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Cannabinoids (CB) are implicated in cardiovascular diseases via the two main receptor subtypes CB1R and CB2R. This study investigated whether cannabinoids regulate the activity of matrix metalloproteases (MMP-2, MMP-9) in vascular smooth muscle cells (VSMCs) and in cells of cardiac origin (H9c2 cell line). The influence of CB1- and CB2 receptor stimulation or inhibition on cell proliferation, apoptosis and glucose uptake was also evaluated. We used four compounds that activate or block CB receptors: arachidonyl-2-chloroethylamide (ACEA)—CB1R agonist, rimonabant—CB1R antagonist, John W. Huffman (JWH133)—CB2R agonist and CB2R antagonist—6-Iodopravadoline (AM630). Treatment of cells with the CB2R agonist JWH133 decreased cytokine activated secretion of proMMP-2, MMP-2 and MMP-9, reduced Fas ligand and caspase-3-mediated apoptosis, normalized the expression of TGF-beta1 and prevented cytokine-induced increase in glucose uptake into the cell. CB1R inhibition with rimonabant showed similar protective properties as the CB2R agonist JWH133, but to a lesser extent. In conclusion, CB1R and CB2R exert opposite effects on cell glucose uptake, proteolysis and apoptosis in both VSMCs and H9c2 cells. The CB2R agonist JWH133 demonstrated the highest protective properties. These findings may pave the way to a new treatment of cardiovascular diseases, especially those associated with extracellular matrix degradation.

The frst May Measurement Month (MMM) campaign, a global blood pressure (BP) screening programme, began in 2017 as an initiative of the International Society of Hypertension.1 Two subsequent annual campaigns have also been completed in consecutive years2,3 and having had to defer activities due to the COVID-19 pandemic in 2020 the fourth campaign was run in 2021, the results of which are currently in press. Since its initiation in 2017, volunteers from more than 100 countries have participated. The aims of MMM have remained consistent from the start-to raise awareness of the importance of the measurement of BP at the individual and population level and to provide a temporary pragmatic solution to the shortfall in BP screening programmes in countries around the world.

Objective The receptor MAS, encoded by Mas1, is expressed in microglia and its activation has been linked to anti-inflammatory actions. However, microglia are involved in several different processes in the central nervous system, including the promotion of angiogenesis. We therefore hypothesized that the receptor MAS also plays a role in angiogenesis via microglia. Approach and results To assess the role of MAS on vascular network development, flat-mounted retinas from 3-day-old wild-type (WT) and -Mas1(-/-) mice were subjected to Isolectin B4 staining. The progression of the vascular front was reduced (- 24%, p < 0.0001) and vascular density decreased (- 38%, p < 0.001) in -Mas1(-/-) compared to WT mice with no change in the junction density. The number of filopodia and filopodia bursts were decreased in -Mas1(-/-) mice at the vascular front (- 21%, p < 0.05; - 29%, p < 0.0001, respectively). This was associated with a decreased number of vascular loops and decreased microglial density at the vascular front in -Mas1(-/-) mice (-32%, p < 0.001; - 26%, p < 0.05, respectively). As the front of the developing vasculature is characterized by reduced oxygen levels, we determined the expression of Mas1 following hypoxia in primary microglia from 3-day-old WT mice. Hypoxia induced a 14-fold increase of Mas1 mRNA expression (p < 0.01). Moreover, stimulation of primary microglia with a MAS agonist induced expression of Notch1 (+ 57%, p < 0.05), Dll4 (+ 220%, p < 0.001) and Jag1 (+ 137%, p < 0.001), genes previously described to mediate microglia/endothelial cell interaction during angiogenesis. Conclusions Our study demonstrates that the activation of MAS is important for microglia recruitment and vascular growth in the developing retina. Foulquier, S (reprint author), Maastricht Univ, Dept Pharmacol Toxicol, POB 616, NL-6200 MD Maastricht, Netherlands, CARIM, Cardiovasc Res Inst Maastricht, Maastricht, Netherlands, Maastricht Univ, Sch Mental Hlth & Neurosci, MH&NS, Maastricht, Netherlands. s.foulquier@maastrichtuniversity.nl

Frontiers in cell and developmental biology 9, 664258 (2021). doi:10.3389/fcell.2021.664258, Published by Frontiers Media, Lausanne

Compound 21 (C21), a selective agonist of angiotensin II type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of the present study was to assess the effect of C21 on thoracic aorta endothelial function in a model of diet-induced obesity (DIO) and to elucidate the potential cross-talk among AT2R, Mas receptor (MasR) and/or bradykinin type 2 receptor (B2R) in this response. Five-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1 mg/kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (HECs; EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels. Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favored the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine (ACh) due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways. In conclusion, C21 favors the interaction among AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.

Aims NOS1AP single-nucleotide polymorphisms (SNPs) correlate with QT prolongation and cardiac sudden death in patients affected by long QT syndrome type 1 (LQT1). NOS1AP targets NOS1 to intracellular effectors. We hypothesize that NOS1AP SNPs cause NOS1 dysfunction and this may converge with prolonged action-potential duration (APD) to facilitate arrhythmias. Here we test (i) the effects of NOS1 inhibition and their interaction with prolonged APD in a guinea pig cardiomyocyte (GP-CMs) LQT1 model; (ii) whether pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from LQT1 patients differing for NOS1AP variants and mutation penetrance display a phenotype compatible with NOS1 deficiency. Methods and results In GP-CMs, NOS1 was inhibited by S-Methyl-L-thiocitrulline acetate (SMTC) or Vinyl-L-NIO hydrochloride (L-VNIO); LQT1 was mimicked by IKs blockade (JNJ303) and β-adrenergic stimulation (isoproterenol). hiPSC-CMs were obtained from symptomatic (S) and asymptomatic (AS) KCNQ1-A341V carriers, harbouring the minor and major alleles of NOS1AP SNPs (rs16847548 and rs4657139), respectively. In GP-CMs, NOS1 inhibition prolonged APD, enhanced ICaL and INaL, slowed Ca2+ decay, and induced delayed afterdepolarizations. Under action-potential clamp, switching to shorter APD suppressed ‘transient inward current’ events induced by NOS1 inhibition and reduced cytosolic Ca2+. In S (vs. AS) hiPSC-CMs, APD was longer and ICaL larger; NOS1AP and NOS1 expression and co-localization were decreased. Conclusion The minor NOS1AP alleles are associated with NOS1 loss of function. The latter likely contributes to APD prolongation in LQT1 and converges with it to perturb Ca2+ handling. This establishes a mechanistic link between NOS1AP SNPs and aggravation of the arrhythmia phenotype in prolonged repolarization syndromes.

The effects of the selective AT 2 R (angiotensin AT2 receptor) agonist, Compound 21 (C21), on abdominal aortic aneurysm formation were investigated in normotensive Wistar rats. Abdominal aortic aneurysm was induced by perfusion of isolated aortic segments with elastase. Treatment with C21 (0.03 mg/kg daily) was started after operation and continued for 14 days. Sham-operated animals and vehicle-treated animals after aneurysm induction served as controls. Aortic diameter, aortic wall distensibility, and pulse propagation velocity were measured via ultrasound. Hemodynamic parameters, aortic tissue protein expression, and serum cytokines were analyzed. On day 14 post aneurysm induction, aortic diameter of vehicle-treated animals was increased 1.6-fold compared with sham-operated rats (2.65±0.05 versus 1.70±0.06 mm; P P P P 2 R stimulation with C21 prevented extracellular matrix degradation, maintained vascular integrity of the aorta and prevented abdominal aortic aneurysm progression.

