Blockade of RAGE suppresses periodontitis-associated bone loss in diabetic mice (original) (raw)
We demonstrated previously that sustained hyperglycemia in C57BL/6 mice inoculated with the periodontal pathogen P. gingivalis resulted in significantly increased alveolar bone loss 2 months after infection (26). To test the hypothesis that interaction of AGEs and/or EN-RAGEs with RAGE on critical cells in diabetic periodontium accounted for, at least in part, exaggerated inflammation and excessive alveolar bone loss, mice were rendered diabetic with streptozotocin and 1 month later were inoculated with P. gingivalis. Commencing immediately after inoculation, mice were treated once daily with intraperitoneal injections of either murine sRAGE or vehicle (MSA) for 2 months. At that time point, alveolar bone loss, defined as the area between the CEJ and BC, in diabetic MSA-treated mice was 6,272 ± 81 pixels (mean ± SE; Figure 1, a and c), significantly greater than that observed in non-diabetic mice (4,234 ± 114 pixels; Figure 1, a and b). Upon administration of sRAGE, diminished bone loss was observed. Alveolar bone loss in diabetic mice receiving sRAGE 25, 35, 50, or 100 μg per day was significantly suppressed (5,437 ± 74, 5129 ± 117, 4,912 ± 39, and 4,879 ± 151 pixels, respectively) when compared with diabetic mice receiving MSA; P values were less than 0.0002 in all cases (Figure 1 a, d, and c, respectively). The effects of sRAGE were dose-dependent. At lower doses of sRAGE (3.5 and 12 μg per day), levels of bone loss were similar to those observed in MSA-treated diabetic mice (6,149 ± 47 and 6,147 ± 79 pixels, respectively; Figure 1a). Because these latter 2 doses of sRAGE were not effective in preventing excessive alveolar bone loss, they are herein noted as sRAGE(–); those higher doses effective in reducing bone loss are noted as sRAGE(+).
Administration of murine sRAGE results in dose-dependent suppression of alveolar bone loss in diabetic mice. (a) Alveolar bone loss. Male C57BL/6J mice were rendered diabetic with streptozotocin or treated with vehicle, citrate buffer alone. One month after documentation of diabetes or control state, all mice were inoculated with P. gingivalis, strain 381, as described in Methods. Immediately after inoculation, diabetic animals (filled bars) were treated with either MSA, 100 μg/day (n = 11), or sRAGE, 100 μg/day (n = 5), sRAGE, 50 μg/day (n = 13), sRAGE, 35 μg/day (n = 12), sRAGE, 25 μg/day (n = 11), sRAGE, 12 μg/day (n = 13), or sRAGE, 3.5 μg/day (n = 12) for 2 months. Similarly, non-diabetic mice (hatched bar) were treated with MSA, 100 μg/day (n = 12). Upon sacrifice, defleshed mandibles were assessed for extent of alveolar bone loss by morphometric analysis. Results are reported as mean arbitrary pixels per condition ± SE. In these studies, 12,210 pixels = 1 mm2. Statistical analyses are as follows: diabetes/MSA vs. non-diabetes/MSA, P = 0.009; diabetes/MSA vs.: sRAGE 25, 35, or 50 μg/day, P < 0.00001; sRAGE 100 μg/day, P < 0.0002; sRAGE, 3.5 μg/day, P = 0.28; and vs. sRAGE, 12 μg/day, P = 0.20. AStatistically significant differences compared with diabetes/MSA. (b–d). Representative photographs of mandibles. The lingual surfaces of posterior teeth are shown in defleshed half mandibles obtained from a non-diabetic mouse treated with MSA (b) and diabetic mice treated with MSA (c) or sRAGE(+) (d). The outlined areas between the CEJ and BC were computer analyzed and measured to evaluate differences in alveolar bone loss between groups. (e) Measurement of glycosylated hemoglobin. At the time of sacrifice, lysates of red blood cells were prepared as described and percent of glycosylated hemoglobin determined. The mean ± SE is reported. Statistical analyses are as follows: non-diabetes/MSA vs. diabetes/MSA, P = 0.005; diabetes/MSA vs. diabetes/sRAGE(+), P = 0.5; and diabetes/sRAGE(+) vs. non-diabetes/MSA, P = 0.005. AStatistically significant differences compared with diabetes/MSA or diabetes/sRAGE (+).
