traditional distinction between monocytes and dendritic cells has become blurred. Within this phenotype report, we provide a concise overview of traditional and recently described markers for the phenotyping of DCs and monocytes in the human system.

Microglia facilitate repair of demyelinated lesions via post-squalene sterol synthesis.

The repair of inflamed, demyelinated lesions as in multiple sclerosis necessitates the clearance of cholesterol-rich myelin debris by microglia/macrophages and the switch from a pro-inflammatory to an anti-inflammatory lesion environment. Subsequently, oligodendrocytes increase cholesterol levels as a prerequisite for synthesizing new myelin membranes. We hypothesized that lesion resolution is regulated by the fate of cholesterol from damaged myelin combined with oligodendroglial sterol synthesis. By integrating expression profiling, genetics, and comprehensive phenotyping, we found that paradoxically sterol synthesis in myelin-phagocytosing microglia/macrophages determines repair of acutely demyelinated lesions. Rather than producing cholesterol, microglia/macrophages synthesized desmosterol, the immediate cholesterol precursor. Desmosterol activated LXR-signaling to resolve inflammation, creating a permissive environment for oligodendrocyte differentiation. Moreover, LXR-target gene products facilitated the efflux of lipid/cholesterol from lipid-laden microglia/macrophages to support remyelination by oligodendrocytes. Consequently, pharmacological stimulation of sterol synthesis boosted repair of demyelinated lesions, suggesting novel therapeutic strategies for myelin repair in multiple sclerosis.

Comprehensive phenotyping of endothelial cells using flow cytometry 1: Murine.

The endothelium forms a selective barrier between circulating blood or lymph and surrounding tissue. Endothelial cells play an essential role in vessel homeostasis, and identification of these cells is critical in vascular biology research. However, characteristics of endothelial cells differ depending on the location and type of blood or lymph vessel. Endothelial cell subsets are numerous and often identified using different flow cytometric markers, making immunophenotyping these cells complex. In part 1 of this two part review series, we present a comprehensive overview of markers for the flow cytometric identification and phenotyping of murine endothelial subsets. These subsets can be distinguished using a panel of cell surface and intracellular markers shared by all endothelial cells in combination with additional markers of specialized endothelial cell types. This review can be used to determine the best markers for identifying and phenotyping desired murine endothelial cell subsets.

Platelet electrical resistance for measuring platelet activation and adhesion in human health and disease

BackgroundThe ability to measure changes in platelet reactivity is important to identify novel aspects of platelet biology and develop targeted therapeutics to prevent bleeding or thrombosis. Current platelet function testing allows for single agonist analysis at a time. The ability to phenotype platelets in a single assay with multiple agonists and adhesion substrates could yield more insights into altered pathways than are feasible with current approaches. We hypothesized platelet electrical resistance (PER) could be used for more comprehensive phenotyping of platelets. MethodsPlatelets were isolated from male and female healthy donors (age 39.6 ± 6.9) and septic patients (age 44.0 ± 13.5). PER 96-well plates were coated with various substrates, including fibrinogen and collagen. Platelets were added to the coated plates in the presence or absence of thrombin or convulxin. Platelet activation and spreading was monitored by measuring changes in electrical impedance. ResultsPlatelets adhesion to fibrinogen and collagen increased impedance. In addition, impedance increased in response to thrombin or convulxin. No changes in impedance were observed in the absence of platelets or when wells were uncoated, indicating changes in impedance were directly due to platelet adhesion and activation. Inhibiting integrin αIIbβ3 decreased impedance when fibrinogen was used as a substrate, consistent with platelet-dependent effects. Platelets from septic patients caused increased impedance compared to healthy donors, demonstrating this assay can be used to assess platelet hyperreactivity. ConclusionPER can be applied as a high throughput tool to measure platelet reactivity in health and disease, where platelet activation is increased.

