N-(Heteroaryl)thiophene sulfonamides as angiotensin AT2 receptor ligands.

Two series of N-(heteroaryl)thiophene sulfonamides, encompassing either a methylene imidazole group or a tert-butylimidazolylacetyl group in the meta position of the benzene ring, have been synthesized. An AT2R selective ligand with a Ki of 42 nM was identified in the first series and in the second series, six AT2R selective ligands with significantly improved binding affinities and Ki values of <5 nM were discovered. The binding modes to AT2R were explored by docking calculations combined with molecular dynamics simulations. Although some of the high affinity ligands exhibited fair stability in human liver microsomes, comparable to that observed with C21 undergoing clinical trials, most ligands displayed a very low metabolic stability with t½ of less than 10 min in human liver microsomes. The most promising ligand, with an AT2R Ki value of 4.9 nM and with intermediate stability in human hepatocytes (t½ = 77 min) caused a concentration-dependent vasorelaxation of pre-contracted mouse aorta.

Novel AT2R agonist, ß-Pro7Ang III, is cardio- and vaso-protective in diabetic spontaneously hypertensive rats.

Stimulation of the angiotensin II type 2 receptor (AT2R) evokes protective effects in various cardiovascular diseases. Thus, this study aimed to investigate the effects of AT2R stimulation, with or without AT1R blockade, in a model of hypertension with concomitant type 1 diabetes mellitus (T1DM). Spontaneously hypertensive rats (SHRs) were given either citrate or a single dose of streptozotocin (STZ; 55 mg/kg, i.p.) to induce diabetes. After 4 weeks of diabetes, animals were administered either a vehicle (saline), AT2R agonist, ß-Pro7Ang III (0.1 mg/kg/day via osmotic mini-pump), AT1R blocker, candesartan (2 mg/kg/day via drinking water), or a combination of both for a further 8 weeks. ß-Pro7Ang III treatment had no effect on blood pressure, but attenuated the significant increase in cardiac interstitial collagen and protein expression of fibrotic and inflammatory markers, and superoxide levels that was evident in diabetic SHRs. These effects were not observed with candesartan, despite its blood pressure lowering effects. Although ß-Pro7Ang III had no effect on aortic fibrosis, it significantly attenuated MCP-1 protein expression and superoxide levels when compared to both the non-diabetic and diabetic SHRs, to a similar extent as candesartan. In both the heart and vasculature, the effects of ß-Pro7Ang III in combination with candesartan were similar to those of ß-Pro7Ang III alone, and superior to candesartan alone. It was concluded that in hypertension with concomitant diabetes, AT2R stimulation with a novel ligand alone, or in combination with AT1R blockade, improved the cardiac and vascular structural changes that were strongly associated with inflammation and oxidative stress, independent of blood pressure regulation.

Long-term dietary nitrate supplementation slows the progression of established atherosclerosis in ApoE-/- mice fed a high fat diet.

BACKGROUND AND AIMS: Atherosclerosis is associated with a reduction in the bioavailability and/or bioactivity of endogenous nitric oxide (NO). Dietary nitrate has been proposed as an alternate source when endogenous NO production is reduced. Our previous study demonstrated a protective effect of dietary nitrate on the development of atherosclerosis in the apoE-/- mouse model. However most patients do not present clinically until well after the disease is established. The aims of this study were to determine whether chronic dietary nitrate supplementation can prevent or reverse the progression of atherosclerosis after disease is already established, as well as to explore the underlying mechanism of these cardiovascular protective effects. METHODS: 60 apoE-/- mice were given a high fat diet (HFD) for 12 weeks to allow for the development of atherosclerosis. The mice were then randomized to (i) control group (HFD + 1 mmol/kg/day NaCl), (ii) moderate-dose group (HFD +1 mmol/kg/day NaNO3), or (iii) high-dose group (HFD + 10 mmol/kg/day NaNO3) (20/group) for a further 12 weeks. A group of apoE-/- mice (n = 20) consumed a normal laboratory chow diet for 24 weeks and were included as a reference group. RESULTS: Long-term supplementation with high dose nitrate resulted in ~ 50% reduction in plaque lesion area. Collagen expression and smooth muscle accumulation were increased, and lipid deposition and macrophage accumulation were reduced within atherosclerotic plaques of mice supplemented with high dose nitrate. These changes were associated with an increase in nitrite reductase as well as activation of the endogenous eNOS-NO pathway. CONCLUSION: Long-term high dose nitrate significantly attenuated the progression of established atherosclerosis in the apoE-/- mice fed a HFD. This appears to be mediated in part through a XOR-dependent reduction of nitrate to NO, as well as enhanced eNOS activation via increased Akt and eNOS phosphorylation.

The single-chain relaxin mimetic, B7-33, maintains the cardioprotective effects of relaxin and more rapidly reduces left ventricular fibrosis compared to perindopril in an experimental model of cardiomyopathy.

The hormone, relaxin (RLX), exerts various organ-protective effects independently of etiology. However, its complex two-chain and three disulphide bonded structure is a limitation to its preparation and affordability. Hence, a single chain-derivative of RLX, B7-33, was developed and shown to retain the anti-fibrotic effects of RLX in vitro and in vivo. Here, we determined whether B7-33 could retain the other cardioprotective effects of RLX, and also compared its therapeutic efficacy to the ACE inhibitor, perindopril. Adult male 129sv mice were subjected to isoprenaline (ISO; 25 mg/kg/day, s.c)-induced cardiomyopathy, then s.c-treated with either RLX (0.5 mg/kg/day), B7-33 (0.25 mg/kg/day; equivalent dose corrected for MW) or perindopril (1 mg/kg/day) from days 7-14 post-injury. Control mice received saline instead of ISO. Changes in animal body weight (BW) and systolic blood pressure (SBP) were measured weekly, whilst cardiomyocyte hypertrophy and measures of vascular dysfunction and rarefaction, left ventricular (LV) inflammation and fibrosis were assessed at day 14 post-injury. ISO-injured mice had significantly increased LV inflammation, cardiomyocyte hypertrophy, fibrosis, vascular rarefaction and aortic contractility in the absence of any changes in BW or SBP at day 14 post-injury. Both B7-33 and RLX equivalently reduced LV fibrosis and normalised the ISO-induced LV inflammation and cardiomyocyte hypertrophy, whilst restoring blood vessel density and aortic contractility. Comparatively, perindopril lowered SBP and the ISO-induced LV inflammation and vascular rarefaction, but not fibrosis or hypertrophy. As B7-33 retained the cardioprotective effects of RLX and provided rapid-occurring anti-fibrotic effects compared to perindopril, it could be considered as a cost-effective cardioprotective therapy.

Relaxin Inhibits the Cardiac Myofibroblast NLRP3 Inflammasome as Part of Its Anti-Fibrotic Actions via the Angiotensin Type 2 and ATP (P2X7) Receptors.

Chronic NLRP3 inflammasome activation can promote fibrosis through its production of interleukin (IL)-1ß and IL-18. Conversely, recombinant human relaxin (RLX) can inhibit the pro-fibrotic interactions between IL-1ß, IL-18 and transforming growth factor (TGF)-ß1. Here, the broader extent by which RLX targeted the myofibroblast NLRP3 inflammasome to mediate its anti-fibrotic effects was elucidated. Primary human cardiac fibroblasts (HCFs), stimulated with TGF-ß1 (to promote myofibroblast (HCMF) differentiation), LPS (to prime the NLRP3 inflammasome) and ATP (to activate the NLRP3 inflammasome) (T+L+A) or benzoylbenzoyl-ATP (to activate the ATP receptor; P2X7R) (T+L+Bz), co-expressed relaxin family peptide receptor-1 (RXFP1), the angiotensin II type 2 receptor (AT2R) and P2X7R, and underwent increased protein expression of toll-like receptor (TLR)-4, NLRP3, caspase-1, IL-1ß and IL-18. Whilst RLX co-administration to HCMFs significantly prevented the T+L+A- or T+L+Bz-stimulated increase in these end points, the inhibitory effects of RLX were annulled by the pharmacological antagonism of either RXFP1, AT2R, P2X7R, TLR-4, reactive oxygen species (ROS) or caspase-1. The RLX-induced amelioration of left ventricular inflammation, cardiomyocyte hypertrophy and fibrosis in isoproterenol (ISO)-injured mice, was also attenuated by P2X7R antagonism. Thus, the ability of RLX to ameliorate the myofibroblast NLRP3 inflammasome as part of its anti-fibrotic effects, appeared to involve RXFP1, AT2R, P2X7R and the inhibition of TLR-4, ROS and caspase-1.

