Human Renin-angiotensin System Research Articles

Galectin-7 dysregulates renin-angiotensin-aldosterone and NADPH oxide synthase pathways in preeclampsia.

Preeclampsia is a life-threatening disorder of pregnancy unique to humans. Poor placentation in the first trimester of pregnancy is widely accepted to be an underlying cause of preeclampsia. Galectin-7 is abnormally elevated in chorionic villous samples and serum from women that subsequently develop pre-term preeclampsia. Administration of exogenous galectin-7 to pregnant mice causes preeclampsia-like features (hypertension, proteinuria), associated with dysregulation of the renin-angiotensin system (RAS). In this study investigated the mechanism by which galectin-7 induces alterations to tissue RAS homeostasis and ROS production. We hypothesized that galectin-7 induces alterations in the production of either placental RAS or NADPH oxidases (or both) to drive the dysregulated RAS and ROS production seen in preeclampsia. Mated female mice (n=5-6/group) received single (embryonic day [E]12/13) or multiple (E8-12) subcutaneous injections of 400μg/kg/day galectin-7 or vehicle control and killed on E13 or E18. Human first trimester placental villous and decidual tissue (n=11) was cultured under 8% oxygen with 1µg/mL galectin-7 or vehicle control for 16h. Galectin-7 administration to pregnant mice impaired placental labyrinth formation, suppressed circulating aldosterone and altered placental RAS (Agt, Renin) and NADPH oxidase (Cyba, Cybb and Icam1) mRNA expression. In vitro, galectin-7 regulated human placental villous RAS (AGT) and NADPH oxidase (CYBA, ICAM1 and VCAM1) mRNA expression. Overall, galectin-7 likely drives hypertension in preeclampsia via its direct regulation of multiple pathways associated with preeclampsia in the placenta. Galectin-7 may therefore be a therapeutic target to improve placental function and prevent preeclampsia.

Myocardial Angiotensin Metabolism in End-Stage Heart Failure

BackgroundThe myocardium exhibits an adaptive tissue-specific renin-angiotensin system (RAS), and local dysbalance may circumvent the desired effects of pharmacologic RAS inhibition, a mainstay of heart failure with reduced ejection fraction (HFrEF) therapy. ObjectivesThis study sought to investigate human myocardial tissue RAS regulation of the failing heart in the light of current therapy. MethodsFifty-two end-stage HFrEF patients undergoing heart transplantation (no RAS inhibitor: n = 9; angiotensin-converting enzyme [ACE] inhibitor: n = 28; angiotensin receptor blocker [ARB]: n = 8; angiotensin receptor neprilysin-inhibitor [ARNi]: n = 7) were enrolled. Myocardial angiotensin metabolites and enzymatic activities involved in the metabolism of the key angiotensin peptides angiotensin 1-8 (AngII) and Ang1-7 were determined in left ventricular samples by mass spectrometry. Circulating angiotensin concentrations were assessed for a subgroup of patients. ResultsAngII and Ang2-8 (AngIII) were the dominant peptides in the failing heart, while other metabolites, especially Ang1-7, were below the detection limit. Patients receiving an ARB component (i.e., ARB or ARNi) had significantly higher levels of cardiac AngII and AngIII (AngII: 242 [interquartile range (IQR): 145.7 to 409.9] fmol/g vs 63.0 [IQR: 19.9 to 124.1] fmol/g; p < 0.001; and AngIII: 87.4 [IQR: 46.5 to 165.3] fmol/g vs 23.0 [IQR: <5.0 to 59.3] fmol/g; p = 0.002). Myocardial AngII concentrations were strongly related to circulating AngII levels. Myocardial RAS enzyme regulation was independent from the class of RAS inhibitor used, particularly, a comparable myocardial neprilysin activity was observed for patients with or without ARNi. Tissue chymase, but not ACE, is the main enzyme for cardiac AngII generation, whereas AngII is metabolized to Ang1-7 by prolyl carboxypeptidase but not to ACE2. There was no trace of cardiac ACE2 activity. ConclusionsThe failing heart contains considerable levels of classical RAS metabolites, whereas AngIII might be an unrecognized mediator of detrimental effects on cardiovascular structure. The results underline the importance of pharmacologic interventions reducing circulating AngII actions, yet offer room for cardiac tissue-specific RAS drugs aiming to limit myocardial AngII/AngIII peptide accumulation and actions.

Regulation of the human placental (pro)renin receptor-prorenin-angiotensin system by microRNAs.

