Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai Institute of Cardiovascular Diseases, Shanghai, China.
Institute of Biomedical Sciences, Fudan University, Shanghai, China.
Clin Transl Med. 2021 May;11(5):e402. doi: 10.1002/ctm2.402.
Post-ischemic angiogenesis is critical for blood flow recovery and ischemic tissue repair. N6-methyladenosine (m6A) plays essential roles in numerous biological processes. However, the impact and connected mechanism of m6A on post-ischemic angiogenesis are not fully understood.
AlkB homolog 5 (ALKBH5) was screened out among several methyltransferases and demethylases involved in dynamic m6A regulation. Cardiac microvascular endothelial cells (CMECs) angiogenesis and WNT family member 5A (WNT5A) stability were analyzed upon ALKBH5 overexpression with adenovirus or knockdown with small interfering RNAs in vitro. The blood flow recovery, capillary, and small artery densities were evaluated in adeno-associated virus (AAV)-ALKBH5 overexpression or ALKBH5 knockout (KO) mice in a hind-limb ischemia model. The same experiments were conducted to explore the translational value of transient silencing of ALKBH5 with adenovirus.
ALKBH5 was significantly upregulated in hypoxic CMECs and led to a global decrease of m6A level. ALKBH5 overexpression further reduced m6A level in normoxic and hypoxic CMECs, impaired proliferation, migration, and tube formation only in hypoxic CMECs. Conversely, ALKBH5 knockdown preserved m6A levels and promoted angiogenic phenotypes in hypoxic but not in normoxic CMECs. Mechanistically, ALKBH5 regulated WNT5A expression through post-transcriptional mRNA modulation in an m6A-dependent manner, which decreased its stability and subsequently impeded angiogenesis in hypoxic CMECs. Furthermore, ALKBH5 overexpression hindered blood flow recovery and reduced CD31 and alpha-smooth muscle actin expression in hind-limb ischemia mice. As expected, ALKBH5-KO mice exhibited improved blood flow recovery, increased capillary, and small artery densities after hind-limb ischemia, and similar beneficial effects were observed in mice with transient adenoviral ALKBH5 gene silencing.
We demonstrate that ALKBH5 is a negative regulator of post-ischemic angiogenesis via post-transcriptional modulation and destabilization of WNT5A mRNA in an m6A-dependent manner. Targeting ALKBH5 may be a potential therapeutic option for ischemic diseases, including peripheral artery disease.
缺血后血管生成对于血流恢复和缺血组织修复至关重要。N6-甲基腺苷（m6A）在许多生物过程中发挥着重要作用。然而，m6A 对缺血后血管生成的影响及其相关机制尚不完全清楚。
在涉及动态 m6A 调节的几种甲基转移酶和去甲基酶中筛选出 AlkB 同源物 5（ALKBH5）。在体外通过腺病毒过表达或小干扰 RNA 敲低分析 ALKBH5 对心脏微血管内皮细胞（CMECs）血管生成和 WNT 家族成员 5A（WNT5A）稳定性的影响。在腺相关病毒（AAV）-ALKBH5 过表达或 ALKBH5 敲除（KO）小鼠的后肢缺血模型中评估血流恢复、毛细血管和小动脉密度。还进行了相同的实验，以探索腺病毒瞬时沉默 ALKBH5 的转化价值。
ALKBH5 在缺氧 CMECs 中显著上调，并导致整体 m6A 水平降低。ALKBH5 过表达进一步降低了常氧和缺氧 CMECs 中的 m6A 水平，仅在缺氧 CMECs 中损害增殖、迁移和管形成。相反，ALKBH5 敲低在缺氧但不在常氧 CMECs 中保留 m6A 水平并促进血管生成表型。机制上，ALKBH5 通过依赖于 m6A 的转录后 mRNA 调节来调节 WNT5A 的表达，从而降低其稳定性并随后抑制缺氧 CMECs 的血管生成。此外，ALKBH5 过表达阻碍了后肢缺血小鼠的血流恢复并降低了 CD31 和 alpha-平滑肌肌动蛋白的表达。正如预期的那样，ALKBH5-KO 小鼠在后肢缺血后表现出更好的血流恢复，增加了毛细血管和小动脉密度，并且在短暂腺病毒 ALKBH5 基因沉默的小鼠中观察到类似的有益效果。
我们证明 ALKBH5 是一种负调节因子，通过依赖于 m6A 的方式对 WNT5A mRNA 进行转录后调节和不稳定化，从而促进缺血后血管生成。靶向 ALKBH5 可能是包括外周动脉疾病在内的缺血性疾病的一种潜在治疗选择。
