Publications by authors named "Robert E Widdop"

95 Publications

Preclinical rodent models of cardiac fibrosis.

Br J Pharmacol 2021 May 10. Epub 2021 May 10.

Cardiovascular Disease Program, Monash Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Clayton, Victoria, Australia.

Cardiac fibrosis (scarring), characterised by an increased deposition of extracellular matrix (ECM) proteins, is a hallmark of most types of cardiovascular disease and plays an essential role in heart failure progression. Inhibition of cardiac fibrosis could improve outcomes in patients with cardiovascular diseases and particularly heart failure. However, pharmacological treatment of the ECM build-up is still lacking. In this context, preclinical models of heart disease are important tools for understanding the complex pathogenesis involved in the development of cardiac fibrosis which in turn could identify new therapeutic targets and the facilitation of antifibrotic drug discovery. Many preclinical models have been used to study cardiac fibrosis and each model provides mechanistic insights into the many factors that contribute to cardiac fibrosis. This review discusses the most frequently used rodent models of cardiac fibrosis and also provides context for the use of particular models of heart failure.
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http://dx.doi.org/10.1111/bph.15450DOI Listing
May 2021

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

Hypertension 2021 Jul 10;78(1):128-137. Epub 2021 May 10.

Cardiovascular Disease Program, Monash Biomedicine Discovery Institute (G.B., K.M.M.C., S.L.W., T.A.G., I.S., A.A.P., L.M.H.K., R.E.W., C.S.S., K.M.D.), Monash University, Melbourne, Victoria, Australia.

[Figure: see text].
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.121.17164DOI Listing
July 2021

Using conformational constraints at position 6 of Angiotensin II to generate compounds with enhanced AT2R selectivity and proteolytic stability.

Bioorg Med Chem Lett 2021 Jul 7;43:128086. Epub 2021 May 7.

Laboratory of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina 45110, Greece; University Research Center of Ioannina (URCI), Institute of Materials Science and Computing, Ioannina, Greece. Electronic address:

The Renin-Angiotensin System (RAS) plays a crucial role in numerous pathological conditions. Two of the critical RAS players, the angiotensin receptors AT1R and AT2R, possess differential functional profiles, although they share high sequence similarity. Although the main focus has been placed on AT1R, several epidemiological studies have evidenced that activation of AT2R could operate as a multimodal therapeutic target for different diseases. Thus, the development of selective AT2R ligands could have a high clinical potential for different therapeutic directions. Furthermore, they could serve as a powerful tool to interrogate the molecular mechanisms that are mediated by AT2R. Based on our recently established high affinity and AT2R selective compound [Y]-AII we developed several analogues through modifying aminoacids located at positions 6 and 7 with various conformationally constrained analogues to enhance both the selectivity and stability. We report the development of high-affinity AT2R binders, which displayed high selectivity for AT2R versus AT1R. Furthermore, all analogues presented enhanced stability in human plasma with respect to the parent hormone Angiotensin II as also [Y]-AII.
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http://dx.doi.org/10.1016/j.bmcl.2021.128086DOI Listing
July 2021

Assessment of renal fibrosis and anti-fibrotic agents using a novel diagnostic and stain-free second-harmonic generation platform.

FASEB J 2021 05;35(5):e21595

Cardiovascular Disease Program, Monash Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Melbourne, VIC, Australia.

Current histological measurement techniques for interstitial collagen, the basis of interstitial fibrosis, are semi-quantitative at best and only provide a ratio of collagen levels within tissues. The Genesis200 imaging system and supplemental image analysis software, FibroIndex from HistoIndex, is a novel, automated platform that uses second-harmonic generation (SHG) for imaging and characterization of interstitial collagen deposition and additional characteristics, in the absence of any staining. However, its ability to quantify renal fibrosis requires investigation. This study compared SHG imaging of renal fibrosis in mice with unilateral ureteric obstruction (UUO), to that of Masson's trichrome staining (MTS) and immunohistochemistry (IHC) of collagen I. Additionally, the platform generated data on collagen morphology and distribution patterns. While all three methods determined that UUO-injured mice underwent significantly increased renal fibrosis after 7 days, the HistoIndex platform additionally determined that UUO-injured mice had a significantly increased collagen-to-tissue cross reticulation ratio (all P < .001 vs sham group). Furthermore, in UUO-injured mice treated with the relaxin family peptide receptor-1 agonists, relaxin (0.5 mg/kg/day) or B7-33 (0.25 mg/kg/day), or angiotensin converting enzyme-inhibitor, perindopril (1 mg/kg/day) over the 7-day period, only the HistoIndex platform determined that the drug-induced prevention of renal fibrosis correlated with significantly reduced collagen fiber thickness and collagen-to-tissue cross reticulation ratio, but increased collagen fiber counts. Relaxin or B7-33 treatment also increased renal matrix metalloproteinase-2 and reduced tissue inhibitor of metalloproteinase-1 levels (all P < .01 vs UUO alone). This study demonstrated the diagnostic value of the HistoIndex platform over currently used staining techniques.
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http://dx.doi.org/10.1096/fj.202002053RRRDOI Listing
May 2021

Combining mesenchymal stem cells with serelaxin provides enhanced renoprotection against 1K/DOCA/salt-induced hypertension.

Br J Pharmacol 2021 03;178(5):1164-1181

Cardiovascular Disease Program, Monash University, Clayton, Victoria, Australia.

Background And Purpose: Fibrosis is a hallmark of chronic kidney disease (CKD) that significantly contributes to renal dysfunction, and impairs the efficacy of stem cell-based therapies. This study determined whether combining bone marrow-derived mesenchymal stem cells (BM-MSCs) with the renoprotective effects of recombinant human relaxin (serelaxin) could therapeutically reduce renal fibrosis in mice with one kidney/deoxycorticosterone acetate/salt (1K/DOCA/salt)-induced hypertension, compared with the effects of the ACE inhibitor, perindopril.

Experimental Approach: Adult male C57BL/6 mice were uni-nephrectomised and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt) for 21 days. Control mice were uni-nephrectomised but received water over the same time period. Sub-groups of 1K/DOCA/salt-injured mice (n = 5-8 per group) were treated with either serelaxin (0.5 mg·kg ·day ) or BM-MSCs (1 × 10 per mouse) alone; both treatments combined (with 0.5 × 10 or 1 × 10 BM-MSCs per mouse); or perindopril (2 mg·kg ·day ) from days 14-21.

Key Results: 1K/DOCA/salt-injured mice developed elevated BP and hypertension-induced renal damage, inflammation and fibrosis. BM-MSCs alone reduced the injury-induced fibrosis and attenuated BP to a similar extent as perindopril. Serelaxin alone modestly reduced renal fibrosis and effectively reduced tubular injury. Strikingly, the combined effects of BM-MSCs (at both doses) with serelaxin significantly inhibited renal fibrosis and proximal tubular epithelial injury while restoring renal architecture, to a greater extent than either therapy alone, and over the effects of perindopril.

Conclusion And Implications: Combining BM-MSCs and serelaxin provided broader renoprotection over either therapy alone or perindopril and might represent a novel treatment for hypertensive CKD.
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http://dx.doi.org/10.1111/bph.15361DOI Listing
March 2021

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

Bioorg Med Chem 2021 01 7;29:115859. Epub 2020 Nov 7.

Department of Medicinal Chemistry, Science for Life Laboratory, BMC, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden; The Beijer Laboratory, Department of Medicinal Chemistry, Uppsala University, BMC, Box 591, 751 24 Uppsala, Sweden. Electronic address:

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 K of 9.3 nM, that demonstrates a high stability in human liver microsomes (t = 62 min) and in human hepatocytes (t = 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 ATR prototype antagonist 3 (C38). Ligand 20 acts as an ATR agonist and caused an ATR 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 ATR 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 ATR ligands were explored by docking calculations combined with molecular dynamics simulations.
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http://dx.doi.org/10.1016/j.bmc.2020.115859DOI Listing
January 2021

Enhancement of glioblastoma multiforme therapy through a novel Quercetin-Losartan hybrid.

Free Radic Biol Med 2020 11 18;160:391-402. Epub 2020 Aug 18.

University of Ioannina, Section of Organic Chemistry and Biochemistry, Department of Chemistry, Ioannina, Greece; University Research Center of Ioannina (URCI), Institute of Materials Science and Computing, Ioannina, Greece. Electronic address:

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor. Maximal surgical resection followed by radiotherapy and concomitant chemotherapy with temozolomide remains the first-line therapy, prolonging the survival of patients by an average of only 2.5 months. There is therefore an urgent need for novel therapeutic strategies to improve clinical outcomes. Reactive oxygen species (ROS) are an important contributor to GBM development. Here, we describe the rational design and synthesis of a stable hybrid molecule tethering two ROS regulating moieties, with the aim of constructing a chemopreventive and anticancer chemical entity that retains the properties of the parent compounds. We utilized the selective ATR antagonist losartan, leading to the inhibition of ROS levels, and the antioxidant flavonoid quercetin. In GBM cells, we show that this hybrid retains the binding potential of losartan to the ATR through competition-binding experiments and simultaneously exhibits ROS inhibition and antioxidant capacity similar to native quercetin. In addition, we demonstrate that the hybrid is able to alter the cell cycle distribution of GBM cells, leading to cell cycle arrest and to the induction of cytotoxic effects. Last, the hybrid significantly and selectively reduces cancer cell proliferation and angiogenesis in primary GBM cultures with respect to the isolated parent components or their simple combination, further emphasizing the potential utility of the current hybridization approach in GBM.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.08.007DOI Listing
November 2020

Single Peptide Backbone Surrogate Mutations to Regulate Angiotensin GPCR Subtype Selectivity.

Chemistry 2020 Aug 21;26(47):10690-10694. Epub 2020 Jul 21.

Department of Chemistry, University of Ioannina, Ioannina, 45110, Greece.

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y] -Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT R/AT R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.
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http://dx.doi.org/10.1002/chem.202000924DOI Listing
August 2020

The anti-fibrotic actions of relaxin are mediated through AT R-associated protein phosphatases via RXFP1-AT R functional crosstalk in human cardiac myofibroblasts.

FASEB J 2020 06 16;34(6):8217-8233. Epub 2020 Apr 16.

Cardiovascular Disease Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia.

