Large-scale identification of human cerebrovascular proteins: Inter-tissue and intracerebral vascular protein diversity

<div><p>The human cerebrovascular system is responsible for regulating demand-dependent perfusion and maintaining the blood-brain barrier (BBB). In addition, defects in the human cerebrovasculature lead to stroke, intracerebral hemorrhage, vascular malformations, and vascular cognitive impairment. The objective of this study was to discover new proteins of the human cerebrovascular system using expression data from the Human Protein Atlas, a large-scale project which allows public access to immunohistochemical analysis of human tissues. We screened 20,158 proteins in the HPA and identified 346 expression patterns correlating to blood vessels in human brain. Independent experiments showed that 51/52 of these distributions could be experimentally replicated across different brain samples. Some proteins (40%) demonstrated endothelial cell (EC)-enriched expression, while others were expressed primarily in vascular smooth muscle cells (VSMC; 18%); 39% of these proteins were expressed in both cell types. Most brain EC markers were tissue oligospecific; that is, they were expressed in endothelia in an average of 4.8 out of 9 organs examined. Although most markers expressed in endothelial cells of the brain were present in all cerebral capillaries, a significant number (21%) were expressed only in a fraction of brain capillaries within each brain sample. Among proteins found in cerebral VSMC, virtually all were also expressed in peripheral VSMC and in non-vascular smooth muscle cells (SMC). Only one was potentially brain specific: VHL (Von Hippel-Lindau tumor suppressor). HRC (histidine rich calcium binding protein) and VHL were restricted to VSMC and not found in non-vascular tissues such as uterus or gut. In conclusion, we define a set of brain vascular proteins that could be relevant to understanding the unique physiology and pathophysiology of the human cerebrovasculature. This set of proteins defines inter-organ molecular differences in the vasculature and confirms the broad heterogeneity of vascular cells within the brain.</p></div

Proteins that show differences between VSMC and non-vascular SMC.

<p>Proteins that show differences between VSMC and non-vascular SMC.</p

Functionally-related groups of brain vascular markers identified from the Human Protein Atlas.

<p>Clusters of functionally related proteins were identified in brain vessels using PANTHER analysis and are listed in groups that contain more than 3 proteins. Proteins related to calcium regulation are also included.</p

Proteins curated from the Human Protein Atlas that are expressed in both EC and VSMC of the brain.

<p>All proteins (n = 135) are predicted to be expressed in both types of the two principle types of vascular cells but not other cells of the central nervous system.</p

Protein interaction networks among vascular proteins identified from the Human Protein Atlas.

<p>We applied the STRING tool (string-db.org; Version 10.0) to human brain EC proteins to identify possible novel networks among molecules. The graphical representation of proteins identified as having at least one connected partner in the EC protein set are presented here. Connections included both physical interactions, co-expression, and text-mined connected proteins, and only interactions that were consider medium to high confidence were included. The same analysis, applied to SMC proteins, yielded only limited protein interactions.</p

Proteins that show differences between VSMC and non-vascular SMC.

<p>Proteins that show differences between VSMC and non-vascular SMC.</p

Curated brain endothelial proteins from the Human Protein Atlas.

<p>Listed proteins (n = 140) were predicted to be expressed only in brain endothelium but not other cell types in the brain. A majority of these proteins are also expressed in non-cerebral smooth muscle.</p

Brain EC proteins displaying intracerebral heterogeneous expression.

<p>Proteins (n = 16) were identified as heterogeneously expressed if in all cortex samples examined, capillary EC displayed highly variable levels of staining.</p

Brain perivascular proteins curated from the Human Protein Atlas.

<p>Proteins to be expressed in brain perivascular cells but excluded from EC and SMC are listed (n = 9). Proteins in perivascular compartments but also in other vascular cell types are listed in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188540#pone.0188540.t001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188540#pone.0188540.t003" target="_blank">3</a>.</p

Structurally related grouping of brain vascular markers identified from the Human Protein Atlas.

<p>Clusters of structurally related proteins were identified in brain vessels and are listed in groups that include the SLC, TRIM, CCDC, ZNF, and OR protein families.</p