CD26 Is Differentially Expressed throughout the Life Cycle of Infantile Hemangiomas and Characterizes the Proliferative Phase.

Infantile hemangiomas (IHs) are benign vascular neoplasms of childhood (prevalence 5-10%) due to the abnormal proliferation of endothelial cells. IHs are characterized by a peculiar natural life cycle enclosing three phases: proliferative (≤12 months), involuting (≥13 months), and involuted (up to 4-7 years). The mechanisms underlying this neoplastic disease still remain uncovered. Twenty-seven IH tissue specimens (15 proliferative and 12 involuting) were subjected to hematoxylin and eosin staining and a panel of diagnostic markers by immunohistochemistry. WT1, nestin, CD133, and CD26 were also analyzed. Moreover, CD31pos/CD26pos proliferative hemangioma-derived endothelial cells (Hem-ECs) were freshly isolated, exposed to vildagliptin (a DPP-IV/CD26 inhibitor), and tested for cell survival and proliferation by MTT assay, FACS analysis, and Western blot assay. All IHs displayed positive CD31, GLUT1, WT1, and nestin immunostaining but were negative for D2-40. Increased endothelial cell proliferation in IH samples was documented by ki67 labeling. All endothelia of proliferative IHs were positive for CD26 (100%), while only 10 expressed CD133 (66.6%). Surprisingly, seven involuting IH samples (58.3%) exhibited coexisting proliferative and involuting aspects in the same hemangiomatous lesion. Importantly, proliferative areas were characterized by CD26 immunolabeling, at variance from involuting sites that were always CD26 negative. Finally, in vitro DPP-IV pharmacological inhibition by vildagliptin significantly reduced Hem-ECs proliferation through the modulation of ki67 and induced cell cycle arrest associated with the upregulation of p21 protein expression. Taken together, our findings suggest that CD26 might represent a reliable biomarker to detect proliferative sites and unveil non-regressive IHs after a 12-month life cycle.

Β-blockers activate autophagy on infantile hemangioma-derived endothelial cells in vitro.

The mechanisms underlying the success of propranolol in the treatment of infantile hemangioma (IH) remain elusive and do not fully explain the rapid regression of hemangiomatous lesions following drug administration. As autophagy is critically implicated in vascular homeostasis, we determined whether ß-blockers trigger the autophagic flux on infantile hemangioma-derived endothelial cells (Hem-ECs) in vitro. MATERIAL AND METHODS: Fresh tissue specimens, surgically removed for therapeutic purpose to seven children affected by proliferative IH, were subjected to enzymatic digestion. Cells were sorted with anti-human CD31 immunolabeled magnetic microbeads. Following phenotypic characterization, expanded Hem-ECs, at P2 to P6, were exposed to different concentrations (50 µM to 150 µM) of propranolol, atenolol or metoprolol alone and in combination with the autophagy inhibitor Bafilomycin A1. Rapamycin, a potent inducer of autophagy, was also used as control. Autophagy was assessed by Lysotracker Red staining, western blot analysis of LC3BII/LC3BI and p62, and morphologically by transmission electron microscopy. RESULTS: Hem-ECs treated with either propranolol, atenolol or metoprolol displayed positive LysoTracker Red staining. Increased LC3BII/LC3BI ratio, as well as p62 modulation, were documented in ß-blockers treated Hem-ECs. Abundant autophagic vacuoles and multilamellar bodies characterized the cytoplasmic ultrastructural features of autophagy in cultured Hem-ECs exposed in vitro to ß-blocking agents. Importantly, similar biochemical and morphologic evidence of autophagy were observed following rapamycin while Bafilomycin A1 significantly prevented the autophagic flux promoted by ß-blockers in Hem-ECs. CONCLUSION: Our data suggest that autophagy may be ascribed among the mechanisms of action of ß-blockers suggesting new mechanistic insights on the potential therapeutic application of this class of drugs in pathologic conditions involving uncontrolled angiogenesis.

Propranolol treatment for infantile hemangioma: a case series of sixty-two patients.

Infantile hemangiomas (IH) complicated by ulceration, disfigurement, functional impairment or life-threatening conditions need early, safe and effective treatment. This study explores the impact of propranolol on complicated IH. We report our experience of 62 patients treated with oral propranolol for complicated IH. The effect of propranolol was assessed using a score on a visual analogue scale integrated with echo, magnetic resonance or endoscopic findings. The average age at the beginning of the treatment was seven months [standard deviation (SD)±8.9], with a median of four months (range 1-53 months). The average age at the end of the treatment was 15 months (SD±8.4), with a median of 13 months (range 7-59 months). The mean treatment length was eight months (SD±3.2). Oral propranolol was successful in 95.2% of the patients in reducing the volume, the intensity of color and the elevation of IH. Statistically significant improvement of IH volume was observed in the first two months of therapy (P≤0.001), and between the second month and the end of the treatment (P<0.05). No significant bradycardia or hypotension occurred. Severe hypoglycemia occurred in one patient. Mild adverse effects were observed in seven patients. Our study demonstrates that propranolol administered orally at 2 to 3 mg/kg/day has a rapid therapeutic effect leading to remarkable shortening of the natural course of IH and it is safe in the majority of patients.