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Dysregulation of nitric oxide (NO) production can cause ischaemic retinal injury and result in blindness. How this dysregulation occurs is poorly understood but thought to be due to an impairment in NO synthase function (NOS) and nitro-oxidative stress. Here we investigated the possibility of correcting this defective NOS activity by supplementation with the cofactor tetrahydrobiopterin, BH 4 . Retinal ischaemia was examined using the oxygen-induced retinopathy model and BH 4 deficient Hph-1 mice used to establish the relationship between NOS activity and BH 4 . Mice were treated with the stable BH 4 precursor sepiapterin at the onset of hypoxia and their retinas assessed 48 h later. HPLC analysis confirmed elevated BH 4 levels in all sepiapterin supplemented groups and increased NOS activity. Sepiapterin treatment caused a significant decrease in neuronal cell death in the inner nuclear layer that was most notable in WT animals and was associated with significantly diminished superoxide and local peroxynitrite formation. Interestingly, sepiapterin also increased inflammatory cytokine levels but not microglia cell number. BH 4 supplementation by sepiapterin improved both redox state and neuronal survival during retinal ischaemia, in spite of a paradoxical increase in inflammatory cytokines. This implicates nitro-oxidative stress in retinal neurones as the cytotoxic element in ischaemia, rather than enhanced pro-inflammatory signalling.

Loss of retinal blood flow autoregulation is an early feature of diabetes that precedes the development of clinically recognizable diabetic retinopathy (DR). Retinal blood flow autoregulation is mediated by the myogenic response of the retinal arterial vessels, a process that is initiated by the stretch‑dependent activation of TRPV2 channels on the retinal vascular smooth muscle cells (VSMCs). Here, we show that the impaired myogenic reaction of retinal arterioles from diabetic animals is associated with a complete loss of stretch‑dependent TRPV2 current activity on the retinal VSMCs. This effect could be attributed, in part, to TRPV2 channel downregulation, a phenomenon that was also evident in human retinal VSMCs from diabetic donors. We also demonstrate that TRPV2 heterozygous rats, a nondiabetic model of impaired myogenic reactivity and blood flow autoregulation in the retina, develop a range of microvascular, glial, and neuronal lesions resembling those observed in DR, including neovascular complexes. No overt kidney pathology was observed in these animals. Our data suggest that TRPV2 dysfunction underlies the loss of retinal blood flow autoregulation in diabetes and provide strong support for the hypothesis that autoregulatory deficits are involved in the pathogenesis of DR.

Despite being first described 45 years ago, the existence of a distinct diabetic cardiomyopathy remains controversial. Nonetheless, it is widely accepted that the diabetic heart undergoes characteristic structural and functional changes in the absence of ischaemia and hypertension, which are independently linked to heart failure progression and are likely to underlie enhanced susceptibility to stress. A prominent feature is marked collagen accumulation linked with inflammation and extensive extracellular matrix changes, which appears to be the main factor underlying cardiac stiffness and subclinical diastolic dysfunction, estimated to occur in as many as 75% of optimally controlled diabetics. Whether this characteristic remodelling phenotype is primarily driven by microvascular dysfunction or alterations in cardiomyocyte metabolism remains unclear. Although hyperglycaemia regulates multiple pathways in the diabetic heart, increased reactive oxygen species (ROS) generation is thought to represent a central mechanism underlying associated adverse remodelling. Indeed, experimental and clinical diabetes are linked with oxidative stress which plays a key role in cardiomyopathy, while key processes underlying diabetic cardiac remodelling, such as inflammation, angiogenesis, cardiomyocyte hypertrophy and apoptosis, fibrosis and contractile dysfunction, are redox sensitive. This review will explore the relative contributions of the major ROS sources (dysfunctional nitric oxide synthase, mitochondria, xanthine oxidase, nicotinamide adenine dinucleotide phosphate oxidases) in the diabetic heart and the potential for therapeutic targeting of ROS signalling using novel pharmacological and non-pharmacological approaches to modify specific aspects of the remodelling phenotype in order to prevent and/or delay heart failure development and progression.

Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is a protein with roles in early development, activation of the transcription factor NF-κB, and production of mitochondrial reactive oxygen species (mROS) that facilitates clearance of intracellular bacteria like Salmonella. ECSIT is also an important assembly factor for mitochondrial complex I. Unlike the murine form of Ecsit (mEcsit), we demonstrate here that human ECSIT (hECSIT) is highly labile. To explore whether the instability of hECSIT affects functions previously ascribed to its murine counterpart, we created a potentially novel transgenic mouse in which the murine Ecsit gene is replaced by the human ECSIT gene. The humanized mouse has low levels of hECSIT protein, in keeping with its intrinsic instability. Whereas low-level expression of hECSIT was capable of fully compensating for mEcsit in its roles in early development and activation of the NF-κB pathway, macrophages from humanized mice showed impaired clearance of Salmonella that was associated with reduced production of mROS. Notably, severe cardiac hypertrophy was manifested in aging humanized mice, leading to premature death. The cellular and molecular basis of this phenotype was delineated by showing that low levels of human ECSIT protein led to a marked reduction in assembly and activity of mitochondrial complex I with impaired oxidative phosphorylation and reduced production of ATP. Cardiac tissue from humanized hECSIT mice also showed reduced mitochondrial fusion and more fission but impaired clearance of fragmented mitochondria. A cardiomyocyte-intrinsic role for Ecsit in mitochondrial function and cardioprotection is also demonstrated. We also show that cardiac fibrosis and damage in humans correlated with low expression of human ECSIT. In summary, our findings identify a role for ECSIT in cardioprotection, while generating a valuable experimental model to study mitochondrial dysfunction and cardiac pathophysiology.

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8–NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.

Pressure overload-induced left ventricular hypertrophy (LVH) is initially adaptive but ultimately promotes systolic dysfunction and chronic heart failure. Whilst underlying pathways are incompletely understood, increased reactive oxygen species generation from Nox2 NADPH oxidases, and metabolic remodelling, largely driven by PPARα downregulation, are separately implicated. Here, we investigated interaction between the two as a key regulator of LVH using in vitro, in vivo and transcriptomic approaches. Phenylephrine-induced H9c2 cardiomyoblast hypertrophy was associated with reduced PPARα expression and increased Nox2 expression and activity. Pressure overload-induced LVH and systolic dysfunction induced in wild-type mice by transverse aortic constriction (TAC) for 7 days, in association with Nox2 upregulation and PPARα downregulation, was enhanced in PPARα−/− mice and prevented in Nox2−/− mice. Detailed transcriptomic analysis revealed significantly altered expression of genes relating to PPARα, oxidative stress and hypertrophy pathways in wild-type hearts, which were unaltered in Nox2−/− hearts, whilst oxidative stress pathways remained dysregulated in PPARα−/− hearts following TAC. Network analysis indicated that Nox2 was essential for PPARα downregulation in this setting and identified preferential inflammatory pathway modulation and candidate cytokines as upstream Nox2-sensitive regulators of PPARα signalling. Together, these data suggest that Nox2 is a critical driver of PPARα downregulation leading to maladaptive LVH.

Background Progenitor endothelial colony forming cells (ECFCs) are critical for vascular homeostasis and hold therapeutic potential for ischaemic cardiovascular disease (CVD). As angiogenic capacity and efficacy within diseased tissues is particularly impacted in diabetic patients, who show high incidence of ischaemic CVD, targeting of critical ECFC pathways in this setting represents an innovative focus towards enhancing intrinsic vasoreparative function. We previously reported that NADPH oxidase 4 (NOX4)-derived reactive oxygen species promote cord blood-derived ECFC (CB-ECFC) pro-angiogenic response, whilst NOX4 overexpression (OE) enhances revascularisation capacity. Here, we aimed to investigate specific influence of NOX4-dependent signalling on CB-ECFC angiogenic dysfunction observed upon exposure to both experimental and clinical diabetes to define whether NOX4 may represent a viable therapeutic target in this context. Methods CB-ECFCs were cultured in high glucose (D-glucose, 25 mmol/L) or control media (5 mmol/L) ± phorbol 12-myristate 13- acetate (PMA, 500 nmol/L) for 72 h with assessment of migratory/tubulogenic capacity and NOX4 mRNA expression (qRT-PCR). Detailed analysis of angiogenic function and signalling (Western blot, RNA sequencing) was performed in CB-ECFCs isolated from donors with gestational diabetes prior to NOX4 plasmid OE to define rescue potential and key mechanistic pathways (network analysis, proteome profiling). Statistical significance was determined using one-way ANOVA with Bonferroni post-host testing or paired/unpaired Student’s t-test, as appropriate. Results PMA-stimulated CB-ECFC migration and tube-forming capacity observed in control cells was suppressed in experimental diabetes in parallel with reduced NOX4 expression and rescued by plasmid NOX4OE. As direct evidence of clinical relevance, CB-ECFCs from gestational diabetic donors showed reduced angiogenic potential associated with attenuated NOX4, eNOS activity and downregulation of key vasoreparative signalling. Furthermore, NOX4OE rescued angiogenic function in chronically diabetic CB-ECFCs via modulation of downstream signalling involving both direct and indirect enhancement of pro-angiogenic protein expression (endoglin/SERPINE1/E2F1) linked to reduced p53 phosphorylation. Conclusions Taken together, these data indicate for the first time that reduced NOX4 expression plays a pivotal role in CB-ECFC angiogenic dysfunction linked with diabetes whilst highlighting NOX4-dependent signalling as a potential target to protect and augment their intrinsic vasoreparative capacity towards addressing current translational barriers.