Are any microRNAs (miRNAs) that target the placental renin-angiotensin system (RAS) in the human placenta suppressed in early gestation? Overall, 21 miRNAs with predicted RAS mRNA targets were less abundant in early versus term placentae and nine were more highly expressed. Regulation of human placental RAS expression could alter placental development and therefore normal pregnancy outcome. The expression of genes encoding prorenin (REN), angiotensinogen, (pro)renin receptor, angiotensin converting enzyme 2, and the angiotensin II type 1 receptor are highest in early gestation, at a time when oxygen tension is at its lowest. Studies have shown that the human placental RAS is sensitive to oxygen, as are some miRNAs that regulate RAS mRNAs. We propose that in early pregnancy, the prevailing low O2 tension, by suppression of levels of miRNAs that target RAS mRNAs, results in increased expression of RAS mRNAs and encoded proteins. As gestation proceeds and the prevailing oxygen tension rises, abundance of these miRNAs increases, and placental RAS mRNA expression is suppressed. The expression of miRNAs was compared in human placentae collected in early (10-11 weeks; n = 7) and mid-gestation (14-18 weeks; n = 8) with placenta collected at term (38-40 weeks; n = 8). Expression of placental miRNAs in women with early (29-35.1 weeks; n = 8) or late-onset pre-eclampsia (PE) (>34-weeks gestation; n = 8) and gestational age matched preterm (31.6-35.1 weeks; n = 8) and term normotensive controls were also compared. Agilent Human miRNA microarray v19 was used to detect up to 2006 miRNAs in four placentae from each group. Statistically different levels of expression were determined and refined using predictive modelling. Placental miRNAs predicted to target RAS mRNAs were identified in three databases. Differences detected on the array were confirmed for some miRNAs by semi-quantitative RT-PCR (qPCR, n = 7-8 for all groups). Two differentially expressed miRNAs that were known to target human renal REN mRNA (miR-181a-5p and miR-663) were transfected into human HTR-8/SVneo trophoblast cells to examine their effect on placental REN expression and prorenin levels. In early gestation placentae, 186 miRNAs were differentially expressed compared with term placentae (109 increased, 77 decreased). Thirty of the differentially expressed miRNAs were predicted to target RAS components. In mid-gestation placentae, 117 miRNAs were differentially expressed compared with term placentae (69 increased, 48 decreased). Of these, 19 had RAS mRNAs as predicted targets. Eight miRNAs that were lower in early gestation and predicted to target RAS mRNAs were confirmed by qPCR. All showed an increase during gestation and could influence the transgestational profile of the human placental RAS. Additionally, on the array, three miRNAs predicted to target RAS mRNAs (miR-892c-3p, miR-378c and miR-514b-3p) were overexpressed in placentae from women with late-onset PE (P = 3.6E-10, P = 1.8E-05, P = 5.3E-06; respectively). miR-663, which suppresses renal REN mRNA expression, was overexpressed in early-onset PE placentae as determined by qRT-PCR analysis (P = 0.014). Transfection of miR-181a-5p and miR-663 into HTR-8/SVneo trophoblast cells suppressed REN mRNA expression (P = 0.05) and prorenin protein production (P = 0.001). Data can be found via GEO accession number GSE109832. Further validation that the differentially expressed miRNAs do indeed directly target RAS mRNAs and affect placental development and function is required. This study is limited by the small sample size. Therefore independent validation in a larger cohort is required. We propose that suppression of miRNAs that target the placental RAS in early gestation is partly responsible for the increase in RAS expression at this time, in order to promote placental development. Later in pregnancy, we have detected overexpression of several miRNAs in placentae from women with PE. These may prove to be biomarkers for early detection of women at risk of developing PE. Since the placenta produces at least two miRNAs that were found in the kidney to target REN mRNA, and that also target placental REN mRNA, the escape of these miRNAs into the maternal circulation in excess amounts could affect maternal renal REN mRNA production and thereby disturb maternal fluid and electrolyte homoeostasis. This work was supported by the National Health and Medical Research Council, Australia (APP1043537). K.G.P. is supported by an Australian Research Council Future Fellowship (FT150100179). C.T.R. is supported by a Lloyd Cox Professorial Research Fellowship from the University of Adelaide. F.Z.M. is supported by a National Heart Foundation Future Leader Fellowship and Baker Heart and Diabetes Institute Fellowship. The authors declare that they have no competing interests.

How Is the Brain Renin–Angiotensin System Regulated?

