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)是一种致命的血管疾病，分子起源不明。虽然单个的microRNAs(MiRNAs)可以适度地控制PH，但相关的miRNAs可能共同调节多个靶基因的“网络”，从而对疾病产生更强大的影响。然而，在活体内对miRNAs的网络生物学研究很少。我们假设miR-130/301家族是一个致病的关键基因，控制着许多靶向通路(除了促增殖信号)，以实现对PH的广泛控制。我们将在体内确定miR-130/301家族与肺血管功能障碍的联系机制--可能通过靶向PPARg和TIMP2来改变血管运动张力和血管僵硬。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中得到检验。目的：1)确定PPARg和ET-1在miR-130/301调控肺血管舒张性张力中的作用。为了证明miR-130/301是否通过调节PPARg和内皮素-1(ET-1)而导致血管收缩，将使用RNAi和腺相关病毒(AAV)转基因到肺血管的系统来调节miR-130/301、PPARg和ET-1，并结合活体肺血管显微镜和压力-流量曲线分析来直接测量血管收缩张力。目的2)通过miR-130/301确定PPARg和TIMP2在控制基质沉积和肺血管僵硬中的重要性。我们推测miR-130/301通过抑制PPARg和TIMP2进一步控制PH的表现，从而增加基质沉积和血管僵硬。我们将在小鼠中调节miR-130/301、PPARg和TIMP2，然后进行生物物理(原子力显微镜)和分子评估肺血管硬度。这些结果可能为miRNA特异性调节肺血管僵硬和通过多个相关的miRNA靶点联合控制PH提供了第一个活体证据。目的3)明确miR-130/301家族成员在整体PH表现上的协调作用。我们推测miR-130/301家族既利用协同作用又利用冗余来促进PH。一个优化的miRNA体内肺血管递送系统将被用来系统地询问每个miR-130/301家族成员对小鼠肺高压表现的个体和综合贡献。这项研究将首次确定基于网络的miRNA生物学在体内控制PH的具体参数，并可能为发展基于miRNA的PH治疗提供必要的指导。意义：我们的建议将miRNA生物学方面的严格专业知识与在活体PH的分子、生物物理和生理研究方面的新技术进步结合在一起。因此，我们的目标是建立miR-130/301作为PH的多方面调节器--提供新的治疗靶点，并可能加速发现其他依赖于复杂的miRNA网络来控制所产生的病理表型的疾病。

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.