Intracellular proteomics and extracellular vesiculomics as a metric of disease recapitulation in 3D-bioprinted aortic valve arrays.

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)-model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)-bioprinted into 96-well arrays. Liquid chromatography-tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput-based bioengineered model systems, and potentiates future drug discovery.

Crosslinking porcine aortic valve by radical polymerization for the preparation of BHVs with improved cytocompatibility, mild immune response, and reduced calcification.

Over one million artificial heart valve transplantations are performed each year due to valvular stenosis or regurgitation. Among them, bioprosthetic heart valves (BHVs) are increasingly being used because of the absence of the need for lifelong anticoagulation. Almost all of the commercial BHVs are treated with Glutaraldehyde (GLUT). As GLUT-treated BHVs are prone to calcification and structural degradation, their durability is greatly reduced with a service life of only 12-15 years. The physiological structure and mechanical properties of the porcine aortic valve (PAV) are closer to that of a human heart valve, so in this study, PAV is used as the model to explore the comprehensive properties of the prepared BHVs by radical polymerization crosslinking method. We found that PAV treated by radical polymerization crosslinking method showed similar ECM stability and biaxial mechanical properties with GLUT-treated PAV. However, radical polymerization crosslinked PAV exhibited better cytocompatibility and endothelialization potential in vitro cell experiment as better anticalcification potential and reduced immune response than GLUT-treated PAV through subcutaneous animal experiments in rats. To conclude, a novel crosslinking method of non-glutaraldehyde fixation of xenogeneic tissues for the preparation of BHVs is expected.

Multiplexed imaging mass spectrometry of the extracellular matrix using serial enzyme digests from formalin-fixed paraffin-embedded tissue sections.

We report a multiplexed imaging mass spectrometry method which spatially localizes and selectively accesses the extracellular matrix on formalin-fixed paraffin-embedded tissue sections. The extracellular matrix (ECM) consists of (1) fibrous proteins, post-translationally modified (PTM) via N- and O-linked glycosylation, as well as hydroxylation on prolines and lysines, and (2) glycosaminoglycan-decorated proteoglycans. Accessing all these components poses a unique analytical challenge. Conventional peptide analysis via trypsin inefficiently captures ECM peptides due to their low abundance, intra- and intermolecular cross-linking, and PTMs. In previous studies, we have developed matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) techniques to capture collagen peptides via collagenase type III digestion, both alone and after N-glycan removal via PNGaseF digest. However, in fibrotic tissues, the buildup of ECM components other than collagen-type proteins, including elastin and glycosaminoglycans, limits efficacy of any single enzyme to access the complex ECM. Here, we have developed a novel serial enzyme strategy to define the extracellular matrix, including PTMs, from a single tissue section for MALDI-IMS applications. Graphical Abstract.

Thinner biological tissues induce leaflet flutter in aortic heart valve replacements.

Valvular heart disease has recently become an increasing public health concern due to the high prevalence of valve degeneration in aging populations. For patients with severely impacted aortic valves that require replacement, catheter-based bioprosthetic valve deployment offers a minimally invasive treatment option that eliminates many of the risks associated with surgical valve replacement. Although recent percutaneous device advancements have incorporated thinner, more flexible biological tissues to streamline safer deployment through catheters, the impact of such tissues in the complex, mechanically demanding, and highly dynamic valvular system remains poorly understood. The present work utilized a validated computational fluid-structure interaction approach to isolate the behavior of thinner, more compliant aortic valve tissues in a physiologically realistic system. This computational study identified and quantified significant leaflet flutter induced by the use of thinner tissues that initiated blood flow disturbances and oscillatory leaflet strains. The aortic flow and valvular dynamics associated with these thinner valvular tissues have not been previously identified and provide essential information that can significantly advance fundamental knowledge about the cardiac system and support future medical device innovation. Considering the risks associated with such observed flutter phenomena, including blood damage and accelerated leaflet deterioration, this study demonstrates the potentially serious impact of introducing thinner, more flexible tissues into the cardiac system.

Pathological Role of Receptor for Advanced Glycation End Products in Calcified Aortic Valve Stenosis.

