Abstract Body (Do not enter title and authors here): Background
Atherosclerosis is driven by macrophage (MΦ)-mediated chronic inflammation and cholesterol accumulation. Fortilin, a multifunctional protein, protects MΦ from apoptosis and promotes atherogenesis, yet its cellular role remains unclear.
Hypothesis
Fortilin deletion in MΦ attenuates atherosclerosis by promoting apoptosis, reducing lipid uptake, and altering intimal cell composition.
Methods
We generated MΦ-specific Fortilin knockout mice (fortilinKO-MΦ-HC) using LysM-Cre crossed with Ldlr–/–Apobec1–/– mice. Aortic root sections were analyzed by multicolor immunofluorescence staining for Mac2 (macrophages), αSMA (vascular smooth muscle cells; VSMCs), and CD31 (endothelial cells; ECs). Separately, CRISPR/Cas9 was used to generate Fortilin-deficient THP-1 macrophages (THP1KO-fortilin). These and control cells (THP1WT-fortilin) were treated with or without oxidized low-density lipoprotein (oxLDL) and assessed by DNA fragmentation assay, MTS proliferation assay, and quantification of CD36 (an oxLDL receptor) using the JESS capillary-based western blot system.
Results
Compared to fortilinWT-MΦ-HC mice, fortilinKO-MΦ-HC mice showed significantly reduced Mac2 positive area (P < 0.0001) and increased αSMA positive area (P < 0.0027), while CD31 positive endothelial cells remained unchanged. In vitro, Fortilin deletion enhanced THP-1 apoptosis: DNA fragmentation was 41% higher at baseline and 112% higher following oxLDL stimulation in THP1KO-fortilin compared to WT cells (P < 0.0001 for both). MTS assays showed significantly reduced proliferation in THP1KO-fortilin at 24, 48, 72, and 96 hours (P < 0.0001 at all time points). CD36, an oxLDL receptor, was markedly decreased in Fortilin-deficient MΦ (P < 0.0001).
Conclusion
Fortilin in MΦ facilitates atherosclerosis by promoting survival, proliferation, and lipid uptake. Its deletion induces apoptosis, suppresses oxLDL uptake, and shifts the cellular composition of intima—reducing MΦ and increasing VSMCs. These findings provide mechanistic insight into Fortilin's role in regulating macrophage function and influencing plaque progression.