Publications by authors named "Chrishan S Samuel"

138 Publications

Comparing the renoprotective effects of BM-MSCs versus BM-MSC-exosomes, when combined with an anti-fibrotic drug, in hypertensive mice.

Biomed Pharmacother 2021 Dec 1;144:112256. Epub 2021 Oct 1.

Cardiovascular Disease Program, Monash Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; Stem Cells and Development Program, Monash Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia. Electronic address:

Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow derived-mesenchymal stromal cells (BM-MSCs) post-transplantation. To address this, we demonstrated that combining BM-MSCs with the anti-fibrotic drug, serelaxin (RLX), enhanced BM-MSC-induced renoprotection in preclinical CKD models. Given the increased interest and manufacturing advantages to using stem cell-derived exosomes (EXO) as therapeutics, this study determined whether RLX could enhance the therapeutic efficacy of BM-MSC-EXO, and compared the renoprotective effects of RLX and BM-MSC-EXO versus RLX and BM-MSCs in mice with hypertensive CKD. Adult male C57BL/6 mice were uninephrectomised, received deoxycorticosterone acetate and given saline to drink (1K/DOCA/salt) for 21 days. Control mice were uninephrectomised and given normal drinking water for the same time-period. Subgroups of 1K/DOCA/salt-hypertensive mice were then treated with either RLX (0.5 mg/kg/day) or BM-MSC-EXO (25 μg/mouse; equivalent to 1-2 × 10 BM-MSCs/mouse) alone; combinations of RLX and BM-MSC-EXO or BM-MSCs (1 × 10/mouse); or the mineralocorticoid receptor antagonist, spironolactone (20 mg/kg/day), from days 14-21. 1K/DOCA/salt-hypertensive mice developed kidney tubular damage, inflammation and fibrosis, and impaired kidney function 21 days post-injury. Whilst RLX alone attenuated the 1K/DOCA/salt-induced fibrosis, BM-MSC-EXO alone only diminished measures of tissue inflammation post-treatment. Comparatively, the combined effects of RLX and BM-MSC-EXO or BM-MSCs demonstrated similar anti-fibrotic efficacy, but RLX and BM-MSCs offered broader renoprotection over RLX and/or BM-MSC-EXO, and comparable effects to spironolactone. Only RLX and BM-MSCs, but not RLX and/or BM-MSC-EXO, also attenuated the 1K/DOCA/salt-induced hypertension. Hence, although RLX improved the renoprotective effects of BM-MSC-EXO, combining RLX with BM-MSCs provided a better therapeutic option for hypertensive CKD.
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http://dx.doi.org/10.1016/j.biopha.2021.112256DOI Listing
December 2021

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 07 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

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

Pulmonary myeloid cell uptake of biodegradable nanoparticles conjugated with an anti-fibrotic agent provides a novel strategy for treating chronic allergic airways disease.

Biomaterials 2021 06 16;273:120796. Epub 2021 Apr 16.

Monash Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Clayton, Victoria, Australia; Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria, Australia. Electronic address:

Asthma (chronic allergic airways disease, AAD) is characterized by airway inflammation (AI), airway remodeling (AWR) and airway hyperresponsiveness (AHR). Current treatments for AAD mainly focus on targeting AI and its contribution AHR, with the use of corticosteroids. However, there are no therapies for the direct treatment of AWR, which can contribute to airway obstruction, AHR and corticosteroid resistance independently of AI. The acute heart failure drug, serelaxin (recombinant human gene-2 relaxin, RLX), has potential anti-remodeling and anti-fibrotic effects but only when continuously infused or injected to overcome its short half-life. To alleviate this limitation, we conjugated serelaxin to biodegradable and noninflammatory nanoparticles (NP-RLX) and evaluated their therapeutic potential on measures of AI, AWR and AHR, when intranasally delivered to a preclinical rodent model of chronic AAD and TGF-β1-stimulated collagen gel contraction from asthma patient-derived myofibroblasts. NP-RLX was preferentially taken-up by CD206-infiltrating and CD68-tissue resident alveolar macrophages. Furthermore, NP-RLX ameliorated the chronic AAD-induced AI, pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), chemokines (CCL2, CCL11) and the pro-fibrotic TGF-β1/IL-1β axis on AWR and resulting AHR, as well as human myofibroblast-induced collagen gel contraction, to a similar extent as unconjugated RLX. Hence, NP-RLX represents a novel strategy for treating the central features of asthma.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120796DOI Listing
June 2021

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

Br J Pharmacol 2021 06 31;178(12):2424-2442. Epub 2021 Mar 31.

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

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 )-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 ·day ), NAC (25 mg·kg ·day ) 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.
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http://dx.doi.org/10.1111/bph.15428DOI Listing
June 2021

Atrial Fibrillation and Obesity: Reverse Remodeling of Atrial Substrate With Weight Reduction.

JACC Clin Electrophysiol 2021 05 24;7(5):630-641. Epub 2021 Feb 24.

Centre for Heart Rhythm Disorders, University of Adelaide, Adelaide, Australia; Department of Cardiology, Royal Adelaide Hospital, Adelaide, Australia. Electronic address:

Objectives: This study sought to evaluate the effect of weight loss on the atrial substrate for atrial fibrillation (AF).

