Cell Migration Is Regulated by AGE-RAGE Interaction in Human Oral Cancer Cells In Vitro

Advanced glycation end products (AGEs) are produced in an irreversible non-enzymatic reaction of carbohydrates and proteins. Patients with diabetes mellitus (DM) are known to have elevated AGE levels, which is viewed as a risk factor of diabetes-related complications. In a clinical setting, it has been shown that patients with oral cancer in conjunction with DM have a higher likelihood of cancer metastasis and lower cancer survival rates. AGE-RAGE (a receptor of AGEs) is also correlated with metastasis and angiogenesis. Recent studies have suggested that the malignancy of cancer may be enhanced by glyceraldehyde-derived AGEs; however, the underlying mechanism remains unclear. This study examined the apparently close correlation between AGE-RAGE and the malignancy of SAS oral cancer cell line. In this study, AGEs increased ERK phosphorylation, enhanced cell migration, and promoted the expression of RAGE, MMP2, and MMP9. Using PD98059, RAGE antibody, and RAGE RNAi to block RAGE pathway resulted in the inhibition of ERK phosphorylation. Cell migration, MMP2 and MMP9 expression were also reduced by this treatment. Our findings demonstrate the importance of AGE-RAGE with regard to the malignancy of oral cancer, and help to explain the poor prognosis of DM subjects with oral cancer.     

High dietary advanced glycation end products are associated with poorer spatial learning and accelerated Aβ deposition in an Alzheimer mouse model

There is growing evidence of the involvement of advanced glycation end products (AGEs) in the pathogenesis of neurodegenerative processes including Alzheimer's disease (AD) and their function as a seed for the aggregation of Aβ, a hallmark feature of AD. AGEs are formed endogenously and exogenously during heating and irradiation of foods. We here examined the effect of a diet high in AGEs in the context of an irradiated diet on memory, insoluble Aβ₄₂, AGEs levels in hippocampus, on expression of the receptor for AGEs (RAGE), and on oxidative stress in the vasculature. We found that AD‐like model mice on high‐AGE diet due to irradiation had significantly poorer memory, higher hippocampal levels of insoluble Aβ₄₂ and AGEs as well as higher levels of oxidative stress on vascular walls, compared to littermates fed an isocaloric diet. These differences were not due to weight gain. The data were further supported by the overexpression of RAGE, which binds to Aβ₄₂ and regulates its transport across the blood–brain barrier, suggesting a mediating pathway. Because exposure to AGEs can be diminished, these insights provide an important simple noninvasive potential therapeutic strategy for alleviating a major lifestyle‐linked disease epidemic.     

Vasa vasorum inside out/outside in communication: a potential role in the patency of saphenous vein coronary artery bypass grafts

The saphenous vein (SV) is the most commonly used conduit for revascularization in patients undergoing coronary artery bypass surgery (CABG). The patency rate of this vessel is inferior to the internal thoracic artery (ITA). In the majority of CABG procedures the ITA is removed with its outer pedicle intact whereas the (human) SV (hSV) is harvested with pedicle removed. The vasa vasorum, a microvessel network providing the adventitia and media with oxygen and nutrients, is more pronounced and penetrates deeper towards the lumen in veins than in arteries. When prepared in conventional CABG the vascular trauma caused when removing the hSV pedicle damages the vasa vasorum, a situation affecting transmural flow potentially impacting on graft performance. In patients, where the hSV is harvested with pedicle intact, the vasa vasorum is preserved and transmural blood flow restored at graft insertion and completion of CABG. By maintaining blood supply to the hSV wall, apart from oxygen and nutrients, the vasa vasorum may also transport factors potentially beneficial to graft performance. Studies, using either corrosion casts or India ink, have shown the course of vasa vasorum in animal SV as well as in hSV. In addition, there is some evidence that vasa vasorum of hSV terminate in the vessel lumen based on ex vivo perfusion, histological and ultrastructural studies. This review describes the preparation of the hSV as a bypass conduit in CABG and its performance compared with the ITA as well as how and why its patency might be improved by harvesting with minimal trauma in a way that preserves an intact vasa vasorum.     

