Identifying the origin and mechanisms of pathological angiogenesis in neuroinflammatory diseases

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). Studies in human MS and its animal model experimental autoimmune encephalomyelitis (EAE) have shown that new abnormal vessels develop in demyelinating plaques through pathological neo-angiogenesis, which exacerbates MS/EAE pathology by contributing to the leakage of serum components and infiltration of immune cells into the CNS. However, the cellular origin and the signaling pathways underlying this pathology remain poorly understood. Here, using single-cell RNA sequencing and validation with in situ hybridization and immunofluorescence, we discovered that neo-angiogenesis is initiated specifically by venous endothelial cells during EAE. Moreover, we identified VEGF-A signaling as the primary driver of neo-angiogenesis in EAE, as treatment with the VEGF-A blocking antibody, bevacizumab, ameliorated the EAE pathology by reducing neo-angiogenesis in vivo. Our findings may lead to the development of novel therapeutics designed to reduce neo-angiogenesis and improve long-term neurological deficits in MS. MOG35-55 EAE was induced in 10–12-week-old TCF/LEF1::H2B::eGFP female Wnt reporter mice in the C57BL/6J background and spinal cords were collected at acute peak (score = 2-4; 16-17 d.p.i.) and early chronic (score ≤ 1; 28-29 d.p.i.) phases of the disease. Age-matched healthy and CFA-immunized mice served as controls. To test whether VEGF-A is necessary to induce neo-angiogenesis in vivo in EAE, EAE-induced wild-type C57BL/6J mice were treated with either the humanized, anti-VEGF-A monoclonal antibody (mAb), bevacizumab, or IgG control antibody. Following single cell dissociation of the spinal cord, endothelial cells were labeled with CD31-APC antibody and FACS for single cell RNA sequencing analysis.