Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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NOTCH3 Regulates Mural Cell Differentiation Of HemSCs
Kyle J. Glithero, MD, Naikhoba C.O. Munabi, BA, Ryan W. England, BS, Alex Kitajewski, BS, Michelle Chang, BS, Jan K. Kitajewski, PhD, Carrie J. Shawber, PhD, June K. Wu, MD.
Columbia University Medical Center, New York, NY, USA.

PURPOSE: Infantile hemangiomas (IHs) are vascular hyperplasias with high flow and have been proposed to originate from hemangioma stem cells (HemSCs). Proper vessel maturation requires interactions between endothelial cells and their surrounding mural cells. IH pathology has been proposed to be partially due to defective mural cell function. Previous studies in our lab showed NOTCH3 inhibition in HemSC resulted in reduced blood flow in a murine IH model as detected by US Doppler. This correlated with reduced vessel diameter when compared to controls. Since Notch3 functions to regulate mural cell maturation, we determined if Notch3 has a role in mural cell differentiation of HemSCs.
METHODS: CD133+ HemSCs were isolated from resected hemangioma specimens. HemSC were transduced with lentivirus encoding a NOTCH3 shRNA (HemSC- shN3), an activated form of NOTCH3 (HemSC-N3IC) or a virus containing a scrambled sequence (HemSC-scr) serving as a control. HemSC-N3IC, HemSC-shN3 and HemSC-scr were grown in mural cell differentiation media. After two weeks, immunofluorescent staining was performed with antibodies against the mural cell markers, neuron-glial antigen 2 (NG2) and alpha smooth muscle actin (α-SMA). Fluorescence was quantified and averaged over multiple areas with ImageJ.
RESULTS: All CD133+ HemSC cell lines expressed low levels of NG2 at baseline (data not shown). When mural cell differentiation was induced, NOTCH3 knockdown upregulated NG2 compared to HemSC-scr (Figure 1). In contrast, NOTCH3 activation decreased NG2 expression. Expression of α-SMA was strongly upregulated in HemSC-scr and Hem-N3IC to a similar level in differentiation medium compared to cell maintained in growth medium (basal) (Figure 2). α-SMA expression was unchanged from basal conditions in HemSC-shN3 cells.
CONCLUSIONS: In HemSCs, perturbations in NOTCH3 level differential altered the expression of mural cell proteins NG2 and α-SMA. In mural cell differentiation conditions NOTCH3 activation strongly downregulated NG2, while having no effect on the expression of α-SMA. In contrast, NOTCH3 inhibition modestly increased NG2 levels, and suppressed the upregulation of α-SMA. Thus, precise regulation of NOTCH3 signaling levels maybe necessary for proper mural cell differentiation of HemSCs.


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