Extracellular Volume in Primary Mitral Regurgitation

ObjectivesThis study used cardiovascular magnetic resonance (CMR) to evaluate whether elevated extracellular volume (ECV) was associated with mitral valve prolapse (MVP) or if elevated ECV was a consequence of remodeling independent of primary mitral regurgitation (MR) etiology. BackgroundReplacement fibrosis in primary MR is more prevalent in MVP; however, data on ECV as a surrogate for diffuse interstitial fibrosis in primary MR are limited. MethodsPatients with chronic primary MR underwent comprehensive CMR phenotyping and were stratified into an MVP cohort (>2 mm leaflet displacement on a 3-chamber cine CMR) and a non-MVP cohort. Factors associated with ECV and replacement fibrosis were assessed. The association of ECV and symptoms related to MR and clinical events (mitral surgery and cardiovascular death) was ascertained. ResultsA total of 424 patients with primary MR (229 with MVP and 195 non-MVP) were enrolled. Replacement fibrosis was more prevalent in the MVP cohort (34.1% vs. 6.7%; p < 0.001), with bi-leaflet MVP having the strongest association with replacement fibrosis (odds ratio: 10.5; p < 0.001). ECV increased with MR severity in a similar fashion for both MVP and non-MVP cohorts and was associated with MR severity but not MVP on multivariable analysis. Elevated ECV was independently associated with symptoms related to MR and clinical events. ConclusionsAlthough replacement fibrosis was more prevalent in MVP, diffuse interstitial fibrosis as inferred by ECV was associated with MR severity, regardless of primary MR etiology. ECV was independently associated with symptoms related to MR and clinical events. (DeBakey Cardiovascular Magnetic Resonance Study [DEBAKEY-CMR]; NCT04281823)

Impaired muscle mitochondrial energetics is associated with uremic metabolite accumulation in chronic kidney disease.

Chronic kidney disease (CKD) causes progressive skeletal myopathy involving atrophy, weakness, and fatigue. Mitochondria have been thought to contribute to skeletal myopathy; however, the molecular mechanisms underlying muscle metabolism changes in CKD are unknown. We employed a comprehensive mitochondrial phenotyping platform to elucidate the mechanisms of skeletal muscle mitochondrial impairment in mice with adenine-induced CKD. CKD mice displayed significant reductions in mitochondrial oxidative phosphorylation (OXPHOS), which was strongly correlated with glomerular filtration rate, suggesting a link between kidney function and muscle mitochondrial health. Biochemical assays uncovered that OXPHOS dysfunction was driven by reduced activity of matrix dehydrogenases. Untargeted metabolomics analyses in skeletal muscle revealed a distinct metabolite profile in CKD muscle including accumulation of uremic toxins that strongly associated with the degree of mitochondrial impairment. Additional muscle phenotyping found CKD mice experienced muscle atrophy and increased muscle protein degradation, but only male CKD mice had lower maximal contractile force. CKD mice had morphological changes indicative of destabilization in the neuromuscular junction. This study provides the first comprehensive evaluation of mitochondrial health in murine CKD muscle to our knowledge and uncovers several unknown uremic metabolites that strongly associate with the degree of mitochondrial impairment.

Membrane-bound sn-1,2-diacylglycerols explain the dissociation of hepatic insulin resistance from hepatic steatosis in MTTP knockout mice

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp−/−) mice and age-weight matched wild-type control mice. Young (10–12-week-old) L-Mttp−/− mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp−/− mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp−/− mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp−/− mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp−/− mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp−/− mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp−/− mice.

Urinary metabolic phenotyping for Alzheimer\u2019s disease

Finding early disease markers using non-invasive and widely available methods is essential to develop a successful therapy for Alzheimer’s Disease. Few studies to date have examined urine, the most readily available biofluid. Here we report the largest study to date using comprehensive metabolic phenotyping platforms (NMR spectroscopy and UHPLC-MS) to probe the urinary metabolome in-depth in people with Alzheimer’s Disease and Mild Cognitive Impairment. Feature reduction was performed using metabolomic Quantitative Trait Loci, resulting in the list of metabolites associated with the genetic variants. This approach helps accuracy in identification of disease states and provides a route to a plausible mechanistic link to pathological processes. Using these mQTLs we built a Random Forests model, which not only correctly discriminates between people with Alzheimer’s Disease and age-matched controls, but also between individuals with Mild Cognitive Impairment who were later diagnosed with Alzheimer’s Disease and those who were not. Further annotation of top-ranking metabolic features nominated by the trained model revealed the involvement of cholesterol-derived metabolites and small-molecules that were linked to Alzheimer’s pathology in previous studies.

