Different Oxidative Stress and Inflammation Patterns of Diseased Left Anterior Descending Coronary Artery versus Internal Thoracic Artery.

BACKGROUND: Oxidative stress and inflammation are typically implied in atherosclerosis pathogenesis and progression, especially in coronary artery disease (CAD). Our objective was to investigate the oxidative stress and inflammation burden directly associated with atherosclerotic plaque in patients with stable coronary disease undergoing coronary artery bypass graft (CABG) surgery. Specifically, markers of oxidative stress and inflammation were compared in blood samples obtained from the atherosclerotic left anterior descending artery (LAD) and blood samples obtained from the healthy left internal thoracic artery (LITA), used as a bypass graft, within the same patient. METHODS: Twenty patients scheduled for off-pump CABG were enrolled. Blood samples were collected from the LITA below anastomosis and the LAD below the stenosis. Samples were analysed for oxidative stress (sNOXdp, H2O2, NO) and inflammation markers (TNFα, IL-6, IL-1ß, IL-10). RESULTS: The analysis showed a significant increase in oxidative stress burden in the LAD as compared to LITA, as indicated by higher sNOX2-dp and H2O2 levels and lower NO levels (p < 0.01). Also, pro-inflammatory cytokines were increased in the LAD as compared to the LITA, as indicated by higher TNFα and IL-6 amounts (p < 0.01). On the other hand, no significant differences could be seen regarding IL-1ß and IL-10 levels between the two groups. CONCLUSIONS: The oxidative stress and inflammatory burden are specifically enhanced in the LAD artery of stable coronary patients compared to systemic blood from the LITA of stable coronary patients.

Association Between NOX2-Mediated Oxidative Stress, Low-Grade Endotoxemia, Hypoalbuminemia, and Clotting Activation in COVID-19.

Low-grade endotoxemia by lipopolysaccharide (LPS) has been detected in COVID-19 and could favor thrombosis via eliciting a pro-inflammatory and pro-coagulant state. The aim of this study was to analyze the mechanism accounting for low-grade endotoxemia and its relationship with oxidative stress and clotting activation thrombosis in COVID-19. We measured serum levels of sNOX2-dp, zonulin, LPS, D-dimer, and albumin in 175 patients with COVID-19, classified as having or not acute respiratory distress syndrome (ARDS), and 50 healthy subjects. Baseline levels of sNOX2-dp, LPS, zonulin, D-dimer, albumin, and hs-CRP were significantly higher in COVID-19 compared to controls. In COVID-19 patients with ARDS, sNOX2-dp, LPS, zonulin, D-dimer, and hs-CRP were significantly higher compared to COVID-19 patients without ARDS. Conversely, concentration of albumin was lower in patients with ARDS compared with those without ARDS and inversely associated with LPS. In the COVID-19 cohort, the number of patients with ARDS progressively increased according to sNOX2-dp and LPS quartiles; a significant correlation between LPS and sNOX2-dp and LPS and D-dimer was detected in COVID-19. In a multivariable logistic regression model, LPS/albumin levels and D-dimer predicted thrombotic events. In COVID-19 patients, LPS is significantly associated with a hypercoagulation state and disease severity. In vitro, LPS can increase endothelial oxidative stress and coagulation biomarkers that were reduced by the treatment with albumin. In conclusion, impaired gut barrier permeability, increased NOX2 activation, and low serum albumin may account for low-grade endotoxemia and may be implicated in thrombotic events in COVID-19.

Atrial natriuretic peptide (ANP) modulates stress-induced autophagy in endothelial cells.

Atrial natriuretic peptide (ANP), a cardiac hormone involved in the regulation of water/sodium balance and blood pressure, is also secreted by endothelial cells, where it exerts protective effects in response to stress. Autophagy is an intracellular self-renewal process involved in the degradation of dysfunctional cytoplasmic elements. ANP was recently reported to act as an extracellular regulator of cardiac autophagy. However, its role in the regulation of endothelial autophagy has never been investigated. Here, we tested the effects of ANP in the regulation of autophagy in human umbilical vein endothelial cells (HUVECs). We found that ANP rapidly increases autophagy and autophagic flux at physiological concentrations through its predominant pathway, mediated by natriuretic peptide receptor type A (NPR-A) and protein kinase G (PKG). We further observed that ANP is rapidly secreted by HUVEC under stress conditions, where it mediates stress-induced autophagy through autocrine and paracrine mechanisms. Finally, we found that the protective effects of ANP in response to high-salt loading or tumor necrosis factor (TNF)-α are blunted by concomitant inhibition of autophagy. Overall, our results suggest that ANP acts as an endogenous autophagy activator in endothelial cells. The autophagy mechanism mediates the protective endothelial effects exerted by ANP.