In Aged Females, the Enhanced Pressor Response to Angiotensin II Is Attenuated By Estrogen Replacement via an Angiotensin Type 2 Receptor-Mediated Mechanism.

[Figure: see text].

Simultaneous targeting of oxidative stress and fibrosis reverses cardiomyopathy-induced ventricular remodelling and dysfunction.

BACKGROUND AND PURPOSE: Oxidative stress and fibrosis are hallmarks of cardiomyopathy-induced heart failure yet are not effectively targeted by current frontline therapies. Here, the therapeutic effects of the anti-oxidant, N-acetylcysteine (NAC), were compared and combined with an acute heart failure drug with established anti-fibrotic effects, serelaxin (RLX), in a murine model of cardiomyopathy. EXPERIMENTAL APPROACH: Adult male 129sv mice were subjected to repeated isoprenaline (25 mg·kg-1 )-induced cardiac injury for five consecutive days and then left to undergo fibrotic healing until Day 14. Subgroups of isoprenaline-injured mice were treated with RLX (0.5 mg·kg-1 ·day-1 ), NAC (25 mg·kg-1 ·day-1 ) or both combined, given subcutaneously via osmotic minipumps from Day 7 to 14. Control mice received saline instead of isoprenaline. KEY RESULTS: Isoprenaline-injured mice showed increased left ventricular (LV) inflammation (~5-fold), oxidative stress (~1-2.5-fold), cardiomyocyte hypertrophy (~25%), cardiac remodelling, fibrosis (~2-2.5-fold) and dysfunction by Day 14 after injury. NAC alone blocked the cardiomyopathy-induced increase in LV superoxide levels, to a greater extent than RLX. Additionally, either treatment alone only partly reduced several measures of LV inflammation, remodelling and fibrosis. In comparison, the combination of RLX and NAC prevented the cardiomyopathy-induced LV macrophage infiltration, remodelling, fibrosis and cardiomyocyte size, to a greater extent than either treatment alone after 7 days. The combination therapy also restored the isoprenaline-induced reduction in LV function, without affecting systolic BP. CONCLUSION AND IMPLICATIONS: These findings demonstrated that the simultaneous targeting of oxidative stress and fibrosis is key to treating the pathophysiology and dysfunction induced by cardiomyopathy.

Relaxin Attenuates Organ Fibrosis via an Angiotensin Type 2 Receptor Mechanism in Aged Hypertensive Female Rats.

Background: The antifibrotic effects of recombinant human relaxin (RLX) in the kidney are dependent on an interaction between its cognate receptor (RXFP1) and the angiotensin type 2 receptor (AT2R) in male models of disease. Whether RLX has therapeutic effects, which are also mediated via AT2R, in hypertensive adult and aged/reproductively senescent females is unknown. Thus, we determined whether treatment with RLX provides cardiorenal protection via an AT2R-dependent mechanism in adult and aged female stroke-prone spontaneously hypertensive rats (SHRSPs). Methods: In 6-month-old (6MO) and 15-month-old ([15MO]; reproductively senescent) female SHRSP, systolic BP (SBP), GFR, and proteinuria were measured before and after 4 weeks of treatment with vehicle (Veh), RLX (0.5 mg/kg per day s.c.), or RLX+PD123319 (AT2R antagonist; 3 mg/kg per day s.c.). Aortic endothelium-dependent relaxation and fibrosis of the kidney, heart, and aorta were assessed. Results: In 6MO SHRSP, RLX significantly enhanced GFR by approximately 25% (P=0.001) and reduced cardiac fibrosis (P=0.01) as compared with vehicle-treated counterparts. These effects were abolished or blunted by PD123319 coadministration. In 15MO females, RLX reduced interstitial renal (P=0.02) and aortic (P=0.003) fibrosis and lowered SBP (13±3 mm Hg; P=0.04) relative to controls. These effects were also blocked by PD123319 cotreatment (all P=0.05 versus RLX treatment alone). RLX also markedly improved vascular function by approximately 40% (P<0.001) in 15MO SHRSP, but this was not modulated by PD123319 cotreatment. Conclusions: The antifibrotic and organ-protective effects of RLX, when administered to a severe model of hypertension, conferred cardiorenal protection in adult and reproductively senescent female rats to a great extent via an AT2R-mediated mechanism.

