Monoamine oxidase contributes to the valvular oxidative stress in patients with mitral valve regurgitation

Background Oxidative stress plays a central role in the pathogenesis of cardiovascular diseases but the role of enzymatic sources of reactive oxygen species (ROS) remains elusive. There is scarce information in the literature regarding valvular oxidative stress. Monoamine oxidases (MAOs), with 2 isoforms, A and B, have emerged as important sources of oxidative stress in the cardiovascular system. Purpose To assess whether MAOs-related oxidative stress occurs in the pathological valves in patients with severe mitral regurgitation (due to valve degeneration and chordae rupture) and surgical indication and its interference with the activation of the renin-angiotensin aldosterone (RAAS) system. Material and methods Samples of mitral valve (n=17) were harvested during the valvular replacement procedure and used for reactive oxygen species (ROS) assessment (immune-fluorescence, spectrophotometry) and MAO mRNA and protein expression (qPCR and immune-fluorescence) measurement. Inflammatory markers, biochemical parameters and echocardiography (GE, Vivid 9, Vivid E95) data were also collected. Results Both MAO isoforms are expressed in the diseased mitral valves, with a predominance of MAO-A isoform. Ex vivo incubation with angiotensin 2 (12 h, 100 nM) of samples obtained from patients without RAAS medication lead to MAO upregulation and high ROS production. MAO-related oxidative stress was mitigated by MAO inhibition with clorgyline (MAO-A inhibitor, 10 microM) and selegyline (MAO-B inhibitor, 10 microM) and also by the angiotensin II receptor type 1 (AT1) antagonist, irbersartan (10 microM). Conclusions Monoamine oxidase is expressed in the pathological mitral valves, regardless the etiology. Its expression and the related-oxidative stress are modulated by angiotensin 2, irbesartan. Whether the latter effect is present in valvular patients treated with RAAS inhibitors is currently under investigation. FUNDunding Acknowledgement Type of funding sources: None.

Discovery of Novel Multi-target Inhibitor of angiotensin type 1 receptor and neprilysin inhibitors from Traditional Chinese Medicine

Angiotensin II type-1 receptor–neprilysin inhibitor (ARNi) is consisted of Angiotensin II type-1 receptor (AT1) antagonist and neprilysin (NEP) inhibitor, which could simultaneously increase the vasodilators of the natriuretic peptides and antagonize vasoconstrictors of Ang II. ARNi has been proved a superior effect and lower risks of death on chronic heart failure (CHF) and hypertension. In this paper, ARNi from Traditional Chinese Medicines (TCM) was discovered based on target combination of AT1 and NEP by virtual screening, biological assay and molecular dynamics (MD) simulations. Two customized strategies of combinatorial virtual screening were implemented to discover AT1 antagonist and NEP inhibitor based on pharmacophore modeling and docking computation respectively. Gyrophoric acid (PubChem CID: 135728) from Parmelia saxatilis was selected as AT1 antagonist and assayed with IC50 of 29.76 μM by calcium influx assay. And 3,5,3′-triiodothyronine (PubChem CID: 861) from Bos taurus domesticus was screened as NEP inhibitor and has a dose dependent inhibitory activity by biochemistry fluorescence assay. Combined with MD simulations, these compounds can generate interaction with the target, key interactive residues of ARG167, TRP84, and VAL108 in AT1, and HIS711 in NEP were also identified respectively. This study designs the combinatorial strategy to discover novel frames of ARNi from TCM, and gyrophoric acid and 3,5,3′-triiodothyronine could provide the clues and revelations of drug design and therapeutic method of CHF and hypertension for TCM clinical applications.

