Extended Data Figure 1. Bulk and single cell transcriptome and proteome profiling of young and aged BECs reveal increased inflammatory signature with aging.

(a) Schematic of flow sorting of CD31+CD45− BECs from mouse cortex and hippocampi. n=6 young and 6 aged biologically independent samples; each sample= 2 biologically independent mice cortex/hippocampi pooled as one sample. There were 1006 significant differentially expressed genes (*q<0.05, Cuffdiff Statistical Package61).

(b) FACS gating strategy to isolate single BECs. PI+ dead cells were excluded. CD11b+ and CD45+ cells were gated to exclude monocytes/macrophages and microglia. CD31+Cd11b−CD45− cells were defined as the BEC population.

(c) Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of CNS cell-type specific markers. n=6 young and 6 aged biologically independent samples. Mean +/− SEM.

(d) FPKM values of leukocyte binding adhesion molecules including Vcam1. n=6 young and 6 aged biologically independent samples. Bars represent mean. Error bars derived from SEM. Specific q values shown are derived from Cuffdiff Statistical Package (*q=0.0015). See Methods and Source Data for details.

(e) FPKM values of tight junction genes. n=6 young and 6 aged biologically independent samples. Mean +/− SEM. q=0.16, *q=0.0013, **q=0.0015, Cuffdiff Statistical Package. See Methods and Source Data for details.

(f) FPKM values of the gene transcripts in murine young and aged CD31+BECs of human plasma proteins that change with age (see Supplementary Table 2 for list of human plasma proteins expressed in murine BECs). n=6 young and 6 aged biologically independent samples. Mean +/− SEM. *q=0.0015, **q=0.021, Cuffdiff Statistical Package. See Methods and Source Data for details.

(g) C57BL6 mice were injected with anti-VCAM1-DL488 or IgG-DL488 isotype control (r.o.) 2 hours before perfusion to label BECs in vivo prior to brain dissociation, staining and FACS.

(h) Flow gating and histogram plots of pooled (n=4 mice/ age group), young or aged hippocampi isolated from healthy mice injected with fluorescently tagged DL488 anti-VCAM1 mAb or IgG-DL488 conjugated isotype control as depicted in (g).

(i) Quantification of CD31+VCAM1+cells isolated from (left) healthy cortex (n=4 mice per age group, individually measured) and (right) 4 technical replicates of hippocampi that are pooled from 4 mice per age group. Mean +/− SEM. *p=0.0015. Two-tailed Student’s t-test.

(j) sVCAM1 ELISA in plasma from young isochronic or heterochronic parabionts following 5 weeks of parabiosis. n=11 mice/group pooled from two independent experiments. **p=0.0031,Two-tailed Student’s t-test. Mean +/− SEM.

(k) Confocal images in the DG of VCAM1, lectin, and Aqp4 of young isochronic or heterochronic parabionts 5 weeks after surgery. Quantification shown in Fig. 1j. Hoechst labels cell nuclei. Scale bar = 100 μm. n= 8 mice in the Young isochronic group and 13 mice in Young heterochronic group from two independent experiments; representative images are shown.

(l) Boxplot of expression levels of classical pan-endothelial and BBB-specific transcripts (n=272 BECs total). Minima, maxima, median, and percentiles are listed in Supplementary Table 3. (n=146 Capillary BECs, n=59 Venous BECs, n=67 Arterial BECs pooled from 8 mice hippocampi).

(m) Overlay of Vcam1 mRNA levels on corresponding coordinate on the Cd31 vs Vcam1 fluorescent intensity plots obtained during FACs sorting.

(n) Validation of the correlation (Spearman’s rho = 0.704) between protein and mRNA levels of 77 single BECs sorted from both Vcam1+ and Vcam1− gates. Scatterplot of Vcam1 fluorescence intensity as measured by FACs and corresponding transcript counts (per million).

(o) tSNE visualization colored by cell identity (aged vs. young) (n=160 young BECs, n=112 aged BECs pooled from 4 mice hippocampi per age group).

(p) Comparison of Vcam1 expression levels in young and aged hippocampal CD31+ BECs collected from the VCAM1+ gate during FACs sorting (bars represent mean and error bars = SD). (n=160 young BECs, n=112 aged BECs pooled from 4 mice hippocampi per age group). *p=0.017. Two-tailed Mann-Whitney test.

(q) Violin plots of mRNA expression levels of Icam1 in all isolated BECs (bottom) and specifically in VCAM1+ enriched BECs (top). Other adhesion molecules, namely Psele and Sele were not found to be expressed in isolated CD31+ BECs. (All BECs: n=160 young BECs, n=112 aged BECs pooled from 4 mice hippocampi per age group; VCAM1+ enriched BECs: n=56 Vcam1+ young BECs, n=44 Vcam1+ Aged BECs pooled from 4 mice hippocampi per age group). Minima, maxima, median, and percentiles are listed in Supplementary Table 3.

(r) Violin plots of tight junction markers in all isolated young and aged BECs. (n=160 young BECs, n=112 aged BECs pooled from 4 mice hippocampi per age group). Minima, maxima, median, and percentiles are listed in Supplementary Table 3.