Abstract Body: Background
Our recently reported that patent foramen ovale (PFO) shunt enables the elevation of total homocysteine (tHcy) in patients’ circulation (Deng, Neurology 2021). However, the underlying molecular mechanism of how tHcy causes PFO-related injury is unclear. Considering that brain vascular endothelial cells are among the first to respond to the alterations in bloodstream, we hypothesized that elevated tHcy acts on brain endothelial cells, opens blood brain barrier (BBB) and leads to further neurovascular injury.

Method
Brain microvascular endothelial cells were exposed to high concentration of tHcy for 72 hr and subjected to RNA sequencing analysis. The viability of the cells was evaluated using MTT method. Additionally, mass spectrometry was employed to measure tHcy levels in both plasma and cerebrospinal fluid (CSF) samples collected from 56 patients with cerebrovascular disease. BBB permeability was assessed by CSF/plasma albumin ratio, a highly reliable clinical indicator of BBB integrity.

Result
Gene set enrichment analysis (GSEA) revealed significant activation of apoptosis in brain endothelial cells following exposure to high tHcy levels (NES: 1.80; p < 0.001) (Figure 1A). Many pro-apoptotic factors, such as Bax, Bak and FAS, were upregulated in response to tHcy (Figure 1B). Consistent with this, cell viability decreased with increasing tHcy concentrations (Figure 1C), suggesting that tHcy induces damages to brain endothelial cells. Supporting this, plasma tHcy levels in stroke patients were well correlated with those in CSF (r = 0.46, p = 0.001) (Figure 1D). Patients with high tHcy in plasma (> 12 μM) demonstrated increased BBB permeability (Figure 1E).

Conclusion
Our findings suggest that elevated circulatory tHcy may injure brain endothelium and increase BBB permeability, potentially contributing to downstream neurovascular dysfunction. The impact of tHcy on BBB and other brain cell types are ongoing to explore its important role in PFO related neurovascular injury.