J Mol Med (2014) 92:441–452 DOI 10.1007/s00109-014-1146-1

REVIEW

The pathobiology of diabetic vascular complications—cardiovascular and kidney disease Stephen P. Gray & Karin Jandeleit-Dahm

Received: 30 January 2014 / Revised: 3 February 2014 / Accepted: 14 March 2014 / Published online: 1 April 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract With the increasing incidence of obesity and type 2 diabetes, it is predicted that more than half of Americans will have diabetes or pre-diabetes by 2020. Diabetic patients develop vascular complications at a much faster rate in comparison to non-diabetic individuals, and cardiovascular risk is increased up to tenfold. With the increasing incidence of diabetes across the world, the development of vascular complications will become an increasing medical burden. Diabetic vascular complications affect the micro- and macrovasculature leading to kidney disease often requiring dialysis and transplantation or cardiovascular disease increasing the risk for myocardial infarction, stroke and amputations as well as leading to premature mortality. It has been suggested that many complex pathways contribute to the pathobiology of diabetic complications including hyperglycaemia itself, the production of advanced glycation end products (AGEs) and interaction with the receptors for AGEs such as the receptor for advanced glycation end products (RAGE), as well as the activation of vasoactive systems such as the renin-angiotensin aldosterone system (RAAS) and the endothelin system. More recently, it has been hypothesised that reactive oxygen species derived from NAD(P)H oxidases (Nox) may represent a common downstream mediator of vascular injury in diabetes. Current standard treatment of care includes the optimization of blood glucose and blood pressure usually including inhibitors of the renin-angiotensin system. Although these interventions are able to delay progression, they fail to prevent the development of complications. Thus, there is an urgent medical need to identify novel targets in diabetic vascular complications which may include the blockade of Nox-derived ROS S. P. Gray (*) : K. Jandeleit-Dahm Diabetes Complications Division, Baker IDI Heart & Diabetes Research Institute, PO Box 6492, St Kilda Rd, Melbourne, VIC 8008, Australia e-mail: [email protected]

formation, as well as blockade of AGE formation and inhibitors of RAGE activation. These strategies may provide superior protection against the deleterious effects of diabetes on the vasculature. Keywords Diabetes . Oxidative stress . RAS . AGEs . Nephropathy . Cardiovascular disease

Introduction It is estimated that 7.7 % of adults in developed countries have type 2 diabetes, and by 2030, this is predicted to increase by 20 % in developed countries and by almost 70 % in developing countries [1]. Diabetes is currently viewed as the major epidemic of this century, with the incidence of new diabetic patients increasing by 50 % over the last 10 years [2, 3]. Individuals with both type 1 and type 2 diabetes are at a greater risk of developing complications of the vascular system [4, 5]. Diabetic patients have an up to tenfold increased risk compared to age-matched non-diabetic patients to experience cardiovascular events [6–8]. Diabetes-associated vascular complications are grouped into two major arms, either affecting the macro-vasculature (large arteries, including aorta, femoral and coronary arteries) or the micro-vasculature (small blood vessels, including capillaries of the eye, kidney and nerves) [4, 9–14]. Macro-vascular complications generally encompass the accelerated development of cardiovascular and peripheral vascular diseases resulting clinically in myocardial infarction, stroke and amputations. The micro-vascular complications of diabetes include eye disease (retinopathy) and kidney disease (nephropathy) as well as neuropathy. In this review, we will largely focus on the development of macro-vascular complications and the development of micro-vascular complications in the kidney.

