Abstract 91: Hemangioma Endothelium Loses VE-CADHERIN After Involution: Implications For Cell Death?

Abstract

Purpose: Infantile hemangiomas (IHs) are the most common benign endothelial cell (EC) tumor of infancy, arising in the first days of life. IHs undergo a predictable natural history, beginning with a rapid proliferative phase, followed by a period of stabilization, and finally involution. Hemangioma endothelial cells (HemECs) are distinct from normal ECs: HemECs express Glut1 and are hyperproliferative. The mechanisms governing the progress from proliferation, to stabilization, and finally to involution is not understood. Our laboratory found, on a preliminary screen, that involuting IHs endothelium had loss of VECADHERIN (VECAD) expression. VECAD is a cell adhesion protein specific to ECs. VECAD plays a key role in angiogenesis and cell cycle progression, as well as EC survival. We thus hypothesize that loss of VECAD in IH endothelial cells contributed to cell death that may contribute to IH involution. Methods: IHs from different stages (proliferating, stable, and involuting) were used in this study. Resected IH samples were clinically evaluated and categorized as proliferating (n=2), stable (n=5), or involuting (n=3) based on age and histological appearance. All samples were from patients who did not receive propranolol or other systemic treatment. Dermal vasculature in neonatal dermal tissues were used as controls (n=4). All specimens were embedded in paraffin and sectioned. Immunofluorescent staining was done using antibodies against VECAD and EC marker, CD31. Apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The mean fluorescent intensity for endothelial VECAD and CD31 were quantified using ImageJ software (NIH) over 18 regions of interests per IH or control sample. Data is presented as a ratio of VECAD/CD31 mean intensity. Statistical analysis with one-way ANOVA and post-hoc t-test was performed with Graphpad and a p-value of <0.05 was considered statistically significant. Results: Proliferating IHs had high cell density with poorly defined vascular spaces; stable IHs had well defined, tightly packed vascular channels, and involuting samples had large vascular channels that were interspersed with fatty infiltrates. Proliferating HemECs had weak CD31 and weak VECAD staining when compared to stable IHs; but relative VECAD/CD31expression ratios were similar to stable IH and control vessels. VECAD expression was significantly descreased in involuting HemECs with the lowest VECAD/CD31 ratio, even as CD31 intensity was comparable to stable IHs. As expected, the number of TUNEL+ ECs were significantly higher in involuting IHs when compared to proliferating or stable IHs (p<0.0001). Conclusion: Loss of VECAD expression in IH endothelium corresponded to IH involution and increased apoptosis in IH endothelium. It is unclear whether loss of VECAD is causative of IH involution; further studies are needed to elucidate the role of VECAD function in EC survival.