Telotristat ethyl reverses myxomatous changes in mice mitral valves.

Rationale: Myxomatous mitral valve degeneration is a common pathological manifestation of mitral valve regurgitation, with or without valvular prolapse. In addition to similarities between naturally occurring and serotonergic valve degeneration, an increasing body of evidence has recently suggested that serotonin signaling is a regulator of degenerative valvulopathies. Studies have found that serotonin can be synthesized locally by valvular cells and serotonin receptors in turn may be activated to promote signaling. Recently, telotristat ethyl (TE) has been introduced as a treatment for carcinoid disease, by selectively inhibiting tryptophan hydroxylase 1, the rate-limiting enzyme in peripheral serotonin synthesis. TE provides a unique tool to test inhibition of serotonin synthesis in vivo, without impacting brain serotonin, to further confirm the role of local serotonin synthesis on heart valves. Objective: To confirm the link between serotonin and myxomatous valvular disease in vivo. Methods and results: A hypertension-induced myxomatous mitral valve disease mouse model was employed to test the effect of TE on valvular degeneration. Circulating serotonin and local serotonin in valve tissues were tested by enzyme immunoassay and immunohistochemistry, respectively. TE was administrated in two modes: (1) parallel with angiotensin II (A2); (2) post A2 treatment. Myxomatous changes were successfully recapitulated in hypertensive mice, as determined by ECM remodeling, myofibroblast transformation, and serotonin signaling activation. These changes were at least partially reversed upon TE administration. Conclusion: This study provides the first evidence of TE as a potential therapeutic for myxomatous mitral disease, either used to prevent or reverse myxomatous degeneration.

The Individual and Combined Effects of Shear, Tension, and Flexure on Aortic Heart Valve Endothelial Cells in Culture.

PURPOSE: The necessity of living engineered heart valves to treat patients with severe heart disease poses a challenge to tissue engineers. To reach such goal it is crucial to fully understand the role and the activities of valvular endothelial cells (VECs) when they face different types of mechanical stimuli. This study focuses on decomposing the roles of different mechanical stimuli on heart valve endothelial surfaces and the response of VECs in terms of morphology and phenotype change. METHODS: This study utilizes soft hydrogel-based scaffolds to use as a substrate for cell culture to mimic heart valve tissue leaflet. VECs were cultured as a monolayer on the gel surface and different types of mechanical stimuli were applied. Finally, the response of cells was investigated in terms of morphology and protein expression changes. RESULTS: Single stimuli introduces actin fibers reorganization in VECs, change in cell morphology, and higher mesenchymal protein expression. On the other hand, combined stimuli application has lower impact on actin fibers reorganization and cell morphology change, with lower mesenchymal protein expression. CONCLUSIONS: When VECs face a single mechanical stimuli, they undergo transdifferentiation and transform into mesenchymal cells. However, when these cells face a combination of mechanical stimuli, the rate of transformation decreases compared to single stimuli applications. This indicates that a single stimulus induces endothelial to mesenchymal transition in VECs while the process is slower under the combination of multiple mechanical stimuli.

Valvular Endothelial Cell Response to the Mechanical Environment-A Review.

Heart valve leaflets are complex structures containing valve endothelial cells, interstitial cells, and extracellular matrix. Heart valve endothelial cells sense mechanical stimuli, and communicate amongst themselves and the surrounding cells and extracellular matrix to maintain tissue homeostasis. In the presence of abnormal mechanical stimuli, endothelial cell communication is triggered in defense and such processes may eventually lead to cardiac disease progression. This review focuses on the role of mechanical stimuli on heart valve endothelial surfaces-from heart valve development and maintenance of tissue integrity to disease progression with related signal pathways involved in this process.

Comparison of Serotonin-Regulated Calcific Processes in Aortic and Mitral Valvular Interstitial Cells.

