RESUMEN
Delivery of antisense oligonucleotides (ASOs) to airway epithelial cells is arduous due to the physiological barriers that protect the lungs and the endosomal entrapment phenomenon, which prevents ASOs from reaching their intracellular targets. Various delivery strategies involving peptide-, lipid-, and polymer-based carriers are being investigated, yet the challenge remains. S10 is a peptide-based delivery agent that enables the intracellular delivery of biomolecules such as GFP, CRISPR-associated nuclease ribonucleoprotein (RNP), base editor RNP, and a fluorescent peptide into lung cells after intranasal or intratracheal administrations to mice, ferrets, and rhesus monkeys. Herein, we demonstrate that covalently attaching S10 to a fluorescently labeled peptide or a functional splice-switching phosphorodiamidate morpholino oligomer improves their intracellular delivery to airway epithelia in mice after a single intranasal instillation. Data reveal a homogeneous delivery from the trachea to the distal region of the lungs, specifically into the cells lining the airway. Quantitative measurements further highlight that conjugation via a disulfide bond through a pegylated (PEG) linker was the most beneficial strategy compared with direct conjugation (without the PEG linker) or conjugation via a permanent thiol-maleimide bond. We believe that S10-based conjugation provides a great strategy to achieve intracellular delivery of peptides and ASOs with therapeutic properties in lungs.
RESUMEN
The lesions observed in AS have been shown to be sex specific, with women presenting extensive fibrotic remodeling while men developing more calcification deposit. We thus aimed to evaluate the influence of sex and sex hormones on the pathophysiology of aortic valve stenosis (AS) in our mouse model of AS. LDLr-/- ApoB100/100 IGF-II+/- mice (n = 210) were separated in six different groups: (1) intact male (IM), (2) intact female (IF), (3) castrated male (CM), (4) ovariectomized females (OF), (5) CM with testosterone supplementation (CMT), and (6) OF with 17ß-estradiol supplementation (OFE). Mice were fed a high-fat/high-sucrose/high-cholesterol diet for 6 months. Hemodynamic progression of AS was followed by transthoracic echocardiography (at 12 and 36 weeks) and analyzed in all mice alive at 36 weeks. Aortic valves were collected for histological and digital droplet PCR* analysis. Increases in peak velocity were comparable in IF and IM (24.2 ± 5.7 vs. 25.8 ± 5.3 cm/s; p = 0.68), but IF presented with less severe AS. Between the three groups of male mice, AS progression was more important in IM (increase in peak velocity: 24.2 ± 5.7 cm/s; p < 0.001) compared to CM (6.2 ± 1.4; p = 0.42), and CMT (15.1 ± 3.5; p = 0.002). In the three groups of female mice, there were no statistical differences in AS progression. Digital PCR analysis revealed an important upregulation of the osteogenic gene RunX2 in IM (p < 0.0001) and downregulation of the pro-calcifying gene ALPL in IF (p < 0.05). Male sex and testosterone play an important role in upregulation of pro-calcifying genes and hemodynamic progression of AS. However, female mice appeared to be protected against calcification, characterized by downregulation of pro-osteogenic genes, but presented a similar AS hemodynamic progression.
Asunto(s)
Estenosis de la Válvula Aórtica , Calcinosis , Animales , Válvula Aórtica , Modelos Animales de Enfermedad , Femenino , Hormonas Esteroides Gonadales , Humanos , Masculino , Ratones , TestosteronaRESUMEN
BACKGROUND: LDLr-/-/ApoB100/100/IGF-II+/- mice are used as a calcific aortic valve disease (CAVD) model. However, normal aortic valve hemodynamics i.e. remotely from CAVD onset and the sex-related differences are poorly known. METHODS AND RESULTS: Four groups of mice, intact males (IM, n = 49) and females (IF, n = 50), castrated males (CxM, n = 79) and ovariectomized females (OxF: 73), underwent a Doppler-echocardiography at 12 weeks of age. Gonadectomy was performed at 8 weeks. Aortic valve assessment using effective orifice area (EOA, using the continuity equation) and peak aortic transvalvular velocity (VPeak) was feasible in 89% of the mice with good to excellent reliability (intraclass correlation coefficients ranging from 0.90 to 0.98, p < 0.001). Mean VPeak was 104 ± 17 cm/s and mean EOA was 1.18*10-2 ± 0.22*10-2 cm2. EOA indexed to body surface area was 1.5 ± 0.3 cm2/m2. The 95th percentile of Vpeak was 132 cm/s and the 5th percentile of indexed EOA was 1.0 cm2/m2. Interestingly, IM had the highest VPeak (114 ± 14 cm/s) vs each of the other groups (CxM: 106 ± 19 cm/s, OxF: 97 ± 13 cm/s and IF: 96 ± 12 cm/s, ANOVA and corrected p < 0.001). This was mostly explained by a higher stroke volume (ANOVA and corrected p < 0.001) in IM compared to other groups. There were no major sex-differences in ventricular systolic function parameters. CONCLUSION: In LDLr-/-/ApoB100/100/IGF-II CAVD mice model, an aortic EOA <0.8*10-2 cm2 (or indexed EOA <1.0cm2/m2), and a peak aortic valve velocity > 132 cm/s may be proposed as thresholds to define CAVD. Intact male mice appear to have higher velocities.
Asunto(s)
Estenosis de la Válvula Aórtica , Válvula Aórtica , Animales , Válvula Aórtica/diagnóstico por imagen , Estenosis de la Válvula Aórtica/diagnóstico por imagen , Apolipoproteína B-100/genética , Femenino , Factor II del Crecimiento Similar a la Insulina , Masculino , Ratones , Reproducibilidad de los ResultadosRESUMEN
Background In calcific aortic valve disease on tricuspid aortic valves (TAVs), men have higher aortic valve calcification and less fibrosis than women. However, little is known in bicuspid aortic valves (BAV). We thus aimed to investigate the impact of age, sex, and valve phenotype (TAVs versus BAVs) on fibro-calcific remodeling in calcific aortic valve disease. Methods and Results We included 2 cohorts: 411 patients who underwent multidetector computed tomography (37% women) for aortic valve calcification density assessment and 138 explanted aortic valves (histological cohort; 50% women). The cohorts were divided in younger (<60 years old) or older patients with BAV (≥60 years old), and TAV patients. In each group, women and men were matched. Women presented less aortic valve calcification density than men in each group of the multidetector computed tomography cohort (all P≤0.01). Moreover, in women, younger patients with BAV had the lowest aortic valve calcification density (both P=0.02). In multivariate analysis, aortic valve calcification density correlated with age (ß estimate±standard error: 6.5±1.8; P=0.0004) and male sex (109.2±18.4; P<0.0001), and there was a trend with TAVs (41.5±23.0; P=0.07). Women presented a higher collagen content than men (77.8±10.8 versus 69.9±12.9%; P<0.001) in the entire cohort. In women, younger patients with BAV had denser connective tissue than TAV and older patients with BAV (both P≤0.05), while no difference was observed between men. Conclusions In calcific aortic valve disease, women had less calcification and more fibrotic remodeling than men, regardless of the phenotype of the valve or age of the patient. Moreover, younger women with BAVs had less valve calcification. Thus, mineralization/fibrosis of the aortic valve is likely to have sex/age-specific mechanisms and be influenced by the valve morphology.