Cell-specific epigenetic changes in atherosclerosis.

Atherosclerosis is a disease of large and medium arteries that can lead to life-threatening cerebrovascular and cardiovascular consequences such as heart failure and stroke and is a major contributor to cardiovascular-related mortality worldwide. Atherosclerosis development is a complex process that involves specific structural, functional and transcriptional changes in different vascular cell populations at different stages of the disease. The application of single-cell RNA sequencing (scRNA-seq) analysis has discovered not only disease-related cell-specific transcriptomic profiles but also novel subpopulations of cells once thought as homogenous cell populations. Vascular cells undergo specific transcriptional changes during the entire course of the disease. Epigenetics is the instruction-set-architecture in living cells that defines and maintains the cellular identity by regulating the cellular transcriptome. Although different cells contain the same genetic material, they have different epigenomic signatures. The epigenome is plastic, dynamic and highly responsive to environmental stimuli. Modifications to the epigenome are driven by an array of epigenetic enzymes generally referred to as writers, erasers and readers that define cellular fate and destiny. The reversibility of these modifications raises hope for finding novel therapeutic targets for modifiable pathological conditions including atherosclerosis where the involvement of epigenetics is increasingly appreciated. This article provides a critical review of the up-to-date research in the field of epigenetics mainly focusing on in vivo settings in the context of the cellular role of individual vascular cell types in the development of atherosclerosis.

Endothelium-restricted endothelin-1 overexpression in type 1 diabetes worsens atherosclerosis and immune cell infiltration via NOX1.

AIMS: NADPH oxidase (NOX) 1 but not NOX4-dependent oxidative stress plays a role in diabetic vascular disease, including atherosclerosis. Endothelin (ET)-1 has been implicated in diabetes-induced vascular complications. We showed that crossing mice overexpressing human ET-1 selectively in endothelium (eET-1) with apolipoprotein E knockout (Apoe-/-) mice enhanced high-fat diet-induced atherosclerosis in part by increasing oxidative stress. We tested the hypothesis that ET-1 overexpression in the endothelium would worsen atherosclerosis in type 1 diabetes through a mechanism involving NOX1 but not NOX4. METHODS AND RESULTS: Six-week-old male Apoe-/- and eET-1/Apoe-/- mice with or without Nox1 (Nox1-/y) or Nox4 knockout (Nox4-/-) were injected intraperitoneally with either vehicle or streptozotocin (55 mg/kg/day) for 5 days to induce type 1 diabetes and were studied 14 weeks later. ET-1 overexpression increased 2.5-fold and five-fold the atherosclerotic lesion area in the aortic sinus and arch of diabetic Apoe-/- mice, respectively. Deletion of Nox1 reduced aortic arch plaque size by 60%; in contrast, Nox4 knockout increased lesion size by 1.5-fold. ET-1 overexpression decreased aortic sinus and arch plaque alpha smooth muscle cell content by ∼35% and ∼50%, respectively, which was blunted by Nox1 but not Nox4 knockout. Reactive oxygen species production was increased two-fold in aortic arch perivascular fat of diabetic eET-1/Apoe-/- and eET-1/Apoe-/-/Nox4-/- mice but not eET-1/Apoe-/-/Nox1y/- mice. ET-1 overexpression enhanced monocyte/macrophage and CD3+ T-cell infiltration ∼2.7-fold in the aortic arch perivascular fat of diabetic Apoe-/- mice. Both Nox1 and Nox4 knockout blunted CD3+ T-cell infiltration whereas only Nox1 knockout prevented the monocyte/macrophage infiltration in diabetic eET-1/Apoe-/- mice. CONCLUSION: Endothelium ET-1 overexpression enhances the progression of atherosclerosis in type 1 diabetes, perivascular oxidative stress, and inflammation through NOX1.

Differential sympathetic response to lesion-induced chronic kidney disease in rabbits.

