High Throughput Plasma Proteomics and Risk of Heart Failure and Frailty in Late Life.

Importance: Heart failure (HF) and frailty frequently coexist and may share a common pathobiology, although the underlying mechanisms remain unclear. Understanding these mechanisms may provide guidance for preventing and treating both conditions. Objective: To identify shared pathways between incident HF and frailty in late life using large-scale proteomics. Design, Setting, and Participants: In this cohort study, 4877 aptamers (Somascan v4) were measured among participants in the community-based longitudinal Atherosclerosis Risk In Communities (ARIC) cohort study at visit 3 (V3; 1993-1995; n = 10 638) and at visit 5 (V5; 2011-2013; n = 3908). Analyses were externally replicated among 3189 participants in the Cardiovascular Health Study (CHS). Data analysis was conducted from February 2022 to June 2023. Exposures: Protein aptamers, measured at study V3 and V5. Main Outcomes and Measures: Outcomes assessed included incident HF hospitalization after V3 and after V5, prevalent frailty at V5, and incident frailty between V5 and visit 6 (V6; 2016-2017; n = 4131). Frailty was assessed using the Fried criteria. Analyses were adjusted for age, gender, race, field center, hypertension, diabetes, smoking status, body mass index, estimated glomerular filtration rate, prevalent coronary heart disease, prevalent atrial fibrillation, and history of myocardial infarction. Mendelian randomization (MR) analysis was performed to assess potential causal effects of candidate proteins on HF and frailty. Results: A total of 4877 protein aptamers were measured among 10 638 participants at V3 (mean [SD] age, 60 [6] years; 4886 [46%] men). Overall, 286 proteins were associated with incident HF after V3 (822 events; P < 1.0 × 10-5), 83 of which were also associated with incident after V5 (336 events; P < 1.7 × 10-4). Among HF-free participants at V5 (n = 3908; mean [SD] age, 75 [5] years; 1861 [42%] men), 48 of 83 HF-associated proteins were associated with prevalent frailty (223 cases; P < 6.0 × 10-4), 18 of which were also associated with incident frailty at V6 (152 cases; P < 1.0 × 10-3). These proteins enriched fibrosis and inflammation pathways and demonstrated stronger associations with incident HF with preserved ejection fraction (HFpEF) than HF with reduced ejection fraction. All 18 proteins were associated with both prevalent frailty and incident HF in CHS. MR identified potential causal effects of several proteins on frailty and HF. Conclusions and Relevance: In this study, the proteins associated with risk of HF and frailty enrich for pathways related to inflammation and fibrosis as well as risk of HFpEF. Several of these proteins could potentially contribute to the shared pathophysiology of frailty and HF.

Circulating Androgen Concentrations and Risk of Incident Heart Failure in Older Men: The Cardiovascular Health Study.

Background Circulating androgen concentrations in men decline with age and have been linked to diabetes and atherosclerotic cardiovascular disease (ASCVD). A similar relationship has been reported for low total testosterone and incident heart failure (HF) but remains unstudied for free testosterone or the more potent androgen dihydrotestosterone (DHT). We hypothesized that total/free testosterone are inversely related, sex hormone-binding globulin is positively related, and total/free DHT bear a U-shaped relationship with incident HF. Methods and Results In a sample of men from the CHS (Cardiovascular Health Study) without atherosclerotic cardiovascular disease or HF, serum testosterone and DHT concentrations were measured by liquid chromatography-tandem mass spectrometry, and sex hormone-binding globulin by immunoassay. Free testosterone or DHT was calculated from total testosterone or total DHT, sex hormone-binding globulin, and albumin. We used Cox regression to estimate relative risks of HF after adjustment for potential confounders. In 1061 men (aged 76±5 years) followed for a median of 9.6 years, there were 368 HF events. After adjustment, lower calculated free testosterone was significantly associated with higher risk of HF (hazard ratio [HR], 1.14 [95% CI, 1.01-1.28]). Risk estimates for total testosterone (HR, 1.12 [95% CI, 0.99-1.26]), total DHT (HR, 1.10 [95% CI, 0.97-1.24]), calculated free dihydrotestosterone (HR, 1.09 [95% CI, 0.97-1.23]), and sex hormone-binding globulin (HR, 1.07 [95% CI, 0.95-1.21]) were directionally similar but not statistically significant. Conclusions Calculated free testosterone was inversely associated with incident HF, suggesting a contribution of testosterone deficiency to HF incidence among older men. Additional research is necessary to determine whether testosterone replacement therapy might be an effective strategy to lower HF risk in older men.