The defective protein level of myosin light chain phosphatase (MLCP) in the isolated saphenous vein, as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM)

We examined the contractile reactivity to 5-hydroxytryptamine (5-HT) in isolated human saphenous vein (SV), as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM) and non-DM (NDM). Vascular rings of endothelium-denuded SV were used for functional and biochemical experiments. The vasoconstrictions caused by 5-HT were significantly greater (hyperreactivity) in the DM group than in the NDM group. RhoA/ROCK pathway is activated by various G-protein-coupled receptor agonists and consequently induces phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin light chain phosphatase (MLCP), which inhibits MLCP activity. In the resting state of the vessels, total tissue protein levels of 5-HT_2A receptor, 5-HT_1B receptor, RhoA, ROCK1, and ROCK2 did not differ between NDM and DM groups. However, the total protein level of MYPT1 was significantly lower in the DM group than in the NDM group. Furthermore, the ratio of P(Thr⁶⁹⁶)-MYPT1 to total MYPT1 was significantly higher in the DM group than in the NDM group. These results suggest that the hyperreactivity to 5-HT in the SV smooth muscle of patients with DM is due to not only enhanced phosphorylation of MLCP but also defective protein level of MLCP. Thus, we reveal for the first time that the defective protein level of MLCP in the DM group can partially explain the poor patency of SV graft harvested from patients with DM.     

Angiotensin II,as well as 5-hydroxytriptamine,is a potent vasospasm inducer of saphenous vein graft for coronary artery bypass grafting in patients with diabetes mellitus

Diabetes mellitus (DM) is an important risk factor for adverse outcomes of coronary artery bypass grafting. The bypass grafts harvested from patients with DM tend to go into spasm after their implantation into the coronary circulation. To clarify the contribution of 5-hydroxytriptamine (5-HT) and angiotensin II (AngII) in the bypass graft spasm, we examined the contractile reactivity to 5-HT or AngII of isolated human endothelium-denuded saphenous vein (SV) harvested from DM and non-DM patients. The 5-HT-induced constriction of the SV was significantly augmented in the DM group than in the non-DM group, which is similar to our previous report. AngII-induced constriction of the SV was also significantly augmented in the DM group than the non-DM group. Especially in the non-DM group, the AngII-induced maximal vasoconstriction was markedly lower than the 5-HT-induced one. Meanwhile, the increasing rates of AngII-induced vasoconstriction in the DM group to the non-DM group were significantly greater than those of 5-HT-induced vasoconstriction. These results indicate that 5-HT is a potent inducer of SV graft spasm in both DM and non-DM patients, while AngII is a potent inducer of SV graft spasm only in patients with DM. Furthermore, the protein level of AngII AT₁ receptor (AT₁R), but not the protein level of 5-HT_2A receptor, in the membrane fraction of the SV smooth muscle cells of DM patients was significantly increased as compared with that of the non-DM patients. These results suggest that the mechanism for hyperreactivity to AngII in the SV from DM patients is due to, at least in part, the increase in the amount of AT₁R on membrane of the SV smooth muscle cells.     

Antioxidant icariside II combined with insulin restores erectile function in streptozotocin‐induced type 1 diabetic rats

Erectile dysfunction (ED) worsens in patients with diabetes mellitus (DM) despite good control of blood glucose level with insulin. Recent studies imply that diabetic vascular stresses (e.g. oxidative stress) persist in spite of glucose normalization, which is defined as metabolic memory. Studies suggest that the interaction between advanced glycation end products (AGEs) and their receptor (RAGE) mediates the development of metabolic memory. To investigate the effects of the antioxidant icariside II plus insulin on erectile function in streptozotocin (STZ)‐ induced type 1 diabetic rats. Fifty 8‐week‐old Sprague‐Dawley rats were randomly distributed into five groups: normal control, diabetic, insulin‐treated diabetic, icariside II‐treated diabetic, and insulin plus icariside II‐treated diabetic. Diabetes was induced by a single intraperitoneal injection of STZ. Eight weeks after induction of diabetes, icariside II was administered by gastric lavage once a day (5 mg/kg) for 6 weeks; and 2–6 units of intermediate‐acting insulin were given to maintain normal glycemia for 6 weeks. The main outcome measures were the ratio of intracavernous pressure (ICP) to mean arterial pressure (MAP); histology of penile endothelial cells and smooth muscle cells; neural nitric oxide synthase, AGEs and RAGE expression; malondialdehyde concentration; superoxide dismutase activity; and apoptosis index. Diabetic rats demonstrated a significantly lower ICP/MAP ratio, reduced penile endothelial cells, reduced smooth muscle cells, increased AGEs and RAGE, and increased apoptosis. Insulin and icariside II monotherapy partially restored erectile function and histological changes. However, the combination therapy group showed significantly better erectile parameters, cytological components and biochemistry, similar to those in the normal control group. These results suggest that, although insulin can effectively control glycemic levels, it does not completely alter the pathological changes in erectile tissues. Better efficacy could be expected with tight glycemic control plus the antioxidant icariside II. The proposed combination therapy might have the potential to eliminate metabolic memory by down‐regulating the AGEs‐RAGE‐oxidative stress axis.     

