Is Infantile Hemangioma a Neuroendocrine Tumor?

Abstract

Infantile hemangioma (IH) is the most common infantile tumor, affecting 5-10% of newborns. Propranolol, a nonselective β-adrenergic receptor (ADRB) antagonist, is currently the first-line treatment for severe IH; however, both its mechanism of action and its main cellular target remain poorly understood. Since betablockers can antagonize the effect of natural ADRB agonists, we postulated that the catecholamine produced in situ in IH may have a role in the propranolol response. By quantifying catecholamines in the IH tissues, we found a higher amount of noradrenaline (NA) in untreated proliferative IHs than in involuted IHs or propranolol-treated IHs. We further found that the first three enzymes of the catecholamine biosynthesis pathway are expressed by IH cells and that their levels are reduced in propranolol-treated tumors. To study the role of NA in the pathophysiology of IH and its response to propranolol, we performed an in vitro angiogenesis assay in which IH-derived endothelial cells, pericytes and/or telocytes were incorporated. The results showed that the total tube formation is sensitive to propranolol only when exogenous NA is added in the three-cell model. We conclude that the IH's sensitivity to propranolol depends on crosstalk between the endothelial cells, pericytes and telocytes in the context of a high local amount of local NA.

Keywords: adrenergic; betablocker; catecholamines; hemangioma; propranolol; telocytes.

Figure 1

Propranolol treatment reduces noradrenaline level in proliferative IHs. (A) Characterization of IHs compared to congenital hemangiomas by GLUT-1 immunofluorescence staining. Red blood cell (white arrows) staining is used as positive control. Endothelial cells in IHs express GLUT-1 but not in congenital hemangiomas. (B) Characterization of IH with AQP1 staining. Telocytes in IH tissue express AQP1 compared to congenital hemangiomas where it is expressed in EC. (C) Characterization of proliferative and involutive IHs by Ki67 immunofluorescence staining. Skin epidermis (left panel) is used as positive control. Numbers of Ki67+ cells are significantly higher in the proliferative IH than in the involutive IH. Scale bar 100 µm. (D) Levels of NA, dopamine and adrenaline measured in IH tissues by ELISA. (E) NA levels in the untreated (NT) proliferative or involutive IH as well as in the propranolol-treated (Treated) IH were measured by ELISA. Data are presented as mean ± SEM. * p < 0.05, *** p < 0.01, **** p < 0.0001. NA: noradrenaline, IH: infantile hemangioma.

Figure 2

Enzymes implicated in catecholamine biosynthesis pathway are expressed in IH tissues and their expressions are decreased by propranolol treatment. (A) Catecholamine biosynthesis pathway is shown. (B) All catecholamine biosynthesis enzymes except PNMT are expressed in IH tissue, enabling them to synthesize dopamine and NA. Expression levels of these enzymes are reduced with propranolol treatment. The adrenal gland is used as a positive control. Expression levels of catecholamine biosynthesis enzymes in untreated patients are different from treated patients. Two representative pictures of 5 untreated patients and 5 propranolol-treated patients are shown. Scale bar size is 100 µm. NA: noradrenaline, IH: infantile hemangioma. (C) Immunostained sections were scored considering the intensity of TH, DDC and DBH in a blinded manner for 5 untreated patients and 5 propranolol-treated patients. Data are represented as mean ± SEM, * p < 0.05, **** p < 0.0001.

Figure 3

Catecholamine biosynthesis enzymes are expressed in IH-EC, -PER and -TC. (A) Immunofluorescence staining of TH, DDC and DBH in IHs. Endothelial cells are characterized by CD34-positive staining, pericytes by α-SMA and telocytes by AQP1 antibodies. Nucleus is stained with DAPI. The white scale bar size is 50 µm, and the yellow scale bar is 10 µm. (B) TH, DDC and DBH expression levels in the different cell types isolated from IHs were evaluated by Western blot. The adrenal gland (AG) is used as a positive control. GAPDH or β-actin are used as loading controls. IH: infantile hemangioma.

Figure 4

A threshold level of NA and a crosstalk between EC, PER and TC are the prerequisite for detecting the antiangiogenic effect of propranolol on our in vitro Matrigel tube formation assay. (A) IH-EC, IH-PER and IH-TC were seeded separately into Matrigel-coated wells. The formations of tube-like structures were imaged 3 h later and quantified using Wimasis image analysis system. (B) Schematic showing the in vitro tube formation assay using two or three different cell types. Each cell type is labeled with a distinct fluorescent probe. Here, EC were labeled in red, PER in blue and TC in green. Tube formations were then surveyed and imaged. The images were finally analyzed with Wimasis image analysis system. (C) Left panel is a representative image illustrating tube formation by IH-EC and IH-PER 3 h after their seeding into Matrigel-coated wells containing propranolol and/or NA. Quantification of total formed tubes is shown in right panel. (D) Left panel is a representative image illustrating tube formation by IH-EC, IH-PER and IH-TC (1:1:1 ratio) at 3 h after their seeding into wells. Quantification of total tubes (right panel) showed that NA promotes total tube numbers and this promotion is abolished if propranolol is added. Data are represented as mean ± SEM, ns: non-significant, * p < 0.05, ** p < 0.01, **** p < 0.0001. IH: infantile hemangioma, EC: endothelial cells, PER: pericytes, TC: telocytes, NA: noradrenaline.

Figure 5

Proliferative IH tissues present some features of neuroendocrine tumors. (A) Immunostaining for Chromogranin A (CgA) counterstained with hemalun. CgA-positive cells appear in brown. Scale bar size is 100µm. While high expression levels of CgA are detected in untreated proliferative IHs, low levels are detected in involutive IHs and other vascular tumors, including rapidly involutive congenital hemangiomas (RICH) and pyogenic granulomas. The adrenal gland used as a positive control. (B,C) Model outlining IH responses to propranolol. (B) Electron microscopy of IH vessel showing endothelial cells surrounded by pericytes and telopodes of telocytes in perivascular space. Scale bar size is 5 µm. (C) IH-EC, -PER and -TC express the first three enzymes involved in catecholamine biosynthesis pathway, enabling them to release NA locally. Our previous data [17,27] demonstrated that beta-adrenergic receptors are present on IH-EC, -PER, -TC and mast cells. It is likely that NA-mediated activation of ADRB in an autocrine and/or paracrine manner results in activation of proangiogenic signaling pathways in different cell types, leading to dramatic growth and development of blood vessels. Propranolol antagonizes a proangiogenic NA effect through binding to ADRB receptors. NA: noradrenaline, IH: infantile hemangioma.