RESUMEN
Valve disease and particularly calcific aortic valve disease (CAVD) and diabetes (DM) are progressive diseases constituting a global health burden for all aging societies (Progress in Cardiovascular Diseases. 2014;56(6):565: Circulation Research. 2021;128(9):1344). Compared to non-diabetic individuals (The Lancet. 2008;371(9626):1800: The American Journal of Cardiology. 1983;51(3):403: Journal of the American College of Cardiology. 2017;69(12):1523), the diabetic patients have a significantly greater propensity for cardiovascular disorders and faster degeneration of implanted bioprosthetic aortic valves. Previously, using an original experimental model, the diabetic-hyperlipemic hamsters, we have shown that the earliest alterations induced by these conditions occur at the level of the aortic valves and, with time these changes lead to calcifications and CAVD. However, there are no pharmacological treatments available to reverse or retard the progression of aortic valve disease in diabetes, despite the significant advances in the field. Therefore, it is critical to uncover the mechanisms of valve disease progression, find biomarkers for diagnosis and new targets for therapies. This review aims at presenting an update on the basic research in CAVD in the context of diabetes. We provide an insight into the accumulated data including our results on diabetes-induced progressive cell and molecular alterations in the aortic valve, new potential biomarkers to assess the evolution and therapy of the disease, advancement in targeted nanotherapies, tissue engineering and the potential use of circulating endothelial progenitor cells in CAVD.
Asunto(s)
Válvula Aórtica/patología , Complicaciones de la Diabetes , Diabetes Mellitus/metabolismo , Enfermedades de las Válvulas Cardíacas/etiología , Enfermedades de las Válvulas Cardíacas/metabolismo , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/ultraestructura , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Biomarcadores , Terapia Combinada , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/ultraestructura , Transición Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Enfermedades de las Válvulas Cardíacas/diagnóstico , Enfermedades de las Válvulas Cardíacas/terapia , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Mediadores de Inflamación/metabolismoRESUMEN
Atherosclerosis, calcific aortic valve disease (CAVD), and bioprosthetic heart valve degeneration (alternatively termed structural valve deterioration, SVD) represent three diseases affecting distinct components of the circulatory system and their substitutes, yet sharing multiple risk factors and commonly leading to the extraskeletal calcification. Whereas the histopathology of the mentioned disorders is well-described, their ultrastructural pathology is largely obscure due to the lack of appropriate investigation techniques. Employing an original method for sample preparation and the electron microscopy visualisation of calcified cardiovascular tissues, here we revisited the ultrastructural features of lipid retention, macrophage infiltration, intraplaque/intraleaflet haemorrhage, and calcification which are common or unique for the indicated types of cardiovascular disease. Atherosclerotic plaques were notable for the massive accumulation of lipids in the extracellular matrix (ECM), abundant macrophage content, and pronounced neovascularisation associated with blood leakage and calcium deposition. In contrast, CAVD and SVD generally did not require vasculo- or angiogenesis to occur, instead relying on fatigue-induced ECM degradation and the concurrent migration of immune cells. Unlike native tissues, bioprosthetic heart valves contained numerous specialised macrophages and were not capable of the regeneration that underscores ECM integrity as a pivotal factor for SVD prevention. While atherosclerosis, CAVD, and SVD show similar pathogenesis patterns, these disorders demonstrate considerable ultrastructural differences.