Comprehensive phenotyping revealed transient startle response reduction and histopathological gadolinium localization to perineuronal nets after gadodiamide administration in rats

Gadolinium based contrast agents (GBCAs) are widely used in clinical MRI since the mid-1980s. Recently, concerns have been raised that trace amounts of Gadolinium (Gd), detected in brains even long time after GBCA application, may cause yet unrecognized clinical consequences. We therefore assessed the behavioral phenotype, neuro-histopathology, and Gd localization after repeated administration of linear (gadodiamide) or macrocyclic (gadobutrol) GBCA in rats. While most behavioral tests revealed no difference between treatment groups, we observed a transient and reversible decrease of the startle reflex after gadodiamide application. Residual Gd in the lateral cerebellar nucleus was neither associated with a general gene expression pathway deregulation nor with neuronal cell loss, but in gadodiamide-treated rats Gd was associated with the perineuronal net protein aggrecan and segregated to high molecular weight fractions. Our behavioral finding together with Gd distribution and speciation support a substance class difference for Gd presence in the brain after GBCA application.

Abstract 13812: Pulmonary Function Predicts Cardiac Function, Structure &amp; Clinical Outcome in Chronic Heart Failure: Results From the Myovasc Study

Introduction: A growing body of evidence suggests a relevance of pulmonary function in the pathophysiology of heart failure (HF). Comprehensive data on the role of forced expiratory volume in 1 second (FEV1) in the development &amp; progression of HF is limited. Hypothesis: Given this background, we hypothesized, that (FEV1) is associated with deteriorated left ventricular (LV) functional as well as structural status, &amp; predicts specific clinical outcome in HF. Methods: Data from the MyoVasc Study (n=3,289) were analysed. Comprehensive clinical phenotyping including body plethysmography for the evaluation of FEV1 &amp; standardized echocardiography were performed during a five-hour investigation in a dedicated study center. Worsening of HF (comprising transition from asymptomatic to symptomatic HF, HF hospitalization, &amp; cardiac death) was assessed during a structured follow-up with subsequent validation &amp; adjudication of endpoints. Results: Information on FEV1 was available in 2,998 individuals (age 35-84 years; 34.1% female, mean FEV1 92.4[% predicted]). In multivariable regression analysis adjusted for age, sex, height, cardiovascular risk factors (CVRF) &amp; clinical profile, FEV1 per-1SD was an independent predictor of reduced LV ejection fraction (β:-1.6[95%CI -2.0;-1.3]), elevated E/E’-ratio (β:0.82[0.64;0.99]), &amp; increased LV mass/Height 2.7 (β:1.6[1.1;2.1]) (for all, P&lt;0.001). During a median follow-up of 2.3 (interquartile range 1.1; 4.0) years, FEV1 per-1SD predicted worsening of HF (hazard ratio(HR) 1.46[1.30;1.65], P&lt;0.001) independent of age, sex, height, CVRF &amp; comorbidities. This finding was consistent among the single components of the primary study endpoint: FEV1 per-1SD predicted an increased risk for transition to symptomatic HF (HR 1.57[1.19;2.06]), HF hospitalization (HR 1.35[1.16;1.57]) &amp; for cardiac death (HR 1.84[1.49;2.26]) (for all, P&lt;0.001). FEV1 per-1SD remained an independent predictor of worsening of HF after additional adjustment for NT-proBNP (HR 1.25[1.08;1.45], P=0.002). Conclusion: Impaired FEV1 is directly linked to the pathophysiology of HF &amp; represents a strong &amp; independent predictor of HF outcome highlighting its potential value to advance risk prediction in HF.

