Mast cells promote atherosclerosis by inducing both an atherogenic lipid profile and vascular inflammation

Accumulating in vitro and in vivo studies have proposed a role for mast cells in the pathogenesis of atherosclerosis. Here, we studied the role of mast cells in lipoprotein metabolism, a key element in the atherosclerotic disease. Male mice deficient in low‐density lipoprotein receptors and mast cells on a Western diet for 26 weeks had significantly less atherosclerotic changes both in aortic sinus (55%, P = 0.0009) and in aorta (31%, P = 0.049), as compared to mast cell‐competent littermates. Mast cell‐deficient female mice had significantly less atherosclerotic changes in aortic sinus (43%, P = 0.011). Furthermore, we found a significant positive correlation between the extent of atherosclerosis and the number of adventitial/perivascular mast cells in aortic sinus of mast cell‐competent mice (r = 0.615, P = 0.015). Serum cholesterol and triglyceride levels were significantly lower in both male (63%, P = 0.0005 and 57%, P = 0.004) and female (73%, P = 0.00009 and 54%, P = 0.007) mast cell‐deficient mice, with a concomitant decrease in atherogenic apoB‐containing particles and serum preβ‐high‐density lipoprotein and phospholipid transfer protein activity in both male (69% and 24%) and female (74% and 54%) mast cell‐deficient mice. Serum soluble intercellular adhesion molecule was decreased in both male (32%, P = 0.004) and female (28%, P = 0.003) mast cell‐deficient mice, whereas serum amyloid A was similar between mast cell‐deficient and competent mice. In conclusion, mast cells participate in the pathogenesis of atherosclerosis in ldlr^?/? mice by inducing both an atherogenic lipid profile and vascular inflammation. J. Cell. Biochem. 109: 615–623, 2010. © 2009 Wiley‐Liss, Inc.     

Proteasome function is required to maintain muscle cellular architecture

BACKGROUND INFORMATION: Protein degradation via the UPS (ubiquitin-proteasome system) plays critical roles in muscle metabolism and signalling pathways. The present study investigates temporal requirements of the UPS in muscle using conditional expression of mutant proteasome beta subunits to cause targeted inhibition of proteasome function. RESULTS AND CONCLUSIONS: The Drosophila GeneSwitch system was used, with analyses of the well-characterized larval somatic body wall muscles. This method acutely disrupts proteasome function and causes rapid accumulation of polyubiquitinated proteins, specifically within the muscle. Within 12 h of transgenic proteasome inhibition, there was a gross disorganization of muscle architecture and prominent muscle atrophy, progressing to the arrest of all co-ordinated movement by 24 h. Progressive muscle architecture changes include rapid loss of sarcomere organization, loss of nuclei spacing/patterning, vacuole formation and the accumulation of nuclear and cytoplasmic aggregates at the ultrastructural level. At the neuromuscular junction, the highly specialized muscle membrane folds of the subsynaptic reticulum were rapidly lost. Within 24 h after transgenic proteasome inhibition, muscles contained numerous autophagosomes and displayed highly elevated expression of the endoplasmic reticulum chaperone GRP78 (glucose-regulated protein of 78 kDa), indicating that the loss of muscle maintenance correlates with induction of the unfolded protein response. Taken together, these results demonstrate that the UPS is acutely required for maintenance of muscle and neuromuscular junction architecture, and provides a Drosophila genetic model to mechanistically evaluate this requirement.     

MiR-216a: a link between endothelial dysfunction and autophagy

Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis.     

Acidocalcisome is required for autophagy in Trypanosoma brucei

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca²⁺ and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.     

Phosphorylation of Atg31 is required for autophagy

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17- Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.     

Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H₂O₂-induced viability loss, which specifically evokes an autophagic response. Exposed to H₂O₂, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.     

Autocrine VEGF signaling is required for vascular homeostasis

Vascular endothelial growth factor (VEGF) is essential for developmental and pathological angiogenesis. Here we show that in the absence of any pathological insult, autocrine VEGF is required for the homeostasis of blood vessels in the adult. Genetic deletion of vegf specifically in the endothelial lineage leads to progressive endothelial degeneration and sudden death in 55% of mutant mice by 25 weeks of age. The phenotype is manifested without detectable changes in the total levels of VEGF mRNA or protein, indicating that paracrine VEGF could not compensate for the absence of endothelial VEGF. Furthermore, wild-type, but not VEGF null, endothelial cells showed phosphorylation of VEGFR2 in the absence of exogenous VEGF. Activation of the receptor in wild-type cells was suppressed by small molecule antagonists but not by extracellular blockade of VEGF. These results reveal a cell-autonomous VEGF signaling pathway that holds significance for vascular homeostasis but is dispensable for the angiogenic cascade.     