Asunto(s)
Enfermedad de la Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/patología , Válvula Aórtica/patología , Aterosclerosis/patología , Bioprótesis , Calcinosis/patología , Prótesis Valvulares Cardíacas , Anciano , Válvula Aórtica/ultraestructura , Enfermedad de la Válvula Aórtica/terapia , Biomarcadores , Bioprótesis/efectos adversos , Diagnóstico Diferencial , Femenino , Prótesis Valvulares Cardíacas/efectos adversos , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Modelos BiológicosRESUMEN
Calcific aortic valve disease (CAVD) is characterized by valvular fibrosis and calcification and driven by differentiating valvular interstitial cells (VICs). Expression data from patient biopsies suggest that transforming growth factor (TGF)-ß1 is implicated in CAVD pathogenesis. However, CAVD models using isolated VICs failed to deliver clear evidence on the role of TGF-ß1. Thus, employing cultures of aortic valve leaflets, we investigated effects of TGF-ß1 in a tissue-based three-dimensional (3-D) CAVD model. We found that TGF-ß1 induced phosphorylation of Mothers against decapentaplegic homolog (SMAD) 3 and expression of SMAD7, indicating effective downstream signal transduction in valvular tissue. Thus, TGF-ß1 increased VIC contents of rough endoplasmic reticulum, Golgi, and secretory vesicles as well as tissue levels of RNA and protein. In addition, TGF-ß1 raised expression of proliferation marker cyclin D1, attenuated VIC apoptosis, and upregulated VIC density. Moreover, TGF-ß1 intensified myofibroblastic VIC differentiation as evidenced by increased α-smooth muscle actin and collagen type I along with diminished vimentin expression. In contrast, TGF-ß1 attenuated phosphorylation of SMAD1/5/8 and upregulation of ß-catenin while inhibiting osteoblastic VIC differentiation as revealed by downregulation of osteocalcin expression, alkaline phosphatase activity, and extracellular matrix incorporation of hydroxyapatite. Collectively, these effects resulted in blocking of valvular tissue calcification and associated disintegration of collagen fibers. Instead, TGF-ß1 induced development of fibrosis. Overall, in a tissue-based 3-D CAVD model, TGF-ß1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of VICs, thus triggering dominant fibrosis. Simultaneously, by inhibiting SMAD1/5/8 activation and canonical Wnt/ß-catenin signaling, TGF-ß1 attenuates osteoblastic VIC differentiation, thus blocking valvular tissue calcification. These findings question a general phase-independent CAVD-promoting role of TGF-ß1.NEW & NOTEWORTHY Employing aortic valve leaflets as a tissue-based three-dimensional disease model, our study investigates the role of transforming growth factor (TGF)-ß1 in calcific aortic valve disease pathogenesis. We find that, by activating Mothers against decapentaplegic homolog 3, TGF-ß1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of valvular interstitial cells, thus triggering dominant fibrosis. Simultaneously, by inhibiting activation of Mothers against decapentaplegic homolog 1/5/8 and canonical Wnt/ß-catenin signaling, TGF-ß1 attenuates apoptosis and osteoblastic differentiation of valvular interstitial cells, thus blocking valvular tissue calcification. These findings question a general phase-independent calcific aortic valve disease-promoting role of TGF-ß1.
Asunto(s)
Estenosis de la Válvula Aórtica/metabolismo , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Calcinosis/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Actinas/metabolismo , Animales , Válvula Aórtica/ultraestructura , Estenosis de la Válvula Aórtica/patología , Apoptosis , Calcinosis/patología , Calcio/metabolismo , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Fibrosis , Ovinos , Proteína smad7/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
One of the leading trends in the modern tissue engineering is the development of new effective methods of decellularization aimed at the removal of cellular components from a donor tissue, reducing its immunogenicity and the risk of rejection. Supercritical CO2 (scCO2)-assisted processing has been proposed to improve the outcome of decellularization, reduce contamination and time costs. The resulting products can serve as personalized tools for tissue-engineering therapy of various somatic pathologies. However, the decellularization of heterogeneous 3D structures, such as the aortic root, requires optimization of the parameters, including preconditioning medium composition, the type of co-solvent, values of pressure and temperature inside the scCO2 reactor, etc. In our work, using an ovine aortic root model, we performed a comparative analysis of the effectiveness of decellularization approaches based on various combinations of these parameters. The protocols were based on the combinations of treatments in alkaline, ethanol or detergent solutions with scCO2-assisted processing at different modes. Histological analysis demonstrated favorable effects of the preconditioning in a detergent solution. Following processing in scCO2 medium provided a high decellularization degree, reduced cytotoxicity, and increased ultimate tensile strength and Young's modulus of the aortic valve leaflets, while the integrity of the extracellular matrix was preserved.
Asunto(s)
Válvula Aórtica/ultraestructura , Estructuras Celulares , Ingeniería de Tejidos/métodos , Animales , Dióxido de Carbono , Células Cultivadas , Módulo de Elasticidad , Matriz Extracelular , Humanos , Células Madre Mesenquimatosas , Ovinos , Resistencia a la TracciónRESUMEN
Calcific aortic stenosis is a disorder that impacts the physiology of heart valves. Fibrocalcific events progress in conjunction with thickening of the valve leaflets. Over the years, these events promote stenosis and obstruction of blood flow. Known and common risk factors are congenital defects, aging and metabolic syndromes linked to high plasma levels of lipoproteins. Inflammation and oxidative stress are the main molecular mediators of the evolution of aortic stenosis in patients and these mediators regulate both the degradation and remodeling processes. Mitochondrial dysfunction and dysregulation of autophagy also contribute to the disease. A better understanding of these cellular impairments might help to develop new ways to treat patients since, at the moment, there is no effective medical treatment to diminish neither the advancement of valve stenosis nor the left ventricular function impairments, and the current approaches are surgical treatment or transcatheter aortic valve replacement with prosthesis.