Imaging Protocol, Feasibility, and Reproducibility of Cardiovascular Phenotyping in a Large Tri-Ethnic Population-Based Study of Older People: The Southall and Brent Revisited (SABRE) Study.

Background: People of South Asian and African Caribbean ethnicities living in UK have a high risk of cardiometabolic disease. Limited data exist regarding detailed cardiometabolic phenotyping in this population. Methods enabling this are widely available, but the practical aspects of undertaking such studies in large and diverse samples are seldom reported.Methods: The Southall and Brent Revisited (SABRE) study is the UK's largest tri-ethnic longitudinal cohort. Over 1,400 surviving participants (58–85 years) attended the 2nd study visit (2008–2011); during which, comprehensive cardiovascular phenotyping, including 3D-echocardiography [3D-speckle-tracking (3D-STE)], computed tomography, coronary artery calcium scoring, pulse wave velocity, central blood pressure, carotid artery ultrasound, and retinal imaging, were performed. We describe the methods used with the aim of providing a guide to their feasibility and reproducibility in a large tri-ethnic population-based study of older people.Results: Conventional echocardiography and all vascular measurements showed high feasibility (>90% analyzable of clinic attendees), but 3D-echocardiography (3DE) and 3D-STE were less feasible (71% 3DE acquisition feasibility and 38% 3D-STE feasibility of clinic attendees). 3D-STE feasibility differed by ethnicity, being lowest in South Asian participants and highest in African Caribbean participants (p < 0.0001). Similar trends were observed in men (P < 0.0001) and women (P = 0.005); however, in South Asians, there were more women with unreadable 3D-images compared to men (67 vs. 58%). Intra- and inter-observer variabilities were excellent for most of conventional and advanced echocardiographic measures. The test-retest reproducibility was good-excellent and fair-good for conventional and advanced echocardiographic measures, respectively, but lower than when re-reading the same images. All vascular measures demonstrated excellent or fair-good reproducibility.Conclusions: We describe the feasibility and reproducibility of detailed cardiovascular phenotyping in an ethnically diverse population. The data collected will lead to a better understanding of why people of South Asian and African Caribbean ancestry are at elevated risk of cardiometabolic diseases.

Comprehensive Phenotyping of Dendritic Cells in Cancer Patients by Flow Cytometry.

Dendritic cells (DCs) play a crucial role in the complex interplay between tumor cells and the immune system. During the elimination phase of cancer immunoediting, immunostimulatory DCs are critical for the control of tumor growth. During the escape phase, regulatory DCs sustain tumor tolerance and contribute to the development of the immunosuppressive tumor microenvironment that characterizes this phase. Moreover, increasing evidence indicates that DCs are also critical for the success of cancer immunotherapy. Hence, there is increasing need to fully characterize DC subsets and their activatory/inhibitory profile in cancer patients. In this review, we describe the role played by different DC subsets in the different phases of cancer immunoediting, the function exerted by different activatory and inhibitory molecules expressed on DC surface, and the cytokines produced by distinct DC subsets, in order to provide an overview on the DC features that may be useful to be assessed when dealing with the flow cytometric characterization of DCs in cancer patients. © 2020 International Society for Advancement of Cytometry.

Comprehensive Phenotyping of Peripheral Blood T Lymphocytes in Healthy Mice.

T lymphocytes play a central role in antigen-specific immune responses. They modulate the function of different immune cells both through a direct contact (receptor binding) and through the secretion of cytokines. At the same time, they are deeply involved in the direct killing of aberrant target cells. T lymphocytes derive from a bone marrow precursor that migrates in the thymus where the main differentiation steps take place. Mature CD4 and CD8 single-positive cells, then, leave the thymus to reach the secondary lymphoid organs. T-cell subsets and their maturation steps can be identified mainly based on the expression of extracellular markers, intracellular transcription factors and cytokine production profiles. In this review, we report, from a cytometric point of view, an overview of the most important T-cell subpopulations and their differentiation state. © 2020 International Society for Advancement of Cytometry.

Global longitudinal strain predicts outcome in chronic heart failure across American Heart Association stages: results from the MyoVasc study

Abstract Background Global longitudinal strain (GLS) demonstrated a superior prognostic value over left ventricular ejection fraction (LVEF) in acute heart failure (HF). Its prognostic value across American Heart Association (AHA) stages of HF – especially under considering of conventional echocardiographic measures of systolic and diastolic function – has not yet been comprehensively evaluated. Purpose To evaluate the prognostic value of GLS for HF-specific outcome across AHA HF stages A to D. Methods Data from the MyoVasc-Study (n=3,289) were analysed. Comprehensive clinical phenotyping was performed during a five-hour investigation in a dedicated study centre. GLS was measured offline utilizing QLab 9.0.1 (PHILIPS, Germany) in participants presenting with sinus rhythm during echocardiography. Worsening of HF (comprising transition from asymptomatic to symptomatic HF, HF hospitalization, and cardiac death) was assessed during a structured follow-up with subsequent validation and adjudication of endpoints. AHA stages were defined according to current guidelines. Results Complete information on GLS was available in 2,400 participants of whom 2,186 categorized to AHA stage A to D were available for analysis. Overall, 434 individuals were classified as AHA stage A, 629 as stage B and 1,123 as stage C/D. Mean GLS increased across AHA stages of HF: it was lowest in stage A (−19.44±3.15%), −18.01±3.46% in stage B and highest in AHA stage C/D (−15.52±4.64%, P for trend &amp;lt;0.0001). During a follow-up period of 3.0 [1.3/4.0] years, GLS denoted an increased risk for worsening of HF after adjustment for age and sex (hazard ratio, HRGLS [per standard deviation (SD)] 1.97 [95% confidence interval 1.73/2.23], P&amp;lt;0.0001) in multivariable Cox regression analysis. After additional adjustment for cardiovascular risk factors, clinical profile, LVEF and E/E' ratio, GLS was the strongest echocardiographic predictor of worsening of HF (HRGLS [per SD] 1.47 [1.20/1.80], P=0.0002) in comparison to LVEF (HRLVEF [per SD] 1.23 [1.02/1.48], P=0.031) and E/E' ratio (HRE/E' [per SD] 1.12 [0.99/1.26], P=0.083). Interestingly, when stratifying for AHA stages, GLS denoted a similar increased risk for worsening of HF in individuals classified as AHA stage A/B (HRGLS [per SD] 1.63 [1.02/2.61], P=0.039) and in those classified as AHA stage C/D (HRGLS [per SD] 1.95 [1.65/2.29], P&amp;lt;0.0001) after adjustment for age and sex. For further evaluation, Cox regression models with interaction analysis indicated no significant interaction for (i) AHA stage A/B vs C/D (P=0.83) and (ii) NYHA functional class &amp;lt;II vs ≥II in individuals classified as AHA stage C/D (P=0.12). Conclusions GLS demonstrated a higher predictive value for worsening of HF than conventional echocardiographic measures of systolic and diastolic function. Interestingly, GLS indicated an increased risk for worsening of HF across AHA stages highlighting its potential value to advance risk prediction in chronic HF. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Center for Cardiovascular Research (DZHK), Center for Translational Vascular Biology (CTVB) of the University Medical Center of the Johannes Gutenberg-University Mainz

Using biomarkers to identify diabetic patients with multiple silent cardiac abnormalities