Background: Whether weight loss can reverse the atrial substrate of obesity is not known.

Methods: Thirty sheep had sustained obesity induced by ad libitum calorie-dense diet over 72 weeks. Animals were randomized to 3 groups: sustained obesity and 15% and 30% weight loss. The animals randomized to weight loss underwent weight reduction by reducing the quantity of hay over 32 weeks. Eight lean animals served as controls. All were subjected to the following: dual-energy x-ray absorptiometry, echocardiogram, cardiac magnetic resonance, electrophysiological study, and histological and molecular analyses (fatty infiltration, fibrosis, transforming growth factor β1, and connexin 43).

Results: Sustained obesity was associated with increased left atrium (LA) pressure (p < 0.001), inflammation (p < 0.001), atrial transforming growth factor β1 protein (p < 0.001), endothelin-B receptor expression (p = 0.04), atrial fibrosis (p = 0.01), epicardial fat infiltration (p < 0.001), electrophysiological abnormalities, and AF burden (p = 0.04). Connexin 43 expression was decreased in the obese group (p = 0.03). In this obese ovine model, 30% weight reduction was associated with reduction in total body fat (p < 0.001), LA pressure (p = 0.007), inflammation (p < 0.001), endothelin-B receptor expression (p = 0.01), atrial fibrosis (p = 0.01), increase in atrial effective refractory period (cycle length: 400 and 300 ms; p < 0.001), improved conduction velocity (cycle length: 400 and 300 ms; p = 0.01), decreased conduction heterogeneity (p < 0.001), and decreased AF inducibility (p = 0.03). Weight loss was associated with a nonsignificant reduction in epicardial fat infiltration in posterior LA (p = 0.34).

Conclusions: Weight loss in an obese ovine model is associated with structural and electrophysiological reverse remodeling and a reduced propensity for AF. This provides evidence for the direct role of obesity in AF substrate and the role of weight reduction in patients with AF.
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http://dx.doi.org/10.1016/j.jacep.2020.11.015DOI 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

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

Front Pharmacol 2020 4;11:1201. Epub 2020 Aug 4.

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

Introduction: The NLRP3 inflammasome produces interleukin (IL)-1β and IL-18, which when chronically activated by transforming growth factor (TGF)-β1, contribute to fibrosis. The recombinant form of the anti-fibrotic hormone, relaxin (RLX), suppresses the pro-fibrotic influence of TGF-β1 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-β1 [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-1β, 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.
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http://dx.doi.org/10.3389/fphar.2020.01201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417934PMC
August 2020

Investigation of molecular mechanisms of experimental compounds in murine models of chronic allergic airways disease using synchrotron Fourier-transform infrared microspectroscopy.

Sci Rep 2020 07 16;10(1):11713. Epub 2020 Jul 16.

Epigenomic Medicine Laboratory, Department of Diabetes, Central Clinical School, Monash University, Alfred Centre, 99 Commercial Road, Melbourne, VIC, 3004, Australia.

The ovalbumin-induced (OVA) chronic allergic airways murine model is a well-established model for investigating pre-clinical therapies for chronic allergic airways diseases, such as asthma. Here, we examined the effects of several experimental compounds with potential anti-asthmatic effects including resveratrol (RV), relaxin (RLN), L-sulforaphane (LSF), valproic acid (VPA), and trichostatin A (TSA) using both a prevention and reversal model of chronic allergic airways disease. We undertook a novel analytical approach using focal plane array (FPA) and synchrotron Fourier-transform infrared (S-FTIR) microspectroscopic techniques to provide new insights into the mechanisms of action of these experimental compounds. Apart from the typical biological effects, S-FTIR microspectroscopy was able to detect changes in nucleic acids and protein acetylation. Further, we validated the reduction in collagen deposition induced by each experimental compound evaluated. Although this has previously been observed with conventional histological methods, the S-FTIR technique has the advantage of allowing identification of the type of collagen present. More generally, our findings highlight the potential utility of S-FTIR and FPA-FTIR imaging techniques in enabling a better mechanistic understanding of novel asthma therapeutics.
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http://dx.doi.org/10.1038/s41598-020-68671-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366655PMC
July 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

Targeting the NLRP3 inflammasome to treat cardiovascular fibrosis.

Pharmacol Ther 2020 05 22;209:107511. Epub 2020 Feb 22.

