Circulating microRNAs and DNA Methylation as Regulators of Direct Oral Anticoagulant Response in Atrial Fibrillation and Key Elements for the Identification of the Mechanism of Action (miR-CRAFT): Study Design and Patient Enrolment.

Direct oral anticoagulants (DOACs) are the standard treatment for thromboembolic protection in atrial fibrillation (AF) patients. Epigenetic modifications, such as DNA methylation and microRNAs, have emerged as potential biomarkers of AF. The epigenetics of DOACs is still an understudied field. It is largely unknown whether epigenetic modifications interfere with DOAC response or whether DOAC treatment induces epigenetic modifications. To fill this gap, we started the miR-CRAFT (Circulating microRNAs and DNA methylation as regulators of Direct Oral Anticoagulant Response in Atrial Fibrillation) research study. In miR-CRAFT, we follow, over time, changes in DNA methylation and microRNAs expression in naïve AF patients starting DOAC treatment. The ultimate goal of miR-CRAFT is to identify the molecular pathways epigenetically affected by DOACs, beyond the coagulation cascade, that are potentially mediating DOAC pleiotropic actions and to propose specific microRNAs as novel circulating biomarkers for DOAC therapy monitoring. We herein describe the study design and briefly present the progress in participant enrolment.

EXPLORATORY ANALYSIS OF URINE PO2 PERI-PROCEDURAL KINETICS AND CI-AKI PROGNOSTIC ABILITIES IN PATIENTS UNDERGOING PCI.

Novel contrast-induced acute kidney injury (CI-AKI) biomarkers are needed to detect earlier and with greater precision the pathophysiological changes in renal medulla associated with kidney damage. We prospectively assessed the kinetics of urine oxygen tension (PO2) in control healthy individuals, and its prognostic ability for CI-AKI in patients undergoing percutaneous coronary intervention (PCI). We enrolled 202 consecutive patients (78% men, mean age 66±10 years) treated with elective or urgent PCI. PO2 was measured using a point-of-care (POC) standard blood gas analyzer at 3 time points (baseline, post -within 3 hours- PCI and at 24 hours post PCI) in urine samples. CI-AKI was defined as an increase of ≥25% or ≥0.5 mg/dl in pre-PCI serum creatinine at 48 hours post PCI. Between baseline and post-PCI measurements, patients without CI-AKI showed a decrease of -37 (36) mmHg in PO2 urine levels whereas patients with CI-AKI showed a decrease of only -23 (38) mmHg. (P=0.014). Using ROC analysis, percentage change in urine PO2 immediately after PCI relative to baseline levels, significantly predicted CI-AKI (AUC 0.804 95%CI 0.717-0.892). A significant drop in urine oxygen tension appears as a normal response of the kidney medulla to an acute insult (contrast media) immediately post PCI with a recovery to baseline levels 24 hours later. Absence or attenuation of this drop in urine oxygen tension could predict CI-AKI earlier and more precisely.

Images of an ectopic balloon expandable valve deployed at the aortic arch level following valve embolization.

An 83-year-old man with severe aortic stenosis underwent implantation of a 29-mm SAPIEN-3 (Edwards Lifesciences) transcatheter aortic valve (TAV) appropriately sized for an aortic annulus area of 543.6 mm2.

Renal artery revascularization is a controversial treatment strategy for renal artery stenosis: A case series and a brief review of the current literature.

Renal artery stenosis (RAS) may cause secondary hypertension, progressive decline in renal function, and cardiac destabilization syndromes including "flash" pulmonary edema, recurrent congestive heart failure, and cerebro-cardiovascular disease. Atherosclerotic lesions, fibromuscular dysplasia, and vasculitides are the pathophysiological basis of the disease. Common therapeutic pathways for RAS include medical therapy and revascularization with or without stenting. Randomized controlled trials evaluating renal revascularization have not reported any advantages of revascularization over medical therapy alone in terms of renal function improvement or prevention of cardiovascular events. However, mounting clinical experience suggests that the best strategy in RAS management is to identify which patients are most likely to benefit from renal artery stenting and to optimize the safety and durability of the procedure. This review presents 3 cases of patients who have undergone renal revascularization and discusses the available clinical evidence for the identification of RAS patients who will potentially respond well to revascularization.

