Age, sex, hormonal status, and anti-inflammatory therapy for atherosclerosis

PROJECT SUMMARY.
A great accomplishment of modern medicine is the increased lifespan of the population worldwide. Unfortunately,
longevity also increased the healthcare burden due to comorbidities of aging such as atherosclerotic cardio-
vascular disease (CVD). Overwhelming evidence indicates that hyperlipidemia is a main driver of CVD, which
promotes an inflammatory response by vasculature-resident macrophages. Other inflammatory cells, chiefly
among them T-cells, are also recruited to growing plaques and play a fundamental role in their remodeling and
susceptibility to rupture. In women, it is well recognized that loss of ovarian hormones at menopause is an
independent risk factor for cardiovascular events. Current therapies, such as statins, lower circulating LDL-
cholesterol and have been clinically demonstrated to decrease mortality in the elderly. Yet, despite the use of
statins for over five decades, atherosclerosis still remains the leading cause of death in men and women globally.
These observations indicate that 1) there are additional, yet unidentified, lipid-independent mechanisms that
contribute to atheroprogression and plaque rupture; and 2) additional therapies are needed to treat ASCVD.
Unpublished preliminary data from our laboratories in mice show that aging in both sexes and loss of estrogen
in females promotes T-cell mediated inflammation. We hypothesize that this age- and menopause-dependent
inflammation exacerbates underlying CVD such that further lowering cholesterol does not provide added benefit.
Here we will test the idea that promoting an anti-inflammatory, pro-resolving environment in the plaque is
atheroprotective. To accomplish this, we will use treat mice with pulsed low-dose RANKL (pRL), which we show
induces plaque resident regulatory CD8+FoxP3+CD25+CTLA4+ T-cells that secrete IL-10. If successful, pRL will
be a novel therapeutic for treating ASCVD, that works by mechanism distinct from therapies currently in use.
Additionally, the proposed studies will deliver two key innovations. First, we will compare atheromata in young
(2-month-old) and old (18-month-old at beginning of intervention, corresponding to ~60-year-old human) male
and female mice. Second, old females will be ovariectomized to mimic human menopause. Thus, in females we
will segregate the effects of aging and menopause on plaque development. We will comprehensively map
athero-prone proximal aorta transcriptomic profiles at a single-cell resolution, focusing on inflammatory cell
clusters, cell-to-cell communication networks, T-cell-mediated dysfunction of smooth muscle cells and
endothelial cells, markers linked to plaque stability/vulnerability, and transcripts encoding predicted secreted
proteins (secretome). The integrated mechanistic data obtained with our unprecedented discovery approach will
have a lasting impact in the field of vascular physiology and pathology because most published murine
atherosclerosis studies have systematically ignored the effects of age, sex, and hormonal status, despite most
CVD patients being older (and post-menopausal). Overall, these outcomes will be the foundation for future
mechanistic studies and may identify novel plaque-secreted biomarkers to assess disease severity.

Grant Number: 1R21AG089957-01
NIH Institute/Center: NIH
Principal Investigator: Angel Baldan