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

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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.