Differential Responses in the Splenic CD4⁺ T Cell Proteome Following Ang II‐Induced Hypertension in VCD‐Treated Menopausal Mice

Abstract

Cycling female mice are protected against Angiotensin II (Ang II) hypertension, and inducing ovarian failure (menopause) eliminates this protection. T lymphocytes are required for the development of Ang II hypertension in male mice, however premenopausal females are resistant to T cell‐induced hypertension. Following menopause (loss of estrogen) resistance is lost, allowing activation of T cell‐induced hypertension in postmenopausal females. The purpose of this study was to identify, in vivo, T cell‐specific proteome responses to Ang II, before and after menopause. 10‐week‐old C57BL/6 female mice received i.p. 4‐vinylcyclohexene diepoxide (VCD) injections for 20 consecutive days to induce ovarian failure (VCD menopause model). Cyclicity was monitored daily via vaginal cytology. Once in menopause, Ang II was infused (800 ng/kg/min) for 14 days, which has been shown to cause an increase in SBP of 25 mmHg. Ang II in VCD‐treated menopausal females (VCD/AngII) resulted in significantly decreased heart rates versus controls (Control 682 ± 5.6 vs. VCD/AngII 592.5 ± 28.5, p < 0.05). Splenic CD4+ T cells were purified via negative immune‐magnetic selection and CD4+ purity was measured via flow cytometry (>90% purity). Proteomic analysis was performed on control, pre‐menopausal/Ang II and VCD/AngII mice (n=4 per group). Protein lysates from the CD4+ T cells were separated by SDS‐PAGE and subjected to in‐gel tryptic digestion followed by tandem mass spectrometry analysis, resulting in 7,123 proteins identified across the entire experiment. Quantitative proteomics were performed via label‐free quantification using Progenesis software‐based extracted ion abundance. Of the 5,857 proteins identified with two or more unique peptides, 474 of the proteins exhibited significant abundance differences between control, Ang II and VCD/Ang II groups as assessed by One‐Way ANOVA statistical analysis (p ≤ 0.05). Gene Ontology (GO) enrichment analysis of these 474 proteins identified 159 GO biological pathways that were significantly overrepresented (p ≤ 0.05). Pathways associated with increased inflammation and reduced inhibition of inflammatory cytokines were among those identified. Using IHC we show that renal macrophage infiltration is significantly increased in VCD/Ang II mice compared to Ang II and control groups. Overall, our data suggests that following Ang II infusion, splenic T cells are differently impacted by the loss of estrogen, leading to an increased pro‐hypertensive cytokine milieu compared to pre‐menopausal Ang II infused mice.Support or Funding InformationR01HL131834 (HLB)Sarver Heart Center Endowment for Women's Health Research (HLB)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.