Fibrosis is a hallmark of several cardiovascular diseases. The relaxin family peptide receptor 1 (RXFP1) agonist, relaxin, has rapidly occurring anti-fibrotic actions which are mediated through RXFP1 and angiotensin II receptor crosstalk on renal and cardiac myofibroblasts. Here, we investigated whether this would allow relaxin to indirectly activate angiotensin II type 2 receptor (AT R)-specific signal transduction in primary human cardiac myofibroblasts (HCMFs). The anti-fibrotic effects of recombinant human relaxin (RLX; 16.8 nM) or the AT R-agonist, Compound 21 (C21; 1 μM), were evaluated in TGF-β1-stimulated HCMFs, in the absence or presence of an RXFP1 antagonist (1 μM) or AT R antagonist (0.1 μM) to confirm RXFP1-AT R crosstalk. Competition binding for RXFP1 was determined. Western blotting was performed to determine which AT R-specific protein phosphatases were expressed by HCMFs; then, the anti-fibrotic effects of RLX and/or C21 were evaluated in the absence or presence of pharmacological inhibition (NSC95397 (1 μM) for MKP-1; okadaic acid (10 nM) for PP2A) or siRNA-knockdown of these phosphatases after 72 hours. The RLX- or C21-induced increase in ERK1/2 and nNOS phosphorylation, and decrease in α-SMA (myofibroblast differentiation) and collagen-I expression by HCMFs was abrogated by pharmacological blockade of RXFP1 or the AT R, confirming RXFP1-AT R crosstalk in these cells. HCMFs were found to express AT R-dependent MKP-1 and PP2A phosphatases, while pharmacological blockade or siRNA-knockdown of either phosphatase also abolished RLX and/or C21 signal transduction in HCMFs (all P < .05 vs RLX or C21 alone). These findings demonstrated that RLX can indirectly activate AT R-dependent phosphatase activity in HCMFs by signaling through RXFP1-AT R crosstalk, which have important therapeutic implications for its anti-fibrotic actions.
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http://dx.doi.org/10.1096/fj.201902506RDOI Listing
June 2020

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

ACS Pharmacol Transl Sci 2020 Feb 23;3(1):76-87. Epub 2020 Jan 23.

Cardiovascular Disease Program, Biomedicine Discovery Institute, Department of Pharmacology, and Department of Physiology, Monash University, Clayton, Victoria 3800 Australia.

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 ATR agonist CGP42112 (CGP) were compared with those of the established ATR 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 ATR stimulation represents novel therapeutic strategies to target fibrotic conditions, particularly in HS states that may be refractory to ATR blockade.
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http://dx.doi.org/10.1021/acsptsci.9b00095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088889PMC
February 2020

Renal functional effects of the highly selective AT2R agonist, β-Pro7 Ang III, in normotensive rats.

Clin Sci (Lond) 2020 04;134(7):871-884

Department of Pharmacology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

Recently, we designed a group of peptides by sequential substitution of the naturally occurring α-amino acid throughout the Ang III peptide sequence with the corresponding β-amino acid. β-Amino acid substitution at the proline residue of Ang III (β-Pro7-Ang III) resulted in a highly selective AT2R ligand, demonstrating remarkable selectivity for the AT2R in both binding and functional studies. To provide additional functional evidence for the suitability of β-Pro7 Ang III as a novel AT2R agonist, we tested effects of acute systemic administration of β-Pro7-Ang III on renal hemodynamic and excretory function in anesthetized normotensive male and female rats. We also compared the natriuretic effects of acute intrarenal administration of native Ang III and β-Pro7-Ang III in the presence of systemic AT1R blockade in anesthetized female rats to allow for the differentiation of systemic versus direct intrarenal natriuretic actions of β-Pro7-Ang III. In both male and female rats, acute systemic administration of β-Pro7-Ang III elicited renal vasodilatation and natriuresis. Notably, greater renal vasodilatory effects were observed in female versus male rats at the highest dose of β-Pro7-Ang III administered. Moreover, intra-renal administration of β-Pro7-Ang III produced significant natriuretic effects in female rats and, like Ang III, evoked AT2R translocation to the apical plasma membrane in renal proximal tubular cells. Taken together, our findings support the use of β-Pro7-Ang III as a novel AT2R agonist and experimental tool for exploring AT2R function and its potential as a therapeutic target. Furthermore, our findings provide further evidence of a sex-specific influence of AT2R stimulation on renal function.
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http://dx.doi.org/10.1042/CS20200153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158249PMC
April 2020

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

Cardiovasc Drugs Ther 2019 12;33(6):687-692

Eastern Health Clinical School, Monash University, Clayton, Australia.

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.
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http://dx.doi.org/10.1007/s10557-019-06921-wDOI Listing
December 2019

AT1R-AT2R-RXFP1 Functional Crosstalk in Myofibroblasts: Impact on the Therapeutic Targeting of Renal and Cardiac Fibrosis.

J Am Soc Nephrol 2019 11 11;30(11):2191-2207. Epub 2019 Sep 11.

Department of Biochemistry and Molecular Biology, and

Background: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (ATR) to ameliorate renal fibrogenesis and . Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (ATR), which is expressed on myofibroblasts along with RXFP1 and ATR, is unknown.

Methods: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts , and in three models of renal- or cardiomyopathy-induced fibrosis .

Results: The ATR blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin RXFP1 and . Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to ATRs but that constitutive ATR-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an ATR agonist (compound 21).

Conclusions: These findings have significant implications for the concomitant use of RXFP1 or ATR agonists with ATR blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.
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http://dx.doi.org/10.1681/ASN.2019060597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830801PMC
November 2019

Morphology and Function of the Lamb Ileum following Preterm Birth.

Front Pediatr 2018 29;6. Epub 2018 Jan 29.

The Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

Background: For infants born moderately/late preterm (32-37 weeks of gestation), immaturity of the intestine has the potential to impact both short- and long-term gastrointestinal function. The aim of this study conducted in sheep was to compare the morphology and smooth muscle contractility of the ileum in term and late preterm lambs.

Materials And Methods: Lambs delivered preterm (132 days gestation;  = 7) or term (147 days gestation;  = 9) were milk-fed after birth and euthanased at 2 days of age. A segment of distal ileum was collected for analysis of the length and cellular composition of the villi and crypts, smooth muscle width and contractility, and mRNA expression of the cell markers Ki67, lysozyme, mucin 2, synaptophysin, chromogranin A, olfactomedin 4, axis inhibition protein 2, and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5).

Results: There was no difference in the proportion of inflammatory, proliferating, apoptotic, enterocyte, or goblet cells between groups, but preterm lambs exhibited a significant upregulation of the stem cell marker LGR5 ( = 0.01). Absolute villus height (term: 1,032 ± 147 µm, preterm: 651 ± 52 µm;  < 0.0001) and crypt depth (term: 153 ± 11 µm, preterm: 133 ± 17 µm;  = 0.01) were significantly shorter in the preterm ileums, with a trend ( = 0.06) for a reduction in muscularis externa width. There was no difference between groups in the contractile response to acetylcholine, but peak contractility in response to bradykinin ( = 0.02) and angiotensin II ( = 0.03) was significantly greater in the preterm lambs.

Conclusion: Findings demonstrate that the crypt-villus units are shorter in the ileum of late preterm offspring, but functionally mature with an equivalent cellular composition and normal contractile response to acetylcholine compared with term offspring. The exaggerated contractility to inflammatory mediators evident in the preterm ileum, however, may be of concern.
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http://dx.doi.org/10.3389/fped.2018.00008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5810295PMC
January 2018

Dapagliflozin attenuates human vascular endothelial cell activation and induces vasorelaxation: A potential mechanism for inhibition of atherogenesis.

Diab Vasc Dis Res 2018 01 4;15(1):64-73. Epub 2017 Oct 4.

2 Eastern Health Clinical School and Department of Medicine, Monash University, Melbourne, VIC, Australia.

Background: Sodium glucose transporter type 2 inhibitors may reduce cardiovascular events in type 2 diabetes. Our study aimed to determine the effect of the sodium glucose transporter type 2 inhibitor dapagliflozin on endothelial cell activation, vasoreactivity and atherogenesis using in vitro and in vivo models and identify associated molecular mechanisms.

Methods: In vitro studies utilised human vascular endothelial cells stimulated with tumour necrosis factor α or hyperglycaemic conditions. In vivo studies were performed in C57Bl/6J mice to evaluate direct vasorelaxation responses evoked by acute dapagliflozin administration and acute vaso-protective effects of dapagliflozin on hyperglycaemia-induced endothelial dysfunction. Adult and aged Apolipoprotein E-deficient mice maintained on a high-fat diet were used to investigate endothelial-dependent vascular reactivity and atherogenesis. Dapagliflozin treatment (1.0 mg/kg/day) was administered for 4 weeks.

Results: In vitro studies demonstrated dapagliflozin-mediated attenuation of tumour necrosis factor α- and hyperglycaemia-induced increases in intercellular adhesion molecule-1, vascular cell adhesion molecule-1, plasminogen activator inhibitor type 1 and NFκB expression. Acute dapagliflozin administration dose-dependently induced endothelium-independent vasorelaxation. Chronic dapagliflozin treatment improved endothelial function and significantly reduced in vivo vascular adhesion molecule and phospho-IκB expression together with macrophage vessel wall infiltration.

Conclusion: These observations identify a potential role for dapagliflozin in the attenuation of atherogenesis and identify anti-inflammatory molecular mechanisms associated with these effects.
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http://dx.doi.org/10.1177/1479164117733626DOI Listing
January 2018

Anti-fibrotic Potential of AT Receptor Agonists.

Front Pharmacol 2017 31;8:564. Epub 2017 Aug 31.

Department of Pharmacology, Cardiovascular Disease Program, Biomedicine Discovery Institute, Monash University, ClaytonVIC, Australia.

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 (ATR) is a novel anti-fibrotic strategy and we have reviewed the published preclinical data relating to the effects of compound 21 (C21), which is the only nonpeptide ATR agonist that is currently available for use in chronic preclinical studies. In particular, the differential influence of ATR on extracellular matrix status in various preclinical fibrotic models is discussed. Collectively, these studies demonstrate that pharmacological ATR stimulation using C21 decreases organ fibrosis, which has been most studied in the setting of cardiovascular and renal disease. In addition, ATR-mediated anti-inflammatory effects may contribute to the beneficial ATR-mediated anti-fibrotic effects seen in preclinical models.
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http://dx.doi.org/10.3389/fphar.2017.00564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583590PMC
August 2017

Potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acid-sensing ion channel 1a.

