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病房婴儿的共同特征，常导致大脑功能受损