The angiotensin II (Ang II) type 2 receptor (AT2R) and the angiotensin-(1–7) (Ang-(1–7)) receptor (MasR) play a cardiovascular protective role by counter-regulating Ang II type 1 receptor (AT1R)-mediated effects, but whether this involves blunting of adrenocortical hormone secretion is unknown. We investigated the presence of AT1R, AT2R, and MasR in aldosterone-producing adenoma (APA), a condition featuring hyperaldosteronism, and in APA-adjacent tissue. The effect of Compound 21 (C21), an AT2R agonist, on CYP11B1 (cortisol synthase) and CYP11B2 (aldosterone synthase) gene expression in NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, was also assessed using the AT1R antagonist irbesartan to ascertain the specificity of C21 effect. We found that the AT1R, AT2R, and MasR were expressed in APA and APA-adjacent tissue, albeit heterogeneously. The gene expression of AT1R and AT2R was lower, and that of the MasR higher in APAs than in APA-adjacent tissue. In steroid-producing NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, C21 was ineffective at nanomolar concentrations, but increased CYP11B1 and CYP11B2 gene expression at micromolar concentrations through AT1R, as this effect was blunted by irbesartan. The scant expression of the AT2R, along with the lack of any effect of C21 at low concentrations on CYP11B2, do not support the contention that the protective arm of renin–angiotensin system (RAS) blunts aldosterone synthase in the normal adrenal cortex and primary aldosteronism.

Die multifaktorielle Genese kardiovaskularer Erkrankungen hat in der Primar- und Sekundarprophylaxe zur Polypharmazie mit evidenzbasierten Therapeutika wie Statinen, Antihypertensiva und Thrombozytenhemmern gefuhrt. Die Anzahl verschriebener Phamaka korreliert umgekehrt mit der Adharenz, was die Effektivitat der Therapie beeintrachtigen kann. Fixe Kombinationspraparate („Polypill“) konnten die Adharenz der Patienten steigern und damit kardiovaskularen Ereignissen vorbeugen. Literaturrecherche in Medline (via PubMed) und The Cochrane Library sowie der Studiendatenbank ClinicalTrials.gov. In den bisher durchgefuhrten Studien zur kardiovaskularen Primarpravention zeigt die Polypill eine uberlegene Kontrolle der Risikofaktoren (Hypertonie, „Low-density-lipoprotein“-Cholesterin [LDL-C]) gegenuber Placebo und gegenuber einer Standardtherapie aus Einzelpraparaten mindestens eine Nichtunterlegenheit. In der Sekundarpravention zeigen Kombinationspraparate vorwiegend Vorteile bei nichtadharenten Patienten bezuglich Blutdruckkontrolle und Reduktion der LDL-C-Konzentration. Der Nachweis, dass die Polypill die kardiovaskulare Morbiditat und Mortalitat gegenuber einer Standardtherapie absenkt, steht aus. Kombinationspraparate sind nach Risiko-Nutzen-Einschatzung als Alternative zur Polypharmazie, insbesondere bei nichtadharenten Patienten in Erwagung zu ziehen. Inwieweit durch die erwiesene Senkung der Risikofaktoren kardiovaskulare Ereignisse verhindert werden konnen, ist Gegenstand laufender Untersuchungen. Limitiert sind die gegenwartigen Polypills durch eine zu geringe Auswahl an Dosierungen der Einzelwirkstoffe, um eine Uber- und Untertherapie in der individuellen Behandlung zu vermeiden.

This study investigated the effect of post-stroke, direct AT2-receptor (AT2R) stimulation with the non-peptide AT2R-agonist compound 21 (C21) on infarct size, survival and neurological outcome after middle cerebral artery occlusion (MCAO) in mice and looked for potential underlying mechanisms. C57/BL6J or AT2R-knockout mice (AT2-KO) underwent MCAO for 30 min followed by reperfusion. Starting 45 min after MCAO, mice were treated once daily for 4 days with either vehicle or C21 (0.03 mg/kg ip). Neurological deficits were scored daily. Infarct volumes were measured 96 h post-stroke by MRI. C21 significantly improved survival after MCAO when compared to vehicle-treated mice. C21 treatment had no impact on infarct size, but significantly attenuated neurological deficits. Expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB) (receptor for BDNF) and growth-associated protein 43 (GAP-43) were significantly increased in the peri-infarct cortex of C21-treated mice when compared to vehicle-treated mice. Furthermore, the number of apoptotic neurons was significantly decreased in the peri-infarct cortex in mice treated with C21 compared to controls. There were no effects of C21 on neurological outcome, infarct size and expression of BDNF or GAP-43 in AT2-KO mice. From these data, it can be concluded that AT2R stimulation attenuates early mortality and neurological deficits after experimental stroke through neuroprotective mechanisms in an AT2R-specific way. Key message • AT2R stimulation after MCAO in mice reduces mortality and neurological deficits.• AT2R stimulation increases BDNF synthesis and protects neurons from apoptosis.• The AT2R-agonist C21 acts protectively when applied post-stroke and peripherally.

Cardiovascular and metabolic disorders, such as hypertension, insulin resistance, dyslipidemia or obesity are linked with chronic low-grade inflammation and dysregulation of the renin-angiotensin system (RAS). Consequently, RAS inhibition by ACE inhibitors or angiotensin AT1 receptor (AT1R) blockers is the evidence-based standard for cardiovascular risk reduction in high-risk patients, including diabetics with albuminuria. In addition, RAS inhibition reduces the new onset of diabetes mellitus. Yet, the high and increasing prevalence of metabolic disorders, and the high residual risk even in properly treated patients, calls for additional means of pharmacological intervention. In the past decade, the stimulation of the angiotensin AT2 receptor (AT2R) has been shown to reduce inflammation, improve cardiac and vascular remodeling, enhance insulin sensitivity and increase adiponectin production. Therefore, a concept of dual AT1R/AT2R modulation emerges as a putative means for risk reduction in cardio-metabolic diseases. The approach employing simultaneous RAS blockade (AT1R) and RAS stimulation (AT2R) is distinct from previous attempts of double intervention in the RAS by dual blockade. Dual blockade abolishes the AT1R-linked RAS almost completely with subsequent risk of hypotension and hypotension-related events, i.e. syncope or renal dysfunction. Such complications might be especially prominent in patients with renal impairment or patients with isolated systolic hypertension and normal-to-low diastolic blood pressure values. In contrast to dual RAS blockade, the add-on of AT2R stimulation does not exert significant blood pressure effects, but it may complement and enhance the anti-inflammatory and antifibrotic/de-stiffening effects of the AT1R blockade and improve the metabolic profile. Further studies will have to investigate these putative effects in particular for settings in which blood pressure reduction is not primarily desired.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic lung disease characterized by scar formation and respiratory insufficiency, which progressively leads to death. Pulmonary hypertension (PH) is a common complication of IPF that negatively impacts clinical outcomes, and has been classified as Group III PH. Despite scientific advances, the dismal prognosis of IPF and associated PH remains unchanged, necessitating the search for novel therapeutic strategies. Accumulating evidence suggests that stimulation of the angiotensin II type 2 (AT2) receptor confers protection against a host of diseases. In this study, we investigated the therapeutic potential of Compound 21 (C21), a selective AT2 receptor agonist in the bleomycin model of lung injury. A single intra-tracheal administration of bleomycin (2.5 mg/kg) to 8-week old male Sprague Dawley rats resulted in lung fibrosis and PH. Two experimental protocols were followed: C21 was administered (0.03 mg/kg/day, ip) either immediately (prevention protocol, BCP) or after 3 days (treatment protocol, BCT) of bleomycin-instillation. Echocardiography, hemodynamic, and Fulton's index assessments were performed after 2 weeks of bleomycin-instillation. Lung tissue was processed for gene expression, hydroxyproline content (a marker of collagen deposition), and histological analysis. C21 treatment prevented as well as attenuated the progression of lung fibrosis, and accompanying PH. The beneficial effects of C21 were associated with decreased infiltration of macrophages in the lungs, reduced lung inflammation and diminished pulmonary collagen accumulation. Further, C21 treatment also improved pulmonary pressure, reduced muscularization of the pulmonary vessels and normalized cardiac function in both the experimental protocols. However, there were no major differences in any of the outcomes measured from the two experimental protocols. Collectively, our findings indicate that stimulation of the AT2 receptor by C21 attenuates bleomycin-induced lung injury and associated cardiopulmonary pathology, which needs to be further explored as a promising approach for the clinical treatment of IPF and Group III PH.

