FOXO1 mediates RANKL-induced osteoclast formation and activity.

We have previously shown that the transcription factor FOXO1 is elevated in conditions with high levels of bone resorption. To investigate the role of FOXO1 in the formation of osteoclasts, we examined mice with lineage-specific deletion of FOXO1 in osteoclast precursors and by knockdown of FOXO1 with small interfering RNA. The receptor activator for NF-κB ligand (RANKL), a principal bone-resorbing factor, induced FOXO1 expression and nuclear localization 2 d after stimulation in bone marrow macrophages and RAW264.7 osteoclast precursors. RANKL-induced osteoclast formation and osteoclast activity was reduced in half in vivo and in vitro with lineage-specific FOXO1 deletion (LyzM.Cre(+)FOXO1(L/L)) compared with matched controls (LyzM.Cre(-)FOXO1(L/L)). Similar results were obtained by knockdown of FOXO1 in RAW264.7 cells. Moreover, FOXO1-mediated osteoclast formation was linked to regulation of NFATc1 nuclear localization and expression as well as a number of downstream factors, including dendritic cell-specific transmembrane protein, ATP6vod2, cathepsin K, and integrin αv. Lastly, FOXO1 deletion reduced M-CSF-induced RANK expression and migration of osteoclast precursors. In the present study, we provide evidence that FOXO1 plays a direct role in osteoclast formation by mediating the effect of RANKL on NFATc1 and several downstream effectors. This is likely to be significant because FOXO1 and RANKL are elevated in osteolytic conditions.

Accuracy of cast posts fabricated by the direct and the indirect techniques.

STATEMENT OF PROBLEM: Patterns for custom cast posts and cores can be fabricated either by the direct or the indirect technique. Which technique is more accurate is unknown. PURPOSE: The purpose of this in vitro study was to investigate the effect of pattern fabrication technique on the accuracy of post fit. MATERIAL AND METHODS: Ten intact extracted premolar teeth with a single canal and similar dimensions received root canal treatment. The teeth were sectioned 2 mm above the cementoenamel junction. A total of 20 cast post and core patterns, 2 for each tooth, were fabricated, 10 with the direct technique and 10 with the indirect technique. Patterns were cast to produce 20 cast post and cores. Each tooth was scanned using a microcomputed tomography (µCT) system with a resolution of 14.5 µm, once with the post of the direct technique and once with the post of the indirect technique. Ct analyzer software was used to calculate the overall space between the post and canal walls and the space areas in 3 different standardized sections. The Student paired t test was used to determine any significant difference in the scores of the groups. RESULTS: The overall space between the canal walls and posts made with the direct technique ranged between 7.86 and 17.39 mm(3), with a mean value of 12.25 mm(3), whereas with the indirect technique, the space ranged between 6.68 and 18.02 mm(3), with a mean of 11.92 mm(3). No significant differences were found between the results of either technique (P>.05). CONCLUSIONS: Within the limitations of this study, neither the indirect nor direct pattern fabrication technique influenced the accuracy of post fitting.

Assessing the Inhospital Complications in Patients Postrenal Transplantation, in a Tertiary Care Center, Riyadh, Saudi Arabia.

Complications of chronic kidney disease (CKD) can range from localized to systemic manifestations that can worsen patients' outcomes. CKD results in irreversible deterioration in renal function, which ultimately progresses to end-stage renal failure and necessitates the need for renal transplantation. Our study aimed to identify patients' complications postrenal transplant during hospitalization and assess the main factors affecting these patients' outcomes and survival rates. This study is a single-centered, retrospective cohort chart review conducted from January 2016 to March 2019. The collected data parameters included patients' characteristics (e.g., gender, age, body mass index), as well as surgical-related details and postoperative complications. Microsoft Excel and IBM SPSS Statistics version 22.0 were used for data entry and analysis. The descriptive statistics were presented as frequency and percentage for the categorical variables (e.g., gender and smoking status), while the mean ± standard deviation was used for numerical variables. A total of 80 posttransplant patients who fulfilled the inclusion criteria were recruited. Urogenital complications were the most commonly seen during the postoperative period, especially developing urinary tract infections by 16%. During our study, the rate of complications was considered minimal and not significant in assessing posttransplant patients.

Five-year survival, performance, and neurodevelopmental outcome following cardiopulmonary resuscitation after pediatric cardiac surgery, preliminary investigation in a single-center experience.