Proc Natl Acad Sci U S A 2017 04 20;114(14):3750-3755. Epub 2017 Mar 20.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia;

Stroke is the second-leading cause of death worldwide, yet there are no drugs available to protect the brain from stroke-induced neuronal injury. Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and a key mediator of acidosis-induced neuronal damage following cerebral ischemia. Genetic ablation and selective pharmacologic inhibition of ASIC1a reduces neuronal death following ischemic stroke in rodents. Here, we demonstrate that Hi1a, a disulfide-rich spider venom peptide, is highly neuroprotective in a focal model of ischemic stroke. Nuclear magnetic resonance structural studies reveal that Hi1a comprises two homologous inhibitor cystine knot domains separated by a short, structurally well-defined linker. In contrast with known ASIC1a inhibitors, Hi1a incompletely inhibits ASIC1a activation in a pH-independent and slowly reversible manner. Whole-cell, macropatch, and single-channel electrophysiological recordings indicate that Hi1a binds to and stabilizes the closed state of the channel, thereby impeding the transition into a conducting state. Intracerebroventricular administration to rats of a single small dose of Hi1a (2 ng/kg) up to 8 h after stroke induction by occlusion of the middle cerebral artery markedly reduced infarct size, and this correlated with improved neurological and motor function, as well as with preservation of neuronal architecture. Thus, Hi1a is a powerful pharmacological tool for probing the role of ASIC1a in acid-mediated neuronal injury and various neurological disorders, and a promising lead for the development of therapeutics to protect the brain from ischemic injury.
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http://dx.doi.org/10.1073/pnas.1614728114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389327PMC
April 2017

Novel approaches for treating hypertension.

F1000Res 2017 27;6:80. Epub 2017 Jan 27.

Department of Pharmacology and Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia.

Hypertension, or high blood pressure, is a prevalent yet modifiable risk factor for cardiovascular disease. While there are many effective treatments available to combat hypertension, patients often require at least two to three medications to control blood pressure, although there are patients who are resistant to such therapies. This short review will briefly update on recent clinical advances and potential emerging therapies and is intended for a cross-disciplinary readership.
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http://dx.doi.org/10.12688/f1000research.10117.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288674PMC
January 2017

Angiotensin AT2-receptor stimulation improves survival and neurological outcome after experimental stroke in mice.

J Mol Med (Berl) 2016 08 16;94(8):957-66. Epub 2016 Mar 16.

Department of Cardiovascular and Renal Research, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark.

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.
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http://dx.doi.org/10.1007/s00109-016-1406-3DOI Listing
August 2016

Decorated self-assembling β(3)-tripeptide foldamers form cell adhesive scaffolds.

Chem Commun (Camb) 2016 Mar;52(24):4549-52

Department of Biochemistry & Molecular Biology, Monash University, Clayton, VIC, Australia.

A popular strategy to control cellular growth and differentiation is the employment of self-assembling peptides as biomaterials. In this study we decorated ultrashort helical N-acetylated β-tripeptides with cell adhesion signals IKVAV and RGD, which spontaneously self-assemble to give nanofibres with multiple signals, and form a bioscaffold that supports the growth of cells.
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http://dx.doi.org/10.1039/c6cc00247aDOI Listing
March 2016

Effect of a Selective Mas Receptor Agonist in Cerebral Ischemia In Vitro and In Vivo.

PLoS One 2015 5;10(11):e0142087. Epub 2015 Nov 5.

Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria, 3800, Australia.

Functional modulation of the non-AT1R arm of the renin-angiotensin system, such as via AT2R activation, is known to improve stroke outcome. However, the relevance of the Mas receptor, which along with the AT2R forms the protective arm of the renin-angiotensin system, as a target in stroke is unclear. Here we tested the efficacy of a selective MasR agonist, AVE0991, in in vitro and in vivo models of ischemic stroke. Primary cortical neurons were cultured from E15-17 mouse embryos for 7-9 d, subjected to glucose deprivation for 24 h alone or with test drugs, and percentage cell death was determined using trypan blue exclusion assay. Additionally, adult male mice were subjected to 1 h middle cerebral artery occlusion and were administered either vehicle or AVE0991 (20 mg/kg i.p.) at the commencement of 23 h reperfusion. Some animals were also treated with the MasR antagonist, A779 (80 mg/kg i.p.) 1 h prior to surgery. Twenty-four h after MCAo, neurological deficits, locomotor activity and motor coordination were assessed in vivo, and infarct and edema volumes estimated from brain sections. Following glucose deprivation, application of AVE0991 (10-8 M to 10-6 M) reduced neuronal cell death by ~60% (P<0.05), an effect prevented by the MasR antagonist. By contrast, AVE0991 administration in vivo had no effect on functional or histological outcomes at 24 h following stroke. These findings indicate that the classical MasR agonist, AVE0991, can directly protect neurons from injury following glucose-deprivation. However, this effect does not translate into an improved outcome in vivo when administered systemically following stroke.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142087PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634944PMC
June 2016

Molecular and cellular mechanisms of glucagon-like peptide-1 receptor agonist-mediated attenuation of cardiac fibrosis.

Diab Vasc Dis Res 2016 Jan 25;13(1):56-68. Epub 2015 Sep 25.

Department of Medicine, Monash University, Melbourne, VIC, Australia

Background: Glucagon-like peptide-1 receptor agonists may have a role in modulation of cardiac fibrosis. Our study aimed to determine the effect of the glucagon-like peptide-1 receptor agonist liraglutide in obesity, hypertension and age-induced murine models of cardiac fibrosis and identify associated molecular mechanisms.