Recently, AT2 receptors have been discovered on the surface of human immunocompetent cells such as monocytes. Data on regulative properties of this receptor on the cellular immune response are poor. We hypothesized that direct stimulation of the AT2 receptor mediates anti-inflammatory responses in these cells. Human monocytic THP-1 and U937 cells were stimulated with lipopolysaccharide (LPS) and the selective AT2 receptor agonist Compound 21 (C21). Expression of pro- and anti-inflammatory cytokines IL-6, IL-10, tumor necrosis factor-alpha (TNF alpha), and IL-1 beta were analyzed on both the transcriptional and the translational level over course of time. Treatment with C21 attenuated the expression of TNF alpha, IL-6, and IL-10 after LPS challenge in both cell lines in a time- and dose-dependent manner. We conclude that selective AT2 receptor stimulation acts anti-inflammatory in human monocytes. Modulation of cytokine response by AT2 receptor activation might be a beneficial and novel treatment concept in inflammatory conditions.

In most situations, the angiotensin AT2-receptor (AT2R) mediates physiological actions opposing those mediated by the AT1-receptor (AT1R), including a vasorelaxant effect. Nevertheless, experimental evidence vastly supports that systemic application of AT2R-agonists is blood pressure neutral. However, stimulation of AT2R locally within the brain or the kidney apparently elicits a systemic blood pressure lowering effect. A systemic effect of AT2R stimulation on blood pressure can also be achieved, when the prevailing effect of continuous background AT1R-stimulation is attenuated by low-dose AT1R blockade. Despite a lack of effect on blood pressure, AT2R stimulation still protects from hypertensive end-organ damage. Current data and evidence therefore suggest that AT2R agonists will not be suitable as future anti-hypertensive drugs, but that they may well be useful for end-organ protection in combination with established anti-hypertensives.

Abstract— Elevated systolic blood pressure (SBP) correlates to cognitive decline and incident dementia. The effects of heart rate (HR), visit to visit HR variation, and visit to visit SBP variation are less well established. Patients without preexisting cognitive dysfunction (N=24 593) were evaluated according to mean SBP, SBP visit to visit variation (coefficient of variation [standard deviation/mean×100%], CV), mean HR, and visit to visit HR variation (HR-CV) in the Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial and the Telmisartan Randomized Assessment Study in ACE Intolerant Subjects with Cardiovascular Disease. Cognitive function was assessed with mini mental state examination. Cognitive dysfunction (fall in mini mental state examination ≤24 points), important cognitive decline (drop of ≥5 points), and cognitive deterioration (drop of >1 point per year or decline to < 24 points) were assessed. SBP and HR were measured over 10.7±2.2 (mean±SD) visits. Mean SBP, mean HR, and SBP-CV were associated with cognitive decline, dysfunction, and deterioration (all P P =0.0030) and mean HR ( P =0.0008) remained predictors for cognitive dysfunction (odds ratios [95% confidence intervals], 1.32 [1.10–1.58] for 5th versus 1st quintile of SBP-CV and 1.40 [1.18–1.66] for 5th versus 1st quintile of mean HR). Similar effects were observed for cognitive decline and deterioration. SBP-CV and mean HR showed additive effects. In conclusion, SBP-CV and mean HR are independent predictors of cognitive decline and cognitive dysfunction in patients at high CV risk. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT 00153101.

Summary Background The generation of thrombin in time is the combined effect of the processes of prothrombin conversion and thrombin inactivation. Measurement of prothrombin consumption used to provide valuable information on hemostatic disorders, but is no longer used, due to its elaborate nature. Objectives Because thrombin generation (TG) curves are easily obtained with modern techniques, we developed a method to extract the prothrombin conversion curve from the TG curve, using a computational model for thrombin inactivation. Methods Thrombin inactivation was modelled computationally by a reaction scheme with antithrombin, α2Macroglobulin and fibrinogen, taking into account the presence of the thrombin substrate ZGGR-AMC used to obtain the experimental data. The model was validated by comparison with data obtained from plasma as well as from a reaction mixture containing the same reactants as plasma. Results The computational model fitted experimental data within the limits of experimental error. Thrombin inactivation curves were predicted within 2 SD in 96% of healthy subjects. Prothrombin conversion was calculated in 24 healthy subjects and validated by comparison with the experimental consumption of prothrombin during TG. The endogenous thrombin potential (ETP) mainly depends on the total amount of prothrombin converted and the thrombin decay capacity, and the peak height is determined by the maximum prothrombin conversion rate and the thrombin decay capacity. Conclusions Thrombin inactivation can be accurately predicted by the proposed computational model and prothrombin conversion can be extracted from a TG curve using this computational prediction. This additional computational analysis of TG facilitates the analysis of the process of disturbed TG.

We describe a family with an autosomal dominant disorder characterized by severe trauma- and surgery-related bleeding. The proband, who experienced life-threatening bleeding during a routine operation, had normal clotting times, but markedly reduced prothrombin consumption. Plasma levels of all coagulation factors and of the main coagulation inhibitors were normal. Thrombin generation at low triggers was severely impaired and mixing experiments suggested the presence of a coagulation inhibitor. Using whole exome sequencing, the underlying genetic defect was identified as the THBD c.1611C>A mutation (p.Cys537Stop), which predicts a truncated form of thrombomodulin that is shed from the vascular endothelium. The patient had decreased expression of endothelium-bound thrombomodulin, but extremely elevated levels of soluble thrombomodulin in plasma, impairing the propagation phase of coagulation via rapid activation of protein C and consequent inactivation of factors Va and VIIIa. The same thrombomodulin mutation has been recently described in an unrelated British family with strikingly similar features.