Calcification is an important pathological process and a common complication of degenerative valvular heart diseases, with higher incidence in aortic versus mitral valves. Two phenotypes of valvular interstitial cells (VICs), activated VICs and osteoblastic VICs (obVICs), synergistically orchestrate this pathology. It has been demonstrated that serotonin is involved in early stages of myxomatous mitral degeneration, whereas the role of serotonin in calcific aortic valve disease is still unknown. To uncover the link between serotonin and osteogenesis in heart valves, osteogenesis of aortic and mitral VICs was induced in vitro. Actin polymerization and serotonin signaling were inhibited using cytochalasin D and serotonin inhibitors, respectively, to investigate the role of cell activation and serotonin signals in valvular cell osteogenesis. To evaluate calcification progress, calcium and collagen deposits along with the expression of protein markers, including the rate-limiting enzyme of serotonin synthesis [tryptophan hydroxylase 1 (TPH1)], were assessed. When exposed to osteogenic culture conditions and grown on soft surfaces, passage zero aortic VICs increased extracellular collagen deposits and obVIC phenotype markers. A more intense osteogenic process was observed in aortic VICs of higher passages, where cells were activated prior to osteogenic induction. For both, TPH1 expression was upregulated as osteogenesis advanced. However, these osteogenic changes were reversed upon serotonin inhibition. This discovery provides a better understanding of signaling pathways regulating VIC phenotype transformation and explains different manifestations of degenerative pathologies. In addition, the discovery of serotonin-based inhibition of valvular calcification will contribute to the development of potential novel therapies for calcific valvular diseases.

Investigating the correlation of muscle function tests and sarcomere organization in C. elegans.

BACKGROUND: Caenorhabditis elegans has been widely used as a model to study muscle structure and function. Its body wall muscle is functionally and structurally similar to vertebrate skeletal muscle with conserved molecular pathways contributing to sarcomere structure, and muscle function. However, a systematic investigation of the relationship between muscle force and sarcomere organization is lacking. Here, we investigate the contribution of various sarcomere proteins and membrane attachment components to muscle structure and function to introduce C. elegans as a model organism to study the genetic basis of muscle strength. METHODS: We employ two recently developed assays that involve exertion of muscle forces to investigate the correlation of muscle function to sarcomere organization. We utilized a microfluidic pillar-based platform called NemaFlex that quantifies the maximum exertable force and a burrowing assay that challenges the animals to move in three dimensions under a chemical stimulus. We selected 20 mutants with known defects in various substructures of sarcomeres and compared the physiological function of muscle proteins required for force generation and transmission. We also characterized the degree of sarcomere disorganization using immunostaining approaches. RESULTS: We find that mutants with genetic defects in thin filaments, thick filaments, and M-lines are generally weaker, and our assays are successful in detecting the functional changes in response to each sarcomere location tested. We find that the NemaFlex and burrowing assays are functionally distinct informing on different aspects of muscle physiology. Specifically, the burrowing assay has a larger bandwidth in phenotyping muscle mutants, because it could pick ten additional mutants impaired while exerting normal muscle force in NemaFlex. This enabled us to combine their readouts to develop an integrated muscle function score that was found to correlate with the score for muscle structure disorganization. CONCLUSIONS: Our results highlight the suitability of NemaFlex and burrowing assays for evaluating muscle physiology of C. elegans. Using these approaches, we discuss the importance of the studied sarcomere proteins for muscle function and structure. The scoring methodology we have developed enhances the utility of  C. elegans as a genetic model to study muscle function.

Shear type and magnitude affect aortic valve endothelial cell morphology, orientation, and differentiation.

Valvular endothelial cells line the outer layer of heart valves and can withstand shear forces caused by blood flow. In contrast to vascular endothelial cells, there is limited amount of research over valvular endothelial cells. For this reason, the exact physiologic behavior of valvular endothelial cells is unclear. Prior studies have concluded that valvular endothelial cells align perpendicularly to the direction of blood flow, while vascular endothelial cells align parallel to blood flow. Other studies have suggested that different ranges of shear stress uniquely impact the behavior of valvular endothelial cells. The goal of this study was to characterize the response of valvular endothelial cell under different types, magnitudes, and durations of shear stress. In this work, the results demonstrated that with increased shear rate and duration of exposure, valvular endothelial cells no longer possessed the traditional cuboidal morphology. Instead through the change in cell circularity and aspect ratio, valvular endothelial cells aligned in an organized manner. In addition, different forms of shear exposure caused the area and circularity of valvular endothelial cells to decrease while inducing mesenchymal transformation validated through αSMA and TGFß1 expression. This is the first investigation showing that valvular endothelial cells alignment is not as straightforward as once thought (perpendicular to flow). Different types and magnitudes of shear induce different local behaviors. This is also the first demonstration of valvular endothelial cells undergoing EndMT without chemical inducers on a soft surface in vitro. Findings from this study provide insights to understanding the pathophysiology of valvular endothelial cells which can potentially propel future artificial engineered heart valves.

