ApoE KO Research Articles

Supplement of exogenous inorganic pyrophosphate inhibits atheromatous calcification in Apolipoprotein E knockout mice

Inorganic pyrophosphate (PPi) is the endogenous inhibitor for vascular calcification (VC). The present study was to investigate the effects of adenosine disodium triphosphate (ADTP) and alendronate sodium (AL), two exogenous PPi sources, on the atheromatous calcification (AC) in Apolipoprotein E knockout (ApoE KO) mice. ApoE KO mice were randomly divided into five groups: ApoE KO group, ApoE KO + ADTP (Low) group, ApoE KO + ADTP (High) group, ApoE KO + AL (Low) group and ApoE KO + AL (High) group. The mice in ApoE KO + ADTP (Low) group and ApoE KO + ADTP (High) group were intraperitoneally injected with ADTP with dose of 0.5 and 1.0 mg/kg/day for 2 months respectively. The mice in ApoE KO + AL (Low) group and ApoE KO + AL (High) group were intraperitoneally injected with AL with dose of 0.6 and 1.2 mg/kg/day for 2 months respectively. The age matched C57 mice were used as control group. All ApoE KO and C57 mice were fed with normal chow throughout the experiment. The calcification was evaluated using von Kossa method. The contents of PPi, triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL) and low density lipoprotein (LDL), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interferon-γ (IFN-γ) and interleukin-10 (IL-10) as well as the activity of alkaline phosphatase (ALP) in serum were measured. The results showed that compared with C57 mice, ApoE KO mice developed severe AC accompanied with high levels of TC, TG, LDL, IL-6, TNF-α and IFN-γ in serum and with low levels of PPi and IL-10 in serum. Both ADTP and AL dose-dependently reduced the AC in ApoE KO mice compared with that of ApoE mice, without affecting the contents of lipid profiles. In addition, ADTP and AL increased the contents of PPi and IL-10 while decreased the contents of TNF-α, IL-6 and IFN-γ in serum of ApoE KO mice, having no affection on ALP activity. The results suggested that ADTP and AL reduced AC in ApoE KO mice by increasing the PPi level and regulating the inflammation.

1602-P: Galectin-3 Inhibition Protects ApoE Knockout Mice against Western Diet–Induced Nonalcoholic Steatohepatitis and Glucose Intolerance

Nonalcoholic Fatty Liver Disease represents a broad spectrum of histological abnormalities ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which may progress to cirrhosis or hepatocellular carcinoma. NASH is characterized by steatosis, hepatic inflammation, hepatocellular ballooning, and fibrosis. Therefore, the aim of this work was to evaluate the effect of modified citrus pectin (MCP), inhibitor of galectin-3, in ApoE KO mice fed a western diet (WD). ApoE KO mice were divided into 3 groups: standard diet-fed (SD) group, WD-fed group, and WD-fed + MCP-treated group (WD+MCP). WD-fed mice displayed increased hepatic triacylglycerides (TAG-290%), and plasma ALT (115%) and AST (130%) compared with SD group. MCP treatment decreased hepatic TAG (30%) in WD-fed mice, plasma ALT and AST were reverted. WD-fed mice also displayed increased hepatic collagen deposition (18%), and MCP treatment inhibited this increase. These results were similar with hydroxyproline concentration in liver. WD-fed mice showed an impaired glucose tolerance compared with SD-fed mice, while MCP treatment improved glucose tolerance in WD-fed mice. Also, WD-fed mice presented increased fasting insulin (140%) compared with SD-fed mice, and MCP treatment reverted this increase by 100%. Gene expression of F4/80 was increased by 76% in the WD group compared with SD group, however had not significant difference between WD+MCP and SD group. Gene expression of matrix metalloproteinase 2 was increased by 5-fold in WD group compared with SD group, while this increase was inhibited with MCP treatment. Also, it was observed an increase in JNK phosphorylation by 3-fold in WD group, and MCP treatment inhibited this increase. Taken together, we confirm WD-fed ApoE KO mice as a suitable experimental model of NASH, and the inhibition of galectin-3 by the treatment with MCP protects ApoE KO mice against the development of NASH and glucose intolerance. Disclosure L. Araujo: None. F. Sucupira: None. C.C.B. Dias: None. A. Amaral: None. J. Camporez: None. Funding São Paulo Research Foundation (FAPESP)

Estradiol Protects Female ApoE KO Mice against Western-Diet-Induced Non-Alcoholic Steatohepatitis.

we evaluated if female apolipoprotein E (ApoE) KO mice were protected against Western-diet (WD)-induced NASH. Female ovariectomized (OVX) ApoE KO mice or sham-operated (SHAM) mice were fed either a WD or a regular chow (RC) for 7 weeks. Additionally, OVX mice fed a WD were treated with either estradiol (OVX + E2) or vehicle (OVX). Whole-body fat, plasma glucose, and plasma insulin were increased and associated with increased glucose intolerance in OVX mice fed a WD (OVX + WD). Plasma and hepatic triglycerides, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) hepatic enzymes were also increased in the plasma of OVX + WD group, which was associated with hepatic fibrosis and inflammation. Estradiol replacement in OVX mice reduced body weight, body fat, glycemia, and plasma insulin associated with reduced glucose intolerance. Treatment also reduced hepatic triglycerides, ALT, AST, hepatic fibrosis, and inflammation in OVX mice. These data support the hypothesis that estradiol protects OVX ApoE KO mice from NASH and glucose intolerance.

Estimation of Mouse Carotid Arterial Wall Shear Stress Using High-Frequency Ultrasound Imaging.

Wall shear stress (WSS) is a crucial hemodynamic factor that promotes atherosclerosis (plaque) development in arteries; although the relationship between WSS and arterial atherosclerosis has been explored in many animal studies, it is not fully understood. No suitable tool, however, exists for rapidly estimating dynamic WSS in small-animal studies. This study proposes a 40-MHz high-frequency ultrasound (HFUS) imaging system for dynamic WSS estimation based on mouse carotid artery blood flow velocity gradient measurements by vector Doppler imaging (VDI). Aliasing reduces the accuracy of Doppler measurements, which can be prevented by increasing the imaging frame rate. Conventionally, imaging is performed at two tilted angles by alternating between the angles; in the proposed method, the frame rate was doubled by imaging at each tilted angle sequentially and by then temporally aligning the sequences based on pulsatile flow characteristics. Velocity estimation using this method had low errors for both a steady-flow straight-tube and pulsatile flow 60%-stenosis phantom. The method was tested for wild-type (WT) C57BL/6 mice at 16 weeks old and apolipoprotein E knockout (ApoE KO) mice at 16 and 24 weeks old; differences in time-averaged and oscillatory WSS were observed, and histology confirmed that the 24-week ApoE KO mice with the highest oscillatory WSS had the greatest plaque formation. The proposed HFUS WSS imaging method can predict the location and extent of plaque development; thus, this method is useful for small-animal studies investigating the WSS effect on atherosclerotic plaque development.

Human ApoE2 Endows Stronger Contractility in Rat Cardiomyocytes Enhancing Heart Function

(1) Background: Apolipoprotein E (ApoE) is a critical plasma apolipoprotein for lipid transport and nonlipid-related functions. Humans possess three isoforms of ApoE (2, 3, and 4). ApoE2, which exhibits beneficial effects on cardiac health, has not been adequately studied. (2) Methods: We investigated the cardiac phenotypes of the humanized ApoE knock-in (hApoE KI) rats and compared to wild-type (WT) and ApoE knock-out (ApoE KO) rats using echocardiography, ultrasound, blood pressure measurements, histology strategies, cell culture, Seahorse XF, cardiomyocyte contractility and intracellular Ca2+ tests, and Western blotting; (3) Results: hApoE2 rats exhibited enhanced heart contractile function without signs of detrimental remodeling. Isolated adult hApoE2 cardiomyocytes had faster and stronger sarcomere contractility because of more mitochondrial energy generation and stimulation-induced fast and elevated intracellular Ca2+ transient. The abundant energy is a result of elevated mitochondrial function via fatty acid β-oxidation. The fast and elevated Ca2+ transient is associated with decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2) and increased expression of cardiac ryanodine receptor 2 (RyR2) conducting a potent Ca2+ release from SR.; (4) Conclusions: Our studies validated the association of polymorphic ApoEs with cardiac health in the rat model, and revealed the possible mechanisms of the protective effect of ApoE2 against heart diseases.

PS-B01-4: ANGIOTENSIN II TYPE1 RECEPTOR INHIBITION IMPROVES VASCULAR SENESCENCE THROUGH IMPROVEMENT OF MITOCHONDRIAL QUALITY CONTROL VIA CRAF/MEK/ERK PATHWAY

Introduction: Prevention of atherosclerotic cardiovascular disease has a problem of residual risk that is insufficient even if lipids are properly lowered by drugs such as statin. It has been reported that lectin-like oxidized low-density lipoprotein scavenger receptor-1 (LOX-1) associated with Angiotensin II Type 1 Receptor (AT1R). Mitochondria play crucial role in vascular senescence and atherosclerosis. We hypothesized receptor association with LOX-1 and AT1R might be a breakthrough to solve residual risk in terms of mitochondrial quality control. Methods: We performed in vitro and in vivo experiments using vascular smooth muscle cell (VSMC), C57BL6 (WT) and apolipoprotein E deficient (ApoE KO) mice. Results: Administration of ox-LDL to VSMC induced mitochondrial dysfunction and cellular senescence accompanied by excessive mitochondrial fission through Drp1 activation. This Drp1 activation was derived from the activation of CRAF/MEK/ERK pathway, which is a downstream of AT1R. AT1R inhibition inactivated ox-LDL-mediated Drp1 activation and retarded mitochondrial dysfunction, excessive mitochondrial fission and cellular senescence, indicating that LDL receptor LOX-1 associates with AT1R and activates CRAF/MEK/ERK/Drp1, leading to induction of mitochondrial fission and resulting in facilitating cellular senescence. AT1R inhibition also induced mitophagy assessed by electron microscopy and immunohistochemistry of LAMP2 and TOMM20. AT1R inhibition-induced mitophagy was not affected by Atg7 knockdown, whereas it was diminished by Rab9 knockdown. Immunohistochemistry showed TOMM20 dots were co-localized to LAMP2 and Rab9 but not LC3. These results suggest that AT1R signal induces mitophagy derived from Rab9-dependent alternative autophagy. Moreover, inhibition of either CRAF or MEK also decreased excessive mitochondrial fission and induced mitophagy, suggesting that AT1R signal followed by CRAF/MEK pathway modulates both mitochondrial dynamics and mitophagy. Taken together, inhibition of AT1R induces dual modification of mitochondrial dynamics and mitophagy through inactivation of CRAF/MEK/ERK axis. Finally, we confirmed that the degree of vascular senescence and the number of fragmented mitochondria were higher in ApoE KO than those of WT mice, and treatment with Candesartan to ApoE KO inhibited vascular senescence through induction of mitophagy and imbalance of mitochondrial dynamics. Conclusion: Receptor association of LOX-1 with AT1R plays a crucial role in cellular/vascular senescence induced by ox-LDL, and AT1R inhibition improves mitochondrial quality control through regulation of mitochondrial dynamics and mitophagy derived from Rab9-dependent alternative autophagy.

PS-BPB05-1: DUAL DISRUPTION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE AND APOE GENE ACCELERATES KIDNEY FIBROSIS AND AGING AFTER INJURY

Objective: Advances in medical science have made it possible to control diseases such as cancer, autoimmune diseases, and infectious diseases, extending the lifespan of affected people around the world. On the other hand, the number of patients with Chronic Kidney Disease (CKD) is increasing worldwide, and hypertension is a key factor. Suppression of CKD is important for extending healthy life expectancy. In this study, we will clarify the effects of the interaction between hypertension and atherosclerosis on renal fibrosis and aging. Design and method: Wild type (WT) mouse, apolipoprotein E-/- (ApoE KO) mouse, and endothelial nitric oxide synthase (eNOS)-/- ; ApoE-/- (DKO) mouse were obtained by crossing eNOS+/- mouse and ApoE+/- mouse. Unilateral ureteral obstruction (UUO) was performed on 8–10 weeks old male mice after blood pressure and lipid profile were measured. Mice were sacrificed 10 days after UUO. The degree of renal tubular injury, fibrosis and kidney senescence were evaluated. Results: DKO mice exhibited the most pronounced blood pressure and lipid abnormalities among the three groups, with a significant elevation of blood pressure, total cholesterol, and low HDL cholesterol compared to WT mice. DKO mice manifested elevated blood pressure, higher total cholesterol and lower HDL than WT mice. Compared with WT mice, ApoE KO and DKO mice showed sustained kidney injury molecule-1 expression and kidney fibrosis was significantly higher in ApoEKO and DKO mice. mRNA expression of genes related to kidney fibrosis was the highest in DKO mice. The mRNA expression of Zinc alpha2 Glycoprotein and heme oxygenase-1 were significantly decreased in DKO mice. Furthermore, mRNA expression of p53, p21 and p16 were increased both in ApoEKO and DKO mice, with DKO mice being the highest. Senescence associated beta-gal positive tubule area was significantly increased in DKO. Increased DNA damage and target of rapamycin autophagy spatial coupling compartments (TASCCs, Sci Transl Med. 2019;11(476):eaav4754. J Clin Invest. 2019;129(11):4797- 4816.) formation was found in DKO mice. Conclusion: Mice at high risk for cardiovascular disease developed kidney fibrosis and aging even in the young mice after injury. Vulnerability to oxidative stress due to endothelial cell dysfunction promotes fibrosis and aging. Managing lifestyle-related diseases including hypertension from a young age is important for CKD prevention.

PS-B12-8: ANGIOTENSIN II TYPE I RECEPTOR SIGNAL REGULATES MITOCHONDRIAL DYNAMICS, CAUSING MUSCULAR MITOCHONDRIAL DYSFUNCTION AND SARCOPENIA UNDER METABOLIC STRESS

Introduction: Metabolic stress, such as oxidized low density lipoprotein (ox-LDL) and advanced glycation end products (AGE) cause atherosclerotic cardiovascular diseases through dysfunction of mitochondria, which undergo quality control by mitochondrial dynamics, fission and fusion. Mitochondria also plays a crucial role in regulating muscular function, and its dysfunction leads to sarcopenia. This study aims to clarify whether and how mitochondrial dynamics are involved in the development of sarcopenia under metabolic stress. Methods: We used human skeletal muscle cells (HSMC) in in vitro experiments and C57BL6 (WT), apolipoprotein E (ApoE) deficient and the double knockout of ApoE and AT1R (DKO) mice in in vivo experiments. Results: Administration of either ox-LDL or AGE to HSMC induced mitochondrial fission through activation of fission factor, Drp1, decreased mitochondrial membrane potential assessed by JC-1 staining and increased reactive oxygen species assessed by Mito-sox Red staining and apoptosis, which were restored by Drp1-specific inhibitor, mdivi-1. Inhibition of angiotensin II type 1 receptor (AT1R) by AT1R blocker also ameliorated excessive mitochondrial fission, its function and oxidative stress induced by either ox-LDL or AGE in HSMC. These results suggest that metabolic stress induces mitochondrial fission and its dysfunction, which may cause sarcopenia. Muscular strength and the ratio of muscle weight to tibia length were lower in ApoE than those in WT. The number of fused mitochondria and mitochondrial function were lower and oxidative stress and the number of apoptotic cell were higher in ApoE KO than those of WT. Muscle strength, the ratio of muscle weight to tibia length, the number of fused mitochondria and its function were higher and the number of apoptotic cell were lower in DKO than those in ApoE KO mice. Taken together, these results suggest that either ox-LDL or AGE causes sarcopenia through AT1R signal-dependent mitochondrial fission and subsequent mitochondrial dysfunction. Conclusion: Either ox-LDL or AGE induces mitochondrial dysfunction through AT1R signal-regulating mitochondrial fission, which causes sarcopenia. Inhibition of AT1R signal may be a potential therapeutic strategy to avoid development of sarcopenia in patients with life style related diseases, such as dyslipidemia and diabetics.

PS-BPB01-5: LOX-1 DEFICIENCY INCREASES RUPTURED ABDOMINAL AORTIC ANEURYSM VIA THINNING OF ADVENTITIAL COLLAGEN

Objective: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a key mediator of inflammation and plays an important role in the pathogenesis of atherosclerosis. Both inflammation and atherosclerosis have been proposed as contributors to abdominal aortic aneurysm (AAA) pathogenesis. However, the role of LOX-1 in AAA pathogenesis remains unknown. We aimed to investigate effects of Olr1, which encodes LOX-1, deletion on angiotensin II (Ang II)-induced AAA in apolipoprotein E knockout (ApoE KO) mice to determine whether LOX-1 deficiency mitigates AAA development. Design and method: Either Ang II (1.44 mg/kg/day) or saline (control) was administered continuously through a subcutaneously implanted osmotic pump for 4 weeks in the following 8-week-old male mice: Apoe-/-Olr1+/+ mice (Ang II, n = 40; saline, n = 10), Apoe-/-Olr1+/- mice (Ang II, n = 30; saline, n = 9), and Apoe-/-Olr1-/- mice (Ang II, n = 39; saline, n = 9). Results: Ang II infusion raised systolic blood pressure by tail-cuff methods in all three groups. Serial non-invasive assessment with ultrasound demonstrated that Ang II gradually increased the maximum area of the suprarenal abdominal aortas leading to AAAs. Although the incidence and expansion rate of AAA were comparable among genotypes, Olr1 deletion significantly increased the severity and rupture rate of AAAs (p = 0.01; incidence of type IV and ruptured AAA was 25% in Apoe-/-Olr1-/- mice vs 5% in Apoe-/-Olr1+/+ mice). Since atherosclerosis is considered a major contributor to the development of AAAs, the extent of the atheroma burden localized in aneurysmal lesions was evaluated by Oil Red O staining. Olr1 deletion did not decrease atherosclerosis formation in the aneurysmal wall. Interestingly, further histopathological analysis revealed that aneurysmal lesions in LOX-1-deficient mice had fewer fibroblasts and myofibroblasts, as well as thinner adventitial collagen, although the degree of elastin fragmentation or disruption was similar between LOX-1-deficient and control mice. These in vivo data suggested that adventitial fibroblasts play a pivotal role in the increased severity of AAA in LOX-1-deficient mice. To further determine the effects of Olr1 deletion on fibroblast differentiation, fibroblasts were isolated from the adventitia of abdominal aortas harvested from 8-week-old Apoe-/-Olr1+/+ and Apoe-/-Olr1-/- mice, and then cultured with Ang II stimulation. An in vitro study confirmed that Olr1 deletion completely abolished the Ang II-induced proliferative activity in the aortic adventitial fibroblasts (p = 0.02). Conclusions: Olr1 deletion may not mitigate aneurysm development but rather increase the vulnerability to rupture by suppressing adventitial fibroblast proliferation and collagen.

Caffeine can alleviate non-alcoholic fatty liver disease by augmenting LDLR expression via targeting EGFR.

Increasing low-density lipoprotein receptor (LDLR) protein levels represents a key strategy for the prevention and treatment. Berberine can reportedly alleviate non-alcoholic fatty liver disease (NAFLD) by increasing the LDLR expression in an ERK1/2 signaling-dependent manner of NAFLD. Studies have shown that caffeine can inhibit fat deposition in the livers of mice; however, caffeine has not been reported to alleviate NAFLD by augmenting the LDLR expression via targeting EGFR. Here, an MTT assay, western blotting, RT-qPCR, immunohistochemistry, and surface plasmon resonance (SPR) analysis were used to investigate the role of caffeine in low-density lipoprotein cholesterol (LDL-C) clearance both in vitro and in vivo. In vitro, we found that caffeine could activate the EGFR-ERK1/2 signaling pathway in HepG2 cells, leading to increased LDLR mRNA and protein expression, and this effect could be inhibited by cetuximab. The SPR assay results have indicated that caffeine may increase the LDLR expression by directly binding to the EGFR extracellular domain and activating the EGFR-ERK1/2 signaling pathway. In vivo, caffeine markedly improved fatty liver and related blood indices in ApoE KO mice with high-fat-diet-induced NAFLD. Consistent with our in vitro results, we found that caffeine could also activate EGFR-ERK1/2 signaling and promote the LDLR expression in ApoE KO mice. In summary, caffeine can enhance the LDLR expression by directly binding to EGFR and activating the EGFR-ERK1/2 signaling pathway. EGFR signaling may represent a novel target for the prevention and treatment of NAFLD.

Structure-activity relationship and biological evaluation of xanthine derivatives as PCSK9 inhibitors for the treatment of atherosclerosis

Developing non-statin small molecules for the treatment of hypercholesterolemia remains challenging. The proprotein convertase subtilisin/kexin type 9 (PCSK9)-targeted therapies have attracted considerable attentions. Forty-five 7030B-C5 derivatives were synthesized and evaluated for the PCSK9 repression activity, taking the PCSK9 transcriptional inhibitor 7030B-C5 as the lead. Structure-activity relationship (SAR) analysis at C8 and N7-position was carried out, and compound 3s and 5r exhibited comparable PCSK9 transcriptional inhibitory activity but much lower cytotoxicity with the therapeutic index (TI) values doubled of that of 7030B-C5. In the in vitro assay, both compounds significantly reduced the level of PCSK9 protein and increased LDL receptor (LDLR) protein level. What's more, both compounds promoted LDL cholesterol (LDL-C) clearance more efficiently than 7030B-C5 in HepG2 cells. Most importantly, compound 3s reduced the atherosclerotic plaque areas with promising lipid-lowing effects in ApoE KO mice with a higher in vivo activity and lower toxicity. The regulatory mechanism of 3s was explored that it might target the transcription factor HNF1α and/or HINFP upstream of PCSK9 transcription, similar to that of 7030B-C5. Thus, 3s was considered as a potential anti-atherosclerosis drug candidate as a novel PCSK9 down-regulatory agent, worthy of further investigations.

Iron Restriction Alleviates Atherosclerosis in ApoE KO Mice: An iTRAQ Proteomic Analysis.

The "iron hypothesis" of atherosclerosis has long been controversial. Several studies have shown that dietary iron restriction or low-iron diets can effectively alleviate atherosclerosis in rabbits and mice. However, the underlying molecular mechanisms of these phenomena remain to be elucidated. In this study, we further evaluated possible correlations between a low-iron diet and atherosclerosis alleviation by using a quantitative proteomic approach. For this purpose, apolipoprotein E knockout (ApoE KO) mice were divided into three groups and fed a normal diet (ND), a high-fat diet (HFD), or a high-fat +low-iron diet (HFD + LI). Our results showed that the HFD-LI improved atherosclerosis by decreasing en face lesions of the aorta and reducing the accumulation of macrophages and disordered smooth muscle cells. HFD-LI also decreased iron levels, serum hepcidin levels and the serum concentration of low-density lipoprotein cholesterol (LDL-C). The use of the isobaric tag for absolute quantification (iTRAQ) proteomic method and subsequent multi-technique molecular validation indicated that many of the proteins involved in atherosclerotic inflammation, vascular remodeling, and focal adhesion had significant changes in their expression among the diet groups. Importantly, the proteins Gal-3 and VCAM1, which are key participants of atherosclerosis pathogenesis, revealed lower expression after a low-iron diet. The present findings widely support the "iron hypothesis" of atherosclerosis. Further studies are suggested to fully understand the implications of these results.

Angiogenic adipokine C1q-TNF-related protein 9 ameliorates myocardial infarction via histone deacetylase 7-mediated MEF2 activation.

C1q/tumor necrosis factor-related protein 9 (CTRP9) is an adipokine and has high potential as a therapeutic target. However, the role of CTRP9 in cardiovascular disease pathogenesis remains unclear. We found CTRP9 to induce HDAC7 and p38 MAPK phosphorylation via tight regulation of AMPK in vascular endothelial cells, leading to angiogenesis through increased MEF2 activity. The expression of CTRP9 and atheroprotective MEF2 was decreased in plaque tissue of atherosclerotic patients and the ventricle of post-infarction mice. CTRP9 treatment inhibited the formation of atherosclerotic plaques in ApoE KO and CTRP9 KO mice. In addition, CTRP9 induced significant ischemic injury prevention in the post-MI mice. Clinically, serum CTRP9 levels were reduced in patients with MI compared with healthy controls. In summary, CTRP9 induces a vasoprotective response via the AMPK/HDAC7/p38 MAPK pathway in vascular endothelial cells, whereas its absence can contribute to atherosclerosis and MI. Hence, CTRP9 may represent a valuable therapeutic target and biomarker in cardiovascular diseases.

Lack of ApoE inhibits ADan amyloidosis in a mouse model of familial Danish dementia

The Apolipoprotein E-ε4 allele (APOE-ε4) is the strongest genetic risk factor for late onset Alzheimer disease (AD). ApoE plays a critical role in amyloid-β (Aβ) accumulation in AD, and genetic deletion of the murine ApoE gene in mouse models results in a decrease or inhibition of Aβ deposition. The association between the presence of ApoE and amyloid in amyloidoses suggests a more general role for ApoE in the fibrillogenesis process. However, whether decreasing levels of ApoE would attenuate amyloid pathology in different amyloidoses has not been directly addressed. Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease characterized by the presence of widespread parenchymal and vascular Danish amyloid (ADan) deposition and neurofibrillary tangles. A transgenic mouse model for FDD (Tg-FDD) is characterized by parenchymal and vascular ADan deposition. To determine the effect of decreasing ApoE levels on ADan accumulation invivo, we generated a mouse model by crossing Tg-FDD mice with ApoE KO mice (Tg-FDD+/-/ApoE-/-). Lack of ApoE results in inhibition of ADan deposition up to 18months of age. Additionally, our results from a genetic screen of Tg-FDD+/-/ApoE-/- mice emphasize the significant role for ApoE in neurodegeneration in FDD via glial-mediated mechanisms. Taken together, our findings suggest that the interaction between ApoE and ADan plays a key role in FDD pathogenesis, in addition to the known role for ApoE in amyloid plaque formation in AD.

LOX-1 deficiency increases ruptured abdominal aortic aneurysm via thinning of adventitial collagen.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a key mediator of inflammation and plays an important role in the pathogenesis of atherosclerosis. Conversely, LOX-1 deficiency has been shown to decrease inflammation and atherosclerosis, both of which have been proposed to contribute to abdominal aortic aneurysm (AAA) pathogenesis. However, the role of LOX-1 in AAA pathogenesis remains unknown. Here, we investigated the effects of Olr1 (which encodes LOX-1) deletion on angiotensin II (Ang II)-induced AAA in apolipoprotein E knockout (ApoE KO) mice to determine whether LOX-1 deficiency mitigates AAA development. To accomplish this, we used serial, non-invasive ultrasound assessment, which revealed that the incidence and expansion rate of AAA were similar regardless of Olr1 deletion. However, Olr1 deletion significantly increased severe AAAs, including ruptured AAAs resulting in death. Oil Red O staining of the harvested aortas showed that the extent of atheroma burden localized in aneurysmal lesions did not differ between LOX-1-deficient and control mice, suggesting that Olr1 deletion did not decrease atheroma burden in the aneurysmal wall. Further histopathological analysis revealed that aneurysmal lesions in LOX-1-deficient mice had fewer fibroblasts and myofibroblasts, as well as thinner adventitial collagen, although the degree of elastin fragmentation or disruption was similar between LOX-1-deficient and control mice. An in vitro study confirmed that the proliferation of adventitial fibroblasts collected from LOX-1-deficient mice was significantly attenuated despite Ang II stimulation. In conclusion, Olr1 deletion may not mitigate aneurysm development but rather increases the vulnerability of rupture by suppressing adventitial fibroblast proliferation and collagen synthesis.

Age-related hearing loss was accelerated by apoptosis of spiral ganglion and stria vascularis cells in ApoE KO mice with hyperglycemia and hyperlipidemia.

Age-related hearing loss (ARHL) is associated with diabetes and/or dyslipidemia in humans. However, the detailed mechanism for the development of ARHL by diabetes and/or dyslipidemia has not been elucidated. In this study, we investigated the etiology of ARHL in apolipoprotein E (ApoE)-deficient mice with diabetes and dyslipidemia. The atherosclerotic CD-STZ (mice fed with a control diet and received an STZ injection), WD-con (mice fed with a western diet), and WD-STZ (mice fed with a western diet and received an STZ injection) mice showed a 2.4-, 4.9-, and 6.8-fold larger area, respectively, occupied by lesions throughout the aorta compared with the CD-con mice. A significantly larger area under the curve (AUC) was observed in the STZ-treated groups than in the non-treated groups based on the oral glucose tolerance test (OGTT). At 20 weeks of age, HbA1c levels were significantly higher in the CD-STZ and WD-STZ mice than in the CD-con and WD-con mice. In all the groups, the auditory brainstem response (ABR) thresholds of the 16-week-old mice were significantly higher compared with those of the 8-week-old mice. In particular, in the WD-STZ mice, the ABR thresholds of the left and right ears reached the maximum decibel peak equivalent sound pressure levels (130 dBpeSPL), which is a sign of deafness. The apoptotic spiral ganglion neurons (SGNs) of the WD-STZ mice were significantly increased compared with those of the other three groups, indicating that SGN apoptosis resulted in hearing loss in STZ-induced diabetic ApoE KO mice fed with a WD. A significant loss of the stria vascularis cells was observed in the WD-STZ group compared with the CD-con mice. In the organ of Corti, few apoptotic hair cells were found in all the groups; however, no significant difference was observed. Therefore, we consider that the reduced hearing ability in the STZ-treated and WD-fed groups was attributed to the damage to the SGN and stria vascularis in the cochlea. Thus, our results indicated that ototoxicity by diabetes and/or dyslipidemia accelerated ARHL in ApoE KO mice, thereby suggesting the importance of appropriate treatment of patients with diabetes and/or dyslipidemia to prevent ARHL.

Role of Translationally Controlled Tumor Protein (TCTP) in the Development of Hypertension and Related Diseases in Mouse Models.

Translationally controlled tumor protein (TCTP) is a multifunctional protein that plays a wide variety of physiological and pathological roles, including as a cytoplasmic repressor of Na,K-ATPase, an enzyme pivotal in maintaining Na+ and K+ ion gradients across the plasma membrane, by binding to and inhibiting Na,K-ATPase. Studies with transgenic mice overexpressing TCTP (TCTP-TG) revealed the pathophysiological significance of TCTP in the development of systemic arterial hypertension. Overexpression of TCTP and inhibition of Na,K-ATPase result in the elevation of cytoplasmic Ca2+ levels, which increases the vascular contractility in the mice, leading to hypertension. Furthermore, studies using an animal model constructed by multiple mating of TCTP-TG with apolipoprotein E knockout mice (ApoE KO) indicated that TCTP-induced hypertension facilitates the severity of atherosclerotic lesions in vivo. This review attempts to discuss the mechanisms underlying TCTP-induced hypertension and related diseases gleaned from studies using genetically altered animal models and the potential of TCTP as a target in the therapy of hypertension-related pathological conditions.

Western Diet-Fed ApoE Knockout Male Mice as an Experimental Model of Non-Alcoholic Steatohepatitis.

One of the consequences of the Western lifestyle and high-fat diet is non-alcoholic fatty liver disease (NAFLD) and its aggressive form, non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma (HCC) and is rapidly becoming the leading cause of end-stage liver disease or liver transplantation. Currently, rodent NASH models lack significant aspects of the full NASH spectrum, representing a major problem for NASH research. Therefore, this work aimed to characterize a fast rodent model with all characteristic features of NASH. Eight-week-old male ApoE KO mice were fed with Western diet (WD), high fatty diet (HFD) or normal chow (Chow) for 7 weeks. Whole-body fat was increased by ~2 times in WD mice and HFD mice and was associated with increased glucose intolerance, hepatic triglycerides, and plasma ALT and plasma AST compared with Chow mice. WD mice also showed increased galectin-3 expression compared with Chow or HFD mice and increased plasma cholesterol compared with Chow mice. WD and HFD displayed increased hepatic fibrosis and increased F4/80 expression. WD mice also displayed increased levels of plasma MCP-1. Hepatic inflammatory markers were evaluated, and WD mice showed increased levels of TNF-α, MCP-1, IL-6 and IFN-γ. Taken together, these data demonstrated that the ApoE KO mouse fed with WD is a great model for NASH research, once it presents the fundamental parameters of the disease, including hepatic steatosis, fibrosis, inflammation, and metabolic syndrome.

Effects of Six Weeks of Hypoxia Exposure on Hepatic Fatty Acid Metabolism in ApoE Knockout Mice Fed a High-Fat Diet.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease with a characteristic of abnormal lipid metabolism. In the present study, we employed apolipoprotein E knockout (ApoE KO) mice to investigate the effects of hypoxia exposure on hepatic fatty acid metabolism and to test whether a high-fat diet (HFD) would suppress the beneficial effect caused by hypoxia treatment. ApoE KO mice were fed a HFD for 12 weeks, and then were forwarded into a six-week experiment with four groups: HFD + normoxia, normal diet (ND) + normoxia, HFD + hypoxia exposure (HE), and ND + HE. The C57BL/6J wild type (WT) mice were fed a ND for 18 weeks as the baseline control. The hypoxia exposure was performed in daytime with normobaric hypoxia (11.2% oxygen, 1 h per time, three times per week). Body weight, food and energy intake, plasma lipid profiles, hepatic lipid contents, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and molecular/biochemical makers and regulators of the fatty acid synthesis and oxidation in the liver were measured at the end of interventions. Six weeks of hypoxia exposure decreased plasma triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) contents but did not change hepatic TG and non-esterified fatty acid (NEFA) levels in ApoE KO mice fed a HFD or ND. Furthermore, hypoxia exposure decreased the mRNA expression of Fasn, Scd1, and Srebp-1c significantly in the HFD + HE group compared with those in the HFD + normoxia group; after replacing a HFD with a ND, hypoxia treatment achieved more significant changes in the measured variables. In addition, the protein expression of HIF-1α was increased only in the ND + HE group but not in the HFD + HE group. Even though hypoxia exposure did not affect hepatic TG and NEFA levels, at the genetic level, the intervention had significant effects on hepatic metabolic indices of fatty acid synthesis, especially in the ND + HE group, while HFD suppressed the beneficial effect of hypoxia on hepatic lipid metabolism in male ApoE KO mice. The dietary intervention of shifting HFD to ND could be more effective in reducing hepatic lipid accumulation than hypoxia intervention.

2-Hydroxypropyl-beta-cyclodextrin Treatment Does Not Induce Atherosclerotic Lesion Regression in Western-Type Diet-Fed Apolipoprotein E Knockout Mice

2-Hydroxypropyl-beta-cyclodextrin (2HPβCD) is able to bind and solubilize unesterified cholesterol and may therefore be able to reverse the deposition of cholesterol in macrophages within the aortic vessel wall, a hallmark of atherosclerotic cardiovascular disease. However, conflicting results regarding the potential of 2HPβCD to induce regression of established atherosclerotic lesions have been described. In the current study, we therefore also investigated the ability of 2HPβCD to stimulate cholesterol removal from macrophage foam cells in vitro and induce the regression of established atherosclerotic lesions in apolipoprotein E knockout (APOE KO) mice. In vitro studies using murine thioglycollate-elicited peritoneal macrophages verified that 2HPβCD is able to induce cholesterol efflux from macrophages in an ATP-binding cassette transporter-independent manner. Switching Western-type-diet-fed APOE KO mice with established atherosclerotic lesions back to a chow diet was associated with a reduction in the hypercholesterolemia extent and an increase in the absolute lesion size and plaque collagen-to-macrophage ratio. Importantly, parallel subcutaneous administration of 2HPβCD was not able to prevent the diet-switch-associated lesion growth or induce atherosclerosis regression. Although in our hands, 2HPβCD does effectively stimulate cellular cholesterol efflux from macrophages, we do not consider it worthwhile to further pursue 2HPβCD as therapeutic moiety in the atherosclerosis regression context.