Aging-related iron deposit prevents the benefits of HRT from late postmenopausal atherosclerosis

Reviewer #1 (Public Review):

In this study, the authors seek to determine the potential role of aging-induced iron accumulation on the effects of hormone replacement therapy (HRT) on atherosclerosis in late postmenopausal women. The authors are commended for the novel and relevant line of investigation and the many complex experiments that they performed. Central to the manuscript, the authors find that high iron levels, in late postmenopausal women as well as in ApoE-/- mice, are associated with reduced expression of the estrogen receptor (ERα). Also, estradiol (E2) treatment in ApoE-/- mice further downregulated ERα expression, but the authors have not sufficiently demonstrated that this occurs in an iron-dependent manner, as the authors have concluded in section 3.3.

The data showed that high iron and E2 treatment trigger the ubiquitin proteasome degradation pathway to degrade ERα via Mdm2-mediated degradation. Interestingly, iron chelation therapy restored ERα expression and attenuated E2-triggered atherosclerosis in late postmenopausal mice. Overall, the authors have concluded that in late postmenopause, iron accumulation prevents the HRT benefit through negative regulation of ERα expression via Mdm2-mediated proteolysis. However, important control groups in the in vivo experiments need to be included to support the conclusions made by the authors and variability in the in vitro experiments diminishes enthusiasm for the findings. Furthermore, in the in vitro experiments, the predominant reduction in ERalpha expression appears to be driven by iron even in the absence of E2, thus making it uncertain how specific these findings are to menopause.

Reviewer #2 (Public Review):

It is believed that the reason why women generally have lower rates of atherosclerotic events than men until menopause is due to the beneficial effects of estrogen on the cardiovascular system. The paper attempts to explain why hormone replacement therapy with estrogen is not effective in preventing atherosclerosis in post-menopausal women. The authors posit that accumulation of iron after menopause inhibits estrogen receptor expression and makes estrogen ineffective. Using mouse model of atherosclerosis and iron overload, they demonstrate that 1)atherosclerosis is increased in overectomized mice 2) estrogen supplement seems to further exacerbate atherosclerosis and this is accompanied by increased total body iron; 3) iron itself causes a decrease in ERa via increased proteasome degradation of Era via E3 ligase MDM2 and 4) iron chelation rescues the protective effects of estrogen in overectomized mice on atherosclerosis progression.

While interesting in terms of hypothesis, I found the manuscript (not the overall themes) but the individual experimental logic difficult to follow with unclear rationale for many of the experiments and timepoints chosen. Moreover the human data supporting these claims are weak in terms of what is shown. The authors only partially achieve their aims as many of the experiments in mice appear incomplete in terms of data shown and transparency. Some important controls are also missing.

This work has important potential to understand the causes of accelerated atherosclerosis in women after menopause and how to better prevent atherosclerosis in women of this age group

Reviewer #3 (Public Review):

This manuscript aims to address whether age-related iron status influences differential effects of estrogen replacement on atherogenesis in postmenopausal females. Specifically, whether age-related iron accumulation reduces estrogen signaling through ERa receptor in relevant cell types (endothelial cells and macrophages). They test this fairly rigorously using in vitro, preclinical and clinical data, and the data is presented logically.

First, the authors demonstrate that in postmenopausal women, there is an inverse correlation between age-related increase in iron levels and age-related decrease in ERa levels in atherosclerotic plaques. This is consistent with the fact that, in ovariectomized (OVX) pro-atherogenic (ApoE KO) mice, there is differential effect of estrogen (E2) on ERa, atherosclerosis, lipid profiles and key biomarkers (ABCA1, eNOS, etc) based on age (early: 16 weeks, versus late: 40 weeks), with young mice responding favorably while older mice responding negatively. Consistent with this, in the older OVX ApoE KO mice, E2 treatment worsened atherosclerosis. Importantly, they point this differential effect of E2 to iron overload in macrophages using ApoE KO; LysM-macrophage-specific Fpn1 KO as E2 now has deleterious effects regardless of age. Next, using ferric ammonium citrate (FAC) to supplement iron and deferiprone (DFP) to chelate iron, they show that iron manipulation impacts the E2 response appropriately in the relevant cell types (endothelial cells and macrophage cells). Then they provide evidence for the MdM2-mediated post-translational regulation of ERa as a mechanism by which iron status impacts differential E2 has differential effects on ERa. Finally, they test the impact of systemic iron chelation on the OVX ApoE KO mice model of atherosclerosis and show that iron chelation attenuates the deleterious effects of E2 in late postmenopausal mice.

Overall the evidence is solid and logically laid out. Given that serum iron levels do not correlate with the rest of the story, inclusion of LysM-macrophage-specific Fpn1 KO provides the key evidence that iron loading in macrophages drives differential effects to E2 in postmenopausal mice. The paper provides evidence that iron levels influence how the relevant cells respond to E2 with clinical implications to hormonal replacement therapy in younger and older postmenopausal women. While this study is limited by a small clinical sample size, it provides an important framework for future studies on the impact of age-related iron status on the response to hormonal replacement therapy.