AGEs induce caspase-mediated apoptosis of rat BMSCs via TNFα production and oxidative stress

Abstract

Diabetic humans and animals exhibit lower bone mass and healing, resulting from diminished bone formation. We have recently reported that type 1 diabetic rats have fewer bone marrow osteoprogenitor cells, and since the formation of advanced glycation end products (AGEs) in bone increases in diabetes, we explored possible mechanisms involved in AGE-induced apoptosis of rat bone marrow stromal cells (BMSCs). BMSCs isolated from 4-month-old rats were exposed to 10-400 mg/ml AGE-BSA for 16 h and apoptosis was quantified with PI/annexin V staining and flow cytometry. Signaling mechanisms were evaluated by preincubating the cells with appropriate inhibitors. The formation of reactive oxygen species (ROS) was quantified by flow cytometric analysis of DCFDA fluorescence and the expression of genes by RT-PCR analysis. AGE-BSA at a concentration of 400 mg/ml increased the apoptosis of BMSCs two-to threefold, an effect completely blocked by a pan-caspase inhibitor. BSA or high concentrations of glucose had no effect. AGE-BSA-induced BMSC apoptosis was attenuated by a p38 inhibitor but not by an NF-kB inhibitor. Treatment with AGE-BSA induced the expression of several pro-apoptotic ligands and receptors, most notably tumor necrosis factor a (TNFa), TRAIL, lymphotoxin alpha, CD40, and TNFR2. Furthermore, AGE-BSA-induced apoptosis was completely blocked by pirfenidone, an inhibitor of TNFa production/secretion. Finally, AGE-BSA increased the production of ROS in BMSCs, and its apoptogenic effect was blocked by the antioxidant N-acetylcysteine (N-acetyl-L-cysteine). Thus, AGE-BSA increases the apoptosis of rat BMSCs via the activation of caspases, involving TNFa production/ secretion, p38 MAPK signaling, and oxidative stress. We propose that increased protein glycation, such as that occurring under hyperglycemia, causes the apoptosis of BMSCs, which might significantly contribute to the development of osteopenia in diabetic animals.

Autophagy protects osteoblasts from advanced glycation end products-induced apoptosis through intracellular reactive oxygen species

“…Many apoptosisrelated signaling pathways participate in this process, including activation of MAPKs and increasing activation of caspase-3, -8, and -9 (Alikhani et al 2007, Weinberg et al 2014. Consistent with previous studies, apoptosis was also induced in AGEs-treated osteoblastic MC3T3-E1 cells in this study.…”

“…Human and animal experiments have shown that AGEs accumulate dramatically in bone tissue during diabetes, and are associated with bone cell dysfunction as well as bone tissue fragility (Santana et al 2003, Yoon et al 2004, Hein et al 2006, Momma et al 2012, Fajardo et al 2014, Hofbauer et al 2016. Several studies have demonstrated that AGEs can induce apoptosis in bone cells through MAPK, p38, caspase-8, and caspase-9 signaling pathways; however, this apoptotic response cannot fully explain the susceptibility of diabetic patients with normal BMD to fracture (Alikhani et al 2007, Weinberg et al 2014, Tanaka et al 2015.…”

Progenitor Cell Dysfunctions Underlie Some Diabetic Complications

“…Predominantly, changes in ROS and hypoxia from neighboring cells in the presence of excessive glucose alter juxtacrine and paracrine signaling to the stem cells. 77,78 Moreover, diabetes causes an influx of inflammatory cells and stimulates adipocyte production, altering the stem cell microenvironment. 79 These molecular changes are addressed in this section.…”

“…Pretreatment of hyperglycemia-exposed bone marrow MSCs with pirfenidone, a tumor necrosis factor-a blocker, and N-acetyl-L-cysteine can counter ROS-induced inflammation and progenitor apoptosis through modulation of AGEs. 78 Taken together, preclinical data suggest that enriched autologous progenitor cell subsets preconditioned in vitro to metabolically withstand a high-glucose, high-ROS environment are a promising approach for treating diabetic complications. In the event where sufficient numbers of critical autologous progenitor subsets cannot be obtained, readily available banked allogenic progenitors from adult bone marrow, adipose tissue, or blood, or from umbilical cord blood or placenta, might serve as alternative sources of cell-based therapy.…”

Etomidate deteriorates the toxicity of advanced glycation end products to human endothelial Eahy926 cells

“…AGE treatment promotes a significant release of activators of caspase-3, including Cyt c (cytochrome c) and ROS (reactive oxygen species) from mitochondria to cytoplasmic compartment, which then activates caspase-3 (Weinberg et al, 2014). To elucidate the apoptosis induced by Glu-BSA and etomidate treatment, we ana- lyzed the Cyt c release, activated caspase 3 level and the lysis of poly-ADP-ribose polymerase (PARP) by caspase 3 with western blot assay.…”