Tumor necrosis factor-alpha mediates diabetes-enhanced apoptosis of matrix-producing cells and impairs diabetic healing

Abstract

Diabetics suffer increased infection followed by increased apoptosis of fibroblasts and bone-lining cells during the healing process. To investigate a potential mechanism, we inoculated Porphyromonas gingivalis into the scalp of type 2 diabetic (db/db) or control mice and inhibited tumor necrosis factor alpha (TNF-alpha) with etanercept. Mice were euthanized at the early phase of infection (21 hours) or during the peak repair of the bacteria-induced wound (8 days). At 21 hours, TNF-alpha inhibition significantly reduced fibroblast apoptosis and caspase-3 activity in both diabetic and normoglycemic mice (P < 0.05). During healing etanercept reduced fibroblast apoptosis and caspase-3 activity by almost 50% in diabetic but not normoglycemic mice (P < 0.05). Concomitantly, etanercept significantly increased fibroblast number by 31% and new matrix formation by 72% in diabetic mice. When bone was examined during healing, administration of the TNF-alpha blocker reduced apoptosis of bone-lining cells by 53%, increased their number by 48%, and enhanced new bone formation by 140% in the diabetic group (P < 0.05). The degree of connective tissue and osseous healing stimulated in the diabetic mice by anti-TNF-alpha treatment was within the range that is physiologically relevant. This enhanced healing may in part be explained by block-ing TNF-alpha-induced apoptosis of critical matrix-producing cells.

Figure 1

TNF-α inhibition reduces fibroblast apoptosis and caspase-3 activity in both diabetic and normoglycemic mice during the early response to bacterial stimulus. Live P. gingivalis was inoculated into the scalp of diabetic (db/db) and normoglycemic (db/+) littermates. TNF-α was inhibited by local and systemic injection of etanercept while control mice received an injection of vehicle alone. Mice were euthanized 21 hours after bacterial inoculation. Fibroblast apoptosis was detected with the TUNEL assay, and caspase-3 activity was measured using a fluorimetric kit. An asterisk indicates a significant difference in mice treated with etanercept compared to vehicle alone (P < 0.05).

Figure 2

TNF-α inhibition reduces fibroblast apoptosis and caspase-3 activity in diabetic but not normoglycemic mice during the healing response to bacteria-induced injury. Formalin-killed P. gingivalis was inoculated into the scalp of diabetic (db/db) and normoglycemic (db/+) littermates. Mice were treated with systemic and local injection of etanercept to inhibit TNF-α or with equivalent vehicle alone. Mice were euthanized on day 8. The TUNEL assay was used to detect fibroblast apoptosis, and a fluorimetric kit was used to measure caspase-3 activity. In A the arrows point to TUNEL-positive fibroblastic cells in a section that was counterstained with nuclear fast red. For B and C an asterisk indicates a significant difference in diabetic mice treated with etanercept compared to vehicle alone (P < 0.05).

Figure 3

Inhibition of TNF-α increases fibroblast density in diabetic but not normoglycemic mice during healing. Formalin-killed P. gingivalis was inoculated into diabetic and normoglycemic mice, which were treated with etanercept as described in Figure 2. Mice were euthanized 8 days after bacterial inoculation. Fibroblast number was counted at ×1000 magnification in H&E-stained sections. An asterisk indicates a significant difference in diabetic mice treated with etanercept compared to vehicle alone (P < 0.05).

Figure 4

Inhibition of TNF increases new matrix formation in diabetic but not normoglycemic mice. Formalin-killed bacteria were inoculated and diabetic and normoglycemic mice were treated with etanercept or vehicle alone as described in Figure 2. A: Newly formed connective tissue matrix was identified in Van Gieson-stained histological sections. B: The area of newly formed matrix on day 8 was measured with computer-assisted image analysis. An asterisk indicates a significant difference in diabetic mice treated with etanercept compared to diabetic mice treated with vehicle alone (P < 0.01). Original magnifications, ×400.

Figure 5

Inhibition of TNF-α reduces apoptosis of bone-lining cells in diabetic mice but not normoglycemic mice. Formalin-killed bacteria were inoculated into diabetic and normoglycemic mice, which were treated with etanercept or vehicle alone as described in Figure 2. Apoptosis of bone-lining cells on day 8 was measured by the TUNEL assay. An asterisk indicates a significant difference in diabetic mice treated with etanercept compared to diabetic mice treated with vehicle alone (P < 0.05).

Figure 6

Inhibition of TNF-α increases the number of normal bone-lining cells in diabetic but not normoglycemic mice. Bacteria were inoculated into diabetic and normoglycemic mice, which were treated with etanercept as described in Figure 2. The number of normal bone-lining cells per mm bone length on day 8 was counted at ×1000 magnification in H&E-stained sections. An asterisk indicates a significant difference in diabetic mice treated with etanercept (P < 0.05).

Figure 7

Inhibition of TNF-α enhances new bone formation in diabetic but not normoglycemic mice. Formalin-killed bacteria were inoculated into diabetic and normoglycemic mice, which were treated with etanercept or vehicle alone as described in Figure 2. A: Newly formed bone matrix on day 8 was identified in Van Gieson-stained sections by its characteristic blue color. B: The area of newly formed bone was measured with computer-assisted image analysis. An asterisk indicates a significant difference in diabetic mice treated with etanercept compared to diabetic mice treated with vehicle alone (P < 0.05). Original magnifications, 400.