Cardiovascular Disease Theme, Monash Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Melbourne, Victoria, Australia. Electronic address:

Cardiovascular fibrosis refers to the scar tissue that develops in the injured heart and blood vessels from an aberrant wound healing response to organ injury or insult. Established fibrosis becomes a hallmark of chronic disease progression and a key contributor to tissue stiffness and dysfunction, which ultimately leads to heart failure. As wound healing and fibrotic responses to myocardial injury are multifactorial processes, current therapies that only target specific contributing factors to disease pathogenesis offer limited overall anti-fibrotic efficacy. As such, recent attention has turned to targeting the body's immune system, which orchestrates the wound healing response to tissue injury. This review focuses on the increasing body of work that has identified the NLRP3 inflammasome, a multiprotein oligomer complex responsible for activation of inflammatory responses via its production of IL-1β and IL-18, as an immune system-initiated facilitator of cardiovascular healing, but also an important contributor to tissue scarring following its persistent activation. The review summarises the factors that can elicit priming and activation of the inflammasome complex, how the activated inflammasome complex contributes to cardiovascular pathophysiology and fibrosis progression, and the molecular mechanisms involved from various cell culture and animal model studies that have utilised genetic deletion or pharmacological inhibition of specific components of the inflammasome. Finally, it outlines currently known and previously unrecognised cardiovascular receptors that may be pharmacologically targeted to ablate the contribution of the NLRP3 inflammasome to cardiovascular diseases characterised by fibrosis, by compounds that may be developed as effective adjunct therapies to current standard of care medication.
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http://dx.doi.org/10.1016/j.pharmthera.2020.107511DOI Listing
May 2020

Inflammasomes-A Molecular Link for Altered Immunoregulation and Inflammation Mediated Vascular Dysfunction in Preeclampsia.

Int J Mol Sci 2020 Feb 19;21(4). Epub 2020 Feb 19.

Cardiovascular Disease Program, Monash Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Victoria 3168, Australia.

Preeclampsia (PE) is a pregnancy-specific multisystem disorder and is associated with maladaptation of the maternal cardiovascular system and abnormal placentation. One of the important characteristics in the pathophysiology of PE is a dysfunction of the placenta. Placental insufficiency is associated with poor trophoblast uterine invasion and impaired transformation of the uterine spiral arterioles to high capacity and low impedance vessels and/or abnormalities in the development of chorionic villi. Significant progress in identifying potential molecular targets in the pathophysiology of PE is underway. The human placenta is immunologically functional with the trophoblast able to generate specific and diverse innate immune-like responses through their expression of multimeric self-assembling protein complexes, termed inflammasomes. However, the type of response is highly dependent upon the stimuli, the receptor(s) expressed and activated, the downstream signaling pathways involved, and the timing of gestation. Recent findings highlight that inflammasomes can act as a molecular link for several components at the syncytiotrophoblast surface and also in maternal blood thereby directly influencing each other. Thus, the inflammasome molecular platform can promote adverse inflammatory effects when chronically activated. This review highlights current knowledge in placental inflammasome expression and activity in PE-affected pregnancies, and consequently, vascular dysfunction in PE that must be addressed as an interdependent interactive process.
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http://dx.doi.org/10.3390/ijms21041406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073120PMC
February 2020

Distinct Redox Signalling following Macrophage Activation Influences Profibrotic Activity.

J Immunol Res 2019 11;2019:1278301. Epub 2019 Nov 11.

Cardiovascular Disease Program, Biomedicine Discovery Institute & Department of Pharmacology, Monash University, VIC 3800, Australia.

Aims: To date, the ROS-generating capacities of macrophages in different activation states have not been thoroughly compared. This study is aimed at determining the nature and levels of ROS generated following stimulation with common activators of M1 and M2 macrophages and investigating the potential for this to impact fibrosis.

Results: Human primary and THP-1 macrophages were treated with IFN-+LPS or IL-4-activating stimuli, and mRNA expression of established M1 (CXCL11, CCR7, IL-1) and M2 (MRC-1, CCL18, CCL22) markers was used to confirm activation. Superoxide generation was assessed by L-012-enhanced chemiluminescence and was increased in both M(IFN-+LPS) and M(IL-4) macrophages, as compared to unpolarised macrophages (M). This signal was attenuated with NOX2 siRNA. Increased expression of the p47phox and p67phox subunits of the NOX2 oxidase complex was evident in M(IFN-+LPS) and M(IL-4) macrophages, respectively. Amplex Red and DCF fluorescence assays detected increased hydrogen peroxide generation following stimulation with IL-4, but not IFN-+LPS. Coculture with human aortic adventitial fibroblasts revealed that M(IL-4), but not M(IFN-+LPS), enhanced fibroblast collagen 1 protein expression. Macrophage pretreatment with the hydrogen peroxide scavenger, PEG-catalase, attenuated this effect.

Conclusion: We show that superoxide generation is not only enhanced with stimuli associated with M1 macrophage activation but also with the M2 stimulus IL-4. Macrophages activated with IL-4 also exhibited enhanced hydrogen peroxide generation which in turn increased aortic fibroblast collagen production. Thus, M2 macrophage-derived ROS is identified as a potentially important contributor to aortic fibrosis.
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http://dx.doi.org/10.1155/2019/1278301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877990PMC
April 2020

The efficacy of pirfenidone in a sheep model of pulmonary fibrosis.

Exp Lung Res 2019 Nov - Dec;45(9-10):310-322. Epub 2019 Nov 25.