Background Aortic stenosis (AS) is highly prevalent in patients with atherosclerotic cardiovascular disease. Advanced glycation end products (AGEs) and the receptor for AGEs (RAGE) play a pivotal role for vascular calcification in atherosclerosis. We hypothesize that the AGEs-RAGE axis could also be involved in the pathophysiological mechanism of calcified AS. Methods and Results A total of 54 patients with calcified AS who underwent aortic valve replacement were prospectively enrolled from 2014 to 2016 (mean age 75.3±7.7 years). Aortic valve specimens were obtained from 47 patients and 16 deceased control subjects without aortic valve disease (mean age 63.2±14.5 years). The valvular expression of RAGE was evaluated by immunohistochemistry. Serum levels of AGEs and soluble RAGE were measured in 50 patients with calcified AS and 70 age-matched and sex-matched control subjects without heart disease. The valvular RAGE expression in patients with calcified AS was higher than controls (P=0.004) and was significantly associated with a decreased ankle-brachial pressure index (P=0.007) and an increased intima-media thickness (P=0.026). RAGE and α-smooth muscle actin were coexpressed and were partially costained with osteocalcin and alkaline phosphatase. The serum levels of AGEs and soluble RAGE were significantly higher in the patients with calcified AS than in the controls (P=0.013 and P<0.001, respectively). Soluble RAGE (inversely) and use of aspirin were independently correlated with changes in left ventricular systolic function after aortic valve replacement (P=0.012 and P=0.002, respectively). Conclusions Our present study suggests that RAGE may play a role in the pathogenesis of calcified AS, which is a prognostic marker in patients with AS after aortic valve replacement.

Age, Sex, and Valve Phenotype Differences in Fibro-Calcific Remodeling of Calcified Aortic Valve.

Background In calcific aortic valve disease on tricuspid aortic valves (TAVs), men have higher aortic valve calcification and less fibrosis than women. However, little is known in bicuspid aortic valves (BAV). We thus aimed to investigate the impact of age, sex, and valve phenotype (TAVs versus BAVs) on fibro-calcific remodeling in calcific aortic valve disease. Methods and Results We included 2 cohorts: 411 patients who underwent multidetector computed tomography (37% women) for aortic valve calcification density assessment and 138 explanted aortic valves (histological cohort; 50% women). The cohorts were divided in younger (<60 years old) or older patients with BAV (≥60 years old), and TAV patients. In each group, women and men were matched. Women presented less aortic valve calcification density than men in each group of the multidetector computed tomography cohort (all P≤0.01). Moreover, in women, younger patients with BAV had the lowest aortic valve calcification density (both P=0.02). In multivariate analysis, aortic valve calcification density correlated with age (ß estimate±standard error: 6.5±1.8; P=0.0004) and male sex (109.2±18.4; P<0.0001), and there was a trend with TAVs (41.5±23.0; P=0.07). Women presented a higher collagen content than men (77.8±10.8 versus 69.9±12.9%; P<0.001) in the entire cohort. In women, younger patients with BAV had denser connective tissue than TAV and older patients with BAV (both P≤0.05), while no difference was observed between men. Conclusions In calcific aortic valve disease, women had less calcification and more fibrotic remodeling than men, regardless of the phenotype of the valve or age of the patient. Moreover, younger women with BAVs had less valve calcification. Thus, mineralization/fibrosis of the aortic valve is likely to have sex/age-specific mechanisms and be influenced by the valve morphology.

Fibrotic aortic valve disease after radiotherapy: an immunohistochemical study in breast cancer and lymphoma patients.

BACKGROUND: Radiation-associated aortic valve (AV) stenosis is frequently seen as a late sequela after thoracic radiotherapy (RT). Although the clinical relationship between thoracic radiotherapy and valvular dysfunction has been established, the process leading to accelerated aortic valve stenosis remains unclear. The aim of this study was to determine whether increased inflammatory cell infiltration, fibrosis, and calcification is present in aortic valves after radiotherapy at the time of aortic valve replacement. METHODS: Stenotic aortic valve specimens from 43 patients were obtained after surgical aortic valve replacement. A total 28 patients had previously undergone radiotherapy for breast cancer or malignant lymphoma. A total 15 patients were included as control. The valve leaflets were assessed by (immuno)histochemistry for inflammatory cell composition (CD3, CD20, CD68, and CD163) and extracellular matrix changes (collagen and calcification). RESULTS: Aortic valve cell density after radiotherapy for lymphoma was markedly decreased when compared with other groups. Irradiated aortic valve show similar (low) degrees of late T and B lymphocyte infiltration as control valves, whereas macrophage marker CD68 was decreased after radiotherapy for breast cancer. Collagen content was increased following radiotherapy. Aortic valves of patients with lymphoma contained significantly less calcified tissue when compared with the other groups. CONCLUSION: High-dose radiation at a young age (patients with lymphoma) results in cell loss and premature fibrotic aortic valve stenosis as opposed to the degenerative calcific stenosis observed in patients with breast cancer. Our findings suggest a possible dose-dependent effect of radiotherapy on aortic valve fibrosis. The active presence of inflammatory cells may be limited to the acute phase after radiotherapy.

