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Degenerative aortic valve disease in combination with diabetes is an increasing burden worldwide. There is growing evidence that particularly small leucine-rich proteoglycans are involved in the development of degenerative aortic valve disease. Nevertheless, the role of these molecules in this disease in the course of diabetes has not been elucidated in detail and previous studies remain controversial. Therefore, the aim of this study is to broaden the knowledge about small leucine-rich proteoglycans in degenerative aortic valve disease and the influence of diabetes and hyperglycaemia on aortic valves and valvular interstitial cells is examined. Analyses were performed using reverse-transcription polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, (immuno)histology and colorimetric assays. We could show that biglycan, but not decorin and lumican, is upregulated in degenerated human aortic valve cusps. Subgroup analysis reveals that upregulation of biglycan is stage-dependent. In vivo, loss of biglycan leads to stage-dependent calcification and also to migratory effects on interstitial cells within the extracellular matrix. In late stages of degenerative aortic valve disease, diabetes increases the expression of biglycan in aortic valves. In vitro, the combinations of hyperglycaemic with pro-degenerative conditions lead to an upregulation of biglycan. In conclusion, biglycan represents a potential link between degenerative aortic valve disease and diabetes.

Introduction: Calcific aortic valve disease (CAVD) is the most common acquired heart valve disease with complex underlying pathomechanisms that are yet not fully understood. Three-dimensional (3D) cell culture models as opposed to conventional two-dimensional (2D) techniques may reveal new aspects of CAVD and serve as a transitional platform between conventional 2D cell culture and in vivo experiments. Methods: Here we report on fabrication and characterization of a novel 3D hydrogel derived from cell-free native aortic valves. A detailed analysis containing protein composition, rheological behavior, cytotoxic and proliferative effects as well as results of 3D cell culture experiments are presented. Moreover, this aortic valve derived hydrogel (AVdH) is compared to commercially available biological extracellular matrix (ECM) components to evaluate and classify AVdH with respect to other currently used ECM solutions, i.e. Collagen type I and Matrigel®. Results: On the biochemical level, a complex composition of native proteins was detected. Using different techniques, including mass spectrometry with Gene Ontology network and enrichment analysis, different fundamental biological functions of AVdH were identified, including peptidase-, peptidase inhibitor-, growth- and binding activity. No cytotoxic effects were detected and AVdH showed positive effects on cell growth and proliferation in vitro when compared to Collagen type I and Matrigel®. Conclusion: These results suggest AVdH as an organotypic ECM supporting sophisticated 3D cell culture model studies, while mimicking the native environment of the aortic valve to a greater level for enhanced in vitro analyses.

Calcific aortic valve disease is an active disease process with lipoprotein deposition, chronic inflammation, and progressive leaflet degeneration. Expression of ectonucleotidases, a group of membrane-bound enzymes that regulate the metabolism of ATP and its metabolites, may coregulate the degeneration process of valvular interstitial cells (VICs). The aim of this study was to investigate the role of the enzymes of the purinergic system in the degeneration process of VICs. Ovine VICs were cultivated in vitro under different prodegenerative conditions and treated with inhibitors of ectonucleoside triphosphate diphosphohydrolase 1 (CD39)/ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and 5'-nucleotidase (CD73), as well as with adenosine and adenosine receptor agonists. Experiments were performed both in 2-dimensional (2-D) and 3-dimensional (3-D) cell-culture models. Our main findings were that VICs continuously release ATP. Inhibition of ATP hydrolyzing enzymes (CD39 and ENPP1) resulted in profound prodegenerative effects with a vigorous up-regulation of CD39, ENPP1, and CD73, as well as TGF-β1 and osteopontin at the gene level. In our 3-D model, the effect was more pronounced than in 2-D monolayers. Increasing adenosine levels, as well as stimulating the adenosine receptors A and A, exhibited strong prodegenerative effects, whereas conversely, lowering adenosine levels by inhibition of CD73 resulted in protective effects against degeneration. Dysregulation of any one of these enzymes plays an important role in the degeneration process of VICs. Stimulation of ATP and adenosine has prodegenerative effects, whereas lowering the adenosine levels exerts a protective effect.-Weber, A., Barth, M., Selig, J. I., Raschke, S., Dakaras, K., Hof, A., Hesse, J., Schrader, J., Lichtenberg, A., Akhyari, P. Enzymes of the purinergic signaling system exhibit diverse effects on the degeneration of valvular interstitial cells in a 3-D microenvironment.

