BACKGROUND Type 2 diabetes impairs the healing process and induces apoptosis of fibroblasts, which are thought to be involved in this process. We investigated the possible mechanisms involved in ...AGEs-induced apoptosis of human dermal fibroblasts. MATERIAL AND METHODS We examined the expression of apoptosis-related proteins in fibroblasts isolated from human diabetic wounds. Human dermal fibroblasts exposed to AGEs were used to study the links among apoptosis, ROS, and NLRP3 inflammasome activation. Signaling mechanisms were evaluated by preincubating the cells with appropriate inhibitors. Cleaved caspase-8, cleaved caspase-3, BAX, Bcl-2, and NLRP3 inflammasome expression were measured by Western blot analysis. ROS generation, cell viability, and cell apoptosis were assessed. RESULTS We observed a higher level of cleaved caspase-8 and cleaved caspase-3 expression in fibroblasts isolated from human diabetic wounds compared with controls. AGEs decreased the proliferation of cells in a concentration-dependent and time-dependent manner. The exposure of fibroblasts to AGEs significantly increased the number of cells in early and late apoptosis stages. AGES-induced human dermal fibroblasts showed high expressions of cleaved caspase3, cleaved caspase8, and Bax. Treatment with AGEs induced the expression of NLRP3, caspase-1, and ASC. AGES-induced apoptosis was blocked by BAY 11-7082, an inhibitor of the NLRP3 inflammasome. AGEs increased the production of ROS in fibroblasts, and its apoptogenic effect was blocked by NAC. CONCLUSIONS AGEs cause apoptosis of fibroblasts by inducing the generation of ROS and activating the NLRP3 inflammasome. In vivo experiments are needed to confirm these results.
Arterial stiffening with increased pulse pressure is a leading risk factor for cardiovascular disease in the elderly. We tested whether ALT-711, a novel nonenzymatic breaker of advanced glycation ...end-product crosslinks, selectively improves arterial compliance and lowers pulse pressure in older individuals with vascular stiffening.
Nine US centers recruited and randomly assigned subjects with resting arterial pulse pressures >60 mm Hg and systolic pressures >140 mm Hg to once-daily ALT-711 (210 mg; n=62) or placebo (n=31) for 56 days. Preexisting antihypertensive treatment (90% of subjects) was continued during the study. Morning upright blood pressure, stroke volume, cardiac output, systemic vascular resistance, total arterial compliance, carotid-femoral pulse wave velocity, and drug tolerability were assessed. ALT-711 netted a greater decline in pulse pressures than placebo (-5.3 versus -0.6 mm Hg at day 56; P=0.034 for treatment effect by repeated-measures ANOVA). Systolic pressure declined in both groups, but diastolic pressure fell less with ALT-711 (P=0.056). Mean pressure declined similarly in both groups (-4 mm Hg; P<0.01 for each group, P=0.34 for treatment effect). Total arterial compliance rose 15% in ALT-711-treated subjects versus no change with placebo (P=0.015 versus ALT-711), an effect that did not depend on reduced mean pressure. Pulse wave velocity declined 8% with ALT-711 (P<0.05 at day 56, P=0.08 for treatment effect). Systemic arterial resistance, cardiac output, and heart rate did not significantly change in either group.
ALT-711 improves total arterial compliance in aged humans with vascular stiffening, and it may provide a novel therapeutic approach for this abnormality, which occurs with aging, diabetes, and isolated systolic hypertension.
The chemical modification of protein by nonenzymatic browning or Maillard reactions increases with age and in disease. Maillard products are formed by reactions of both carbohydrate- and ...lipid-derived intermediates with proteins, leading to formation of advanced glycation and lipoxidation end-products (AGE/ALEs). These modifications and other oxidative modifications of amino acids increase together in proteins and are indicators of tissue aging and pathology. In this review, we describe the major pathways and characteristic products of chemical modification of proteins by carbohydrates and lipids during the Maillard reactions and identify major intersections between these pathways. We also describe a new class of intracellular sulfhydryl modifications, Cys-AGE/ALEs, that may play an important role in regulatory biology and represent a primitive link between nonenzymatic and enzymatic chemistry in biological systems.
Background: Periodontal disease is a major complication of type 1 diabetes mellitus (T1DM). The aim of the present study is to investigate the relationship between glycated hemoglobin and circulating ...levels of interleukin (IL)‐6, IL‐8, and C‐X‐C motif chemokine ligand 5 (CXCL5) in non‐smoking patients suffering from T1DM, with and without periodontitis. In addition, to determine the effect of advanced glycation end products (AGE) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) on IL‐6, IL‐8, and CXCL5 expression by THP‐1 monocytes and OKF6/TERT‐2 cells.
