Fibrose-assoziierte Biomarker – Einfluss der Behandlung mit Serelaxin im murinen Nierenfibrose-Modell und prädiktiver Aussagewert in der pulmonalen Hypertonie

Die Fibrose stellt aufgrund ihrer hohen Prävalenz in der Bevölkerung, der schlechten Prognose für die Patienten, sowie der komplexen Pathophysiologie ein sehr wichtiges Forschungsgebiet mit einem hohen Bedarf an neuen Therapeutika dar. Serelaxin zeigte in diversen Indikationen bereits eine organprotektive Wirkung, welche unter anderem auf die antifibrotischen Eigenschaften der Substanz zurückzuführen sind. Hier könnte der second messenger cGMP und die abhängige Proteinkinase cGKI eine entscheidende Rolle spielen. Auch in der Therapie der pulmonalen Hypertonie sind cGMP-modulierende Pharmaka, wie z. B. Inhibitoren der Phosphodiesterase 5 oder Stimulatoren der löslichen Guanylatzyklase, im Einsatz. Aufgrund der schlechten Prognose ist hier der Bedarf an weiteren innovativen Pharmaka als auch neuen prognostischen Markern – beispielsweise Fibrose-assoziierten Biomarkern – hoch. Ziel der Arbeit war es, einerseits Erkenntnisse über cGKI – eine cGMP-abhängige Proteinkinase – und deren Substrate in der renalen Fibrose unter Einfluss von Serelaxin zu gewinnen. Dies wurde in Wildtyp- sowie cGKI-KO Mäusen mithilfe der unilateralen Ureterligation untersucht, welche ein etabliertes Modell für die tubulointerstitielle Nierenfibrose darstellt. Zusätzlich wurde getestet, ob durch die Gabe des PDE5-Inhibitors Zaprinast ähnliche bzw. additive antifibrotische Effekte in der Niere erzielt wurden. In einer weiteren Untersuchung wurde anschließend an Patienten mit pulmonaler Hypertonie die klinische Relevanz ausgewählter Fibrose-assoziierter Biomarker – der MMP/TIMP-Verhältnisse – evaluiert, indem ein biologischer Marker identifiziert wurde, der mit dem Krankheitsverlauf sowie der Prognose assoziiert ist und zugleich mit den antifibrotischen Effekten von Serelaxin verknüpft ist. Im renalen Nierenfibrose-Modell waren die cGMP-Konzentrationen im fibrotischen Nierengewebe nach 7-tägiger Behandlung mit Serelaxin, Zaprinast sowie der Kombination beider Pharmaka erhöht. In WT Mäusen konnten ähnliche über die NO-cGMP-abhängige Proteinkinase cGKI vermittelte antifibrotische Effekte in allen drei Behandlungsgruppen beobachtet werden. Die Verabreichung von Serelaxin führte hierbei vermutlich über die Hemmung der Smad2- und ERK1-Phosphorylierung zu einer Reduzierung von Bestandteilen der extrazellulären Matrix (z. B. Kollagene, Fibronektin). Die 7-tägige Behandlung mit dem Phosphodiesterase-5-Hemmer Zaprinast resultierte in einer tendenziell verringerten ERK1- und ERK2-Phosphorylierung ohne Einfluss auf die Smad2-Phosphorylierung. Zusätzlich wurde mit Zaprinast die antifibrotische Wirkung über cGKI-unabhängige Mechanismen vermittelt. Die Kombination der beiden cGMP-erhöhenden Therapieoptionen war nicht antifibrotisch wirksamer als die jeweiligen Einzelsubstanzen. Matrix-Metalloproteinasen – Proteine, die für den Umbau der extrazellulären Matrix verantwortlich sind – wurden nur durch die 7-tägige Serelaxin-Behandlung positiv moduliert, indem das in der Fibrose stark erhöhte Angebot an MMP2 reduziert wurde. Die in der Fibrose nahezu unveränderte MMP9-Proteinexpression wurde nach Serelaxin-Gabe lediglich vermehrt in die aktive Form überführt. Weiterhin wurde in Patienten mit pulmonaler Hypertonie die klinische Relevanz von Matrix-Metalloproteinasen (MMP2, MMP9) und deren Inhibitoren (TIMP1, TIMP4) untersucht. Dazu wurde in einem Patientenkollektiv mit idiopathischer pulmonalarterieller Hypertonie (n=24) sowie pulmonaler Hypertonie aufgrund einer Linksherzinsuffizienz (n=11) nachgewiesen, dass durch den Fibrose-assoziierten Biomarker MMP2/TIMP4 ein Zusammenhang mit der Hämodynamik (mPAP, PVR) sowie der rechtsventrikulären Funktion (TAPSE) gezeigt werden konnte. Zusätzlich war MMP2/TIMP4 in Patienten mit iPAH prädiktiv für das klinische Outcome (klinische Verschlechterung, Tod). Die Ergebnisse dieser Arbeit haben also gezeigt, dass Serelaxin in der Nierenfibrose über die NO-cGMP-cGKI-Signalkaskade antifibrotisch wirkt, unter anderem durch die Modulation der Matrix-Metalloproteinasen. Zusätzlich haben sich Matrix-Metalloproteinasen und deren Inhibitoren bei Patienten mit pulmonaler Hypertonie, einer kardiovaskulären Erkrankung charakterisiert durch fibrotischen Gewebeumbau, als prognostisch wertvoll erwiesen