Importantly, the beneficial effects of sRAGE in suppressing alveolar bone loss were independent of the level of glycemia. Mean levels of glycosylated hemoglobin (note that these moieties are early glycation products, not AGEs) were not significantly different in sRAGE(+)-treated diabetic mice than levels observed in MSA-treated diabetic animals (9.55 ± 0.34% vs. 9.28 ± 0.14%, respectively; Figure 1e).
Since cytokines and MMPs are implicated in the soft tissue injury and bone loss that are pathognomonic of periodontal disease (9–10, 30–32), we assessed the levels of these mediators in gingival tissue. We first studied levels of MMP protein in gingival extracts. Compared with non-diabetic mice treated with MSA, diabetic mice demonstrated an approximately 7.3-fold increase in levels of MMP-9 as determined by immunoblotting (Figure 2a, lanes 1 and 2, respectively). In sRAGE(+)-treated diabetic animals, levels of MMP-9 were reduced 47% (Figure 2a, lane 4). In contrast, in sRAGE(–)-treated mice, levels of MMP-9 were reduced only 19% (Figure 2a, lane 3). We similarly assessed levels of MMP-3 in gingival extracts. An approximately 3.9-fold increase in levels of MMP-3 by immunoblotting was observed in diabetic mice compared with non-diabetic controls, both receiving MSA (Figure 2b, lanes 2 and 1, respectively). In the presence of effective doses of sRAGE, levels of MMP-3 were reduced 70% in diabetic gingival extracts (Figure 2b, lane 4) and 45% in diabetic mice treated with the lower doses of sRAGE (Figure 2b, lane 3).
Administration of sRAGE to diabetic mice results in diminished levels of MMP protein and activity. (a, b) Immunoblotting. (a) MMP-9. Gingival tissue extracts were prepared as described and total protein (150 ng) was subjected to electrophoresis on Tris-glycine gels. The contents of the gels were transferred to nitrocellulose membranes, and immunoblotting was performed using mouse monoclonal anti-MMP 9 (2 μg/mL). Densitometry was performed using ImageQuant. Molecular weight markers (kDa) are indicated at the right of the immunoblot. In these experiments, intensity of the band obtained from gingival extract of non-diabetic MSA-treated animals was arbitrarily defined as 1. These experiments were performed 3 times with analogous results. (b) MMP-3. These experiments were performed as described in a; final concentration of anti-MMP-3 IgG was 1 μg/mL. (c) Zymography. At sacrifice, gingival extracts were prepared as described. Two hundred twenty nanograms of total protein per sample was subjected to chromatography onto gels containing gelatin (0.1%); bands representing MMP-2 are indicated. Densitometric analysis was performed and is demonstrated in the inset. Bands from non-diabetic mice treated with MSA were arbitrarily defined as 1. These experiments were performed 3 times with analogous results.
Consistent with these observations, MMP-2 activity, as assessed in gingival extracts by zymography, was enhanced 3.4-fold in diabetic, MSA-treated mice compared with non-diabetic controls (Figure 2c and inset, lanes 2 and 1, respectively). MMP-2 activity was reduced 50% in mice treated with higher, effective doses of sRAGE, but only 17% in mice treated with lower doses of sRAGE (Figure 2c and inset, lanes 4 and 3, respectively).
TNF-α is an important proinflammatory cytokine associated with tissue destruction in periodontitis (30–32). Levels of TNF-α in gingival tissue extracts from diabetic MSA-treated mice (n = 9) were significantly increased compared with levels observed in non-diabetic MSA-treated controls (n = 10; 6.4 ± 0.29 vs. 4.7 ± 0.32 ng/μg tissue; P = 0.00003). In diabetic sRAGE(+)-treated mice (n = 8), levels of TNF-α were diminished 40% compared with MSA-treated diabetic animals (3.8 ± 0.58 vs. 6.4 ± 0.29 ng/μg tissue; P = 0.001). Indeed, levels of TNF-α in diabetic sRAGE(+)-treated mice were comparable to those in non-diabetic animals. In contrast, however, in sRAGE(–)-treated diabetic mice (n = 11) levels of TNF-α were only suppressed 20% compared with MSA-treated diabetic mice (5.1 ± 0.3 vs. 6.4 ± 0.29 ng/μg tissue; P = 0.07).
