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

项目总结 基于细胞的治疗是促进中风康复的一种很有前途的治疗方法。多个研究小组发现，在实验动物模型中，骨髓间充质干细胞(MSCs)可以改善中风的康复。尽管结果令人振奋，但MSCs如何促进复苏的机制仍不清楚。大多数静脉注射(IV)的MSCs(一种通常在实验和初步临床研究中使用的方法)被困在肺部，而其余的MSCs迁移到肝脏和脾，只有少数细胞到达大脑。研究表明，虽然MSCs在肺部的存活时间很短，但它们的有益效果可持续数周，这表明一些肺细胞被MSCs修饰，以传递针对炎症的内分泌效应，并提供营养反应。其他人试图在静脉注射后改善MSCs的肺通道，而没有考虑到这种包裹实际上可能是MSC介导的益处的贡献因素。因此，本项目的主要目的是研究静脉注射MSCs后肺-脑串扰在脑康复中的重要性。我们的初步数据显示，MSCs直接与肺内皮细胞(ECs)相互作用，增加了脑源性神经营养因子(BDNF)等神经营养因子的释放。我们还发现，暴露于MSCs的肺内皮细胞中BDNF mRNA的表达显著增加，MSC治疗后中风小鼠血浆中的BDNF水平也增加。BDNF通过酪氨酸受体激酶B(TrkB)发挥作用。因此，我们假设MSCs被包裹在肺内皮细胞中，释放作为关键的肺-脑串扰介质的BDNF，并通过促进卒中后神经元的健康和营养作用来给予卒中后神经保护。我们将追求以下目标：(1)利用体外卒中模型，阐明肺内EC-MSC的相互作用是肺产生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，我们将采用炎症和抗炎刺激下的原代肺EC-MSC共培养，并对培养的皮质神经元(NAIVE与TrkB缺乏)进行介质转移实验，并使用选择性TrkB受体拮抗剂(ANA-12)和BDNF中和抗体进行抑制剂研究。为了明确BDNF的来源，我们将使用RNA干扰(RNAi)研究，并在肺内皮细胞和/或骨髓间充质干细胞中沉默BDNF，以进行共培养和介质转移研究。(2)我们将在体内确定静脉注射MSC治疗后肺内皮细胞释放的“愈合性”BDNF，介导肺-脑串扰，促进卒中后功能恢复。为了确定这一点，我们将通过气管内注射BDNF siRNA来进行肺组织特异性的BDNF体内沉默，并使用这些小鼠来研究缺血性中风后IV MSCs的功能恢复。考虑到目前卒中治疗方案的局限性，我们建议的研究具有两方面的优势：1)探索MSCs如何介导肺-脑串扰以促进卒中康复的新机制(由BDNF介导)；2)阐明可能导致新的卒中治疗方法的新的靶向途径。

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.