A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, for the degree of Doctor of Philosophy. Johannesburg, South Africa September 2017. / Although brachial blood pressure (BP) is a well-recognized risk factor for predicting cardiovascular events, aspects of aortic BP may enhance risk prediction. Pulse pressure (PP) is amplified from the aorta to peripheral arteries and variations in differences between brachial and aortic PP (PP amplification) are determined by factors that influence either the aortic forward (Pf) or backward (Pb)(reflected) pressure waves. Although aortic Pb may be more important than Pf in mediating cardiovascular risk, the best approach to assessing backward wave function (augmentation pressures [Pa] and index [AIx] or wave separation analysis); the relative impact of aortic Pb versus Pf on cardiovascular damage; and whether the ability of aortic-to-brachial PP amplification (PPamp) to add to risk prediction reflects backward or forward wave effects, is uncertain. In the present thesis I therefore first assessed in 808 community participants whether gender influences relations between Pa or AIx and left ventricular mass (LVM), a well-accepted end-organ measure. Aortic haemodynamics were determined using radial applanation tonometry and SphygmoCor software and LVM from echocardiography. In men, both AIx derived from Pa/central aortic PP (Pa/PPc) (p<0.01) and AIx derived from the second peak/first peak (P2/P1) of the aortic pulse wave (p<0.0005) were associated with LVM. In contrast, in women neither AIx derived from Pa/PPc (p=0.08) nor AIx derived from P2/P1 (p=0.17) were associated with LVM. Both the strength of the correlations (p<0.001 and p<0.0005) and the slope of the AIx-LVM relationships (p=0.001 and p<0.0005) were greater in men as compared to women. Therefore, in the present study I show that AIx is independently associated with LVMI in men, but not in women. I subsequently evaluated whether in women, measures of aortic systolic pressure augmentation (Pa or AIx) underestimate the effects of reflected waves on cardiovascular risk or whether Pb plays little role in cardiovascular risk prediction. In the same community sample I therefore evaluated sex-specific contributions of reflected (Pb and the reflection index [RI]) versus augmented (Pa and AIx) pressure wave indices to iii variations in PPc (n=1185, 65.0% women), and LVM (n=793, 64.9% women). Aortic Pb and Pf were determined using wave separation analysis. In both women and in men, independent of confounders, RI and Pb contributed more than Pf, whilst Pa and AIx contributed less than incident wave pressure (Pi) to variations in PPc (p<0.0001 for comparison of partial r values). In both men and in women Pb contributed more than Pf (p<0.05) to variations in LVM. Although in men Pa (partial r=0.33, p<0.0001) contributed to a similar extent as Pi ((partial r=0.34, p<0.0001) to variations in LVMI, in women Pa (partial r=0.05, p=0.36) failed to contribute to LVM, whilst Pi was significantly associated with LVM (partial r=0.30, p<0.0001). Similar results were obtained with AIx as opposed to Pa in the regression models. Therefore, in both women