Important protective effects of toll like receptor 3 and downstream interferon regulating factors on vein graft remodelling

Abstract

Purpose: Toll like receptors (TLRs) are key regulators of innate immunity and are highly involved in vein graft remodelling. Activation of most TLRs results in MyD88 and NFκB signalling and induction of inflammatory cytokines. In vascular disease models this leads to pro-inflammatory profiles resulting in augmentation of lesion formation and remodelling. TLR3 however, has protective effects on vascular diseases, although the molecular mechanism is not yet fully clarified. Interestingly, TLR3 is unique among the TLRs since signalling occurs in a MyD88 independent manner; via activation of TRIF and key interferon regulating factors IRF3 and IRF7 resulting in induction of type I interferons. The aim of this study was to investigate the role of TLR3 pathway constituents on vein graft remodelling. Methods: This study was performed in compliance with Dutch government guidelines and principles of laboratory animal care (NIH Publication no. 85-23 revised 1985). Donor caval veins were engrafted in carotid arteries of recipient Tlr3-/-, Irf3-/-, Irf7-/- and control C57BL/6 mice (n=7-10/group). 28 days after surgery vein grafts were harvested and analyzed for vessel wall morphology, cell content and cytokine RNA levels. Results: Tlr3-/- mice developed substantially larger vein grafts than the control mice (155%, p<0.001). Also Irf3-/- and Irf7-/- mice showed increased vein graft thickening (Irf3-/-; 39%, p=0.185, Irf7-/-; 68% p=0.003). The lumen area did not deviate between these strains and their control mice, resulting in outward remodelling in all three strains (Tlr3-/-; 52%, p<0.001, Irf3-/-; 26%, p=0.081, Irf7-/-; 42%, p=0.049). Since macrophages produce large amounts of TLRs, we looked at the influx of these cells in the vein grafts. Tlr3-/-, Irf3-/- and Irf7-/- mice showed significant more influx of macrophages than the control mice. The relative percentage smooth muscle cells and collagen in the vein graft walls did not deviate between the three strains and controls. RNA analysis of vein grafts revealed that pro-inflammatory cytokines TNFα and CCL2 were significantly increased in the Tlr3-/- mice, whereas Irf3-/- and Irf7-/- mice showed no increases in gene expression compared to controls. Conclusions: The TLR3 pathway seems to be protective for vein graft remodelling since Tlr3-/-, as well as the key downstream factors Irf3-/- and Irf7-/- vein grafts show increased vessel wall thickening and outward remodelling. This increased remodelling in the knock out mice is the result of a pro-inflammatory response as reflected by the increase in macrophages and cytokines in the vein graft wall.