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Estenosis de la Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Calcinosis/metabolismo , Mitocondrias Cardíacas/fisiología , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/ultraestructura , Estenosis de la Válvula Aórtica/diagnóstico , Estenosis de la Válvula Aórtica/epidemiología , Estenosis de la Válvula Aórtica/cirugía , Autofagia , Membrana Basal/ultraestructura , Progresión de la Enfermedad , Células Endoteliales/patología , Humanos , Inflamación , Lípidos/análisis , Óxido Nítrico Sintasa de Tipo III/fisiología , Estrés Oxidativo , Terapias en Investigación , Respuesta de Proteína DesplegadaRESUMEN
Improved thromboresistance of mechanical valves is desired to decrease the risk of thromboembolism and thrombosis and reduce the dosage of anticoagulation with a vitamin K antagonist (e.g., warfarin). For several mechanical valves, design-related features are responsible for their improved thromboresistance. However, it remains unclear whether material-related features provide a practical level of thromboresistance to mechanical valves. Here, we studied the effect of a bileaflet valve made of poly(ether ether ketone) (PEEK) with a poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-grafted surface (PEEK-g-PMPC). PMPC is a well-known thromboresistant polymeric material. A short-term (<26 h) porcine aortic valve replacement model using neither an anticoagulant nor an antiplatelet agent showed that the PEEK-g-PMPC valve opened and closed normally with an allowable transvalvular gradient. Unlike an untreated PEEK valve, no thrombus formed on the PEEK-g-PMPC valves on gross anatomy examination in addition to the absence of traveled thrombi in the kidney and lung tissues. Material (PEEK-g-PMPC)-related thromboresistance appeared to decrease the risk of thromboembolism and thrombosis for patients with mechanical valves. However, thromboresistance of the PEEK-g-PMPC valve requires improvement because fibrous fouling was still observed on the leaflet. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1052-1063, 2019.
Asunto(s)
Válvula Aórtica/cirugía , Implantación de Prótesis de Válvulas Cardíacas , Cetonas/farmacología , Fosforilcolina/análogos & derivados , Polietilenglicoles/farmacología , Ácidos Polimetacrílicos/farmacología , Trombosis/terapia , Animales , Válvula Aórtica/efectos de los fármacos , Válvula Aórtica/ultraestructura , Benzofenonas , Materiales Biocompatibles/farmacología , Modelos Animales de Enfermedad , Riñón/citología , Pulmón/citología , Masculino , Ensayo de Materiales , Fosforilcolina/farmacología , Polímeros , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Porcinos , Porcinos EnanosRESUMEN
AIMS: Calcific aortic valve stenosis (CAVS) is characterized by a fibrocalcific process. Studies have shown an association between CAVS and the activation of platelets. It is believed that shear stress associated with CAVS promotes the activation of platelets. However, whether platelets actively participate to the mineralization of the aortic valve (AV) and the progression of CAVS is presently unknown. To identify the role of platelets into the pathobiology of CAVS. METHODS AND RESULTS: Explanted control non-mineralized and mineralized AVs were examined by scanning electron microscope (SEM) for the presence of activated platelets. In-depth functional assays were carried out with isolated human valve interstitial cells (VICs) and platelets as well as in LDLR-/- apoB100/100 IGFII (IGFII) mice. Scanning electron microscope and immunogold markings for glycoprotein IIb/IIIa (GPIIb/IIIa) revealed the presence of platelet aggregates with fibrin in endothelium-denuded areas of CAVS. In isolated VICs, collagen-activated platelets induced an osteogenic programme. Platelet-derived adenosine diphosphate induced the release of autotaxin (ATX) by VICs. The binding of ATX to GPIIb/IIIa of platelets generated lysophosphatidic acid (LysoPA) with pro-osteogenic properties. In IGFII mice with CAVS, platelet aggregates were found at the surface of AVs. Administration of activated platelets to IGFII mice accelerated the development of CAVS by 2.1-fold, whereas a treatment with Ki16425, an antagonist of LysoPA receptors, prevented platelet-induced mineralization of the AV and the progression of CAVS. CONCLUSIONS: These findings suggest a novel role for platelets in the progression of CAVS.