The presence of angiotensin type 2 (AT2) receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT2 receptors including their presence in mitochondria and their role in the induction of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor agonist, Compound 21 (C21), penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped cell death, displayed activation of the mitochondrial apoptotic pathway, i.e. down-regulation of the Bcl-2 protein, induction of the Bax protein and activation of caspase-3. All quiescent SK-UT-1 cells died within 5 days after treatment with a single dose of C21. C21 was devoid of cytotoxic effects in proliferating SK-UT-1 cells and in quiescent HutSMC. Our results point to a new, unique approach enabling the elimination non-cycling uterine leiomyosarcoma cells providing that they over-express the AT2 receptor. Abbreviations: Ang, II, angiotensin II; AT1, angiotensin type 1; AT2, angiotensin type 2; ATIP, AT2 receptor interacting proteins; C21, Compound 21; DMEM, Dulbecco’s modified Eagle’s medium; 18F-FDG, [18F]2-fluoro-2-deoxy-D-glucose; HutSMC, human uterine smooth muscle cells; LC–MS, liquid chromatography–mass spectrometry; LDH, lactate dehydrogenase; RAS, renin–angiotensin system; RT, reverse transcription; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling

et al., [Aims]: Polyunsaturated fatty n-3 acids (PUFAs) have been reported to exhibit antiarrhythmic properties. However, the mechanisms of action remain unclear. We studied the electrophysiological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on IKs, and on the expression and location of Kv7.1 and KCNE1. [Methods and results]: Experiments were performed using patch-clamp, western blot, and sucrose gradient techniques in COS7 cells transfected with Kv7.1/KCNE1 channels. Acute perfusion with both PUFAs increased Kv7.1/KCNE1 current, this effect being greater for DHA than for EPA. Similar results were found in guinea pig cardiomyocytes. Acute perfusion of either PUFA slowed the activation kinetics and EPA shifted the activation curve to the left. Conversely, chronic EPA did not modify Kv7.1/KCNE1 current magnitude and shifted the activation curve to the right. Chronic PUFAs decreased the expression of Kv7.1, but not of KCNE1, and induced spatial redistribution of Kv7.1 over the cell membrane. Cholesterol depletion with methyl-β-cyclodextrin increased Kv7.1/KCNE1 current magnitude. Under these conditions, acute EPA produced similar effects than those induced in non-cholesterol-depleted cells. A ventricular action potential computational model suggested antiarrhythmic efficacy of acute PUFA application under IKr block. [Conclusions]: We provide evidence that acute application of PUFAs increases Kv7.1/KCNE1 through a probably direct effect, and shows antiarrhythmic efficacy under IKr block. Conversely, chronic EPA application modifies the channel activity through a change in the Kv7.1/KCNE1 voltage-dependence, correlated with a redistribution of Kv7.1 over the cell membrane. This loss of function may be pro-arrhythmic. This shed light on the controversial effects of PUFAs regarding arrhythmias., This work was supported by grants from CICYT (SAF2010-14916 and SAF2013-45800-R to C.V.; BFU2011-23268 and CSD2008-00005 to A.F.) and FIS (PI11/02459, RD06/0014/0006, and RD12/0042/0019 to C.V.). C.M. and M.G. hold FPI grants. N.C. and A.d.l.C. hold Juan de la Cierva and RIC contracts, respectively.

The aim of this study was to evaluate the effect of compound 21 (C21), a selective AT2 receptor agonist, on diabetic nephropathy and the potential additive effect of C21, when associated with losartan treatment, on the development of albuminuria and renal fibrosis in Zucker diabetic fatty (ZDF) rats. The experiments lasted 15 wk (from 5 to 20 wk of age) and were performed in 40 ZDF rats and 12 control lean rats. ZDF rats were divided into 4 groups: 1) 9 rats were treated with losartan; 2) 10 rats were treated with C21; 3) 9 rats were treated with losartan plus C21; and 4) 12 rats were maintained without any treatment. ZDF rats showed an increase in blood glucose level, albuminuria, renal fibrosis, macrophage infiltration, and TNF-α expression and a reduction of glomerular nephrin expression compared with control lean rats. C21 treatment reduced renal glomerular, tubulointerstitial, and perivascular fibrosis, and macrophage infiltration and TNF-α expression in ZDF rats. C21 treatment caused a decrease in albuminuria in ZDF rats up to 11 wk of age. Losartan decreased macrophage infiltration, TNF-α expression, and renal glomerular and perivascular fibrosis, restored glomerular nephrin expression, but did not affect tubulointerstitial fibrosis. Losartan treatment caused a decrease in albuminuria in ZDF rats up to 15 wk of age. At the end of the protocol, only the combination of C21 plus losartan significantly reduced albuminuria in ZDF rats. These data demonstrate that C21 has beneficial effects on diabetic nephropathy, suggesting the combination of C21 and losartan as a novel pharmacological tool to slow the progression of nephropathy in type II diabetes.

Background: Cardiovascular diseases remain the predominant cause of death worldwide, with the prevalence of heart failure continuing to increase. Despite increased knowledge of the metabolic alterations that occur in heart failure, novel therapies to treat the observed metabolic disturbances are still lacking. Methods: Mice were subjected to pressure overload by means of angiotensin-II infusion or transversal aortic constriction. MicroRNA-146a was either genetically or pharmacologically knocked out or genetically overexpressed in cardiomyocytes. Furthermore, overexpression of dihydrolipoyl succinyltransferase (DLST) in the murine heart was performed by means of an adeno-associated virus. Results: MicroRNA-146a was upregulated in whole heart tissue in multiple murine pressure overload models. Also, microRNA-146a levels were moderately increased in left ventricular biopsies of patients with aortic stenosis. Overexpression of microRNA-146a in cardiomyocytes provoked cardiac hypertrophy and left ventricular dysfunction in vivo, whereas genetic knockdown or pharmacological blockade of microRNA-146a blunted the hypertrophic response and attenuated cardiac dysfunction in vivo. Mechanistically, microRNA-146a reduced its target DLST—the E2 subcomponent of the α-ketoglutarate dehydrogenase complex, a rate-controlling tricarboxylic acid cycle enzyme. DLST protein levels significantly decreased on pressure overload in wild-type mice, paralleling a decreased oxidative metabolism, whereas DLST protein levels and hence oxidative metabolism were partially maintained in microRNA-146a knockout mice. Moreover, overexpression of DLST in wild-type mice protected against cardiac hypertrophy and dysfunction in vivo. Conclusions: Altogether we show that the microRNA-146a and its target DLST are important metabolic players in left ventricular dysfunction.

Aims Erectile dysfunction (ED) is associated with cardiovascular risk factors as elevated systolic blood pressure (SBP), resting high heart rate (HR), and endothelial dysfunction and predicts cardiovascular events. However, the interaction between high HR and SBP and the development of ED remains unclear. Methods and Results We evaluated 1015 male patients enrolled in the ED substudy of ONTARGET and TRANSCEND, examining the influence of mean HR and mean SBP obtained over all study visits (mean 10.91.4 study visits) and their interaction with ED. In patients without pre-existing ED, new onset ED was detected in 29% of patients below, and 41% of patients above, the median of mean HR (OR 1.72, 95% CI 1.8-2.5, p=0.0047). In patients with pre-existing ED, high HR had no add-on effect. With or without pre-existing ED, high SBP had no influence after adjustment for covariates (OR 1.03, 95% CI 0.66-1.59, p=0.91). In a continuous model, it was shown that effects of high HR were prominent at low Kolner (Cologne) Evaluation of Erectile Function (KEED) score baseline values and in the presence of SBP above the median. Conclusions In patients at risk for cardiovascular events, high HR is associated with ED, whereas the effect of high SBP was not significant. High resting HR might represent a cardiovascular risk indicator. Whether HR represents a potential treatment target to improve ED in high-risk individuals must be scrutinized in prospective trials.