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The precise mechanisms leading to the development of vascular complications in diabetes remain to be fully determined. It has been suggested that a range of mechanisms contribute to the development of a diabetes-associated vascular disease, including glucose itself as well as glucosedependent mechanisms, such as the formation of advanced glycation end products (AGEs) and the activation of vasoactive hormonal systems involved in blood pressure regulation such as the renin-angiotensin-aldosterone system (RAAS) and the endothelin system. More recently, is has been postulated that NAD(P)H oxidase (Nox) expression and activity, in addition to a breakdown in the nitric oxide synthase (NOS) signalling pathway [12, 15–20], may be common downstream pathway where many of the other pathways converge and ultimately lead to inflammation and fibrosis as well as vascular remodelling. In this present review, we will focus on three key pathways involved in the development of diabetic vascular complications. Firstly, we will discuss the renin-angiotensin system (RAS) which is involved in blood pressure control and regulation of vascular tone. Its effector hormone angiotensin II (AngII) which also mediates multiple effects on inflammation and fibrosis. Secondly, we will discuss the enhanced formation of advanced glycation end products (AGEs) in diabetes and their interaction with RAGE. Lastly, we will provide current evidence about the role of the family of NAD(P)H oxidases (Nox) which are involved in reactive oxygen species production and redox balance.

Diabetic micro-vascular complications Nephropathy Diabetic nephropathy represents the major cause for end-stage renal failure in the western world often requiring dialysis or transplantation. In 2010, more than 117,000 patients started dialysis for end-stage renal disease, with up to 44 % of these patients being diabetic [21, 22]. According to the World Health Organisation in 2012 >300 million people suffered from diabetes and that the increased all-cause mortality of diabetic patients will be end-stage renal disease [2, 21, 23]. Underlying renal disease is also a major risk factor for the development of a macro-vascular disease in diabetes leading to heart attacks and strokes [24]. The development and progression of diabetic nephropathy is complex due to the many cell types that are present within the kidney and the various physiological and molecular processes that occur within the kidney. High glucose concentrations induce specific cellular effects, which affect all cell types within the kidney, including podocytes, mesangial cells, endothelial cells, smooth muscle cells as well as the tubular cells including those of the collecting ducts [25].

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Clinically, diabetic nephropathy is characterised by the onset of micro-albuminuria which can further progress to macro-albuminuria or overt proteinuria and a subsequent decline in glomerular filtration rate, ultimately leading to uremia [25]. During these stages, the kidney undergoes a variety of changes including hypertrophy, characterised by an enlargement of the kidney through both hyperplasia and hypertrophy [26]. Furthermore, the kidney undergoes functional changes, and a period of hyperfiltration is often followed by a progressive decline in renal function. Diabetes is often associated with hypertension, and indeed, in the context of concomitant hypertension and diabetes, kidney disease is often accelerated [27, 28]. It has been shown that the intrarenal RAAS is activated in diabetes and contributes to many of the changes observed in diabetic nephropathy [29]. The classical signs of diabetic nephropathy include mesangial expansion due to the accumulation of extracellular matrix, glomerular hypertrophy and macrophage infiltration [30, 31]. In more advanced stages of kidney disease, tubulointerstitial injury is increased and is closely associated with the decline in renal function. In fact, recent reports are demonstrating that tubular injury is a better predictor of end-stage renal disease than classical markers which include albuminuria [32–35]. The current standard treatments of care include the control of systemic blood pressure and intraglomerular hypertension, involving the use of angiotensin converting enzyme (ACE) inhibitors [36] and angiotensin II (AngII) receptor antagonists [37] as well as the optimisation of glucose control. However, while these first-line therapies are effective at slowing the progression of diabetic nephropathy, they do not prevent or reverse the disease. Thus, a greater understanding of the disease process and focus on the early stages of disease development are needed in order to prevent disease development and progression [38]. Cardiovascular disease Diabetes is associated with increased cardiovascular disease (CVD) rates, increasing the risk for myocardial infarction, stroke and peripheral amputation [39, 40]. CVD accounts for more than half the mortality of diabetic patients and equates to an approximately threefold to tenfold increased risk of cardiovascular events in diabetes compared with the general population [41]. Atherosclerosis is a complex multi-cellular process involving the endothelium and smooth muscle cells but also circulating platelets and immune cells which interact and ultimately lead to the development of the characteristic ‘fatty streak’. Later in the disease process, more advanced and complex plaques are formed. These plaques may become unstable and rupture. Indirect markers of plaque instability include a larger number and increased size of necrotic cores as well as intraplaque haemorrhage and a thin fibrous cap. A ruptured plaque may lead to acute thrombus formation

