Raelene Pickering

Genetic Ace2 deficiency accentuates vascular inflammation and atherosclerosis in the ApoE knockout mouse.

Rationale: Angiotensin-converting enzyme (ACE)2 opposes the actions of angiotensin (Ang) II by degrading it to Ang 1-7. Objective: Given the important role of Ang II/Ang 1-7 in atherogenesis, we investigated the impact of ACE2 deficiency on the development of atherosclerosis. Methods and Results: C57Bl6, Ace2 knockout (KO), apolipoprotein E (ApoE) KO and ApoE/Ace2 double KO mice were followed until 30 weeks of age. Plaque accumulation was increased in ApoE/Ace2 double KO mice when compared to ApoE KO mice. This was associated with increased expression of adhesion molecules and inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, and an early increase in white cell adhesion across the whole aortae on dynamic flow assay. In the absence of a proatherosclerotic (ApoE KO) genotype, ACE2 deficiency was also associated with increased expression of these markers, suggesting that these differences were not an epiphenomenon. ACE inhibition prevented increases of these markers and atherogenesis in ApoE/ACE2 double KO mice. Bone marrow macrophages isolated from Ace2 KO mice showed increased proinflammatory responsiveness to lipopolysaccharide and Ang II when compared to macrophages isolated from C57Bl6 mice. Endothelial cells isolated from Ace2 KO mice also showed increased basal activation and elevated inflammatory responsiveness to TNF-&agr;. Similarly, selective inhibition of ACE2 with MLN-4760 also resulted in a proinflammatory phenotype with a physiological response similar to that observed with exogenous Ang II (10−7 mol/L). Conclusions: Genetic Ace2 deficiency is associated with upregulation of putative mediators of atherogenesis and enhances responsiveness to proinflammatory stimuli. In atherosclerosis-prone ApoE KO mice, these changes potentially contribute to increased plaque accumulation. These findings emphasize the potential utility of ACE2 repletion as a strategy to reduce atherosclerosis.

Candesartan attenuates diabetic retinal vascular pathology by restoring glyoxalase-I function.

OBJECTIVE Advanced glycation end products (AGEs) and the renin-angiotensin system (RAS) are both implicated in the development of diabetic retinopathy. How these pathways interact to promote retinal vasculopathy is not fully understood. Glyoxalase-I (GLO-I) is an enzyme critical for the detoxification of AGEs and retinal vascular cell survival. We hypothesized that, in retina, angiotensin II (Ang II) downregulates GLO-I, which leads to an increase in methylglyoxal-AGE formation. The angiotensin type 1 receptor blocker, candesartan, rectifies this imbalance and protects against retinal vasculopathy. RESEARCH DESIGN AND METHODS Cultured bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRP) were incubated with Ang II (100 nmol/l) or Ang II+candesartan (1 μmol/l). Transgenic Ren-2 rats that overexpress the RAS were randomized to be nondiabetic, diabetic, or diabetic+candesartan (5 mg/kg/day) and studied over 20 weeks. Comparisons were made with diabetic Sprague-Dawley rats. RESULTS In BREC and BRP, Ang II induced apoptosis and reduced GLO-I activity and mRNA, with a concomitant increase in nitric oxide (NO•), the latter being a known negative regulator of GLO-I in BRP. In BREC and BRP, candesartan restored GLO-I and reduced NO•. Similar events occurred in vivo, with the elevated RAS of the diabetic Ren-2 rat, but not the diabetic Sprague-Dawley rat, reducing retinal GLO-I. In diabetic Ren-2 rats, candesartan reduced retinal acellular capillaries, inflammation, and inducible nitric oxide synthase and NO•, and restored GLO-I. CONCLUSIONS We have identified a novel mechanism by which candesartan improves diabetic retinopathy through the restoration of GLO-I.

Dicarbonyl stress in the absence of hyperglycemia increases endothelial inflammation and atherogenesis similar to that observed in diabetes.

The deleterious effects of high glucose levels and enhanced metabolic flux on the vasculature are thought to be mediated by the generation of toxic metabolites, including reactive dicarbonyls like methylglyoxal (MG). In this article, we demonstrate that increasing plasma MG to levels observed in diabetic mice either using an exogenous source (1% in drinking water) or generated following inhibition, its primary clearance enzyme, glyoxalase-1 (with 50 mg/kg IP bromobenzyl-glutathione cyclopentyl diester every second day), was able to increase vascular adhesion and augment atherogenesis in euglycemic apolipoprotein E knockout mice to a similar magnitude as that observed in hyperglycemic mice with diabetes. The effects of MG appear partly mediated by activation of the receptor for advanced glycation end products (RAGE), as deletion of RAGE was able to reduce inflammation and atherogenesis associated with MG exposure. However, RAGE deletion did not completely prevent inflammation or vascular damage, possibly because the induction of mitochondrial oxidative stress by dicarbonyls also contributes to inflammation and atherogenesis. Such data would suggest that a synergistic combination of RAGE antagonism and antioxidants may offer the greatest utility for the prevention and management of diabetic vascular complications.