The tricuspid valve also maladapts as shown in sheep with biventricular heart failure.

Over 1.6 million Americans suffer from significant tricuspid valve leakage. In most cases this leakage is designated as secondary. Thus, valve dysfunction is assumed to be due to valve-extrinsic factors. We challenge this paradigm and hypothesize that the tricuspid valve maladapts in those patients rendering the valve at least partially culpable for its dysfunction. As a first step in testing this hypothesis, we set out to demonstrate that the tricuspid valve maladapts in disease. To this end, we induced biventricular heart failure in sheep that developed tricuspid valve leakage. In the anterior leaflets of those animals, we investigated maladaptation on multiple scales. We demonstrated alterations on the protein and cell-level, leading to tissue growth, thickening, and stiffening. These data provide a new perspective on a poorly understood, yet highly prevalent disease. Our findings may motivate novel therapy options for many currently untreated patients with leaky tricuspid valves.

Pluronic gel-based burrowing assay for rapid assessment of neuromuscular health in C. elegans.

Whole-organism phenotypic assays are central to the assessment of neuromuscular function and health in model organisms such as the nematode C. elegans. In this study, we report a new assay format for engaging C. elegans in burrowing that enables rapid assessment of nematode neuromuscular health. In contrast to agar environments that pose specific drawbacks for characterization of C. elegans burrowing ability, here we use the optically transparent and biocompatible Pluronic F-127 gel that transitions from liquid to gel at room temperature, enabling convenient and safe handling of animals. The burrowing assay methodology involves loading animals at the bottom of well plates, casting a liquid-phase of Pluronic on top that solidifies via a modest temperature upshift, enticing animals to reach the surface via chemotaxis to food, and quantifying the relative success animals have in reaching the chemoattractant. We study the influence of Pluronic concentration, gel height and chemoattractant choice to optimize assay performance. To demonstrate the simplicity of the assay workflow and versatility, we show its novel application in multiple areas including (i) evaluating muscle mutants with defects in dense bodies and/or M-lines (pfn-3, atn-1, uig-1, dyc-1, zyx-1, unc-95 and tln-1), (ii) tuning assay conditions to reveal changes in the mutant gei-8, (iii) sorting of fast burrowers in a genetically-uniform wild-type population for later quantitation of their distinct muscle gene expression, and (iv) testing proteotoxic animal models of Huntington and Parkinson's disease. Results from our studies show that stimulating animals to navigate in a dense environment that offers mechanical resistance to three-dimensional locomotion challenges the neuromuscular system in a manner distinct from standard crawling and thrashing assays. Our simple and high throughput burrowing assay can provide insight into molecular mechanisms for maintenance of neuromuscular health and facilitate screening for therapeutic targets.

Força muscular e habilidade de locomoção em indivíduos pós-acidente vascular encefálico crônico / Fuerza muscular y habilidad de locomoción en individuos post-accidente cerebrovascular crónico / Muscle strength and locomotion ability in individuals with chronic stroke

RESUMO O objetivo do estudo foi verificar se existem diferenças na força muscular dos membros inferiores (MMII) e na habilidade de locomoção de indivíduos pós-acidente vascular encefálico (AVE) crônico, classificados como deambuladores comunitários ou não comunitários. Foi realizado um estudo transversal em 60 indivíduos pós-AVE crônico, divididos em deambuladores comunitários (n=33) e não comunitários (n=27) pela velocidade de marcha. A força muscular de sete grupos musculares bilaterais de MMII foi avaliada por meio do teste do esfigmomanômetro modificado e habilidade de locomoção pelo ABILOCO. Estatísticas descritivas foram utilizadas para caracterizar a amostra, e o teste t de Student para amostras independentes, a fim de comparar os dois grupos de indivíduos pós-AVE. Observou-se que os deambuladores comunitários apresentaram maiores valores de força muscular para a maioria dos grupos musculares de MMII (−0,973≥t≥−3,189; p≤0,04), e na habilidade de locomoção (t=−2,841; p=0,006). Os indivíduos pós-AVE crônico deambuladores comunitários possuem maior força muscular de MMII e mais habilidade de locomoção em comparação aos deambuladores não comunitários. Sugere-se que a avaliação fisioterapêutica de indivíduos pós-AVE inclua, além da mensuração da força muscular de MMII e seu tratamento, a mensuração da percepção da habilidade de locomoção, para análises da evolução do paciente e da eficácia da conduta terapêutica.

