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NOTCH3 KNOCKDOWN IN HEMANGIOMA STEM CELLS ALTERS HEMANGIOMA FORMATION IN A MURINE MODEL
Presenter: Alvin Wong
Co-Authors: Thirumoorthi A; Andrews J; Hardy K; England R; Shawber CJ; Kitajewski J; Wu JK
Columbia University

Background: Infantile hemangiomas (IHs) are thought to originate from hemangioma stem cells (HemSC), though IH pathogenesis is poorly understood. Our recent work showed that NOTCH3 is highly expressed in HemSCs and may play a role in IH progression. In cultured HemSCs, NOTCH3 knockdown perturbed adipogenesis and enhanced endothelial cell differentiation. Using a murine model of IH, we compared the development of IHs from control HemSCs (HemSC-scr) to HemSCs with Notch3 shRNA (HemSC-shN3).

Materials and Methods: HemSCs were isolated from resected IH specimens using CD133+ magnetic bead selection. HemSCs were infected by lentiviruses containing either a control scrambled sequence (scr), or shRNA for NOTCH3 (shN3). NOTCH3 knockdown was assessed with qRT-PCR for NOTCH3 transcripts. Both HemSC-scr and HemSC-shN3 were re-suspended in matrigel and implanted subcutaneously into immunodeficient mice. IH implants were followed by weekly Doppler USG studies. The mice were sacrificed at weeks 2 and 3, and implants were removed for histological and immunohistochemical analyses.

Results: NOTCH3 knockdown in HemSC-shN3 was confirmed to be 50% compared to the control by qRT-PCR. In the HemSC-scr control, Doppler USG showed blood flow within the matrigel by week 2. By contrast, flow was not observed in the HemSC-sh N3 mouse (Fig 1). Both control and shN3 showed development of CD31+/Glut1+ vessels and adipocytes. Adipocyte size was decreased in HemSC-shN3 compared to the control (p < 0.05) and the vasculature was ectatic (Fig 2).

Conclusion: In the IH murine model, NOTCH3 knockdown resulted in dilated vessels, though flow was undetectable by Doppler. This suggests that reduced expression of NOTCH3 led to formation of larger vessel lumens with slower flow. NOTCH3 knockdown also led to decreased adipocyte size in the IH implants. These results were consistent with our in vitro findings of perturbed adipogenesis and enhanced endothelial cell differentiation. By using a murine IH model, we have created a method to track IH development in real time using sonic imaging, which can be used to monitor the effects of potential therapeutics.


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