Activation of the Renin-Angiotensin System Mediates the Effects of Dietary Salt Intake on Atherogenesis in the Apolipoprotein E Knockout Mouse

Dietary salt intake is a major determinant of the activation state of renin-angiotensin-aldosterone system. Given the important role of the renin-angiotensin-aldosterone system in plaque accumulation, we investigated its role in the development of atherogenesis associated with sodium intake in apolipoprotein E knockout mice. Six-weeks of a low-salt diet (containing 0.03% sodium) resulted in a 4-fold increase in plaque accumulation in apolipoprotein E knockout mice when compared with mice receiving normal chow (containing 0.30% sodium). This was associated with activation of the renin-angiotensin-aldosterone system, increased vascular expression of adhesion molecules and inflammatory cytokines, and increased adhesion of labeled leukocytes across the whole aorta on a dynamic flow assay. These changes were blocked with the angiotensin-converting enzyme inhibitor perindopril (2 mg/kg per day). A high-salt diet (containing 3% sodium) attenuated vascular inflammation and atherogenesis, associated with suppression of the renin-angiotensin-aldosterone system, although systolic blood pressure levels were modestly increased (5±1 mmHg). Constitutive activation of the renin-angiotensin-aldosterone system in angiotensin-converting enzyme 2 apolipoprotein E knockout mice was also associated with increased atherosclerosis and vascular adhesion, and this was attenuated by a high-salt diet associated with suppression of the renin-angiotensin-aldosterone system. By contrast, a low-salt diet failed to further activate the renin-angiotensin-aldosterone system or to increase atherosclerosis in angiotensin-converting enzyme 2 apolipoprotein E knockout mice. Together, these data validate a relationship between salt-mediated renin-angiotensin-aldosterone system activation and atherogenesis, which may partly explain the inconclusive or paradoxical findings of recent observational studies, despite clear effects on blood pressure.

Alagebrium Reduces Glomerular Fibrogenesis and Inflammation Beyond Preventing RAGE Activation in Diabetic Apolipoprotein E Knockout Mice

Advanced glycation end products (AGEs) are important mediators of diabetic nephropathy that act through the receptor for AGEs (RAGE), as well as other mechanisms, to promote renal inflammation and glomerulosclerosis. The relative contribution of RAGE-dependent and RAGE-independent signaling pathways has not been previously studied in vivo. In this study, diabetic RAGE apoE double-knockout (KO) mice with streptozotocin-induced diabetes were treated with the AGE inhibitor, alagebrium (1 mg/kg/day), or the ACE inhibitor, quinapril (30 mg/kg/day), for 20 weeks, and renal parameters were assessed. RAGE deletion attenuated mesangial expansion, glomerular matrix accumulation, and renal oxidative stress associated with 20 weeks of diabetes. By contrast, inflammation and AGE accumulation associated with diabetes was not prevented. However, treatment with alagebrium in diabetic RAGE apoE KO mice reduced renal AGE levels and further reduced glomerular matrix accumulation. In addition, even in the absence of RAGE expression, alagebrium attenuated cortical inflammation, as denoted by the reduced expression of monocyte chemoattractant protein-1, intracellular adhesion molecule-1, and the macrophage marker cluster of differentiation molecule 11b. These novel findings confirm the presence of important RAGE-independent as well as RAGE-dependent signaling pathways that may be activated in the kidney by AGEs. This has important implications for the design of optimal therapeutic strategies for the prevention of diabetic nephropathy.

Osteoprotegerin promotes vascular fibrosis via a TGF-β1 autocrine loop.

BACKGROUND This study was designed to evaluate the potential role of osteoprotegerin (OPG) in arterial fibrosis. METHODS Aortic samples were analyzed after in vivo treatment of ApoE(-/-) mice with recombinant human OPG. Mouse vascular smooth muscle cells (VSMC) were exposed in vitro to recombinant OPG and analyzed for markers of inflammation and fibrosis, such as fibronectin, collagen I, III, IV and transforming growth factor-β1 (TGF-β1). Conversely, the potential modulation of endogenous OPG expression and release by VSMC was analyzed in response to different pro-atherosclerotic cytokines, TGF-β1, platelet derived growth factor (PDGF) and angiogensin II (Ang II). RESULTS In vivo treatment with human OPG induced signs of fibrosis and up-regulated the arterial expression of TGF-β1. Consistently, in vitro treatment of VSMC with human OPG induced the expression of fibronectin, collagen type I, III, IV, metalloprotein-2 (MMP-2) and MMP-9, as well as of TGF-β1. On the other hand, exposure to recombinant TGF-β1 promoted the expression/release of endogenous OPG and mediated the increase of OPG release induced by PDGF and Ang II in VSMC. CONCLUSIONS Taken together, these data support a pathogenic role for OPG in the development and progression of atherosclerotic lesions and suggest the existence of a vicious circle between TGF-β1 and OPG.