Racial and Ethnic Disparities in Transthyretin Cardiac Amyloidosis.

Purpose of Review: Transthyretin amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease that disproportionately affects older adults and people of African descent. This review discusses current knowledge regarding racial and ethnic disparities in the diagnosis and management of ATTR-CM. Recent Findings: Historically, ATTR-CM was thought to be a rare cause of heart failure. Recent evidence has shown that ATTR-CM is more common among older adults, men, and people of African descent. In addition, significant geographic variation exists in the identification of amyloid cardiomyopathy. Despite the high burden of ATTR-CM among Black individuals, most clinical data for ATTR-CM are from North America and Europe. Moreover, only a minority of clinical trial participants thus far have been Black patients. In addition to racial differences, socioeconomic disparities may be further compounded by the potentially prohibitive cost and limited accessibility of disease-modifying ATTR therapies. Summary: ATTR-CM is an important cause of heart failure that disproportionately affects people of African descent. Efforts to promote earlier identification of ATTR-CM in general practice will likely improve clinical outcomes for all groups. Future trials should strive to enroll a higher proportion of Black patients. Furthermore, enhanced efforts are warranted to improve treatment accessibility among racial and ethnic minority groups that may be more likely to be affected by ATTR-CM.

Proteomic Evaluation of the Comorbidity-Inflammation Paradigm in Heart Failure With Preserved Ejection Fraction: Results From the PROMIS-HFpEF Study.

BACKGROUND: A systemic proinflammatory state has been hypothesized to mediate the association between comorbidities and abnormal cardiac structure/function in heart failure with preserved ejection fraction (HFpEF). We conducted a proteomic analysis to investigate this paradigm. METHODS: In 228 patients with HFpEF from the multicenter PROMIS-HFpEF study (Prevalence of Microvascular Dysfunction in Heart Failure With Preserved Ejection Fraction), 248 unique circulating proteins were quantified by a multiplex immunoassay (Olink) and used to recapitulate systemic inflammation. In a deductive approach, we performed principal component analysis to summarize 47 proteins known a priori to be involved in inflammation. In an inductive approach, we performed unbiased weighted coexpression network analyses of all 248 proteins to identify clusters of proteins that overrepresented inflammatory pathways. We defined comorbidity burden as the sum of 8 common HFpEF comorbidities. We used multivariable linear regression and statistical mediation analyses to determine whether and to what extent inflammation mediates the association of comorbidity burden with abnormal cardiac structure/function in HFpEF. We also externally validated our findings in an independent cohort of 117 HFpEF cases and 30 comorbidity controls without heart failure. RESULTS: Comorbidity burden was associated with abnormal cardiac structure/function and with principal components/clusters of inflammation proteins. Systemic inflammation was also associated with increased mitral E velocity, E/e' ratio, and tricuspid regurgitation velocity; and worse right ventricular function (tricuspid annular plane systolic excursion and right ventricular free wall strain). Inflammation mediated the association between comorbidity burden and mitral E velocity (proportion mediated 19%-35%), E/e' ratio (18%-29%), tricuspid regurgitation velocity (27%-41%), and tricuspid annular plane systolic excursion (13%) (P<0.05 for all), but not right ventricular free wall strain. TNFR1 (tumor necrosis factor receptor 1), UPAR (urokinase plasminogen activator receptor), IGFBP7 (insulin-like growth factor binding protein 7), and GDF-15 (growth differentiation factor-15) were the top individual proteins that mediated the relationship between comorbidity burden and echocardiographic parameters. In the validation cohort, inflammation was upregulated in HFpEF cases versus controls, and the most prominent inflammation protein cluster identified in PROMIS-HFpEF was also present in HFpEF cases (but not controls) in the validation cohort. CONCLUSIONS: Proteins involved in inflammation form a conserved network in HFpEF across 2 independent cohorts and may mediate the association between comorbidity burden and echocardiographic indicators of worse hemodynamics and right ventricular dysfunction. These findings support the comorbidity-inflammation paradigm in HFpEF.

A flow cytometric analysis of macrophage- nanoparticle interactions in vitro: induction of altered Toll-like receptor expression.

