Polybacterial Periodontal Pathogens Alter Vascular and Gut BH4/nNOS/NRF2-Phase II Enzyme Expression

Abstract

Periodontal disease is a highly prevalent chronic inflammatory disease and is associated with complex microbial infection in the subgingival cavity. Recently, American Heart Association supported a century old association between periodontal disease and atherosclerotic vascular disease. We have recently shown that polybacterial periodontal infection led to aortic atherosclerosis and modulation of lipid profiles; however the underlying mechanism(s) has not been yet demonstrated. Altered nitric oxide (NO) synthesis and tetrahydrobiopterin (BH4), a cofactor for nitric oxide synthases (NOS) has long been shown to be associated with vascular dysfunction and gastrointestinal motility disorders. We sought to examine the mechanism of periodontal infection leading to altered vascular and gastrointestinal smooth muscle relaxation, focusing on the BH4/nNOS pathways. In addition, we also have investigated how the antioxidant system (NRF2-Phase II enzyme expression) in vascular and GI specimens is altered by oral infection. Eight week old male ApoEnull mice were either sham-infected or infected orally for 16 weeks with a mixture of major periodontal bacteria Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia to induce experimental periodontitis. Serum, vascular (mesenteric), stomach, and colon specimens were collected at the end of periodontal pathogen infection. Bacterial infection induced significant (p<0.05) reductions in the levels of BH4,in ratio of BH4:BH2+B and also in nitric oxide levels compared to sham-infected controls. In addition, we identified a significant (p<0.05) reduction in eNOS dimerization, nNOS dimerization and protein expression of BH4 biosynthesis enzymes; GCH-1, DHFR and NRF2 & Phase II enzymes in infected mice versus controls in both mesenteric artery and colon tissues. However, we found no differences in nNOS/BH4 protein expression in stomach tissues of infected and sham-infected mice. This suggests that a polybacterial infection can cause significant changes in the vascular and colonic BH4/nNOS/NRF2 pathways which might lead to impaired vascular relaxation and colonic motility.

Fig 1. Reduced NO and elevated oxidized biopterin levels in serum from mice with periodontitis versus controls.

(A) Oral polybacterial infection significantly reduced serum NO levels (μmoles) and (B) elevated oxidized biopterins levels thus reducing BH₄ bioavailability when compared to control mice. C = ApoE^null male mice; PD = ApoE^null male mice with periodontitis (PD). Values are mean ± SE (n = 4 mice per group). Statistical significance was determined by Tukey test. *p < 0.05 compared with controls.

Fig 2. Polybacterial infection alters the vascular BH₄/eNOS/nNOS pathway.

(A) Polybacterial infection reducedeNOS protein expression, (B) dimerization of eNOS, (C) nNOSα protein expressionand (D) dimerization of nNOS and (E) GCH-1 levels, but not (F) DHFR (data not shown) in vascular mesenteric arteries compared to mesenteric arteries of control mice. C = ApoE^null males; PD = ApoE^null mice with periodontitis (PD). Values are mean ± SE (n = 4). Statistical significance was determined by Tukey test. *p < 0.05 compared with controls.

Fig 3. Polybacterial infection reduces vascular NRF2/antioxidant enzymes expression.

(A) Polybacterial infection reduced mesenteric vascular protein expression of antioxidant enzymes Nrf2, (B) GCSc and (C) HO-1. GCSm, however, was not affected (data not shown) C = ApoE^null males; PD = ApoE^null males with periodontitis (PD). Values are mean ± SE (N = 4). Statistical significance was determined by Tukey test. *p < 0.05 compared with controls.

Fig 4. Impairment of BH₄/nNOS/ NRF2 pathway in proximal colon tissue from infected mice compared to controls.

(A) Polybacterial infection reducednNOSα protein expression, (B) dimerization of nNOSα, and (D) DHFR but not (C) GCH-1 expression in proximal colon. Polybacterial infection also reduced antioxidant enzyme protein expression in proximal colon as evidenced by (E) Nrf2 and (F) GCSc compared to controls. GCSm, however, was not affected (data not shown). C = ApoE^null males; PD = ApoE^null male mice with periodontitis (PD). Values are mean ± SE (N = 4). Statistical significance was determined by Tukey test. *p < 0.05 compared with controls.