N-(Methyloxycarbonyl)thiophene sulfonamides as high affinity AT2 receptor ligands.

A series of meta-substituted acetophenone derivatives, encompassing N-(alkyloxycarbonyl)thiophene sulfonamide fragments have been synthesized. Several selective AT2 receptor ligands were identified, among those a tert-butylimidazole derivative (20) with a Ki of 9.3 nM, that demonstrates a high stability in human liver microsomes (t½ = 62 min) and in human hepatocytes (t½ = 194 min). This methyloxycarbonylthiophene sulfonamide is a 20-fold more potent binder to the AT2 receptor and is considerably more stable in human liver microsomes, than a previously reported and broadly studied structurally related AT2R prototype antagonist 3 (C38). Ligand 20 acts as an AT2R agonist and caused an AT2R mediated concentration-dependent vasorelaxation of pre-contracted mouse aorta. Furthermore, in contrast to imidazole derivative C38, the tert-butylimidazole derivative 20 is a poor inhibitor of CYP3A4, CYP2D6 and CYP2C9. It is demonstrated herein that smaller alkyloxycarbonyl groups make the ligands in this series of AT2R selective compounds less prone to degradation and that a high AT2 receptor affinity can be retained after truncation of the alkyloxycarbonyl group. Binding modes of the most potent AT2R ligands were explored by docking calculations combined with molecular dynamics simulations.

Relaxin Can Mediate Its Anti-Fibrotic Effects by Targeting the Myofibroblast NLRP3 Inflammasome at the Level of Caspase-1.

INTRODUCTION: The NLRP3 inflammasome produces interleukin (IL)-1ß and IL-18, which when chronically activated by transforming growth factor (TGF)-ß1, contribute to fibrosis. The recombinant form of the anti-fibrotic hormone, relaxin (RLX), suppresses the pro-fibrotic influence of TGF-ß1 and toll-like receptor (TLR)-4 on NLRP3 inflammasome priming and activity in human cardiac myofibroblasts and mice with cardiomyopathy. However, whether RLX also modulates components of the myofibroblast NLRP3 inflammasome remains unknown. METHODS AND RESULTS: Stimulation of a human dermal fibroblast (HDF) cell line with TGF-ß1 [5 ng/ml; to promote myofibroblast (HDMF) differentiation], LPS (100 ng/ml; to prime the NLRP3 inflammasome) and ATP (5 mM; to activate the NLPR3 inflammasome) (T+L+A) significantly increased NLRP3 inflammasome priming and activity after 8 and 72 h; and α-SMA expression (myofibroblast differentiation) and collagen-I deposition after 72 h. siRNA-induced knock-down of NLRP3 inflammasome priming components (NLRP3, ASC, caspase-1) in T+L+A-stimulated HDMFs for 24 h, completely knocked-down each component after 72 h. RLX (100 ng/ml) administration to T+L+A-stimulated HDMFs after control, NLRP3 or ASC siRNA transfection, equivalently suppressed IL-1ß, pro-IL-18, α-SMA, and collagen-I protein levels (by 40%-50%; all p<0.05 vs. T+L+A) after 72 h, as determined by Western blotting. These RLX-induced effects were abrogated by siRNA knock-down of caspase-1. CONCLUSION: The anti-fibrotic actions of RLX appear to require modulation of caspase-1 within the myofibroblast NLRP3 inflammasome.

Serelaxin and the AT2 Receptor Agonist CGP42112 Evoked a Similar, Nonadditive, Cardiac Antifibrotic Effect in High Salt-Fed Mice That Were Refractory to Candesartan Cilexetil.

Fibrosis is involved in the majority of cardiovascular diseases and is a key contributor to end-organ dysfunction. In the current study, the antifibrotic effects of recombinant human relaxin-2 (serelaxin; RLX) and/or the AT2R agonist CGP42112 (CGP) were compared with those of the established AT1R antagonist, candesartan cilexetil (CAND), in a high salt-induced cardiac fibrosis model. High salt (HS; 5%) for 8 weeks did not increase systolic blood pressure in male FVB/N mice, but CAND treatment alone significantly reduced systolic blood pressure from HS-induced levels. HS significantly increased cardiac interstitial fibrosis, which was reduced by either RLX and/or CGP, which were not additive under the current experimental conditions, while CAND failed to reduce HS-induced cardiac fibrosis. The antifibrotic effects induced by RLX and/or CGP were associated with reduced myofibroblast differentiation. Additionally, all treatments inhibited the HS-induced elevation in tissue inhibitor of matrix metalloproteinases-1, together with trends for increased MMP-13 expression, that collectively would favor collagen degradation. Furthermore, these antifibrotic effects were associated with reduced cardiac inflammation. Collectively, these results highlight that either RXFP1 or AT2R stimulation represents novel therapeutic strategies to target fibrotic conditions, particularly in HS states that may be refractory to AT1R blockade.

