Restoration of the ER stress response protein TDAG51 in hepatocytes mitigates NAFLD in mice.

Endoplasmic reticulum stress is associated with insulin resistance and the development of nonalcoholic fatty liver disease. Deficiency of the endoplasmic reticulum stress response T-cell death-associated gene 51 (TDAG51) (TDAG51-/-) in mice promotes the development of high-fat diet (HFD)-induced obesity, fatty liver, and hepatic insulin resistance. However, whether this effect is due specifically to hepatic TDAG51 deficiency is unknown. Here, we report that hepatic TDAG51 protein levels are consistently reduced in multiple mouse models of liver steatosis and injury as well as in liver biopsies from patients with liver disease compared to normal controls. Delivery of a liver-specific adeno-associated virus (AAV) increased hepatic expression of a TDAG51-GFP fusion protein in WT, TDAG51-/-, and leptin-deficient (ob/ob) mice. Restoration of hepatic TDAG51 protein was sufficient to increase insulin sensitivity while reducing body weight and fatty liver in HFD fed TDAG51-/- mice and in ob/ob mice. TDAG51-/- mice expressing ectopic TDAG51 display improved Akt (Ser473) phosphorylation, post-insulin stimulation. HFD-fed TDAG51-/- mice treated with AAV-TDAG51-GFP displayed reduced lipogenic gene expression, increased beta-oxidation and lowered hepatic and serum triglycerides, findings consistent with reduced liver weight. Further, AAV-TDAG51-GFP-treated TDAG51-/- mice exhibited reduced hepatic precursor and cleaved sterol regulatory-element binding proteins (SREBP-1 and SREBP-2). In vitro studies confirmed the lipid-lowering effect of TDAG51 overexpression in oleic acid-treated Huh7 cells. These studies suggest that maintaining hepatic TDAG51 protein levels represents a viable therapeutic approach for the treatment of obesity and insulin resistance associated with nonalcoholic fatty liver disease.

TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

OBJECTIVE: Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. METHODS AND RESULTS: Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. CONCLUSIONS: Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

Endoplasmic reticulum stress plays an important role in cardiovascular disease (CVD) and atherosclerosis. We aimed to assess the ability of 4-phenylbutyrate (4-PBA), a small chemical chaperone administered via drinking water, to reduce atherosclerotic lesion size in chow-fed apolipoprotein (Apo) e-/- mice and to identify mechanisms that contribute to its antiatherogenic effect. Chow-fed 17-wk-old female Apoe-/- mice treated with 4-PBA-supplemented drinking water for 5 wk exhibited smaller lesions as well as increased plasma levels of heat shock protein (HSP) 25, the mouse homolog of human HSP27, compared with controls. In addition, 4-PBA inhibited cell death and increased HSP27 expression as measured by real-time PCR and immunoblotting, as well as induced nuclear localization of its transcription factor, heat shock factor 1, in human monocyte/macrophage (THP-1) cells. Furthermore, HSP27 small interfering RNA diminished the protective effect of 4-PBA on THP-1 macrophage attachment and differentiation. In summary, drinking water containing 4-PBA attenuated early lesion growth in Apoe-/- mice fed a chow diet and increased expression of HSP25 and HSP27 in macrophages and HSP25 in the circulation of Apoe-/- mice. Given that increased expression of HSP27 is inversely correlated with CVD risk, our findings suggest that 4-PBA protects against the early stages of atherogenesis in part by enhancing HSP27 levels, leading to inhibition of both macrophage cell death and monocyte-macrophage differentiation.-Lynn, E. G., Lhoták, S., Lebeau, P., Byun, J. H., Chen, J., Platko, K., Shi, C., O'Brien, E. R., Austin, R. C. 4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

IL-6 mediates ER expansion during hyperpolarization of alternatively activated macrophages.

Although recent evidence has shown that IL-6 is involved in enhanced alternative activation of macrophages toward a profibrotic phenotype, the mechanisms leading to their increased secretory capacity are not fully understood. Here, we investigated the effect of IL-6 on endoplasmic reticulum (ER) expansion and alternative activation of macrophages in vitro. An essential mediator in this ER expansion process is the IRE1 pathway, which possesses a kinase and endoribonuclease domain to cleave XBP1 into a spliced bioactive molecule. To investigate the IRE1-XBP1 expansion pathway, IL-4/IL-13 and IL-4/IL-13/IL-6-mediated alternative programming of murine bone marrow-derived and human THP1 macrophages were assessed by arginase activity in cell lysates, CD206 and arginase-1 expression by flow cytometry, and secreted CCL18 by ELISA, respectively. Ultrastructural intracellular morphology and ER biogenesis were examined by transmission electron microscopy and immunofluorescence. Transcription profiling of 128 genes were assessed by NanoString and Pharmacological inhibition of the IRE1-XBP1 arm was achieved using STF-083010 and was verified by RT-PCR. The addition of IL-6 to the conventional alternative programming cocktail IL-4/IL-13 resulted in increased ER and mitochondrial expansion, profibrotic profiles and unfolded protein response-mediated induction of molecular chaperones. IRE1-XBP1 inhibition substantially reduced the IL-6-mediated hyperpolarization and normalized the above effects. In conclusion, the addition of IL-6 enhances ER expansion and the profibrotic capacity of IL-4/IL-13-mediated activation of macrophages. Therapeutic strategies targeting IL-6 or the IRE1-XBP1 axis may be beneficial to prevent the profibrotic capacity of macrophages.

Anti-GRP78 autoantibodies induce endothelial cell activation and accelerate the development of atherosclerotic lesions.

The 78-kDa glucose-regulated protein (GRP78) is an ER molecular chaperone that aids in protein folding and secretion. However, pathological conditions that cause ER stress can promote the relocalization of GRP78 to the cell surface (csGRP78), where it acts as a signaling receptor to promote cancer progression. csGRP78 also possesses antigenic properties, leading to the production of anti-GRP78 autoantibodies, which contribute to tumor growth. In contrast, the presence and role of anti-GRP78 autoantibodies in atherosclerosis is unknown. Here, we show that atherosclerotic-prone ApoE-/- mice develop circulating anti-GRP78 autoantibodies that bind to csGRP78 on lesion-resident endothelial cells. Moreover, GRP78-immunized ApoE-/- mice exhibit a marked increase in circulating anti-GRP78 autoantibody titers that correlated with accelerated lesion growth. Mechanistically, engagement of anti-GRP78 autoantibodies with csGRP78 on human endothelial cells activated NF-κB, thereby inducing the expression of ICAM-1 and VCAM-1, a process blocked by NF-κB inhibitors. Disrupting the autoantibody/csGRP78 complex with enoxaparin, a low-molecular-weight heparin, reduced the expression of adhesion molecules and attenuated lesion growth. In conclusion, anti-GRP78 autoantibodies play a crucial role in atherosclerosis development, and disruption of the interaction between anti-GRP78 autoantibodies and csGRP78 represents a therapeutic strategy.

Sialidase down-regulation reduces non-HDL cholesterol, inhibits leukocyte transmigration, and attenuates atherosclerosis in ApoE knockout mice.

Atherosclerosis is a complex disease that involves alterations in lipoprotein metabolism and inflammation. Protein and lipid glycosylation events, such as sialylation, contribute to the development of atherosclerosis and are regulated by specific glycosidases, including sialidases. To evaluate the effect of the sialidase neuraminidase 1 (NEU1) on atherogenesis, here we generated apolipoprotein E (ApoE)-deficient mice that express hypomorphic levels of NEU1 (Neu1hypoApoe-/-). We found that the hypomorphic NEU1 expression in male Apoe-/- mice reduces serum levels of very-low-density lipoprotein (VLDL) and LDL cholesterol, diminishes infiltration of inflammatory cells into lesions, and decreases aortic sinus atherosclerosis. Transplantation of Apoe-/- bone marrow (BM) into Neu1hypoApoe-/- mice significantly increased atherosclerotic lesion development and had no effect on serum lipoprotein levels. Moreover, Neu1hypoApoe-/- mice exhibited a reduction in circulating monocyte and neutrophil levels and had reduced hyaluronic acid and P-selectin adhesion capability on monocytes/neutrophils and T cells. Consistent with these findings, administration of a sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, had a significant anti-atherogenic effect in the Apoe-/- mice. In summary, the reduction in NEU1 expression or function decreases atherosclerosis in mice via its significant effects on lipid metabolism and inflammatory processes. We conclude that NEU1 may represent a promising target for managing atherosclerosis.

Autoantibodies against the cell surface-associated chaperone GRP78 stimulate tumor growth via tissue factor.

Tumor cells display on their surface several molecular chaperones that normally reside in the endoplasmic reticulum. Because this display is unique to cancer cells, these chaperones are attractive targets for drug development. Previous epitope-mapping of autoantibodies (AutoAbs) from prostate cancer patients identified the 78-kDa glucose-regulated protein (GRP78) as one such target. Although we previously showed that anti-GRP78 AutoAbs increase tissue factor (TF) procoagulant activity on the surface of tumor cells, the direct effect of TF activation on tumor growth was not examined. In this study, we explore the interplay between the AutoAbs against cell surface-associated GRP78, TF expression/activity, and prostate cancer progression. First, we show that tumor GRP78 expression correlates with disease stage and that anti-GRP78 AutoAb levels parallel prostate-specific antigen concentrations in patient-derived serum samples. Second, we demonstrate that these anti-GRP78 AutoAbs target cell-surface GRP78, activating the unfolded protein response and inducing tumor cell proliferation through a TF-dependent mechanism, a specific effect reversed by neutralization or immunodepletion of the AutoAb pool. Finally, these AutoAbs enhance tumor growth in mice bearing human prostate cancer xenografts, and heparin derivatives specifically abrogate this effect by blocking AutoAb binding to cell-surface GRP78 and decreasing TF expression/activity. Together, these results establish a molecular mechanism in which AutoAbs against cell-surface GRP78 drive TF-mediated tumor progression in an experimental model of prostate cancer. Heparin derivatives counteract this mechanism and, as such, represent potentially appealing compounds to be evaluated in well-designed translational clinical trials.

Endoplasmic Reticulum Stress and Ca2+ Depletion Differentially Modulate the Sterol Regulatory Protein PCSK9 to Control Lipid Metabolism.

Accumulating evidence implicates endoplasmic reticulum (ER) stress as a mediator of impaired lipid metabolism, thereby contributing to fatty liver disease and atherosclerosis. Previous studies demonstrated that ER stress can activate the sterol regulatory element-binding protein-2 (SREBP2), an ER-localized transcription factor that directly up-regulates sterol regulatory genes, including PCSK9 Given that PCSK9 contributes to atherosclerosis by targeting low density lipoprotein (LDL) receptor (LDLR) degradation, this study investigates a novel mechanism by which ER stress plays a role in lipid metabolism by examining its ability to modulate PCSK9 expression. Herein, we demonstrate the existence of two independent effects of ER stress on PCSK9 expression and secretion. In cultured HuH7 and HepG2 cells, agents or conditions that cause ER Ca2+ depletion, including thapsigargin, induced SREBP2-dependent up-regulation of PCSK9 expression. In contrast, a significant reduction in the secreted form of PCSK9 protein was observed in the media from both thapsigargin- and tunicamycin (TM)-treated HuH7 cells, mouse primary hepatocytes, and in the plasma of TM-treated C57BL/6 mice. Furthermore, TM significantly increased hepatic LDLR expression and reduced plasma LDL concentrations in mice. Based on these findings, we propose a model in which ER Ca2+ depletion promotes the activation of SREBP2 and subsequent transcription of PCSK9. However, conditions that cause ER stress regardless of their ability to dysregulate ER Ca2+ inhibit PCSK9 secretion, thereby reducing PCSK9-mediated LDLR degradation and promoting LDLR-dependent hepatic cholesterol uptake. Taken together, our studies provide evidence that the retention of PCSK9 in the ER may serve as a potential strategy for lowering LDL cholesterol levels.

Characterization of Proliferating Lesion-Resident Cells During All Stages of Atherosclerotic Growth.

BACKGROUND: Monocyte recruitment leads to accumulation of macrophage foam cells and contributes to atherosclerotic lesion growth. Recent studies have reported that lesion-resident macrophages can proliferate and represent a major cellular component during lesion development. This study was designed to assess whether the rate of macrophage proliferation changes during well-established stages of lesion growth and to characterize other populations of proliferating cells within these lesions. METHODS AND RESULTS: Using murine models of atherosclerosis (Apoe(-/-) and LDLr(-/-) mice) and human coronary artery lesions, in situ proliferation of lesion-resident cells at different stages of growth was assessed by staining for Ki67 and bromodeoxyuridine (BrdU). In early lesions, close to half of all actively growing macrophages were proliferating in situ. BrdU pulse labeling allowed for accurate identification of in situ proliferating macrophages compared to those derived from monocyte recruitment. Local macrophage proliferation declined as lesions advanced. Interestingly, intimal inflammatory cell infiltrates containing proliferating T lymphocytes were identified during the active phase of lesion growth and correlated with apoptotic cell death. Inflammatory cell infiltrates were completely resolved in advanced lesions and replaced with the necrotic core. CONCLUSIONS: Our findings indicate that atherosclerotic lesions contain locally proliferating macrophages primarily during early and intermediate stages of lesion growth. Furthermore, T-lymphocyte-enriched inflammatory cell infiltrates represent a novel subset of proliferating cells within the atherosclerotic lesion that correlate with apoptosis and precede the necrotic core. These findings have novel implications in understanding the pathogenesis of atherosclerosis and may implicate proliferating T lymphocytes as a contributing factor to lesion progression and stability.

Salicylate improves macrophage cholesterol homeostasis via activation of Ampk.

Atherosclerosis stems from imbalances in lipid metabolism and leads to maladaptive inflammatory responses. The AMP-activated protein kinase (Ampk) is a highly conserved serine/threonine kinase that regulates many aspects of lipid and energy metabolism, although its specific role in controlling macrophage cholesterol homeostasis remains unclear. We sought to address this question by testing the effects of direct Ampk activators in primary bone marrow-derived macrophages from Ampk ß1-deficient (ß1(-/-)) mice. Macrophages from Ampk ß1(-/-) mice had enhanced lipogenic capacity and diminished cholesterol efflux, although cholesterol uptake was unaffected. Direct activation of Ampk ß1 via salicylate (the unacetylated form of aspirin) or A-769662 (a small molecule activator), decreased the synthesis of FAs and sterols in WT but not Ampk ß1(-/-) macrophages. In lipid-laden macrophages, Ampk activation decreased cholesterol content (foam cell formation) and increased cholesterol efflux to HDL and apoA-I, effects that occurred in an Ampk ß1-dependent manner. Increased cholesterol efflux was also associated with increased gene expression of the ATP binding cassette transporters, Abcg1 and Abca1. Moreover, in vivo reverse cholesterol transport was suppressed in mice that received Ampk ß1(-/-) macrophages compared with the WT control. Our data highlight the therapeutic potential of targeting macrophage Ampk with new or existing drugs for the possible reduction in foam cell formation during the early stages of atherosclerosis.

Novel function of Oncostatin M as a potent tumour-promoting agent in lung.

Oncostatin M is a leukocyte product that has been reported to have anti-proliferative effects directly on melanoma and other cancer cell lines in vitro. However, its function(s) in cancers in vivo appears complex and its roles in cancer growth in lungs are unknown. Here, we show that OSM promotes marked growth of tumour cells in mouse lungs. Local pulmonary administration of adenovirus vector expressing mouse OSM (AdOSM) induced >13-fold increase in lung tumour burden of ectopically delivered B16-F10 melanoma cells in C57BL/6 mice. AdOSM caused increases in tumour size (14 days post-challenge), whereas control vector (Addel70) did not. AdOSM had no such action in C57BL/6 mice deficient in the OSM receptor beta chain (OSMRß-/-), indicating that these effects required OSMRß expression on non-tumour cells in the recipient mice. AdOSM induced elevated levels of chemokines and inflammatory cells in the bronchoalveolar lavage (BAL) fluid, elevated arginase-1 mRNA levels (60-fold), and increased arginase-1+immunostaining macrophage numbers in lungs. Adherent BAL cells collected from AdOSM-treated mice expressed elevated arginase-1 activity. In contrast to AdOSM-induced effects, pulmonary over-expression of IL-1ß (AdIL-1ß) induced neutrophil accumulation and iNOS mRNA, but did not modulate tumour burden. AdOSM also increased lung tumour load (>50-fold) upon ectopic administration of Lewis lung carcinoma (LLC) cells in vivo. However, in vitro, neither recombinant OSM nor AdOSM infection stimulated B16-F10 or LLC cell growth directly. We conclude that pulmonary over-expression of OSM promotes tumour growth, and does so through altering the local lung environment with accumulation of M2 macrophages.

Underactivation of the adiponectin-adiponectin receptor 1 axis in clear cell renal cell carcinoma: implications for progression.