Abstract Background Primary prevention of cardiovascular events in people with Type 2 diabetes mellitus (T2DM) needs to improve due to risk of cardiovascular events. A major problem in T2DM is the high incidence of silent yet potentially lethal cardiac abnormalities, namely myocardial ischaemia (MI), left ventricular hypertrophy (LVH), left ventricular systolic dysfunction (LVSD), left ventricular diastolic dysfunction (LVDD), and left atrial enlargement (LAE). All these independently predict cardiovascular events and mortality. There is not a single study with comprehensive enough cardiac phenotyping to document the prevalence of all the aforementioned 5 cardiac abnormalities. To improve primary prevention of cardiovascular disease in diabetes, we need to first identify the type of silent cardiac abnormality and treat accordingly. However cardiac phenotyping in all people with T2DM would be prohibitively expensive. Purpose Our study examines the prevalence of all 5 cardiac abnormalities, and the accuracy of biomarkers in identifying them in a cohort of patients with well-controlled T2DM and blood pressure (BP) with no known cardiovascular symptom or disease. Methods This is a cross-sectional study of randomly selected patients with T2DM with no known cardiovascular symptom or disease, clinic BP or average 24-hour BP ≤140/80mmHg, and HbA1c ≤64mmol/mol. Patients with renal impairment, atrial fibrillation, moderate to severe valvular heart disease were excluded. All participants underwent transthoracic echocardiogram, electrocardiogram and dobutamine stress echocardiogram (DSE). Those who did not tolerate DSE or whose DSE was inconclusive, a myocardial perfusion scan or computed tomography coronary angiogram was done. Biomarkers such as BNP, NT-proBNP, high-sensitivity cardiac Troponin I (hs-cTnI), and high sensitivity cardiac Troponin T (hs-cTnT) were measured. Results Of 246 participants (mean age 66 years, 63% male), 141 (57.3%) had silent cardiac abnormalities. 90 (36.6%) had 1 cardiac abnormality, 44 (17.9%) had 2 cardiac abnormalities and 7 (2.8%) had 3 cardiac abnormalities. The most prevalent abnormality was LAE, n=106 (43.1%); followed by LVH, n=71 (28.9%); LVDD, n=13 (5.3%); MI, n=8 (3.3%); LVSD, n=1 (0.4%). Both NT-proBNP and hs-cTnI performed best in detecting silent cardiac abnormalities with p-values of 0.02 and 0.0004, and AUC 0.66 and 0.68 respectively. Increasing NT-proBNP (p=0.002) and hs-cTnI (p=0.002) levels correlated to increasing number of concomitant cardiac abnormalities. Our key new finding is that biomarkers identify those with multiple silent cardiac abnormalities. Conclusion BNP and high-sensitivity cardiac Troponin appear to identify those with multiple silent cardiac abnormalities which may make them useful screening tests so that cardiac investigations are focused on this high-risk subset, with a view to intensifying potential therapies on this subset to reduce the cardiotoxic effect of diabetes. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Chief Scientist Office

Comprehensive Metabolic Phenotyping Refines Cardiovascular Risk in Young Adults.

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. 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. 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. 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.

Age-induced mitochondrial DNA point mutations are inadequate to alter metabolic homeostasis in response to nutrient challenge.

Mitochondrial dysfunction is frequently associated with impairment in metabolic homeostasis and insulin action, and is thought to underlie cellular aging. However, it is unclear whether mitochondrial dysfunction is a cause or consequence of insulin resistance in humans. To determine the impact of intrinsic mitochondrial dysfunction on metabolism and insulin action, we performed comprehensive metabolic phenotyping of the polymerase gamma (PolG) D257A “mutator” mouse, a model known to accumulate supraphysiological mitochondrial DNA (mtDNA) point mutations. We utilized the heterozygous PolG mutator mouse (PolG+/mut) because it accumulates mtDNA point mutations ~ 500‐fold > wild‐type mice (WT), but fails to develop an overt progeria phenotype, unlike PolGmut/mut animals. To determine whether mtDNA point mutations induce metabolic dysfunction, we examined male PolG+/mut mice at 6 and 12 months of age during normal chow feeding, after 24‐hr starvation, and following high‐fat diet (HFD) feeding. No marked differences were observed in glucose homeostasis, adiposity, protein/gene markers of metabolism, or oxygen consumption in muscle between WT and PolG+/mut mice during any of the conditions or ages studied. However, proteomic analyses performed on isolated mitochondria from 12‐month‐old PolG+/mut mouse muscle revealed alterations in the expression of mitochondrial ribosomal proteins, electron transport chain components, and oxidative stress‐related factors compared with WT. These findings suggest that mtDNA point mutations at levels observed in mammalian aging are insufficient to disrupt metabolic homeostasis and insulin action in male mice.

