Micro-RNA-126 Reduces the Blood Thrombogenicity in Diabetes Mellitus via Targeting of Tissue Factor

Abstract

Objective: Diabetes mellitus involves vascular inflammatory processes and is a main contributor to cardiovascular mortality. Notably, heightened levels of circulating tissue factor (TF) account for the increased thrombogenicity and put those patients at risk for thromboembolic events. Here, we sought to investigate the role of micro-RNA (miR)-driven TF expression and thrombogenicity in diabetes mellitus.

Approach and results: Plasma samples of patients with diabetes mellitus were analyzed for TF protein and activity as well as miR-126 expression before and after optimization of the antidiabetic treatment. We found low miR-126 levels to be associated with markedly increased TF protein and TF-mediated thrombogenicity. Reduced miR-126 expression was accompanied by increased vascular inflammation as evident from the levels of vascular adhesion molecule-1 and fibrinogen, as well as leukocyte counts. With optimization of the antidiabetic treatment miR-126 levels increased and thrombogenicity was reduced. Using a luciferase reporter system, we demonstrated miR-126 to directly bind to the F3-3'-untranslated region, thereby reducing TF expression both on mRNA and on protein levels in human microvascular endothelial cells as well as TF mRNA and activity in monocytes.

Conclusions: Circulating miR-126 exhibits antithrombotic properties via regulating post-transcriptional TF expression, thereby impacting the hemostatic balance of the vasculature in diabetes mellitus.

Keywords: diabetes mellitus; endothelial cells; monocytes; thromboplastin; untranslated regions.

Figure 1.

High micro-RNA (miR)-126 expression is associated with reduced tissue factor (TF) protein and TF-mediated thrombogenicity in patients with diabetes mellitus. A, Expression of miR-126 in both groups with either low or high miR-126 expression. Differences in (B) TF protein and (C) TF activity in plasma depending on low or high miR-126 plasma expression. D, Levels of D-dimers in the patients of both groups. Data are expressed as mean±SEM. n= 46, for D-dimers n=40, *P<0.05, ***P<0.0001.

Figure 2.

Micro-RNA (miR)-126 is associated with reduced vascular inflammation in patients with diabetes mellitus. A, Expression levels of the inflammatory proteins vascular adhesion molecule (VCAM)-1, (B) endothelin, (C) E-selectin, (D) leukocyte count as well as (E) fibrinogen in patients with either low or high miR-126 expression. Data are expressed as mean±SEM. n= 46, *P<0.05.

Figure 3.

The optimized antidiabetic treatment is associated with upregulation of micro-RNA (miR)-126 levels and a reduction in tissue factor (TF)–mediated thrombogenicity. A, Fasting blood glucose levels, (B) miR-126 expression, and (C) TF activity in all patients before and after optimizing of anti-diabetic treatment. Changes in miR-126 expression (D), TF activity (E), and fasting blood glucose levels (F) on optimized antidiabetic treatment in both, patients with low and high initial miR-126 expression on admission. Data are expressed as mean±SEM. n= 46, *P<0.05, **P<0.01, ***P<0.0001.

Figure 4.

Increased full-length tissue factor (flTF) and alternatively spliced (as) TF expression in endothelial cells after stimulation with tumor necrosis factor (TNF)-α. Human microvascular endothelial cells were cultured for 24 h and then treated with 10 ng/mL TNFα. Gene expression was determined for (A) alternatively spliced TF, (B) flTF, and (C) miR-126 2 and 6 h after stimulation with TNFα. GAPDH was used as a house-keeping gene. Data are represented as mean±SEM. *P<0.05, n≥3. D, A representative Western blot shows protein expression 6 and 24 h post TNFα stimulation. GAPDH was used as loading control (n=3, performed in duplicates).

Figure 5.

Micro-RNA (miR)-126 reduces the expression of both alternatively spliced tissue factor (asTF) and full-length (fl) TF in human microvascular endothelial cells (HMEC) after stimulation with tumor necrosis factor (TNF)-α. HMECs were cultured for 24 h and then transfected with a control mimic or a miR-126 mimic (A–C) as well as an inhibitor control or anti–miR-126 (D–F). The transfection efficiency was confirmed by real-time TaqMan polymerase chain reaction (A and D). Twenty-four hours post transfection, the cells were stimulated with 10 ng/mL TNFα. Gene expression was determined for asTF (B and E) and flTF (C and F) 2 h after stimulation with TNFα. GAPDH was used as house-keeping gene. Data are represented as mean±SEM. *P<0.05, **P<0.01, ***P<0.0001, n≥3. Representative western blots show the protein expression of flTF and asTF after transfection with miR-126 (G) or anti–miR-126 (H) 6 h post TNFα stimulation. GAPDH was used as loading control (n=3).

Figure 6.

Micro-RNA (miR)-126 decreases tissue factor (TF) transcription and TF-depending FXa generation in THP-1 cells. THP-1 cells were grown over night and then transfected with either a control mimic or a miR-126 mimic for 24 h. The cells were left untreated or stimulated with lipopolysaccharide (LPS; 10 μg/mL) for 2 or 6 h. Subsequently, miR-126 transfection efficiency (A), alternatively spliced (as) TF mRNA levels (B), full-length (fl) TF mRNA levels (C) or the TF activity (D) were assessed using real-time TaqMan polymerase chain reaction or a FX chromogenic assay, respectively. Data are represented as mean±SEM. **P<0.01, ***P<0.001, n≥6.

Figure 7.

Micro-RNA (miR)-126 targets the 3′-untranslated region (UTR) of the F3 transcript. A, Human embryonic kidney 293 (HEK) cells were cultured for 24 h and then cotransfected with a mock plasmid and 200 μmol/L control mimic or 200 μmol/L miR-126 mimic as well as with a tissue factor (TF)-3′-UTR harboring plasmid and 200 μmol/L control mimic, 200 μmol/L miR-126 mimic or 200 μmol/L miR-19b mimic. Twenty-four hours post transfection, the luciferase activity was measured. B, HEK cells were transfected with the TF-3′-UTR plasmid and decreasing amounts of a miR-126 mimic (200, 20, or 2 μmol/L). Subsequently, the luciferase activity was measured. *P<0.05, n=3. C, Predicted heteroduplex of miR-126 and the 3′-UTR of the F3 (TF) transcript in relation to the known proposed miR-binding sites in the human TF transcript. n.s. indicates not significant.