Indoxyl sulfate promotes vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A through oxidative stress

We previously demonstrated that indoxyl sulfate (IS), a uremic toxin, induces aortic calcification in hypertensive rats and induces oxidative stress and the expression of osteoblast-specific proteins in vascular smooth muscle cells. This study aimed to clarify whether IS stimulates senescence of cultured human aortic smooth muscle cells (HASMCs) and aorta in Dahl salt-sensitive hypertensive rats and whether AST-120, an oral sorbent, prevents senescence of aorta in subtotally nephrectomized uremic rats. IS increased the mRNA expression of p53 and p21 in HASMCs, whereas it did not change that of p16 and retinoblastoma protein (pRb). The IS-induced expression of p53 and p21 was suppressed by N-acetylcysteine, an antioxidant. IS promoted protein expression of p53, p21, and senescence-associated β-galactosidase (SA-β-gal) activity in HASMCs, and N-acetylcysteine and pifithrin-α,p-nitro, a p53 inhibitor, blocked these effects. IS upregulated prelamin A, a hallmark of vascular smooth muscle cell senescence, and downregulated FACE1/Zempste24 protein expression in HASMCs, and N-acetylcysteine suppressed these effects. Administration of IS to hypertensive rats increased expression of SA-β-gal, p53, p21, prelamin A, and oxidative stress markers such as 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in the cells embedded in the calcification area of arcuate aorta. Further, the uremic rat model showed positive staining for SA-β-gal, p53, p21, prelamin A, 8-OHdG, and MDA in the cells embedded in the calcification area of arcuate aorta, whereas AST-120 reduced the expression of these biomarkers. Taken together, IS accelerates vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A and downregulation of FACE1 through oxidative stress.     

Indoxyl sulfate, a uremic toxin, promotes cell senescence in aorta of hypertensive rats

We demonstrated that administration of indoxyl sulfate, a uremic toxin, promotes aortic calcification in hypertensive rats. This study aimed to clarify if indoxyl sulfate could contribute to cell senescence in the aorta of hypertensive rats. The rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS). After 32 weeks, their arcuate aortas were excised for histological and immunohistochemical analysis. Cell senescence was evaluated by immunohistochemistry of senescence-associated β-galactosidase (SA-β-gal), and senescence-related proteins such as p16^INK4a, p21^WAF1/CIP1, p53 and retinoblastoma protein (Rb). Both DH and DH + IS rats showed significantly higher systolic blood pressure than DN and DN + IS rats, respectively. Serum indoxyl sulfate levels were significantly higher in DN + IS and DH + IS rats than in DN and DH rats, respectively. In aorta, DH rats showed significantly increased aortic calcification and wall thickness, and increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the calcification area of arcuate aorta as compared with DN rats. More notably, DH + IS rats showed significantly increased aortic calcification and wall thickness, and significantly increased expression of SA-β-gal, p16^INK4a, p21^WAF1/CIP1, p53 and Rb in the cells embedded in the calcification area as compared with DH rats. In conclusion, indoxyl sulfate promotes cell senescence with aortic calcification and expression of senescence-related proteins in hypertensive rats.     

Endoplasmic reticulum stress plays a role in the advanced glycation end product-induced inflammatory response in endothelial cells

Aims

Both advanced glycation end products (AGEs) and endoplasmic reticulum (ER) stress play important roles in the development of various diseases. This study aimed to clarify the consequence of AGE-induced ER stress and its underlying mechanisms in human umbilical venous endothelial cells (HUVECs).

Main methods

AGE-induced ER stress was assessed by the increased expression and activation of the ER stress marker proteins GRP78, IRE1α and JNK, which were detected using Western blot. NF-κB translocation was revealed using Western blot and immunofluorescent staining in IRE1α-knockdown HUVECs. The mechanism of AGE-induced ER stress was also explored by inhibiting the effect of reactive oxygen species (ROS) using NADPH oxidase 4 (Nox4) siRNA and the antioxidant reduced glutathione (GSH). The cellular ROS level was measured using flow cytometry.

