Introduction
Uncontrolled hyperglycaemia has long been recognized as a major risk factor in the development of vascular complications in diabetic patients [
1]. Diabetic macrovascular complications are involved in ischemic heart disease [
2], peripheral vascular disease [
3] and thromboembolic stroke [
4]. These complications are major contributors to the morbidity and mortality associated with diabetes [
5]. Another important diabetic feature are vascular lesions which involves impaired endothelial-dependent vasomotor responses and significant alterations in endothelial growth, survival and migration induced by exposure of vascular endothelial cells to high glucose [
6‐
8]. A causal link between diabetic hyperglycaemia and the development of macrovascular complications has been established early in the disease onset and tight glucose control in diabetic patients have been shown to reduce the progression of disease [
9].
Endothelial cells play critical roles in vascular biology, being both the protective inner lining of vessels and the local site for oxygen delivery to all tissues. Endothelial damage or dysfunction is considered a critical initiator of large vessel diseases such as atherosclerosis [
8]. An intact endothelial cell monolayer modulates local haemostasis and thrombolysis and provides a non-permeable barrier protecting vascular smooth muscle cells (VSMCs) from circulating growth-promoting factors [
10]. Vascular endothelial cell proliferation and migration are vital in many physiological and pathological processes, such as angiogenesis and healing of the injured endothelium [
11].
It has been shown in previous investigations that high glucose exposure in human umbilical vein cells (HUVEC) impairs endothelial function such as insulin signalling [
12] and expression of a host of proteins involved in thrombosis and blood viscosity [
13]. A number of in vitro studies focused on the effects of high glucose concentration on growth and survival of various types of endothelial cells (EC), including HUVECs [
7,
14], human pulmonary artery EC [
15], human dermal microvascular EC [
16], aortic EC [
17] and retinal EC [
18,
19]. However, conflicting results in EC properties under high glucose [
20,
21] make the interpretation of these results difficult. These conflicting reports may be explained by differences in species, macrovascular ECs versus microvascular ECs, or changes in experimental conditions, but most likely reflects fundamental differences in EC cell type.
Epidemiological studies show that high-density lipoprotein (HDL) is antiatherogenic and an independent protective factor for coronary artery diseases [
22]. HDL and its major protein constituent apolipoprotein A-1 (apoA-1) play major roles in mediating reverse cholesterol transport (RCT), an important atheroprotective mechanism. HDL has many other functions, including removal or detoxification of oxidized sterols/phospholipids and its anti-inflammatory, antioxidant and antithrombotic activities [
23‐
25]. HDL also exhibits potent endothelial protective and reparative capabilities [
10,
24]. HDL has been shown to promote endothelial cell migration and protect it from cellular apoptosis as well as elevate nitric oxide (NO) production through increases in endothelial NO synthase (eNOS) expression and activity [
10,
26]. However, the efficiency of HDL on high glucose-triggered dysfunction in ECs and the related signalling pathways is still to be fully elucidated.
The present study aimed to determine if HDL can attenuate dextrose-induced high glucose impaired HUVEC proliferation and migration as well as the activation of the key transcription factors ERK, p38 and Akt that regulate these functions. We used dextrose as this is the most common, naturally occurring isoform of glucose. We report that dextrose inhibits EC migration, proliferation and the phosphorylation of ERK, p38 and Akt. Coincubation with HDL completely mitigates these effects. Our findings provide a greater understanding of the endothelial protective effects of HDL, with implications for the treatment of diabetic vascular complications.
Discussion
We report that HDL is able to attenuate high glucose-impaired endothelial cell function and signalling. Our studies show that dextrose-induced high glucose significantly suppresses the important endothelial functions of proliferation and migration, as well as the related ERK, p38 and Akt signalling pathways. Coincubation with HDL is able rescue these impairments to normo-glucose levels. These findings have implications for the therapeutic modulation of endothelial repair by HDL in diabetes.
The cardio-protective role of HDL has been observed for decades [
22‐
25], but there are gaps in knowledge regarding its effect on HUVEC proliferative and the related cell signalling pathway in the settling of early high glucose insult. The protein kinase Akt is a multifunctional regulator of cell growth and survival [
30,
31]. Akt is primarily activated when the threonine 308 (Thr
308) and serine 473 (Ser
473) residues are phosphorylated by PI3-K, which in turn then activate Akt serine/threonine kinase activity. In this study, the level of phosphorylated Akt at Ser
473 was significantly inhibited by dextrose-induced high glucose, but rescued by coincubation with HDL. Pre-treatment of HUVECs with the Akt inhibitor LY294002 almost completely inhibited HDL-induced Akt phosphorylation providing further evidence that this is mediated by Akt. These findings are consistent with other studies that have found reconstituted HDL (rHDL) augments Akt phosphorylation [
26]. In the early stages of a high glucose insult, suppression of the activation of Akt likely plays an important role in the impairment of cell survival [
32]. It has been shown that hyperglycaemic exposure results in decreased viability and attenuated proliferation of endothelial cells and this is the result of downregulation of platelet-derived growth factor C and its receptor [
33]. The activation of Akt is downstream of this axis. Therefore, the maintenance of Akt by HDL in high glucose suggests HDL may play a role in improved endothelial integrity in hyperglycaemia. This concept is consistent with previous studies [
6,
34].
