Postolow Fabiana, Fediuk Jena, Nolette Nora, Hinton Martha, Dakshinamurti Shyamala
Department of Pediatrics, University of Manitoba, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada.
Department of Physiology, University of Manitoba, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada ; Biology of Breathing Group, Manitoba Institute of Child Health, 715 McDermot Avenue, Winnipeg, MB R3E 3P4 Canada.
Fibrogenesis Tissue Repair. 2015 Nov 1;8:20. doi: 10.1186/s13069-015-0037-6. eCollection 2015.
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.
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.
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.
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.
新生儿持续性肺动脉高压（PPHN）的特征是血管收缩和肺血管重塑。人们认为重塑是对物理或化学刺激的一种反应，这些刺激包括促有丝分裂的炎症介质，如血栓素。我们的目的是在组织培养中，研究缺氧和血栓素信号在体外和体内对PPHN及对照新生儿肺动脉（PA）心肌细胞的表型定向、细胞周期进入和增殖的影响。
为了研究缺氧和血栓素对心肌细胞生长的协同作用，将血清喂养的第一代新生猪PA心肌细胞随机分为常氧（21% O₂）或缺氧（10% O₂）培养3天，每天添加血栓素模拟物U46619（10⁻⁹至10⁻⁵ M）或稀释剂。通过MTT法检测细胞存活情况。为了确定慢性血栓素暴露（与全血清相比）对动脉重塑激活的影响，通过3天的缺氧暴露（FiO₂ 0.10）在新生仔猪中诱导PPHN；对照组为3日龄常氧仔猪和0日龄仔猪。将第三代PA进行分割，并在生理缓冲液、Ham's F-12培养基（存在或不存在10%胎牛血清）或含有10⁻⁶ M U46619的培养基中培养3天。通过³H-胸腺嘧啶核苷摄取测量DNA合成，通过³H-亮氨酸摄取测量蛋白质合成，通过Ki67免疫染色测量增殖。通过碘化丙啶染色后对动脉中膜细胞核进行激光扫描细胞术研究细胞周期进入情况。通过对中膜进行肌球蛋白重链和结蛋白免疫染色确定表型定向，并通过激光扫描细胞术进行定量。
收缩性和合成性心肌细胞亚群对血栓素刺激有不同反应。U46619降低了合成性和收缩性心肌细胞的增殖。在所有培养条件下，PPHN动脉的蛋白质合成均减少。用U46619处理的补充血清的PA中，G1/G0期心肌细胞减少，S期和G2/M期增加。当血清剥夺时，与U46619一起孵育的PPHN PA显示细胞周期进入停滞（G0/G1期增加，S期和G2/M期减少），且收缩性表型标志物丰度增加。
我们得出结论，血栓素不会在PPHN PA中引发表型去分化和增殖激活。暴露于血栓素会触发细胞周期退出和心肌细胞向收缩性表型定向。