The renin–angiotensin system (RAS) is one of the most studied physiological pathways controlling arterial blood pressure (BP). The longevity of RAS blockers as effective antihypertensive therapies evidences the continued clinical importance of the RAS. New components of the RAS and novel mechanisms continue to be discovered with regularity. For example, the RAS has recently been implicated in metabolic control and its association with metabolic pathways.1–3 Moreover, the RAS is one of the most complex systems because of the interaction between circulating and tissue RAS, among RAS in different tissues, and the activity of counter-regulatory peptides and receptors, vis-a-vis Ang-II/angiotensin type 1 (AT1) receptor versus Ang-(1–7)/Mas. The intricate neural circuitry between cardiovascular, fluid homeostasis, and metabolic control regions, some of which have the capacity for the independent generation and action of angiotensin peptides further complicates our understanding of the brain RAS. Instead of an all-encompassing review of the brain RAS, which can be found elsewhere, this article will focus primarily on investigations into how RAS activity in the brain is regulated.4 There have been numerous studies examining the transcriptional mechanisms regulating expression of the angiotensinogen (AGT) gene. A vast majority of these studies have focused on hepatocytes and renal proximal tubule. In the brain, AGT is expressed in astrocytes and in some neurons, particularly in regions of the brain controlling cardiovascular and metabolic function.5–7 Expression of AGT in glial cells is of clear importance as overexpression of the human RAS in glial cells results in hypertension, and its removal from glia significantly blunts hypertension in mice.8,9 Similarly, transgenic rats with decreased glial AGT exhibit severe defects in water homeostasis and a blunting of hypertension when bred with a rat model of increased brain Ang-II.10 It has been widely presumed that …

Vitamin D depletion does not affect key aspects of the preeclamptic phenotype in a transgenic rodent model for preeclampsia

Maternal vitamin D deficiency is proposed as a risk factor for preeclampsia in humans. We tested the hypothesis that vitamin D depletion aggravates and high supplementation ameliorates the preeclampsia phenotype in an established transgenic rat model of human renin-angiotensin system–mediated preeclampsia. Adult rat dams, transgenic for human angiotensinogen (hAGT) and mated with male rats transgenic for human renin (hREN), were fed either vitamin D-depleted chow (VDd) or enriched chow (VDh) 2 weeks before mating and during pregnancy. Mean blood pressure was recorded by tail-cuff, and 24-hour urine samples were collected in metabolic cages at days 6 and 18 of gestation. Rats were sacrificed at day 21 of gestation. Depleted dams (VDd) had negligible serum 25-hydroxyvitamin D2+3 levels (mean ± SEM; 2.95 ± 0.45 nmol/l vs. VDh 26.20 ± 2.88 nmol/l, P = .01), but in both groups, levels of 1,25(OH)2D3 remained below detection level of 25 pmol/l. Dietary vitamin D depletion did not aggravate hypertension (mean ± SEM BP, day 20 of gestation: 151.38 ± 5.65 mmHg VDd vs. 152.00 ± 4.10 mmHg VDh) or proteinuria. Fetal anthropometrics were similar between the groups, whereas VDd displayed lower placental:fetal weight ratios (0.15 vs. 0.16 g/g, P = .01) and increased sFlt-1/PlGF ratio. Expression of hREN was lower in placenta of VDd dams (0.82 ± 0.44 AU vs. 1.52 ± 0.15 AU, P = .04). Expression of key vitamin D metabolizing enzymes was unchanged. Dietary vitamin D intervention did not alter key aspects of the preeclampsia phenotype using the transgenic rodent model of human renin-angiotensin system–mediated pre-eclampsia, plausibly due to altered vitamin D metabolism or excretion in the transgenic rats.

Structure-based design and optimization of antihypertensive peptides to obtain high inhibitory potency against both renin and angiotensin I-converting enzyme

The human renin–angiotensin system (RAS) plays an essential role in regulating blood pressure and systemic vascular resistance. Renin and angiotensin I-converting enzyme (ACE) are two key enzymes in RAS and have long been recognized as attractive antihypertensive targets. Here, a synthetic strategy was proposed integrating quantitative structure–activity relationship (QSAR), molecular dynamics (MD) simulation and binding free energy analysis to discover novel dual renin and ACE peptidic inhibitors. With the strategy a number of candidates were generated virtually, from which eight promising peptides were selected and synthesized for biological assay. Consequently, three peptides (RYLP, YTAWVP and YRAWVL) were successfully identified to have satisfactory inhibitory profile against both renin and ACE with IC50 values of <1 mM and <10 μM, respectively. Structural analysis and energetic dissection revealed different binding modes of peptide to renin and ACE; a peptide only inserts its C-terminus into the active site of ACE, whereas the whole peptide packs tightly against renin. In addition, when limited to structural diversity it is hard to reconcile the renin and ACE inhibitory activities of short peptides such as dipeptides. These findings can be used to guide peptide optimization with improved biological activity.

Similarities and differences of X and Y chromosome homologous genes, SRY and SOX3, in regulating the renin-angiotensin system promoters.

