Comparative transcriptome analysis of inner blood-retinal barrier and blood-brain barrier in rats

Abstract

Although retinal microvessels (RMVs) and brain microvessels (BMVs) are closely related in their developmental and share similar blood-neural barriers, studies have reported markedly different responses to stressors such as diabetes. Therefore, we hypothesized that RMVs and BMVs will display substantial differences in gene expression levels even though they are of the same embryological origin. In this study, both RMVs and BMVs were mechanically isolated from rats. Full retinal and brain tissue samples (RT, BT) were collected for comparisons. Total RNA extracted from these four groups were processed on Affymetrix rat 2.0 microarray Chips. The transcriptional profiles of these tissues were then analyzed. In the present paper we looked at differentially expressed genes (DEGs) in RMVs (against RT) and BMVs (against BT) using a rather conservative threshold value of ≥ ± twofold change and a false discovery rate corrected for multiple comparisons (p < 0.05). In RMVs a total of 1559 DEGs were found, of which 1004 genes were higher expressed in RMVs than in RT. Moreover, 4244 DEGs between BMVs and BT were identified, of which 1956 genes were ≥ twofold enriched in BMVs. Using these DEGs, we comprehensively analyzed the actual expression levels and highlighted their involvement in critical functional structures in RMVs and BMVs, such as junctional complex, transporters and signaling pathways. Our work provides for the first time the transcriptional profiles of rat RMVs and BMVs. These results may help to understand why retina and brain microvasculature show different susceptibilities to stressors, and they might even provide new insight for pharmacological interventions.

Figure 1

Principal component analysis (PCA) performed on whole transcriptome genes identified from RMVs, BMVs, RT and BT of rats. The first component (PC1) with a variance of 95% is on the X-axis and the second component (PC2) with a variance of 3% is on the Y-axis. Each dot represents one tissue sample. RMVs, retinal microvessels; BMVs, brain microvessels; RT, retinal tissue; BT, brain tissue.

Figure 2

Gene expression comparison between retinal microvessels (RMVs) and retinal tissue (RT) in rats (n = 6). (A) Volcano plot for the RMVs versus RT whole transcriptomes. The red dots indicate the genes that are significantly higher expressed (fold change [FC] ≥ 2.0 and adjusted p < 0.05) in RMVs compared to RT, while blue dots indicate the lower expressed genes (FC < -2.0 and adjusted p < 0.05). (B) Hierarchical cluster analysis for genes that are significantly enriched (FC > 2.0 and adjusted p < 0.05) in RMVs. (C,D) Top 15 gene ontology (GO) biological processes (BP) and top 12 PANTHER pathways identified from the RMVs enriched genes. All the biological processes shown are ranked by enrichment score with a Bonferroni adjusted p < 0.05.

Figure 3

Gene expression comparison between brain microvessels (BMVs) and brain tissue (BT) in rats (n = 6). (A) Volcano plot for the BMVs versus BT whole transcriptomes. The red dots indicate the genes that are significantly higher expressed (fold change [FC] ≥ 2.0 and adjusted p < 0.05) in BMVs compared to BT, while blue dots indicate the lower expressed genes (FC ≤ -2.0 and adjusted p < 0.05) in BMVs. (B) Hierarchical cluster analysis for genes that are significantly enriched (FC > 2.0 and adjusted p < 0.05) in BMVs. (C,D) Top 15 gene ontology (GO) biological processes (BP) and 12 PANTHER pathways identified from the BMVs enriched genes. All the biological processes shown are ranked by enrichment score with a Bonferroni adjusted p < 0.05.

Figure 4

Enrichment of 6 target genes in retina microvessels (RMVs) and brain microvessels (BMVs) over the respective full tissues. (A) Fold changes obtained in the semiquantitative RT-PCR (qRT-PCR) measurements in RMVs (open bars) and BMVs (filled bars). Gja5, gap junction alpha-5 protein (also termed Cx40); Slc2A1, solute carrier family 2 member 1; Slc7A1, solute carrier family 7 member 1; Slc38A5, solute carrier family 38 member 5; Abcc4, ATP binding cassette subfamily C member 4; Pdgfrb, platelet derived growth factor receptor beta. (B) Agreement of results obtained by microarray measurements (abscissa) and qRT-PCR measurements (ordinate). In this graph enrichment is indicated as log₂ fold changes. The regression line and the corresponding correlation coefficient show the significant agreement of data obtained by the two methods.

Figure 5

Gene expression comparison between RMVs and BMVs in rats (n = 6). (A) Venn diagram showing the numbers of genes that are significantly higher expressed in microvessels (MVs) obtained from the retina (RMVs, 1,004 genes) and from the brain (BMVs, 1956 genes). A total of 854 genes were commonly enriched in both, RMVs and BMVs. (B) Top 15 gene ontology (GO) biology processes (BP) identified from the 854 common genes. (C) Top 13 PANTHER pathways identified from the 854 common genes. All the biological processes shown are ranked by enrichment score with a Bonferroni adjusted p < 0.05 level of significance.