The Human Blood-Nerve Barrier Transcriptome

Abstract

The blood-nerve barrier (BNB), formed by tight junction-forming microvessels within peripheral nerve endoneurium, exists to regulate its internal microenvironment essential for effective axonal signal transduction. Relatively little is known about the unique human BNB molecular composition. Such knowledge is crucial to comprehend the relationships between the systemic circulation and peripheral nerves in health, adaptations to intrinsic or extrinsic perturbations and alterations that may result in disease. We performed RNA-sequencing on cultured early- and late-passage adult primary human endoneurial endothelial cells and laser-capture microdissected endoneurial microvessels from four cryopreserved normal adult human sural nerves referenced to the Genome Reference Consortium Human Reference 37 genome browser, using predefined criteria guided by known transcript or protein expression in vitro and in situ. We identified 12881 common transcripts associated by 125 independent biological networks, defined as the normal adult BNB transcriptome, including a comprehensive array of transporters and specialized intercellular junctional complex components. These identified transcripts and their interacting networks provide insights into peripheral nerve microvascular morphogenesis, restrictive barrier formation, influx and efflux transporters with relevance to understanding peripheral nerve homeostasis and pharmacology, including targeted drug delivery and the mediators of leukocyte trafficking in peripheral nerves during normal immunosurveillance.

Figure 1

Sample characteristics. A representative digital light photomicrograph of a normal adult sural nerve plastic-embedded axial section, depicting the three peripheral nerve compartments is shown (A), with normal appearing endoneurial microvessels shown at higher magnification (B). A representative digital fluorescent photomicrograph of a normal sural nerve axial cryosection shows UEA-1-positive (green) endoneurial and epineurial vessels (C). A representative phase contrast digital photomicrograph shows confluent pHEndEC monolayers in culture (D), with a higher magnification image demonstrating their spindle shape without intercellular gaps (E). High sensitivity digital electropherograms following ribosomal RNA depletion demonstrate high quality cDNA for RNA sequencing from laser-capture microdissected endoneurial microvessels from a single normal adult (F) and P3 pHEndECs (G). Scale bars: A = 100 µm, B = 10 µm, C = 500 µm, D = 200 µm, E = 100 µm.

Figure 2

Human BNB transcriptome. Venn diagrams depict the human BNB defined as transcripts expressed by both the In Vitro BNB and In Situ BNB, with numbers indicating the number of transcripts identified based on predefined criteria (A). Hierarchical clustering (B) demonstrates transcripts that are equally expressed in vitro and in situ (a), highly expressed in situ (b), expressed in situ only (c), highly expressed in vitro (d) and expressed in vitro only (e).

Figure 3

Validation of the human BNB transcriptome. Merged digital photomicrographs of sural nerve endoneurial microvessels (UEA-1 FITC-positive, green) in situ show BNB endothelial cell expression (yellow-green, yellow or orange co-localization dependent of the relative fluorescent intensity of protein marker in red) of transporters (ABCA8, ABCB1, AQP1, SLC1A1, SLC2A1, SLC3A2, SLC5A6, SLC16A1 and SLC19A2), cytoskeletal proteins (ACTG1, CALD1, FLNA, MYO10), cell membrane proteins (CAV1, CD44, CD63, ESAM), junctional complex proteins (CDH5, CDH6, CLDN4, CLDN5, CTNNA1, GJA1, LIN7A, MPDZ, PCDH1, TJP1, VEZT, ZYX), vascular endothelial cell secreted protein CTGF, and chemokine receptor CXCR4. Positive expression of AQP1, CALD1, CAV1, CD44, CD63, FLNA, MYO10 and SLC2A1 (red) by cells that surround and are in direct contact with endothelial cells (most likely pericytes), and ABCA8 by cells close to but not in direct contact with endothelial microvessels (most likely Schwann cells) are also observed. Nuclei are identified in blue. Original magnification 400X.

Figure 4

Human BNB Biological Process. PANTHER generated pie charts and bar histograms demonstrate the diversity of human BNB biologic processes and the number of genes implicated in each process.

Figure 5

Human BNB Molecular Function. PANTHER generated pie charts and bar histograms demonstrate human BNB molecular function and the number of genes involved with each function.

Figure 6

Human BNB Cellular Component. PANTHER generated pie charts and bar histograms demonstrate human BNB cellular component and the number of genes that contribute to each component.

Figure 7

Human BNB Protein Class. PANTHER generated pie charts and bar histograms demonstrate the diversity of human BNB protein classes and the number of genes classified in each group.