Tesfahun Desta

Diabetes Enhances Periodontal Bone Loss through Enhanced Resorption and Diminished Bone Formation

Using a ligature-induced model in type-2 Zucker diabetic fatty (ZDF) rat and normoglycemic littermates, we investigated whether diabetes primarily affects periodontitis by enhancing bone loss or by limiting osseous repair. Diabetes increased the intensity and duration of the inflammatory infiltrate (P < 0.05). The formation of osteoclasts and percent eroded bone after 7 days of ligature placement was similar, while four days after removal of ligatures, the type 2 diabetic group had significantly higher osteoclast numbers and activity (P < 0.05). The amount of new bone formation following resorption was 2.4- to 2.9-fold higher in normoglycemic vs. diabetic rats (P < 0.05). Diabetes also increased apoptosis and decreased the number of bone-lining cells, osteoblasts, and periodontal ligament fibroblasts (P < 0.05). Thus, diabetes caused a more persistent inflammatory response, greater loss of attachment and more alveolar bone resorption, and impaired new bone formation. The latter may be affected by increased apoptosis of bone-lining and PDL cells.

Cytokine Profiling of Macrophages Exposed to Porphyromonas gingivalis, Its Lipopolysaccharide, or Its FimA Protein

ABSTRACT To characterize the roles of Porphyromonas gingivalis and its components in the disease processes, we investigated the cytokine profile induced by live P. gingivalis, its lipopolysaccharides (LPS), and its major fimbrial protein, fimbrillin (FimA). Using cytokine antibody arrays, we found that P. gingivalis LPS and FimA induced a similar profile of cytokine expression when exposed to mouse peritoneal macrophages but that this profile differed significantly in response to live P. gingivalis. In vitro, mouse peritoneal macrophages were stimulated to produce interleukin-6 (IL-6), granulocyte colony-stimulating factor, and lymphotactin by live P. gingivalis, but not by P. gingivalis LPS or FimA, while RANTES, gamma interferon, IL-17, vascular cell adhesion molecule 1 (VCAM-1), and vascular endothelial growth factor were induced by P. gingivalis LPS or FimA, but not by live P. gingivalis. In vivo, IL-6 mRNA was strongly induced only by live P. gingivalis while monocyte chemoattractant protein 1 mRNA was strongly induced only by P. gingivalis LPS and FimA in mouse calvarial scalp, further confirming the differences of cytokine profile induced in vitro. Cytokine antibody arrays using toll-like receptor 2 (TLR2)- and TLR4-deficient macrophages revealed that most of the cytokines induced by P. gingivalis LPS or FimA signal through TLR2, while most of cytokines induced by live P. gingivalis signal through both TLR2 and TLR4. Interestingly, the activation of TLR2 by live P. gingivalis inhibited the release of RANTES, VCAM-1, and IL-1α from mouse peritoneal macrophages. A tumor necrosis factor alpha enzyme-linked immunosorbent assay further confirmed that P. gingivalis LPS and FimA activate mouse peritoneal macrophages via TLR2. These results indicate that host immune cells sense live P. gingivalis and its components differently, which translates into the expression of different inflammatory cytokine profiles.

Diabetes-Enhanced Tumor Necrosis Factor-α Production Promotes Apoptosis and the Loss of Retinal Microvascular Cells in Type 1 and Type 2 Models of Diabetic Retinopathy

Retinal microvascular cell loss plays a critical role in the pathogenesis of diabetic retinopathy. To examine this further, type 1 streptozotocin-induced diabetic rats and type 2 Zucker diabetic fatty rats were treated by intravitreal injection of the tumor necrosis factor-specific inhibitor pegsunercept, and the impact was measured by analysis of retinal trypsin digests. For type 2 diabetic rats, the number of endothelial cells and pericytes positive for diabetes-enhanced activated caspase-3 decreased by 81% and 86%, respectively, when treated with pegsunercept (P < 0.05). Similarly, the number of diabetes-enhanced terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive endothelial cells and pericytes decreased by 81% and 67% respectively when treated with pegsunercept (P < 0.05). Diabetes-increased activated caspase-3- and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive microvascular cell numbers were both reduced by 81% and 80%, respectively, in pegsunercept-treated type 1 diabetic rats (P < 0.05). Inhibition of tumor necrosis factor reduced type 1 diabetes-enhanced pericyte ghost formation by 87% and the number of type 2 diabetes-enhanced pericyte ghosts by 62% (P < 0.05). Similarly, increased acellular capillary formation caused by type 1 and type 2 diabetes was reduced by 68% and 67%, respectively, when treated with pegsunercept (P < 0.05). These results demonstrate a previously unrecognized role of tumor necrosis factor-alpha in promoting the early pathogenesis of diabetic retinopathy leading to loss of retinal microvascular cells and demonstrate the potential therapeutic benefit of modulating its activity.