Methods: There were 104 participants in the study: 19 healthy volunteers, 23 patients with periodontitis, 28 patients with T1DM, and 34 patients with T1DM and periodontitis. Levels of blood glucose/glycated hemoglobin (International Federation of Clinical Chemistry IFCC) were determined by high‐performance liquid chromatography. Levels of IL‐6, IL‐8, and CXCL5 in plasma were determined by enzyme‐linked immunosorbent assay (ELISA). In vitro stimulation of OKF6/TERT‐2 cells and THP‐1 monocytes was performed with combinations of AGE and P. gingivalis LPS. Changes in expression of IL‐6, IL‐8, and CXCL5 were monitored by ELISA and real‐time polymerase chain reaction.
Results: Patients with diabetes and periodontitis had higher plasma levels of IL‐8 than patients with periodontitis alone. Plasma levels of IL‐8 correlated significantly with IFCC units, clinical probing depth, and attachment loss. AGE and LPS, alone or in combination, stimulated IL‐6, IL‐8, and CXCL5 expression in both OKF6/TERT‐2 cells and THP‐1 monocytes.
Conclusions: Elevated plasma levels of IL‐8 potentially contribute to the cross‐susceptibility between periodontitis and T1DM. P. gingivalis LPS and AGE in combination caused significantly greater expression of IL‐6, IL‐8, and CXCL5 from THP‐1 monocytes and OKF6/TERT‐2 cells than LPS alone.
OBJECTIVE—S100A12 and fibroblast growth factor 23 are biomarkers of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). We tested the hypothesis that human ...S100/calgranulin would accelerate cardiovascular disease in mice subjected to CKD.
APPROACH AND RESULTS—A bacterial artificial chromosome of the human S100/calgranulin gene cluster containing the genes and regulatory elements for S100A8, S100A9, and S100A12 was expressed in C57BL/6J mouse (hBAC-S100) to generate a novel humanized mouse model. CKD was induced by ureteral ligation, and hBAC-S100 mice and wild-type mice were studied after 10 weeks of chronic uremia. hBAC-S100 mice with CKD showed increased fibroblast growth factor 23 in the hearts, left ventricular hypertrophy, diastolic dysfunction, focal cartilaginous metaplasia, and calcification of the mitral and aortic valve annulus together with aortic valve sclerosis. This phenotype was not observed in wild-type mice with CKD or in hBAC-S100 mice lacking the receptor for advanced glycation end products with CKD, suggesting that the inflammatory milieu mediated by S100/receptor for advanced glycation end products promotes pathological cardiac hypertrophy in CKD. In vitro, inflammatory stimuli including interleukin-6, tumor necrosis factor-α, lipopolysaccarides, or serum from hBAC-S100 mice upregulated fibroblast growth factor 23 mRNA and protein in primary murine neonatal and adult cardiac fibroblasts.
CONCLUSIONS—Myeloid-derived human S100/calgranulin is associated with the development of cardiac hypertrophy and ectopic cardiac calcification in a receptor for advanced glycation end products–dependent manner in a mouse model of CKD. We speculate that fibroblast growth factor 23 produced by cardiac fibroblasts in response to cytokines may act in a paracrine manner to accelerate left ventricular hypertrophy and diastolic dysfunction in hBAC-S100 mice with CKD.
Background: Advanced glycation end products (AGEs) are involved in the inflammatory process and are considered to be etiologic factors of diabetic periodontitis. The purpose of this study is to ...investigate the capability of N‐phenacylthiazolium bromide (PTB), a glycated cross‐link breaker, in the modulation of periodontitis in various disease phases.
Methods: Mitogenesis and cytotoxicity of human periodontal ligament cells (hPDLCs) undergoing PTB treatment were evaluated in vitro. In vivo biomodulation was investigated by systemically administering PTB in the induction, progression, and recovery phases of ligature‐induced periodontitis in rats, with the results evaluated by microcomputed tomography, histology, immunohistochemistry of the AGE and AGE receptor (RAGE), and gene expression of tumor necrosis factor‐α (TNF‐α), RAGE, periostin, fibronectin, and type I collagen.
Results: Significantly promoted mitogenesis and reduced cytotoxicity of hPDLCs were noted with 0.05 to 0.1 mM PTB treatment at 24 hours. Systemic PTB administration significantly reduced periodontal bone loss, AGE deposition, and expressions of TNF‐α and RAGE but elevated the periostin level in all three phases of periodontitis.
Conclusion: PTB inhibits the induction and progression of periodontitis and facilitates its recovery via improving cellular viability and inhibiting the AGE–RAGE axis.
