Mechanisms mediating age-dependent inhibition of cerebrovascular MLCK activity and contractility by chronic hypoxia

慢性缺氧对脑血管 MLCK 活性和收缩力的年龄依赖性抑制的介导机制

• 批准号: 9072345
• 负责人: William J. Pearce
• 金额: $ 19.15万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9072345
• 关键词: Acclimatization; Address; Adult; Age; Anemia; Animal Experiments; Animals; Arteries; Asthma; Attenuated; Binding; Biological Assay; Blood Vessels; Calcium; Cardiovascular system; Carotid Arteries; Cerebral Ischemia; Cerebral hemisphere hemorrhage; Cerebrum; Chronic; Clinical Management; Computers; Contractile Proteins; Custom; Data; Depressed mood; Down-Regulation; Drug usage; Enzymes; Evaluation; Exhibits; Fetus; Freezing; Genetic Transcription; Genetic Translation; Gestational Diabetes; Harvest; Homeostasis; Human; Hypoxia; Hypoxic-Ischemic Brain Injury; Immunoblotting; In Situ; Infant; Injection of therapeutic agent; Internal carotid artery structure; Laboratories; Ligase; MYH11 gene; Measurement; Measures; Mediating; Messenger RNA; Methods; MicroRNAs; Molecular; Morbidity - disease rate; Myosin Heavy Chains; Myosin Light Chain Kinase; Myosin Light Chains; Myosin Regulatory Light Chains; Neonatal; Neurologic; Operative Surgical Procedures; Organ Culture Techniques; Pathology; Pathway interactions; Phosphorylation; Placental Insufficiency; Pre-Eclampsia; Proteins; RNA Binding; Regulation; Risk; Role; Sheep; Small Interfering RNA; Smooth Muscle; Speed; Stress; Testing; Tissues; Transcript; Transfection; Translations; Ubiquitin; Ubiquitination; Uterus; Vascular remodeling; Work; age effect; age related; alpha Actin; attenuation; base; cerebral artery; cerebrovascular; developmental plasticity; extracellular; fetal; fetus hypoxia; improved; in utero; in vivo; intraventricular hemorrhage; knock-down; member; mortality; neonatal hypoxic-ischemic brain injury; neonate; non-muscle myosin; novel; postnatal; programs; protein degradation; protein expression; research study; response; ubiquitin ligase; voltage

Cardiovascular instability is a common feature of NICU infants that often leads to compromised cerebral autoregulation, hypoxic-ischemic brain injury, and intraventricular hemorrhage. Our recent work suggests that postnatal cardiovascular instability involves depressed function of Myosin Light Chain Kinase (MLCK), the rate- limiting enzyme responsible for initiation and regulation of vascular contraction. Because rates of mRNA transcription for MLCK vary little with age and hypoxia, our results implicate changes in mRNA translation, MLCK degradation, and MLCK activity as the main mechanisms that govern neonatal MLCK function. First, we will examine effects of micro-RNAs on MLCK translation. Numerous micro-RNAs are induced by hypoxia and influence contractile protein expression directly through binding to transcripts, and indirectly by influencing smooth muscle differentiation. To explore these mechanisms we have developed surgical methods that enable the in vivo adenoviral transfection of pre-term fetal lambs, in utero. This approach offers unprecedented opportunities to explore the molecular roles of micro-RNAs in fetal responses to hypoxic stress, particularly as related to regulation of MLCK function. Second, we will examine the roles of ubiquitination and protein degradation in fetal vascular responses to hypoxia. Despite the recognized importance of ubiquitination, it has not been studied in fetal lambs, their cerebral arteries or their responses to hypoxia. Our findings demonstrate that expression of some ubiquitin ligases is age-dependent and for others is potently upregulated by chronic hypoxia. These results advance the novel idea that changes in protein degradation are intimately involved in fetal vascular adaptation to chronic hypoxia. Third, we will examine effects of hypoxia on MLCK activity, in situ. Using novel methods to measure high-speed transients in cytosolic calcium and myosin light chain phosphorylation in whole arteries, we have found that MLCK velocity is enhanced by chronic hypoxia in fetal but not adult arteries. Our confocal methods further suggest that colocalization of MLCK with its substrate is stronger in fetal than adult arteries, and is significantly altered by chronic hypoxia, suggesting a new role for MLCK compartmentalization in regulation of fetal cerebrovascular contractility. Overall, further study of the mechanisms identified by our recent work promises to reveal multiple important new features of the molecular, cellular, and tissue level regulation of MLCK function, and offers new understanding of how these mechanisms might be leveraged to improve clinical management of postnatal cardiovascular instability.

心血管不稳定是新生儿重症监护室婴儿的常见特征， 自动调节、缺氧缺血性脑损伤和脑室内出血。我们最近的研究表明， 出生后心血管不稳定涉及肌球蛋白轻链激酶（MLCK）功能低下， 负责启动和调节血管收缩的限制酶。因为mRNA MLCK的转录随年龄和缺氧变化不大，我们的结果暗示了mRNA翻译的变化， MLCK降解和MLCK活性是控制新生儿MLCK功能的主要机制。第一、 我们将研究micro-RNA对MLCK翻译的影响。低氧诱导大量的micro-RNA 并通过与转录本结合直接影响收缩蛋白表达，并通过影响间接影响收缩蛋白表达 平滑肌分化为了探索这些机制，我们开发了手术方法， 能够在子宫内对早产胎羊进行体内腺病毒转染。这种方法提供 前所未有的机会来探索微RNA在胎儿缺氧应激反应中的分子作用， 特别是与MLCK功能的调节有关。其次，我们将研究泛素化的作用， 胎儿血管对缺氧反应中的蛋白质降解。尽管公认的重要性， 泛素化，尚未在胎羊、其脑动脉或其对缺氧的反应中进行研究。我们 研究结果表明，一些泛素连接酶的表达是年龄依赖性的，而另一些则是有效的。 在慢性缺氧中上调。这些结果提出了新的想法，即蛋白质降解的变化是 密切参与胎儿血管对慢性缺氧的适应。第三，我们将研究缺氧对 MLCK活性，原位。使用新方法测量胞浆钙和肌球蛋白的高速瞬变 在整个动脉的轻链磷酸化，我们已经发现，MLCK的速度增加， 胎儿动脉缺氧，而成人动脉无缺氧。我们的共聚焦方法进一步表明，MLCK与 它的底物在胎儿动脉中比成人动脉中更强，并且被慢性缺氧显著改变，这表明 MLCK区室化在调节胎儿脑血管收缩力中的新作用总体而言，进一步 对我们最近工作所确定的机制的研究有望揭示多个重要的新特征， MLCK功能的分子、细胞和组织水平的调节，并提供了新的理解， 这些机制可能被用来改善产后心血管不稳定的临床管理。