Sage Journals: Discover world-class research

Abstract

In healthy individuals, the vascular endothelium regulates an intricate balance of factors that maintain vascular homeostasis and normal arterial function. Functional disruption of the endothelium is known to be an early event that underlies the development of subsequent cardiovascular disease (CVD) including atherosclerosis and coronary heart disease. In addition, the rising global epidemic of type 2 diabetes is a significant problem conferring a significantly higher risk of CVD to individuals in whom endothelial dysfunction is also notable. This review first summarises the role of endothelium in health and explores and evaluates the impact of diabetes on endothelial function. The characteristic features of insulin resistance and other metabolic disturbances that may underlie long-term changes in vascular endothelial function (metabolic memory) are described along with proposed cellular, molecular and epigenetic mechanisms. Through understanding the underlying mechanisms, novel targets for future therapies to restore endothelial homeostasis and ‘drive’ a reparative cellular phenotype are explored.

Keywords

Diabetes endothelial function endothelial dysfunction metabolic memory microRNA novel therapies

Introduction

Largely owing to the vast quantities of energy-dense food consumed in the Western world, coupled with increasingly sedentary lifestyles, diabetes mellitus (DM) is considered to be a public health problem of epidemic proportions.^1,2 In the United Kingdom alone, 2.9 million people are diagnosed with the disease, with an estimated 850,000 more undiagnosed.³ DM is recognised as an independent risk factor for cardiovascular disease (CVD),⁴ even when under glycaemic control. With up to 75% of mortality in diabetic patients arising from CVD,⁵ this is an issue of utmost importance. Endothelial cell (EC) dysfunction is associated with both diabetes and the pathogenesis of CVD.⁶ Establishing the causative mechanisms linking these factors could provide essential insights and inform novel therapeutics.

The aim of this review is to explore the relationship between DM and EC function/dysfunction, focussing predominantly on cellular and molecular mechanisms as opposed to clinical manifestations.

Physiological role of endothelium

Forming a vast interface between the blood and surrounding tissues, the endothelium forms a monolayer comprising the innermost lining of blood vessels. Advances over recent decades have revealed the complexity of this semi-permeable membrane and its key role in maintaining vascular homeostasis.⁷ While facilitating the passage of substances such as nutrients and leukocytes across the vessel wall, the endothelium secretes numerous mediators necessary for normal vascular functioning including those that regulate vascular tone, coagulation, modulate immune responses and control vascular cell growth.⁸

Arguably, the most significant endothelium-derived mediator is nitric oxide (NO), which plays multiple roles in preventing atheroma formation.⁹ Produced by the action of endothelial nitric oxide synthase (eNOS), NO diffuses into neighbouring vascular smooth muscle cells (SMC), activating guanylyl cyclase and producing cyclic guanosine monophosphate (cGMP) and activating kinases responsible for vascular relaxation.⁸ Vasodilation via NO synthesis is well documented in vitro and in vivo.^10–12 Additionally, NO inhibits platelet aggregation, SMC proliferation and nuclear transcription of leukocyte-adhesion molecules including vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM). Arterial wall shear stress¹³ and coupling of agonists such as acetylcholine and bradykinin to endothelial-bound receptors⁸ are potent stimuli for NO synthesis. Although these stimuli act via different intracellular mechanisms, each increases NO production via enhanced expression of eNOS. Other important vasodilatory factors are endothelium-derived hyperpolarizing factor (EDHF) and prostacyclin (PGI₂), the latter also playing a role in platelet inhibition.⁸

Conversely, the endothelium is able to secrete the potent vasoconstrictor endothelin (ET-1), which through its pro-inflammatory and mitogenic effects augments the pathogenesis of CVD.¹⁴ Other endothelial-derived vasoconstrictors include prostaglandin H₂ (PGH₂), thromboxane A₂ (TXA₂) and reactive oxygen species (ROS). EC-bound angiotensin-converting enzyme (ACE) catalyses production of the vasoconstrictive angiotensin II, and ACE inhibitors are commonly used to decrease blood pressure.¹⁵ Under physiological conditions, the endothelium maintains a fine balance between anti- and pro-thrombotic states. While NO, prostacyclin and thrombomodulin favour blood fluidity and are dominant under basal conditions, ‘activated’ endothelium drives an opposing pro-thrombotic state. Under these conditions, plasminogen activator inhibitor-1 and thromboxane, together with von Willebrand Factor (vWF), a key constituent of the coagulation cascade, are deleterious.^8,16

Through NO production, the endothelium plays a crucial role in preventing leukocyte adhesion; however, when ‘activated’, expression of adhesion molecules aid the passage of leukocytes across the vascular wall promoting a pro-inflammatory environment. Under these conditions, EC produce pro-inflammatory cytokines, including tumour necrosis factor-alpha (TNF-α), which further augment inflammation.¹⁷

Finally, the endothelium plays a key role in angiogenesis initiated by tissue growth factors, particularly vascular endothelial growth factor (VEGF). The VEGF receptor is coupled to activation of mitogen-activated protein kinase (MAPK) signalling, promoting angiogenesis by increasing nuclear transcription of relevant genes. This mechanism is counteracted by the anti-angiogenic factors angiostatin and thrombospondin, which suppress angiogenesis unless required.⁸

Endothelial dysfunction

Endothelial dysfunction refers to inability of the endothelium to regulate vascular homeostasis, and essentially describes ‘tipping’ of the physiological balance in favour of vasoconstrictive, pro-inflammatory and pro-thrombotic effects⁵ that promote atherosclerosis. As such, abnormalities in endothelial function are detected early in the development of CVD, often before symptoms are clinically evident.¹⁶

Given the vast range of vasoprotective effects of NO, the term endothelial dysfunction generally refers to reduced NO bioavailability, through decreased eNOS expression.¹⁶ Procedures employed to evaluate endothelial dysfunction measure blunted vasodilatory responses to certain agonists.⁵ Indeed, by this method, impaired endothelial-dependent vasodilation in patients with confirmed CVD,^18–21 and importantly in those who carry risk factors for future CVD events,²² has been demonstrated. This suggests that endothelial dysfunction is the commonality by which these risk factors, particularly those associated with the metabolic syndrome, lead to CVD development. This point is underscored by reports that endothelial dysfunction appears to reverse when lifestyle changes and modulatory drug therapies are implemented.²³ DM is one of the aforementioned risk factors whose association with endothelial dysfunction will now be evaluated.