Key findings

AGEs time- and dose-dependently enhanced the expression of GRP78 and increased the phosphorylation of IRE1α and its downstream signal JNK in HUVECs. siRNA-induced IRE1α down-regulation suppressed AGE-induced NF-κB p65 nuclear translocation. Inhibiting the ROS production using Nox4 siRNA or antagonizing ROS using GSH reduced cellular ROS level and attenuated AGE-induced GRP78 expression and IRE1α and JNK activation.

Significance

This study confirms that AGE-induced ER stress in HUVECs focuses on the ER stress-enhanced inflammatory response through JNK and NF-κB activation. It further reveals the involvement of ROS in the AGE-induced ER stress mechanism.     

IGF-I enhances cellular senescence via the reactive oxygen species-p53 pathway

Cellular senescence is characterized by growth arrest, enlarged and flattened cell morphology, the expression of senescence-associated β-galactosidase (SA-β-gal), and by activation of tumor suppressor networks. Insulin-like growth factor-I (IGF-I) plays a critical role in cellular growth, proliferation, tumorigenesis, and regulation of aging. In the present study, we show that IGF-I enhances cellular senescence in mouse, rat, and human primary cells in the confluent state. IGF-I induced expression of a DNA damage marker, γH2AX, the increased levels of p53 and p21 proteins, and activated SA-β-gal. In the confluent state, an altered downstream signaling of IGF-I receptor was observed. Treatment with a reactive oxygen species (ROS) scavenger, N-acetylcystein (NAC) significantly suppressed induction of these markers, indicating that ROS are involved in the induction of cellular senescence by IGF-I. In p53-null mouse embryonic fibroblasts, the IGF-I-induced augmentation of SA-β-gal and p21 was inhibited, demonstrating that p53 is required for cellular senescence induced by IGF-I. Thus, these data reveal a novel pathway whereby IGF-I enhances cellular senescence in the ROS and p53-dependent manner and may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging.     

Aging of the cells: Insight into cellular senescence and detection Methods

Cellular theory of aging states that human aging is the result of cellular aging, in which an increasing proportion of cells reach senescence. Senescence, from the Latin word senex, means “growing old,” is an irreversible growth arrest which occurs in response to damaging stimuli, such as DNA damage, telomere shortening, telomere dysfunction and oncogenic stress leading to suppression of potentially dysfunctional, transformed, or aged cells. Cellular senescence is characterized by irreversible cell cycle arrest, flattened and enlarged morphology, resistance to apoptosis, alteration in gene expression and chromatin structure, expression of senescence associated- β-galactosidase (SA-β-gal) and acquisition of senescence associated secretory phenotype (SASP). In this review paper, different types of cellular senescence including replicative senescence (RS) which occurs due to telomere shortening and stress induced premature senescence (SIPS) which occurs in response to different types of stress in cells, are discussed. Biomarkers of cellular senescence and senescent assays including BrdU incorporation assay, senescence associated- β-galactosidase (SA-β-gal) and senescence-associated heterochromatin foci assays to detect senescent cells are also addressed.     

缬沙坦对人肾小球系膜细胞糖基化终产物受体表达的影响

目的：本实验探讨缬沙坦对糖基化终产物诱导的人肾小球系膜细胞氧化应激水平及糖基化终产物受体（RAGE）表达的影响。方法：体外常规培养人肾小球系膜细胞,运用糖基化修饰的牛血清白蛋白（AGE-BSA）和缬沙坦进行干预,流式细胞术检测细胞内活性氧（ROS）,RT-PCR法检测NADPH氧化酶的亚基p47^phox的mRNA表达,RT-PCR和细胞免疫化学法检测RAGE的表达量。结果：缬沙坦干预组人肾小球系膜细胞的ROS产生量、NADPH氧化酶的亚基p47^phox mRNA表达量、RAGE表达量均低于AGE-BSA组（P〈0．05）,且缬沙坦的抑制作用呈浓度和时间依赖性。结论：缬沙坦可能通过降低氧化应激水平来抑制RAGE的表达。     

A novel improved therapy strategy for diabetic nephropathy

Diabetic nephropathy (DN), is a disorder that causes significant morbidity and mortality. Studies on the pathological mechanisms of DN reveal that advanced glycation end products (AGEs) play an important role in the pathogenesis of DN through interacting with receptors for advanced glycation end products (RAGE), which activate a series of intracellular signaling pathways. AGEs and RAGE have therefore been considered to be two potential key targets. Although multiple studies have been made for anti-DN therapy against AGEs or RAGE, the results have been disappointing due to poor effectiveness or to side effects in clinical practice. In this hypothesis article, we propose a novel treatment based on a dual-target approach. A kind of multi-functional intelligent nanoparticle is constructed, which has a core-shell nanoparticle structure to load the dual-target drugs (AGEs inhibitors and RAGE inhibitors), and has a functional “RAGE analog” to be used as “bait” to catch AGEs and target them to the kidney. Owing to its advantages of having a dual-target, synergistic effect and high efficiency, the proposition may have potential applications in DN therapy.     

A novel improved therapy strategy for diabetic nephropathy: Targeting AGEs

Diabetic nephropathy (DN), is a disorder that causes significant morbidity and mortality. Studies on the pathological mechanisms of DN reveal that advanced glycation end products (AGEs) play an important role in the pathogenesis of DN through interacting with receptors for advanced glycation end products (RAGE), which activate a series of intracellular signaling pathways. AGEs and RAGE have therefore been considered to be two potential key targets. Although multiple studies have been made for anti-DN therapy against AGEs or RAGE, the results have been disappointing due to poor effectiveness or to side effects in clinical practice. In this hypothesis article, we propose a novel treatment based on a dual-target approach. A kind of multi-functional intelligent nanoparticle is constructed, which has a core-shell nanoparticle structure to load the dual-target drugs (AGEs inhibitors and RAGE inhibitors), and has a functional "RAGE analog" to be used as "bait" to catch AGEs and target them to the kidney. Owing to its advantages of having a dual-target, synergistic effect and high efficiency, the proposition may have potential applications in DN therapy.     

Repeated stimulation by LPS promotes the senescence of DPSCs via TLR4/MyD88-NF-κB-p53/p21 signaling

Dental pulp stem cells (DPSCs), one type of mesenchymal stem cells, are considered to be a type of tool cells for regenerative medicine and tissue engineering. Our previous studies found that the stimulation with lipopolysaccharide (LPS) might introduce senescence of DPSCs, and this senescence would have a positive correlation with the concentration of LPS. The β-galactosidase (SA-β-gal) staining was used to evaluate the senescence of DPSCs and immunofluorescence to show the morphology of DPSCs. Our findings suggested that the activity of SA-β-gal has increased after repeated stimulation with LPS and the morphology of DPSCs has changed with the stimulation with LPS. We also found that LPS bound to the Toll-like receptor 4 (TLR4)/myeloid differentiation factor (MyD) 88 signaling pathway. Protein and mRNA expression of TLR4, MyD88 were enhanced in DPSCs with LPS stimulation, resulting in the activation of nuclear factor-κB (NF-κB) signaling, which exhibited the expression of p65 improved in the nucleus while the decreasing of IκB-α. Simultaneously, the expression of p53 and p21, the downstream proteins of the NF-κB signaling, has increased. In summary, DPSCs tend to undergo senescence after repeated stimulation in an inflammatory microenvironment. Ultimately, these findings may lead to a new direction for cell-based therapy in oral diseases and other regenerative medicines.     