The renin-angiotensin system (RAS) is subject to sex-specific modulation by hormones and gene products. However, sex differences in the balance between the vasoconstrictor/proliferative ACE/ANG II/AT1 axis, and the vasodilator/antiproliferative ACE2/ANG-(1-7)/MAS axis are poorly known. Data in the rat have suggested the male-specific Y-chromosome gene Sry to contribute to balance between these two axes, but why the testis-determining gene has these functions remains unknown. A combination of in silico genetic/protein comparisons, functional luciferase assays for promoters of the human RAS, and RNA-Seq profiling in rat were used to address if regulation of Sry on the RAS is conserved in the homologous X-chromosome gene, Sox3. Both SRY and SOX3 upregulated the promoter of Angiotensinogen (AGT) and downregulated the promoters of ACE2, AT2, and MAS, likely through overlapping mechanisms. The regulation by both SRY and SOX3 on the MAS promoter indicates a cis regulation through multiple SOX binding sites. The Renin (REN) promoter is upregulated by SRY and downregulated by SOX3, likely through trans and cis mechanisms, respectively. Sry transcripts are found in all analyzed male rat tissues including the kidney, while Sox3 transcripts are found only in the brain and testis, suggesting that the primary tissue for renin production (kidney) can only be regulated by SRY and not SOX3. These results suggest that SRY regulation of the RAS is partially shared with its X-chromosome homolog SOX3, but SRY gained a sex-specific control in the kidney for the rate-limiting step of the RAS, potentially resulting in male-specific blood pressure regulation.

Methylation of promoter regions of genes of the human intrauterine Renin Angiotensin system and their expression.

The intrauterine renin angiotensin system (RAS) is implicated in placentation and labour onset. Here we investigate whether promoter methylation of RAS genes changes with gestation or labour and if it affects gene expression. Early gestation amnion and placenta were studied, as were term amnion, decidua, and placenta collected before labour (at elective caesarean section) or after spontaneous labour and delivery. The expression and degree of methylation of the prorenin receptor (ATP6AP2), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1), and two proteases that can activate prorenin (kallikrein, KLK1, and cathepsin D, CTSD) were measured by qPCR and a DNA methylation array. There was no effect of gestation or labour on the methylation of RAS genes and CTSD. Amnion and decidua displayed strong correlations between the percent hypermethylation of RAS genes and CTSD, suggestive of global methylation. There were no correlations between the degree of methylation and mRNA abundance of any genes studied. KLK1 was the most methylated gene and the proportion of hypermethylated KLK1 alleles was lower in placenta than decidua. The presence of intermediate methylated alleles of KLK1 in early gestation placenta and in amnion after labour suggests that KLK1 methylation is uniquely dynamic in these tissues.

Evaluating hormonal mechanisms of vitamin D receptor agonist therapy in diabetic kidney disease: the VALIDATE-D study

BackgroundInsufficient vitamin D status and increased renin-angiotensin system (RAS) activity have been associated with renal-vascular disease and nephropathy in diabetes. Accumulating evidence indicates that vitamin D receptor (VDR) activation lowers unfavorable RAS activity; however, more human intervention studies evaluating whether this mechanism could influence diabetic kidney disease are needed. We previously reported that both vitamin D levels and genetic variation at the VDR predict human RAS activity, and that vitamin D therapy can lower RAS activity in non-diabetics. The VALIDATE-D study is a randomized, placebo-controlled, intervention study designed to extend these findings by evaluating whether direct VDR activation in diabetes lowers circulating and local renal-vascular tissue RAS activity (Aims 1 and 2) in a manner similar to the action of ACE inhibitors (Aim 3).Methods/DesignForty subjects with type 2 diabetes, microalbuminuria, and without chronic kidney disease will be recruited to undergo detailed assessment of the RAS before and after randomization to calcitriol 0.75 mcg/day or placebo. Primary analyses will evaluate whether calcitriol therapy reduces circulating and renal-vascular tissue-RAS activity in comparison to placebo. All subjects will thereafter be treated with lisinopril and followed for 3.5 months to evaluate whether combination therapy (calcitriol + lisinopril vs. placebo + lisinopril) additively or synergistically improves renal-vascular function, and lowers proteinuria.DiscussionThe VALIDATE-D study is the first human intervention study to evaluate whether direct VDR activation can lower the human RAS in diabetes, compared to the effect of an ACE inhibitor, and whether this mechanism can translate to clinically relevant endpoints for diabetic kidney disease. The outcomes of VALIDATE-D will have major implications for the recommendation of vitamin D supplementation for the primary prevention of kidney complications in diabetes.Trial registrationClinicalTrials.gov, NCT01635062