Endothelial dysfunction in DM

Current evidence links endothelial dysfunction to type 1 DM (T1DM) and type 2 DM (T2DM), through demonstration of impaired endothelial-dependent vasodilation.^24–26 Despite many proposed mechanisms for this relationship, the definitive pathogenesis remains unclear, possibly because diabetes patients usually display multiple homeostatic imbalances alongside the typically described hyperglycaemia. Such disturbances induce endothelial dysfunction independently of the presence of diabetes, indicating a multifactorial aetiology rather than hyperglycaemia per se.^27–29 The following sections will evaluate these factors.

Selective insulin resistance

In addition to its role in glucose homeostasis, insulin activates intracellular signalling pathways critical for maintenance of healthy endothelium, the most vasoprotective of which is the phosphoinositide-3 kinase (PI3-K)/Akt pathway (Figure 1). Through this pathway, enhanced eNOS expression and activation (phosphorylation) are mediated.^30,31 In contrast, activation of the MAPK/extracellular signal-regulated kinase (ERK) pathway, promotes expression of ET-1 and a less beneficial outcome of cellular proliferation.³² In physiological circumstances, the PI3-K pathway predominates to regulate normal vasomotor control,³³ but it is well accepted that in insulin resistance that characterises T2DM, there is a selective deficiency of the PI3-K pathway, while signalling via the MAPK pathway is unaffected.^34,35 Moreover, this selective resistance promotes hyperinsulinaemia, resulting in further stimulation of MAPK signalling.³⁶ The outcome therefore is that of pro-atherogenic signalling that is clearly detrimental to the vasculature.³⁷ This signalling deficiency has been demonstrated experimentally in obese Zucker rats, in which defective PI3-K/Akt signalling consequently resulted in decreased NO bioavailability.³⁴

Figure 1.

Intracellular insulin signalling pathways: Upon binding to its receptor, insulin initiates two key intracellular signalling pathways. Activation of PI3-Kinase dominates under physiological conditions resulting in a net anti-atherogenic effect. In insulin resistance, selective deficiency in the PI3-kinase pathway leads to a dominant MAP kinase pathway, whose effects are predominantly pro-atherogenic.

Pro-inflammatory signalling

T2DM is associated with a state of chronic systemic inflammation; elevated levels of circulating inflammatory markers are observed in patients with diabetes and obesity^38,39 and are believed to confer a pathological EC phenotype. A diverse range of harmful stimuli have been shown to contribute to vascular complications in diabetes. These include pro-inflammatory cytokines, chemokines, adhesion molecules and transcription factors (summarised in Table 1) and have been reviewed recently.⁴⁰

Table 1.

Inflammatory components of diabetes complications.

Inflammatory cytokines

Interleukins IL-1, IL-6, IL-18

Tumour necrosis factor-alpha (TNF-α)

C-reactive protein (CRP)

Adhesion molecules

Intercellular adhesion molecule 1 (ICAM-1)

Vascular cell adhesion molecule-1 (VCAM1)

E-selectin

Chemokines

CCL-2 (MCP-1)

CX3CL1 (fractalkine)

CCL5 (RANTES)

Transcription factors

NFκB

Toll-like receptors

TLR2

TLR4

Profibrotic cytokines

Transforming growth factor beta (TGF-β)

Connective tissue growth factor (CTGF)

Key categories of inflammatory mediators associated with vascular complications of diabetes, indicating the principal components of each category. There is widespread evidence for inflammatory cytokines, adhesion molecules, chemokines and transcription factors. More recent data support roles for profibrotic cytokines and toll-like receptors that appear to mediate responses to a number of ‘diabetogenic’ stimuli (reviewed in Forbes and Cooper⁴⁰).

Animal models of obesity have identified adipose tissue as a site of inflammatory cytokine production, with accompanying elevated plasma levels of TNF-α.^41,42 TNF-α activates nuclear factor κB (NFκB), a transcription factor able to further stimulate expression of inflammatory genes.⁴³ NFκB is also activated by free fatty acids (FFA) and the receptor for advanced glycation end-products (RAGE), both of which are characteristic of the diabetic milieu.¹⁶ Evidence suggests that overexpression of either TNF-α or IκB kinase mediates the development of insulin resistance. Obese mice with null mutations in the TNF-α gene display significantly improved insulin sensitivity.⁴⁴ Indeed, NFκB that is permanently present in the cell could be viewed as the ‘first responder’ to many inflammatory signals due to rapid activation of its downstream pathways. More recently, new pharmacological roles for the salicylate anti-inflammatory drugs have emerged regarding their ability to decrease insulin resistance via inhibition of IκB kinase. Preliminary results from the TINSAL-T2D (Targeting INflammation Using SALsalate in T2DM) clinical trial advocates their use in glucose-lowering, presumably as a consequence of improved insulin sensitivity.⁴⁵ The association of insulin resistance with inflammation implies that the latter adversely impairs the NO-producing PI3-K/Akt pathway, ultimately reducing NO bioavailability. Exposure of cultured EC to TNF-α leads to impaired eNOS expression,⁴⁶ inferring that reduced bioavailability of NO nullifies its otherwise significant anti-inflammatory role, thus augmenting formation of inflammatory atherosclerotic lesions.

