Single-cell dissection of the human cerebrovasculature in health and disease

Abstract

SummaryDespite the importance of the blood-brain barrier in maintaining normal brain physiology and in understanding neurodegeneration and CNS drug delivery, human cerebrovascular cells remain poorly characterized due to their sparsity and dispersion. Here, we perform the first single-cell characterization of the human cerebrovasculature using both ex vivo fresh-tissue experimental enrichment and post mortem in silico sorting of human cortical tissue samples. We capture 31,812 cerebrovascular cells across 17 subtypes, including three distinct subtypes of perivascular fibroblasts as well as vasculature-coupled neurons and glia. We uncover human-specific expression patterns along the arteriovenous axis and determine previously uncharacterized cell type-specific markers. We use our newly discovered human-specific signatures to study changes in 3,945 cerebrovascular cells of Huntington’s disease patients, which reveal an activation of innate immune signaling in vascular and vasculature-coupled cell types and the concomitant reduction to proteins critical for maintenance of BBB integrity. Finally, our study provides a comprehensive resource molecular atlas of the human cerebrovasculature to guide future biological and therapeutic studies.

Molecular atlas of the human brain vasculature at the single-cell level

“…The MHC class II signature was upregulated in all pathologies according to the pattern described above showing highest expression levels at the level of large veins, veins>large arteries and arteries (Figure 5c,g,h, Extended Data Figures 32 and 28 ). We observed a partial overlap of the MHC class II and peripheral signatures and of the BBB dysfunction module with a common predominance in large caliber vessels, but a more widespread/stronger expression of the peripheral signature and BBB dysfunction module in angiogenic capillaries (Extended Data Figures 33,34), in line with the previously-observed links between BBB dysregulation and innate immune activation 44 . Together, these data suggest that pathological CNS ECs upregulate MHC class II receptors in brain tumors and brain vascular malformations.…”

“…In brain pathologies, we found that the MHC class II signature was highest in brain vascular malformations (CAV>AVM), the extra-axial brain tumor MEN, the intra-axial secondary brain tumor MET, followed by the intra-axial primary brain tumors HEM>LGG>GBM, TL and TLadjCAV and the fetal brain, grossly following the peripheral signature expression gradient whereas the CNS signature showed an inverse trend The MHC class II signature was upregulated in all pathologies according to the pattern described above showing highest expression levels at the level of large veins, veins>large arteries and arteries (Figure 5c,g,h, Extended Data Figures 32 and 28 ). We observed a partial overlap of the MHC class II and peripheral signatures and of the BBB dysfunction module with a common predominance in large caliber vessels, but a more widespread/stronger expression of the peripheral signature and BBB dysfunction module in angiogenic capillaries (Extended Data Figures 33,34), in line with the previously-observed links between BBB dysregulation and innate immune activation 44 . Together, these data suggest that pathological CNS ECs upregulate MHC class II receptors in brain tumors and brain vascular malformations.…”

“…Comparison of the CNS-specific properties between fetal, adult and pathological ECs revealed CNS properties in the fetal and adult brains as well as an alteration of the CNS signature and an acquisition of the peripheral signature in pathologies (Figure 4a-f, Extended Data Figures 23-25). When comparing the CNS signature between pathological and adult/control brain ECs, we found downregulation of SLC2A1 which is known to be dysregulated in neurodegenerative conditions 44 , as well as of the lipid transporter MFSD2A, which is expressed in brain ECs and restricts caveolae-mediated transcytosis at the BBB 63,65,66 , and the BBB marker CLDN5 (Figure 4d-f, Supplementary Table 9), thus suggesting BBB alteration in pathologies 16 . Comparing CNS and peripheral signature expression across entities revealed that the CNS signature was highest in the TL, followed by intra-axial primary brain tumors and fetal brain (LGG>fetal brain>GBM), brain vascular malformations (TLadjCAV>CAV>AVM), intra-axial secondary brain tumor MET, extraaxial brain tumor MEN and the intra-axial primary brain tumor HEM, whereas the peripheral signature followed an inversed pattern (Figure 4e,f, Extended Data Figure 25e,f).…”

“…To broadly map the neurovasculature and to address the NVU across different brain entities, 44,116 fetal, 47,305 adult/control and 121,436 pathological unsorted EC and PVC transcriptomes from 33 patients were pooled, clustered, annotated using known marker genes and comparison to public datasets 22,28,33 and visualized (Figure 1a, Extended Data Figure 3c,e). We identified 18 major cell types, including all known human brain vascular and perivascular, and other tissue-derived cell types in the human brain (Extended Data Figures 3e-g,4).…”

“…Next, to further address EC heterogeneity across different brain entities at the single-cell level, we pooled, batch-corrected [35][36][37][38] , clustered and visualized all fetal (21,512), adult/control (47,652) and pathological (166,549) sorted brain EC transcriptomes from 38 patients (Figure 2a-d, Extended Data Figure 7a-e). Brain vascular ECs are organized along the human brain arteriovenous axis, referred to as zonation 22,[39][40][41][42][43][44] . To address arteriovenous zonation, endothelial clusters were biologically annotated using top-ranking marker genes/DEGs in fetal, adult and pathological ECs, and we identified 44 clusters (Figure 2e,f, Extended Data Figure 7a-e,h, Supplementary Table 7) that we then grouped into 14 major EC subtypes for further downstream analysis (Figure 2a-d).…”