Orientador: Aparecido Antonio Camacho / Resumo: A cardiomiopatia arritmogênica do ventrículo direito (CAVD) é uma doença de origem genética, muito prevalente em cães da raça Boxer. Histologicamente ocorre atrofia dos cardiomiócitos do ventrículo direito (VD) e consequente infiltração fibroadiposa, levando ao aparecimento de arritmias ventriculares. Mesmo sendo as alterações miocárdicas microscópicas em sua maioria, estudos recentes têm identificado disfunção contrátil do ventrículo direito, mesmo em estágios iniciais da doença em humanos, tanto por técnicas convencionais de ecocardiografia quanto por técnicas mais avançadas, como no caso do speckle tracking. Por isso, o objetivo deste estudo foi avaliar se há disfunção miocárdica de VD detectável pela ecocardiografia convencional e pela técnica de speckle tracking bidimensional em cães da raça Boxer portadores da CAVD. Para tanto, utilizou-se cães da raça Boxer com idade superior a quatro anos, distribuídos em dois grupos de acordo com o resultado da avaliação Holter (GC: < 50 EVs/24h, n=11 e GD: > 100 EVs/24h, n=11). Os animais foram submetidos a ecocardiografia e avaliou-se diâmetro e comprimento interno do VD em sístole e diástole, encurtamento fracional da área do VD (FAC), excursão sistólica do plano anular da tricúspide (TAPSE), velocidade miocárdica sistólica do ânulo lateral da tricúspide derivada do Doppler tecidual (S’) e deformação (strain) e taxa de deformação (strain rate) longitudinal global da parede livre do VD, derivados da análise por speckle tracking. As... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disease with a high prevalence in Boxer dogs. Histologically, right ventricle (RV) cardiomyocytes’ atrophy occurs, and thus fibrofatty infiltration, triggering ventricular arrhythmias. Although the myocardial changes are mostly microscopic, recent studies in human beings have identified right ventricular systolic dysfunction even in the early stages of ARVC, both by conventional echocardiographic techniques and advanced methods, such as speckle tracking. Therefore, the aim of this study was to investigate if RV myocardial dysfunction is present in Boxers affected by ARVC, evaluated by conventional echocardiography and bidimensional speckle tracking technique. For this purpose, Boxers older than four years were recruited, and based on 24-hour Holter recording were divided in groups: GC:< 50 VPC/24h (n=11), or GD: > 100 VPC/24h (n=11). The following echocardiographic parameters were analyzed: RV internal dimension during systole and diastole; RV fractional area change (FAC); tricuspid annular plane systolic excursion (TAPSE); myocardial systolic velocity of lateral tricuspid annulus (S’), and global longitudinal strain and strain rate. Groups were compared by Student’ t-test or Mann-Whitney test according distribution, and correlation between conventional and speckle tracking echocardiographic variables, as well as number and severity of the ventricular arrhythmias were accomplished by the Pearson test, assumin... (Complete abstract click electronic access below) / Mestre