A Novel Epigenetic Drug-Eluting Balloon Angioplasty Device: Evaluation in a Large Animal Model of Neointimal Hyperplasia.

PURPOSE: Drug-eluting balloon catheters (DEBc) coated with paclitaxel (PTX) have been associated with potential safety concerns. An efficacious but less toxic balloon coating may reduce these outcomes. We evaluated a novel DEBc, Epi-Solve, coated with metacept-3 (MCT-3), a member of the histone deacetylase inhibitor (HDACi) class of epigenetic agents, in a large animal model of neointimal hyperplasia (NIH). METHODS: Plain balloon angioplasty (PABA) catheters were ultrasonically coated with MCT-3 to generate Epi-Solve DEBc. An ovine model of NIH formation was established utilising partial left common carotid artery (LCA) ligation. Twenty-eight days post neointima (NI) induction, PABA, Epi-Solve or PTX-coated DEBc were deployed at the site of induced NI formation. Twenty-eight days post-intervention, ligated vessels were evaluated for attenuation of NI formation, gene expression profiles and immunohistochemical analysis. RESULTS: Epi-Solve DEBc demonstrated attenuation of NIH over no intervention and a trend to inhibition of NIH over PABA. Gene expression analysis and immunohistochemical studies identified significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA. CONCLUSIONS: Epi-Solve is a novel HDACi-coated DEBc which demonstrates significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA in an ovine model and may afford endothelial protection.

Serelaxin inhibits the profibrotic TGF-ß1/IL-1ß axis by targeting TLR-4 and the NLRP3 inflammasome in cardiac myofibroblasts.

The recombinant form of the peptide hormone relaxin, serelaxin (RLX), mediates its anti-fibrotic actions by impeding the profibrotic activity of cytokines including TGF-ß1 and IL-1ß. As IL-1ß can be produced by the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domains-containing protein 3 (NLRP3) inflammasome, this study determined whether RLX targeted the inflammasome to inhibit the profibrotic TGF-ß1/IL-1ß axis in primary human cardiac myofibroblasts (HCMFs) in vitro and in mice with isoproterenol (ISO)-induced cardiomyopathy in vivo. HCMFs stimulated with TGF-ß1 (5 ng/ml), LPS (100 ng/ml), and ATP (5 mM) (T+L+A) for 8 h, to induce the NLRP3 inflammasome, demonstrated significantly increased protein expression of markers of NLRP3 priming (NLRP3, apoptosis-associated speck-like protein containing a C-terminal caspase-recruitment domain, procaspase-1) and activity (IL-1ß, IL-18). After 72 h, there was significantly increased neuronal NOS (nNOS), TLR-4, procaspase-1, myofibroblast differentiation, and collagen-I deposition. These measures, along with interstitial TGF-ß1 expression and collagen deposition, were also increased in the left ventricle (LV) of ISO-injured mice 14 d postinjury. RLX [16.8 nM (100 ng/ml) in vitro; 0.5 mg/kg per day in vivo] inhibited T+L+A- and ISO-induced TLR-4 expression, NLRP3 priming, IL-1ß, IL-18, myofibroblast differentiation, and interstitial collagen deposition at the time points studied, via the promotion of nNOS; with the NLRP3- and IL-1ß-inhibitory effects of RLX in HCMFs being abrogated by pharmacological blockade of nNOS or TLR-4. Comparatively, the small molecule NLRP3 inhibitor, N-{[(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)amino]carbonyl}-4-(1-hydroxy-1-methylethyl)-2-furansulfonamide (1 µM in vitro, 10 mg/kg/d in vivo), inhibited components of the NLRP3 inflammasome in vitro and in vivo and ISO-induced interstitial LV fibrosis in vivo but did not affect nNOS, TLR-4, myofibroblast differentiation, or myofibroblast-induced collagen deposition. Hence, RLX can inhibit the TGF-ß1/IL-1ß axis via a nNOS-TLR-4-NLRP3 inflammasome-dependent mechanism on cardiac myofibroblasts.-Cáceres, F. T., Gaspari, T. A., Samuel, C. S., Pinar, A. A. Serelaxin inhibits the profibrotic TGF-ß1/IL-1ß axis by targeting TLR-4 and the NLRP3 inflammasome in cardiac myofibroblasts.