Colocalization of Erythrocytes and Vascular Calcification in Human Atherosclerosis: A Systematic Histomorphometric Analysis.

Background Intimal calcification typically develops in advanced atherosclerosis, and microcalcification may promote plaque progression and instability. Conversely, intraplaque hemorrhage and erythrocyte extravasation may stimulate osteoblastic differentiation and intralesional calcium phosphate deposition. The presence of erythrocytes and their main cellular components (membranes, hemoglobin, and iron) and colocalization with calcification has never been systematically studied. Methods and Results We examined three types of diseased vascular tissue specimens, namely, degenerative aortic valve stenosis ( n = 46), atherosclerotic carotid artery plaques ( n = 9), and abdominal aortic aneurysms ( n = 14). Biomaterial was obtained from symptomatic patients undergoing elective aortic valve replacement, carotid artery endatherectomy, or aortic aneurysm repair, respectively. Serial sections were stained using Masson-Goldner trichrome, Alizarin red S, and Perl's iron stain to visualize erythrocytes, extracelluar matrix and osteoid, calcium phosphate deposition, or the presence of iron and hemosiderin, respectively. Immunohistochemistry was employed to detect erythrocyte membranes (CD235a), hemoglobin or the hemoglobin scavenger receptor (CD163), endothelial cells (CD31), myofibroblasts (SMA), mesenchymal cells (osteopontin), or osteoblasts (periostin). Our analyses revealed a varying degree of intraplaque hemorrhage and that the majority of extravasated erythrocytes were lysed. Osteoid and calcifications also were frequently present, and erythrocyte membranes were significantly more prevalent in areas with calcification. Areas with extravasated erythrocytes frequently contained CD163-positive cells, although calcification also occurred in areas without CD163 immunosignals. Conclusion Our findings underline the presence of extravasated erythrocytes and their membranes in different types of vascular lesions, and their association with areas of calcification suggests an active role of erythrocytes in vascular disease processes.

Duration of interventricular septal shift toward the left ventricle is associated with poor clinical outcome in precapillary pulmonary hypertension: A cardiac magnetic resonance study.

BACKGROUND: Right ventricular pressure overload results in interventricular septal shift toward the left ventricle in patients with precapillary pulmonary hypertension (PH). We aimed to investigate the predictive role of the duration of septal curvature configuration during the cardiac cycle, as expressed by the novel marker curvature duration index (CDi) in precapillary PH. METHODS: This was a prospective study. All patients underwent cardiac magnetic resonance (CMR). CDi was defined by the number of CMR frames in which septal curvature configuration toward left ventricle is observed *100/total number of frames per cardiac cycle. Time from enrollment to first clinical failure event (death, hospitalization due to PH, and disease progression) was recorded. RESULTS: The study included 36 patients with precapillary PH. During a median follow-up of 20 months (IQR 4-37 months), 14 clinical failure events were observed. Survival ROC analysis showed that the optimal cutoff value of CDi, which predicted clinical failure, was 67%. Kaplan-Meier survival analysis showed that CDi≥67% was associated with a 9.4-fold increase in the risk for clinical failure. Addition of CDi to baseline models including six-minute walk test distance (c-statistic = 0.65 vs. c-statistic = 0.79), NT-proBNP (c-statistic = 0.72 vs. c-statistic = 0.83), and WHO functional class (c-statistic = 0.76 vs. c-statistic = 0.81) improved risk stratification. CONCLUSION: Ventricular septal shift toward the left ventricle lasting for more than the two thirds of the cardiac cycle is associated with worse prognosis in precapillary PH.

Increased Lymphangiogenesis and Lymphangiogenic Growth Factor Expression in Perivascular Adipose Tissue of Patients with Coronary Artery Disease.