Identification of pyridinoline,a collagen crosslink,as a novel intrinsic ligand for the receptor for advanced glycation end-products (RAGE)

Advanced glycation end-products (AGEs) elicit inflammatory responses via the receptor for AGEs (RAGE) and participate in the pathogenesis of diabetic complications. An earlier study showed that 3-hydroxypyridinium (3-HP), a common moiety of toxic AGEs such as glyceraldehyde-derived pyridinium (GLAP) and GA-pyridine, is essential for the interaction with RAGE. However, the physiological significance of 3-HP recognition by RAGE remains unclear. We hypothesized that pyridinoline (Pyr), a collagen crosslink containing the 3-HP moiety, could have agonist activity with RAGE. To test this hypothesis, we purified Pyr from bovine achilles tendons and examined its cytotoxicity to rat neuronal PC12 cells. Pyr elicited toxicity to PC12 cells in a concentration-dependent manner, and this effect was attenuated in the presence of either the anti-RAGE antibody or the soluble form of RAGE. Moreover, surface plasmon resonance-based analysis showed specific binding of Pyr to RAGE. These data indicate that Pyr is an intrinsic ligand for RAGE.

Abbreviations: AGEs: advanced glycation end-products; RAGE: receptor for advanced glycation end-products; DAMPs: damage-associated molecular patterns; PRR: pattern recognition receptor; TLR: toll-like receptor; GLAP: glyceraldehyde-derived pyridinium; 3-HP: 3-hydroxypyridinium; Pyr: pyridinoline; HFBA: heptafluorobutyric acid; GST: glutathione S-transferase; SPR: surface plasmon resonance; ECM: extracellular matrix; EMT: epithelial to mesenchymal transition     

AMPK induces vascular smooth muscle cell senescence via LKB1 dependent pathway

Vascular cells have a limited lifespan with limited cell proliferation and undergo cellular senescence. The functional changes associated with cellular senescence are thought to contribute to age-related vascular disorders. AMP-activated protein kinase (AMPK) has been discussed in terms of beneficial or harmful effects for aging-related diseases. However, the detailed functional mechanisms of AMPK are largely unclear. An aging model was established by stimulating vascular smooth muscle cell (VSMC) with adriamycin. Adriamycin progressively increased the mRNA and protein expressions of AMPK. The phosphorylation levels of LKB1 and acetyl-CoA carboxylase (ACC), the upstream and downstream of AMPK, were dramatically increased by adriamycin stimulation. The expressions of p53 and p21, which contribute to vascular senescence, were also increased. Inhibition of AMPK diminished senescence-associated β-galactosidase (SA-β-gal) staining, and restored VSMC proliferation. Cytosolic translocation of LKB1 by adriamycin could be a mechanism for AMPK activation in senescence. Furthermore, p53 siRNA and p21 siRNA transfection attenuated adriamycin-induced SA-β-gal staining. These results suggest that LKB1 dependent AMPK activation elicits VSMC senescence and p53–p21 pathway is a mediator of LKB1/AMPK-induced senescence.     

MicroRNA-21介导非对称性二甲基精氨酸诱导的内皮细胞衰老

目的：观察非对称性二甲基精氨酸（ADMA）对内皮细胞中microRNA-21（miR-21）表达的影响,探讨microRNA-21在ADMA诱导的内皮细胞衰老中的作用。方法：人脐静脉内皮细胞（HUVEC）与10uM的ADMA孵育48小时后收集细胞提取总RNA及蛋白,荧光定量实时RT—PCR检测miR-21表达,Westernblot检测超氧化物歧化酶2（SOD2）表达,衰老相关半乳糖苷酶（SA-β-gal）染色鉴定衰老的内皮细胞;然后HUVEC与miR-21抑制剂转染6小时后继续与10uM的ADMA孵育48小时留取细胞按上述方法检测相关指标。结果：HUVEC与ADMA孵育后miR-21表达量明显增加（P〈0．01）,同时衰老的内皮细胞数量增多（P〈0．05）,而SOD2表达减少（P〈0．01）;MiR-21抑制剂转染HUVEC后ADMA诱导的miR-21表达明显减少,同时衰老的内皮细胞减少,而SOD2表达明显增加（所有P〈0．01）。结论：ADMA诱导了HUVEC中miR-21表达及细胞衰老,miR-21介导了ADMA诱导的内皮细胞衰老作用,其机制可能与其抑制SOD2表达有关。     