Left ventricular (LV) remodeling is the main reason for the development of progressive cardiac dysfunction after myocardial infarction (MI). This study investigated whether stimulation of the angiotensin type 2 receptor is able to ameliorate post-MI cardiac remodeling and what the underlying mechanisms may be. MI was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with the angiotensin type 2 receptor agonist compound 21 ( 0.03 mg/kg) was started 6 hours post-MI and continued for 6 weeks. Hemodynamic parameters were measured by echocardiography and intracardiac catheter. Effects on proteolysis were studied in heart tissue and primary cardiac fibroblasts. Compound 21 significantly improved systolic and diastolic functions, resulting in improved ejection fraction (71.2±4.7% versus 53.4±7.0%; P P P P P

Aldosterone is involved in various cardiovascular pathologies, including hypertension, heart failure, atherosclerosis and fibrosis. Mineralocorticoid receptor (MR)-dependent and -independent, genomic and non-genomic processes mediate its complex effects. Spironolactone and eplerenone, both MR antagonists, are the only commercially available compounds targeting directly the actions of aldosterone. However, due to the poor selectivity (spironolactone), low potency (eplerenone) and the fact that only MR-dependent effects of aldosterone can be inhibited, these drugs have limited clinical use. An attractive approach to abolish potentially all of aldosterone-mediated pathologies is the inhibition of aldosterone synthase. This review summarizes current knowledge on the complex effects mediated by aldosterone, potential advantages and disadvantages of aldosterone inhibition and novel directions in the development of aldosterone synthase inhibitors.

Anticoagulation by a standard dosage of an inhibitor of thrombin generation presupposes predictable pharmacokinetics and pharmacodynamics of the anticoagulant. We determined the inter-individual variation of the effect on thrombin generation of a fixed concentration of direct and antithrombin-mediated inhibitors of thrombin and factor Xa. Thrombin generation was determined by calibrated automated thrombinography in platelet-poor plasma from 44 apparently healthy subjects which was spiked with fixed concentrations of otamixaban, melagatran, unfractionated heparin, dermatan sulfate and pentasaccharide. The variability of the inhibitory effect of the different anticoagulants within the population was determined using the coefficient of variation, i.e. the standard deviation expressed as a percentage of the mean. The inter-individual coefficients of variation of the endogenous thrombin potential and peak height before inhibition were 18% and 16%, respectively and became 20%-24% and 24%-43% after inhibition. The average inhibition of endogenous thrombin potential and peak height (ETP, peak) brought about by the anticoagulants was respectively: otamixaban (27%, 83%), melagatran (56%, 63%), unfractionated heparin (43%, 58%), dermatan sulfate (68%, 57%) and pentasaccharide (25%, 67%). This study demonstrates that the addition of a fixed concentration of any type of anticoagulant tested causes an inhibition that is highly variable from one individual to another. In this respect there is no difference between direct inhibitors of thrombin and factor Xa and heparin(-like) inhibitors acting on the same factors.

To obtain a thrombin generation (TG) curve from the conversion of added fluorogenic substrate, thrombin concentrations are to be derived from the observed velocity of increase of fluorescence (dF/dt). The relation between velocity and thrombin concentration varies during the experiment because substrate is consumed and because fluorescence is not linear with the concentration of product. Here we review the techniques that we developed to: A: Transform the fluorescence trace into the "ideal" trace that would be seen if substrate consumption and non-linearity of fluorescence would not play a role. B: Subtract the contribution of alpha M-2-thrombin so as to obtain the course of free thrombin. C: Calculate the velocity of prothrombin conversion from the course of free thrombin. D: Fit a smooth curve through the points obtained in a TG experiment

The (pro)renin receptor ((P)RR) and Wnt signalling are both involved in different diseases ranging from cardiac and renal end-organ damage to cancer. (P)RR function involves signalling via the transcription factor promyelocytic leukemia zinc finger protein (PLZF) as well as the furin-mediated generation of vacuolar proton-translocating ATPase (V-ATPase)-associated and soluble (P)RR isoforms. Recently, the (P)RR was described as adaptor protein of Wnt (co)receptors. The aim of this study was to analyse the contribution of these distinct (P)RR functions to Wnt signalling. Using Tcf/Lef reporter gene systems in HEK293T and HepG2 cells and quantification of endogenous axin2 mRNA and protein levels in HEK293T cells we were able to demonstrate that full-length (P)RR acts as a repressor of Wnt signalling in a system preactivated either by Wnt3a stimulation or by constitutively active β-catenin. These repressive effects are mediated by Dvl but are independent of the mutation status of β-catenin. Furthermore, the V-ATPase complex, but not PLZF translocation or renin enzymatic activity, is necessary for the induction of Tcf/Lef-responsive genes by Wnt3a. Our data indicate interference of (P)RR and Wnt cascades, a fact that has to be considered concerning pathophysiology of cardio-renal and oncological entities as well as in drug development programs targeting (P)RR or Wnt pathways.

Radiotherapy of intrathoracic and chest wall tumors may lead to exposure of the heart to ionizing radiation, resulting in radiation-induced heart diseases (RIHD). The main manifestations of RIHD become apparent many years after treatment and include cardiomyopathy and accelerated atherosclerosis. This study examines the role of the kallikrein-kinin system (KKS) in RIHD by investigating the cardiac radiation response in a kininogen-deficient Brown Norway Katholiek (BN/Ka) rat model. BN/Ka rats and wild-type Brown Norway (BN) rats were exposed to local heart irradiation with a single dose of 18 Gy or 24 Gy and were observed for 3 to 6 months. Examinations included in vivo and ex vivo cardiac function, histopathology, gene and protein expression measurements, and mitochondrial swelling assays. Upon local heart irradiation, changes in in vivo cardiac function were significantly less in BN/Ka rats. For instance, a single dose of 24 Gy caused a 35% increase in fractional shortening in BN rats compared with a 16% increase in BN/Ka rats. BN rats, but not BN/Ka rats, showed a 56% reduction in cardiac numbers of CD2-positive cells, and a 57% increase in CD68-positive cells, together with a 52% increase in phosphorylation of extracellular signal–regulated kinase 1/2 (Erk1/2). Local heart irradiation had similar effects on histopathology, mitochondrial changes, and left ventricular mRNA levels of NADPH oxidases in the two genotypes. These results suggest that the KKS plays a role in the effects of radiation on cardiac function and recruitment of inflammatory cells. The KKS may have these effects at least in part by altering Erk1/2 signaling. Cancer Res; 72(19); 4984–92. ©2012 AACR.

AIMS/HYPOTHESIS: Angiotensin II is well-recognised to be a key mediator in driving the pathological events of diabetes-associated atherosclerosis via signalling through its angiotensin II type 1 receptor (AT1R) subtype. However, its actions via the angiotensin II type 2 receptor (AT2R) subtype are still poorly understood. This study is the first to investigate the role of the novel selective AT2R agonist, Compound 21 (C21) in an experimental model of diabetes-associated atherosclerosis (DAA).METHODS: Streptozotocin-induced diabetic Apoe-knockout mice were treated with vehicle (0.1 mol/l citrate buffer), C21 (1 mg/kg per day), candesartan cilexetil (4 mg/kg per day) or C21 + candesartan cilexetil over a 20 week period. In vitro models of DAA using human aortic endothelial cells and monocyte cultures treated with C21 were also performed. At the end of the experiments, assessment of plaque content and markers of oxidative stress, inflammation and fibrosis were conducted.RESULTS: C21 treatment significantly attenuated aortic plaque deposition in a mouse model of DAA in vivo, in association with a decreased infiltration of macrophages and mediators of inflammation, oxidative stress and fibrosis. On the other hand, combination therapy with C21 and candesartan (AT1R antagonist) appeared to have a limited additive effect in attenuating the pathology of DAA when compared with either treatment alone. Similarly, C21 was found to confer profound anti-atherosclerotic actions at the in vitro level, particularly in the setting of hyperglycaemia. Strikingly, these atheroprotective actions of C21 were completely blocked by the AT2R antagonist PD123319.CONCLUSIONS/INTERPRETATION: Taken together, these findings provide novel mechanistic and potential therapeutic insights into C21 as a monotherapy agent against DAA.