RESUMEN El objetivo del estudio fue verificar si existen diferencias en la fuerza muscular de los miembros inferiores (MMII) y en la habilidad de locomoción de individuos post-accidente cerebrovascular encefálico (ACV) crónico, clasificados como deambuladores comunitarios o no comunitarios. Se realizó un estudio transversal en 60 individuos post-ACV crónico, divididos en deambuladores comunitarios (n = 33) y no comunitarios (n = 27) por la velocidad de marcha. La fuerza muscular de siete grupos musculares bilaterales de MMII fue evaluada por medio de la prueba del esfigmomanómetro modificado, y la habilidad de locomoción por el ABILOCO. Las estadísticas descriptivas se utilizaron para caracterizar la muestra, y la prueba t de Student para muestras independientes con el fin de comparar los dos grupos de sujetos post-ACV. Se observó que los deambuladores comunitarios presentaron mayores valores de fuerza muscular para la mayoría de los grupos musculares de MMII (−0,973≥t≥−3,189; p≤0,04), y en la habilidad de locomoción (t=−2,841; p=0,006). Los individuos post-ACV crónico deambuladores comunitarios poseen mayor fuerza muscular de MMII y más habilidad de locomoción en comparación a los deambuladores no comunitarios. Se sugiere que la evaluación fisioterapéutica de individuos post-ACV incluya, además de la medición de la fuerza muscular de MMII y su tratamiento, la medición de la percepción de la habilidad de locomoción, para análisis de la evolución del paciente y de la eficacia de la conducta terapéutica.

ABSTRACT The objective of this study was to verify if there are differences in the lower-limb muscle strength (LL) and in the locomotion ability among post-stroke patients classified as community or non-community ambulators. A cross-sectional study was conducted in 60 post-chronic stroke subjects, divided into community (n=33) and non-community (n=27) ambulators by gait speed. The muscle strength of seven bilateral muscle groups of LL was evaluated through the modified sphygmomanometer test and locomotion ability through ABILOCO. Descriptive statistics were used to characterize the sample, and Student's t-test was used for independent samples to compare the two groups of post-stroke individuals. We observed that community ambulators had higher values of muscle strength for most muscle groups of LL (−0.973≥t≥3.189; p≤0.04), and in the locomotion ability (t=−2.841; p=0.006). Community ambulators showed higher LL muscle strength and better locomotion ability compared with non-community ambulators. Physiotherapeutic evaluation of post-stroke individuals should include, besides the measurement of LL muscle strength and its treatment, the measurement of the perception of locomotion ability to analyze the evolution of the patient and the efficacy of the therapeutic behavior.

Characterization of jet injection efficiency with mouse cadavers.

Needle-free drug delivery is highly sought after for reduction in sharps waste, prevention of needle-stick injuries, and potential for improved drug dispersion and uptake. Whilst there is a wealth of literature on the array of different delivery methods, jet injection is proposed as the sole candidate for delivery of viscous fluids, which is especially relevant with the advent of DNA-based vaccines. The focus of this study was therefore to assess the role of viscosity and jet configuration (i.e. stand-off relative to the skin) upon injection efficiency for a fixed spring-loaded system (Bioject ID Pen). We performed this assessment in the context of mouse cadavers and found that the dominant factor in determining success rates was the time from euthanasia, which was taken as a proxy for the stiffness of the underlying tissue. For overall injection efficiency, ANOVA tests indicated that stiffness was highly significant (P <  < 0.001), stand-off was moderately significant (P < 0.1), and viscosity was insignificant. In contrast, both viscosity and standoff were found to be significant (P < 0.01) when evaluating the percentage delivered intradermally. Using high-resolution micro-computed tomography (µ-CT), we also determined the depth and overall dispersion pattern immediately after injection.

The effect of physiological stretch and the valvular endothelium on mitral valve proteomes.

IMPACT STATEMENT: This work is important to the field of heart valve pathophysiology as it provides new insights into molecular markers of mechanically induced valvular degeneration as well as the protective role of the valvular endothelium. These discoveries reported here advance our current knowledge of the valvular endothelium and how its removal essentially takes valve leaflets into an environmental shock. In addition, it shows that static conditions represent a mild pathological state for valve leaflets, while 10% cyclic stretch provides valvular cell quiescence. These findings impact the field by informing disease stages and by providing potential new drug targets to reverse or slow down valvular change before it affects cardiac function.

Osteogenesis inducers promote distinct biological responses in aortic and mitral valve interstitial cells.

Both aortic and mitral valves calcify in pathological conditions; however, the prevalence of aortic valve calcification is high whereas mitral valve leaflet calcification is somewhat rare. Patterns of valvular calcification may differ due to valvular architecture, but little is known to that effect. In this study, we investigated the intrinsic osteogenic differentiation potential of aortic versus mitral valve interstitial cells provided minimal differentiation conditions. For the assessment of calcification at the cellular level, we used classic inducers of osteogenesis in stem cells: ß-glycerophosphate (ß-Gly), dexamethasone (Dex), and ascorbate (Asc). In addition to proteomic analyses, osteogenic markers and calcium precipitates were evaluated across treatments of aortic and mitral valve cells. The combination of ß-Gly, Asc, and Dex induced aortic valve interstitial cells to synthesize extracellular matrix, overexpress osteoblastic markers, and deposit calcium. However, no strong evidence showed the calcification of mitral valve interstitial cells. Mitral cells mainly responded to Asc and Dex by cell activation. These findings provide a deeper understanding of the physiological properties of aortic and mitral valves and tendencies for calcific changes within each valve type, contributing to the development of future therapeutics for heart valve diseases.

Correlation between valvular interstitial cell morphology and phenotypes: A novel way to detect activation.

Valvular interstitial cells (VICs) constitute the major cell population in heart valves. Quiescent fibroblastic VICs are seen in adult healthy valves. They become activated myofibroblastic VICs during development, in diseased valves and in vitro. 2D substrate stiffness within a 5-15 kPa range along with high passage numbers promote VIC activation in vitro. In this study, we characterize VIC quiescence and activation across a 1-21 kPa range of substrate stiffness and passages. We define a cell morphology characterization system for VICs as they transform. We hypothesize that VICs show distinct morphological characteristics in different activation states and the morphology distribution varies with substrate stiffness and passage number. Four VIC morphologies - tailed, spindle, rhomboid and triangle - account for the majority of VIC in this study. Using α-smooth muscle actin (α-SMA), non-muscle myosin heavy chain B (SMemb) and transforming growth factor ß (TGF-ß) as activation markers for validation, we developed a system where we categorize morphology distribution of VIC cultures, to be potentially used as a non-destructive detection method of activation state. We also show that this system can be used to force stiffness-induced deactivation. The reversibility in VIC activation has important implications in in vitro research and tissue engineering.

A microfluidic cardiac flow profile generator for studying the effect of shear stress on valvular endothelial cells.

To precisely investigate the mechanobiological responses of valvular endothelial cells, we developed a microfluidic flow profile generator using a pneumatically-actuated micropump consisting of microvalves of various sizes. By controlling the closing pressures and the actuation times of these microvalves, we modulated the magnitude and frequency of the shear stress to mimic mitral and aortic inflow profiles with frequencies in the range of 0.8-2 Hz and shear stresses up to 20 dyn cm-2. To demonstrate this flow profile generator, aortic inflow with an average of 5.9 dyn cm-2 shear stress at a frequency of 1.2 Hz with a Reynolds number of 2.75, a Womersley number of 0.27, and an oscillatory shear index (OSI) value of 0.2 was applied to porcine aortic valvular endothelial cells (PAVECs) for mechanobiological studies. The cell alignment, cell elongation, and alpha-smooth muscle actin (αSMA) expression of PAVECs under perfusion, steady flow, and aortic inflow conditions were analyzed to determine their shear-induced cell migration and trans-differentiation. In this morphological and immunocytochemical study, we found that the PAVECs elongated and aligned themselves perpendicular to the directions of the steady flow and the aortic inflow. In contrast, under perfusion with a fluidic shear stress of 0.47 dyn cm-2, the PAVECs elongated and aligned themselves parallel to the direction of flow. The PAVECs exposed to the aortic inflow upregulated their αSMA-protein expression to a greater degree than those exposed to perfusion and steady flow. By comparing these results to those of previous studies of pulsatile flow, we also found that the ratio of positive to negative shear stress plays an important role in determining PAVECs' trans-differentiation and adaptation to flow. This microfluidic cardiac flow profile generator will enable future valvular mechanobiological studies to determine the roles of magnitude and frequency of shear stresses.

A Three-Dimensional Collagen-Elastin Scaffold for Heart Valve Tissue Engineering.

