Plasma natriuretic peptide concentrations rise in response to either atrial or ventricular wall stretch and have been found to be useful in the diagnosis and assessment of patients with congestive cardiac failure. Although previous studies have suggested that plasma natriuretic peptides may offer some prognostic information in patients with valvular heart disease, it is unclear whether concentrations reflect disease severity and how plasma concentrations vary across different valve lesions. The aim of this research was to identify the factors that affect natriuretic peptide releases in valvular heart disease (VHD) and to investigate whether natriuretic peptides can be used in clinical practice to identify those patients who may benefit from early intervention. Plasma brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) concentrations were measured in patients with normal left ventricular (LV) systolic function and isolated VHD (mitral regurgitation, MR, n=33; aortic regurgitation, AR, n=39; aortic stenosis, AS, n=34; mitral stenosis, MS, n=30), and age and sex matched controls (n=39) immediately prior to exercise stress echocardiography. Peptide levels were compared against age and sex matched controls and against markers of severity for each valve lesions and across different valve lesions. Compared to controls, patients with all types of VHD had elevated plasma BNP concentrations [(MR median 35(inter quartile range 23-52), AR 34(22-45), AS 31(22-60), MS 58(34-90); controls 24(16-33) pg/mL; p < 0.01 for all]. LV end diastolic volume index varied by valve lesion; [MR (mean ± standard deviation 77±14), AR (91±28), AS (50±17), MS (43±11), controls (52±13) mL/m2; p < 0.0001]. There were no associations between LV volume and BNP. Left atrial (LA) area index varied [MR (18±4cm2/m2), AR (12±2), AS (11±3), MS (19±6), controls (11±2); p < 0.0001], but correlated with plasma BNP concentrations: MR (r=0.42,p=0.02), MS (r=0.86,p < 0.0001), AR (r=0.53,p=0.001), AS (r=0.52, p=0.002). Higher plasma BNP concentrations were associated with increased pulmonary artery pressure and reduced exercise capacity. Despite adverse cardiac remodelling, 81(60%) patients had a BNP concentration within the normal range. In patients with MS BNP was strongly associated with left atrial area index (r=0.86; p < 0.0001) and a BNP level of greater than 2 times the upper limit of normal identified patients who fulfilled guideline criteria for intervention (Area under the curve (AUC) 0.87 [0.74,0.99], p =0.006) and lower exercise capacity (AUC 0.82 [0.67,0.97]; p=0.004). In AR patients significant remodelling could occur whilst BNP remained within the normal range and in general BNP appeared less useful in assessing disease severity. However raised levels of BNP was associated with more severe AR as assessed by left ventricular outflow tract:AR Jet area ratio (r=0.43 p=0.0007). AR patients with an abnormal BNP had signs of early LV dysfunction on exercise with a lower LV longitudinal strain rate post exercise compared to AR patients with a normal BNP (0.68±0.31 vs. 1.06±0.45 1/sec; p=0.02). In MR patients, higher plasma BNP concentrations were associated with larger left atrial area index (r=0.42, p=0.02), higher pulmonary artery pressure (r=0.53, p=0.002) and a lower exercise time (r=-0.60, p=0.0002). BNP was not associated with any marker of left ventricular size or function in MR. These findings suggest that despite significant LV remodelling, plasma BNP concentrations are often normal in patients with VHD. Consequently, plasma BNP concentrations should be interpreted with caution when assessing patients with VHD. However natriuretic peptide levels offer complementary information to the standard assessment of patients with VHD and an unexplained finding of an elevated BNP in an otherwise asymptomatic patient should prompt further investigation.

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

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.

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

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.