Relationship between levels of advanced glycation end products and their soluble receptor and adverse outcomes in adults with type 2 diabetes

OBJECTIVE This study explored whether activation of the receptor for advanced glycation end products (RAGE) is implicated in the development of diabetes complications. RESEARCH DESIGN AND METHODS A case-cohort study was performed in 3,763 participants with prevalent diabetes in the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial. The hazard ratios (HRs) for death, major cardiovascular events, and new or worsening nephropathy were derived using Cox regression models, and the ability of sRAGE and AGE levels to reclassify the risk of nephropathy was assessed. RESULTS After adjustment for a range of possible confounders and other risk factors, sRAGE levels were associated with all-cause mortality (HR 1.11 for a 1-SD increase of log sRAGE [95% CI 1.00–1.22]; P = 0.045) and new or worsening nephropathy (HR 1.20 for a 1-SD increase of log sRAGE [95% CI 1.02–1.41]; P = 0.032). Circulating AGE levels were also independently associated with new or worsening nephropathy (HR 1.21 for a 1-SD increase [95% CI 1.08–1.36]; P = 0.001). Both markers also significantly improved the accuracy with which the 5-year risk of new or worsening nephropathy could be predicted (net reclassification index in continuous model, 0.25 for sRAGE and 0.24 for AGE levels). CONCLUSIONS In adults with type 2 diabetes, increased levels of sRAGE are independently associated with new or worsening kidney disease and mortality over the next 5 years. Higher levels of AGE are also associated with an increased risk of adverse renal outcomes. The AGE/RAGE axis may be of importance in the prevention and management of diabetes complications.

Angiotensin-converting enzyme 2 regulates renal atrial natriuretic peptide through angiotensin-(1-7)

Deficiency of ACE2 (angiotensin-converting enzyme 2), which degrades Ang (angiotensin) II, promotes the development of glomerular lesions. However, the mechanisms explaining why the reduction in ACE2 is associated with the development of glomerular lesions have still to be fully clarified. We hypothesized that ACE2 may regulate the renoprotective actions of ANP (atrial natriuretic peptide). The aim of the present study was to investigate the effect of ACE2 deficiency on the renal production of ANP. We evaluated molecular and structural abnormalities, as well as the expression of ANP in the kidneys of ACE2-deficient mice and C57BL/6 mice. We also exposed renal tubular cells to AngII and Ang-(1-7) in the presence and absence of inhibitors and agonists of RAS (renin-angiotensin system) signalling. ACE2 deficiency resulted in increased oxidative stress, as well as pro-inflammatory and profibrotic changes. This was associated with a down-regulation of the gene and protein expression on the renal production of ANP. Consistent with a role for the ACE2 pathway in modulating ANP, exposing cells to either Ang-(1-7) or ACE2 or the Mas receptor agonist up-regulated ANP gene expression. This work demonstrates that ACE2 regulates renal ANP via the generation of Ang-(1-7). This is a new mechanism whereby ACE2 counterbalances the renal effects of AngII and which explains why targeting ACE2 may be a promising strategy against kidney diseases, including diabetic nephropathy.

Cultivation of E. coli carrying a plasmid-based Measles vaccine construct (4.2 kbp pcDNA3F) employing medium optimisation and pH-temperature induction techniques

BackgroundPlasmid-based measles vaccines offer great promises over the conventional fertilised egg method such as ease of manufacture and mimic wild-type intracellular antigen expression. The increasing number of clinical trials on plasmid-based measles vaccines has triggered the need to make more in less time.ResultsIn this work, we investigated the process variables necessary to improve the volumetric and specific yields of a model plasmid-based measles vaccine (pcDNA3F) harboured in E. coli DH5α. Results from growth medium optimisation in 500 mL shake flasks by response surface methodology (RSM) generated a maximum volumetric yield of 13.65 mg/L which was 1.75 folds higher than that of the base medium. A controlled fed-batch fermentation employing strategic glycerol feeding and optimised growth conditions resulted in a remarkable pcDNA3F volumetric yield of 110 mg/L and a specific yield of 14 mg/g. In addition, growth pH modification and temperature fluctuation between 35 and 45°C were successfully employed to improve plasmid production.ConclusionProduction of a high copy number plasmid DNA containing a foreign gene of interest is often hampered by the low plasmid volumetric yield which results from the over expression of foreign proteins and metabolic repressors. In this work, a simple bioprocess framework was employed and successfully improved the production of pcDNA3F.

Is there a role for plant-made vaccines in the prevention of HIV/AIDS?

Although educational programs have had some impact, immunization against HIV will be necessary to control the AIDS pandemic. To be effective, vaccination will need to be accessible and affordable, directed against multiple antigens, and delivered in multiple doses. Plant‐based vaccines that are heat‐stable and easy to produce and administer are suited to this type of strategy. Pilot studies by a number of groups have demonstrated that plant viral expression systems can produce HIV antigens in quantities that are appropriate for use in vaccines. In addition, these plant‐made HIV antigens have been shown to be immunogenic. However, given the need for potent cross‐clade humoral and T‐cell immunity for protection against HIV, and the uncertainty surrounding the efficacy of protein subunit vaccines, it is most likely that plant‐made HIV vaccines will find their niche as booster immunizations in prime‐boost vaccination schedules.