Characterization of isolated brain microvessels (BMVs)
Mouse (Figure 1A-E) and human (Figure 1F-I) BMVs isolated using a modified separation protocols described  were characterized by immunofluorescence staining for the endothelial antigen CD31/PECAM-1, the basement membrane component collagen IV and the astrocyte (end-feet) marker glial fibrillary acidic protein (GFAP). The luminal surface of endothelial cells was visualized using lectin GSL-1, which binds mouse endothelial glycocalyx. BMVs exhibited a strong immunoreactivity for CD31/PECAM-1, which co-localized with the luminal endothelial marker GSL-1 (Figure 1 A-C), as well as a weak and ‘spotty’ immunoreactivity for GFAP, co-localized with the abluminal marker collagen IV (Figure 1 D-I). The majority of vessels in analyzed samples measured less than 20 mm in diameter.
The enrichment of cell-specific markers in BMVs in comparison to vessel-depleted brain parenchyma, was also analyzed using targeted nanoLC-MS/MS. A relative BMV enrichment of specified proteins was presented in Log2 ratio in Figure 2. Endothelial cell markers coagulation factor VIII-related antigen (F8), E-selectin (Sele), E-cadherin (Cdh1) and Pecam1/CD31 showed 4 to 8-fold enrichment in BMV preparations compared to vessel-depleted brain parenchyma; brain-endothelial cell-specific Slc2a1/glucose transporter (Glut1) showed over 60-fold enrichment in BMVs. Pericyte markers, platelet-derived growth factor receptor beta (Pdgfrb), desmin (Des), smooth muscle actin Acta2, and CD13 (aminopeptidase N/Anpep), were also enriched in BMVs (2 to 6-fold), whereas astrocyte markers glial fibrillary acidic protein (Gfap), protein S100-beta (S100β), electrogenic sodium bicarbonate cotransporter 1 (Slc4a4), and aquaporin-4 (Aqp4) were 2 to 4-fold enriched in brain parenchyma compared to BMVs. This data confirms that BMV isolation protocol used in this study yielded endothelial cell- and pericyte-enriched microvessels with significant depletion of astrocytes, although remnants of the astrocytic end-feet could still be detected by in-situ immunofluorescence.
RNA-seq Datasets: Comparability and Validation
Isolated mouse and human BMVs and organs were subjected to RNA-seq analyses; an enrichment of RMT receptors in BMVs was compared to peripheral organs and the whole brain (without vascular depletion).
RNA-seq data generated in this study for human (total) brain and lung were compared with public RNA-seq data which showed strong correlation (Pearson correlation coefficient of 0.96) (Supplementary Figure 1A). Comparison of biological replicates (brain total, lung total and brain vessels) analyzed in this study also showed high correlation (Pearson correlation coefficients ranging between 0.94 and 0.97, Supplementary Figure 1B). These analyses confirmed internal reproducibility and comparability of the RNA-seq data generated in this study with available ‘benchmark’ external datasets.
Further quality control of the dataset was performed by analyzing a relative enrichment of endothelial or BBB-specific gene transcripts in BMVs compared to (total) brain. Gene transcripts encoding endothelial cell-specific genes Glut-1, E-selectin, CD31, tight-junction protein 1, ABC transporter ABCG2, enzymes alkaline phosphatase (ALP) and g-glutamyl transpeptidase (g-GTP) were highly enriched in BMVs compared to the total brain in both mouse and human samples (Figure 3). In contrast, the transcript abundances of the astrocyte-specific glutamate transporters GLAST1 and SLC1A6 were similar in BMVs and the brain (Figure 3), suggesting that BMV preparations contain some astrocyte ‘contamination’, as also shown by immunofluorescence analyses.
The expression of RMT genes in isolated human BMVs, brain and lung
The expression levels of genes encoding RMT receptors in isolated human BMVs, total brain and lung tissues are listed in Table 2. The rank order of RMT receptor transcript abundance in human BMVs was LRP1> SLC3A2/CD98hc > CDC50A/TMEM30A > INSR > TFRC > LDLR > IGF1R > LEPR > LRP8 (Table 2; Figure 4A). LRP1, CD98 and CDC50A were expressed at similar levels, and were significantly higher than other RMT receptors studied (Figure 4A).
INSR showed higher abundance (enrichment) in isolated human BMVs compared to either the brain or the lung (Table 2). SLC3A2/CD98hc, CDC50/TMEM30A and IGF1R were expressed at similar levels in brain vessels, brain and lung; whereas TFRC, LRP1, LDLR and LEPR were comparatively highly enriched in the lung (Table 2).
