Upregulation of miR-221 and -222 in response to increased extracellular signal-regulated kinases 1/2 activity exacerbates neointimal hyperplasia in diabetes mellitus

Abstract

Background and aims: Diabetes is associated with accelerated arterial intimal thickening that contributes to the increased cardiovascular disease seen in this population. In healthy arteries, intimal thickening is inhibited by elevated levels of the cyclin-dependent kinase inhibitor, p27^Kip1, and intimal thickening is promoted by activation of the mammalian Target of Rapamycin to promote degradation of p27^Kip1 protein. Recently, we reported that two microRNAs, miR-221 and -222, which promote intimal thickening via down-regulation of mRNA encoding p27^Kip1, are elevated in the arteries of diabetic patients. To determine if these miRNAs are critical to the increased intimal thickening under diabetic conditions, we examined the regulation of p27^Kip1in a mouse model of diabetes.

Methods: Comparisons of p27^Kip1 signaling in NONcNZO10 mice fed a diabetogenic versus control diet were performed using immunochemistry and real-time PCR.

Results: Vascular smooth muscle cells and arteries of diabetic mice exhibited decreased levels of p27^Kip1 that derived from destabilization of p27^Kip1 mRNA in an extracellular signal response kinase-1/2 (ERK-1/2) dependent manner. The activity of ERK-1/2 is increased in the arteries of diabetic mice and promotes an increase in miR-221 and -222. Inhibition of miR-221 and -222 restores normal levels of p27^Kip1 mRNA and protein in the arteries of diabetic mice and reduces intimal thickening following wire injury.

Conclusions: These data suggest diabetes is accompanied by increases in arterial miR-221 and -222 expression that promotes intimal thickening. Inhibition of the increased miR-221 and -222 may be efficacious in the prevention of the cardiovascular complications of diabetes.

Keywords: Diabetes mellitus; Intimal thickening; miR-221; miR-222; p27(Kip1).

Figure 1. p27^Kip1 levels are reduced in the arteries of DM mice in response to elevated miR-221 and -222

ND and DM mice were treated with a perivascular Pluronic gel containing non-targeting oligonucleotides (NT) or 2’OMe-miR-222 (AS, n=3 for all groups). Relative levels of (A) miR-221 and -222 levels and (B) p27Kip1 mRNA in the femoral arteries of ND and DM mice. Data is normalized to the ND NT group. † p < 0.05. (C) p27^Kip1 protein levels in the aortae of ND and DM mice. † p < 0.05.

Figure 2. Intimal thickening following wire injury is increased in DM mice in a miR-221 and -222 dependent manner

ND and DM mice underwent femoral artery wire injury coupled with perivascular implantation of a Pluronic gel with either non-targeting oligonucleotide (NT) or 2’OMe-miR-222 (AS). (A–D) Representative micrographs of injured femoral arteries from ND treated with NT (A, n = 8), AS (B, n = 5), and DM mice treated with NT (C, n = 11) and AS (D, n = 6). (E) Intima to media ratio for ND and DM mice. Bar p < 0.05.

Figure 3. DM-VSMCs exhibit increased proliferation and migration coupled with a relative resistance to rapamycin

(A) Phosphorylation of Akt at threonine 308 residue (p-Akt) is reduced in DM-VSMCs compared to ND-VSMCs following stimulation with 1 pmol/L of insulin. P-Akt is normalized to total Akt. The upper panel shows a representative Western blot and the lower panel shows the densitometric analysis. + p < 0.05. (B) Basal phosphorylation of IRS-1 at serine 307 residue and p70^S6kinase at tyrosine 421 and serine 424 residues in serum starved DM-VSMCs and ND-VSMCs. (C) Percent of BrdU positive VSMCs following serum starvation overnight (U) and stimulation with 20% serum/DMEM with rapamycin (10 nmol/L) or vehicle (0). Bar p < 0.05. (D) Migration of VSMCs toward 10 ng/mL PDGF in a modified Boyden chamber. VSMCs were incubated in normal media with vehicle (Veh) or 10 nM rapamycin (RPM) overnight prior to seeding in the chamber. All studies were performed in triplicate. Data is expressed as percentage of ND-VSMCs treated with vehicle. Bar p < 0.05.

Figure 4. Regulation of p27^Kip1 by the mTOR pathway is lost under diabetic conditions

(A) Representative Western blots for p27^Kip1, β-actin, and 4E-BP1 in ND and DM mice undergoing femoral artery wire injury, coupled with intraperitoneal injection of either vehicle or rapamycin. The contralateral artery underwent a sham procedure. (B) Densitometry of Western blots for p27^Kip1 for ND (n=4 for vehicle, n=6 for rapamycin) and DM (n=3 for both groups) mice. Data is presented as mean of the percentage of p27^Kip1 in the injured artery compared to the sham. This results in a reduction of the number of comparisons to 6. (C) Representative Western blots for p27^Kip1 and β-actin in ND-VSMCs and DM-VSMCs. VSMCs were serum starved overnight (U), stimulated with DMEM/20% FBS supplemented with the indicated dose of rapamycin (RPM). (D) Densitometry of Western blots for p27^Kip1 for ND-VSMCs and DM-VSMCs treated as indicated in C. All studies were performed in triplicate. Bar p < 0.05. (E) p27^Kip1 protein levels in the aortae of ND and DM mice (n=4 for both groups) as measured by ELISA assay. † p < 0.05. (F) p27^Kip1 mRNA in the aortae of ND and DM mice (n=3 for both groups).

Figure 5. Increased ERK-1/2 activity promotes increased miR-221 and -222 as well as p27^Kip1 mRNA destabilization

(A) Ratio of phosphorylated to total ERK-1/2 in the aortae of DM (n=3) and ND (n=4) mice as measured by ELISA. Bar p < 0.05. (B) Half-life of p27^Kip1 mRNA in ND-VSMCs and DM-VSMCs treated with vehicle (Veh) and PD98059 (PD). Data are presented as mean of triplicate samples ± standard error of the mean. † p < 0.05. (C) Half-life of p27^Kip1 mRNA in ND-VSMCs and DM-VSMCs transfected with non-targeting control oligonucleotides (NT) or siRNA targeting ERK 1/2 (ERK siRNA). Data are presented as mean of triplicate samples ± standard error of the mean. † p < 0.05. (D) Relative levels of miR-221 and -222 in serum stimulated ND-VSMCs and DM-VSMCs treated with vehicle or U0126 (50 µmol/L) overnight. Data are normalized to the vehicle treated samples. Bar p < 0.05. (E) Relative p27^Kip1 mRNA levels in the aortae of ND mice treated with vehicle (ND, n=3) or U0126 (ND+EI, n=5) and DM mice treated with vehicle (DM, n=4) or U0126 (DM+EI, n=4). Data are normalized to ND. Bar p < 0.05. (F) Relative p27^Kip1 protein levels in the aortae of ND mice treated with vehicle (ND, n=4) or U0126 (ND+EI, n=4) and DM mice treated with vehicle (DM, n=4) or U0126 (DM+EI, n=6). Bar p < 0.05.