5894The biased angiotensin receptor 1-ligand TRV023 attenuates atherosclerosis through selective inhibition of Gq-mediated signaling

Introduction Antagonists of the angiotensin II receptor type 1 (AT1) belong to the most successful treatments of cardiovascular disease. Besides G-protein-independent β-arrestin-mediated signaling, AT1 preferencially signals via G-proteins of the families Gq and G13. Conventional AT1-antagonists equally inhibit all of these pathways, even though the beneficial effects are generally attributed to the inhibition of Gq only. While data on β-arrestin signaling in the vascular disease context are conflicting, we found an excessive pathological remodeling and dramatic exacerbation of atherosclerosis upon interference with G13-mediated signaling. Against this background, selective inhibition of the Gq-pathway seems desirable. Thus, based on the novel concept of ligand-biased signaling, we aimed to identify biased AT1-antagonists that selectively inhibit the Gq-pathway, while allowing for continued signaling through G13. Methods and results We systematically profiled known AT1-ligands and identified the angiotensin analogue Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH (TRV023) as a Gq-selective AT1-antagonist: TRV023 has recently been described as a β-arrestin-biased ligand; however its signaling properties regarding distinct G-protein isoforms have never been defined. Indeed, using bioluminescence reporter assays, we found that TRV023 inhibits Gq-signaling at least as potently as losartan and telmisartan, but unlike the conventional antagonists, it does not impair β-arrestin-signaling (Fig. 1A). Intriguingly, while the conventional antagonists effectively inhibited induction of a G13 luciferase reporter, TRV023 did not significantly interfere with G13-mediated signaling, indicating Gq-selective antagonistic effects. According to our hypothesis, these G-protein-biased signaling properties could render TRV023 superior to conventional antagonists in the treatment of vascular disease. In order to test this hypothesis, we exposed ApoE-deficient mice to one of two different atherosclerosis models: A novel model for accelerated atherosclosis based on partial carotid ligation and western diet, or a conventional atherosclerosis model based on 12 weeks of western diet. Using osmotic pumps, mice were continuously treated with vehicle control, TRV023 or losartan in doses that proved equipotent with respect to blood pressure lowering effects. In line with our hypothesis, TRV023 but not losartan significantly attenuated plaque development in both atherosclerosis models (Fig. 1B). Figure 1 Conclusion Our studies have identified TRV023 as a Gq-biased AT1-antagonist and indicate that selective inhibition of AT1-dependent Gq-mediated signaling by biased ligands may be a promising approach for the treatment of atherosclerosis. Hence, TRV023 qualifies as a drug candidate, particularly since it has already proven to be safe and well-tolerable in clinical phase I and II trials in the context of acute heart failure. Acknowledgement/Funding Else Kröner-Fresenius-Foundation

Mixed Vehicle Emissions Induces Angiotensin II and Cerebral Microvascular Angiotensin Receptor Expression in C57Bl/6 Mice and Promotes Alterations in Integrity in a Blood-Brain Barrier Coculture Model

Exposure to traffic-generated pollution is associated with alterations in blood-brain barrier (BBB) integrity and exacerbation of cerebrovascular disorders. Angiotensin (Ang) II signaling through the Ang II type 1 (AT1) receptor is known to promote BBB disruption. We have previously reported that exposure to a mixture of gasoline and diesel vehicle engine emissions (MVE) mediates alterations in cerebral microvasculature of C57Bl/6 mice, which is exacerbated through consumption of a high-fat (HF) diet. Thus, we investigated the hypothesis that inhalation exposure to MVE results in altered central nervous system microvascular integrity mediated by Ang II-AT1 signaling. Three-month-old male C57Bl/6 mice were placed on an HF or low-fat diet and exposed via inhalation to either filtered air (FA) or MVE (100 μg/m3 PM) 6 h/d for 30 days. Exposure to HF+MVE resulted in a significant increase in plasma Ang II and expression of AT1 in the cerebral microvasculature. Results from a BBB coculture study showed that transendothelial electrical resistance was decreased, associated with reduced expression of claudin-5 and occludin when treated with plasma from MVE+HF animals. These effects were attenuated through pretreatment with the AT1 antagonist, Losartan. Our BBB coculture showed increased levels of astrocyte AT1 and decreased expression of aryl hydrocarbon receptor and glutathione peroxidase-1, associated with increased interleukin-6 and transforming growth factor-β in the astrocyte media, when treated with plasma from MVE-exposed groups. Our results indicate that inhalation exposure to traffic-generated pollutants results in altered BBB integrity, mediated through Ang II-AT1 signaling and inflammation, which is exacerbated by an HF diet.

