Metformin alleviates hyperglycemia-induced endothelial impairment by downregulating autophagy via the Hedgehog pathway
Creators: name: Niu, Chao
name: Chen, Zhiwei
name: Kim, Kyoung Tae
name: Sun, Jia
name: Xue, Mei
name: Chen, Gen
name: Li, Santie
name: Shen, Yingjie
name: Zhu, Zhongxin
name: Wang, Xu
name: Liang, Jiaojiao
name: Jiang, Chao
name: Cong, Weitao
name: Jin, Litai
name: Li, Xiaokun
Publisher: name: Taylor & Francis
Year: 2019
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Science Policy
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: 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 <i>ATG7</i> 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, <i>GLI</i>-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, <i>GLI1</i> 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 <i>BNIP3</i> 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. <b>Abbreviations:</b> 3-MA = 3-methyladenine; 8×GLI BS-FL = 8×GLI-binding site firefly luciferase; AAV = adeno-associated virus; AAV-<i>Cdh5</i>-sh-<i>Atg7</i> = AAV vectors carrying shRNA against murine <i>Atg7</i> under control of murine <i>Cdh5</i> promoter; AAV-<i>Cdh5</i>-sh-<i>Gli1</i> = AAV vectors carrying shRNA against murine <i>Gli1</i> under control of murine <i>Cdh5</i> promoter; AAV-<i>Cdh5-Gli1</i> = AAV vectors carrying murine <i>Gli1</i> cDNA under the control of murine <i>Cdh5</i> core promoter; ACAC = acetyl-CoA carboxylase; Ad-<i>BNIP3</i> = adenoviruses harboring human <i>BNIP3`;</i> Ad-<i>GLI1</i> = adenoviruses harboring human <i>GLI1;</i> Ad-sh-<i>ATG7</i> = adenoviruses harboring shRNA against human <i>ATG7;</i> Ad-sh-<i>BNIP3</i> = adenoviruses harboring shRNA against human <i>BNIP3;</i> Ad-sh-<i>GLI</i> = adenoviruses harboring shRNA against human <i>GLI;</i> AGEs = advanced glycation end products; ATG = autophagy-related; <i>atg7</i><sup>flox/flox</sup> mice = mice bearing an <i>Atg7</i><sup>flox</sup> allele, in which exon 14 of the <i>Atg7</i> gene is flanked by 2 loxP sites; BafA1 = bafilomycin A<sub>1</sub>; 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 = <i>Renilla</i> luciferase; SHH = sonic hedgehog; shRNA = short hairpin RNA; sh-<i>PRKAA1/2</i> = short hairpin RNA against human <i>PRKAA1/2;</i> 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; <i>Tek-Cre</i> (+) mice = a mouse strain expressing Cre recombinase under the control of the promoter/enhancer of <i>Tek</i>, in a pan-endothelial fashion; TUNEL = terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling