Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood–brain barrier in acute ischemic stroke

Abstract

Blood–brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database (https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

Anti-osteopontin therapy leads to improved edema and infarct size in a murine model of ischemic stroke

“…We showed previously that osteopontin is highly expressed in the extracellular space and in the major NVU cells (endothelial cells, pericytes, astrocytes and microglia) both in acute human and murine ischemic stroke lesions. We also showed that osteopontin unfolds deleterious effects on BBB function in a mouse model of acute ischemic stroke, further supporting brain edema formation and increasing the risk for hemorrhagic transformation 12 . CD44, a receptor for osteopontin 16 , which is a crucial osteopontin signaling pathway member, was also significantly upregulated in all major NVU cell types in acute murine ischemic stroke based on our recently published transcriptomic dataset 12 .…”

“…We also showed that osteopontin unfolds deleterious effects on BBB function in a mouse model of acute ischemic stroke, further supporting brain edema formation and increasing the risk for hemorrhagic transformation 12 . CD44, a receptor for osteopontin 16 , which is a crucial osteopontin signaling pathway member, was also significantly upregulated in all major NVU cell types in acute murine ischemic stroke based on our recently published transcriptomic dataset 12 . Interestingly, CD44 has been reported to promote detrimental effects in cerebral ischemia 15 and impairment of BBB integrity in vitro 17 , 18 .…”

“…Osteopontin as a potential therapeutic target was confirmed in human stroke specimen from acute phases. As osteopontin was upregulated in multiple NVU cell types that co-regulate the BBB function, we hypothesized a critical role of osteopontin in BBB dysfunction in stroke, that was also suggested by earlier reports 12 – 14 . In our previous work, we therefore targeted osteopontin using an anti-osteopontin IgG in the early acute phase in a murine model of transient middle cerebral artery occlusion (tMCAO).…”

“…In vitro rescue using anti-osteopontin IgG was performed on primary mouse brain endothelial cells (MBMEC) subjected to oxygen glucose deprivation (OGD) to mimic stroke in vitro. Upon anti-osteopontin therapy, we demonstrated improved cerebral edema and BBB recovery, which was associated with improved neurological outcome and survival 12 . However, we did not observe an improvement in infarct size.…”

“…We have recently identified osteopontin as a novel therapeutic target in ischemic stroke. Utilizing our recently introduced EPAM-ia method 12 , we isolated major NVU cell-types post ischemic stroke from mice and subjected them to RNA sequencing and bioinformatic analyses. Using this dataset ( https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke ; NCBI GEO ID GSE163752) and using UpSet analysis (visualized at: http://vcg.github.io/upset/ submitting the JSON file link— https://raw.githubusercontent.com/SGD2020/mcao/master/mcao.json in the data address bar), we identified osteopontin (Spp1 gene product) as a target regulated in all major NVU cell types.…”