Pathways linking atherosclerosis to aortic stenosis

Cardiovascular disease is the most common cause of death world-wide where atherosclerosis is the main culprit and aortic valve disease accounts about two percent of all CVD deaths. Atherosclerosis is a lipid and inflammation driven disease that share many features with aortic valve stenosis (AVS). Globally, the prevalence of AVS has been estimated to over 10 million patients and the incidence to over 12 500 new cases annually which is likely increasing due to increased longevity, yet no medical treatment is available. A link between atherosclerosis and AVS has previously been established by overlapping prevalence and common pathobiological hallmarks including lipid infiltration, inflammation, and calcification. Recent genetic studies have demonstrated several loci in which single nucleotide polymorphisms are associated with both diseases. However, there is also evidence pointing to separate etiologies including disease specific genetic risk factors, histopathological differences, and isolated clinical presentation. The aim of this thesis was to establish the interplay between atherosclerosis and AVS. A physiologic part was covered in Article I, specific mechanisms in Article II-IV and molecular epidemiology in Article IV. In Article I, arterial stiffness was determined in a cohort with ascending aortic dilatation and/or aortic valve disease before and after cardiac surgery. Arterial stiffness correlates with atherosclerotic cardiovascular disease and aggravates the increased left ventricular stress in AVS. Cardio-ankle vascular index (CAVI) measures arterial stiffness from the heart to the ankle and was lower in subjects with AVS compared with aortic regurgitation and ascending aortic dilation, before surgery, despite being older. In contrast, aortic stiffness assessed by carotid femoral pulse wave velocity (cfPWV) was not different between the groups. After surgery, CAVI but not cfPWV increased in patients with AVS but remained unchanged in patients undergoing aortic surgery. Age, diabetes, lower body mass index, decreased ejection time and lower preoperative CAVI was associated with an increased CAVI after surgery. The results suggest that AVS may mask an increased arterial stiffness if peripheral arteries are included in the measurement. Also, ejection time emerged as an important variable to account for when measuring arterial stiffness in aortic valve disease patients. Future work should aim to establish if arterial stiffness may be used to risk-stratify AVS patients. In Article II, the impact of a single nucleotide polymorphism (SNP) within FADS1 on aortic valve gene expression and fatty acid composition was identified. Fatty acid desaturase (FADS)1 and FADS2 encode rate limiting enzymes in the metabolism of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) and the SNP within this locus is associated with lower risk of both AVS and CAD. The SNP rs17547 was associated with FADS2 mRNA expression in calcified aortic valve tissue and the enzymatic activity of both FADS1 and FADS2. In addition, the aortic valve omega-3 PUFA docosahexaenoic acid proportion was higher in non-calcified compared with calcified tissue and positively correlated with the SNP. The results indicate that the protective effects of the SNP might be mediated via an increased DHA proportion in the aortic valve and/or possibly via downstream mediators from DHA such as specialized pro-resolving mediators which have been shown to dampen inflammation. Further pathophysiological evidence of shared pathways between CAD and AVS was obtained in Article III. The presence of antiphospholipid antibodies (aPL) in the general population is higher in patients with a recent myocardial infarction. Positivity for antibodies against β2-glycoprotein I and/or cardiolipin of IgG isotype was identified to be 8-fold higher in AVS patients compared with matched controls. In aortic valve tissue, aPL positivity was associated with downregulated interferon pathways and upregulated pathways related to mechanosensory signaling. Importantly, the differentially expressed genes could predict resilient (healthy), thickened (fibrotic) and calcified aortic valve tissue with high accuracy using supervised machine learning models suggesting a tight relationship between aPL related genes and local disease progression. The overall results imply that aPL IgG in the general population (without rheumatic disease) could be a risk factor for AVS and may potentially be used guide AVS precision medicine. In Article IV, CAD associated gene expression in aortic valve tissue was identified. First, the prevalence of CAD in a contemporary surgical tricuspid AVS cohort was established at 49% and was associated with claudication, smoking, male sex, and diabetes. An exploratory analysis of aortic valve transcriptomic data from 74 patients revealed that severe CAD, affecting 2 or 3 vessel territories, was associated with the most prominent difference in gene expression. The differentially expressed genes were primarily found in non-calcified tissue and were enriched in pathways related to oxidative stress, inflammation, and lipids. Furthermore, a supervised machine learning model could predict if aortic valve tissue stemmed from patients with severe CAD, at high accuracy. The most important gene predictors of severe CAD could further be used to predict atherosclerotic or macroscopically normal carotid artery tissue. The results suggest that AVS patients with concomitant severe CAD exhibit more atherosclerosis related mechanisms in non-calcified tissue, ultimately leading to a common end-stage disease with severe AVS. In summary, the results in this thesis demonstrate that AVS may be a cause of masked systemic arterial stiffness. Furthermore, pathways related to fatty acid metabolism and aPL are implicated in the pathophysiology of AVS and patients with severe CAD exhibit upregulated pathways related to atherosclerosis in the aortic valve. Collectively, pathways linking and differentiating aortic valve and vascular atherosclerotic disease were unraveled which open up for novel precision treatment regiments to halt AVS