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resulting in heart attacks and stroke [42]. The initiation steps of atherosclerosis development are still not fully determined; however, endothelial dysfunction seems to play a significant role. The endothelium is important for the maintenance of vascular homeostasis, ensuring a balance is maintained between vasoactive factors such as AngII and nitric oxide (NO) controlling its permeability, adhesiveness and integrity [43]. In the diabetic setting, this equilibrium is disrupted allowing for infiltration of oxLDL and lipids as well as immune cells including macrophages and T-cells into the vascular wall thus contributing to the formation of foam cell and fatty streak formation [28, 44–48]. Mechanisms of vascular complications in diabetes Renin-Angiotensin Aldosterone System (RAAS) Over the last decade, it has been increasingly appreciated that the renin-angiotensin-aldosterone system plays a pivotal role in the development of diabetic vascular complications and that this system is more complex than initially anticipated and still needs to be fully explored. The effector hormone of the RAAS is the vasoconstrictor molecule AngII which is a key regulator of mean arterial blood pressure and vascular tone. It has been thought to be the primary contributor to the development of diabetic vascular complications. However, it is also becoming more evident that a reduction in the vasodilatory factors of the RAAS such as angiotensin 1–7 may play just as a significant role [49]. The biological effects of AngII are mediated via two specific receptor subtypes, the angiotensin type 1 (AT1) receptor and the angiotensin type 2 (AT2) receptors [50]. The AT1 receptor is widely expressed across the vascular system, with the AT2 receptor predominately being expressed in the embryo; however, it can be significantly upregulated in the adult under pathological conditions [51–53]. AT1 knockout (KO) mice tend to have a lower blood pressure [54]. In the presence of diabetes, there is an attenuated development of renal disease with a reduction in renal inflammation and fibrosis, indicating that the AT1 receptor plays a pivotal role in the development of renal injury in diabetes [54]. These findings are supported by a number of studies examining pharmacological interventions of the RAAS, which have demonstrated end organ protection in diabetic complications [37]. These agents appear to block a large number of effects of AngII including vasoactive effects as well as the production of proinflammatory mediators and pro-fibrotic growth factors as well as extracellular matrix accumulation. Furthermore, RAAS blockade attenuates albuminuria/proteinuria through effects on growth factors such as VEGF as well as via effects on podocyte structure and function [36, 37, 55]. The role of the AT2 receptor in diabetic complications remains to be fully defined. Although there is very low

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expression of the AT2 receptor under the normal physiological setting in the adult vasculature, under pathological conditions such as in hypertension or diabetes the expression of the AT2 receptor is significantly upregulated [56, 57]. Classically, in most contexts the AT2 receptor appeared to act as a functional antagonist to the AT1 receptor via protection against reactive oxygen species (ROS)-mediated damage [58]; however, in the context of diabetes, there is increasing data to suggest that the AT2R may have similar actions to that of the AT1 receptor. These include the induction of cytokines such as VEGF, which is particularly important in the diabetic kidney and eye as well as promoting macrophage infiltration, a critical process in atherosclerosis development. It has been suggested that the AT2 receptor also mediates NFκB-dependent pathways, including activation of RANTES and MCP-1 [59–62]. In a recent study performed by our group, using a streptozotocin (STZ)-induced type 1 diabetes model, both an AT2 receptor antagonist and genetic deletion of the AT2 receptor were associated with reduced plaque formation suggesting that the suppression of the AT2 receptor leads to a reduction in the development of a macro-vascular disease [63]. Furthermore, AT2 blockade demonstrated a reduction in vascular hypertrophy and fibrosis in the model of AngII infusion [64]. In contrast, other studies have shown that blockade of the AT2 receptor can lead to a greater susceptibility to vascular injury [65, 66]. In addition, ventricular hypertrophy induced by pressure overload was not observed in AT2 KO mice [67]. These studies indicate that the role of the AT2 receptor particularly in the context of diabetes is highly complex and yet to be fully determined. More recently, a role for ACE2 has been suggested in the development of diabetic nephropathy. It has been hypothesised that ACE2 may be able to mitigate the negative effects of AngII [68]. Previously, studies have shown that the ACE2-mediated production of Ang1-7 counteracts the Ang production by ACE in such a way that mice with a global deletion of ACE2 have more renal injury in response to RAS activation [69]. More recently, studies in diabetic mice found that the global deletion of ACE2 or the pharmacological inhibition of ACE2 exacerbates the development of diabetic nephropathy [70, 71]. However, there remains uncertainty about the mechanisms involved [72]. In turn, podocytespecific overexpression of the human ACE2 was able to attenuate the development of diabetic nephropathy [73], highlighting that ACE2 activation could be a new target in mitigating the effects of diabetes on nephropathy. Advanced Glycation End Products (AGEs) AGEs are a chemically heterogeneous group of compounds that represent irreversible post-translational modifications resulting from a chemical reaction between reducing sugars or sugar-derived products and amino groups on proteins,