Involvement of cyclic guanosine monophosphate-dependent protein kinase I in renal antifibrotic effects of serelaxin

Introduction: Kidney fibrosis has shown to be ameliorated through the involvement of cyclic guanosine monophosphate (cGMP) and its dependent protein kinase I (cGKI). Serelaxin, the recombinant form of human relaxin-II, increases cGMP levels and has shown beneficial effects on kidney function in acute heart failure patients. Antifibrotic properties of serelaxin are supposed to be mediated via relaxin family peptide receptor 1 and subsequently enhanced nitric oxide/ cGMP to inhibit transforming growth factor 1)) (TGFI)) signaling. This study examines the involvement of cGKI in the antifibrotic signaling of serelaxin. Methods and Results: Kidney fibrosis was induced by unilateral ureteral obstruction in wildtype (WT) and cGKI knock-out (KO) mice. After 7 days, renal antifibrotic effects of serelaxin were assessed. Serelaxin treatment for 7 days significantly increased cGMP in the kidney of WT and cGKI-KO. In WT, renal fibrosis was reduced through decreased accumulation of collagenl A1, total collagen, and fibronectin. The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and-9 were positively modulated after treatment. Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased. However, these effects were not observed in cGKI-KO. Conclusion: Antifibrotic renal effects of serelaxin are mediated via cGMP/cGKI to inhibit Smad2- and ERK1-dependent TGF-13 signaling and increased PDE5a phosphorylation

Involvement of cyclic guanosine monophosphate-dependent protein kinase I in renal antifibrotic effects of serelaxin

Introduction: Kidney fibrosis has shown to be ameliorated through the involvement of cyclic guanosine monophosphate (cGMP) and its dependent protein kinase I (cGKI). Serelaxin, the recombinant form of human relaxin-II, increases cGMP levels and has shown beneficial effects on kidney function in acute heart failure patients. Antifibrotic properties of serelaxin are supposed to be mediated via relaxin family peptide receptor 1 and subsequently enhanced nitric oxide/ cGMP to inhibit TGF-β signaling. This study examines the involvement of cGKI in the antifibrotic signaling of serelaxin. Methods and Results: Kidney fibrosis was induced by unilateral ureter obstruction in wildtype (WT) and cGKI knock-out (KO) mice. After 7 days, renal antifibrotic effects of serelaxin were assessed. Serelaxin treatment for 7 days significantly increased cGMP in the kidney of WT and cGKI-KO. In WT, renal fibrosis was reduced through decreased accumulation of collagen1A1, total collagen and fibronectin. The profibrotic CTGF as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and -9 were positively modulated after treatment. Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased. However, these effects were not observed in cGKI-KO. Conclusion: Antifibrotic renal effects of serelaxin are mediated via cGMP/cGKI to inhibit Smad2- and ERK1 -dependent TGF-β signaling and increased PDE5a phosphorylation

Cyclic Nucleotide Signalling in Kidney Fibrosis

Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure

Differences in the renal antifibrotic cGMP/cGKI-dependent signaling of serelaxin, zaprinast, and their combination