RAGE-mediated MAPK activation by food-derived AGE and non-AGE products

Investigating the cellular effects of food compounds formed by heat treatment during processing, we recently demonstrated the expression of the receptor for advanced glycation endproducts (RAGE) and the p44/42 MAP kinase activation by casein-N(epsilon )-(carboxymethyl)lysine (casein-CML), a food-derived AGE, in the intestinal cell line Caco-2. In this work, we report a Caco-2 p44/42 MAP kinase activation by bread crust and coffee extract. After identification, quantification, and synthesis of two key compounds formed in association with the process-induced heat impact applied to bread dough and coffee beans, those compounds, namely the AGE pronyl-glycine and the non-AGE N-methylpyridinium, were also demonstrated for the first time to activate the p44/42 MAP kinase through binding to RAGE in Caco-2 cells. Blocking of RAGE by an antagonistic antibody and expression of C-terminally truncated RAGE resulted in a reduced Caco-2- and HEK-293-MAP kinase activation. These findings unequivocally point to a RAGE-mediated activating effect of chemically defined food-derived, thermally generated products, both, AGEs and non-AGEs, on cellular signal transduction pathways involved in inflammatory response and cellular proliferation.     

Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation

We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC.     

RAGE expression and AGE-induced MAP kinase activation in Caco-2 cells.

RAGE (receptor for advanced glycation end products) is a multiligand cell surface molecule of the immunoglobulin superfamily. It was originally described as a receptor for protein adducts formed by glycoxidation (AGEs) that accumulate in diseases such as diabetes and renal failure. Performing RT-PCR and Western blot analysis we intended to determine RAGE expression in the human colon adenocarcinoma cell line Caco-2. Moreover, Caco-2 cells were incubated in the presence of AGEs. Since RAGE ligation triggers the p21(ras) signal transduction pathway the activation state of p44/42 (ERK1/2) MAP kinases was determined. Here we demonstrate for the first time that Caco-2 cells express RAGE and that administration of the food-derived casein-linked AGE N(epsilon)-(carboxymethyl)lysine (Cas-CML) results in Caco-2 p44/42 (ERK1/2) MAP kinase activation.     

Advanced glycation end‐products activate extracellular signal‐regulated kinase via the oxidative stress‐EGF receptor pathway in renal fibroblasts

Advanced glycation end‐products (AGEs), epidermal growth factor receptor (EGFR), reactive oxygen species (ROS), and extracellular signal‐regulated kinases (ERK) are implicated in diabetic nephropathy (DN). Therefore, we asked if AGEs‐induced ERK protein phosphorylation and mitogenesis are dependent on the receptor for AGEs (RAGE)–ROS–EGFR pathway in normal rat kidney interstitial fibroblast (NRK‐49F) cells. We found that AGEs (100 µg/ml) activated EGFR and ERK1/2, which was attenuated by RAGE short‐hairpin RNA (shRNA). AGEs also increased RAGE protein and intracellular ROS levels while RAGE shRNA and N‐acetylcysteine (NAC) attenuated AGEs‐induced intracellular ROS. Hydrogen peroxide (5–25 µM) increased RAGE protein level while activating both EGFR and ERK1/2. Low‐dose hydrogen peroxide (5 µM) increased whereas high‐dose hydrogen peroxide (100 µM) decreased mitogenesis at 3 days. AGEs‐activated EGFR and ERK1/2 were attenuated by an anti‐oxidant (NAC) and an EGFR inhibitor (Iressa). Moreover, AGEs‐induced mitogenesis was attenuated by RAGE shRNA, NAC, Iressa, and an ERK1/2 inhibitor (PD98059). In conclusion, it was found that AGEs‐induced mitogenesis is dependent on the RAGE–ROS–EGFR–ERK1/2 pathway whereas AGEs‐activated ERK1/2 is dependent on the RAGE–ROS–EGFR pathway in NRK‐49F cells. J. Cell. Biochem. 109: 38–48, 2010. © 2009 Wiley‐Liss, Inc.     