Gbb/BMP signaling is required to maintain energy homeostasis in Drosophila

The coordination of animal growth and development requires adequate nutrients. During times of insufficient food, developmental progression is slowed and stored energy is utilized to ensure that cell and tissue survival are maintained. Here, we report our finding that the Gbb/BMP signaling pathway, known to play an important role in many developmental processes in both vertebrates and invertebrates, is critical in the Drosophila larval fat body for regulating energy homeostasis. Animals with mutations in the Drosophila BMP-5,7 orthologue, glass bottom boat (gbb), or in its signaling components, display phenotypes similar to nutrient-deprived and Tor mutant larvae. These phenotypes include a developmental delay with reduced overall growth, a transparent appearance, and altered total lipid, glucose and trehalose levels. We find that Gbb/BMP signaling is required in the larval fat body for maintaining proper metabolism, yet interestingly, following nutrient deprivation larvae in turn show a loss of BMP signaling in fat body cells indicating that Gbb/BMP signaling is a central player in homeostasis. Finally, despite strong phenotypic similarities between nutrient-compromised animals and gbb mutants, distinct differences are observed in the expression of a group of starvation responsive genes. Overall, our results implicate Gbb/BMP signaling as a new pathway critical for positive regulation of nutrient storage and energy homeostasis during development.     

EGFR-dependent suppression of synaptic autophagy is required for neuronal circuit development

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Autophagy is required for lipid homeostasis during dark-induced senescence

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon-starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) subjected to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming toward lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants.

Autophagy is required for the mobilization of membrane lipid components and lipid droplet dynamics during extended darkness in Arabidopsis.     

Acidocalcisome is required for autophagy in Trypanosoma brucei

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca²⁺ and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.     

Activation of autophagy is required for muscle homeostasis during physical exercise

Skeletal muscle fibers of collagen VI null (Col6a12/2) mice show signs of degeneration due to a block in autophagy, leading to the accumulation of damaged mitochondria and excessive apoptosis. Attempts to induce autophagic flux by subjecting these mutant mice to long-term or shorter bursts of physical activity are unsuccessful (see Grumati, et al., pp. 1415–23). In normal mice, the induction of autophagy in the skeletal muscles post-exercise is able to prevent the accumulation of damaged organelles and maintain cellular homeostasis. Thus, these studies provide an important connection between autophagy and exercise physiology.     

Activation of autophagy is required for muscle homeostasis during physical exercise

Skeletal muscle fibers of collagen VI null (Col6a1?/?) mice show signs of degeneration due to a block in autophagy, leading to the accumulation of damaged mitochondria and excessive apoptosis. Attempts to induce autophagic flux by subjecting these mutant mice to long-term or shorter bursts of physical activity are unsuccessful (see Grumati, et al., pp. 1415–23). In normal mice, the induction of autophagy in the skeletal muscles post-exercise is able to prevent the accumulation of damaged organelles and maintain cellular homeostasis. Thus, these studies provide an important connection between autophagy and exercise physiology.     

Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle

The autophagy–lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. It has been reported that a diverse collection of extracellular matrix constituents, including decorin, collagen VI, laminin α2, endorepellin, and endostatin, can modulate autophagic signaling pathways. However, the association between autophagy and perlecan in muscle homeostasis remains unclear. The mechanical unloading of perlecan-deficient soleus muscles resulted in significantly decreased wet weights and cross-section fiber area compared with those of control mice. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This was accompanied by a decrease in autophagic substrates, such as p62, and an increase in LC3II levels. Furthermore, perlecan deficiency caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPKα. Our findings suggested that perlecan inhibits the autophagic process through the activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.     

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.     

Resveratrol attenuates vascular endothelial inflammation by inducing autophagy through the cAMP signaling pathway

Inflammation participates centrally in all stages of atherosclerosis (AS), which begins with inflammatory changes in the endothelium, characterized by expression of the adhesion molecules. Resveratrol (RSV) is a naturally occurring phytoalexin that can attenuate endothelial inflammation; however, the exact mechanisms have not been thoroughly elucidated. Autophagy refers to the normal process of cell degradation of proteins and organelles, and is protective against certain inflammatory injuries. Thus, we intended to determine the role of autophagy in the antiinflammatory effects of RSV in human umbilical vein endothelial cells (HUVECs). We found that RSV pretreatment reduced tumor necrosis factor α (TNF/TNFα)-induced inflammation and increased MAP1LC3B2 (microtubule-associated protein 1 light chain 3 β 2) expression and SQSTM1/p62 (sequestosome 1) degradation in a concentration-dependent manner. A bafilomycin A₁ (BafA1) challenge resulted in further accumulation of MAP1LC3B2 in HUVECs. Furthermore, autophagy inhibitors 3-methyladenine (3-MA), chloroquine as well as ATG5 and BECN1 siRNA significantly attenuated RSV-induced autophagy, which, subsequently, suppressed the downregulation of RSV-induced inflammatory factors expression. RSV also increased cAMP (cyclic adenosine monophosphate) content, the expression of PRKA (protein kinase A) and SIRT1 (sirtuin 1), as well as the activity of AMPK (AMP-activated protein kinase). RSV-induced autophagy in HUVECs was abolished in the presence of inhibitors of ADCY (adenylyl cyclase, KH7), PRKA (H-89), AMPK (compound C), or SIRT1 (nicotinamide and EX-527), as well as ADCY, PRKA, AMPK, and SIRT1 siRNA transfection, indicating that the effects of RSV on autophagy induction were dependent on cAMP, PRKA, AMPK and SIRT1. In conclusion, RSV attenuates endothelial inflammation by inducing autophagy, and the autophagy in part was mediated through the activation of the cAMP-PRKA-AMPK-SIRT1 signaling pathway.     

