Metformin alleviates hyperglycemia-induced endothelial impairment by downregulating autophagy via the Hedgehog pathway
Metformin alleviates hyperglycemia-induced endothelial impairment by downregulating autophagy via the Hedgehog pathway
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二甲双胍通过 Hedgehog 通路下调自噬来减轻高血糖引起的内皮损伤
DOI：
10.1080/15548627.2019.1569913
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发表时间：
2019-05-04
期刊：
影响因子：
13.3
通讯作者：
Li, Xiaokun
中科院分区：
文献类型：
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作者：
Niu, Chao;Chen, Zhiwei;Li, Xiaokun
摘要 关于糖尿病条件下内皮细胞 (EC) 巨自噬/自噬调节的研究非常有限。临床证据证实二甲双胍具有内皮保护作用，但其潜在机制仍不清楚。我们的目的是研究二甲双胍是否通过自噬机制发挥其对高血糖诱导的内皮损伤的保护作用。 db/db小鼠接受玻璃体内注射二甲双胍治疗。将人脐静脉内皮细胞 (HUVEC) 在正常葡萄糖 (NG, 5.5 mM) 或高葡萄糖 (HG, 33 mM) 培养基中在存在或不存在二甲双胍的情况下培养 72 小时。我们观察到二甲双胍对糖尿病视网膜血管和培养的 HUVEC 中高血糖触发的自噬体合成有明显的抑制作用，同时恢复了高血糖受损的 Hedgehog (Hh) 通路活性。具体而言，db/db 小鼠视网膜血管 EC 和培养的 HUVEC 中 ATG7 的缺失表明自噬在高血糖诱导的内皮功能障碍中具有有害作用。用 Hh 通路抑制剂 GANT61 进行预处理，消除了二甲双胍介导的自噬和内皮保护作用的下调。此外，HUVEC 和视网膜脉管系统中的 GLI 家族（Hh 通路的转录因子）敲低表明，二甲双胍介导的 Hh 通路激活对高血糖激活的自噬的下调是 GLI1 依赖性的。从机制上讲，GLI1 敲低引发的自噬与 BNIP3 的上调有关，从而破坏了 BECN1/Beclin 1 和 BCL2 的关联。 BNIP3 过表达或 BNIP3 RNAi 进一步证实了 BNIP3 在 BECN1 从 BCL2 解离中的作用。综上所述，二甲双胍在高血糖条件下的内皮保护作用可能部分归因于其通过 Hh 通路激活下调自噬的作用。缩写：3-MA = 3-甲基腺嘌呤； 8×GLI BS-FL = 8×GLI结合位点萤火虫荧光素酶； AAV = 腺相关病毒； AAV-Cdh5-sh-Atg7 = 在鼠类 Cdh5 启动子控制下携带针对鼠类 Atg7 的 shRNA 的 AAV 载体； AAV-Cdh5-sh-Gli1 = 在鼠类 Cdh5 启动子控制下携带针对鼠类 Gli1 的 shRNA 的 AAV 载体； AAV-Cdh5-Gli1 = 在鼠类 Cdh5 核心启动子控制下携带鼠类 Gli1 cDNA 的 AAV 载体； ACAC = 乙酰辅酶A羧化酶； Ad-BNIP3 = 携带人类 BNIP3 的腺病毒； Ad-GLI1 = 携带人 GLI1 的腺病毒； Ad-sh-ATG7 = 含有针对人 ATG7 的 shRNA 的腺病毒； Ad-sh-BNIP3 = 含有针对人 BNIP3 的 shRNA 的腺病毒； Ad-sh-GLI = 含有针对人 GLI 的 shRNA 的腺病毒； AGEs = 晚期糖基化终产物； ATG = 自噬相关； atg7flox/flox 小鼠 = 携带 Atg7flox 等位基因的小鼠，其中 Atg7 基因的外显子 14 两侧有 2 个 loxP 位点； BafA1=巴弗洛霉素A1； BECN1 = 贝克林 1； CDH5/VE-钙粘蛋白 = 钙粘蛋白 5； CASP3=半胱天冬酶3； CASP8=半胱天冬酶8； CASP9=半胱天冬酶9； ECs = 内皮细胞； GAPDH = 3-磷酸甘油醛脱氢酶； GCL = 神经节细胞层； GFP-LC3B = 绿色荧光蛋白标记的 LC3B； HG = 高血糖； Hh = 刺猬； HHIP = 刺猬蛋白相互作用蛋白； HUVEC = 人脐静脉内皮细胞； IB4=异凝集素B4； INL = 内核层； ip = 腹膜内； MAP1LC3/LC3 = 微管相关蛋白 1 轻链 3； MAN=甘露醇； MET = 二甲双胍； NG = 正常血糖； ONL = 外核层； p-ACAC = 磷酸化乙酰辅酶A羧化酶； PECAM1/CD31=血小板/内皮细胞粘附分子1； PRKAA1/2 = 蛋白激酶 AMP 激活的催化亚基 α 1/2； p-PRKAA1/2 = 磷酸化 PRKAA1/2； PTCH1 = 修补 1； RAPA=雷帕霉素； RL = 海肾荧光素酶； SHH = 音速刺猬； shRNA = 短发夹 RNA； sh-PRKAA1/2 = 针对人 PRKAA1/2 的短发夹 RNA；乱序 shRNA = 乱序短发夹 RNA 作为靶标特异性短发夹 RNA 的阴性对照，其核苷酸组成与输入序列相同，且与所选生物数据库的任何 mRNA 均不匹配； SMO = 平滑、卷曲类受体； sqRT-PCR = 半定量 RT-PCR； TEK/Tie2 = TEK 受体酪氨酸激酶； Tek-Cre (+) 小鼠 = 在 Tek 启动子/增强子控制下以泛内皮方式表达 Cre 重组酶的小鼠品系； TUNEL = 末端脱氧核苷酸转移酶 dUTP 介导的缺口末端标记
ABSTRACT Studies regarding macroautophagic/autophagic regulation in endothelial cells (ECs) under diabetic conditions are very limited. Clinical evidence establishes an endothelial protective effect of metformin, but the underlying mechanisms remain unclear. We aimed to investigate whether metformin exerts its protective role against hyperglycemia-induced endothelial impairment through the autophagy machinery. db/db mice were treated with intravitreal metformin injections. Human umbilical vein endothelial cells (HUVECs) were cultured either in normal glucose (NG, 5.5 mM) or high glucose (HG, 33 mM) medium in the presence or absence of metformin for 72 h. We observed an obvious inhibition of hyperglycemia-triggered autophagosome synthesis in both the diabetic retinal vasculature and cultured HUVECs by metformin, along with restoration of hyperglycemia-impaired Hedgehog (Hh) pathway activity. Specifically, deletion of ATG7 in retinal vascular