Abstract:

Angiotensin II (Ang II) is an octapeptide that regulates water and salt reabsorption by the kidneys

and the constriction of arterioles, causing an increase in blood pressure. The effect of AngII is mediated

through angiotensin II receptors (ATR1and ATR2), which are classes of G protein-coupled receptors that

are responsible for regulating various signal transduction pathways. Previous studies have shown that

Ang II can bind to ATR1 receptor in T-cells and increase T-cell activation. The mechanism of activation

in T-cell is not well characterized. In this study, the effect of Ang II on CD4+ T cells was investigated

using RNA Sequencing (RNA seq) and Western Blot Analysis.

A publicly available Gene Expression Omnibus (GSM number 4816125) dataset was obtained

from the National Center for Biotechnology Information (NCBI). Analysis of genes affected in T cells

from mice treated with Ang II showed that 176 were downregulated and 192 were upregulated genes (p-

value <0.05). Using the Database for Annotation, Visualization and Integrated Discovery (DAVID),

several signaling pathways were found to be affected such as the cytokine, NF-κB, and IL-17 signaling

pathway. In addition, a possible dephosphorylation of Protein Kinase B (AKT) by the upregulation of

Protein Phosphatase 2A (PP2A) was identified. To determine if there was a dephosphorylation of AKT, a

western blot analysis was performed. Analysis revealed that the level of phosphorylation of AKT slightly

decreased with AngII treatment, supporting the RNA Seq data. This study shows that AngII can affect

multiple signaling pathways in CD4+ T, including AKT signaling.

Characterization of Normalized Data. (A) Whisker plot of normalized data, (B) density plot of distribution, (C) Multi-Dimensional Scaling (MDS), and (D) Principal Component Analysis (PCA).

Heatmap of Differentially Expressed Genes between Ang II Treated and Control Groups. Heatmaps were generated with IDEP v0.94 to show the differentially expressed genes.

GO analysis of Differentially Expressed Genes. (A) Cellular components and (B) biological processes were analyzed.

Cytokines Affected in CD4+ T Cells by Ang II. (A) Following the Ang II treatment, TNF α was downregulated. (B) Additionally, the Ang II treatment resulted in IL-17 being upregulated.

Effect of Ang II on MCHI and Antigen Processing and Presentation. (A) the antigen processing and presentation genes of the CD4 T cell where, notably, MCHI is seen as downregulated. (B) The relative expression of MCHI is shown with control compared to Ang II, with MCHI being downregulated.

Effect of Ang II on NF-κB and T Cell Receptor Signaling Pathway. (A) The T Cell receptor signaling pathway is shown where AKT is affected by Ang II. (B)The relative expression of both IKKα and IκB is also shown. Here, IKKα is upregulated and IκB is downregulated.

PI3K AKT Signaling Pathway and PP2A. (A) The PI3K AKT signaling pathway is shown. (B) In this pathway, Protein Phosphatase 2A (PP2A) is present and shown as upregulated.

pAKT and AKT Western Blot Analysis. (A) The top band showcases the phosphorylated AKT (pAKT) western blot results while the bottom band showcases the AKT western blot results. (B) This graph shows the quantified results of the AKT western blots.