Shear-mediated ALK5 expression regulates endothelial activation.

Calcific aortic valve disease affects the aortic side of the valve, exposed to low magnitude multidirectional ("disturbed) blood flow, more than it affects the ventricular side, exposed to high magnitude uniaxial flow. Overt disease is preceded by endothelial dysfunction and inflammation. Here we investigate the potential role of the transforming growth factor-ß (TGF-ß) receptor ALK5 in this process. Although ECs are always subject to shear stress due to blood flow, and their responses to shear stress are important in healthy valve development and homeostasis, low magnitude multidirectional flow can induce pathophysiological changes. Previous work has shown ALK5 to be an important mechanosensor. ALK5 transduces mechanically sensed signals via the activation of the SMAD2/3 transcriptional modulators. However, it is currently unclear precisely how ALK5-mediated shear stress responses translate into pathological changes under conditions of chronically disturbed flow. Here, we demonstrate that ALK5 mechanosensory signalling influences flow-induced endothelial leukocyte adhesion and paracellular permeability. Low magnitude multidirectional flow resulted in downregulation of the receptor, accompanied by increased SMAD2 phosphorylation, in human umbilical vein endothelial cell (HUVEC) monolayers. These changes correlated with elevated monocyte adhesion and significantly increased transendothelial transport of an albumin-sized tracer. These effects were abolished by inhibition of ALK5 kinase activity. Analysis of ALK5 expression patterns in porcine aortic valve tissue corroborated the findings from cell-based experiments. Together, these results suggest that ALK5 has a role in shear stress-associated cardiovascular disease pathology, emphasising the importance of further mechanistic investigations and supporting it as a potential therapeutic target.

Coronary vasodilation mediated by T cells expressing choline acetyltransferase.

CD4+ T cells expressing choline acetyltransferase (ChAT) have recently been shown to cause a drop in systemic blood pressure when infused into mice. The aim of this study was to determine if ChAT-expressing T cells could regulate coronary vascular reactivity. Preconstricted segments of epicardial and intramyocardial porcine coronary arteries relaxed in response to Jurkat T cells (JT) that overexpressed ChAT (JTChAT cells). The efficacy of the JTChAT cells was similar in epicardial and intramyocardial vessels with a maximum dilator response to 3 × 105 cells/mL of 38.0 ± 6.7% and 38.7 ± 7.25%, respectively. In contrast, nontransfected JT cells elicited a weak dilator response, followed by a weak contraction. The response of JTChAT cells was dependent on the presence of the endothelial cells. In addition, the response could be significantly reduced by Nω-nitro-l-arginine methyl ester (l-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the presence of indomethacin. JTChAT cells, but not JT cells, increased the expression of phosphorylated endothelial nitric oxide synthase (eNOS). JTChAT cells contained significantly greater levels of acetylcholine compared with JT cells; however, the nonselective muscarinic antagonist atropine and the M1 receptor antagonist pirenzepine both failed to block the dilator effect of JTChAT cells. Exogenously added acetylcholine induced only a weak relaxation (∼10%) at low concentrations, which became a contractile response at higher concentrations. These data illustrate the capacity for cells that express ChAT to regulate coronary vascular reactivity, via mechanisms that are dependent on interaction with the endothelium and in part mediated by the release of nitric oxide.NEW & NOTEWORTHY This study shows ChAT-expressing T cells can induce vasodilation of the blood vessel in the coronary circulation and that this effect relies on a direct interaction between T cells and the coronary vascular endothelium. The study establishes a potential immunomodulatory role for T cells in the coronary circulation. The present findings offer an additional possibility that a deficiency of ChAT-expressing T cells could contribute to reduced coronary blood flow and ischemic events in the myocardium.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Interferons Are Pro-Inflammatory Cytokines in Sheared-Stressed Human Aortic Valve Endothelial Cells.

