Introduction: Natriuretic peptide receptor 1 (NPR1) is a membrane-bound guanylate cyclase activated by atrial and brain natriuretic peptides (ANP and BNP), playing a central role in blood pressure (BP) regulation via vasorelaxation and modulation of intravascular volume. Published data suggest that NPR1 loss-of-function variants, L1034F and E967K, localized to the guanylyl cyclase domain, exhibit a partial decrease in NPR1 activation in response to ANP and BNP. In this study, we report a newly identified variant, I751N, associated with elevated blood pressure; however, the impact of this variant on NPR1 function remains unclear. Research Questions: Does the NPR1 variant I751N affect the function of NPR1? If it does, what are the underlying mechanisms? Methods: We performed genetic association analysis for BP using whole-exome sequencing data from 1,252,240 individuals. The functional characterization of NPR1 variants was examined using flow cytometry, cyclic guanosine monophosphate (cGMP) assays, western blotting, and internalization assay in HEK293 cells overexpressing either the wild-type (WT) NPR1 or the variants E967K and I751N. Results: The NPR1 I751N variant was significantly associated with higher systolic BP ( p = 2.9e-11; effect size = +5.3 mmHg per allele; alternative allele frequency = 0.02%). Flow cytometry confirmed that NPR1 cell surface expression was comparable across WT and both variant-expressing HEK293 stable cell lines. cGMP assays demonstrated partial loss of signaling with E967K and near-complete loss with I751N. The I751N protein had a lower molecular weight (~75–90 kDa) than WT and E967K (~119 kDa), suggesting truncation or degradation due to the introduction of a polar residue (N, Asparagine) in a predicted hydrophobic region. Furthermore, biosensor-labeled NPR1 antibody experiments revealed accelerated ligand internalization in NPR1-I751N cells compared to NPR1-WT cells. Conclusion: The NPR1-I751N variant encodes a truncated receptor that rapidly internalizes ligands without triggering downstream signaling, suggesting that this variant may function as a decoy receptor, akin to NPR3. These findings reveal new mechanisms by which the NPR1-I751N variant impacts BP in individuals carrying this mutation.

Background: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling and elevated pulmonary artery pressure that leads to right ventricular (RV) failure. Platelet-derived growth factor (PDGF) signaling contributes to PAH pathogenesis. Research question: To understand the contribution of PDGF signaling on PAH disease progression, using a fully human monoclonal antibody (REGN13335) that binds and neutralizes PDGF with high affinity. Methods: REGN13335 treatment was initiated in 3 independent PAH rodent models (Sugen-Hypoxia [SuHx] mouse, monocrotaline rat, SuHx rat) to study the impact of PDGF inhibition on RV systolic pressure (RVSP). Utilizing the SuHx rat PAH model, we conducted a longitudinal multimodal imaging study using micro-ultrasound (US) and cardiac MRI to evaluate the therapeutic potential of REGN13335 on RV remodeling and dysfunction. Arterial oxygen saturation was measured to evaluate pulmonary health. RV gene expression analysis was performed to evaluate cardiac stress biomarkers (Nppb, Myh7, Gdf15) and PDGF receptor (PDGFR-β) expression. Results: REGN13335 (10-25 mg/kg/wk) significantly lowered RVSP in 3 rodent models of PAH. Longitudinal US imaging was conducted in the SuHx rat PAH model and revealed significant RV hypertrophy, chamber dilation, and reduced pulmonary artery flow. Sildenafil, a standard of care (SOC) treatment in PAH, attenuated RV dysfunction in this model suggesting its translatability to study RV function in PAH. REGN13335 treatment significantly attenuated RV remodeling, limiting increases in wall thickness compared to isotype control at wk 4 and wk 8. Importantly, REGN13335 preserved cardiac output (61% improvement compared to controls [p<0.0001)] at wk 4; 40% improvement at wk 8 [p<0.01]; Figure). MRI measurements at wk 4 showed REGN13335 treatment preserved RV fractional area change by 28% (p<0.05) compared to SuHx isotype control treated rats (Figure). REGN13335 treatment preserved heart rate and arterial oxygen saturation in SuHx rats. Also, REGN13335 prevented transcriptional upregulation of Pdgfrb and reduced cardiac stress biomarkers (Nppb, Myh7, and Gdf15) in SuHx rat RV tissue. Conclusion: Longitudinal cardiac imaging revealed RV dysfunction in the SuHx rat model of PAH. Treatment with SOC or REGN13335 improved RV hypertrophy, systolic function and output. REGN13335 may represent a novel therapeutic approach to address RV remodeling and dysfunction in patients with PAH.

