Canine myxomatous mitral valve disease (MMVD) is the most common cardiac disease in dogs affecting all breeds, and it shares many similarities with the equivalent human disease. From the only transcriptomic report for canine MMVD published in 2006, serotonin signalling was identified as a contributing factor and has been widely studied since. Two transcriptomic profiling studies in human MMVD have also identified oxidative stress response and bone morphogenic protein signalling contributing to disease pathology. All studies at the transcriptional level have identified a variety of biological functions in MMVD suggesting dynamic extracellular matrix (ECM) remodelling processes are on-going. Moreover, cellular changes found in MMVD are somewhat reminiscent of the events seen in early heart valve, suggesting possible re-activation of signalling pathways of which those driving development and endothelial-to-mesenchymal transition (EndoMT) are particularly interesting. EndoMT, in which endothelial cells change their identity to mesenchymal phenotype and migrate into the cardiac jelly underneath the endothelium, is a crucial mechanism in valvulogenesis. Whether or not gene regulation of EndoMT and valve development also plays a role in MMVD is unknown. In this study, the MMVD cellular changes in the Cavalier King Charles Spaniel (CKCS), a breed with the highest prevalence, earliest onset, and rapid progression of the disease, was investigated. Secondly, transcriptional profiling was conducted using the latest canine microarray chips, a single affected breed (CKCSs), stringent sample quality control and statistical thresholds, with quantitative polymerase chain reaction (Q-PCR) for data validation. After transcriptional mapping, multi-platform in silico analysis was conducted to identify relationship between differentially expressed genes and their relevant biological functions. Next, a comparison study using immunohistochemistry was performed on different severities of myxomatous valves to localize the proteins of interest. Finally, to model the transcriptional factors and their downstream targets, mitral valve endothelial cell (MVEC) clones were derived from the canine normal mitral valves for future in vitro studies. Cellular changes of MMVD between CKCS and non-CKCS populations showed no difference in their distribution, number and phenotypic markers. Global genomic expression analysis identified similar (inflammation, up-regulation of serotonin receptor and bone morphogenic protein) and novel biological functions (epithelial-to-mesenchymal transition) compared to the previous study in 2006. Key transcriptional factors and genes associated with EndoMT including SNAI1, TAGLN, ACTA2, ACTG2, HAS2, and CTNNB1 were found up-regulated, and NID1, LAMA2, CDH5 were down-regulated in the MMVD group. In myxomatous mitral valves, increased expression of HAS2 in myofibroblasts, SNAI1 expression in endothelial cells, and co-expression of CDH5 and α-smooth muscle actin (α-SMA) also suggested the presence of EndoMT compared to normal valves. Nevertheless, there is also evidence of EndoMT in normal valves (α-SMA positive endothelial cells) which might suggest contribution to life-long valve re-modelling. In addition, there was a decreased expression of microRNAs associated with modulation of extracellular matrix transcripts, including miR-23, miR-29, and miR-218, indicating epigenetic regulation in MMVD. Based on the cellular changes, MMVD in CKCS appears to be representative of MMVD in all breeds and the early-onset of MMVD in that breed does not lead to different end-stage pathology. Novel biological functions such as EndoMT, were identified by transcriptional profiling, and by using powerful bioinformatic tools providing insight into understanding gene regulation in MMVD. Furthermore, a relationship between developmental biology processes and MMVD pathogenesis was established, with a likely important role for epigenetics in disease pathogenesis.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-08T14:29:39Z No. of bitstreams: 2 MARILIA AVILA VALANDRO ok.pdf: 1044698 bytes, checksum: 07606147cdffad848dc4db44d0162dd3 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-08T14:30:12Z (GMT) No. of bitstreams: 2 MARILIA AVILA VALANDRO ok.pdf: 1044698 bytes, checksum: 07606147cdffad848dc4db44d0162dd3 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2016-09-08T14:30:12Z (GMT). No. of bitstreams: 2 MARILIA AVILA VALANDRO ok.pdf: 1044698 bytes, checksum: 07606147cdffad848dc4db44d0162dd3 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2016-03-29 / A endocardiose valvar (EV) é a cardiopatia mais prevalente na espécie canina, capaz de alterar o balanço autonômico pela ativação crônica do sistema nervoso simpático, relacionado ao risco de morte súbita e pior prognóstico. Diversos programas de treinamento com caminhada foram eficazes no restabelecimento do equilíbrio autonômico em seres humanos cardiopata, verificados a luz da variabilidade da frequência cardíaca (VFC). Dessa forma, objetivou-se avaliar o efeito de oito semanas de caminhada, realizadas três vezes por semana, durante 30 a 50 minutos, de moderada intensidade (60 a 80% da frequência cardíaca máxima), sobre a função autonômica cardíaca de cães com EV, utilizando a VFC no domínio do tempo e da frequência como ferramenta. Para tanto, 20 cães com EV foram divididos em dois grupos: grupo controle - não treinado (GC, n=9) e grupo treinamento (GT, n=11), e avaliados nos momentos basal (T0), quatro semanas (T1) e oito semanas (T2). No domínio do tempo, a variável média rMSSD (raiz quadrada da média ao quadrado das diferenças sucessivas entre os intervalos NN) foi maior no GT em quatro (155,5+42,07) e oito semanas (199,8+83,54) em relação ao GC (91,17+35,79 e 88,17+57,51) (p<0,05). No domínio da frequência, a variável High Frequency (HF) foi a mais representativa, e apresentou aumento no GT (30950+25810) após quatro semanas quando comparado ao GC (19090+23210) (p<0,05) e dentro do grupo GT após oito semanas de treinamento (40300+33870) em relação à avaliação basal (29340+20950) (p<0,05). As demais variáveis não sofreram influências do programa de treinamento proposto. Esses resultados demonstram que o treinamento físico com a utilização de caminhadas foi capaz de alterar a VFC, indicando uma maior participação parassimpática em cães com EV. / Valvular endocardiosis is the most prevalent cardiopathy in canine specie. This disease is able to change autonomic balance, which