Abstract
Aims: FK506-binding protein like (FKBPL) and its therapeutic peptide mimetic, AD-01, are crucial negative regulators of angiogenesis, yet the underlying mechanisms involved in hypoxia- and/or inflammation-induced cardiovascular disease (CVD) remain unclear. We investigated whether FKBPL regulates endothelial dysfunction induced by hypoxia or inflammation and if AD-01 can maintain endothelial cell function and vascular integrity under these conditions.
Methods and Results: Hindlimb ischemia was induced in mice by ligating the proximal and distal ends of the right femoral artery, and, after three days, the gastrocnemius muscle was collected for immunofluorescence staining, and RNA extraction. A 3D in vitro microfluidics model of endothelial cell function was developed to determine the cell migration and FKBPL-mediated mechanisms following treatments with: (i) 24 mM FKBPL targeted siRNA, (ii) 1 mM hypoxia inducible factor (HIF-1)α activator (DMOG), (iii) 50% (v/v) macrophage conditioned media (MCM), ± 100 nM AD-01. Here, we demonstrated that FKBPL expression is substantially downregulated in mice with hindlimb ischemia (p<0.05, protein; p<0.001, mRNA), correlating with reduced neovascularization and altered vascular adhesion molecule expression. In our real-time advanced 3D microfluidics endothelial cell model, hypoxia suppressed FKBPL (p<0.05) and VE-cadherin (p<0.001), leading to increased endothelial cell number and migration (p<0.001), which was restored by AD-01 treatment (p<0.01). Under inflammatory conditions, FKBPL (p<0.01) and HIF-1α (p<0.05) expression were elevated, correlating with increased endothelial cell migration (p<0.05). Unlike hypoxia, AD-01 did not affect endothelial cell migration in inflammation but normalized FKBPL (p<0.001), HIF-1α (p<0.05) and CD31 (P<0.05), expression. Proteomic analysis revealed that AD-01 treatment in hypoxia promoted vascular integrity and stimulated tissue remodelling proteins by increasing the expression of collagen alpha-1(XIX) chain and junctional cadherin associated-5 (JCAD) proteins.
Conclusions: FKBPL represents an important novel mechanism in hypoxia and inflammation-induced angiogenesis. The FKBPL-based therapeutic peptide, AD-01, could be a viable treatment option for CVD-related endothelial cell dysfunction.