Sex Differences in Lymphatic Response to Estrogen Shape Atherosclerosis in High-Risk Mice

Mona Mesples^1,2Élizabeth Lacroix^1,2Nolwenn Tessier^1,2Maya Farhat^1,2Andreea Milasan^1,2Sara Babran^1,2Cristina Fernandez¹Tally Latendresse¹Justin Benoit¹Valérie Long^1,3Julie Guillemette¹Sami El Khakani¹Azadeh Alikashani¹Charles-Alexandre Leblanc¹Marie-Ève Higgins¹Vanessa Durocher-Granger¹Céline Fiset^1,3Catherine Martel^1,2*

• ¹Institut de Cardiologie de Montréal, Université de Montréal, Montréal, Canada
• ²Department of Medecine, Faculty of Medecine, Universite de Montreal, Montreal, Canada
• ³Faculty of Pharmacy, Universite de Montreal, Montreal, Canada

Atherosclerosis, the cholesterol-driven inflammatory process underlying cardiovascular disease (CVD), remains the leading cause of death in high-income countries despite major advances in risk factor management. This underscores the urgent need for therapies that directly target plaque development and progression. Recent evidence has uncovered an important role for lymphatic vessels in cardiovascular health: by facilitating reverse cholesterol transport, lymphatics help clear excess cholesterol from arterial walls and influence atherosclerosis from its earliest stages to advanced disease. Enhancing lymphatic pumping before atherogenesis limits plaque formation, while restoring lymphatic function in established atherosclerosis reduces lesion size and promotes stabilization. CVD risk rises sharply after menopause, and lymphedema studies suggest that women experience a more pronounced age-related decline in lymphatic pumping than men, pointing to a potential link with hormonal fluctuations. Hormonal changes throughout life—whether due to aging, therapeutic interventions, or personal choice—are key determinants of CVD vulnerability. Yet, how these changes affect lymphatic transport in individuals predisposed to CVD remains unexplored. In this study, age-matched Ldlr⁻/⁻ males and ovariectomized females— in which estrogen levels were reduced to mimic the decline observed during menopause— were treated with 17β-estradiol (E2) to assess the impact of hormone therapy on in vivo lymphatic function and atherosclerosis. In males, E2 reduced lesion burden and improved lymphatic transport without increasing the expression of key lymphatic endothelial and muscle cell genes involved in vessel integrity and function. In females, estrogen receptor α—but not estrogen receptor β—was critical for lymphatic vessel function, and its downregulation reduced Flt4 mRNA abundance, a gene essential for lymphatic growth and pumping. E2 impaired lymphatic function in ovariectomized females; however, enhancing lymphatic transport beforehand prevented this effect and reduced atherosclerotic plaque formation. Our findings reveal that estrogens modulate lymphatic function and atherosclerosis differently according to sex and baseline hormonal status. These results suggest that lymphatic function may contribute to the interplay between hormonal changes and cardiovascular risk, supporting the development of more targeted therapeutic strategies for populations undergoing hormonal transitions.