Quality Control of Vasculome Gene Expression
Two levels of quality control were assessed. First, quality and integrity of RNA samples were tested with standard NanoDrop and Bioanalyzer approaches to ensure sufficient RNA concentrations, 260/280 ratios, 28 s/18 s ratios and RNA integrity number (RIN) scores. Second, the quality of microarray hybridization was also assessed by manually checking the distribution of hybridization signals, percentage of positive signals, ratio of 3′ to 5′ end of housekeeping genes (GAPDH and β-Actin), and applying principal component analysis to identify potential outliers. All samples passed these various checkpoints. For the final data sets, RNA from n
5 mice were pooled per microarray, and n
3 independent microarrays were used for each group (see Methods).
Next, we asked whether our vasculome was contaminated by parenchymal cells. For the brain vasculome, we compared our data with gene markers for different brain cells in public GEO GSE13379 datasets 
, that contain gene expression profiles for neurons, astrocytes and oligodendrocytes. This analysis demonstrated that genes known to be representative of neurons, astrocytes and oligodendrocytes had extremely low expression levels (signals <50,) in our brain vasculome, whereas gene markers of endothelial cells had much higher expression levels than others (). These data suggest that our brain vasculome is endothelial-specific and not contaminated by surrounding parenchymal brain cells. Another check of endothelial purity was performed using RT-PCR to assess the expression of gene markers in the vasculome from brain, heart and kidney glomeruli (Table S1
). Compared to corresponding whole organ tissue, each vasculome had higher expression of endothelial markers (≥ 3 fold), whereas gene expression levels were enriched for neuron and astrocyte markers in whole brain samples; myocyte markers in heart tissue samples; and kidney tubular markers in kidney tissue samples, respectively (Figure S1
). Taken together, these analyzes suggest that the various organ vasculomes were not overtly contaminated with parenchymal genes.
Differential expression of cell-type specific markers in brain vasculome versus other cell types in brain.
Brain Vasculome Specific Genes and Enriched Pathways
Although the microarrays revealed a large amount of data, we only focused on genes whose maximal expression values across all microarrays were greater than 200. Based on these criteria, we identified 3,557 genes expressed in brain endothelial cells. Next, we asked whether this brain vasculome differed from patterns found in our comparative heart and kidney glomerular vasculomes. Applying criteria of p<0.01, fold change ≥ 4, and maximal expression value across all samples >200, we identified 318 probes corresponding to 243 genes found to be highly expressed in brain endothelial cells, 143 probes corresponding to 110 genes highly expressed in heart endothelial cells, and 114 probes corresponding to 81 genes highly expressed in kidney glomerular endothelial cells. A heat-map analysis demonstrated that each vasculome was highly organ-specific. Gene expression patterns in the brain vasculome significantly differed from those in heart or kidney glomeruli ().
The vasculome of mouse brain is unique and different from those found in mouse heart and kidney.
In the brain vasculome, as expected, blood-brain barrier genes were easily detected. These included occludin (OCLN) and claudin-5 (CLDN5), two major components of tight junctions in the blood-brain barrier. Another known feature of cerebral endothelium is the presence of glutamate receptors that influence barrier function 
. In our datasets, both ionotrophic and metabtrophic glutamate receptors (Gria2, Gria3, Grin2b and Grm5 for AMPA2, AMPA3, NMDA2B and mGluR5 respectively) were specifically expressed in the vasculome from mouse brain but not heart and kidney glomeruli. Overactivation of glutamate receptors may cause excitotoxicity in neuronal compartments. Similarly, activation of NMDA or mGluR5 receptors could also mediate vascular responses caused by hyperhomocysteinemia and nitrosative stress in brain endothelial cells 
. The glutamate transporter Slc1a1 (solute carrier family 1, member 1), known as EAAC1/EAAT3, was also enriched in the brain vasculome. In neurons, this transporter plays a key role in regulating synaptic glutamate kinetics. But vascular functions may also exist, since brain endothelial cells co-cultured with astrocytes displayed a polarized brain-to-blood transport of glutamate, suggesting that transporter responses in brain microvessels may participate in the regulation of potentially excitotoxic amino acid concentrations 
. Overall, the brain vasculome demonstrated many mediators commonly found in neural systems. For example, semaphorins comprise a family of factors that control neurite extension and axon guidance 
. Here, we saw that Sema3C and Sema4D were enriched in the vasculome of brain compared to heart and kidney. Both Sema3C and Sema4D have been implicated in angiogenesis regulation 
, so their involvement in brain vascular homeostasis may also be important. Finally, another parallel with neural signaling may also be underscored by the detection of CamKIIα (calcium/calmodulin-stimulated protein kinase II alpha) 
, indicating that common responses to intracellular calcium fluxes may occur in both neuronal and non-neuronal systems in the brain.
