Isolation and Characterization of Porcine Endocardial Endothelial Cells

Abstract

The heart contains diverse endothelial cell types. We sought to characterize the endocardial endothelial cells (EECs), which line the chambers of the heart. EECs are relatively understudied, yet their dysregulation can lead to various cardiac pathologies. Due to the lack of commercial availability of these cells, we reported our protocol for isolating EECs from porcine hearts and for establishing an EEC population through cell sorting. In addition, we compared the EEC phenotype and fundamental behaviors to a well-studied endothelial cell line, human umbilical vein endothelial cells (HUVECs). The EECs stained positively for classic phenotypic markers such as CD31, von Willebrand Factor, and vascular endothelial (VE) cadherin. The EECs proliferated more quickly than HUVECs at 48 h (1310 ± 251 cells vs. 597 ± 130 cells, p = 0.0361) and at 96 h (2873 ± 257 cells vs. 1714 ± 342 cells, p = 0.0002). Yet EECs migrated more slowly than HUVECs to cover a scratch wound at 4 h (5% ± 1% wound closure vs. 25% ± 3% wound closure, p < 0.0001), 8 h (15% ± 4% wound closure vs. 51% ± 12% wound closure, p < 0.0001), and 24 h (70% ± 11% wound closure vs. 90% ± 3% wound closure, p < 0.0001). Finally, the EECs maintained their endothelial phenotype by positive expression of CD31 through more than a dozen passages (three populations of EECs showing 97% ± 1% CD31⁺ cells in over 14 passages). In contrast, the HUVECs showed significantly reduced CD31 expression over high passages (80% ± 11% CD31⁺ cells over 14 passages). These important phenotypic differences between EECs and HUVECs highlight the need for researchers to utilize the most relevant cell types when studying or modeling diseases of interest.

Keywords: behavior; cell isolation; endocardial; endothelial cells; phenotype.

FIG. 1.

Brightfield images of HUVECs and EECs. Scale bar = 200 μm. HUVEC, human umbilical vein endothelial cell; EEC, endocardial endothelial cell.

FIG. 2.

Immunofluorescence staining of EECs. In Row A, the EECs were stained for von Willebrand Factor. In Row B, the EECs were stained for VE Cadherin. In Row C, the EECs were stained for CD31. In Row D, the EECs were stained for F-actin. Scale bar in Rows A, C, and D = 50 μm. Scale bar in Row B = 100 μm. Color images are available online.

FIG. 3.

Brightfield images of HUVECs (top row) and EECs (bottom row) during the scratch wound migration assay at 0, 4, and 24 h. The images shown also demonstrate the capability of the ImageJ wound healing size tool plugin, which outlined the edges of the wound created and calculated the size of the wound. Scale bar = 200 μm.

FIG. 4.

Graph comparing the migration rates of HUVECs and EECs at 0, 4, 8, and 24 h. n = 5. ****p < 0.0001. Color images are available online.

FIG. 5.

Graph comparing the proliferation rates of HUVECs at passage 5 and EECs at passage 5 calculated from live cell staining at 0, 24, 48, 72, and 96 h. n = 3. *p between 0.01 and 0.05; ****p < 0.0001. Color images are available online.

FIG. 6.

Flow cytometry results evaluating protein expression of CD31 between three populations of EECs and a population of HUVECs across passages. Expression of CD31 across the samples can be seen at passage 4 (graph on left) and passage 11 (graph on right). Color images are available online.

FIG. 7.

Graph of percentage of cells in each sample that positively expressed CD31 in the flow cytometry analysis from passage 4 through passage 15. Graph compares the HUVEC population to the three populations of EECs. ^#p between 0.0001 and 0.0003 for comparison of HUVECs with each EEC population. Color images are available online.