Oxidative stress

Oxidative stress is commonly implicated as an important unifying mechanism in conferring endothelial injury (see Figure 2). The term refers to the accumulation of ROS such as superoxide anion (O₂^·−), when production exceeds the capability of the anti-oxidant system to remove them.⁴⁷ A key adverse effect of oxidative stress is diminished NO bioavailability, either through direct degradation by ROS, or alterations in the functional capacity of eNOS. Direct reaction of NO with O₂^·− yields peroxynitrite (ONOO⁻), a highly potent oxidant capable of altering the functions of multiple intracellular molecules, including those related directly to the NO pathway.⁴⁸ Additionally, ONOO⁻ serves to uncouple eNOS from its critical co-factor tetrahydrobiopterin (BH₄), resulting in preferential formation of O₂^·− in the place of NO and hence further exacerbation of the problem.⁴⁹ In the setting of insulin resistance, experimental evidence has implicated endogenous ONOO⁻ as a down-regulator of the PI3-K/Akt pathway.^50,51

Figure 2.

Production of peroxynitrite in endothelial cells: In healthy cells, eNOS catalyses synthesis of NO from l-arginine. The factors listed (bullet-pointed box) produce O₂^·−, which reacts with NO to produce ONOO⁻. This uncouples eNOS from BH₄, which enhances O₂^·− production, and increased ONOO⁻.

Aside from uncoupled eNOS, significant sources of O₂^·− include xanthine oxidase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondria.⁴⁷ Mitochondria produce negligible ROS in physiological circumstances, but greatly increase O₂^·− output when high glucose concentrations increase the proton gradient within the electron transport chain.⁵² Recent evidence has also implicated mitochondrial dynamics, namely, increased mitochondrial fission and fragmentation, as contributing factors to ROS production in DM.⁵³

Anti-oxidant therapy

Anti-oxidant therapies aiming to reduce the detrimental effects of ROS have so far yielded conflicting results. Studies of diabetic vessels in vivo have demonstrated improvements in NO-mediated vasodilation in response to anti-oxidants such as superoxide dismutase (SOD).^54,55 Additionally, the anti-oxidant properties of vitamin C have been shown to effectively augment endothelial-dependent vasodilatation in several studies performed on human forearm vessels, in the setting of diabetes and hypertension.^56–58 Despite promising experimental data, large-scale clinical trials investigating the effects of anti-oxidant vitamins in patients with diabetes failed to report any benefits.^59,60 Although conducting clinical trials is a logical progression following experimental evidence, the discrepancies in these results may indicate that the benefits of anti-oxidant therapies are too short term to affect mortality outcomes. The vastly greater sample size and follow-up period in clinical trials indicates that these results should take precedent when interpreting the effects of anti-oxidant therapy. However, further experimental evidence regarding the longer term outcomes of anti-oxidant therapies is required.

Protein kinase C

Protein kinase C beta (PKCβ) is an endothelial isoform of the serine/threonine kinase family. A host of evidence implicated this enzyme as a chief contributor to endothelial dysfunction observed in DM.⁶¹ PKCβ is activated by diacylglycerol (DAG), which is typically produced subsequent to ligand-receptor binding.⁶² However, in conditions of high circulating FFA and hyperglycaemia, a novel route for the chronic activation of DAG has been proposed, via de novo synthesis from glucose.⁶³ Significantly increased levels of PKC and DAG have been identified in animal models of diabetes.⁶³ Upon activation, PKC induces several intracellular effects (Figure 3), many of which have been experimentally identified using PKC inhibitors such as ruboxistaurin.^43,64 Such alterations include amplified expression of ET-1, VCAM and ICAM.⁶² PKC also activates vascular NADPH oxidase, a significant source of O₂^·− and subsequent oxidative stress in the endothelium. Indeed, inhibition of PKC has been shown to diminish O₂^·− in the diabetic vasculature.⁶⁵ A trial in healthy humans indicated that PKC inhibition prevented hyperglycaemia-induced impairment of endothelial-dependent vasodilation.⁶⁶ Disappointingly, a later trial by the same authors showed that this benefit was not apparent in T2DM patients,⁶⁷ suggesting that further work is required to delineate the role of PKC in T2DM. Additional effects of PKC in the endothelium include activation of NFκB, alterations in eNOS expression and inhibition of the PI3-K signalling pathway.⁴³

Figure 3.

Activation and effects of protein kinase C (PKC): High levels of free fatty acids and hyperglycaemia activate DAG, which in turn activates PKC. This increases expression of ET-1, VCAM, ICAM, NFκB and NADPH oxidase. NADPH oxidase activation results in decreased NO bioavailability. PKC may have a role in PI3-kinase pathway inhibition. Ruboxistaurin inhibits PKC activation.

Hyperglycaemia

The hallmark of DM, chronic hyperglycaemia, has been implicated in the development of endothelial dysfunction via four principal mechanisms: PKC activation, activation of the hexosamine and polyol pathways and formation of advanced glycation end-products (AGEs).⁶⁸ These pathways are believed to mediate vascular dysfunction through the unifying mechanism of ROS overproduction, most notably increases in O₂^·−.⁶⁹ Briefly, the polyol and hexosamine pathways display low affinity for glucose in the physiological state; however, intracellular hyperglycaemia leads to shunting of glucose through these pathways, with resultant increases in PKC activation, changes in gene expression and protein function.⁶⁸ AGEs initially arise from intracellular hyperglycaemia and subsequent non-enzymatic reactions, causing alterations in both intra- and extracellular proteins, as well as modifications to the extracellular matrix.⁷⁰ Additionally, activation of RAGE on EC influences ROS production and leads to NFκB activation, which induces atherogenic gene expression.^71,72 Collectively, these mechanisms have been shown to increase the proton gradient across the inner mitochondrial membrane, consequently increasing O₂^·− as previously discussed. SOD-enabled inhibition of mitochondrial ROS is capable of inhibiting increases in all these mechanisms.⁶⁸

