Thromboxane promotes smooth muscle phenotype commitment but not remodeling of hypoxic neonatal pulmonary artery.

BACKGROUND: Persistent pulmonary hypertension of the newborn (PPHN) is characterized by vasoconstriction and pulmonary vascular remodeling. Remodeling is believed to be a response to physical or chemical stimuli including pro-mitotic inflammatory mediators such as thromboxane. Our objective was to examine the effects of hypoxia and thromboxane signaling ex vivo and in vitro on phenotype commitment, cell cycle entry, and proliferation of PPHN and control neonatal pulmonary artery (PA) myocytes in tissue culture. METHODS: To examine concurrent effects of hypoxia and thromboxane on myocyte growth, serum-fed first-passage newborn porcine PA myocytes were randomized into normoxic (21 % O2) or hypoxic (10 % O2) culture for 3 days, with daily addition of thromboxane mimetic U46619 (10(-9) to 10(-5) M) or diluent. Cell survival was detected by MTT assay. To determine the effect of chronic thromboxane exposure (versus whole serum) on activation of arterial remodeling, PPHN was induced in newborn piglets by a 3-day hypoxic exposure (FiO2 0.10); controls were 3 day-old normoxic and day 0 piglets. Third-generation PA were segmented and cultured for 3 days in physiologic buffer, Ham's F-12 media (in the presence or absence of 10 % fetal calf serum), or media with 10(-6) M U46619. DNA synthesis was measured by (3)H-thymidine uptake, protein synthesis by (3)H-leucine uptake, and proliferation by immunostaining for Ki67. Cell cycle entry was studied by laser scanning cytometry of nuclei in arterial tunica media after propidium iodide staining. Phenotype commitment was determined by immunostaining tunica media for myosin heavy chain and desmin, quantified by laser scanning cytometry. RESULTS: Contractile and synthetic myocyte subpopulations had differing responses to thromboxane challenge. U46619 decreased proliferation of synthetic and contractile myocytes. PPHN arteries exhibited decreased protein synthesis under all culture conditions. Serum-supplemented PA treated with U46619 had decreased G1/G0 phase myocytes and an increase in S and G2/M. When serum-deprived, PPHN PA incubated with U46619 showed arrested cell cycle entry (increased G0/G1, decreased S and G2/M) and increased abundance of contractile phenotype markers. CONCLUSIONS: We conclude that thromboxane does not initiate phenotypic dedifferentiation and proliferative activation in PPHN PA. Exposure to thromboxane triggers cell cycle exit and myocyte commitment to contractile phenotype.

Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling.

In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms regulating thromboxane-mediated actin polymerization are potential targets for future PPHN pharmacotherapy.

AMPK and FoxO1 regulate catalase expression in hypoxic pulmonary arterial smooth muscle.

BACKGROUND: Hypoxia and reactive oxygen species (ROS) including H(2)O(2) play major roles in triggering and progression of pulmonary vascular remodeling in persistent pulmonary hypertension. Catalase (CAT), the major endogenous enzyme scavenging H(2)O(2), is regulated in a tissue- and context-specific manner. OBJECTIVE: To investigate mechanisms by which hypoxia and H(2)O(2) regulate catalase expression, and the role of AMPK-FoxO pathway, in neonatal porcine pulmonary artery smooth muscle (PASMC). DESIGN/METHODS: PASMC were grown in hypoxia (10% O(2)) or normoxia (21% O(2)) for 72 hr. We measured catalase activity and lipid peroxidation; CAT, FoxO1, and FoxO3a expression by qPCR; protein contents of CAT, FoxOs, p-AMPK, p-AKT, p-JNK, p-ERK1/2 in whole lysates, and FoxOs in nuclear extracts, by immunoblot; and FoxO-1 nuclear localization by immunocytochemistry, quantified by laser scanning cytometry. RESULTS: Hypoxia upregulated CAT transcription, content and activity, by increasing CAT transcription factors FoxO1 and FoxO3a mRNA, and promoting nuclear translocation of FoxO1. However, lipid peroxidation increased in hypoxic PASMC. Among candidate FoxO regulatory kinases, hypoxia activated AMPK, and decreased p-Akt and ERK1/2. AMPK activation increased FoxO1 (total and nuclear) and CAT, while AMPK inhibition inhibited FoxO1 and CAT, but not FoxO3a. Exogenous H(2)O(2) decreased p-AMPK and increased p-AKT in hypoxic PASMC. This decreased active FoxO1, and reduced mRNA and protein content of CAT. Hypoxic induction of CAT, AKT inhibition (LY294002), or addition of PEG-catalase partly ameliorated the H(2)O(2) -mediated loss of nuclear FoxO1. CONCLUSIONS: Hypoxia induces catalase expression, though this adaptation is insufficient to protect PASMC from hypoxia-induced lipid peroxidation. This occurs via hypoxic activation of AMPK, which promotes nuclear FoxO1 and thus catalase expression. Exogenous ROS may downregulate cellular antioxidant defenses; H(2)O(2) activates survival factor Akt, decreasing nuclear FoxO1 and thus catalase.

Thromboxane-induced actin polymerization in hypoxic pulmonary artery is independent of Rho.

Actin polymerization (APM), regulated by Rho GTPases, promotes myocyte force generation. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes. We compared basal and agonist-induced APM in myocytes from PA and descending aorta (Ao), under hypoxic and normoxic conditions. We also examined effects of thromboxane challenge on force generation and cytoskeletal assembly in resistance PA and renal arteries from neonatal swine with persistent pulmonary hypertension (PPHN) induced by 72-h normobaric hypoxia, compared with age-matched controls. Synthetic and contractile phenotype myocytes from neonatal porcine PA or Ao were grown in hypoxia (10% O(2)) or normoxia (21% O(2)) for 7 days, then challenged with 10(-6) M thromboxane mimetic U46619. F/G actin ratio was quantified by laser-scanning cytometry and by cytoskeletal fractionation. Thromboxane receptor (TP) G protein coupling was measured by immunoprecipitation and probing for Gαq, G12, or G13, RhoA activation by Rhotekin-RBD affinity precipitation, and LIM kinase (LIMK) and cofilin phosphorylation by Western blot. Isometric force to serial concentrations of U46619 was measured in muscular pulmonary and renal arteries from PPHN and control swine; APM was quantified in fixed contracted vessels. Contractile PA myocytes exhibit marked Rho-dependent APM in hypoxia, with increased active RhoA and LIMK phosphorylation. Their additional APM response to U46619 challenge is independent of RhoA, reflecting decreased TP association with G12/13 in favor of Gαq. In contrast, hypoxic contractile Ao myocytes polymerize actin modestly and depolymerize to U46619. Both basal APM and the APM response to U46619 are increased in PPHN PA. APM corresponds with increased force generation to U46619 challenge in PPHN PA but not renal arteries.