Vascular tone

The degree of constriction experienced by a blood vessel relative to its maximally dilated state is referred to as the vascular tone, which is modulated by the release of endothelium-derived relaxing and constricting factors⁽ Reference Rubanyi ^{4 )}. The vasoprotective effects of a healthy endothelium are vasodilation, suppression of smooth muscle cell growth and inhibition of inflammatory responses, in which the vasodilators act against the effects of endothelium-derived vasoconstrictors. Vascular tone is maintained by homeostasis between the vasodilators such as nitric oxide (NO^∙), prostacyclin (PGI2) and endothelium-derived hyperpolarising factor and vasoconstrictors, including endothelin-1 (ET-1), angiotensin II (Ang II), thromboxane A2 (TXA2) and platelet-activating factor released by the endothelial cells⁽ Reference Shimokawa, Yasutake and Fujii ^{5 )}. Damage to the endothelium creates an imbalance between vasodilation and vasoconstriction, resulting in the initiation of a cascade of events such as increased endothelial permeability, platelet aggregation, leucocyte adhesion and generation of cytokines⁽ Reference Rajendran, Rengarajan and Thangavel ^{6 )}.

Coagulation and fibrinolysis

Endothelial cells have major roles in regulating haemostatic balance, preventing the activation of thrombin and inhibiting platelet adhesion, thereby mediating anticoagulant activity⁽ Reference Chan and Paredes ^{7 )}. These cells secrete an integral membrane protein, thrombomodulin, which causes a reduction in blood coagulation along with the assistance of platelet tissue factor, plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator⁽ Reference Michiels ^{8 )}. The release of endothelial products by thrombin has a critical role in coagulation, inflammation and vascular homeostasis, which is largely arbitrated by the thrombin receptor, also known as protease-activated receptor-1 (PAR-1). The activation of PAR-1 mediates intracellular signalling pathways by (a) enhancement in the production of NO^∙ and PGI2, which induce vasodilation and regulation of platelet activation; (b) stimulating tissue plasminogen activator (tPA), which binds to fibrin and mediates effective fibrinolysis; and (c) activation of Weibel–Palade bodies that release von Willebrand factor (vWF), angiopoietin-2 and P-selectin, which are involved in the modulation of inflammatory response⁽ Reference Verhamme and Hoylaerts ^{9 )}.

Cell growth and differentiation

The synchronised regulation of vasculogenesis or de novo differentiation of bone marrow-derived endothelial progenitor cells (EPC), that is, angioblasts into endothelial cells followed by angiogenesis, is essential for the development of the vascular system. Angiogenesis is the major mechanism of vascularisation involving endothelial cell sprouting, vessel branching and intussusceptions of the vasculature from the pre-existing blood vessels⁽ Reference Patan ^{10 )}. Vascular endothelial growth factor (VEGF) secreted by the endothelial cells is known to be the main growth factor specific for the vascular endothelium, and hence expression of VEGF receptors has been considered a critical regulator of endothelial cell development⁽ Reference Zachary ^{11 )}. Expression of growth factors such as fibroblast growth factor, heparin-binding epidermal growth factor, platelet-derived growth factor (PDGF) and transforming growth factor β in addition to tyrosine kinase receptors tie-2 (tek) and angiopoietins (Ang-1 and Ang-2) assists in the vascular development and promotes angiogenesis, a major event in embryonic development, regeneration and wound healing⁽ Reference Olsson, Dimberg and Kreuger ^{12 )}.

Adhesion and permeability

Endothelial cells produce specific adhesion molecules, namely E-selectin, intracellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), for the regulation of cell adhesion and permeability⁽ Reference Biegelsen and Loscalzo ^{13 )}. The intact endothelium expresses low levels of these adhesion molecules, and upon activation, over-expression of these molecules has been reported, which play a role in maintaining endothelial barrier integrity and also mediate paracellular and transcellular migration⁽ Reference Carman and Springer ^{14 )}. In addition, leucocyte passage from blood to underlying tissues involves the interaction between leucocyte carbohydrate ligand and endothelial E- and P-selectins. Further, firm adhesion mediated by the interactions between leucocyte integrins and endothelial adhesion molecules and their up-regulation increases endothelial permeability⁽ Reference Rahman, Anwar and Malik ^{15 )}. Moreover, binding and accumulation of circulating cells such as platelets, leucocytes and erythrocytes to the endothelial cells restricts blood flow⁽ Reference Mestas and Ley ^{16 )}.

Hyperglycaemia

Increased blood glucose level, the hallmark of diabetes, is a major risk factor for endothelial dysfunction-mediated vascular complications. In hyperglycaemia, endothelial dysfunction is triggered by the inhibition of the glycolytic enzyme, d-glyceraldehyde 3-phosphate dehydrogenase. This causes accumulation of glycolytic intermediates leading to the activation of major metabolic pathways such as the protein kinase C (PKC) pathway, the hexosamine pathway and the sorbitol pathway⁽ Reference De Vriese, Verbeuren and Van de Voorde ^{24 )}, in addition to the generation of methylglyoxal and advanced glycation end products (AGE).

Accumulated evidence shows altered phenotype of endothelial cell including apoptosis⁽ Reference Zanetti, Stocca and Dapas ^{25 )} through the activation of caspases, increased reactive oxygen species (ROS) and inflammatory markers⁽ Reference Bucciarelli, Pollreisz and Kebschull ^{26 )} because of increased glucose concentration in the blood, altered metabolic activity with increased levels of lipid peroxidation products⁽ Reference Banumathi, Sheikpranbabu and Haribalaganesh ^{27 )} and enhanced intracellular calcium levels⁽ Reference Wang, Peng and Zhang ^{28 )}. In addition, high glucose also triggers the production of vasoconstrictors such as prostanoids and thromboxanes, resulting in vascular contractility. Further alteration in insulin signalling mediates an increase in the levels of vasoactive peptides such as Ang II and ET-1, contributing to endothelial dysfunction⁽ Reference Vanhoutte, Shimokawa and Tang ^{29 )}.

Insulin resistance

Under physiological conditions, insulin delivery is the rate-limiting step in insulin-stimulated glucose uptake in the skeletal muscle. This is mediated by the involvement of endothelial cells, which regulate the capillary recruitment and glucose uptake⁽ Reference Kubota, Kubota and Kumagai ^{30 )}. Insulin resistance is associated with the impairment of GLUT-4 translocation in fat tissues and muscles with reduced NO^∙ production, this weakens the insulin signalling and glucose uptake, mediating vasodilation⁽ Reference Kim, Koh and Quon ^{31 )}. Attenuation of Akt phosphorylation in response to insulin⁽ Reference Gogg, Smith and Jansson ^{32 )} has been reported in the microvascular endothelial cells in T2D models resulting from impairment of the phosphoinositide 3-kinase-protein kinase B (PI3K-Akt) pathway.

