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 (Figure 1B), which binds mouse endothelial glycocalyx. BMVs exhibited a strong immunoreactivity for CD31/PECAM-1 (Figure 1 A & C), continuous staining with the abluminal marker collagen IV, as well as a weak and ‘spotty’ immunoreactivity for GFAP (Figure 1 E & G). 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), VE-cadherin (Cdh5) 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).
When RNA-seq data generated in this study for human (total) brain and lung were compared with public RNA-seq data, a strong correlation (Pearson correlation coefficient of 0.96) (Supplementary Figure 1A & 1B) was observed. 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 1C, 1D & 1E). 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 genes Glut-1, VE-cadherin, E-selectin, CD31, tight-junction protein 1 (TJP1)/ZO1, occludin (OCLN), ABC transporter ABCG2 and 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 3A). Pericyte marker genes similarly were enriched in brain vessels relative to total brain tissues (Figure 3B); while the transcript abundances of the astrocyte-specific glutamate transporters GLAST1 and SLC1A6 were higher in the brain than in the BMV (Figure 3B).
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 1. The SLC2A1/GLUT-1 was the most abundant among the receptors/transporters studied in human BMVs (Table 1). The rank order of other RMT receptor transcript abundance in human BMVs was LRP1> SLC3A2/CD98hc > CDC50A/TMEM30A > INSR > TFRC > LDLR > IGF1R > LEPR > LRP8 = IGF2R (Table 1; Figure 4A). LRP1, SLC3A2/CD98hc and CDC50A were expressed at similar levels, and were significantly higher than all other RMT receptors studied (Figure 4A).
INSR showed higher abundance (enrichment) in isolated human BMVs compared to either the brain or the lung (Table 1). SLC3A2/CD98hc, CDC50/TMEM30A and IGF1R were expressed at similar levels in brain vessels, brain and lung; whereas TFRC, LRP1, LDLR, IGF2R and LEPR were comparatively highly enriched in the lung (Table 1).
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, and whole tissue extracts of the brain, liver and spleen (Table 2; Fig. 4B). The gene showing the highest abundance and selectivity in isolated mouse BMVs was SLC2A1/GLUT1 (Table 2). Among other putative RMT receptors/transporters, the rank order in abundance in mouse BMVs was LRP1 > IGF1R = SLC3A2/CD98hc > CDC50A = LRP8 = TFRC = INSR > LDLR = IGF2R> LEPR (Table 2; Fig. 4B).
SLC2A1, IGF1R, INSR and LRP8 were distinctly enriched in mouse BMVs compared to the brain, lung vessels and peripheral tissues examined in this study (Table 2). TFRC showed high transcript abundance in both BMVs and the spleen; whereas SLC3A2/CD98hc showed high transcript abundance in the spleen and lung vessels. CDC50A was enriched in the brain, liver and lung vessels (Table 2). IGF2R and LEPR showed relative enrichment in lung microvessels; while LDLR was highly expressed in liver, spleen and lung vessels (Table 2).
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 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 [49-51].
Comparative analyses suggested that the endothelial enrichment in SLC2A1, TfR, INSR, SLC3A2/CD98hc and LRP8 is largely responsible for the high abundance of these genes observed in the isolated mouse BMVs [50,51]; 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. , which mapped 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 than TfR in BEC . Human BMVs analyzed in this study appeared to have lower than expected expression of SLC2A1, 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 [49-51]. 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 of human and mouse were compared across species (Table 3). The abundance of receptor transcripts was compared across species using normalized transcripts per million (TPM) counts (Table 3).
The expression levels of SLC2A1, TFRC, LRP1, LRP8, IGF1R, IGF2R, CDC50A/TMEM30A and SLC3A2/CD98hc were all significantly higher in mouse BMVs compared to human BMVs (Table 3). The expression levels of LRP8, LDLR and LEPR were not significantly different in BMVs between two species (Table 3). LRP1, LRP8 and CDC50A/TMEM30A were more enriched in the mouse compared to human brain (Table 3).
The structural genes (GAPDH and TUBB4A), and other genes (S100B and ABCA2) were similarly expressed in mouse and human BMVs and the brain (Table 3).
IGF1R, LRP1 and TfR protein expression in human and mouse BMVs
Some of the above findings were validated at the protein expression level using the same BMV preparations that were analyzed by RNA-seq. Due to limited amount of BMV samples and based on the availability of species cross-reactive antibodies, three receptors were chosen against which antibodies/ligand were developed and tested as BBB-carriers in clinical (TfR, LRP-1) or advanced preclinical (IGF1R) studies. All three RMT receptors showed higher transcript abundance in mouse BMVs by RNA-seq. The protein expression of these RMT receptors was analyzed by Western blot (WesTM) and immunodetection.
TfR protein expression was higher in mouse BMVs compared to human BMVs by WesTM analyses (Figure 5 A&B; t-test p<0.05) (normalized to β-actin). TfR was detected by immunofluorescence in both mouse (Figure 5 C-E) and human (Figure 5 F-H) BMVs.
The protein levels of LRP1 were also higher in mouse BMVs compared to human BMVs by WesTM analyses (Figure 6A&B; t-test p<0.05) (normalized to β-actin); strong immunofluorescence was detected in BMVs from both species around cell nuclei and borders and, occasionally, overlapping with collagen IV immunofluorescence (Figure 6 D-E, H-I).
Western blot (WesTM) analysis demonstrated significantly lower expression of IGF1R in human compared to mouse BMVs (Figure 7A&B; t-test p<0.0001) (normalized to β-actin). Immunofluorescence (Figure 7C-G) and immunohistochemistry (Figure 7H-I) analyses demonstrated a strong expression of IGF1R in both mouse (Figure 7 C-D) and human (Figure 7 E-I) BMVs, often observed as punctate, vesicular immunoreactivity in vessel walls. Immunostaining studies could not detect differences in IGF1R expression between mouse and human BMVs.