自噬在肝稳态调节作用中的研究进展

肝脏是人体最大的消化腺,也是最主要的代谢器官。自20世纪60年代,人们在肝脏溶酶体的研究中提出"自噬"这一概念时,就发现肝脏内的营养水平与激素影响自噬活动。近年来的研究表明,自噬不仅是正常的生理过程,也参与许多病理过程的调节。本文介绍了自噬在健康肝脏中维持稳态的作用,旨在为肝脏生理学及自噬失调相关疾病的治疗提供新思路。     

Rac3, but not Rac1, promotes ox-LDL induced endothelial dysfunction by downregulating autophagy

The endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis, which can lead to oxidative stress and inflammation. The role of autophagy in the process of atherosclerosis has drawn increasing attention. The human umbilical vein endothelial cells (HUVECs), whose Ras-related C3 botulinum toxin substrate 1 (Rac1) and Rac3 was knockdown, were used to detect whether the possible molecular mechanisms of Rac1 and Rac3 for anti-inflammatory in endothelial cells was effected by downregulation of autophagy. The HUVECs were incubated with ox-LDL. The inflammatory factors and autophagy proteins were evaluated to ascertain and compare the effect of Rac1 and Rac3 on autophagy. Then, 3-methyladenine (3-MA) as an inhibiter of autophagy was used to detect whether the effect of Rac1 and Rac3 was related to autophagy. ox-LDL-induced cell dysfunction in HUVECs was determined by testing the formation of foam cells, the expression of nuclear factor (NF)-κB and nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 and NF-κB p65 and other inflammatory factors, the release of reactive oxygen species by oxidative stress and the dysfunction of the cytomembrane. And ApoE^−/− mice on a high-fat diet were used as an animal model to detect the effect of Rac1 and Rac3 in vivo. The results showed that when Rac1 and Rac3 were decreased in HUVECs, the cell dysfunction caused by ox-LDL was inhibited. If 3-MA was used to inhibit autophagy in Rac1 and Rac3 knockdown cells, the injury induced by ox-LDL on the cells was recovered. These results indicated that the effect of Rac1 and Rac3 was combined with ox-LDL, which was related to inhibition of autophagy. The effect of Rac3 was more significant than that of Rac1.     

Programmed autophagy in the fat body of Aedes aegypti is required to maintain egg maturation cycles

Autophagy plays a pivotal role by allowing cells to recycle cellular components under conditions of stress, starvation, development and cancer. In this work, we have demonstrated that programmed autophagy in the mosquito fat body plays a critical role in maintaining of developmental switches required for normal progression of gonadotrophic cycles. Mosquitoes must feed on vertebrate blood for their egg development, with each gonadotrophic cycle being tightly coupled to a separate blood meal. As a consequence, some mosquito species are vectors of pathogens that cause devastating diseases in humans and domestic animals, most importantly malaria and Dengue fever. Hence, deciphering mechanisms to control egg developmental cycles is of paramount importance for devising novel approaches for mosquito control. Central to egg development is vitellogenesis, the production of yolk protein precursors in the fat body, the tissue analogous to a vertebrate liver, and their subsequent specific accumulation in developing oocytes. During each egg developmental cycle, the fat body undergoes a developmental program that includes previtellogenic build-up of biosynthetic machinery, intense production of yolk protein precursors, and termination of vitellogenesis. The importance of autophagy for termination of vitellogenesis was confirmed by RNA interference (RNAi) depletions of several autophagic genes (ATGs), which inhibited autophagy and resulted in untimely hyper activation of TOR and prolonged production of the major yolk protein precursor, vitellogenin (Vg). RNAi depletion of the ecdysone receptor (EcR) demonstrated its activating role of autophagy. Depletion of the autophagic genes and of EcR led to inhibition of the competence factor, betaFTZ-F1, which is required for ecdysone-mediated developmental transitions. Moreover, autophagy-incompetent female mosquitoes were unable to complete the second reproductive cycle and exhibited retardation and abnormalities in egg maturation. Thus, our study has revealed a novel function of programmed autophagy in maintaining egg maturation cycles in mosquitoes.