Heat shock protein 90 is downregulated in calcific aortic valve disease.

BACKGROUND: Calcific aortic valve disease (CAVD) is an atheroinflammatory process; finally it leads to progressive calcification of the valve. There is no effective pharmacological treatment for CAVD and many of the underlying molecular mechanisms remain unknown. We conducted a proteomic study to reveal novel factors associated with CAVD. METHODS: We compared aortic valves from patients undergoing valvular replacement surgery due to non-calcified aortic insufficiency (control group, n = 5) to a stenotic group (n = 7) using two-dimensional difference gel electrophoresis (2D-DIGE). Protein spots were identified with mass spectrometry. Western blot and immunohistochemistry were used to validate the results in a separate patient cohort and Ingenuity Pathway Analysis (IPA) was exploited to predict the regulatory network of CAVD. RESULTS: We detected an upregulation of complement 9 (C9), serum amyloid P-component (APCS) and transgelin as well as downregulation of heat shock protein (HSP90), protein disulfide isomerase A3 (PDIA3), annexin A2 (ANXA2) and galectin-1 in patients with aortic valve stenosis. The decreased protein expression of HSP90 was confirmed with Western blot. CONCLUSIONS: We describe here a novel data set of proteomic changes associated with CAVD, including downregulation of the pro-inflammatory cytosolic protein, HSP90.

Heterogeneous multi-laminar tissue constructs as a platform to evaluate aortic valve matrix-dependent pathogenicity.

Designing and constructing controlled in vitro cell culture platforms is imperative toward pinpointing factors that contribute to the development of calcific aortic valve disease. A 3D, laminar, filter paper-based cell culture system that was previously established as a method of analyzing valvular interstitial cell migration and protein expression was adapted here for studying the impact of specific extracellular matrix proteins on cellular viability and calcification proclivity. Hydrogels incorporating hyaluronan and collagen I, two prevalent valvular extracellular matrix proteins with altered pathological production, were designed with similar mechanics to parse out effects of the individual proteins on cell behavior. Laminar constructs containing varying combinations of discrete layers of collagen and hyaluronan were assembled to mimic native and pathological valve compositions. Proteinaceous and genetic expression patterns pertaining to cell viability and calcific potential were quantified via fluorescent imaging. A significant dose-dependency was observed, with increased collagen content associated with decreased viability and increased calcific phenotype. These results suggest that extracellular composition is influential in calcific aortic valve disease progression and will be key toward development of future tissue-engineered or pharmaceutical calcific aortic valve treatments. STATEMENT OF SIGNIFICANCE: Calcific aortic valve disease (CAVD), a widespread heart valve disorder, is characterized by fibrotic leaflet thickening and calcific nodule formation. This pathological remodeling is an active process mediated by the valvular interstitial cells (VICs). Currently, the only treatment available is surgical replacement of the valve - a procedure associated with significant long-term risk and morbidity. Development of effective alternate therapies is hindered by our poor understanding of CAVD etiology. Previous work has implicated the composition and mechanics of the extracellular matrix in the progression of CAVD. These individual factors and their magnitude of influence have not been extensively explored - particularly in 3D systems. Here, we have bridged this gap in understanding through the employment of a heterogeneous 3D filter-paper culture system.

Non-Destructive Reflectance Mapping of Collagen Fiber Alignment in Heart Valve Leaflets.