and in men, Pb is more closely associated with PPc and LVM than Pf, but indices of aortic pressure augmentation markedly underestimate these effects, particularly in women. As the relative impact of aortic Pb as compared to Pf on cardiovascular damage independent of brachial BP is uncertain, in 1174 participants from a community sample I subsequently assessed the relative impact of Pb and Pf on variations in LVM (n=786), aortic pulse wave velocity (PWV)(n=1019), carotid intima-media thickness (IMT)(n=578), transmitral early-to-late LV diastolic velocity (E/A)(n=779) and estimated glomerular filtration rate (eGFR)(n=1174). Independent of mean arterial pressure and confounders, PPc and both Pb and Pf were associated with end-organ measures or damage (p<0.05 to <0.0001). With adjustments for brachial PP and confounders, Pb remained directly associated with LVM (partial r=0.10, p<0.01), PWV (partial r=0.28, p<0.0001), and IMT (partial r=0.28, p<0.0001), and inversely associated with E/A (partial r=-0.31, p<0.0001) and eGFR (partial r=-0.14, p<0.0001). Similar relations were noted with the presence of end-organ damage (p<0.05 to <0.0001). In contrast, with adjustments for brachial PP and confounders, Pf no longer retained direct relations with LVM, PWV, and IMT or inverse relations with E/A and eGFR. Adjustments for Pb, but not Pf diminished brachial PP-independent relationships between PPc and end-organ measures. Thus, although both Pf and Pb contribute to end-organ measures and damage, independent of brachial iv BP, the impact of aortic BP is accounted for largely by Pb. PPamp is independently associated with cardiovascular outcomes. However, the aortic functional change most likely to account for this effect is uncertain. In 706 community participants I subsequently aimed to identify the aortic functional change that accounts for relations between PPamp and LVM. In multivariate models with the inclusion of brachial PP, 1/PPamp (partial r=0.12, p<0.005), Pb (partial r=0,09, p<0.05), and aortic PWV (partial r=0.09, p<0.05) were independently associated with LVMI. Similarly, in multivariate models with the inclusion of brachial PP, 1/PPamp (p<0.005), Pb (p<0.01), and aortic PWV (p<0.01) were independently associated with LV hypertrophy (LVH). With adjustments for Pb, the brachial PP-independent relationships between 1/PPamp and LVMI or LVH were abolished (p>0.08 for both). However, adjustments for PWV failed to modify brachial PP-independent relations between 1/PPamp and LVMI or LVH. Hence, independent relations between PPamp and LVM or LVH are largely accounted for by Pb. In conclusion, in the present thesis I show that the use of augmented pressures underestimates the impact of reflected pressure wave effects on end-organs, particularly in women; that brachial BP-independent relations between aortic BP and end organs is determined largely by Pb and that relations between PPamp and end organ measures is largely accounted for by Pb. These findings add to our understanding of the adverse effects of aortic functional changes on the cardiovascular system and suggest cost-effective approaches to add to risk prediction. / LG2018

