Age-Related Decline in NCKX4-Mediated Calcium Clearance Accelerates Aortic Remodeling and Drives Early Vascular Aging

Aging is the primary nonmodifiable risk factor for cardiovascular diseases (CVDs), with older women facing a greater risk of CVDs than age-matched men. Vascular smooth muscle cells (VSMCs) dysfunction and impaired calcium (Ca2+) handling are recognized as central contributors to arterial stiffening and calcification. However, the molecular and functional determinants of Ca2+ clearance in vascular aging remains a topic of ongoing research. We identify the (Na+)-sodium/Ca2+-calcium (K+)-potassium-dependent exchanger 4 (NCKX4) as a critical functional regulator of VSMCs Ca2+ clearance and vascular integrity. We demonstrate that NCKX4 (coded by Slc24A4) expression is markedly reduced in aorta of aged (72-78 weeks) mice, with a pronounced decline in females. Functional assays revealed impaired Ca2+ clearance in both aged and Nckx4-/- VSMCs, which was accompanied by increased calcification. Histomorphometric analyses of young Nckx4-/- mice revealed fragmentation of elastic fibers, collagen accumulation, wall thickening, and extracellular matrix (ECM) remodeling, all hallmarks of vascular aging that closely resembled those of aged wild-type mice. Transcriptomic profiling of VSMCs showed that loss of NCKX4 alters pathways linked to Ca2+-integrin signaling, ECM turnover, and mineralization, including dysregulation of protective anchorage integrins, microfibril-stabilizing, osteogenic drivers and pro-fibrotic integrins. These findings support a model in which impaired Ca2+ clearance promotes maladaptive inside-out integrin signaling, disrupting VSMCs anchorage, ECM homeostasis, and mineralization processes. Collectively, our results establish NCKX4 as a previously unrecognized determinant of vascular aging, whose decline accelerates premature arterial remodeling and calcification. This study positions NCKX4 as a potential mechanistic link between age, sex-dependent vulnerability, and vascular stiffening, with implications for novel therapeutic strategies targeting Ca2+ handling in CVDs prevention.