PGC-1α limits angiotensin II-induced rat vascular smooth muscle cells proliferation via attenuating NOX1-mediated generation of reactive oxygen species

AngII (angiotensin II)-induced excessive ROS (reactive oxygen species) generation and proliferation of VSMCs (vascular smooth muscle cells) is a critical contributor to the pathogenesis of atherosclerosis. PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-1α] is involved in the regulation of ROS generation, VSMC proliferation and energy metabolism. The aim of the present study was to investigate whether PGC-1α mediates AngII-induced ROS generation and VSMC hyperplasia. Our results showed that the protein content of PGC-1α was negatively correlated with an increase in cell proliferation and migration induced by AngII. Overexpression of PGC-1α inhibited AngII-induced proliferation and migration, ROS generation and NADPH oxidase activity in VSMCs. Conversely, Ad-shPGC-1α (adenovirus-mediated PGC-1α-specific shRNA) led to the opposite effects. Furthermore, the stimulatory effect of Ad-shPGC-1α on VSMC proliferation was significantly attenuated by antioxidant and NADPH oxidase inhibitors. Analysis of several key subunits of NADPH oxidase (Rac1, p22^phox, p40^phox, p47^phox and p67^phox) and mitochondrial ROS revealed that these mechanisms were not responsible for the observed effects of PGC-1α. However, we found that overexpression of PGC-1α promoted NOX1 degradation through the proteasome degradation pathway under AngII stimulation and consequently attenuated NOX1 (NADPH oxidase 1) expression. These alterations underlie the inhibitory effect of PGC-1α on NADPH oxidase activity. Our data support a critical role for PGC-1α in the regulation of proliferation and migration of VSMCs, and provide a useful strategy to protect vessels against atherosclerosis.     

Park7 interacts with p47phox to direct NADPH oxidase-dependent ROS production and protect against sepsis

Inappropriate inflammation responses contribute to mortality during sepsis. Through Toll-like receptors (TLRs), reactive oxygen species (ROS) produced by NADPH oxidase could modulate the inflammation responses. Parkinson disease (autosomal recessive, early onset) 7 (Park7) has a cytoprotective role by eliminating ROS. However, whether Park7 could modulate inflammation responses and mortality in sepsis is unclear. Here, we show that, compared with wild-type mice, Park7^−/− mice had significantly increased mortality and bacterial burdens in sepsis model along with markedly decreased systemic and local inflammation, and drastically impaired macrophage phagocytosis and bacterial killing abilities. Surprisingly, LPS and phorbol-12-myristate-13-acetate stimulation failed to induce ROS and proinflammatory cytokine production in Park7^−/− macrophages and Park7-deficient RAW264.7 cells. Through its C-terminus, Park7 binds to p47^phox, a subunit of the NADPH oxidase, to promote NADPH oxidase-dependent production of ROS. Restoration of Park7 expression rescues ROS production and improves survival in LPS-induced sepsis. Together, our study shows that Park7 has a protective role against sepsis by controlling macrophage activation, NADPH oxidase activation and inflammation responses.     

p47phox-Nox2-dependent ROS Signaling Inhibits Early Bone Development in Mice but Protects against Skeletal Aging

Bone remodeling is age-dependently regulated and changes dramatically during the course of development. Progressive accumulation of reactive oxygen species (ROS) has been suspected to be the leading cause of many inflammatory and degenerative diseases, as well as an important factor underlying many effects of aging. In contrast, how reduced ROS signaling regulates inflammation and remodeling in bone remains unknown. Here, we utilized a p47^phox knock-out mouse model, in which an essential cytosolic co-activator of Nox2 is lost, to characterize bone metabolism at 6 weeks and 2 years of age. Compared with their age-matched wild type controls, loss of Nox2 function in p47^phox−/− mice resulted in age-related switch of bone mass and strength. Differences in bone mass were associated with increased bone formation in 6-week-old p47^phox−/− mice but decreased in 2-year-old p47^phox−/− mice. Despite decreases in ROS generation in bone marrow cells and p47^phox-Nox2 signaling in osteoblastic cells, 2-year-old p47^phox−/− mice showed increased senescence-associated secretory phenotype in bone compared with their wild type controls. These in vivo findings were mechanistically recapitulated in ex vivo cell culture of primary fetal calvarial cells from p47^phox−/− mice. These cells showed accelerated cell senescence pathway accompanied by increased inflammation. These data indicate that the observed age-related switch of bone mass in p47^phox-deficient mice occurs through an increased inflammatory milieu in bone and that p47^phox-Nox2-dependent physiological ROS signaling suppresses inflammation in aging.     

