Department of Medicine and Surgery, University of Parma, Parma, Italy.
Pediatric Surgery, Ospedale dei Bambini of Parma, University Hospital of Parma, Parma, Italy.
Vascul Pharmacol. 2022 Oct;146:107110. doi: 10.1016/j.vph.2022.107110. Epub 2022 Sep 11.
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 β-blockers trigger the autophagic flux on infantile hemangioma-derived endothelial cells (Hem-ECs) in vitro.
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.
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 β-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 β-blocking agents. Importantly, similar biochemical and morphologic evidence of autophagy were observed following rapamycin while Bafilomycin A1 significantly prevented the autophagic flux promoted by β-blockers in Hem-ECs.
Our data suggest that autophagy may be ascribed among the mechanisms of action of β-blockers suggesting new mechanistic insights on the potential therapeutic application of this class of drugs in pathologic conditions involving uncontrolled angiogenesis.
探讨普萘洛尔治疗婴幼儿血管瘤（IH）的作用机制，及其在药物治疗后迅速消退的原因。自噬在血管稳态中起关键作用，本研究旨在确定β受体阻滞剂是否能诱导体外培养的婴幼儿血管内皮细胞（Hem-ECs）发生自噬。
为治疗目的，从 7 名患有增殖性 IH 的儿童中手术切除新鲜组织标本，进行酶消化。使用抗人 CD31 免疫标记的磁性微珠对细胞进行分选。经表型鉴定后，在 P2 至 P6 时，将扩增的 Hem-ECs 暴露于不同浓度（50 μM 至 150 μM）的普萘洛尔、阿替洛尔或美托洛尔单独及与自噬抑制剂巴弗洛霉素 A1 联合处理。雷帕霉素作为自噬的有效诱导剂也被用作对照。通过 Lysotracker Red 染色、LC3BII/LC3BI 和 p62 的 Western blot 分析以及透射电镜观察来评估自噬。
用普萘洛尔、阿替洛尔或美托洛尔处理的 Hem-ECs 显示出阳性的 Lysotracker Red 染色。在β受体阻滞剂处理的 Hem-ECs 中，观察到 LC3BII/LC3BI 比值增加和 p62 调节。体外培养的 Hem-ECs 暴露于β受体阻滞剂后，细胞质超微结构特征表现为大量自噬空泡和多层体。重要的是，雷帕霉素也观察到类似的生化和形态学自噬证据，而巴弗洛霉素 A1 可显著阻止β受体阻滞剂在 Hem-ECs 中诱导的自噬流。
本研究结果表明，自噬可能是β受体阻滞剂作用机制之一，为该类药物在涉及失控性血管生成的病理情况下的潜在治疗应用提供了新的机制见解。
