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

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, p27Kip1, and intimal thickening is promoted by activation of the mammalian Target of Rapamycin to promote degradation of p27Kip1 protein. Recently, we reported that two microRNAs, miR-221 and -222, which promote intimal thickening via down-regulation of mRNA encoding p27Kip1, 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 p27Kip1in a mouse model of diabetes. METHODS: Comparisons of p27Kip1 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 p27Kip1 that derived from destabilization of p27Kip1 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 p27Kip1 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.

Elevation of miR-221 and -222 in the internal mammary arteries of diabetic subjects and normalization with metformin.

Diabetes is a major risk factor for cardiovascular disease and is associated with increased intimal thickening and accelerated vascular smooth muscle cell (VSMC) proliferation. We measured the expression of two microRNAs that promote intimal thickening, miR-221/222, and mRNA encoding a downstream target, p27(Kip1), in internal mammary artery (IMA) segments collected from 37 subjects undergoing coronary artery bypass grafting. The segments were stratified into three groups: non-diabetic subjects (ND), diabetic subjects not on metformin (DMMet-), and diabetic subjects on metformin (DMMet+). The DMMet- group exhibited a significant increase in miR-221/222 and decrease in p27(Kip1) mRNA compared to both the ND and DMMet+ groups. miR-221/222 levels inversely correlated with metformin dose. VSMCs isolated from the IMAs of the DMMet- group proliferate at a faster rate than those of the ND and DMMet+ groups. Further studies into the importance of miR-221/222 in the increased intimal thickening observed in diabetic subjects is warranted.

Elevated serum bone morphogenetic protein 4 in patients with chronic kidney disease and coronary artery disease.

Chronic kidney disease (CKD) is associated with increased coronary artery disease (CAD) and coronary artery calcification. We hypothesized that the osteogenic factor, bone morphogenetic protein-4 (sBMP-4), is elevated in subjects with both CKD and CAD. Serum was collected from 79 subjects undergoing diagnostic angiography and stratified according to CAD and CKD status. Subjects with both CAD and CKD had significantly elevated sBMP-4 compared to those with only one or no disease. sBMP-4 continued to be associated with the presence of both diseases after adjustment for other risk factors. To determine if sBMP-4 is associated with coronary artery calcification, we compared coronary artery calcium scores (CAC) to sBMP-4 in 22 subjects. A positive correlation between CAC and sBMP-4 was seen. In conclusion, sBMP-4 is elevated in patients with both CAD and CKD and positively correlates with CAC, suggesting a role for sBMP-4 in the increased CAD seen in CKD patients.

Rapamycin regulates endothelial cell migration through regulation of the cyclin-dependent kinase inhibitor p27Kip1.

Rapamycin is a macrolide antibiotic that inhibits vascular smooth muscle cell proliferation and migration and that is used clinically on drug-eluting stents to inhibit in-stent restenosis. Although inhibition of cell migration is an asset in preventing restenosis, it also leads to impaired stent endothelialization, a significant limitation of current drug-eluting stent technology that necessitates prolonged antiplatelet therapy. We measured the ability of rapamycin to inhibit the migration of human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAEC) toward the chemoattractant vascular endothelial cell growth factor. Although acute administration of rapamycin had no effect, exposure for 24 h inhibited HUVEC and HCAEC migration. Disruption of the mTORC2 via small interfering RNA was also effective in inhibiting HCAEC migration. Treatment of HCAECs for this period with rapamycin produced an increase in the cyclin-dependent kinase inhibitor p27(Kip), through a decrease in the targeting of the protein for degradation by phosphorylation at Thr(187). ECs isolated from a knock-in mouse expressing p27(Kip1) with a mutation of this residue to an alanine, blocking this phosphorylation, exhibited reduced migration compared with wild-type controls. Silencing of p27(Kip1) with small interfering RNA blocked the effects of rapamycin on migration and tube formation as well as RhoA activation and cytoskeletal reorganization. We conclude that prolonged exposure of ECs to rapamycin increases p27(Kip1) and in turn inhibits RhoA activation, blocking cell migration and differentiation. These data elucidate the molecular mechanism underlying regulation of p27(Kip1) protein and cell migration by rapamycin.

Sera from patients with diabetes do not alter the effect of mammalian target of rapamycin inhibition on smooth muscle cell proliferation.

Clinical studies of drug-eluting stents delivering the mammalian target of rapamycin (mTOR) inhibitor, rapamycin (Sirolimus), have demonstrated a reduced efficacy for these devices in patients with diabetes, which suggests that the mTOR pathway may cease to be dominant in mediating the vascular response to injury under diabetic conditions. We hypothesized that changes in serum composition accompanying diabetes may reduce the role of mTOR in mediating the vascular response to injury. We measured the ability of a median dose of rapamycin (10 nM) to inhibit the proliferation of human coronary artery smooth muscle cells (huCASMCs) stimulated with serum obtained from donors with diabetes (n = 14) and without diabetes (n = 16). In an additional analysis, we compared the effects of rapamycin on huCASMCs stimulated with the serum of donors with metabolic syndrome (n = 15) versus those without (n = 7). There was no difference in the effect of rapamycin on huCASMC proliferation after stimulation with serum from either donors with diabetes or donors with metabolic syndrome compared with the respective controls. We conclude that the changes in the serum composition common to diabetes and metabolic syndrome are insufficient to diminish the role of mTOR in the progression of cardiovascular disease.

Isolation of endothelial cells and vascular smooth muscle cells from internal mammary artery tissue.

Analyses of vascular smooth muscle cell and endothelial cell function through tissue culture techniques are often employed to investigate the underlying mechanisms regulating cardiovascular disease. As diseases such as diabetes mellitus and chronic kidney disease increase a patient's risk of cardiovascular disease, the development of methods for examining the effects of these diseases on vascular smooth muscle cells and endothelial cells is needed. Commercial sources of endothelial cells and vascular smooth muscle cells generally provide minimal donor information and are in limited supply. This study was designed to determine if vascular smooth muscle cells and endothelial cells could be isolated from human internal mammary arteries obtained from donors undergoing coronary artery bypass graft surgery. As coronary artery bypass graft surgery is a commonly performed procedure, this method would provide a new source for these cells that when combined with the donor's medical history will greatly enhance our studies of the effects of complicating diseases on vascular biology. Internal mammary artery tissue was obtained from patients undergoing coronary artery bypass graft surgery. Through a simple method employing two separate tissue digestions, vascular smooth muscle cells and endothelial cells were isolated and characterized. The isolated vascular smooth muscle cells and endothelial cells exhibited the expected morphology and were able to be passaged for further analysis. The vascular smooth muscle cells exhibited positive staining for α-smooth muscle actin and the endothelial cells exhibited positive staining for CD31. The overall purity of the isolations was > 95%. This method allows for the isolation of endothelial cells and vascular smooth muscle cells from internal mammary arteries, providing a new tool for investigations into the interplay of vascular diseases and complicating diseases such as diabetes and kidney disease.