Proteolytic Processing of Angiotensin-I in Human Blood Plasma

In mammalian species, except humans, N-terminal processing of the precursor peptide angiotensin I (ANG-1-10) into ANG-2-10 or ANG-3-10 was reported. Here we hypothesize that aminopeptidase-generated angiotensins bearing the same C-terminus as ANG-1-10 are also present in humans. We demonstrate the time dependent generation of ANG-2-10, ANG-3-10, ANG-4-10, ANG-5-10 and ANG-6-10 from the precursor ANG-1-10 by human plasma proteins. The endogenous presence of ANG-4-10, ANG-5-10 and ANG-6-10 in human plasma was confirmed by an immuno-fluorescence assay. Generation of ANG-2-10, ANG-3-10 and ANG-4-10 from ANG-1-10 by immobilized human plasma proteins was sensitive to the cysteine/serine protease inhibitor antipain. The metal ion chelator EDTA inhibited Ang-6-10-generation. Incubation of the substrates ANG-3-10, ANG-4-10 and ANG-5-10 with recombinant aminopeptidase N (APN) resulted in a successive N-terminal processing, finally releasing ANG-6-10 as a stable end product, demonstrating a high similarity concerning the processing pattern of the angiotensin peptides compared to the angiotensin generating activity in plasma. Recombinant ACE-1 hydrolyzed the peptides ANG-2-10, ANG-3-10, ANG-4-10 and ANG-5-10 into ANG-2-8, ANG-3-8, ANG-4-8 and ANG-5-8. Since ANG-2-10 was processed into ANG-2-8, ANG-4-8 and ANG-5-8 by plasma proteases the angiotensin peptides bearing the same C-terminus as ANG-1-10 likely have a precursor function in human plasma. Our results confirm the hypothesis of aminopeptidase mediated processing of ANG-1-10 in humans. We show the existence of an aminopeptidase mediated pathway in humans that bypasses the known ANG-1-8-carboxypeptidase pathway. This expands the knowledge about the known human renin angiotensin system, showing how efficiently the precursor ANG-1-10 is used by nature.

Local renin-angiotensin system in the thyroid – myth or fact?

Beside the basic control system associated with the hypothalamus-hypophysis-thyroid axis and thyrotropin, a growing evidence suggests that thyroid function is influenced by a vast spectrum of locally produced factors. Renin-angiotensin system (RAS) is considered as one of the most powerful and effective regulatory systems in humans. In the “classical” cascade of RAS, angiotensinogen is enzymatically conversed to the most active element of RAS – angiotensin (Ang) II. Ang II exerts its effects via two main receptors: AT1R and AT2R. The understanding of RAS biological function was significantly changed by the discovery of new enzymatic cascades leading to the production of molecules with different than Ang II functional properties – for instance Ang (1-7), molecule reacting with its specific receptor – Mas. Beside the growing complexity of RAS-structure, diverse expression patterns of RAS components in various tissues formed a basis for a concept of “local RAS” in many organs including brain and immune system. It was shown that many organ-specific effects of hormonal factors, including the influence of thyroid hormones on cardiovascular system, were dependent on the modulation of local RAS components. However, very little is known about the role of RAS in the control of thyroid function. In our immunohistochemical analysis of operative specimens we observed for the first time expression of RAS components in human thyroid. Interestingly, although our recently published findings suggest a direct regulatory influence of thyroid function on the phenotype and activity of immune cells in humans, no studies regarding the influence of human RAS on the course of such common inflammatory process as thyroiditis are available. Flow Cytometry analyses performed in our laboratory on thyroid fine needle aspiration biopsy material allowed us to assess quantitatively and phenotypically the immune cell populations of local thyroid blood. The comparison of results obtained with paired peripheral blood and local thyroid blood samples suggested differences in the structure of antigen presenting cell populations, e.g. monocytes, known to be the main source of RAS components in the immune system. Interestingly, RAS positive immune cells have been shown to correlate with clinical course of various inflammatory and malignant diseases. In conclusion, better knowledge about local RAS in the thyroid seems to be of great importance for the understanding of both: thyroid physiology and pathological processes associated with inflammatory and malignant disorders of this gland.

Molecular recognition and regulation of human angiotensin-I converting enzyme (ACE) activity by natural inhibitory peptides

Angiotensin-I converting enzyme (ACE), a two-domain dipeptidylcarboxypeptidase, is a key regulator of blood pressure as a result of its critical role in the renin-angiotensin-aldosterone and kallikrein-kinin systems. Hence it is an important drug target in the treatment of cardiovascular diseases. ACE is primarily known for its ability to cleave angiotensin I (Ang I) to the vasoactive octapeptide angiotensin II (Ang II), but is also able to cleave a number of other substrates including the vasodilator bradykinin and N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a physiological modulator of hematopoiesis. For the first time we provide a detailed biochemical and structural basis for the domain selectivity of the natural peptide inhibitors of ACE, bradykinin potentiating peptide b and Ang II. Moreover, Ang II showed selective competitive inhibition of the carboxy-terminal domain of human somatic ACE providing evidence for a regulatory role in the human renin-angiotensin system (RAS).