Calcific aortic valve disease (CAVD) is an athero-inflammatory process. Growing evidence supports the inflammation-driven calcification model, mediated by cytokines such as interferons (IFNs) and tumor necrosis factor (TNF)-α. Our goal was investigating IFNs' effects in human aortic valve endothelial cells (VEC) and the potential differences between aortic (aVEC) and ventricular (vVEC) side cells. The endothelial phenotype was analyzed by Western blot, qPCR, ELISA, monocyte adhesion, and migration assays. In mixed VEC populations, IFNs promoted the activation of signal transducers and activators of transcription-1 and nuclear factor-κB, and the subsequent up-regulation of pro-inflammatory molecules. Side-specific VEC were activated with IFN-γ and TNF-α in an orbital shaker flow system. TNF-α, but not IFN-γ, induced hypoxia-inducible factor (HIF)-1α stabilization or endothelial nitric oxide synthase downregulation. Additionally, IFN-γ inhibited TNF-α-induced migration of aVEC. Also, IFN-γ triggered cytokine secretion and adhesion molecule expression in aVEC and vVEC. Finally, aVEC were more prone to cytokine-mediated monocyte adhesion under multiaxial flow conditions as compared with uniaxial flow. In conclusion, IFNs promote inflammation and reduce TNF-α-mediated migration in human VEC. Moreover, monocyte adhesion was higher in inflamed aVEC sheared under multiaxial flow, which may be relevant to understanding the initial stages of CAVD.

Hypoxia-mediated regulation of the secretory properties of mitral valve interstitial cells.

The sophisticated function of the mitral valve depends to a large extent on its extracellular matrix (ECM) and specific cellular components. These are tightly regulated by a repertoire of mechanical stimuli and biological pathways. One potentially important stimulus is hypoxia. The purpose of this investigation is to determine the effect of hypoxia on the regulation of mitral valve interstitial cells (MVICs) with respect to the synthesis and secretion of extracellular matrix proteins. Hypoxia resulted in reduced production of total collagen and sulfated glycosaminoglycans (sGAG) in cultured porcine MVICs. Increased gene expression of matrix metalloproteinases-1 and -9 and their tissue inhibitors 1 and 2 was also observed after incubation under hypoxic conditions for up to 24 h. Hypoxia had no effect on MVIC viability, morphology, or phenotype. MVICs expressed hypoxia-inducible factor (HIF)-1α under hypoxia. Stimulating HIF-1α chemically caused a reduction in the amount of sGAG produced, similar to the effect observed under hypoxia. Human rheumatic valves had greater expression of HIF-1α compared with normal or myxomatous degenerated valves. In conclusion, hypoxia affects the production of certain ECM proteins and expression of matrix remodeling enzymes by MVICs. The effects of hypoxia appear to correlate with the induction of HIF-1α. This study highlights a potential role of hypoxia and HIF-1α in regulating the mitral valve, which could be important in health and disease.NEW & NOTEWORTHY This study demonstrates that hypoxia regulates extracellular matrix secretion and the remodeling potential of heart valve interstitial cells. Expression of hypoxia-induced factor-1α plays a role in these effects. These data highlight the potential role of hypoxia as a physiological mediator of the complex function of heart valve cells.

Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.

RATIONALE: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Expression of smooth muscle cell markers and co-activators in calcified aortic valves.

AIMS: Similar risk factors and mediators are involved in calcific aortic stenosis (CAS) and atherosclerosis. Since normal valves harbour a low percentage of smooth muscle cells (SMCs), we hypothesize that the SMC phenotype participates in the pathogenesis of CAS. METHOD AND RESULTS: We analysed 12 normal and 22 calcified aortic valves for SMC markers and the expression of co-activators of SMC gene expression, myocardin and myocardin-related transcription factors (MRTF-A/B). Transforming growth factor ß (TGFß1) was used to upregulate SMC markers and co-activators in valve interstitial cells (VICs) and transmission electron microscopy (TEM) was used to detect the presence of SMC in atypical regions of the valve leaflets. Smooth muscle cell markers and co-activators, myocardin, MRTF-A, and MRTF-B, demonstrated an increased incidence and aberrant expression around calcified nodules in all 22 calcified valves as well as in surface and microvessel endothelial cells. Smooth muscle cell markers and MRTF-A were significantly increased in calcified valves. Transforming growth factor ß1 (TGFß1) (10 ng/mL) was able to significantly upregulate the expression of some SMC markers and MRTF-A in VICs. Transmission electron microscopy of the fibrosa layer of calcified valves demonstrated the presence of bundles of SMCs and smooth muscle-derived foam cells. CONCLUSION: Smooth muscle cell markers and co-activators, myocardin and MRTFs, were aberrantly expressed in calcified valves. Transforming growth factor ß1 was able to significantly upregulate SMC markers and MRTF-A in VICs. Transmission electron microscopy unequivocally identified the presence of SMCs in calcified regions of valve leaflets. These findings provide evidence that the SMC phenotype plays a role in the development of CAS.

Shear stress and VEGF enhance endothelial differentiation of human adipose-derived stem cells.