Empagliflozin: Primus Inter Pares Among Sodium-Glucose Cotransporter-2 Inhibitors?

ABSTRACT: Sodium/glucose cotransporter-2 (SGLT2) inhibitors are a novel class of antidiabetic medications which have proved capable of providing breakthrough cardiovascular (CV) benefits in a variety of clinical scenarios, including patients with heart failure or obesity, irrespective of diabetic status. Several SGLT2 inhibitors are available, but the most prominent ones are canagliflozin, dapagliflozin, and empagliflozin. Several studies have focused on empagliflozin and its effects on the risk of heart failure incidence and recurrences. Most recently, empagliflozin has been recently tested in patients with recent myocardial infarction in the EMPAgliflozin on Hospitalization for Heart Failure and Mortality in Patients With aCuTe Myocardial Infarction randomized trial, with apparently ambiguous findings. The present viewpoint succinctly illustrates the main features of SGLT2 inhibitors as a pharmacologic class, their ever expanding role as a CV medication, and the comparative effectiveness of different individual SGLT2 inhibitors, explicitly commenting on the recent data on empagliflozin in patients with acute myocardial infarction. The reader will find in this article a poignant perspective on this novel avenue for CV prevention and treatment, which greatly expands the management armamentarium of CV practitioners. Indeed, we make the case that SGLT2 inhibitors have a clearly favorable class effect, with differences between individual agents mainly suitable for personalization of care and minimization of side effects.

Exposure to serum from exclusive heated tobacco product smokers induces mTOR activation and fibrotic features in human cardiac stromal cells.

Chronic smokers have increased risk of fibrosis-related atrial fibrillation. The use of heated-tobacco products (HTPs) is increasing exponentially, and their health impact is still uncertain. We aim to investigate the effects of circulating molecules in exclusive HTP chronic smokers on the fibrotic behavior of human atrial cardiac stromal cells (CSCs). CSCs were isolated from atrial tissue of elective cardiac surgery patients, and exposed to serum lots from young healthy subjects, stratified in exclusive HTP smokers, tobacco combustion cigarette (TCC) smokers, or nonsmokers (NS). CSCs treated with TCC serum displayed impaired migration and increased expression of pro-inflammatory cytokines. Cells cultured with HTP serum showed increased levels of pro-fibrotic markers, and reduced expression of connexin-43. Both TCC and HTP sera increased collagen release and reduced secretion of angiogenic protective factors from CSCs, compared to NS serum. Paracrine support to tube-formation by endothelial cells and to viability of cardiomyocytes was significantly impaired. Treatment with sera of both smokers groups impaired H2O2/NO release balance by CSCs and reduced early phosphorylation of several pathways compared to NS serum, leading to mTOR activation. Cotreatment with rapamycin was able to reduce mTOR phosphorylation and differentiation into aSMA-positive myofibroblasts in CSCs exposed to TCC and HTP sera. In conclusion, the circulating molecules in the serum of chronic exclusive HTP smokers induce fibrotic behavior in CSCs through activation of the mTOR pathway, and reduce their beneficial paracrine effects on endothelial cells and cardiomyocytes. These results point to a potential risk for cardiac fibrosis in chronic HTP users.

Exploring the landscape of academic retractions in medicine: a comprehensive umbrella review.