Resolvin E1 attenuates doxorubicin-induced cardiac fibroblast senescence: A key role for IL-1β

Cardiac fibroblasts (CFs) undergo senescence in reaction to different stressors, leading to a poor prognosis of cardiac disease. Doxorubicin (Doxo) is an antineoplastic drug with strong cardiotoxic effects, which induces IL-1β secretion and thus, triggers a potent pro-inflammatory response. Doxo induces CFs senescence; however, the mechanisms are not fully understood. Different pharmacological strategies have been used to eliminate senescent cells by inducing their apoptosis or modifying their secretome. However, Resolvin E1 (RvE1), a lipid derivative resolutive mediator with potent anti-inflammatory effects has not been used before to prevent CFs senescence.CFs were isolated from adult male C57BL/6J mice and subsequently stimulated with Doxo, in the presence or absence of RvE1. Senescence-associated β-galactosidase activity (SA-β-gal), γ-H2A.X, p53, p21, and senescence-associated secretory phenotype (SASP) were evaluated. The involvement of the NLRP3 inflammasome/interleukin-1 receptor (IL-1R) signaling pathway on CFs senescence was studied using an NLRP3 inhibitor (MCC950) and an endogenous IL-1R antagonist (IR1A).Doxo is able to trigger CFs senescence, as evidenced by an increase of γ-H2A.X, p53, p21, and SA-β-gal, and changes in the SASP profile. These Doxo effects were prevented by RvE1. Doxo triggers IL-1β secretion, which was dependent on NLRP3 activation. Doxo-induced CFs senescence was partially blocked by MCC950 and IR1A. In addition, IL-1β also triggered CFs senescence, as evidenced by the increase of γ-H2A.X, p53, p21, SA-β-gal activity, and SASP. All these effects were also prevented by RvE1 treatment.ConclusionThese data show the anti-senescent role of RvE1 in Doxo-induced CFs senescence, which could be mediated by reducing IL-1β secretion.     

Cell-Free Culture Supernatant of Probiotic Lactobacillus fermentum Protects Against H2O2-Induced Premature Senescence by Suppressing ROS-Akt-mTOR Axis in Murine Preadipocytes

Information regarding cellular anti-senescence attributes of probiotic bacteria vis-à-vis modulation of senescence-associated secretory phenotype (SASP) and mTOR signaling is very limited. The present study assessed anti-senescence potential of secretory metabolites of probiotic Lactobacillus fermentum (Lact. fermentum) using H₂O₂-induced model of senescence in 3T3-L1 preadipocytes. Application of H₂O₂-induced cellular senescence characterized by increased cell size and SA-β-gal activity, activation of SASP and reactive oxygen species (ROS), DNA damage response and induction of cell cycle inhibitors (p53/p21^WAF1/p16^INK4a). Further, a robust stimulation of the PI3K/Akt/mTOR pathway and AMPK signaling was also observed in H₂O₂-treated cells. However, exposure of cells to cell-free supernatant of Lact. fermentum significantly attenuated phosphorylation of PI3K/Akt/mTOR pathway and alleviated senescence markers p53, p21^WAF1, SA-β-gal, p38MAPK, iNOS, cox-2, ROS, NF-κB, and DNA damage response. These results provide evidence that secretory metabolites of Lact. fermentum can mitigate the development as well as severity of stress-induced senescence thereby indicating its utility for use as anti-aging or age-delaying agent.