Anti-fibrotic Potential of AT2 Receptor Agonists.

There are a number of therapeutic targets to treat organ fibrosis that are under investigation in preclinical models. There is increasing evidence that stimulation of the angiotensin II type 2 receptor (AT2R) is a novel anti-fibrotic strategy and we have reviewed the published in vivo preclinical data relating to the effects of compound 21 (C21), which is the only nonpeptide AT2R agonist that is currently available for use in chronic preclinical studies. In particular, the differential influence of AT2R on extracellular matrix status in various preclinical fibrotic models is discussed. Collectively, these studies demonstrate that pharmacological AT2R stimulation using C21 decreases organ fibrosis, which has been most studied in the setting of cardiovascular and renal disease. In addition, AT2R-mediated anti-inflammatory effects may contribute to the beneficial AT2R-mediated anti-fibrotic effects seen in preclinical models.

Direct AT2 receptor stimulation is athero-protective and stabilizes plaque in apolipoprotein E-deficient mice.

BACKGROUND: The angiotensin II type 2 receptor (AT2R) has been suggested to have an athero-protective role, however no studies have investigated the effect of direct stimulation of this receptor in atherosclerosis. Thus this study aimed to determine the effect of direct AT2R stimulation in setting of atherosclerosis, using the known AT2R agonist, CGP42112. METHODS AND RESULTS: Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high fat (21%) diet for 16 weeks, with subcutaneous infusions of CGP42112 (1, 5 or 10 µg/kg/min) administered via osmotic mini-pumps in the final 4 weeks. CGP42112 treatment at all doses significantly improved endothelial function (p<0.001) when compared to acetylcholine mediated-vasorelaxation in aorta taken from vehicle-treated ApoE(-/-) mice. In aortic segments adjacent to those used for vascular reactivity studies, CGP42112 treatment at all doses concomitantly increased eNOS immunoreactivity and protein levels whilst superoxide (O2(-)) production was significantly (p<0.01) decreased compared to levels measured in aorta from vehicle-treated ApoE(-/-) mice. Moreover, CGP42112 (1 µg/kg/min) treatment significantly attenuated (p<0.05) atherosclerotic lesion progression (assessed as both lipid deposits and luminal encroachment in thoracic aorta and aortic arch) and significantly increased plaque stability in the brachiocephalic artery, a region normally prone to rupture. Both the vaso- and athero-protective effects of CGP42112 (1 µg/kg/min) were reversed with co-infusion of the AT2R antagonist, PD123319, but not the MasR antagonist, A779. CONCLUSION: For the first time we have shown that direct stimulation of the AT2R improves endothelial function, reduces atherosclerotic lesion progression and mediates plaque stability with these effects at least partly due to restoration of nitric oxide bioavailability.

Chronic NaHS Treatment Is Vasoprotective in High-Fat-Fed ApoE(-/-) Mice.

Hydrogen sulfide is emerging as an important mediator of vascular function that has antioxidant and cytoprotective effects. The aim of this study was to investigate the role of endogenous H2S and the effect of chronic exogenous H2S treatment on vascular function during the progression of atherosclerotic disease. ApoE(-/-) mice were fed a high-fat diet for 16 weeks and treated with the H2S donor NaHS or the cystathionine- γ -lyase (CSE) inhibitor D,L-propargylglycine (PPG), to inhibit endogenous H2S production for the final 4 weeks. Fat-fed ApoE(-/-) mice displayed significant aortic atherosclerotic lesions and significantly impaired endothelial function compared to wild-type mice. Importantly, 4 weeks of NaHS treatment significantly reduced vascular dysfunction and inhibited vascular superoxide generation. NaHS treatment significantly reduced the area of aortic atherosclerotic lesions and attenuated systolic blood pressure. Interestingly, inhibiting endogenous, CSE-dependent H2S production with PPG did not exacerbate the deleterious vascular changes seen in the untreated fat-fed ApoE(-/-) mice. The results indicate NaHS can improve vascular function by reducing vascular superoxide generation and impairing atherosclerotic lesion development. Endogenous H2S production via CSE is insufficient to counter the atherogenic effects seen in this model; however exogenous H2S treatment has a significant vasoprotective effect.