Loganin alleviates testicular damage and germ cell apoptosis induced by AGEs upon diabetes mellitus by suppressing the RAGE/p38MAPK/NF-κB pathway

Diabetes mellitus (DM) damages male reproduction at multiple levels, such as endocrine secretion, spermatogenesis and penile erection. We herein investigated the protective effects and mechanism of loganin targeting the advanced glycation end products (AGEs)/receptor for AGEs (RAGE)/p38 mitogen-activated protein kinase (p38MAPK)/NF-κB signalling pathway. Loganin relieved the general DM symptoms and decreased the blood glucose level of KK-Ay DM mice. Haematoxylin-eosin staining demonstrated that loganin ameliorated testicular histology and function and enhanced the activities of testis-specific markers lactate dehydrogenase (LDH), acid phosphatase (ACP) and gamma-glutamyl transferase (γ-GT). Loganin also showed evident anti-oxidative stress, anti-apoptotic and anti-inflammatory effects on DM-induced reproductive damage by restoring glutathione (GSH) level and superoxide dismutase (SOD) activity, as well as reducing reactive oxygen species (ROS) level and Bax/Bcl-2 ratio in vivo and in vitro. Western blotting exhibited that loganin significantly inhibited the AGEs/RAGE/p38MAPK/NF-κB signalling pathway. Acridine orange and ethidium bromide staining (AOEB) and Western blotting showed that loganin in combination with inhibitors of RAGE, p38MAPK and NF-κB exerted stronger anti-apoptotic effects on AGE-induced GC-2 cell damage compared with loganin alone. In conclusion, loganin can protect against DM-induced reproductive damage, probably by suppressing the AGEs/RAGE/p38MAPK/NF-κB pathway.     

Recurrent BRAF kinase fusions in melanocytic tumors offer an opportunity for targeted therapy

BRAF is the most prevalent oncogene and an important therapeutic target in melanoma. In some cancers, BRAF is activated by rearrangements that fuse its kinase domain to 5′ partner genes. We examined 848 comparative genomic hybridization profiles of melanocytic tumors and found copy number transitions within BRAF in 10 tumors, of which six could be further characterized by sequencing. In all, the BRAF kinase domain was fused in‐frame to six N‐terminal partners. No other mutations were identified in melanoma oncogenes. One of the seven melanoma cell lines without known oncogenic mutations harbored a similar BRAF fusion, which constitutively activated the MAP kinase pathway. Sorafenib, but not vemurafenib, could block MAP kinase pathway activation and proliferation of the cell line at clinically relevant concentrations, whereas BRAF^V600E mutant melanoma cell lines were significantly more sensitive to vemurafenib. The patient from whom the cell line was derived showed a durable clinical response to sorafenib.     

V1b vasopressin receptor trafficking and signaling: Role of arrestins,G proteins and Src kinase

The signaling pathway of G protein‐coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V_1b subtype (V_1bR) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of β‐arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V_1bR‐mediated MAP kinase pathway. Using MEF cells Knocked‐out for β‐arrestins 1 and 2, we demonstrated that both β‐arrestins 1 and 2 play a fundamental role in internalization and recycling of V_1bR with a rapid and transient V_1bR‐β‐arrestin interaction in contrast to a slow and long‐lasting β‐arrestin recruitment of the V₂ vasopressin receptor subtype (V₂R). Using V_1bR‐V₂R chimeras and V_1bR C‐terminus truncations, we demonstrated the critical role of the V_1bR C‐terminus in its interaction with β‐arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation‐independent manner. In parallel, V_1bR MAP kinase activation was dependent on arrestins and Src‐kinase but independent on G proteins. Interestingly, Src interacted with hV_1bR at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V_1bR involving both arrestins and Src kinase family.      