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lipids and nucleic acids [74]. In the presence of a chronic elevated glucose, the production of AGEs is accelerated, largely due in part to the enhanced metabolism of glucose known as glycolysis, resulting in the formation of a number of reactive products, which interact with protein residues to rapidly form AGEs [75]. Early reactive intermediates such as methyglyoxal, glyoxal and 3-deoxyglucasone are precursors for AGE formation and are highly reactive as well as leading to elevations in reactive oxygen species [76, 77]. The effects of AGEs in the vasculature are both receptor dependent and independent and are associated with an increased vascular stiffness via an increased vascular AGE accumulation. Currently, the best-described receptor is termed receptor for advanced glycation end products (RAGE) [78, 79]. The precise role of AGE receptors in normal physiology is unclear but may involve signalling and clearance; however, under specific disease states, upregulation of AGE receptors including RAGE occurs. Ligand binding of AGEs to RAGE is associated with increased inflammation, macrophage and T-lymphocyte infiltration as well as vascular remodelling, leading to accelerated atherosclerosis [80–84]. Furthermore, there is evidence that urinary AGE concentrations could act as a biomarker for diabetic kidney disease in humans [85–87]. Binding of AGEs to RAGE induces the activation of a cellular signalling cascade including vascular adhesion molecule 1 (VCAM-1), E-Selectin, VEGF and pro-inflammatory cytokines including IL1β, IL-6, TNFα and signalling molecules, such as NF-κB [88–92]. AGE/RAGE interaction is also able to stimulate the production of oxidative stress through NAD(P)H oxidase (covered later in this review), in addition to stimulating macrophage recruitment and accumulation into the vascular wall [93]. In diabetic individuals with complications, studies have demonstrated increased tissue and circulating concentrations of RAGE including a soluble form of the receptor (soluble RAGE), which are predictive of both cardiovascular events [94–97] and all-cause mortality [97, 98]. Transgenic mice with overexpression of RAGE demonstrate accelerated kidney disease in both the diabetic and nondiabetic contexts [99]. The administration of RAGEneutralising antibodies in rodent models of diabetes has also shown protection against renal complications [45, 93, 100–102]. The role of dietary AGEs remains controversial, but there have been reports that a high AGE diet can mimic some of the changes observed in diabetic nephropathy [103]. Inhibitors of AGE formation in human studies, including vitamin B supplementation, had only modest effects [104, 105] or even contributed to the enhancement of renal disease in diabetic patients [106]. In animal studies, therapies lowering AGE formation and accumulation have been shown to reduce renal injury and cardiovascular disease [45, 107–117]. One of the newer therapies is Alagebrium (ALT-711), which has been shown in vitro to cleave AGE crosslinks as well as to