Renal fibrosis is an important factor for end-stage renal failure. However, only few therapeutic options for its treatment are established. Zaprinast, a phosphodiesterase 5 inhibitor, and serelaxin, the recombinant form of the naturally occurring hormone relaxin, are differently acting modulators of cyclic guanosine monophosphate (cGMP) signaling. Both agents enhance cGMP availability in kidney tissue. These substances alone or in combination might interfere with the development of kidney fibrosis. Therefore, we compared the effects of combination therapy with the effects of monotherapy on renal fibrosis. Renal fibrosis was induced by unilateral ureteral obstruction (UUO) for 7 days in wild-type (WT) and cGKI knockout (KO) mice. Renal antifibrotic effects were assessed after 7 days. In WT, zaprinast and the combination of zaprinast and serelaxin significantly reduced renal interstitial fibrosis assessed by alpha-SMA, fibronectin, collagen1A1, and gelatinases (MMP2 and MMP9). Intriguingly in cGKI-KO, mRNA and protein expression of fibronectin and collagen1A1 were reduced by zaprinast, in contrast to serelaxin. Gelatinases are not regulated by zaprinast. Although both substances showed similar antifibrotic properties in WT, they distinguished in their effect mechanisms. In contrast to serelaxin which acts both on Smad2 and Erk1, zaprinast did not significantly diminish Erk1/2 phosphorylation. Interestingly, the combination of serelaxin/zaprinast achieved no additive antifibrotic effects compared to the monotherapy. Due to antifibrotic effects of zaprinast in cGKI-KO, we hypothesize that additional cGKI-independent mechanisms are supposed for antifibrotic signaling of zaprinast

Plasma MMP2/TIMP4 Ratio at Follow-up Assessment Predicts Disease Progression of Idiopathic Pulmonary Arterial Hypertension

Purpose Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are of particular interest in the remodeling processes of pulmonary hypertension. The aim of this study was to investigate MMP/TIMP ratios of selected biomarkers (MMP2, MMP9, TIMP1, TIMP4) at follow-up examination (V2) and their prognostic value in patients with idiopathic pulmonary arterial hypertension (iPAH). Methods Blood samples were taken from iPAH patients during right heart catheterization at diagnosis (V1, from 2003 to 2012) and first follow-up examination (V2). MMP2, MMP9, TIMP1, and TIMP4 plasma levels at V2 were determined by ELISA. Coincident with sample collection hemodynamic, laboratory, and clinical parameters were acquired. Additionally, death and clinical worsening (CW) events were listed until July 2015. Results MMP2/TIMP1 and MMP9/TIMP1 did not correlate with hemodynamic and clinical parameters. MMP2/TIMP4 showed a good correlation with mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance, estimated glomerular filtration rate (eGFR), and tricuspid annular plain systolic excursion (TAPSE). MMP9/TIMP4 shows good correlation with mPAP and eGFR. MMP2/TIMP4 showed significant results in the receiver operating characteristics analysis predicting death (AUC = 0.922; p = 0.005) and CW event (AUC = 0.818; p = 0.026). Patients above the cut-off values had a significantly higher probability to die or experience CW, respectively, estimated by log-rank test (p = 0.010 for death; p = 0.032 for CW). Conclusions MMP2/TIMP4 ratio was detected as a marker of disease severity and right ventricular function as well as a predictor for survival and time to clinical worsening and therefore might help for guidance of disease progression in iPAH patients at V2

Fast TWIST with iterative reconstruction improves diagnostic accuracy of AVM of the hand

Very high temporal and spatial resolution is mandatory for the diagnosis of arteriovenous malformations (AVM) of the hand. Until now, magnetic resonance imaging (MRI) has not fulfilled both requirements simultaneously. This study presents how the combination of a very fast TWIST MRI (time-resolved angiography with interleaved stochastic trajectories) sequence and iterative reconstructions optimizes temporal as well as spatial resolution. 11 patients were examined at a 3-T MRI scanner with two different TWIST protocols: the standard and the study protocol, acquiring a data set every 5.57 s and 1.44 s respectively. The study data was retrospectively iteratively reconstructed with different regularization factors (0.001, 0.002, 0.004, 0.008). Results were compared using the sign-test. P-values<0.05 were regarded statistically significant. With a low amount of contrast medium, the temporal resolution of the study protocol enabled the differentiation of arteries from veins in all patients whereas the signal-to-noise ratio (SNR) deteriorated. Depending on the regularization factors, SNR, delineation of arterial feeders and non-involved hand and interdigital arteries, as well as artefact levels varied. Overall, iterative reconstruction with regularization factor 0.004 achieved the best results, consequently showing the ability of MRI as a reliable diagnostic method in AVMs of the hand