BACKGROUND AND AIM: Children who suffer cardiopulmonary arrest (CPA) after cardiac surgery frequently survive with return of spontaneous circulation. However, their neurodevelopmental outcomes and performance are still unclear. The aim of this study is to evaluate the midterm neurodevelopmental outcome and overall performance of children who survived CPA following cardiac surgery. MATERIALS AND METHODS: In this cohort study, we followed-up children who received cardiopulmonary resuscitation (CPR) post cardiac surgery during 2012-2013. We assessed their 5-year survival, functional, and neurodevelopmental outcomes using two performance scales: Pediatric Cerebral Performance Category (PCPC) and Pediatric Overall Performance Category (POPC). Both scales ranged from 1 for normal to 6 for brain death/death. We compared CPR group with a matching group (1:1) that had similar characteristics and conditions but no CPR. RESULTS: Out of 758 postoperative cardiac children, 15 (2%) children had 19 episodes of CPA. Their median age was 10 months (0.5-168). Survival rates were 12/15 (80%) on hospital discharge and 10/15 (66%) after 5 years. Among 12 survivors, two patients (17%) scored 6, one (8%) scored 4, five (42%) scored 2, and four (33%) scored 1 on both PCPC and POPC. The median PCPC and POPC scores were [2, (interquartile range: 1-6) and 1, (interquartile range: 1-3, p = 0.018] for CPR and matching group, respectively. Regression analysis identifies duration of CPR, number of CPR session, and late-occurring CPA as risk factors for poor outcome. CONCLUSION: Two-thirds of children requiring CPR post cardiac surgery survived after 5 years. Their neurodevelopmental and functional evaluation demonstrated worse outcome in comparison with their matching cases. CPR duration, number of CPA events, and late CPA were risk factors for poor outcome. Rehabilitation and special education programs might be needed for these groups of children with special needs.

FOXO1 expression in keratinocytes promotes connective tissue healing.

Wound healing is complex and highly orchestrated. It is well appreciated that leukocytes, particularly macrophages, are essential for inducing the formation of new connective tissue, which requires the generation of signals that stimulate mesenchymal stem cells (MSC), myofibroblasts and fibroblasts. A key role for keratinocytes in this complex process has yet to be established. To this end, we investigated possible involvement of keratinocytes in connective tissue healing. By lineage-specific deletion of the forkhead box-O 1 (FOXO1) transcription factor, we demonstrate for the first time that keratinocytes regulate proliferation of fibroblasts and MSCs, formation of myofibroblasts and production of collagen matrix in wound healing. This stimulation is mediated by a FOXO1 induced TGFß1/CTGF axis. The results provide direct evidence that epithelial cells play a key role in stimulating connective tissue healing through a FOXO1-dependent mechanism. Thus, FOXO1 and keratinocytes may be an important therapeutic target where healing is deficient or compromised by a fibrotic outcome.

FOXO1 Regulates Bacteria-Induced Neutrophil Activity.

Neutrophils play an essential role in the innate immune response to microbial infection and are particularly important in clearing bacterial infection. We investigated the role of the transcription factor FOXO1 in the response of neutrophils to bacterial challenge with Porphyromonas gingivalis in vivo and in vitro. In these experiments, the effect of lineage-specific FOXO1 deletion in LyzM.Cre+FOXO1L/L mice was compared with matched littermate controls. FOXO1 deletion negatively affected several critical aspects of neutrophil function in vivo including mobilization of neutrophils from the bone marrow (BM) to the vasculature, recruitment of neutrophils to sites of bacterial inoculation, and clearance of bacteria. In vitro FOXO1 regulated neutrophil chemotaxis and bacterial killing. Moreover, bacteria-induced expression of CXCR2 and CD11b, which are essential for several aspects of neutrophil function, was dependent on FOXO1 in vivo and in vitro. Furthermore, FOXO1 directly interacted with the promoter regions of CXCR2 and CD11b. Bacteria-induced nuclear localization of FOXO1 was dependent upon toll-like receptor (TLR) 2 and/or TLR4 and was significantly reduced by inhibitors of reactive oxygen species (ROS and nitric oxide synthase) and deacetylases (Sirt1 and histone deacetylases). These studies show for the first time that FOXO1 activation by bacterial challenge is needed to mobilize neutrophils to transit from the BM to peripheral tissues in response to infection as well as for bacterial clearance in vivo. Moreover, FOXO1 regulates neutrophil function that facilitates chemotaxis, phagocytosis, and bacterial killing.

Sustained, localized salicylic acid delivery enhances diabetic bone regeneration via prolonged mitigation of inflammation.