O RDW é um índice hematológico utilizado para avaliação quantitativa da anisocitose eritrocitária, sendo útil na diferenciação das causas de quadros anêmicos. Atualmente, na medicina humana, o RDW tem sido considerado como um importante fator prognóstico em quadros de ICC, mesmo na ausência de anemia. A CRP é considerada a principal proteína de fase aguda sendo amplamente utilizada como marcadora de inflamação. O objetivo deste estudo foi determinar se há diferença nos valores do RDW e da concentração da CRP em cães com DVDCM com e sem ICC, bem como nos diferentes estágios (A, B1, B2, C e D) da doença, segundo a classificação da ACVIM, comparados a cães de raças predispostas, mas sem a doença cardíaca, e associações entre as variáveis. Esta pesquisa constituiu um estudo clínico observacional prospectivo transversal de caráter exploratório, realizada em cães com DVDCM. Para tal, foram selecionados 141 cães, de até 20kg, machos e fêmeas. O grupo com ICC e sem ICC apresentaram menor número de hemácias, hemoglobina e hematócrito em relação ao grupo controle (P&lt;0,001). O índice RDW não diferiu entre os grupos estudados quando foram alocados quanto ao estadiamento ACVIM (P&#61; 0,25), quanto à presença de ICC (P&#61; 0,146), ou de remodelamento cardíaco (P&#61;0,078). Quanto à CRP, os grupos C e D (C: 2.251 ng/dL, 962,05-5.292,5 ng/dL e D: 3.628,4 ng/dL, 4.1592,3-6.254,5 ng/dL) apresentaram concentração maior que o grupo controle (1.685,5 ng/dL, 1.045,45-3.795,5 ng/dL). Quanto ao estadiamento da DVDCM os grupos diferiram entre si (P&#61;0,014) porém, não foi possível identificar quais grupos apresentaram diferença. O grupo com ICC (2.939,7-1.304,25-5.908,8 ng/dL) apresentou maior concentração de CRP em relação ao grupo controle (1.685 ng/dL, 1.1015,45-3.795,5 ng/dL) (P&#61;0,03). O grupo com remodelamento (4.916,66&plusmn;6.102,14) apresentou maior concentração de CRP em relação ao grupo sem remodelamento (2.510,67&plusmn;3,26) (P&#61;0,009). Houve correlação positiva fraca entre CRP e número total de leucócitos (r&#61;0,197; P&#61;0,019). Conclui-se que apesar de não ter sido detectada diferença significativa para o RDW houve tendência a valores maiores nos cães com remodelamento comparados aos sem remodelamento. A concentração de CRP foi maior nos cães com remodelamento cardíaco em relação àqueles sem remodelamento e nos cães com ICC em relação aos cães sem ICC e àqueles de raças predispostas, mas sem alterações cardíacas. A dosagem sérica de CRP juntamente com outros marcadores e exames complementares já utilizados rotineiramente, poderá ser útil na avaliação e acompanhamento de pacientes portadores de DVDCM / RDW is a hematological index used for anisocytosis quantitative assesment, and useful to diferenciate anemic states. In human medicine, it has been considered an important prognostic factor in heart failure (HF), even in the absence of anemia. CRP is considered the main acute phase protein, and it is widely used as a marker of inflammation. An exploratory prospective cross sectional study was conducted aiming to determine whether there is a difference in RDW and CRP concentration in dogs with chronic degenerative mitral valve disease (CDMVD) with and without HF, presence of remodeling, as well as between the different disease stages (A, B1, B2, C e D - ACVIM), compared to dogs of predisposed breeds, but without heart disease. For this study, 141 dogs, up to 20kg, males and females were distributed in groups in order to achieve the main golas. Dogs with and without HF presented smaller number of red blood cells, hemoglobin and hematocrit compared to control group (P&lt;0.001). RDW was not diferente between groups for CDMVD stages (P&#61; 0.25), regarding HF presence (P&#61; 0.146), or cardiac remodeling (P&#61;0.078). Groups C and D (C: 2,251 ng/dL, 962.05-5,292.5 ng/dL e D: 3,628.4 ng/dL, 4,1592.3-6,254.5 ng/dL) presented higher CRP concentration compared to control (1,685.5 ng/dL, 1,045.45-3,795.5 ng/dL). There was a difference in CRP concentration between groups, however, it was not possible to identify the differing groups (P&#61;0.014). CRP concentration was higher in HF group (2,939.7-1,304.25-5,908.8 ng/dL) compared to control (1,685 ng/dL, 1,1015.45-3,795.5 ng/dL) (P&#61;0.03). CRP concentration was higher in cardiac remodeling group (4,916.66&plusmn;6,102.14) in relation to the group without remodeling (2,510.67&plusmn;3.26) (P&#61;0.009). There was a weak positive correlation between CRP and total leucocyte count (r&#61;0.197; P&#61;0.019). We conclude that although we could not identify a significative difference for RDW, there was a tendency to increased values in dogs with cardiac remodeling. CRP concentration was higher in cardiac remodeling group and in HF group compared to control. CRP serum together with other markers and auxiliary exames already routinely used may provide useful information for assessment and follow up of dogs with CDMVD