Faculty of Veterinary and Agricultural Science, The University of Melbourne, Parkville, Australia.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease with unknown cause. While the drugs nintedanib and pirfenidone have been approved for the treatment of IPF, they only slow disease progression and can induce several side-effects, suggesting that there is still an unmet need to develop new efficacious drugs, and interventions strategies, to combat this disease. We have recently developed a sheep model of pulmonary fibrosis for the preclinical testing of novel anti-fibrotic drugs. The aim of this study was to assess the effects of pirfenidone to ascertain its suitability as a benchmark for comparing other novel therapeutics in this sheep model. To initiate localized fibrosis, sheep were given two infusions of bleomycin (0.6 U/ml per infusion), a fortnight apart, to a specific lung segment. The contralateral lung segment in each sheep was infused with saline to act as an internal control. Two weeks after the final bleomycin infusion, either pirfenidone or methylcellulose (vehicle control) were administered orally to sheep twice daily for 5 weeks. Results showed that sheep treated with pirfenidone had improved lung function, ameliorated fibrotic pathology, lower numbers of active myofibroblasts, and reduced extra cellular matrix deposition when compared with the relevant measurements obtained from control sheep treated with vehicle. This study showed that pirfenidone can attenuate bleomycin-induced pulmonary fibrosis in sheep, and can therefore be used as a positive control to assess other novel therapeutics for IPF in this model.
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http://dx.doi.org/10.1080/01902148.2019.1695019DOI Listing
May 2020

Serelaxin inhibits the profibrotic TGF-β1/IL-1β axis by targeting TLR-4 and the NLRP3 inflammasome in cardiac myofibroblasts.

FASEB J 2019 12 5;33(12):14717-14733. Epub 2019 Nov 5.

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

The recombinant form of the peptide hormone relaxin, serelaxin (RLX), mediates its anti-fibrotic actions by impeding the profibrotic activity of cytokines including TGF-β1 and IL-1β. As IL-1β 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-β1/IL-1β axis in primary human cardiac myofibroblasts (HCMFs) and in mice with isoproterenol (ISO)-induced cardiomyopathy . HCMFs stimulated with TGF-β1 (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-1β, 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-β1 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) ; 0.5 mg/kg per day ] inhibited T+L+A- and ISO-induced TLR-4 expression, NLRP3 priming, IL-1β, IL-18, myofibroblast differentiation, and interstitial collagen deposition at the time points studied, the promotion of nNOS; with the NLRP3- and IL-1β-inhibitory effects of RLX in HCMFs being abrogated by pharmacological blockade of nNOS or TLR-4. Comparatively, the small molecule NLRP3 inhibitor, -{[(1,2,3,5,6,7-hexahydro--indacen-4-yl)amino]carbonyl}-4-(1-hydroxy-1-methylethyl)-2-furansulfonamide (1 μM , 10 mg/kg/d ), inhibited components of the NLRP3 inflammasome and and ISO-induced interstitial LV fibrosis but did not affect nNOS, TLR-4, myofibroblast differentiation, or myofibroblast-induced collagen deposition. Hence, RLX can inhibit the TGF-β1/IL-1β axis 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-β1/IL-1β axis by targeting TLR-4 and the NLRP3 inflammasome in cardiac myofibroblasts.
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http://dx.doi.org/10.1096/fj.201901079RRDOI 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

Profiling histone modifications in the normal mouse kidney and after unilateral ureteric obstruction.

Am J Physiol Renal Physiol 2019 09 3;317(3):F606-F615. Epub 2019 Jul 3.

Department of Nephrology, Royal Melbourne Hospital, Parkville, Victoria, Australia.

Posttranslational modification of nucleosomal histones is a major determinant of chromatin structure and gene activity. In the present study, we hypothesized that unilateral ureteric obstruction (UUO), a widely used model of tubulointerstitial injury, would be associated with a distinct pattern of histone modifications (marks) in the kidney. Mass spectrometry was used to profile 63 different histone marks in normal mouse kidneys and those after 10 days of UUO. A subsequent histochemical analysis further examined examples of specific marks that changed significantly after UUO for which antisera are available. Histone marks were much more widely distributed and abundant in the normal kidney than is usually appreciated. Although aggregate analysis of the mass spectrometry results revealed net differences between control and UUO groups, residue-specific variations were subtle. Of the 16/63 significant changes ( < 0.05), only 8 changes were quantitatively different by >5%. Nevertheless, we identified several that are not usually examined in the kidney, including marks in the globular domain of core histones (H3:K79), linker histones (H1.4), and histone variants (H3.1:K27 and H3.3:K27). In several cases, there were complementary changes in different marks on the same amino acid. Using H3:K79ME2 as an example, mark enrichment was heterogeneous but largely colocalized with active transcription in a subset of tubular pathology. In conclusion, our study highlights the importance of unbiased screening in examining histone marks. Simultaneous changes in multiple marks on the same amino acid indicate a coordinated histone mark signature. The heterogeneous enrichment of marks, even within the same tubule, highlights the importance of regulatory context.
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http://dx.doi.org/10.1152/ajprenal.00262.2019DOI Listing
September 2019

The Use of Live Cell Imaging and Automated Image Analysis to Assist With Determining Optimal Parameters for Angiogenic Assay .

Front Cell Dev Biol 2019 10;7:45. Epub 2019 Apr 10.

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

Testing angiogenic potential and function of cells in culture is important for the understanding of the mechanisms that can modulate angiogenesis, especially when discovering novel anti- or pro-angiogenic therapeutics. Commonly used angiogenic assays include tube formation, proliferation, migration, and wound healing, and although well-characterized, it is important that methodology is standardized and reproducible. Human endothelial progenitor cells (EPCs) are critical for post-natal vascular homeostasis and can be isolated from human peripheral blood. Endothelial colony forming cells (ECFCs) are a subset of EPCs and are of interest as a possible therapeutic target for hypoxic diseases such as kidney disease, as they have a high angiogenic potential. However, once ECFCs are identified in culture, the exact timing of passaging has not been well-described and the optimal conditions to perform angiogenic assays such as seeding density, growth media (GM) concentrations and end-points of these assays is widely varied in the literature. Here, we describe the process of isolating, culturing and passaging ECFCs from patients with end-stage renal disease (ESRD), aided by image analysis. We further describe optimal conditions, for human bladder endothelial cells (hBECs), challenged in angiogenic assays and confirm that cell density is a limiting factor in accurately detecting angiogenic parameters. Furthermore, we show that GM along is enough to alter the angiogenic potential of cells, seeded at the same density. Lastly, we report on the success of human ECFCs in angiogenic assays and describe the benefits of live-cell imaging combined with time-lapse microscopy for this type of investigation.
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http://dx.doi.org/10.3389/fcell.2019.00045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468051PMC
April 2019

Serelaxin enhances the therapeutic effects of human amnion epithelial cell-derived exosomes in experimental models of lung disease.

Br J Pharmacol 2019 07 7;176(13):2195-2208. Epub 2019 May 7.

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

Background And Purpose: There is growing interest in stem cell-derived exosomes for their therapeutic and regenerative benefits given their manufacturing and regulatory advantages over cell-based therapies. As existing fibrosis impedes the viability and efficacy of stem cell/exosome-based strategies for treating chronic diseases, here we tested the effects of the anti-fibrotic drug, serelaxin, on the therapeutic efficacy of human amnion epithelial cell (AEC)-derived exosomes in experimental lung disease.

Experimental Approach: Female Balb/c mice were subjected to either the 9.5-week model of ovalbumin and naphthalene (OVA/NA)-induced chronic allergic airway disease (AAD) or 3-week model of bleomycin (BLM)-induced pulmonary fibrosis; then administered increasing concentrations of AEC-exosomes (5 μg or 25μg), with or without serelaxin (0.5mg/kg/day) for 7-days. 1x10 AECs co-administered with serelaxin over the corresponding time-period were included for comparison in both models, as was pirfenidone-treatment of the BLM model. Control groups received saline/corn oil or saline, respectively.

Key Results: Both experimental models presented with significant tissue inflammation, remodelling, fibrosis and airway/lung dysfunction at the time-points studied. While AEC-exosome (5 μg or 25μg)-administration alone demonstrated some benefits in each model, serelaxin was required for AEC-exosomes (25μg) to rapidly normalise chronic AAD-induced airway fibrosis and airway reactivity, and BLM-induced lung inflammation, epithelial damage and subepithelial/basement membrane fibrosis. Combining serelaxin with AEC-exosomes (25μg) also demonstrated broader protection compared to co-administration of serelaxin with 1x10 AECs or pirfenidone.

Conclusions And Implications: Serelaxin enhanced the therapeutic efficacy of AEC-exosomes in treating basement membrane-induced fibrosis and related airway dysfunction.
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http://dx.doi.org/10.1111/bph.14666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555854PMC
July 2019

Relaxin and fibrosis: Emerging targets, challenges, and future directions.

Mol Cell Endocrinol 2019 05 14;487:66-74. Epub 2019 Feb 14.

Cardiovascular Disease Theme, Monash Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Clayton, VIC, Australia; Central Clinical School, Monash University, Prahran, VIC, Australia. Electronic address:

The peptide hormone relaxin is well-known for its anti-fibrotic actions in several organs, particularly from numerous studies conducted in animals. Acting through its cognate G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), serelaxin (recombinant human relaxin) has been shown to consistently inhibit the excessive extracellular matrix production (fibrosis) that results from the aberrant wound-healing response to tissue injury and/or chronic inflammation, and at multiple levels. Furthermore, it can reduce established scarring by promoting the degradation of aberrant extracellular matrix components. Following on from the review that describes the mechanisms and signaling pathways associated with the extracellular matrix remodeling effects of serelaxin (Ng et al., 2019), this review focuses on newly identified tissue targets of serelaxin therapy in fibrosis, and the limitations associated with (se)relaxin research.
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http://dx.doi.org/10.1016/j.mce.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475456PMC
May 2019

iPSC- and mesenchymoangioblast-derived mesenchymal stem cells provide greater protection against experimental chronic allergic airways disease compared with a clinically used corticosteroid.

FASEB J 2019 05 15;33(5):6402-6411. Epub 2019 Feb 15.

Monash Biomedicine Discovery Institute Monash University, Clayton, Victoria, Australia.