Celastrol Prevents Atherosclerosis via Inhibiting LOX-1 and Oxidative Stress

Celastrol is a triterpenoid compound extracted from the Chinese herb Tripterygium wilfordii Hook F. Previous research has revealed its anti-oxidant, anti-inflammatory, anti-cancer and immunosuppressive properties. Here, we investigated whether celastrol inhibits oxidized low-density lipoprotein (oxLDL) induced oxidative stress in RAW 264.7 cells. In addition, the effect of celastrol on atherosclerosis in vivo was assessed in apolipoprotein E knockout (apoE^−/−) mouse fed a high-fat/high-cholesterol diet (HFC). We found that celastrol significantly attenuated oxLDL-induced excessive expression of lectin-like oxidized low density lipoprotein receptor-1(LOX-1) and generation of reactive oxygen species (ROS) in cultured RAW264.7 macrophages. Celastrol also decreased IκB phosphorylation and degradation and reduced production of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor (TNF)-α and IL-6. Celastrol reduced atherosclerotic plaque size in apoE^−/− mice. The expression of LOX-1 within the atherosclerotic lesions and generation of superoxide in mouse aorta were also significantly reduced by celastrol while the lipid profile was not improved. In conclusion, our results show that celastrol inhibits atherosclerotic plaque developing in apoE^−/− mice via inhibiting LOX-1 and oxidative stress.     

Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

Background

Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy.

Methods and Results

HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS.

Conclusion

VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease.     

MicroRNA-590 Inhibits Lipoprotein Lipase Expression and Prevents Atherosclerosis in apoE Knockout Mice

Recent studies have suggested that miR-590 may play critical roles in cardiovascular disease. This study was designed to determine the effects of miR-590 on lipoprotein lipase (LPL) expression and development of atherosclerosis in apolipoprotein E knockout (apoE^−/−) mice and explore the potential mechanisms. En face analysis of the whole aorta revealed that miR-590 significantly decreased aortic atherosclerotic plaque size and lipid content in apoE^−/− mice. Double immunofluorescence staining in cross-sections of the proximal aorta showed that miR-590 agomir reduced CD68 and LPL expression in macrophages in atherosclerotic lesions. MiR-590 agomir down-regulated LPL mRNA and protein expression as analyzed by RT-qPCR and western blotting analyses, respectively. Consistently, miR-590 decreased the expression of CD36 and scavenger receptor A1 (SRA1) mRNA and protein. High-performance liquid chromatography (HPLC)analysis confirmed that treatment with miR-590 agomir reduced lipid levels either in plasma orinabdominal cavity macrophages of apoE^−/− mice. ELISA analysis showed that miR-590 agomir decreased plasma levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β)and interleukin-6 (IL-6). In contrast, treatment with miR-590 antagomir prevented or reversed these effects. Taken together, these results reveal a novel mechanism of miR-590 effects, and may provide new insights into the development of strategies for attenuating lipid accumulation and pro-inflammatory cytokine secretion.     

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47^phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47^phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47^phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47^phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22^phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47^phox−/− coronary microvascular cells. Compared with wild-type p47^phox cDNA transfected cells, the single mutation of S379A completely blocked p47^phox membrane translocation, binding to p22^phox and endothelial O₂^⨪ production in response to acute stimulation of PKC. p47^phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47^phox conformational changes and NADPH oxidase-dependent superoxide production by cells.     

