A20 and TNIP-3 Reduce NF-κB-Mediated Paracrine Responses to Hypoxia/Hyperglycemia-Induced Endothelial Senescence

Highlights: What are the main findings? Hypoxia, alone or combined with hyperglycemia, induces endothelial cell senescence without activating the classical pro-inflammatory SASP. This condition is associated with the upregulation of A20 and TNIP-3, suggesting a deviation from canonical senescence programs. What are the implications of the main findings? The non-canonical senescence profile observed under hypoxia indicates that endothelial senescence may be more heterogeneous than previously recognised. The functional significance of A20 and TNIP-3 upregulation in this context remains to be clarified and represents an important direction for future studies. Background: Hypoxia and ageing both involve impaired oxygen delivery, leading to oxidative damage, and endothelial cell (EC) dysfunction. In the presence of chronic hyperglycemia, these effects are amplified, accelerating EC senescence and vascular impairment. Methods: We assessed key mediators of inflammatory signalling and senescence, as well as transcriptional regulators responsive to oxidative stress in ECs exposed to high glucose (30.5 mmol/L) for 72 h under either normoxia (21% O2) or prolonged (16 h) hypoxia (2% O2) followed by 2 h of reoxygenation. Results: ECs exposed to high glucose and hypoxia developed a senescent phenotype, as indicated by increased expression of p21 and p16, and elevated β-galactosidase staining. Interestingly, hypoxia-induced senescence did not coincide with the classical senescence-associated secretory phenotype (SASP). Compared to normoxia, ECs exposed to hypoxia, particularly under high-glucose conditions, showed reduced NF-κB-driven proinflammatory secretome (MCP-1, IL-6, IL-8), downregulation of the NF-κB p50 subunit, and simultaneous upregulation of the angiogenic factor VEGF-A with downregulation of YAP-1, a key regulator of cell survival. Notably, we observed a strong upregulation of A20 and TNIP-3, two well-characterized negative regulators of NF-κB signalling. Conclusions: Hypoxia-induced senescence did not trigger a typical inflammatory SASP. Although ECs enter a senescent state, they activate an anti-inflammatory response, suppressing NF-κB signalling and increasing the expression of its inhibitors, A20 and TNIP-3. This may reflect a non-canonical senescence response whose functional significance remains to be determined.