The suitability of iodinated Angiotensin-(1-7) peptides as pharmacological tools

INTRODUCTION: The heptapeptide Angiotensin-(1-7) [(Ang-(1-7)] is a biologically active component of the Renin-Angiotensin System. Pharmacological studies often involve Ang-(1-7) radioactively labelled with (125)I. Given the small size of the original peptide, we investigated whether introduction of a rather bulky iodine label interferes with the biological activity of Ang-(1-7). METHODS: Ang-(1-7) was labelled with nonradioactive iodine with the chloramine-T method. The reaction products were separated on HPLC and analysed with mass spectrometry. The products were tested for biological activity in two ways: The ability of labelled Ang-(1-7) to block Ang II-induced contraction in rat aortic rings was tested in an organ bath setup. The affinity of labelled angiotensin for ACE in rat plasma was examined in vitro. RESULTS: Iodination of Angiotensin-(1-7) resulted in two main products: monoiodinated and diiodinated Ang-(1-7) that could be easily separated on HPLC. In an organ bath experiment, monoiodinated Ang-(1-7) blocked Ang II responses identical to the native compound, whereas diiodinated Ang-(1-7) had lost its ability to block Ang II responses. Likewise, monoiodinated Ang-(1-7) had retained its affinity for ACE, while the affinity of diiodinated Ang-(1-7) was greatly reduced. DISCUSSION: Monoiodinated Ang-(1-7) has a biological activity identical to the native compound, whereas this is lost in diiodinated Ang-(1-7). Therefore, only the monoiodinated radioactive form seems suited for pharmacological studies

The Novel Compound SUL-138 Counteracts Endothelial Cell and Kidney Dysfunction in Sepsis by Preserving Mitochondrial Function

Sepsis is defined as a dysregulated host response leading to organ dysfunction, which may ultimately result in the patient’s death. Mitochondrial dysfunction plays a key role in developing organ dysfunction in sepsis. In this study, we explored the efficacy of the novel mitochondrial protective compound, SUL-138, in sepsis models in HUVECs and mice. In LPS-challenged HUVECs, SUL-138 preserved mitochondrial membrane potential and oxygen consumption and limited mitochondrial oxidative stress, resulting in increased survival at 48 h. Further, SUL-138 dampened the LPS-induced expression of IL-1β, but not of NLRP3, and IL-18 in HUVECs. Sepsis in mice induced by cecal ligation and puncture (CLP) led to a lower mitochondrial membrane potential and increased levels of mitochondrial oxidative stress in the kidney, which SUL-138 limited. In addition, SUL-138 mitigated the CLP-induced increase in kidney dysfunction markers NGAL and urea. It dampened the rise in kidney expression of IL-6, IL-1β, and ICAM-1, but not TNF-α and E-selectin. Yet, SUL-138 limited the increase in plasma levels of IL-6 and TNF-α of CLP mice. These results demonstrate that SUL-138 supports mitochondrial function, resulting in a limitation of systemic inflammation and preservation of kidney function

Adventitial dissection:a simple and effective way to reduce radial artery spasm in coronary bypass surgery

<p>OBJECTIVE: Over the last two decades, the radial artery (RA) has become a routinely used conduit for coronary artery bypass graft surgery. One potential disadvantage of the radial artery is its higher susceptibility to vasospasm compared with other arterial grafts. We investigated whether adventitial dissection of the radial artery can reduce vasoconstriction and increase free blood flow.</p><p>METHODS: Following harvesting, the adventitia of the radial artery was dissected using coronary scissors. Surplus distal radial artery segments (n = 35) with and without adventitial dissection of patients undergoing coronary artery bypass surgery were collected and pairwise assessment of vasoreactivity to potassium chloride, U46619 and acetylcholine was performed in organ bath experiments. Free blood flow was measured before and after adventitial dissection.</p><p>RESULTS: Full curve and maximal vasoconstriction of the RA to potassium chloride (P = 0.015 and 0.001) and U46619 (P = 0.048 and 0.001) was significantly reduced after adventitial dissection compared with non-adventitial dissected radial arteries. Endothelium-dependent relaxation to acetylcholine of adventitial dissected radial arteries was significantly increased (P = 0.006) compared with non-adventitial dissected radial arteries. Maximal vasorelaxation to acetylcholine was significantly increased for adventitial dissected radial arteries compared with non-adventitial dissected radial arteries (P = 0.018). Free blood flow was significantly increased after adventitial dissection (P = 0.037).</p><p>CONCLUSION: The adventitial dissected radial artery is less susceptible to vasoconstriction and more prone to vasorelaxation ex vivo and shows an increased free blood flow. Therefore, we suggest adventitial dissection of the radial artery graft to reduce vasospasm for arterial revascularization in coronary artery bypass surgery.</p>

Troubleshooting methods for microarray gene expression analysis in the onset of diabetic kidney disease

<p>Introduction: Microarrays have become the standard technique for discovering new genes involved in the development of (kidney) disease. Diabetic nephropathy is a frequent complication of diabetes and is characterized by renal fibrosis. As the pathways leading to fibrosis are initiated early in diabetes and in the current study, we aimed at identifying genes associated with renal fibrosis in the first week after induction of diabetes in the rat streptozotocin (STZ) model. Methods: Conventional microarray analysis methods comparing gene expression to a common reference are not very suitable for time series as gene lists for all time point are very heterogeneous. We therefore sought an analysis technique that would allow us to easily find genes that we either substantially up or down regulated during the first week of diabetes. In the new method, the normalized expression of individual genes was plotted in time. Subsequently, the area under the curve (AUC) was calculated to quantify the overall level of changes in expression of individual genes. Results: AUCs for all genes were plotted in a histogram showing a normal distribution with a mean of close to 0, indicating no change in expression for the majority of genes. Genes with AUCs outside 3 standard deviations of the mean were considered significantly different from control. Discussion: Using this technique, a total of 290 genes were found to be significantly changed in the first week of diabetes. Data on a subset of genes were confirmed by real-time PCR, indicating the validity of the employed new analysis method. (C) 2013 Elsevier Inc. All rights reserved.</p>