Collagen fibers are the primary structural elements that define many soft-tissue structure and mechanical function relationships, so that quantification of collagen organization is essential to many disciplines. Current tissue-level collagen fiber imaging techniques remain limited in their ability to quantify fiber organization at macroscopic spatial scales and multiple time points, especially in a non-contacting manner, requiring no modifications to the tissue, and in near real-time. Our group has previously developed polarized spatial frequency domain imaging (pSFDI), a reflectance imaging technique that rapidly and non-destructively quantifies planar collagen fiber orientation in superficial layers of soft tissues over large fields-of-view. In this current work, we extend the light scattering models and image processing techniques to extract a critical measure of the degree of collagen fiber alignment, the normalized orientation index (NOI), directly from pSFDI data. Electrospun fiber samples with architectures similar to many collagenous soft tissues and known NOI were used for validation. An inverse model was then used to extract NOI from pSFDI measurements of aortic heart valve leaflets and clearly demonstrated changes in degree of fiber alignment between opposing sides of the sample. These results show that our model was capable of extracting absolute measures of degree of fiber alignment in superficial layers of heart valve leaflets with only general a priori knowledge of fiber properties, providing a novel approach to rapid, non-destructive study of microstructure in heart valve leaflets using a reflectance geometry.

Increased mesenchymal podoplanin expression is associated with calcification in aortic valves.

BACKGROUND AND AIM OF THE STUDY: Calcific aortic valve disease (CAVD) is a progressive disease starting from mild valvular sclerosis and progressing to severe aortic stenosis (AS) with calcified valves. The origin of the calcification is proposed to be mesenchymal cells which have differentiated towards an osteoblastic phenotype. Podoplanin is a glycoprotein expressed in the endothelium of lymphatic vessels and in osteoblasts and osteocytes, mesenchymal cells, as well as in many carcinomas and aortic atherosclerotic lesions. In CAVD, its expression has been evaluated only as a marker of the lymphatic vasculature. MATERIALS AND METHODS: We determined podoplanin expression in human aortic valves in four patient groups: control (C, n=7), aortic regurgitation (AR, n=8), aortic regurgitation and fibrosis (AR + f, n=15) and AS (n=49) by immunohistochemistry and quantitative real-time PCR (RT-PCR). RESULTS: Immunohistochemically, podoplanin expression was significantly increased in AR + f and AS groups when compared with the control and AR groups and the level of expression positively correlated with the extent of calcification and vascularity. Podoplanin mRNA levels were 1.7-fold higher in the AS group as compared with the control group (P=.05). Podoplanin-positivity was present not only in lymphatic vessel endothelium but also in osteoblasts, osteocytes, chondrocytes, macrophages and extracellular matrix. The majority of the podoplanin-positivity was in spindle cells with a myofibroblastic phenotype, often associated with calcifications. Tricuspid valves had more calcification-associated podoplanin than bi/unicuspid valves (median 1.52 vs 1.16, P<.001). CONCLUSIONS: CAVD is characterized by an increased expression of podoplanin; this is associated with the differentiation of valvular interstitial cells into calcium-producing, myofibroblast-like cells. In addition, tricuspid valves express relatively more podoplanin than bi/unicuspid valves.

Mechanical behavior of bovine pericardium treated with hyaluronic acid derivative for bioprosthetic aortic valves.

We studied the mechanical behavior of bovine pericardium (BP) after anticalcification treatment using hyaluronic acid (HA) derivative. To simulate the physiological environment and stimulate the calcification process, the BP samples were immersed into simulated body fluid solution. We conducted scanning electron microscopy with energy dispersive X-ray spectrometry, and uniaxial mechanical tests of HA-treated and non-treated samples. Although our microstructural analyses indicated that the HA treatment actually prevents the formation of calcium phosphate deposits, the mechanical tests show significant increase of stiffness of the HA-treated samples. Using data from our mechanical tests as input parameters, we performed finite element (FE) computer simulations to estimate how this increase in the BP stiffness affects the stress distribution in the bioprosthetic leaflet. Although the maximum stress observed during the closing phase of the membrane in vivo is below the experimental yield stress in all cases we analyzed, our FE results indicate that increase of BP stiffness due to HA anticalcification treatment results in higher risk of disruption and failure of the leaflets in bioprosthetic heart valves. Since our FE results indicate that the commissure and the fixed edge are the regions that withstand the highest mechanical stresses during the closing phase, new designs of the valve might be efficient to enhance the endurance of the prosthesis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2273-2280, 2019.

Surface biofunctionalization of the decellularized porcine aortic valve with VEGF-loaded nanoparticles for accelerating endothelialization.