Hyperinsulinemia

Metabolic insulin resistance is usually accompanied by increased levels of circulating insulin or hyperinsulinemia which impairs endothelial function⁽ Reference Arcaro, Cretti and Balzano ^{33 )}. Imbalance in PI3K- and mitogen-activated protein kinase (MAPK)-dependent functions of insulin and down-regulation of endothelial nitric oxide synthase (eNOS) has been evidenced in hyperinsulinemia⁽ Reference Muniyappa, Iantorno and Quon ^{34 )}. Elevated insulin levels stimulate VCAM-1 and E-selectin through the MAPK-dependent pathway and ET-1 secretion, causing pronounced abnormal vascular function⁽ Reference Cusi, Maezono and Osman ^{35 )}. ET-1 induces NAD(P)H oxidase (NOX) expression with increased generation of superoxide anion (_∙O₂ ^–), as reported in rat aortic endothelium⁽ Reference Duerrschmidt, Wippich and Goettsch ^{36 )}. Hyperinsulinemia also contributes to cellular hypertrophy with increased synthesis and accumulation of elements of the extracellular matrix, thereby altering the structure of the vessel wall⁽ Reference Potenza, Gagliardi and Nacci ^{37 )}.

Obesity and dyslipidaemia

Metabolic disorders including obesity, hypertension and lipid abnormalities linking atherosclerosis cluster with insulin resistance. Dyslipidaemia and obesity are the major risk factors for cardiovascular events in diabetes⁽ Reference Bakker, Eringa and Sipkema ^{38 )}. It has been demonstrated that obesity induces endothelial dysfunction before the development of insulin resistance⁽ Reference Erdei, Toth and Pasztor ^{39 )}. In T2D, the reduced endothelium-dependent vasodilation is mediated by the attenuation of NO^∙ production, decreased sensitivity to NO^∙ decreased Akt phosphorylation and enhanced vasoconstrictor tone⁽ Reference Okon, Chung and Rauniyar ^{40 –} Reference Bagi, Koller and Kaley ^{42 )}. The abnormal lipids affect endothelial cells directly, by the activation of lectin-like oxidised LDL receptor, stimulation of NOX-dependent _∙O₂ ^– formation and release of cytokines, culminating in apoptosis⁽ Reference Shin, Kim and Kim ^{43 )}.

Oxidative stress

Increased oxidative stress has been reported to be a common feature of endothelial dysfunction often associated with NOX, xanthine oxidase (XO), aldehyde oxidase and glucose oxidase, the enzymes involved in the generation of ROS.

Endoplasmic reticulum stress

ER stress known as unfolded protein responses (UPR) has a crucial role in the pathogenesis of diabetes, contributing to pancreatic β-cell loss and insulin resistance. Vascular endothelial cells of hyperglycaemic subjects are characterised by altered rough endoplasmic reticulum (rER) and protein folding, which leads to ER stress⁽ Reference Bakker, Eringa and Sipkema ^{38 ,} Reference Sheikh-Ali, Sultan and Alamir ^{72 )}. As reported by Sheikh-Ali et al. ⁽ Reference Sheikh-Ali, Sultan and Alamir ^{72 )}, hyperglycaemia-induced ER stress in endothelial cells may be initiated through different stress signalling molecules of the glucose metabolism such as pyruvate, glycerol and dihydroxyacetone. The accumulation of unfolded or misfolded proteins in the ER initially restores the normal function of ER by halting translation, degrading misfolded proteins and activating signalling pathways to increase molecular chaperones involved in protein folding. Sustained UPR has been implicated in the initiation of several cell death pathways mediated by the activation of caspase-12⁽ Reference Nakagawa, Zhu and Morishima ^{73 )}, apoptosis signal-regulating kinase 1, the c-Jun N-terminal kinase (JNK) pathway⁽ Reference Nishitoh, Matsuzawa and Tobiume ^{74 )}, the protein kinase RNA-like-like endoplasmic reticulum kinase (PERK) pathway and C/ERB homologous protein (CHOP) pathway⁽ Reference Ma, Brewer and Diehl ^{75 )}. Provoked ER stress increases the activity of JNK and catalytic IκB kinase subunits and induces inflammation associated with impairment of insulin receptor substrate 1 (IRS-1) signalling, thus linking diabetes and vascular endothelial dysfunction⁽ Reference Olsson, Dimberg and Kreuger ^{12 ,} Reference Ozcan, Cao and Yilmaz ^{76 )}.

Diabetes-mediated structural alterations in endothelial cells

Under normal physiological conditions, endothelial cells show stable interaction with smooth muscle surface and scanty organelles with flat nucleus, separated from the underlying connective tissues by a thin, extracellular matrix of tissues named as ‘basal lamina’, which functions in heterotypic tissue interactions. In addition, normal endothelial cells are actively engaged in transcytosis, a process by which the transcellular transfer of molecules across the endothelium takes place⁽ Reference Langille and Adamson ^{86 )}.

The ultrastructure of the endothelial cell and the extracellular matrix tends to be altered under hyperglycaemia. A significant enrichment of biosynthetic organelles such as golgi complex in the aortic and capillary endothelial cells, abundance of rER in aortic arch of retinal venules and femoral artery have been reported⁽ Reference Popov ^{87 )}. Further predominant thickening of endothelial cell basal lamina accounting to impairment in the circulation of metabolic products between tissues⁽ Reference Dokken ^{88 )} has been reported (Fig. 3). Hyperglycaemia also mediates endothelial dysfunction by causing endothelial barrier injury leading to hyperpermeability and plasma leakage⁽ Reference Simionescu ^{89 )}.

Fig. 3

Structural modifications in endothelial cells under hyperglycaemia. Hyperglycaemia induced thickening of endothelial basal lamina impairs the transport of metabolic products and nutrients between blood and tissues. Further, hyperglycaemia induces telomere shortening, DNA fragmentation, intracellular calcium enhancement leading to mitochondrial calcium overload causing mitochondrial DNA fragmentation and alteration in membrane potential. A colour figure is available in the online version of the paper.

Metformin

Metformin, an antidiabetic drug, significantly improved endothelium-dependent vasodilator response. Reduction in the levels of endothelial markers such as vWF, VCAM-1, tPA, PAI-1 and ICAM-1⁽ Reference Mather, Verma and Anderson ^{90 )} have been reported to be the mechanism underlying the protective effects of metformin. In addition, long-term treatment with metformin improved endothelial function and decreased inflammatory response⁽ Reference de Jager, Kooy and Schalkwijk ^{91 )}.

Glucagon-like peptide-1 receptor agonists

GLP-1 receptor agonists mimic GLP-1 and increase incretin effect in patients with T2D. They stimulate the release of insulin and improve the endothelial function through direct vascular action⁽ Reference Pratley and Gilbert ^{92 )}. Recent report showed that exenatide, a GLP-1 receptor agonist, reverted glucose- and lipid-induced endothelial dysfunction. Exenatide activated eNOS and NO^∙ production in endothelial cells, in addition to induced vasorelaxation and reduced endothelial dysfunction in arterioles⁽ Reference Koska, Sands and Burciu ^{93 )}. In obesity and pre-diabetic patients, exenatide treatment showed a significant change in inflammation and oxidative stress status. Effects of exenatide have also improved postprandial vascular endothelial dysfunction in T2D patients⁽ Reference Torimoto, Okada and Mori ^{94 )}.

Phosphodiesterase-5 inhibitors

PDE5 is a cytosolic enzyme that primarily functions to degrade cGMP and induce vasoconstriction. PDE5 inhibitors up-regulate eNOS transcription and increase NO^∙ release, causing long-term vasodilator effects. They are also reported for improving endothelium-dependent vasorelaxation in diabetic rats and for their potential to reduce glomerular hypertension in diabetic nephropathy⁽ Reference Thompson ^{95 )}. Sildenafil, a PDE5 inhibitor, attenuates diabetic nephropathy in Otsuka Long-Evans Tokushima Fatty rats⁽ Reference Kuno, Iyoda and Shibata ^{96 )}.

Dipeptidyl peptidase-4 inhibitor

Endothelial cells are the main endogenous sources of DPP4, an enzyme involved in inactivating active GLP-1 (7–36) to GLP-1 (9–36). The inhibitors of DPP-4 have been used in the treatment of T2D⁽ Reference Pratley and Gilbert ^{92 )}. DPP4 interacts with pro-inflammatory pathways and impairs endothelial function through incretin-dependent and incretin-independent mechanisms. It has been reported that the DPP-4 inhibitor des-fluoro-sitagliptin enhanced acetylcholine-induced endothelium-dependent vasodilatation in mouse aortic rings⁽ Reference Matsubara, Sugiyama and Sugamura ^{97 )}. Vildagliptin, another DPP-4 inhibitor, improved endothelial function, as indicated by measurement of forearm blood flow during acetylcholine infusion in T2D patients⁽ Reference van Poppel, Netea and Smits ^{98 )}.

Sodium–glucose co-transporter inhibitors

A newly developed therapeutic strategy for the treatment of diabetes is the use of the SGLT2 inhibitors, which offer a novel insulin-independent approach for the control of hyperglycaemia and are currently in phase 2 and 3 clinical trials. These inhibitors target the SGLT2, the main GLUT in the kidney that is also responsible for the reabsorption of >90 % of the glucose in the kidney⁽ Reference Rieg, Masuda and Gerasimova ^{99 )}. SGLT2 inhibition reduces the reabsorption of glucose and therefore enhances urinary glucose excretion preventing glucotoxicity and consequently decreasing both fasting and postprandial hyperglycaemia. SGLT2 inhibitor therapy has been reported to reverse glucose-induced vascular dysfunction by reducing glucotoxicity, oxidative stress, inflammation and restoring insulin signalling, thereby reversing endothelial dysfunction⁽ Reference Oelze, Kroller-Schon and Welschof ^{100 )}. Empagliflozin has been reported to cause reduction in cellular glucotoxicity, prevention of oxidative stress, AGE signalling and inflammation via NOX inhibition, decreased AGE precursor and methylglyoxal thereby maintaining the normal endothelial function in STZ-animal T1D model⁽ Reference Oelze, Kroller-Schon and Welschof ^{100 )}. In addition, SGLT-2 inhibitors improved the vascular architecture in connective tissue participating in arterial stiffening and regulated NO^∙-dependent relaxation in mouse pulmonary arteries⁽ Reference Han, Cho and Ayon ^{101 )}.

Exogenous nitrate treatment

Administration of exogenous NO^∙ in the form of organic nitrates rather than improving vascular function leads to an increased formation of ONOO^– mediating adverse effects. In particular, nitroglycerin induces clinical tolerance, oxidative stress and endothelial dysfunction similar to other nitrates such as isosorbide-5-mononitrate (ISMN) and isosorbide dinitrate. These are more likely to induce endothelial dysfunction and increase oxidative stress via activation of the vascular NOX. In contrast, pentaerythritol tetranitrate (PETN) reduces oxidative stress in vascular tissue through its interaction with the powerful antioxidant enzyme heme oxygenase-1 (HO-1). Regulation of cardiac oxidative stress and lipid peroxidation in STZ-induced diabetic rats treated with PETN treatment proved a clear correlation between NOX activity, endothelial dysfunction and nitrate resistance⁽ Reference Wenzel, Oelze and Coldewey ^{102 )}. In STZ-induced rats, PTEN treatment prevented the activation of NOX, inhibited XO-mediated _∙O₂ ^– production, eNOS uncoupling and thereby reduced oxidative stress, which was not the case with ISMN. In addition, PETN modified the metabolism of glucose in diabetic rats via the AMP-activated protein kinase (AMPK) pathway⁽ Reference Schuhmacher, Oelze and Bollmann ^{103 )}.

Exercise

Moderate exercise tends to be the stimulator of NO^∙ release, and it influences endothelial function, which in turn reduces the cardiovascular profile in diabetic patients⁽ Reference Hambrecht, Wolf and Gielen ^{104 )}. It has been reported that exercise training improved the vascular endothelial function in patients with long-term T1D⁽ Reference Fuchsjager-Mayrl, Pleiner and Wiesinger ^{105 )}. Currently, the beneficial effects of exercise training in T2D patients have been well established. Participation in regular physical activity maintained glucose metabolism, improved energy metabolism and insulin sensitivity and delayed T2D by attenuating the lipid profile, blood pressure and cardiovascular events⁽ Reference Colberg, Sigal and Fernhall ^{106 ,} Reference Boule, Haddad and Kenny ^{107 )}. Lee et al. ⁽ Reference Lee, Park and Zhang ^{108 )} demonstrated that exercise recovered eNOS phosphorylation, increased antioxidant enzymes such as superoxide dismutase (SOD)-1 and SOD-2, increased NO^∙ bioavailability and also decreased pro-inflammatory cytokines such as TNF-α and IL-6, in the T2D mouse model.

Antioxidant therapy

Antioxidants have been accounted to improve endothelial dysfunction in diabetes by re-coupling eNOS and mitochondrial function, by increasing the activity of _∙O₂ ^– scavenging enzymes or by decreasing vascular NOX activity.

Nitric oxide signalling

NO^∙ production in endothelial cells is mainly regulated by the constitutive expression of eNOS in the presence of its substrate l-arginine and cofactors such as NADPH, FAD, FMN and BH₄ ⁽ Reference Alderton, Cooper and Knowles ^{131 )}. eNOS is regulated at multiple sites by phosphorylation of serine (Ser), threonine (Thr) and tyrosine (Tyr) residues by Akt kinase, cyclic AMP (cAMP)-dependent PKA and AMPK in endothelial cells⁽ Reference Mount, Kemp and Power ^{132 ,} Reference Barbosa, Luciano and Marques ^{133 )}. Further, the activation of IRS-1 by insulin subsequently phosphorylates eNOS through PI3K/Akt signalling pathways⁽ Reference Muniyappa, Montagnani and Koh ^{134 )}. eNOS is activated in response to fluid shear stress and numerous agonists via cellular events such as increased intracellular calcium, interaction with substrate and cofactors, protein phosphorylation and cellular factors such as VEGF, IGF and so on. Dysregulation of these processes attenuates eNOS expression and reduces NO^∙ levels, a characteristic of numerous pathophysiological disorders, including diabetes. Under conditions of insulin resistance, IRS-1 mutation reduces the insulin-stimulated eNOS phosphorylation in endothelial cells⁽ Reference Federici, Pandolfi and De Filippis ^{135 )} and impairs NO^∙ bioavailability⁽ Reference Jiang, Lin and Clemont ^{136 )} through PI3k/Akt signalling. In addition, NOX localised in the endothelium serves as a major source of _∙O₂ ^–, and its interaction with eNOS leads to uncoupling, which is implicated in diabetic complications⁽ Reference Asaba, Tojo and Onozato ^{137 )}. OONO^– formed by the reaction of NO^∙ with _∙O₂ ^– reduces the availability of NO^∙, causing induction of VCAM-1, ICAM-1 and E-selectin in endothelial cells⁽ Reference Beckman and Koppenol ^{138 )}. The activated endothelium expresses chemotactic factors such as monocyte chemoattractant protein-1 (MCP-1) and other proinflammatory cytokines such as macrophage colony-stimulating factor and TNF-β. Expression of these factors contributes to the development of inflammation within the arterial wall and promotes atherogenesis.

Modulation of NO^∙ and the role of antioxidants in restoring endothelial dysfunction is gaining momentum. Polyphenol antioxidants are being investigated for enhancement of the release of NO^∙ from the endothelium to assess the possibility of reversing endothelial dysfunction caused by decreased production of NO^∙ in diabetes. Polyphenols are considered to be the most effective antioxidants. One of the most studied polyphenols, resveratrol, is present in red grapes and other fruits. It has been reported to restore the IRS-1/Akt/eNOS signalling pathway in endothelial cells under palmitate-induced insulin resistance⁽ Reference Liu, Jiang and Zhang ^{139 )}. Further, resveratrol is effective in restoring the vascular functions mediated through eNOS in diabetic rats⁽ Reference Arrick, Sun and Patel ^{140 )}.

Machha et al. ⁽ Reference Machha, Achike and Mustafa ^{141 )} reported modulation of endothelium-derived NO^∙ bioavailability in aortic tissues of diabetic rats by quercetin, a flavonol. Recently, the effect of quercetin against high-glucose-induced toxicity mediated by sirtuin 1-dependent eNOS and intracellular cGMP expression in bone marrow-derived EPC⁽ Reference Zhao, Du and Chen ^{142 )} has been reported. The vasoprotective role of quercetin has also been evidenced by phosphorylating eNOS at Ser1179 through cAMP/PKA signalling and enhancing the production of NO^{∙ (} Reference Li, Sun and Han ^{143 )}.

Gallic acid, a trihydroxy benzoic acid, restored eNOS activity in glomerular endothelial cells of diabetic rats through oxidative stress reduction in diabetic nephropathy⁽ Reference Tian, Tang and Liu ^{144 ,} Reference Tian, Gan and Li ^{145 )}. Daidzein, a phyto-oestrogen, reversed changes in vascular reactivity through NO^∙ and PG-related pathways, and attenuated oxidative stress in aortic tissue of diabetic rats⁽ Reference Roghani, Vaez Mahdavi and Jalali-Nadoushan ^{146 )}. Other polyphenolic compounds such as epigallocatechin gallate (EGCG), hesperetin, α-linolenic acid, ferulic acid and catechin hydrate have also been reported to reverse endothelial dysfunction through the activation of the PI3K/Akt/eNOS pathway, as demonstrated in both in vitro and in vivo diabetic models⁽ Reference Yin, Qi and Song ^{147 –} Reference Bhardwaj, Khanna and Balakumar ^{151 )}.

ADMA, the endogenous NOS inhibitor, is disposed to be one of the causative factors in endothelial dysfunction. Tang et al.⁽ Reference Tang, Hu and Chen ^{152 )} demonstrated that EGCG, a polyphenolic catechin, attenuated endothelial dysfunction in diabetic models by decreasing ADMA level via increasing DDAH activity. In addition, silibinin, a flavonolignan, markedly improved endothelial function in T2D mice by reducing circulating and vascular ADMA levels⁽ Reference Li Volti, Salomone and Sorrenti ^{153 )}.

The protective role of berberine, an alkaloid, has been reported to increase the expression of AMPK and eNOS, whereas it down-regulates NOX, thereby ameliorating endothelial dysfunction in palmitate-exposed endothelial cells⁽ Reference Zhang, Wang and Li ^{154 )}. Wang et al.⁽ Reference Wang, Huang and Lam ^{155 )} showed that berberine attenuated high-glucose-induced endothelial dysfunction through the activation of AMPK/eNOS signalling, mediating NO^∙ and cGMP production, and endothelium-dependent vasodilatation.

Vascular endothelial growth factor-mediated angiogenesis

VEGF, a potent pro-angiogenic growth factor, and its receptors in endothelial cells promote angiogenesis and cell proliferation⁽ Reference Ferrara ^{156 )}. Under diabetic conditions, elevated VEGF level acts as a pathological angiogenic stimulus leading to ocular neovascularisation (retinopathy) and nephropathy, whereas low levels of VEGF activity lead to cardiomyopathy and peripheral neuropathy because of insufficient angiogenesis⁽ Reference Wirostko, Wong and Simo ^{157 )}. The relationship between VEGF and vascular complications is an intricate process, particularly in patients with diabetes. This situation provides an insight into the complicated interplay between VEGF, VEGF receptors and the resulting systemic effects on microvascular and macrovascular diseases⁽ Reference Wirostko, Wong and Simo ^{157 )}.

In diabetic retinopathy, VEGF receptors are up-regulated in retinal endothelial cells. Increased VEGF favours the proteolysis of endothelial basement membrane, which is the limiting step in angiogenesis, leading to retinopathy. In addition, a direct correlation between VCAM-1 and VEGF in the vitreous fluid of retinopathy patients has been reported⁽ Reference Simo and Hernandez ^{158 )}. VEGF and its receptor expression levels are critical in maintaining normal glomerular podocytes and renal tubular function. VEGF expressed in glomerular podocytes activates VEGF receptor 2 on glomerular capillary endothelial cells, regulating endothelial fenestrations and permeability⁽ Reference Schrijvers, Flyvbjerg and De Vriese ^{159 )}. Hyperglycaemia together with ROS elevates VEGF level and increases glomerular permeability, hyperfiltration and hypertrophy contributing to diabetic nephropathy⁽ Reference Nakagawa, Kosugi and Haneda ^{160 )}.

Diabetic macrovascular complications such as cardiomyopathy are characterised by impaired angiogenesis. Cardiomyopathy, an inability of the heart to circulate blood throughout the body, has been reported with reduced myocardial VEGF and its receptor expressions. In addition, it results in decreased capillary density accompanied by decreased myocardial perfusion and progressive left ventricular dysfunction⁽ Reference Yoon, Uchida and Masuo ^{161 )}. VEGF has an important role in nerve regeneration, and its regulation reflects the functional state of peripheral nerves. It protects the dorsal root ganglia against diabetes-induced neurotoxicity. The reduced levels of VEGF under hyperglycaemia lead to late-onset motor neuron degeneration⁽ Reference Verheyen, Peeraer and Lambrechts ^{162 )}. Under hyperglycaemic conditions, impaired wound healing mediated by down-regulation of VEGF receptors results in impaired angiogenesis, which in turn is the cause for EPC dysfunction, low platelet count, reduced granulation and defective lymphatic vasculature⁽ Reference Verheul and Pinedo ^{163 )}.