Herein we combine chemical and mechanical stimulation to investigate the effects of vascular endothelial growth factor (VEGF) and physiological shear stress in promoting the differentiation human adipose derived stem cells (ADSCs) into endothelial cells. ADSCs were isolated and characterized; endothelial differentiation was promoted by culturing confluent cells in 50 ng/ml VEGF under physiological shear stress for up to 14 days. Afterwards, endothelial cells were seeded onto collagen or acellular aortic valve matrices and exposed to four culture conditions: shear stress + VEGF; shear stress - VEGF; static + VEGF and static - VEGF. After 7 days, phenotype was investigated. ADSCs subjected to shear stress and VEGF express a comprehensive range of specific endothelial markers (vWF, eNOS and FLT-1 after 7 days and CD31, FLk-1 and VE-cadherin after 14 days) and maintain the phenotype when seeded onto scaffolds. Our protocol proved to be an efficient source of endothelial-like cells for tissue engineering based on autologous ADSC.

Side-specific mechanical properties of valve endothelial cells.

Aortic valve endothelial cells (ECs) function in vastly different levels of shear stress. The biomechanical characteristics of cells on each side of valve have not been investigated. We assessed the morphology and mechanical properties of cultured or native valve ECs on intact porcine aortic valve cusps using a scanning ion conductance microscope (SICM). The autocrine influence of several endothelial-derived mediators on cell compliance and the expression of actin were also examined. Cells on the aortic side of the valve are characterized by a more elongated shape and were aligned along a single axis. Measurement of EC membrane compliance using the SICM showed that the cells on the aortic side of intact valves were significantly softer than those on the ventricular side. A similar pattern was seen in cultured cells. Addition of 10(-6) M of the nitric oxide donor sodium nitroprusside caused a significant reduction in the compliance of ventricular ECs but had no effect on cells on the aortic side of the valve. Conversely, endothelin-1 (10(-10)-10(-8) M) caused an increase in the compliance of aortic cells but had no effect on cells on the ventricular side of the valve. Aortic side EC compliance was also increased by 10(-4) M of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester. Immunofluorescent staining of actin filaments revealed a great density of staining in ECs on the ventricular surface. The expression of actin and the relative membrane compliance of ECs on both side of the valve were not affected by ventricular and aortic patterns of flow. This study has shown side-specific differences in the biomechanics of aortic valve ECs. These differences can have important implications for valve function.

Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification.

The accumulation of calcified material in cardiovascular tissue is thought to involve cytochemical, extracellular matrix and systemic signals; however, its precise composition and nanoscale architecture remain largely unexplored. Using nano-analytical electron microscopy techniques, we examined valves, aortae and coronary arteries from patients with and without calcific cardiovascular disease and detected spherical calcium phosphate particles, regardless of the presence of calcific lesions. We also examined lesions after sectioning with a focused ion beam and found that the spherical particles are composed of highly crystalline hydroxyapatite that crystallographically and structurally differs from bone mineral. Taken together, these data suggest that mineralized spherical particles may play a fundamental role in calcific lesion formation. Their ubiquitous presence in varied cardiovascular tissues and from patients with a spectrum of diseases further suggests that lesion formation may follow a common process. Indeed, applying materials science techniques to ectopic and orthotopic calcification has great potential to lend critical insights into pathophysiological processes underlying calcific cardiovascular disease.

Side-specific endothelial-dependent regulation of aortic valve calcification: interplay of hemodynamics and nitric oxide signaling.

Arterial endothelial cells maintain vascular homeostasis and vessel tone in part through the secretion of nitric oxide (NO). In this study, we determined how aortic valve endothelial cells (VEC) regulate aortic valve interstitial cell (VIC) phenotype and matrix calcification through NO. Using an anchored in vitro collagen hydrogel culture system, we demonstrate that three-dimensionally cultured porcine VIC do not calcify in osteogenic medium unless under mechanical stress. Co-culture with porcine VEC, however, significantly attenuated VIC calcification through inhibition of myofibroblastic activation, osteogenic differentiation, and calcium deposition. Incubation with the NO donor DETA-NO inhibited VIC osteogenic differentiation and matrix calcification, whereas incubation with the NO blocker l-NAME augmented calcification even in 3D VIC-VEC co-culture. Aortic VEC, but not VIC, expressed endothelial NO synthase (eNOS) in both porcine and human valves, which was reduced in osteogenic medium. eNOS expression was reduced in calcified human aortic valves in a side-specific manner. Porcine leaflets exposed to the soluble guanylyl cyclase inhibitor ODQ increased osteocalcin and α-smooth muscle actin expression. Finally, side-specific shear stress applied to porcine aortic valve leaflet endothelial surfaces increased cGMP production in VEC. Valve endothelial-derived NO is a natural inhibitor of the early phases of valve calcification and therefore may be an important regulator of valve homeostasis and pathology.