Abstract 387: Individual Biochemical Characterization of the Renin-Angiotensin-System by RAS-Fingerprinting in Healthy Volunteers Treated with RAS-Blockers

The Renin-Angiotensin-System (RAS) is critically involved in the regulation of important physiological functions including blood pressure and fluid balance. It is constituted by multiple enzymes giving rise to a meshwork of effector peptides, which mediate their functions through binding to specific receptor molecules. Therefore, the modification of angiotensin peptide levels represents a common strategy for the treatment of RAS-associated diseases and is frequently achieved by the pharmacologic regulation of enzymes taking part in angiotensin metabolism. We developed a highly sensitive method, which allows the simultaneous absolute quantification of up to 10 different angiotensin peptides in human plasma and whole blood (RAS-Fingerprinting). The measurement of RAS-Fingerprints provides unique insights into the physiology of the human RAS and therefore represents a powerful tool for the patient specific evaluation of this physiologically important peptide hormone system. Beside the precise quantification of angiotensin peptides in plasma and whole blood, RAS-Fingerprints analyze the biochemical hardware of the RAS at a previously unachieved level of detail and accuracy. During the development and validation of our LC-MS/MS based method, comprehensive datasets showing multiple applications for RAS-Fingerprinting have been generated. Investigation of healthy volunteers revealed that a patient specific state of the RAS exists, which is defined by a unique composition of RAS enzyme activities that might have crucial effects on the outcome of RAS targeted therapies in individual patients. RAS-Fingerprinting in healthy volunteers treated with an Angiotensin-Receptor-Blockers, ACE-Inhibitor or Renin-Inhibitor revealed that the human RAS reacts to different drugs in a very dynamic and specific way. Therefore, RAS-Fingerprinting could contribute to the development of innovative therapeutic approaches affecting the RAS. The implementation of RAS-Fingerprinting into clinical studies would substantially enhance our understanding of the human RAS and could lead to the development of personalized treatment schemes in the management of RAS-associated diseases in the near future.

Abstract 291: RAS Fingerprinting: Patient-Specific Evaluation of the Renin-Angiotensin-System in Plasma by LC-MS/MS-Based Multiplex Quantification of Angiotensin Peptides

The Renin-Angiotensin-System (RAS) is critically involved in the regulation of important physiological functions including blood pressure and fluid balance. It is constituted by multiple enzymes giving rise to a meshwork of effector peptides, which mediate their functions through binding to specific receptor molecules. Therefore, the modification of angiotensin peptide levels represents a common strategy for the treatment of RAS-associated diseases and is frequently achieved by the pharmacologic regulation of enzymes taking part in angiotensin metabolism. We developed a highly sensitive method, which allows the simultaneous absolute quantification of up to 10 different angiotensin peptides in human plasma and whole blood (RAS-Fingerprinting). Either the measurement of in vivo RAS-Fingerprints (immediate sample stabilization) or heparin derived ex vivo RAS-Fingerprints in plasma or whole blood, provide unique insights into the biochemical constitution of the human RAS and therefore represent powerful tools for the patient specific evaluation of this physiologically important peptide hormone system. During the development and validation of a new LC-MS/MS based method for angiotensin quantification, comprehensive datasets for various applications of RAS-Fingerprinting have been generated. RAS-Fingerprints were measured in blood of healthy volunteers during the treatment with different RAS-blockers, which provided deep insights in the physiology and regulation of the human RAS and revealed surprising differences between individual patients. With the help of RAS-Fingerprinting, we were able to re-draw the picture of the human RAS at a previously unachieved level of detail and accuracy. Due to the availability of new selection criteria for pharmacologic screens, RAS-Fingerprinting could contribute to the development of innovative therapeutic approaches affecting the RAS. The extensive utilization of RAS-Fingerprinting in clinical studies will substantially enhance our understanding of the human RAS and could lead to the development of personalized treatment schemes in the management of RAS-associated diseases in the near future.

High glucose potentiates and renin-angiotensin blockade downregulates LPS-induced tissue factor expression in human mononuclear cells

BackgroundIntimate links connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally generated Angiotensin (ANG) II, the effector arm of the Renin Angiotensin System (RAS). The RAS, in turn, plays a pathophysiological role in diabetes, a proinflammatory state to which elevated glucose, the disease hallmark, contributes by activating key signalling pathways and increasing the cellular content of RAS components. AimsTo evaluate the effect of high glucose concentrations on TF antigen (Ag) expression and procoagulant activity (PCA) in lipopolysaccharide(LPS)-primed human mononuclear cell(MNC)s and to test whether pharmacological RAS blockade modifies that pattern. MethodsLPS-activated MNCs exposed to increasing D-glucose (from 5.5 to 50mM) in absence or presence of aliskiren, a renin inhibitor, zofenopril, an ANG converting enzyme inhibitor, and olmesartan, an ANGII type I receptor blocker. PCA was assessed by one-stage clotting assay and TF antigen expression by ELISA. ResultsIncreasing ambient glucose (range 5.5–50mM) potentiated LPS-induced PCA and TF Ag expression. Aliskiren, zofenopril and olmesartan downregulated those responses but the efficacy of the former decreased by ascending drug concentration while both zofenopril and olmesartan showed an opposite behaviour. TF Ag expression modulation by RAS blockade was stronger in 50 than 5mM ambient glucose. ConclusionsHigh glucose potentiates the procoagulant action of LPS in human MNCs and RAS blockers downregulate that response possibly as a reflection of the underlying involvement of the system in that mechanism.