Diabetes is a metabolic disorder caused by insulin resistance and/or deficiency and impairs bone quality and bone healing due to altered gene expression, reduced vascularization, and prolonged inflammation. No effective treatments for diabetic bone healing are currently available, and most existing treatments do not directly address the diabetic complications that impair bone healing. We recently demonstrated that sustained and localized delivery of salicylic acid (SA) via an SA-based polymer provides a low-cost approach to enhance diabetic bone regeneration. Herein, we report mechanistic studies that delve into the biological action and local pharmacokinetics of SA-releasing polymers shown to enhance diabetic bone regeneration. The results suggest that low SA concentrations were locally maintained at the bone defect site for more than 1 month. As a result of the sustained SA release, a significantly reduced inflammation was observed in diabetic animals, which in turn, yielded reduced osteoclast density and activity, as well as increased osteoblastogenesis. Based upon these results, localized and sustained SA delivery from the SA-based polymer effectively improved bone regeneration in diabetic animals by affecting both osteoclasts and osteoblasts, thereby providing a positive basis for clinical treatments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2595-2603, 2016.

FOXO1 differentially regulates both normal and diabetic wound healing.

Healing is delayed in diabetic wounds. We previously demonstrated that lineage-specific Foxo1 deletion in keratinocytes interfered with normal wound healing and keratinocyte migration. Surprisingly, the same deletion of Foxo1 in diabetic wounds had the opposite effect, significantly improving the healing response. In normal glucose media, forkhead box O1 (FOXO1) enhanced keratinocyte migration through up-regulating TGFß1. In high glucose, FOXO1 nuclear localization was induced but FOXO1 did not bind to the TGFß1 promoter or stimulate TGFß1 transcription. Instead, in high glucose, FOXO1 enhanced expression of serpin peptidase inhibitor, clade B (ovalbumin), member 2 (SERPINB2), and chemokine (C-C motif) ligand 20 (CCL20). The impact of high glucose on keratinocyte migration was rescued by silencing FOXO1, by reducing SERPINB2 or CCL20, or by insulin treatment. In addition, an advanced glycation end product and tumor necrosis factor had a similar regulatory effect on FOXO1 and its downstream targets and inhibited keratinocyte migration in a FOXO1-dependent manner. Thus, FOXO1 expression can positively or negatively modulate keratinocyte migration and wound healing by its differential effect on downstream targets modulated by factors present in diabetic healing.

Osteoblast Lineage Cells Play an Essential Role in Periodontal Bone Loss Through Activation of Nuclear Factor-Kappa B.

Bacterial pathogens stimulate periodontitis, the most common osteolytic disease in humans and the most common cause of tooth loss in adults. Previous studies identified leukocytes and their products as key factors in this process. We demonstrate for the first time that osteoblast lineage cells play a critical role in periodontal disease. Oral infection stimulated nuclear localization of NF-κB in osteoblasts and osteocytes in the periodontium of wild type but not transgenic mice that expressed a lineage specific dominant negative mutant of IKK (IKK-DN) in osteoblast lineage cells. Wild-type mice were also susceptible to bacteria induced periodontal bone loss but transgenic mice were not. The lack of bone loss in the experimental group was linked to reduced RANKL expression by osteoblast lineage cells that led to diminished osteoclast mediated bone resorption and greater coupled new bone formation. The results demonstrate that osteoblast lineage cells are key contributors to periodontal bone loss through an NF-κB mediated mechanism.

Foxo1 inhibits diabetic mucosal wound healing but enhances healing of normoglycemic wounds.

Re-epithelialization is an important part in mucosal wound healing. Surprisingly little is known about the impact of diabetes on the molecular events of mucosal healing. We examined the role of the transcription factor forkhead box O1 (Foxo1) in oral wounds of diabetic and normoglycemic mice with keratinocyte-specific Foxo1 deletion. Diabetic mucosal wounds had significantly delayed healing with reduced cell migration and proliferation. Foxo1 deletion rescued the negative impact of diabetes on healing but had the opposite effect in normoglycemic mice. Diabetes in vivo and in high glucose conditions in vitro enhanced expression of chemokine (C-C motif) ligand 20 (CCL20) and interleukin-36γ (IL-36γ) in a Foxo1-dependent manner. High glucose-stimulated Foxo1 binding to CCL20 and IL-36γ promoters and CCL20 and IL-36γ significantly inhibited migration of these cells in high glucose conditions. In normal healing, Foxo1 was needed for transforming growth factor-ß1 (TGF-ß1) expression, and in standard glucose conditions, TGF-ß1 rescued the negative effect of Foxo1 silencing on migration in vitro. We propose that Foxo1 under diabetic or high glucose conditions impairs healing by promoting high levels of CCL20 and IL-36γ expression but under normal conditions, enhances it by inducing TGF-ß1. This finding provides mechanistic insight into how Foxo1 mediates the impact of diabetes on mucosal wound healing.