What does good care look like to people living with congenital heart disease in the 21st century? Qualitative online, asynchronous discussion forums.

OBJECTIVES: As part of a wider study, our aim was to elicit perspectives of people with congenital heart disease (CHD) and/or their parents/carers about their experiences of healthcare and what is important to them when receiving care. DESIGN AND SETTING: A qualitative study involving a series of closed, asynchronous, online discussion forums underpinned by an interpretivist framework and set up and moderated by three patient charities via their Facebook pages. PARTICIPANTS: People with CHD and parents/carers of people with CHD from the UK. RESULTS: Five forums were run for 12-24 weeks across the three charities, and 343 participants signed up to the forums. Four linked themes related to processes of care were identified following thematic analysis of the transcripts: relationships and communication; access and coordination; experience of discrete episodes of care and psychological support. These impacted how care was experienced and, for some patients, outcomes of CHD and its treatment as well as broader health outcomes. In addition, context relating to stages of the patient journey was described, together with patient-related factors such as patients' knowledge and expertise in their own condition. CONCLUSIONS: People with CHD and their parents/carers want individualised, person-centred care delivered within an appropriately resourced, multidisciplinary service. Although examples of excellent care were provided it is evident that, from the perspective of patients and parents/carers, some National Health Service Standards for people with CHD were not being met.

Exogenous and endogenous nitric oxide eluting polylactic acid-based nanofibrous scaffolds for enhancing angiogenesis of diabetic wounds.

Delayed wound healing is a major complication that diabetic patients suffer from due to high microbial infection susceptibility, high diabetic wound alkalinity, a low lymphangiogenesis rate, and a high inflammation rate, resulting in severe gangrene. Hence, this study aims to develop a multifunctional adhesive nanofibrous patch to promote the wound healing process. Phenytoin, sildenafil citrate, and/or nitric oxide-eluting nanoparticles were incorporated separately within the polylactic acid nanofibrous layer. Polylactic acid was fabricated in the form of highly porous nanofibrous matrices that resemble the natural structure of skin tissues in order to act as scaffolds that help cell migration and proliferation. A polylactic acid nanofibrous layer incorporating phenytoin was designed to stimulate fibroblast proliferation and inhibit inflammation. Another polylactic acid nanofibrous layer was loaded either with nitric oxide-eluting nanoparticles or sildenafil as a pro-angiogenic layer that can supply tissues with nitric oxide gas either exogenously or endogenously, respectively. The developed nanofibrous layers were in-vitro evaluated through different physicochemical, mechanical, and biological approaches. Finally, the efficiency of the prepared single multilayered patch was tested using an in-vivo alloxan-induced diabetic rats' model, which proved that the patches were able to release the incorporated cargos in a controlled manner, enhancing the wound healing process.

Atrioventricular valve development: new perspectives on an old theme.

Atrioventricular valve development commences with an EMT event whereby endocardial cells transform into mesenchyme. The molecular events that induce this phenotypic change are well understood and include many growth factors, signaling components, and transcription factors. Besides their clear importance in valve development, the role of these transformed mesenchyme and the function they serve in the developing prevalve leaflets is less understood. Indeed, we know that these cells migrate, but how and why do they migrate? We also know that they undergo a transition to a mature, committed cell, largely defined as an interstitial fibroblast due to their ability to secrete various matrix components including collagen type I. However, we have yet to uncover mechanisms by which the matrix is synthesized, how it is secreted, and how it is organized. As valve disease is largely characterized by altered cell number, cell activation, and matrix disorganization, answering questions of how the valves are built will likely provide us with information of real clinical relevance. Although expression profiling and descriptive or correlative analyses are insightful, to advance the field, we must now move past the simplicity of these assays and ask fundamental, mechanistic based questions aimed at understanding how valves are "built". Herein we review current understandings of atrioventricular valve development and present what is known and what isn't known. In most cases, basic, biological questions and hypotheses that were presented decades ago on valve development still are yet to be answered but likely hold keys to uncovering new discoveries with relevance to both embryonic development and the developmental basis of adult heart valve diseases. Thus, the goal of this review is to remind us of these questions and provide new perspectives on an old theme of valve development.