Metformin Improves Endothelial Function and Reduces Blood Pressure in Diabetic Spontaneously Hypertensive Rats Independent from Glycemia Control: Comparison to Vildagliptin

Metformin confers vascular benefits beyond glycemia control, possibly via pleiotropic effects on endothelial function. In type-1-diabetes-mellitus (T1DM-)patients metformin improved flow-mediated dilation but also increased prostaglandin(PG)-F-2 alpha, a known endothelial-contracting factor. To explain this paradoxical finding we hypothesized that metformin increased endothelial-vasodilator mediators (e.g. NO and EDHF) to an even larger extent. Spontaneously-hypertensive-rats (SHR) display impaired endothelium-dependent relaxation (EDR) involving contractile PGs. EDR was studied in isolated SHR aortas and the involvement of PGs, NO and EDHF assessed. 12-week metformin 300 mg/kg/day improved EDR by up-regulation of NO and particularly EDHF; it also reduced blood pressure and increased plasma sulphide levels (a proxy for H2S, a possible mediator of EDHF). These effects persisted in SHR with streptozotocin (STZ)-induced T1DM. Vildagliptin (10 mg/kg/day), targeting the incretin axis by increasing GLP-1, also reduced blood pressure and improved EDR in SHR aortas, mainly via the inhibition of contractile PGs, but not in STZ-SHR. Neither metformin nor vildagliptin altered blood glucose or HbA(1c). In conclusion, metformin reduced blood pressure and improved EDR in SHR aorta via up-regulation of NO and particularly EDHF, an effect that was independent from glycemia control and maintained during T1DM. A comparison to vildagliptin did not support effects of metformin mediated by GLP-1

Low sodium diet inhibits the local counter-regulator effect of angiotensin-(1-7) on angiotensin II

Objective The heptapeptide angiotensin-(1-7) [Ang-(1-7)] has been identified as a versatile, endogenous inhibitor of the renin-angiotensin system (RAS). As the therapeutic response to exogenous RAS inhibitors, such as AT, receptor antagonists, is altered by changes in salt intake, we investigated the effect of a low, normal and high sodium diet on the antagonism of Ang II by Ang-(1-7). The role of angiotensin receptor subtypes and the endothelium was assessed. Methods Male Wistar rats received a normal sodium (0.3% NaCl), high sodium (2.0% NaCl) or low sodium (0.05% NaCl) diet for 10 days. Vascular responses were assessed ex vivo in thoracic aortic rings in the presence of the nitric oxide (NO) inhibitor N-G-monomethyl-L-arginine (L-NMMA) to avoid aspecific vasodilator effects of Ang-(1-7). Results After a normal or high salt diet, Ang-(1-7) significantly decreased maximal Ang II-induced vascular constrictions by 40-50%. After a low salt diet this noncompetitive antagonism disappeared. The AT(2) receptor antagonist PD 123319 and the Ang-(1-7) receptor antagonist A779 attenuated the effect of Ang-(1-7) found in rats fed with a normal or high sodium diet. Further, removal of endothelium and pretreatment with the prostaglandin synthesis inhibitor indomethacin (10(-5) mol/l) abolished the non-competitive antagonism by Ang-(1-7). Conclusion Ang-(1-7) elicits a specific, endothelium-dependent and non-competitive antagonism of Ang II, which involves AT(2) and Ang-(1-7) receptors but is independent of NO production. This non-competitive antagonism of Ang-(1-7) is abolished by a low sodium intake in normotensive rats, suggesting that it serves as a negative feedback towards Ang 11 in response to an altered sodium intake. (C) 2004 Lippincott Williams Wilkins

Selective Hepatic <i>Cbs</i> Knockout Aggravates Liver Damage, Endothelial Dysfunction and ROS Stress in Mice Fed a Western Diet

Cystathionine-β-synthase (CBS) is highly expressed in the liver, and deficiencies in Cbs lead to hyperhomocysteinemia (HHCy) and disturbed production of antioxidants such as hydrogen sulfide. We therefore hypothesized that liver-specific Cbs deficient (LiCKO) mice would be particularly susceptible to the development of non-alcoholic fatty liver disease (NAFLD). NAFLD was induced by a high-fat high-cholesterol (HFC) diet; LiCKO and controls were split into eight groups based on genotype (con, LiCKO), diet (normal diet, HFC), and diet duration (12 weeks, 20 weeks). LiCKO mice displayed intermediate to severe HHCy. Plasma H2O2 was increased by HFC, and further aggravated in LiCKO. LiCKO mice fed an HFC diet had heavier livers, increased lipid peroxidation, elevated ALAT, aggravated hepatic steatosis, and inflammation. LiCKO mice showed decreased L-carnitine in the liver, but this did not result in impaired fatty acid oxidation. Moreover, HFC-fed LiCKO mice demonstrated vascular and renal endothelial dysfunction. Liver and endothelial damage correlated significantly with systemic ROS status. In conclusion, this study demonstrates an important role for CBS in the liver in the development of NAFLD, which is most probably mediated through impaired defense against oxidative stress