Experimental and human autopsy studies have associated adventitial lymphangiogenesis with atherosclerosis. An analysis of perivascular lymphangiogenesis in patients with coronary artery disease is lacking. Here, we examined lymphangiogenesis and its potential regulators in perivascular adipose tissue (PVAT) surrounding the heart (C-PVAT) and compared it with PVAT of the internal mammary artery (IMA-PVAT). Forty-six patients undergoing coronary artery bypass graft surgery were included. Perioperatively collected C-PVAT and IMA-PVAT were analyzed using histology, immunohistochemistry, real time PCR, and PVAT-conditioned medium using cytokine arrays. C-PVAT exhibited increased PECAM-1 (platelet endothelial cell adhesion molecule 1)-positive vessel density. The number of lymphatic vessels expressing lymphatic vessel endothelial hyaluronan receptor-1 or podoplanin was also elevated in C-PVAT and associated with higher inflammatory cell numbers, increased intercellular adhesion molecule 1 (ICAM1) expression, and fibrosis. Significantly higher expression of regulators of lymphangiogenesis such as vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor-3 was observed in C-PVAT compared to IMA-PVAT. Cytokine arrays identified angiopoietin-2 as more highly expressed in C-PVAT vs. IMA-PVAT. Findings were confirmed histologically and at the mRNA level. Stimulation of human lymphatic endothelial cells with recombinant angiopoietin-2 in combination with VEGF-C enhanced sprout formation. Our study shows that PVAT surrounding atherosclerotic arteries exhibits more extensive lymphangiogenesis, inflammation, and fibrosis compared to PVAT surrounding a non-diseased vessel, possibly due to local angiopoietin-2, VEGF-C, and VEGF-D overexpression.

Long-term impact of acute kidney injury on prognosis in patients with acute myocardial infarction.

BACKGROUND: Little evidence exists regarding the long-term impact of acute kidney injury (AKI) during index hospitalisation for acute myocardial infarction (AMI). We prospectively assessed the long-term prognostic significance of the occurrence of in-hospital AKI in a multicentre cohort of patients admitted with AMI. METHODS: Data were obtained from 518 AMI patients with a median follow-up of 5.6 (IQR 4.6-6.5) years. Patients were followed up regarding the occurrence of death, major adverse cardiovascular events (MACE), and any deterioration in kidney function. RESULTS: From the study cohort, 84 patients (16%) had developed AKI at discharge during index hospitalisation. 96 patients died during follow-up, MACE occurred in 90 patients, and 30 patients showed evidence of deterioration in kidney function. Patients with AKI at hospital discharge had a three-fold increased mortality risk (HR 3.2, 95% CI 2.1-4.8; P < 0.001). This association was independent of possible confounding by variables that could influence prognosis (HR 1.9 95% CI 1.1-3.2; P = 0.028) evident only up to three years during follow-up. During long-term follow-up, patients with AKI during their index hospitalisation had a significantly (P = 0.027) higher incidence of MACE (26%) than those who did not develop AKI (15%). Patients with AKI had a higher incidence of deteriorating kidney function (10%) than those without AKI (5%) during follow-up, but this difference was not significant (P = 0.124). CONCLUSIONS: Our findings emphasise in addition to the need for appropriate long term follow-up in such patients, an increased mortality and morbidity during the first three years after the index event.

Lysed Erythrocyte Membranes Promote Vascular Calcification.

BACKGROUND: Intraplaque hemorrhage promotes atherosclerosis progression, and erythrocytes may contribute to this process. In this study we examined the effects of red blood cells on smooth muscle cell mineralization and vascular calcification and the possible mechanisms involved. METHODS: Erythrocytes were isolated from human and murine whole blood. Intact and lysed erythrocytes and their membrane fraction or specific erythrocyte components were examined in vitro using diverse calcification assays, ex vivo by using the murine aortic ring calcification model, and in vivo after murine erythrocyte membrane injection into neointimal lesions of hypercholesterolemic apolipoprotein E-deficient mice. Vascular tissues (aortic valves, atherosclerotic carotid artery specimens, abdominal aortic aneurysms) were obtained from patients undergoing surgery. RESULTS: The membrane fraction of lysed, but not intact human erythrocytes promoted mineralization of human arterial smooth muscle cells in culture, as shown by Alizarin red and van Kossa stain and increased alkaline phosphatase activity, and by increased expression of osteoblast-specific transcription factors (eg, runt-related transcription factor 2, osterix) and differentiation markers (eg, osteopontin, osteocalcin, and osterix). Erythrocyte membranes dose-dependently enhanced calcification in murine aortic rings, and extravasated CD235a-positive erythrocytes or Perl iron-positive signals colocalized with calcified areas or osteoblast-like cells in human vascular lesions. Mechanistically, the osteoinductive activity of lysed erythrocytes was localized to their membrane fraction, did not involve membrane lipids, heme, or iron, and was enhanced after removal of the nitric oxide (NO) scavenger hemoglobin. Lysed erythrocyte membranes enhanced calcification to a similar extent as the NO donor diethylenetriamine-NO, and their osteoinductive effects could be further augmented by arginase-1 inhibition (indirectly increasing NO bioavailability). However, the osteoinductive effects of erythrocyte membranes were reduced in human arterial smooth muscle cells treated with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide or following inhibition of NO synthase or the NO receptor soluble guanylate cyclase. Erythrocytes isolated from endothelial NO synthase-deficient mice exhibited a reduced potency to promote calcification in the aortic ring assay and after injection into murine vascular lesions. CONCLUSIONS: Our findings in cells, genetically modified mice, and human vascular specimens suggest that intraplaque hemorrhage with erythrocyte extravasation and lysis promotes osteoblastic differentiation of smooth muscle cells and vascular lesion calcification, and also support a role for erythrocyte-derived NO.

Manual Versus Mechanical Compression of the Radial Artery After Transradial Coronary Angiography: The MEMORY Multicenter Randomized Trial.

OBJECTIVES: The aim of this study was to compare manual versus mechanical compression of the radial artery after coronary angiography via transradial access regarding radial artery occlusion (RAO), access-site bleeding complications, and duration of hemostasis. BACKGROUND: Hemostasis of the radial artery after sheath removal can be achieved either by manual compression at the puncture site or by using a mechanical hemostasis device. Because mechanical compression exerts a more stable, continuous pressure on the artery, it could be hypothesized that it is more effective compared with manual compression regarding hemostasis time, bleeding, and RAO risks. METHODS: A total of 589 patients undergoing diagnostic coronary angiography by transradial access with a 5-F sheath were randomized in a 1:1 ratio to receive either manual or mechanical patent hemostasis of the radial artery. Radial artery patency was evaluated by color duplex ultrasonography 24 h after the procedure. The primary endpoint was early RAO at 24 h. Secondary endpoints included access-site bleeding complications and duration of hemostasis. RESULTS: Thirty-six (12%) early RAOs occurred in the manual group, and 24 (8%) occurred in the mechanical group (p = 0.176). There were no significant differences between the 2 groups regarding access-site bleeding complications (hematoma, 52 [17%] vs. 50 [18%]; p = 0.749; bleedings, 8 [3%] vs. 9 [3%]; p = 1.000). Duration of hemostasis was significantly shorter in the manual group (22 ± 34 min vs. 119 ± 72 min with mechanical compression; p < 0.001). CONCLUSIONS: Manual and mechanical compression resulted in similar rates of early RAO, although the total duration of hemostasis was significantly shorter in the manual group.

C-terminal fragment of agrin (CAF) levels predict acute kidney injury after acute myocardial infarction.

BACKGROUND: Patients with acute myocardial infarction are at high risk for acute kidney injury. Novel biomarkers that can predict acute kidney injury in AMI may allow timely interventions. C-terminal fragment of agrin (CAF), a proteoglycan of the glomerular and tubular basement membrane, have been recently associated with rapid renal function deterioration and proximal tubular dysfunction. It is unknown whether elevated CAF levels may serve as a novel AKI biomarker in patients presenting with AMI. METHODS: In 436 persons enrolled in a multicenter prospective observational cohort study of patients with acute myocardial infarction, we measured plasma and urine levels of several kidney injury biomarkers including CAF, neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18) and cystatin-C.The relationship between biomarker levels at baseline and the development of AKI and long-term mortality were analyzed after adjustment for demographic and clinical variables. RESULTS: AKI incidence was up to 15% during hospitalization. The predictive accuracy for AKI of urinary CAF was similar to NGAL and superior to other tested kidney injury biomarkers. In a multivariate model that included all possible confounding variables only urinary CAF continued to be an independent marker for AKI (OR 1.35 95%CI 1.05 -1.74). During the 2 years follow-up, only plasma CAF levels remained a significant independent predictor of mortality (OR 2.5 95%CI 1.02-6.2; P = 0.04). CONCLUSIONS: Elevated CAF levels are associated with AKI in patients with acute myocardial infarction. Our study provides preliminary evidence that CAF levels may predict AKI and mortality after AMI in low risk patients with relative preserved kidney function at baseline.

Differences between perivascular adipose tissue surrounding the heart and the internal mammary artery: possible role for the leptin-inflammation-fibrosis-hypoxia axis.

AIM: The factors mediating the paracrine effects of perivascular adipose tissue (PVAT) in atherosclerosis are largely unknown. The adipokine leptin has been implicated in the increased cardiovascular risk in obesity and may locally promote neointima formation independently of circulating leptin levels. In patients with established coronary artery disease, we examined the expression of leptin as well as of its possible inducers in 'cardiac' PVAT surrounding the aortic root and coronary arteries (C-PVAT), and compared it to the PVAT surrounding the internal mammary artery (IMA-PVAT), a vessel resistant to atherosclerosis. METHODS AND RESULTS: Tissue specimens collected from male patients undergoing coronary artery bypass surgery were processed for real-time PCR, ELISA, in situ hybridization, and immunohistochemistry analysis. Leptin protein expression was elevated in C-PVAT compared to IMA-PVAT, independent of serum leptin levels. Compared to IMA-PVAT, C-PVAT exhibited more pronounced angiogenesis and inflammation, as indicated by significantly higher numbers of PECAM1-positive vessels and CD68-positive macrophages, and was characterized by a greater extent of fibrosis and hypoxia. Increased expression of hypoxia-inducible factor-1α and Fos-like antigen (FOSL)2, factors known to enhance leptin gene transcription, was observed in C-PVAT. As a proof of concept, exposure of human adipocytes to chemical hypoxia resulted in significantly increased FOSL2 and leptin mRNA levels. CONCLUSIONS: A higher degree of local tissue hypoxia and up-regulation of leptin expression in the perivascular adipose tissue, along with increased vascularization, inflammation, and fibrosis, may contribute to the increased atherosclerotic plaque burden in the coronary arteries compared to the IMA.

Spot urine albumin to creatinine ratio outperforms novel acute kidney injury biomarkers in patients with acute myocardial infarction.

BACKGROUND: Acute kidney injury (AKI) is a frequent complication in patients hospitalized for acute myocardial infarction (AMI), and is associated with in-hospital and long-term morbidity and mortality. We prospectively assessed the diagnostic performance of spot urine albumin to creatinine ratio (uACR) in an adequately sized multicenter cohort of patients admitted to hospital with AMI. We further compared uACR to novel renal injury associated biomarkers regarding their diagnostic ability. METHODS: We enrolled 805 consecutive patients presenting with acute ST-elevation and non-ST elevation AMI. Patients were assessed for presence of AKI at 48h post-admission and at hospital discharge using the Acute Kidney Injury Network (AKIN), the Acute Dialysis Quality Initiative [Risk, Injury and Failure (RIFLE)] criteria and the Kidney Disease: Improving Global Outcomes (KDIGO) criteria. Blood and urine sampling for neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), cystatin-C, and uACR assessment was performed during admission. RESULTS: The predictive accuracy of uACR was good (Area Under the Curve (AUC), 0.725; 95% CI 0.676-0.774) and was better compared to urine NGAL (P=0.007), urine (P<0.001) and plasma Cystatin-C (P=0.001). ROC analysis identified concentrations of ≥66.7µg/mg as having the best diagnostic accuracy. The use of uACR exhibited good discriminating ability independent to possible cofounders and additive regarding the use of novel biomarkers. CONCLUSIONS: The use of uACR can easily be applied in the clinical setting, allows for robust risk assessment and offers the potential to improve the management of AMI patients at risk for acute kidney injury.

Biomarkers of the extracellular matrix and of collagen fragments.