     

Inactivation of heat shock factor Hsf4 induces cellular senescence and suppresses tumorigenesis in vivo

Studies suggest that Hsf4 expression correlates with its role in cell growth and differentiation. However, the role of Hsf4 in tumorigenesis in vivo remains unexplored. In this article, we provide evidence that absence of the Hsf4 gene suppresses evolution of spontaneous tumors arising in p53- or Arf-deficient mice. Furthermore, deletion of hsf4 alters the tumor spectrum by significantly inhibiting development of lymphomas that are normally observed in the majority of mice lacking p53 or Arf tumor suppressor genes. Using mouse embryo fibroblasts deficient in the hsf4 gene, we have found that these cells exhibit reduced proliferation that is associated with induction of senescence and senescence-associated β-galactosidase (SA-β-gal). Cellular senescence in hsf4-deficient cells is associated with the increased expression of the cyclin-dependent kinase inhibitors, p21 and p27 proteins. Consistent with the cellular senescence observed in vitro, specific normal tissues of hsf4(-/-) mice and tumors that arose in mice deficient in both hsf4 and p53 genes exhibit increased SA-β-gal activity and elevated levels of p27 compared with wild-type mice. These results suggest that hsf4 deletion-induced senescence is also present in vivo. Our results therefore indicate that Hsf4 is involved in modulation of cellular senescence, which can be exploited during cancer therapy.     

Material basis,effect, and mechanism of ethanol extract of Pinellia ternata tubers on oxidative stress-induced cell senescence

BackgroundThe tuber of Pinellia ternata has been used for a thousand years in China. P. ternata possessed the activities of anti-emetic, sedative-hypnotic, anti-cancer, anti-asthmatic, anti-tussive, and anti-inflammatory. It is the representative of expectorant medicines in Traditional Chinese Medicine (TCM). Phlegm is the pathological product and a new pathogenic factor of the metabolite, which is analogous to the damage of oxidative stress.PurposeThe objectives of the study were to investigate the protective activity and mechanism of ethanol extract of P. ternata tubers (PTE) and its main constituents on oxidative stress-induced cell senescence.MethodsH₂O₂ and AAPH were used to establish cellular senescence models. The anti-aging effects of PTE and its components were evaluated by SA-β-gal staining, flow cytometry, scanning electron microscope (SEM), and multiple microplate reader, the molecular mechanisms of them were investigated by qRT-PCR and Western Blot.ResultsWe found PTE exhibited the apparent effect on cell senescence, evidenced by inhibiting senescence β-Galactosidase (SA-β-gal) expression, lipofuscin accumulation, cell cycle arrest at the G2/M phase, oxidative damage and apoptosis, and increasing telomerase activity. Their mechanisms were related to increase expressions of SIRT1, forkhead box 3a (Foxo3a), Bcl-2, active regulator of SIRT1, RPS19BP1 (AROS), and Hu antigen R (HuR), but decrease Bax, p53 and deleted in breast cancer 1 (DBC1) levels. Furthermore, adenosine, and succinic acid, as the critical substances in PTE, could also inhibit SA-β-gal expression and cell cycle arrest, down-regulate the expression of Bax, and up-regulate Bcl-2, SirT1, and Foxo3a.ConclusionsWe have demonstrated that PTE slows down oxidative stress-induced cell senescence, and adenosine and succinic acid are the key active components.     

Mechanistic targeting of advanced glycation end-products in age-related diseases

Glycative stress, caused by the accumulation of cytotoxic and irreversibly-formed sugar-derived advanced glycation end-products (AGEs), contributes to morbidity associated with aging, age-related diseases, and metabolic diseases. In this review, we summarize pathways leading to formation of AGEs, largely from sugars and glycolytic intermediates, and discuss detoxification of AGE precursors, including the glyoxalase system and DJ-1/Park7 deglycase. Disease pathogenesis downstream of AGE accumulation can be cell autonomous due to aggregation of glycated proteins and impaired protein function, which occurs in ocular cataracts. Extracellular AGEs also activate RAGE signaling, leading to oxidative stress, inflammation, and leukostasis in diabetic complications such as diabetic retinopathy. Pharmaceutical agents have been tested in animal models and clinically to diminish glycative burden. We summarize existing strategies and point out several new directions to diminish glycative stress including: plant-derived polyphenols as AGE inhibitors and glyoxalase inducers; improved dietary patterns, particularly Mediterranean and low glycemic diets; and enhancing proteolytic capacities of the ubiquitin-proteasome and autophagy pathways that are involved in cellular clearing of AGEs.     

