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.