Impaired wound healing in mouse models of diabetes is mediated by TNF-α dysregulation and associated with enhanced activation of forkhead box O1 (FOXO1)

Aims/hypothesisThe role of TNF-α in impaired wound healing in diabetes was examined by focusing on fibroblasts.MethodsSmall excisional wounds were created in the db/db mice model of type 2 diabetes and normoglycaemic littermates, and in a streptozotocin-induced type 1 diabetes mouse model and control mice. Fibroblast apoptosis was measured by the TUNEL assay, proliferation by detection of proliferating cell nuclear antigen, and forkhead box O1 (FOXO1) activity by DNA binding and nuclear translocation. TNF-α was specifically inhibited by pegsunercept.ResultsDiabetic wounds had increased TNF-α, fibroblast apoptosis, caspase-3/7 activity and activation of the pro-apoptotic transcription factor FOXO1, and decreased proliferating cell nuclear antigen positive fibroblasts (p < 0.05). TNF-α inhibition improved healing in the diabetic mice and increased fibroblast density. This may be explained by a decrease in fibroblast apoptosis and increased proliferation when TNF-α was blocked (p < 0.05). Although decreased fibroblast proliferation and enhanced FOXO1 activity were investigated in type 2 diabetes, they may also be implicated in type 1 diabetes. In vitro, TNF-α enhanced mRNA levels of gene sets related to apoptosis and Akt and p53 but not mitochondrial or cell-cycle pathways. FOXO1 small interfering RNA reduced gene sets that regulate apoptosis, Akt, mitochondrial and cell-cycle pathways. TNF-α also increased genes involved in inflammation, cytokine, Toll-like receptor and nuclear factor-kB pathways, which were significantly reduced by FOXO1 knockdown.Conclusions/interpretationThese studies indicate that TNF-α dysregulation in diabetic wounds impairs healing, which may involve enhanced fibroblast apoptosis and decreased proliferation. In vitro, TNF-α induced gene sets through FOXO1 that regulate a number of pathways that could influence inflammation and apoptosis.

FOXO1 Plays an Important Role in Enhanced Microvascular Cell Apoptosis and Microvascular Cell Loss in Type 1 and Type 2 Diabetic Rats

OBJECTIVE To investigate early events leading to microvascular cell loss in diabetic retinopathy. RESEARCH DESIGN AND METHODS FOXO1 was tested in vivo by DNA binding activity and by nuclear translocation in microvascular cells in retinal trypsin digests. In vivo studies were undertaken in STZ-induced diabetic rats and Zucker diabetic fatty rats using the tumor necrosis factor (TNF)-specific blocker, pegsunercept, or by inhibiting FOXO1 with RNAi. Microvascular cell apoptosis, formation of pericyte ghosts, and acellular capillaries were measured. Upstream and downstream effects of high-glucose–induced FOXO1 were tested on rat microvascular endothelial cells (RMECs) by small-interfering RNA (siRNA) in vitro. RESULTS DNA binding or nuclear translocation of FOXO1, which was reduced by TNF inhibition, was elevated in type 1 and type 2 diabetic retinas. Diabetes stimulated microvascular cell apoptosis; pericyte ghost and acellular capillary development was inhibited by FOXO1 siRNA. High glucose in vitro decreased FOXO1 phosphorylation and DNA binding activity and decreased Akt phosphorylation in RMECs. High-glucose–stimulated FOXO1 DNA binding activity was mediated through TNF-α and formation of reactive oxygen species (ROS), while inhibitors of TNF and ROS and FOXO1 siRNA reduced high-glucose–enhanced RMEC apoptosis. The caspase-3/7 activity and capacity of high glucose to increase mRNA levels of several genes that regulate RMEC activation and apoptosis were knocked down by FOXO1 siRNA. CONCLUSIONS FOXO1 plays an important role in rat retinal microvascular cell loss in type 1 and type 2 diabetic rats and can be linked to the effect of high glucose on FOXO1 activation.

Altered Fibroblast Proliferation and Apoptosis in Diabetic Gingival Wounds

Although it is known that diabetes impairs oral wound healing, relatively little is known about the cellular parameters affected, particularly in connective tissue. This study investigated the hypothesis that diabetes impairs connective tissue formation in healing gingiva, and that impaired healing is associated with factors that decrease fibroblast numbers. Full-thickness wounds were created in the palatal gingiva of type 1 and type 2 diabetic and normoglycemic mice. Five days after wounding, diabetic mice had less epithelial wound coverage, less new connective tissue formation, and reduced fibroblast density (p < 0.05). This occurred with increased numbers of caspase-3- and TUNEL-positive fibroblasts, decreased fibroblast proliferation, increased nuclear translocation of the pro-apoptotic transcription factor FOXO1, and increased numbers of polymorphonuclear leukocytes, all of which were significant (p < 0.05). The results suggest that diabetes may decrease fibroblast numbers through increased apoptosis and reduced proliferation, both of which may be mediated through increased activation of FOXO1.