Thrombin is a pivotal player in the coagulation system. In clotting blood a transient wave of thrombin appears after a lag time. Clotting occurs at the start of the wave. The amount of thrombin formed reflects the function of the hemostatic system much better than the clotting time does: "The more thrombin the less bleeding but the more thrombosis, the less thrombin the more bleeding but the less thrombosis" has been shown to hold for congenital and acquired tendencies to venous thrombosis and bleeding and under all variants of antithrombotic treatment. The situation with arterial thrombosis is less clear. Calibrated automated thrombinography (CAT) allows quantitative assessment of the thrombin generation (TG) curve in platelet poor as well as in platelet rich plasma. Procedures to measure TG in whole blood and at the point of care are under development. TG measurement in platelet rich plasma underlines the close cooperation between platelets and the clotting system and challenges the traditional division between primary and secondary hemostases.

Background— Epidemiological data suggest that sex independently contributes to cardiovascular risk. Clinical trials are often hampered by the enrollment of few female patients. Methods and Results— The Ongoing Telmisartan Alone and in Combination With Ramipril Global End Point Trial (ONTARGET) and the parallel Telmisartan Randomized Assessment Study in ACE Intolerant Subjects With Cardiovascular Disease (TRANSCEND) included a large proportion of female patients (9378 female versus 22 168 male patients). Differences in male and female patients enrolled in ONTARGET/TRANSCEND were analyzed for the primary 4-fold end point (composite of cardiovascular death, myocardial infarction, stroke, or admission to hospital for heart failure), a secondary 3-fold end point (cardiovascular death, myocardial infarction, stroke), and individual components of the primary composite. Baseline characteristics included age, ethnicity, body mass index, physical activity, tobacco use, alcohol consumption, formal education, clinical diagnosis for study entry, patient history, and concomitant medication. Patients were followed up until death or the end of the study (median, 56 months). Compared with male patients, female patients had a 19% significantly lower risk for the 4-fold end point and 21% for the 3-fold end point (after adjustment for study, treatment, and the above baseline values). Similarly, the adjusted risk for cardiovascular death (17%) and myocardial infarction (22%), but not for stroke and hospitalization for heart failure, was also significantly lower in women. Diabetic female patients were characterized by a higher risk for acute myocardial infarction compared with diabetic male patients, whereas alcohol consumption resulted in significantly lower risk in women. Conclusions— In our analysis made up of 70.3% male and 29.7% female patients, an ≈20% lower risk for the combined cardiovascular end points in female patients was observed despite treatment with cardioprotective agents. This difference was driven primarily by a significantly lower incidence of myocardial infarction. Thus, we demonstrate in a large interventional trial that sex greatly affects the occurrence of cardiovascular events in patients with vascular disease or high-risk diabetes mellitus. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00153101.

BACKGROUND The calibrated automated thrombogram (CAT) assay in plasma is a versatile tool to investigate patients with hypo- or hypercoagulable phenotypes. The objective was to make this method applicable for whole blood measurements. METHODS Thin-layer technology and the use of a rhodamine 110–based thrombin substrate appear to be essential for a reliable thrombin generation (TG) assay in whole blood. Using this knowledge we developed a whole blood CAT-based assay. RESULTS We demonstrated that the whole blood CAT-based assay is a sensitive and rapid screening test to assess function of the hemostatic system under more nearly physiological conditions than the TG assay in plasma. Under conditions of low tissue factor concentration (0.5 pmol/L) and 50% diluted blood, the intraassay CV of the thrombogram parameters, endogenous thrombin potential and thrombin peak height, were 6.7% and 6.5%, respectively. The respective interassay CVs were 12% and 11%. The mean interindividual variation (SD) of 40 healthy volunteers was 633 (146) nmol · min/L for the endogenous thrombin potential and 128 (23) nmol/L for the thrombin peak. Surprisingly, erythrocytes contributed more than platelets to the procoagulant blood cell membranes necessary for optimal TG. Statistically significant (P < 0.001) and potentially clinically significant correlations were observed between circulating factor-VIII concentrations in blood of hemophilia A patients and endogenous thrombin potential (r = 0.62) and thrombin peak height (r = 0.58). CONCLUSIONS We have developed a reliable method to measure TG in whole blood. The assay can be performed with a drop of blood and may provide a useful measurement of TG under more physiological conditions than plasma.

Although various effective treatments for hypertension are available, novel therapies to reduce elevated blood pressure, improve blood-pressure control, treat resistant hypertension, and reduce the associated cardiovascular risk factors are still required. A novel angiotensin-receptor blocker (ARB) was approved in 2011, and additional compounds are in development or being tested in clinical trials. Several of these agents have innovative mechanisms of action (an aldosterone synthase inhibitor, a natriuretic peptide agonist, a soluble epoxide hydrolase inhibitor, and an angiotensin II type 2 receptor agonist) or dual activity (a combined ARB and neutral endopeptidase inhibitor, an ARB and endothelin receptor A blocker, and an endothelin-converting enzyme and neutral endopeptidase inhibitor). In addition, several novel fixed-dose combinations of existing antihypertensive agents were approved in 2010-2011, including aliskiren double and triple combinations, and an olmesartan triple combination. Upcoming fixed-dose combinations are expected to introduce calcium-channel blockers other than amlodipine and diuretics other than hydrochlorothiazide. Finally, device-based approaches to the treatment of resistant hypertension, such as renal denervation and baroreceptor activation therapy, have shown promising results in clinical trials. However, technical improvements in the implantation procedure and devices used for baroreceptor activation therapy are required to address procedural safety concerns.

Purpose of review Research about the angiotensin AT2 receptor (AT2R) has been hampered in the past by the lack of a specific and selective agonist with in-vivo stability. Such an eagerly awaited agonist, compound 21, has recently become available, giving momentum to AT2R research which so far has resulted in 14 original publications. This article is intending to review those publications which address AT2R function by direct in-vivo stimulation instead of indirect approaches such as receptor blockade or genetic alteration of AT2R expression. Recent findings Studies reviewed in this article looked at the effect of AT2R stimulation in disease models of hypertension, renal disease, stroke, Alzheimer's disease and myocardial infarction. AT2R stimulation does not have an antihypertensive effect, but promoted tissue protection in all models in which it was tested. Antiinflammation and antiapoptosis seem important features of the AT2R underlying improved outcome in experimental disease models. Summary Availability of nonpeptidic, orally active AT2R agonists will facilitate future AT2R research and hopefully contribute to the clarification of many still open questions regarding AT2R signalling and function. Furthermore, AT2R agonists represent a potential novel class of drugs and are expected to enter a phase I clinical study in 2012.

Background/Aims: Putative in vitro-in vivo correlations of pharmacokinetic (PK) parameters are regarded as a prerequisite to filter hits derived from high-throughput screening (HTS) approaches for subsequent murine in vivo PK studies. Methods: In this study, we assessed stabilities in rat and human microsomes of 121 compounds from an early, academic drug discovery programme targeting the (pro)renin receptor and correlated the respective data with single-dose, in vivo PK parameters of 22 hits administered intravenously in rats. Results: After transformation of in vitro half-lives to predicted in vivo hepatic clearances, r2 regarding in vitro-in vivo clearance correlations were 0.31 and 0.27 for the rat and human species, respectively. Conclusions: Our data concerning structurally diverse real-world compounds indicate that microsomal stability testing is not a tool to triage early compounds for in vivo PK testing.

Introduction Circulating microparticles are reported to play a role in cancer hypercoagulability. The procoagulant properties of microparticles derive from the amount of tissue factor and/or phosphatidylserine that they can expose. The aim of our study is to assess the procoagulant activity, including microparticles’ activity, in the plasma of newly diagnosed cancer patients with a simple assay, easy to implement in the laboratory. Material and methods Newly diagnosed cancer patients (n = 31) before the start of anticoagulant or chemotherapy were compared to matched controls. We used a thrombin generation assay in four conditions: 1: addition of 1pM tissue factor and 4 μM procoagulant phospholipids, 2: without any trigger, 3 and 4: addition of tissue factor or procoagulant phospholipids alone respectively. Results When we added only phospholipids, so that thrombin generation is dependent upon endogenous tissue factor, the lag time was significantly shorter in cancer patients. When we added only tissue factor, i.e. made the results dependent upon phospholipids, the endogenous thrombin potential, the peak, and the velocity index were significantly higher and the time-to-peak was significantly shorter. This suggests that the plasma of cancer patients contained a higher activity of endogenous phospholipids and/or tissue factor which may be borne by microparticles. Conclusion This new simple methodology can demonstrate a procoagulant activity in the plasma of newly diagnosed cancer patients which can be explained by higher procoagulant phospholipids and tissue factor activity and thus, brings potentially useful information that current coagulation tests cannot provide.

Calibrated automated thrombin generation assay was adapted to measure thrombin generation in platelet rich plasma from mice. Vena cava phlebotomy appeared the best technique for blood sampling. The concentration-effect curves of tissue factor and platelet count have been determined. Corn trypsin inhibitor 2 mu M inhibits contact activation effectively. Recombinant human thrombomodulin does not inhibit thrombin generation in mouse plasma but activated protein C (20 nM) does. Thrombin generation was dose dependently diminished by low molecular weight heparin and increased by high concentrations of exogenous factor VIII i.e. the assay can detect both hypo-and hypercoagulability.

Background: Interaction of murine Gas6 with the platelet Gas6 receptors Tyro3, Axl and Mer (TAM) plays an important role in arterial thrombus formation. However, a role for Gas6 in human platelet activation has been questioned. Objective: To determine the role of Gas6 in human and murine platelet activation and thrombus formation. Methods and Results: Gas6 levels appeared to be 20-fold higher in human plasma than in platelets, suggesting a predominant role of plasma-derived Gas6. Human Gas6 synergizes with ADP-P2Y(12) by enhancing and prolonging the phosphorylation of Akt. Removal of Gas6 from plasma impaired ADP-induced platelet aggregation. Under flow conditions, absence of human Gas6 provoked gradual platelet disaggregation and integrin alpha(IIb)beta(3) inactivation. Recombinant human Gas6 reversed the effects of Gas6 removal. In mouse blood, deficiency in Gas6 or in one of the TAM receptors led to reduced thrombus formation and increased disaggregation, which was completely antagonized by external ADP. In contrast, collagen-induced platelet responses were unchanged by the absence of Gas6 in both human and mouse systems. Conclusions: The ADP-P2Y(12) and Gas6-TAM activation pathways synergize to achieve persistent alpha(IIb)beta(3) activation and platelet aggregation. We postulate a model of thrombus stabilization in which plasma Gas6, by signaling via the TAM receptors, extends and enhances the platelet-stabilizing effect of autocrine ADP, particularly when secretion becomes limited.

Background. Thrombin generation (TG) in plasma can be monitored continuously with a fluorogenic thrombin substrate using calibrated automated thrombinography ( CAT). In the presence of low concentrations of a reversible direct thrombin inhibitor (DTI), CAT shows an unexpected effect: the endogenous thrombin potential (ETP) increases at low concentrations of the inhibitor to subsequently decrease concentration dependently at higher concentrations (> approximately 100 nM). Objectives. To find an explanation for this phenomenon, we measured the concentrations of free thrombin and alpha(2)-macroglobulin- thrombin complex (alpha 2MT) with a sub-sampling technique in the presence of AR-H067637, a selective DTI. Results. At all concentrations of the DTI there was a gradual dose-dependent decrease in the concentration of free, not-inhibited thrombin but a transient increase in free alpha 2MT due to competition of thrombin and alpha 2MT for the inhibitor. Because the CAT technique uses an algorithm to subtract alpha 2MT activity from the total amidolytic activity, this transient increase in alpha 2MT activity is not subtracted and erroneously attributed to thrombin itself. Conclusions. This study explains the spurious increase in ETP observed at low DTI concentrations. The results obtained in plasma were corroborated by observations in a thrombin generating system reconstituted with purified factors. In practise, the effect of DTIs on TG can be reliably evaluated from the area under the curve till time-to-peak.

The transforming and tumor growth–promoting properties of Axl, a member of the Tyro3, Axl, and Mer (TAM) family of receptor tyrosine kinases (TAMRs), are well recognized. In contrast, little is known about the role of the TAMR ligand growth arrest–specific gene 6 (Gas6) in tumor biology. By using Gas6-deficient (Gas6−/−) mice, we show that bone marrow–derived Gas6 promotes growth and metastasis in different experimental cancer models, including one resistant to vascular endothelial growth factor inhibitors. Mechanistic studies reveal that circulating leukocytes produce minimal Gas6. However, once infiltrated in the tumor, leukocytes up-regulate Gas6, which is mitogenic for tumor cells. Consistent herewith, impaired tumor growth in Gas6−/− mice is rescued by transplantation of wild-type bone marrow and, conversely, mimicked by transplantation of Gas6−/− bone marrow into wild-type hosts. These findings highlight a novel role for Gas6 in a positive amplification loop, whereby tumors promote their growth by educating infiltrating leukocytes to up-regulate the production of the mitogen Gas6. Hence, inhibition of Gas6 might offer novel opportunities for the treatment of cancer.