Role of hypoxia inducible factor HIF-1α in heart valves.

The 2016 Albert Lasker Basic Medical Research Award and subsequently the 2019 Nobel Prize in Physiology or Medicine were awarded to William Kaelin, Jr., Sir Peter Ratcliffe, and Gregg Semenza for their work on how cells sense and adapt to hypoxic conditions. Their work showed that the changes in gene expression, cell metabolism, and tissue remodelling that occur in response to low oxygen concentrations are orchestrated by the transcription factor, hypoxia inducible factor-1α (HIF-1α). While the effects mediated by HIF-1α have been widely studied, its role in heart valves has only recently been investigated. These studies have shown that HIF-1α expression is evident in mechanisms that regulate the structure and function of heart valves. These include embryonic development, the regulation of the extracellular matrix, angiogenesis and the initiation of the calcification process. This review provides a background on the role and function of HIF-1α in response to hypoxia and a discussion of the available evidence of its involvement in the regulation of heart valves in health and disease.

In vitro cholesterol lowering activity of Ganoderma australe mycelia based on mass spectrometry, synchrotron Fourier-transform infrared analysis and liver-spheroid bioactivity.

Mycelia were cultivated from a Thai wild mushroom identified as Ganoderma australe based on polymerase chain reaction (PCR) and morphological analyses. The mycelial extracts were examined for their active ingredients using a liquid chromatography-tandem mass spectrometry (LC‒MS/MS) method. This revealed the presence of lovastatin and tentative compounds including p-coumaric, nicotinamide, gamma-aminobutyric acid, choline, nucleosides, amino acids, and saccharides. The extracts had an inhibitory effect on the activity of HMG-CoA reductase in a concentration-dependent manner. At 2.5 mg/mL, the G. australe extracts did not interfere with the viability of HepG2 spheroids, but their biochemical composition was altered as determined by Fourier-transform infrared (FTIR) spectroscopy. The lipid profile of the spheroids treated with the mycelial extract was distinct from that of the control and the 5 µM lovastatin treatment, corresponding with the production of cholesterol by the spheroids. The mycelia of G. australe increased the percentage of high-density lipoprotein (HDL) production to 71.35 ± 2.74%, compared to the control and lovastatin-treated spheroids (33.26 ± 3.15% and 32.13 ± 3.24%, respectively). This study revealed the superior effect of natural compound mixtures to pure lovastatin, and the potential use of Thailand's wild G. australe as a functional food to prevent or alleviate hypercholesterolemia.

Valvulogenesis of a living, innervated pulmonary root induced by an acellular scaffold.

Heart valve disease is a major cause of mortality and morbidity worldwide with no effective medical therapy and no ideal valve substitute emulating the extremely sophisticated functions of a living heart valve. These functions influence survival and quality of life. This has stimulated extensive attempts at tissue engineering "living" heart valves. These attempts utilised combinations of allogeneic/ autologous cells and biological scaffolds with practical, regulatory, and ethical issues. In situ regeneration depends on scaffolds that attract, house and instruct cells and promote connective tissue formation. We describe a surgical, tissue-engineered, anatomically precise, novel off-the-shelf, acellular, synthetic scaffold inducing a rapid process of morphogenesis involving relevant cell types, extracellular matrix, regulatory elements including nerves and humoral components. This process relies on specific material characteristics, design and "morphodynamism".

Impact of γ-irradiation on extracellular matrix of porcine pulmonary valves.

BACKGROUND: The extracellular matrix plays an important role in heart valve function. To improve the processing of porcine pulmonary valves for clinical use, we have studied the influence of cryopreservation, decellularization, and irradiation on extracellular matrix components. METHODS: Decellularization was carried out followed by DNAseI/RNAseA digestion and isotonic washout. Valves were cryopreserved in 10% DMSO/10% fetal bovine serum, and then subjected to 25-40 kGy γ-radiation. Extracellular matrix constituents were evaluated by histologic staining, immunohistochemistry, transmission electron microscopy, and liquid chromatography/mass spectrometry. RESULTS: Histologic, immunohistochemical, ultrastructural, and biochemical analyses demonstrated a marked reduction in the expression of extracellular matrix components particularly in the valves that had been γ-irradiated following decellularization and cryopreservation. In this group, histology and immunohistochemistry showed an obvious reduction in staining for chondroitin sulphates, versican, hyaluronan, and collagens. Transmission electron microscopy revealed the smallest fibril diameter of collagen, shortest D-period, and loss of compactness of collagen fiber packaging and fragmentation of elastic fibers. Biochemical analysis showed loss of collagen and elastin crosslinks. Decellularization followed by cryopreservation showed some reduction in staining for collagens and versican, smaller diameter, shorter D-period in collagen fibers, and ridges in elastic fibers. Cryopreservation alone showed minimal changes in ECM staining intensity, collagen, and elastin ultrastructure and biochemistry. CONCLUSION: γ-Irradiated valves that have been decellularized and cryopreserved produces significant changes in the expression of ECM components, thus providing useful information for improving valve preparation for clinical use and also some indication as to why irradiated human heart valves were not clinically successful.