The impact of antihypertensive treatment on the progression of cardiac dysfunction and aortic stenosis

L'hypertension artérielle et la sténose aortique (SA) font partie des maladies cardiovasculaires qui sont le plus répandues dans les pays à revenus élevés. La SA affecte 2% des adultes de plus de 65 ans et son incidence augmente avec l'âge. L'hypertension est une comorbidité qui affecte de 30% à 80% des patients atteints de SA. Elle a un impact sur le développement et la progression de la SA. Chez les patients atteints des deux maladies, le ventricule gauche (VG) fait face à une double charge ce qui accélère son remodelage et sa perte de fonction. Ainsi, l'hypertension est associée à un mauvais pronostic chez les patients atteints de SA. Jusqu'à aujourd'hui, il n'y a pas de traitement pharmacologique pour arrêter ou réduire la progression de la SA, la dysfonction systolique du VG et le remodelage ventriculaire conséquents à la maladie. Quelques études ont montré que les bloqueurs des récepteurs de l'angiotensine (BRA), qui sont un des traitements principaux de l'hypertension, peuvent avoir un impact bénéfique sur les mécanismes physiopathologiques de la SA et aussi un effet protecteur contre la dysfonction systolique du VG. Peu d'études ont évalué l'impact des BRA sur la progression de la SA et la dysfonction systolique du VG simultanément. De plus, aucun essai clinique n'a étudié les effets des traitements hypertenseurs par les BRA sur la progression de la SA ainsi que sur la dysfonction systolique du VG causée par ces deux maladies. Par conséquent, l'hypothèse principale de ce projet de maîtrise est que les BRA réduisent la dysfonction systolique du VG chez les patients hypertendus atteints de SA. L'objectif principal est de déterminer l'impact des traitements hypertenseurs (BRA) sur la progression de la dysfonction systolique du VG chez les patients hypertendus atteints de SA.Hypertension and aortic stenosis (AS) are two cardiovascular diseases with elevated prevalence in high-income countries. AS affects 2% of adults over 65 years old and its incidence increases with age. Hypertension is a comorbidity that affects 30% to 80% of patients with AS. It has an impact on the development and the progression of AS. In patients with both diseases, the left ventricle (LV) faces a double load, accelerating its remodeling and function impairment. Thus, hypertension is linked to worse clinical outcomes and prognosis in those patients. To date, there is no pharmacological treatment to stop or reduce the progression of AS, LV systolic dysfunction, or LV remodeling produced by this disease. Some previous studies have shown that angiotensin receptor blockers (ARBs), one of the first-line treatments of hypertension, may have a beneficial impact on the physiopathological mechanism of AS and a protective effect on LV systolic function as well. Few studies have evaluated the impact of ARBs on AS progression and LV systolic dysfunction at the same time. Moreover, no clinical trial has studied the effect of antihypertensive treatment by ARBs on the AS progression and the LV systolic dysfunction caused by both diseases. Therefore, the principal hypothesis of this master project is that the ARBs reduce the LV systolic dysfunction in hypertensive patients with AS. The principal objective of this pilot study is to determine the impact of antihypertensive treatment with ARBs on the progression of LV systolic dysfunction in hypertensive patients with AS

Risk factors for coronary, aortic arch and carotid calcification; The Rotterdam Study

This study was performed to examine the association of cardiovascular risk factors with calcification in the coronary arteries, aortic arch and carotid arteries, assessed by multislice computed tomography (MSCT). This study was embedded in the Rotterdam Study, a population-based study in subjects aged 55 years and over. From October 2003 until December 2004, subjects were invited to undergo a MSCT scan. Coronary, aortic arch and carotid calcification were quantified according to the Agatston score. Analyses were performed in the first 1003 subjects. Age and current smoking were the strongest independent risk factors for arterial calcification. The odds ratio (OR) for age in women, irrespective of the vessel bed, was 1.1 (P<0.001) and in men it was 1.2 with aortic arch and 1.1 with carotid calcification (both P<0.001). Current smoking was associated with aortic arch calcification with an OR of 3.5 in women and of 4.7 in men (both P<0.001); and with carotid calcifi

Primary Cardiovascular Disease Prevention: Risk Factors Control vs. Imaging Subclinical Atherosclerosis

The burden of cardiovascular disease in developed countries has shown dramatic improvements over the last 50 years, largely due the identification and control of major risk factors including, smoking hypertension and high cholesterol. However, due to the significant increase in obesity and diabetes CVD incidence rates will not reduce as far over over the next years. Risk prediction in asymptomatic individuals remains a major challenge. Primary preventive treatment is currently based on the assessment of individual's global risk mainly through screening of conventional risk factors and their treatment with lifestyle intervention and pharmacotherapy, often based on multivariate risk equations, and yet a large proportion of CVD still occurs in individuals who are classified as carrying low- or intermediate-risk according to the risk scores. Atherosclerosis is the most common pathophysiologic process underlying CVD, often after a prolonged asymptomatic phase during which it may be possible to modify the course of the disease. Unlike conventional probabilistic risk scores, non-invasive imaging techniques such as carotid intima-media thickness (CIMT) along with plaque assessment (Figure 2), measured by B-mode ultrasound, and coronary calcium scoring (CAC) detected by CT scan have the advantage of direct visualization of the consequences of atherosclerosis on the arterial system. We consider the proposal that imaging of subclinical atherosclerosis is superior to risk equations as it directly identifies the disease and can effectively predict the risk of future CV events in low- and intermediate-risk individuals. In addition, imaging can improve the adherence to guidelines based treatment in patients and their physicians

Effects of an Angiotensin II Type 1 Receptor Blocker on Cardiovascular Calcification

Aims: Three types of cardiovascular calcification are commonly found in humans: arterial calcification, intimal calcification, and calcific aortic valve disease. Very little is known about the mechanisms driving cardiovascular calcification despite serious clinical implications and a clear association with morbidity and mortality. Indeed, it is even unclear whether the same factors are involved in arterial, intimal, and valvular calcification. The objective of this study was to elucidate the effects of an angiotensin II type 1 receptor blocker (ARB) on the progression of cardiovascular calcification in male New Zealand White rabbits. Where appropriate, statins were examined in conjunction and in combination with ARBs. Methods and Results: In vivo and ex vivo techniques were used to assess overall disease burden and the extent of calcification including magnetic resonance imaging, micro-computed tomography, histology, and immunohistochemistry. ARB administration significantly inhibited progression of arterial calcification (2.80 ± 1.17 versus 0.01 ± 0.01 % calcified tissue in Cholesterol and ARB-treated, respectively; P \u3c 0.05), but not intimal or valvular calcification. ARB treatment significantly reduced atherosclerotic lesion area when delivered alone (95.50 ± 1.94 versus 61.61 ± 10.17 % lesion area in Cholesterol and ARB-treated, respectively; P \u3c 0.05), but not when combined with statin therapy (92.39 ± 3.25 % in ARB+Statin; P \u3c 0.05 when compared to ARB monotherapy). Finally, ARB-treated animals had significantly increased valvular calcium. Conclusions: This study provides evidence that ARBs robustly inhibit arterial calcification and is the first to suggest ARBs as a novel treatment option for those at risk for cardiovascular calcification. It also suggests that ARBs may not be beneficial for those at risk for intimal or valvular calcification. These disparate results suggest that the three types of cardiovascular calcification are distinct from one another and provides impetus to further examine the underlying molecular mechanisms at play in these debilitating disease processes

Aortic valve calcification : in vivo and ex vivo evaluation

Aortic valve calcification (AVC) or thickening is found in around one fifth of the general population between 65-75 years of age and increasingly thereafter. The process of aortic valve thickening and calcification is not only an aging (wear and tear) process of the valve leaflets. It is now considered to be closely related to atherosclerosis. The presence and the degree of AVC have been shown to have prognostic value in patients with cardiovascular diseases and in the general population. Despite its importance, there is no widely agreed upon scoring system that objectively quantifies AVC. The aim of this project was to investigate different methods for evaluating AVC, using transthoracic and transoesophageal echocardiography (TTE and TOE), intra-operative assessment of the valve (IOS) and ex vivo evaluation based on computed tomography (CT) of the excised aortic valves and based on valve weight. A 5-grade scoring system was used for the visual assessment of AVC on real-time and still TTE and TOE images of the aortic valve, as well as intra-operatively. Computer-based greyscale measurement (GSM) software was used to obtain a quantitative-ultrasoundbased AVC measure, which was compared with the visual AVC score from TTE and TOE, whereas IOS was used as the gold-standard method. We also aimed at identifying the most suitable ex vivo conditions and CT parameters for optimal AVC scanning by CT in a calcium hydroxyapatite (CaHA) phantom study. The TTE and TOE AVC scores and IOS were compared to the weight and the CT CaHA mineral mass (MM) index of the explanted aortic valves. The study cohorts were recruited among patients undergoing aortic valve replacement because of aortic valve disease and/or ascending aorta aneurysm. In Study I, which included 185 patients, we showed that the visual evaluation of AVC using real-time TTE images yielded better correlations with IOS than did quantitative still frame measures based on GSM (r = 0.83 vs 0.64, respectively). In Study II, AVC scores based on TTE and TOE realtime images from 169 patients showed strong correlations with IOS (r = 0.83 and 0.82, respectively). GSM-based measures correlated less well with IOS, even for TOE (r = 0.52). In Study II, we also showed that TOE was more accurate than TTE in diagnosing the aortic valve phenotype. In the CTphantom-based, methodological Study III, we identified optimal CT scanning and reconstruction parameters, as well as the most suitable medium (normal saline) for ex vivo tissue CT-based calcium scoring. In Study IV, 155 operatively explanted aortic valves were weighed and scanned ex vivo by CT, and CaHA MM was measured. The CaHA MM exhibited a strong correlation with valve weight (r = 0.91), whereas AVC scores based on TTE, TOE and IOS showed weaker correlations. Conversely, echocardiographic and intra-operative AVC evaluation showed a better correlation with haemodynamic parameters compared with ex vivo CT AVC scoring. In conclusion, real-time echocardiographic images are crucial for accurate AVC scoring, regardless of whether TTE or TOE is used. Echocardiographic AVC scoring was as accurate as intraoperative assessment, according to valve weight and ex vivo CT. For ex vivo calcium scoring by CT, in addition to scanning and reconstruction parameters, using saline as the surrounding medium seems to be important. TOE is more accurate than TOE in detecting the bicuspid aortic valve