INTRODUCTION: The escalating trend of academic article retractions over the last decades raises concerns about scientific integrity, but heterogeneity in retractions and reasons for them pose a major challenge. We aimed to comprehensively overview systematic reviews focusing on retractions in the biomedical literature. EVIDENCE ACQUISITION: We abstracted salient features and bibliometric details from shortlisted articles. The Joanna Briggs Institute (JBI) Checklist for Systematic Reviews and Research Syntheses was used for validity appraisal. EVIDENCE SYNTHESIS: A total of 11 reviews were included, published between 2016 and 2023, and reporting on a total of 1851 retracted studies. Several major reasons for retractions were identified, spanning both misconduct (e.g., falsification, duplication, plagiarism) and non-misconduct issues (e.g., unreliable data, publishing problems). Correlates include author-related factors (number of authors, nationality) and journal-related factors (impact factor), with repeat offenders contributing significantly. Impacts of retractions is profound, affecting scholarly credibility, public trust, and resource utilization. CONCLUSIONS: In order to prevent retractions and amend their adverse effects, rigorous and transparent reporting standards, enhanced training in research ethics, strengthened peer review processes, and the establishment of collaborative and integrated research integrity offices are proposed.

Antiphospholipid Syndrome: Insights into Molecular Mechanisms and Clinical Manifestations.

Antiphospholipid syndrome (APS) is a complex systemic autoimmune disorder characterized by a hypercoagulable state, leading to severe vascular thrombosis and obstetric complications. The 2023 ACR/EULAR guidelines have revolutionized the classification and understanding of APS, introducing broader diagnostic criteria that encompass previously overlooked cardiac, renal, and hematologic manifestations. Despite these advancements, diagnosing APS remains particularly challenging in seronegative patients, where traditional tests fail, yet clinical symptoms persist. Emerging non-criteria antiphospholipid antibodies offer promising new diagnostic and management avenues for these patients. Managing APS involves a strategic balance of cardiovascular risk mitigation and long-term anticoagulation therapy, though the use of direct oral anticoagulants remains contentious due to varying efficacy and safety profiles. This article delves into the intricate pathogenesis of APS, explores the latest classification criteria, and evaluates cutting-edge diagnostic tools and therapeutic strategies.

Safety and efficacy studies of CRISPR-Cas9 treatment of sickle cell disease highlights disease-specific responses.

Fetal hemoglobin (HbF) reactivation expression through CRISPR-Cas9 is a promising strategy for the treatment of sickle cell disease (SCD). Here, we describe a genome editing strategy leading to reactivation of HbF expression by targeting the binding sites (BSs) for the lymphoma-related factor (LRF) repressor in the γ-globin promoters. CRISPR-Cas9 treatment in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny. LRF BS disruption did not impair HSPC engraftment and differentiation but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared with HD cells. We detected off-target activity and chromosomal rearrangements, particularly in SCD samples (likely because of the higher overall editing efficiency) but did not impact the target gene expression and HSPC engraftment and differentiation. Transcriptomic analyses showed that the editing procedure results in the up-regulation of genes involved in DNA damage and inflammatory responses, which was more evident in SCD HSPCs. This study provides evidence of efficacy and safety for an editing strategy based on HbF reactivation and highlights the need of performing safety studies in clinically relevant conditions, i.e., in patient-derived HSPCs.

Towards the Fifth Pillar for the Treatment of Heart Failure with Reduced Ejection Fraction: Vericiguat in Older and Complex Patients.

Heart failure with reduced ejection fraction (HFrEF) represents an emerging epidemic, particularly affecting frail, older, and multimorbid patients. Current therapy for the management of HFrEF includes four different classes of disease-modifying drugs, commonly referred to as 'four pillars', which target the neurohormonal system that is overactivated in HF and contributes to its progression. These classes of drugs include ß-blockers, inhibitors of the renin-angiotensin-aldosterone system, mineralocorticoid receptor antagonists, and sodium-glucose co-transporter-2 (SGLT2) inhibitors. Unfortunately, these agents cannot be administered as frequently as needed to older patients because of poor tolerability and comorbidities. In addition, although these drugs have dramatically increased the survival expectations of patients with HF, their residual risk of rehospitalization and death at 5 years remains considerable. Vericiguat, a soluble guanylate cyclase (sGC) stimulator, was reported to exert beneficial effects in patients with worsening HF, including older subjects, reducing the rate of both hospitalizations and deaths, with limited adverse effects and drug interaction. In this narrative review, we present the current state of art on vericiguat, with a particular focus on elderly and frail patients.

Oleuropein, a Component of Extra Virgin Olive Oil, Improves Liver Steatosis and Lobular Inflammation by Lipopolysaccharides-TLR4 Axis Downregulation.