The processes of endothelial cell proliferation and migration are crucial to both neovascularisation and a successful response to vascular injury [
9]. High glucose induced endothelial dysfunction is known to not only involve impaired endothelial cell proliferation but also cell migration. ERK and Akt (i.e. MAPK) pathways promote endothelial cell proliferation and migration in response to various extracellular stimuli [
35]. Major subfamilies of structurally related MAPKs have been identified in mammalian cells, including ERK1/2 MAPK, p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPKs) [
11]. It has been shown that HDL-stimulated endothelial cell migration is driven by the activation of Src kinase, PI3K-kinase and p44/42 MAP kinase [
26]. The current study now shows that HDL rescues the high-glucose-impaired HUVECs migration. Consistent with this we found that HDL reversed the inhibition of p-38 activation in high glucose, a key transcription factor for the promotion of cell migration. The use of a specific p38 inhibitor in this study attenuated the induction of p-p38 by HDL in high glucose, confirming the importance of its role. These results demonstrate a role for HDL in the rescue of high glucose-impaired cell migration and proliferation. In support of our findings, a recent study [
36] found rHDL was able to restore angiogenesis in a diabetic murine model of hind limb ischemia. This was shown in vitro to be mediated by the scavenger receptor (SR-BI) which led to the activation of the PI3 K/Akt signalling pathway. On the flip side, dysfunctional HDL isolated from diabetic patients, has diminished capacity to stimulate HUVECs proliferation and migration [
37]. These investigators showed that dysfunctional HDL induced Akt phosphorylation initially but this was attenuated with time as SR-BI was down regulated by the dysfunctional lipoprotein. Glycated HDL was also shown to attenuate NO production and increase reactive oxygen/nitrogen species in human aortic endothelial cells [
38]. Other properties of HDL were also compromised when HDL is glycated, including its antioxidant and anti-inflammatory properties [
39].
Clinical studies using infusions of reconstituted HDL (rHDL, apoA-1 + phospholipid) have already demonstrated promising findings in diabetic patients. For example, a single infusion of rHDL into type 2 diabetes mellitus (T2DM) patients was found to reduce platelet activation [
40]. Other studies have found that rHDL infusions in T2DM patients elevate circulating endothelial progenitor cell number [
41] and increase the anti-inflammatory properties of endogenous HDL [
42]. These studies report that the kinetics of an rHDL infusion are such that there is a steady increase in circulating HDL-cholesterol (50%) and apoA-1 concentrations (> twofold) during the 4 h infusion period. Then out to 72 h post-infusion, the concentration of apoA-1 drops by ~ 50% while the HDL-cholesterol remains elevated. These studies suggest that in the context of diabetes, infusions of rHDL are likely to provide beneficial effects on the endothelium. Whilst the half-life of apoA-1 and HDL-cholesterol may be viewed as relatively short, it must sufficient to impart significant changes during that time.
This interaction between HDL, SR-BI, endothelial function and its signalling pathways may have important implications not only in CVD but also in cancer [
43]. Depending on the cancer type, SR-BI expression can correlate with survival rates. SR-BI activation by HDL play a critical role in signalling that stimulates endothelial cell proliferation and migration, important for tumour growth. HDL have been shown to activate Akt and ERK1/2 pathways in breast cancer while knockdown and pharmacological inhibition of SR-BI resulted in a decrease in these pathways [
44].
In conclusion, we show that HDL protects endothelial cells from high glucose-impaired cell proliferation and migration. Additionally, HDL rescues high glucose-impaired activation of ERK, p38 and Akt signalling pathways. These findings with HDL suggest that it could be considered as a future therapeutic target to protect against diabetic vascular complications.
Authors’ contributions
XC conducted the experiments, analysed and interpreted the results and wrote the manuscript. MD provided experimental support and was a major contributor in writing the manuscript. PJP provided intellectual input. CAB was a major contributor to intellectual input, writing and editing the manuscript. SJN provided intellectual and editorial input. All authors read and approved the final manuscript.