Defining the complex biology of the miR-130/301 family in pulmonary hypertension

描述（申请人提供）：肺动脉高压（PH）是一种分子起源不明的致死性血管疾病。虽然单个microrna （mirna）可以适度地控制PH，但相关的mirna可能共同调节多个靶基因“网络”，从而对疾病产生更强大的影响。然而，mirna的网络生物学在体内的研究却很少。我们假设miR-130/301家族是一个致病关键，控制了许多靶通路（除了促增殖信号），从而广泛控制ph。我们将确定miR-130/301家族与体内肺血管功能障碍的联系机制——通过可能靶向ppar和TIMP2来改变血管舒张性和血管僵硬。我们还将描述miRNA家族成员的个体作用及其在体内的协同作用。因此，被认为在miRNA生物学中活跃的冗余/协同效应将首次在体内PH中进行检测。目的1)确定PPARg和内皮素-1对miR-130/301调控肺血管舒缩张力的重要性。为了证明miR-130/301是否通过调节PPARg和内皮素-1 （ET-1）导致血管收缩，我们将在小鼠中使用RNAi和腺相关病毒（AAV）转基因系统向肺血管输送miR-130/301、PPARg和ET-1，同时使用活体肺血管显微镜和压力-流量曲线分析来直接测量血管舒缩张力。目的2)通过miR-130/301确定PPARg和TIMP2在控制基质沉积和肺血管刚度中的重要性。我们假设miR-130/301通过抑制ppar和TIMP2进一步控制PH表现，从而增加基质沉积和血管刚度。我们将在小鼠中调节miR-130/301， ppar和TIMP2，然后进行生物物理（原子力显微镜）和肺血管刚度的分子评估。研究结果为miRNA特异性调控肺血管硬度和通过多个相关miRNA靶点联合调控PH提供了第一个活体证据。目的3)定义miR-130/301家族成员对PH整体表现的协同作用。我们假设miR-130/301家族通过协同作用和冗余来促进PH。一个优化的miRNA在体内向肺血管输送的系统将被系统地用于询问每个miR-130/301家族成员对小鼠PH表现的个体和整体贡献。这项研究将首次定义体内控制PH的miRNA生物学的特定网络参数，并可能成为开发基于miRNA的PH治疗的必要指南。意义：我们的提案结合了miRNA生物学的严格专业知识和体内PH的分子，生物物理和生理研究的新技术进展。因此，我们的目标是建立miR-130/301作为PH的多方面调节剂-提供新的治疗靶点，并可能加速发现依赖复杂miRNA网络控制所产生的病理表型的其他疾病。

DESCRIPTION (provided by applicant): Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. While individual microRNAs (miRNAs) can control PH modestly, related miRNAs may conspire to regulate multiple target gene "networks" for a more robust influence on disease. Yet, the network biology of miRNAs has been poorly explored in vivo. We hypothesize that the miR-130/301 family is a pathogenic lynchpin controlling numerous target pathways (beyond pro-proliferative signaling) for broad control over PH. We will define mechanisms connecting the miR-130/301 family to pulmonary vascular dysfunction in vivo - via possible targeting of PPARg and TIMP2 to alter vasomotor tone and vascular stiffness. We will also delineate individual actions of miRNA family members and their coordinated effects in vivo. Thus, the redundant/ synergistic effects thought to be active in miRNA biology will be examined for the first time in PH in vivo. Specific Aims: Aim 1) Determine the importance of PPARg and endothelin-1 for control of pulmonary vasomotor tone by miR-130/301. To prove whether miR-130/301 causes vasoconstriction via regulating PPARg and endothelin-1 (ET-1), miR-130/301, PPARg, and ET-1 will be modulated in mice using systems of RNAi and adeno-associated viral (AAV) transgene delivery to the pulmonary vessels, along with intravital pulmonary vascular microscopy and pressure-flow curve analyses to measure vasomotor tone directly. Aim 2) Determine the importance of PPARg and TIMP2 for control of matrix deposition and pulmonary vascular stiffness by miR-130/301. We postulate that miR-130/301 further controls PH manifestation via repressing both PPARg and TIMP2, thus increasing matrix deposition and vascular stiffness. We will modulate miR-130/301, PPARg, and TIMP2 in mice, followed by biophysical (atomic force microscopy) and molecular assessment of pulmonary vascular stiffness. Results could provide the first evidence in vivo of miRNA-specific regulation of pulmonary vascular stiffness and combinatorial control of PH via multiple related miRNA targets. Aim 3) Define the coordinated actions of the miR-130/301 family members on overall PH manifestation. We postulate that the miR-130/301 family employs both synergism and redundancy to promote PH. An optimized system of miRNA delivery to the pulmonary vasculature in vivo will be used systematically to interrogate the individual versus integrated contributions of each miR-130/301 family member to PH manifestation in mice. This study will be the first to define the specific network-based parameters of miRNA biology in control of PH in vivo and could be a necessary guide in developing miRNA-based therapies of PH. Significance: Our proposal incorporates a rigorous expertise in miRNA biology with new technological advancements in the molecular, biophysical, and physiological study of PH in vivo. Thus, we aim to establish miR-130/301 as a multi-faceted regulator of PH -- offering new therapeutic targets and perhaps accelerating discovery in other diseases that rely upon complex miRNA networks for control of resultant pathophenotypes.