Gut-dysbiosis-induced lipopolysaccharides (LPS) translocation into systemic circulation has been suggested to be implicated in nonalcoholic fatty liver disease (NAFLD) pathogenesis. This study aimed to assess if oleuropein (OLE), a component of extra virgin olive oil, lowers high-fat-diet (HFD)-induced endotoxemia and, eventually, liver steatosis. An immunohistochemistry analysis of the intestine and liver was performed in (i) control mice (CTR; n = 15), (ii) high-fat-diet fed (HFD) mice (HFD; n = 16), and (iii) HFD mice treated with 6 µg/day of OLE for 30 days (HFD + OLE, n = 13). The HFD mice developed significant liver steatosis compared to the controls, an effect that was significantly reduced in the HFD + OLE-treated mice. The amount of hepatocyte LPS localization and the number of TLR4+ macrophages were higher in the HFD mice in the than controls and were lowered in the HFD + OLE-treated mice. The number of CD42b+ platelets was increased in the liver sinusoids of the HFD mice compared to the controls and decreased in the HFD + OLE-treated mice. Compared to the controls, the HFD-treated mice showed a high percentage of intestine PAS+ goblet cells, an increased length of intestinal crypts, LPS localization and TLR4+ expression, and occludin downregulation, an effect counteracted in the HFD + OLE-treated mice. The HFD-fed animals displayed increased systemic levels of LPS and zonulin, but they were reduced in the HFD + OLE-treated animals. It can be seen that OLE administration improves liver steatosis and inflammation in association with decreased LPS translocation into the systemic circulation, hepatocyte localization of LPS and TLR4 downregulation in HFD-induced mouse model of NAFLD.

CAR, mGPS and hs-mGPS: What is among them the best gero-biomarker for age-related diseases? And for what clinical application?

The identification of biomarkers linked to the onset, progression, and prevention of age-related diseases (ARD), in the era of personalized medicine, represents the best goal of geroscience. Geroscience has the fundamental role of exploring and identifying the biological mechanisms of aging to suggest interventions capable of stopping/delaying the many pathological conditions and disabilities related to age. Therefore, it has become its key priority, as well as that of clinical practice and research, based on identifying and validating a range of biomarkers, geromarkers, which can be used to diagnostic, prognostic, or predictive clinical purposes. Indeed, geromarkers have, the potential to predict ARD trajectories and facilitate targeted interventions to slow down the related disabilities. Here our attention is paid to the inflammatory indexes (CAR, mGPS, hs-mGPS) linked to the relationship between the plasma levels of two inflammatory analytes, the typical positive protein of the acute phase, and the negative one, i.e. c-reactive protein (CRP) and albumin, respectively. These indexes allow us to understand the magnitude of the two main mechanisms predicted to influence the aging process, including inflammation and immunosenescence, as well as the degree of ARD severity. Evidence on their relationship with ARD is widely reported and discussed, to understand which can represent the best ARD geromarker, and its clinical application.

Prediction of 30-day mortality after surgery for infective endocarditis using risk scores: Insights from a European multicenter comparative validation study.

BACKGROUND: It remains unclear today whether risk scores created specifically to predict early mortality after cardiac operations for infective endocarditis (IE) outperform or not the European System for Cardiac Operative Risk Evaluation II (EuroSCORE II). METHODS: Perioperative data and outcomes from a European multicenter series of patients undergoing surgery for definite IE were retrospectively reviewed. Only the cases with known pathogen and without missing values for all considered variables were retained for analyses. A comparative validation of EuroSCORE II and 5 specific risk scores for early mortality after surgery for IE-(1) STS-IE (Society of Thoracic Surgeons for IE); (2) PALSUSE (Prosthetic valve, Age ≥70, Large intracardiac destruction, Staphylococcus spp, Urgent surgery, Sex (female), EuroSCORE ≥10); (3) ANCLA (Anemia, New York Heart Association class IV, Critical state, Large intracardiac destruction, surgery on thoracic Aorta); (4) AEPEI II (Association pour l'Étude et la Prévention de l'Endocardite Infectieuse II); (5) APORTEI (Análisis de los factores PROnósticos en el Tratamiento quirúrgico de la Endocarditis Infecciosa)-was carried out using calibration plot and receiver-operating characteristic curve analysis. Areas under the curve (AUCs) were compared 1:1 according to the Hanley-McNeil's method. The agreement between APORTEI score and EuroSCORE II of the 30-day mortality prediction after surgery was also appraised. RESULTS: A total of 1,012 patients from 5 European university-affiliated centers underwent 1,036 cardiac operations, with a 30-day mortality after surgery of 9.7%. All IE-specific risk scores considered achieved better results than EuroSCORE II in terms of calibration; AEPEI II and APORTEI score showed the best performances. Despite poor calibration, EuroSCORE II overcame in discrimination every specific risk score (AUC, 0.751 vs 0.693 or less, P = .01 or less). For a higher/lesser than 20% expected mortality, the agreement of prediction between APORTEI score and EuroSCORE II was 86%. CONCLUSION: EuroSCORE II discrimination for 30-day mortality after surgery for IE was higher than 5 established IE-specific risk scores. AEPEI II and APORTEI score showed the best results in terms of calibration.

