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.

Salsalate reduces atherosclerosis through AMPKß1 in mice.

OBJECTIVE: Salsalate is a prodrug of salicylate that lowers blood glucose in people with type 2 diabetes. AMP-activated protein kinase (AMPK) is an αßγ heterotrimer which inhibits macrophage inflammation and the synthesis of fatty acids and cholesterol in the liver through phosphorylation of acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), respectively. Salicylate binds to and activates AMPKß1-containing heterotrimers that are highly expressed in both macrophages and liver, but the potential importance of AMPK and ability of salsalate to reduce atherosclerosis have not been evaluated. METHODS: ApoE-/- and LDLr-/- mice with or without (-/-) germline or bone marrow AMPKß1, respectively, were treated with salsalate, and atherosclerotic plaque size was evaluated in serial sections of the aortic root. Studies examining the effects of salicylate on markers of inflammation, fatty acid and cholesterol synthesis and proliferation were conducted in bone marrow-derived macrophages (BMDMs) from wild-type mice or mice lacking AMPKß1 or the key AMPK-inhibitory phosphorylation sites on ACC (ACC knock-in (KI)-ACC KI) or HMGCR (HMGCR-KI). RESULTS: Salsalate reduced atherosclerotic plaques in the aortic roots of ApoE-/- mice, but not ApoE-/- AMPKß1-/- mice. Similarly, salsalate reduced atherosclerosis in LDLr-/- mice receiving wild-type but not AMPKß1-/- bone marrow. Reductions in atherosclerosis by salsalate were associated with reduced macrophage proliferation, reduced plaque lipid content and reduced serum cholesterol. In BMDMs, this suppression of proliferation by salicylate required phosphorylation of HMGCR and the suppression of cholesterol synthesis. CONCLUSIONS: These data indicate that salsalate suppresses macrophage proliferation and atherosclerosis through an AMPKß1-dependent pathway, which may involve HMGCR phosphorylation and cholesterol synthesis. Since rapidly-proliferating macrophages are a hallmark of atherosclerosis, these data indicate further evaluation of salsalate as a potential therapeutic agent for treating atherosclerotic cardiovascular 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.

Pcsk9 knockout exacerbates diet-induced non-alcoholic steatohepatitis, fibrosis and liver injury in mice.

The fatty acid translocase, also known as CD36, is a well-established scavenger receptor for fatty acid (FA) uptake and is abundantly expressed in many metabolically active tissues. In the liver, CD36 is known to contribute to the progression of non-alcoholic fatty liver disease and to the more severe non-alcoholic steatohepatitis, by promoting triglyceride accumulation and subsequent lipid-induced endoplasmic reticulum (ER) stress. Given the recent discovery that the hepatocyte-secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) blocks CD36 expression, we sought to investigate the role of PCSK9 in liver fat accumulation and injury in response to saturated FAs and in a mouse model of diet-induced hepatic steatosis. METHODS: In this study, we investigated the role of PCSK9 on the uptake and accumulation of FAs, as well as FA-induced toxicity, in a variety of cultured hepatocytes. Diet-induced hepatic steatosis and liver injury were also assessed in Pcsk9 -/- mice. RESULTS: Our results indicate that PCSK9 deficiency in cultured hepatocytes increased the uptake and accumulation of saturated and unsaturated FAs. In the presence of saturated FAs, PCSK9 also protected cultured hepatocytes from ER stress and cytotoxicity. In line with these findings, a metabolic challenge using a high-fat diet caused severe hepatic steatosis, ER stress inflammation and fibrosis in the livers of Pcsk9 -/- mice compared to controls. Given that inhibition of CD36 ablated the observed accumulation of lipid in vitro and in vivo, our findings also highlight CD36 as a strong contributor to steatosis and liver injury in the context of PCSK9 deficiency. CONCLUSIONS: Collectively, our findings demonstrate that PCSK9 regulates hepatic triglyceride content in a manner dependent on CD36. In the presence of excess dietary fats, PCSK9 can also protect against hepatic steatosis and liver injury. LAY SUMMARY: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein known to reduce the abundance of receptors on the surface of liver cells charged with the task of lipid uptake from the circulation. Although PCSK9 deficiency is known to cause lipid accumulation in mice and in cultured cells, the toxicological implications of this observation have not yet been reported. In this study, we demonstrate that PCSK9 can protect against cytotoxicity in cultured liver cells treated with a saturated fatty acid and we also show that Pcsk9 knockout mice develop increased liver injury in response to a high-fat diet.

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.

Loss-of-function PCSK9 mutants evade the unfolded protein response sensor GRP78 and fail to induce endoplasmic reticulum stress when retained.

The proprotein convertase subtilisin/kexin type-9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low-density lipoprotein receptor (LDLR). As such, loss-of-function (LOF) PCSK9 variants that fail to exit the endoplasmic reticulum (ER) increase hepatic LDLR levels and lower the risk of developing CVD. The retention of misfolded protein in the ER can cause ER stress and activate the unfolded protein response (UPR). In this study, we investigated whether a variety of LOF PCSK9 variants that are retained in the ER can cause ER stress and hepatic cytotoxicity. Although overexpression of these PCSK9 variants caused an accumulation in the ER of hepatocytes, UPR activation or apoptosis was not observed. Furthermore, ER retention of endogenous PCSK9 via splice switching also failed to induce the UPR. Consistent with these in vitro studies, overexpression of PCSK9 in the livers of mice had no impact on UPR activation. To elucidate the cellular mechanism to explain these surprising findings, we observed that the 94-kDa glucose-regulated protein (GRP94) sequesters PCSK9 away from the 78-kDa glucose-regulated protein (GRP78), the major activator of the UPR. As a result, GRP94 knockdown increased the stability of GRP78-PCSK9 complex and resulted in UPR activation following overexpression of ER-retained PCSK9 variants relative to WT secreted controls. Given that overexpression of these LOF PCSK9 variants does not cause UPR activation under normal homeostatic conditions, therapeutic strategies aimed at blocking the autocatalytic cleavage of PCSK9 in the ER represent a viable strategy for reducing circulating PCSK9.

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.

Liver-specific ATP-citrate lyase inhibition by bempedoic acid decreases LDL-C and attenuates atherosclerosis.

Despite widespread use of statins to reduce low-density lipoprotein cholesterol (LDL-C) and associated atherosclerotic cardiovascular risk, many patients do not achieve sufficient LDL-C lowering due to muscle-related side effects, indicating novel treatment strategies are required. Bempedoic acid (ETC-1002) is a small molecule intended to lower LDL-C in hypercholesterolemic patients, and has been previously shown to modulate both ATP-citrate lyase (ACL) and AMP-activated protein kinase (AMPK) activity in rodents. However, its mechanism for LDL-C lowering, efficacy in models of atherosclerosis and relevance in humans are unknown. Here we show that ETC-1002 is a prodrug that requires activation by very long-chain acyl-CoA synthetase-1 (ACSVL1) to modulate both targets, and that inhibition of ACL leads to LDL receptor upregulation, decreased LDL-C and attenuation of atherosclerosis, independently of AMPK. Furthermore, we demonstrate that the absence of ACSVL1 in skeletal muscle provides a mechanistic basis for ETC-1002 to potentially avoid the myotoxicity associated with statin therapy.

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.

GRP78 and CHOP modulate macrophage apoptosis and the development of bleomycin-induced pulmonary fibrosis.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78(+/-) , Chop(-/-) and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78(+/-) mice were strongly protected from bleomycin-induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL-positive cells were observed in Grp78(+/-) mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78(+/-) mice was also strongly positive for cleaved caspase-3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop(-/-) mice resulted in increased quasi-static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase-1-positive macrophages that were negative for cleaved caspase-3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78- and CHOP-mediated macrophage apoptosis was found to protect against bleomycin-induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78-mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

Deficiency of TDAG51 protects against atherosclerosis by modulating apoptosis, cholesterol efflux, and peroxiredoxin-1 expression.

BACKGROUND: Apoptosis caused by endoplasmic reticulum (ER) stress contributes to atherothrombosis, the underlying cause of cardiovascular disease (CVD). T-cell death-associated gene 51 (TDAG51), a member of the pleckstrin homology-like domain gene family, is induced by ER stress, causes apoptosis when overexpressed, and is present in lesion-resident macrophages and endothelial cells. METHODS AND RESULTS: To study the role of TDAG51 in atherosclerosis, male mice deficient in TDAG51 and apolipoprotein E (TDAG51(-/-)/ApoE(-/-)) were generated and showed reduced atherosclerotic lesion growth (56 ± 5% reduction at 40 weeks, relative to ApoE(-/-) controls, P<0.005) and necrosis (41 ± 4% versus 63 ± 8% lesion area in TDAG51(-/-)/ApoE(-/-) and ApoE(-/-), respectively; P<0.05) without changes in plasma levels of lipids, glucose, and inflammatory cytokines. TDAG51 deficiency caused several phenotypic changes in macrophages and endothelial cells that increase cytoprotection against oxidative and ER stress, enhance PPARγ-dependent reverse cholesterol transport, and upregulate peroxiredoxin-1 (Prdx-1), an antioxidant enzyme with antiatherogenic properties (1.8 ± 0.1-fold increase in Prdx-1 protein expression, relative to control macrophages; P<0.005). Two independent case-control studies found that a genetic variant in the human TDAG51 gene region (rs2367446) is associated with CVD (OR, 1.15; 95% CI, 1.07 to 1.24; P=0.0003). CONCLUSIONS: These findings provide evidence that TDAG51 affects specific cellular pathways known to reduce atherogenesis, suggesting that modulation of TDAG51 expression or its activity may have therapeutic benefit for the treatment of CVD.

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.

Loss of TDAG51 results in mature-onset obesity, hepatic steatosis, and insulin resistance by regulating lipogenesis.

Regulation of energy metabolism is critical for the prevention of obesity, diabetes, and hepatic steatosis. Here, we report an important role for the pleckstrin homology-related domain family member, T-cell death-associated gene 51 (TDAG51), in the regulation of energy metabolism. TDAG51 expression was examined during adipocyte differentiation. Adipogenic potential of preadipocytes with knockdown or absence of TDAG51 was assessed. Weight gain, insulin sensitivity, metabolic rate, and liver lipid content were also compared between TDAG51-deficient (TDAG51(-/-)) and wild-type mice. In addition to its relatively high expression in liver, TDAG51 was also present in white adipose tissue (WAT). TDAG51 was downregulated during adipogenesis, and TDAG51(-/-) preadipocytes exhibited greater lipogenic potential. TDAG51(-/-) mice fed a chow diet exhibited greater body and WAT mass, had reduced energy expenditure, displayed mature-onset insulin resistance (IR), and were predisposed to hepatic steatosis. TDAG51(-/-) mice had increased hepatic triglycerides and SREBP-1 target gene expression. Furthermore, TDAG51 expression was inversely correlated with fatty liver in multiple mouse models of hepatic steatosis. Taken together, our findings suggest that TDAG51 is involved in energy homeostasis at least in part by regulating lipogenesis in liver and WAT, and hence, may constitute a novel therapeutic target for the treatment of obesity and IR.