BACKGROUND: Nanoparticles exhibit unique physiochemical characteristics that provide the basis for their utilization. The diversity of potential and actual applications compels a thorough understanding regarding the consequences of their containment within the cellular environment. PURPOSE: This paper presents a flow cytometric examination of the biologic effects associated with the internalization of citrate-buffered silver (Ag) nanoparticles (NP) by the murine macrophage cell line, RAW264.7. MATERIALS AND METHODS: Cells were cultured with varying concentrations of citrate-buffered Ag nanoparticle and analyzed for changes in cellular volume, fluorescence emissions, and surface receptor expression. RESULTS: Notable changes in side scatter (SSC) signal occurred following the phagocytosis of citrate-buffered Ag NP representative of the 10 nm, 50 nm, and 100 nm particle size by cultured RAW 264.7 cells. A characteristic associated with the internalization of all the citrated Ag NP sizes tested, was the detection of emitted infra-red and near-infrared wavelength emissions. This characteristic consistently permitted the detection of 10 nm, 50 nm, and 100 nm Ag NP particles internalized within the RAW cells by flow cytometry. A functional distinction between monocyte subsets within the RAW 264.7 cell line was noted as Ag NP are taken up by the F4/80+ subset of cells within the culture. Further, the internalization of Ag NP by the cells resulted in an increased cell surface expression of the Toll-like receptor (TLR) 3, but not TLR4. CONCLUSION: Taken together, these results implicate the more mature macrophage in the ingestion of Ag NP; and an influence upon at least one of the Toll receptors present in macrophages following exposure to Ag NP. Further, our flow cytometric approach presents a potentially viable detection method for the identification of occult Ag NP material using an indicator cell line.

Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells.

Exposure to nanosilver found in food- and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food- and cosmetics-related chemicals including nanosilver.

Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by a flow cytometric in vitro micronucleus assay.

Two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, and flow cytometry techniques were evaluated as tools for rapid screening of potential genotoxicity of food-related nanosilver. Comparative genotoxic potential of 20 nm silver was evaluated in HepG2 and Caco2 cell cultures by a flow cytometric-based in vitro micronucleus assay. The nanosilver, characterized by the dynamic light scattering, transmission electron microscopy and inductively coupled plasma-mass spectrometry analysis, showed no agglomeration of the silver nanoparticles. The inductively coupled plasma-mass spectrometry and transmission electron microscopy analysis demonstrated the uptake of 20 nm silver by both cell types. The 20 nm silver exposure of HepG2 cells increased the concentration-dependent micronucleus formation sevenfold at 10 µg ml(-1) concentration in attached cell conditions and 1.3-fold in cell suspension conditions compared to the vehicle controls. However, compared to the vehicle controls, the 20 nm silver exposure of Caco2 cells increased the micronucleus formation 1.2-fold at a concentration of 10 µg ml(-1) both in the attached cell conditions as well as in the cell suspension conditions. Our results of flow cytometric in vitro micronucleus assay appear to suggest that the HepG2 cells are more susceptible to the nanosilver-induced micronucleus formation than the Caco2 cells compared to the vehicle controls. However, our results also suggest that the widely used in vitro models, HepG2 and Caco2 cells and the flow cytometric in vitro micronucleus assay are valuable tools for the rapid screening of genotoxic potential of nanosilver and deserve more careful evaluation.

Hydroxyurea induces fetal hemoglobin by the nitric oxide-dependent activation of soluble guanylyl cyclase.

Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of gamma-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced gamma-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.

Fetal hemoglobin modulation during human erythropoiesis: stem cell factor has "late" effects related to the expression pattern of CD117.

A cytokine-screening assay of cultured peripheral blood cells obtained using immune rosetting and separation of progenitors was developed to identify determinants of fetal hemoglobin (HbF) modulation during adult erythropoiesis. Among the 12 erythroid growth-promoting cytokines tested, stem cell factor (SCF) at a concentration of 50 ng/mL resulted in the most significant increase in cell proliferation and HbF content. The average HbF/hemoglobin A (HbA) ratio was 30.9% +/- 18.7% in cultures containing SCF compared with 4.1% +/- 2.2% in those grown with erythropoietin (EPO) alone (P = 8.5E-8). To further investigate the hemoglobin-modulating effects of SCF, we examined the surface expression pattern of the SCF receptor, CD117, among maturing erythroblasts. CD117 expression increased during the first week of culture and peaked on culture days 7 to 9. After culture day 9, the level of CD117 declined to lower levels. The rise in CD117 expression to high levels mirrored that of the transferrin receptor (CD71), and the subsequent reduction in CD117 was inversely related to increases in expression of glycophorin A. SCF-related increases in the HbF/HbA ratio correlated with the expression pattern of CD117. SCF added during days 7 to 14 resulted in a more pancellular distribution of HbF on day 14 compared with the heterocellular distribution present in cultures supplemented with SCF on days 0 to 7. A significant SCF-mediated increase in HbF was also measured using progenitors derived from cord blood. These results suggest that the HbF response to SCF is greatest at the late progenitor stage as a function of surface CD117 expression.