NOTCH3 Regulates Hemangioma Stem Cell Differentiation and Alters the Vascular Phenotype in an Infantile Hemangioma Murine Model
Kyle J. Glithero, MD, Naikhoba C.O. Munabi, BA, Michelle M. Chang, BS, Jan K. Kitajewski, PhD, Carrie J. Shawber, PhD, June K. Wu, MD.
Columbia University, New York, NY, USA.
Infantile hemangiomas (IHs) are high flow vascular tumors that have been proposed to originate from hemangioma stem cells (HemSCs). Proper vessel maturation requires interactions between endothelial cells and their surrounding mural cells. Previous work by our lab has shown that HemSCs in IH tissues and culture express NOTCH3, suggesting a role for NOTCH3 in IH pathobiology. Previously, we demonstrated that NOTCH3 knockdown in HemSCs resulted in reduced US Doppler detectable blood flow in a murine IH model and this correlated with a reduction in vessel caliber. NOTCH3 is necessary for pericyte-endothelial cell communication and proper vessel development. We hypothesized that NOTCH3 functions in mural and endothelial cell differentiation of HemSCs.
CD133+ HemSCs were isolated from resected hemangioma specimens. HemSC were transduced with lentivirus encoding a NOTCH3 shRNA (HemSC-shN3), or a virus containing a scrambled sequence (HemSC-scr). Successful NOTCH3 knockdown was confirmed by RT-PCR. HemSC-shN3 and HemSC-scr were grown in mural cell differentiation, endothelial cell differentiation, or basal growth media. After two weeks, immunofluorescent staining was performed with antibodies against αSMA (mural cells) or GLUT1/CD31 (endothelial cells). HemSC-shN3 and HemSC-scr suspended in matrigel were injected subcutaneously into immunodeficient mice. After 3 weeks, the matrigel implants were paraffin embedded, and sections stained with antibodies against GLUT1 and αSMA. Staining was quantified over multiple areas using Adobe Photoshop.
During in vitro endothelial cells differentiation, HemSCs, with NOTCH3 knockdown had increased CD31 (p < 0.05), and decreased GLUT1 expression (p < 0.001) relative to control HemSC (Figure 1). In mural cell differentiation media, HemSCs with NOTCH3 knockdown failed to induce αSMA expression relative to control (p < 0.01; Figure 2). In the IH murine model, a decrease in GLUT1+ vessels was observed in Notch3 knockdown HemSC implants (p < 0.02, Figure 3). However, αSMA+ cell coverage of vessels was unchanged between NOTCH3 knockdowns and control.
In HemSCs, knockdown of NOTCH3 altered mural and endothelial cell differentiation in vitro and changed the vessel phenotype in a murine model of IH in vivo. These changes correlated with normalization of capillaries. Thus, NOTCH3 inhibition may disrupt abnormal vasculature development in IH and is a potential therapeutic target.
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