Targeting LOXL2 against arterial stiffening in post-menopausal females

Introduction: Cardiovascular disease (CVD) is the number one cause of death for both men and women. Even though pre-menopausal women are relatively protected from CVD, a disproportionate increase in CVD risk and incidence occurs after menopause. Aortic stiffening, as occurs in aging, is predictive of major adverse cardiovascular events. The hormonal shift, particularly loss of estrogen, present in post-menopausal women has also been widely studied; however, it is increasingly clear that estrogen loss alone is not responsible for the elevated CVD risk. Our goal is to identify and develop novel therapeutic targets against this vascular stiffening. Here we propose LOXL2, a key matrix remodeling enzyme, as a potential therapeutic target. In this study, we examined if the depletion of LOXL2 improves or even reverses vascular stiffening in post-menopausal females. We also studied the effect of sex hormones in LOXL2 activation. Methods and Results: Female C57Bl/6J mice and Loxl2 +/- mice were used. Female mice underwent ovariectomy (OVX) or sham surgery at 10 weeks old. Body weight, pulse wave velocity (PWV, the gold-standard index of in vivo arterial stiffening), and blood pressure (BP) were measured noninvasively from 3 to 18 months old. Loxl2 +/- OVX mice presented significantly improved arterial stiffening compared to WT OVX at all ages. Loxl2 +/- sham showed improved PWV at 18 months old. BP increased with age and was similar for all groups. Body weight gain was decreased in Loxl2 +/- OVX females compared to OVX WT. Mice were dissected at 4 and 18 months old. Intact (ECM+cells) and decellularized (ECM only) aortic rings were subjected to uniaxial tensile testing. At 4 months old, sham and OVX females’ results were similar for both genotypes. At 18 months old, Loxl2 deletion reduced stiffening in intact rings of OVX and sham females. Decellularized aortic rings presented similar results for all groups. Wire myography showed that contractile response to phenylephrine was improved in Loxl2 +/- OVX. The impaired endothelial-dependent relaxation present at 18 months old sham was amended in Loxl2 +/- females. Western blotting on aortic tissue revealed an increasing trend in LOXL2 processing in OVX WT females compared to sham WT mice at 4 months old. Next, we studied the modulation of LOXL2 via sex hormones using human aortic smooth muscle cells. Dihydrotestosterone (DHT) and estradiol (E2) were modulated to mimic the hormonal levels of young and post-menopausal women. After 16h incubation, culture media and cells were collected. qPCR and western blotting both showed increased LOXL2 mRNA and protein when mimicking post-menopausal hormonal levels compared to control, menstruating women, and E2 levels only. Conclusion: Loxl2 depletion significantly improved vascular stiffness and body weight gain in OVX females. This is supported by the reduced stiffening showed in intact aortic rings via tensile testing, as well as improved contractility via wire myography. LOXL2 processing is increased in WT OVX compared to WT sham. LOXL2 is modulated by sex hormones in vascular smooth muscle cells, with significant increase when mimicking post-menopausal women’ hormonal levels. LOXL2 is a potential feasible target against arterial aging after menopause. Future studies will continue to: 1) determine the molecular mechanisms underlaying this improvement of vascular stiffening when targeting LOXL2, 2) continue to investigate LOXL2 activation via sex hormones, and 3) study the role of hormonal shift to androgen signaling in old females in arterial aging. This work was supported by NIH R01HL148112, an award from the American Heart Association (AHA 24PRE1196370), the DC Women's Board, and Dr. Santhanam lab members This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.