ABSTRACT Background Glucagon-like peptide-1 (GLP-1) therapies are routinely used for glycaemic control in diabetes and their emerging cardiovascular actions have been a major recent research focus. In addition to GLP-1 receptor activation, the metabolically-inactive breakdown product, GLP-1(9–36)amide, also appears to exert notable cardiovascular effects, including protection against acute cardiac ischaemia. Here, we specifically studied the influence of GLP-1(9–36)amide on chronic post-myocardial infarction (MI) remodelling, which is a major driver of heart failure progression. Methods Adult female C57BL/6 J mice were subjected to permanent coronary artery ligation or sham surgery prior to continuous infusion with GLP-1(9–36)amide or vehicle control for 4 weeks. Results Infarct size was similar between groups with no effect of GLP-1(9–36)amide on MI-induced cardiac hypertrophy, although modest reduction of in vitro phenylephrine-induced H9c2 cardiomyoblast hypertrophy was observed. Whilst echocardiographic systolic dysfunction post-MI remained unchanged, diastolic dysfunction (decreased mitral valve E/A ratio, increased E wave deceleration rate) was improved by GLP-1(9–36)amide treatment. This was associated with modulation of genes related to extracellular matrix turnover (MMP-2, MMP-9, TIMP-2), although interstitial fibrosis and pro-fibrotic gene expression were unaltered by GLP-1(9–36)amide. Cardiac macrophage infiltration was also reduced by GLP-1(9–36)amide together with pro-inflammatory cytokine expression (IL-1β, IL-6, MCP-1), whilst in vitro studies using RAW264.7 macrophages revealed global potentiation of basal pro-inflammatory and tissue protective cytokines (e.g. IL-1β, TNF-α, IL-10, Fizz1) in the presence of GLP-1(9–36)amide versus exendin-4. Conclusions These data suggest that GLP-1(9–36)amide confers selective protection against post-MI remodelling via preferential preservation of diastolic function, most likely due to modulation of infiltrating macrophages, indicating that this often overlooked GLP-1 breakdown product may exert significant actions in this setting which should be considered in the context of GLP-1 therapy in patients with cardiovascular disease.

IntroductionDiabetes is characterised by hyperglycaemia, which increases reactive oxygen species (ROS) production in endothelial cells (ECs). The consequent microvascular dysfunction drives progression of cardiovascular complications, such as cardiac remodelling and heart failure. NADPH oxidases are enzymes whose main function is to generate ROS and which are important in the development of cardiovascular disease. NOX4, in particular, is highly expressed in ECs and increased by hyperglycaemia. The aim of this project was therefore to identify the precise role of NOX4 in ECs under hyperglycaemic conditions with a specific role on ROS signalling and paracrine communication with fibroblasts, as key regulators of adverse cardiovascular remodelling in diabetes.MethodsHuman aortic endothelial cells (HAoECs), with or without NOX4 siRNA knockdown (KD) were treated with normal (NG, 5.5 mM) or high glucose (HG, 25 mM) for up to 5 days. Cells were assessed for changes in mRNA and protein expression (real time (RT-PCR and Western blot, respectively; relative to GAPDH/HPRT/HSP90 and HPRT, respectively), and superoxide generation (DHE fluorescence), whilst conditioned media from NOX4-modified HAoECs with/without HG was incubated with NIH 3 T3 fibroblasts to interrogate effects on EC paracrine signalling.ResultsAfter 5 days of HG treatment, HAoECs exhibited increased NOX4 mRNA expression (NG 1.01±0.06, HG 1.27±0.06; n=9, p<0.05), which was reflected at protein level. This was associated with upregulation of antioxidant (e.g. NRF2: NG 0.91±0.04, HG 1.22±0.04; n=6, p<0.05) and proinflammatory genes (e.g. IL-6: NG 1.03±0.13, HG 2.59±0.58; n=6, p<0.05) and elevated superoxide production after 2 but not 5 days (262±12 vs. 338±11 arbitrary units; n=4, p<0.05). Of note, NOX4 KD under HG conditions (25 mM for 2 days) increased mRNA expression of endogenous antioxidants (e.g. NRF2: NG 1.90±0.06, HG 2.21±0.06; SOD1: NG 0.89±0.01, HG 1.39±0.08; NQO1: NG 0.79±0.02, HG 1.025±0.06; n=3, p<0.05) whilst normalising the HG-induced increase in superoxide production. Moreover, conditioned media from HG-treated HAoECs increased TGF-β induced differentiation, which was ablated following NOX4 KD (e.g. α-SMA: scrambled control 1.31±0.04, NOX4 KD 0.94±0.07; CTGF: scrambled control 1.36±0.08, NOX4 KD 1.13±0.03; n=3, p<0.05).ConclusionsHG-induced NOX4 expression modulates ROS signalling in ECs, thereby altering paracrine regulation of fibroblast differentiation. EC-localised NOX4 is therefore likely to make a significant contribution to adverse cardiovascular remodelling in diabetes.

BackgroundThe characteristic structural and functional cardiac abnormalities which occur in diabetes, including fibrosis, inflammation and microvascular remodelling, and result in increased susceptibility to cardiovascular stress are largely mediated by reactive oxygen species (ROS) generation. In this study we aimed to investigate the specific contribution of endothelial Nox4 NADPH oxidase, a major source of cardiovascular ROS, to adverse cardiac remodelling in experimental diabetes.MethodsDiabetes was induced in gene-modified mice with endothelial-specific Nox4 overexpression (Tg) and wild-type (WT) littermate controls (10–12 weeks old; n=8–12 per group) by streptozotocin injection (STZ, 50 mg/kg i.p. for 5 consecutive days). After 6 months, analyses of cardiac function (echocardiography), glucose metabolism (blood glucose, HbA1c) and myocardial gene expression (qPCR) were performed.ResultsEndothelial Nox4 overexpression had no effect on blood glucose or HbA1c levels in control or diabetic Tg animals. Tg control mice demonstrated impaired basal diastolic function (E/A ratio: WT 1.6±0.09 vs Tg 1.4±0.08, p<0.05) which was not affected by experimental diabetes, whilst WT STZ mice showed significant diastolic dysfunction versus controls (E/A ratio: control 1.6±0.09 versus STZ 1.3±0.04, p<0.05). Systolic function remained similar between groups. Consistent with these functional data, CTGF and MMP2 expression were increased (p<0.05) in Tg control animals, but were not affected by STZ, whereas CTGF was elevated in WT STZ animals versus controls (p<0.05). Expression of eNOS (p<0.05) and CD31 (p<0.01) was also increased in Tg versus WT controls suggestive of vascular remodelling. Interestingly, expression of antioxidant genes in response to STZ diabetes was more highly increased in Tg versus WT animals (compared to controls, p<0.05): SOD1 (WT 31±6.3%, Tg 78±24%), SOD2 (WT 37±10%, Tg 63±23%), PRDX1 (WT 55±14%, Tg 139±68%) and catalase (WT 24±14%, Tg 85±28%). This may at least partly explain protection of Tg STZ animals against further diastolic dysfunction.ConclusionTaken together, these data indicate that while increased endothelial Nox4 expression may impair basal cardiac function, it protects against adverse cardiac remodelling and dysfunction seen in experimental diabetes, via modification of key antioxidant signalling pathways, thus highlighting a key role for this major Nox isoform in the diabetic heart.