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

描述（由申请人提供）：肺动脉高压（PH）是一种致命的血管疾病，其分子起源不明。虽然单个microRNAs（miRNAs）可以适度控制PH，但相关的miRNAs可能共同调节多个靶基因“网络”，对疾病产生更强大的影响。然而，miRNAs的网络生物学在体内的研究却很少。我们假设miR-130/301家族是一个致病的关键，控制许多靶向途径（除了促增殖信号传导），广泛控制PH。我们将定义miR-130/301家族与体内肺血管功能障碍的机制-通过可能的PPARg和TIMP 2靶向改变血管紧张度和血管硬度。我们还将描述miRNA家族成员的个体作用及其在体内的协调作用。因此，被认为在miRNA生物学中具有活性的冗余/协同效应将首次在体内PH中进行检查。具体目的：目的1）确定PPARg和内皮素-1对于通过miR-130/301控制肺血管紧张素的重要性。为了证明miR-130/301是否通过调节PPARg和内皮素-1（ET-1）引起血管收缩，将使用RNAi和腺相关病毒（AAV）转基因递送至肺血管的系统在小鼠中调节miR-130/301、PPARg和ET-1，沿着活体肺血管显微镜检查和压力-流量曲线分析以直接测量血管紧张度。目的2）通过miR-130/301确定PPARg和TIMP 2对于控制基质沉积和肺血管僵硬的重要性。我们推测miR-130/301通过抑制PPARg和TIMP 2进一步控制PH表现，从而增加基质沉积和血管硬度。我们将在小鼠中调节miR-130/301、PPARg和TIMP 2，然后进行肺血管硬度的生物物理学（原子力显微镜）和分子评估。结果可以提供体内miRNA特异性调节肺血管硬度和通过多个相关miRNA靶点组合控制PH的第一个证据。目的3）确定miR-130/301家族成员对总体PH表现的协调作用。我们假设miR-130/301家族采用协同作用和冗余来促进PH。将系统地使用体内miRNA递送至肺血管的优化系统来询问每个miR-130/301家族成员对小鼠PH表现的个体贡献与综合贡献。这项研究将是第一个定义特定的网络为基础的参数的miRNA生物学控制PH在体内，并可能是一个必要的指导，在开发基于miRNA的治疗PH. Significance：我们的建议结合了严格的专业知识，在miRNA生物学的分子，生物物理学和生理学研究PH在体内的新技术进步。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，并可能加速依赖复杂的miRNA网络控制所产生的病理表型的其他疾病的发现。

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.