Chronic kidney disease (CKD) is associated with greater sympathetic nerve activity but it is unclear if this is a kidney-specific response or due to generalized stimulation of sympathetic nervous system activity. To determine this, we used a rabbit model of CKD in which quantitative comparisons with control rabbits could be made of kidney sympathetic nerve activity and whole-body norepinephrine spillover. Rabbits either had surgery to lesion 5/6th of the cortex of one kidney by electro-lesioning and two weeks later removal of the contralateral kidney, or sham lesioning and sham nephrectomy. After three weeks, the blood pressure was statistically significantly 20% higher in conscious rabbits with CKD compared to rabbits with a sham operation, but their heart rate was similar. Strikingly, kidney nerve activity was 37% greater than in controls, with greater burst height and frequency. Total norepinephrine spillover was statistically significantly lower by 34%, and kidney baroreflex curves were shifted to the right in rabbits with CKD. Plasma creatinine and urine output were elevated by 38% and 131%, respectively, and the glomerular filtration rate was 37% lower than in sham-operated animals (all statistically significant). Kidney gene expression of fibronectin, transforming growth factor-ß, monocyte chemotactic protein1, Nox4 and Nox5 was two- to eight-fold greater in rabbits with CKD than in control rabbits. Overall, the glomerular layer lesioning model in conscious rabbits produced a moderate, stable degree of CKD characterized by elevated blood pressure and increased kidney sympathetic nerve activity. Thus, our findings, together with that of a reduction in total norepinephrine spillover, suggest that kidney denervation, rather than generalized sympatholytic treatments, may represent a preferable management for CKD associated hypertension.

A novel synthetic small molecule DMFO targets Nrf2 in modulating proinflammatory/antioxidant mediators to ameliorate inflammation.

Inflammation is a protective immune response against invading pathogens, however, dysregulated inflammation is detrimental. As the complex inflammatory response involves multiple mediators, including the involvement of reactive oxygen species, concomitantly targeting proinflammatory and antioxidant check-points may be a more rational strategy. We report the synthesis and anti-inflammatory/antioxidant activity of a novel indanedione derivative DMFO. DMFO scavenged reactive oxygen species (ROS) in in-vitro radical scavenging assays and in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. In acute models of inflammation (carrageenan-induced inflammation in rat paw and air pouch), DMFO effectively reduced paw oedema and leucocyte infiltration with an activity comparable to diclofenac. DMFO stabilised mast cells (MCs) in in-vitro A23187 and compound 48/80-induced assays. Additionally, DMFO stabilised MCs in an antigen (ovalbumin)-induced MC degranulation model in-vivo, without affecting serum IgE levels. In a model of chronic immune-mediated inflammation, Freund's adjuvant-induced arthritis, DMFO reduced arthritic score and contralateral paw oedema, and increased the pain threshold with an efficacy comparable to diclofenac but without being ulcerogenic. Additionally, DMFO significantly reduced serum TNFα levels. Mechanistic studies revealed that DMFO reduced proinflammatory genes (IL1ß, TNFα, IL6) and protein levels (COX2, MCP1), with a concurrent increase in antioxidant genes (NQO1, haem oxygenase 1 (HO-1), Glo1, Nrf2) and protein (HO-1) in LPS-stimulated macrophages. Importantly, the anti-inflammatory/antioxidant effect on gene expression was absent in primary macrophages isolated from Nrf2 KO mice suggesting an Nrf2-targeted activity, which was subsequently confirmed using siRNA transfection studies in RAW macrophages. Therefore, DMFO is a novel, orally-active, safe (even at 2 g/kg p.o.), a small molecule which targets Nrf2 in ameliorating inflammation.

Protective Effect of let-7 miRNA Family in Regulating Inflammation in Diabetes-Associated Atherosclerosis.

The let-7 miRNA family plays a key role in modulating inflammatory responses. Vascular smooth muscle cell (SMC) proliferation and endothelial cell (EC) dysfunction are critical in the pathogenesis of atherosclerosis, including in the setting of diabetes. Here we report that let-7 levels are decreased in diabetic human carotid plaques and in a model of diabetes-associated atherosclerosis, the diabetic ApoE-/- mouse. In vitro platelet-derived growth factor (PDGF)- and tumor necrosis factor-α (TNF-α)-induced vascular SMC and EC activation was associated with reduced let-7 miRNA expression via Lin28b, a negative regulator of let-7 biogenesis. Ectopic overexpression of let-7 in SMCs inhibited inflammatory responses including proliferation, migration, monocyte adhesion, and nuclear factor-κB activation. The therapeutic potential of restoring let-7 levels using a let-7 mimic was tested: in vitro in SMCs using an endogenous anti-inflammatory lipid (lipoxin A4), ex vivo in murine aortas, and in vivo via tail vein injection in a 24-h murine model. Furthermore, we delivered let-7 mimic to human carotid plaque ex vivo and observed significant changes to the secretome in response to let-7 therapy. Restoration of let-7 expression could provide a new target for an anti-inflammatory approach in diabetic vascular disease.