Purpose: Low molecular weight carbonyl compounds, such as the α-ketoaldehydes methylglyoxal (MGO) and glyoxal (GO), are formed under hyperglycemic conditions and behave as advanced glycation end ...product (AGE) precursors. They form adducts on proteins, thereby inducing cellular dysfunctions involved in chronic complications of diabetes. Methods and main findings: Nontoxic concentrations of GO or MGO altered the PDGF-induced PDGFRβ-phosphorylation, ERK1/2-activation, and nuclear translocation, and the subsequent proliferation of mesenchymal cells (smooth muscle cells and skin fibroblasts). This resulted mainly from inhibition of the intrinsic tyrosine kinase of PDGFRβ and in part from altered PDGF-BB binding to PDGFRβ. Concomitantly, the formation of AGE adducts (Nεcarboxymethyl-lysine and Nεcarboxyethyl-lysine) was observed on immunoprecipitated PDGFRβ. Arginine and aminoguanidine, used as carbonyl scavengers, reversed the inhibitory effect and the formation of AGE adducts on PDGFRβ. AGE-PDGFRβ adducts were also detected by anti-AGE antibodies in PDGFRβ immunopurified from aortas of diabetic (streptozotocin-treated) compared to nondiabetic apolipoprotein E-null mice. Mass spectrometry analysis of aortas demonstrated increased AGE formation in diabetic specimens. Conclusions: these data indicate that MGO and GO induce desensitization of PDGFRβ that helps to reduce mesenchymal cell proliferation.--Cantero, A.-V., Portero-Otín, M., Ayala, V., Auge, N., Sanson, M., Elbaz, M., Thiers, J-C., Pamplona, R., Salvayre, R., Nègre-Salvayre, A. Methylglyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor-β: implications for diabetic atherosclerosis.
Background: The effect of advanced glycation end products (AGEs) on gingival inflammation has not been fully elucidated. This study aims to investigate the hypothesis that AGEs may enhance the ...expression of matrix metalloproteinase‐1 (MMP‐1) of human gingival fibroblasts (HGFs) and to explore whether the signal pathway receptor for AGE (RAGE)/nuclear factor‐κB (NF‐κB) are involved in the expression of MMP‐1 in HGFs.
Methods: Cultured HGFs from 12 healthy gingival human tissue samples were coincubated with AGEs for the detection of MMP‐1 protein and mRNA. Thirty‐six gingival samples were collected and treated for the determination of RAGE, NF‐κB, and MMP‐1 mRNA level in gingival connective tissues from the participants with chronic periodontitis, diabetes‐associated periodontitis, and healthy controls. Enzyme‐linked immunosorbent assay and real‐time fluorescence reverse transcription‐polymerase chain reaction were used for the measurement of protein and mRNA level, respectively. In addition, clinical periodontal parameters were also checked.
Results: AGEs strongly induced MMP‐1 mRNA and protein expression in HGFs and in a time‐ and concentration‐dependent manner (P <0.05). In gingival connective tissue, the level of both RAGE mRNA and NF‐κB mRNA were higher in patients with periodontitis than in healthy controls (P <0.05). There was significant correlation between the level of RAGE mRNA and NF‐κB mRNA (R2 = 0.90, P <0.05).
Conclusions: Accumulation of AGEs may upregulate the expression of MMP‐1 by HGFs, which may play a role in the development of diabetes‐associated periodontitis, and RAGE/NF‐κB pathway may be involved in metabolism of MMP‐1 in HGFs.
Background: Diabetes is known to impair wound healing and deteriorate the periodontal condition. There is limited information about the patterns and events associated with periodontal wound repair. ...In this study, we evaluate the dynamics of periodontal wound repair using micro‐computed tomography (microCT) and immunohistochemistry.
Methods: Thirty‐six male rats were used, and diabetes was induced by streptozotocin. The maxillary first molars were extracted, and a tooth‐associated osseous defect was created in the extraction area. Animals were sacrificed after 7, 14, and 21 days. Volumetry and distribution of bone trabeculae were evaluated by microCT imaging. The patterns of healing and collagen alignment were evaluated by histology. Advanced glycation end‐product (AGE) deposition and expression of the receptor for AGEs (RAGE), tartrate‐resistant acid phosphatase, and proliferating cell nuclear antigen were evaluated by histochemical and immunohistochemical staining.
Results: Diabetic animals demonstrated a significantly reduced bone volume and trabecular number as well as thinner trabeculae and more trabecular separation in osseous defects. The early stage was characterized by significantly reduced cellular proliferation and prolonged active inflammation without evident bone resorption, whereas delayed recovery of collagen realignment, matrix deposition, and bone turnover was noted in later stages. Although AGEs and RAGE were present during healing in diabetes and controls, a stronger and more persistent level of expression was observed in the group with diabetes
Conclusions: Diabetes significantly delayed osseous defect healing by augmenting inflammation, impairing proliferation, and delaying bone resorption. The AGE–RAGE axis can be activated under metabolic disturbance and inflammation.
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