The original intention for building a tissue-engineered heart valve (TEHV) was to simulate a normal heart valve and overcome the insufficiency of the commonly used heart valve replacement in the clinic. The endothelialization of the TEHV is very important as the endothelialized TEHV can decrease platelet adhesion and delay the valvular calcification decline process. In this work, we encapsulated vascular endothelial growth factor (VEGF) into polycaprolactone (PCL) nanoparticles. Then, through the Michael addition reaction, PCL nanoparticles were introduced onto the decellularized aortic valve to prepare a hybrid valve. The encapsulation efficiency of the PCL nanoparticles for VEGF was up to 82%, and the in vitro accumulated release rate was slow without an evident initial burst release. In addition, the hybrid valve had a decreased hemolysis ratio and possessed antiplatelet adhesion capacity, and it was able to promote the adhesion and proliferation of endothelial cells, covering the surface with a dense cell layer to accelerate endothelialization. An experiment involving the subcutaneous implant in SD rats showed that at week 8, lots of blood capillaries were formed in the hybrid valve. Mechanics performance testing indicated that the mechanical property of the hybrid valve was partly improved. Taken together, we applied a nano-drug controlled release system to fabricate TEHV, and provide an approach for the biofunctionalization of the TEHV scaffold for accelerating endothelialization.

Simultaneous accurate quantification of HO-1, CD39, and CD73 in human calcified aortic valves using multiple enzyme digestion - filter aided sample pretreatment (MED-FASP) method and targeted proteomics.

Several proteins such as membrane-associated ectonucleotidases: ecto-5'-nucleotidase (E5NT/CD73) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), and intracellular heme oxygenase-1 (HO-1) may contribute to protection from inflammation-related diseases such as calcific aortic valve stenosis (CAS). Accurate quantification of these proteins could contribute to better understanding of the disease mechanisms and identification of biomarkers. This report presents development and validation of quantification method for E5NT/CD73, ENTPD1/CD39 and HO-1. The multiplexed targeted proteomic assay involved antibody-free, multiple-enzyme digestion, filter-assisted sample preparation (MED-FASP) strategy and a nanoflow liquid chromatography/mass spectrometry under multiple reaction monitoring mode (LC-MRM/MS). The method developed presented high sensitivity (LLOQ of 5 pg/mL for each of the analytes) and accuracy that ranged from 92.0% to 107.0%, and was successfully applied for the absolute quantification of HO-1, CD39 and CD73 proteins in homogenates of human calcified and non-calcified valves. The absolute CD39 and CD73 concentrations were lower in calcified aortic valves (as compared to non-stenotic ones) and were found to be: 1.16 ± 0.39 vs. 3.15 ± 0.37 pmol/mg protein and 1.94 ± 0.21 vs. 2.39 ± 0.39 pmol/mg protein, respectively, while the quantity of HO-1 was elevated in calcified valves (10.72 ± 1.18 vs. 4.28 ± 0.42 amol/mg protein). These results were consistent but more reproducible as compared to immunoassays. In conclusion, multiplexed quantification of HO-1, CD39 and CD73 proteins by LC-MRM/MS works well in challenging human tissues such as aortic valves. This analysis confirmed the relevance of these proteins in pathogenesis of CAS and could be extended to other biomedical investigations.

Periodontal Bacterial DNA and Their Link to Human Cardiac Tissue: Findings of a Pilot Study.

BACKGROUND: The aim of this pilot study was to detect correlations of microbiological DNA, inflammatory proteins, and infection parameters in patients with periodontal disease (PD) and valvular heart disease (VHD). METHODS: A perioperative comprehensive dental examination for the investigation of periodontal status, including sampling of specific subgingival bacteria, was performed in 10 patients with indication for surgery of aortic valve stenosis with or without concomitant myocardial revascularization. Standard protocol biopsies were taken from right atrium (A), left septal myocardium (M), and aortic valve (V). Eleven periodontal pathogens DNA in oral and cardiac tissue samples (A/M/V) were analyzed using polymerase chain reaction. For cardiac tissue samples, Western blot analysis of LPS-binding protein (LBP), immunohistochemical (IHC) detection of LBP-big42, LPS-binding protein receptor (CD14), and macrophages (CD68), as well as inflammation scoring measurement were performed. RESULTS: Periodontitis was present in all patients with severe intensity in 7, moderate in 2 and mild in one patient. Same bacterial DNA was detected in A, M, and V in different distribution, and detection was more often in atrium than in myocardium or valve tissue. Morphological investigation revealed increased extracellular inflammatory cell migration. In IHC markers of LBP, CD68 and CD14 showed positive findings for all patients in atrium and myocardium. CONCLUSION: Our results demonstrate the presence of oral bacterial DNA in human cardiac tissue, as well as inflammatory markers potentially indicating connection of PD and VHD. Further investigation is necessary to confirm these preliminary data.