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Estenosis de la Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Plaquetas/metabolismo , Calcinosis/metabolismo , Osteogénesis , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/ultraestructura , Apolipoproteína B-100/metabolismo , Progresión de la Enfermedad , Humanos , Integrina beta3/metabolismo , Lisofosfolípidos/metabolismo , Ratones , Microscopía Electrónica de Rastreo/métodos , Hidrolasas Diéster Fosfóricas/metabolismo , Glicoproteína IIb de Membrana Plaquetaria/metabolismoRESUMEN
BACKGROUND: Aortic regurgitation is a prevalent, detrimental complication of left ventricular assist devices (LVADs). The altered hemodynamics of LVADs results in aortic valves (AVs) having distinct mechanical stimulation. Our hypothesis was that the altered AV hemodynamics modulates the valve cells and matrix, resulting in changes in valvular mechanical properties that then can lead to regurgitation. METHODS: AVs were collected from 16 LVAD and 6 non-LVAD patients at time of heart transplant. Standard demographic and preoperative data were collected and comparisons between the two groups were calculated using standard statistical methods. Samples were analyzed using biaxial mechanical tensile testing, mass spectrometry-based proteomics, and transmission electron microscopy to assess ultrastructure. RESULTS: The maximum circumferential leaflet strain in LVAD patients was less than in non-LVAD patients (0.35 ± 0.10MPa versus 0.52 ± 0.18 MPa, p = 0.03) with a trend of reduced radial strain (p = 0.06) and a tendency for the radial strain to decrease with increasing LVAD duration (p = 0.063). Numerous proteins associated with actin and myosin, immune signaling and oxidative stress, and transforming growth factor ß were increased in LVAD patients. Ultrastructural analysis showed a trend of increased fiber diameter in LVAD patients (46.2 ± 7.2 nm versus 45.1 ± 6.9 nm, p = 0.10), but no difference in fiber density. CONCLUSIONS: AVs in LVAD patients showed decreased compliance and increased expression of numerous proteins related to valve activation and injury compared to non-LVAD patients. Further knowledge of AV changes leading to regurgitation in LVAD patients and the pathways by which they occur may provide an opportunity for interventions to prevent and/or reverse this detrimental complication.
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Insuficiencia de la Válvula Aórtica/etiología , Válvula Aórtica/ultraestructura , Insuficiencia Cardíaca/cirugía , Corazón Auxiliar/efectos adversos , Hemodinámica/fisiología , Estrés Oxidativo/fisiología , Proteómica/métodos , Válvula Aórtica/metabolismo , Válvula Aórtica/fisiopatología , Insuficiencia de la Válvula Aórtica/diagnóstico , Insuficiencia de la Válvula Aórtica/fisiopatología , Citocinas/metabolismo , Femenino , Insuficiencia Cardíaca/metabolismo , Humanos , Masculino , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Estudios Retrospectivos , Resistencia a la TracciónRESUMEN
INTRODUCTION: Cross-linking and anti-calcification of prosthetic heart valves have been continuously improved to prevent degeneration and calcification. However, non-calcific structural deteriorations such as cuspal dehiscences along the stent still require further analysis. MATERIAL AND METHOD: Based upon the previous analysis of an explanted valve after 7 years, a fresh commercial aortic valve was embedded in poly(methyl methacrylate) (PMMA) and cut into slices to ensure the detailed observation of the assembly and material structures. A pericardial patch embossed to provide the adequate shape of the cusps was investigated after paraffin embedding and appropriate staining. The microstructural damages that occurred during manufacturing process were identified and evaluated by light microscopy, polarized microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: The wavy collagen bundles, the key structure of the pericardium patch, were damaged to a great extent at suture sites along the stent and in the compressed areas around the stent post. The fixation of the embossed pericardium patch along the plots of the stent aggravated the microstructural modifications. The damages mainly appeared as the elimination of collagen bundle waviness and delamination between the bundles. CONCLUSION: Considering the modes of failure of the explant, the damages to the collagen bundles may identify the vulnerable sites that play an important role in the cusp dehiscence of heart valve implants. Such information is important to the manufacturers. Recommendations to prevent in vivo cusp dehiscence can therefore be formulated.