is related to sudden death and worse prognostic, through chronic activation of sympathetic nervous system. Looking at heart rate variability, various walk training plans were efficient for the autonomic balance reestablishment in cardiophats people. In this way, this study focused on evaluate the effect of 8 weeks walking plan on cardiac autonomic function of dogs with valvular endocardiosis. These walking plans were consisted of moderated intensity walking (reaching from 60 to 80% of maximum heart rate) during 30 to 50 minutes, three times a week. Heart rate variability was analyzed by utilizing time and frequency domain as a tool. Thus, 20 dogs with valvular endocardiosis were divided into two groups: Control group, with no training (CG, n=9), and Training group (TG, n=11). They were evaluated at basal moment (T0), 4 weeks (T1), and 8 weeks (T2). At time domain, only the medium variable rMSSD (the root mean square of successive differences between the square NN intervals) was higher in the TG in four (155.5+42.07) and eight weeks (199.8+83.54) than CG (91.17+35.79 and 88.17+57.51) (p<0.05). At frequency domain, the high frequency variable was the most representative and after four weeks, it showed higher on TG (30950+25810) than CG (19090+23210) (p<0.05). After eight weeks, within TG there was higher frequency (40300+33870) when compared to basal evaluation (29340+20950) (p<0.05). The proposed training program did not affect the other variables. These results suggest that the physical training with utilization of walking plans were able to change the heart rate variability what indicated a higher parasimpathetic participation of dogs with valvular endocardiosis.

Myxomatous mitral valve disease (MMVD) is the single most common acquired cardiac disease of dogs, and is a disease of significant veterinary importance. It also bears close similarities to mitral valve prolapse in humans and therefore is a disease of emerging comparative interest. Realising the importance of collagen fibres in mitral heart valves and considering the paramount significance of myxomatous mitral valve disease, a better understanding of the pathogenesis of MMVD is essential. Thus, this study was designed to investigate the changes in collagen molecules, including fibril structure, fibril orientation, d-spacing, collagen density, collagen content, thermal stability, and the status of mature and immature crosslinks. A combination of biophysical and biochemical tools such as x-ray diffraction, neutron diffraction, HPLC were utilised in order to fulfil the objectives. Biochemical assay of hydroxyproline revealed a 10% depletion of collagen in mildly affacted (grade I and II) leaflets, while a 20% depletion of fibrillar collagen was revealed by mapping the collagen fibrils onto the anatomy of cardiac leaflets using x-ray data. Differential scanning calorimetry showed that there were no significant differences in the onset temperature of denaturation of collagen between the healthy and affected leaflets. However, in affected areas of leaflets, the enthalpy of denaturation significantly dropped by 20%. In the affected regions, neutron diffraction results showed an increase in the immature reducible cross-links though the low number of the samples can be considered a limiting factor in this regard. However, the HPLC results showed a 25% decrease in the number of mature cross-links. Additionally, the recently introduced imaging technologies to biology and medicine such as differential enhancing imaging (DEI) and coherent anti-Stokes Raman scattering spectroscopy (CARS) were, to the author’s best knowledge, applied for the first time to this disease. In doing so, this thesis furthers our understanding of the pathogenesis of MMVD, especially in relation to the collagen. The thesis provides new findings about MMVD and demonstrates the potential of biophysical tools for studying similar conditions.

Myxomatous mitral valve disease (MMVD) is one of the most common degenerative cardiac diseases affecting humans and dogs; however, its pathogenesis is not completely understood. This study focussed on developing tissue-engineered fibrin based canine mitral valve constructs, which can be used as an in vitro platform to study the pathogenesis of MMVD. Prior to three dimensional (3D) construct fabrication, primary canine mitral valve endothelial cells (VECs) and valve interstitial cells (VICs) were isolated, cultured and characterized utilising a variety of techniques. Moreover, preliminary experiments were carried out to optimise the purity of VEC cultures. It is uncertain if canine MMVD is initiated by long term shear stress damage to the valve endothelium or from abnormalities of VICs. To investigate both hypotheses, three types of models were produced using fibrin/based 3D culture techniques: healthy VEC-VIC co-culture (Type 1); healthy VEC-diseased VIC co-culture (Type 2); healthy VEC-VIC co-culture with endothelial damage during culture (Type 3). Histological examination demonstrated partial native tissue-like morphology of the 3D constructs. Results suggest that current static cultured constructs express MMVD markers irrespective of using healthy or diseased VICs. Simple mechanical stimulation was found to regulate VIC activity in the 3D models. Endothelial damage resulting in VIC phenotypic activation (a change typically observed in MMVD), and decreased mechanical tension appeared to be a negative regulator of this effect. Moreover, there appears to be heterogeneity in the activated VIC population. Additionally, distinct advanced glycation end product (AGE) carboxymethyllysine (CML) expression was found in canine MMVD valves, which suggesting this biochemical compound (known to affect long living protein) might be a putative regulator of MMVD pathogenesis. The role of CML in MMVD can be further investigated utilizing current 3D static mitral valve construct model in future studies. Lastly a prototype dynamic tubular construct and a customised bioreactor system were developed. Preliminary data suggest the feasibility of tubular construct fabrication and endothelialisation, which provides foundation for future dynamic conditioning experiments and will allow examination of the role of endothelial shear stress in triggering MMVD. In summary, this project successfully developed fibrin based canine mitral valve constructs. It is believed they are promising models for MMVD research, allowing new insights in understanding MMVD pathogenesis.