Introduction: Activation of natriuretic peptide receptor 1 (NPR1) regulates vascular tone, lowers venous pressures, and effects natriuresis and diuresis, which may have therapeutic benefit in heart failure with preserved ejection fraction (HFpEF). REGN5381, an investigational monoclonal antibody agonist of NPR1, has the potential to overcome the limitations of previous recombinant natriuretic peptide (NP) infusions that were limited by their short half-life. Research question: What are the safety, tolerability, and hemodynamic effects of REGN5381 in patients with HFpEF (NCT05353166)? Methods: Patients aged 18–75 years, with New York Heart Association class II/III heart failure with left ventricular ejection fraction ≥50% and N-terminal pro B-type natriuretic peptide (NT-proBNP) >300 pg/mL, were randomized 1:1 to a single intravenous injection of REGN5381 (30 mg, 100 mg, 300 mg) or placebo. Key hemodynamic inclusion criteria were pulmonary capillary wedge pressure (PCWP) ≥15 mmHg and right atrial pressure >5 mmHg upon right heart catheterization on Day 1. Safety and tolerability were assessed, including change from baseline in PCWP, systemic blood pressure (BP), and biomarkers. Results: Thirty-six patients received REGN5381 30 mg (n=3), 100 mg (n=3), 300 mg (n=12), or placebo (n=18). Mean (SD) change from baseline 6 h post-infusion in PCWP for 100 mg and 300 mg was −9.3 (1.4) and −7.7 (2.1), respectively, and −3.3 (2.4) for placebo (Fig. 1). Baseline mean (SD) systolic BP was 141.1 (20.3), 128.3 (12.6), and 134.8 (8.8) mmHg in the placebo, 100 mg, and 300 mg dose groups, respectively, with changes from baseline 6 h post-infusion of −0.6 (11.2), −5.7 (6.4), and −8.5 (13.7) mmHg, respectively. No effect of REGN5381 on cardiac output, no effect on systemic BP beyond 24 h, and no effects on renal function or biomarkers including NP, were observed. Most reported treatment-emergent adverse events (TEAEs) were mild (Table). One patient (8.3%) had a serious TEAE (respiratory tract procedural complication and hemoptysis), and 2 (16.7%) had mild treatment-related TEAEs (increased liver enzymes and dizziness) in the 300 mg dose group. No deaths were reported. Conclusions: REGN5381 was generally well-tolerated and improved PCWP with no persistent drops in systemic BP or cardiac output in patients with HFpEF. A reduction in PCWP with REGN5381 suggests efficacy in cardiopulmonary decongestion. These data support future clinical studies of REGN5381 in patients with HFpEF.

Background: The cardiac hormones atrial natriuretic peptide and brain natriuretic peptide activate natriuretic peptide receptor 1 (NPR1), leading to the generation of cyclic guanosine monophosphate. Subsequently, reduction of blood pressure (BP) occurs via vasodilation and decreasing intravascular volume. Research questions: 1) What are the safety, tolerability, and pharmacodynamics of REGN7544, an investigational first-in-class NPR1-blocking monoclonal antibody? 2) What are the physiological effects and therapeutic potential of NPR1 signaling blockade? Methods: This phase 1, randomized, double-blind, placebo-controlled trial included 10 dose cohorts of healthy adults (NCT05970718). Participants received REGN7544 or placebo in single doses of 3–1000 mg IV or 100–600 mg SC. Eighty adults (aged 18–55 years; 59% male) were dosed with REGN7544 (IV, n=42; SC, n=18) or placebo (n=20). The primary endpoint was the incidence and severity of treatment-emergent adverse events (TEAEs). Results: REGN7544 administration increased BP by approximately 5–10 mmHg above baseline as measured by ambulatory BP monitoring. The magnitude of the BP effect appeared to plateau at higher doses; however, its duration was apparently dose-dependent and persisted for ≥4 weeks. REGN7544 was associated with increases in N-terminal pro-brain natriuretic peptide and with decreases in hemoglobin concentration, both consistent with the expansion of plasma volume. As further evidence of volume expansion, exposure to REGN7544 was associated with increases in body weight and a decrease in natriuresis. The incidence of moderate-to-severe TEAEs for REGN7544-treated adults was comparable to placebo for each cohort. No serious adverse events related to REGN7544 were reported. Conclusions: REGN7544 was generally well-tolerated at doses up to 1000 mg IV and 600 mg SC. REGN7544 durably increased BP to a plateau (5–10 mmHg), with body weight and biomarker results suggesting increased plasma volume. These data support the development of REGN7544 for conditions characterized by hypovolemia and/or hypotension, such as postural orthostatic tachycardia syndrome and sepsis-associated hypotension.

