Diabetes increases the likelihood of fracture, interferes with fracture healing and impairs angiogenesis. The latter may be significant due to the critical nature of angiogenesis in fracture healing. Although it is known that diabetes interferes with angiogenesis the mechanisms remain poorly defined. We examined fracture healing in normoglycemic and streptozotocin-induced diabetic mice and quantified the degree of angiogenesis with antibodies to three different vascular markers, CD34, CD31 and Factor VIII. The role of diabetes-enhanced inflammation was investigated by treatment of the TNFα-specific inhibitor, pegsunercept starting 10 days after induction of fractures. Diabetes decreased both angiogenesis and VEGFA expression. The reduced angiogenesis and VEGFA expression in diabetic fractures was rescued by specific inhibition of TNF in vivo. In addition, the TNF inhibitor rescued the negative effect of diabetes on endothelial cell proliferation and endothelial cell apoptosis. The effect of TNFα in vitro was enhanced by high glucose and an advanced glycation endproduct to impair microvascular cell proliferation and stimulate apoptosis. The effect of TNF, high glucose and an AGE was mediated by the transcription factor FOXO1, which increased expression of p21 and caspase-3. These studies indicate that inflammation plays a major role in diabetes-impaired angiogenesis in endochondral bone formation through its effect on microvascular endothelial cells and FOXO1.

Type 1 diabetes impairs fracture healing. We tested the hypothesis that diabetes affects chondrocytes to impair fracture healing through a mechanism that involves the transcription factor FOXO1. Type 1 diabetes was induced by streptozotocin in mice with FOXO1 deletion in chondrocytes (Col2α1Cre+.FOXO1L/L) or littermate controls (Col2α1Cre−.FOXO1L/L) and closed femoral fractures induced. Diabetic mice had 77% less cartilage and 30% less bone than normoglycemics evaluated histologically and by micro-computed tomography. Both were reversed with lineage-specific FOXO1 ablation. Diabetic mice had a threefold increase in osteoclasts and a two- to threefold increase in RANKL mRNA or RANKL-expressing chondrocytes compared with normoglycemics. Both parameters were rescued by FOXO1 ablation in chondrocytes. Conditions present in diabetes, high glucose (HG), and increased advanced glycation end products (AGEs) stimulated FOXO1 association with the RANKL promoter in vitro, and overexpression of FOXO1 increased RANKL promoter activity in luciferase reporter assays. HG and AGE stimulated FOXO1 nuclear localization, which was reversed by insulin and inhibitors of TLR4, histone deacetylase, nitric oxide, and reactive oxygen species. The results indicate that chondrocytes play a prominent role in diabetes-impaired fracture healing and that high levels of glucose, AGEs, and tumor necrosis factor-α, which are elevated by diabetes, alter RANKL expression in chondrocytes via FOXO1.

Chondrocytes play an essential role in fracture healing by producing cartilage, which forms an anlage for endochondral ossification that stabilizes the healing fracture callus. More recently it has been appreciated that chondrocytes have the capacity to produce factors that may affect the healing process. We examined the role of chondrocytes in angiogenesis during fracture healing and the role of the transcription factor forkhead box‐O 1 (FOXO1), which upregulates wound healing in soft tissue. Closed fractures were induced in experimental mice with lineage‐specific FOXO1 deletion by Cre recombinase under the control of a collagen‐2α1 promoter element (Col2α1Cre+FOXO1L/L) and Cre recombinase negative control littermates containing flanking loxP sites (Col2α1Cre–FOXO1L/L). Experimental mice had significantly reduced CD31+ new vessel formation. Deletion of FOXO1 in chondrocytes in vivo suppressed the expression of vascular endothelial growth factor‐A (VEGFA) at both the protein and mRNA levels. Overexpression of FOXO1 in chondrocytes in vitro increased VEGFA mRNA levels and VEGFA transcriptional activity whereas silencing FOXO1 reduced it. Moreover, FOXO1 interacted directly with the VEGFA promoter and a deacetylated FOXO1 mutant enhanced VEGFA expression whereas an acetylated FOXO1 mutant did not. Lastly, FOXO1 knockdown by siRNA significantly reduced the capacity of chondrocytes to stimulate microvascular endothelial cell tube formation in vitro. The results indicate that chondrocytes play a key role in angiogenesis which is FOXO1 dependent and that FOXO1 in chondrocytes regulates a potent angiogenic factor, VEGFA. These studies provide new insight into fracture healing given the important role of vessel formation in the fracture repair process. © 2018 American Society for Bone and Mineral Research.

Osteocyte plays an essential role in bone metabolism by regulating osteoblast and osteoclast activities. Dysfunction or apoptosis of osteocyte will severely endanger the bone homeostasis and result in bone diseases such as osteoporosis. Osteoporosis has been considered as one of the diabetes complications; however, the mechanism is still to be discovered. Advanced glycation end products (AGEs), as the main pathogenic factor of diabetes mellitus, have the capacity to induce osteocyte apoptosis thus sabotaging bone homeostasis. Here, we examined the role of AGE during osteocyte apoptosis and how this effect would affect osteocyte's regulation of osteoblast and osteoclast. Mouse osteocyte-like MLO-Y4 cells were used to study the properties of osteocyte and to examine its biological and pathological function. MTT assay and Annexin V assay showed that AGE significantly induce MLO-Y4 cell apoptosis. qPCR and Western blot results have shown that AGE upregulates proapoptotic gene p53 and its downstream target gene Bax, which leads to enhanced activation of caspase-3, thus inducing apoptosis in MLO-Y4 cells. Increased expression of sclerostin and RANKL in osteocytes has shown that AGE induces osteocyte dysfunction thus severely damaging the bone homeostasis by decreasing osteoblast and increasing osteoclast activities. Furthermore, the role of the transcription factor FOXO1, which is intensely associated with apoptosis, has been determined. Western blot has shown that AGE significantly decreases Akt activities. Immunofluorescence has shown that AGE promotes FOXO1 nuclei localization and enhances FOXO1 expression. Silencing of FOXO1 suppressed AGE-enhanced apoptosis; mRNA and protein expressions of cleaved caspase-3, sclerostin, and RANKL were downregulated as well. Moreover, exogenous FOXO1 increased caspase-3 mRNA levels and caspase-3 transcriptional activity. Lastly, ChIP assay has established the capacity of FOXO1 binding directly on the caspase-3, sclerostin, and RANKL promoter region in AGE environment, providing the mechanism of the AGE-induced osteocyte apoptosis and dysfunction. Our results have shown that FOXO1 plays a crucial role in AGE-induced osteocyte dysfunction and apoptosis through its regulation of caspase-3, sclerostin, and RANKL. This study provides new insight into diabetes-enhanced risk of osteoporosis given the critical role of AGE in the pathogenesis of diabetes and the essential part of osteocyte in bone metabolism.