Serelaxin as a potential treatment for renal dysfunction in cirrhosis: Preclinical evaluation and results of a randomized phase 2 trial

<div><p>Background</p><p>Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension.</p><p>Methods and findings</p><p>To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 μg/kg/d and then 60 min at 30 μg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow.</p><p>Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; <i>p <</i> 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study’s main limitations were the relatively small sample size and stable, well-compensated population.</p><p>Conclusions</p><p>Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01640964" target="_blank">NCT01640964</a></p></div

A topical hydrogel with deferiprone and gallium-protoporphyrin targets bacterial iron metabolism and has antibiofilm activity

Accepted manuscript posted online 10 April 2017Many infectious diseases are associated with multidrug-resistant (MDR) bacteria residing in biofilms that require high antibiotic concentrations. While oral drug delivery is frequently ineffective, topical treatments have the potential to deliver higher drug concentrations to the infection site while reducing systemic side effects. This study determined the antibiofilm activity of a surgical wound gel loaded with the iron chelator deferiprone (Def) and the heme analogue gallium-protoporphyrin (GaPP), alone and in combination with ciprofloxacin. Activity against MDR Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Acinetobacter johnsonii biofilms was assessed in the colony biofilm and artificial wound model by enumeration of CFU and correlative light/electron microscopy. While Staphylococcus biofilms were equally susceptible to GaPP and Def-GaPP gels (log10 reduction of 3.8 and 3.7, respectively), the Def-GaPP combination was crucial for significant activity against P. aeruginosa biofilms (log10 reduction of 1.3 for GaPP and 3.3 for Def-GaPP). When Def-GaPP gel was combined with ciprofloxacin, the efficacy exceeded the activity of the individual compounds. Def-GaPP delivered in a surgical wound gel showed significant antibiofilm activity against different MDR strains and could enhance the gel's wound-healing properties. Moreover, Def-GaPP indicated a potentiation of ciprofloxacin. This antibiofilm strategy has potential for clinical utilization as a therapy for topical biofilm-related infections.Katharina Richter, Nicky Thomas, Jolien Claeys, Jonathan McGuane, Clive A. Prestidge, Tom Coenye, Peter-John Wormald, Sarah Vreugd

Relaxin regulates vascular wall remodeling and passive mechanical properties in mice

Administration of recombinant human relaxin (rhRLX) to conscious rats increases global arterial compliance, and small renal arteries (SRA) isolated from these rats demonstrate increased passive compliance. Here we characterize relaxin-induced vascular remodeling and examine its functional relevance. SRA and external iliac arteries (EIA) were examined in rhRLX-treated (1.0 μg/h for 5 days) and relaxin knockout mice. Arterial geometric remodeling and compositional remodeling were quantified using immunohistochemical and biochemical techniques. Vascular mechanical properties were quantified using an ex vivo preparation wherein pressure-diameter data were obtained at various axial lengths. Compared with vehicle-treated mice, SRA from rhRLX-treated mice showed outward geometric remodeling (increased unstressed wall area and wall-to-lumen area ratio), increased smooth muscle cell (SMC) density, reduction in collagen-to-total protein ratio, and unchanged elastin-to-tissue dry weight ratio. Compared with wild-type mice, relaxin knockout mice exhibited the opposite pattern: decreased unstressed wall area and wall-to-lumen area ratio, decreased SMC density, and increased collagen-to-total protein ratio. Although tissue biaxial strain energy of SRA was not different between rhRLX- and vehicle-treated groups at low-to-physiological circumferential and axial strains, it was lower for the rhRLX-treated group at the highest circumferential strain. In contrast to SRA, relaxin administration was not associated with any vascular remodeling or changes in passive mechanics of EIA. Thus relaxin induces both geometric and compositional remodeling in vessel-specific manner. Relaxin-induced geometric remodeling of SRA is responsible for the increase in passive compliance under low-to-physiological levels of circumferential and axial strains, and compositional remodeling becomes functionally relevant only under high circumferential strain.This project was supported by National Heart, Lung, and Blood Institute and McGinnis Chair Endowment Funds Research Grant R01-HL-067937. D. O. Debrah was supported by National Heart, Lung, and Blood Institute Predoctoral Fellowship Award F31-HL-079882

