Age-related decline in NCKX4-mediated calcium clearance accelerates aortic remodelling and drives early vascular ageing

Ageing is the primary non-modifiable 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 vascular calcification. However, the molecular and functional determinants of Ca²⁺ clearance that drive vascular ageing remain poorly understood. We identify the (Na⁺)-sodium/Ca²⁺-calcium (K⁺)-potassium-dependent exchanger 4 (NCKX4) as a critical functional regulator of VSMC Ca²⁺ clearance and vascular integrity. Expression of NCKX4 (encoded by Slc24A4) was significantly reduced in aortae from aged (72-78 weeks) mice. Aged-related loss of NCKX4 impaired Ca²⁺ clearance function and increased Ca²⁺-phosphate mineralization. Notably, young (12-15 weeks) Nckx4-/- mice exhibited elastic fibre fragmentation, collagen accumulation, arterial wall thickening and extracellular matrix (ECM) remodelling, which are hallmarks of vascular ageing that closely resembled those observed in aged wild-type mice. Transcriptomic profiling revealed that loss of NCKX4 alters pathways associated with Ca²⁺-integrin signalling, ECM turnover and mineralization, including dysregulation of anchorage integrins, microfibril-stabilizing components, osteogenic drivers and profibrotic integrins. Collectively, these findings demonstrate that impaired Ca²⁺ clearance promotes maladaptive inside-out integrin signalling, disrupting VSMC anchorage, ECM homeostasis and mineralization. Our results establish NCKX4 as a previously unrecognized determinant of vascular ageing, and its decline accelerates premature arterial remodelling and calcification. This study positions NCKX4 as a mechanistic link between age-dependent vascular vulnerability and arterial stiffening, with implications for novel therapeutic strategies targeting Ca²⁺ handling to prevent CVDs. KEY POINTS: Dysfunctional calcium (Ca²⁺) handling in vascular smooth muscle cells (VSMCs) increases susceptibility to cardiovascular diseases (CVDs), vascular calcification, stiffness and aortic remodelling. The K⁺-dependent Na⁺/Ca²⁺ exchanger NCKX4 mediates high-capacity Ca²⁺ clearance extrusion, thereby maintaining VSMC Ca² ⁺ homeostasis. NCKX4 (encoded by Slc24a4) expression markedly declines in the aorta and VSMCs of aged (72-78 weeks) mice. Genetic deletion or age-related loss of NCKX4 impairs Ca²⁺ clearance, leading to enhanced VSMCs calcification, premature arterial remodelling through disruption of Ca²⁺-mediated integrin-ECM signalling. NCKX4 is a newly identified mechanistic driver of vascular ageing and a potential therapeutic target for early detection, preservation of vascular integrity and mitigation of age-related CVDs risk.