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reduce their accumulation [118]. In animal studies, Alagebrium has also been shown to be effective in preventing and delaying the progression of kidney disease in models of diabetic nephropathy [114]. In the clinical setting, the administration of Alagebrium demonstrated cardiovascular benefits, such as a reduction in arterial pulse pressure, improved largeartery compliance and endothelial function [119, 120]. A smaller clinical trial performed with Alagebrium in type1 diabetic patients was terminated early (ClinicalTrial.gov NCT00739687) due to a lack of financial support. Another approach is the manipulation of the enzyme glyoxalase-1 (Glo-1), which is responsible for the removal of the highly reactive and toxic AGE precursor, methyglyoxal, and also lowers the tissue accumulation of AGEs [121, 122]. This approach has also been shown to translate into functional and structural benefits for diabetic neuropathy [123] and retinopathy [124]. As such, approaches to increase the activity of Glo-1 are an active area of research as it proves a potential therapeutic target in the treatment of diabetic micro-vascular complications, including in the eye, nerve and kidney. NAD(P)H Oxidase (Nox) There are many sources of ROS, however, only NAD(P)H oxidases (Nox) produce ROS as its primary function [125, 126]. Within the human vasculature, four isoforms of the Nox family have been detected, including Nox1, Nox2, Nox4 and Nox5 as well as subunits such as p47phox, p22phox and rac-1. The role of Nox5, which is absent in mice and rats, but present in humans and upregulated in disease, remains relatively unknown [127, 128]. The (patho) physiological functions of these Nox isoforms remain to be fully explored. The effects of ROS derived from Nox appear to modulate redox-sensitive MAP kinases (ERK1/2, p38MAP kinase, JNK), pro-inflammatory kinases (ERK5), involved in protein synthesis, cell cycle progression and cell proliferation, tyrosine kinases (c-Src, EGFR, PI3K) and transcription factors including those that have been extensively linked to inflammation (NFκB, AP-1 and HIF-1). ROS also promote a proinflammatory state through the direct activation of adhesion molecule expression, by inducing pro-inflammatory gene activation and by reducing NO availability [129]. Interaction between •O2− and endothelial NO leads to peroxynitrate (ONOO–) formation and loss of the beneficial actions of NO (which mediate endothelium-dependent vasodilation) [130, 131] further implicating the redox balance as being a key mediator in vascular pathology, particularly in the context of diabetes. Basal Nox1 activity within the vascular wall is much lower in comparison to other members of the NAD(P)H family, such as Nox4. Nox4 is highly expressed at baseline and is present within all cell types of the vascular system [17, 132]. Nox1, Nox2 and Nox5 produce superoxide, but it has been reported

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that Nox4 produces predominantly H2O2 at a constant rate. Under pathological conditions such as in hypertension, in balloon injury models as well as in response to proatherogenic stimuli, such as LDL and AngII, the rate of superoxide and H2O2 production via the Nox isoforms is increased [61, 133–136]. Nox5 differs from the other isoforms, as its activity is independent of other additional subunits and appears to be mediated by both calcium and AngII [137, 138]. Previous studies have shown a role for Nox1 in hypertension and in neointima formation [139–141]. Deletion of the p47phox subunit of Nox1 leads to an attenuation of atherosclerosis in the ApoE−/− mouse, suggesting that targeting Nox1 may be effective at reducing atherosclerosis. These findings are consistent with previous work demonstrating a beneficial role for the targeted disruption of Nox1 in attenuating a macro-vascular disease in the non-diabetic setting [142, 143]. Our own studies have demonstrated a predominant role for Nox1-derived ROS in diabetes-associated atherosclerosis development by using genetic Nox1-/y mice on the atherosclerosis-prone ApoE−/− background and rendered diabetic by streptozotocin injections [20]. Genetic deletion of Nox4 did not attenuate plaque formation in this model. [20]. In high-fat feeding studies, genetic deletion of Nox2 mice reduced atherosclerosis development in conjunction with decreased aortic ROS production [144] demonstrating a Nox2mediated response in the development of atherosclerosis. A role for Nox2 has also been suggested in the pathogenesis of type 1 diabetes as Nox2 deficiency decreased β-cell destruction by reducing ROS production and eventual β-cell apoptosis [145]. However, it needs to be noted that the primary role of Nox2 is in the innate immune defence against bacterial infection. Human Nox2 mutations result in chronic granulomatous disease (CGD) with variable reductions in Nox2 activity. Recently, we demonstrated that in STZ diabetic Nox2−/− mice, there was an increased susceptibility to bacterial infections and increased mortality [20]. Even when placed on antibiotics, deletion of Nox2 did not provide renoprotective effects in diabetic mice [146]. These results suggest that targeting Nox2 in diabetes is not a favourable approach, considering that diabetes per se is already associated with an increased susceptibility to infections. The role of Nox4 in vascular disease remains an area of contentious debate with some studies reporting a protective role, while others report a detrimental role for Nox4 [147–153]. Endothelial dysfunction is considered an early step in the development of atherosclerosis, and increases in H2O2 in diabetes may be associated with vasodilation but may also be mediators in the process of endothelial dysfunction [154–156]. A recent study has demonstrated a potential protective role for Nox4-derived H2O2 in vascular injury during femoral artery ligation [157]. Furthermore, it was shown that Nox4−/− animals had impaired acetylcholine-induced relaxation in aortic segments removed from animals treated with