Imaging calcification in aortic stenosis

BACKGROUND Aortic stenosis is a common and potentially fatal condition in which fibro-calcific changes within the valve leaflets lead to the obstruction of blood flow. Severe symptomatic stenosis is an indication for aortic valve replacement and timely referral is essential to prevent adverse clinical events. Calcification is believed to represent the central process driving disease progression. 18F-Fluoride positron emission tomography computed tomography (PET-CT) and CT aortic valve calcium scoring (CT-AVC) quantify calcification activity and burden respectively. The overarching aim of this thesis was to evaluate the applications of these techniques to the study and management of aortic stenosis. METHODS AND RESULTS REPRODUCIBILITY The scan-rescan reproducibility of 18F-fluoride PET-CT and CT-AVC were investigated in 15 patients with mild, moderate and severe aortic stenosis who underwent repeated 18F-fluoride PET-CT scans 3.9±3.3 weeks apart. Modified techniques enhanced image quality and facilitated clear localization of calcification activity. Percentage error was reduced from ±63% to ±10% (tissue-to-background ratio most-diseased segment (MDS) mean of 1.55, bias -0.05, limits of agreement - 0·20 to +0·11). Excellent scan-rescan reproducibility was also observed for CT-AVC scoring (mean of differences 2% [limits of agreement, 16 to -12%]). AORTIC VALVE CALCIUM SCORE: SINGLE CENTRE STUDY Sex-specific CT-AVC thresholds (2065 in men and 1271 in women) have been proposed as a flow-independent technique for diagnosing severe aortic stenosis. In a prospective cohort study, the impact of CT-AVC scores upon echocardiographic measures of severity, disease progression and aortic valve replacement (AVR)/death were examined. Volunteers (20 controls, 20 with aortic sclerosis, 25 with mild, 33 with moderate and 23 with severe aortic stenosis) underwent CT-AVC and echocardiography at baseline and again at either 1 or 2-year time-points. Women required less calcification than men for the same degree of stenosis (p<0.001). Baseline CT-AVC measurements appeared to provide the best prediction of subsequent disease progression. After adjustment for age, sex, peak aortic jet velocity (Vmax) ≥ 4m/s and aortic valve area (AVA)<1 cm2, the published CT-AVC thresholds were the only independent predictor of AVR/death (hazard ratio = 6.39, 95% confidence intervals, 2.90-14.05, p<0.001). AORTIC VALVE CALCIUM SCORE: MULTICENTRE STUDY CT-AVC thresholds were next examined in an international multicenter registry incorporating a wide range of patient populations, scanner vendors and analysis platforms. Eight centres contributed data from 918 patients (age 77±10, 60% male, Vmax 3.88±0.90 m/s) who had undergone ECG-gated CT within 3 months of echocardiography. Of these 708 (77%) had concordant echocardiographic assessments, in whom our own optimum sex-specific CT-AVC thresholds (women 1377, men 2062 AU) were nearly identical to those previously published. These thresholds provided excellent discrimination for severe stenosis (c-statistic: women 0.92, men 0.88) and independently predicted AVR and death after adjustment for age, sex, Vmax ≥4 m/s and AVA <1 cm2 (hazards ratio, 3.02 [95% confidence intervals, 1.83-4.99], p<0.001). In patients with discordant echocardiographic assessments (n=210), CT-AVC thresholds predicted an adverse prognosis. BICUSPID AORTIC VALVES Within the multicentre study, higher continuity-derived estimates of aortic valve area were observed in patients with bicuspid valves (n=68, 1.07±0.35 cm) compared to those with tri-leaflet valves (0.89±0.36 cm p<0.001,). This was despite no differences in measurements of Vmax (p=0.152), or CT-AVC scores (p=0.313). The accuracy of AVA measurments in bicuspid valves was therefore tested against alternative markers of disease severity. AVA measurements in bicuspid valves demonstrated extremely weak associations with CT-AVC scores (r2=0.08, p=0.02) and failed to correlate with downstream markers of disease severity in the valve and myocardium and against clinical outcomes. AVA measurements in bicuspid patients also failed to independently predict AVR/death after adjustment for Vmax ≥4 m/s, age and gender. In this population CT-AVC thresholds (women 1377, men 2062 AU) again provided excellent discrimination for severe stenosis. CONCLUSIONS Optimised 18F-fluoride PET-CT scans quantify and localise calcification activity, consolidating its potential as a biomarker or end-point in clinical trials of novel therapies. CT calcium scoring of aortic valves is a reproducible technique, which provides diagnostic clarity in addition to powerful prediction of disease progression and adverse clinical events

