Mechanisms of embryonic vascular mural cell differentiation and the onset of blood flow regulation

胚胎血管壁细胞分化的机制和血流调节的开始

• 批准号: RGPIN-2019-07176
• 负责人: Childs, Sarah
• 金额: $ 3.06万
• 依托单位: University of Calgary
• 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=739342
• 关键词: Mechanisms; embryonic; vascular; mural; cell

Blood vessels respond to increased or decreased metabolic needs by regulating blood flow, adjusting to ensure tissues and organs receive adequate blood supply. The multi-layered vascular wall has two key cell types, the inner endothelial lining and the external `mural' layer. Pericytes are single mural cells that associate with smaller vessels on the vascular tree, while vascular Smooth Muscle Cells (vSMCs) form layers on larger vessels. In embryonic development, once the endothelial lining of the vessel assembles, it immediately is subjected to fluid flow forces. `Naked' vessels can only passively carry flow, but as mural cells adhere and differentiate, they begin to contract and relax to ensure steady blood supply. In preliminary data we have determined an early vSMC transcriptome and discovered mechanisms of paracrine signalling leading to vSMC differentiation. We have developed Computational Fluid Dynamics analysis of blood flow. We have developed methods to quantitate vessel diameter changes in vivo in response to pharmacological agents and we have determined the timing of both marker expression and of functional contraction and relaxation responses. These preliminary data and novel techniques now poise us to ask questions at the junction of developmental biology and vascular function. In our NSERC program we hypothesize that blood flow and vascular mural cells each influence each other during development through multiple mechanisms. Aim 1: Probe how mural cell attachment to the developing endothelium controls blood flow Here we study the interacting effects of vSMC and pericyte differentiation on blood flow. We integrate computational fluid dynamics analysis of vessel architecture with live imaging data of mural cells over different developmental stages from the period before mural cells associate with endothelium through the stages where they become contractile. We genetically ablate mural cells to understand differences in vessel architecture. We design constructs for optogenetic activation of contraction in vSMCs. Aim 2: Understand the role of flow-sensitive, mechanosensory pathways in vSMC differentiation Here we test the relative roles of mechanosensation and signalling pathways reliant on blood flow on mural cell differentiation probing the function of 3 mechanosensory proteins and a circulating BMP on smooth muscle differentiation. Aim 3: Determine how Ca2+ dynamics change with mural cell differentiation and how spatial control of Ca2+ affects the vascular network. This aim will investigate the molecular mechanisms of functional vSMC differentiation by understanding how vascular tone develops through the regulation of Ca2+ dynamics using the live reporter GCamp5G expressed in pericytes or vSMCs to determine how Ca2+ handling changes over the period when mural cells gain contractile function.

血管通过调节血流来应对代谢需求的增加或减少，以确保组织和器官获得充足的血液供应。多层血管壁有两种关键的细胞类型，内层内皮细胞和外部“壁”层。周细胞是单一的壁细胞，与血管树上的小血管相关联，而血管平滑肌细胞（vSMCs）在大血管上形成层状。在胚胎发育过程中，一旦血管的内皮层组装起来，它立即受到流体流动力的影响。“裸”血管只能被动地输送血液，但随着附壁细胞的粘附和分化，它们开始收缩和放松，以确保稳定的血液供应。在初步数据中，我们已经确定了早期vSMC转录组，并发现了导致vSMC分化的旁分泌信号传导机制。我们发展了计算流体动力学来分析血流。我们已经开发了一些方法来量化体内对药物反应的血管直径变化，我们已经确定了标记物表达和功能性收缩和舒张反应的时间。这些初步数据和新技术现在使我们能够在发育生物学和血管功能的交界处提出问题。在我们的NSERC项目中，我们假设血流和血管壁细胞在发育过程中通过多种机制相互影响。目的1：探讨壁细胞附着于发育中的内皮细胞对血流的调控作用。本文研究了血管内皮细胞与周细胞分化对血流的相互作用。我们将血管结构的计算流体动力学分析与壁细胞在不同发育阶段的实时成像数据结合起来，从壁细胞与内皮细胞结合之前的时期到壁细胞收缩的阶段。我们通过基因切除壁细胞来了解血管结构的差异。我们设计了用于vSMCs光遗传激活收缩的构建体。目的2：了解血流敏感、机械感觉通路在vSMC分化中的作用在这里，我们测试了依赖血流的机械感觉和信号通路在壁细胞分化中的相对作用，探测了3种机械感觉蛋白和循环BMP在平滑肌分化中的功能。目的3：确定Ca2+动态如何随壁细胞分化而变化，以及Ca2+的空间控制如何影响血管网络。该目的将研究功能性vSMC分化的分子机制，通过使用周细胞或vSMC中表达的活报告基因GCamp5G来了解血管张力是如何通过Ca2+动力学调控而发展的，以确定壁细胞获得收缩功能时Ca2+处理的变化。