Characterization of the denaturation and renaturation of human plasma vitronectin II. Investigation into the mechanism of formation of multimers

Unfolding and refolding of plasma vitronectin appear irreversible under near physiological conditions, with rearrangements of disulfides and self-association to a multimeric form observed as prominent structural alterations which accompany denaturation. A mechanism for the folding reactions of vitronectin has been proposed (Zhuang, P., Blackburn, M. NPeterson, C. B. (1996) J. Biol. Chem. 270, 14323-14332) in which vitronectin acquires a partially folded intermediate structure which is highly prone to oligomerize into a multimeric form. Strongly oxidizing conditions adopted for refolding from urea were effective at preventing disulfide rearrangement which disrupts distal disulfides near the C terminus of the protein. Prohibiting disulfide rearrangement under these conditions, however, was not sufficient to achieve reversibility in folding. In contrast, variations in the ionic strength of the refolding medium affect the partitioning of species so that refolded monomers are obtained at high ionic strength, and self-association is precluded. The effects of ionic strength on the partially folded intermediate in the vitronectin folding pathway appear to favor intramolecular hydrophobic collapse to form a stable hydrophobic core for the monomer versus intermolecular hydrophobic interactions which stabilize multimeric vitronectin. Although both ionic and hydrophobic interactions presumably contribute to subunit interfaces within the multimer, the basic heparin-binding region near the C terminus of the protein does not provide binding interactions which are important for self-association of vitronectin

A heart team’s perspective on interventional mitral valve repair: Percutaneous clip implantation as an important adjunct to a surgical mitral valve program for treatment of high-risk patients

ObjectiveSurgical mitral valve repair carries an elevated perioperative risk in the presence of severely reduced ventricular function and relevant comorbidities. We sought to assess the feasibility of catheter-based mitral valve repair using a clip-based percutaneous edge-to-edge repair system in selected patients at high surgical risk with mitral regurgitation grade 3 or worse.MethodsBetween 2002 and January 2011, 202 consecutive patients without prior mitral valve surgery (age 75 ± 9 years; 63% were male) with symptomatic functional (65%), degenerative (27%), or mixed (8%) mitral regurgitation were treated with a percutaneous clip system for approximation of the anterior and posterior mitral leaflets. Risk for mitral valve surgery was considered high in terms of a mean logistic European System for Cardiac Operative Risk Evaluation of 44% (range, 21%–54%). Preprocedural left ventricular ejection fraction was 35% or less in 36% of patients. An interdisciplinary heart team of cardiologists and cardiac surgeons discussed all patients.ResultsPercutaneous clip implantation was successful in 186 patients (92%). Patients were treated with 1 clip (n = 125; 62%), 2 clips (n = 64; 32%), or 3 or more clips (n = 7; 3%). Reduction in mitral regurgitation from pre- to postprocedure was significant (P < .0001) and remained stable within the first 12 months in the majority of patients. Thirty-day mortality was 3.5% (7/202 patients). Hospital stay was 12 ± 10 days, and median intensive care unit stay was 1 day (range, 0–45 days). Eleven patients required surgical valve repair/replacement at a median of 38 days (0–468 days) after percutaneous clip implantation.ConclusionsClip-based percutaneous mitral valve repair is a safe, low-risk, and effective therapeutic option in symptomatic patients with a high risk for surgery and does not exclude later surgical repair

Rationale and design for the development of a novel nitroxyl donor in patients with acute heart failure

Hospitalisation for acute heart failure remains a major public health problem with high prevalence, morbidity, mortality, and cost. Prior attempts to develop new therapies for this condition have not been successful. Nitroxyl (HNO) plays a unique role in cardiovascular physiology by direct post‐translational modification of thiol residues on target proteins, specifically SERCA2a, phospholamban, the ryanodine receptor and myofilament proteins in cardiomyocytes. In animal models, these biological effects lead to vasodilatation, increased inotropy and lusitropy, but without tachyphylaxis, pro‐arrhythmia or evidence of increased myocardial oxygen demand. BMS‐986231 is an HNO donor being developed as a therapy for heart failure, and initial studies in patients with heart failure support the potential clinical value of these physiological effects. In this manuscript, we describe the ongoing phase II development programme for BMS‐986231, which consists of three related randomised placebo‐controlled clinical trials, StandUP‐AHF, StandUP‐Imaging and StandUP‐Kidney, which are designed to provide evidence of tolerability and efficacy as well as confirm the anticipated physiological effects in patients with heart failure with reduced ejection fraction. These studies will set the stage for the further study of BMS‐986231 in future phase III clinical trials

Identification and validation of G protein-coupled receptors modulating flow-dependent signaling pathways in vascular endothelial cells

Vascular endothelial cells are exposed to mechanical forces due to their presence at the interface between the vessel wall and flowing blood. The patterns of these mechanical forces (laminar vs. turbulent) regulate endothelial cell function and play an important role in determining endothelial phenotype and ultimately cardiovascular health. One of the key transcriptional mediators of the positive effects of laminar flow patterns on endothelial cell phenotype is the zinc-finger transcription factor, krüppel-like factor 2 (KLF2). Given its importance in maintaining a healthy endothelium, we sought to identify endothelial regulators of the KLF2 transcriptional program as potential new therapeutic approaches to treating cardiovascular disease. Using an approach that utilized both bioinformatics and targeted gene knockdown, we identified endothelial GPCRs capable of modulating KLF2 expression. Genetic screening using siRNAs directed to these GPCRs identified 12 potential GPCR targets that could modulate the KLF2 program, including a subset capable of regulating flow-induced KLF2 expression in primary endothelial cells. Among these targets, we describe the ability of several GPCRs (GPR116, SSTR3, GPR101, LGR4) to affect KLF2 transcriptional activation. We also identify these targets as potential validated targets for the development of novel treatments targeting the endothelium. Finally, we highlight the initiation of drug discovery efforts for LGR4 and report the identification of the first known synthetic ligands to this receptor as a proof-of-concept for pathway-directed phenotypic screening to identify novel drug targets

Effects of a Novel Nitroxyl Donor in Acute Heart Failure The STAND-UP AHF Study

Objectives: The primary objective was to identify well-tolerated doses of cimlanod in patients with acute heart failure (AHF). Secondary objectives were to identify signals of efficacy, including biomarkers, symptoms, and clinical events. Background: Nitroxyl (HNO) donors have vasodilator, inotropic and lusitropic effects. Bristol-Myers Squibb-986231 (cimlanod) is an HNO donor being developed for acute heart failure (AHF). Methods: This was a phase IIb, double-blind, randomized, placebo-controlled trial of 48-h treatment with cimlanod compared with placebo in patients with left ventricular ejection fraction ≤40% hospitalized for AHF. In part I, patients were randomized in a 1:1 ratio to escalating doses of cimlanod or matching placebo. In part II, patients were randomized in a 1:1:1 ratio to either of the 2 highest tolerated doses of cimlanod from part I or placebo. The primary endpoint was the rate of clinically relevant hypotension (systolic blood pressure &lt;90 mm Hg or patients became symptomatic). Results: In part I (n = 100), clinically relevant hypotension was more common with cimlanod than placebo (20% vs. 8%; relative risk [RR]: 2.45; 95% confidence interval [CI]: 0.83 to 14.53). In part II (n = 222), the incidence of clinically relevant hypotension was 18% for placebo, 21% for cimlanod 6 μg/kg/min (RR: 1.15; 95% CI: 0.58 to 2.43), and 35% for cimlanod 12 μg/kg/min (RR: 1.9; 95% CI: 1.04 to 3.59). N-terminal pro–B-type natriuretic peptide and bilirubin decreased during infusion of cimlanod treatment compared with placebo, but these differences did not persist after treatment discontinuation. Conclusions: Cimlanod at a dose of 6 μg/kg/min was reasonably well-tolerated compared with placebo. Cimlanod reduced markers of congestion, but this did not persist beyond the treatment period. (Evaluate the Safety and Efficacy of 48-Hour Infusions of HNO (Nitroxyl) Donor in Hospitalized Patients With Heart Failure [STANDUP AHF]; NCT03016325

Haemodynamic effects of the nitroxyl donor cimlanod (BMS-986231) in chronic heart failure: a randomized trial

Aims Nitroxyl provokes vasodilatation and inotropic and lusitropic effects in animals via post-translational modification of thiols. We aimed to compare effects of the nitroxyl donor cimlanod (BMS-986231) with those of nitroglycerin (NTG) or placebo on cardiac function in patients with chronic heart failure with reduced ejection fraction (HFrEF). Methods and results In a randomized, multicentre, double-blind, crossover trial, 45 patients with stable HFrEF were given a 5 h intravenous infusion of cimlanod, NTG, or placebo on separate days. Echocardiograms were done at the start and end of each infusion period and read in a core laboratory. The primary endpoint was stroke volume index derived from the left ventricular outflow tract at the end of each infusion period. Stroke volume index with placebo was 30 ± 7 mL/m2 and was lower with cimlanod (29 ± 9 mL/m2; P = 0.03) and NTG (28 ± 8 mL/m2; P = 0.02). Transmitral E-wave Doppler velocity on cimlanod or NTG was lower than on placebo and, consequently, E/e′ (P = 0.006) and E/A ratio (P = 0.003) were also lower. NTG had similar effects to cimlanod on these measurements. Blood pressure reduction was similar with cimlanod and NTG and greater than with placebo. Conclusion In patients with chronic HFrEF, the haemodynamic effects of cimlanod and NTG are similar. The effects of cimlanod may be explained by venodilatation and preload reduction without additional inotropic or lusitropic effects. Ongoing trials of cimlanod will further define its potential role in the treatment of heart failure