Lung-Brain Crosstalk as a target of cell therapy in ischemic stroke

项目摘要 基于细胞的治疗代表了一种有前途的治疗方法，以提高中风恢复。各种研究小组已经发现，骨髓来源的间充质干细胞（MSC）在实验动物模型中改善中风恢复。尽管结果令人鼓舞，但MSC如何增强恢复的机制仍不清楚。大多数静脉内（IV）施用的MSC（通常在实验和初始临床研究中使用的方法）被截留在肺中，而剩余的MSC迁移到肝和脾，只有少数细胞到达脑。研究表明，虽然MSC在肺中的寿命很短，但它们的有益作用可持续数周，这表明一些肺细胞被MSC修饰，以提供靶向炎症的内分泌效应并提供营养反应。其他人已经尝试在IV施用后改善MSC的肺通道，而没有考虑到这种截留实际上可能是MSC介导的益处的贡献因素。因此，本项目的主要目标是研究在脑恢复中IV施用MSC后肺-脑串扰的重要性。我们的初步数据显示，MSC直接与肺内皮细胞（EC）相互作用，从而增加神经营养因子如脑源性神经营养因子（BDNF）的释放。我们还发现暴露于MSC后肺EC中BDNF mRNA表达的稳健增加以及MSC治疗后中风小鼠血浆中BDNF水平的增加。BDNF通过酪氨酸受体激酶B（Trk B）发挥其作用。因此，我们假设，MSC包埋在肺总理EC释放BDNF，作为一个关键的肺-脑串扰介质，并赋予中风后的神经保护，促进神经元的健康和营养中风后。本研究的主要目的是：（1）利用体外脑卒中模型，阐明肺EC-MSC相互作用是肺产生BDNF的来源，有利于脑神经元的完整性。为了确定肺释放的BDNF在神经元健康中的因果作用，我们将在炎症和抗炎刺激下采用原代肺EC-MSC共培养物，并对培养的皮质神经元（幼稚与TrkB缺陷）进行培养基转移实验，再加上使用选择性TrkB受体拮抗剂（ANA-12）和BDNF中和抗体的抑制剂研究。特别是为了确定BDNF的来源，我们将使用RNA干扰（RNAi）研究并沉默肺EC和/或MSC中的BDNF以进行共培养和培养基转移研究。(2)我们将确定在体内IV MSC治疗后从肺EC释放的“愈合”BDNF介导肺-脑串扰并改善中风后的功能恢复。为了确定这一点，我们将通过脑内施用BDNF siRNA进行BDNF的肺特异性体内沉默，并使用这些小鼠研究缺血性中风后IV MSC的功能恢复。考虑到目前中风可用治疗方案的局限性，我们提出的研究的优势有两个方面：1）探索MSC如何介导肺-脑串扰以促进中风恢复的新机制（由BDNF介导），以及2）阐明可能导致中风新治疗的新靶向途径。

PROJECT SUMMARY Cell based therapies represent a promising therapeutic approach to enhance stroke recovery. Various research groups have found that bone marrow derived mesenchymal stromal cells (MSCs) improve stroke recovery in experimental animal models. Despite promising results, mechanisms of how MSCs enhance recovery remain unclear. The majority of intravenously (IV) administered MSCs (an approach normally used experimentally and in initial clinical studies), are entrapped in the lungs, while remaining MSCs migrate to liver and spleen, only few cells reach the brain. Studies have shown that while MSCs are short-lived in lungs, their beneficial effects extend for weeks, suggesting that some lung cells are modified by MSCs to deliver endocrine effects that target inflammation and provide trophic responses. Others have tried to improve lung passage of MSCs after IV administration, without considering that this entrapment could in fact be the contributing factor to MSC-mediated benefit. Hence, the main goal of this project is to study the importance of lung-brain crosstalk after IV administration of MSCs in brain recovery. Our preliminary data show that MSCs interact directly with lung endothelial cells (ECs), which increases the release of neurotrophins such as brain-derived neurotrophic factor (BDNF). We also find a robust increase in BDNF mRNA expression in lung ECs upon exposure to MSCs as well as an increase in BDNF levels in plasma from stroke mice after MSC treatment. BDNF exerts its actions through tyrosine receptor kinase B (TrkB). Hence, we hypothesize that MSCs entrapped in lungs prime ECs to release BDNF that acts as a key lung-brain crosstalk mediator, and imparts neuroprotection after stroke by promoting neuronal health and trophicity after stroke. We will pursue the following aims: (1) Using in vitro model of stroke, we will elucidate that lung EC-MSC interaction is the source of BDNF produced in lungs, which benefits brain neuronal integrity. To establish a causal role of lung-released BDNF in neuronal health, we will employ primary lung EC-MSC co-cultures under inflammatory and anti-inflammatory stimulus and perform media transfer experiments on cultured cortical neurons (naïve vs TrkB-deficient), coupled with inhibitor studies using selective TrkB receptor antagonist (ANA-12) and BDNF neutralizing antibody. Specifically to pin down the source of BDNF, we will use RNA-interference (RNAi) studies and silence BDNF in lung EC and/or MSCs to perform co- cultures and media transfer studies. (2) We will determine that “healing” BDNF released from lung ECs after IV MSC treatment in vivo, mediates lung-brain crosstalk and improves functional recovery after stroke. To determine this, we will perform lung specific in vivo silencing of BDNF by intratracheal administration of BDNF siRNA and use these mice to investigate functional recovery from IV MSCs after ischemic stroke. Considering the current limitations in available treatment options for stroke, the advantages of our proposed studies are two- fold: 1) Exploring novel mechanisms (mediated by BDNF) on how MSCs mediate lung-brain crosstalk to promote stroke recovery, and 2) Elucidating a novel targetable pathway that may lead to new treatments for stroke.