Since most of the body's extracellular matrix (ECM) is composed of collagen and elastin, we believe the choice of these materials is key for the future and promise of tissue engineering. Once it is known how elastin content of ECM guides cellular behavior (in 2D or 3D), one will be able to harness the power of collagen-elastin microenvironments to design and engineer stimuli-responsive tissues. Moreover, the implementation of such matrices to promote endothelial-mesenchymal transition of primary endothelial cells constitutes a powerful tool to engineer 3D tissues. Here, we design a 3D collagen-elastin scaffold to mimic the native ECM of heart valves, by providing the strength of collagen layers, as well as elasticity. Valve interstitial cells (VICs) were encapsulated in the collagen-elastin hydrogels and valve endothelial cells (VECs) cultured onto the surface to create an in vitro 3D VEC-VIC co-culture. Over a seven-day period, VICs had stable expression levels of integrin ß1 and F-actin and continuously proliferated, while cell morphology changed to more elongated. VECs maintained endothelial phenotype up to day five, as indicated by low expression of F-actin and integrin ß1, while transformed VECs accounted for less than 7% of the total VECs in culture. On day seven, over 20% VECs were transformed to mesenchymal phenotype, indicated by increased actin filaments and higher expression of integrin ß1. These findings demonstrate that our 3D collagen-elastin scaffolds provided a novel tool to study cell-cell or cell-matrix interactions in vitro, promoting advances in the current knowledge of valvular endothelial cell mesenchymal transition.

Survival analysis of ART restorations in primary molars of preschool children: 1 year follow-up / Análise de sobrevivência de restaurações ART em molares decíduos de crianças com idade pré-escolar: acompanhamento de 1 ano

Among the minimally invasive approaches available today, the atraumatic restorative treatment (ART) has demonstrated promising results both in the primary and permanent dentition. Objective: To evaluate the survival of Class I ART restorations in preschoolers with two Brazilian brands of glass ionomer cements (GIC) in comparison with a reference GIC. Material and method: The cavities of 49 preschool children (three to five years) with carious lesions in the posterior teeth (N=81) were filled by two experienced pediatric dentists according to the ART technique. The Brazilian GICs Maxxion-R (MR) and Vitro-Fil LC (VF), and the reference GIC Ketac-Molar (KM) were placed in a randomly pre-established sequence. Restorations were evaluated after 6 and 12 months by another investigator. Scores 0 and 1 were considered successful, while scores 3-9 were classified as failures. Kaplan-Meier survival analysis and the log-rank test were performed (p<0.05). Result: No statistically significant differences in survival rates of the tested GIC were observed after 12 months. Conclusion: The clinical performance the low-cost Brazilian GICs MR and VF observed after 12 months suggests that they may be an alternative for Class I ART restorations to safeguard the natural exfoliation of primary teeth. However, until further studies involving a larger number of restorations and longer follow-up periods are conducted, reference GIC such as KM should continue to be the material of choice for ART restorations.

Dentre as abordagens minimamente invasivas atualmente disponíveis, o tratamento restaurador atraumático (ART) demonstra resultados promissores tanto na dentadura decídua quanto permanente. Objetivo: Avaliar a sobrevivência de restaurações ART Classe I, em pré-escolares, com duas marcas brasileiras de cimentos de ionômeros de vidro (CIV) em comparação com um CIV de referência. Material e método: Cavidades de 49 crianças pré-escolares (três a cinco anos de idade) com lesões cariosas nos dentes posteriores (N = 81) foram preenchidas por dois odontopediatras experientes, de acordo com a técnica ART. Os CIV brasileiros Maxxion-R (MR) e Vitro-Fil LC (VF) e o CIV de referência, Ketac-Molar (KM), foram inseridos em uma sequência pré-estabelecida aleatoriamente. As restaurações foram avaliadas após 6 e 12 meses por outro pesquisador. As pontuações 0 e 1 foram consideradas bem-sucedidas, enquanto as pontuações 3-9 foram classificadas como falhas. Foram aplicadas a análise de sobrevivência de Kaplan-Meier e o teste log-rank (p <0,05). Resultado: Não foram observadas diferenças estatisticamente significativas nas taxas de sobrevivência dos CIV testados após 12 meses. Conclusão: O desempenho clínico dos CIV brasileiros MR e VF, observado após 12 meses, sugere que estes podem ser uma alternativa para restaurações ART Classe I para proteger a esfoliação natural dos dentes decíduos. No entanto, até que sejam realizados estudos adicionais envolvendo um maior número de restaurações e períodos de acompanhamento mais longos, os CIV de referência, como o KM, devem continuar sendo o material de escolha para as restaurações ART.

