RESUMEN
BACKGROUND: Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. METHODS: Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. RESULTS: Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. CONCLUSIONS: PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.
Asunto(s)
Hipertensión Arterial Pulmonar , Arteria Pulmonar , Remodelación Vascular , Animales , Humanos , Ratas , Ratones , Masculino , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/genética , Hipertensión Arterial Pulmonar/fisiopatología , Hipertensión Arterial Pulmonar/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Modelos Animales de Enfermedad , Ratas Sprague-Dawley , Proliferación Celular , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Quinasas de Proteína Quinasa Activadas por MitógenosRESUMEN
Exercise as a lifestyle modification is a frontline therapy for nonalcoholic fatty liver disease (NAFLD), but how components of exercise attenuate steatosis is unclear. To uncouple the effect of increased muscle mass from weight loss in obesity, myostatin knockout mice were bred on a lean and obese db/db background. Myostatin deletion increases gastrocnemius (Gastrocn.) mass and reduces hepatic steatosis and hepatic sterol regulatory element binding protein 1 (Srebp1) expression in obese mice, with no impact on adiposity or body weight. Interestingly, hypermuscularity reduces hepatic NADPH oxidase 1 (Nox1) expression but not NADPH oxidase 4 (Nox4) in db/db mice. To evaluate a deterministic function of Nox1 on steatosis, Nox1 knockout mice were bred on a lean and db/db background. NOX1 deletion significantly attenuates hepatic oxidant stress, steatosis, and Srebp1 programming in obese mice to parallel hypermuscularity, with no improvement in adiposity, glucose control, or hypertriglyceridemia to suggest off-target effects. Directly assessing the role of NOX1 on SREBP1, insulin (Ins)-mediated SREBP1 expression was significantly increased in either NOX1, NADPH oxidase organizer 1 (NOXO1), and NADPH oxidase activator 1 (NOXA1) or NOX5-transfected HepG2 cells versus ?-galactosidase control virus, indicating superoxide is the key mechanistic agent for the actions of NOX1 on SREBP1. Metabolic Nox1 regulators were evaluated using physiological, genetic, and diet-induced animal models that modulated upstream glucose and insulin signaling, identifying hyperinsulinemia as the key metabolic derangement explaining Nox1-induced steatosis in obesity. GEO data revealed that hepatic NOX1 predicts steatosis in obese humans with biopsy-proven NAFLD. Taken together, these data suggest that hypermuscularity attenuates Srebp1 expression in db/db mice through a NOX1-dependent mechanism.NEW & NOTEWORTHY This study documents a novel mechanism by which changes in body composition, notably increased muscle mass, protect against fatty liver disease. This mechanism involves NADPH oxidase 1 (NOX1), an enzyme that increases superoxide and increases insulin signaling, leading to increased fat accumulation in the liver. NOX1 may represent a new early target for preventing fatty liver to stave off later liver diseases such as cirrhosis or liver cancer.