Heart valves from polyester fibers: a preliminary 6-month in vivo study.

Transcatheter aortic valve implantation (TAVI) has become a popular alternative technique to surgical valve replacement for critical patients. Biological valve tissue has been used in TAVI procedures for over a decade, with over 150,000 implantations to date. However, with only 6 years of follow up, little is known about the long-term durability of biological tissue. Moreover, the high cost of tissue harvesting and chemical treatment procedures favor the development of alternative synthetic valve leaflet materials. In that context, textile polyester [polyethylene terephthalate (PET)] could be considered as an interesting candidate to replace the biological valve leaflets in TAVI procedures. However, no result is available in the literature about the behavior of textile once in contact with biological tissue in the valve position. The interaction of synthetic textile material with living tissues should be comparable to biological tissue. The purpose of this preliminary work is to compare the in vivo performances of various woven textile PET valves over a 6-month period in order to identify favorable textile construction features. In vivo results indicate that fibrosis as well as calcium deposit can be limited with an appropriate material design.

miR-92a: A Novel Potential Biomarker of Rapid Aortic Valve Calcification.

BACKGROUND AND AIM OF THE STUDY: The study aim was to compare the tissular expression of microRNAs (miRs) in bicuspid and tricuspid valves, and to evaluate their use as potential novel biomarkers of aortic valve calcification in bicuspid valves. METHODS: A prospective single-center observational study was conducted on stenotic bicuspid and tricuspid human aortic valves. According to their potential role in valve vascular and valvular calcification, a decision was taken to include miR- 92a, miR-141, and miR-223 in this analysis. A real-time quantitative polymerase chain reaction was used to measure the expression of each miR, using U6 and Cel-miR-39 as endogenous and exogenous gene controls, respectively. RESULTS: Among a total of 47 human calcified aortic valves collected, 30 (63.8%) were tricuspid valves. The mean preoperative transvalvular gradient was 50.8 mmHg (range: 37-89 mmHg), with no significant difference between bicuspid and tricuspid valves (50 mmHg versus 51.2 mmHg; p = 0.729). The mean aortic valve area was 0.79 cm2 (range: 0.33-1.3 cm2), again with no significant difference between the two groups (p = 0.34). The level of miR-92a expression was twofold higher in bicuspid valves compared to tricuspid valves (0.38 versus 0.17; p = 0.016), but no significant difference in miR-141 and miR-223 expression was observed between the two groups (p = 0.68 and p = 0.35, respectively). A positive correlation was observed between miR-92a expression and mean preoperative transvalvular gradient (r = 0.3257, p = 0.04). CONCLUSIONS: miR-92a is overexpressed in calcified bicuspid aortic valves, and may serve as a potential biomarker of rapid aortic valve calcification. Further studies based on these results may be designed to correlate the relative expression of miR-92a in the serum with its tissular expression in AS.

Extraction of Total RNA from Calcified Human Heart Valves for Gene Expression Analysis.

BACKGROUND: The isolation of high-quality RNA is an important first step in gene expression studies. However, difficult tissue disruption, low cell content and low RNA content makes consistent RNA extraction from human aortic valve tissue a challenging task. METHODS: A protocol has been developed for the successful isolation of high-quality RNA from human aortic valve samples by optimizing RNA extraction protocols based on a comparison of commercial kits. RESULTS: Guanidinium thiocyanate-phenolchloroform extraction was found to be a prerequisite for successful purification. Two protocols based on this extraction were further optimized. RNA quality and quantity were assessed spectrophotometrically, using a Bioanalyzer and by PCR analysis of several housekeeping genes. Optimized parameters included storage in RNAlater™, DNase digestion, the amount of tissue, homogenization time, and freezing of tissue after homogenization. CONCLUSIONS: The modified protocol for fatty and fibrous tissue achieved satisfactory results for gene expression analysis of human aortic valve samples.

