Quantification of fibroblast growth factor 23 and N-terminal pro-B-type natriuretic peptide to identify patients with atrial fibrillation using a high-throughput platform: A validation study.

BACKGROUND: Large-scale screening for atrial fibrillation (AF) requires reliable methods to identify at-risk populations. Using an experimental semi-quantitative biomarker assay, B-type natriuretic peptide (BNP) and fibroblast growth factor 23 (FGF23) were recently identified as the most suitable biomarkers for detecting AF in combination with simple morphometric parameters (age, sex, and body mass index [BMI]). In this study, we validated the AF model using standardised, high-throughput, high-sensitivity biomarker assays. METHODS AND FINDINGS: For this study, 1,625 consecutive patients with either (1) diagnosed AF or (2) sinus rhythm with CHA2DS2-VASc score of 2 or more were recruited from a large teaching hospital in Birmingham, West Midlands, UK, between September 2014 and February 2018. Seven-day ambulatory ECG monitoring excluded silent AF. Patients with tachyarrhythmias apart from AF and incomplete cases were excluded. AF was diagnosed according to current clinical guidelines and confirmed by ECG. We developed a high-throughput, high-sensitivity assay for FGF23, quantified plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) and FGF23, and compared results to the previously used multibiomarker research assay. Data were fitted to the previously derived model, adjusting for differences in measurement platforms and known confounders (heart failure and chronic kidney disease). In 1,084 patients (46% with AF; median [Q1, Q3] age 70 [60, 78] years, median [Q1, Q3] BMI 28.8 [25.1, 32.8] kg/m2, 59% males), patients with AF had higher concentrations of NT-proBNP (median [Q1, Q3] per 100 pg/ml: with AF 12.00 [4.19, 30.15], without AF 4.25 [1.17, 15.70]; p < 0.001) and FGF23 (median [Q1, Q3] per 100 pg/ml: with AF 1.93 [1.30, 4.16], without AF 1.55 [1.04, 2.62]; p < 0.001). Univariate associations remained after adjusting for heart failure and estimated glomerular filtration rate, known confounders of NT-proBNP and FGF23. The fitted model yielded a C-statistic of 0.688 (95% CI 0.656, 0.719), almost identical to that of the derived model (C-statistic 0.691; 95% CI 0.638, 0.744). The key limitation is that this validation was performed in a cohort that is very similar demographically to the one used in model development, calling for further external validation. CONCLUSIONS: Age, sex, and BMI combined with elevated NT-proBNP and elevated FGF23, quantified on a high-throughput platform, reliably identify patients with AF. TRIAL REGISTRATION: Registry IRAS ID 97753 Health Research Authority (HRA), United Kingdom.

Data-driven discovery and validation of circulating blood-based biomarkers associated with prevalent atrial fibrillation.

AIMS: Undetected atrial fibrillation (AF) is a major health concern. Blood biomarkers associated with AF could simplify patient selection for screening and further inform ongoing research towards stratified prevention and treatment of AF. METHODS AND RESULTS: Forty common cardiovascular biomarkers were quantified in 638 consecutive patients referred to hospital [mean ± standard deviation age 70 ± 12 years, 398 (62%) male, 294 (46%) with AF] with known AF or ≥2 CHA2DS2-VASc risk factors. Paroxysmal or silent AF was ruled out by 7-day ECG monitoring. Logistic regression with forward selection and machine learning algorithms were used to determine clinical risk factors, imaging parameters, and biomarkers associated with AF. Atrial fibrillation was significantly associated with age [bootstrapped odds ratio (OR) per year = 1.060, 95% confidence interval (1.04-1.10); P = 0.001], male sex [OR = 2.022 (1.28-3.56); P = 0.008], body mass index [BMI, OR per unit = 1.060 (1.02-1.12); P = 0.003], elevated brain natriuretic peptide [BNP, OR per fold change = 1.293 (1.11-1.63); P = 0.002], elevated fibroblast growth factor-23 [FGF-23, OR = 1.667 (1.36-2.34); P = 0.001], and reduced TNF-related apoptosis-induced ligand-receptor 2 [TRAIL-R2, OR = 0.242 (0.14-0.32); P = 0.001], but not other biomarkers. Biomarkers improved the prediction of AF compared with clinical risk factors alone (net reclassification improvement = 0.178; P < 0.001). Both logistic regression and machine learning predicted AF well during validation [area under the receiver-operator curve = 0.684 (0.62-0.75) and 0.697 (0.63-0.76), respectively]. CONCLUSION: Three simple clinical risk factors (age, sex, and BMI) and two biomarkers (elevated BNP and elevated FGF-23) identify patients with AF. Further research is warranted to elucidate FGF-23 dependent mechanisms of AF.

PR3 and elastase alter PAR1 signaling and trigger vWF release via a calcium-independent mechanism from glomerular endothelial cells.

Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH(2) and SLIGKV-NH(2,) respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.

Neutrophil serine proteases mediate inflammatory cell recruitment by glomerular endothelium and progression towards dysfunction.

BACKGROUND: Neutrophil recruitment into glomerular tissues and reduced capillary wall integrity has been implicated in the development of vasculitic glomerulonephritis (VGN). This study investigated the stages and mechanisms through which neutrophil serine proteases (SPs), proteinase 3 (PR3) or elastase contribute to endothelial dysfunction. METHODS: Protease-induced damage to endothelium and adhesion molecule upregulation was measured by viability assays and ELISA. Neutrophil/platelet adhesion to human glomerular and umbilical vein endothelium was assessed using in vitro adhesion assays. RESULTS: PR3 and elastase (1 µg/mL, 2 h) significantly induced neutrophil adhesion to endothelial cells (EnC) whilst PR3 also enhanced platelet-EnC interactions. This neutrophil adhesion was associated with enhanced P-selectin expression and required CXCL8 receptor involvement, and could be inhibited by blocking the P-selectin ligand PSGL-1. SPs induced damage in a time- and dose-dependent fashion, decreasing cell monolayer integrity followed by cell membrane integrity, inducing caspase-3 activation and p21 cleavage. However, SPs caused significant EnC damage with increasing concentrations and prolonged exposures. CONCLUSION: Neutrophil SPs induce a pro-adhesive phenotype in glomerular endothelium primarily by inducing neutrophil and platelet adhesion that transits to dysfunction after high/prolonged exposures. Dysregulated release of these enzymes within glomeruli may contribute to injury during diseases such as VGN.

Docosahexaenoic acid inhibits the adhesion of flowing neutrophils to cytokine stimulated human umbilical vein endothelial cells.

The (n-3) PUFA, DHA, is widely thought to posses the ability to modulate the inflammatory response. However, its modes of interaction with inflammatory cells are poorly understood. In particular, there are limited data on the interactions of DHA with vascular endothelium, the cells that regulate the traffic of leukocytes from the blood into inflamed tissue. Using human umbilical vein endothelial cells (EC) cultured in a flow-based adhesion assay and activated with TNFα, we tested whether supplementing human umbilical vein EC with physiologically achievable concentrations of DHA would inhibit the recruitment of flowing neutrophils. DHA caused a dose-dependent reduction in neutrophil recruitment to the EC surface, although cells that became adherent were activated and could migrate across the human umbilical vein EC monolayer normally. Using EPA as an alternative supplement had no effect on the levels of neutrophil adhesion in this assay. Analysis of adhesion receptor expression by qPCR demonstrated that DHA did not alter the transcriptional activity of human umbilical vein EC. However, DHA did significantly reduce E-selectin expression at the human umbilical vein EC surface without altering the total cellular pool of this adhesion receptor. Thus, we have identified a novel mechanism by which DHA alters the trafficking of leukocytes during inflammation and demonstrate that this involves disruption of intracellular transport mechanisms used to present adhesion molecules on the surface of cytokine-stimulated EC.

Omega-3 Fatty acids and inflammation: novel interactions reveal a new step in neutrophil recruitment.

Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-alpha, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D(2) (PGD(2)) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD(3). This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD(2) receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD(2) signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.

Cellular pathology of atherosclerosis: smooth muscle cells promote adhesion of platelets to cocultured endothelial cells.

Although platelets do not ordinarily bind to endothelial cells (EC), pathological interactions between platelets and arterial EC may contribute to the propagation of atheroma. Previously, in an in vitro model of atherogenesis, where leukocyte adhesion to EC cocultured with smooth muscle cells was greatly enhanced, we also observed attachment of platelets to the EC layer. Developing this system to specifically model platelet adhesion, we show that EC cocultured with smooth muscle cells can bind platelets in a process that is dependent on EC activation by tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1. Recapitulating the model using EC alone, we found that a combination of TGF-beta1 and TNF-alpha promoted high levels of platelet adhesion compared with either agent used in isolation. Platelet adhesion was inhibited by antibodies against GPIb-IX-V or alpha(IIb)beta3 integrin, indicating that both receptors are required for stable adhesion. Platelet activation during interaction with the EC was also essential, as treatment with prostacyclin or theophylline abolished stable adhesion. Confocal microscopy of the surface of EC activated with TNF-alpha and TGF-beta1 revealed an extensive matrix of von Willebrand factor that was able to support the adhesion of flowing platelets at wall shear rates below 400 s(-1). Thus, we have demonstrated a novel route of EC activation which is relevant to the atherosclerotic microenvironment. EC activated in this manner would therefore be capable of recruiting platelets in the low-shear environments that commonly exist at points of atheroma formation.