NOX4-derived reactive oxygen species limit fibrosis and inhibit proliferation of vascular smooth muscle cells in diabetic atherosclerosis.

Smooth muscle cell (SMC) proliferation and fibrosis contribute to the development of advanced atherosclerotic lesions. Oxidative stress caused by increased production or unphysiological location of reactive oxygen species (ROS) is a known major pathomechanism. However, in atherosclerosis, in particular under hyperglycaemic/diabetic conditions, the hydrogen peroxide-producing NADPH oxidase type 4 (NOX4) is protective. Here we aim to elucidate the mechanisms underlying this paradoxical atheroprotection of vascular smooth muscle NOX4 under conditions of normo- and hyperglycaemia both in vivo and ex vivo. Following 20-weeks of streptozotocin-induced diabetes, Apoe(-/-) mice showed a reduction in SM-alpha-actin and calponin gene expression with concomitant increases in platelet-derived growth factor (PDGF), osteopontin (OPN) and the extracellular matrix (ECM) protein fibronectin when compared to non-diabetic controls. Genetic deletion of Nox4 (Nox4(-/)(-)Apoe(-/-)) exacerbated diabetes-induced expression of PDGF, OPN, collagen I, and proliferation marker Ki67. Aortic SMCs isolated from NOX4-deficient mice exhibited a dedifferentiated phenotype including loss of contractile gene expression, increased proliferation and ECM production as well as elevated levels of NOX1-associated ROS. Mechanistic studies revealed that elevated PDGF signalling in NOX4-deficient SMCs mediated the loss of calponin and increase in fibronectin, while the upregulation of NOX1 was associated with the increased expression of OPN and markers of proliferation. These findings demonstrate that NOX4 actively regulates SMC pathophysiological responses in diabetic Apoe(-/-) mice and in primary mouse SMCs through the activities of PDGF and NOX1.

Differential effects of NOX4 and NOX1 on immune cell-mediated inflammation in the aortic sinus of diabetic ApoE-/- mice.

Oxidative stress and inflammation are central mediators of atherosclerosis particularly in the context of diabetes. The potential interactions between the major producers of vascular reactive oxygen species (ROS), NADPH oxidase (NOX) enzymes and immune-inflammatory processes remain to be fully elucidated. In the present study we investigated the roles of the NADPH oxidase subunit isoforms, NOX4 and NOX1, in immune cell activation and recruitment to the aortic sinus atherosclerotic plaque in diabetic ApoE(-/-) mice. Plaque area analysis showed that NOX4- and NOX1-derived ROS contribute to atherosclerosis in the aortic sinus following 10 weeks of diabetes. Immunohistochemical staining of the plaques revealed that NOX4-derived ROS regulate T-cell recruitment. In addition, NOX4-deficient mice showed a reduction in activated CD4(+) T-cells in the draining lymph nodes of the aortic sinus coupled with reduced pro-inflammatory gene expression in the aortic sinus. Conversely, NOX1-derived ROS appeared to play a more important role in macrophage accumulation. These findings demonstrate distinct roles for NOX4 and NOX1 in immune-inflammatory responses that drive atherosclerosis in the aortic sinus of diabetic mice.

Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling.

