The anti-fibrotic actions of relaxin are mediated through AT2 R-associated protein phosphatases via RXFP1-AT2 R functional crosstalk in human cardiac myofibroblasts.

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 (AT2 R)-specific signal transduction in primary human cardiac myofibroblasts (HCMFs). The anti-fibrotic effects of recombinant human relaxin (RLX; 16.8 nM) or the AT2 R-agonist, Compound 21 (C21; 1 µM), were evaluated in TGF-ß1-stimulated HCMFs, in the absence or presence of an RXFP1 antagonist (1 µM) or AT2 R antagonist (0.1 µM) to confirm RXFP1-AT2 R crosstalk. Competition binding for RXFP1 was determined. Western blotting was performed to determine which AT2 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 AT2 R, confirming RXFP1-AT2 R crosstalk in these cells. HCMFs were found to express AT2 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 AT2 R-dependent phosphatase activity in HCMFs by signaling through RXFP1-AT2 R crosstalk, which have important therapeutic implications for its anti-fibrotic actions.

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

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

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

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.

Immune Mechanisms and Related Targets for the Treatment of Fibrosis in Various Organs.

Inflammation and fibrosis are two interrelated disease pathologies with several overlapping components. Three specific cell types, namely macrophages, T helper cells, and myofibroblasts, play important roles in regulating both processes. Following tissue injury, an inflammatory stimulus is often necessary to initiate tissue repair, where cytokines released from infiltrating and resident immune and inflammatory cells stimulate the proliferation and activation of extracellular matrix-producing myofibroblasts. However, persistent tissue injury drives an inappropriate pro-fibrotic response. Additionally, activated myofibroblasts can take on the role of traditional antigen-presenting cells, secrete pro-inflammatory cytokines, and recruit inflammatory cells to fibrotic foci, amplifying the fibrotic response in a vicious cycle. Moreover, inflammatory cells have been shown to play contradictory roles in the initiation, amplification, and resolution of fibrotic disease processes. The central role of the inflammasome molecular platform in contributing to fibrosis is only beginning to be fully appreciated. In this review, we discuss the immune mechanisms that can lead to fibrosis, the inflammasomes that have been implicated in the fibrotic process in the context of the immune response to injury, and also discuss current and emerging therapies that target inflammasome-induced collagen deposition to treat organ fibrosis.

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

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-ß1-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-1ß, IL-6, TNF-α), chemokines (CCL2, CCL11) and the pro-fibrotic TGF-ß1/IL-1ß 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.

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

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-1 ·day-1 ) or BM-MSCs (1 × 106 per mouse) alone; both treatments combined (with 0.5 × 106 or 1 × 106 BM-MSCs per mouse); or perindopril (2 mg·kg-1 ·day-1 ) 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.

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

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

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

[Figure: see text].

Assays for Measuring the Role of MIF in NLRP3 Inflammasome Activation.

Hallmarks of NLRP3 inflammasome activation include the cleavage and secretion of the mature forms of caspase-1, IL-1ß, and IL-18 and aggregation of ASC into "specks." We have previously shown that macrophage migratory inhibitory factor (MIF) directly regulates activation of the NLRP3 inflammasome, inhibiting the release of interleukin (IL)-1α, IL-1ß, and IL-18. Here we present protocols for studying activation of the NLRP3 inflammasome in human and mouse macrophages and peripheral blood mononuclear cells (PBMCs). These protocols can also be applied to different cell types, such as fibroblasts, neutrophils, endothelial cells, and epithelial cells, although further optimization may be required for each. We also cover the stimulation of macrophages with established NLRP3 inflammasome activators.

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

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

Targeting the NLRP3 inflammasome to treat cardiovascular fibrosis.

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-1ß 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.

Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation.

Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside the immune system. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6, and IL-1 family cytokines. Here, we show that inhibition of MIF regulates the release of IL-1α, IL-1ß, and IL-18, not by affecting transcription or translation of these cytokines, but via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation.