Ursodeoxycholic Acid (UDCA) Exerts Anti-Atherogenic Effects by Inhibiting RAGE Signaling in Diabetic Atherosclerosis

A naturally occurring bile acid, ursodeoxycholic acid (UDCA), is known to alleviate endoplasmic reticulum (ER) stress at the cellular level. However, the detailed action mechanisms of UDCA in atherosclerosis are not fully understood. In this study, we demonstrated whether UDCA exerts anti-atherogenic activity in diabetic atherosclerosis by targeting ER stress and “receptor for advanced glycation endproduct” (RAGE) signaling. UDCA markedly reduced ER stress, RAGE expression, and pro-inflammatory responses [including NF-κB activation and reactive oxygen species (ROS) production] induced in endothelial cells (ECs) by high glucose (HG). In particular, UDCA inhibited HG-induced ROS production by increasing the Nrf2 level. In macrophages, UDCA also blocked HG-induced RAGE and pro-inflammatory cytokine expression and inhibited foam cell formation via upregulation of the ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1. In the diabetic mouse model, UDCA inhibited atheromatous plaque formation by decreasing ER stress, and the levels of RAGE and adhesion molecules. In conclusion, UDCA exerts an anti-atherogenic activity in diabetic atherosclerosis by targeting both ER stress and RAGE signaling. Our work implicates UDCA as a potential therapeutic agent for prevention or treatment of diabetic atherosclerosis.     

Endoplasmic reticulum stress is implicated in retinal inflammation and diabetic retinopathy

Diabetic retinopathy is a chronic low-grade inflammatory disease; however, the mechanisms remain elusive. In the present study, we demonstrated that endoplasmic reticulum (ER) stress was activated in the retina in animal models of diabetes and oxygen-induced retinopathy (OIR). Induction of ER stress by tunicamycin resulted in significantly increased expression of inflammatory molecules in the retina. Inhibition of ER stress by chemical chaperone 4-phenyl butyric acid ameliorated inflammation in cultured human retinal endothelial cells exposed to hypoxia, and in the retinas of diabetic and OIR mice. These findings indicate that ER stress is a potential mediator of retinal inflammation in diabetic retinopathy.     

Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3

It is well known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box-binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a VEGF receptor and PI3K/Akt-dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Overexpression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization, and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3, and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.     

Down-regulation of PERK enhances resistance to ionizing radiation

Although, ionizing radiation (IR) has been implicated to cause stress in endoplasmic reticulum (ER), how ER stress signaling and major ER stress sensors modulate cellular response to IR is unclear. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein which initiates unfolded protein response (UPR) or ER stress signaling when ER homeostasis is disturbed. Here, we report that down-regulation of PERK resulted in increased clonogenic survival, enhanced DNA repair and reduced apoptosis in irradiated cancer cells. Our study demonstrated that PERK has a role in sensitizing cancer cells to IR.     

Flow-activated chloride channels in vascular endothelium. Shear stress sensitivity, desensitization dynamics, and physiological implications

Although activation of outward rectifying Cl(-) channels is one of the fastest responses of endothelial cells (ECs) to shear stress, little is known about these channels. In this study, we used whole-cell patch clamp recordings to characterize the flow-activated Cl(-) current in bovine aortic ECs (BAECs). Application of shear stress induced rapid development of a Cl(-) current that was effectively blocked by the Cl(-) channel antagonist 5-nitro-2-(3-phenopropylamino)benzoic acid (100 microM). The current initiated at a shear stress as low as 0.3 dyne/cm(2), attained its peak within minutes of flow onset, and saturated above 3.5 dynes/cm(2) approximately 2.5-3.5-fold increase over pre-flow levels). The Cl(-) current desensitized slowly in response to sustained flow, and step increases in shear stress elicited increased current only if the shear stress levels were below the 3.5 dynes/cm(2) saturation level. Oscillatory flow with a physiological oscillation frequency of 1 Hz, as occurs in disturbed flow zones prone to atherosclerosis, failed to elicit the Cl(-) current, whereas lower oscillation frequencies led to partial recovery of the current. Nonreversing pulsatile flow, generally considered protective of atherosclerosis, was as effective in eliciting the current as steady flow. Measurements using fluids of different viscosities indicated that the Cl(-) current is responsive to shear stress rather than shear rate. Blocking the flow-activated Cl(-) current abolished flow-induced Akt phosphorylation in BAECs, whereas blocking flow-sensitive K(+) currents had no effect, suggesting that flow-activated Cl(-) channels play an important role in regulating EC flow signaling.     

Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway

Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.     

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.     

Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress

The ability to respond to perturbations in endoplasmic reticulum (ER) function is a fundamentally important property of all cells, but ER stress can also lead to apoptosis. In settings of chronic ER stress, the associated apoptosis may contribute to pathophysiological processes involved in a number of prevalent diseases, including neurodegenerative diseases, diabetes, atherosclerosis and renal disease. The molecular mechanisms linking ER stress to apoptosis are the topic of this review, with emphases on relevance to pathophysiology and integration and complementation among the various apoptotic pathways induced by ER stress.     

ER stress is associated with reduced ABCA-1 protein levels in macrophages treated with advanced glycated albumin - reversal by a chemical chaperone

ATP-binding cassette transporter A1 mediates the export of excess cholesterol from macrophages, contributing to the prevention of atherosclerosis. Advanced glycated albumin (AGE-alb) is prevalent in diabetes mellitus and is associated with the development of atherosclerosis. Independently of changes in ABCA-1 mRNA levels, AGE-alb induces oxidative stress and reduces ABCA-1 protein levels, which leads to macrophage lipid accumulation. These metabolic conditions are known to elicit endoplasmic reticulum (ER) stress. We sought to determine if AGE-alb induces ER stress and unfolded protein response (UPR) in macrophages and how disturbances to the ER could affect ABCA-1 content and cholesterol efflux in macrophages. AGE-alb induced a time-dependent increase in ER stress and UPR markers. ABCA-1 content and cellular cholesterol efflux were reduced by 33% and 47%, respectively, in macrophages treated with AGE-alb, and both were restored by treatment with 4-phenyl butyric acid (a chemical chaperone that alleviates ER stress), but not MG132 (a proteasome inhibitor). Tunicamycin, a classical ER stress inductor, also impaired ABCA-1 expression and cholesterol efflux (showing a decrease of 61% and 82%, respectively), confirming the deleterious effect of ER stress in macrophage cholesterol accumulation. Glycoxidation induces macrophage ER stress, which relates to the reduction in ABCA-1 and in reverse cholesterol transport, endorsing the adverse effect of macrophage ER stress in atherosclerosis. Thus, chemical chaperones that alleviate ER stress may represent a useful tool for the prevention and treatment of atherosclerosis in diabetes.     

Different fatty acids inhibit apoB100 secretion by different pathways: unique roles for ER stress, ceramide, and autophagy

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.     

Ursolic acid induces ER stress response to activate ASK1–JNK signaling and induce apoptosis in human bladder cancer T24 cells

Here we studied the cellular mechanisms of ursolic acid's anti-bladder cancer ability by focusing on endoplasmic reticulum stress (ER stress) signaling. We show that ursolic acid induces a significant ER stress response in cultured human bladder cancer T24 cells. ER stress inhibitor salubrinal, or PERK silencing, diminishes ursolic acid-induced anti-T24 cell effects. Salubrinal inhibits ursolic acid-induced CHOP expression, Bim ER accumulation and caspase-3 activation in T24 cells. Ursolic acid induces IRE1–TRAF2–ASK1 signaling complex formation to activate pro-apoptotic ASK1–JNK signaling. We suggest that ER stress contributes to ursolic acid's effects against bladder cancer cells.     

Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin

Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.     

A potential strategy for treating atherosclerosis: improving endothelial function via AMP-activated protein kinase

Endothelial dysfunction is caused by many factors, such as dyslipidemia, endoplasmic reticulum(ER) stress, and inflammation.It has been demonstrated that endothelial dysfunction is the initial process of atherosclerosis. AMP-activated protein kinase(AMPK) is an important metabolic switch that plays a crucial role in lipid metabolism and inflammation. However, recent evidence indicates that AMPK could be a target for atherosclerosis by improving endothelial function. For instance, activation of AMPK inhibits the production of reactive oxygen species induced by mitochondrial dysfunction, ER stress, and NADPH oxidase. Moreover, activation of AMPK inhibits the production of pro-inflammatory factors induced by dyslipidemia and hyperglycemia and restrains production of perivascular adipose tissue-released adipokines. AMPK activation prevents endothelial dysfunction by increasing the bioavailability of nitric oxide. Therefore, we focused on the primary risk factors involved in endothelial dysfunction, and summarize the features of AMPK in the protection of endothelial function, by providing signaling pathways thought to be important in the pathological progress of risk factors.     

Sterculic acid antagonizes 7-ketocholesterol-mediated inflammation and inhibits choroidal neovascularization

Sterculic acid is a cyclopropene fatty acid with numerous biological activities. In this study we demonstrate that sterculic acid is a potent inhibitor of endoplasmic reticulum (ER) stress and related inflammation caused by 7-ketocholesterol (7KCh). 7KCh is a highly toxic oxysterol suspected in the pathogenesis of various age-related diseases such as atherosclerosis, Alzheimer's disease and age-related macular degeneration. Sterculic acid demonstrated to be 5-10 times more effective than other anti-inflammatory fatty acids at inhibiting 7KCh-mediated inflammatory responses in cultured cells. In vivo, sterculic acid was effective at inhibiting the formation of choroidal neovascularization (CNV) in the laser-injury rat model. Our data suggests that sterculic acid may be useful in treating CNV in certain forms of age-related macular degeneration.     

内质网应激预处理对人正常肝细胞缺氧再复氧损伤的保护作用

目的:研究内质网应激预处理对人肝细胞缺氧复氧损伤的保护作用。方法:将培养的人肝细胞分为4组:正常对照(C)组、细胞缺氧复氧损伤(H/R)组、内质网应激(ER)组、内质网应激预处理(ERP+H/R)组。收集各组细胞,以流式细胞仪检测细胞凋亡,Western-bloting及RT-PCR检测内质网应激特异蛋白GRP78表达水平,并通过透射电镜观察各组细胞超微结构改变。结果:ERP+H/R组细胞凋亡率明显低于H/R组(P<0.05),ER及ERP+H/R组GRP78蛋白表达明显高于H/R组(P<0.05)。结论:内质网应激预处理对肝细胞缺氧复氧损伤具有明显的保护作用,内质网应激特异性蛋白GRP78可能在肝细胞缺氧复氧损伤中作为一种关键性的保护蛋白出现。     

Aspirin prevents adhesion of T lymphoblasts to vascular smooth muscle cells

In the development of atherosclerosis, inflammatory cells adhere to and migrate into the vascular walls by interacting with vascular smooth muscle cells. To investigate the mechanism of aspirin’s anti-atherogenic activity, we examined whether aspirin inhibits the adhesion of lymphocytes to human aortic smooth muscle cells (AoSMC). Aspirin inhibited T-cell adhesion to AoSMC activated by interleukin 1β (IL-1β) in a dose-dependent manner. Antibodies to the adhesion molecules ICAM-1 or VCAM-1, but not to E-selectin, prevented T-cell adhesion. ICAM-1 and VCAM-1 expression stimulated by IL-1β was reduced by the treatment with aspirin, whereas the expression of E-selectin was unaffected. Nuclear factor κB (NF-κB) activity was enhanced by IL-1β and reduced by aspirin, indicating that decreased ICAM-1 and VCAM-1 expression was due to reduced NF-κB activity.Thus, aspirin inhibits the adhesion of Jurkat T cells to IL-1β-activated AoSMC by reducing NF-κB activity and decreasing expression of ICAM-1 and VCAM-1, and may prevent the development of atherosclerosis.     