Reversal from endothelial dysfunction, evidenced by a few polyphenol antioxidants, is effected by down-regulation of angiogenesis mediated by VEGF. Curcumin, a well-known antioxidant, inhibits VEGF expression in STZ-induced diabetic retina and kidney, thereby mediating vascular protection⁽ Reference Mrudula, Suryanarayana and Srinivas ^{164 –} Reference Sawatpanich, Petpiboolthai and Punyarachun ^{166 )}. Chlorogenic acid found abundantly in coffee significantly decreased VEGF levels and thereby reduced retinal vascular hyperpermeability and leakage in the diabetic retinopathy⁽ Reference Shin, Sohn and Park ^{167 )} rat model.

VEGF mediates the activation of PKC, and its translocation to endothelial cell membranes triggers the angiogenesis process⁽ Reference Wang, Pampou and Fujikawa ^{168 )}. The antiangiogenic activity of curcumin was demonstrated in human retinal endothelial cells reported to be caused by a reduction in glucose-induced mRNA levels of VEGF expression, and it also inhibits PKCβII translocation induced by VEGF⁽ Reference Premanand, Rema and Sameer ^{169 )}. The protective role of paeonol has been studied against high-glucose-induced vascular endothelial cell injury in a co-culture model system with vascular smooth muscle cells. Restored vascular homeostasis was evidenced by down-regulation of VEGF and PDGF-B, rat sarcoma (Ras), phosphorylated rapidly accelerated fibrosarcoma (p-Raf) and phosphorylated extracellular signal-regulated kinases (p-ERK) protein expressions in the vascular smooth muscle cells⁽ Reference Chen, Dai and Wang ^{170 )}. Scutellarein, a flavone found in Scutellaria lateriflora, suppresses VEGF and cell proliferation⁽ Reference Gao, Zhu and Tang ^{171 )} in high-glucose-treated human retinal endothelial cells. Resveratrol down-regulated VEGF/fetal liver kinase-1 (Flk-1) (VEGF receptor-2) expression and thereby modulated hyperpermeability and junction disruption in glomerular endothelial cells. It also ameliorates high-glucose-induced hyperpermeability mediated by overexpressed caveolin-1 in aortic endothelial cells⁽ Reference Tian, Zhang and Ye ^{172 )}.

R-(+)- α-lipoic acid inhibits VEGF-stimulated proliferation in microvascular and macrovascular endothelial cell isolated from the diabetic patients independent of their vascular origin. It reduces both apoptosis and proliferation of retinal endothelial cells through the activation of Akt and retinoblastoma protein transcription factor (E2F-1)⁽ Reference Artwohl, Muth and Kosulin ^{173 )}. Hyperglycaemia and increased AGE in diabetes cause inactivation of the VEGF receptors such as Flk-1 and hence altered VEGF expression, affecting endothelial growth and migration⁽ Reference Simons ^{174 )}. VEGF is vital in promoting collateral vessel formation after ischaemic events and plays a key role in wound healing⁽ Reference Ferrara ^{156 )}. Defective wound healing in diabetic patients is often associated with impaired VEGF expression resulting in failure to form the vasculature⁽ Reference Lerman, Galiano and Armour ^{175 )}. VEGF also mediates the survival of immature vessels through Flk-1 via the PI3K/Akt pathway⁽ Reference Naruse, Rask-Madsen and Takahara ^{176 )}. In addition to cell growth, VEGF inhibits apoptosis through the Akt pathway⁽ Reference Park, Kim and Lim ^{177 )}. Nakamura et al.⁽ Reference Nakamura, Naruse and Kobayashi ^{178 )} demonstrated the antiapoptotic potential of VEGF in EPC isolated from cord blood through the activation of Akt signalling. The abnormal cross talk between VEGF-A and NO^∙ pathways is fuelled by the hyperglycaemia-induced oxidative stress⁽ Reference Tufro and Veron ^{179 )}.

Yang et al. reported that the elevated glucose down-regulated VEGF through attenuation of p42/44 MAPK phosphorylation with the activation of mitochondrial apoptosis pathway mediated by ROS and Ca leading to endothelial dysfunction. Restoration of VEGF explains its role in apoptosis inhibition with a decrease in the B-cell lymphoma 2-associated X protein:B-cell lymphoma 2 (Bax:Bcl-2) ratio, thereby inhibiting caspase-3 activation⁽ Reference Yang, Mo and Gong ^{180 )}.

With cultured human glomerular endothelial cells under high-glucose and vascular endothelial growth factor receptor 1 (VEGFR1) inhibition, induced apoptosis and oxidative stress are mediated by the suppression of PI3K-Akt phosphorylation⁽ Reference Yang, Lim and Kim ^{181 )}. T2D mice with VEGFR1 inhibition showed apoptosis of the glomerular cells with albuminuria, mesangial matrix expansion and inflammatory cell infiltration, with the inactivation of eNOS-NOx signalling. The uncoupling of the VEGF–NO^∙ axis in the glomeruli resulting from eNOS inactivation has been reported in glomerular endothelial dysfunction⁽ Reference Nakagawa, Sato and Kosugi ^{182 )}. Tian et al.⁽ Reference Tian, Gan and Li ^{145 )} confirmed the improvement of glomerular pathological changes in diabetic rats through oxidative stress reduction and VEGF–NO^∙ axis recovery and prevention of glomerular endothelial dysfunction by propyl gallate.

Endoplasmic reticulum stress pathway

ER is the organelle in which proteins fold and attain their native conformation and post-translational modifications such as N-linked glycosylation, disulfide bond formation, lipidation, hydroxylation and oligomerisation⁽ Reference Benyair, Ron and Lederkremer ^{183 )}. Any perturbation in the protein folding machinery leads to the accumulation of unfolded/misfolded proteins and activates UPR. Elevation in levels of glucose, oxidised phospholipids and homocysteines disturbs the endothelial cells and trigger ER stress⁽ Reference Luo, He and Zhang ^{184 ,} Reference Gharavi, Gargalovic and Chang ^{185 )}. As a counter mechanism to maintain ER homeostasis, UPR up-regulates ER chaperons and foldases, and activates endoplasmic reticulum-associated degradation and also down-regulates the expression of secretory proteins, thereby reducing the accumulation of unfolded proteins⁽ Reference Friedlander, Jarosch and Urban ^{186 )}. ER stress sensors such as PERK, inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) play a central role in the initiation and regulation of UPR⁽ Reference Parmar and Schroder ^{187 )}. However, when the stress is beyond the holding capacity of UPR, it up-regulates IRE1, which activates JNK signalling and caspase-12⁽ Reference Yoneda, Imaizumi and Oono ^{188 )}; in addition to CHOP activation via phosphorylation⁽ Reference Xu, Bailly-Maitre and Reed ^{189 )}, these events culminate in the suppression of the antiapoptotic protein Bcl-2, rendering the cells to undergo apoptosis⁽ Reference McCullough, Martindale and Klotz ^{190 )}.

The ER stress responses has been implicated in the endothelial dysfunction observed in T1D, T2D and obesity⁽ Reference Chen, Wang and Li ^{191 ,} Reference Tsiotra and Tsigos ^{192 )}. Experimental conditions mimicking hyperglycaemia with supra-physiological dextrose concentrations and over-expression of exogenous GLUT-1 increased the ER stress in human umbilical vein endothelial cells (HUVEC)⁽ Reference Sheikh-Ali, Sultan and Alamir ^{72 )}. It has also been evaluated that ER stress activates IRE1 via JNK phosphorylation. It perturbs insulin signalling in endothelial cells by inhibiting IRS-1 and reducing NO production⁽ Reference Bakker, Eringa and Sipkema ^{38 )}. PERK mediated hyperglycaemia-induced endothelial inflammation and retinal vascular leakage in T1D mouse model⁽ Reference Chen, Wang and Li ^{191 )}, and endothelial cell apoptosis under high-glucose conditions have been reported⁽ Reference Zhang, Fu and Xu ^{193 )}.

Experimental evidence spotlights ER stress to be a major causative factor for neurodegenerative diseases in diabetes⁽ Reference Sheikh-Ali, Sultan and Alamir ^{72 ,} Reference Kaufman ^{194 )}. For instance, ER stress is a potential mediator of inflammation in diabetic retinopathy⁽ Reference Li, Wang and Yu ^{195 )}, which has been well documented with the experiment of Chen et al.⁽ Reference Chen, Wang and Li ^{191 )}. Moreover, diabetic retinopathy-associated distorted retinal angiogenesis has been linked to ER stress⁽ Reference Salminen, Kauppinen and Hyttinen ^{196 ,} Reference Wang, Park and Duh ^{197 )}. Hence, inhibition of ER stress and its specific mediators has been acknowledged to be a potential target for vascular complications under hypeglycaemia. Antioxidants such as α-tocopherol and ascorbic acid did not ameliorate glucose-induced ER stress condition⁽ Reference Sheikh-Ali, Sultan and Alamir ^{198 )}. Research studies focusing on endothelial ER stress under hyperglycaemic conditions are limited, and only few polyphenolic compounds are reported to overcome the UPR. Recent research with human endothelial cells by Song et al. indicated provoking of ER stress with increased ROS production in hyperglycaemia. Mangiferin, a polyphenolic compound, effectively inhibited ER stress-associated oxidative stress by reducing ROS production and by attenuating IRE1 phosphorylation⁽ Reference Song, Li and Hou ^{199 )}.

Inflammasome is a multiprotein intracellular complex and an innate immune system receptor that regulates the activation of caspase-1 and induces inflammation under stress conditions⁽ Reference Guo, Callaway and Ting ^{200 )}. The important components of the inflammasome complex include the nucleotide-binding domain and leucine-rich repeat-containing proteins (also known as NOD (nucleotide oligomerisation domain)-like receptors (NLR))⁽ Reference Takeuchi and Akira ^{201 )}. Upon sensing stress stimuli, the relevant NLR activates caspase-1, which subsequently functions to cleave the proinflammatory IL-1 cytokines into their active forms: IL-1β and IL-18. It has been reported that ER-activated inflammasome formation evokes endothelial inflammation and apoptosis, playing a critical role in the onset of endothelial dysfunction⁽ Reference Mohamed, Hafez and Fairaq ^{202 )}. Mangiferin, a xanthonoid, has been reported to reduce the expression of thioredoxin-interacting protein (TXNIP) and its NOD-like receptor protein 3 (NLRP3) inflammasome. It also inhibited ER stress-associated IRE1α phosphorylation and regulated endothelial homeostasis in an AMPK-dependent manner⁽ Reference Song, Li and Hou ^{199 )}.

Polyphenolic compound combinations such as quercetin, luteolin and EGCG alleviate palmitate-induced ER stress in endothelial cells. They were found to maintain the endothelial homeostasis by inhibiting ER stress-associated TXNIP and NLRP3 inflammasome activation, and they restored mitochondrial function and protected cells against inflammation and apoptosis⁽ Reference Wu, Xu and Li ^{203 )}. Few other compounds such as ilexgenin A⁽ Reference Li, Yang and Chen ^{204 )}, astragaloside IV and cycloastragenol⁽ Reference Zhao, Li and Zhao ^{205 )} have been reported to improve endothelial function under palmitate-induced ER stress in an AMPK-dependent manner and by inhibiting TXNIP/NLRP3 inflammasome pathway.

Experimental ER stress has been induced with pharmacological agents such as thapsigargin, tunicamycin, A23187 and brefeldin A⁽ Reference Natsume, Ito and Satsu ^{206 )}. The contemplated possible involvement of ER stress in endothelial dysfunction has been studied by our group in tunicamycin-induced HUVEC. Tunicamycin blocks N-glycosylation of proteins and causes an extensive protein misfolding, thus activating UPR in the endothelial cells⁽ Reference Reiling, Clish and Carette ^{207 )}. The protective effect of quercetin against the activation of ER stress was attributed to the up-regulation of markers such as 78kDa glucose-regulated protein (GRP78), a molecular chaperone and CHOP in unresolved diabetic and experimental ER stress conditions⁽ Reference Szegezdi, Logue and Gorman ^{208 )}. Quercetin pre-treatment decreased tunicamycin-induced ER stress marker expression in HUVEC⁽ Reference Suganya, Bhakkiyalakshmi and Suriyanarayanan ^{209 )}. Poly ADP ribose polymerase (PARP), the end point of a number of cell signalling pathways, is known to control many physiological and pathological outcomes and tends to mediate immunity and inflammation⁽ Reference Cuzzocrea ^{210 )}. In addition, it has been demonstrated that quercetin re-establishes ER homeostasis by reducing caspase-3 and caspase-dependent PARP cleavage and normalising the antioxidant enzymes SOD1 and catalase (CAT) under ER stress⁽ Reference Suganya, Bhakkiyalakshmi and Suriyanarayanan ^{209 )}. Further, thapsigargin-induced ER calcium depletion due to inhibition of the calcium-ATPase has been restored with resveratrol in the endothelium⁽ Reference Buluc and Demirel-Yilmaz ^{211 )}, and it also prevented retinal vascular degeneration induced by tunicamycin⁽ Reference Li, Wang and Huang ^{212 )}.

Inflammatory pathway

In addition to the mediators of vasomotor functions, vascular endothelial cells release inflammatory mediators that are forerunners in the initiation, amplification and resolution of the inflammatory response. Endothelial cells are more prone to various stresses including glucose toxicity, which stimulates the secretion of proinflammatory cytokines and adhesion factors⁽ Reference Du, Edelstein and Rossetti ^{213 )}. Endothelial dysfunction focusing vascular inflammation has been implicated in the induction of vasoconstrictors, adhesion molecules, Ang II and ET-1⁽ Reference Durier, Fassot and Laurent ^{214 )}. ROS tends to be the competent stimulator of ET-1, which in turn activates NOX and XO, worsening the cellular oxidative stress contributing to vascular remodelling and endothelial dysfunction⁽ Reference Gao and Mann ^{215 ,} Reference Amiri, Virdis and Neves ^{216 )}.

In addition, hyperglycaemia activates NF-κB, the master regulator and major proinflammatory transcription factor that controls multiple proinflammatory and proatherosclerotic targets in endothelial cells, and thus activates inflammation⁽ Reference Rahman and Fazal ^{217 )}. The regulation of NF-κB is primarily associated with the IκB family of transcription factor inhibitor proteins, and its phosphorylation implies the most important step in its activation⁽ Reference Viatour, Merville and Bours ^{218 )}. At the downstream, NF-κB activates the transcription of proinflammatory genes including TNF-α, IL-1, IL-8, E-selectin, VCAM-1 and ICAM-1 in vascular endothelial cells, as reviewed by Xiao et al.⁽ Reference Xiao, Liu and Wang ^{219 )}, and it also promotes the expression of MCP-1 and adhesion of leucocyte and monocytes to the endothelial cells, which is followed by their infiltration and differentiation into macrophages under hyperglycaemic conditions⁽ Reference Takaishi, Taniguchi and Takahashi ^{220 )}.

It has been well documented that under conditions of insulin resistance and T2D, inflammation has been reflected with an increase in TNF-α, IL-6, PAI-1, ET-1 and high-sensitive C-reactive protein relating it to endothelial dysfunction⁽ Reference Natali, Toschi and Baldeweg ^{221 )}. In addition, it has been explicated that NEFA-induced ROS activates NF-κB, inhibitor of kappaB kinase (IKK)α and impairs insulin-stimulated activation of eNOS and NO^∙ production in endothelial cells⁽ Reference Zhang, Dellsperger and Zhang ^{222 )}.

Many polyphenolic compounds have been reported to ameloriate endothelial dysfunction by inhibiting inflammatory mediators. A recent study with palmitate-induced insulin resistance model reveals that resveratrol suppressed IKKβ/NF-κB phosphorylation, TNF-α and IL-6 production and restored the IRS-1/Akt/eNOS signalling pathway in endothelial cells⁽ Reference Liu, Jiang and Zhang ^{139 )}. Resveratrol has been reported to block TNF-α-induced activation of NF-κB in coronary arterial endothelial cells and to inhibit inflammatory mediators⁽ Reference Csiszar, Smith and Labinskyy ^{223 )}, exerting its effect through action on (IKK) cascade, thereby attributing to its antioxidant properties.

Ferulic acid combined with astragaloside IV is known to protect vascular endothelial dysfunction in STZ-induced diabetic rats by promoting the release of NO^∙ and eNOS, and inhibiting the hyper-stimulation of MCP-1, TNF-α and NF-κB P65 in aorta⁽ Reference Yin, Qi and Song ^{147 )}. The administration of the flavonoids such as EGCG, quercetin and delphinidin increased the bioactivity of NO^∙ and prevented endothelial cell apoptosis by modulating inflammatory pathways⁽ Reference Dayoub, Andriantsitohaina and Clere ^{224 )}. Curcumin inhibits proinflammatory cytokines, TNF-α, ICAM-1, NOX2 and cyclo-oxygenase-2 expressions and reduces leucocyte–endothelium interaction in diabetes-induced vascular inflammatory models⁽ Reference Wongeakin, Bhattarakosol and Patumraj ^{225 –} Reference Rungseesantivanon, Thenchaisri and Ruangvejvorachai ^{227 )}. The report of Mahmoud et al. confirmed that quercetin confers protection to diabetes-induced vasoconstriction, leading to low-grade inflammation with concomitant reduction in serum levels of both TNF-α, CRP and inhibition of NF-κB in aorta⁽ Reference Mahmoud, Hassan and El Bassossy ^{228 )}.

In addition, increased adhesion of monocytes to bovine aortic endothelial cells (BAEC) and expression of VCAM-1 in response to TNF-α treatment was reversed by pre-exposure with hesperetin⁽ Reference Rizza, Muniyappa and Iantorno ^{148 )}. In a clinical study, hesperetin treatment increased flow-mediated dilation with the reduction in circulating inflammatory biomarkers such as CRP, serum amyloid A protein and soluble E-selectin⁽ Reference Rizza, Muniyappa and Iantorno ^{148 )}. Yamagata et al. reported increased expression of ICAM-1 and VCAM1 in the endothelial cells under exposure to high glucose and TNF-α; further, the treatment with apigenin significantly inhibited the expression of adhesion molecules and also IKKα and IKKi, thereby mediating the protection against atherosclerotic vascular diseases⁽ Reference Yamagata, Miyashita and Matsufuji ^{229 )}.

The molecular link between the activation of PKC and up-regulation of VCAM-1 expression under hyperglycaemic conditions involving NF-κB activation has been studied⁽ Reference Tuttle and Anderson ^{230 )}. EGCG has been reported to inhibit the vascular inflammation in hyperglycaemia by suppression of the PKC/NF-κB signalling pathway, and it also prevented monocyte adhesion in HUVEC cells⁽ Reference Yang, Han and Chen ^{231 )}.

MAPK signalling was reported to regulate the high-glucose-induced inflammatory cytokines⁽ Reference Kim, Kim and Kim ^{232 )}. A study with emodin (3-methyl-1, 6, 8-trihydroxyanthraquinone), an anthraquinone in HUVEC, reported the inhibition of glucose-induced phosphorylation of ERK 1/2 and p38 MAPK, thereby protecting the cells against inflammation⁽ Reference Gao, Zhang and Li ^{233 )}. Brazilin markedly inhibited the high-glucose-induced MAPK/ERK signal transduction pathway, thereby inhibiting the phosphorylation of extracellular signal-regulated kinase and transcription factor NF-κB in HUVEC cells⁽ Reference Jayakumar, Chang and Lin ^{234 )}. The report of Kim et al.⁽ Reference Kim, Kim and Kim ^{232 )} demonstrated that hesperidin, naringenin and resveratrol reduced high-glucose-induced ICAM-1 expression via the p38 MAPK signalling pathway, contributing to the inhibition of monocyte adhesion to endothelial cells. In STZ-induced diabetic rats and high-glucose-induced HUVEC cells, α-linolenic acid treatment decreased the expression of P-selectin, ICAM-1 and neutrophil adhesion via Akt phosphorylation⁽ Reference Zhang, Li and Li ^{150 )}.

In high-glucose-induced vascular inflammation in HUVEC and mouse models, increased vascular permeability, monocyte adhesion, expressions of CAM, formation of ROS and activation of NF-κB, with induced expressions of MCP-1 and IL-8, were ameliorated not only by emodin-6-O-β-d-glucoside⁽ Reference Lee, Ku and Lee ^{235 )} but also by other polyphenols such as fisetin, orientin and naringin model, thereby reducing diabetic complications and atherosclerosis⁽ Reference Xiong, Wang and Zhang ^{236 –} Reference Ku, Kwak and Bae ^{238 )}. A recent study with three structurally related polyphenols such as baicalin, baicalein and wogonin inhibited endothelial cell barrier disruption, suggesting its protection against vascular inflammatory diseases⁽ Reference Ku and Bae ^{239 )}. Chronic resveratrol treatment in T2D rats showed a vasoprotective effect through the inhibition of inflammation mediators such as IL-1β and IL-6, decreasing the circulating vWF levels, recovered vascular permeability in both carotid artery and thoracic aorta. In addition, resveratrol showed an inhibitory effect against NF-κB p65, proinflammatory mediators including TNF-α, ICAM-1 and MCP-1 in endothelial cell lines⁽ Reference Zheng, Zhu and Chang ^{240 )}.