Quantification and comparison of the mechanical properties of four human cardiac valves.

OBJECTIVE: Although having the same ability to permit unidirectional flow within the heart, the four main valves-the mitral valve (MV), aortic (AV), tricuspid (TV) and pulmonary (PV) valves-experience different loading conditions; thus, they exhibit different structural integrity from one another. Most research on heart valve mechanics have been conducted mainly on MV and AV or an individual valve, but none quantify and compare the mechanical and structural properties among the four valves from the same aged patient population whose death was unrelated to cardiovascular disease. METHODS: A total of 114 valve leaflet samples were excised from 12 human cadavers whose death was unrelated to cardiovascular disease (70.1±3.7years old). Tissue mechanical and structural properties were characterized by planar biaxial mechanical testing and histological methods. The experimental data were then fitted with a Fung-type constitutive model. RESULTS: The four valves differed substantially in thickness, degree of anisotropy, and stiffness. The leaflets of the left heart (the AV leaflets and the anterior mitral leaflets, AML) were significantly stiffer and less compliant than their counterparts in the right heart. TV leaflets were the most extensible and isotropic, while AML and AV leaflets were the least extensible and the most anisotropic. Age plays a significant role in the reduction of leaflet stiffness and extensibility with nearly straightened collagen fibers observed in the leaflet samples from elderly groups (65years and older). CONCLUSIONS: Results from 114 human leaflet samples not only provided a baseline quantification of the mechanical properties of aged human cardiac valves, but also offered a better understanding of the age-dependent differences among the four valves. It is hoped that the experimental data collected and the associated constitutive models in this study can facilitate future studies of valve diseases, treatments and the development of interventional devices. STATEMENT OF SIGNIFICANCE: Most research on heart valve mechanics have been conducted mainly on mitral and aortic valves or an individual valve, but none quantify and compare the mechanical and structural properties among the four valves from the same relatively healthy elderly patient population. In this study, the mechanical and microstructural properties of 114 leaflets of aortic, mitral, pulmonary and tricuspid valves from 12 human cadaver hearts were mechanically tested, analyzed and compared. Our results not only provided a baseline quantification of the mechanical properties of aged human valves, but a age range between patients (51-87years) also offers a better understanding of the age-dependent differences among the four valves. It is hoped that the obtained experimental data and associated constitutive parameters can facilitate studies of valve diseases, treatments and the development of interventional devices.

Determination of residual dimethylsulfoxide in cryopreserved cardiovascular allografts.

Dimethylsulfoxide (DMSO) is a solvent which protects the structure of allografts during the cryopreservation and thawing process. However, several toxic effects of DMSO in patients after transplantation of cryopreserved allografts have been described. The aim of this study is to determine the residual DMSO in the cardiovascular allografts after thawing and preparation of cryopreserved allografts for clinical application following guidelines of the European Pharmacopoeia for DMSO detection. Four types of EHB allografts (aortic valve-AV, pulmonary valve-PV, descending thoracic aorta-DA, and femoral artery-FA) are cryopreserved using as cryoprotecting solution a 10% of DMSO in medium 199. Sampling is carried out after thawing, after DMSO dilution and after delay of 30 min from final dilution (estimated delay until allograft implantation). After progressive thawing in sterile water bath at 37-42 °C (duration of about 20 min), DMSO dilution is carried out by adding consecutively 33, 66 and 200 mL of saline. Finally, tissues are transferred into 200 mL of a new physiologic solution. Allograft samples are analysed for determination of the residual DSMO concentration using a validated Gas Chromatography analysis. Femoral arteries showed the most important DMSO reduction after the estimated delay: 92.97% of decrease in the cryoprotectant final amount while a final reduction of 72.30, 72.04 and 76.29% in DMSO content for AV, PV and DA, was found, respectively. The residual DMSO in the allografts at the moment of implantation represents a final dose of 1.95, 1.06, 1.74 and 0.26 mg kg-1 in AV, PV, DA and FA, respectively, for men, and 2.43, 1.33, 2.17 and 0.33 mg kg-1 for same tissues for women (average weight of 75 kg in men, and 60 kg in women). These results are seriously below the maximum recommended dose of 1 g DMSO kg-1 (Regan et al. in Transfusion 50:2670-2675, 2010) of weight of the patient guaranteeing the safety and quality of allografts.