Pro‐inflammatory angiogenesis is mediated by p38 MAP kinase

Chronic inflammation is tightly linked to diseases associated with endothelial dysfunction including aberrant angiogenesis. To better understand the endothelial role in pro‐inflammatory angiogenesis, we analyzed signaling pathways in continuously activated endothelial cells, which were either chronically exposed to soluble TNF or the reactive oxygen species (ROS) generating H₂O₂, or express active transmembrane TNF. Testing in an in vitro capillary sprout formation assay, continuous endothelial activation increased angiogenesis dependent on activation of p38 MAP kinase, NADPH oxidase, and matrix metalloproteinases (MMP). p38 MAP kinase‐ and MMP‐9‐dependent angiogenesis in our assay system may be part of a positive feed forward autocrine loop because continuously activated endothelial cells displayed up‐regulated ROS production and subsequent endothelial TNF expression. The pro‐angiogenic role of the p38 MAP kinase in continuously activated endothelial cells was in stark contrast to the anti‐angiogenic activity of the p38 MAP kinase in unstimulated control endothelial cells. In vivo, using an experimental prostate tumor, pharmacological inhibition of p38 MAP kinase demonstrated a significant reduction in tumor growth and in vessel density, suggesting a pro‐angiogenic role of the p38 MAP kinase in pathological angiogenesis in vivo. In conclusion, our results suggest that continuous activation of endothelial cells can cause a switch of the p38 MAP kinase from anti‐angiogenic to pro‐angiogenic activities in conditions which link oxidative stress and autocrine TNF production. J. Cell. Physiol. 226: 800–808, 2011. © 2010 Wiley‐Liss, Inc.     

Retracted: Receptor for advanced glycation end products reveals a mechanism regulating thyroid hormone secretion through the SIRT1/Nrf2 pathway

Advanced glycation end products (AGEs) play a causative role in the complications involved with diabetes mellitus (DM). Nowadays, DM with hypothyroidism (DM-hypothyroidism) is indicative of an ascended tendency in the combined morbidity. In this study, we examine the role of the receptor (RAGE) played for AGEs in thyroid hormone (TH) secretion via the silent information regulator 1 (SIRT1)/nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) pathway. Blood samples were collected from patients with type 2 DM (T2DM)-hypothyroidism and from patients with T2DM, followed by detection of serum AGEs level. The underlying regulatory mechanisms of RAGE were analyzed in association with the treatment of high glucose, siRNA against RAGE, AGE, SIRT1, or Nrf2 vector in normal immortalized thyroid Nthy-ori 3-1 cells. Serum of patients with T2DM-hypothyroidism indicated promoted levels of AGEs vs those with just T2DM. Both AGEs and high glucose triggered cellular damage, increased oxidative stress, as well as displayed a decreased survival rate along with TH secretion in the Nthy-ori 3-1 cells. Moreover, AGEs and high glucose also led to RAGE upregulation, both SIRT1 and NRF2 downregulation, and the decreased expression of TH secretion–related proteins in Nthy-ori 3-1 cells. Notably, these alternations induced by the AGEs can be reserved by silencing RAGE or upregulating either SIRT1 or Nrf2, indicating a mechanism of regulating TH secretion through the SIRT1/Nrf2 pathway. Collectively, our data proposed that AGEs and high glucose exerted a potent effect on cellular damage and TH deficiency in Nthy-ori 3-1 cells through the RAGE upregulation as well as SIRT1/Nrf2 pathway inactivation. This mechanism may underlie the occurrence of DM-hypothyroidism.     

Advanced Glycation End Product (AGE)-Receptor for AGE (RAGE) Signaling and Up-regulation of Egr-1 in Hypoxic Macrophages

Receptor for advanced glycation end product (RAGE)-dependent signaling has been implicated in ischemia/reperfusion injury in the heart, lung, liver, and brain. Because macrophages contribute to vascular perturbation and tissue injury in hypoxic settings, we tested the hypothesis that RAGE regulates early growth response-1 (Egr-1) expression in hypoxia-exposed macrophages. Molecular analysis, including silencing of RAGE, or blockade of RAGE with sRAGE (the extracellular ligand-binding domain of RAGE), anti-RAGE IgG, or anti-AGE IgG in THP-1 cells, and genetic deletion of RAGE in peritoneal macrophages, revealed that hypoxia-induced up-regulation of Egr-1 is mediated by RAGE signaling. In addition, the observation of increased cellular release of RAGE ligand AGEs in hypoxic THP-1 cells suggests that recruitment of RAGE in hypoxia is stimulated by rapid production of RAGE ligands in this setting. Finally, we show that mDia-1, previously shown to interact with the RAGE cytoplasmic domain, is essential for hypoxia-stimulated regulation of Egr-1, at least in part through protein kinase C βII, ERK1/2, and c-Jun NH₂-terminal kinase signaling triggered by RAGE ligands. Our findings highlight a novel mechanism by which an extracellular signal initiated by RAGE ligand AGEs regulates Egr-1 in a manner requiring mDia-1.     