A great body of evidence has shown that extracellular matrix (ECM) alterations are present in the major types of cardiac diseases: ischemic heart disease, heart disease associated with pressure overload, heart disease associated with volume overload, and intrinsic myocardial disease or cardiomyopathy. Collagen, type I and III, is the principal structural protein found in the myocardium and its pro- or telopeptides are released into the circulation during the course of cardiovascular diseases. Therefore, these peptides may reflect collagen synthesis and break-down and also represent a much more useful tool to address ECM changes from a distance. Clinical trials have been performed during recent years to examine the usage of these peptides as diagnostic or prognostic biomarkers in heart failure (HF) patients. This review aims to summarize published data concerning cardiac ECM and its circulating biomarkers. Studies that focused on collagen metabolism related biomarkers in patients with HF are analyzed. Finally, limitations associated with the clinical use of the aforementioned biomarkers are also discussed.

Validation of a new risk score to predict contrast-induced nephropathy after percutaneous coronary intervention.

Contrast-induced nephropathy (CIN) is a frequent, potentially lethal complication of percutaneous coronary interventions (PCIs). We prospectively validated the diagnostic performance of a simple CIN risk score in a large multicenter international cohort of patients who underwent PCI. About 2,882 consecutive patients treated with elective or urgent PCI were enrolled. A simple CIN risk score was calculated for all patients by allocating points according to a prespecified scale (pre-existing renal disease = 2; metformin use = 2; previous PCI = 1; peripheral arterial disease = 2; and injected volume of contrast medium ≥300 ml = 1). CIN was defined as an increase, compared with baseline, of serum creatinine by ≥25%, or by ≥0.5 mg/dl, 48 hours after PCI. CIN occurred in 15.7% of the study population. The predictive accuracy of the CIN risk score was good (c-statistic 0.741, 95% confidence interval 0.713 to 0.769). Receiver-operating characteristic analysis identified a score of ≥3 as having the best diagnostic accuracy. Examination of the performance of the proposed risk score using different definitions of CIN yielded a robust predictive ability. The score exhibited good discrimination (area under the curve ≥0.700) across all predefined subgroups of the study population. Compared with 2 previously published risk scores for CIN, our score demonstrated higher discriminative ability and resulted in a net reclassification improvement and an integrated discrimination improvement (p <0.001). In conclusion, the new risk score can easily be applied in the setting of urgent or elective PCI, allows for robust risk assessment and offers the potential to improve the peri-interventional management of patients at risk for CIN.

Erythrocyte membrane cholesterol and lipid core growth in a rabbit model of atherosclerosis: modulatory effects of rosuvastatin.

BACKGROUND: Lipid core expansion is partly responsible for the conversion of a stable atherosclerotic lesion to a rupture-prone plaque. Intraplaque hemorrhage contributes to the accumulation of cholesterol within unstable plaques. In the present study, we investigated, using a rabbit model of atherosclerosis, the extent to which diet-induced increases in cholesterol content of erythrocyte membranes (CEM) contribute to lipid core expansion and the modulatory effect of rosuvastatin use. METHODS AND RESULTS: Rabbits fed with atherogenic diet (0.75% cholesterol) for 5 months exhibited advanced atherosclerotic lesions (mean plaque area, 0.39 ± 0.03 mm(2)), and lipid core size was associated with the concentration-time integral (CTI) of CEM levels (r=0.567, P=0.004) independent of other established predictors of lipid core size. Further experiments were performed by feeding rabbits atherogenic diet (1% cholesterol) for 3 months, followed by either normal diet or normal diet plus rosuvastatin for the next 3 months. Although no differences were observed in total plaque area between both groups, administration of rosuvastatin was associated with significantly smaller lipid cores, fewer macrophages within the lipid core, less microvessels as well as with lower CTI of CEM levels compared to normal diet alone. Moreover, intraplaque erythrocyte membranes covered a smaller lipid core area in rabbits under rosuvastatin plus normal diet as opposed to rabbits under diet alone. CONCLUSIONS: Increased CEM levels, induced by high-cholesterol diet, are associated with lipid core growth. Ingestion of a potent HMG-CoA reductase inhibitor (rosuvastatin) may decrease CEM levels, and this effect may contribute to regression of the lipid core.