The protective effect of genistein was proven in the human aortic endothelial cells under exposure to high glucose by its inhibitory action on monocytes, and suppression of MCP-1 and IL-8⁽ Reference Babu, Si and Fu ^{241 )}. The combination of quercetin, EGCG and curcumin attenuated high-glucose-induced membrane fluidity and transmembrane potential of HUVEC cells by reducing the AGE products formed and limiting the release of pro-inflammatory factors such as MCP-1, thereby preventing chronic inflammation⁽ Reference Margina, Gradinaru and Manda ^{242 )}.

Nuclear factor-E2-related factor 2-mediated antioxidant pathway

Nrf2 is a transcription factor that is perhaps the most prominent cellular defence mechanism against oxidative stress. Under basal conditions, Nrf2 is primarily localised in the cytoskeleton as a complex with Kelch-like erythroid cell-derived protein with cap (n) collar homology [ECH]-associated protein 1 (Keap1). Oxidative stress causes the dissociation of Nrf2 from Keap1 and is found to bind with antioxidant response elements (ARE) in the nucleus, promoting the transcription of a number of endogenous protective genes including antioxidant genes, phase II detoxification enzyme genes and molecular chaperones. Nrf2 endorses cellular redox homeostasis via its downstream activation of several intracellular antioxidants such as γ-glutamine cysteine synthase, SOD, CAT, glutathione reductase, glutathione peroxidase, peroxiredoxin and thioredoxin reductase, and phase II detoxifying enzymes such as HO-1, glutathione S-transferase, NAD(P)H quinone oxidoreductase 1; and also proteins involved in detoxifying xenobiotics and neutralising ROS⁽ Reference Cho, Reddy and Kleeberger ^{243 ,} Reference Zhang ^{244 )}.

Several protein kinases such as MAPK, PKC, PI3K, glycogen synthase kinase 3 beta (GSK3b) and casein kinase 2 are reported to be involved in Nrf2 regulation, playing a role in its phosphorylation⁽ Reference Chen, Varner and Rao ^{245 )}. In addition, the cross talk between Nrf2 and ATF6 has been demonstrated in endothelial cells⁽ Reference Afonyushkin, Oskolkova and Philippova ^{246 )}. The Nrf2/ARE pathway regulates DDAH in order to reduce ADMA and PPAR-γ to increase eNOS and its phosphorylation⁽ Reference Luo, Aslam and Welch ^{247 )}. Several studies have reported that Nrf2 acts as a defence mechanism against diabetes-mediated complications. Zhong et al. ⁽ Reference Zhong, Mishra and Kowluru ^{248 )} reported impairment of Nrf2-Keap1 in endothelial cells that were exposed to high glucose and also in retinas from donors with diabetic retinopathy. In vivo models of high-fat-diet Nrf2 ^–/– and Nrf2 ^+/+ mice explained the increase of vascular ROS in Nrf2 ^–/– than in Nrf2 ^+/+ mice and the adaptive activation of the Nrf2-ARE pathway confirming the endothelial protection under diabetes⁽ Reference Ungvari, Bailey-Downs and Gautam ^{249 )}. It has also reported that patients with diminished Nrf2 activity had a higher incidence of T2D and cardiovascular problems⁽ Reference Suh, Shenvi and Dixon ^{250 )}. In STZ-induced diabetic Nrf2 knockdown mice, the diminished oxidative defence causes reduced renal function leading to diabetic nephropathy⁽ Reference Jiang, Huang and Lin ^{251 )}. Activation of insulin signalling through the PI3K/Akt/mTOR (mechanistic target of rapamycin)/Nrf2/GCLc (glutamate-l-cysteine ligase; catalytic subunit) pathway affords significant protection in human brain endothelial cells against hyperglycaemia by maintaining cellular redox balance⁽ Reference Okouchi, Okayama and Alexander ^{252 )}. In addition, the Nrf2 knockout mouse model confirmed the role of Nrf2 in hyperglycaemia-induced ROS production and in cardiomyocytes and renal tissue injury⁽ Reference Yoh, Hirayama and Ishizaki ^{253 )}. Attention is being drawn to uncover the protective mechanism of polyphenolic compounds against diabetes-mediated endothelial dysfunction via Nrf2 activation.

Resveratrol confers a protective effect against high-glucose-induced oxidative stress in endothelial cells and vasoprotective effect in high-fat-diet mice, through the Nrf2 pathway⁽ Reference Ungvari, Bagi and Feher ^{254 )}. Sulforaphane decreases the ROS production and restores myogenic response in T2D mesenteric arterioles through the Nrf2 pathway⁽ Reference Velmurugan, Sundaresan and Gupta ^{255 )}. HUVEC treated with eriodictyol showed the up-regulation of HO-1 through ERK/Nrf2/ARE-dependent pathways, in which ERK aids in the translocation of Nrf2 in the nucleus for HO-1 activation⁽ Reference Lee, Yang and Son ^{256 )}.

Moreover, Nrf2 activation inhibits NF-κB through reduced ROS-mediated IKK activation and inhibits the degradation of IκB. Interaction between these two pathways maintains the homeostasis, in which oxidative stress causes the imbalance in the Nrf2–NF-κB axis⁽ Reference Ganesh Yerra, Negi and Sharma ^{257 )}. Sulforaphane also suppressed hyperglycaemia-induced ROS and metabolic dysfunction in human microvascular endothelial cell via Nrf2 activation⁽ Reference Xue, Qian and Adaikalakoteswari ^{258 )}. Sulforaphane has been also reported to enhance Nrf2-mediated antioxidant expression in experimental models of diabetic neuropathy through Nrf2 and NF-κB modulation⁽ Reference Negi, Kumar and Sharma ^{259 )}.

The molecular mechanisms of polyphenol antioxidants with emphasis on their potential in improvement of endothelial function are summarised in Table 1.

Table 1

The molecular mechanism of polyphenol antioxidants in modulating endothelial dysfunction under diabetes

eNOS, endothelial nitric oxide synthase; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; AMPK, AMP-activated protein kinase; TXNIP, thioredoxin-interacting protein; NLRP3, NOD-like receptor protein 3; ER, endoplasmic reticulum; IRE1, inositol-requiring enzyme 1; HSP90, heat shock protein 90; NO^∙, nitric oxide; VEGF, vascular endothelial growth factor; PKC, protein kinase C; NOX2, NADPH oxidase isoform 2; SOD, superoxide dismutase; CAT, catalase; COX-2, cyclo-oxygenase 2; IRS-1, insulin receptor substrate 1; DDAH, dimethylarginine dimethylaminohydrolase; ADMA, asymmetric dimethylarginine; MCP-1, monocyte chemoattractant protein-1; Nrf2, nuclear factor-E2-related factor 2; ARE, antioxidant response elements; ET-1, endothelin-1; PERK, protein kinase RNA-like endoplasmic reticulum kinase; PDGF-B, platelet-derived growth factor B; cGMP, cyclic GMP; CHOP, C/ERB homologous protein; SIRT1, sirtuin 1; Ang 2, angiopoietin 2; HO-1, heme oxygenase-1; cAMP, cyclic AMP; vWF, von Willebrand factor; AMPK, AMP-activated protein kinase; MDA, malondialdehyde; THP-1, engineered human reporter monocytes; iNOS, inducible nitric oxide synthase; GCLc, glutamate-cysteine ligase, catalytic subunit; GCLM, glutamate-cysteine ligase, modifier subunit; GSH-Px, glutathione peroxidase.