Identifying Behavioral Phenotypes and Heterogeneity in Heart Valve Surface Endothelium.

Heart valvular endothelial cells (VECs) are distinct from vascular endothelial cells (ECs), but have an uncertain context within the spectrum of known endothelial phenotypes, including lymphatic ECs (LECs). Profiling the phenotypes of the heart valve surface VECs would facilitate identification of a proper seeding population for tissue-engineered valves, as well as elucidate mechanisms of valvular disease. Porcine VECs and porcine aortic ECs (AECs) were isolated from pig hearts and characterized to assess known EC and LEC markers. A transwell migration assay determined their propensity to migrate toward vascular endothelial growth factor, an angiogenic stimulus, over 24 h. Compared to AECs, Flt-1 was expressed on almost double the percentage of VECs, measured as 74 versus 38%. The expression of angiogenic EC markers CXCR4 and DLL4 was >90% on AECs, whereas VECs showed only 35% CXCR4+ and 47% DLL4+. AECs demonstrated greater migration (71.5 ± 11.0 cells per image field) than the VECs with 30.0 ± 15.3 cells per image field (p = 0.032). In total, 30% of VECs were positive for LYVE1+/Prox1+, while these markers were absent in AECs. In conclusion, the population of cells on the surface of heart valves is heterogeneous, consisting largely of nonangiogenic VECs and a subset of LECs. Previous studies have indicated the presence of LECs within the interior of the valves; however, this is the first study to demonstrate their presence on the surface. Identification of this unique endothelial mixture is a step forward in the development of engineered valve replacements as a uniform EC seeding population may not be the best option to maximize transplant success.

3-Dimensional spatially organized PEG-based hydrogels for an aortic valve co-culture model.

Physiologically relevant in vitro models are needed to study disease progression and to develop and screen potential therapeutic interventions for disease. Heart valve disease, in particular, has no early intervention or non-invasive treatment because there is a lack of understanding the cellular mechanisms which lead to disease. Here, we establish a novel, customizable synthetic hydrogel platform that can be used to study cell-cell interactions and the factors which contribute to valve disease. Spatially localized cell adhesive ligands bound in the scaffold promote cell growth and organization of valve interstitial cells and valve endothelial cells in 3D co-culture. Both cell types maintained phenotypes, homeostatic functions, and produced zonally localized extracellular matrix. This model extends the capabilities of in vitro research by providing a platform to perform direct contact co-culture with cells in their physiologically relevant spatial arrangement.

A three-dimensional co-culture model of the aortic valve using magnetic levitation.

The aortic valve consists of valvular interstitial cells (VICs) and endothelial cells (VECs). While these cells are understood to work synergistically to maintain leaflet structure and valvular function, few co-culture models of these cell types exist. In this study, aortic valve co-cultures (AVCCs) were assembled using magnetic levitation and cultured for 3 days. Immunohistochemistry and quantitative reverse-transcriptase polymerase chain reaction were used to assess the maintenance of cellular phenotype and function, and the formation of extracellular matrix. AVCCs stained positive for CD31 and α-smooth muscle actin (αSMA), demonstrating that the phenotype was maintained. Functional markers endothelial nitric oxide synthase (eNOS), von Willebrand factor (VWF) and prolyl-4-hydroxylase were present. Extracellular matrix components collagen type I, laminin and fibronectin also stained positive, with reduced gene expression of these proteins in three dimensions compared to two dimensions. Genes for collagen type I, lysyl oxidase and αSMA were expressed less in AVCCs than in 2-D cultures, indicating that VICs are quiescent. Co-localization of CD31 and αSMA in the AVCCs suggests that endothelial-mesenchymal transdifferentiation might be occurring. Differences in VWF and eNOS in VECs cultured in two and three dimensions also suggests that the AVCCs possibly have anti-thrombotic potential. Overall, a co-culture model of the aortic valve was designed, and serves as a basis for future experiments to understand heart valve biology.

Age-related changes in aortic valve hemostatic protein regulation.

OBJECTIVE: Although valvular endothelial cells have unique responses compared with vascular endothelial cells, valvular regulation of hemostasis is not well-understood. Heart valves remodel throughout a person's lifetime, resulting in changes in extracellular matrix composition and tissue mechanical properties that may affect valvular endothelial cell hemostatic function. This work assessed valvular endothelial cell regulation of hemostasis in situ and in vitro as a function of specimen age. APPROACH AND RESULTS: Porcine aortic valves were assigned to 1 of 3 age groups: Young (YNG) (6 weeks); Adult (ADT) (6 months); or Elderly (OLD) (2 years). Histological examination of valves showed that secreted thrombotic/antithrombotic proteins localize at the valve endothelium and tissue interior. Gene expression and immunostains for von Willebrand factor (VWF), tissue factor pathway inhibitor, and tissue plasminogen activator in YNG porcine aortic valve endothelial cells were higher than they were for OLD, whereas plasminogen activator inhibitor 1 levels in OLD were higher than those for YNG and ADT. Histamine-stimulated YNG porcine aortic valve endothelial cells released higher concentrations of VWF proteins than OLD, and the fractions of VWF-140 fragments was not different between age groups. A calcific aortic valve disease in vitro model using valvular interstitial cells confirmed that VWF in culture significantly increased valvular interstitial cell nodule formation and calcification. CONCLUSIONS: Hemostatic protein regulation in aortic valve tissues and in valvular endothelial cells changes with age. The presence of VWF and other potential hemostatic proteins increase valvular interstitial cell calcification in vitro. Therefore, the increased capacity of elderly valves to sequester the hemostatic proteins, together with age-associated loss of extracellular matrix organization, warrants investigation into potential role of these proteins in the formation of calcific nodules.

Hyaluronan turnover and hypoxic brown adipocytic differentiation are co-localized with ossification in calcified human aortic valves.

The calcification process in aortic stenosis has garnered considerable interest but only limited investigation into selected signaling pathways. This study investigated mechanisms related to hypoxia, hyaluronan homeostasis, brown adipocytic differentiation, and ossification within calcified valves. Surgically explanted calcified aortic valves (n=14) were immunostained for markers relevant to these mechanisms and evaluated in the center (NodCtr) and edge (NodEdge) of the calcified nodule (NodCtr), tissue directly surrounding nodule (NodSurr); center and tissue surrounding small "prenodules" (PreNod, PreNodSurr); and normal fibrosa layer (CollFibr). Pearson correlations were determined between staining intensities of markers within regions. Ossification markers primarily localized to NodCtr and NodEdge, along with markers related to hyaluronan turnover and hypoxia. Markers of brown adipocytic differentiation were frequently co-localized with markers of hypoxia. In NodCtr and NodSurr, brown fat and ossification markers correlated with hyaluronidase-1, whereas these markers, as well as hypoxia, correlated with hyaluronan synthases in NodEdge. The protein product of tumor necrosis factor-α stimulated gene-6 strongly correlated with ossification markers and hyaluronidase in the regions surrounding the nodules (NodSurr, PreNodSurr). In conclusion, this study suggests roles for hyaluronan homeostasis and the promotion of hypoxia by cells demonstrating brown fat markers in calcific aortic valve disease.