Deficiency of TLR4 homologue RP105 aggravates outward remodeling in a murine model of arteriovenous fistula failure

Abstract

Arteriovenous access dysfunction is a major cause of morbidity for hemodialysis patients. The pathophysiology of arteriovenous fistula (AVF) maturation failure is associated with inflammation, impaired outward remodeling (OR) and intimal hyperplasia. RP105 is a critical physiologic regulator of TLR4 signaling in numerous cell types. In the present study, we investigated the impact of RP105 on AVF maturation, and defined cell-specific effects of RP105 on macrophages and vascular smooth muscle cells (VSMCs). Overall, RP105^-/- mice displayed a 26% decrease in venous OR. The inflammatory response in RP105^-/- mice was characterized by accumulation of anti-inflammatory macrophages, a 76% decrease in pro- inflammatory macrophages, a 70% reduction in T-cells and a 50% decrease in MMP-activity. In vitro, anti-inflammatory macrophages from RP105^-/- mice displayed increased IL10 production, while MCP1 and IL6 levels secreted by pro-inflammatory macrophages were elevated. VSMC content in RP105^-/- AVFs was markedly decreased. In vitro, RP105^-/- venous VSMCs proliferation was 50% lower, whereas arterial VSMCs displayed a 50% decrease in migration, relative to WT. In conclusion, the impaired venous OR in RP105^-/- mice could result from of a shift in both macrophages and VSMCs towards a regenerative phenotype, identifying a novel relationship between inflammation and VSMC function in AVF maturation.

Figure 1

Effect of RP105 deficiency on AVF maturation in vivo. (a) Quantification of morphometric parameters. Decrease in vessel circumference (outward remodeling) in RP105 deficient mice was observed 14 days after AVF creation, compared to WT. Lumen and intimal hyperplasia did not differ between RP105^−/− and WT mice. (b) Histological staining of venous outflow tract 14 days after surgery. Weigert elastine staining was used to determine histomorphometrical parameters of the vessel. Circumference (internal elastic lamina area) was used to quantify outward remodeling (red line). Intimal hyperplasia (green arrow) measured as a difference between luminal area (blue line) and vessel circumference. αSMA staining shows area of intimal hyperplasia 14 days after AVF creation. (#) intimal hyperplasia; P < 0.05; n = 11 per group. Bar = 200 μm; 100x magnification.

Figure 2

Effect of RP105 deficiency on VSMCs proliferation in vivo. Quantification (a) and immunofluorescent staining (b) of αSMA⁺/Ki67⁺ cells (white arrows) revealed reduction in number of proliferating VSMCs in AVF lesions of RP105^−/− mice compared to WT 14 days after AVF surgery. n = 11 per group. Bar = 100 μm.

Figure 3

In vitro cultured arterial and venous SMCs. (a) Microscopic representation of morphological difference between cultured arterial and venous VSMCs. Bar = 100 μm; 200x magnification. (b) VSMCs phenotype after 14 days in culture was confirmed by the stable mRNA expression levels of smooth muscle α-actin (SMA), myosin heavy chain (MHC) and calponin. (c) Stable increase in EphB4 mRNA levels was decreased in venous VSMCs isolated from WT and RP105^−/− mice. ^*P < 0.05; n = 3.

Figure 4

Difference in mRNA expression levels between arterial and venous VSMCs in vitro. (a) RP105, (b) MD1, (c) TLR4. The relative expression normalized to GAPDH. ^*P < 0.05; n = 3.

Figure 5

Functional difference between arterial and venous VSMCs in vitro. Reduction in proliferation rate was limited to VSMCs isolated from RP105^−/− veins. Decrease in migration of VSMCs isolated from RP105^−/− mice was restricted to arterial cells only. Proliferation rate and migration were measured over 16 h time period. (b) Venous VSMCs isolated from WT and RP105^−/− mice produce significantly higher amounts of inflammatory cytokines IL6 and MCP1. Cells were maintain in culture for 14 days. ^*P < 0.05; n = 3.

Figure 6

Effects of RP105 deficiency on inflammatory response in vivo. Quantification and immunohistochemical staining of (a) MAC3⁺/CCR2⁺  macrophages and MAC3⁺/CD206⁺ macrophages (white arrows) and (b) CD3⁺ T-lymphocytes (black arrows) in AVF lesions 14 days after surgery. Decrease in cell number of pro-inflammatory macrophages and upregulation in anti-inflammatory macrophages upon RP105 deletion was observed. Bar = 100 μm. Number of CD3 T-lymphocytes was reduced in RP105 as compared to WT. Bar = 50 μm; 400x magnification. ^*P < 0.05; n = 11 per group.

Figure 7

In vivo near-infrared biofluorescent imaging and quantitative analysis of MMP activity. (a) Quantitative analysis of fluorescent intensity showed decrease in MMP activity in RP105 mice, as compared to WT. (b) Visual representation of near-infrared signal from active MMPs. Accumulation of green color can be seen in the anastomotic region 24 h after injection of MMPSenseTM 680 probe. ^*P < 0.05; n = 4 per group.

Figure 8

Effect of RP105 deficiency on macrophage function. (a) Bone marrow-derived macrophages from RP105 primed towards pro-inflammatory phenotype secrete increased levels of MCP1 and IL6 as compared to WT control mice. (b) Anti-inflammatory macrophages from RP105 secrete increased levels of repair associated cytokine IL10. ^*P < 0.05; n = 3.

Figure 9

RP105 expression in human AVF. RP105 is highly expressed within the venous neointimal lesions of human AVF n = 4.