Elucidating the role of MCP-1/VCAM-1 during re-endothelialisation of circulating progenitor cells in denuded human umbilical artery

Abstract

Endothelial denudation is a key-initiating event in the pathophysiology of cardiovascular disease and the development of intimal hyperplasia (IH), yet human ex vivo platforms that enable controlled investigation of early endothelial repair responses remain limited. In particular, the mechanisms governing chemokine-mediated recruitment of circulating cells to denuded vascular surfaces are not fully defined. This study aimed to establish a reproducible human umbilical artery (HUA) ex vivo model and used it as a platform to investigate the role of monocyte chemoattractant protein-1 (MCP-1) in peripheral blood mononuclear cells (PBMC) recruitment and adhesion-associated intracellular signalling following endothelial injury. An optimised enzymatic protocol was developed to generate consistent endothelial removal while preserving extracellular matrix integrity, validated by histology, immunofluorescence and glycosaminoglycan (GAG) assessment. Using this denuded HUA platform under dynamic rocking conditions, the effect of MCP-1 supplementation (20 ng/mL) on PBMC recruitment was examined. Although MCP-1-treated samples exhibited numerically higher PBMC recruitment, particularly at 2 hours post-incubation, these differences were not statistically significant under the experimental conditions tested. To explore the intracellular signalling context associated with MCP-1-mediated recruitment and adhesion stabilisation, smooth muscle cells (SMC)-PBMC in vitro co-culture experiments were performed and phosphorylation of focal adhesion kinase (FAK) at Tyr397 was assessed as a marker of integrin-associated adhesion signalling. Vascular cell adhesion molecule-1 (VCAM- 1) inhibition attenuated recruitment-associated responses and altered pFAK (Tyr397) levels. This suggests a potential involvement of adhesion-associated signalling during cellular recruitment following endothelial injury. Furthermore, denuded HUA segments were maintained in prolonged culture for up to 21 days to model ex vivo IH development. However, the vessels demonstrated modest, non-progressive intima-media thickening with limited proliferative activity, indicating adaptive remodelling rather than aggressive IH progression. In summary, this thesis establishes a human ex vivo vascular platform for investigating cellular responses to endothelial injury and vascular repair. Collectively, the findings provide insight into PBMC recruitment dynamics following endothelial denudation and support further investigation of chemokine-mediated recruitment strategies for promoting re-endothelialisation following vascular intervention.