Methods: C57Bl/6J mice on a high-fat diet and C57Bl/6J mice on a normal chow diet treated with angiotensin II were used to induce obesity and hypertension-mediated cardiac fibrosis, respectively. C57Bl/6J mice 20 months old were used to study age-induced cardiac fibrosis. Liraglutide treatment of 30 µg/kg/day-300 µg/kg s.c. twice daily was administered for 4 weeks.

Results: Liraglutide treatment attenuated obesity, hypertension and age-induced increases in interstitial cardiac fibrosis and expression of inflammatory and oxidative stress markers.

Conclusions: These observations identify a potential role for liraglutide in the prevention of cardiac fibrosis and identify molecular mechanisms associated with these effects.
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http://dx.doi.org/10.1177/1479164115605000DOI Listing
January 2016

PcTx1 affords neuroprotection in a conscious model of stroke in hypertensive rats via selective inhibition of ASIC1a.

Neuropharmacology 2015 Dec 29;99:650-7. Epub 2015 Aug 29.

Department of Pharmacology, Monash University, Clayton, Vic 3800, Australia. Electronic address:

Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and plays a major role in neuronal injury following cerebral ischemia. Evidence that inhibition of ASIC1a might be neuroprotective following stroke was previously obtained using "PcTx1 venom" from the tarantula Psalmopeous cambridgei. We show here that the ASIC1a-selective blocker PcTx1 is present at only 0.4% abundance in this venom, leading to uncertainty as to whether the observed neuroprotective effects were due to PcTx1 blockade of ASIC1a or inhibition of other ion channels and receptors by the hundreds of peptides and small molecules present in the venom. We therefore examined whether pure PcTx1 is neuroprotective in a conscious model of stroke via direct inhibition of ASIC1a. A focal reperfusion model of stroke was induced in conscious spontaneously hypertensive rats (SHR) by administering endothelin-1 to the middle cerebral artery via a surgically implanted cannula. Two hours later, SHR were treated with a single intracerebroventricular (i.c.v.) dose of PcTx1 (1 ng/kg), an ASIC1a-inactive mutant of PcTx1 (1 ng/kg), or saline, and ledged beam and neurological tests were used to assess the severity of symptomatic changes. PcTx1 markedly reduced cortical and striatal infarct volumes measured 72 h post-stroke, which correlated with improvements in neurological score, motor function and preservation of neuronal architecture. In contrast, the inactive PcTx1 analogue had no effect on stroke outcome. This is the first demonstration that selective pharmacological inhibition of ASIC1a is neuroprotective in conscious SHRs, thus validating inhibition of ASIC1a as a potential treatment for stroke.
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http://dx.doi.org/10.1016/j.neuropharm.2015.08.040DOI Listing
December 2015

β-Pro7Ang III is a novel highly selective angiotensin II type 2 receptor (AT2R) agonist, which acts as a vasodepressor agent via the AT2R in conscious spontaneously hypertensive rats.

Clin Sci (Lond) 2015 Sep 8;129(6):505-13. Epub 2015 May 8.

Departments of Pharmacology, Monash University, Melbourne, VIC 3800, Australia.

We have previously shown that individual β-amino acid substitution in angiotensin (Ang) II reduced Ang II type 1 receptor (AT1R) but not Ang II type 2 receptor (AT2R)-binding and that the heptapeptide Ang III exhibited greater AT2R:AT1R selectivity than Ang II. Therefore, we hypothesized that β-amino-acid-substituted Ang III peptide analogues would yield highly selective AT2R ligands, which we have tested in binding and functional vascular assays. In competition binding experiments using either AT1R- or AT2R-transfected human embryonic kidney (HEK)-293 cells, novel β-substituted Ang III analogues lacked appreciable AT1R affinity, whereas most compounds could fully displace (125)I-Sar(1)Ile(8) Ang II from AT2R. The rank order of affinity at AT2R was CGP42112 > Ang III > β-Pro(7) Ang III=Ang II > β-Tyr(4) Ang III ≥ PD123319 >> β-Phe(8) Ang III >> β Arg(2) Ang III=β-Val(3) Ang III >> β-Ile(5) Ang III. The novel analogue β-Pro(7) Ang III was the most selective AT2R ligand tested, which was >20,000-fold more selective for AT2R than AT1R. IC50 values at AT2R from binding studies correlated with maximum vasorelaxation in mouse aortic rings. Given that β-Pro(7) Ang III was an AT2R agonist, we compared β-Pro(7) Ang III and native Ang III for their ability to reduce blood pressure in separate groups of conscious spontaneously hypertensive rats. Whereas Ang III alone increased mean arterial pressure (MAP), β-Pro(7) Ang III had no effect. During low-level AT1R blockade, both Ang III and β-Pro(7) Ang III, but not Ang II, lowered MAP (by ∼30 mmHg) at equimolar infusions (150 pmol/kg/min for 4 h) and these depressor effects were abolished by the co-administration of the AT2R antagonist PD123319. Thus, β-Pro(7) Ang III has remarkable AT2R selectivity determined in binding and functional studies and will be a valuable research tool for insight into AT2R function and for future drug development.
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http://dx.doi.org/10.1042/CS20150077DOI Listing
September 2015

Structural determinants for binding to angiotensin converting enzyme 2 (ACE2) and angiotensin receptors 1 and 2.

Front Pharmacol 2015 30;6. Epub 2015 Jan 30.