The airway remodeling (AWR) associated with chronic allergic airways disease (AAD)/asthma contributes to irreversible airway obstruction. This study compared and combined the antiremodeling and other effects of induced pluripotent stem cell and mesenchymoangioblast-derived mesenchymal stem cells (MCA-MSCs) with the corticosteroid dexamethasone (Dex) in experimental chronic AAD/asthma. Female BALB/c mice subjected to 11 wk of ovalbumin (Ova)-induced chronic AAD were intranasally administered MCA-MSCs (1 × 10 cells/mouse; once weekly on wk 10 and 11), Dex (0.5 mg/ml; once daily for 2 wk), or both combined. MCA-MSC detection and changes in airway inflammation (AI), AWR, and airway hyperresponsiveness (AHR) were measured at the end of wk 11. Mice with chronic AAD had significant AI, goblet cell metaplasia, epithelial damage/thickening, aberrant TGF-β1 levels, subepithelial myofibroblast accumulation, airway/lung fibrosis, and AHR (all < 0.001 healthy controls). MCA-MSCs were detected in the lungs up to 5-7 d postadministration and demonstrated modest anti-inflammatory but striking antifibrotic effects against Ova-induced AAD, effectively decreasing AHR by 70-75% (all < 0.05 Ova alone). In comparison, Dex predominantly demonstrated anti-inflammatory effects, decreasing AHR by ∼30%. Combining MCA-MSCs with Dex provided equivalent protection to that offered by either therapy alone. MCA-MSCs reduce chronic AAD-induced AWR and AHR to a greater extent than Dex and may act as a suitable adjunct therapy to corticosteroid treatment of asthma.-Royce, S. G., Mao, W., Lim, R., Kelly, K., Samuel, C. S. iPSC- and mesenchymoangioblast-derived mesenchymal stem cells provide greater protection against experimental chronic allergic airways disease compared with a clinically used corticosteroid.
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http://dx.doi.org/10.1096/fj.201802307RDOI Listing
May 2019

Atypical cannabinoid ligands O-1602 and O-1918 administered chronically in diet-induced obesity.

Endocr Connect 2019 Mar;8(3):203-216

Institute for Health and Sport, Victoria University, St Albans campus, Melbourne, Victoria, Australia.

Atypical cannabinoid compounds O-1602 and O-1918 are ligands for the putative cannabinoid receptors G protein-coupled receptor 55 and G protein-coupled receptor 18. The role of O-1602 and O-1918 in attenuating obesity and obesity-related pathologies is unknown. Therefore, we aimed to determine the role that either compound had on body weight and body composition, renal and hepatic function in diet-induced obesity. Male Sprague-Dawley rats were fed a high-fat diet (40% digestible energy from lipids) or a standard chow diet for 10 weeks. In a separate cohort, male Sprague-Dawley rats were fed a high-fat diet for 9 weeks and then injected daily with 5 mg/kg O-1602, 1 mg/kg O-1918 or vehicle (0.9% saline/0.75% Tween 80) for a further 6 weeks. Our data demonstrated that high-fat feeding upregulates whole kidney G protein receptor 55 expression. In diet-induced obesity, we also demonstrated O-1602 reduces body weight, body fat and improves albuminuria. Despite this, treatment with O-1602 resulted in gross morphological changes in the liver and kidney. Treatment with O-1918 improved albuminuria, but did not alter body weight or fat composition. In addition, treatment with O-1918 also upregulated circulation of pro-inflammatory cytokines including IL-1α, IL-2, IL-17α, IL-18 and RANTES as well as plasma AST. Thus O-1602 and O-1918 appear not to be suitable treatments for obesity and related comorbidities, due to their effects on organ morphology and pro-inflammatory signaling in obesity.
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http://dx.doi.org/10.1530/EC-18-0535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391900PMC
March 2019

Relaxin and extracellular matrix remodeling: Mechanisms and signaling pathways.

Mol Cell Endocrinol 2019 05 17;487:59-65. Epub 2019 Jan 17.

Research Service, VA Nebraska-Western Iowa Health Care System, Departments of Internal Medicine and Biochemistry & Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA. Electronic address:

Fibrosis is associated with accumulation of excess fibrillar collagen, leading to tissue dysfunction. Numerous processes, including inflammation, myofibroblast activation, and endothelial-to-mesenchymal transition, play a role in the establishment and progression of fibrosis. Relaxin is a peptide hormone with well-known antifibrotic properties that result from its action on numerous cellular targets to reduce fibrosis. Relaxin activates multiple signal transduction pathways as a mechanism to suppress inflammation and myofibroblast activation in fibrosis. In this review, the general mechanisms underlying fibrotic diseases are described, along with the current state of knowledge regarding cellular targets of relaxin. Finally, an overview is presented summarizing the signaling pathways activated by relaxin and other relaxin family peptide receptor agonists to suppress fibrosis.
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http://dx.doi.org/10.1016/j.mce.2019.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384500PMC
May 2019

Understanding relaxin signalling at the cellular level.

Mol Cell Endocrinol 2019 05 25;487:24-33. Epub 2018 Dec 25.