Metabolic memory: role of epigenetics

The mechanisms described suggest routes by which environmental factors such as diet-induced hyperglycaemia may induce endothelial dysfunction, and it would be easy to believe that correcting these imbalances would promptly normalise endothelial function. This is not the case. Indeed, follow-up trials succeeding the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) propose the existence of a so-called metabolic memory, in which the effects of either prolonged or transient changes in glycaemia persist long after these have been re-adjusted.^73–75 Not only does this suggest that intensive glycaemic control cannot completely reverse pre-established hyperglycaemia-induced vascular complications, but importantly showed that the benefits of intensive therapy were maintained for many years despite a return to often inferior glycaemic control.⁷³ Such observations have been confirmed in vitro and in vivo, including EC-specific studies.^76–78 Together, these data imply that heritable alterations in cell phenotype may be induced by diabetogenic stimuli, particularly hyperglycaemia. While this was once thought to be due to mutations in mitochondrial DNA,⁶⁸ more recent evidence has implicated epigenetic mechanisms as the underlying cause of this phenomenon–alterations in gene expression without changes in DNA sequence.⁷⁹ Indeed, epigenetic studies are shedding new light on how environmental factors influence gene expression and the associated susceptibility to T2DM and increased CVD.⁷⁹ Three highly connected pathways provide the foundation for epigenetic theories, namely, post-translational histone modifications, DNA methylation and non-coding RNA-based mechanisms, detailed studies of which are clearly warranted to investigate endothelial-specific mechanisms that may inform novel therapeutic targets.

Histone modifications

Post-translational modifications of histones in chromatin alter the conformation of DNA, providing or denying access to specific sites that enhance or retard gene transcription, respectively. Histone modifications (e.g. acetylation/deacetylation; methylation/demethylation) are regulated by a family of enzymes that are responsible for their transfer [histone acetyltransferases (HATs), histone methyltransferases (HMTases)] or removal [histone deacetylases (HDAC), histone demethylases (HDM)].⁷⁹ Several studies have been performed specifically in ECs, where changes in the expression and activity of these enzymes have been linked to diabetic complications. For example, specific histone methylations are associated with the chronic inflammatory phenotype in EC exposed to high glucose concentrations.⁸⁰ Transient exposure of human and bovine endothelium to hyperglycaemia led to transcriptional activation of inflammatory NFκB-dependent promoters that were sustained even following restoration of normoglycaemia.^80,81

DNA methylation

Studies examining the role of DNA methylation in diabetes and vascular complications are few, although altered patterns of DNA methylation have been associated with atherosclerosis⁸² and in the endothelium itself.⁸³ Both global and gene-specific methylation is believed to contribute to the metabolic memory of diabetic complications. Covalent methylation of cytosine residues is a stable DNA modification that persists throughout experimental processing,⁸⁴ providing a robust platform on which to base future investigations. Akin to modifications in histones, DNA methylation can confer persistent alterations in gene expression and a clear understanding of the complex mechanisms involved will be of immense value. High fat feeding of obese rats has been reported to induce hypermethylation of particular genes,⁸⁵ so it is reasonable to consider that this phenomenon may also exist in human diabetic individuals, and specifically at the level of the endothelium.

microRNAs

Another area of intense interest in which gene expression can be post-transcriptionally modified is by microRNAs (miRs), the most widely studied of the non-coding RNAs. miRs are short RNAs that inhibit expression of a range of target proteins through mRNA degradation or translational repression. As regards ECs, in vitro studies have exposed a complex network of miRs with regulatory roles in proliferation and migration (miR-320,⁸⁶ miR-503,⁸⁷ miR221/222^88–90), inflammation (miR-10a,⁹¹ miR-126 ⁹²) and angiogenesis (miR221/222,⁹³ miR-126,⁹⁴ miR-210,⁹⁵ miR-21⁹⁶). These roles may be divergent in physiological circumstances, for example, specific miRs are capable of exerting protective, pro-angiogenic effects,^94,95 while others confer more unfavourable anti-angiogenic properties.^93,96 Furthermore, a distinct signature of miR dysregulation has been proposed to exist in DM,⁹⁷ whereby high glucose conditions may induce over or under expression of detrimental or beneficial miRs, respectively, thereby contributing to the development of vascular complications. In such a way, upregulation of miR-320 was observed in EC exposed to hyperglycaemia, with a resultant reduction in cellular proliferation and migration.⁸⁶ Importantly, inhibition of the overexpressed miRs restored EC regenerative functions.⁸⁶

The stable and consistent levels of plasma miRs in healthy individuals has identified them as potential biomarkers for diseases such as T2DM,⁹⁸ not only for evaluating disease progression but also to detect risk of future disease development. In this way, plasma profiling of T2DM patients has revealed altered levels of miRs, which may contribute to a pathophysiological state.⁹⁹ Highly reported miRs that appear to modulate EC function either beneficially or detrimentally are presented in Table 2.

Table 2.

Principal endothelial microRNAs and their involvement in cell function.

Micro-RNA	Reference	Functional endothelial cell effects	Specific targets	Effect of high-glucose conditions and/or T2DM
miR-320	Wang et al.⁸⁶	↓ proliferation, ↓ migration, ↓ angiogenesis	VEGF, FGF, IGF-1	Up-regulated *Reduced levels observed in plasma of T2DM patients (99)
miR-503	Caporali et al.⁸⁷	↓ proliferation, ↓ migration, ↓ angiogenesis, ↓ cell cycle progression	CDC, CCNE1	Up-regulated
miR-221/222	Togliatto et al.,⁸⁸ Liu et al.,⁸⁹ Li et al.,⁹⁰ Dentelli et al.⁹³	↓ proliferation, ↓ migration, ↓ angiogenesis, ↑ apoptosis	Ckit, p27kip1, p57kip2	Up-regulated/down-regulated *Conflicting results
miR-126	Wang et al.,⁹⁴ Fish et al.⁹²	↓ inflammation,↑ angiogenesis	VCAM1, SDF-1, PAK1, SPRED1, PI3KR2	Not reported *Reduced levels observed in plasma of T2DM patients (99)
miR-210	Fasanaro et al.⁹⁵	↑ angiogenesis	Ephrin A3	Not reported
miR-21	Sabatel et al.⁹⁶	↑ inflammation, ↓ angiogenesis	ERK, PTEN, RhoB, PPARα, Bcl2, PDCD4	Not reported *Reduced levels observed in plasma of T2DM patients (99)
miR-10a	Fang et al.⁹¹	↓ inflammation	HOXA, MAP3K7, bTRC	Not reported

VEGF: vascular endothelial growth factor; FGF: fibroblast growth factor; IGF: insulin-like growth factor; VCAM: vascular cell adhesion molecule; SDF: stromal cell–derived factor; PAK1: P21-activated kinase 1; SPRED1: sprouty-related protein 1; PI3K2: phosphoinositol-3 kinase regulatory subunit 2; ERK: extracellular signal-regulated kinase; PTEN: phosphatase and tensin homolog; PPAR: peroxisome proliferators activated receptor; PDCD4: programmed cell death 4; HOXA: homeobox A1; MAP3K7: mitogen-activated protein kinase 7; bTRC: β-transducin repeat-containing gene.