Department of Biochemistry and Molecular Biology, Monash University Clayton, VIC, Australia.

Angiotensin converting enzyme 2 (ACE2) is a zinc carboxypeptidase involved in the renin-angiotensin system (RAS) and inactivates the potent vasopressive peptide angiotensin II (Ang II) by removing the C-terminal phenylalanine residue to yield Ang1-7. This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule at the Mas receptor and potentially other receptors. Given the growing complexity of RAS and level of cross-talk between ligands and their corresponding enzymes and receptors, the design of molecules with selectivity for the major RAS binding partners to control cardiovascular tone is an on-going challenge. In previous studies we used single β-amino acid substitutions to modulate the structure of Ang II and its selectivity for ACE2, AT1R, and angiotensin type 2 (AT2R) receptor. We showed that modification at the C-terminus of Ang II generally resulted in more pronounced changes to secondary structure and ligand binding, and here, we further explore this region for the potential to modulate ligand specificity. In this study, (1) a library of 47 peptides derived from the C-terminal tetrapeptide sequence (-IHPF) of Ang II was synthesized and assessed for ACE2 binding, (2) the terminal group requirements for high affinity ACE2 binding were explored by and N- and C-terminal modification, (3) high affinity ACE2 binding chimeric AngII analogs were then synthesized and assessed, (4) the structure of the full-length Ang II analogs were assessed by circular dichroism, and (5) the Ang II analogs were assessed for AT1R/AT2R selectivity by cell-based assays. Studies on the C-terminus of Ang II demonstrated varied specificity at different residue positions for ACE2 binding and four Ang II chimeric peptides were identified as selective ligands for the AT2 receptor. Overall, these results provide insight into the residue and structural requirements for ACE2 binding and angiotensin receptor selectivity.
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http://dx.doi.org/10.3389/fphar.2015.00005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311625PMC
February 2015

Compound 21, a selective agonist of angiotensin AT2 receptors, prevents endothelial inflammation and leukocyte adhesion in vitro and in vivo.

Br J Pharmacol 2016 Feb 29;173(4):729-40. Epub 2015 Jun 29.

Vascular Pharmacology, Baker IDI Heart and Diabetes Institute, Melbourne, Vic., Australia.

Background And Purpose: Angiotensin AT2 receptors are upregulated in disease states such as atherosclerosis and blockade of the AT2 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 AT2 receptor stimulation with Compound 21 (C21) on the leukocyte adhesion cascade in vitro, right through to plaque formation in vivo.

Experimental Approach: Effects of C21 on TNFα-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 Results: C21 attenuated TNFα-induced: monocyte adhesion to cultured HUVECs, adhesion molecule expression and abolished TNFα-induced ROS production. TNFα-induced NFκB 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 TNFα and IL-6 mRNA expression in M1 macrophages. The anti-inflammatory effects of C21 were abolished by an AT2 receptor antagonist confirming that the effects of C21 were AT2 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 Implications: C21 prevented TNFα-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 AT2 receptors.
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http://dx.doi.org/10.1111/bph.13063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742292PMC
February 2016

Differential phenotypes of tissue-infiltrating T cells during angiotensin II-induced hypertension in mice.

PLoS One 2014 11;9(12):e114895. Epub 2014 Dec 11.

Department of Pharmacology, Monash University, Clayton, Victoria, Australia.

Hypertension remains the leading risk factor for cardiovascular disease (CVD). Experimental hypertension is associated with increased T cell infiltration into blood pressure-controlling organs, such as the aorta and kidney; importantly in absence of T cells of the adaptive immune system, experimental hypertension is significantly blunted. However, the function and phenotype of these T cell infiltrates remains speculative and undefined in the setting of hypertension. The current study compared T cell-derived cytokine and reactive oxygen species (ROS) production from normotensive and hypertensive mice. Splenic, blood, aortic, kidney and brain T cells were isolated from C57BL/6J mice following 14-day vehicle or angiotensin (Ang) II (0.7 mg/kg/day, s.c.) infusion. T cell infiltration was increased in aorta, kidney and brain from hypertensive mice. Cytokine analysis in stimulated T cells indicated an overall Th1 pro-inflammatory phenotype, but a similar proportion (flow cytometry) and quantity (cytometric bead array) of IFN-γ, TNF-α, IL-4 and IL-17 between vehicle- and Ang II- treated groups. Strikingly, elevated T cell-derived production of a chemokine, chemokine C-C motif ligand 2 (CCL2), was observed in aorta (∼6-fold) and kidney in response to Ang II, but not in brain, spleen or blood. Moreover, T cell-derived ROS production in aorta was elevated ∼3 -fold in Ang II-treated mice (n = 7; P<0.05). Ang II-induced hypertension does not affect the overall T cell cytokine profile, but enhanced T cell-derived ROS production and/or leukocyte recruitment due to elevated CCL2, and this effect may be further amplified with increased infiltration of T cells. We have identified a potential hypertension-specific T cell phenotype that may represent a functional contribution of T cells to the development of hypertension, and likely several other associated vascular disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114895PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263711PMC
December 2015

Sex- and age-related differences in the chronic pressure-natriuresis relationship: role of the angiotensin type 2 receptor.

Am J Physiol Renal Physiol 2014 Oct 27;307(8):F901-7. Epub 2014 Aug 27.