We also assessed levels of IL-6 in gingival extracts, because it has been implicated in a number of proinflammatory events, including bone-resorptive activity (33, 34). Even though levels of IL-6 in gingival tissue, in general, demonstrated some variability, an approximately 1.7-fold increase in the level of IL-6 was noted in diabetic MSA-treated mice (n = 5) compared with non-diabetic controls (n = 7; 4.98 ± 1.4 vs. 3.00 ± 0.7 ng/μg gingival tissue; P = 0.34). A trend toward decreased levels of IL-6 was noted in sRAGE(+)-treated diabetic mice (n = 6; 3.98 ± 1.1 ng/μg tissue) compared with MSA-treated diabetic mice (P = 0.84). In contrast, levels of gingival IL-6 were not decreased in sRAGE(–)-treated mice (n = 6; 5.53 ± 1.7 ng/μg tissue) compared with MSA-treated diabetic animals (P = 0.48).
Since the promoter of RAGE is enriched in elements tightly linked to the inflammatory response, such as NF-κB (35), and since sRAGE prevents access of ligand to the cell-surface receptor, we hypothesized that decreased levels of RAGE itself might ensue upon administration of sRAGE. Consistent with these concepts, immunoblotting of gingival extracts revealed increased RAGE protein in diabetic compared with non-diabetic MSA-treated mice (Figure 3a); levels of tissue RAGE were suppressed, however, in mice treated with sRAGE (Figure 3a).
Immunoblotting for RAGE (a) and EN-RAGEs (b) and immunohistochemistry for AGEs (c–h). At sacrifice, gingival tissue was removed and was either prepared for immunoblotting as described above or fixed in buffered formalin (10%). Paraffin-embedded sections, 5 μm thick, were prepared for immunohistochemistry. Immunoblotting was performed with rabbit anti-murine RAGE IgG (4.5 μg/mL) or rabbit anti–EN-RAGE IgG (2 μg/mL) as above. Molecular weight markers (kDa) are indicated on the right side of the immunoblot. Immunohistochemistry is shown for a representative section from a diabetic MSA-treated mouse using nonimmune rabbit IgG (1 μg/mL in c) or affinity-purified anti-AGE IgG (1 μg/mL in d–g). Samples are as follows: non-diabetes/MSA, d; diabetes/MSA, e; diabetes/sRAGE(–), f; and diabetes/sRAGE(+), g. Scale bar: 45 μm. In h, the results of quantitative analysis of the immunohistochemistry, performed as described above, are shown.
Both AGEs and/or EN-RAGEs are putative ligands promoting RAGE activation. We thus assessed their levels in gingival tissue. Compared with non-diabetic mice, levels of EN-RAGEs were increased ≈2-fold in diabetic MSA-treated mice (Figure 3b). Significant suppression of EN-RAGE levels was observed in gingival tissue of mice treated with sRAGE(–) or sRAGE(+) (Figure 3b). Furthermore, consistent with earlier observations (26), gingival tissue from diabetic MSA-treated mice demonstrated enhanced accumulation of AGEs compared with non-diabetic MSA-treated controls, especially within the vascular structures and surrounding epithelial and connective tissues (Figure 3, e and d, respectively, and Figure 3h). Accumulation of epithelial/connective tissue and vascular AGE was significantly suppressed in diabetic sRAGE(+) mice; indeed these levels were not significantly different than those observed in non-diabetic MSA-treated mice (Figure 3, g and d, respectively, and Figure 3h). In contrast, accumulation of gingival AGE retrieved from sRAGE(–)-treated diabetic mice was not different than that retrieved from diabetic MSA-treated mice (Figure 3, f and e, respectively, and Figure 3h).