Effect of miRs on aspects of EC function, together with known specific molecular targets of each miR. Where reported, changes in miR expression in EC subjected to high-glucose conditions or altered plasma levels in patients with T2DM are documented.

In summary, while the study of epigenetic mechanisms associated with diabetes and its complications has gained momentum in recent years, there is still much to be learned. Moreover, the complexity of these interactions will pose considerable challenges for their elucidation.

Current therapies for DM

Most current treatment options for reducing T2DM-related cardiovascular risk focus on controlling metabolic imbalances such as hyperglycaemia, hypertension and raised levels of FFA. While insulin is the obvious treatment of choice in T1DM, first line action for T2DM is through lifestyle changes. The large-scale Look AHEAD (Action for Health in Diabetes) trial¹⁰⁰ reported, however, that while weight loss in DM patients conferred multiple health benefits, cardiovascular risk remained unchanged.¹⁰¹ Oral hypoglycaemic drugs aim to correct the pathophysiological features that lead to T2DM generally by targeting the key characteristic of insulin resistance. Essentially, these drugs act by either increasing insulin production (insulin secretagogues such as sulphonylureas, glinides and more recently incretin mimetics) or increasing target tissue sensitivity (insulin sensitisers such as metformin and thiazolidinediones).¹⁰² While there is genuine optimism that new oral therapies may also contribute to reducing cardiovascular risk, a systematic review showed metformin to be the only current agent with such potential.¹⁰³ While able to reduce levels of glycosylated haemoglobin and microvascular disease to some degree, oral antidiabetic drugs still fall short of ameliorating cardiovascular complications in the short or medium term, and importantly have been implicated in increased mortality from hypoglycaemia¹⁰⁴ or myocardial infarction.¹⁰⁵ Nevertheless, trials have highlighted the importance of early glycaemic intervention to reduce vascular complications, likely due to the effects of metabolic memory.

Approach to future therapies

This review has highlighted the critical involvement of endothelial dysfunction in the pathogenesis of vascular disease in DM, in which diabetic mediators inflict a pro-atherogenic, inflammatory phenotype, often with limited or delayed reversibility. Owing to the silent nature of insulin resistance and hence an inevitable problem in rapid diagnosis, early intervention is difficult. Manipulation or inhibition of some of the described mechanisms may provide novel avenues for therapeutic intervention. However, the transition from this experimental stage through to production of safe and effective drugs is not easy, and requires results from valid and reliable large-scale clinical trials. Such clinical trials have already revealed disappointing outcomes regarding the use of anti-oxidants in alleviating oxidative stress;^59,60 however, they should not deter further anti-oxidant therapy trials. It is likely that a ‘prevention rather than cure’ approach is preferable in the management of ROS, with the enzymatic sources of superoxide anion a promising target for future therapeutics.¹⁶ Other mechanisms meriting further investigation include PKC inhibition⁶⁵ and the previously mentioned use of salicylate anti-inflammatory drugs.⁴⁵

Rapidly accumulating evidence supports the notion that miRs are cell-type specific and differentially expressed, and may actually play a causative role in diabetes and its related vascular complications.⁹⁷ Recent evidence suggests that miRs can be communicated between cells and specifically, EC-derived plasma miRs are reportedly able to regulate the biological functions of not only other EC, but also vascular SMC and leukocytes (reviewed in Yamakuchi¹⁰⁶). In future, understanding and exploiting these mechanisms has potential to be translated into defining clinical biomarkers and elucidating specific molecular targets for therapeutics. In the longer term, these may be of value in reversing the devastating outcomes attributed to endothelial dysfunction in diabetes by erasing persistent metabolic memory by ‘re-setting’ the epigenetic cell signature.

Conclusion

Recent advances in knowledge surrounding metabolic memory and the role of epigenetics have provided exciting new avenues for future research. However, there is still much to learn regarding their roles in diabetic vascular complications, and specifically on the EC. To date, in vitro studies of metabolic memory in cultured EC have largely focused on changes in gene expression in response to hyperglycaemia.⁸⁰ However, this review has highlighted the contribution of a complex array of factors other than glucose in the pathogenesis of endothelial dysfunction. For this reason, it is apparent that identifying the presence of a diabetic metabolic memory might be most accurately observed in EC derived from human diabetic vessels. In such a way, we have previously identified inherent functional differences between vascular SMC cultured from patients with and without T2DM,¹⁰⁷ such data offering an explanation for increased venous bypass graft stenosis after revascularisation surgery in these patients. Identification of any sustained functional differences in T2DM-EC may provide further justification for the aforementioned graft failure. Such information would potentially create a foundation, upon which therapeutics to ameliorate endothelial dysfunction could be developed.

Footnotes

Declaration of conflicting interests

The authors declare no conflict of interest.

Funding

Anna C Roberts undertook this work as part of an Intercalated Bachelor of Science degree in Cardiovascular Medicine, funded by the Alumni Footsteps Fund at the University of Leeds.

References

Wang

Luben

Khaw

K-T

. Dietary energy density predicts the risk of clinically incident type 2 diabetes: the EPIC-Norfolk study. Diabetes Care 2008; 31: 2120–2125.

Wilmot

Edwardson

Achana

. Sedentary time in adults and the association with diabetes, cardiovascular disease and death: systematic review and meta-analysis. Diabetologia 2012; 55: 2895–2905.