Using the Fisher’s exact test to probe GO (Gene Ontology) and KEGG databases, many signaling and regulatory pathways were found to be enriched in the brain vasculome (). These included transporter activity, cell adhesion molecules (CAMs), and the Wnt signaling pathway. Besides well-known brain endothelial transporters such as Glut1 (glucose transporter type 1, or slc2a1, solute carrier family 2, member 1) and P-gp (multidrug resistence poly-glycoprotein, or Abcb1a, ATP-binding cassette, sub-family B, member1A), high levels of transferrin (Tf), transferrin receptor (TfR) and exporter ferroportin (slc40a1) were also detected in the brain vasculome. For cell adhesion molecules, NrCAM (neuronal cell adhesion molecule) appeared to be enriched in the brain vasculome. NrCAM was originally known as a neuron-specific gene required for axon guidance and organization of neural circuitry 
. However, NrCAM has recently been discovered in dermal and umbilical venous endothelium as well, with potential function in angiogenesis regulation and stress response in endothelial cells 
. The presence of NrCAM in our initial draft of the brain vasculome but not the heart or glomerular vasculome, further suggest close interactions and potential crosstalk between vascular systems and the organ milieu they inhabit. Similar enrichment in membrane proteins was found in the neurexin network. We detected the expression of neurexin and neuroligin in our mouse brain vasculome. In particular, neurexin-1 showed high level of expression in the brain vasculome rather than heart and kidney vasculomes. Once again, expression of neural-related guidance systems in the brain vasculome suggests crosstalk and signaling functions between blood vessels and brain parenchyma 
Enriched pathways detected in the vasculome of mouse brain.
In addition to physiologic pathways that underlie normal function, pathophysiologic pathways related to inflammation were also expressed in the brain vasculome. In the context of brain injury and neurodegeneration, cytokines and chemokines comprise a key network for regulating inflammation. In this vasculome project, 236 probes were screened for 150 cytokines/chemokines. Overall, low signals were detected for most cytokines/chemokines (<50). Applying the criteria of signal intensity >200, only 17 probes for 11 cytokines/chemokines were expressed in the normal mouse brain vasculome - ccl3, ccl9, ccl27, csf1, cxcl12 (SDF1), kitl, pdgfb, pglyrp1, ptn, socs7 and tgfb2 (). Compared to heart and kidney glomeruli, ccl3 (chemokine (C-C motif) ligand 3), ccl27 (chemokine (C-C motif) ligand 27) and pglyrp1 peptidoglycan recognition protein 1) appeared to be enriched in the brain vasculome (). Examination of the existing literature suggested that these may be relevant hits. Ccl3 is released by stimulated brain endothelial cells 
, and it has been reported that it may be elevated in brain vessels of Alzheimer’s disease patients 
. CCL27 is well known for mediating skin inflammation but has also been detected in the brain 
. Unlike other chemokines, CCL27 has both secreted and nuclear targeting forms that directly modulate transcription of many response genes, thus any involvement of this factor in the brain vasculome could potentially act as a potent amplifier of inflammation 
. PGLYRPs (or PGRPs, peptidoglycan recognition proteins) have four isoforms, PGLYRP1-4, that function in antibacterial immunity and inflammation 
. PGLYRP1 can bind with the key stress response proteins such as Hsp70 and S100A4 to trigger cytotxicity for antibacterial activity 
. Expression pglyrp1 in the brain have been reported, but its endothelial function is currently unknown 
List of cytokines/chemokines expressed in the vasculome of mouse brain.
Another inflammatory example was found in pathways involved in leukocyte transendothelial migration (). The brain vasculome-enriched genes in this pathway included Ncf1 (neutrophil cytosolic factor1, or p47 phox), Prkcb (protein kinase C, beta) and Prkcc (protein kinase C, gamma). Ncf1 is a subunit of NADPH oxidase, a critical enzyme for ROS production in injured or diseased vascular systems 
. It was reported that Ncf1 mediated the Abeta42 and RAGE ligation induced ROS production and downstream ERK1/2 phosphorylation and cPLA2 (cytosolic phospholipase A2) phosphorylation in cerebral endothelial cells 
. The PKC family is known to regulate the phosphorylation and uptake of SLC6 family of neurotransmitter transporters 
, also reported to be present in brain endothelial cells and regulate the blood-brain barrier 
. Whether Prkcb and Prkcc in the brain vasculome contribute to disease phenomena involved in cerebral ischemia, brain injury and neurodegeneration remains to be fully elucidated.
Protein-protein interaction (PPI) networks in the vasculome of mouse brain.