OBJECTIVE: Oxidative stress is considered a hallmark of atherosclerosis. In particular, the superoxide-generating type 1 NADPH oxidase (NOX1) has been shown to be induced and play a pivotal role in early phases of mouse models of atherosclerosis and in the context of diabetes mellitus. Here, we investigated the role of the most abundant type 4 isoform (NOX4) in human and mouse advanced atherosclerosis. APPROACH AND RESULTS: Plaques of patients with cardiovascular events or established diabetes mellitus showed a surprising reduction in expression of the most abundant but hydrogen peroxide (H2O2)-generating type 4 isoform (Nox4), whereas Nox1 mRNA was elevated, when compared with respective controls. As these data suggested that NOX4-derived reactive oxygen species may convey a surprisingly protective effect during plaque progression, we examined a mouse model of accelerated and advanced diabetic atherosclerosis, the streptozotocin-treated ApoE(-/-) mouse, with (NOX4(-/-)) and without genetic deletion of Nox4. Similar to the human data, advanced versus early plaques of wild-type mice showed reduced Nox4 mRNA expression. Consistent with a rather protective role of NOX4-derived reactive oxygen species, NOX4(-/-) mice showed increased atherosclerosis when compared with wild-type mice. Deleting NOX4 was associated with reduced H2O2 forming activity and attenuation of the proinflammatory markers, monocyte chemotratic protein-1, interleukin-1ß, and tumor necrosis factor-α, as well as vascular macrophage accumulation. Furthermore, there was a greater accumulation of fibrillar collagen fibres within the vascular wall and plaque in diabetic Nox4(-/-)ApoE(-/-) mice, indicative of plaque remodeling. These data could be replicated in human aortic endothelial cells in vitro, where Nox4 overexpression increased H2O2 and reduced the expression of pro-oxidants and profibrotic markers. Interestingly, Nox4 levels inversely correlated with Nox2 gene and protein levels. Although NOX2 is not constitutively active unlike NOX4 and forms rather superoxide, this opens up the possibility that at least some effects of NOX4 deletion are mediated by NOX2 activation. CONCLUSIONS: Thus, the appearance of reactive oxygen species in atherosclerosis is apparently not always a nondesirable oxidative stress, but can also have protective effects. Both in humans and in mouse, the H2O2-forming NOX4, unlike the superoxide-forming NOX1, can act as a negative modulator of inflammation and remodeling and convey atheroprotection. These results have implications on how to judge reactive oxygen species formation in cardiovascular disease and need to be considered in the development of NOX inhibitory drugs.

Are reactive oxygen species still the basis for diabetic complications?

Despite the wealth of pre-clinical support for a role for reactive oxygen and nitrogen species (ROS/RNS) in the aetiology of diabetic complications, enthusiasm for antioxidant therapeutic approaches has been dampened by less favourable outcomes in large clinical trials. This has necessitated a re-evaluation of pre-clinical evidence and a more rational approach to antioxidant therapy. The present review considers current evidence, from both pre-clinical and clinical studies, to address the benefits of antioxidant therapy. The main focus of the present review is on the effects of direct targeting of ROS-producing enzymes, the bolstering of antioxidant defences and mechanisms to improve nitric oxide availability. Current evidence suggests that a more nuanced approach to antioxidant therapy is more likely to yield positive reductions in end-organ injury, with considerations required for the types of ROS/RNS involved, the timing and dosage of antioxidant therapy, and the selective targeting of cell populations. This is likely to influence future strategies to lessen the burden of diabetic complications such as diabetes-associated atherosclerosis, diabetic nephropathy and diabetic retinopathy.

AT2R agonist, compound 21, is reno-protective against type 1 diabetic nephropathy.

The hemodynamic and nonhemodynamic effects of angiotensin II on diabetic complications are considered to be primarily mediated by the angiotensin II type 1 receptor subtype. However, its biological and functional effect mediated through the angiotensin II type 2 receptor subtype is still unclear. Activation of the angiotensin II type 2 receptors has been postulated to oppose angiotensin II type 1 receptor-mediated actions and thus attenuate fibrosis. This study aimed to elucidate the reno-protective role of the novel selective angiotensin II type 2 receptor agonist, Compound 21, in an experimental model of type 1 diabetic nephropathy. Compound 21 treatment significantly attenuated diabetes mellitus-induced elevated levels of cystatin C, albuminuria, mesangial expansion, and glomerulosclerosis in diabetic mice. Moreover, Compound 21 markedly inhibited the expression of various proteins implicated in oxidative stress, inflammation, and fibrosis, in association with decreased extracellular matrix production. These findings demonstrate that monotherapy of Compound 21 is protective against the progression of experimental diabetic nephropathy by inhibiting renal oxidative stress, inflammation, and fibrosis.

Role of bone-marrow- and non-bone-marrow-derived receptor for advanced glycation end-products (RAGE) in a mouse model of diabetes-associated atherosclerosis.

