Blockade of VCAM1 or VLA4 preserves cerebrovasculature and prevents cognitive decline late after stroke

Abstract

Infarct-induced neurodegeneration occurs chronically after stroke, doubling the risk of dementia. Endothelial vascular cell adhesion molecule 1 (VCAM1) facilitates blood-brain barrier opening and immune cell diapedesis by binding very late antigen 4 (VLA4) on immune cells. We hypothesized that vascular dysfunction persists after stroke and contributes to chronic neuroinflammation and cognitive decline via signaling through the VLA4/VCAM1 axis. We used adult (3-5 month old) and middle-aged (10 month old) C57BL/6J male & female mice and a permanent middle cerebral artery occlusion stroke model. Sham surgery consisted of an identical procedure without occlusion of the artery. We quantified vascular integrity using blood vessel length, pericyte coverage of vasculature, tight junctions, and extravascular fibrinogen leakage by immunostaining. Cognitive testing was performed using both Barnes maze and novel object recognition prior to stroke, and 1 and 6 weeks after stroke, and replicated in both male and female mice. We utilized anti-VCAM1, anti-VLA4 or isotype control antibodies to block VCAM1 or VLA4 function, and then to confirm mechanisms we utilized single cell RNA sequencing on immune and endothelial cells, aptamer-based plasma proteomics, and additional immunostaining for vascular integrity. Mouse brains exhibited signs of persistent vascular dysfunction and loss of blood-brain barrier integrity at 8 weeks after stroke, compared to sham animals. We observed reduced ZO-1 tight junction and pericyte coverage of vasculature, and increased extravascular fibrinogen. Mice with stroke also developed a cognitive deficit in both Barnes maze and novel object by 6 weeks. Treatment with anti-VCAM1 or anti-VLA4 resulted in mice with stroke performing as well as sham mice treated with isotype control antibody on both the Barnes maze and novel object recognition tasks. Anti-VCAM1 and anti-VLA4 both increased expression of blood-brain barrier maintenance genes in brain endothelial cells, while only minimally altering immune cell gene expression. Immune cell infiltration was reduced by anti-VCAM1 but not anti-VLA4 in tissue sections. In contrast to this, both antibodies increased blood vessel length and pericyte vascular coverage. Finally, extravascular fibrinogen was reduced by both antibody treatments in multiple brain regions. Together, our findings establish the VLA4/VCAM1 axis as a promising target to preserve vascular integrity and prevent cognitive decline late after stroke. Our data is consistent with a model where blocking either VCAM1 or VLA4 chronically after stroke promotes new blood vessel growth and maturation and restores the blood-brain barrier to prevent infarct-induced neurodegeneration.