Energy-sensing pathways, normally coordinated by 5' AMP-activated protein kinase (AMPK), are dysregulated in renal cell carcinoma (RCC). Obesity can accentuate the pre-existing pro-tumorigenic metabolic machinery in RCC cells through its associated obesogenic hormonal milieu, characterized by lower circulating levels of adiponectin. In RCC patients, low adiponectin levels associate clinically with more aggressive disease. We investigated the adiponectin signaling pathway in RCC, focusing on adiponectin receptor 1 (AdipoR1) and associated activation of AMPK. AdipoR1 protein in RCC and normal surrounding renal tissues was determined by Western blot analysis and immunohistochemistry. Anti-tumorigenic effects of adiponectin in RCC cells in vitro were investigated via VEGF and MMP ELISA and invasion assays. Using in vivo models of RCC, the effect of AdipoR1-knockdown (shRNA) on tumor latency, growth and dissemination were determined. AdipoR1 protein was significantly reduced in clear cell RCC specimens. Adiponectin treatment inhibited VEGF, MMP-2 and MMP-9 secretion and activity and invasive and migratory capacities of RCC cells. AMPKα1-knockdown (shRNA) attenuated adiponectin's effects. In cells stably expressing AdipoR1-specific shRNA, AMPK activation by adiponectin was significantly reduced compared to cells expressing control shRNA. In vivo, AdipoR1 knockdown increased the growth, dissemination and angiogenesis of RCC. These findings suggest that deficiencies in the entire adiponectin hormonal axis (the hormone and its receptor) result in underactivation of AMPK leading to increased angiogenic and invasive capacities of RCC. The established link between obesity and RCC can therefore be further explained by the adiponectin deficiency in obese individuals together with reduced AdipoR1 protein in RCC.

Decreased endogenous production of hydrogen sulfide accelerates atherosclerosis.

BACKGROUND: Cystathionine γ-lyase (CSE) produces hydrogen sulfide (H2S) in the cardiovascular system. The deficiency of CSE in mice leads to a decreased endogenous H2S level, an age-dependent increase in blood pressure, and impaired endothelium-dependent vasorelaxation. To date, there is no direct evidence for a causative role of altered metabolism of endogenous H2S in atherosclerosis development. METHODS AND RESULTS: Six-week-old CSE gene knockout and wild-type mice were fed with either a control chow or atherogenic paigen-type diet for 12 weeks. Plasma lipid profile and homocysteine levels, blood pressure, oxidative stress, atherosclerotic lesion size in the aortic roots, cell proliferation, and adhesion molecule expression were then analyzed. CSE-knockout mice fed with atherogenic diet developed early fatty streak lesions in the aortic root, elevated plasma levels of cholesterol and low-density lipoprotein cholesterol, hyperhomocysteinemia, increased lesional oxidative stress and adhesion molecule expression, and enhanced aortic intimal proliferation. Treatment of CSE-knockout mice with NaHS, but not N-acetylcysteine or ezetimibe, inhibited the accelerated atherosclerosis development. Double knockout of CSE and apolipoprotein E gene expression in mice exacerbated atherosclerosis development more than that in the mice with only apolipoprotein E or CSE knockout. CONCLUSIONS: Endogenously synthesized H2S protects vascular tissues from atherogenic damage by reducing vessel intimal proliferation and inhibiting adhesion molecule expression. Decreased endogenous H2S production predisposes the animals to vascular remodeling and early development of atherosclerosis. The CSE/H2S pathway is an important therapeutic target for protection against atherosclerosis.

Cystathionine protects against endoplasmic reticulum stress-induced lipid accumulation, tissue injury, and apoptotic cell death.

Cystathionine (R-S-(2-amino-2-carboxyethyl)-l-homocysteine) is a non-proteinogenic thioether containing amino acid. In mammals, cystathionine is formed as an intermediate of the transsulfuration pathway by the condensation of serine and homocysteine (Hcy) in a reaction catalyzed by cystathionine ß-synthase (CBS). Cystathionine is subsequently converted to cysteine plus ammonia and α-ketobutyrate by the action of cystathionine γ-lyase (CGL). Pathogenic mutations in CBS result in CBS-deficient homocystinuria (HCU) which, if untreated, results in mental retardation, thromboembolic complications and connective tissue disorders. Currently there is no known function for cystathionine other than serving as an intermediate in transsulfuration and to date, the possible contribution of the abolition of cystathionine synthesis to pathogenesis in HCU has not been investigated. Using both mouse and cell-culture models, we have found that cystathionine is capable of blocking the induction of hepatic steatosis and kidney injury, acute tubular necrosis, and apoptotic cell death by the endoplasmic reticulum stress inducing agent tunicamycin. Northern and Western blotting analysis indicate that the protective effects of cystathionine occur without any obvious alteration of the induction of the unfolded protein response. Our data constitute the first experimental evidence that the abolition of cystathionine synthesis may contribute to the pathology of HCU and that this compound has therapeutic potential for disease states where ER stress is implicated as a primary initiating pathogenic factor.

