Angiotensin 1-7 (Ang 1-7) is a product of the renin-angiotensin system (RAS) that can reduce blood pressure and fibrosis. For more than a decade Mas has been viewed as the receptor for Ang 1-7, however, there are lingering doubts regarding the nature of the interaction between the heptapeptide and Mas. To date, no one has pharmacologically characterized Ang 1-7 binding in tissue membrane preparations. In this study we show that 125 I-Ang 1-7 binds to a site for which Ang 1-7 has low (μmolar) affinity, suggesting that the addition of an iodine molecule to the tyrosine in position 4 of Ang 1-7 drastically changes the characteristics of this peptide making it unsuitable for characterization of Ang 1-7 receptors. 125 I-Ang 1-7 binds with moderately high affinity (10-20 nM) and saturability to a binding site in rat liver membranes that is displaceable by 127 I-Ang 1-7 (iodoAng 1-7) at nanomolar concentrations. This binding is also displaceable by inhibitors of metalloproteases. To test the hypothesis that 125 I-Ang 1-7 binds to a peptidase, a series of peptidase inhibitors were evaluated for their ability to inhibit 125 I-Ang 1-7 binding. The non-selective matrix metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) inhibitors marimastat, batimastat, and GM-6001, which inhibit MMP1,2,3,7,8,13,14 and ADAM 9,17, partially inhibited 125 I-Ang 1-7 binding with nanomolar affinities, similar to their IC 50 values for MMPs, suggesting that 125 I-Ang 1-7 binds to MMPs and/or ADAMs as well as other tissue elements. Further testing with the specific MMP-2 (ARP100), MMP-9 and ADAM-17 inhibitors indicate that 125 I-Ang 1-7 is not binding to these peptidases. Continuing studies are underway to establish conditions under which the 125 I-Ang 1-7 ligand is not metabolized by peptidases, while retaining the ability to bind to peptidases is underway to enable the identification of the specific peptidase(s) or other binding moieties to which 125 I-Ang 1-7 binds to determine their potential therapeutic importance as a druggable targets.

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system (RAS) that exhibits central actions to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been considered the receptor for Ang 1‐7, however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has demonstrated high affinity (low nanomolar KD) binding of a preparation of 125/127I‐Ang 1‐7 in liver membranes, however, this binding is not pharmacologically specific in that the IC50 of Ang 1‐7 is in the micromolar range and all angiotensin peptides compete for 125/127I‐Ang 1‐7 binding equivalently. 125/127I‐Ang 1‐7 binding to liver, kidney, brain and testes membrane preparations from both rats and mice has also failed to demonstrate a high affinity, pharmacologically specific binding site for 125/127I‐Ang 1‐7. Considering that rapid degradation of Ang 1‐7 could be adversely affecting the binding assay, we tested different peptidase inhibitors (JMV‐390, O‐phenanthroline and a proprietary combination of peptidase inhibitors Biotools®) to prevent degradation of the radioligand. Although we were able to reduce Ang 1‐7 degradation with these inhibitors (~80% preservation), we also reduced the total and specific binding of 125I‐Ang 1‐7. Indeed, competition binding assays for 125I‐Ang‐(1–7) with JMV‐390, O‐phenanthroline and Biotools® showed that 125I‐Ang‐(1–7) and all the inhibitors compete for the same binding site with JMV‐390 having a nanomolar IC50. It is important to note that the radioligand used for receptor binding studies contains an iodine125 on the tyrosine in position four of Ang 1‐7, and may have different characteristics for enzymatic metabolism. To assess this possibility, we determined the metabolism of 127I‐Ang 1‐7 (non‐radioactive iodine isotope) and indeed, the metabolism of Ang 1‐7 and 127I‐Ang 1‐7 in liver appear to differ, with 127I‐Ang 1‐7 being more resistant to metabolism. In addition, competition binding assays showed that 127I‐Ang 1‐7 is ~1000‐fold more potent than Ang 1‐7 in competing for 125I‐Ang 1‐7 binding. In conclusion, our results suggest that 125I‐Ang‐(1–7) may be binding to a peptidase rather than a receptor and the addition of the iodine molecule to the tyrosine in position four attenuates its degradation. Studies are underway to identify this peptidase and determine its potential therapeutic importance as a druggable target.Support or Funding InformationCardiovascular Neuroscience Research Fund 337667 Speth (PI) 03/31/15‐12/31/18 ‐ Nova Southeastern Univ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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We are documenting this to express our appreciation for the recent publication of the research article entitled Hyperinsulinemia Impairs coronary collateral circulation in patients with Chronic Total Coronary Occlusion (CTO) by Zou et al. 1 The authors did fantastic work, and the findings of the study have important clinical relevance as they highlight the effects of hyperinsulinemia on the development of collateral blood vessels in patients with CTO.While the introduction highlights conditions that impede collateral development, such as diabetes and metabolic syndrome, other potential confounding variables or limits that could affect the study's findings in ultimately finding the essential association of hyperinsulinemia and collateral circulation in patients with CTO would be beneficial. Addressing these limitations up front allows readers to grasp the study's potential consequences and generalizability.The study tells us that higher fasting insulin levels are associated with poor collateral circulation, which will help us in the future in the management of patients with ischemic heart diseases.We really appreciate the way the author has explained the relationship between hyperinsulinemia and poor collaterals; this can also be used as a prognostic factor in people with ischemic heart disease. Although further studies should be done to investigate the consideration of hyperinsulinemia as a prognostic marker. Adding on, some targeted approaches to reducing insulin levels can provide a direction towards greater therapeutic value.The study recognises that the sample size is limited. A larger sample size would give significant statistical strength to the study.The author correlates the two variables in a systemic way, but a potent stimulator, vascular endothelial growth factor (VEGF), is underemphasized. As insulin regulates VEGF gene expression and is an angiogenic mediator that causes vascularization in the myocardium specifically via the insulin receptor and PI3K/Akt pathway. 2 The authors investigated the role of hyperinsulinemia on collateral circulation in patients with CTO. However, the available evidence demonstrates that even this type of collateral flow is insufficient to prevent additional ischemia episodes in functional CTOs, pointing to the necessity of revascularization. 3 Hence, in the larger context, the prognostic significance of hyperinsulinemia in saving lives is not established.The study does not explain whether reducing insulin levels or addressing hyperinsulinemia could improve collateral flow.The research was conducted at a single hospital, which may limit the general representation of the population. If multiple hospital patients were involved in the study, then the impacts would be of greater significance.