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AngII, indicating a potential defect in NO bioavailability. Furthermore, endothelial-specific overexpression of Nox4 has been shown to enhance vasodilation and reduce blood pressure in vivo [158]. Very little is known about the potential role of Nox1 in diabetic nephropathy. Increased renal expression of Nox1 has been reported in hypertension-associated forms of renal disease such as in models of Ang infusion and in Dahl saltsensitive hypertensive rats [159, 160]. More recently, Nox1 was suggested to play a role in the modulation of renal oxidative stress and redox-dependent signalling of c-Src, p38 mitogen-activated protein kinase (MAPK), JNK, and focal adhesion kinase [161]. However, in a recent study performed by our group, Nox1 deletion in STZ diabetic ApoE−/− mice did not attenuate ROS formation or renal injury [162]. Because Nox4 is highly expressed within the kidney, it has been postulated to play a more predominant role in the pathogenesis of diabetic nephropathy [148, 149, 163]. It has been shown that during early stages of diabetes, Nox4-derived ROS was associated with increased renal and glomerular hypertrophy and contributed to increased fibronectin expression, and both were attenuated by targeted anti-sense oligonucleotides directed to Nox4 [149]. Induction of Nox4-derived ROS can be stimulated by AngII in mesangial cells [164]. Recent studies have suggested that high glucose increases Nox4 expression and contributes to increased ROS generation in the mouse’s proximal and distal tubules [150, 165] and activates pro-fibrotic processes via Nox4 sensitive, p38MAP kinase-dependent pathways. Other reports have demonstrated that Nox4-dependent ROS production and Akt phosphorylation led to the accumulation of fibronectin in proximal tubular epithelial cells [166]. These findings implicated Nox4mediated ROS production as a potential molecular mechanism underlying fibrosis in diabetic nephropathy [150]. Our own study has suggested that the genetic deletion or pharmacological inhibition of Nox4 prevents the development of diabetic nephropathy [162]. However, previous studies using genetic deletion of Nox4 appear to show different phenotypic outcomes in relation to diabetic nephropathy development. For example, the study by Babelova et al. used a global and inducible Nox4 knockout mouse, albeit on the C57 background, which is known to be less susceptible to the development of diabetic nephropathy [147]. It needs to be considered that there are differences in genetic background and duration of diabetes in these studies. In addition, these studies did not demonstrate an increase in Nox4 expression in response to diabetes. However, other studies have shown an increased Nox4 expression in diabetic nephropathy and that short-term administration of anti-sense oligonucleotides targeted towards Nox4 are able to attenuate renal and glomerular hypertrophy as well as reduce fibrosis within the diabetic kidney [149]. Our own studies have shown a significant role for Nox4 in diabetic nephropathy [162].