RAGE (receptor for advanced glycation end-products) is expressed on multiple cell types implicated in the progression of atherosclerosis and plays a role in DAA (diabetes-associated atherosclerosis). The aim of the present study was to determine the relative role of either BM (bone marrow)- or non-BM-derived RAGE in the pathogenesis of STZ (streptozotocin)-induced DAA. Male ApoE (apolipoprotein E)-null (ApoE-/-:RAGE+/+) and ApoE:RAGE-null (ApoE-/-:RAGE-/-) mice at 7 weeks of age were rendered diabetic with STZ. At 8 weeks of age, ApoE-/- and ApoE-/-:RAGE-/- control and diabetic mice received BM from either RAGE-null or RAGE-bearing mice, generating various chimaeras. After 10 and 20 weeks of diabetes, mice were killed and gene expression and atherosclerotic lesion formation were evaluated respectively. Deletion of RAGE in either the BM cells or non-BM cells both resulted in a significant attenuation in DAA, which was associated with reduced VCAM-1 (vascular cell adhesion molecule-1) expression and translated into reduced adhesion in vitro. In conclusion, the results of the present study highlight the importance of both BM- and non-BM-derived RAGE in attenuating the development of DAA.

Genetic targeting or pharmacologic inhibition of NADPH oxidase nox4 provides renoprotection in long-term diabetic nephropathy.

Diabetic nephropathy may occur, in part, as a result of intrarenal oxidative stress. NADPH oxidases comprise the only known dedicated reactive oxygen species (ROS)-forming enzyme family. In the rodent kidney, three isoforms of the catalytic subunit of NADPH oxidase are expressed (Nox1, Nox2, and Nox4). Here we show that Nox4 is the main source of renal ROS in a mouse model of diabetic nephropathy induced by streptozotocin administration in ApoE(-/-) mice. Deletion of Nox4, but not of Nox1, resulted in renal protection from glomerular injury as evidenced by attenuated albuminuria, preserved structure, reduced glomerular accumulation of extracellular matrix proteins, attenuated glomerular macrophage infiltration, and reduced renal expression of monocyte chemoattractant protein-1 and NF-κB in streptozotocin-induced diabetic ApoE(-/-) mice. Importantly, administration of the most specific Nox1/4 inhibitor, GKT137831, replicated these renoprotective effects of Nox4 deletion. In human podocytes, silencing of the Nox4 gene resulted in reduced production of ROS and downregulation of proinflammatory and profibrotic markers that are implicated in diabetic nephropathy. Collectively, these results identify Nox4 as a key source of ROS responsible for kidney injury in diabetes and provide proof of principle for an innovative small molecule approach to treat and/or prevent chronic kidney failure.

NADPH oxidase, NOX1, mediates vascular injury in ischemic retinopathy.

AIMS: Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity. RESULTS: Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831. INNOVATION: Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. CONCLUSIONS: Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies.

Targeting the upregulation of reactive oxygen species subsequent to hyperglycemia prevents type 1 diabetic cardiomyopathy in mice.

Cardiac oxidative stress is an early event associated with diabetic cardiomyopathy, triggered by hyperglycemia. We tested the hypothesis that targeting left-ventricular (LV) reactive oxygen species (ROS) upregulation subsequent to hyperglycemia attenuates type 1 diabetes-induced LV remodeling and dysfunction, accompanied by attenuated proinflammatory markers and cardiomyocyte apoptosis. Male 6-week-old mice received either streptozotocin (55mg/kg/day for 5 days), to induce type 1 diabetes, or citrate buffer vehicle. After 4 weeks of hyperglycemia, the mice were allocated to coenzyme Q10 supplementation (10mg/kg/day), treatment with the angiotensin-converting-enzyme inhibitor (ACE-I) ramipril (3mg/kg/day), treatment with olive oil vehicle, or no treatment for 8 weeks. Type 1 diabetes upregulated LV NADPH oxidase (Nox2, p22(phox), p47(phox) and superoxide production), LV uncoupling protein UCP3 expression, and both LV and systemic oxidative stress (LV 3-nitrotyrosine and plasma lipid peroxidation). All of these were significantly attenuated by coenzyme Q10. Coenzyme Q10 substantially limited type 1 diabetes-induced impairments in LV diastolic function (E:A ratio and deceleration time by echocardiography, LV end-diastolic pressure, and LV -dP/dt by micromanometry), LV remodeling (cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis), and LV expression of proinflammatory mediators (tumor necrosis factor-α, with a similar trend for interleukin IL-1ß). Coenzyme Q10's actions were independent of glycemic control, body mass, and blood pressure. Coenzyme Q10 compared favorably to improvements observed with ramipril. In summary, these data suggest that coenzyme Q10 effectively targets LV ROS upregulation to limit type 1 diabetic cardiomyopathy. Coenzyme Q10 supplementation may thus represent an effective alternative to ACE-Is for the treatment of cardiac complications in type 1 diabetic patients.