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Agradecemos a compreensão. on 2018-05-22T11:07:47Z (GMT) / Submitted by Michelli Fenerich (michellifenerich@hotmail.com) on 2018-05-23T00:06:49Z No. of bitstreams: 1 Dissertação Michelli Fenerich.pdf: 839583 bytes, checksum: 4eb04bc52d571c22b951ce3b11e0a705 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-05-23T16:47:59Z (GMT) No. of bitstreams: 1 fenerich_m_me_jabo.pdf: 839583 bytes, checksum: 4eb04bc52d571c22b951ce3b11e0a705 (MD5) / Made available in DSpace on 2018-05-23T16:47:59Z (GMT). No. of bitstreams: 1 fenerich_m_me_jabo.pdf: 839583 bytes, checksum: 4eb04bc52d571c22b951ce3b11e0a705 (MD5) Previous issue date: 2018-04-12 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cardiomiopatia arritmogênica do ventrículo direito (CAVD) é uma doença de origem genética, muito prevalente em cães da raça Boxer. Histologicamente ocorre atrofia dos cardiomiócitos do ventrículo direito (VD) e consequente infiltração fibroadiposa, levando ao aparecimento de arritmias ventriculares. Mesmo sendo as alterações miocárdicas microscópicas em sua maioria, estudos recentes têm identificado disfunção contrátil do ventrículo direito, mesmo em estágios iniciais da doença em humanos, tanto por técnicas convencionais de ecocardiografia quanto por técnicas mais avançadas, como no caso do speckle tracking. Por isso, o objetivo deste estudo foi avaliar se há disfunção miocárdica de VD detectável pela ecocardiografia convencional e pela técnica de speckle tracking bidimensional em cães da raça Boxer portadores da CAVD. Para tanto, utilizou-se cães da raça Boxer com idade superior a quatro anos, distribuídos em dois grupos de acordo com o resultado da avaliação Holter (GC: < 50 EVs/24h, n=11 e GD: > 100 EVs/24h, n=11). Os animais foram submetidos a ecocardiografia e avaliou-se diâmetro e comprimento interno do VD em sístole e diástole, encurtamento fracional da área do VD (FAC), excursão sistólica do plano anular da tricúspide (TAPSE), velocidade miocárdica sistólica do ânulo lateral da tricúspide derivada do Doppler tecidual (S’) e deformação (strain) e taxa de deformação (strain rate) longitudinal global da parede livre do VD, derivados da análise por speckle tracking. As variáveis foram comparadas entre os grupos pelo teste t de Student e de Mann-Whitney e a correlação entre variáveis ecocardiográficas convencionais e de speckle tracking e o número e severidade das arritmias pelo teste de correlação de Pearson, com nível de significância de 5%. Os grupos não foram estatisticamente diferentes e não houve correlação entre os dados. Os resultados da pesquisa demonstraram que a ecocardiografia, tanto na avaliação do VD por índices sistólicos convencionais quanto por aqueles derivados da técnica de speckle tracking não foram capazes de detectar disfunção miocárdica do VD em Boxers portadores de CAVD. / Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disease with a high prevalence in Boxer dogs. Histologically, right ventricle (RV) cardiomyocytes’ atrophy occurs, and thus fibrofatty infiltration, triggering ventricular arrhythmias. Although the myocardial changes are mostly microscopic, recent studies in human beings have identified right ventricular systolic dysfunction even in the early stages of ARVC, both by conventional echocardiographic techniques and advanced methods, such as speckle tracking. Therefore, the aim of this study was to investigate if RV myocardial dysfunction is present in Boxers affected by ARVC, evaluated by conventional echocardiography and bidimensional speckle tracking technique. For this purpose, Boxers older than four years were recruited, and based on 24-hour Holter recording were divided in groups: GC:< 50 VPC/24h (n=11), or GD: > 100 VPC/24h (n=11). The following echocardiographic parameters were analyzed: RV internal dimension during systole and diastole; RV fractional area change (FAC); tricuspid annular plane systolic excursion (TAPSE); myocardial systolic velocity of lateral tricuspid annulus (S’), and global longitudinal strain and strain rate. Groups were compared by Student’ t-test or Mann-Whitney test according distribution, and correlation between conventional and speckle tracking echocardiographic variables, as well as number and severity of the ventricular arrhythmias were accomplished by the Pearson test, assuming a level of significance at 5%. Groups were considered similar, and there was no correlation among the evaluated parameters. Our findings suggesting that the echocardiographic evaluation of RV, either by conventional and speckle tracking techniques, were not able to detect systolic myocardial dysfunction in Boxers affected by ARVC. / FAPESP: 16/14800-7