The gold standard treatment for patients with AVD is surgical replacement of the aortic valve with either mechanical or fixed tissue prostheses. These implants have a limited lifespan and are associated with serious adverse events. Patient autologous tissue engineered heart valves (TEHVs) offer a solution. Vital to the development of a TEHV is determining a source of donor tissue(s) that most closely mimics the native valve tissue. In pursuit of determining an alternative cell source for patient autologous TEHVs we compared a number of phenotypic and genotypic characteristics of atrial fibroblasts, dermal fibroblasts and differentiated bone marrow-derived progenitor cells (BMCs) and made a comparison to valvular interstitial cells (VICS). We demonstrate that while VICs share some phenotypic similarities with fibroblasts and BMCs, they also possess unique characteristics and demonstrate differential mRNA expression of key regulatory pathways that may influence their phenotype. / October 2016

Calcific aortic valvular disease (CAVD) is the most common valvular pathology in the developed world. CAVD results in calcifications forming on the aortic valve leaflets, inhibiting proper closure and causing complications of stenosis and regurgitation. Although, the mechanisms behind the disease initiation are unknown, it is believed to be a cell-mediated phenomenon, and not the result of passive degradation of the valve as once believed due to the increased prevalence with age. Currently, there are no pharmaceutical options for the prevention or reversal of calcifications, the only treatment option is complete valve replacement, an imperfect solution. Hindering the development of potential therapeutics is that currently there are no adequate animal models which replicate the calcification and cell death seen in disease explanted valves. An in vitro model has been develop where valvular interstitial cells (VICs), the main cell type of the valve, are seeded at high density into tissue culture polystyrene dishes and cultured with TGF-β1. This results in VICs activating to the myofibroblast phenotype and forming cell aggregates. Due to currently unknown mechanisms, apoptosis occurs within the center of the aggregates and calcification ensues. Although simplistic, this model has been used to show that rate and frequency of aggregation is affected by cellular tension; conditions of high tension increase aggregation response, while conditions of low tension prevent aggregation and calcification from occurring. It is important to note; however, that despite its wide usage, the current model is limited as the aggregation and subsequent calcification are random occurrences and are not consistent across literature where same conditions for control samples are used. The motivation of the presented work is two-fold. First, high intracellular tension has been suggested as one of the mechanisms leading to disease in the valve. Despite the clear and important role of cell tension, VIC tension has never before been measured in a dynamic environment. The ways in which dynamic stimulation affects individual VIC tension is not known. In aim one, a method is developed to allow for long-term cyclic stretch of VICs with measurement of cell traction force. It was found that cyclic stretch decreased cell tension in cells with high prestress and increased cell tension for conditions of low prestress. Combined, these findings indicate a homeostatic cellular tension which is dependent upon the mechanical environment. In the second aim, a novel method for creating VIC aggregates is validated. Micro-contact printing, essentially “stampingâ€� of a protein in a defined pattern, is used to create circular aggregates on polyacrylamide gels. This method allows for the separation of the aggregation from the subsequent calcification, an improvement over the current in vitro model. The method is then used to explore the role of the distribution of tension in the initiation of disease

Vascular calcification is a prominent cardiovascular condition found worldwide. This condition is predominantly found in the elderly population, and patients who suffer from chronic kidney disease, due to an imbalance of serum phosphate and calcium levels. For many years, vascular calcification was believed to be a passive pathological process which develops with ageing and/or lifestyle. Little has been documented about the disease until the 20th century, when interest in cardiovascular research grew amongst scientists. Indeed, vascular calcification underpins severe clinical outcomes and cardiovascular diseases have been labelled the global leading cause of death. Calcific aortic valve diseases (CAVD) is a progressive degenerative condition characterised by the development of lipo-calcification around the aortic valve leaflets leading to severe aortic stenosis and aortic regurgitation, which may ultimately lead to heart failure. At present there are no pharmaceutical therapies that can stop its progression and its molecular mechanisms are not fully understood. Recent findings have suggested that vascular smooth muscle cell (VSMC) calcification shares many common features with physiological skeletogenesis via the release of matrix vesicles (MVs), which are specialised structures that initiate mineralisation during bone formation. The ability for MVs to nucleate calcium and phosphate highly depend on their protein composition, as this may vary depending on active cell signalling and the microenvironment. This mechanism involving MV-regulated calcification has yet to be examined in CAVD. In this study, examined whether calcium and/or phosphate regulate VIC-derived MVs to induce calcification in the aortic valve. I used a primary rat valve interstitial cell (VIC) model, coupled with stenotic human valve tissues to characterise and study the mechanisms underpinning CAVD. X-ray fluorescence and diffraction analysis showed the mineral found in calcified human aortic valves to be hydroxyapatite (HA), the main component in bone. Additional imaging studies employing transmission electron microscopy (TEM) revealed particles that were similar in size and morphology to skeletal MVs. To further characterise VIC-derived MVs in vitro, I harvested MVs from rat VICs, and subsequently studied their protein composition using Isobaric tag for relative and absolute quantitation (iTRAQ) mass spectrometry. The data obtained from the proteomics analysis was compared to previous published studies on MV proteins derived from osteoblasts and VSMCs. The results showed the upregulation of numerous calcification regulators in MVs isolated from all 3 cell types, in particular, the Annexin family, which are known calcium binding proteins. Further studies conducted with Annexin 6, an established calcium regulator in arterial calcification, revealed its colocalisation with MV-enriched areas in calcified human aortic valve tissue suggesting it may play an important role in calcium regulation during CAVD.

