Metformin improves the angiogenic potential of human CD34⁺ cells co-incident with downregulating CXCL10 and TIMP1 gene expression and increasing VEGFA under hyperglycemia and hypoxia within a therapeutic window for myocardial infarction

Abstract

Background: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with diabetes mellitus (DM). To identify the most effective treatment for CVD, it is paramount to understand the mechanism behind cardioprotective therapies. Although metformin has been shown to reduce CVD in Type-2 DM clinical trials, the underlying mechanism remains unexplored. CD34(+) cell-based therapies offer a new treatment approach to CVD. The aim of this study was to investigate the effect of metformin on the angiogenic properties of CD34(+) cells under conditions mimicking acute myocardial infarction in diabetes.

Methods: CD34(+) cells were cultured in 5.5 or 16.5 mmol/L glucose ± 0.01 mmol/L metformin and then additionally ± 4 % hypoxia. The paracrine function of CD34(+) cell-derived conditioned medium was assessed by measuring pro-inflammatory cytokines, vascular endothelial growth factor A (VEGFA), and using an in vitro tube formation assay for angiogenesis. Also, mRNA of CD34(+) cells was assayed by microarray and genes of interest were validated by qRT-PCR.

Results: Metformin increased in vitro angiogenesis under hyperglycemia-hypoxia and augmented the expression of VEGFA. It also reduced the angiogenic-inhibitors, chemokine (C-X-C motif) ligand 10 (CXCL10) and tissue inhibitor of metalloproteinase 1 (TIMP1) mRNAs, which were upregulated under hyperglycemia-hypoxia. In addition metformin, increased expression of STEAP family member 4 (STEAP4) under euglycemia, indicating an anti-inflammatory effect.

Conclusions: Metformin has a dual effect by simultaneously increasing VEGFA and reducing CXCL10 and TIMP1 in CD34(+) cells in a model of the diabetic state combined with hypoxia. Therefore, these angiogenic inhibitors are promising therapeutic targets for CVD in diabetic patients. Moreover, our data are commensurate with a vascular protective effect of metformin and add to the understanding of underlying mechanisms.

Fig. 1

Expression of pro-angiogenic factor VEGFA in CD34⁺ cell-derived conditioned media. The levels of VEGFA were assayed in three independent biological replicates using the MSD technique. Results are presented as ±SEM and were statistically analyzed using one-way ANOVA followed by Fisher’s LSD test. Data for effects of hypoxia and hyperglycemia were compared with control (5.5 mmol/L), and data for cells treated with metformin were compared with the corresponding metformin-untreated condition. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Effect of CM from CD34⁺ cells exposed to different conditions on in vitro angiogenic assays. a HUVEC were plated on Matrigel with CM from 2.0 × 10⁵ CD34⁺ cells treated with euglycemia, euglycemia and hypoxia, hyperglycemia or hyperglycemia and hypoxia in the presence and absence of metformin. EBM-2 medium containing the VEGF inhibitor sunitinib (14 µmol/L) was used as a negative control for the assay. The highlighted image shows the greatest tube length, which was achieved in HUVEC incubated with CM-derived from CD34⁺ cells treated with hyperglycemia and hypoxia in the presence of metformin. b Tube length at 6 h was expressed as a percentage of the tube length of HUVEC treated with 5.5 mmol/L glucose CM (n = 3); *P < 0.05 compared pairwise, i.e., condition with metformin versus metformin-untreated condition

Fig. 3

Summary of the effect of metformin on CD34⁺ cells incubated with hyperglycemia–hypoxia. CD34⁺ cells were treated with hyperglycemia–hypoxia versus euglycemia (a), and hyperglycemia–hypoxia with metformin versus metformin untreated-condition (b). Green shading indicates downregulation, orange shading indicate upregulation and gray shading indicate unchanged gene expression. In vitro tube formation (angiogenesis), green shading indicates inhibition, whereas orange shading indicates activation