Introduction: Natriuretic peptide receptor 1 (NPR1) activation by atrial and brain natriuretic peptides (ANPs and BNPs) reduces BP through vasorelaxation and decreased intravascular volume. Blocking NPR1 to counteract the effects of ANPs and BNPs is expected to raise BP. Hypothesis: We hypothesized that the human monoclonal NPR1-blocking antibody REGN7544 raises BP, possibly via inhibition of vasorelaxation and increased intravascular volume. Methods: REGN7544, developed using VelocImmune ® technology, was assessed for binding kinetics, NPR1 inhibition, and structural basis of inhibition. Pharmacodynamics were assessed in telemetered normotensive and hypotensive (ANP overexpression by hydrodynamic DNA delivery) humanized-NPR1 ( NPR1 hu/hu ) mice and normotensive cynomolgus monkeys. Vessel reactivity assays were performed using mesenteric vessels isolated from NPR1 hu/hu mice to determine functional effect of NPR1 blockade on ANP-induced vasorelaxation. Reversal of the REGN7544 effect on BP in NPR1 hu/hu mice was performed using SOC BP-lowering agents. Results: REGN7544 binds to human and monkey NPR1, independent of ANP/BNP, with subnanomolar affinity. Ligand-induced NPR1 activation was noncompetitively inhibited by REGN7544. Dose-dependent SBP increased in normotensive NPR1 hu/hu mice after one dose of REGN7544; a high dose achieved a 10–15 mmHg rise for up to 28 d with no further increase following repeat dosing. SBP increased by 30–40 mmHg in hypotensive NPR1 hu/hu mice. A dose-dependent and persistent SBP increase was observed in monkeys following a single high dose of REGN7544, causing a 15–20 mmHg rise for up to 28 d, along with a transient N-terminal pro-ANP increase and hematocrit decrease (suggesting hemodilution). REGN7544 significantly inhibited ANP-induced relaxation of precontracted mesenteric vessels isolated from NPR1 hu/hu mice confirming direct modulation of vascular tone. The BP increase caused by REGN7544 in NPR1 hu/hu mice was reversed by SOC BP-lowering agents, indicating a specific reversal agent would not be required. Conclusions: These data demonstrated that the high-affinity NPR1-blocking antibody REGN7544 increased SBP in normotensive and hypotensive NPR1 hu/hu mice and normotensive monkeys, possibly via inhibited vasorelaxation and altered blood volume. REGN7544 has potential to be a selective and long-acting treatment for hypervolemic and hypotensive disorders, including postural orthostatic tachycardia syndrome and sepsis-induced hypotension.

ABSTRACT:Neutrophils interact with the external milieu in both tissue and blood microenvironments and are emerging as important regulators of blood coagulation. In this study, we explored whether complement induces neutrophil extracellular trap (NET) formation and related blood coagulation using human donor-derived neutrophils. Complement C1q induces NETosis in lipopolysaccharide (LPS) O127-primed neutrophils, whereas LPS alone does not induce NETosis. Bulk RNA sequencing revealed a unique LPS-driven altered neutrophil state, and complement sensitivity for NETosis was found to be transcriptionally dependent. Using an arrayed CRISPR knockout screen in the neutrophil-like differentiated HL60 cells, we identified that SCARF1 and complement receptor 3 are required for C1q-NETosis. Given NETs contain procoagulatory components such as DNA and histones, we investigated whether C1q-related NETs influenced blood coagulation. LPS+C1q-NETs were associated with reduced coagulation activity compared with LPS treatment alone. We further found that LPS upregulated tissue factor expression and coagulation-related activity in neutrophils. Furthermore, neutrophils secrete anticoagulant proteins, including protein C and tissue factor pathway inhibitors, during C1q-mediated NET formation that functionally regulate NET-related coagulation. C1q-NETs also activate the coagulation factors XII and XI, facilitating both intrinsic coagulation and kallikrein-dependent bradykinin production. This study elucidates how NETs regulate both procoagulatory and anticoagulatory components that may influence the pathophysiology of disease.

