Discovery and pharmacological characterisation of angiotensin-(1-7) receptors and identification of their importance in diabetes mellitus

The renin-angiotensin system (RAS) is known to be the main regulator of blood pressure and fluid balance. Within the RAS, angiotensin (Ang)-(1-7) is known to have cardiovascular protective effects. It represents the opponent of the often detrimental Ang II, which is known to stimulate the angiotensin receptor type 1 (AT1), causing negative effects such as a pathological rise in blood pressure. Beside the well accepted fact that the G-protein coupled receptor Mas is a receptor for the heptapeptide, it was not possible to characterise the ligand/receptor pharmacology or to identify further receptors, since there was a lack in the understanding of the initial intracellular signalling pathways stimulated by Ang-(1-7). In this study, cyclic adenosine monophosphate (cAMP) was identified as a second messenger stimulated by Ang-(1-7). The heptapeptide elevates cAMP concentration in various cell lines, as well as in Mas transfected HEK293 cells, confirming that Mas is a functional receptor for Ang-(1-7). Even more important, MrgD was identified as a second receptor for the peptide, while AT2 could be excluded to be targeted by the heptapeptide. It was also examined, if there are any changes in the intracellular signalling if the first amino acid of the peptide is decarboxylated. The receptor fingerprint for Ala1 -Ang-(1-7) was discovered, and the consequences for pharmacodynamics characterised. The dose-response curves were clearly different from the curves generated with Ang-(1-7). They showed a much lower EC50 and a bell-shaped curve for Ala1 -Ang-(1-7). Furthermore, pharmacological proof was provided that both, Mas and MrgD, are functional receptors for Ala1 -Ang-(1-7). Interestingly, it was also discovered that the AT2 receptor blocker PD123319 is not AT2 specific, but can also block the effects of Ang-(1-7) and Ala1 -Ang(-1-7) in Mas and MrgDtransfected and in primary cells. This raised the question whether the selective nonpeptidic AT2 receptor agonist, Compound 21 (C21), is also unspecific and stimulates Mas and MrgD too. This hypothesis was supported by the fact that the chemical structure of C21 is similar to the Mas receptor specific, non-peptidic agonist AVE0991. Using cAMP and downstream molecules as readouts, pharmacological proof that Mas and MrgD are functional receptors for C21 was generated. The last part of the study examined the role of Ang-(1-7) and its receptors in diabetes mellitus (DM). Previous studies demonstrated that the ACE2/ Ang-(1–7)/ Mas axis has beneficial effects on glucose homeostasis, but the underlying mechanisms remained unknown. The effects of Ang-(1–7) and its receptor Mas on the function of β-cells were investigated. Islets isolated from Mas-deficient and wild-type mice were stimulated with Ang-(1–7) or its antagonists and effects on insulin secretion were determined. It was found that Ang-(1–7) was able to increase the insulin secretion from wild-type islets, but not from islets derived from Mas deficient animals. Interestingly, Ang-(1-7) antagonist DPro, but not A779 could block the Ang-(1-7) mediated effects indicating the involvement of another Ang-(1-7) receptor. However, the heptapeptide did not affect the insulin gene expression or the excitation-secretion coupling, but increased intracellular cAMP involving exchange protein activated directly by cAMP (EPAC), leading to a higher insulin secretion by the β-cells. Ang-(1–7) was also applied to normo-glycaemic mice for 14 days using osmotic pumps. The effects of the heptapeptide in vivo had only marginal effects on glucose tolerance in wild-type mice. However, Ang-(1-7) had improved the insulin secretion in islets isolated from these mice. Interestingly, although less pronounced than in wild-types, Ang-(1–7) still affected insulin secretion in islets derived from Mas deficient mice. The effect of Ang- (1-7) in mice with STZ-induced diabetes was marginal, as in normo-glycaemic mice. Taken together, these results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for Ang-(1-7), and by excluding AT2 as a receptor for the heptapeptide. Furthermore, the identification of Ala1 -Ang-(1-7) as a peptide with specific pharmacodynamic properties can be used as a basis for the design of more potent and efficient Ang-(1-7) analogues, which can be useful in therapeutic interventions in a rapidly growing number of diseases. The proof that C21 and PD123319 are not AT2 receptor specific as generally assumed, but also interact with the two Ang-(1- 7) receptors, Mas and MrgD, might be an explanation for the partial overlap in beneficial effects of both compounds. Thus, the better understanding of the interaction of small molecules like C21 with their receptors, lays the foundation for the development of small molecules which stimulate all or just one of the Ang-(1-7) receptors, which may be beneficial in diseases like diabetes mellitus. Since it could be shown that Ang-(1–7) plays a significant role in the regulation of insulin secretion from mouse islets in vitro and in vivo, mainly, but not exclusively, by Mas-dependent signalling, modulating the accessory pathway of insulin secretion via increase in cAMP, makes clear that Ang-(1-7) and its receptors are very promising therapeutic targets