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death.

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca(2+)-independent phospholipase A(2) (iPLA(2)ß), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.

Induction of the unfolded protein response after monocyte to macrophage differentiation augments cell survival in early atherosclerotic lesions.

Endoplasmic reticulum (ER) stress causes macrophage cell death within advanced atherosclerotic lesions, thereby contributing to necrotic core formation and increasing the risk of atherothrombotic disease. However, unlike in advanced lesions, the appearance of dead/apoptotic macrophages in early lesions is less prominent. Given that activation of the unfolded protein response (UPR) is detected in early lesion-resident macrophages and can enhance cell survival against ER stress, we investigated whether UPR activation occurs after monocyte to macrophage differentiation and confers a cytoprotective advantage to the macrophage. Human peripheral blood monocytes were treated with monocyte colony-stimulating factor to induce macrophage differentiation, as assessed by changes in ultrastructure and scavenger receptor expression. UPR markers, including GRP78, GRP94, and spliced XBP-1, were induced after macrophage differentiation and occurred after a significant increase in de novo protein synthesis. UPR activation after differentiation reduced macrophage cell death by ER stress-inducing agents. Further, GRP78 overexpression in macrophages was sufficient to reduce ER stress-induced cell death. Consistent with these in vitro findings, UPR activation was observed in viable lesion-resident macrophages from human carotid arteries and from the aortas of apoE(-/-) mice. However, no evidence of apoptosis was observed in early lesion-resident macrophages from the aortas of apoE(-/-) mice. Thus, our findings that UPR activation occurs during macrophage differentiation and is cytoprotective against ER stress-inducing agents suggest an important cellular mechanism for macrophage survival within early atherosclerotic lesions.

Mouse models of cystathionine beta-synthase deficiency reveal significant threshold effects of hyperhomocysteinemia.

Untreated cystathionine beta-synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 microM), with thrombosis as the major cause of morbidity. Treatment with vitamins and diet leads to a dramatic reduction in thrombotic events, even though patients often still have severe elevations in tHcy (>80 microM). To understand the difference between extreme and severe hyperhomocysteinemia, we have examined two mouse models of CBS deficiency: Tg-hCBS Cbs(-/-) mice, with a mean serum tHcy of 169 microM, and Tg-I278T Cbs(-/-) mice, with a mean tHcy of 296 microM. Only Tg-I278T Cbs(-/-) animals exhibited strong biological phenotypes, including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in mean survival time. Metabolic profiling of serum and liver reveals that Tg-I278T Cbs(-/-) mice have significantly elevated levels of free oxidized homocysteine but not protein-bound homocysteine in serum and elevation of all forms of homocysteine and S-adenosylhomocysteine in the liver compared to Tg-hCBS Cbs(-/-) mice. RNA profiling of livers indicate that Tg-I278T Cbs(-/-) and Tg-hCBS Cbs(-/-) mice have unique gene signatures, with minimal overlap. Our results indicate that there is a clear pathogenic threshold effect for tHcy and bring into question the idea that mild elevations in tHcy are directly pathogenic.

Immunohistochemical detection of N-homocysteinylated proteins in humans and mice.

N-homocysteinylation of epsilon-amino group of protein lysine residues by homocysteine (Hcy) thiolactone has been implicated in vascular disease in humans. We have previously generated polyclonal rabbit anti-N-Hcy-protein IgG antibodies that specifically recognize the Nepsilon-Hcy-Lys epitope on N-homocysteinylated proteins. The present work was undertaken to examine the utility of these antibodies for the immunohistochemical detection of N-homocysteinylated proteins in biological samples. We found that the rabbit antibody specifically detected N-Hcy-protein in a dot-blot assay, that the signal resulting from the reaction of the antibody with N-Hcy-protein depended on the amount of the antigen, and that the sensitivity of the assay was protein-dependent. The rabbit anti-N-Hcy-protein IgG also specifically detected Nepsilon-Hcy-Lys epitopes in human tissues, as shown by positive immunohistochemical staining of myocardium and aorta samples from cardiac surgery patients, and a lack of staining when the antibody was pre-adsorbed with N-Hcy-albumin. We also observed increased immunohistochemical staining for N-Hcy-proteins in aortic lesions from ApoE-/- mice with hyperhomocysteinemia induced by a high methionine diet, relative to ApoE-/- mice fed a control chow diet. In conclusion, polyclonal rabbit anti-N-Hcy-protein antibody can detect and monitor N-homocysteinylated proteins in human and mouse tissues with good sensitivity and specificity.