Asunto(s)
Válvula Aórtica/ultraestructura , Bioprótesis , Prótesis Valvulares Cardíacas , Pericardio/ultraestructura , Manejo de Especímenes/efectos adversos , Animales , Válvula Aórtica/patología , Calcinosis/prevención & control , Bovinos , Colágeno/ultraestructura , Reactivos de Enlaces Cruzados/química , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Adhesión en Parafina , Pericardio/anatomía & histología , Pericardio/patología , Adhesión en Plástico/métodos , Polimetil Metacrilato/química , Falla de Prótesis , Manejo de Especímenes/métodos , StentsRESUMEN
Patients with end-stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4-fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8-fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP-1, and LAMP-2 and a concomitant up-regulation of the Annexin family of calcium-binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC-derived MVs (51.9-fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up-regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC-derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co-localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC-derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD.
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Anexina A6/metabolismo , Estenosis de la Válvula Aórtica/metabolismo , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Calcinosis/metabolismo , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Hipercalcemia/etiología , Fallo Renal Crónico/complicaciones , Anciano , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Válvula Aórtica/ultraestructura , Estenosis de la Válvula Aórtica/etiología , Estenosis de la Válvula Aórtica/genética , Estenosis de la Válvula Aórtica/patología , Calcinosis/etiología , Calcinosis/genética , Calcinosis/patología , Calcio/metabolismo , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Matriz Extracelular/ultraestructura , Vesículas Extracelulares/ultraestructura , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hipercalcemia/diagnóstico , Fallo Renal Crónico/diagnóstico , Masculino , Microscopía Electrónica de Transmisión , Mapas de Interacción de Proteínas , Proteómica/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Valve dystrophic calcification is a common disorder affecting normophosphatemic subjects. Here, cultured aortic valve interstitial cells (AVICs) were treated 3 to 28 days with phosphate (Pi) concentrations spanning the normal range in humans (0.8, 1.3, and 2.0 mM) alone or supplemented with proinflammatory stimuli to assess possible priming of dystrophic-like calcification. Compared with controls, spectrophotometric analyses revealed marked increases in calcium amounts and alkaline phosphatase activity for 2.0-mM-Pi-containing cultures, with enhancing by proinflammatory mediators. Ultrastructurally, AVICs treated with low/middle Pi concentrations showed an enormous endoplasmic reticulum (ER) enclosing organelle debris, so apparently executing a survival-related atypical macroautophagocytosis, consistently with ultracytochemical demonstration of ER-associated acid phosphatase activity and decreases in autophagosomes and immunodetectable MAP1LC3. In contrast, AVICs cultured at 2.0-mM Pi underwent mineralization due to intracellular release and peripheral layering of phospholipid-rich material acting as hydroxyapatite nucleator, as revealed by Cuprolinic Blue and von Kossa ultracytochemical reactions. Lack of immunoblotted caspase-3 cleaved form indicated apoptosis absence for all cultures. In conclusion, fates of cultured AVICs were crucially driven by Pi concentration, suggesting that serum Pi levels just below the upper limit of normophosphatemia in humans may represent a critical watershed between macroautophagy-associated cell restoring and procalcific cell death.
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Válvula Aórtica/citología , Válvula Aórtica/patología , Calcinosis/patología , Fosfatos/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/ultraestructura , Autofagia , Calcinosis/metabolismo , Calcio/metabolismo , Bovinos , Supervivencia Celular , Células Cultivadas , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Retículo Endoplásmico/ultraestructuraRESUMEN
OBJECTIVES: Endothelial cells harbor many antigenic determinants that may be targets for the immune system. The aim of this study was to determine the immunologic effects of decellularization, using 3 different methods, on xenograft rejection. MATERIALS AND METHODS: In a sterile plate containing phosphate-buffered saline, fresh sheep aortic heart valves were decellularized using 3 different enzymatic methods: with 900 µg/mL of collagenase at 40°C (method A), with 450 µg/mL of collagenase at 4°C (method B), and with 900 µg/mL of collagenase at 4°C (method C). Intact and decellularized valves were implanted subdermally into inbred male albino rabbits and extracted after 21 days (extra valve pieces were also extracted after 60 days, as control samples, for assessing chronic rejection). Valves were histologically analyzed for inflammatory cell infiltration. Subendothelial structure integrity was determined using surface electron microscope. RESULTS: No inflammatory cell infiltration was seen around the decellularized valve with method A, and no subendothelial structure change was observed by surface electron microscope. Infiltration of immune cells involved in rejection was not seen around valves decellularized with method B, although the subendothelial structure was relatively preserved and valve stiffness was increased. With method C, we observed a foreign body-type reaction around the intact valve and the decellularized valve. CONCLUSIONS: Method A is considered the optimal method of decellularization in our study, as this method significantly reduced the immune response to xenograft tissue, while maintaining subendothelial tissue.