Heart failure (HF) is a major contributor to global morbidity and mortality. While distinct clinical subtypes, defined by etiology and left ventricular ejection fraction, are well recognized, their genetic determinants remain inadequately understood. In this study, we report a genome-wide association study of HF and its subtypes in a sample of 1.9 million individuals. A total of 153,174 individuals had HF, of whom 44,012 had a nonischemic etiology (ni-HF). A subset of patients with ni-HF were stratified based on left ventricular systolic function, where data were available, identifying 5,406 individuals with reduced ejection fraction and 3,841 with preserved ejection fraction. We identify 66 genetic loci associated with HF and its subtypes, 37 of which have not previously been reported. Using functionally informed gene prioritization methods, we predict effector genes for each identified locus, and map these to etiologic disease clusters through phenome-wide association analysis, network analysis and colocalization. Through heritability enrichment analysis, we highlight the role of extracardiac tissues in disease etiology. We then examine the differential associations of upstream risk factors with HF subtypes using Mendelian randomization. These findings extend our understanding of the mechanisms underlying HF etiology and may inform future approaches to prevention and treatment.

Introduction: Heart failure with reduced ejection fraction (HFrEF) causes high morbidity and mortality despite available therapies, indicating an unmet medical need. Activation of natriuretic peptide receptor 1 (NPR1) regulates vascular tone, lowers venous pressures, and affects natriuresis and diuresis, which may have therapeutic benefit. Recombinant natriuretic peptide infusions, though promising, were limited by short duration of effect. REGN5381, an investigational NPR1 agonist, has shown hemodynamic effects without adverse systemic hypotension along with sustained bioavailability after a single-dose in preclinical studies and a first-in-human study (NCT04506645). Research question: How does REGN5381 impact safety, tolerability, and hemodynamic parameters in patients with HFrEF? Methods: Patients aged 18–75 years, with New York Heart Association class II/III heart failure with left ventricular ejection fraction ≥20% and <50%, were enrolled in this phase 2a, double-blind study (NCT05353166) and received single intravenous infusions of REGN5381 at doses between 10 mg and 300 mg or placebo. Patients taking sacubitril-valsartan were excluded. Safety was monitored as treatment-emergent adverse events (TEAEs). Pulmonary capillary wedge pressure (PCWP), systemic blood pressure (BP), and biomarkers such as N-terminal pro B-type natriuretic peptide (NT-proBNP) were measured at baseline and at 2, 4, and 6 h post-infusion. Results: Twenty-three patients received REGN5381 10 mg (n=3), 30 mg (n=3), 100 mg (n=6), or 300 mg (n=4); 7 received placebo. Compared with placebo, numeric reductions in PCWP were observed with the 100 mg and 300 mg doses at 6 h post-infusion (Fig. 1). Baseline mean (SD) systolic BP was 130.6 (17.9), 118.7 (9.2), and 123.0 (30.0) mmHg for the placebo, 100 mg, and 300 mg groups, with changes from baseline 6 h post-infusion of −1.3 (2.6), −5.7 (7.3), and −7.5 (15.6) mmHg. No apparent differences or persistent trends in NT-proBNP and systemic BP reductions among dose groups were noted. TEAEs were observed in 7 patients (Table). Two severe serious TEAEs occurred in the 300 mg group, ischemic hepatitis and cardiac failure, which was possibly related to treatment and resulted in death 10 days after study drug administration. Conclusions: In this small phase 2 trial, REGN5381 was generally well tolerated. However, more data are needed to confirm its safety. The numeric reduction in PCWP suggests REGN5381 may aid in heart failure decongestion.