Aims Donor heart shortage leads to increasing use of left ventricular assist device (LVAD) as bridge‐to‐transplant or destination therapy. Prolonged LVAD support is associated with aortic valve insufficiency, representing a relevant clinical problem in LVAD patients. Nevertheless, the impact of LVAD support on inflammation, remodelling, and chondro‐osteogenic differentiation of the aortic valve is still not clearly understood. The aim of the study is to evaluate the impact of LVAD support on structural and molecular alterations of the aortic valve. Methods and results During heart transplantation, aortic valves of 63 heart failure patients without (n = 22) and with LVAD support (n = 41) were collected and used for analysis. Data on clinical course as well as echocardiographic data were analysed. Calcification and markers of remodelling, chondro‐osteogenic differentiation, and inflammation were evaluated by computed tomography, by mRNA analysis and by histology and immunohistochemistry. Expression of inflammation markers of the LVAD group was analysed with regard to levels of C‐reactive protein and driveline infections. Calcium accumulation and mRNA expression of determined markers were correlated with duration of LVAD support. Data were also analysed relating to aortic valve opening and aortic valve insufficiency. There was no difference in the frequency of cardiovascular risk factors or comorbidities between the patient groups. Expression of matrix metalloproteinase‐9 (P = 0.007), alpha‐smooth muscle actin (P = 0.045), and osteopontin (P = 0.003) were up‐regulated in aortic valves of LVAD patients. Histological appearance of the aortic valve was similar in patients with or without LVAD, and computed tomography‐based analysis not yet revealed significant difference in tissue calcification. Expression of interferon gamma (P = 0.004), interleukin‐1 beta (P < 0.0001), and tumour necrosis factor alpha (P = 0.04) was up‐regulated in aortic valves of LVAD patients without concomitant inflammatory cell infiltration and independent from unspecific inflammation. Expression of matrix metalloproteinase‐2 (P = 0.038) and transforming growth factor beta (P = 0.0504) correlated negatively with duration of LVAD support. Presence of aortic valve insufficiency led to a significantly higher expression of interferon gamma (P = 0.007) in LVAD patients. There was no alteration in the determined markers in relation to aortic valve opening in LVAD patients. Conclusions Left ventricular assist device support leads to signs of early aortic valve degeneration independent of support duration. Thus, the aortic valve of patients with LVAD support should be closely monitored, particularly in patients receiving destination therapy as well as in the prospect of using aortic valves of LVAD patients as homografts in case of bridge‐to‐transplant therapy.

A comparison of different techniques for increasing interstitial repopulation of detergent dECM revealed that an additional limited trypsin treatment was most effective. Nevertheless, a complete interstitial repopulation of decellularized heart valves remains a challenging endeavour. Additional experimental fine-tuning may improve the in vitro results of heart valve tissue engineering.

Calcific aortic valve disease is the most common valvular heart disease in industrialized countries. Pulsatile pressure, sheer and bending stress promote initiation and progression of aortic valve degeneration. The aim of this work is to establish an ex vivo model to study the therein involved processes. Ovine aortic roots bearing aortic valve leaflets were cultivated in an elaborated bioreactor system with pulsatile flow, physiological temperature, and controlled pressure and pH values. Standard and pro-degenerative treatment were studied regarding the impact on morphology, calcification, and gene expression. In particular, differentiation, matrix remodeling, and degeneration were also compared to a static cultivation model. Bioreactor cultivation led to shrinking and thickening of the valve leaflets compared to native leaflets while gross morphology and the presence of valvular interstitial cells were preserved. Degenerative conditions induced considerable leaflet calcification. In comparison to static cultivation, collagen gene expression was stable under bioreactor cultivation, whereas expression of hypoxia-related markers was increased. Osteopontin gene expression was differentially altered compared to protein expression, indicating an enhanced protein turnover. The present ex vivo model is an adequate and effective system to analyze aortic valve degeneration under controlled physiological conditions without the need of additional growth factors.

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