Date of Award

2009

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Deepak Narayan

Second Advisor

John Persing

Third Advisor

Murat Gunel

Abstract

Introduction: Hemangiomas of infancy (HOI) are the most common benign tumor of childhood. Initially thought to be composed entirely of endothelial cells, it has recently been shown that mesenchymal stem cells reside within these tumors. We propose that hemangiomas represent mesenchymal stem cell tumors of pericyte origin, as demonstrated by expression of pericytic markers (NG2, PDGFR-β, and DLK), neural crest origin (expression of nestin and sox10), expression of factors that play a role in the maintenance of stem cell pluripotency (Oct4, Sox2, Nanog, C-myc, and piRNAs), and a microRNA expression profile suggestive of mesenchymal stem cells. Methods: Quantitative RT-PCR was performed on 19 hemangioma specimens (4 proliferating, 10 plateau, 5 involuting) analyzing transcription factors (Oct4, Sox2, C-myc, and Nanog) known to regulate stem cell pluripotency. Transcription factors RB, IGF2, CTCF, BORIS, DLK, and CDX-2 were also examined. PiRNA analysis was performed on 7 hemangioma specimens to investigate the role of these small RNA transcripts that interact with Piwi proteins expressed in the germline and stem cells. MicroRNA microarray analysis was performed on 9 hemangioma specimens. MicroRNA pathway analysis was performed using Ingenuity Pathway Analysis software and MetaCore Software. Freshly resected hemangioma specimens were cultured in embryonic stem cell media with and without recombinant human basic fibroblast growth factor (rhbFGF), recombinant human transforming growth factor β (rhTGF-β), and 17-β estradiol. Results: All hemangiomas expressed factors RB, Oct-4, Sox-2, Nanog, C-myc, DLK, IGF-2, and CTCF at higher levels than endothelial cell controls. DLK, a gene that functions as a negative regulator of adipocyte differentiation, is increased in hemangiomas more than 105 orders of magnitude compared to control endothelial cells. MiRNA-195, which is known to target DLK, is also upregulated in hemangiomas. PiRNA analysis revealed that hemangiomas do not contain piRNA transcripts. A microRNA microarray analysis using adult and neonatal dermal endothelial cells as controls indicated that microRNAs associated with mesenchymal stem cells (miR-143, miR320a, miR320c, let-7c) were expressed in 9 samples studied. Hemangioma growth in culture was not observed in embryonic stem cell media with or without supplementation with bFGF and TGF-β. Growth was observed in standard culture media with 17-β estradiol supplementation. Conclusion: Hemangioma specimens in all stages of growth express transcription factors and genes known to play a role in the maintenance of stem cell pluripotency at a level greater than control endothelial cells. Specifically, transcription factors Oct-4, Sox-2, Nanog, and C-myc are increased. Furthermore miR-143, miR320a, miR320c, and let-7c (all microRNAs identified in mesenchymal stem cells) are upregulated in hemangioma tissue in all stages of growth. DLK, a gene that functions as a negative regulator of adipocyte differentiation, is increased in hemangiomas compared to controls. The downregulation of this gene may lead to transformation of mesenchymal stem cells into adipose tissue. Future experiments are necessary to confirm this role.

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