An Evaluation of Open Source Unit Testing Tools Suitable for Data Warehouse Testing

Verification and validation are two important processes in the software system lifecycle. Despite the importance of these processes, a recent survey has shown that testing of data warehouse systems is currently neglected. The survey participants named besides others modest budget and the lack of appropriate tools as potential reasons for this circumstance. In order to verify these reasons, the paper at hand presents an evaluation of unit testing tools suitable for data warehouse testing. To address the modest budget problem, the range of evaluation candidates is limited to no charge, open source solutions, namely AnyDbTest, BI.Quality, DbFit, DbUnit, NDbUnit, SQLUnit, TSQLUnit, and utPLSQL. The evaluation follows the IEEE 14102-2010 guidelines for evaluation and selection of computeraided software engineering tools in order to guarantee benefits from a practioners’ as well as scientific point of view. It results in a detailed overview of how the testing tools meet criteria such as different testing functionalities. At least one tool, namely DbFit, can be identified as a promising candidate with regard to the requirements

Decarboxylation of Ang-(1–7) to Ala1-Ang-(1–7) leads to significant changes in pharmacodynamics

The heptapeptide angiotensin (Ang)–(1–7) is part of the beneficial arm of the renin-angiotensin system. Ang-(1–7) has cardiovascular protective effects, stimulates regeneration, and opposes the often detrimental effects of Ang II. We recently identified the G protein-coupled receptors Mas and MrgD as receptors for the heptapeptide. Ala1-Ang-(1–7) (Alamandine), a decarboxylated form of Ang-(1–7), has similar vasorelaxant effects, but has been described to only stimulate MrgD. Therefore, this study aimed to characterise the consequences of the lack of the carboxyl group in amino acid 1 on intracellular signalling and to identify the receptor fingerprint for Ala1-Ang-(1–7). In primary endothelial and mesangial cells, Ala1-Ang-(1–7) elevated cAMP concentration. Dose response curves generated with Ang-(1–7) and Ala1-Ang-(1–7) significantly differed from each other, with a much lower EC50 and a bell-shape curve for Ala1-Ang-(1–7). We provided pharmacological proof that both, Mas and MrgD, are functional receptors for Ala1-Ang-(1–7). Consequently, in primary mesangial cells with genetic deficiency in both receptors the heptapeptide failed to increase cAMP concentration. As we previously described for Ang-(1–7), the Ala1-Ang-(1–7)-mediated cAMP increase in Mas/MrgD-transfected HEK293 cells and primary cells were blocked by the AT2 receptor blocker, PD123319. The very distinct dose-response curves for both heptapeptides could be explained by in silico modelling, electrostatic potential calculations, and an involvement of Galpha i for higher concentrations of Ala1-Ang-(1–7). Our results identify Ala1-Ang-(1–7) as a peptide with specific pharmacodynamic properties and build the basis for the design of more potent and efficient Ang-(1–7) analogues for therapeutic interventions in a rapidly growing number of diseases

Heterogeneous expression and functional relevance of the ubiquitin carboxyl-terminal hydrolase L1 in melanoma

The expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is deregulated in human cancer cells with tumor inhibiting or promoting functions. Due to less knowledge on the role of UCHL1 in melanoma progression, the expression pattern and function of UCHL1 as well as the deregulated signaling pathways were characterized. A large number of melanoma cell lines, tissue microarrays of melanoma lesions and control tissues were analyzed for UCHL1 expression using PCR, Western blot and/or immunohistochemistry. The analysis revealed that melanocyte cultures, 24 of 331 melanoma lesions, two of 18 short-term cultures and two of 19 melanoma cell lines tested, respectively, heterogeneously expressed UCHL1. The low frequency of UCHL1 expression in melanoma cells was due to gene silencing by promoter DNA hypermethylation. Using different transfection models an enzyme activity-dependent growth promoting function of UCHL1 via the activation of the mitogen-activated protein kinase signaling pathway was found in melanoma cells. Under oxygen stress a dose-dependent effect of UCHL1 was detected, which was mediated by a dynamic modification of the PI3K-Akt signaling. Thus, the aberrant UCHL1 expression in melanoma cells is linked to dynamic changes in growth properties and signal transduction cascades suggesting that UCHL1 provides a novel marker and/or therapeutic target at least for a subset of melanoma patients

Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1–7)

The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1 h, 3 h, 6 h, and 9 h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1 h, 119 after 3 h, 31 after 6 h, and 86 after 9 h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1 h and 3 h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7) treatment. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells

Further intracellular proteins and signaling pathways regulated by angiotensin-(1-7) in human endothelial cells.

In 2016, Meinert et al. (doi: 10.1016/j.jprot.2015.09.020) published the first 25 proteins in a protein array regulated in Human Umbilical Vein Endothelial Cells (HUVEC) by the heptapeptide angiotensin (Ang)-(1-7) and the first 10 intracellular signaling cascades at different time points. This supporting data article shows further proteins and pathways stimulated by Ang-(1-7) in human endothelial cells at time points of 1 h, 3 h, 6 h, and 9 h. HUVECs were stimulated with Ang-(1-7), and regulated proteins were identified via antibody microarray. Bioinformatics software IPA was used for association of regulated proteins to metabolic pathways

Further intracellular proteins and signaling pathways regulated by angiotensin-(1–7) in human endothelial cells

In 2016, Meinert et al. (doi: 10.1016/j.jprot.2015.09.020) published the first 25 proteins in a protein array regulated in Human Umbilical Vein Endothelial Cells (HUVEC) by the heptapeptide angiotensin (Ang)-(1–7) and the first 10 intracellular signaling cascades at different time points. This supporting data article shows further proteins and pathways stimulated by Ang-(1–7) in human endothelial cells at time points of 1 h, 3 h, 6 h, and 9 h. HUVECs were stimulated with Ang-(1–7), and regulated proteins were identified via antibody microarray. Bioinformatics software IPA was used for association of regulated proteins to metabolic pathways

Further intracellular proteins and signaling pathways regulated by angiotensin-(1-7) in human endothelial cells.

In 2016, Meinert et al. (doi: 10.1016/j.jprot.2015.09.020) published the first 25 proteins in a protein array regulated in Human Umbilical Vein Endothelial Cells (HUVEC) by the heptapeptide angiotensin (Ang)-(1-7) and the first 10 intracellular signaling cascades at different time points. This supporting data article shows further proteins and pathways stimulated by Ang-(1-7) in human endothelial cells at time points of 1 h, 3 h, 6 h, and 9 h. HUVECs were stimulated with Ang-(1-7), and regulated proteins were identified via antibody microarray. Bioinformatics software IPA was used for association of regulated proteins to metabolic pathways

Neprilysin-dependent neuropeptide Y cleavage in the liver promotes fibrosis by blocking NPY-receptor 1

Development of liver fibrosis is paralleled by contraction of hepatic stellate cells (HSCs), the main profibrotic hepatic cells. Yet, little is known about the interplay of neprilysin (NEP) and its substrate neuropeptide Y (NPY), a potent enhancer of contraction, in liver fibrosis. We demonstrate that HSCs are the source of NEP. Importantly, NPY originates majorly from the splanchnic region and is cleaved by NEP in order to terminate contraction. Interestingly, NEP deficiency (Nep−/−) showed less fibrosis but portal hypertension upon liver injury in two different fibrosis models in mice. We demonstrate the incremental benefit of Nep−/− in addition to AT1R blocker (ARB) or ACE inhibitors for fibrosis and portal hypertension. Finally, oral administration of Entresto, a combination of ARB and NEP inhibitor, decreased hepatic fibrosis and portal pressure in mice. These results provide a mechanistic rationale for translation of NEP-AT1R-blockade in human liver fibrosis and portal hypertension