South Australian Health and Medical Research Institute, Adelaide, SA, Australia.
College of Medicine and Public Health, Flinders University, Adelaide, SA, Australia.
Diabetologia. 2021 Jun;64(6):1402-1411. doi: 10.1007/s00125-021-05414-6. Epub 2021 Mar 2.
AIMS/HYPOTHESIS: 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).
CONCLUSIONS/INTERPRETATION: 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.
目的/假设：糖尿病是澳大利亚土著人群的主要负担，其疾病和血管并发症发生率较高。糖尿病血管并发症与缺血驱动的血管生成受损有关。微小 RNA（miRNA）是调节血管生成的关键因素。高密度脂蛋白胆固醇（HDL-c）水平与发生糖尿病并发症的风险呈负相关，HDL 可以携带 miRNA。HDL-miRNA 谱在疾病状态下不同，并且可以作为具有生物活性信号分子作用的生物标志物。最近的研究表明，糖尿病环境中的 HDL 功能失调，失去其血管保护作用，变得更具动脉粥样硬化形成作用。我们试图确定 HDL 相关 miRNA 谱和 HDL 功能是否可预测澳大利亚土著人群糖尿病血管并发症的严重程度。
从没有糖尿病的土著参与者（“健康”）、患有 2 型糖尿病（“T2DM”）和患有糖尿病相关大血管并发症（具体为外周动脉疾病，“T2DM+Comp”）的参与者的血浆样本中分离 HDL。为了评估 HDL 的血管生成能力，用人冠状动脉内皮细胞用人血白蛋白（PBS，阳性对照）或分离的 HDL 处理，然后暴露于高葡萄糖（25mmol/L）条件下。用实时定量 PCR 检测 HDL 级分、血浆和处理的人冠状动脉内皮细胞中两种抗血管生成 miRNA（miR-181c-5p 和 miR-223-3p）和一种促血管生成 miRNA（miR-27b-3p）的表达水平。使用 Matrigel 管形成测定法评估体外内皮小管形成。
令人惊讶的是，我们发现糖尿病并发症的澳大利亚土著人群的 HDL 中的抗血管生成 miRNA miR-181c-5p 水平比健康人群（100±121%，p<0.05）和 T2DM 人群（82±77%，p<0.05）高 14 倍（1454±1346%）。有趣的是，我们观察到 HDL 相关的 miR-181c-5p 水平与疾病严重程度呈正相关（p=0.0020）。在高葡萄糖条件下，用 rHDL、健康 HDL 和 T2DM HDL 处理的细胞增加了小管（rHDL：136±8%，p<0.01；健康 HDL：128±6%，p<0.01；T2DM HDL：124±5%，p<0.05）和分支点（rHDL：138±8%，p<0.001；健康 HDL：128±6%，p<0.01；T2DM HDL：127±5%，p<0.01）的数量，并伴有缺氧血管生成转录因子 HIF1A 的 mRNA 水平升高（rHDL：140±10%，p<0.01；健康 HDL：136±8%，p<0.01；T2DM HDL：133±9%，p<0.05）。然而，用 T2DM+Comp HDL 处理的细胞没有观察到这种血管生成能力的增加（小管数量：113±6%，p=0.32；分支点：113±5%，p=0.28；HIF1A：117±6%，p=0.43），这可能归因于细胞中 miR-181c-5p 水平的升高（T2DM+Comp HDL：136±7%比 PBS：100±9%，p<0.05）。
总之，患有糖尿病并发症的澳大利亚土著人群的 HDL 具有降低的血管生成能力。这种损伤与抗血管生成 miR-181c-5p 的表达增加有关。这些发现为更好地告知疾病严重程度的临床诊断提供了依据，并有可能纳入靶向、个性化的 HDL-miRNA 干预治疗，以预防澳大利亚土著人群中糖尿病血管并发症的进一步发展或逆转。
