Heart valve structures open and close during the cardiac cycle to provide unidirectional blood flow through the heart, critical for efficient cardiovascular function. Valve dysfunction results in either incomplete opening or incomplete closure of the valve. Both types of valve dysfunction decrease efficiency of blood flow, increasing the load on the myocardium and leading to secondary heart disease such as pathological hypertrophy and heart failure. There are currently no effective treatments to prevent or slow the progression of valve disease, and there are no pharmacological treatments for advanced valve disease. Although most valve disease is associated with aging, increasing evidence suggests that valve disease often has origins in development. Congenital valvuloseptal defects affect many newborns, ranging from life-threatening malformations requiring immediate repair to more subtle, often undiagnosed defects that increase susceptibility to valve disease later in life. Therefore, an improved understanding of the mechanisms of heart valve formation and maintenance of adult valves may serve as an important step in improving valve disease treatment options. In this work, the mechanisms of normal valve development and the role of Sox9 in developing and mature valves are further studied. The temporal and spatial expression of extracellular matrix genes and proteins are examined throughout normal murine valve development. Sox9 function in the processes of valve development and valve maintenance is examined using mouse models of conditional Sox9 loss-of-function. Heart valve phenotypes in mice with reduced Sox9 function are examined throughout development and in adult mice with resultant calcific valve disease. The possible causative mechanisms of calcific valve disease in mice with reduced Sox9 function are further investigated by identification of novel possible targets of Sox9 transcriptional regulation. Together these studies improve our understanding of heart valve development, characterize a model of heart valve calcification with genetic etiology, and identify and characterize novel targets of Sox9.

The objectives of this thesis are: a) To examine racial/ethnic differences in coronary artery calcification (CAC) and CAD, between symptomatic South Asians and Caucasians, matched for age, gender and conventional cardiovascular risk factors, b) To assess, using a meta-analysis model, the natural history of and stability of measurements of coronary artery calcium scoring (CACs) based on data collected from two large published trials: St Francis and EBEAT, c) To investigate the prevalence of coronary artery calcification in individuals with CT evidence for AVC, mitral valve calcification (MAC) or of both of them (AVC+MAC), d) To assess any potential association between premature CAD (<55 years in first-degree male relatives and <65 years in first-degree female relatives) and CAC in a large cohort of asymptomatic individuals. We found that coronary artery calcification is more extensive and diffuse in symptomatic patients of South Asian ethnic origin as compared to Caucasians, despite similar conventional risk factors for CAD. This is more evident in those >50 years of age, suggesting potential genetic or other risk factors yet to be determined. The natural history of coronary artery calcification was overtime progression in the majority of subjects, irrespective of gender. The higher variability in RCA measurements could be related to the low baseline CACs or exaggerated movement of the right side atrioventricular ring, whereas those for LCA brances are influenced by the branch allocation of the CACs. Valve calcification is not isolated but involve also and the coronary arteries. The presence of calcification in the aortic valve or combined aortic and mitral valves predicted coronary artery calcification. Additionally patients in whom both valves have become calcified tend to have severe coronary artery calcification. And finally, there is no relationship between the prevalence and extent of coronary artery calcification and the presence of family history of coronary heart disease in asymptomatic individuals with none of the conventional risk factors for atherosclerosis.

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.

Calcific aortic valve disease is highly prevalent, especially in the elderly. Currently, the exact mechanism of the calcification process is not completely understood, limiting our ability to prevent or cure the disease. Ex vivo investigations, however, have provided evidence that the aortic valve's biological response is sensitive to mechanical forces, including fluid shear stresses, leading to the hypothesis that adverse fluid shear stress environment play a role in leading to valve calcification. This thesis seeks to investigate this hypothesis. A method for performing experimental measurement of time-varying shear stress on aortic valve leaflets under physiologic flow conditions was first developed, based on the Laser Doppler Velocimetry technique, and was systematically validated. This method was then applied to both the aortic surface and the ventricular surface of a normal tricuspid the aortic valve, and then on a congenital bicuspid aortic valve, using suitable in vitro valve models and an in vitro pulsatile flow loop. It was found that in the tricuspid valve, the peak shear stress on the aortic surface under adult resting condition was approximately 15-19 dyn/cm². Aortic surface shear stresses were elevated during mid- to late-systole, with the development of the sinus vortex, and were low during all other instances. Aortic surface shear stresses were observed to increase with increasing stroke volume and with decreasing heart rate. On the ventricular surface, shear stresses had a systolic peak of approximately 64-71 dyn/cm² under adult resting conditions. During late systole, due to the Womersley effect, shear stresses were observed to reverse in direction to a substantial magnitude for a substantial period of time. Further, it was found that a moderately stenotic bicuspid aortic valve can experience excessive unsteadiness in shear stress experienced by its leaflets, most likely due to the turbulent forward flow resulting from the stenosis, and due to the skewed forward flow. To demonstrate that the measured shear stresses can have an effect on the aortic valve biology, ex vivo experiments were performed in specific to determine the effects of these various shear stress characteristics on the biological response of porcine aortic valve leaflets, using the cone and plate bioreactor. It was found that unsteady shear stress measured in the bicuspid valve resulted in increased calcium accumulation. Further, it was found that low shear stresses and high frequency shear stresses resulted in increased calcium accumulation. Thus, shear stress was found to affect aortic valve pathology, and low and unsteady fluid shear stresses can enhance pathology.