Atypical Expression of Smooth Muscle Markers and Co-activators and Their Regulation in Rheumatic Aortic and Calcified Bicuspid Valves.

Objective: We have previously reported that human calcified aortic cusps have abundant expression of smooth muscle (SM) markers and co-activators. We hypothesised that cells in bicuspid aortic valve (BAV) cusps and those affected by rheumatic heart valve (RHV) disease may follow a similar phenotypic transition into smooth muscle cells, a process that could be regulated by transforming growth factors (TGFs). Aims: Cusps from eight patients with BAV and seven patients with RHV were analysed for early and late SM markers and regulators of SM gene expression by immunocytochemistry and compared to healthy aortic valves from 12 unused heart valve donors. The ability of TGFs to induce these markers in valve endothelial cells (VECs) on two substrates was assessed. Results: In total, 7 out of 8 BAVs and all the RHVs showed an increased and atypical expression of early and late SM markers α-SMA, calponin, SM22 and SM-myosin. The SM marker co-activators were aberrantly expressed in six of the BAV and six of the RHV, in a similar regional pattern to the expression of SM markers. Additionally, regions of VECs, and endothelial cells lining the vessels within the cusps were found to be positive for SM markers and co-activators in three BAV and six RHV. Both BAVs and RHVs were significantly thickened and HIF1α expression was prominent in four BAVs and one RHV. The ability of TGFßs to induce the expression of SM markers and myocardin was greater in VECs cultured on fibronectin than on gelatin. Fibronectin was shown to be upregulated in BAVs and RHVs, within the cusps as well as in the basement membrane. Conclusion: Bicuspid aortic valves and RHVs expressed increased numbers of SM marker-positive VICs and VECs. Concomittantly, these cells expressed MRTF-A and myocardin, key regulators of SM gene expression. TGFß1 was able to preferentially upregulate SM markers and myocardin in VECs on fibronectin, and fibronectin was found to be upregulated in BAVs and RHVs. These findings suggest a role of VEC as a source of cells that express SM cell markers in BAVs and RHVs. The similarity between SM marker expression in BAVs and RHVs with our previous study with cusps from patients with aortic stenosis suggests the existance of a common pathological pathway between these different pathologies.

Profiling Genome-Wide DNA Methylation Patterns in Human Aortic and Mitral Valves.

Cardiac valves exhibit highly complex structures and specialized functions that include dynamic interactions between cells, extracellular matrix (ECM) and their hemodynamic environment. Valvular gene expression is tightly regulated by a variety of mechanisms including epigenetic factors such as histone modifications, RNA-based mechanisms and DNA methylation. To date, methylation fingerprints of non-diseased human aortic and mitral valves have not been studied. In this work we analyzed the differential methylation profiles of 12 non-diseased aortic and mitral valve tissue samples (in matched pairs). Analysis of methylation data [reduced representation bisulfite sequencing (RRBS)] of 16,101 promoters genome-wide revealed 584 differentially methylated (DM) promoters, of which 13 were reported in endothelial mesenchymal trans-differentiation (EMT), 37 in aortic and mitral valve disease and 7 in ECM remodeling. Both functional classification as well as network analysis showed that the genes associated with the DM promoters were enriched for WNT-, Cadherin-, Endothelin-, PDGF-, HIF-1 and VEGF- signaling implicated in valvular physiology and pathophysiology. Additional enrichment was detected for TGFB-, NOTCH- and Integrin- signaling involved in EMT as well as ECM remodeling. This data provides the first insight into differential regulation of human aortic and mitral valve tissue and identifies candidate genes linked to DM promoters. Our work will improve the understanding of valve biology, valve tissue engineering approaches and contributes to the identification of relevant drug targets.