Identifying Behavioral Phenotypes and Heterogeneity in Heart Valve Surface Endothelium

Abstract

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.

Maturation of heart valve cell populations during postnatal remodeling

“…The VEC subset, which is characterized by classic endothelial markers, seems to correspond to endothelial cells described on the aortic side of porcine aortic valves described with higher Vwf and Selp expression, but we show that these cells can be found on both sides of the valve leaflets. Lymph-VECs are exclusively located away from pulsatile blood flow in a similar proportion to that reported in adult porcine aortic valves (Blancas et al, 2016). The presence of VICs with a lymphatic gene expression signature in developing valves has not been previously reported, and the specific functions of these cells in valve remodeling and homeostasis is unknown.…”

Pathogenic lymphangiogenesis promotes autoimmune valvular carditis

“…We also identified lymphatic vessels in human RHD valves. This work substantiates previous studies reporting individual instances of lymphatic vessels in canine, pig, and human mitral valves in a variety of disease states [37][38][39][40][41][42][43] . Similar to these studies, the lymphatic vessels localized closer to the atrial surface of the valve in both health and disease.…”

Proteomic profiling of concurrently isolated primary microvascular endothelial cells, pericytes, and vascular smooth muscle cells from adult mouse heart

“…While our isolation protocol focuses on microvessels, it does not distinguish between arterioles, capillaries, and venules. For example, Lyve-1 (lymphatic vessel endothelial hyaluronan receptor-1) is predominantly associated with expression in the lymphatic endothelium; however, it does have a broader distribution; it has been reported in ECs of the lung, spleen, liver and the heart 67 , including the heart valve 68 , and has been suggested to be involved in uptake and degradation of waste products, including hyaluronan, from the blood and lymph 67 , especially during development 69 . The concern related to the mixed nature of our cell populations can be addressed using single-cell analysis methods, such as scRNAseq or single-cell mass cytometry (scCyTOF), which allows separation of cells from different vascular segment.…”