Finally, a prominent network that was enriched in the brain vasculome comprised the Wnt pathway (). Wnt is known to regulate neuronal stem cells, neurogenesis and neuroplasticity 
. But recently, Wnt signaling has been reported to also participate in the development of CNS vasculature, blood-brain barrier formation, and the protection of endothelial cells after injury 
. In our draft of the brain vasculome, β-cantenin (CTNNB1) was presented in the hub position of the Wnt protein-protein interaction network, along with brain endothelial-specific genes Axin2, MAPK10 (mitogen-activated protein kinase 10) and Lef1 (lymphoid enhancer binding factor 1). Axin2 is a transcriptional target of active Wnt signaling that also serves to autoregulate and repress the pathway by promoting β-cantenin degradation 
; In the conditional transgenic mice overexpressing β-Catenin, Axin2 is one of the antagonists changed in the brain 
. MAPK10 was originally described in neurons but it was recently reported to also mediate endothelial migration via eNOS 
. And Lef1 is the specific transcriptional factor in the downstream effectors of the Wnt pathway 
. A critical role of Wnt signaling in cell-cell communication can also be seen because its central hub β-Catenin also serves in the protein-protein interaction network for adherens junctions () for brain endothelial cells, linking specifically with the brain vasculome genes of Igf1r (insulin-like growth factor 1 receptor), Tgfbr1 (transforming growth factor, beta receptor 1) and Lef1 in this network.
Angiogenesis and the Brain Vasculome
In terms of functional networks, angiogenesis should comprise a central part of any vasculome. Probing the GO database revealed a dense protein-protein interaction network for angiogenesis-related genes in the brain vasculome, with hub positions occupied by β-catenin, Rtn4, HIF-1α, Mapk14, Notch1, Ptk2 (protein tyrosine kinase 2, also called focal adhesion kinase 1) and Tgfbr2 (). As described in above, β-catenin is highly expressed in the brain and in the hub positions of other pathways, connecting angiogenesis with these pathways, including Wnt pathway and adherens junctions. Also, Rtn4 (also called Nogo) was highly expressed in the brain. Rtn4 produces 3 isoforms (Nogo-A, Nogo-B, Nogo-C) that may play overlapping roles in vascular as well as neuronal systems in the CNS. Nogo-A is a well-characterized inhibitor of axonal growth and repair 
, whereas Nogo-B is already known to be highly expressed in endothelial cells 
. Nogo-B regulates vascular homeostasis and remodeling, in part by controlling endothelial cell migration, macrophage infiltration, leukocyte transmigration, and overall inflammation response after tissue ischemia and injury 
. Overall, Nogo-B may be protective since it is lost after injury 
Another angiogeneis gene with high expression levels in the brain vasculome is Gpx-1 (glutathione peroxidase 1), an intracellular antioxidant enzyme that converts hydrogen peroxide to water 
. Various studies with Gpx-1 transgenic mice suggest that this mediator may be neuroprotective against amyloid toxicity 
, Parkinsons-related pathologies 
, ischemia-reperfusion 
, or trauma 
. These underlying mechanisms of vascular neuroprotection may broadly include amelioration of cell death, suppression of astrocyte and microglia activation, preservation of BBB function, and a reduction of inflammatory infiltration 
. Furthermore, Gpx-1 may also contribute to CNS recovery, in part by interacting with hypoxia inducible factor 1 (HIF-1) and its target genes such as VEGF to regulate the angiogenesis process for tissue repair 
. Gpx-1-deficient mice show decreased recruitment and activation of endothelial progenitor cells after ischemic injury, leading to impaired angiogenesis and revascularization 
Within the CNS context, VEGF signaling may be especially important because this mediator may participate in both angiogenesis as well as neurogenesis 
. Comparison of VEGF signaling pathways showed that these were highly conserved across all three vasculomes in brain, heart and kidney glomeruli (). This may not be surprising since VEGF-mediated angiogenesis may be commonly required network regardless of organ systems. However, it is worth noting that there were two VEGF signaling mediators that appeared to be specifically expressed in the brain vasculome - Prkcb and Prkcc. These two signals were also identified in the leukocyte transendothelial migration network of the brain vasculome (see previous section). Thus, it is possible that particular brain vasculome-specific components may critically influence how the CNS responds to injury and disease. Angiogenesis is a physiological process involving the growth of new blood vessels. This phenomenon is vital not only for organ development but also for tissue repair and wound healing. Insofar as the brain vasculome may be a critical component of CNS plasticity and remodeling, these angiogenesis networks may represent a rich database to probe for potential mechanisms and targets for neurorecovery after stroke, brain injury or neurodegeneration.