Anti-Angiogenic miR-222, miR-195, and miR-21a Plasma Levels in T1DM Are Improved by Metformin Therapy, Thus Elucidating Its Cardioprotective Effect: The MERIT Study

Abstract

Type 1 diabetes (T1DM) is associated with increased cardiovascular disease (CVD) and reduced life expectancy. We thus hypothesized that anti-angiogenic miRs are increased in T1DM, and the cardioprotective effect of metformin is mediated via reducing those miRs. In an open label, case-controlled study, 23 T1DM patients without CVD were treated with metformin for eight weeks (TG), matched with nine T1DM patients on standard treatment (SG) and 23 controls (CG). Plasma miR-222, miR-195, miR-21a and miR-126 were assayed by real-time RT-qPCR. The results were correlated with: endothelial function (RHI), circulating endothelial progenitor cells (cEPCs) (vascular repair marker, CD45^dimCD34⁺VEGFR2⁺ cells) and circulating endothelial cells (cECs) (vascular injury marker, CD45^dimCD34⁺CD133⁻CD144⁺ cells). miR-222, miR-195 and miR-21a were higher in T1DM than CG; p = 0.009, p < 0.0001, p = 0.0001, respectively. There was an inverse correlation between logmiR-222 and logRHI (p < 0.05) and a direct correlation between logmiR-222 and logCD34⁺ (p < 0.05) in TG. Metformin reduced miR-222, miR-195 and miR-21a levels in TG; p = 0.007, p = 0.002 p = 0.0012, respectively. miRs remained unchanged in SG. miR-126 was similar in all groups. There was a positive association between changes in logmiR-222 and logcECs after metformin in TG (p < 0.05). Anti-angiogenic miRs are increased in T1DM. Metformin has cardioprotective effects through downregulating miR-222, miR-195 and miR-21a, beyond improving glycemic control.

Keywords: T1DM; anti-angiogenic; metformin; miRs.

Figure 1

(a–c) A comparison of miR-222, miR-195 and miR-21a levels in plasma for all groups. At baseline (TG V1), miR-222, miR-195 and miR-21a levels were significantly upregulated in the TG compared to healthy controls (CG). The levels of miR-222, miR-195 and miR-21a were all significantly reduced in the TG after metformin therapy (TG V2). The results are presented as the mean ± SEM. Within the TG and SG, the comparison was analyzed using the paired Student t-test. An unpaired Student t-test was used to compare the healthy controls and patients’ groups. Treatment group pre-metformin (TG V1), treatment group post-metformin (TG V2), standard group pre-observation (SG V1) and standard group post-observation (SG V2). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

The significant correlation between levels of circulating miR-222 and indices of vascular health in patients with T1DM at baseline. LogmiR-222 was inversely correlated with endothelial function logRHI measured by the reactive hyperemia index (RHI). This relationship was demonstrated by Pearson correlation.

Figure 3

The significant correlation between levels of circulating miR-222 (logmiR222) and CD34⁺ cells (log CD34⁺) at baseline. CD34⁺ were analyzed by flow cytometry. The relationship was demonstrated by Pearson correlation.

Figure 4

A direct correlation between the change (log delta) in levels of circulating miR-222 and the change (log delta) of circulating endothelial cells (cECs). cECs are defined by FACS as CD45^dim, CD34⁺, CD 133⁻, CD144⁺ and delta cECs, an index of vascular damage. This relationship was demonstrated by Pearson correlation.

Figure 5

A comparison of the plasma levels of miR-126 (per mL) in all groups. The results are presented as the mean ± SEM. Treatment group pre-metformin (TG V1), treatment group post-metformin (TG V2), standard group pre-observation (SG V1) and standard group post-observation (SG V2).

Figure 6

A heatmap showing the pathways altered by miR-222, miR-195 and miR-21a. The heatmap was generated with the DIANA miRPATH software, based on experimentally-validated miRNA interactions derived from the DIANA-TarBase. Axes on the attached dendrograms depict hierarchical clustering results for miRs and their pathways, respectively.