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, 3010, Australia. Electronic address:

The peptide hormone relaxin mediates many biological actions including anti-fibrotic, vasodilatory, angiogenic, anti-inflammatory, anti-apoptotic, and organ protective effects across a range of tissues. At the cellular level, relaxin binds to the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1) to activate a variety of downstream signal transduction pathways. This signalling cascade is complex and also varies in diverse cellular backgrounds. Moreover, RXFP1 signalling shows crosstalk with other receptors to mediate some of its physiological functions. This review summarises known signalling pathways induced by acute versus chronic treatment with relaxin across a range of cell types, it describes RXFP1 crosstalk with other receptors, signalling pathways activated by other ligands targeting RXFP1, and it also outlines physiological relevance of RXFP1 signalling outputs. Comprehensive understanding of the mechanism of relaxin actions in fibrosis, vasodilation, as well as organ protection, will further support relaxin's clinical potential.
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http://dx.doi.org/10.1016/j.mce.2018.12.017DOI Listing
May 2019

Pharmacological inhibition of the NLRP3 inflammasome reduces blood pressure, renal damage, and dysfunction in salt-sensitive hypertension.

Cardiovasc Res 2019 03;115(4):776-787

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

Aims: Renal inflammation, leading to fibrosis and impaired function is a major contributor to the development of hypertension. The NLRP3 inflammasome mediates inflammation in several chronic diseases by processing the cytokines pro-interleukin (IL)-1β and pro-IL-18. In this study, we investigated whether MCC950, a recently-identified inhibitor of NLRP3 activity, reduces blood pressure (BP), renal inflammation, fibrosis and dysfunction in mice with established hypertension.

Methods And Results: C57BL6/J mice were made hypertensive by uninephrectomy and treatment with deoxycorticosterone acetate (2.4 mg/day, s.c.) and 0.9% NaCl in the drinking water (1K/DOCA/salt). Normotensive controls were uninephrectomized and received normal drinking water. Ten days later, mice were treated with MCC950 (10 mg/kg/day, s.c.) or vehicle (saline, s.c.) for up to 25 days. BP was monitored by tail-cuff or radiotelemetry; renal function by biochemical analysis of 24-h urine collections; and kidney inflammation/pathology was assessed by real-time PCR for inflammatory gene expression, flow cytometry for leucocyte influx, and Picrosirius red histology for collagen. Over the 10 days post-surgery, 1K/DOCA/salt-treated mice became hypertensive, developed impaired renal function, and displayed elevated renal levels of inflammatory markers, collagen and immune cells. MCC950 treatment from day 10 attenuated 1K/DOCA/salt-induced increases in renal expression of inflammasome subunits (NLRP3, ASC, pro-caspase-1) and inflammatory/injury markers (pro-IL-18, pro-IL-1β, IL-17A, TNF-α, osteopontin, ICAM-1, VCAM-1, CCL2, vimentin), each by 25-40%. MCC950 reduced interstitial collagen and accumulation of certain leucocyte subsets in kidneys of 1K/DOCA/salt-treated mice, including CD206+ (M2-like) macrophages and interferon-gamma-producing T cells. Finally, MCC950 partially reversed 1K/DOCA/salt-induced elevations in BP, urine output, osmolality, [Na+], and albuminuria (each by 20-25%). None of the above parameters were altered by MCC950 in normotensive mice.

Conclusion: MCC950 was effective at reducing BP and limiting renal inflammation, fibrosis and dysfunction in mice with established hypertension. This study provides proof-of-concept that pharmacological inhibition of the NLRP3 inflammasome is a viable anti-hypertensive strategy.
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http://dx.doi.org/10.1093/cvr/cvy252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432065PMC
March 2019

The therapeutic effect of mesenchymal stem cells on pulmonary myeloid cells following neonatal hyperoxic lung injury in mice.

Respir Res 2018 06 8;19(1):114. Epub 2018 Jun 8.

Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.

Background: Exposure to high levels of oxygen (hyperoxia) after birth leads to lung injury. Our aims were to investigate the modulation of myeloid cell sub-populations and the reduction of fibrosis in the lungs following administration of human mesenchymal stem cells (hMSC) to neonatal mice exposed to hyperoxia.

Method: Newborn mice were exposed to 90% O (hyperoxia) or 21% O (normoxia) from postnatal days 0-4. A sub-group of hyperoxia mice were injected intratracheally with 2.5X10 hMSCs. Using flow cytometry we assessed pulmonary immune cells at postnatal days 0, 4, 7 and 14. The following markers were chosen to identify these cells: CD45 (leukocytes), Ly6CLy6G (granulocytes), CD11bCD11c (macrophages); macrophage polarisation was assessed by F4/80 and CD206 expression. hMSCs expressing enhanced green fluorescent protein (eGFP) and firefly luciferase (fluc) were administered via the trachea at day 4. Lung macrophages in all groups were profiled using next generation sequencing (NGS) to assess alterations in macrophage phenotype. Pulmonary collagen deposition and morphometry were assessed at days 14 and 56 respectively.