Diabetes

. What is diabetes? http://www.diabetes.org.uk/Guide-to-diabetes/Introduction-to-diabetes/What_is_diabetes/ (accessed 17 October 2012).

International Diabetes Federation. Diabetes: a major risk factor. http://www.cvd.idf.org (accessed 17 October 2012).

Zou

M-H

. Molecular insights and therapeutic targets for diabetic endothelial dysfunction. Circulation 2009; 120: 1266–1286.

Feener

King

. Endothelial dysfunction in diabetes mellitus: role in cardiovascular disease. Heart Fail Monit 2001; 1: 74–82.

Michiels

. Endothelial cell functions. J Cell Physiol 2003; 196: 430–443.

Levick

. An introduction to cardiovascular physiology. 4th ed. London: Arnold Publishers, 2003.

Yasa

Turkseven

. Vasoprotective effects of nitric oxide in atherosclerosis. FABAD J Pharm Sci 2005; 30: 41–53.

10.

Tronc

Wassef

Esposito

. Role of NO in flow-induced remodeling of the rabbit common carotid artery. Arterioscler Thromb Vasc Biol 1996; 16: 1256–1262.

11.

Gardiner

Compton

Bennett

. Control of regional blood flow by endothelium-derived nitric oxide. Hypertension 1990; 15: 486–492.

12.

Rees

Palmer

RMJ

Schulz

. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 1990; 101: 746–752.

13.

Buga

Gold

Fukuto

. Shear stress-induced release of nitric oxide from endothelial cells grown on beads. Hypertension 1991; 17: 187–193.

14.

Böhm

Pernow

. The importance of endothelin-1 for vascular dysfunction in cardiovascular disease. Cardiovasc Res 2007; 76: 8–18.

15.

Varin

Mulder

Tamion

. Improvement of endothelial function by chronic angiotensin-converting enzyme inhibition in heart failure. Circulation 2000; 102: 351–356.

16.

Tabit

Chung

Hamburg

. Endothelial dysfunction in diabetes mellitus: molecular mechanisms and clinical implications. Rev Endocr Metab Disord 2010; 11: 61–74.

17.

Sumpio

Riley

Dardik

. Cells in focus: endothelial cell. Int J Biochem Cell Biol 2002; 34: 1508–1512.

18.

Förstermann

Mügge

Alheid

. Selective attenuation of endothelium-mediated vasodilation in atherosclerotic human coronary arteries. Circ Res 1988; 62: 185–190.

19.

Werns

Walton

Hsia

. Evidence of endothelial dysfunction in angiographically normal coronary arteries of patients with coronary artery disease. Circulation 1989; 79: 287–291.

20.

Bossaller

Habib

Yamamoto

. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5’-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest 1987; 79: 170–174.

21.

Freiman

Mitchell

Heistad

. Atherosclerosis impairs endothelium-dependent vascular relaxation to acetylcholine and thrombin in primates. Circ Res 1986; 58: 783–789.

22.

Vita

Treasure

Nabel

. Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease. Circulation 1990; 81: 491–497.

23.

Widlansky

Gokce

Keaney

JJF

. The clinical implications of endothelial dysfunction. J Am Coll Cardiol 2003; 42: 1149–1160.

24.

McVeigh

Brennan

Johnston

. Impaired endothelium-dependent and independent vasodilation in patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 1992; 35: 771–776.

25.

Johnstone

Creager

Scales

. Impaired endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus. Circulation 1993; 88: 2510–2516.

26.

Williams

Cusco

Roddy

M-A

. Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J Am Coll Cardiol 1996; 27: 567–574.

27.

Pasimeni

Ribaudo

Capoccia

. Non-invasive evaluation of endothelial dysfunction in uncomplicated obesity: relationship with insulin resistance. Microvasc Res 2006; 71: 115–120.

28.

Steinberg

Chaker

Leaming

. Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance. J Clin Invest 1996; 97: 2601–2610.

29.

Hamburg

Larson

Vita

. Metabolic syndrome, insulin resistance, and brachial artery vasodilator function in Framingham offspring participants without clinical evidence of cardiovascular disease. Am J Cardiol 2008; 101: 82–88.

30.

Kuboki

Jiang

Takahara

. Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo. Circulation 2000; 101: 676–681.

31.

Montagnani

Chen

Barr

. Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser1179. J Biol Chem 2001; 276: 30392–30398.

32.

Ferri

Pittoni

Piccoli

. Insulin stimulates endothelin-1 secretion from human endothelial cells and modulates its circulating levels in vivo. J Clin Endocrinol Metab 1995; 80: 829–835.

33.

Taniguchi

Emanuelli

Kahn

. Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 2006; 7: 85–96.

34.

Jiang

Lin

Y-W

Clemont

. Characterization of selective resistance to insulin signaling in the vasculature of obese Zucker (fa/fa) rats. J Clin Invest 1999; 104: 447–457.

35.

Cusi

Maezono

Osman

. Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle. J Clin Invest 2000; 105: 311–320.

36.

Montagnani

Golovchenko

Kim

. Inhibition of phosphatidylinositol 3-kinase enhances mitogenic actions of insulin in endothelial cells. J Biol Chem 2002; 277: 1794–1799.

37.

Wheatcroft

Williams

Shah

. Pathophysiological implications of insulin resistance on vascular endothelial function. Diabet Med 2003; 20: 255–268.

38.

Vozarova

Weyer

Hanson

. Circulating interleukin-6 in relation to adiposity, insulin action, and insulin secretion. Obes Res 2001; 9: 414–417.

39.

Festa

D’Agostino

Howard

. Chronic subclinical inflammation as part of the insulin resistance syndrome. Circulation 2000; 102: 42–47.

40.

Forbes

Cooper

. Mechanisms of diabetic complications. Physiol Rev 2013; 93: 137–188.

41.

Hotamisligil

Shargill

Spiegelman

. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993; 259: 87–91.

42.

Sethi

Hotamisligil

. The role of TNFα in adipocyte metabolism. Semin Cell Dev Biol 1999; 10: 19–29.

43.