Calcific aortic valve disease (CAVD) is one of the most common causes of cardiovascular disease in North America. Mechanical factors have been closely linked to the pathogenesis of CAVD and may contribute to the disease by actively regulating the mechanobiology of valve interstitial cells (VICs). Mechanical forces affect VIC function through interactions between the VIC and the extracellular matrix (ECM). Studies have shown that the transfer of mechanical stimulus during cell-ECM interaction depends on the local material properties at hierarchical length scales encompassing tissue, cell and cytoskeleton. In this thesis, biomechanical tools were developed and applied to investigate hierarchical cell-ECM interactions, using VICs and valve tissue as a model system. Four topics of critical importance to understanding VIC-ECM interactions were studied: focal biomechanical material properties of aortic valve tissue; viscoelastic properties of VICs; transduction of mechanical deformation from the ECM to the cytoskeletal network; and the impact of altered cell-ECM interactions on VIC survival. To measure focal valve tissue properties, a micropipette aspiration (MA) method was implemented and validated. It was found that nonlinear elastic properties of the top layer of a multilayered biomaterial can be estimated by MA by using a pipette with a diameter smaller than the top layer thickness. Using this approach, it was shown that the effective stiffness of the fibrosa layer is greater than that of the ventricularis layer in intact aortic valve leaflets (p<0.01). To characterize the viscoelastic properties of VICs, an inverse FE method of single cell MA was developed and compared with the analytical half-space model. It was found that inherent differences in the half-space and FE models of single cell MA yield different cell viscoelastic material parameters. However, under particular experimental conditions, the parameters estimated by the half-space model are statistically indistinguishable from those predicted by the FE model. To study strain transduction from the ECM to cytoskeleton, an improved texture correlation algorithm and a uniaxial tension release device were developed. It was found that substrate strain fully transfers to the cytoskeletal network via focal adhesions in live VICs under large strain tension release. To study the effects of cell-ECM interactions on VIC survival, two mechanical stimulus systems that can simulate the separate effects of cell contraction and cell monolayer detachment were developed. It was found that cell sheet detachment and disrupted cell-ECM signaling is likely responsible for the apoptosis of VICs grown in culture on thin collagen matrices, leading to calcification. The studies presented in this thesis refine existing biomechanical tools and provide new experimental and analytical tools with which to study cell-ECM interactions. Their application resulted in an improved understanding of hierarchical valve biomechanics, mechanotransduction, and mechanobiology.

Calcific aortic valve disease (CAVD) is one of the most common causes of cardiovascular disease in North America. Mechanical factors have been closely linked to the pathogenesis of CAVD and may contribute to the disease by actively regulating the mechanobiology of valve interstitial cells (VICs). Mechanical forces affect VIC function through interactions between the VIC and the extracellular matrix (ECM). Studies have shown that the transfer of mechanical stimulus during cell-ECM interaction depends on the local material properties at hierarchical length scales encompassing tissue, cell and cytoskeleton. In this thesis, biomechanical tools were developed and applied to investigate hierarchical cell-ECM interactions, using VICs and valve tissue as a model system. Four topics of critical importance to understanding VIC-ECM interactions were studied: focal biomechanical material properties of aortic valve tissue; viscoelastic properties of VICs; transduction of mechanical deformation from the ECM to the cytoskeletal network; and the impact of altered cell-ECM interactions on VIC survival. To measure focal valve tissue properties, a micropipette aspiration (MA) method was implemented and validated. It was found that nonlinear elastic properties of the top layer of a multilayered biomaterial can be estimated by MA by using a pipette with a diameter smaller than the top layer thickness. Using this approach, it was shown that the effective stiffness of the fibrosa layer is greater than that of the ventricularis layer in intact aortic valve leaflets (p<0.01). To characterize the viscoelastic properties of VICs, an inverse FE method of single cell MA was developed and compared with the analytical half-space model. It was found that inherent differences in the half-space and FE models of single cell MA yield different cell viscoelastic material parameters. However, under particular experimental conditions, the parameters estimated by the half-space model are statistically indistinguishable from those predicted by the FE model. To study strain transduction from the ECM to cytoskeleton, an improved texture correlation algorithm and a uniaxial tension release device were developed. It was found that substrate strain fully transfers to the cytoskeletal network via focal adhesions in live VICs under large strain tension release. To study the effects of cell-ECM interactions on VIC survival, two mechanical stimulus systems that can simulate the separate effects of cell contraction and cell monolayer detachment were developed. It was found that cell sheet detachment and disrupted cell-ECM signaling is likely responsible for the apoptosis of VICs grown in culture on thin collagen matrices, leading to calcification. The studies presented in this thesis refine existing biomechanical tools and provide new experimental and analytical tools with which to study cell-ECM interactions. Their application resulted in an improved understanding of hierarchical valve biomechanics, mechanotransduction, and mechanobiology.