The cardiovascular system is an essential component of mammalian biology. It is a complex network of various tissues and structures with unique functions. The function of the cardiovascular system is to supply nutrients including oxygen to the various cells, tissues and organs within the body, and remove waste products from them. Given the importance of this role, it is not surprising that there are countless regulatory mechanisms at the molecular, cellular and tissue levels that are required to support this functional system. Perturbations in parts of this system are likely to lead to abnormalities, and thus give rise to cardiovascular-related diseases. Despite the currently expanding list of genes reported to be involved in a variety of cardiovascular-related diseases, including calcific aortic valve disease (CAVD), the functions and associated pathways of these factors in both normal and pathological physiology have yet to be fully understood, such as at the transcriptomic level. In this thesis, a genome-wide transcriptomic atlas of the healthy mammalian cardiovascular system was generated using the sheep as a large animal model. This atlas was generated using RNA-seq, with the aim of further understanding normal gene expression patterns in the context of the known physiology of healthy mammalian tissues. Through this work, I identified novel gene networks and detailed functional clustering of co-expressed genes with region-specific expression and specialised cardiovascular roles. One interesting cluster was highly expressed in the cardiac valves, and shared genes found in physiological bone development, such as bone morphogenetic protein 4 (BMP4), collagen type I alpha 2 (COL1A2), Sry homeobox 8 (SOX8) and bone gamma-carboxyglutamate protein (BGLAP), some of which have been implicated in vascular calcification. Further to this work, I studied the expression profiles of these key cardiovascular genes during development in the sheep from foetal to adult stages. In addition, I investigated the gene expression patterns of various key vascular calcification genes. These studies showed differential expression of genes in the different cardiovascular tissues, demonstrating transcriptional differences between these different tissues known to have different functions. CAVD involves progressive valve leaflet thickening and severe calcification, resulting in impaired leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to examine the mechanisms of CAVD. However, to date, no published studies have utilised cell lines to investigate this process Thus, in this project, I generated and evaluated the calcification potential of an immortalised cell line derived from sheep aortic VICs (SAVICs). This novel large animal in vitro model of CAVD was demonstrated to calcify under high calcium and phosphate conditions. Changes in the expression of key calcification genes during VIC calcification was also observed, including increased mRNA expression of bone markers Runt-related transcription factor 2 (RUNX2) and sodium-dependent phosphate transporter 1 (PiT1), and a concomitant decrease in matrix Gla protein (MGP) mRNA expression. In addition, the role of extracellular nucleotides and their receptors (P2 receptors), which have been previously shown to be important in bone and vascular calcification, were investigated using SAVICs in vitro. This study has shown that extracellular nucleotides, particularly adenosine 5’-triphosphate (ATP) and uridine 5’-triphosphate (UTP) and other agonists of P2 receptors, reduced VIC calcification in vitro. Moreover, the cutting-edge gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), was successfully applied to generate large animal models of cardiovascular-related diseases. In this project, I applied the CRISPR/Cas9 technology to edit ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and fibrillin 1 (FBN1) to generate two models of vascular calcification and Marfan Syndrome (MFS), respectively. In the ENPP1-edited animals, soft tissue calcification has been observed in the biallelic mutant and homozygous pigs. In this project, I have developed a range of novel in vitro and in vivo tools to advance the study of cardiovascular disease. These studies demonstrate that large animal models are highly valuable in the field of cardiovascular biology. The in vivo and in vitro experimental models described should facilitate detailed analysis of cardiovascular molecular biology and ultimately lead to therapies which will minimise the morbidity and mortality currently arising from cardiovascular pathology.