BACKGROUND: Calcific aortic valve disease (CAVD) is the most prevalent heart valve disease in the developed world, claiming almost 17,000 deaths annually in the United States. The lack of noninvasive therapeutics to slow or halt the disease warrants the need for further understanding of the pathobiological mechanisms of CAVD. A tri-laminar structure of aortic valve determines the biomechanical properties of its leaflets. Valvular endothelial cells (VECs) and interstitial cells (VICs) are responsible for valve structural integrity. Traditional two-dimensional culture conditions spontaneously activate the pathological differentiation of VICs making in vitro studies challenging. A monolayered three-dimensional (3D) hydrogel platform was recently developed as a novel in vitro culture system to study the phenotypic changes of VICs leading to microcalcification (early stages of calcification). This system, however, did not fully recapitulate the microenvironment of native valve tissues because of the lack of individual layer representations and endothelial coverage. Bioprinting technology, which allows precise and integrated positioning of cells, matrix, and biomolecules, may provide an innovative approach toward building a more biologically relevant 3D culture platform. OBJECTIVE: This study aims to lay the groundwork for building a multilayered 3D-bioprinted culture platform to study CAVD by first validating the use of bioprinting in monolayered cell-laden 3D hydrogel constructs. METHODS: Human VICs were isolated from patients undergoing valve replacement surgeries at Brigham and Women’s Hospital (Boston, MA) according to Institutional Review Board (IRB) protocols. VICs were expanded in culture medium containing growth factors for up to 6 passages and then encapsulated in hydrogels using 3D bioprinting technology. After encapsulation, VIC-laden 3D constructs were cultured in either normal or osteogenic conditions for 21 days. Microcalcification, cell proliferation, and cell apoptosis were evaluated using fluorescent staining and confocal microscopy. Results were compared with results from VIC-laden hydrogels made manually. RESULTS: An increase in microcalcification was observed throughout bioprinted VIC-laden hydrogel constructs cultured in osteogenic conditions for 21 days, whereas normal conditions developed negligible calcification signals. Cell proliferation and apoptosis were not significantly different between normal and osteogenic groups in bioprinted hydrogels. Cell-free hydrogels did not exhibit any microcalcification. Overall, bioprinted hydrogels showed less nonspecific background staining than handmade hydrogels, thus providing a better means for quantitative assessments of 3D culture platforms. CONCLUSION: Based on bioprinting technology, an improved monolayered cell-laden hydrogel platform was successfully established as a first step toward building an in vitro multilayered disease model for studying the pathobiological mechanisms of CAVD. The results in this study were consistent with current literature that proposes calcification as a cell-dependent, apoptotic-independent, and proliferation-independent pathway. / 2019-07-13T00:00:00Z

Bisher existiert keine nicht-invasive/ nicht-operative Therapie der Aortenklappenstenose. Als wichtiger Zeitpunkt für eine präventive Maßnahme, zur Verhinderung der Ausbildung einer hömodynamisch relevanten Aortenklappenstenose, kann das Stadium der Aortenklappensklerose angesehen werden. Dieses frühe Erkrankungsstadium verfügt über zahlreiche pathophysiologische Parallelen zur Atherosklerose, für die eine positive Rolle der Prävention durch körperliche Aktivität erwiesen ist. Ziel dieser Arbeit war die Durchführung der Sekundärprävention der kalzifizierenden Aortenklappenerkrankung durch körperliches Training. Um mögliche Effekte dieser Intervention zu eruieren, wurden LDLR-/--Mäuse mit bereits bestehenden pathologischen Aortenklappenveränderungen über einen Zeitraum von 16 Wochen körperlichem Training unterzogen. Durch morphologische, serumanalytische, immunhistochemische und Genexpressionsanalysen konnte abschließend eine Quantifizierung der Effekte körperlichen Trainings - in der Zielsetzung der Sekundärprävention - realisiert werden.

