Human primary lung endothelial cells in culture - PubMed (original) (raw)

Human primary lung endothelial cells in culture

Suzy A A Comhair et al. Am J Respir Cell Mol Biol. 2012 Jun.

Abstract

Pulmonary endothelial functions are critical to maintain the low pressure of the pulmonary circulation and effective diffusion capacity of the lung. To investigate pulmonary endothelial cell biology in healthy or diseased lungs, we developed methods to harvest and culture pure populations of primary pulmonary arterial endothelial cells and microvascular endothelial cells from human lung explanted at time of transplantation or from donor lungs not used in transplantation. The purity and characteristics of cultured endothelial cells is ascertained by morphologic criteria using phase contrast and electron microscopy; phenotypic expression profile for endothelial specific proteins such as endothelial nitric oxide synthase, platelet/endothelial cell adhesion molecule, and von Willbrand factor; and endothelial function assays such as Dil-acetylated low-density lipoprotein uptake and tube formation. This detailed method provides researchers with the ability to establish cells for molecular, genetic, and biochemical investigation of human pulmonary vascular diseases.

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Figures

Figure 1.

Figure 1.

Morphology of endothelial cells and smooth muscle cells. Phase-contrast microscopic analysis of primary pulmonary arterial endothelial cells shows a rosette cobblestone morphology (arrow) (A). Confluent pulmonary arterial endothelial cells (B) and microvascular endothelial cells (C) at passage 5 demonstrating the characteristic cobblestone appearance of endothelial cells. Phase-contrast microscopic of primary pulmonary arterial smooth muscle cells (D). Endothelial cells and smooth muscle cells shown in this figure were initiated from pulmonary arterial hypertension (PAH) lungs. There were no morphological differences between endothelial cells or smooth muscle cells derived from PAH or control lungs.

Figure 2.

Figure 2.

Ultrastructure of control and PAH pulmonary arterial endothelial cells and PAH lung tissue. Electromicroscopy of culture control (A, D) and PAH pulmonary arterial endothelial cells (B, E) and PAH lung tissue (C, F). Ultrastructure detail of endothelial cells in vitro (A, B, D, E) and in vivo (C, F) (scale bar: 1 μm). Weibel-Palade bodies in cultured endothelial cell (E) and endothelial cell of lung tissue (F) (arrows) (scale bar: 250 nm). M = mitochondria; N = nucleus; RBC = red blood cells.

Figure 3.

Figure 3.

Phenotype of primary pulmonary arterial endothelial cells. Characterization of pulmonary arterial cells ascertained by morphologic and immunohistochemistry analyses of endothelial markers (CD31, von Willebrand factor). (A) Immunofluorescence for von Willebrand factor (green) and (B) CD31 (brown) by immunohistochemistry. Representative image is from PAH cells. Hence, control pulmonary arterial endothelial cells have the same phenotype.

Figure 4.

Figure 4.

Endothelial cell types in vessels and plexiform lesions in PAH. Sequential sections for hematoxylin and eosin (H&E), Helix pomatia (red), Griffonia simplicifolia (green), and vWF (purple). H. pomatia binds to endothelium of muscular artery, whereas G. simplicifolia binds to endothelium of microvessels. Red blood cells also bind G. simplicifolia. White arrows and black arrows indicate capillaries. M = smooth muscle; V = vascular lumen of large muscular artery. Scale bar: 100 μm.

Figure 5.

Figure 5.

Lectin binding of microvascular endothelial cells (MVECs) and pulmonary artery endothelial cells (PAECs) in vitro. (A_–_D) Immunofluorescent H. pomatia (red) binds PAECs in vitro (A) but not MVECs (C). G. simplicifolia (green) binds MVECs in culture (D) but not to PAECs (B). Representative image is from PAH cells. Hence, control PAECs have the same phenotype.

Figure 6.

Figure 6.

Endothelial nitric oxide synthase (eNOS) expression in PAECs and MVECs. (A, B) Immunohistochemistry shows strong eNOS staining in the muscular vessels where the staining in alveolar capillaries (arrows) is absent. The plexiform lesion (A) is lined with endothelial cells that are mostly positive for eNOS, but some cells are negative. (C, D) Sequential section confirms endothelial cells by CD31 and in plexiform lesions. (E) Western blot analysis shows that control and PAH PAECs express eNOS, whereas MVECs have undetectable or weak expression of eNOS. C = capillaries; M = smooth muscle; V = vessel.

Figure 7.

Figure 7.

Flow cytometric analysis of endothelial cell surface antigen expression on PAECs. Confluent cell cultures were harvested by trypsinization and stained for cell surface expression of endothelial cell specific markers CD31 and vascular endothelial growth factor receptor 2. (A) Representative profiles for each staining is shown. Gray histograms indicate staining with isotype-matched control antibodies. (B) Purity of primary PAECs, as measured by %CD31-positive cells, increases during passaging of the cells. Cells were more than 95% CD31 positive by passage 4. Passage 1, n = 7; passage 2, n = 14; passage 3, n = 15; passage 4, n = 13; passage 5, n = 31. (C) The smooth muscle cells cultured from pulmonary arteries stained positive for smooth muscle cell α-actin and negative for CD31. Representative profiles for each staining is shown. Gray histograms indicate staining with isotype-matched control antibodies. Representative image is from PAH cells. Control pulmonary arterial endothelial cells have the same phenotype.

Figure 8.

Figure 8.

Gene expression analysis. Hierarchical clustering of gene expression array data from PAECs (n = 14), MVECs (n = 5), and pulmonary artery smooth muscle cells (PASMCs) (n = 4) demonstrates that PAECs and MVECs have closely related gene expression signatures but are distinctly different from PASMCs. Color coding denotes relative gene expression on a continuous scale from blue (lowest expression), through yellow (similar expression levels) to red (highest).

Figure 9.

Figure 9.

Functional assays. (A) Accumulation of acetylated low-density lipoprotein (Dil-Ac-LDL) by primary PAECs (passage 5). (B) Capillary-like tubule formation produced by primary PAECs 8 hours after plating onto Matrigel. Representative image is from control cells. Hence, PAH PAECs have the same phenotype.

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