Kidneys are intricate three-dimensional structures in the body, yet the spatial and molecular principles of kidney health and disease remain inadequately understood. We generated high-quality datasets for 81 samples, including single-cell, single-nuclear, spot-level (Visium) and single-cell resolution (CosMx) spatial-RNA expression and single-nuclear open chromatin, capturing cells from healthy, diabetic and hypertensive diseased human kidneys. Combining these data, we identify cell types and map them to their locations within the tissue. Unbiased deconvolution of the spatial data identifies the following four distinct microenvironments: glomerular, immune, tubule and fibrotic. We describe the complex organization of microenvironments in health and disease and find that the fibrotic microenvironment is able to molecularly classify human kidneys and offers an improved prognosis compared to traditional histopathology. We provide a comprehensive spatially resolved molecular roadmap of the human kidney and the fibrotic process, demonstrating the clinical utility of spatial transcriptomics. A spatial transcriptomic analysis of healthy kidneys and those from individuals with chronic kidney disease characterizes the fibrotic microenvironment and highlights features that could be used to predict prognosis.

AIMS:We sought to characterize circulating protein biomarkers associated with cardiogenic shock (CS) using highly multiplex proteomic profiling. METHODS AND RESULTS:This analysis employed a cross-sectional case-control study design using a biorepository of patients admitted to a cardiac intensive care unit between 2017 and 2020. Cases were patients adjudicated to have CS, and controls were those presenting for cardiac critical care without shock, including subsets of patients with isolated hypotension or heart failure (HF). The Olink platform was used to analyse 359 biomarkers with Bonferroni correction. The analysis included 239 patients presenting for cardiac critical care (69 cases with CS, 170 non-shock controls). A total of 63 biomarkers (17.7%) were significantly associated with CS after Bonferroni correction compared with all controls. Of these, nine biomarkers remained significantly associated with CS when separately cross-validated in subsets of controls presenting with isolated hypotension and HF: cathepsin D, fibroblast growth factor (FGF)-21 and -23, growth differentiation factor (GDF)-15, insulin-like growth factor-binding protein-1, N-terminal pro-B-type natriuretic peptide, osteopontin, oncostatin-M-specific receptor subunit beta (OSMR), and soluble ST2 protein (sST2). Four biomarkers were identified as providing complementary information for CS diagnosis with development of a multi-marker model: sST2, FGF-23, CTSD, and GDF-15. CONCLUSION:In this pilot study of targeted proteomic profiling in CS, we identified nine biomarkers significantly associated with CS when cross-validated against non-shock controls including those with HF or isolated hypotension, illustrating the potential application of a targeted proteomic approach to identify novel candidates that may support the diagnosis of CS.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure and cardiac transplantation. We report a genome-wide association study and multi-trait analysis of DCM (14,256 cases) and three left ventricular traits (36,203 UK Biobank participants). We identified 80 genomic risk loci and prioritized 62 putative effector genes, including several with rare variant DCM associations (MAP3K7, NEDD4L and SSPN). Using single-nucleus transcriptomics, we identify cellular states, biological pathways, and intracellular communications that drive pathogenesis. We demonstrate that polygenic scores predict DCM in the general population and modify penetrance in carriers of rare DCM variants. Our findings may inform the design of genetic testing strategies that incorporate polygenic background. They also provide insights into the molecular etiology of DCM that may facilitate the development of targeted therapeutics.

Heart failure is a leading cause of morbidity and mortality1,2. Elevated intracardiac pressures and myocyte stretch in heart failure trigger the release of counter-regulatory natriuretic peptides, which act through their receptor (NPR1) to affect vasodilation, diuresis and natriuresis, lowering venous pressures and relieving venous congestion3-8. Recombinant natriuretic peptide infusions were developed to treat heart failure but have been limited by a short duration of effect9,10. Here we report that in a human genetic analysis of over 700,000 individuals, lifelong exposure to coding variants of the NPR1 gene is associated with changes in blood pressure and risk of heart failure. We describe the development of REGN5381, an investigational monoclonal agonist antibody that targets the membrane-bound guanylate cyclase receptor NPR1. REGN5381, an allosteric agonist of NPR1, induces an active-like receptor conformation that results in haemodynamic effects preferentially on venous vasculature, including reductions in systolic blood pressure and venous pressure in animal models. In healthy human volunteers, REGN5381 produced the expected haemodynamic effects, reflecting reductions in venous pressures, without obvious changes in diuresis and natriuresis. These data support the development of REGN5381 for long-lasting and selective lowering of venous pressures that drive symptomatology in patients with heart failure.