Table 2.

Summary of studies investigating the effects of metformin on EPCs.

-	Study	EPC Subtype	Main Findings
Early EPCs and FACS Isolated EPCs	Ahmed et al., 2016 [79]	Cells were isolated from the peripheral blood of T1D patients. EPCs quantification by FACS (circulating EPCs were defined as CD45dim CD34+VEGFR-2+ cells and cECs as CD45dim, CD133−, CD34+, and CD144+ cells). Also, isolation of proangiogenic cells and CFU-Hill’s (early EPCs).	Metformin (1000 mg/bid) treatment of T1D patients for 8 weeks improved circulating EPCs, proangiogenic cells, CFU-Hill’s colony numbers and function.
	Chen et al., 2010 [80]	Early EPCs (CFU) isolated by selective plating, and cells isolated by FACS (CD45low/CD34+/VEGFR2+).	Metformin alone (500-2500 mg/day in patients), and metformin + gliclazide (a sulfonylurea) combination treatment improved the number and function of EPCs in patients with T2D over 16 weeks (short term hypoglycemic treatment).
	Dore et al., 2018 [81]	CD34+ cells were isolated by CD34+ microbead antibody protocol and counted by FACS from 42 patients with T2D diagnosed within 10 years. Also, isolation of CFU-Hill’s colonies.	Saxigliptin for 12 weeks added to concurrent metformin therapy increased CD31+ numbers (reflecting a mature circulating pool of endothelial cells) but not CD34+. Double positive cells for CD34 and CXCR4 were increased with dual treatments.
	Dallaglio et al., 2014 [84]	FACS enumeration of CD45- Sca1+ CD34+ CD31+ EPCs in peripheral blood and visceral white adipose tissue of obese mice receiving a high-fat diet (0.5mg metformin/ml orally).	EPCs were increased in high-fat diet fed mice, and treatments with metformin reduced these numbers in white adipose tissue but had no effect on peripheral blood EPCs only in obese mice.
	Yu et al., 2016* [82]	Bone marrow Sca-1+ Flk-1+ EPCs were isolated from the blood of type 1 diabetes mice and treated with metformin (250 mg/kg/d, intragastric) by FACS. Also, selective plating of EPCs on vitronectin coated plates.	Metformin significantly increased the number of Sca-1+ Flk-1+ EPCs in diabetic mice improved angiogenesis and wound closure in vivo. Metformin increased the expression of phosphorylated-AMPK and eNOS and the generation of NO in diabetic EPCs. In vitro experiments: Cultured bone marrow EPCs were treated with high glucose media (33 mM) with or without metformin (2 mM) for 24 h and compared to controls cultured in normal glucose media (5.5 mM). Metformin (2 mM) induced AMPK and eNOS phosphorylation and the generation of NO.
	Han et al., 2016 [83]	Genetically hyperglycaemic db/db mice were treated with metformin (250 mg/kg/day intragastric) for 2 weeks. FACS enumerates Sca-1+ and Flk-1+ cells. Also, selective plating on vitronectin coated plates.	Treatment with metformin improved wound closure and capillary formation in vivo. EPCs numbers were reduced in db/db mice, and treatments with metformin partially restored these numbers. In mice treated with metformin, tube formation ability of EPCs was induced in addition to an increase in NO generation and a reduction in intracellular oxygen concentration and TSP-1.
Late EPCs	Li et al., 2015* [85]	Bone marrow EPCs isolated from healthy rats by selective plating. Colonies appeared at day 7 and reached confluency at day 14.	Metformin (1mM) regulates EPCs differentiation to endothelial cells through autophagy and AMPK-eNOS-NO pathways. Treatment with metformin resulted in an increased expression of CD31 and vWF, increased phosphorylation of AMPK, and decreased expression of mTOR and p70S6K phosphorylation. Metformin concentrations between 2-10 mM inhibited EPC proliferation following 48 h of treatment, while concentrations between 0.5-1mM had no effect on proliferation.
	Li et al., 2017* [86]	Late EPCs isolated by selective plating on collagen coated plates.	Metformin treatment (10 mM) of healthy EPCs reduced the expression of matrix metalloproteinase-2 and 9, and decreased EPCs migration.

Note: *Attention is drawn to the high concentrations of metformin that were used in some of the listed in vitro studies. Peak plasma levels of metformin, when used clinically in humans, are unlikely to be higher than 20 μM, although higher levels of 50 μM might be initially achieved in the portal circulation immediately after absorption [87]. With a half-life of between 4-5 h and administered either once daily or maximally thrice, studies on the effects of metformin should ideally be performed with concentrations no higher than 20 μM.