Protein phosphorylation on tyrosine restores expression and glycosylation of cyclooxygenase-2 by 2-deoxy-D-glucose-caused endoplasmic reticulum stress in rabbit articular chondrocyte

2-deoxy-D-glucose(2DG)-caused endoplasmic reticulum (ER) stress inhibits protein phosphorylation at tyrosine residues. However, the accurate regulatory mechanisms, which determine the inflammatory response of chondrocytes to ER stress via protein tyrosine phosphorylation, have not been systematically evaluated. Thus, in this study, we examined whether protein phosphorylation at tyrosine residues can modulate the expression and glycosylation of COX-2, which is reduced by 2DG-induced ER stress. We observed that protein tyrosine phosphatase (PTP) inhibitors, sodium orthovanadate (SOV), and phenylarsine oxide (PAO) significantly decreased expression of ER stress inducible proteins, glucose-regulated protein 94 (GRP94), and CCAAT/enhancer-binding-protein- related gene (GADD153), which was induced by 2DG. In addition, we demonstrated that SOV and PAO noticeably restored the expression and glycosylation of COX-2 after treatment with 2DG. These results suggest that protein phosphorylation of tyrosine residues plays an important role in the regulation of expression and glycosylation during 2DG-induced ER stress in rabbit articular chondrocytes.     

Role of shear stress direction in endothelial mechanotransduction

Fluid shear stress due to blood flow can modulate functions of endothelial cells (ECs) in blood vessels by activating mechano-sensors, signaling pathways, and gene and protein expressions. Laminar shear stress with a definite forward direction causes transient activations of many genes that are atherogenic, followed by their down-regulation; laminar shear stress also up-regulates genes that inhibit EC growth. In contrast, disturbed flow patterns with little forward direction cause sustained activations of these atherogenic genes and enhancements of EC mitosis and apoptosis. In straight parts of the arterial tree, laminar shear stress with a definite forward direction has anti-atherogenic effects. At branch points, the complex flow patterns with little net direction are atherogenic. Thus, the direction of shear stress has important physiological and pathophysiological effects on vascular ECs.     

Phosphatidylethanolamines modified by γ-ketoaldehyde (γKA) induce endoplasmic reticulum stress and endothelial activation

Peroxidation of plasma lipoproteins has been implicated in the endothelial cell activation and monocyte adhesion that initiate atherosclerosis, but the exact mechanisms underlying this activation remain unclear. Lipid peroxidation generates lipid aldehydes, including the γ-ketoaldehydes (γKA), also termed isoketals or isolevuglandins, that readily modify the amine headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde modification of PE could mediate some of the proinflammatory effects of lipid peroxidation. We found that PE modified by γKA (γKA-PE) induced THP-1 monocyte adhesion to human umbilical cord endothelial cells. γKA-PE also induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in human umbilical cord endothelial cells. To determine the structural requirements for γKA-PE activity, we tested several related compounds. PE modified by 4-oxo-pentanal induced THP-1 adhesion, but N-glutaroyl-PE and C(18:0)N-acyl-PE did not, suggesting that an N-pyrrole moiety was essential for cellular activity. As the N-pyrrole headgroup might distort the membrane, we tested the effect of the pyrrole-PEs on membrane parameters. γKA-PE and 4-oxo-pentanal significantly reduced the temperature for the liquid crystalline to hexagonal phase transition in artificial bilayers, suggesting that these pyrrole-PE markedly altered membrane curvature. Additionally, fluorescently labeled γKA-PE rapidly internalized to the endoplasmic reticulum (ER); γKA-PE induced C/EBP homologous protein CHOP and BiP expression and p38 MAPK activity, and inhibitors of ER stress reduced γKA-PE-induced C/EBP homologous protein CHOP and BiP expression as well as EC activation, consistent with γKA-PE inducing ER stress responses that have been previously linked to inflammatory chemokine expression. Thus, γKA-PE is a potential mediator of the inflammation induced by lipid peroxidation.