Nicola Scott

Background: LCZ696 (sacubitril/valsartan) antagonizes the renin-angiotensin system while simultaneously augmenting the natriuretic peptides (NPs). Inhibition of phosphodiesterase 9 inhibition (PDE9i), which hydrolyses NP-generated cGMP may be a more specific means of enhancing NP bioactivity. The objective of the present study was to compare for the first time effects of LCZ696 and PDE9i+valsartan in experimental heart failure (HF) and investigate combination PDE9i+LCZ696.

Methods and results: Seven sheep received, on separate days, intravenous boli of (1) LCZ696, (2) PDE9i+valsartan, (3) PDE9i+LCZ696, and (4) vehicle control, during mild HF and severe HF. Compared with control, LCZ696 and PDE9i+LCZ696 induced similar increases in plasma NPs (greater increments in severe HF), whereas PDE9i+valsartan reduced NP levels. Circulating cGMP was elevated following LCZ696 and PDE9i+valsartan, and further increased with combination PDE9i+LCZ696. All active treatments increased plasma renin and angiotensin II (greater increments in severe HF). Plasma aldosterone was unchanged. Plasma endothelin-1 was elevated by LCZ696 and PDE9i+LCZ696, but not PDE9i+valsartan. Active treatments reduced arterial and left atrial pressure and peripheral resistance and increased cardiac output (more pronounced in severe-HF). PDE9i+LCZ696 induced greater hemodynamic changes relative to LCZ696 and PDE9i+valsartan. Treatments had minimal renal effect in mild HF. In severe HF, treatments elevated urine cGMP in association with significant increases in creatinine clearance and diuresis and natriuresis, effects especially prominent with PDE9i+LCZ696.

Conclusions: LCZ696 and PDE9i+valsartan have similar beneficial hemodynamic and renal effects in HF, with combined PDE9i+LCZ696 producing additional improvements. Overall, effects were significantly greater in severe HF. Findings suggest PDE9i might serve as a replacement for neprilysin inhibition or as an adjunct therapy to LCZ696.

Abstract Osteocrin (OSTN) is an endogenous protein sharing structural similarities with the natriuretic peptides [NPs; atrial (ANP), B‐type (BNP) and C‐type (CNP) NP], which are hormones known for their crucial role in maintaining pressure/volume homeostasis. Osteocrin competes with the NPs for binding to the receptor involved in their clearance (NPR‐C). In the present study, having identified, for the first time, the major circulating form of OSTN in human and ovine plasma, we examined the integrated haemodynamic, endocrine and renal effects of vehicle‐controlled incremental infusions of ovine proOSTN (83–133) and its metabolism in eight conscious normal sheep. Incremental i.v . doses of OSTN produced stepwise increases in circulating concentrations of the peptide, and its metabolic clearance rate was inversely proportional to the dose. Osteocrin increased plasma levels of ANP, BNP and CNP in a dose‐dependent manner, together with concentrations of their intracellular second messenger, cGMP. Increases in plasma cGMP were associated with progressive reductions in arterial pressure and central venous pressure. Plasma cAMP, renin and aldosterone were unchanged. Despite significant increases in urinary cGMP levels, OSTN administration was not associated with natriuresis or diuresis in normal sheep. These results support OSTN as an endogenous ligand for NPR‐C in regulating plasma concentrations of NPs and associated cGMP‐mediated bioactivity. Collectively, our findings support a role for OSTN in maintaining cardiovascular homeostasis. What is the central question of this study? What role does osteocrin (OSTN) have in integrated cardiovascular, renal and neurohumoral function in normal health? What is the main finding and its importance? Osteocrin is structurally similar to natriuretic peptides (NPs) that play a crucial role in cardiovascular homeostasis and binds to the NP receptor‐C (NPR‐C). Stepped doses of OSTN raised plasma atrial NP, B‐type NP, C‐type NP and cGMP and reduced arterial and venous pressures. Collectively, these findings provide strong evidence that increased NP levels result from competitive displacement from NPR‐C, not from increased NP secretion, suggesting that OSTN might play a role in maintaining cardiovascular homeostasis.

Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R = 0.85-0.90) and plasma (SPEED R = 0.84; SDC R = 0.76, S-Trap R = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired.

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide. Despite recent advances in clinical management, reoccurence of heart failure after AMI remains high, in part because of the limited capacity of cardiac tissue to repair after AMI-induced cell death. Growth factor-based therapy has emerged as an alternative AMI treatment strategy. Understanding the underlying mechanisms of growth factor cardioprotective and regenerative actions is important. This review focuses on the function of different growth factors at each stage of the cardiac repair process. Recent evidence for growth factor therapy in preclinical and clinical trials is included. Finally, different delivery strategies are reviewed with a view to providing workable strategies for clinical translation. Plain language summary Acute myocardial infarction is a serious, life-threatening disease. Current treatments for acute myocardial infarction are unsatisfactory, and new treatments are required. Growth factors are promising treatments for myocardial infarction. It is intriguing to understand how growth factors provide cardioprotective benefits. This article describes the various growth factors used to treat myocardial infarction and how they are delivered to the infarcted heart.

One-quarter of patients with acute decompensated heart failure (ADHF) experience acute kidney injury (AKI)-an abrupt reduction or loss of kidney function associated with increased long-term mortality. There is a critical need to identify early and real-time markers of AKI in ADHF; however, to date, no protein biomarkers have exhibited sufficient diagnostic or prognostic performance for widespread clinical uptake. We aimed to identify novel protein biomarkers of AKI associated with ADHF by quantifying changes in protein abundance in the kidneys that occur during ADHF development and recovery in an ovine model. Relative quantitative protein profiling was performed using sequential window acquisition of all theoretical fragment ion spectra-mass spectrometry (SWATH-MS) in kidney cortices from control sheep ( = 5), sheep with established rapid-pacing-induced ADHF ( = 8), and sheep after ~4 weeks recovery from ADHF ( = 7). Of the 790 proteins quantified, we identified 17 candidate kidney injury markers in ADHF, 1 potential kidney marker of ADHF recovery, and 2 potential markers of long-term renal impairment (differential abundance between groups of 1.2-2.6-fold, adjusted < 0.05). Among these 20 candidate protein markers of kidney injury were 6 candidates supported by existing evidence and 14 novel candidates not previously implicated in AKI. Proteins of differential abundance were enriched in pro-inflammatory signalling pathways: glycoprotein VI (activated during ADHF development; adjusted < 0.01) and acute phase response (repressed during recovery from ADHF; adjusted < 0.01). New biomarkers for the early detection of AKI in ADHF may help us to evaluate effective treatment strategies to prevent mortality and improve outcomes for patients.

BACKGROUND Acute decompensated heart failure (ADHF) is associated with deterioration in renal function-an important risk factor for poor outcomes. Whether ADHF results in permanent kidney damage/dysfunction is unknown. METHODS AND RESULTS We investigated for the first time the renal responses to the development of, and recovery from, ADHF using an ovine model. ADHF development induced pronounced hemodynamic changes, neurohormonal activation, and decline in renal function, including decreased urine, sodium and urea excretion, and creatinine clearance. Following ADHF recovery (25 days), creatinine clearance reductions persisted. Kidney biopsies taken during ADHF and following recovery showed widespread mesangial cell prominence, early mild acute tubular injury, and medullary/interstitial fibrosis. Renal transcriptomes identified altered expression of 270 genes following ADHF development and 631 genes following recovery. A total of 47 genes remained altered post-recovery. Pathway analysis suggested gene expression changes, driven by a network of inflammatory cytokines centered on IL-1 beta (interleukin 1 beta), lead to repression of reno-protective eNOS (endothelial nitric oxide synthase) signaling during ADHF development, and following recovery, activation of glomerulosclerosis and reno-protective pathways and repression of proinflammatory/fibrotic pathways. A total of 31 dysregulated genes encoding proteins detectable in urine, serum, and plasma identified potential candidate markers for kidney repair (including CNGA3 [cyclic nucleotide gated channel subunit alpha 3] and OIT3 [oncoprotein induced transcript 3]) or long-term renal impairment in ADHF (including ACTG2 [actin gamma 2, smooth muscle] and ANGPTL4 [angiopoietin like 4]). CONCLUSIONS In an ovine model, we provide the first direct evidence that an episode of ADHF leads to an immediate decline in kidney function that failed to fully resolve after approximate to 4 weeks and is associated with persistent functional/structural kidney injury. We identified molecular pathways underlying kidney injury and repair in ADHF and highlighted 31 novel candidate biomarkers for acute kidney injury in this setting.

Simple Summary Human heart failure (HF) is a complex clinical syndrome that can be caused by a variety of diseases. While long-term high blood pressure and heart attacks are major contributing factors, there can be many diseases of the heart and circulation that contribute to the development of HF. Although there have been salutary improvements in the medical management of HF over the last 30 years, ongoing ill effects of living with the syndrome and the persistently high death rates mean there is an irrefutable need for new and improved treatment options. Well-characterized animal models have contributed, and continue to contribute, much to the advancement of clinical care. This review will summarize the main large animal models of HF developed to date. Studies utilizing these large animal models are an essential step leading to the development of novel pharmaceutical and device-based therapies before they can undergo definitive clinical trials. This review will discuss the various benefits of different large animal models of HF and highlight some key deficiencies to date. There is clearly a need for ongoing development of clinically relevant large animal models of HF. Heart failure (HF) is the final common end point of multiple metabolic and cardiovascular diseases and imposes a significant health care burden worldwide. Despite significant improvements in clinical management and outcomes, morbidity and mortality remain high and there remains an indisputable need for improved treatment options. The pathophysiology of HF is complex and covers a spectrum of clinical presentations from HF with reduced ejection fraction (HFrEF) (<= 40% EF) through to HF with preserved EF (HFpEF), with HFpEF patients demonstrating a reduced ability of the heart to relax despite an EF maintained above 50%. Prior to the last decade, the majority of clinical trials and animal models addressed HFrEF. Despite growing efforts recently to understand underlying mechanisms of HFpEF and find effective therapies for its treatment, clinical trials in patients with HFpEF have failed to demonstrate improvements in mortality. A significant obstacle to therapeutic innovation in HFpEF is the absence of preclinical models including large animal models which, unlike rodents, permit detailed instrumentation and extensive imaging and sampling protocols. Although several large animal models of HFpEF have been reported, none fulfil all the features present in human disease and few demonstrate progression to frank decompensated HF. This review summarizes well-established models of HFrEF in pigs, dogs and sheep and discusses attempts to date to model HFpEF in these species.

Phosphodiesterase-9 (PDE9) reduces natriuretic peptide (NP) signaling and may be involved in the pathophysiology of heart failure (HF). This study investigated for the first time the integrated hemodynamic, endocrine, and renal effects of phosphodiesterase-9 inhibition (PDE9-I). A total of 8 normal sheep and 8 sheep with pacing-induced HF received incremental intravenous boluses of PDE9-I (30, 100, and 300 mg PF-04749982 at 1-h intervals). PDE9-I dose-dependently increased plasma cyclic guanosine monophosphate (cGMP) in normal sheep (p < 0.05) while concurrently reducing circulating atrial natriuretic peptide levels (p < 0.01). Similar trends were evident in HF, resulting in significant elevations in the cGMP/NP ratio in both states (p < 0.001 and p < 0.05, respectively). PDE9-I also produced progressive falls in arterial pressure (HF: p < 0.001), atrial pressure (Normal: p < 0.001; HF: p < 0.001), and peripheral resistance (HF: p < 0.001), and transiently increased cardiac output at the top dose (Normal: p < 0.05; HF: p < 0.001). Inhibition of PDE9 had a negligible effect on circulating hormones at the lower doses, but post-high dose, acutely increased renin activity (Normal: p < 0.001; HF: p < 0.05), vasopressin (Normal: p < 0.001; HF: p < 0.01), and cyclic adenosine monophosphate (HF: p < 0.001). Plasma aldosterone increased briefly after high-dose PDE9-I in normal sheep, and fell following the top dose in HF. PDE9-I dose-dependently increased urinary cGMP in both states (both p < 0.001). In HF, this was associated with increases in urine volume (p < 0.01), sodium excretion (p < 0.01), and creatinine clearance (p < 0.001). PDE9-I improves NP efficacy in conjunction with beneficial hemodynamic and renal effects in experimental HF. These results support a role for PDE9 in HF pathophysiology and suggest its inhibition may constitute a novel therapeutic approach to this disease.

Background/aim: Patients with breast cancer and metabolic syndrome have poorer outcomes. We aimed to develop and characterise an apolipoprotein E-null/aromatase knockout (ApoE-/-/ArKO) mouse model of breast cancer with metabolic syndrome to aid research of the mechanisms behind poor prognosis.

Materials and methods: Wild-type, ApoE-/- and ApoE-/-/ArKO mice were orthotopically implanted with EO771 murine breast cancer cells. Tumour growth was monitored and tumours investigated for pathological features such as cancer-associated adipocytes, hypoxia and cancer cell proliferation.

Results: Tumours from ApoE-/-/ArKO mice were significantly more proliferative than those from wild-type mice (p=0.003), and exhibited reduced expression of insulin-like growth factor binding protein-5 (p=0.002). However, ApoE-/-/ArKO mice also had a reduced rate of metastasis compared to wild-type and ApoE-/- mice. Tumour hypoxia and the number of cancer-associated adipocytes did not differ.

Conclusion: The ApoE-/-/ArKO model with EO771 breast cancer provides a novel mouse model to investigate the effects of metabolic syndrome on aspects of breast tumour biology.