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Válvula Aórtica/trasplante , Bioprótesis , Implantación de Prótesis de Válvulas Cardíacas/instrumentación , Prótesis Valvulares Cardíacas , Ingeniería de Tejidos/métodos , Animales , Válvula Aórtica/inmunología , Válvula Aórtica/ultraestructura , Colagenasas/metabolismo , Células Endoteliales/inmunología , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Supervivencia de Injerto , Implantación de Prótesis de Válvulas Cardíacas/efectos adversos , Xenoinjertos , Masculino , Diseño de Prótesis , Conejos , Oveja Doméstica , Factores de TiempoRESUMEN
Decellularized porcine aortic valve conduits (AVCs) implanted in a Vietnamese Pig (VP) experimental animal model were matched against decellularized and then cryopreserved AVCs to assess the effect of cryopreservation on graft hemodynamic performance and propensity to in vivo repopulation by host's cells. VPs (n = 12) underwent right ventricular outflow tract substitution using AVC allografts and were studied for 15-month follow-up. VPs were randomized into two groups, receiving AVCs treated with decellularization alone (D; n = 6) or decellularization/cryopreservation (DC; n = 6), respectively. Serial echocardiography was carried out to follow up hemodynamic function. All explanted AVCs were processed for light and electron microscopy. No signs of dilatation, progressive stenosis, regurgitation, and macroscopic calcification were echocardiographically observed in both D and DC groups. Explanted D grafts exhibited near-normal features, whereas the presence of calcification, inflammatory infiltrates, and disarray of elastic lamellae occurred in some DC grafts. In the unaltered regions of AVCs from both groups, almost complete re-endothelialization was observed for both valve cusps and aorta walls. In addition, side-by-side repopulation by recipient's fibroblasts, myofibroblasts, and smooth muscle cells was paralleled by ongoing tissue remodeling, as revealed by the ultrastructural identification of typical canals of collagen fibrillogenesis and elastogenesis-related features. Incipient neo-vascularization and re-innervation of medial and adventitial tunicae of grafted aortic walls were also detected for both D and DC groups. Cryopreservation did not affect post-implantation AVC hemodynamic behavior and was topically propensive to cell repopulation and tissue renewal, although graft deterioration including calcification was present in several areas. Thus, these preliminary data provide essential information on feasibility of decellularization and cryopreservation coupling in the perspective of treatment optimization and subsequent clinical trials using similarly treated human allografts as innovative heart valve substitutes.
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Aorta/trasplante , Válvula Aórtica/trasplante , Bioprótesis , Implantación de Prótesis Vascular/instrumentación , Prótesis Vascular , Criopreservación , Implantación de Prótesis de Válvulas Cardíacas/instrumentación , Prótesis Valvulares Cardíacas , Aloinjertos , Animales , Aorta/fisiopatología , Aorta/ultraestructura , Válvula Aórtica/fisiopatología , Válvula Aórtica/ultraestructura , Implantación de Prótesis Vascular/efectos adversos , Proliferación Celular , Ecocardiografía , Supervivencia de Injerto , Implantación de Prótesis de Válvulas Cardíacas/efectos adversos , Hemodinámica , Microscopía Electrónica de Transmisión , Modelos Animales , Complicaciones Posoperatorias/patología , Complicaciones Posoperatorias/fisiopatología , Porcinos , Factores de TiempoRESUMEN
The hallmarks of calcific aortic valve disease (CAVD) are the significant quantitative and qualitative changes that occur in the extracellular matrix (ECM), which ultimately lead to increased leaflet stiffness and obstruction of left ventricular outflow. Mounting evidence suggests that ECM remodeling not only contribute to valve cell dysfunction but also alter certain cell signaling pathways responsible for the initiation and progression of the disease state. Matrix metalloproteinases (MMPs), collectively called matrixins, are a family of enzymes known to participate in numerous ECM remodeling events during embryonic development and in disease. The aim of the present study was to evaluate whether changes in MMP-9 expression might be involved in the pathophysiology of CAVD. For this purpose, we have analyzed a total of 19 pathologic valves from patients who underwent aortic valve replacement for calcific aortic stenosis. Microscopically, the cusp tissue showed diffuse fibrosis, neovascularization, and abnormal ECM remodeling with collagen disorganization and mineralization. Western blot and immunohistochemical analyses have been performed on both the areas overlying and remote from the mineral deposits. Protein expression data evidenced a significant upregulation of MMP-9 in the calcified lesion area. Consistent with these observations, immunohistochemistry demonstrated that MMP-9 protein was almost exclusively localized near or around the mineralized nodules, whereas was generally quite weak or absent in areas devoid of any calcification. Our data suggest that MMP-9 may play a key role in CAVD probably by promoting the fibrotic and procalcific remodeling of the ECM.