Neutral endopeptidase inhibitors blunt kidney fibrosis by reducing myofibroblast formation

Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor β (TGF-β) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a ‘deep serial analysis of gene expression (Deep SAGE)’ transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.

Candesartan ameliorates the functional renal disorder in diabetic nephropathy induced by streptozotocin

Background Diabetic nephropaty (DN) occurs in approximately 40% of patients with diabetes mellitus, and is the most common cause of end-stage renal disease. The mechanisms of DN are not very clear more recently, but the renin-angiotensin system (RAS) plays an important role. It has been known that rennin-angiotensin system blockers have a renal protective effect. The present study was undertaken to evaluate the effects of the candesartan on functional renal tests in streptozotocin (STZ) induced DN in rats. Methods DM was induced by a single intraperitoneal injection (ip) injection of STZ (60 mg/kg). In order to develop DN the animals were left in diabetic condition during 4 weeks. The DM rats were randomly assigned to the two experimental groups (STZ and STZ+CAN). To estimate the symptoms and signs of DN, the STZ group of rats was left without treatment. For assessment of the effects of the AT1 antagonist, candesartan (CAN) (5 mg/kg/day) was administered from week 4 to week 12. Renal function was assessed by determination of serum creatinine, blood urea nitrogen (BUN) test, 24-hour urine volume and urine albumin. Results The administration of STZ have caused diabetes mellitus (DM) with symptoms and signs of DN including poor general condition, body weight loss, as well as abnormalities of serum and urinary renal function tests. In STZ group of rats, these symptoms have been more distinctly 8 and 12 weeks after administration of STZ. Conclusion The candesartan treatment, although not completely but to a great extent ameliorates the functional renal disorder induced by STZ and may be used as a first line drug in preventing DN.

Abstract 48: Age-associated Imbalance of Vasorin/TGF-β1 Signaling in VSMC Facilitates Collagen Production

Collagen deposition, a hallmark of arterial aging that resembles post-injury arterial restenosis, is perpetrated by angiotensin II (Ang II) signaling in arterial wall. Collagen aggregation at sites of arterial injury is regulated by the coordinated signaling of pro-fibrotic TGF-β1 and anti-fibrotic vasorin within VSMCs. The Ang II/TGF-β1/vasorin signaling relationship within VSMCs with aging, however, remains unknown. In vivo studies in old vs. young FXBN rats show that aortic transcription and translation of vasorin markedly decrease with aging. In vitro studies in VSMCs isolated from old vs. young aortae. Ang II-associated reduction of vasorin protein abundance in young VSMCs and age-associated changes in vasorin protein levels are reversed by the AT1 antagonist, Losartan (Los) (Figure). Dual immunolabeling and co-immunoprecipitation demonstrate that the co-incidence and physical interaction of vasorin and TGF-β1 within VSMCs are significantly decreased with aging. Importantly, exposure of young VSMCs to Ang II that increases p-SMAD2/3 and collagen type I production, mimicking old cells, and this effect is abolished or substantially mitigated by Los treatment, overexpression of ectopic vasorin, or exogenous recombinant human-vasorin protein. In contrast, exposure of old VSMCs to Los decreases p-SMAD2/3 and collagen type I production.Thus, an imbalance of the Ang II/TGF-β1/vasorin signaling cascade, a feature of the aged arterial wall, enhances the collagen production by VSMCs. Maintaining this signaling balance is a novel measure to retard adverse extracellular matrix remodeling, a determinant of arterial stiffening with aging. (MW and GP co-first authors)