Role of angiotensin-converting enzyme 2 in cardiac hypertrophy induced by nitric oxide synthase inhibition

Angiotensin-converting enzyme 2 (ACE2) generates angiotensin-(1-7) [Ang-(1-7)], a peptide highlighted as exerting a pivotal role in cardiovascular remodeling. Moreover, the ACE2/Ang-(1-7)/Mas axis directly activates endothelial nitric oxide (NO) synthase and NO generation in the heart. However, the role of ACE2 in cardiovascular remodeling induced by persistent inhibition of NO under chronic activation of the renin-angiotensin system (RAS) remains poorly understood. Chimeric hypertensive mice that exhibit activation of the human RAS were produced by mating human renin (hRN) and human angiotensinogen (hANG) transgenic mice. Persistent NO inhibition with NG-nitro-L-arginine methyl ester (L-NAME) was started at 8 weeks of age for 4 weeks. After administration of L-NAME, blood pressure (BP) markedly increased in the chimeric mice (hRN/hANG-Tg), whereas wild-type mice (C57BL/6J) showed little increase in BP. Cardiovascular remodeling with enhanced oxidative stress in hRN/hANG-Tg was markedly accelerated by NO inhibition compared with that in wild-type mice. Moreover, ACE2 mRNA expression and activity in cardiac tissue were markedly reduced in L-NAME-treated hRN/hANG-Tg. Co-administration of an angiotensin II type 1 (AT1) receptor blocker (ARB), olmesartan, inhibited L-NAME-induced cardiovascular remodeling and improved the reduction in cardiac ACE2. The preventive effect of olmesartan on cardiac hypertrophy was blunted by co-administration of a selective Ang-(1-7) antagonist, [D-Ala7]-Ang-(1-7). Our findings demonstrate that cardiovascular remodeling induced by persistent NO inhibition was enhanced in hRN/hANG-Tg. An ARB, olmesartan, blunted cardiac remodeling induced by NO inhibition with RAS activation partially through the ACE2/Ang-(1-7)/Mas axis in addition to directly through its classical ACE/Ang II/AT1 receptor axis-blocking action.

Temporary Treatment With AT1 Receptor Blocker, Valsartan, From Early Stage of Hypertension Prevented Vascular Remodeling

The present study examined the inhibitory action of temporary treatment with an angiotensin type 1 (AT(1)) receptor blocker (ARB) on vascular remodeling using hypertensive mice with overexpression of the human renin (hRN) and angiotensinogen (hANG) genes. hRN/hANG transgenic mice (hRN/hANG-Tg) were treated with an ARB, valsartan, from 4 weeks of age. In some mice, valsartan treatment was stopped at 8 weeks of age (temporary treatment). Inflammatory vascular injury was induced by polyethylene-cuff placement around the femoral artery at the age of 10 weeks. Compared with wild-type (WT) mice, hRN/hANG-Tg showed higher blood pressure (BP) and enhancement of oxidative stress and medial thickening even before cuff placement. Inflammatory vascular remodeling and oxidative stress after cuff placement were further enhanced in hRN/hANG-Tg. Temporary treatment with valsartan continuously lowered BP even after cessation of administration, and inhibited these changes. In contrast, administration of hydralazine lowered BP to a similar level to that with valsartan, but did not inhibit medial thickening and inflammatory vascular remodeling. In contrast to the valsartan treatment, BP immediately increased to the untreated level after cessation of hydralazine. These results indicate that temporary ARB treatment leads to prolonged effect of BP lowering and prevents vascular remodeling in hypertensive mice induced by activation of the human renin-angiotensin system. The inhibitory action of valsartan is not due to the BP lowering but is at least in part due to a decrease in oxidative stress and inflammation.