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Estenosis de la Válvula Aórtica/diagnóstico , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Calcinosis/diagnóstico , Matriz Extracelular/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Disfunción Ventricular Izquierda/diagnóstico , Válvula Aórtica/ultraestructura , Matriz Extracelular/ultraestructura , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasa 9 de la Matriz/genética , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Remodelación VentricularRESUMEN
Over the last 10 years, calcifying nanoparticles (CNP) have attracted attention as structures detected. together with many other nanostructures in biopsies from patients operated for the correction of aortic valve malformations. The results of the present work performed with the use of high-resolution transmission and scanning electron microscopes agree on the whole with the data of other authors. Some new findings include CNP adhesion to collagen fibers and specifically-shaped, shallow invaginations or craters at their surface. The possible pathophysiological mechanisms that promote involvement of CNP in the development of the disease are considered.
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Estenosis de la Válvula Aórtica/etiología , Válvula Aórtica/ultraestructura , Nanopartículas Calcificantes/metabolismo , Calcinosis/complicaciones , Colágeno/química , Anciano , Anciano de 80 o más Años , Válvula Aórtica/metabolismo , Válvula Aórtica/cirugía , Estenosis de la Válvula Aórtica/diagnóstico , Estenosis de la Válvula Aórtica/metabolismo , Calcinosis/diagnóstico , Calcinosis/metabolismo , Femenino , Prótesis Valvulares Cardíacas , Humanos , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana EdadRESUMEN
BACKGROUND AND AIM OF THE STUDY: Cryopreservation allows for the long-term storage of aortic homografts, but a high risk of calcification and degeneration is often observed following their transplantation. The study aim was to investigate the cryoprotective effect of trehalose on aortic valve homografts preserved in liquid nitrogen, and to determine the optimal trehalose concentration for such purpose. METHODS: Aortic valve homografts obtained from New Zealand White rabbits were processed using different protectants. Samples were assigned at random to four groups: a control group treated with dimethyl sulfoxide (DMSO; 0.1 mol/l), and test groups A, B and C, which were treated with 0.1 mol/l trehalose + 0.1 mol/l DMSO, 0.2 mol/l trehalose + 0.1 mol/l DMSO, or 0.3 mol/l trehalose + 0.1 mol/l DMSO, respectively, as protectant. Samples in each group were allocated randomly to three subgroups and cryopreserved for 12, 15, and 18 months, respectively. After thawing, apoptosis of the cryopreserved homograft samples was evaluated using immunohistochemistry, semi-quantitative RT-PCR and Western blotting methods. The viability of the tissue cells was assessed by monitoring glucose utilization capacity. RESULTS: The apoptosis assay showed that, among the four groups and at all time points, the expression of caspase-3 was lowest in test groups A and B and highest in the DMSO group. In comparison, the glucose metabolic rates of test groups A and B were highest, while rates for test group C and the control (DMSO) group ranked second and third. CONCLUSION: When aortic homografts are preserved in liquid nitrogen, the cryoprotective effect of trehalose combined with DMSO was superior to that of DMSO alone. The optimal trehalose concentration to cryoprotect rabbit aortic homografts was between 0.1 and 0.2 mol/l.