Results: At day 4, hyperoxia increased the number of pulmonary Ly6CLy6G granulocytes and F4/80CD206 macrophages but decreased F4/80CD206 macrophages. At days 7 and 14, hyperoxia increased numbers of CD45 leukocytes, CD11bCD11c alveolar macrophages and F4/80CD206 macrophages but decreased F4/80CD206 macrophages. hMSCs administration ameliorated these effects of hyperoxia, notably reducing numbers of CD11bCD11c and F4/80CD206 macrophages; in contrast, F4/80CD206 macrophages were increased. Genes characteristic of anti-inflammatory 'M2' macrophages (Arg1, Stat6, Retnla, Mrc1, Il27ra, Chil3, and Il12b) were up-regulated, and pro-inflammatory 'M1' macrophages (Cd86, Stat1, Socs3, Slamf1, Tnf, Fcgr1, Il12b, Il6, Il1b, and Il27ra) were downregulated in isolated lung macrophages from hyperoxia-exposed mice administered hMSCs, compared to mice without hMSCs. Hydroxyproline assay at day 14 showed that the 2-fold increase in lung collagen following hyperoxia was reduced to control levels in mice administered hMSCs. By day 56 (early adulthood), hMSC administration had attenuated structural changes in hyperoxia-exposed lungs.

Conclusions: Our findings suggest that hMSCs reduce neonatal lung injury caused by hyperoxia by modulation of macrophage phenotype. Not only did our cell-based therapy using hMSC induce structural repair, it limited the progression of pulmonary fibrosis.
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http://dx.doi.org/10.1186/s12931-018-0816-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994120PMC
June 2018

Hexarelin treatment preserves myocardial function and reduces cardiac fibrosis in a mouse model of acute myocardial infarction.

Physiol Rep 2018 05;6(9):e13699

School of Biomedical Science, University of Queensland, Brisbane, Australia.

Ischemic heart disease (IHD) is a leading cause of morbidity and mortality worldwide. Growth hormone secretagogues (GHS) have been shown to improve cardiac function in models of IHD. This study determined whether hexarelin (HEX), a synthetic GHS, preserves cardiac function and morphology in a mouse model of myocardial infarction (MI). MI was induced by ligation of the left descending coronary artery in C57BL/6J mice followed by vehicle (VEH; n = 10) or HEX (0.3 mg/kg/day; n = 11) administration for 21 days. MI-injured and sham mice (treated with VEH; n = 6 or HEX; n = 5) underwent magnetic resonance imaging for measurement of left ventricular (LV) function, mass and infarct size at 24 h and 14 days post-MI. MI-HEX mice displayed a significant improvement (P < 0.05) in LV function compared with MI-VEH mice after 14 days treatment. A significant decrease in LV mass, interstitial collagen and collagen concentration was demonstrated with chronic HEX treatment (for 21 days), accompanied by a decrease in TGF-β1 expression, myofibroblast differentiation and an increase in collagen-degrading MMP-13 expression levels. Furthermore, heart rate variability analysis demonstrated that HEX treatment shifted the balance of autonomic nervous activity toward a parasympathetic predominance and sympathetic downregulation. This was combined with a HEX-dependent decrease in troponin-I, IL-1β and TNF-α levels suggestive of amelioration of cardiomyocyte injury. These results demonstrate that GHS may preserve ventricular function, reduce inflammation and favorably remodel the process of fibrotic healing in a mouse model of MI and hold the potential for translational application to patients suffering from MI.
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http://dx.doi.org/10.14814/phy2.13699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5949285PMC
May 2018

Serelaxin as a novel therapeutic opposing fibrosis and contraction in lung diseases.

Pharmacol Ther 2018 07 12;187:61-70. Epub 2018 Feb 12.

Biomedicine Discovery Institute, Monash University, Melbourne, Australia; Department of Pharmacology, School of Biomedical Sciences, Monash University, Melbourne, Australia. Electronic address:

The most common therapies for asthma and other chronic lung diseases are anti-inflammatory agents and bronchodilators. While these drugs oppose disease symptoms, they do not reverse established structural changes in the airways and their therapeutic efficacy is reduced with increasing disease severity. The peptide hormone, relaxin, is a Relaxin Family Peptide Receptor 1 (RXFP1) receptor agonist with unique combined effects in the lung that differentiates it from these existing therapies. Relaxin has previously been reported to have cardioprotective effects in acute heart failure as well anti-fibrotic actions in several organs. This review focuses on recent experimental evidence of the beneficial effects of chronic relaxin treatment in animal models of airways disease demonstrating inhibition of airway hyperresponsiveness and reversal of established fibrosis, consistent with potential therapeutic benefit. Of particular interest, accumulating evidence demonstrates that relaxin can also acutely oppose contraction by reducing the release of mast cell-derived bronchoconstrictors and by directly eliciting bronchodilation. When used in combination, chronic and acute treatment with relaxin has been shown to enhance responsiveness to both glucocorticoids and β-adrenoceptor agonists respectively. While the mechanisms underlying these beneficial actions remain to be fully elucidated, translation of these promising combined preclinical findings is critical in the development of relaxin as a novel alternative or adjunct therapeutic opposing multiple aspects of airway pathology in lung diseases.
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http://dx.doi.org/10.1016/j.pharmthera.2018.02.004DOI Listing
July 2018
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