Assessment of aortic stenosis using modern non-invasive imaging techniques

Introduction. Aortic stenosis is characterised both by progressive narrowing of the valve and the hypertrophic response of the left ventricle. The purpose of this thesis was to study the contribution of inflammation and calcification to valve narrowing using Positron Emission and Computed Tomography (PET/CT) and to investigate the hypertrophic response using cardiovascular magnetic resonance (CMR). Methods. PET/CT studies. Patients with aortic sclerosis and mild, moderate and severe stenosis were prospectively compared to matched control subjects. Aortic valve severity was determined by echocardiography. Calcification and inflammation in the aortic valve and coronary arteries were assessed by sodium 18-­‐fluoride (18F-­‐NaF) and 18-­‐fluorodeoxyglucose (18F-­‐FDG) uptake using PET. CMR studies. Consecutive patients with moderate or severe aortic stenosis undergoing CMR were enrolled into a registry. Patients who received gadolinium contrast were categorised into absent, mid-­‐ wall or infarct patterns of late gadolinium enhancement (LGE) by blinded independent observers. Patients follow-­‐up was completed using patient questionnaires, source record data and the National Strategic Tracing Scheme. After excluding those patients with concomitant triggers to LV remodeling, the extent and patterns of hypertrophy were investigated based upon measurements of indexed LV mass, indexed LV volume and the relative wall mass. Results. PET/CT studies. 121 subjects (20 controls; 20 aortic sclerosis; 25 mild, 33 moderate and 23 severe aortic stenosis) were studied. Quantification of tracer uptake within the valve demonstrated excellent inter-­‐observer reproducibility with no biases and limits of agreement of ±0.21 (18F-­‐NaF) and ±0.13 (18F-­‐FDG) for maximum tissue-­‐to-­‐background ratios (TBR). Activity of both tracers was higher in patients with aortic stenosis than control subjects (18F-­‐NaF: 2.87±0.82 vs 1.55±0.17; 18F-­‐ FDG: 1.58±0.21 vs 1.30±0.13; both P<0.001). 18F-­‐NaF uptake displayed a progressive rise with valve severity (r2=0.540, P<0.001) with a more modest increase observed for 18F-­‐FDG (r2=0.218; P<0.001). Amongst patients with aortic stenosis, 91% had increased 18F-­‐NaF (>1.97) and 35% increased 18F-­‐ FDG (>1.63) uptake. Increased 18F-­‐NaF uptake was also observed in the coronary arteries in a subset of patients with atherosclerosis. These patients (n=40) had higher rates of prior cardiovascular events (p=0.016) and angina (p=0.023), and higher Framingham risk scores (p=0.011). CMR studies. 143 patients (aged 68±14 years; 97 male) were followed up for 2.0±1.4 years and 27 died. Compared to those with no LGE (n=49), univariate analysis revealed that patients with mid-­‐wall fibrosis (n=54) had an eight-­‐fold increase in all-­‐cause mortality despite similar aortic stenosis severity and coronary artery disease burden. Patients with an infarct pattern (n=40) had a six-­‐fold increase. Mid-­‐wall fibrosis (HR 5.35 [95% CI 1.16-­‐24.56]; P=0.03) emerged as an independent predictor of all cause mortality by multivariate analysis. The pattern of LV remodelling was studied in 91 patients (61±21 years; 57 male) and displayed wide variation comprising normal ventricular geometry (n=11), concentric remodelling (n=11), asymmetric remodelling (n=11), concentric hypertrophy (n=34), asymmetric hypertrophy (n=14) and LV decompensation (n=10). The magnitude of the hypertrophic response was unrelated to the severity of aortic valve narrowing. Conclusions. Modern imaging techniques have provided important insights in to the pathology underlying aortic stenosis and suggest that valvular calcification and myocardial fibrosis have a key role. Both represent important potential targets for future therapeutic interventions