Generation and characterization of a conditional eNOS knock out mouse model for cell-specific reactivation of eNOS in gain-of-function studies.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) in the vessel wall regulates blood pressure and cardiovascular hemodynamics. In this study, we generated conditional eNOS knock out (KO) mice characterized by a duplicated/inverted exon 2 flanked with two pairs of loxP regions (eNOSinv/inv); a Cre-recombinase activity induces cell-specific reactivation of eNOS, as a result of a flipping of the inverted exon 2 (eNOSfl). This work aimed to test the efficiency of the Cre-mediated cell-specific recombination and the resulting eNOS expression/function. As proof of concept, we crossed eNOSinv/inv mice with DeleterCrepos (DelCrepos) mice, expressing Cre recombinase in all cells. We generated heterozygous eNOSfl/inv or homozygous eNOSfl/fl mice, and eNOSinv/inv littermate mice. We found that both eNOSfl/fl and eNOSfl/inv mice express eNOS and the overall expression level depends on the number of mutated alleles, while eNOSinv/inv mice did not show any eNOS expression. Vascular endothelial function was restored in eNOSfl/fl and eNOSfl/inv mice, as determined by ACh-dependent vasodilation of aortic rings. Cre-dependent reactivation of eNOS in eNOSfl/fl and eNOSfl/inv mice rescued eNOSinv/inv (phenotypically global eNOS KO) mice from hypertension. These findings demonstrate that eNOS expression is restored in eNOSfl/fl mice at comparable physiological levels of WT mice, and its functional activity is independent on the number of the reactivated alleles. Therefore, eNOSinv/inv mice are a useful model for studying the effects of conditional reactivation of eNOS and gene dosage effects in specific cells for gain-of-function studies.

Outcomes with ibrutinib in patients with chronic lymphocytic leukaemia: Results from the German multicentre REALITY study.

OBJECTIVES: To assess treatment adherence, effectiveness and safety outcomes of patients with chronic lymphocytic leukaemia (CLL) receiving ibrutinib in a real-world setting. METHODS: Patients enrolled in REALITY were ≥18 years with a confirmed diagnosis of CLL and were receiving ibrutinib as a first-line (1L), 2L or ≥3L therapy. Treatment retention, adherence, progression-free survival (PFS), overall survival (OS) and time to next therapy were assessed at 1 and 2 years overall, by typology and by cytogenetic subgroups. PFS and OS were analysed using Kaplan-Meier methods. RESULTS: Exactly 302 patients were enrolled across 57 sites in Germany, from January 2017 to July 2021. One-year retention rates were 69.9% overall (primary endpoint), 77.9% for 1L patients, and 77.6%/78.8% for high-risk patients with del17p/TP53. At 2 years, PFS/OS rates were 77.8%/90.7% overall (1L, 82.7%/90.4%), and were consistent across cytogenetic subgroups. PFS rates were higher for 1L versus ≥3L patients. Patients with the low-acceptance/low-control typology at baseline were less likely to retain treatment at 1 year versus the high-acceptance/high-control typology. No new safety signals were observed. CONCLUSIONS: The REALITY study provides further evidence of the effectiveness and safety of ibrutinib in patients with CLL in a real-world setting, particularly in earlier treatment lines.

Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure.

BACKGROUND: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. METHODS: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. RESULTS: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. CONCLUSIONS: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.