Myocardial infarction (MI) is a major condition causing heart failure (HF). After MI, the renin angiotensin system (RAS) and its signalling octapeptide angiotensin II (Ang II) interferes with cardiac injury/repair via the AT1 and AT2 receptors (AT1R, AT2R). Our study aimed at deciphering the mechanisms underlying the link between RAS and cellular components of the immune response relying on a rodent model of HF as well as HF patients. Flow cytometric analyses showed an increase in the expression of CD4(+) AT2R(+) cells in the rat heart and spleen post-infarction, but a reduction in the peripheral blood. The latter was also observed in HF patients. The frequency of rat CD4(+) AT2R(+) T cells in circulating blood, post-infarcted heart and spleen represented 3.8 ? 0.4%, 23.2 ? 2.7% and 22.6 ? 2.6% of the CD4(+) cells. CD4(+) AT2R(+) T cells within blood CD4(+) T cells were reduced from 2.6 ? 0.2% in healthy controls to 1.7 ? 0.4% in patients. Moreover, we characterized CD4(+) AT2R(+) T cells which expressed regulatory FoxP3, secreted interleukin-10 and other inflammatory-related cytokines. Furthermore, intramyocardial injection of MI-induced splenic CD4(+) AT2R(+) T cells into recipient rats with MI led to reduced infarct size and improved cardiac performance. We defined CD4(+) AT2R(+) cells as a T cell subset improving heart function post-MI corresponding with reduced infarction size in a rat MI-model. Our results indicate CD4(+) AT2R(+) cells as a promising population for regenerative therapy, via myocardial transplantation, pharmacological AT2R activation or a combination thereof.? 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Background and PurposeAngiotensin AT(2) receptors are upregulated in disease states such as atherosclerosis and blockade of the AT(2) receptors exacerbates plaque formation. Direct stimulation of these receptors is anti-atherogenic but the mechanisms and pathways involved remain unknown. We examined the effect of direct AT(2) receptor stimulation with Compound 21 (C21) on the leukocyte adhesion cascade in vitro, right through to plaque formation in vivo. Experimental ApproachEffects of C21 on TNF-induced inflammation were assessed in human umbilical vein endothelial cells (HUVECs), activation of monocytes, polarisation of monocyte-derived macrophages and in intact mouse aortae. Key ResultsC21 attenuated TNF-induced: monocyte adhesion to cultured HUVECs, adhesion molecule expression and abolished TNF-induced ROS production. TNF-induced NFB translocation from the cytoplasm to the nucleus, essential for cytokine production, was prevented by C21. C21 did not influence monocyte activation or macrophage polarisation but did reduce TNF and IL-6 mRNA expression in M1 macrophages. The anti-inflammatory effects of C21 were abolished by an AT(2) receptor antagonist confirming that the effects of C21 were AT(2) receptor-mediated. Also, leukocyte adhesion and cytokine gene expression, induced by high-fat diet (HFD), was attenuated in ApoE(-/-) mice treated with C21. Plaque size and stability were improved with C21 treatment with increased smooth muscle cell composition and decreased lipid size, compared with HFD-saline treated mice. Conclusion and ImplicationsC21 prevented TNF-induced and HFD-induced vascular inflammation in vitro and in vivo. Our data provide strong evidence that the anti-atherosclerotic actions of C21 were due to vascular anti-inflammatory effects, mediated by AT(2) receptors. Linked ArticlesThis article is part of a themed section on Inflammation: maladies, models, mechanisms and molecules. To view the other articles in this section visit

Diabetes is a common and rapidly growing disease that affects more than 380 million people worldwide and is an established risk factor for cardiovascular disease with differential effects on women compared to men. While the general population of women, particularly young women, has more favourable cardiovascular risk profiles than men, this protective effect has been shown to be lost or even reversed in diabetic women. Several studies have demonstrated a significant diabetes-associated excess risk of cardiovascular disease in women. Sex-specific differences in risk factors associated with diabetes and their management may be responsible for the relative excess cardiovascular risk in women with diabetes. Diabetic women need intensive treatment in order to optimize management of cardiovascular risk factors. Further studies are needed to elucidate the mechanisms underlying the excess cardiovascular risk in diabetic women in order to tailor prevention and treatment strategies.

The hemodynamic and nonhemodynamic effects of angiotensin II on diabetic complications are considered to be primarily mediated by the angiotensin II type 1 receptor subtype. However, its biological and functional effect mediated through the angiotensin II type 2 receptor subtype is still unclear. Activation of the angiotensin II type 2 receptors has been postulated to oppose angiotensin II type 1 receptor–mediated actions and thus attenuate fibrosis. This study aimed to elucidate the reno-protective role of the novel selective angiotensin II type 2 receptor agonist, Compound 21, in an experimental model of type 1 diabetic nephropathy. Compound 21 treatment significantly attenuated diabetes mellitus–induced elevated levels of cystatin C, albuminuria, mesangial expansion, and glomerulosclerosis in diabetic mice. Moreover, Compound 21 markedly inhibited the expression of various proteins implicated in oxidative stress, inflammation, and fibrosis, in association with decreased extracellular matrix production. These findings demonstrate that monotherapy of Compound 21 is protective against the progression of experimental diabetic nephropathy by inhibiting renal oxidative stress, inflammation, and fibrosis.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro . In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis. Abbreviations: ACE, angiotensin-converting enzyme; AngII, angiotensin II; AT1R, angiotensin type 1 receptor; AT2R, angiotensin type 2 receptor; BBB, blood–brain barrier; BDNF, brain-derived neurotrophic factor; C21, Compound 21; CFA, complete Freund's adjuvant; CNS, central nervous system; DMEM, Dulbecco's modified Eagle's medium; EAE, experimental autoimmune encephalomyelitis; IB-4, isolectin B4; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MS, multiple sclerosis; MOG, myelin oligodendrocyte glycoprotein; MOG-EAE, myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis; NF-κB, nuclear factor κB; OPC, oligodendrocyte precursor cell; PFA, paraformaldehyde; p.i., post-immunization; RAS, renin–angiotensin system; TCR, T-cell receptor; TNF, tumour necrosis factor

Individuals with blood group O have a higher bleeding risk than non-O blood groups. This could be explained by the lower levels of FVIII and von Willebrand Factor (VWF) levels in O individuals. We investigated the relationship between blood groups, thrombin generation (TG), prothrombin activation and thrombin inactivation. Plasma levels of VWF, FVIII, antithrombin, fibrinogen, prothrombin and alpha(2)Macroglobulin (alpha M-2) levels were determined. TG was measured in platelet rich (PRP) and platelet poor plasma (PPP) of 217 healthy donors and prothrombin conversion and thrombin inactivation were calculated. VWF and FVIII levels were lower (75% and 78%) and alpha M-2 levels were higher (125%) in the O group. TG is 10% lower in the O group in PPP and PRP. Less prothrombin was converted in the O group (86%) and the thrombin decay capacity was lower as well. In the O group, alpha M-2 plays a significantly larger role in the inhibition of thrombin (126%). In conclusion, TG is lower in the O group due to lower prothrombin conversion, and a larger contribution of alpha M-2 to thrombin inactivation. The former is unrelated to platelet function because it is similar in PRP and PPP, but can be explained by the lower levels of FVIII.

Cardiac electrophysiology has evolved into an important subspecialty in cardiovascular medicine. This is in part due to the significant advances made in our understanding and treatment of heart rhythm disorders following more than a century of scientific discoveries and research. More recently, the rapid development of technology in cellular electrophysiology, molecular biology, genetics, computer modelling, and imaging have led to the exponential growth of knowledge in basic cardiac electrophysiology. The paradigm of evidence-based medicine has led to a more comprehensive decision-making process and most likely to improved outcomes in many patients. However, implementing relevant basic research knowledge in a system of evidence-based medicine appears to be challenging. Furthermore, the current economic climate and the restricted nature of research funding call for improved efficiency of translation from basic discoveries to healthcare delivery. Here, we aim to (i) appraise the broad challenges of translational research in cardiac electrophysiology, (ii) highlight the need for improved strategies in the training of translational electrophysiologists, and (iii) discuss steps towards building a favourable translational research environment and culture.