Tie2-Cre-Induced Inactivation of Non-Nuclear Estrogen Receptor-α Signaling Abrogates Estrogen Protection Against Vascular Injury

Abstract

Using the Cre-loxP system, we generated the first mouse model in which estrogen receptor-α non-nuclear signaling was inactivated in endothelial cells. Estrogen protection against mechanical vascular injury was impaired in this model. This result indicates the pivotal role of endothelial estrogen receptor-α non-nuclear signaling in the vasculoprotective effects of estrogen.

Keywords: E2, 17β-estradiol; ECGM, endothelial cell growth medium; ER, estrogen receptor; ERαKI/KI, estrogen receptor-αknock-in/knock-in; LVEDD, left ventricular end-diastolic diameter; NOS, nitric oxide synthase; PI3K, phosphatidylinositol 3-kinase; PLA, proximity ligation assay; Vo2, oxygen consumption; cDNA, complementary deoxyribonucleic acid; eNOS, endothelial nitric oxide synthase; endothelial cells; estrogen receptor-α; non-nuclear signaling; tissue-specific regulation.

Graphical abstract

Figure 1

Mouse Model With Tie2-Cre-Induced Inactivation of ERα Non-Nuclear Signaling (A) A schematic diagram of gene targeting strategy. A single alteration was introduced into the mouse estrogen receptor-α (ERα) exon 4 by replacing Arg263 with alanine. (B) Wild-type ERα and mutated ERα expressions in isolated estrogen receptor-α^{knock-in/knock-in} (ERα^KI/KI)Tie2^Cre endothelial cells with 17β-estradiol (E2) stimulation. (C) Nitric oxide synthase (NOS) activities in isolated ERα^KI/KITie2^Cre endothelial cells with E2 stimulation. (D) Hematoxylin-eosin (H&E) staining, proximity ligation assay (PLA) of p85α and ERα, and CD31 immunohistochemical staining in the carotid arteries treated with E2. Sections were counterstained with hematoxylin. Positive PLA signals are revealed by red dots; red arrow: positive PLA signals on endothelial cell area, black arrow: positive PLA signals on nonendothelial cell area. Scale bars = 20 μm. (E) Quantification results for PLA-positive cells. Data in B, C, and E were analyzed by the unpaired Student's t-test. ∗P < 0.05. Data are presented as mean ± SEM.

Figure 2

ERα Genomic Signaling Is Preserved in ERα^KI/KITie2^Cre+ Mice (A) Representative uterine morphologies of ERα^KI/KITie2^Cre mice after ovariectomy (OVX) and OVX+E2 supplementation. (B) ERα^KI/KITie2^Cre mice uterine weights (ERα^KI/KITie2^Cre−, n = 7-8; ERα^KI/KITie2^Cre+, n = 8). (C) Fertility analysis of ERα^KI/KITie2^Cre female mice. Litter sizes are shown as the number of pups per litter. (D) Endothelial estrogen response element–containing gene expressions by E2 stimulation. Data in B and D were analyzed by 2-way analysis of variance for “genotype × estrogen status” interaction. Data in C were analyzed by the unpaired Student's t-test. ∗P < 0.05. Data are presented as mean ± SEM. Abbreviations as in Figure 1.

Figure 3

Baseline Metabolic Characteristics and Serum E2 Concentration of ERα^KI/KITie2^Cre Mice (A) Oxygen consumption (VO2) normalized to body weight per hour for 24 hours. The graph demonstrates average VO2 during the light (day) and dark (night) phases. (B) Locomotor activity normalized to body weight per hour for 24 hours. The graph depicts average locomotor activity during the light (day) and dark (night) phases. The unpaired Student's t-test was performed separately in each phase. (C) Serum E2 concentrations in nonovariectomy ERα^KI/KITie2^Cre mice (ERα^KI/KITie2^Cre−, n = 5; ERα^KI/KITie2^Cre+, n = 9). Data are presented as mean ± SEM. Abbreviations as in Figure 1.

Figure 4

Assessments of Intimal Hyperplasia, Medial Thickening, and Fibrosis After Wire Injury (A) The protocol of the wire injury model. (B) Representative Elastica van Gieson (EVG) staining and (C) Masson’s trichrome (MT) staining images of carotid arteries 14 days after wire injury. Quantification results of (D) the intima-to-media ratio, (E) medial area (×10³ μm²) (n = 4-8 for each group), and (F) fibrosis (area stained with blue green) in the medial layer (n = 5-13 in each group). P_trend derived from comparison of the trends using 2-way analysis of variance for “genotype × estrogen status” interaction. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 in comparison between ERα^KI/KITie2^Cre− and ERα^KI/KITie2^Cre+ mice by Bonferroni’s post hoc correction for multiple comparisons. †P < 0.05 in comparison between E2+ and E2-; scale bar of upper panel = 64 μm, lower panel = 8 μm. Data are presented as mean ± SEM. E2+/- indicates with/without E2 pellet implantation. Abbreviations as in Figure 1.

Figure 5

Assessments of Re-Endothelialization in the Carotid Arteries 7 Days After Wire Injury (A) Representative images of Evans blue staining of carotid arteries. (B) Quantification results for the re-endothelialization area (%) determined by Evans blue staining (deendothelialized areas are stained with blue) (n = 4-6 in each group). (C) Representative images of CD31 immunohistochemical staining. (D) Quantification results for the endothelial coverage (% of luminal lining) (n = 5 in each group). P_trend derived from comparison of the trends using 2-way analysis of variance for “genotype × estrogen status” interaction. ∗P < 0.05 in comparison between ERα^KI/KITie2^Cre− and ERα^KI/KITie2^Cre+ mice by Bonferroni’s post hoc correction for multiple comparisons. †P < 0.05 in comparison between E2+ and E2-; scale bar of upper panel = 1 mm, lower panel = 32 μm and 64 μm. Data are presented as mean ± SEM. E2+/- indicates with/without E2 pellet implantation. Abbreviations as in Figure 1.

Figure 6

Assessments of Inflammation in the Carotid Arteries 7 Days After Wire injury (A) Representative images of CD45 immunohistochemical staining. (B) Quantification results for the ratio of CD45 positive area/adventitial area (%) (n = 4-6 in each group); P_trend derived from comparison of the trends using 2-way analysis of variance for “genotype × estrogen status” interaction. ∗P < 0.05 in comparison between ERα^KI/KITie2^Cre− and ERα^KI/KITie2^Cre+ mice by Bonferroni’s post hoc correction for multiple comparisons. †P < 0.05 in comparison between E2+ and E2-; scale bars = 100 μm and 200 μm. E2+/- indicates with/without E2 pellet implantation. Data are presented as mean ± SEM. Abbreviations as in Figure 1.