Vasoprotective and atheroprotective effects of angiotensin (1-7) in apolipoprotein E-deficient mice.

OBJECTIVE: To evaluate the effectiveness of long-term angiotensin (Ang) (1-7) treatment to inhibit the progression of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. METHODS AND RESULTS: Ang (1-7) is a heptapeptide fragment that has been proposed to counterregulate the Ang II proatherogenic effects. The effect of long-term 4-week Ang (1-7) treatment on both inhibition of atherosclerotic lesion development and improvement of endothelial function was examined in apolipoprotein E(-/-) mice that had been fed an atherogenic high-fat (21%) diet for 16 weeks. Chronic Ang (1-7) treatment significantly improved endothelial function, an effect reversed with either angiotensin type 2 (AT(2)) or Mas receptor blockade. In these vessels, Ang (1-7) treatment significantly decreased superoxide production and increased endothelial nitric oxide synthase immunoreactivity when compared with vehicle treatment. These effects were blocked by both AT(2) and Mas receptor antagonists. Lesion development, assessed as both fatty deposits (oil red O) and intima to media ratio, was also significantly decreased with Ang (1-7) treatment compared with respective controls. Cotreatment with either AT(2) or Mas receptor antagonists reversed Ang (1-7)-mediated reduction in lesion development. CONCLUSIONS: Long-term Ang (1-7) treatment caused both vasoprotection, via improvement in endothelial function, and atheroprotection, with a reduction in lesion progression in a model of atherosclerosis. These effects appear to be mediated by the restoration of nitric oxide bioavailability and involve a complex interaction of both Mas and AT(2) receptors.

Effect of tocopheryl phosphate on key biomarkers of inflammation: Implication in the reduction of atherosclerosis progression in a hypercholesterolaemic rabbit model.

1. Many studies have evaluated the effectiveness of alpha-tocopherol (vitamin E) in the development and progression of cardiovascular diseases, with conflicting results reported on the protective effect of this anti-oxidant. 2. The present study examined the effectiveness of a novel tocopheryl phosphate mixture (TPm) compared with that of alpha-tocopherol (TA) on key pro-inflammatory markers involved in atherogenesis, including interleukin (IL)-1beta, IL-6, IL-8, plasminogen activator inhibitor-1, tumour necrosis factor-alpha and C-reactive protein (CRP), as well as vascular function and lesion development in rabbits fed a 2% cholesterol diet. 3. Treatment with TPm, incorporated into the rabbit food at four doses ranging from 60 to 360 mg/kg chow, resulted in a significant reduction in plasma levels of all pro-inflammatory cytokines and biomarkers that appeared to be somewhat dose dependent. Conversely, treatment with TA, at a dose equivalent to the highest dose of TPm used, only decreased plasma levels of CRP, IL-6 and IL-8. Both TPm and TA treatment significantly improved vascular function to a similar extent, although TPm was more effective in reducing lesion development. 4. The reduction in these key pro-inflammatory markers appears to follow the improvement in the atherogenic state of the animals, indicating that the anti-inflammatory properties of TPm may be potentially beneficial in inflammatory disease states.

AT2 receptors: functional relevance in cardiovascular disease.

The renin angiotensin system (RAS) is intricately involved in normal cardiovascular homeostasis. Excessive stimulation by the octapeptide angiotensin II contributes to a range of cardiovascular pathologies and diseases via angiotensin type 1 receptor (AT1R) activation. On the other hand, tElsevier Inc.he angiotensin type 2 receptor (AT2R) is thought to counter-regulate AT1R function. In this review, we describe the enhanced expression and function of AT2R in various cardiovascular disease settings. In addition, we illustrate that the RAS consists of a family of angiotensin peptides that exert cardiovascular effects that are often distinct from those of Ang II. During cardiovascular disease, there is likely to be an increased functional importance of AT2R, stimulated by Ang II, or even shorter angiotensin peptide fragments, to limit AT1R-mediated overactivity and cardiovascular pathologies.