O objetivo desse estudo foi determinar se os índices de função, volume (VAE) e área (AAE) do átrio esquerdo (AE) podem ser utilizados para avaliar a gravidade da degeneração valvar crônica de mitral (DVCM) em cães, bem como diagnosticar a insuficiência cardíaca congestiva (ICC) nestes pacientes. A hipótese é que o aumento dos volumes e decréscimo da função atrial esquerda possa estar associado com a gravidade da DVCM e também com a ICC. Oitenta cães foram incluídos em um estudo clínico transversal observacional e prospectivo, sendo agrupados de acordo com a gravidade DVCM. Os cães foram igualmente distribuídos nos grupos A, B1, B2 e C + D, de acordo com o estadiamento para DVCM proposto pelo American College of Veterinary Internal Medicine. A mudança fracional da área (FAC) e a fração de ejeção atrial esquerda (FEAE) foram calculados com as seguintes equações: FAC total = 100 x (AAEmáxima AAEmínima) / AAEmáxima, FAC passiva = 100 x (AAEmáxima AAEpa) / AAEmáxima e FAC ativa = 100 x (AAEpa AAEminima) / AAEpa, cujas as mensurações foram realizadas no corte apical quatro câmaras; e FEAE Total = 100 x (VAEmáxima VAEmínima) / VAEmáxima, FEAE passiva = 100 x (VAEmáxima VAEpa) / VAEmáxima e FEAE ativa = 100 x (VAEpa VAEminima) / VAEpa, calculadas por meio do método biplanar área-comprimento, no corte apical quatro e duas câmaras. A FAC total, FAC ativa, FEAE total e FEAE ativa foram significativamente menores nos pacientes do grupo C+D do que as observadas nos demais grupos. Também foi observado que com o agravamento da DVCM ocorreu o aumento do VAEmáximo/kg, VAEpa/kg, VAEmínimo/kg, AAEmáximo/m2, AAEpa/m2, AAEmínimo/m2. Os volumes do AE, bem como suas áreas, apresentaram grande acurácia e aumentaram a capacidade para diagnosticar a ICC nos cães do DVCM. Conclui-se que a função atrial esquerda total e ativa é reduzida nos cães com ICC secundária a DVCM quando comparados aos cães saudáveis e naqueles com DVCM sem ICC. Além disso, os volumes e as áreas atriais podem ser utilizados para diagnosticar a ICC nesses pacientes / This study aimed to determine whether left atrial (LA) function indices, volume (LAV) and area (LAA) can be used to assess severity of mitral valve chronic degeneration (MVCD) in dogs, as well as to diagnose congestive heart failure (CHF) in these patients. The hypothesis stated that the increase in left atrial volumes and decrease in function are associated with the severity of MVCD and also with CHF. Eigthy dogs were included in a cross sectional prospective observational clinical study, grouped according to the severity of MVCD based on clinical signs and echocardiographic evaluation. Dogs were equitatively distribuited among groups A, B1, B2 and C + D, according to MVCD staging proposed by the American College of Veterinary Internal Medicine. The fractional area change (FAC) and left atrial ejection fraction (LAEF) were calculated based on the following equations: total FAC = 100 x (LAAmaximum LAAminimum) / LAAmaximum, passive FAC = 100 x (LAAmaximum LAApa) / LAAmaximum and active FAC ativa = 100 x (LAApa LAAminimum) / LAApa, which measurements were performed in four chamber apical view; and total LAEF = 100 x (LAVmaximum LAVminimum) / LAVmaximum, passive LAEF = 100 x (LAVmaximum LAVpa) / LAVmáximum and active LAEF = 100 x (LAVpa LAVminimum) / VLAVpa , calculated by biplanar arealength method in four and two-chambers apical view. The total and active FAC, total LAEF and active LAEF were significantly smaller in pacients from group C+D than the observed in the other groups. With increasing severity of MVCD there was increase in LAVmaximum/kg, LAVpa/kg, LAVminimum/kg, LAAmaximum/m2, LAApa/m2, LAAminimum/m2. LA volumes, as well as LA areas had a good performance as diagnostic methods, with high accuracy and increased capacity for heart failure detection in dogs with MVCD. In conclusion, left atrial total and active function are reduced in dogs with HF secondary to MVCD when compared with healthy dogs and dogs with MVCD without HF. Moreover, atrial volumes and areas can be used to diagnose HF in these patients

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.