Comparative effects of different antiplatelet strategies in carriers of CYP2C19 loss-of-function alleles: a network meta-analysis.

BACKGROUND: Carriers of cytochrome 2C19 (CYP2C19) loss-of-function (LoF) alleles treated with clopidogrel have impaired drug metabolism, resulting in reduced active metabolite levels, high platelet reactivity (HPR), and an increased risk of thrombotic events. Several alternative antiplatelet therapies have been proposed to overcome HPR in these patients, but their comparative effects remain poorly explored. METHODS: Randomized controlled trials (RCTs) comparing different oral antiplatelet therapies in carriers of CYP2C19 LoF alleles undergoing percutaneous coronary interventions (PCI) were included. A frequentist network meta-analysis was conducted to estimate mean difference (MD) or odds ratios and 95% confidence intervals (CI). The primary outcome was platelet reactivity assessed by VerifyNow and reported as P2Y12 reaction unit (PRU). The secondary outcome was the rate of HPR. Standard dose of clopidogrel (75 mg daily) was used as a reference treatment. RESULTS: A total of 12 RCTs testing 6 alternative strategies (i.e. clopidogrel 150 mg, prasugrel 3.75 mg, 5 mg, and 10 mg, ticagrelor 90 mg bid, and adjunctive cilostazol 100 mg bid) were included in the network. Compared with standard-dose clopidogrel, the greatest reduction in PRU was observed with prasugrel 10 mg (MD -127.91; 95% CI -141.04; -114.78) and ticagrelor 90 mg bid (MD -124.91; 95% CI -161.78; -88.04), followed by prasugrel 5 mg (MD -76.33; 95% CI -98.01; -54.65) and prasugrel 3.75 mg (MD -73.00; 95% CI -100.28; -45.72). Among other strategies, adjunctive cilostazol (MD -42.64; 95% CI -64.72; -20.57) and high-dose clopidogrel (MD -32.11; 95% CI -51.33; -12.90) were associated with a modest reduction in PRU compared with standard-dose clopidogrel. CONCLUSION: Among carriers of CYP2C19 LoF alleles undergoing PCI, standard-dose prasugrel or ticagrelor are most effective in reducing platelet reactivity, while double-dose clopidogrel and additional cilostazol showed modest effects. Reduced-dose of prasugrel may represent a balanced strategy to overcome HPR without a significant increase in bleeding. The clinical implications of these pharmacodynamic findings warrant further investigation.

ECMO Alone Versus ECPELLA in Patients Affected by Cardiogenic Shock: The Multicenter EVACS Study.

The objective was to investigate the outcomes of concomitant venoarterial extracorporeal membrane oxygenation (ECMO) and left ventricular unloading with Impella (ECPELLA) compared with ECMO alone to treat patients affected by cardiogenic shock. Data from patients needing mechanical circulatory support from 4 international centers were analyzed. Of 438 patients included, ECMO alone and ECPELLA were adopted in 319 (72.8%) and 119 (27.2%) patients, respectively. Propensity score matching analysis identified 95 pairs. In the matched cohort, 30-day mortality rates in the ECMO and ECPELLA were 49.5% and 43.2% ( P = 0.467). The incidences of complications did not differ significantly between groups ( P = 0.877, P = 0.629, P = 1.000, respectively). After a median follow-up of 0.18 years (interquartile range 0.02-2.55), the use of ECPELLA was associated with similar mortality compared with ECMO alone (hazard ratio 0.81, 95% confidence interval 0.54-1.20, P = 0.285), with 1-year overall survival rates of 51.3% and 46.6%, for ECPELLA and ECMO alone, respectively. ECMO alone and ECPELLA are both effective strategies in patients needing mechanical circulatory support for cardiogenic shock, showing similar rates of early and mid-term survival.