Mechanical Properties and Composition of the Basal Leaflet-Annulus Region of the Tricuspid Valve.

The Tricuspid valve (TV) annulus is a transition structure from the leaflets to the myocardium, with 3 different annulus segments corresponding to the TV leaflets, which includes both basal leaflets and bordering myocardium. The objective of this study was to understand TV annulus mechanical properties and correlate it to the biological composition. The uniaxial testing of the annulus segments from ten porcine TVs was performed to measure Young's modulus (E) and extensibility (εT). Western blotting and histology were executed. The septal annulus E value (208.7 ± 67.2 kPa) was statistically greater (p < 0.01) than that of the anterior (92.0 ± 66.8 kPa) and the posterior annulus segment (136.8 ± 56.9 kPa) (p < 0.05), respectively. εT among the 3 segments were equivalent (p values < 0.05). Western blotting and histology indicated that collagen was greatest along the septal annulus segment, which is correlated to E values. Collagen fibers from the leaflets inserted into the myocardium and faded out. Collagen content explains greater E and suture strength in the surgical annulus repair and larger resistance to annulus dilation in the septal annulus as compared with other segments. This study elucidates new knowledge of mechanical properties of the basal leaflet-annulus region of the TV annulus, which can be useful for future TV repair techniques.

Phenotype Transformation of Aortic Valve Interstitial Cells Due to Applied Shear Stresses Within a Microfluidic Chip.

Despite valvular heart diseases constituting a significant medical problem, the acquisition of information describing their pathophysiology remains difficult. Due to valvular size, role and location within the body, there is a need for in vitro systems that can recapitulate disease onset and progression. This study combines the development of an in vitro model and its application in the mechanical stimulation of valvular cell transformation. Specifically, porcine aortic valvular interstitial cells (PAVIC) were cultured on polydimethylsiloxane microfluidic devices with or without exposure to shear stresses. Mechanobiological responses of valvular interstitial cells were evaluated at shear stresses ranging from 0 to 4.26 dyn/cm2. When flow rates were higher than 0.78 dyn/cm2, cells elongated and aligned with the flow direction. In addition, we found that shear stress enhanced the formation of focal adhesions and up-regulated PAVIC transformation, assessed by increased expression of α-smooth muscle actin and transforming growth factor ß. This study reveals a link between the action of shear forces, cell phenotype transformation and focal adhesion formation. This constitutes the first step towards the development of co-cultures (interstitial-endothelial cells) on organ-on-a-chip devices, which will enable studies of the signaling pathways regulating force-induced valvular degeneration in microtissues and potential discovery of valvular degeneration therapies.

A survey of membrane receptor regulation in valvular interstitial cells cultured under mechanical stresses.

Degenerative valvular diseases have been linked to the action of abnormal forces on valve tissues during each cardiac cycle. It is now accepted that the degenerative behavior of valvular cells can be induced mechanically in vitro. This approach of in vitro modeling of valvular cells in culture constitutes a powerful tool to study, characterize, and develop predictors of heart valve degeneration in vivo. Using such in vitro systems, we expect to determine the exact signaling mechanisms that trigger and mediate propagation of degenerative signals. In this study, we aim to uncover the role of mechanosensing proteins on valvular cell membranes. These can be cell receptors and triggers of downstream pathways that are activated upon the action of cyclical tensile strains in pathophysiological conditions. In order to identify mechanosensors of tensile stresses on valvular interstitial cells, we employed biaxial cyclic strain of valvular cells in culture and quantitatively evaluated the expression of cell membrane proteins using a targeted protein array and interactome analyses. This approach yielded a high-throughput screening of all cell surface proteins involved in sensing mechanical stimuli. In this study, we were able to identify the cell membrane proteins which are activated during physiological cyclic tensile stresses of valvular cells. The proteins identified in this study were clustered into four interactomes, which included CC chemokine ligands, thrombospondin (adhesive glycoproteins), growth factors, and interleukins. The expression levels of these proteins generally indicated that cells tend to increase adhesive efforts to counteract the action of mechanical forces. This is the first study of this kind used to comprehensively identify the mechanosensitive proteins in valvular cells.