Elevated HDL-bound miR-181c-5p level is associated with diabetic vascular complications in Australian Aboriginal...

Diabetes is a major burden on Australia's Indigenous population, with high rates of disease and vascular complications. Diabetic vascular complications are associated with impaired ischaemia-driven angiogenesis. MicroRNAs (miRNAs) are key players in the regulation of angiogenesis. HDL-cholesterol (HDL-c) levels are inversely associated with the risk of developing diabetic complications and HDL can carry miRNAs. HDL-miRNA profiles differ in disease states and may present as biomarkers with the capacity to act as bioactive signalling molecules. Recent studies have demonstrated that HDL becomes dysfunctional in a diabetic environment, losing its vasculo-protective effects and becoming more pro-atherogenic. We sought to determine whether HDL-associated miRNA profiles and HDL functionality were predictive of the severity of diabetic vascular complications in Australia's Indigenous population.

HDL was isolated from plasma samples from Indigenous participants without diabetes ('Healthy'), with type 2 diabetes mellitus ('T2DM') and with diabetes-associated macrovascular complications (specifically peripheral artery disease, 'T2DM+Comp'). To assess HDL angiogenic capacity, human coronary artery endothelial cells were treated with PBS, reconstituted HDL (rHDL, positive control) or isolated HDL and then exposed to high-glucose (25 mmol/l) conditions. The expression levels of two anti-angiogenic miRNAs (miR-181c-5p and miR-223-3p) and one pro-angiogenic miRNA (miR-27b-3p) were measured in the HDL fraction, plasma and treated human coronary artery endothelial cells by quantitative real-time PCR. In vitro endothelial tubule formation was assessed using the Matrigel tubulogenesis assay.

Strikingly, we found that the levels of the anti-angiogenic miRNA miR-181c-5p were 14-fold higher (1454 ± 1346%) in the HDL from Aboriginal people with diabetic complications compared with both the Healthy (100 ± 121%, p < 0.05) and T2DM (82 ± 77%, p < 0.05) groups. Interestingly, we observed a positive correlation between HDL-associated miR-181c-5p levels and disease severity (p = 0.0020). Under high-glucose conditions, cells treated with rHDL, Healthy HDL and T2DM HDL had increased numbers of tubules (rHDL: 136 ± 8%, p < 0.01; Healthy HDL: 128 ± 6%, p < 0.01; T2DM HDL: 124 ± 5%, p < 0.05) and branch points (rHDL: 138 ± 8%, p < 0.001; Healthy HDL: 128 ± 6%, p < 0.01; T2DM HDL: 127 ± 5%, p < 0.01) concomitant with elevations in mRNA levels of the key hypoxia angiogenic transcription factor HIF1A (rHDL: 140 ± 10%, p < 0.01; Healthy HDL: 136 ± 8%, p < 0.01; T2DM HDL: 133 ± 9%, p < 0.05). However, this increase in angiogenic capacity was not observed in cells treated with T2DM + Comp HDL (tubule numbers: 113 ± 6%, p = 0.32; branch points: 113 ± 5%, p = 0.28; HIF1A: 117 ± 6%, p = 0.43), which could be attributed to the increase in cellular miR-181c-5p levels (T2DM + Comp HDL: 136 ± 7% vs PBS: 100 ± 9%, p < 0.05).

In conclusion, HDL from Aboriginal people with diabetic complications had reduced angiogenic capacity. This impairment is associated with an increase in the expression of anti-angiogenic miR-181c-5p. These findings provide the rationale for a new way to better inform clinical diagnosis of disease severity with the potential to incorporate targeted, personalised HDL-miRNA intervention therapies to prevent further development of, or to reverse, diabetic vascular complications in Australian Aboriginal people.