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 that drive vascular aging remain poorly understood. We identify the (Na+)-sodium/Ca2+-calcium (K+)-potassium-dependent exchanger 4 (NCKX4) as a crucial functional regulator of VSMCs Ca2+ clearance and vascular integrity. NCKX4 (encoded by Slc24A4) expression was significantly reduced in aortas from aged (72-78 weeks) mice, with a pronounced decline in females. Aged-related loss of NCKX4 impaired Ca2+ clearance function and increased calcium-phosphate mineralization. Young (12-15 weeks) 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 showed that loss of NCKX4 alters pathways linked to Ca2+-integrin signaling, ECM turnover and mineralization, including dysregulation of anchorage integrins, microfibril-stabilizing, osteogenic drivers and pro-fibrotic integrins. These findings reveals that an impaired Ca2+ clearance promotes maladaptive inside-out integrin signaling, disrupting VSMCs anchorage, ECM homeostasis and mineralization. Our results establish NCKX4 as a previously unrecognized determinant of vascular aging, whose decline accelerates premature arterial remodeling and calcification. This study therefore, 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. Overall design: RNA-seq profiling of aortic tissue from young (Y, 12-15 weeks) and aged (A, 72-78 weeks) female wild-type (WT) and their knockdown Slc24a4 (Nckx4-/-) mice.