Exaggeration of Focal Cerebral Ischemia in Transgenic Mice Carrying Human Renin and Human Angiotensinogen Genes

We examined the possibility that activation of the human brain renin-angiotensin system is involved in enhancement of ischemic brain damage using chimeric transgenic mice with human renin (hRN) and human angiotensinogen (hANG) genes. Chimeric (hRN/hANG-Tg) mice were generated by mating of hRN and hANG transgenic mice. Permanent occlusion of the middle cerebral artery (MCA) by an intraluminal filament technique induced focal ischemic brain lesions. hRN/hANG-Tg mice showed higher angiotensin II levels in the plasma and brain. The ischemic brain area at 24 hours after MCA occlusion was significantly enlarged in hRN/hANG-Tg mice with an enhanced neurological deficit compared to that in wild-type, hRN-Tg and hANG-Tg mice. The reduction of cerebral blood flow in the periphery region of the MCA territory after MCA occlusion was markedly exaggerated in hRN/hANG-Tg mice. Superoxide anion production in the brain and arteries was also increased significantly in hRN/hANG-Tg mice even before MCA occlusion and was further enhanced after MCA occlusion. Treatment with an AT(1) receptor blocker, valsartan (3.0 mg/kg per day), for 2 weeks significantly reduced the ischemic brain area and improved the neurological deficit after MCA occlusion in hRN/hANG-Tg mice, similar to those in wild-type, hRN-Tg, and hANG-Tg mice, with restoration of cerebral blood flow in the peripheral region and decreases in superoxide anion production and blood pressure. These results indicate that activation of the human renin-angiotensin system exaggerates ischemic brain damage mainly through stimulation of the AT(1) receptor and marked reduction of cerebral blood flow and enhanced oxidative stress.

Absence of Peroxisome Proliferator-Activated Receptor-α Abolishes Hypertension and Attenuates Atherosclerosis in the Tsukuba Hypertensive Mouse

Peroxisome proliferator-activated receptor-alpha is widely distributed in the vasculature where it is believed to exert pleiotropic antiatherogenic effects. Its role in the regulation of blood pressure is still unresolved; however, some evidence suggests that it may affect the renin-angiotensin system. We investigated its role in angiotensin II-induced hypertension in the Tsukuba hypertensive mouse (THM). This is a model of hypertension and atherosclerosis because of high angiotensin II and aldosterone levels as a result of the transgenic expression of the entire human renin-angiotensin system. Making the THM animals deficient in Peroxisome proliferator-activated receptor-alpha (THM/PPARKO) totally abolished hypertension and myocardial hypertrophy. This was accompanied by a reduction in plasma human active renin in THM/PPARKO mice compared with THM animals from 3525+/-128 mU/L to 1910+/-750 mU/L (P<0.05) and by a normalization of serum aldosterone (1.6+/-0.29 nmol/L versus 3.4+/-0.69 nmol/L; P=0.003). In the THM/PPARKO mice, the extent of atherosclerosis at the aortic sinus after a 12-week period on an atherogenic diet was decreased by >80%. In addition, the spontaneous formation of foam cells from peritoneal macrophages, a blood pressure-independent event, was reduced by 92% in the THM/PPARKO mice, suggesting protection from the usual oxidative stress in these animals, possibly because of lower prevailing angiotensin II levels. Finally, chronic fenofibrate treatment further elevated blood pressure in THM animals but not in THM/PPARKO animals. Taken together, these data indicate that peroxisome proliferator-activated receptor-alpha may regulate the renin-angiotensin system. They raise the possibility that its activation may aggravate hypertension and hasten atherosclerosis in the context of an activated renin-angiotensin system.

Potential of Gene Therapy Strategy for the Treatment of Hypertension

Hypertension is one of the most important risk factors for cardiovascular diseases (CVDs). CVD extends across all populations and represents the leading cause of morbidity and mortality in industrialized countries. Hypertension increases the risk of peripheral arterial disease, cardiomyopathy, stroke, and renal failure. The disease affects &50 million Americans and goes undetected in at least one third of the population. In spite of the tremendous progress made by pharmacotherapy in the management and control of hypertension, there has been a steady escalation in its prevalence during the last decade. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau and that novel and innovative approaches must be sought for the treatment, control, and possible cure of hypertension. As a result, efforts of our group and those of many others have been diverted to explore the use of gene transfer and gene therapy strategies for a long-term control of hypertension. We have argued that gene therapy offers potentially significant improvements and benefits over the use of traditional pharmacotherapy: (1) noncompliance by patients can be significantly reduced or even eliminated because of the fact that a single treatment involving gene transfer could remain effective for months or even years; (2) side effects associated with pharmacotherapy can be minimized as a result of specific targeting of a therapeutic gene in a given tissue and optimally influencing cardiovascular pathophysiology on an individual patient basis; and (3) with its potential to produce long-term beneficial outcomes in end-organ damage, the gene therapy strategy could lead to a cure of hypertension and its related pathophysiology. It is noteworthy to mention that current pharmacotherapies are often unable to reverse end-organ damage associated with hypertension. In order for the gene therapy for hypertension to be successful, one must provide conceptual support of its efficacy in …