Rask-Madsen

King

. Mechanisms of disease: endothelial dysfunction in insulin resistance and diabetes. Nat Clin Pract Endocrinol Metab 2007; 3: 46–56.

44.

Uysal

Wiesbrock

Marino

. Protection from obesity-induced insulin resistance in mice lacking TNF-[alpha] function. Nature 1997; 389: 610–614.

45.

Endocrine Today. TINSAL-T2D: salsalate lowered glucose in patients with type 2 diabetes, http://www.endocrinetoday.com/pda.aspx (accessed 12 November 2012).

46.

Zhang

Patel

. Proinflammatory cytokines downregulate gene expression and activity of constitutive nitric oxide synthase in porcine pulmonary artery endothelial cells. Res Commun Mol Pathol Pharmacol 1997; 96: 71–87.

47.

Forstermann

. Oxidative stress in vascular disease: causes, defense mechanisms and potential therapies. Nat Clin Pract Cardiovasc Med 2008; 5: 338–349.

48.

Pacher

Beckman

Liaudet

. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007; 87: 315–424.

49.

Sasaki

Yamashita

Takaya

. Augmentation of vascular remodeling by uncoupled endothelial nitric oxide synthase in a mouse model of diabetes mellitus. Arterioscler Thromb Vasc Biol 2008; 28: 1068–1076.

50.

Song

. Reactive nitrogen species induced by hyperglycemia suppresses Akt signaling and triggers apoptosis by upregulating phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in an LKB1-dependent manner. Circulation 2007; 116: 1585–1595.

51.

Nomiyama

Igarashi

Taka

. Reduction of insulin-stimulated glucose uptake by peroxynitrite is concurrent with tyrosine nitration of insulin receptor substrate-1. Biochem Biophys Res Commun 2004; 320: 639–647.

52.

Edelstein

Dimmeler

. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest 2001; 108: 1341–1348.

53.

Shenouda

Widlansky

Chen

. Altered mitochondrial dynamics contributes to endothelial dysfunction in diabetes mellitus. Circulation 2011; 124: 444–453.

54.

Voinea

Georgescu

Manea

. Superoxide dismutase entrapped-liposomes restore the impaired endothelium-dependent relaxation of resistance arteries in experimental diabetes. Eur J Pharmacol 2004; 484: 111–118.

55.

Diederich

Skopec

Diederich

. Endothelial dysfunction in mesenteric resistance arteries of diabetic rats: role of free radicals. Am J Physiol 1994; 266: H1153–H1161.

56.

Taddei

Virdis

Ghiadoni

. Vitamin C improves endothelium-dependent vasodilation by restoring nitric oxide activity in essential hypertension. Circulation 1998; 97: 2222–2229.

57.

Ting

Timimi

Haley

. Vitamin C improves endothelium-dependent vasodilation in forearm resistance vessels of humans with hypercholesterolemia. Circulation 1997; 95: 2617–2622.

58.

Timimi

Ting

Haley

. Vitamin C improves endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus. J Am Coll Cardiol 1998; 31: 552–557.

59.

Heart Protection Study Collaborative Group. MRC/BHF Heart Protection Study of antioxidant vitamin supplementation in 20 536 high-risk individuals: a randomised placebo-controlled trial. Lancet 2002; 360: 23–33.

60.

Lonn

Yusuf

Hoogwerf

. Effects of vitamin E on cardiovascular and microvascular outcomes in high-risk patients with diabetes: results of the HOPE study and MICRO-HOPE substudy. Diabetes Care 2002; 25: 1919–1927.

61.

Das Evcimen

King

. The role of protein kinase C activation and the vascular complications of diabetes. Pharmacol Res 2007; 55: 498–510.

62.

Rask-Madsen

King

. Proatherosclerotic mechanisms involving protein kinase C in diabetes and insulin resistance. Arterioscler Thromb Vasc Biol 2005; 25: 487–496.

63.

Inoguchi

Xia

Kunisaki

. Insulin’s effect on protein kinase C and diacylglycerol induced by diabetes and glucose in vascular tissues. Am J Physiol 1994; 267: E369–E379.

64.

Mehta

Sheetz

Price

. Selective PKC beta inhibition with ruboxistaurin and endothelial function in type-2 diabetes mellitus. Cardiovasc Drugs Ther 2009; 23: 17–24.

65.

Hink

Mollnau

. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res 2001; 88: e14–e22.

66.

Beckman

Goldfine

Gordon

. Inhibition of protein kinase Cβ prevents impaired endothelium-dependent vasodilation caused by hyperglycemia in humans. Circ Res 2002; 90: 107–111.

67.

Beckman

Goldfine

Goldin

. Inhibition of protein kinase Cβ does not improve endothelial function in type 2 diabetes. J Clin Endocrinol Metab 2010; 95: 3783–3787.

68.

Brownlee

. Biochemistry and molecular cell biology of diabetic complications. Nature 2001; 414: 813–820.

69.

Nishikawa

Edelstein

. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000; 404: 787–790.

70.

Goldin

Beckman

Schmidt

. Advanced glycation end products. Circulation 2006; 114: 597–605.

71.

Wautier

M-P

Chappey

Corda

. Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE. Am J Physiol Endocrinol Metab 2001; 280: E685–E694.

72.

Schmidt

Hori

Brett

. Cellular receptors for advanced glycation end products. Implications for induction of oxidant stress and cellular dysfunction in the pathogenesis of vascular lesions. Arterioscler Thromb 1994; 14: 1521–1528.

73.

DCCT/EDIC Research Group. Retinopathy and nephropathy in patients with type 1 diabetes four years after a trial of intensive therapy. N Engl J Med 2000; 342: 381–389.

74.

DCCT/EDIC Study Research Group. Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes. N Engl J Med 2005; 353: 2643–2653.

75.

The epidemiology of Diabetes Interventions and Complications. Welcome to the EDIC study website, https://edic.bsc.gwu.edu/en (accessed 21 November 2012).

76.

Roy

Sala

Cagliero

. Overexpression of fibronectin induced by diabetes or high glucose: phenomenon with a memory. Proc Natl Acad Sci U S A 1990; 87: 404–408.

