Elastic Fiber Biology - The Role of Elastin in Tissue Development and Integrity

弹性纤维生物学 - 弹性蛋白在组织发育和完整性中的作用

• 批准号: RGPIN-2019-05119
• 负责人: Davis, Elaine
• 金额: $ 2.33万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738074
• 关键词: Elastic; Fiber; Biology; Role; Elastin

Elastic fibers are present in the extracellular matrix of many tissues, but are most prevalent in tissues that undergo repetitive stretch and recoil, such as blood vessels, lung and skin. At maturity, elastin synthesis is negligible and there is little, if any, turnover of the protein. The lack of elastin synthesis in the adult makes it clear that proper formation of elastic fibers during development is critical, as these structures must remain intact and functional for a lifetime. To better understand the function of elastin, an elastin gene (Eln) knockout mouse was made. Since elastin is essential for the function of the aorta and lungs, we predicted that these mice would not be viable due to aortic rupture or respiratory failure. Surprisingly, mice without elastin are born and can breathe, but they die postnatally - not from vascular rupture, but from occlusion of the aorta due to overproliferation of the vascular smooth muscle cells (vSMCs). Over the past funding period, we made a new elastin mouse model that expresses a reduced level of elastin (ElnR). Using this hypomorph mouse model, we propose to determine how the dosage level of elastin affects tissue development and integrity. In our first aim, we will determine the dosage levels of elastin being produced in ElnR/+, ElnR/R and ElnR/- mouse aortas, and characterize the ultrastructure of the aorta and lung in these mice. Based on the ultrastructure of the elastin-null (Eln-/-) mouse lung, which has arrested alveolar development, we hypothesize that Eln-/- mice are systemically hypoxic, and that this contributes to the abnormal proliferation of the vSMCs seen in these mice. Our preliminary data has shown that hypoxia-inducible factor-1 alpha (HIF-1a) is significantly upregulated in the Eln-/- aorta, indicating that the aortic wall is indeed hypoxic. In our second aim, we will test our hypothesis in vivo and in vitro. For our in vivo studies, we will determine the expression and protein levels of HIF-1a and ETS-1, a transcription factor induced by the activity of HIF-1a, in Eln+/+, ElnR/R, ElnR/- and Eln-/- mouse aortas. Additional in vivo and in vitro studies will explore the expression, protein levels and localization of vSMC differentiation markers and TGF-beta-PI3K/Akt signaling molecules, a signaling pathway induced by hypoxia. Ultimately, our results will provide novel data relating hypoxia to elastic fiber assembly, and support our long-term goal of elucidating the role of elastin in tissue development and homeostasis. The dissemination of our research will provide the field with novel insights on elastic fiber function in several tissues, and will be important to Canada as abnormal development and/or destruction of elastic fibers in the aorta and lungs cause many diseases that affect Canadians. Thus, the identification of novel regulators of elastogenic cells has the potential to lead to the development of better disease treatments, especially in collaboration with bioengineers.

弹性纤维存在于许多组织的细胞外基质中，但在经历重复拉伸和回缩的组织中最普遍，例如血管、肺和皮肤。在成熟期，弹性蛋白的合成是可以忽略不计的，并且蛋白质的周转很少（如果有的话）。在成人中缺乏弹性蛋白合成清楚地表明，在发育过程中弹性纤维的适当形成是至关重要的，因为这些结构必须保持完整和功能终身。为了更好地了解弹性蛋白的功能，制备了弹性蛋白基因（Eln）敲除小鼠。由于弹性蛋白对主动脉和肺的功能至关重要，我们预测这些小鼠将由于主动脉破裂或呼吸衰竭而无法存活。令人惊讶的是，没有弹性蛋白的小鼠出生后可以呼吸，但它们出生后死亡-不是由于血管破裂，而是由于血管平滑肌细胞（vSMC）过度增殖导致的主动脉闭塞。在过去的资助期间，我们制作了一种新的弹性蛋白小鼠模型，表达了降低水平的弹性蛋白（ElnR）。使用这种亚形态小鼠模型，我们建议确定弹性蛋白的剂量水平如何影响组织发育和完整性。在我们的第一个目标中，我们将确定ElnR/+，ElnR/R和ElnR/-小鼠腹主动脉中产生的弹性蛋白的剂量水平，并表征这些小鼠主动脉和肺的超微结构。基于弹性蛋白缺失（Eln-/-）小鼠肺的超微结构，其已经阻止了肺泡发育，我们假设Eln-/-小鼠全身缺氧，并且这有助于在这些小鼠中观察到的vSMC的异常增殖。我们的初步数据表明，缺氧诱导因子-1 α（HIF-1a）在Eln-/-主动脉中显著上调，表明主动脉壁确实缺氧。在我们的第二个目标中，我们将在体内和体外测试我们的假设。对于我们的体内研究，我们将确定HIF-1a和ETS-1（由HIF-1a活性诱导的转录因子）在Eln+/+、ElnR/R、ElnR/-和Eln-/-小鼠睾丸中的表达和蛋白水平。另外的体内和体外研究将探索vSMC分化标志物和TGF-β-PI 3 K/Akt信号传导分子（缺氧诱导的信号传导途径）的表达、蛋白水平和定位。最终，我们的研究结果将提供有关缺氧与弹性纤维组装的新数据，并支持我们阐明弹性蛋白在组织发育和稳态中的作用的长期目标。我们研究的传播将为该领域提供关于几种组织中弹性纤维功能的新见解，并且对加拿大很重要，因为主动脉和肺中弹性纤维的异常发育和/或破坏会导致许多影响加拿大人的疾病。因此，识别新型弹性细胞调节因子有可能开发出更好的疾病治疗方法，特别是与生物工程师合作。