Abstract Body: Introduction: Cerebral Amyloid Angiopathy (CAA) is characterized by the progressive accumulation of amyloid-beta (Aβ) in leptomeningeal vessels and penetrating arterioles, which predisposes patients to intracerebral hemorrhage. Recent studies have found that the secreted cytokine osteopontin (OPN) aids the formation of Aβ plaques. We hypothesized that the pro-fibrotic function of OPN remodels the perivascular extracellular matrix (ECM), thereby facilitating the deposition of Aβ on the cerebrovascular wall leading to CAA.

Methods: We performed single-cell RNA sequencing in blood vessel-enriched preparations of whole brains from young (6 months) and aged (18 months) Tg2576 and age-matched wild-type littermate mice. Using the 10x Genomics Cell Ranger, Seurat, and CellChatDB analysis packages in R studio, we identified significant cell-cell interaction pairs and ligand-receptor pairs in the OPN signaling pathway. To probe the pro-fibrotic function of OPN, we cultured primary human central nervous system (CNS) fibroblasts. These cells were then treated with OPN protein (10 ug/ml, 24 hrs) and imaged for ECM proteins. Fibroblasts receiving OPN stimulation or vehicle control were then treated with Aβ_1-40 conjugated with HiLyte Fluor 555 (HF555) to determine if altered ECMs affected the accumulation of Aβ_1-40-HF555. Two or Three-way ANOVA with Bonferroni correction were used for statistical comparison.

Results: We found that OPN signaling was significantly upregulated in the brains of young Tg2576 mice before the onset of Aβ deposition in the brain, and that this upregulation was predominantly seen in CNS fibroblasts. At the transcriptomic level, CNS fibroblasts were the primary predicted senders of OPN signaling. The predicted receivers of OPN signaling were neurovascular-associated cells. The contributors to the OPN signaling network involved OPN receptor (CD44), and integrins. In CNS fibroblast culture, extracellular fibronectin deposition was significantly (p = 0.0003, 95% C.I. = [-37744, -9736] upregulated by OPN stimulation, compared to vehicle-treated cells. Additionally, Aβ_1-40-HF555 deposition on the ECM was significantly (F (1, 1144) = 25.98, P<0.0001) increased after OPN treatment in CNS fibroblasts.

Conclusions: The present study suggests that the upregulation of OPN signaling leads to pathological vascular ECM remodeling that is conducive to the deposition of Aβ_1-40. This remodeling may be perpetuated by CNS fibroblasts in the early development of CAA.