A Deficiency of Herp,an Endoplasmic Reticulum Stress Protein,Suppresses Atherosclerosis in ApoE Knockout Mice by Attenuating Inflammatory Responses

Herp was originally identified as an endoplasmic reticulum (ER) stress protein in vascular endothelial cells. ER stress is induced in atherosclerotic lesions, but it is not known whether Herp plays any role in the development of atherosclerosis. To address this question, we generated Herp- and apolipoprotein E (apoE)-deficient mice (Herp^−/−; apoE^−/− mice) by crossbreeding Herp^−/− mice and apoE^−/− mice. Herp was expressed in the endothelial cells and medial smooth muscle cells of the aorta, as well as in a subset of macrophages in the atherosclerotic lesions in apoE^−/− mice, while there was no expression of Herp in the Herp^−/−; apoE^−/− mice. The doubly deficient mice developed significantly fewer atherosclerotic lesions than the apoE^−/− mice at 36 and 72 weeks of age, whereas the plasma levels of cholesterol and triglycerides were not significantly different between the strains. The plasma levels of non-esterified fatty acids were significantly lower in the Herp^−/−; apoE^−/− mice when they were eight and 16 weeks old. The gene expression levels of ER stress response proteins (GRP78 and CHOP) and inflammatory cytokines (IL-1β, IL-6, TNF-α and MCP-1) in the aorta were significantly lower in Herp^−/−; apoE^−/− mice than in apoE^−/− mice, suggesting that Herp mediated ER stress-induced inflammation. In fact, peritoneal macrophages isolated from Herp-deficient mice and RAW264.7 macrophages in which Herp was eliminated with a siRNA expressed lower levels of mRNA for inflammatory cytokines when they were treated with tunicamycin. Herp deficiency affected the major mediators of the unfolded protein response, including IRE1 and PERK, but not ATF6. These findings suggest that a deficiency of Herp suppressed the development of atherosclerosis by attenuating the ER stress-induced inflammatory reactions.     

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22^phox, creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H₂O₂ production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22^phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O₂⁻ generation. In contrast, Nox4 released H₂O₂ and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.The family of NADPH oxidases consists of seven members termed Nox/Duox that differ in their tissue expression profiles, modes of activation, reactive oxygen species (ROS) outputs, and physiological functions. Understanding their distinguishing features is a prerequisite for rational inhibitor design and thus targeted intervention in ROS-mediated pathophysiologies (4). The coexpression of different Nox isoforms, each with potentially distinct functional profiles, in the same cell type necessitates a more discriminating approach than application of pan-Nox inhibitors. Detailed structure-function studies are necessary to identify unique regions and their impact with respect to catalytic function or localization of the enzyme. All Nox/Duox enzymes share a Nox backbone with six predicted transmembrane domains and an intracellular carboxyl-terminal domain which harbors FAD and NADPH binding sites. Nox5 and Duox1/2 enzymes contain additional structural elements such as amino terminal EF-hand motifs, a hallmark of their regulation by the intracellular calcium concentration (13, 30).The founding member of the NADPH oxidase family, the phagocyte oxidase, consists of membrane-bound Nox2 in a complex with the smaller subunit p22^phox (3). Heterodimerization of these two proteins is required for maturation and translocation of the enzyme complex to the plasma membrane or to intracellular vesicles. The Nox family members Nox1, Nox3, and Nox4 follow this paradigm (1, 14, 21, 25, 31). Heterodimer formation and association of the Nox/p22^phox complex at particular cellular membranes is essential for catalytic activity, i.e., for ROS generation. Nox2, and to a lesser degree Nox1 and Nox3, remain dormant under resting conditions and rely on stimulus-dependent translocation and assembly of oxidase components such as p47^phox and p67^phox, or NoxO1 and NoxA1 in the case of Nox1 and Nox3 (16). These steps, together with activation and translocation of the GTPase Rac, ultimately lead to the assembled, catalytically active oxidase and to ROS generation.Nox4 differs from the usual theme of multimeric assembly of active NADPH oxidases found in Nox1 to Nox3 (21, 22, 28, 32). Constitutive H₂O₂ production by Nox4 localized at perinuclear vesicles has been reported (1, 21, 28). Since NADPH oxidases catalyze the one-electron reduction of molecular oxygen to superoxide anion, the current dogma suggests that Nox4 generates intracellular superoxide. The superoxide produced will then dismutate rapidly to H₂O₂, diffusing from the cell into the extracellular milieu. Cytosolic proteins, which regulate the activity of Nox1 to Nox3 by binding to the carboxyl-terminal domains of Nox1 to Nox3, seem to be irrelevant for Nox4 function. The membrane-bound subunit p22^phox is to date the only known protein associated with Nox1 to Nox4. Heterodimerization, translocation, and enzymatic function of these oxidases require p22^phox. Recent structure-function analyses of complexes between Nox2 or Nox4 and the subunit p22^phox documented specific regions and amino acid residues in p22^phox necessary for complex formation and oxidase activity (35, 37). Interestingly, a p22^phox mutant (p22^phox Y121H) is capable of distinguishing between Nox1 to Nox3 and Nox4 by forming a functional complex only with Nox4, further suggesting unique structural features in Nox4 (35).In this study, we expand structure-function analysis of the oxidase complex by comparing Nox4/Nox2 chimeric enzymes with respect to NADPH oxidase activity, type of reactive oxygen species produced, requirement for additional oxidase components, and detailed subcellular localization.     

The anti-inflammatory effects of exercise training promote atherosclerotic plaque stabilization in apolipoprotein E knockout mice with diabetic atherosclerosis

Physical exercise is the cornerstone of cardiovascular disease treatment. The present study investigated whether exercise training affects atherosclerotic plaque composition through the modification of inflammatoryrelated pathways in apolipoprotein E knockout (apoE^−/−) mice with diabetic atherosclerosis. Forty-five male apoE^−/− mice were randomized into three equivalent (n=15) groups: control (CO), sedentary (SED), and exercise (EX). Diabetes was induced by streptozotocin administration. High-fat diet was administered to all groups for 12 weeks. Afterwards, CO mice were euthanatized, while the sedentary and exercise groups continued high-fat diet for 6 additional weeks. Exercising mice followed an exercise program on motorizedtreadmill (5 times/week, 60 min/session). Then, blood samples and atherosclerotic plaques in the aortic root were examined. A considerable (P<0.001) regression of the atherosclerotic lesions was observed in the exercise group (180.339±75.613×10³µm²) compared to the control (325.485±72.302×10³ µm²) and sedentary (340.188±159.108×10³µm²) groups. We found decreased macrophages, matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-8 and interleukin-6 (IL-6) concentrations (P<0.05) in the atherosclerotic plaques of the exercise group. Compared to both control and sedentary groups, exercise training significantly increased collagen (P<0.05), elastin (P<0.001), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) (P<0.001) content in the atherosclerotic plaques. Those effects paralleled with increased fibrous cap thickness and less internal elastic lamina ruptures after exercise training (P<0.05), while body-weight and lipid parameters did not significantly change. Plasma MMP-2 and MMP-3 concentrations in atherosclerotic tissues followed a similar trend. From our study we can conclude that exercise training reduces and stabilizes atherosclerotic lesions in apoE^−/− mice with diabetic atherosclerosis. A favorable modification of the inflammatory regulators seems to explain those beneficial effects.Key words: diabetes, atherosclerosis, exercise, matrix metalloproteinases, plaque stability.     

Chronic Over-Expression of Heat Shock Protein 27 Attenuates Atherogenesis and Enhances Plaque Remodeling: A Combined Histological and Mechanical Assessment of Aortic Lesions

Aims

Expression of Heat Shock Protein-27 (HSP27) is reduced in human coronary atherosclerosis. Over-expression of HSP27 is protective against the early formation of lesions in atherosclerosis-prone apoE^−/− mice (apoE^−/−HSP27^o/e) - however, only in females. We now seek to determine if chronic HSP27 over-expression is protective in a model of advanced atherosclerosis in both male and female apoE^−/− mice.

Methods and Results

After 12 weeks on a high fat diet, serum HSP27 levels rose more than 16-fold in male and female apoE^−/−HSP27^o/e mice, although females had higher levels than males. Relative to apoE^−/− mice, female apoE^−/−HSP27^o/e mice showed reductions in aortic lesion area of 35% for en face and 30% for cross-sectional sinus tissue sections – with the same parameters reduced by 21% and 24% in male cohorts; respectively. Aortic plaques from apoE^−/−HSP27^o/e mice showed almost 50% reductions in the area occupied by cholesterol clefts and free cholesterol, with fewer macrophages and reduced apoptosis but greater intimal smooth muscle cell and collagen content. The analysis of the aortic mechanical properties showed increased vessel stiffness in apoE^−/−HSP27^o/e mice (41% in female, 34% in male) compare to apoE^−/− counterparts.

Conclusions

Chronic over-expression of HSP27 is atheroprotective in both sexes and coincides with reductions in lesion cholesterol accumulation as well as favorable plaque remodeling. These data provide new clues as to how HSP27 may improve not only the composition of atherosclerotic lesions but potentially their stability and resilience to plaque rupture.     

A lincRNA-DYNLRB2-2/GPR119/GLP-1R/ABCA1-dependent signal transduction pathway is essential for the regulation of cholesterol homeostasis

Accumulated evidence shows that G protein-coupled receptor 119 (GPR119) plays a key role in glucose and lipid metabolism. Here, we explored the effect of GPR119 on cholesterol metabolism and inflammation in THP-1 macrophages and atherosclerotic plaque progression in apoE^−/− mice. We found that oxidized LDL (Ox-LDL) significantly induced long intervening noncoding RNA (lincRNA)-DYNLRB2-2 expression, resulting in the upregulation of GPR119 and ABCA1 expression through the glucagon-like peptide 1 receptor signaling pathway. GPR119 significantly decreased cellular cholesterol content and increased apoA-I-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. In vivo, apoE^−/− mice were randomly divided into two groups and infected with lentivirus (LV)-Mock or LV-GPR119 for 8 weeks. GPR119-treated mice showed decreased liver lipid content and plasma TG, interleukin (IL)-1β, IL-6, and TNF-α levels, whereas plasma levels of apoA-I were significantly increased. Consistent with this, atherosclerotic lesion development was significantly inhibited by infection of apoE^−/− mice with LV-GPR119. Our findings clearly indicate that, Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression, which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophage-derived foam cells. Moreover, GPR119 decreased lipid and serum inflammatory cytokine levels, decreasing atherosclerosis in apoE^−/− mice. These suggest that GPR119 may be a promising candidate as a therapeutic agent.     

Macrophage-derived apoESendai suppresses atherosclerosis while causing lipoprotein glomerulopathy in hyperlipidemic mice

Lipoprotein glomerulopathy (LPG) is a renal disease often accompanied by dyslipidemia and increased serum apoE levels. apoE_Sendai (Arg145Pro), a rare mutant based on the apoE3 sequence carrying an apoE2 charge, causes LPG in humans and transgenic mice, but its effects on the artery wall are unknown. Macrophage expression of apoE_Sendai may also directly influence renal and arterial homeostasis. We investigated the effects of macrophage-expressed apoE_Sendai in apoE^−/− mice with or without LDL receptor (LDLR). Murine bone marrow transduced to express apoE2, apoE3, or apoE_Sendai was transplanted into lethally irradiated mice. Macrophage apoE_Sendai expression reduced aortic lesion size and inflammation by 32 and 28%, respectively, compared with apoE2 in apoE^−/− recipients. No differences in lesion size or inflammation were found between apoE_Sendai and apoE3 in apoE^−/− recipients. Macrophage apoE_Sendai expression also reduced aortic lesion size by 18% and inflammation by 29% compared with apoE2 in apoE^−/−/LDLR^−/− recipients. Glomerular lesions compatible with LPG with increased mesangial matrix, extracellular lipid accumulation, and focal mesangiolysis were only observed in apoE^−/−/LDLR^−/− mice expressing apoE_Sendai. Thus, macrophage expression of apoE_Sendai protects against atherosclerosis while causing lipoprotein glomerulopathy. This is the first demonstration of an apoprotein variant having opposing effects on vascular and renal homeostasis.     

18F-FDG PET Imaging of Murine Atherosclerosis: Association with Gene Expression of Key Molecular Markers

Aim

To study whether ¹⁸F-FDG can be used for in vivo imaging of atherogenesis by examining the correlation between ¹⁸F-FDG uptake and gene expression of key molecular markers of atherosclerosis in apoE^−/− mice.

Methods

Nine groups of apoE^−/− mice were given normal chow or high-fat diet. At different time-points, ¹⁸F-FDG PET/contrast-enhanced CT scans were performed on dedicated animal scanners. After scans, animals were euthanized, aortas removed, gamma counted, RNA extracted from the tissue, and gene expression of chemo (C-X-C motif) ligand 1 (CXCL-1), monocyte chemoattractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, cluster of differentiation molecule (CD)-68, osteopontin (OPN), lectin-like oxidized LDL-receptor (LOX)-1, hypoxia-inducible factor (HIF)-1α, HIF-2α, vascular endothelial growth factor A (VEGF), and tissue factor (TF) was measured by means of qPCR.

Results

The uptake of ¹⁸F-FDG increased over time in the groups of mice receiving high-fat diet measured by PET and ex vivo gamma counting. The gene expression of all examined markers of atherosclerosis correlated significantly with ¹⁸F-FDG uptake. The strongest correlation was seen with TF and CD68 (p<0.001). A multivariate analysis showed CD68, OPN, TF, and VCAM-1 to be the most important contributors to the uptake of ¹⁸F-FDG. Together they could explain 60% of the ¹⁸F-FDG uptake.

Conclusion

We have demonstrated that ¹⁸F-FDG can be used to follow the progression of atherosclerosis in apoE^−/− mice. The gene expression of ten molecular markers representing different molecular processes important for atherosclerosis was shown to correlate with the uptake of ¹⁸F-FDG. Especially, the gene expressions of CD68, OPN, TF, and VCAM-1 were strong predictors for the uptake.     

Endothelial Dysfunction in Tristetraprolin-deficient Mice Is Not Caused by Enhanced Tumor Necrosis Factor-α Expression

Cardiovascular events are important co-morbidities in patients with chronic inflammatory diseases like rheumatoid arthritis. Tristetraprolin (TTP) regulates pro-inflammatory processes through mRNA destabilization and therefore TTP-deficient mice (TTP^−/− mice) develop a chronic inflammation resembling human rheumatoid arthritis. We used this mouse model to evaluate molecular signaling pathways contributing to the enhanced atherosclerotic risk in chronic inflammatory diseases. In the aorta of TTP^−/− mice we observed elevated mRNA expression of known TTP targets like tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α, as well as of other pro-atherosclerotic mediators, like Calgranulin A, Cathepsin S, and Osteopontin. Independent of cholesterol levels TTP^−/− mice showed a significant reduction of acetylcholine-induced, nitric oxide-mediated vasorelaxation. The endothelial dysfunction in TTP^−/− mice was associated with increased levels of reactive oxygen and nitrogen species (RONS), indicating an enhanced nitric oxide inactivation by RONS in the TTP^−/− animals. The altered RONS generation correlates with increased expression of NADPH oxidase 2 (Nox2) resulting from enhanced Nox2 mRNA stability. Although TNF-α is believed to be a central mediator of inflammation-driven atherosclerosis, genetic inactivation of TNF-α neither improved endothelial function nor normalized Nox2 expression or RONS production in TTP^−/− animals. Systemic inflammation caused by TTP deficiency leads to endothelial dysfunction. This process is independent of cholesterol and not mediated by TNF-α solely. Thus, other mediators, which need to be identified, contribute to enhanced cardiovascular risk in chronic inflammatory diseases.     

HIF-1α Activation by Intermittent Hypoxia Requires NADPH Oxidase Stimulation by Xanthine Oxidase

Hypoxia-inducible factor 1 (HIF-1) mediates many of the systemic and cellular responses to intermittent hypoxia (IH), which is an experimental model that simulates O₂ saturation profiles occurring with recurrent apnea. IH-evoked HIF-1α synthesis and stability are due to increased reactive oxygen species (ROS) generated by NADPH oxidases, especially Nox2. However, the mechanisms by which IH activates Nox2 are not known. We recently reported that IH activates xanthine oxidase (XO) and the resulting increase in ROS elevates intracellular calcium levels. Since Nox2 activation requires increased intracellular calcium levels, we hypothesized XO-mediated calcium signaling contributes to Nox activation by IH. We tested this possibility in rat pheochromocytoma PC12 cells subjected to IH consisting alternating cycles of hypoxia (1.5% O₂ for 30 sec) and normoxia (21% O₂ for 5 min). Kinetic analysis revealed that IH-induced XO preceded Nox activation. Inhibition of XO activity either by allopurinol or by siRNA prevented IH-induced Nox activation, translocation of the cytosolic subunits p47^phox and p67^phox to the plasma membrane and their interaction with gp91^phox. ROS generated by XO also contribute to IH-evoked Nox activation via calcium-dependent protein kinase C stimulation. More importantly, silencing XO blocked IH-induced upregulation of HIF-1α demonstrating that HIF-1α activation by IH requires Nox2 activation by XO.     

CCR2 and CD44 Promote Inflammatory Cell Recruitment during Fatty Liver Formation in a Lithogenic Diet Fed Mouse Model

Non-alcoholic fatty liver disease (NAFLD) is a common disease with a spectrum of presentations. The current study utilized a lithogenic diet model of NAFLD. The diet was fed to mice that are either resistant (AKR) or susceptible (BALB/c and C57BL/6) to hepatitis followed by molecular and flow cytometric analysis. Following this, a similar approach was taken in congenic mice with specific mutations in immunological genes. The initial study identified a significant and profound increase in multiple ligands for the chemokine receptor CCR2 and an increase in CD44 expression in susceptible C57BL/6 (B6) but not resistant AKR mice. Ccr2^−/− mice were completely protected from hepatitis and Cd44^−/− mice were partially protected. Despite protection from inflammation, both strains displayed similar histological steatosis scores and significant increases in serum liver enzymes. CD45⁺CD44⁺ cells bound to hyaluronic acid (HA) in diet fed B6 mice but not Cd44^−/− or Ccr2^−/− mice. Ccr2^−/− mice displayed a diminished HA binding phenotype most notably in monocytes, and CD8⁺ T-cells. In conclusion, this study demonstrates that absence of CCR2 completely and CD44 partially reduces hepatic leukocyte recruitment. These data also provide evidence that there are multiple redundant CCR2 ligands produced during hepatic lipid accumulation and describes the induction of a strong HA binding phenotype in response to LD feeding in some subsets of leukocytes from susceptible strains.     

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22^phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22^phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.     

SYK regulates macrophage MHC-II expression via activation of autophagy in response to oxidized LDL

Adaptive immunity, which plays an important role in the development of atherosclerosis, is mediated by major histocompatibility complex (MHC)-dependent antigen presentation. In atherosclerotic lesions, macrophages constitute an important class of antigen-presenting cells that activate adaptive immune responses to oxidized low-density lipoprotein (OxLDL). It has been reported that autophagy regulates adaptive immune responses by enhancing antigen presentation to MHC class II (MHC-II). In a previous study, we have demonstrated that SYK (spleen tyrosine kinase) regulates generation of reactive oxygen species (ROS) and activation of MAPK8/JNK1 in macrophages. Because ROS and MAPK8 are known to regulate autophagy, in this study we investigated the role of SYK in autophagy, MHC-II expression and adaptive immune response to OxLDL. We demonstrate that OxLDL induces autophagosome formation, MHC-II expression, and phosphorylation of SYK in macrophages. Gene knockout and pharmacological inhibitors of NOX2 and MAPK8 reduced OxLDL-induced autophagy. Using bone marrow-derived macrophages isolated from wild-type and myeloid-specific SYK knockout mice, we demonstrate that SYK regulates OxLDL-induced ROS generation, MAPK8 activation, BECN1-BCL2 dissociation, autophagosome formation and presentation of OxLDL-derived antigens to CD4⁺ T cells. ldlr^−/− syk^−/− mice fed a high-fat diet produced lower levels of IgG to malondialdehyde (MDA)-LDL, malondialdehyde-acetaldehyde (MAA)-LDL, and OxLDL compared to ldlr^−/− mice. These results provide new insights into the mechanisms by which SYK regulates MHC-II expression via autophagy in macrophages and may contribute to regulation of adaptive immune responses in atherosclerosis.