Hyperhomocysteinemia induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice.

A causal relationship between diet-induced hyperhomocysteinemia (HHcy) and accelerated atherosclerosis has been established in apolipoprotein E-deficient (apoE(-/-)) mice. However, it is not known whether the proatherogenic effect of HHcy in apoE(-/-) mice is independent of hyperlipidemia and/or deficiency of apoE. In this study, a comprehensive dietary approach using C57BL/6J mice was used to investigate whether HHcy is an independent risk factor for accelerated atherosclerosis or dependent on additional dietary factors that increase plasma lipids and/or inflammation. C57BL/6J mice at 4 wk of age were divided into 6 dietary groups: chow diet (C), chow diet + methionine (C+M), western-type diet (W), western-type diet + methionine (W+M), atherogenic diet (A), or atherogenic diet + methionine (A+M). After 2, 10, 20, or 40 wk on the diets, mice were sacrificed, and the levels of total plasma homocysteine, cysteine, and glutathione, as well as total plasma cholesterol and triglycerides were analyzed. Aortic root sections were examined for atherosclerotic lesions. HHcy was induced in all groups supplemented with methionine, compared to diet-matched control groups. Plasma total cholesterol was significantly increased in mice fed the W or A diet. However, the W diet increased LDL/IDL and HDL levels, while the A diet significantly elevated plasma VLDL and LDL/IDL levels without increasing HDL. No differences in plasma total cholesterol levels or lipid profiles were observed between methionine-supplemented groups and the diet-matched control groups. Early atherosclerotic lesions containing macrophage foam cells were only observed in mice fed the A or A + M diet. Furthermore, lesion size was significantly larger in the A + M group compared to the A group at 10 and 20 wk; however, mature lesions were never observed even after 40 wk on these diets. The presence of lymphocytes, increased hyaluronan staining, and the expression of endoplasmic reticulum (ER) stress markers were also increased in atherosclerotic lesions from the A + M group. Taken together, these results suggest that HHcy does not independently cause atherosclerosis in C57BL/6J mice even in the presence of increased total plasma lipids induced by the W diet. However, HHcy can accelerate atherosclerotic lesion development under dietary conditions that increase plasma VLDL levels and/or inflammation.

Peroxynitrite causes endoplasmic reticulum stress and apoptosis in human vascular endothelium: implications in atherogenesis.

OBJECTIVE: Peroxynitrite, a potent oxidant generated by the reaction of NO with superoxide, has been implicated in the promotion of atherosclerosis. We designed this study to determine whether peroxynitrite induces its proatherogenic effects through induction of endoplasmic reticulum (ER) stress. METHODS AND RESULTS: Human vascular endothelial cells treated with Sin-1, a peroxynitrite generator, induced the expression of the ER chaperones GRP78 and GRP94 and increased eIF2alpha phosphorylation. These effects were inhibited by the peroxynitrite scavenger uric acid. Sin-1 caused the depletion of ER-Ca2+, an effect known to induce ER stress, resulting in the elevation of cytosolic Ca2+ and programmed cell death (PCD). Sin-1 treatment was also found, via 3-nitrotyrosine and GRP78 colocalization, to act directly on the ER. Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented Sin-1-induced PCD. Consistent with these in vitro findings, 3-nitrotyrosine was observed and colocalized with GRP78 in endothelial cells of early atherosclerotic lesions from apolipoprotein E-deficient mice. CONCLUSIONS: Peroxynitrite is an ER stress-inducing agent. Its effects include the depletion of ER-Ca2+, a known mechanism of ER stress induction. The observation that 3-nitrotyrosine-containing proteins colocalize with markers of ER stress within early atherosclerotic lesions suggests that peroxynitrite contributes to atherogenesis through a mechanism involving ER stress.