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.

Calcific aortic valve disease (CAVD) is a highly prevalent and progressive disorder that ultimately causes gradual narrowing of the left ventricular outflow orifice with ensuing devastating hemodynamic effects on the heart. Calcific mineral accumulation is the hallmark pathology defining this process; however, fibrotic extracellular matrix (ECM) remodeling that leads to extensive deposition of fibrous connective tissue and distortion of the valvular microarchitecture similarly has major biomechanical and functional consequences for heart valve function. Significant advances have been made to unravel the complex mechanisms that govern these active, cell-mediated processes, yet the interplay between fibrosis and calcification and the individual contribution to progressive extracellular matrix stiffening require further clarification. Specifically, we discuss (1) the valvular biomechanics and layered ECM composition, (2) patterns in the cellular contribution, temporal onset, and risk factors for valvular fibrosis, (3) imaging valvular fibrosis, (4) biomechanical implications of valvular fibrosis, and (5) molecular mechanisms promoting fibrotic tissue remodeling and the possibility of reverse remodeling. This review explores our current understanding of the cellular and molecular drivers of fibrogenesis and the pathophysiological role of fibrosis in CAVD.

No description available.

Abstract Calcific aortic valve disease (CAVD) represents a disease spectrum, ranging from mild aortic valve sclerosis to severe obstructive aortic stenosis (AS), associated with a high risk of myocardial infarction and cardiovascular death. It is a common disease in the Western countries, and with their aging populations, its prevalence is likely to increase. Today, CAVD is recognized as an actively regulated disease. Mechanical stress and endothelial injury are the initiating factors, followed by lipid accumulation and oxidation, leading to inflammation, fibrosis and calcification. Ultimately, the progressive calcification hinders the normal valvular function and obstructs the flow of blood through the valve. The only effective treatment for symptomatic AS is aortic valve replacement. The trials with pharmacological treatments, mainly with anti-atherosclerotic drugs, have not been successful in slowing the progression of the disease. This study was aimed to identify differentially expressed transcripts, and molecular markers taking part in the pathophysiology behind CAVD. In particular, factors related to the renin-angiotensin system, and the apelin – APJ pathway, were investigated during the development of CAVD. In addition, the expressions of granzymes and perforin, as well as podoplanin, were studied in different stages of CAVD. It was demonstrated that these molecules are expressed in aortic valves and dysregulated in AS. These results can help to clarify the mechanisms driving CAVD, thus being potential targets for pharmacological therapy. Furthermore, the studied molecules may reflect the stage and possible subgroups of CAVD. / Tiivistelmä Aorttaläpän ahtauma edustaa tautijatkumoa, joka alkaa lievästä aorttaläpän paksuuntumisesta eli aorttaskleroosista ja jatkuu vaikeaan aorttaläpän kalkkeutuneeseen ahtaumaan eli aorttastenoosiin, johon liittyy korkea sydäninfarktin ja sydän- ja verisuonitatutiperäisen kuoleman riski. Aorttaläpän ahtauma on yleinen tauti länsimaissa, ja väestön ikääntyessä sen esiintyvyys on luultavimmin lisääntymässä. Nykyään aorttaläpän ahtauman tiedetään olevan aktiivisesti säädelty tauti. Mekaaninen rasitus ja endoteelivaurio käynnistävät tautiprosessin, läppäkudokseen kertyy lipidejä ja ne hapettuvat, mikä johtaa tulehdukseen, sidekudoksen lisääntymiseen ja kalkkeutumiseen. Lopulta etenevä kalkkeutuminen heikentää läpän normaalia toimintaa ja estää veren normaalia virtausta sydämestä aorttaan. Ainoa tehokas hoito oireiseen aorttastenoosiin on aorttaläpän korvausleikkaus. Lääkehoitoina on kokeiltu erityisesti ateroskleroosin hoitoon käytettäviä lääkkeitä, mutta niillä ei ole onnistuttu estämään taudin etenemistä. Tässä väitöskirjatyössä tutkittiin molekyylejä ja biokemiallisia reittejä, jotka liittyvät reniini-angiotensiinijärjestelmään ja apeliini-APJ-reittiin. Lisäksi tutkittiin grantsyymien ja perforiinin sekä podoplaniinin ilmentymistä aorttaläpän ahtauman eri kehitysvaiheissa. Tulosten perusteella näitä tekijöitä ilmennetään aorttaläpässä ja niiden määrä on muuttunut kalkkeutuneessa läpässä. Tulokset auttavat osaltaan ymmärtämään aorttaläpän ahtaumaan ja kalkkeutumiseen johtavia mekanismeja, joita voidaan hyödyntää uusia lääkehoidon kohteita suunniteltaessa. Tutkitut molekulaariset tekijät voivat kuvastaa aortan ahtaumataudin vaiheita ja mahdollisia alaryhmiä.

Introduction and Objectives Calcific aortic valve disease (CAVD) is the most common valvular disease, accounting for 50% of all valve disorders and is the third most common cardiovascular disease following coronary disease and hypertension.[1,2] Currently, there is no pharmacological agent capable of reversing or slowing down the progression of CAVD and treatment of severe cases consists of surgical repair or valve replacement[2]. Hence, there is a crucial need for earlier detection using predictive biomarkers that will allow for preventative intervention as opposed to post-symptomatic disease treatment or management. Namely, one target of particular interest is the expression of matrix metalloproteinases (MMPs) (specifically MMP-1, -2, and -9) which are upregulated in CAVD prior to calcification events and have been previously shown to serve as an attractive molecular imaging target.1–3 The primary objective of this study is to assess the feasibility of detecting biomarkers of CAVD by various in vivo imaging modalities, such as PET and echocardiography. In addition, this study assesses disease progression in various mouse strains to qualify an appropriate CAVD animal model. Methods In vivo and ex vivo imaging of C57Bl/6 and ApoE-/- (n = 8 per strain cohort) mouse models are used to link unique features of matrix remodelling with CAVD progression. At baseline and longitudinal follow-up (4, 8, and 12 months), in vivo hemodynamic impairment is assessed through echocardiography, and calcification and MMP activity are measured using PET with a series of radiotracers: [18F]NaF for calcification, [18F]BR351 for the molecular targets of MMP-2 and -9, and [18F]FMBP with molecular target specificity for MMP-13. Following imaging, aortic valve (AV) tissue is harvested, sectioned, and analyzed for calcification, inflammatory markers, collagen types, and MMP activity in AV leaflets. Tracer autoradiography, immunofluorescence, and in situ zymography are used to confirm in vivo imaging results with improved resolution and quantification in valves. Histological sample preparation, experimentation, and analyses are then repeated in human AV tissue samples for relative comparison of biomarker expression in animal models. Results Echocardiography suggests positive signs of disease progression in experimental animal models. In comparison to WT, ApoE-/- mice show: increased peak velocity (p<0.0001), decreased aortic valve area (p<0.001), and irregular valve dynamics. [18F]NaF PET imaging shows expected bone uptake and low calcium-burden in young and WT animals. [18F]FMBP shows increased uptake in the valve area of diseased models at later timepoints, 1.530 compared to <0.001 %ID/g (p<0.005), in disease vs control animals respectively. Furthermore, confirmation of sought-after biomarkers has also been assessed by analysis of various histological sample preparations including the presence of leaflet calcification, upregulation of MMP-2, -9, and -13, matrix remodelling, lipids, inflammatory markers, and activated MMP expression. Conclusion Findings from this study suggest that molecular imaging techniques using target-specific radiotracers, as well as echocardiography for assessment of hemodynamic impairment, are feasible solutions in predicting disease onset in CAVD specific animal models.