Molecular mechanisms of vascular senescence and calcification

Vascular aging increases the cardiovascular disease risk, including vascular calcification (VC). A key event in VC is the vascular smooth muscle cells (VSMCs) trans-differentiation into an osteogenic phenotype, which is facilitated by senescence. High Mobility Group Box-1 (HMGB1) plays a role in senescence and the senescence-associated secretory phenotype (SASP), but its involvement in vascular aging and calcification remains unclear. Preliminary results from our lab showed that aortas from old mice had significantly lower HMGB1 protein levels compared with young animals. Senescent human aortic smooth muscle cells (HASMCs) displayed reduced HMGB1 expression and no protein translocation. HASMCs silenced for HMGB1 (shB1/HASMCs) had larger nuclei and lower proliferation compared to control cells (shCTR/HASMCs). Herein, we found that shB1/HASMCs had elevated p21 levels and senescence-associated β-galactosidase activity, and showed an anti-inflammatory SASP profile, with reduced IL-6, IL-1β, IL-8, and MCP-1 compared with shCTR/HASMCs. HMGB1 silencing also modulated the release of extracellular matrix (ECM) proteins involved in tissue mineralization and increased the expression of osteoblastic inducers. These observations were confirmed comparing aortas from Hmgb1+/+/Hmgb1+/- mice. Furthermore, shB1/HASMCs grown in hyperphosphatemic conditions exhibited enhanced calcification and lower inflammation than control cells and, transcriptomics revealed changes in biomineralization, cell fate commitment, immune response, and tissue migration pathways. Aortas from Hmgb1+/- mice treated with Vitamin D showed increased calcification and diminished IL-6 compared with Hmgb1+/+ mice. Finally, HMGB1 content was found lower in aortas isolated from older healthy individuals and chronic kidney disease patients, and HMGB1 levels negatively correlated with calcium content in abdominal aortic aneurysms patients. Our findings demonstrate that nuclear HMGB1 downregulation during vascular aging promotes senescence, an anti-inflammatory SASP, ECM disruption and the expression of calcification modulators that promote VSMCs osteoblastic trans-differentation and eventually VC. Also, HMGB1 loss may serve as a VSMC senescence and calcification marker.