Propranolol enhanced adipogenesis instead of induction of apoptosis of hemangiomas stem cells

Abstract

Propranolol has been widely used in treating infantile hemangiomas (IHs). But recurrence of IHs was found in some cases on cessation of propranolol treatment. The other is that Chinese individuals reacted to propranolol differently from American Whites. Whether the difference of sensitivity is due to the β adrenoceptor (β-AR) expression pattern of hemangioma initiating cells remains unclear. In the present study, we isolated hemangioma-derived stem cells (hemSCs) from proliferative IHs and analyzed the biological characteristics and β-AR expression pattern of hemSCs by immunostaining, Western blotting and multilineage differentiation assay as well. We also tested the effects of propranolol on hemSCs by evaluating VEGF expression, proliferation and apoptosis related parameters. Our results indicated that CD133(+) hemSCs located pre-vascular in proiferative IH tissues. Both β1 and β2-AR were expressed, while β2-AR was dominant on hemSCs. Propranolol at 100-150 μM inhibited proliferation of hemSCs, not did 50 μM. Propranolol down-regulated VEGF expression of hemSCs, instead of inducing apoptosis. The adipogenic potential was enhanced by propranolol. Therefore, our current results suggested propranolol could not induce apoptosis of hemSCs, but played a curative role though suppressing VEGF synthesis and enhancement of adipogenesis of hemSCs. Our results might partially provide the insight of mechanism of relapse in some cases on cessation of propranolol treatment.

Keywords: Propranolol; hemangioma-stem cells; relapse; β adrenoceptor.

Figure 1

HE staining and immunofluorescence staining of proliferating IHs. (A) Microvessels were enriched in the proliferating IHs. Immunofluorescence staining showed CD133 positive (Green) hemSCs located pre-vascular (B). Part of CD133 positive (Green) hemSCs co-expressed Glut1 (C, red), α-SMA (D, red), VEGF (E, red) and vimentin (F, red). The nuclei were counterstained with DAPI (Blue).

Figure 2

Biological characteristics of hemSCs. A: The morphology of CD133⁺ hemSCs. B-F: The CD133⁺ hemSCs expressed Glut-1, Vimentin, α-SMA and VEGF. G: The growth curve revealed that hemSCs proliferated sluggishly compared with HUVECs. H: Western blot showed hemSCs expressed both β1-AR and β2-AR with a dominance of β2-AR, while HUVECs expressed β1-AR and β2-AR equally. I, J: Adipogenic and osteogenic differentiation potential of hemSCs were confirmed by Oil red O staining and alizarin red S staining.

Figure 3

Proliferation of propranolol-treated HemSCs was analyzed by MTT. The cell proliferation was inhibited by 100-150 μM propranolol from 48 hours to 96 hours (P < 0.05). However, 50 μM propranolol stimulated the proliferation of hemSCs, even there was no statistically difference compared with control (P > 0.05).

Figure 4

Western blotting showing expression of VEGF and apoptosis related proteins in HemSCs. Compared with the control groups, VEGF expression was down-regulated after 96 hours’ propranolol incubation. On the other hand, expression of anti-apoptotic protein (Bcl-2) and pro-apoptotic proteins (caspase 3 and caspase 9) was not significantly up-regulated or down-regulated.

Figure 5

Adipogenic and osteogenic differentiation potential of hemSCs after propranolol treatment. A: On the 5^th day after differentiation, robust lipid vacuoles were observed in the cytoplasm of hemSCs, compared with that of control groups. B: Calcium deposit was reduced after propranolol treatment, compared with that of control groups. C: RT-PCR also indicated the up-regulation of adipogenic genes and down-regulation of osteogenic genes.