NOX2 as a Biomarker of Academic Performance: Evidence from University Students during Examination.

BACKGROUND: Cortisol levels, oxidative stress, and lower cerebral performance seem to be closely related. This study aimed to evaluate the question of whether exam stress affected oxidative stress and endothelial function parameters in the salivary samples of students. METHODS: A total of 114 healthy students were recruited. All students were subjected to a 21-item DASS questionnaire to assess perceived stress. Cortisol levels, biomarkers of oxidative stress, and endothelial function were evaluated at T0, during the semester, and T1, in the morning before the exam, in saliva samples. In vitro, HUVECs were stimulated with cortisol, and oxidative stress and endothelial function parameters were evaluated. RESULTS: At T1, cortisol levels were significantly increased compared with the levels during the semester. Moreover, exam results correlated inversely with the DASS score at T1. In addition, NOX2, H2O2 and endothelin-1 significantly increased, while NO bioavailability decreased. In vitro, HUVECs treatment with human cortisol determined the increase of oxidative stress and the decrease of endothelial function, in association with impaired eNOS phosphorylation. CONCLUSION: NOX2-mediated oxidative stress is a mechanism that could mediate cortisol-induced transient endothelial dysfunction during academic examination. Therefore, strategies to monitor or modulate oxidative stress could help students to reduce the impact of examination-related stress.

Comparative diagnostic accuracy of transcranial Doppler and contrast-enhanced transthoracic echocardiography for the diagnosis of patent foramen ovale and atrial septal defect.

BACKGROUND: Recognition of right-to-left shunt is crucial in the work-up of patients with suspected patent foramen ovale (PFO) or atrial septal defect (ASD). While transesophageal echocardiography (TEE) remains the gold standard diagnostic tool for the anatomic assessment of PFO/ASD, transcranial Doppler (TCD) and contrast-enhanced transthoracic echocardiogram (CE-TTE) hold the promise of providing minimally invasive yet accurate clinical details. Their comparative accuracy remains however debated. METHODS: We conducted a retrospective observational study leveraging our extensive institutional experience with systematic TCD and CE-TTE in patients with suspected PFO/ASD. Several measures of diagnostic test accuracy were computed, with point estimates and 95% confidence intervals, when applicable. RESULTS: A total of 1358 patients were included, with age 48±14 years and 772 (58%) women. Tests were performed for diagnostic purposes in 797 (58.6%) and during follow-up in 740 (54.5%). A PFO was eventually diagnosed in 1038 (77.9%) patients, and an ASD in 60 (4.5%). Agreement between TCD and CE-TTE occurred in 1309 (85.2%) cases, with TCD yielding worse findings than CE-TTE in 91 (5.9%) patients, and vice versa in 137 (8.9%), yielding a Cohen kappa of 78.6% (95% CI: 76.3-81.1%) and a highly significant P value at McNemar test (P<0.001). After dichotomization, and using TCD as benchmark, CE-TTE yielded sensitivity 96.9%, specificity 95.1%, area under the curve 92.1%, and P=0.249. Similar findings were obtained when focusing only on diagnostic tests or follow-up ones (Cohen kappa respectively 74.0% [70.2-77.1%], P<0.001 and 80.3% [76.4-84.3%], P<0.001). Notably, Valsalva was necessary to disclose the presence of shunt during TCD in 487 (31.7%) patients and during CE-TTE in 482 (31.4%) cases. Finally, performance of TCD and CE-TTE in a subset of patients eventually undergoing TTE was quite similar. CONCLUSIONS: The diagnostic accuracy of CE-TTE appears favorable, and this imaging test may identify patients who may be missed if only TCD is used to screen patients with suspected PFO/ASD. Accordingly, CE-TTE is recommended as an adjunct diagnostic modality for all patients with a high pre-test probability of PFO/ASD and right-to-left shunt.