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Válvula Aórtica/efectos de los fármacos , Bioprótesis , Criopreservación/métodos , Crioprotectores/farmacología , Prótesis Valvulares Cardíacas , Trehalosa/farmacología , Aloinjertos , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/trasplante , Válvula Aórtica/ultraestructura , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Citoprotección , Dimetilsulfóxido/farmacología , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Glucosa/metabolismo , Implantación de Prótesis de Válvulas Cardíacas/instrumentación , Conejos , Factores de Tiempo , Supervivencia Tisular/efectos de los fármacosRESUMEN
The atrioventricular valve leaflets (mitral and tricuspid) are different from the semilunar valve leaflets (aortic and pulmonary) in layered structure, ultrastructural constitution and organization, and leaflet thickness. These differences warrant a comparative look at the bending properties of the four types of leaflets. We found that the moment-curvature relationships in atrioventricular valves were stiffer than in semilunar valves, and the moment-curvature relationships of the left-side valve leaflets were stiffer than their morphological analog of the right side. These trends were supported by the moment-curvature curves and the flexural rigidity analysis (EI value decreased from mitral, tricuspid, aortic, to pulmonary leaflets). However, after taking away the geometric effect (moment of inertia I), the instantaneous effective bending modulus E showed a reversed trend. The overall trend of flexural rigidity (EI: mitral > tricuspid > aortic > pulmonary) might be correlated with the thickness variations among the four types of leaflets (thickness: mitral > tricuspid > aortic > pulmonary). The overall trend of the instantaneous effective bending modulus (E: mitral < tricuspid < aortic < pulmonary) might be correlated to the layered fibrous ultrastructures of the four types of leaflets, of which the fibers in mitral and tricuspid leaflets were less aligned, and the fibers in aortic and pulmonary leaflets were highly aligned. We also found that, for all types of leaflets, moment-curvature relationships are stiffer in against-curvature (AC) bending than in with-curvature bending (WC), which implies that leaflets tend to flex toward their natural curvature and comply with blood flow. Lastly, we observed that the leaflets were stiffer in circumferential bending compared with radial bending, likely reflecting the physiological motion of the leaflets, i.e., more bending moment and movement were experienced in radial direction than circumferential direction.
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Válvula Aórtica/fisiología , Válvula Mitral/fisiología , Válvula Pulmonar/fisiología , Animales , Válvula Aórtica/ultraestructura , Fenómenos Biomecánicos , Microscopía Electrónica de Rastreo , Válvula Mitral/ultraestructura , Válvula Pulmonar/ultraestructura , PorcinosRESUMEN
PURPOSE: To determine the best method for decellularisation of aortic valve conduits (AVCs) that efficiently removes the cells while preserving the extracellular matrix (ECM) by examining the valvular and conduit sections separately. MATERIAL/METHODS: Sheep AVCs were decellularised by using three different protocols: detergent-based (1% SDS+1% SDC), detergent and enzyme-based (Triton+EDTA+RNase and DNase), and enzyme-based (Trypsin+RNase and DNase) methods. The efficacy of the decellularisation methods to completely remove the cells while preserving the ECM was evaluated by histological evaluation, scanning electron microscopy (SEM), hydroxyproline analysis, tensile test, and DAPI staining. RESULTS: The detergent-based method completely removed the cells and left the ECM and collagen content in the valve and conduit sections relatively well preserved. The detergent and enzyme-based protocol did not completely remove the cells, but left the collagen content in both sections well preserved. ECM deterioration was observed in the aortic valves (AVs), but the ultrastructure of the conduits was well preserved, with no media distortion. The enzyme-based protocol removed the cells relatively well; however, mild structural distortion and poor collagen content was observed in the AVs. Incomplete cell removal (better than that observed with the detergent and enzyme-based protocol), poor collagen preservation, and mild structural distortion were observed in conduits treated with the enzyme-based method. CONCLUSIONS: The results suggested that the detergent-based methods are the most effective protocols for cell removal and ECM preservation of AVCs. The AVCs treated with this detergent-based method may be excellent scaffolds for recellularisation.
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Válvula Aórtica/química , Matriz Extracelular/química , Andamios del Tejido/química , Animales , Válvula Aórtica/citología , Válvula Aórtica/ultraestructura , Fenómenos Químicos , Detergentes/química , Matriz Extracelular/ultraestructura , Irán , Fenómenos Mecánicos , Oveja DomésticaRESUMEN
Calcified heart valves display a significant imbalance in tissue content of trace and essential elements. The valvular calcification is an age-related process and there are data suggesting involvement of lipids. We studied elemental composition and lipid distribution in three distinct regions of calcified human aortic valves, representing successive stages of the calcific degeneration: normal, thickened (early lesion) and calcified (late lesion), using SR-µXRF (Synchrotron Radiation Micro X-Ray Fluorescence) for elemental composition and Oil Red O (ORO) staining for demonstration of lipids. Two-dimensional SR-µXRF maps and precise point spectra were compared with histological stainings on consecutive valve sections to prove topographical localization and colocalization of the examined elements and lipids. In calcified valve areas, accumulation of calcium and phosphorus was accompanied by enhanced concentrations of strontium and zinc. Calcifications preferentially developed in lipid-rich areas of the valves. Calcium concentration ratio between lipid-rich and lipid-free areas was not age-dependent in early lesions, but showed a significant increase with age in late lesions, indicating age-dependent intensification of lipid involvement in calcification process. The results suggest that mechanisms of calcification change with progression of valve degeneration and with age.