The blood-brain barrier (BBB) is crucial for the health of the brain and is often compromised in neurological diseases such as ischemic stroke. Reperfusion injury following ischemic stroke, due to a neurovascular barrier that is damaged after recanalization of an occluded vessel, is a major risk factor that expands brain damage and needs to be addressed therapeutically. Induced pluripotent stem cell (iPSC)-derived BECs can be used to explore novel mechanisms to enhance angiogenic potential and maintain robust CNS barrier properties promoting functional recovery after disease. This project investigates the mechanisms by which anti-miR-23b enhances the angiogenic and barrier properties of human BECs, and how it augments their ability to repair damaged blood vessels, both in vitro and following ischemia/stroke in vivo. We have found that anti-miR-23b promotes expression and proper localization of TJ proteins and induces formation of a paracellular endothelial barrier, through an anti-miR lentiviral screen in BECs. We have successfully differentiated human iPSCs into BECs exhibiting improved BBB properties, by introducing anti-miR-23b during differentiation. We will test the hypothesis that anti-miR-23b enhances the angiogenic potential and barrier properties of iPSC-derived BECs and promotes repair of the damaged endothelium in vitro and after ischemia and stroke in vivo. We will first examine the mechanisms by which anti-miR-23b enhances the angiogenic potential and structural barrier properties of human BECs, by regulating a network of cellular factors important in EC function. We will validate miR-23b targets, identified by miR-23b RIP sequence analysis, and determine whether miR-23b regulates these mRNA targets directly or indirectly. We will test if the anti-miR-23b-mediated augmentation of angiogenic potential and endothelial barrier function are dependent on increased miR-23b target protein expression. We will then characterize the effects of anti-miR-23b on iPSC-derived BECs in vitro with respect to angiogenesis and paracellular or transcellular barrier properties, using BECs cultured alone or in combination with human pericytes and/or astrocytes. Moreover, we will test if anti-miR-23b expression is sufficient to rescue BBB deficits produced in vitro under oxygen and glucose deprivation conditions. Finally, we will determine if anti- miR-23b overexpression in vivo in CNS blood vessels, using a viral delivery method, enhances BBB repair and reduces stroke volume, tissue injury, immune cell infiltration and neurological deficits in the transient middle cerebral artery occlusion mouse model for ischemic stroke. These experiments will provide both in vitro and in vivo proof-of-concept for using anti-miR-23b to generate BECs with robust barrier properties, which may have the capacity to repair damaged blood vessels following ischemic stroke. We predict that novel strategies to repair BBB injury following stroke will translate to other neurological disorders with BBB dysfunction, such as multiple sclerosis (MS).
Reperfusion brain injury due to dysfunction of the blood-brain barrier, after recanalization of an occluded vessel following ischemic stroke, is a major risk factor that needs to be addressed therapeutically. This project will investigate the development of micro-RNA-based methods to generate, from induced pluripotent stem cells (iPSCs), human brain endothelial cells (BECs) that exhibit robust angiogenic potential and barrier properties in the absence of pericytes or astrocytes. We have identified anti-miR-23b as a microRNA that promotes endothelial barrier properties, and have used it to successfully generate in vitro human BECs having robust barrier properties. We will characterize these cells extensively and test the relevance of this approach in vitro and in vivo as a potential treatment for ischemic stroke.