Calcific aortic valve sclerosis is characterized by focal lesions in the valve leaflet. These lesions are rich in myofibroblasts that express α-SMA and cause fibrosis. Lesions tend to occur in regions of the leaflet that are subjected to large bending loads, suggesting a mechanobiological basis for myofibrogenic differentiation and valve pathogenesis. In this thesis, a bioreactor was developed to study the effect of physiological loading on myofibrogenic differentiation of valve interstitial cells. Cyclic loading of native porcine aortic valve leaflets ex vivo resulted in increased α-SMA expression, predominantly in the fibrosa and spongiosa (similar to sclerotic leaflets). Cofilin, an actin-binding protein, was also upregulated by loading, suggesting it plays a role in mechanically-induced myofibrogenesis. Similarly, loading of a tissue engineered aortic valve leaflet model resulted in increased α-SMA transcript and protein expression. These data support an integral role for mechanical stimuli in myofibrogenic differentiation and sclerosis in the aortic valve.

Calcific aortic valve sclerosis is characterized by focal lesions in the valve leaflet. These lesions are rich in myofibroblasts that express α-SMA and cause fibrosis. Lesions tend to occur in regions of the leaflet that are subjected to large bending loads, suggesting a mechanobiological basis for myofibrogenic differentiation and valve pathogenesis. In this thesis, a bioreactor was developed to study the effect of physiological loading on myofibrogenic differentiation of valve interstitial cells. Cyclic loading of native porcine aortic valve leaflets ex vivo resulted in increased α-SMA expression, predominantly in the fibrosa and spongiosa (similar to sclerotic leaflets). Cofilin, an actin-binding protein, was also upregulated by loading, suggesting it plays a role in mechanically-induced myofibrogenesis. Similarly, loading of a tissue engineered aortic valve leaflet model resulted in increased α-SMA transcript and protein expression. These data support an integral role for mechanical stimuli in myofibrogenic differentiation and sclerosis in the aortic valve.

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Congenital heart disease (CHD) is a risk factor for premature cardiovascular complications. Great advances have occurred in the past years leading to the identification of several genes essential for proper cardiac formation such as GATA4/5/6 mutated in some individuals with CHD. GATA6 is a zinc finger transcription factor whose presence is crucial for early embryonic development. GATA6 is expressed in many cell types of the heart including myocardial, endocardial, neural crest, and vascular smooth muscle. In human, mutations in GATA6 result in variable cardiac phenotypes. The objective of this thesis was to determine the roles that GATA6 play in the different cell types of the heart and to elucidate the molecular basis of the cardiac defects associated with Gata6 haploinsufficiency. For this, a combination of cell and molecular techniques were used in vitro and in vivo. First, we show that Gata6 heterozygozity leads to RL-type bicuspid aortic valve (BAV)- the most common CHD affecting 2% of the population. GATA6-dependent BAV is the result of disruption of valve remodeling and extracellular matrix composition in Gata6 haploinsufficient mice. Cell-specific inactivation of one Gata6 allele from Isl-1 positive cells, but not from endothelial or neural crest cells, recapitulates the phenotype of Gata6 heterozygous mice revealing an essential role for GATA6 in secondary heart field myocytes during valvulogenesis. We further uncovered a role for GATA6 as an important regulator of the cardiac conduction system and revealed that GATA6 expression regulates the activity of the cardiac pacemaker. GATA6 exerts its role via regulation of the cross-talk among the different cell types of the SAN. Lastly, some CHDs are characterized by abnormalities of both the limbs and the heart such as the Holt Oram syndrome (caused by mutation in TBX5 transcription factor). The molecular basis for limb-heart defects remain poorly understood. In the course of this work, we discovered that Gata6 haploinsufficiency resulted in a partially penetrant polysyndactyly (extra digits fused together) phenotype. Together, the data provide novel molecular and cellular insight into GATA6 role in normal and pathologic heat development. Our results also suggest that GATA6 should be added to the list of genes whose mutations are potentially associated with heart and limb abnormalities. Better knowledge of the molecular basis of CHD is a prerequisite for the development of diagnostic and therapeutic strategies to improve care of individuals with congenital heart disease.

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