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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE316510 | GEO | 2026/01/21

REPOSITORIES: GEO

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Project description:Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular ageing. Sox9 (SRY-Box Transcription Factor 9) is a master regulator of chondrogenesis, also expressed in the vasculature, that has been implicated in vascular smooth muscle cell (VSMC) osteo-chondrogenic conversion. Here, we investigated the relationship between vascular ageing, calcification and Sox9-driven ECM regulation in VSMCs. Immunohistochemistry in human aortic samples showed that Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16. Analysis of Sox9 expression in vitro showed it was mechanosensitive with increased expression and nuclear translocation in senescent cells and on stiff matrices. Manipulation of Sox9 via overexpression and depletion, combined with atomic force microscopy (AFM) and proteomics, revealed that Sox9 regulates ECM stiffness and organisation by orchestrating changes in collagen expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs whereby senescent cells plated onto ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (LH3) was identified as a Sox9 target, and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles (EVs) and Sox9 promoted EV secretion, leading to increased LH3 deposition within the ECM. These findings identify cellular senescence and Sox9 as a key regulators of ECM stiffness during VSMC ageing and highlight a crucial role for ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle whereby cellular senescence and increased ECM stiffening promote Sox9 expression which drives further ECM modifications that act to accelerate vascular stiffening and cellular senescence.

Project description:Background: Ectopic vascular calcifications represent a major clinical problem associated with cardiovascular disease and mortality. However, the mechanisms underlying pathological vascular calcifications are largely unknown hampering the development of therapies to tackle this life threatening medical condition. Results: In order to gain insight into the genes and mechanisms driving this pathological calcification process we analyzed the transcriptional profile of calcifying vascular smooth muscle cells (C-VSMCs). These profiles were compared to differentiating osteoblasts, cells that constitute their physiological calcification counterparts in the body. Overall the transcriptional program of C-VSMC and osteoblasts did not overlap. Several genes, some of them relevant for bone formation, were distinctly modulated by C-VSMCs which did not necessarily lose their smooth muscle cell markers while calcifying. Bioinformatics gene clustering and correlation analysis disclosed limited bone-related mechanisms being shared by two cell types. Extracellular matrix (ECM) and biomineralization genes represented common denominators between pathological vascular and physiological bone calcifications. These genes constitute the strongest link between these cells and represent potential drivers for their shared end-point phenotype. Conclusions: the analyses support the hypothesis that VSMC trans-differentiate into C-VSMCs keeping their own identity while using mechanisms that osteoblasts use to mineralize. The data provide novel insights into groups of genes and biological processes shared in MSC and VSMC osteogenic differentiation. The distinct gene regulation between C-VSMC and osteoblasts might hold clues to find cell-specific pathway modulations, opening the possibility to tackle undesired vascular calcifications without disturbing physiologic bone formation and vice versa. Total RNA obtained from hMSC and hVSMC cultured in osteogenic differentiation medium supplemented with 1.8 mM Ca2+ for 0, 2, 8, 12 or 25 days respectively. For each timepoint 3 replicates were used, with exception for day 0 where 4 replicates were collected.

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

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