Abstract Body (Do not enter title and authors here): Background
Macrophages (MΦ) cause atherosclerotic lesions, sustain a pro-inflammatory microenvironment, driving plaque instability; while vascular smooth muscle cells (VSMCs) stabilize the plaque by producing collagens and other extracellular matrices in the intima. Fortilin is a 20-kDa protein regulating diverse cellular processes and is important for macrophage survival. The role of fortilin in atherosclerosis remains incompletely understood.
Hypothesis
Fortilin reduces the number of VSMC, possibly through decrease in an atheroprotective cytokine.
Methods
Cross-sectioned aortic root tissue from fortilin knockout (fortilin^+/-) and wild type (fortilin^+/+) mice under LysM/Cre promoter control and on Ldlr^-/-Apobec1^-/- hypercholesterolemic (HC) background (KO and WT, respectively) were subjected to imaging mass cytometry (IMC). Separately, THP1 MΦ or primary human MΦ lacking fortilin (THP1^KO-fortilin or phMΦ^sh-fortilin), and their controls (THP1^WT-fortilin or phMΦ^sh-control) were stimulated with oxidized-LDL (oxLDL) and subjected to further analysis. Umbilical cord derived mesenchymal stem cells (UCDMSCs) were incubated in presence of conditioned media (CM) from THP1^KO-fortilin or THP1^WT-fortilin and the extracted RNA was subjected to RT-qPCR.
Results
IMC analysis indicated that VSMC formation were higher in KO than WT mice intima (WT vs. KO = 159 vs. 1069 cells/mm², P<0.0001). Fortilin expression in VSMCs and endothelial cells (ECs) did not differ between WT and KO mice. Regardless of oxLDL stimulation, THP1^KO-fortilin and phMΦ^sh-fortilin expressed higher VSMC (αSMA, SM22α and CNN1) and lower MΦ (Mac2, CD68) marker genes and protein levels compared to THP1^WT-fortilin and phMΦ^sh-control. The higher levels of VSMC marker genes in THP1^KO-fortilin was due to secretion of higher TGF-β1 (WT vs. KO = 311 vs 726 pg/mL, P<0.0001) in CM. Incubation of UCDMSCs in THP1^KO-fortilin CM led to significant increase in differentiation (CD45, CD34, and CD14) and VSMC marker genes, and reduced stem cell marker genes (CD73, CD90, and CD105) as compared to THP1^WT-fortilin CM. Incubating UCDMSCs in TGF-β1-depleted THP1^KO-fortilin CM led to complete abolishment of differentiation of the cells to VSMCs, indicating the key role of TGF-β1.
Conclusion
Fortilin in MΦ promotes atherosclerosis by inhibiting secretion of TGF-β1, preventing transdifferentiation of MΦ to VSMC, and differentiation of MSCs to VSMCs. Therefore, fortilin functions as a key molecule driving HC-induced atherosclerogenesis.