Advanced Glycation End-Products Induce Apoptosis in Pancreatic Islet Endothelial Cells via NF-κB-Activated Cyclooxygenase-2/Prostaglandin E2 Up-Regulation

Microvascular complications eventually affect nearly all patients with diabetes. Advanced glycation end-products (AGEs) resulting from hyperglycemia are a complex and heterogeneous group of compounds that accumulate in the plasma and tissues in diabetic patients. They are responsible for both endothelial dysfunction and diabetic vasculopathy. The aim of this study was to investigate the cytotoxicity of AGEs on pancreatic islet microvascular endothelial cells. The mechanism underlying the apoptotic effect of AGEs in pancreatic islet endothelial cell line MS1 was explored. The results showed that AGEs significantly decreased MS1 cell viability and induced MS1 cell apoptosis in a dose-dependent manner. AGEs dose-dependently increased the expressions of cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase in MS1 cells. Treatment of MS1 cells with AGEs also resulted in increased nuclear factor (NF)-κB-p65 phosphorylation and cyclooxygenase (COX)-2 expression. However, AGEs did not affect the expressions of endoplasmic reticulum (ER) stress-related molecules in MS1 cells. Pretreatment with NS398 (a COX-2 inhibitor) to inhibit prostaglandin E₂ (PGE₂) production reversed the induction of cleaved caspase-3, cleaved PARP, and MS1 cell viability. Moreover, AGEs significantly increased the receptor for AGEs (RAGE) protein expression in MS1 cells, which could be reversed by RAGE neutralizing antibody. RAGE Neutralizing antibody could also reverse the induction of cleaved caspase-3 and cleaved PARP and decreased cell viability induced by AGEs. These results implicate the involvement of NF-κB-activated COX-2/PGE₂ up-regulation in AGEs/RAGE-induced islet endothelial cell apoptosis and cytotoxicity. These findings may provide insight into the pathological processes within the pancreatic islet microvasculature induced by AGEs accumulation.     

GRP78 protects CHO cells from ribosylation

d-Ribose (Rib), a reactive glycation compound that exists in organisms, abnormally increases in the urine of diabetic patients and can yield large amounts of advanced glycation end products (AGEs), leading to cell dysfunction. However, whether cellular proteins are sensitive to this type of glycation is unknown. In this study, we found that cellular AGEs accumulate in Chinese hamster ovary (CHO) cells with increased Rib concentration and administration time. Mass spectrum analysis of isolated AGE-modified proteins from cell lysates showed that glucose-regulated protein 78?kD (GRP78) is one of the main ribosylated proteins. Co-immunoprecipitation assays further confirmed the interaction between AGEs and GRP78. Compared with d-glucose (Glc), Rib produced much more AGEs in cells. In kinetic studies, the first order rate constant of LDH released from CHO cells incubated with Rib was nearly 8-fold higher than that of Glc, suggesting that Rib is highly cytotoxic. Immunofluorescent co-localization analysis manifested partial superimposition of AGEs and GRP78, which were distributed throughout the endoplasmic reticulum. Western blotting showed that the expression of GRP78 is up-regulated and then down-regulated in CHO cells during Rib treatment. In the presence of Rib, the suppression of GRP78 expression either with transfected siRNA or with the inhibitor (-)-epigallocatechin gallate (EGCG) dramatically increased AGE levels and decreased cell viability compared with these parameters in the control groups. GRP78 overexpression decreased AGE levels and rescued the cells from Rib-induced cytotoxicity. These data indicate that GRP78 plays a role in preventing Rib-induced CHO cell cytotoxicity.