ECs of db/db mice and cultured HUVECs indicated a detrimental role of autophagy in hyperglycemia-induced endothelial dysfunction. Pretreatment with GANT61, a Hh pathway inhibitor, abolished the metformin-mediated downregulation of autophagy and endothelial protective action. Furthermore, GLI-family (transcription factors of the Hh pathway) knockdown in HUVECs and retinal vasculature revealed that downregulation of hyperglycemia-activated autophagy by the metformin-mediated Hh pathway activation was GLI1 dependent. Mechanistically, GLI1 knockdown-triggered autophagy was related to upregulation of BNIP3, which subsequently disrupted the association of BECN1/Beclin 1 and BCL2. The role of BNIP3 in BECN1 dissociation from BCL2 was further confirmed by BNIP3 overexpression or BNIP3 RNAi. Taken together, the endothelial protective effect of metformin under hyperglycemia conditions could be partly attributed to its role in downregulating autophagy via Hh pathway activation. Abbreviations: 3-MA = 3-methyladenine; 8×GLI BS-FL = 8×GLI-binding site firefly luciferase; AAV = adeno-associated virus; AAV-Cdh5-sh-Atg7 = AAV vectors carrying shRNA against murine Atg7 under control of murine Cdh5 promoter; AAV-Cdh5-sh-Gli1 = AAV vectors carrying shRNA against murine Gli1 under control of murine Cdh5 promoter; AAV-Cdh5-Gli1 = AAV vectors carrying murine Gli1 cDNA under the control of murine Cdh5 core promoter; ACAC = acetyl-CoA carboxylase; Ad-BNIP3 = adenoviruses harboring human BNIP3`; Ad-GLI1 = adenoviruses harboring human GLI1; Ad-sh-ATG7 = adenoviruses harboring shRNA against human ATG7; Ad-sh-BNIP3 = adenoviruses harboring shRNA against human BNIP3; Ad-sh-GLI = adenoviruses harboring shRNA against human GLI; AGEs = advanced glycation end products; ATG = autophagy-related; atg7flox/flox mice = mice bearing an Atg7flox allele, in which exon 14 of the Atg7 gene is flanked by 2 loxP sites; BafA1 = bafilomycin A1; BECN1 = beclin 1; CDH5/VE-cadherin = cadherin 5; CASP3 = caspase 3; CASP8 = caspase 8; CASP9 = caspase 9; ECs = endothelial cells; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GCL = ganglion cell layer; GFP-LC3B = green fluorescent protein labelled LC3B; HG = high glucose; Hh = Hedgehog; HHIP = hedgehog interacting protein; HUVECs = human umbilical vein endothelial cells; IB4 = isolectin B4; INL = inner nuclear layer; i.p. = intraperitoneal; MAP1LC3/LC3 = microtubule-associated protein 1 light chain 3; MAN = mannitol; MET = metformin; NG = normal glucose; ONL = outer nuclear layer; p-ACAC = phosphorylated acetyl-CoA carboxylase; PECAM1/CD31= platelet/endothelial cell adhesion molecule 1; PRKAA1/2 = protein kinase AMP-activated catalytic subunits alpha 1/2; p-PRKAA1/2 = phosphorylated PRKAA1/2; PTCH1 = patched 1; RAPA = rapamycin; RL = Renilla luciferase; SHH = sonic hedgehog; shRNA = short hairpin RNA; sh-PRKAA1/2 = short hairpin RNA against human PRKAA1/2; scrambled shRNA = the scrambled short hairpin RNA serves as a negative control for the target-specific short hairpin RNA, which has the same nucleotide composition as the input sequence and has no match with any mRNA of the selected organism database; SMO = smoothened, frizzled class receptor; sqRT-PCR = semi-quantitative RT-PCR; TEK/Tie2 = TEK receptor tyrosine kinase; Tek-Cre (+) mice = a mouse strain expressing Cre recombinase under the control of the promoter/enhancer of Tek, in a pan-endothelial fashion; TUNEL = terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling