Injury In Apolipoprotein E-deficient Mice Research Articles

NAD+ Protects against Hyperlipidemia-induced Kidney Injury in Apolipoprotein E-deficient Mice.

<p>Background: Hyperlipidemia is an independent risk factor for kidney injury. Several studies have shown that nicotinamide adenine dinucleotide (NAD+) is an important coenzyme involved in normal body metabolism. Therefore, this study aimed to investigate the possible protective effects of NAD+ against hyperlipidemia-induced kidney injury in apolipoprotein Edeficient (ApoE-/-) mice. <p> Methods: Twenty-five eight-week-old male ApoE-/- mice were randomly assigned into four groups: normal diet (ND), ND supplemented with NAD+ (ND+NAD+), high-fat diet (HFD), and HFD supplemented with NAD+ (HFD+NAD+). The mice were subjected to their respective diets for a duration of 16 weeks. Blood samples were obtained from the inferior vena cava, collected in serum tubes, and stored at -80°C until use. Kidney tissues was fixed in 10% formalin and then embedded in paraffin for histological evaluation. The remainder of the kidney tissues was snapfrozen in liquid nitrogen for Western blot analysis. <p> Results: Metabolic parameters (total cholesterol, triglycerides, low-density lipoprotein-cholesterol, creatinine, and blood urea nitrogen) were significantly higher in the HFD group compared to the other groups. Histological analysis revealed prominent pathological manifestations in the kidneys of the HFD group. The HFD+NAD+ group showed increased levels of oxidative stress markers (NRF2 and SOD2) and decreased levels of NOX4 compared to the HFD group. Furthermore, the HFD group exhibited higher levels of TGF-β, Smad3, Collagen I, Collagen III, Bax, and Bak compared to the other groups. NAD+ supplementation in the HFD+NAD+ group significantly increased the levels of SIRT3, HO-1, Bcl-2, and Bcl-xL compared to the HFD group. Additionally, NF-κB protein expression was higher in the HFD group than in the HFD+NAD+ group. <p> Conclusion: These findings demonstrated that NAD+ may hold potential as a clinical treatment for kidney injury caused by hyperlipidemia.</p>.

Deletion of angiotensin-converting enzyme 2 exacerbates renal inflammation and injury in apolipoprotein E-deficient mice through modulation of the nephrin and TNF-alpha-TNFRSF1A signaling.

BackgroundThe renin-angiotensin system (RAS) has been implicated in atherosclerotic lesions and progression to chronic kidney diseases. We examined regulatory roles of angiotensin-converting enzyme 2 (ACE2) in the apolipoprotein E (ApoE) knockout (KO) kidneys.MethodsThe 3-month-old wild-type, ApoEKO, ACE2KO and ApoE/ACE2 double-KO (DKO) mice in a C57BL/6 background were used. The ApoEKO mice were randomized to daily deliver either Ang II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. We examined changes in pro-inflammatory cytokines, renal ultrastructure, and pathological signaling in mouse kidneys.ResultsDownregulation of ACE2 and nephrin levels was observed in ApoEKO kidneys. Genetic ACE2 deletion resulted in modest elevations in systolic blood pressure levels and Ang II type 1 receptor expression and reduced nephrin expression in kidneys of the ApoE/ACE2 DKO mice with a decrease in renal Ang-(1-7) levels. These changes were linked with marked increases in renal superoxide generation, NADPH oxidase (NOX) 4 and proinflammatory factors levels, including interleukin (IL)-1beta, IL-6, IL-17A, RANTES, ICAM-1, Tumor necrosis factor-alpha (TNF-alpha) and TNFRSF1A. Renal dysfunction and ultrastructure injury were aggravated in the ApoE/ACE2 DKO mice and Ang II-infused ApoEKO mice with increased plasma levels of creatinine, blood urea nitrogen and enhanced levels of Ang II in plasma and kidneys. The Ang II-mediated reductions of renal ACE2 and nephrin levels in ApoEKO mice were remarkably rescued by rhACE2 supplementation, along with augmentation of renal Ang-(1-7) levels. More importantly, rhACE2 treatment significantly reversed Ang II-induced renal inflammation, superoxide generation, kidney dysfunction and adverse renal injury in ApoEKO mice with suppression of the NOX4 and TNF-alpha-TNFRSF1A signaling. However, rhACE2 had no effect on renal NOX2 and TNFRSF1B expression and circulating lipid levels.ConclusionsACE2 deficiency exacerbates kidney inflammation, oxidative stress and adverse renal injury in the ApoE-mutant mice through modulation of the nephrin, NOX4 and TNF-alpha-TNFRSF1A signaling. While rhACE2 supplementation alleviates inflammation, renal dysfunction and glomerulus injury in the ApoE-mutant mice associated with upregulations of Ang-(1-7) levels and nephrin expression and suppression of the TNF-alpha-TNFRSF1A signaling. Strategies aimed at enhancing the ACE2/Ang-(1-7) actions may have important therapeutic potential for atherosclerotic renal injury and kidney diseases.

Effect of interleukin-6 receptor blockage on renal injury in apolipoprotein E-deficient mice

Hyperlipidemia has been demonstrated to be associated with renal disease, yet the mechanism of renal injury is still poorly understood. Inflammation that occurs with the hyperlipidemia has been considered to play an important role in development of glomerular injury. In the present study, we investigated the role of interleukin-6 (IL-6), a key inflammatory molecule, on renal injury in apolipoprotein E-deficient (ApoE(-/-)) mice with severe hypercholesterolemia. The 6-wk-old mice were fed a high-fat diet and administered weekly rat anti-IL-6 receptor monoclonal antibody (MR16-1), control rat IgG, or saline for a total of 4 wk. We examined histopathological changes in the kidney and urinary excretion of protein and albumin. Saline- and IgG-treated mice showed remarkable proteinuria at 10 wk of age, whereas MR16-1-treated mice exhibited significantly lower levels. Renal histopathology of saline- and IgG-treated mice revealed striking lipid deposits and foam cells in the glomerular tuft, juxtaglomerular area, and arteriolar wall along with range of mesangial cell proliferation and matrix expansion. Notably, the severity of lipid deposits and mesangial cell proliferation were significantly reduced in MR16-1-treated mice. Immunohistochemistry demonstrated that mesangial IL-6 expression was dramatically reduced in MR16-1-treated mice compared with IgG-treated mice. Blocking the IL-6 receptor prevented progression of proteinuria and renal lipid deposit, as well as the mesangial cell proliferation associated with severe hyperlipoproteinemia. These results clearly demonstrate that IL-6 plays an essential role in the pathogenesis of hyperlipidemia-induced glomerular injury in ApoE(-/-) mice and suggests the usefulness of anti-IL-6 receptor antibody in treatments for hyperlipidemia-induced organ damage.

Tumor Necrosis Factor-α and Its Receptors 1 and 2

In healthy hearts, tumor necrosis factor-α (TNF-α) concentration is low, and TNF-α is mainly located in endothelium and resident mast cells.1 TNF-α receptors (TNFRs) 1 (TNFR1) and 2 (TNFR2) are expressed on most cardiac cells, including cardiomyocytes.2 During myocardial ischemia, preformed TNF-α is released within minutes from resident mast cells and macrophages.3 With persistent ischemia, TNF-α also originates from cardiomyocytes.4 In rats after myocardial infarction (MI), TNFR1 density is increased for 10 days, whereas TNFR2 density remains unchanged.5 In contrast, TNFR1 and TNFR2 are both downregulated in the failing heart, whereas soluble TNFRs are increased6 owing to proteolytic cleavage of cardiomyocyte TNFR and release from circulating exosome-like vesicles.7 Increased soluble TNFRs decrease TNF-α bioactivity while at the same time prolonging its half-life.8 Article p 1386 TNF-α contributes to both reversible (contractile dysfunction4,9) and irreversible (MI) injury.1 Preischemic treatment with TNF-α antibodies10 or soluble TNFR1,11 permanent TNF-α knockout,12 or knockout of TNFR1 but not TNFR213 all reduce infarct size. The latter finding highlights the functional difference of TNFR1 and TNFR2 activation for ischemia/reperfusion injury: Only TNFR1 signaling is detrimental (Figure). Figure. Signaling cascade activated (→) or inhibited (⊣) by TNFR1 (red) or TNFR2 (blue) activation. sTNFR1 indicates soluble TNFR1; sTNFR2, soluble TNFR2; S1-P, sphingosine-1-phosphate; NF-κB, nuclear factor-κB; SOCS3, suppressor of cytokine signaling 3; ROS, reactive oxygen species; Akt-P, phosphorylated Akt, a homologue of the transforming v-Akt; NADPH-Ox, NADPH oxidase; STAT3-P, phosphorylated signal transducer and activator of transcription; Jnk-P, phosphorylated c-jun N-terminal kinase; p38-P, phosphorylated mitogen-activated protein kinase; Erk-P, phosphorylated extracellular signal-regulated kinase; PKC, protein kinase C; CamK, calcium/calmodulin-dependent kinase; MMP-2/9, matrix metalloproteinases 2 and 9; and IL-6, interleukin 6. Red indicates proteins involved in apoptosis; purple, proteins involved in growth; green, …

Cigarette Smoke Exposure Promotes Arterial Thrombosis and Vessel Remodeling after Vascular Injury in Apolipoprotein E-Deficient Mice

Background: Cigarette smoking is a major risk factor for the development of cardiovascular disease. However, in terms of the vessel wall, the underlying pathomechanisms of cigarette smoking are incompletely understood, partly due to a lack of adequate in vivo models. Methods: Apolipoprotein E-deficient mice were exposed to filtered air (sham) or to cigarette mainstream smoke at a total particulate matter (TPM) concentration of 600 µg/l for 1, 2, 3, or 4 h, for 5 days/week. After exposure for 10 ± 1 weeks, arterial thrombosis and neointima formation at the carotid artery were induced using 10% ferric chloride. Results: Mice exposed to mainstream smoke exhibited shortened time to thrombotic occlusion (p < 0.01) and lower vascular patency rates (p < 0.001). Morphometric and immunohistochemical analysis of neointimal lesions demonstrated that mainstream smoke exposure increased the amount of α-actin-positive smooth muscle cells (p < 0.05) and dose-dependently increased the intima-to-media ratio (p < 0.05). Additional analysis of smooth muscle cellsin vitro suggested that 10 µg TPM/ml increased cell proliferation without affecting viability or apoptosis, whereas higher concentrations (100 and 500 µg TPM/ml) appeared to be cytotoxic. Conclusions: Taken together, these findings suggest that cigarette smoking promotes arterial thrombosis and modulates the size and composition of neointimal lesions after arterial injury in apolipoprotein E-deficient mice.

Macrophage Depletion by Clodronate Liposomes Suppresses Neointimal Formation After Carotid Artery Injury in Apolipoprotein E-Deficient Mice

Background and Objectives: Clodronate liposomes deplete phagocytic cells, thereby suppressing inflammation after vascular injury. We compared the effect of clodronate liposomes on macrophage depletion and neointimal formation in apolipoprotein E-deficient mice [ApoE (-) mice]. Materials and Methods: ApoE (-) mice were randomly assigned to the clodronate liposomes group (Clodronate Group, n=7) and the vehicle liposomes group (Control Group, n=7). Clodronate (0.1 mL/10 g) was injected via the tail vein starting 2 days (d-2) before left common carotid artery injury. Results: The percentage of blood monocytes was subsequently decreased after clodronate injection (14.0±7.4% at baseline, 6.8±4.9% at 24 hours and 0.7±0.3% at 1 week after the clodronate liposome injection). The percentage of macrophages in the plaque area was significantly lower in the clodronate group at week 2 (32.0±6.5 vs. 68.7± 7.6%, respectively, p<0.05) and at week 4 (37.3±8.5 vs. 62.6±9.4%, respectively, p<0.05). The interleukin (IL)-6 and tumor necrosis factor (TNF)-α concentrations were significantly decreased in the clodronate group at week 4 (12.3±2.5 vs. 22.9±3.5 pg/mL, respectively, p<0.05 for IL-6 and 16.6±2.2 vs. 43.6±6.1 pg/mL, respectively, p<0.05 for TNF-α). The plaque volume was significantly greater in the control group at week 2 (0.345±0.063 vs. 0.153±0.053 mm 2 , respectively, p<0.05) and at week 4 (0.320±0.027 vs. 0.167±0.070 mm 2 , respectively, p<0.05). Conclusion: Intravenous administration of clodronate liposomes depleted monocytes and macrophages, and so this reduced the inflammatory markers and neointimal formation in ApoE (-) mice. (Korean Circ J 2008;38:244-249)

The Involvement of CXCR4 in the Inhibitory Effects of Salvianolic Acid B on Smooth Muscle Cell Proliferation

CXCR4, the unique receptor for stromal cell-derived factor-1 alpha (SDF-1 alpha), has been recently implicated in the development of neoitimal formation after vascular injury in apolipoprotein E-deficient mice. In the present study we investigated a pure compound salvianolic acid B from Salvia militorrhiza, a common Chinese herbal medicine, about its inhibitory effect on rat vascular smooth muscle cells (VSMCs) for cell proliferation, CXCR4 expression, cellular migration and its related signaling activities. Our results demonstrated that SDF-1alpha-induced cell proliferation can be significantly inhibited by salvianolic acid B. The quantitative analysis by real time PCR indicated a potent inhibitory effects of salvianolic acid on CXCR4 mRNA expression. The translational activities of cell proliferation-related proteins, such as Raf-1, MEK, ERK, CXCR4 and FAK, were all inhibited by 42%, 36%, 43%, 31% and 37%, respectively. Cell migration potentiated by SDF-1alpha was also attenuated by salvianolic acid B. In the aspect of apoptosis, salvianolic acid B can induce VSMCs apoptosis via cpspase 3 up-regulation and Bcl-2 down-regulation. In summary, considering the effects on cell proliferation and migration, salvianolic acid B may provide an alternative therapeutic remedy in preventing balloon injury-induced neoitimal formation.

Exercise Training Leads To A significant Reduction In Luminal Stenosis And Neointimal Formation After Vascular Injury In Apolipoprotein E-deficient Mice

Exercise Training Leads To A significant Reduction In Luminal Stenosis And Neointimal Formation After Vascular Injury In Apolipoprotein E-deficient Mice

Exercise Training Leads To A significant Reduction In Luminal Stenosis And Neointimal Formation After Vascular Injury In Apolipoprotein E-deficient Mice

Exercise Training Leads To A significant Reduction In Luminal Stenosis And Neointimal Formation After Vascular Injury In Apolipoprotein E-deficient Mice

1102-53 CX3CR1 gene disruption is not associated with decreased neointima formation after carotid wire injury in apolipoprotein-E deficient mice

1102-53 CX3CR1 gene disruption is not associated with decreased neointima formation after carotid wire injury in apolipoprotein-E deficient mice

Stabilization of atherosclerotic plaques by blockade of macrophage migration inhibitory factor after vascular injury in apolipoprotein E-deficient mice.

