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

Heart valve dysfunction is a relatively common condition in the population, whereas significant heart valve disease is more unusual. The cause of different types of heart valve disease depends on which valve is concerned. Rheumatic heart valve disease, has for a long time been considered to constitute a post-inflammatory condition. During the 1990s it was also shown that the so-called non-rheumatic or degenerative tricuspid aortic stenosis, comprised signs of inflammation. In this study, 118 patients (the VOCIN study group) referred to the University Hospital for preoperative investigation due to significant heart valve disease, were examined regarding signs of inflammation. Twenty-nine aortic valves from patients with significant aortic stenosis were divided into tricuspid and bicuspid aortic valves. The bicuspid aortic stenotic valves revealed signs of inflammation to a similar extent as the tricuspid valves. However, the tricuspid and bicuspid valves differed regarding distribution of calcification. In contrast, inflammation was not a predominant feature in 15 aortic and mitral valves from patients with significant heart valve regurgitation. Gross valvular pathology consistent with rheumatic aortic stenosis was found in 10 patients. These valves revealed a somewhat lower degree of inflammatory cell infiltration, but on the whole, there were no substantial differences when compared to non-rheumatic aortic stenotic valves. They did, however, reveal a similar distribution of calcification as the bicuspid, non-rheumatic aortic valves. The VOCIN study group was compared to an age- and gender matched control group with regard to history and signs of rheumatic disease. There was not any increased prevalence of clinical manifestations of non-cardiac inflammatory disease in patients with significant heart valve disease, when compared to healthy control subjects. However, patients with heart valve disease had significantly increased serum levels of inflammatory markers compared to controls. The increase in inflammatory markers remained significant even in the subgroup of non-rheumatic aortic stenosis devoid of coronary artery disease. These results indicate that a systemic inflammatory component is associated with stenotic, non-rheumatic heart valve disease. The similarities between different forms of calcific aortic valve disease indicate a similar pathogenesis. The question is raised whether aortic stenosis is one disease, mainly caused by a general and non-specific response to dynamic tissue stress due to an underlying malformation of the valve.

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.

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

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<0,001). O índice RDW não diferiu entre os grupos estudados quando foram alocados quanto ao estadiamento ACVIM (P= 0,25), quanto à presença de ICC (P= 0,146), ou de remodelamento cardíaco (P=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=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=0,03). O grupo com remodelamento (4.916,66±6.102,14) apresentou maior concentração de CRP em relação ao grupo sem remodelamento (2.510,67±3,26) (P=0,009). Houve correlação positiva fraca entre CRP e número total de leucócitos (r=0,197; P=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<0.001). RDW was not diferente between groups for CDMVD stages (P= 0.25), regarding HF presence (P= 0.146), or cardiac remodeling (P=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=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=0.03). CRP concentration was higher in cardiac remodeling group (4,916.66±6,102.14) in relation to the group without remodeling (2,510.67±3.26) (P=0.009). There was a weak positive correlation between CRP and total leucocyte count (r=0.197; P=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

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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