Chromosome 2 Fragment Substitutions in Dahl Salt-Sensitive Rats and RNA Sequencing Identified Enpep and Hs2st1 as Vascular Inflammatory Modulators

Abstract

Chromosome 2 introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt-sensitive (SS) background (SS-chromosome 2^BN/Mcwi; consomic S2^B) reduced blood pressure and vascular inflammation under a normal-salt diet (NSD). We hypothesized that BN chromosome 2 contains anti-inflammatory genes that could reduce blood pressure and vascular inflammation in rats fed NSD or high-salt diet (HSD). Four- to 6-week old male SS and congenic rats containing the BN chromosome 2 distal portion (SS.BN-[rs13453786-rs66377062]/Aek; S2^Ba) and middle segment (SS.BN-[rs106982173-rs65057186]/Aek; S2^Bb) were fed NSD or HSD (4% NaCl) up to age 12 to 13 weeks. Systolic blood pressure determined by telemetry was higher in SS rats fed HSD versus NSD. Systolic blood pressure was lower in both congenic rats than in SS under NSD, but similar under HSD versus SS. Reactive oxygen species generation using dihydroethidium staining, expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and immune cell infiltration by immunofluorescence demonstrated that S2^Ba rats present less inflammation under NSD and more under HSD versus SS rats. RNA sequencing and reverse transcription-quantitative PCR identified 2 differentially expressed genes encoded within BN chromosome 2 distal portion that could act as regulators of vascular inflammation. These were downregulated glutamyl aminopeptidase (Enpep) that was anti-inflammatory under NSD and upregulated heparan sulfate 2-O-sulfotransferase 1 (Hs2st1) that was proinflammatory under HSD. In conclusion, 2 differentially expressed genes encoded within introgressed BN chromosome 2 distal fragment were identified: Enpep associated with reduced vascular inflammation under NSD, and Hs2st1, associated with increased vascular inflammation under HSD.

Keywords: blood pressure; genomics; glutamyl aminopeptidase; heparan sulfate 2-O-sulfotransferase 1; transcriptome.

Figure 1.. Map of rat chromosome 2 of S2^B congenic strains.

Chromosome 2 of Dahl salt-sensitive (SS) and congenic S2^Ba and S2^Bb rats with reference single nucleotide polymorphism (RefSNP) makers and their position in megabases (Mb) in the rat (Rattus norvegicus) genome assembly version rn6.0 are presented. SS and introgressed Brown Norway intervals are indicated in white and black, respectively. Hatched regions are in putative recombination intervals. All other chromosomes (1, 3–20, X and Y) are fixed for the SS genotype.

Figure 2.. Systolic blood pressure and Kaplan-Meier seizure-free survival curves.

A. Systolic blood pressure (BP) was measured by telemetry in Dahl salt-sensitive (SS) and congenic S2^Ba and S2^Bb rats fed a normal salt diet (NSD) or after 6 weeks high salt diet (HSD). Daytime and nighttime are highlighted by open and close horizontal bars, respectively. B. The incidence of stroke manifesting as seizures was determined in congenic and SS rats fed HSD. Data are presented as means ± SEM, n = 5–8 in A and n=30–36 in B. Data were analyzed by two-way ANOVA for repeated measures followed by a Student-Newman-Keuls post hoc test in A and by Log-Rank test followed by the Holm-Sidak test for multiple comparisons in B. *P<0.05 and **P<0.01 vs. SS.

Figure 3.. Introgression of Brown Norway chromosome 2 distal fragment was associated with lower vascular inflammation under normal salt diet and greater vascular inflammation under high salt diet.

Reactive oxygen species generation by dihydroethidium (DHE) staining (A), vascular cell adhesion molecule (VCAM)-1 (B) and monocyte chemoattractant protein (MCP)-1 expression (C) in aortic media and monocyte/macrophage (MoMΦ, D) and T cell infiltration using immunofluorescence (E) were determined in Dahl salt-sensitive (SS) and congenic S2^Ba and S2^Bb rats fed normal salt diet (NSD) and high salt diet (HSD). Data are presented as means ± SEM, n = 5–9 in A, 5–8 in B and E, 5–6 in C and D. Data were analyzed using one-way ANOVA followed by a Student-Newman-Keuls post hoc test in A-D, and Kruskal-Wallis test with Dunn’s multiple comparison post hoc test in E. *P<0.05 and **P<0.001 vs. SS, †P<0.05 vs. S2^Ba.

Figure 4.. Congenic rats present different gene expression profiles under both diets.

Venn diagrams showing the number of differentially expressed (DE) genes determined by small (microRNAs) and total RNA sequencing (messenger RNAs [mRNAs] and non-coding RNAs [ncRNAs]) in the aorta of congenic S2^Ba (A and B) and S2^Bb (A and C) vs. Dahl salt-sensitive (SS) rats under normal-salt diet (NSD) and high-salt diet (HSD). DE genes encoded in the whole genome and the introgressed BN chromosome 2 distal and middle fragments are shown in A, B and C, respectively. n = 5–6. The number of DE genes and microRNAs were identified with fold change >1.3 and false discovery rate <0.05.

Figure 5.. Validation of differentially expressed genes encoded in chromosome 2 distal fragment by reverse transcription-quantitative PCR.

Differentially expressed genes encoded within introgressed Brown Norway chromosome 2 distal portion revealed by RNA sequencing (RNA-seq) in thoracic aorta of congenic S2^Ba rats under normal salt diet (NSD) or high salt diet (HSD) were reanalyzed by reverse transcription-quantitative PCR (RT-qPCR) in Dahl salt-sensitive (SS) and congenic S2^Ba and S2^Bb fed a NSD or NSD. The mRNA expression was normalized by ribosomal protein S16 (Rps16) mRNA levels and expressed as fold change over control. Data are presented as means ± SEM, n = 6–9 for centrosome-associated protein E (Cenpe) and 5–9 for Unc-5 netrin receptor C (Unc5c) RT-qPCR and 5–6 for all the other data. RNA-seq data were analyzed using an ANOVA one-way-like test in EdgeR based on generalized linear models with threshold of fold change >1.3 and false discovery rate <0.05. All RT-qPCR data except those of Cenpe were analyzed using two-way ANOVA followed by a Student-Newman-Keuls post hoc test. Cenpe RT-qPCR data were analyzed using a Kruskal-Wallis one-way ANOVA on Ranks followed by a Dunn’s multiple comparison post hoc test. *P, q<0.05 and **P, q<0.001 vs. respective SS, †P, q<0.05 and ††P, q<0.001 vs. respective NSD and ‡P, q<0.05 and ‡‡P, q<0.001 vs. respective S2^Ba group. Aimp1, aminoacyl tRNA synthetase complex-interacting multifunctional protein 1; Bcl10, B-cell CLL/lymphoma 10; Ctbs, chitobiase; Ddah1, dimethylarginine dimethylaminohydrolase 1; Enpep, glutamyl aminopeptidase; H2afz, H2A histone family member Z; Hs2st1, heparan sulfate 2-O-sulfotransferase 1; and Pla2g12a, phospholipase A2, group XIIA.