Effect of the interaction between MiR‐200b‐3p and DNMT3A on cartilage cells of osteoarthritis patients

The aim of this research is to explore the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of osteoarthritis (OA) cartilage cells. Quantitative RT‐PCR was performed to analyse the expression of miR‐200b‐3p, DNMT3A, MMP1, MMP3, MMP9, MMP13 and COL II in normal and OA cartilage tissues. The dual‐luciferase reporter assay and Western blot assay were conducted to confirm the targeting relationship between miR‐200b‐3p and DNMT3A. We also constructed eukaryotic expression vector to overexpress miR‐200b‐3p and DNMT3A. We detected the expression level of MMPs and COL II in stable transfected cartilage cells using RT‐PCR and Western blot. Cell proliferation and apoptosis were evaluated using the MTS, pellet culture and Hoechst 33342 staining method. Finally, we explored the effect of miR‐200b‐3p targeting DNMT3A on the proliferation and apoptosis of OA cartilage cells. The results of RT‐PCR indicated that both miR‐200b‐3p and COL II were down‐regulated in OA cartilage tissues, while the expression of DNMT3A and MMPs was up‐regulated in OA cartilage tissues. The expressions of DNMT3A, MMPs and COL II detected by Western blot showed the same trend of the results of RT‐PCR. The dual‐luciferase reporter assay and Western blot assay confirmed the targeting relationship between miR‐200b‐3p and DNMT3A. In overexpressed miR‐200b‐3p cartilage cells, DNMT3A and MMPs were significantly down‐regulated, COL II was significantly up‐regulated, cell viability was enhanced and apoptosis rate was decreased (P < 0.05). In overexpressed DNM3T cartilage cells, MMPs were significantly up‐regulated, COL II was significantly down‐regulated, cell viability was weakened and apoptosis rate was increased (P < 0.05). MiR‐200b‐3p inhibited the secretion of MMPs, promoted the synthesis of COL II and enhanced the growth and proliferation of OA cartilage cells through inhibiting the expression of DNMT3A.     

Role of Spm–Cer‐S1P signalling pathway in MMP‐2 mediated U46619‐induced proliferation of pulmonary artery smooth muscle cells: protective role of epigallocatechin‐3‐gallate

During remodelling of pulmonary artery, marked proliferation of pulmonary artery smooth muscle cells (PASMCs) occur s , which contributes to pulmonary hypertension. Thromboxane A₂ (TxA₂) has been shown to produce pulmonary hypertension. The present study investigates the inhibitory effect of epigallocatechin‐3‐gallate (EGCG) on the TxA₂ mimetic, U46619‐induced proliferation of PASMCs. U46619 at a concentration of 10 nM induces maximum proliferation of bovine PASMCs. Both pharmacological and genetic inhibitors of p³⁸MAPK, NF‐κB and MMP‐2 significantly inhibit U46619‐induced cell proliferation. EGCG markedly abrogate U46619‐induced p³⁸MAPK phosphorylation, NF‐κB activation, proMMP‐2 expression and activation, and also the cell proliferation. U46619 causes an increase in the activation of sphingomyelinase (SMase) and sphingosine kinase (SPHK) and also increase sphingosine 1 phosphate (S1P) level. U46619 also induces phosphorylation of ERK1/2, which phosphorylates SPHK leading to an increase in S1P level. Both pharmacological and genetic inhibitors of SMase and SPHK markedly inhibit U46619‐induced cell proliferation. Additionally, pharmacological and genetic inhibitors of MMP‐2 markedly abrogate U46619‐induced SMase activity and S1P level. EGCG markedly inhibit U46619‐induced SMase activity, ERK1/2 and SPHK phosphorylation and S1P level in the cells. Overall, Sphingomyeline–Ceramide–Sphingosine‐1‐phosphate (Spm–Cer–S1P) signalling axis plays an important role in MMP‐2 mediated U46619‐induced proliferation of PASMCs. Importantly, EGCG inhibits U46619 induced increase in MMP‐2 activation by modulating p³⁸MAPK–NFκB pathway and subsequently prevents the cell proliferation. Copyright © 2015 John Wiley & Sons, Ltd.     

