Background Menopause is associated with an increase in the prevalence and severity of hypertension in women. Although premenopausal females are protected against T cell-dependent immune activation ...and development of angiotensin II (Ang II) hypertension, this protection is lost in postmenopausal females. Therefore, the current study hypothesized that specific CD4
T cell pathways are regulated by sex hormones and Ang II to mediate progression from premenopausal protection to postmenopausal hypertension. Methods and Results Menopause was induced in C57BL/6 mice via repeated 4-vinylcyclohexene diepoxide injections, while premenopausal females received sesame oil vehicle. A subset of premenopausal mice and all menopausal mice were infused with Ang II for 14 days (Control, Ang II, Meno/Ang II). Proteomic and phosphoproteomic profiles of CD4
T cells isolated from spleens were examined. Ang II markedly increased CD4
T cell protein abundance and phosphorylation associated with DNA and histone methylation in both premenopausal and postmenopausal females. Compared with premenopausal T cells, Ang II infusion in menopausal mice increased T cell phosphorylation of MP2K2, an upstream regulator of ERK, and was associated with upregulated phosphorylation at ERK targeted sites. Additionally, Ang II infusion in menopausal mice decreased T cell phosphorylation of TLN1, a key regulator of IL-2Rα and FOXP3 expression. Conclusions These findings identify novel, distinct T cell pathways that influence T cell-mediated inflammation during postmenopausal hypertension.
There is extensive evidence that the immune system is required for the development of angiotensin II (Ang II) induced hypertension in males. In contrast, before entering menopause, we have shown that ...females are protected from T cell‐mediated Ang II hypertension. Following menopause, female protection from T cell‐mediated Ang II hypertension is lost; in the absence of T cells, systolic blood pressure (SBP) responses to Ang II in Rag‐1−/− postmenopausal mice were similar to those seen in premenopausal mice (SBP Δ12 ± 2 mmHg). After adoptive transfer of CD3+ T cells, Ang II significantly increased SBP in postmenopausal females (SBP Δ28 ± 3 mmHg; p<0.05). Thus, we hypothesize that the loss of estrogen in postmenopausal mice eliminates female protection to T cell‐mediated hypertension. Further, we examined if premenopausal protection from T cell‐mediated hypertension was due to T cell‐specific estrogen receptor alpha (ERα) signaling. CD3+ T cells were purified from ERα null mice (ERαKO) and adoptively transferred into cycling, female Rag‐1−/− mice (no T/B cells). Ang II was infused via osmotic mini‐pump for 14 days (490 ng/kg/min). Similar to our previous studies, cycling female Rag‐1−/− mice were resistant to T cell‐mediated Ang II hypertension following T cell transfer from wild type mice (SBP CD3+WT/Ang Δ6 ± 5 mmHg). Additionally, there was no significant difference in SBP when ERαKO T cells were adoptively transferred (SBP CD3+ ERαKO/Ang Δ5 ± 4 mmHg). Flow cytometric analysis of renal CD4+, CD8+, and regulatory Foxp3+ T cells identified that in the absence of ERα, there was an increase in expression of costimulatory receptor CD28 in all three T cell subsets (CD3+ ERαKO/Ang vs. CD3+WT/Ang: CD4+ 138%, CD8+ 144%, Foxp3+ 141%; p<0.05). Our studies suggest that loss of estrogen (menopause) increases female susceptibility to T cell‐mediated hypertension. However, loss of T cell‐specific ERα signaling does not seem to play a key role in premenopausal protection from T cell‐mediated Ang II hypertension.
Support or Funding Information
1. T32HL007249
2. R01HL131834
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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 Information
R01HL131834 (HLB)Sarver Heart Center Endowment for Women's Health Research (HLB)
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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