Estrogen effects on vascular inflammation are age dependent: role of estrogen receptors

Abstract

Objective: 17β-Estradiol (E2) offers cardiovascular protection in young female animals and postmenopausal women. In contrast, randomized trials of menopausal hormones performed in older women have shown harm or no cardiovascular benefit. We hypothesize that E2 effects on vascular inflammation are age dependent.

Approach and results: Young (10 weeks) and aged (52 weeks) female C57BL/6 mice were used as source for primary cultures of bone marrow-derived macrophages (BMMs) and vascular smooth muscle cells (VSMCs). E2 pretreatment of cells derived from young mice attenuated C-reactive protein (CRP)-induced expression of inflammatory mediators. In contrast, E2 pretreatment of cells from aged mice did not alter (BMMs) or paradoxically exaggerated (VSMCs) inflammatory mediator response to CRP. Using E2 receptor (ER) knockout mice, we demonstrated that E2 regulates inflammatory response to CRP in BMMs via ERα and in VSMCs via ERβ. BMMs derived from aged (versus young) mice expressed significantly less ERα mRNA and protein. A selective ligand of the novel ER GPR30 reproduced the E2 effects in BMMs and VSMCs. Unlike in young mice, E2 did not reduce neointima formation in ligated carotid arteries of aged CRP transgenic mice.

Conclusions: E2 attenuates inflammatory response to CRP in BMMs and VSMCs derived from young but not aged mice and reduces neointima formation in injured carotid arteries of young but not aged CRP transgenic mice. ERα expression in BMMs is greatly diminished with aging. These data suggest that vasoprotective effects of E2 are age dependent and may explain the vasotoxic effects of E2 seen in clinical trials of postmenopausal women.

Keywords: aging; c-reactive protein; estrogen; vascular system injuries.

Figure 1

17β-estradiol (E2) treatment in aged C-reactive protein transgenic mice (CRPtg) enhances neointima formation. The bar graphs show the cross-sectional areas of the neointima and media (and the neointima/media cross-section ratios) of carotid arteries harvested from 50-54 wk old CRPtg mice 28 days after carotid artery ligation. Mice underwent OVX 1 wk prior to carotid artery ligation and were implanted with subcutaneous E2 or vehicle pellets prior to artery ligation. The bars and whiskers are means±SEMs. The number of mice is indicated above the bar. Representative light micrographs of injured arteries from vehicle and E2 treated mice are shown.

Figure 2

Effects of C-reactive protein (CRP, 50 μg·mL⁻¹) and 17β-estradiol (E2, 10⁻⁷ M) alone versus E2 pretreatment followed by CRP (E2+CRP) on mRNA expression of proinflammatory mediators in bone marrow macrophages (BMMs, A) and vascular smooth muscle cells (VSMCs, B) derived from young and aged female C57BL/6 mice. (C) Effects of CRP (50 μg·mL⁻¹) alone and 17β-estradiol (E2, 10⁻⁷ M) pretreatment prior to CRP administration (E2+CRP) on protein expression of proinflammatory mediators in BMMs derived from young and aged female C57BL/6 mice. Results shown are mean±SEM. *; P<0.05 vs. respective vehicle group (Veh). #; P<0.05 vs. respective CRP group.

Figure 3

Effects of C-reactive protein (CRP, 50 μg·mL⁻¹) alone and 17β-estradiol (E2, 10⁻⁷ mol) pretreatment prior to CRP administration (E2+CRP) on mRNA expression of proinflammatory mediators in bone marrow macrophages (BMMs, A) and vascular smooth muscle cells (VSMCs, B) derived from young female ERα knockout and ERß knockout mice. Results shown are mean±SEM. *; P<0.05 vs. respective vehicle group (Veh). #; P<0.05 vs. respective CRP group.

Figure 4

Effects of C-reactive protein (CRP, 50 μg·mL⁻¹) alone and pretreatment with 17β-estradiol (E2, 10⁻⁷ M), nonselective ER antagonist ICI (10⁻⁶ M) in addition to E2 (A), selective ERα agonist PPT (10⁻⁷ M), selective ERß agonist DPN (10⁻⁷ M), and selective ERα antagonist MPP (10⁻⁵ M) in addition to E2 (B) prior to CRP administration on mRNA expression of proinflammatory mediators in bone marrow macrophages (BMMs) derived from young female C57BL/6 mice. Effects of CRP alone and pretreatment with E2 or selective G-protein coupled estrogen receptor agonist (G1, 10⁻⁷ M) prior to CRP administration on mRNA expression of proinflammatory mediators in BMMs (C) and vascular smooth muscle cells (VSMCs, D) derived from young and BMMs (E) derived from aged female C57BL/6 mice. Results shown are mean±SEM. *; P<0.05 vs. respective vehicle group (Veh). #; P<0.05 vs. respective CRP group.

Figure 5

mRNA expression (left) and protein expression (middle) of ERα in bone marrow macrophages (BMMs, A) and vascular smooth muscle cells (VSMCs, B) derived from young and aged female C57BL/6 mice. mRNA expression of ERβ in cells derived from young and aged female C57BL/6 mice is shown (right). Results shown are mean±SEM. *; P<0.05 vs. young mice.