Proliferating Infantile Hemangioma Tissues and Primary Cell Lines Express Markers Associated with Endothelial-to-Mesenchymal Transition

Abstract

Background: We have previously shown that the endothelium of the microvessels of infantile hemangioma (IH) exhibits a hemogenic endothelium phenotype and proposed its potential to give rise to mesenchymal stem cells, similar to the development of hematopoietic cells. This endothelial-to-mesenchymal transition (Endo-MT) process involves the acquisition of a migratory phenotype by the endothelial cells, similar to epithelial-to-mesenchymal transition that occurs during neural crest development. We hypothesized that proliferating IH expresses Endo-MT-associated proteins and investigated their expression at the mRNA, protein, and functional levels.

Methods: Immunohistochemical staining of paraffin-embedded sections of proliferating IH samples from 10 patients was undertaken to investigate the expression of the Endo-MT proteins Twist1, Twist2, Snail1, and Slug. Transcriptional analysis was performed for the same markers on proliferating IH tissues and CD34⁺ and CD34^- cells from proliferating IH-derived primary cell lines. Adipogenic and osteogenic differentiation plasticity was determined on the CD34-sorted fractions.

Results: The endothelium of the microvessels and the cells within the interstitium of proliferating IH tissues expressed Twist1, Twist2, and Slug proteins. Twist1 was also expressed on the pericyte layer of the microvessels, whereas Snail1 was not expressed. Both CD34⁺ and CD34^- populations from the IH-derived primary cell lines underwent adipogenic and osteogenic differentiation.

Conclusions: The expression of Endo-MT-associated proteins Twist1, Twist2, and Slug by both the endothelium of the microvessels and cells within the interstitium, and Twist1 on the pericyte layer of the microvessels of proliferating IH, suggest the presence of a process similar to Endo-MT. This may enable a tightly controlled primitive endothelium of proliferating IH to acquire a migratory mesenchymal phenotype with the ability to migrate away, providing a plausible explanation for the development of a fibrofatty residuum observed during involution of IH.

Fig. 1.

Representative 3,3´-diaminobenzidine immunohistochemical staining on proliferating infantile hemangioma tissue samples demonstrating the expression of Twist1 (A, brown), Twist2 (B, brown), and Slug (C, brown) but not Snail1 (D, brown), on the endothelium of the microvessels (thin arrows) and the cells within the interstitium (arrowheads). Twist1 was also expressed on the pericyte layer (thick arrows) of the microvessels. Nuclei were counterstained with hematoxylin (blue). Original magnification: ×400.

Fig. 2.

Representative immunofluorescence immunohistochemical staining on proliferating infantile hemangioma tissue samples demonstrating the expression of Twist1 (A, red) localized to the CD34⁺ (green) endothelium (thin arrows), the concentric pericyte layer (thick arrows), and the cells within the interstitium (arrowheads). Twist2 (B, green) was localized to the vWF⁺ (red) endothelium (thin arrows) and cells within the interstitium (arrowheads). Slug (C, red) was localized to the CD34⁺ (green) endothelium (thin arrows) and cells within the interstitium (arrowheads). Snail1 (D, green) was localized to few cells within the interstitium that also expressed Slug (red) which was widely expressed by the endothelium (thin arrows) and other cells within the interstitium (arrowheads). All slides were counterstained with 4´, 6´-diamino-2-phenylindole (blue). Original magnification: ×400.

Fig. 3.

NanoString mRNA analysis confirming transcript expression of Twist1, Twist2, Snail1, and Slug in proliferating infantile hemangioma (IH) tissue samples, and both the CD34⁺ and CD34⁻ proliferating IH-derived primary cell lines, relative to the housekeeping gene GusB. Levels of statistical significance are shown above the graphs (*P < 0.05; **P < 0.01).

Fig. 4.

Mesenchymal differentiation assays of proliferating infantile hemangioma–derived primary cell lines demonstrating lipid droplets stained with Oil Red O in CD34⁺ (A, red, arrows) and CD34⁻ (B, red, arrows) cellular fractions for adipogenesis relative to the 3T3 positive control (C, red, arrows), and calcium deposits stained with Alizarin Red in CD34⁺ (D, red, arrows) and CD34⁻ (E, red, arrows) cellular fractions for osteogenesis relative to the 3T3 positive control (F, red, arrows). Original magnification: ×400.