Mani Alikhani

Diabetes-enhanced Inflammation and Apoptosis—Impact on Periodontal Pathology

Diabetes, particularly type 2 diabetes, is a looming health issue with many ramifications. Because diabetes alters the cellular microenvironment in many different types of tissues, it causes myriad untoward effects, collectively referred to as ’diabetic complications’. Two cellular processes affected by diabetes are inflammation and apoptosis. This review discusses how diabetes-enhanced inflammation and apoptosis may affect the oral environment. In particular, dysregulation of tumor necrosis factor and the formation of advanced glycation products, both of which occur at higher levels in diabetic humans and animal models, potentiate inflammatory responses and induce apoptosis of matrix-producing cells. The enhanced loss of fibroblasts and osteoblasts through apoptosis in diabetics could contribute to limited repair of injured tissue, particularly when combined with other known deficits in diabetic wound-healing. These findings may shed light on diabetes-enhanced risk of periodontal diseases.

Advanced Glycation End Products Enhance Expression of Pro-apoptotic Genes and Stimulate Fibroblast Apoptosis through Cytoplasmic and Mitochondrial Pathways

Both aging and diabetes are characterized by the formation of advanced glycation end products (AGEs). Both exhibit other similarities including deficits in wound healing that are associated with higher rates of fibroblast apoptosis. In order to investigate a potential mechanism for enhanced fibroblast apoptosis in diabetes and aged individuals, experiments were carried out to determine whether the predominant advanced glycation end product in skin, N-ϵ-(carboxymethyl) lysine (CML)-collagen, could induce fibroblast apoptosis. In vivo experiments established that CML-collagen but not unmodified collagen induced fibroblast apoptosis and that apoptosis was dependent upon caspase-3, -8, and -9 activity. In vitro experiments demonstrated that CML-collagen but not control collagen induced a time- and dose-dependent increase in fibroblast apoptosis. By use of blocking antibodies, apoptosis was shown to be mediated through receptor for AGE signaling. AGE-induced apoptosis was largely dependent on the effector caspase, caspase-3, which was activated through both cytoplasmic (caspase-8-dependent) and mitochondrial (caspase-9) pathways. CML-collagen had a global effect of enhancing mRNA levels of pro-apoptotic genes that included several classes of molecules including ligands, receptors, adaptor molecules, mitochondrial proteins, and others. However, the pattern of expression was not identical to the pattern of apoptotic genes induced by tumor necrosis factor α.

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 Functions as a Master Switch That Regulates Gene Expression Necessary for Tumor Necrosis Factor-induced Fibroblast Apoptosis

Tumor necrosis factor-α (TNF-α) is a potent pro-inflammatory and pro-apoptotic mediator that plays an important role in several normal and disease processes. TNF-induced cell death is one of the principal mechanisms by which cells are removed. Although TNF-mediated apoptosis has been the subject of intense investigation, the transcriptional mechanisms through which it promotes apoptosis are not well understood and, paradoxically, the archetypal TNF-induced nuclear factor NFκB is anti-apoptotic. To identify a potential master transcriptional regulator of apoptosis, we examined an array of TNF-α-activated transcription factors. Fork-head box class-O 1 (FOXO1) was strongly activated, which was confirmed in vitro and in vivo by electrophoretic mobility shift assay. The central importance of FOXO1 was established in experiments with small inhibitory RNA (siRNA) that specifically silenced FOXO1. When FOXO1 was silenced, fibroblast apoptosis was reduced 76%. Other siRNAs that partially inhibited FOXO1 expression were proportionately effective in reducing apoptosis. Transcriptional profiling was then carried out in conjunction with siRNA to establish mechanisms by which FOXO1 modulated apoptosis. In the absence of FOXO1, TNF-α failed to up-regulate a large number of pro-apoptotic gene families including ligands, receptors, adapter molecules, mitochondrial proteins, and caspases. siRNA silencing also blocked down-regulation of anti-apoptotic genes. These results indicate that TNF induces activation of the FOXO1 transcription factor, which acts as a master switch to control apoptosis.