Macrophage migration inhibitory factor (MIF), a cytokine that controls cell-mediated inflammatory responses, is upregulated in atherogenesis; however, its functional contribution to lesion development has not been evaluated. We studied the role of MIF on neointima lesion formation after wire-induced injury of carotid arteries in apolipoprotein E-deficient (apoE(-/-)) mice. Immunohistochemistry revealed that MIF expression was detectable in endothelial cells before injury and upregulated in smooth muscle cells (SMCs) 24 hours after endothelial denudation. Three weeks after injury, MIF was predominantly found in endothelial cells and macrophage-derived foam cells. Neutralizing MIF with a monoclonal antibody resulted in a marked reduction of neointimal macrophages and inhibited transformation of macrophages into foam cells. Conversely, the content of SMCs and of collagen in the neointima were increased, amounting to a slight but not significant reduction in neointima and media size after 3 weeks of MIF monoclonal antibody treatment. Notably, serum levels of the cytokines IL-2, IL-4, IL-6, IL-10, and tumor necrosis factor were increased in MIF monoclonal antibody-treated mice. In vitro flow assays revealed that MIF pretreatment of aortic endothelium enhanced monocyte recruitment and that the monocyte arrest induced by oxidized LDL is mediated by endothelial MIF, as shown by monoclonal antibody inhibition. Inhibition of MIF resulted in a shift in the cellular composition of neointimal plaques toward a stabilized phenotype with reduced macrophage/foam cell content and increased SMC content. This might be attributable to a reduction of monocyte recruitment mediated by endothelial MIF.

Single injection of P-selectin or P-selectin glycoprotein ligand-1 monoclonal antibody blocks neointima formation after arterial injury in apolipoprotein E-deficient mice.

Emerging data suggest that P-selectin, by controlling adhesion of white blood cells, may be important in limiting the response to vascular injury. We tested the hypothesis that transient inhibition of P-selectin with either anti-P-selectin monoclonal antibody (mAb) or anti-P-selectin glycoprotein ligand-1 (PSGL-1) mAb would reduce neointima formation in the setting of carotid denudation injury in atherosclerosis-prone apolipoprotein E-/- mice. Neointima formation at 28 days was reduced significantly, by 50% or 80%, by a single injection on the day of injury of 100 or 200 microg P-selectin mAb RB 40.34 and by 55% by a single injection of 100 microg PSGL-1 mAb 4RA10 (P< or =0.005). In addition, there was a significant reduction in neointimal macrophage content. These findings demonstrate that transient P-selectin or PSGL-1 blockade at the time of arterial injury significantly limits plaque macrophage content and neointima formation in a dose-dependent manner after carotid denudation injury in apolipoprotein E-/- mice.

115 Stabilization of atherosclerotic plaque by blockade of macrophage migration inhibitory factor after arterial injury in apolipoprotein E-deficient mice

115 Stabilization of atherosclerotic plaque by blockade of macrophage migration inhibitory factor after arterial injury in apolipoprotein E-deficient mice

Aortic atherosclerotic plaque injury in apolipoprotein E deficient mice

The acute platelet response and chronic smooth muscle cell (SMC) proliferation following aortic injury in apolipoprotein E-deficient mice was investigated. The purpose of this study was to evaluate whether thrombus formation would occur following plaque injury, to determine the type of thrombus that developed, and to evaluate SMC proliferation. Aortic injury was performed by squeezing the aorta between forceps. The response to injury reflects the findings primarily associated with plaque disruption. An attempt was made to exclude the use of injured vascular segments that showed marked injury to the media to minimize the effects that medial SMCs may have in thrombus formation. Acute and chronic experiments following injury were terminated at 30 min and at 2 weeks, respectively. Injury in normal and heterozygous mice and nonplaque injury in apolipoprotein E-deficient mice were accompanied by endothelial denudation. In apolipoprotein E-deficient mice, plaque injury, which released plaque contents, foam cells and fragments of foam cells, was followed by thrombus formation that contained degranulating platelets mixed with fibrin. Large platelet-fibrin aggregates were in close contact with disrupted plaques and were mixed with foam cell debris. In addition, small thrombi were in nonplaque areas following plaque disruptions. These thrombi were not associated with injury to the media and most likely represent a heightened thrombogenicity associated with plaque disruption. At 2 weeks following injury, a thickened neointima was present in both wild type and mutant mice. Lipid filled cells were seen only in the media but not in the intima of apo E−/−vessels at 2 weeks. The results suggest that plaque injury in homozygous apolipoprotein E-deficient mice promotes platelet-fibrin thrombus formation and that these thrombi are primarily associated with disrupted plaque contents. The results also suggest that the platelet response and SMC proliferation induced by aortic injury are not altered by hyperlipidemia caused by apolipoprotein E deficiency.