NOTCH3 regulates stem-to–mural cell differentiation in infantile hemangioma
Andrew K. Edwards, Kyle Glithero, Peter Grzesik, Alison A. Kitajewski, Naikhoba C.O. Munabi, Krista Hardy, Qian Kun Tan, Michael Schonning, Thaned Kangsamaksin, Jan K. Kitajewski, Carrie J. Shawber, June K. Wu
Andrew K. Edwards, Kyle Glithero, Peter Grzesik, Alison A. Kitajewski, Naikhoba C.O. Munabi, Krista Hardy, Qian Kun Tan, Michael Schonning, Thaned Kangsamaksin, Jan K. Kitajewski, Carrie J. Shawber, June K. Wu
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Research Article Vascular biology

NOTCH3 regulates stem-to–mural cell differentiation in infantile hemangioma

Abstract

Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to–mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRβ+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRβlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to–mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.

Authors

Andrew K. Edwards, Kyle Glithero, Peter Grzesik, Alison A. Kitajewski, Naikhoba C.O. Munabi, Krista Hardy, Qian Kun Tan, Michael Schonning, Thaned Kangsamaksin, Jan K. Kitajewski, Carrie J. Shawber, June K. Wu

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Figure 4

NOTCH3 is necessary for HemSC mural cell differentiation.

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NOTCH3 is necessary for HemSC mural cell differentiation. (A–C) Assessme...
(A–C) Assessment of NOTCH3 knockdown (N3KD) in HemSCs. (A) Relative NOTCH3 transcript levels in control HemSCs (Scr), and N3KD HemSCs determined by qRT-PCR and normalized by BACTIN. Representative data from 4 independent N3KD HemSC populations and matching Scr HemSC populations are presented. Error bars represent ± SD. *P < 0.007, Student’s t test. (B) Representative data of Western analysis from 2 independent N3KD and matching Scr HemSC lysates probed with antibodies against NOTCH3 or α-tubulin as a loading control. (C) Relative HEYL and HES1 transcript levels in Scr and N3KD HemSCs determined by qRT-PCR and normalized by BACTIN expression. Representative data presented from 3 independent HemSC populations done in duplicate. Error bars represent ± SD. **P < 0.0007, ***P < 0.05, Student’s t test. (D) Scr HemSCs (n = 3) or N3KD HemSCs (n = 3) cocultured with ECFCs in growth media were stained for αSMA at days 0, 5, and 9. Scale bars: 50 μm. (E) Day 3, 5, 7, and 9 lysates from Scr and N3KD HemSC/ECFC cocultures were subjected to Western analysis, and probed with antibodies against αSMA or α-tubulin as a loading control. Ratio of αSMA band intensity normalized by α-tubulin band intensity presented below. Representative data from 3 Scr and N3KD HemSC populations and experiments perform in duplicate for each HemSC population. αSMA, α smooth muscle actin; ECFC, endothelial colony-forming cell; HemSC, hemangioma stem cell; Scr, scrambled.