Zika Virus Infection Results in Biochemical Changes Associated With RNA Editing, Inflammatory and Antiviral Responses in Aedes albopictus.

Rapid and significant range expansion of both the Zika virus (ZIKV) and its Aedes vector species has resulted in the declaration of ZIKV as a global health threat. Successful transmission of ZIKV by its vector requires a complex series of interactions between these entities including the establishment, replication and dissemination of the virus within the mosquito. The metabolic conditions within the mosquito tissues play a critical role in mediating the crucial processes of viral infection and replication and represent targets for prevention of virus transmission. In this study, we carried out a comprehensive metabolomic phenotyping of ZIKV infected and uninfected Ae. albopictus by untargeted analysis of primary metabolites, lipids and biogenic amines. We performed a comparative metabolomic study of infection state with the aim of understanding the biochemical changes resulting from the interaction between the ZIKV and its vector. We have demonstrated that ZIKV infection results in changes to the cellular metabolic environment including a significant enrichment of inosine and pseudo-uridine (Ψ) levels which may be associated with RNA editing activity. In addition, infected mosquitoes demonstrate a hypoglycemic phenotype and show significant increases in the abundance of metabolites such as prostaglandin H2, leukotriene D4 and protoporphyrinogen IX which are associated with antiviral activity. These provide a basis for understanding the biochemical response to ZIKV infection and pathology in the vector. Future mechanistic studies targeting these ZIKV infection responsive metabolites and their associated biosynthetic pathways can provide inroads to identification of mosquito antiviral responses with infection blocking potential.

Comprehensive platelet phenotyping supports the role of platelets in the pathogenesis of acute venous thromboembolism - results from clinical observation studies.

BackgroundThe pathogenesis of arterial and venous thrombosis is in large part interlaced. How much platelet phenotype relates to acute venous thromboembolism (VTE) independent of the underlying cardiovascular profile is presently poorly investigated.MethodsPlatelet count and mean platelet volume (MPV), platelet aggregation in whole blood and platelet rich plasma (PRP), platelet-dependent thrombin generation (TG) and platelet surface activation markers were measured under standardized conditions. Machine learning was applied to identify the most relevant characteristics associated with VTE from a large array (N = 58) of clinical and platelet-related variables.FindingsVTE cases (N = 159) presented with lower platelet count and MPV vs controls (N = 140). Whole blood aggregation showed shorter collagen/Epinephrine closure times in cases, particularly within acetylsalicylic acid (ASA) users. Within ASA users, higher PRP aggregation after adenosine diphosphate (ADP), epinephrine, collagen and arachidonic acid was observed in cases vs controls. Within non-ASA and/or subjects on anticoagulants, cases presented with lower aggregation after ADP and collagen vs controls. Lower platelet-dependent TG, higher CD63 on resting and lower PAC-1 expression after collagen/ADP in-vitro stimulated platelets further characterized VTE cases vs controls, independent of therapy. Lasso regression analysis identified 26 variables associated with VTE of which 69% were platelet-related.InterpretationComprehensive phenotyping of platelet function identified a large proportion of low responders to ASA in VTE cases. Lower platelet-dependent TG and lower platelet reactivity after ex-vivo stimulation characterized the “platelet exhausted syndrome” in cases. Finally, from a large array of covariates including clinical risk factors, platelet biomarkers comprised 69% of all selected variables differentiating VTE cases vs controls.FundingGerman Federal Ministry of Education and Research, CTH-Mainz and Bayer AG.