Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico.
Am J Physiol Heart Circ Physiol. 2020 Feb 1;318(2):H470-H483. doi: 10.1152/ajpheart.00629.2019. Epub 2020 Jan 10.
Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCβ and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCβ and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCβ inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in salineperfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCβ-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCβ in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCβ activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure. This research demonstrates a novel contribution of PKCβ and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.
活性氧（ROS）、线粒体功能障碍和过度的血管收缩是慢性缺氧（CH）诱导新生儿肺动脉高压的重要因素。基于蛋白激酶 Cβ（PKCβ）和线粒体氧化应激参与多种心血管和代谢紊乱的证据，我们假设 PKCβ 和线粒体 ROS（mitoROS）信号通路参与增强暴露于 CH 的新生大鼠的肺血管收缩。为了验证这一假设，我们检测了 PKCβ 抑制剂 LY-333531、ROS 清除剂 1-氧代-2,2,6,6-四甲基-4-羟基哌啶（TEMPOL）以及线粒体抗氧化剂甲硫氨酸（MitoQ）和（2-（2,2,6,6-四甲基哌啶-1-氧基-4-基氨基）-2-氧代乙基）三苯基膦氯化物（MitoTEMPO）对来自 2 周龄对照和 CH（12 天暴露，0.5 大气压）大鼠的盐灌注肺（原位）或加压肺动脉的血管收缩反应的影响。来自 CH 大鼠的肺表现出更大的基础张力和对 9,11-二去氧-9α,11α-甲氧基环氧前列腺素 F（U-46619）的血管收缩敏感性。LY-333531 和 TEMPOL 减弱了 CH 的这些作用，而对对照动物的肺没有影响。与对照大鼠相比，来自新生 CH 大鼠的分离肺动脉的基础张力也升高，而 LY-333531 和靶向线粒体的抗氧化剂均可抑制这种张力。使用靶向线粒体的 ROS 指示剂 MitoSOX 评估线粒体 ROS 产生的其他实验表明，PKCβ 激活剂佛波醇 12-肉豆蔻酸 13-乙酸可通过药理学刺激控制新生儿肺动脉平滑肌细胞（PASMC）中的 PKCβ-线粒体氧化剂信号通路。最后，我们发现，通过对亚细胞部分进行 Western 印迹分析，CH 会增加肺血管中定位于线粒体的 PKCβ。我们得出结论，PKCβ 激活导致来自新生大鼠的 PASMC 产生 mitoROS。此外，该信号轴可能解释了 CH 暴露后肺血管收缩敏感性的增强。这项研究证明了 PKCβ 和线粒体活性氧信号在慢性低氧新生儿中增强肺血管收缩反应的新作用。结果提供了一种潜在的机制，即慢性缺氧增加了基础和激动剂诱导的肺动脉平滑肌张力，这可能导致新生儿肺动脉高压。