Receptor for advanced glycation end products (RAGE) deficiency attenuates the development of atherosclerosis in diabetes.

OBJECTIVE: Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE(-/-) model of accelerated atherosclerosis. RESEARCH DESIGN AND METHODS: ApoE(-/-) and RAGE(-/-)/apoE(-/-) double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis. RESULTS: Although diabetic apoE(-/-) mice showed increased plaque accumulation (14.9 +/- 1.7%), diabetic RAGE(-/-)/apoE(-/-) mice had significantly reduced atherosclerotic plaque area (4.9 +/- 0.4%) to levels not significantly different from control apoE(-/-) mice (4.3 +/- 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-kappaB subunit p65, VCAM-1, and MCP-1; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox, p47phox, and rac-1. Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE(-/-) mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGE(-/-)/apoE(-/-) mice. CONCLUSIONS: This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications.

Lack of the antioxidant enzyme glutathione peroxidase-1 accelerates atherosclerosis in diabetic apolipoprotein E-deficient mice.

BACKGROUND: Recent clinical studies have suggested a major protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx1) in diabetes-associated atherosclerosis. We induced diabetes in mice deficient for both GPx1 and apolipoprotein E (ApoE) to determine whether this is merely an association or whether GPx1 has a direct effect on diabetes-associated atherosclerosis. METHODS AND RESULTS: ApoE-deficient (ApoE-/-) and ApoE/GPx1 double-knockout (ApoE-/- GPx1-/-) mice were made diabetic with streptozotocin and aortic lesion formation, and atherogenic pathways were assessed after 10 and 20 weeks of diabetes. Aortic proinflammatory and profibrotic markers were determined by both quantitative reverse-transcription polymerase chain reaction analysis after 10 weeks of diabetes and immunohistochemical analysis after 10 and 20 weeks of diabetes. Sham-injected nondiabetic counterparts served as controls. Atherosclerotic lesions within the aortic sinus region, as well as arch, thoracic, and abdominal lesions, were significantly increased in diabetic ApoE-/- GPx1-/- aortas compared with diabetic ApoE-/- aortas. This increase was accompanied by increased macrophages, alpha-smooth muscle actin, receptors for advanced glycation end products, and various proinflammatory (vascular cell adhesion molecule-1) and profibrotic (vascular endothelial growth factor and connective tissue growth factor) markers. Quantitative reverse-transcription polymerase chain reaction analysis showed increased expression of receptors for advanced glycation end products (RAGE), vascular cell adhesion molecule-1, vascular endothelial growth factor, and connective tissue growth factor. Nitrotyrosine levels were significantly increased in diabetic ApoE-/- GPx1-/- mouse aortas. These findings were observed despite upregulation of other antioxidants. CONCLUSIONS: Lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE-/- mice. Our study provides evidence of a protective role for GPx1 and establishes GPx1 as an important antiatherogenic therapeutic target in patients with or at risk of diabetic macrovascular disease.

Accelerated nephropathy in diabetic apolipoprotein e-knockout mouse: role of advanced glycation end products.