Association of RAGE gene polymorphism with circulating AGEs level and paraoxonase activity in relation to macro-vascular complications in Indian type 2 diabetes mellitus patients

Background and aims

Sustained interaction of advanced glycation end products (AGEs) with their receptor RAGE and subsequent signaling plays an important role in the development of diabetic complications. Genetic variation of RAGE gene may be associated with the development of vascular complications in type 2 diabetes mellitus (T2DM).

Objectives

The present study aimed to explore the possible association of RAGE gene polymorphisms namely − 374T/A, − 429T/C and G82S with serum level of AGEs, paraoxonase (PON1) activity and macro-vascular complications (MVC) in Indian type 2 diabetes mellitus patients (T2DM).

Methods

A total of 265 diabetic patients, including DM without any complications (n = 135), DM-MVC (n = 130) and 171 healthy individuals were enrolled. Genotyping of RAGE variants were assessed by polymerase chain reaction-restriction fragment length polymorphism. Serum AGEs were estimated by ELISA and fluorometrically. and PON1 activity was assessed spectrophotometrically.

Results

Of the three examined SNPs, association of − 429T/C polymorphism with MVC in T2DM was observed (OR = 3.001, p = 0.001) in the dominant model. Allele ‘A’ of − 374T/A polymorphism seems to confer better cardiac outcome in T2DM. Patients carrying C allele (− 429T/C) and S allele (G82S) had significantly higher AGEs levels. − 429T/C polymorphism was also found to be associated with low PON1 activity. Interaction analysis revealed that the risk of development of MVC was higher in T2DM patients carrying both a CC genotype of − 429T/C polymorphism and a higher level of AGEs (OR = 1.343, p = 0.040).

Conclusion

RAGE gene polymorphism has a significant effect on AGEs level and PON1 activity in diabetic subjects compared to healthy individuals. Diabetic patients with a CC genotype of − 429T/C are prone to develop MVC, more so if AGEs levels are high and PON1 activity is low.     

Temporal PTEN inactivation causes proliferation of saphenous vein smooth muscle cells of human CABG conduits

Internal mammary artery (IMA) coronary artery bypass grafts (CABG) are remarkably resistant to intimal hyperplasia (IH) as compared to saphenous vein (SV) grafts following aorto-coronary anastomosis. The reason behind this puzzling difference still remains an enigma. In this study, we examined the effects of IGF-1 stimulation on the PI3K-AKT/PKB pathway mediating proliferation of smooth muscle cells (SMCs) of IMA and SV origin and the specific contribution of phosphatase and tensin homologue (PTEN) in regulating the IGF-1-PI3K-AKT/PKB axis under these conditions. Mitogenic activation with IGF-1, time-dependently stimulated the phosphorylation of PI3K and AKT/PKB in the SV SMCs to a much greater extent than the IMA. Conversely, PTEN was found to be significantly more active in IMA SMCs. Transient overexpression of PTEN in SMCs of SV and IMA inhibited AKT/PKB activity and upstream of AKT/PKB, caused a reduction of IGF-1 receptors. Downstream, PTEN overexpression in SV SMCs induced the transactivation of tumour suppressor protein p53 by down-regulating the expression of its inhibitor MDM2. However, PTEN overexpression had no significant effect on MDM2 and p53 expression in IMA SMCs. PTEN overexpression inhibited IGF-1-induced SMC proliferation in both SV and IMA. PTEN suppression, induced by siRNA transfection of IMA SMCs diminished the negative regulation of PI3K-PKB signalling leading to greater proliferative response induced by IGF-1 stimulation. Thus, we show for the first time that early inactivation of PTEN in SV SMCs leads to temporally increased activity of the pro-hyperplasia PI3K-AKT/PKB pathway leading to IH-induced vein graft occlusion. Therefore, modulation of the PI3K-AKT/PKB pathway via PTEN might be a novel and effective strategy in combating SV graft failure following CABG.