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The role of the newest member of the Nox family, Nox5 in vascular disease in diabetes is largely unknown. As rodents lack Nox5, delineating the role of Nox5 in vascular function is limited. In vitro experiments and analysis of human biopsy samples have shown an increased expression of Nox5 in coronary arteries of patients with coronary artery disease (CAD) [137, 138, 167–171]. PDGF stimulated proliferation of vascular smooth muscle cells appears to be Nox5dependent, which is an important step in the development of a vascular disease, including atherosclerosis particularly in the context of diabetes [168]. Recent in vitro studies have indicated that Nox5 can activate eNOS, ultimately leading to an enhanced peroxynitrite formation and thus contributing to endothelial dysfunction [170]. A direct link between Nox5 and diabetes-associated vascular disease has yet to be

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established. However, as Nox5 contains calcium-sensitive EF hands and phosphorylation sites for both CAMKII and PKC, this suggests that it is involved within the AngII/AT1 signalling cascade [138, 169, 172]. In fact, it has been demonstrated that in human endothelial cells, AngII is able to increase the expression of Nox5, at both the protein and mRNA level, and that small interfering RNA directed towards Nox5 blunted these AngII-induced changes [138]. Furthermore, in silico analysis of the Nox5 gene promoter region identified the existence of AP-1, NFκB and STAT1/ STAT3 regulatory sites, all important downstream targets involved in vascular disease development in diabetes. Chromatin immunoprecipitation demonstrated a physical interaction between the STAT1/STAT3 and AP-1 proteins with Nox5 [172]. These results suggest a highly significant role for Nox5 in a vascular disease, particularly in the context of diabetes where there is an upregulation of the RAS signalling cascade. A recent study reporting a transgenic mouse expressing a fully functional Nox5 in podocytes displayed findings reminiscent of diabetic nephropathy and suggests a potential involvement in diabetic nephropathy [173]. However, a role for Nox5 in other diabetes-associated vascular complications in vivo has yet to be established.

Concluding statement

Fig. 1 Schematic diagram highlighting the complex interactions between the three major pathways involved in diabetes-mediated vascular disease, the renin-angiotensin system (RAS), the formation of advanced glycation end products (AGEs) and its receptor RAGE and oxidative stress derived from isoforms of the NAD(P)H oxidase (Nox) family. Experimental evidence has demonstrated that hyperglycaemia can increase all three pathways, leading to increased inflammation and fibrosis, contributing to vascular disease development and progression. It has also been postulated that there may be an interaction between each of these pathways

The incidence of diabetes is increasing, and as a consequence the incidence of diabetes-associated micro- and macrovascular complications is also increasing. The pathophysiologic mechanisms leading to accelerated vascular disease in diabetes are still not fully understood but appear to be complex involving the interactions of glucose-dependent pathways and activation of vasoactive hormonal pathways. Furthermore, there is increasing evidence to suggest that reactive oxygen formation derived from NAD(P)H oxidase isoforms is a common downstream mediator of these pathways leading to the activation of pro-inflammatory and pro-fibrotic growth factors, cytokines and chemokines (Fig. 1). Ultimately, this results in the activation of a range of signalling pathways and transcription factors linked to the activation of inflammation, remodelling and fibrosis. Similar pathways appear to operate in the micro- and macro-vasculatures. While current therapies, such as optimisation of glucose and BP control as well as targeted intervention of the RAAS are effective in slowing the progression of vascular complications, they do not prevent them, and there is no cure. There has been good experimental evidence for a vasculoprotective role for AGE-lowering therapies and/or RAGE antagonists; however, this has not resulted in a clinical treatment as yet. Targeting ROS derived from Nox isoforms may provide a superior and more effective treatment of diabetic vascular complications. Continuous basic research into the mechanisms of vascular complications in diabetes will

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identify novel targets. However, it is unlikely that one universal mechanism in the development of vascular complications will be identified; instead, interactions between multiple pathways exist, making targeting such systems increasingly challenging. Acknowledgments KJD is supported by a NHMRC Senior Research Fellowship and SPG is supported by a Australian Diabetes Society Early Career Fellowship. Disclosure The authors declare that they have no conflict of interests.

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