Department of Physiology, Monash University, Clayton, Victoria, Australia;

Sex hormones regulate the renin-angiotensin system. For example, estrogen enhances expression of the angiotensin type 2 receptor. We hypothesized that activation of the angiotensin type 2 receptor shifts the chronic pressure-natriuresis relationship leftward in females compared with males and that this effect is lost with age. Mean arterial pressure was measured by radiotelemetry in adult (4 mo old) and aged (14 mo old) wild-type and angiotensin type 2 receptor knockout male and female mice. Chronic pressure-natriuresis curves were constructed while mice were maintained on a normal-salt (0.26%) diet and following 6 days of high salt (5.0%) diet. Mean arterial pressure was lower in adult wild-type females than males (88 ± 1 and 97 ± 1 mmHg, respectively), a difference that was maintained with age, but was absent in adult knockout mice. In wild-type females, the chronic pressure-natriuresis relationship was shifted leftward compared with knockout females, an effect that was lost with age. In males, the chronic pressure-natriuresis relationship was not influenced by angiotensin type 2 receptor deficiency. Compared with age-matched females, the chronic pressure-natriuresis relationships in male mice were shifted rightward. Renal expression of the angiotensin type 2 receptor was fourfold greater in adult wild-type females than males. With age, the angiotensin type 2 receptor-to-angiotensin type 1 receptor balance was reduced in females. Conversely, in males, angiotensin receptor expression did not vary significantly with age. In conclusion, the angiotensin type 2 receptor modulates the chronic pressure-natriuresis relationship in an age- and sex-dependent manner.
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http://dx.doi.org/10.1152/ajprenal.00288.2014DOI Listing
October 2014

Role of inflammation and the angiotensin type 2 receptor in the regulation of arterial pressure during pregnancy in mice.

Hypertension 2014 Sep 16;64(3):626-31. Epub 2014 Jun 16.

From the Department of Physiology (K.M.M., L.M.H., K.M.D.) and Department of Pharmacology (Z.W., R.E.W., A.V.), Monash University, Clayton, Victoria, Australia; and Baker Medical Research Institute, Melbourne, Victoria, Australia (C.T.).

During normal pregnancy the renin-angiotensin system is activated, yet pregnant women are resistant to the pressor effects of angiotensin II. Our aim was to determine the role of the angiotensin type 2 receptor (AT2R) in the regulation of arterial pressure, natriuresis, and immune cell infiltration during pregnancy. Mean arterial pressure was measured via telemetry, and flow cytometry was used to enumerate immune cell infiltration in 14-week-old wild-type and AT2R knockout mice during gestation. In wild-type mice, mean arterial pressure decreased during gestation, reaching a nadir at gestational day 9 (-6±2 mm Hg) and returned to near preconception levels during late gestation. In AT2R-deficient mice, the midgestational decrease in mean arterial pressure was absent. Furthermore, mean arterial pressure was significantly increased during late gestation compared with wild-type mice (≈10 mm Hg). As expected, circulating immune cell activation was suppressed during pregnancy. However, this response was absent in AT2R-deficient mice. While renal immune cell infiltration was similar between the genotypes, there was a significant T cell phenotypic switch toward a proinflammatory T-helper 1 phenotype in AT2R-deficient mice. These data indicate that the AT2R plays an important role in arterial pressure regulation and may modulate T cell activation and renal cytokine production during pregnancy. Therefore, deficits in AT2R expression may contribute to pregnancy-induced hypertension and thus represents a potential therapeutic target.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.114.03189DOI Listing
September 2014

Serelaxin is a more efficacious antifibrotic than enalapril in an experimental model of heart disease.

Hypertension 2014 Aug;64(2):315-22

Relaxin is a naturally occurring peptide hormone that mediates systemic hemodynamic and renal adaptive changes during pregnancy and abrogates aberrant scar tissue formation (fibrosis) in diverse pathogeneses. However, its efficacy relative to renin–angiotensin system blockade, the most effective antifibrotic strategy currently available, is not known. We compared the individual versus combined antifibrotic effects of serelaxin (a recombinant form of human gene-2 relaxin) and the angiotensin-converting enzyme inhibitor enalapril, in preventative (started before injury) and therapeutic (treatment of established fibrosis) strategies, in a mouse model of isoprenaline-induced cardiac injury (at 17 days). Changes in systolic blood pressure, organ hypertrophy, and tissue remodeling/fibrosis were assessed. Pretreatment with serelaxin (0.5 mg/kg per day via subcutaneous administration) alone reduced cardiac fibrosis to a greater extent than enalapril (200 mg/L via drinking water; equivalent to 48 mg/kg per day) alone (P<0.05 versus enalapril alone). Additionally, the combined effects of serelaxin and enalapril reduced cardiac fibrosis by at least 2-fold compared with enalapril alone, when administered preventatively or therapeutically; by suppressing transforming growth factor-β1 expression and phosphorylation of Smad2 (an intracellular regulator of transforming growth factor-β1 activity; both P<0.05 versus enalapril alone) to a greater extent. The effects of serelaxin were independent of blood pressure, while enalapril lowered systolic blood pressure in the model studied. These findings suggest that serelaxin alone and in combination with an angiotensin-converting enzyme inhibitor more effectively ameliorates fibrosis than angiotensin-converting enzyme inhibition alone in the diseased heart, in a clinically relevant experimental scenario.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.114.03594DOI Listing
August 2014
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