The expression of RMT genes in isolated mouse BMVs, lung microvessels, brain, liver and spleen
RNA-seq analyses were performed on isolated mouse BMVs, lung vessels, brain, liver and spleen (Table 3). The gene showing the highest abundance in isolated mouse BMVs was LRP1 (Table 3; Figure 4B), similar to what was observed in isolated human BMVs. The rank order in RMT receptor transcript abundance in mouse BMVs was LRP1 > IGF1R = SLC3A2/CD98hc > CDC50A = LRP8 = TFRC = INSR > LDLR > LEPR (Table 3; Fig. 4B).
IGF1R, INSR and LRP8 were distinctly enriched in mouse BMVs compared to the brain, lung vessels and peripheral tissues examined in this study (Table 3). TFRC showed high transcript abundance in both BMVs and the spleen; whereas CD98hc showed high transcript abundance in the spleen and lung vessels. CDC50A was enriched in the brain, liver and lung vessels (Table 3). LEPR showed relative enrichment in lung microvessels; while LDLR was highly expressed in liver, spleen and lung vessels (Table 3).
Cellular source of RMT receptor transcripts enriched in mouse and human BMVs
Comparisons of normalized RNA abundance of RMT receptors in human and mouse BMVs (from the current study) and available public datasets obtained from the brain endothelial cells, astrocytes and neurons, as well as the whole brain and lung from corresponding species are shown in Supplementary figure 2A&B. Data included in these analyses were obtained by single-cell sequencing of freshly isolated cells from the mouse brain vascular segments  and from the fetal and adult human cortex [47-49].
Comparative analyses suggested that the endothelial enrichment in TfR, INSR, SLC3A2/CD98hc and LRP8 is largely responsible for the high abundance of these genes observed in isolated mouse BMVs [48,49]; observed LRP1 expression in mouse BMVs appears to originate from its abundance in pericytes and astrocytes; whereas observed expression levels of IGF1R, CDC50A and SLC2A1/Glut-1 may originate from either one or all three cell types forming the neurovascular unit (NVU). A recent publication by Kalucka et al., 2020 , mapping single-cell transcriptome atlas of murine endothelial cells, identified IGF1R, TfR, LRP8 and SLC2A1 as highly enriched in BEC compared to endothelial cells from all other tissues; IGF1R transcript was 3-fold more abundant TfR in BEC . Human BMVs analyzed in this study appeared to have lower than expected expression of TfR, LDLR, LRP8 and IGF1R compared to the endothelial expression of these genes derived from the single cell sequencing of the fetal and adult human cortex [47-49]. Similarly to what was observed with mouse BMVs, high LRP1 expression observed in human BMVs does not appear to originate from endothelial cells.
Species differences in the expression of RMT receptors in isolated human and mouse BMVs
The expression patterns of RMT receptors in BMVs and the whole brain were compared across species (Table 4; Figure 4C). The abundance of receptor transcripts (normalized read counts) were first normalized to the levels of tubulin expressed in the respective samples to obtain the ratios for each species as described in the Data Analysis section. The ratios of each gene expression were then compared between species (Table 4).
The expression levels of TFRC, LRP1, LRP8 and IGF1R were significantly higher in mouse BMVs compared to human BMVs (Table 4; Fig. 4C). The expression levels of LRP1, LRP8 and LDLR were significantly higher in the mouse compared the human brain (Table 4); whereas LEPR expression was higher in the human brain (Table 4).
IGF1R, LRP1 and TfR protein expression in human and mouse BMVs
The protein expression of RMT receptors IGF1R, LRP1 and TfR, was analyzed by Western blot and immunodetection. Western blot (WesTM analysis) demonstrated lower expression of IGF1R in human compared to mouse BMVs (Figure 5A&B) (normalized to β-actin). Immunofluorescence (Figure 5C-G) and immunohistochemistry (Figure 5H-I) analyses demonstrated strong expression of IGF1R in both mouse (Figure 5 C-D) and human (Figure 5 E-I) BMVs, often observed as punctate, vesicular immunoreactivity in vessel walls. Immunostaining studies could not detect apparent differences in IGF1R expression between mouse and human BMVs.
The protein levels of LRP1 were slightly higher in mouse BMVs compared to human BMVs by WesTM analyses (Figure 6A&B); strong immunofluorescence was detected in BMVs from both species around cell nuclei and borders and, occasionally, overlapping with collagen IV immunofluorescence (Figure 6 C-H).
TfR protein expression was higher in mouse BMVs compared to human BMVs by WesTM analyses (Figure 7 A&B). TfR was detected by immunofluorescence in both mouse (Figure 7 C-E) and human (Figure 7 F-H) BMVs.