Functions for platelet factor 4 (PF4/CXCL4) and its receptors in fibroblast-myofibroblast transition and fibrotic failure of arteriovenous fistulas (AVFs)

Background: Over 60% of End Stage Renal Disease (ESRD) patients are relying on hemodialysis (HD) to survive, and the arteriovenous fistula (AVF) is the preferred vascular access method for HD. However approximately half of all newly created AVF fail to mature and cannot be used without a salvage procedure. We have recently demonstrated an association between AVF maturation failure and post-operative fibrosis, while our RNA-seq study also revealed that veins that ultimately failed during AVF maturation had elevated levels of platelet factor 4 (PF4/CXCL4). However, a link between these two findings was yet to be established. Methods: In this study, we investigated potential mechanisms between PF4 levels and fibrotic remodeling in veins. We compared the local expression of PF4 and fibrosis marker integrin ss 6 (ITGB6) in veins that successfully underwent maturation with that in veins that ultimately failed to mature. We also measured the changes of expression level of asmooth muscle actin (aSMA/ACTA2) and collagen (Col1/COL1A1) in venous fibroblasts upon various treatments, such as PF4 pharmacological treatment, alteration of PF4 expression, and blocking of PF4 receptors. Results: We found that PF4 is expressed in veins and co-localizes with aSMA. In venous fibroblasts, PF4 stimulates expression of aSMA and Col1 via different pathways. The former requires integrins a v ss 5 and a 5 ss 1, while chemokine receptor CXCR3 is needed for the latter. Interestingly, we also discovered that the expression of PF4 is associated with that of ITGB6, the ss subunit of integrin a v ss 6. This integrin is critical for the activation of the major fibrosis factor TGF ss, and overexpression of PF4 promotes activation of the TGF ss pathway. Conclusions: These results indicate that upregulation of PF4 may cause venous fibrosis both directly by stimulating fibroblast differentiation and expression of extracellular matrix (ECM) molecules and indirectly by facilitating the activation of the TGF ss pathway.