77.

Engerman

Kern

. Progression of incipient diabetic retinopathy during good glycemic control. Diabetes 1987; 36: 808–812.

78.

Chan

P-S

Kanwar

Kowluru

. Resistance of retinal inflammatory mediators to suppress after reinstitution of good glycemic control: novel mechanism for metabolic memory. J Diabetes Complications 2010; 24: 55–63.

79.

Reddy

Natarajan

. Epigenetic mechanisms in diabetic vascular complications. Cardiovasc Res 2011; 90: 421–429.

80.

El-Osta

Brasacchio

Yao

. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia. J Exp Med 2008; 205: 2409–2417.

81.

Brasacchio

Okabe

Tikellis

. Hyperglycemia induces a dynamic cooperativity of histone methylase and demethylase enzymes associated with gene-activating epigenetic marks that coexist on the lysine tail. Diabetes 2009; 58: 1229–1236.

82.

Hiltunen

Turunen

Häkkinen

. DNA hypomethylation and methyltransferase expression in atherosclerotic lesions. Vasc Med 2002; 7: 5–11.

83.

Chan

Fish

D’Abreo

. The cell-specific expression of endothelial nitric-oxide synthase: a role for DNA methylation. J Biol Chem 2004; 279: 35087–35100.

84.

Fradin

Bougnères

. T2DM: why epigenetics? J Nutr Metab 2011; 2011: 647514 (17 pp.).

85.

Jiang

Zhang

Liu

. Hypermethylation of hepatic glucokinase and L-type pyruvate kinase promoters in high-fat diet–induced obese rats. Endocrinology 2011; 152: 1284–1289.

86.

Wang

Qian

Zhang

. MicroRNA-320 expression in myocardial microvascular endothelial cells and its relationship with insulin-like growth factor-1 in type 2 diabetic rats. Clin Exp Pharmacol Physiol 2009; 36: 181–188.

87.

Caporali

Meloni

Völlenkle

. Deregulation of microRNA-503 contributes to diabetes mellitus–induced impairment of endothelial function and reparative angiogenesis after limb ischemia. Circulation 2011; 123: 282–291.

88.

Togliatto

Trombetta

Dentelli

. MIR221/MIR222-driven post-transcriptional regulation of P27KIP1 and P57KIP2 is crucial for high-glucose- and AGE-mediated vascular cell damage. Diabetologia 2011; 54: 1930–1940.

89.

Liu

Cheng

Yang

. Cell-specific effects of miR-221/222 in vessels: molecular mechanism and therapeutic application. J Mol Cell Cardiol 2012; 52: 245–255.

90.

Song

Y-H

. microRNA-221 regulates high glucose-induced endothelial dysfunction. Biochem Biophys Res Commun 2009; 381: 81–83.

91.

Fang

Shi

Manduchi

. MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro. Proc Natl Acad Sci U S A 2010; 107: 13450–13455.

92.

Fish

Santoro

Morton

. miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell 2008; 15: 272–284.

93.

Dentelli

Rosso

Orso

. microRNA-222 controls neovascularization by regulating signal transducer and activator of transcription 5A expression. Arterioscler Thromb Vasc Biol 2010; 30: 1562–1568.

94.

Wang

Aurora

Johnson

. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell 2008; 15: 261–271.

95.

Fasanaro

D’Alessandra

Di Stefano

. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem 2008; 283: 15878–15883.

96.

Sabatel

Malvaux

Bovy

. MicroRNA-21 exhibits antiangiogenic function by targeting RhoB expression in endothelial cells. PLoS One 2011; 6: e16979.

97.

Shantikumar

Caporali

Emanueli

. Role of microRNAs in diabetes and its cardiovascular complications. Cardiovasc Res 2012; 93: 583–593.

98.

Gilad

Meiri

Yogev

. Serum microRNAs are promising novel biomarkers. PLoS One 2008; 3: e3148.

99.

Zampetaki

Kiechl

Drozdov

. Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes. Circ Res 2010; 107: 810–817.

100.

Look AHEAD (Action for Health in Diabetes). Look AHEAD, https://www.lookaheadtrial.org/public/home.cfm (accessed 23 November 2012).

101.

US Department of Health and Human Services. Weight loss does not lower heart disease risk from type 2 diabetes, http://www.nih.gov/news/health/oct2012/niddk-19.htm (accessed 23 November 2012).

102.

Cuny

Guerci

Cariou

. New avenues for the pharmacological management of type 2 diabetes: an update. Ann Endocrinol 2012; 73: 459–468.

103.

Selvin

Bolen

Yeh

. Cardiovascular outcomes in trials of oral diabetes medications: a systematic review. Arch Intern Med 2008; 168: 2070–2080.

104.

Desouza

Bolli

Fonseca

. Hypoglycemia, diabetes, and cardiovascular events. Diabetes Care 2010; 33: 1389–1394.

105.

Nissen

Wolski

. Rosiglitazone revisited: an updated meta-analysis of risk for myocardial infarction and cardiovascular mortality. Arch Intern Med 2010; 170: 1191–1201.

106.

Yamakuchi

. MicroRNAs in vascular biology. Int J Vasc Med 2012; 2012: 794898.

107.

Madi

Riches

Warburton

. Inherent differences in morphology, proliferation, and migration in saphenous vein smooth muscle cells cultured from nondiabetic and type 2 diabetic patients. Am J Physiol Cell Physiol 2009; 297: C1307–C1317.

Cellular and molecular mechanisms of endothelial dysfunction in diabetes

Abstract

Keywords

Introduction

Physiological role of endothelium

Endothelial dysfunction

Endothelial dysfunction in DM

Selective insulin resistance

Pro-inflammatory signalling

Oxidative stress

Anti-oxidant therapy

Protein kinase C

Hyperglycaemia

Metabolic memory: role of epigenetics

Histone modifications

DNA methylation

microRNAs

Current therapies for DM

Approach to future therapies

Conclusion

Footnotes

Declaration of conflicting interests

Funding

References