Receptor for advanced glycation endproducts mediates pro-atherogenic responses to periodontal infection in vascular endothelial cells

Abstract

Objective: A link between periodontal infections and an increased risk for vascular disease has been demonstrated. Porphyromonas gingivalis, a major periodontal pathogen, localizes in human atherosclerotic plaques, accelerates atherosclerosis in animal models and modulates vascular cell function. The receptor for advanced glycation endproducts (RAGE) regulates vascular inflammation and atherogenesis. We hypothesized that RAGE is involved in P. gingivalis's contribution to pro-atherogenic responses in vascular endothelial cells.

Methods and results: Murine aortic endothelial cells (MAEC) were isolated from wild-type C57BL/6 or RAGE-/- mice and were infected with P. gingivalis strain 381. P. gingivalis 381 infection significantly enhanced expression of RAGE in wild-type MAEC. Levels of pro-atherogenic advanced glycation endproducts (AGEs) and monocyte chemoattractant protein 1 (MCP-1) were significantly increased in wild-type MAEC following P. gingivalis 381 infection, but were unaffected in MAEC from RAGE-/- mice or in MAEC infected with DPG3, a fimbriae-deficient mutant of P. gingivalis 381. Consistent with a role for oxidative stress and an AGE-dependent activation of RAGE in this setting, both antioxidant treatment and AGE blockade significantly suppressed RAGE gene expression and RAGE and MCP-1 protein levels in P. gingivalis 381-infected human aortic endothelial cells (HAEC).

Conclusion: The present findings implicate for the first time the AGE-RAGE axis in the amplification of pro-atherogenic responses triggered by P. gingivalis in vascular endothelial cells.

Figure 1. RAGE levels by immunoblotting in MAEC isolated from C57BL/6 mice

P. gingivalis 381 (Pg 381) significantly increased RAGE expression in C57BL/6 MAEC 6 hours after infection (a) compared to the non-infected cells (NI) (p=0.0122, n=3). At 24 hours (b) the magnitude of the increase was smaller (p=0.0128, n=3). Infection with the fimbriae-deficient mutant DPG3 also caused a significant increase in RAGE expression compared to NI at 6 hours (p=0.0108, n=3), but had no effect at 24 hours (p=0.7634, n=3)

Figure 2. MCP-1 production by ELISA in MAEC isolated from C57BL/6 or RAGE−/− mice

Infection of C57BL/6 MAEC with P. gingivalis 381 (Pg 381) significantly increased secretion of MCP-1 6 hours after infection (a) compared to non-infected cells (NI) or DPG3-infected cells (p<0.0001 for both, n=4). At 24 hours (b) the magnitude of the increase in C57BL/6 MAEC was smaller (p=0.0026 and p=0.0096 compared to NI or DPG3-infected cells, respectively, n=3). DPG3 infection caused a minor MCP-1 increase in C57BL/6 MAEC compared to NI which was statistically significant at 6 hours (p=0.0010, n=4), but not at 24 hours (p=0.2816, n=3). Consistent with an important role for RAGE in this setting, infection with either strain did not affect MCP-1 production in RAGE−/− MAEC.

Figure 3. AGE production by ELISA in MAEC isolated from C57BL/6 or RAGE−/− mice

(a) Infection of C57BL/6 MAEC with P. gingivalis 381 (Pg 381) significantly increased AGE epitopes compared to non-infected (NI) and to DPG3-infected cells at 6 hours (p=0.0003 for both, n=3). In RAGE−/− MAEC P. gingivalis infection did not affect AGE production. (b) Similarly, 24 hours following infection of C57BL/6 MAEC with Pg 381, AGE epitopes were significantly increased compared to NI and to DPG3-infected cells (p=0.0041 and p=0.0046, respectively, n=3). Again, infection had no effect on AGE production in RAGE−/− MAEC.

Figure 4. Effect of AGE blockade and antioxidant treatment on infection-elicited MCP-1 production in HAEC

To directly assess the effect of AGE ligands on P. gingivalis 381-elicited MCP-1 upregulation, we specifically blocked these species using the AGE inhibitor aminoguanidine (AG, 200 μmol/L) or anti-AGE IgG (15 μg/ml). Both blocking agents significantly suppressed MCP-1 production in HAEC 6 hours following infection with P. gingivalis 381 (Pg 381); p=0.0111 and p=0.0039, respectively, n=4 for both. Non-specific IgG antibody (15 μg/ml) 20sed as control had no effect (p=0.9251, n=3). Further, antioxidant treatment using N-acetyl-L-cysteine (NAC, 10 mmol/L), or diphenylene iodonium (DPI, 30 μmol/L) also significantly suppressed MCP-1 production; p=0.0002 and p=0.0006, respectively, n=3 for both.

Figure 5. RAGE mRNA and protein levels, by Real Time PCR and immunoblotting, in HAEC

(a) P. gingivalis 381 (Pg 381) significantly increased RAGE mRNA in HAEC 2 hours after infection compared to the non-infected cells (NI) (0.0077, n=4), while infection with the fimbriae-deficient mutant DPG3 had no such effect (p=0.3804, n=4). The results were similar on the protein level (b); p=0.0007 and p=0.3301, respectively, n=3 for all. (c) Consistent with a role for AGE and ROS generation in RAGE upregulation following P. gingivalis 381 infection, pretreatment with aminoguanidine (AG, 200 μmol/L), N-acetyl-L-cysteine (NAC, 10 mmol/L), or diphenylene iodonium (DPI, 30 μmol/L) significantly suppressed RAGE mRNA (p= 0.0017, p=0.0038 and p=0.0103, respectively, n=4 for all). This effect was demonstrated on the protein level as well (d); p<0.0001 and n=3 for all.