Hyperlipidemia not only may be relevant to cardiovascular disease in diabetes but may also play a role in the development and progression of diabetic nephropathy. Furthermore, there is increasing evidence that advanced glycation end products (AGE) play an important role in diabetic renal disease. The objectives of this study were first to characterize renal injury in diabetic apolipoprotein E knockout (apo E-KO) mice and second to explore the role of AGE in the development and progression of renal disease in this model. Diabetes was induced by injection of streptozotocin in 6-wk-old apo E-KO mice. Diabetic animals received no treatment or treatment with the inhibitor of AGE formation aminoguanidine (1 g/kg per d) or the cross-link breaker [4,5-dimethyl-3-(2-oxo2-phenylethyl)-thiazolium chloride] ALT-711, which cleaves preformed AGE (20 mg/kg per d) for 20 wk. Nondiabetic apo E-KO mice as well as nondiabetic and diabetic C57BL/6 mice served as controls. Compared with nondiabetic apo E-KO mice, induction of diabetes in apo E-KO mice resulted in accelerated renal injury characterized by albuminuria and glomerular and tubulointerstitial injury. These abnormalities were associated with increased expression of collagen type I and type IV and transforming growth factor-beta1 (TGF-beta1), increased alpha-smooth muscle actin immunostaining and macrophage infiltration, and increased serum and renal AGE. The two treatments, which attenuated renal AGE accumulation in a disparate manner, were associated with less albuminuria, structural injury, macrophage infiltration, TGF-beta1, and collagen expression. The accelerated renal injury that was observed in diabetic apo E-KO mice was attenuated by approaches that inhibit renal AGE accumulation.

Irbesartan but not amlodipine suppresses diabetes-associated atherosclerosis.

BACKGROUND: It remains controversial whether specific blockade of the renin-angiotensin system confers superior antiatherosclerotic effects over other antihypertensive agents in diabetes. Therefore, the aim of this study was to compare equihypotensive doses of the angiotensin II subtype 1 (AT1) receptor blocker irbesartan with the calcium antagonist amlodipine on diabetes-induced plaque formation in the apolipoprotein E (apoE)-null mouse and to explore molecular and cellular mechanisms linked to vascular protection. METHODS AND RESULTS: Diabetes was induced by injection of streptozotocin in 6-week-old apoE-null mice. Diabetic animals were randomized to no treatment, irbesartan, or amlodipine for 20 weeks. Diabetes was associated with an increase in plaque area and complexity in the aorta in association with a significant increase in aortic AT1 receptor expression, cellular proliferation, collagen content, macrophage- and alpha-smooth muscle actin-positive cell infiltration, as well as an increased expression of platelet-derived growth factor-B (PDGF-B), monocyte chemoattractant protein-1 (MCP-1), and vascular cell adhesion molecule-1 (VCAM-1). Irbesartan but not amlodipine treatment attenuated the development of atherosclerosis, collagen content, cellular proliferation, and macrophage infiltration as well as diabetes-induced AT1 receptor, PDGF-B, MCP-1, and VCAM-1 overexpression in the aorta despite similar blood pressure reductions by both treatments. CONCLUSIONS: Diabetes-associated atherosclerosis is ameliorated by AT1 receptor blockade but not by calcium channel antagonism, providing further evidence for the vascular renin-angiotensin system playing a pivotal role in the development and acceleration of atherosclerosis in diabetes.

Imatinib attenuates diabetes-associated atherosclerosis.

OBJECTIVE: Diabetes is associated with accelerated atherosclerosis, the major factor contributing to increased mortality and morbidity in the diabetic population. The molecular mechanisms by which diabetes promotes atherosclerosis are not fully understood. Platelet-derived growth factor has been shown to play a major role in the pathology of vascular diseases, but whether it plays a role in atherosclerosis associated with diabetes remains unknown. The aims of this study were to assess whether platelet-derived growth factor-dependent pathways are involved in the development of diabetes-induced atherosclerosis and to determine the effects of platelet-derived growth factor receptor antagonism on this disorder. METHODS AND RESULTS: Diabetes was induced by injection of streptozotocin in 6-week-old apolipoprotein E knockout mice. Diabetic animals received treatment with a tyrosine kinase inhibitor that inhibits platelet-derived growth factor action, imatinib (STI-571, 10 mg/kg per day), or no treatment for 20 weeks. Nondiabetic apolipoprotein E knockout mice served as controls. Induction of diabetes was associated with a 5-fold increase in plaque area in association with an increase in aortic platelet-derived growth factor-B expression and platelet-derived growth factor-beta receptor phosphorylation as well as other prosclerotic and proinflammatory cytokines. Imatinib treatment prevented the development of atherosclerotic lesions and diabetes-induced inflammatory cytokine overexpression in the aorta. CONCLUSIONS: Tyrosine kinase inhibition with imatinib appears to be a novel therapeutic option to retard the development of atherosclerosis, specifically in the context of diabetes.