Effect of micro-osteoperforations on the rate of tooth movement

INTRODUCTION Our objectives were to study the effect of micro-osteoperforations on the rate of tooth movement and the expression of inflammatory markers. METHODS Twenty adults with Class II Division 1 malocclusion were divided into control and experimental groups. The control group did not receive micro-osteoperforations, and the experimental group received micro-osteoperforations on 1 side of the maxilla. Both maxillary canines were retracted, and movement was measured after 28 days. The activity of inflammatory markers was measured in gingival crevicular fluid using an antibody-based protein assay. Pain and discomfort were monitored with a numeric rating scale. RESULTS Micro-osteoperforations significantly increased the rate of tooth movement by 2.3-fold; this was accompanied by a significant increase in the levels of inflammatory markers. The patients did not report significant pain or discomfort during or after the procedure, or any other complications. CONCLUSIONS Micro-osteoperforation is an effective, comfortable, and safe procedure to accelerate tooth movement and significantly reduce the duration of orthodontic treatment.

Foxo1, a Novel Regulator of Osteoblast Differentiation and Skeletogenesis

Skeletogenesis depends on the activity of bone-forming cells derived from mesenchymal cells. The pathways that control mesenchymal cell differentiation are not well understood. We propose that Foxo1 is an early molecular regulator during mesenchymal cell differentiation into osteoblasts. In mouse embryos, Foxo1 expression is higher in skeletal tissues, while Foxo1 silencing has a drastic impact on skeletogenesis and craniofacial development, specially affecting pre-maxilla, nasal bone, mandible, tibia, and clavicle. Similarly, Foxo1 activity and expression increase in mouse mesenchymal cells under the influence of osteogenic stimulants. In addition, silencing Foxo1 blocks the expression of osteogenic markers such as Runx2, alkaline phosphatase, and osteocalcin and results in decreased culture calcification even in the presence of strong osteogenic stimulants. Conversely, the expression of these markers increases significantly in response to Foxo1 overexpression. One mechanism through which Foxo1 affects mesenchymal cell differentiation into osteoblasts is through regulation of a key osteogenic transcription factor, Runx2. Indeed, our results show that Foxo1 directly interacts with the promoter of Runx2 and regulates its expression. Using a tibia organ culture model, we confirmed that silencing Foxo1 decreases the expression of Runx2 and impairs bone formation. Furthermore, our data reveals that Runx2 and Foxo1 interact with each other and cooperate in the transcriptional regulation of osteoblast markers. In conclusion, our in vitro, ex vivo, and in vivo results strongly support the notion that Foxo1 is an early molecular regulator in the differentiation of mesenchymal cells into osteoblast.

TNF-α in vivo stimulates apoptosis in fibroblasts through caspase-8 activation and modulates the expression of pro-apoptotic genes

Apoptosis of matrix producing cells is common among many inflammatory diseases. The goal of the present study was to examine the apoptotic effects of tumor necrosis factor‐α (TNF‐α) on fibroblastic cells in vivo and to investigate the role of different caspases in this process. This was accomplished in vivo by subcutaneous injection of TNF‐α in mice. The direct effects of TNF‐α on fibroblast apoptosis were studied in vitro with normal diploid human fibroblasts. The results indicate that TNF‐α in vivo induces apoptosis of fibroblasts. By RNase protection assay, we demonstrated that TNF‐α stimulates expression of 12 apoptotic genes. Fluorometric studies demonstrated that TNF‐α in vivo predominantly increased caspase‐8 and ‐3 activity and by use of specific inhibitors, the activation of caspase‐3 was shown to be initiated by caspase‐8 with only a minor contribution from caspase‐9. Thus, TNF‐α acts to modulate the expression of many genes that favors apoptosis of fibroblastic cells, which is dependent mostly upon signaling through caspase‐8.