Activation of the Acquired Immune Response Reduces Coupled Bone Formation in Response to a Periodontal Pathogen

Osteoimmunolgy involves the interaction of the immune system with skeletal elements. This interaction can lead to the formation of osseous lesions. To investigate how the acquired immune response could contribute to osteolytic lesions, we injected the periodontal pathogen Porphyromonas gingivalis adjacent to calvarial bone with or without prior immunization against the bacterium. Activation of the acquired immune response increased osteoclastogenesis and decreased coupled bone formation. The latter was accompanied by an increase in nuclear translocation of the transcription factor FOXO1 in vivo, increased apoptosis of bone-lining cells measured by the TUNEL assay and number of activated caspase-3 positive cells and a decrease in bone lining cell density. Further studies were conducted with MC3T3 osteoblastic cells. Apoptosis and increased FOXO1 DNA binding activity were induced when a combination of cytokines was tested, IL-β, TNF-α, and IFN-γ. Knockdown of FOXO1 by small interfering RNA significantly reduced cytokine stimulated apoptosis, cleaved caspase-3/7 activity and decreased mRNA levels of the proapoptotic genes, TNF-α, FADD, and caspase-3, -8, and -9. These results indicate that activation of the acquired immunity by a periodontal pathogen reduces the coupling of bone formation and resorption. This may occur by enhancing bone lining cell apoptosis through a mechanism that involves increased FOXO1 activation. These studies give insight into inflammatory bone diseases such as periodontal disease and arthritis were the formation of lytic lesions occurs in conjunction with deficient bone formation and activation of an acquired immune response.

Inhibition of Experimental Periodontitis by a Topical Boron-based Antimicrobial

AN0128 is a boron-containing compound with antibacterial and anti-inflammatory properties. To test its potential effectiveness in treating periodontal disease, we induced experimental periodontitis in the rat by placing ligatures and assessed the impact of AN0128 and positive and negative controls by micro-CT and histologic measurements. The formation of an inflammatory infiltrate was measured in hematoxylin-and-eosin-stained sections. Daily application of AN0128 (1%) compared with controls reduced bone loss by 38 to 44% (P < 0.05), while vehicle alone had no effect (P > 0.05). The reduction in bone loss with AN0128 was similar to that achieved with a NSAID, ketorolac, and Total toothpaste containing triclosan. AN0128 also reduced the level of gingival inflammation 42% compared with the ligature only (P < 0.05), whereas vehicle alone had no effect (P > 0.05). The results indicate that AN0128 significantly reduces the formation of an inflammatory infiltrate and reduces bone loss, measured histologically and by micro-CT.

Immunization Enhances Inflammation and Tissue Destruction in Response to Porphyromonas gingivalis

ABSTRACT It is well established that host-bacterium interactions play a critical role in the initiation and progression of periodontal diseases. By the use of inhibitors, it has been shown that mediators associated with the innate immune response significantly contribute to the disease process. Less is known regarding the role of the acquired immune response. To investigate mechanisms by which the acquired immune response to Porphyromonas gingivalis could affect connective tissue, we used a well-documented calvarial model to study host-bacterium interactions. Injection of P. gingivalis stimulated gamma interferon, interleukin 6, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 expression as determined by real-time PCR. Prior immunization against P. gingivalis significantly enhanced the mRNA levels of these cytokines and chemokines. Similarly, immunization significantly increased and prolonged the formation of a polymorphonuclear leukocyte and mononuclear cell infiltrate (P < 0.05). In addition, the area of connective tissue destruction, osteoclastogenesis, bone loss, mRNA expression of proapoptotic genes, and degree of fibroblast apoptosis were increased in immunized mice (P < 0.05). These results indicate that activation of the acquired immunity by P. gingivalis increases the inflammatory and destructive responses which occur in part through up-regulating the innate immune response and enhancing osteoclastogenesis and fibroblast apoptosis.

Fibroblast apoptosis induced by Porphyromonas gingivalis is stimulated by a gingipain and caspase-independent pathway that involves apoptosis-inducing factor

Porphyromonas gingivalis is an oral bacterium that causes pathology in a number of dental infections that are associated with increased fibroblast cell death. Studies presented here demonstrated that P. gingivalis stimulates cell death by apoptosis rather than necrosis. Unlike previous studies apoptosis was induced independent of proteolytic activity and was also independent of caspase activity because a pancaspase inhibitor, Z‐VAD‐fmk, had little effect. Moreover, P. gingivalis downregulated caspase‐3 mRNA levels and caspase‐3 activity. The consequence of this downregulation was a significant reduction in tumour necrosis factor‐α‐induced apoptosis, which is caspase‐3‐dependent. Immunofluorescence and immunoblot analysis revealed P. gingivalis‐induced translocation of apoptosis‐inducing factor (AIF) from the cytoplasm to the nucleus. siRNA studies were undertaken and demonstrated that P. gingivalis stimulated cell death was significantly reduced when AIF was silenced (P < 0.05). Treatment of human gingival fibroblasts with H‐89, a protein kinase A inhibitor that blocks AIF activation also reduced P. gingivalis‐induced apoptosis (P < 0.05). These results indicate that P. gingivalis causes fibroblast apoptosis through a pathway that involves protein kinase A and AIF, is not dependent upon bacterial proteolytic activity and is also independent of the classic apoptotic pathways involving caspase‐3.