Effect of Phenotype Selection on Genome Size Variation in Two Species of Diptera

Genome size varies widely across organisms yet has not been found to be related to organismal complexity in eukaryotes. While there is no evidence for a relationship with complexity, there is evidence to suggest that other phenotypic characteristics, such as nucleus size and cell-cycle time, are associated with genome size, body size, and development rate. However, what is unknown is how the selection for divergent phenotypic traits may indirectly affect genome size. Drosophila melanogaster were selected for small and large body size for up to 220 generations, while Cochliomyia macellaria were selected for 32 generations for fast and slow development. Size in D. melanogaster significantly changed in terms of both cell-count and genome size in isolines, but only the cell-count changed in lines which were maintained at larger effective population sizes. Larger genome sizes only occurred in a subset of D. melanogaster isolines originated from flies selected for their large body size. Selection for development time did not change average genome size yet decreased the within-population variation in genome size with increasing generations of selection. This decrease in variation and convergence on a similar mean genome size was not in correspondence with phenotypic variation and suggests stabilizing selection on genome size in laboratory conditions

Relaxin increases human endothelial progenitor cell NO and migration and vasculogenesis in mice

The ovarian peptide hormone, relaxin, circulates during pregnancy, contributing to profound maternal vasodilation through endothelial and nitric oxide (NO)-dependent mechanisms. Circulating numbers of bone marrow-derived endothelial cells (BMDECs), which facilitate angiogenesis and contribute to repair of vascular endothelium, increase during pregnancy. Thus, we hypothesized that relaxin enhances BMDEC NO production, circulating numbers, and function. Recombinant human relaxin-2 (rhRLX) stimulated PI3K/Akt B-dependent NO production in human BMDECs within minutes, and activated BMDEC migration that was inhibited by L-N(G)-nitroarginine methyl ester. In BMDECs isolated from relaxin/insulin-like family peptide receptor 2 gene (Rxfp2) knockout and wild-type mice, but not Rxfp1 knockout mice, rhRLX rapidly increased NO production. Similarly, rhRLX increased circulating BMDEC number in Rxfp2 knockout and wild-type mice, but not Rxfp1 knockout mice as assessed by colony formation and flow cytometry. Taken together, these results indicate that relaxin effects BMDEC function through the RXFP1 receptor. Finally, both vascularization and incorporation of GFP-labeled BMDECs were stimulated in rhRLX-impregnated Matrigel pellets implanted in mice. To conclude, relaxin is a novel regulator of BMDECs number and function, which has implications for angiogenesis and vascular remodeling in pregnancy, as well as therapeutic potential in vascular disease.This work was supported by Gatorade Research (M.S.S.), National Institutes of Health grants R01 HL067937 (K.P.C.), R01 HD037067 (A.I.A.), and R01 EY12601 and EY07739 (M.B.G.)

Identification and Characterization of Small RNA Markers of Age in the Blow Fly Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae)

Blow fly development is important in decomposition ecology, agriculture, and forensics. Much of the impact of these species is from immature samples, thus knowledge of their development is important to enhance or ameliorate their effects. One application of this information is the estimation of immature insect age to provide temporal information for death investigations. While traditional markers of age such as stage and size are generally accurate, they lack precision in later developmental stages. We used miRNA sequencing to measure miRNA expression, throughout development, of the secondary screwworm, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae) and identified 217 miRNAs present across the samples. Ten were identified to be significantly differentially expressed in larval samples and seventeen were found to be significantly differentially expressed in intrapuparial samples. Twenty-eight miRNAs were identified to be differentially expressed between sexes. Expression patterns of two miRNAs, miR-92b and bantam, were qPCR-validated in intrapuparial samples; these and likely food-derived miRNAs appear to be stable markers of age in C. macellaria. Our results support the use of miRNAs for developmental markers of age and suggest further investigations across species and under a range of abiotic and biotic conditions

Adenovirus-mediated delivery of relaxin reverses cardiac fibrosis

We have evaluated the effectiveness of systemic adenovirally delivered mouse relaxin on reversing fibrosis in a transgenic murine model of fibrotic cardiomyopathy due to β2-adrenergic receptor (β2AR) overexpression. Recombinant adenoviruses expressing green fluorescent protein (Ad-GFP), rat relaxin (Ad-rRLN) and mouse relaxin (Ad-mRLN) were generated and Ad-rRLN and Ad-mRLN were demonstrated to direct the expression of bioactive relaxin peptides in vitro. A single systemic injection of Ad-mRLN resulted in transgene expression in the liver and bioactive relaxin peptide in the plasma. Ad-mRLN, but not Ad-GFP, treatment reversed the increased left ventricular collagen content in β2AR mice to control levels without affecting collagen levels in other heart chambers or in the lung and kidney. Hence a single systemic injection of adenovirus producing mouse relaxin reverses cardiac fibrosis without adversely affecting normal collagen levels in other organs and establishes the potential for the use of relaxin gene therapy for the treatment of cardiac fibrosis