Lipopolysaccharides Indirectly Stimulate Apoptosis and Global Induction of Apoptotic Genes in Fibroblasts

Following Gram-negative bacterial infection there is a reduction in matrix-producing cells. The goal of the present study was to examine the apoptotic effects of lipopolysaccharide (LPS) on fibroblastic cells and to investigate the role that the host response plays in this reaction. This was accomplished in vivo by subcutaneous inoculation of LPS in wild type and TNFR1-/-R2-/- mice. The direct effects of LPS on fibroblast apoptosis was studied in vitro with normal diploid human fibroblasts. The results indicate that LPS in vivo induces apoptosis of fibroblasts. By RNA profiling we demonstrated that LPS stimulates global expression of apoptotic genes and down-regulates anti-apoptotic genes. Fluorometric studies demonstrated that LPS in vivo significantly increased caspase-8 and caspase-3 activity and by use of specific inhibitors, the activation of caspase-3 was shown to be initiated by caspase-8 with no contribution from caspase-9. In vitro studies demonstrated that LPS did not induce apoptosis of fibroblasts, whereas tumor necrosis factor (TNF) did. In addition, the pattern of apoptotic gene expression induced by TNF in vitro was nearly identical to that induced by LPS in vivo, as measured by RNase protection assay. Moreover, pre-treatment of cells with TNF greatly enhanced apoptosis induced by a second stimulation with TNF 24 h later, suggesting that the global induction of pro-apoptotic genes was functionally significant. Thus, LPS acts to modulate the expression of a large number of genes that favor apoptosis of fibroblastic cells that is dependent upon activation of caspase-8 and is largely mediated by TNF.

The transcription factor ST18 regulates proapoptotic and proinflammatory gene expression in fibroblasts

Suppression of tumorigenicity 18 (ST18) and the homologues neural zinc‐finger protein‐3 (NZF3) and myelin transcription factor 3 (Myt3) are transcription factors with unknown function. Previous studies have established that they repress transcription of a synthetic reporter construct consisting of the consensus sequence AAAGTTT linked to the thymidine kinase promoter. In addition, ST18 exhibits significantly reduced expression in breast cancer and breast cancer cell lines. We report here for the first time evidence that ST18 mediates tumor necrosis factor (TNF)‐α induced mRNA levels of proapoptotic and proinflammatory genes in fibroblasts by mRNA profiling and silencing with ST18 small interfering RNA (siRNA). Gene set enrichment analysis and mRNA profiling support this conclusion by identifying several apoptotic and inflammatory pathways that are downregulated by ST18 siRNA. In addition, ST18 siRNA reduces TNF‐induced fibroblast apoptosis and caspase3/7 activity. Fibroblasts that overexpress ST18 by transient transfection exhibit significantly increased apoptosis and increased expression of TNF‐α, interleukin (IL)‐1α, and IL‐6. In addition, cotransfection of ST18 and a TNF‐α or IL‐1α reporter construct demonstrates that ST18 overexpression in fibroblasts significantly enhanced promoter activity of these genes. Taken together, these studies demonstrate that the transcription factor ST18/NZF3 regulates the mRNA levels of proapoptotic and proinflammatory genes in revealing a previously unrecognized function.— Yang, J., Siqueira, M. F., Behl, Y., Alikhani, M., and Graves, D. T. The transcription factor ST18 regulates proapoptotic and proinflammatory gene expression in fibroblasts. FASEB J. 22, 3956–3967 (2008)

Apoptotic Effects of LPS on Fibroblasts are Indirectly Mediated through TNFR1

During periods of periodontal attachment loss, one of the most significant cellular changes is a decrease in the number of fibroblasts. We previously demonstrated that LPS induces apoptosis of fibroblastic cells in vivo, largely through TNF-α. We conducted in vivo experiments by subcutaneous inoculation of LPS in wild-type, TNFR1−/−R2−/−, TNFR1−/−, and TNFR2−/− mice to identify which TNF receptors are involved and the specific caspase pathway activated. LPS stimulated apoptosis through TNFR1 but not TNFR2, which was accompanied by the induced expression of 12 apoptotic genes. Fluorometric studies demonstrated that LPS in vivo significantly increased caspase-8 and caspase-3 activity, which was also dependent on TNF receptor signaling. By the use of specific caspase inhibitors, caspases-3 and -8 were shown to play an important role in LPS-induced apoptosis in vivo. Thus, LPS acts through TNFR1 to modulate the expression of apoptotic genes and activate caspases-3 and -8.