19.3 Vascular complications of dysglycaemia

The dysglycaemic cardiovascular continuum is defined as a state of long-lasting insulin resistance, compensatory hyperinsulinaemia, and early glucometabolic impairment clustering with concomitant cardiovascular risk and the development of macrovascular disease prior to the diagnosis of type 2 diabetes. There is indeed a strong biological relation between hyperglycaemia, insulin resistance, and vascular disease.

The dysglycaemic cardiovascular continuum is defined as a state of long-lasting insulin resistance, compensatory hyperinsulinaemia, and early glucometabolic impairment clustering with concomitant cardiovascular risk and the development of macrovascular disease prior to the diagnosis of type 2 diabetes (T2D). There is indeed a strong biological relation between hyperglycaemia, insulin resistance, and vascular disease.¹ In patients with T2D, high glucose levels trigger endothelial inflammation and mitochondrial oxidative stress, and impair nitric oxide (NO) availability, a key marker of vascular health. This leads to the development of macro- and microvascular complications.¹ However, the detrimental effects of glucose already occur with glycaemic levels below the threshold for the diagnosis of T2D. This is explained by the concept of a ‘dysglycaemic cardiovascular continuum’ across the spectrum of prediabetes, diabetes, and cardiovascular risk (Figure 19.3.1).²

High glucose levels affect vascular homeostasis by altering the balance between NO bioavailability and reactive oxygen species (ROS).^3,4 Indeed, hyperglycaemia-induced generation of superoxide anion (O₂⁻) inactivates NO to form peroxynitrite (ONOO⁻), a powerful oxidant which easily penetrates across phospholipid membranes thereby suppressing the activity of scavenger enzymes and endothelial NO synthase (Figure 19.3.2). In the vasculature of individuals with diabetes, oxidative stress is capable of activating an array of cellular pathways including polyol and hexosamine flux, advanced glycation end products, protein kinase C, and nuclear factor kappa B (NF-κB)-mediated vascular inflammation.^5,6 Protein kinase C activation leads to increased ROS generation via activation of the adaptor p66^Shc and NADPH oxidase signalling (Figure 19.3.2).⁷ The p66^Shc adaptor protein functions as a redox enzyme implicated in mitochondrial ROS generation and translation of oxidative signals into apoptosis.⁸ Notably, p66^Shc gene expression is increased in peripheral blood. Mononuclear cells obtained from patients with T2D correlates with plasma 8-isoprostane levels, an in vivo marker of oxidative stress.⁹ In the vessel wall, protein kinase C-dependent ROS production contributes to the atherosclerotic process by triggering vascular inflammation.¹⁰ Indeed, ROS lead to upregulation and nuclear translocation of NF-κB subunit p65 and, hence, transcription of proinflammatory genes encoding monocyte chemoattractant protein 1 (MCP-1), selectins, vascular cell adhesion molecule 1 (VCAM-1), and intracellular cell adhesion molecule 1 (ICAM-1). This latter event facilitates adhesion of monocytes to the vascular endothelium, rolling, and diapedesis in the subendothelium with subsequent formation of foam cells.³ Secretion of interleukin 1 and tumour necrosis factor alpha (TNFα) from active macrophages maintains upregulation of adhesion molecules by enhancing NF-κB signalling in the endothelium and also promotes smooth muscle cell growth and proliferation. Consistently, inhibition of the protein kinase Cβ₂ isoform blunts VCAM-1 upregulation in human endothelial cells upon glucose exposure.¹⁰ Mitochondrial ROS also increase intracellular levels of the glucose metabolite methylglyoxal and synthesis of advanced glycation end products.^11,12 Generation of advanced glycation end products leads to cellular dysfunction by eliciting activation of their receptor which in turn activates ROS-sensitive biochemical pathways.⁵

Insulin resistance is a major feature of T2D and develops in multiple organs including skeletal muscle, liver, adipose tissue, and heart.¹³ Obesity plays a pivotal role in this phenomenon providing an important link between T2D and fat accumulation.¹⁴ In subjects with obesity or T2D, the increase in free fatty acids activate Toll-like receptor 4 leading to NF-κB nuclear translocation and subsequent upregulation of inflammatory genes IL-6 and TNFα. On the other hand, two important kinases, c-Jun N-terminal kinase and protein kinase C, phosphorylate the insulin receptor substrate 1, thus blunting its downstream targets phosphoinositide 3-kinase and Akt. This results in downregulation of glucose transporter GLUT-4 and, hence, insulin resistance.^15,16 Although insulin resistance has been attributed to adipocyte-derived inflammation, recent evidence is overturning the ‘adipocentric paradigm’.¹⁷ Indeed, inflammation and macrophage activation seem to primarily occur in non-adipose tissue in obesity. This concept is supported by the notion that suppression of inflammation in the vasculature prevents insulin resistance in other organs and prolongs lifespan, suggesting that blockade of vascular inflammation and oxidative stress may be a promising approach to prevent metabolic disorders.¹⁸

Deregulation of factors involved in coagulation and platelet activation accounts for the increased risk of coronary events in patients with T2D.¹⁹ Insulin resistance increases plasminogen activator inhibitor 1 and fibrinogen and reduces tissue plasminogen activator levels. Hyperinsulinaemia and low-grade inflammation induce tissue factor expression in monocytes of patients with T2D leading to increased tissue factor procoagulant activity and thrombin generation.²⁰ Emerging evidence has shown that microparticles, vesicles released in the circulation from various cell types following activation or apoptosis, are increased in patients with diabetes and predict cardiovascular outcome.²¹ Microparticles from patients with T2D have been shown to increase coagulation activity in endothelial cells. Moreover, microparticles carrying tissue factor promote thrombus formation at sites of injury, representing a novel and additional mechanism of coronary thrombosis in diabetes.²² Among the factors contributing to the prothrombotic state in patients with diabetes, platelet hyper-reactivity is of major relevance.²³ Hyperglycaemia alters platelet Ca²⁺ homeostasis leading to cytoskeleton abnormalities and increased secretion of proaggregant factors. Moreover, upregulation of glycoproteins Ib and IIb/IIIa triggers thrombus formation via interaction with von Willebrand factor and fibrin molecules.²⁴

Emerging evidence indicates that epigenetic changes, including chromatin remodelling and microRNAs, may contribute to explain gene–environment interactions and subsequent dysregulation of key oxidant and inflammatory pathways involved in the vascular disease phenotype related to diabetes.²⁵ Epigenetics refers to heritable changes in gene expression without altering DNA sequences.²⁶ Epigenetic variations may be classified into three main categories: (1) DNA methylation, (2) post-translational histone modifications, and (3) RNA-based mechanisms including microRNAs and long non-coding RNAs. DNA methylation is an important repressor of gene transcription. Post-translational modification of histone tails (acetylation/methylation) also represents a key component in the epigenetic regulation of genes. Several enzymes have been implicated in plastic alterations of chromatin upon physiological and pathological conditions.²⁷ DNA and histone methyltransferases, as well as histone acetyltransferases, orchestrate a fine balance between activating and inhibitory epigenetic signatures. A growing body of evidence suggests that epigenetic changes occurring at the level of DNA and histone-binding promoter of pro-oxidant and proinflammatory genes are associated with diabetes. Specifically, methylation and acetylation are critical epigenetic marks modulated by the hyperglycaemic environment. In vitro studies demonstrated that methylation of the p65/NFkB promoter by the ROS-dependent methyltransferase Set7 is indeed the mechanism whereby vascular inflammation is triggered in this setting.²⁸ The expression of Set7 has recently been found to be increased in peripheral blood monocytes isolated from patients with T2D.²⁹ T2D patients showed Set7-dependent histone-3 methylation on the NF-κB p65 promoter and this epigenetic signature was associated with upregulation of NF-κB p65, subsequent transcription of oxidant genes (iNOS and COX-2), and increased plasma levels of ICAM-1 and MCP-1. Consistently, Set7 expression significantly correlated with oxidative marker 8-isoPGF2α and impaired flow-mediated dilation of the brachial artery.²⁹ Epigenetic regulation of the mitochondrial adaptor p66^Shc, a key enzyme involved in mitochondrial ROS generation, may significantly contribute to endothelial dysfunction in the context of diabetes.³⁰ MicroRNAs are a newly identified class of small non-coding RNAs emerging as key players in the pathogenesis of diabetes-induced vascular complications.³¹ These small non-coding RNAs trigger vascular disease due to diabetes by regulating gene expression at the post-transcriptional level. Microarray studies have shown an altered profile of microRNA expression in subjects with T2D.^32,33 Indeed, patients with diabetes display a significant deregulation of microRNAs involved in angiogenesis, vascular repair, endothelial homeostasis, and cardiac remodelling.^32,33,34 Altogether, these studies suggest that the removal of epigenetic marks of oxidant and inflammatory genes may represent a promising option to prevent endothelial dysfunction and, hence, vascular complications in people with T2D.

The metabolic syndrome is defined as a cluster of risk factors for cardiovascular disease and T2D including raised blood pressure, dyslipidaemia (high triglycerides and low high-density lipoprotein cholesterol), elevated plasma glucose, and central obesity. Although there is agreement that the metabolic syndrome deserves attention, there has been an active debate concerning the terminology and diagnostic criteria related to its definition.³⁵ However, the medical community agrees that the term metabolic syndrome is appropriate to represent the combination of multiple risk factors. Although metabolic syndrome does not include established risk factors (i.e. age, gender, and smoking) such patients have a twofold increase in risk of cardiovascular disease and a fivefold increase to develop T2D.

Oxidative stress plays a major role in the development of dysglycaemia-induced vascular complications. Accumulation of ROS in the vasculature is responsible for the activation of detrimental biochemical pathways, microRNA deregulation, release of microparticles, and epigenetic changes contributing to vascular inflammation and ROS generation. In this setting, mechanism-based therapeutic changes are in high demand. Specifically, inhibition of key enzymes involved in dysglycaemia-induced vascular damage or activation of pathways improving insulin sensitivity may represent promising approaches. Moreover, the progressive identification of a complex scenario driven by epigenetic changes that modulate transcription of ROS-generating and proinflammatory genes may represent an attractive opportunity to dampen oxidative stress and vascular inflammation, and hence to prevent cardiovascular complications in patients with diabetes.