Arteriosclerosis, Thrombosis, and Vascular Biology BRIEF REPORT
Spontaneous Atherosclerosis in Aged LCATDeficient Hamsters With Enhanced Oxidative Stress Mengmeng Guo,* Zongyu Liu,* Yitong Xu, Ping Ma, Wei Huang , Mingming Gao, Yuhui Wang, George Liu, Xunde Xian OBJECTIVE: LCAT (lecithin cholesterol acyltransferase) deficiency results in severe low HDL (high-density lipoprotein). Although whether LCAT is pro- or antiatherosclerosis was in debate in mouse studies, our previous study clearly shows that LCAT deficiency (LCAT−/−) in hamster accelerates atherosclerotic development on high-fat diet. However, unlike in hypercholesterolemia and hypertriglyceridemia, whether LCAT deficiency could lead to spontaneous atherosclerosis has not been studied yet in animal models. We, therefore, sought to investigate the atherosclerosis in LCAT−/− hamsters on standard laboratory diet and explore the potential underlying mechanisms.
Downloaded from http://ahajournals.org by on October 3, 2020
APPROACH AND RESULTS: Young (16 months) male and female wild-type and LCAT−/− hamsters on standard laboratory diet were used. Compared with age- and sex-matched wild-type hamsters, LCAT−/− hamsters showed a complete loss of plasma HDL and an increase in triglyceride by 2- to 8-folds at different stages of age. In aged LCAT−/− hamsters, the lesion areas at the aortic roots were ≈40×104 μm3 in males and 18×104 μm3 in females, respectively, which were consistent with the en face plaques observed in male (1.2%) and (1.5%) female groups, respectively. The results of plasma malondialdehyde measurement showed that malondialdehyde concentrations were markedly elevated to 54.4 μmol/L in males and 30 μmol/L in females, which are significantly associated with the atherosclerotic lesions. CONCLUSIONS: Our study demonstrates the development of spontaneous atherosclerotic lesions in aged male and female LCAT−/− hamsters with higher plasma oxidative lipid levels independent of plasma total cholesterol levels, further confirming the antiatherosclerotic role of LCAT. Key Words: atherosclerosis ◼ esterification ◼ hypercholesterolemia ◼ lipoprotein lipase ◼ malondialdehyde
L
CAT (lecithin cholesterol acyltransferase) is a key secretory enzyme largely synthesized in liver to catalyze the esterification of free cholesterol (FC) in circulation, thus participating in the reverse cholesterol transport from peripheral tissues to liver.1 Loss-of-function mutations in Lcat gene in human cause familial LCAT deficiency (FLD), which is characterized by complete loss of LCAT activity in plasma and impaired FC esterification, then leading to dyslipidemia-related disease.2,3 Recently, Oldoni et al4 reported that carotid intima-media thickness was differentially affected by the mutations with complete and partial loss of LCAT activity in humans, depending
on the esterification of FC carried on apoB (apolipoprotein B)-containing lipoproteins. However, whether LCAT deficiency contributes to the development of atherosclerosis has not been conclusive in other multiple population studies.5–7 Although more evidence still needs to be collected to clarify the relationship between LCAT and atherosclerosis, rhLCAT (recombinant human LCAT) has been shown to significantly correct lipid metabolism disorders in mouse models and patients with FLD with well tolerance and improve in vitro HDL (high-density lipoprotein)-mediated function of endothelium in acute coronary syndrome, suggesting that LCAT replacement
Correspondence to: Xunde Xian, Institute of Cardiovascular Sciences, Peking University, NO. 38 Xueyuan Rd, Haidian District, Beijing, China 100191. Email [email protected] *These authors contributed equally to this article. The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.120.315265. For Sources of Funding and Disclosures, see page xxx. © 2020 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb
Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.315265
December 2020 1
Brief Report - AL
Guo et al
LCAT Deficiency and Spontaneous Atherosclerosis
Nonstandard Abbreviation and Acronyms
Highlights
ABCA1 ATP-binding cassette transporter ACAT acyl-CoA:cholesterol acyltransferase apoB apolipoprotein B apoE apolipoprotein E FC free cholesterol FLD familial LCAT deficiency GPIHBP1 glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 HDL high-density lipoprotein HDL-C HDL-cholesterol LCAT lecithin cholesterol acyltransferase LDL low-density lipoprotein LPL lipoprotein lipase rhLCAT recombinant human LCAT SR-B1 scavenger receptor class membrane I VCAM-1 vascular cell adhesion protein 1 VLDL very low-density lipoprotein WT wild-type α-SMA α-smooth muscle actin
• LCAT−/− hamsters display complete loss of HDL (high-density lipoprotein) and elevated triglyceride levels on standard laboratory diet. • Spontaneous atherosclerotic lesions are developed in aged male and female LCAT−/− hamsters on standard laboratory diet independent of total cholesterol levels. • Aged male and female LCAT−/− hamsters on standard laboratory diet exhibit increased plasma malondialdehyde levels that are associated with the severity of atherosclerosis.
Downloaded from http://ahajournals.org by on October 3, 2020
could be a potential therapeutic approach for human disease caused by LCAT deficiency.8–10 Given that the heterogeneity of samples in human studies and the difficulty in obtaining the tissues with atherosclerotic lesions, animal models have been widely used to study the relationship between LCAT and atherosclerosis. However, both human LCAT transgenic or LCAT-deficient mouse models showed contradictory results.11–14 In view of the lack of CETP in mice15 and the presence of apoB48 in VLDL (very low-density lipoprotein) caused by the same apoB editing enzyme in small intestine as well as in the liver,16 which lead to an overt difference in metabolic profiles between human and mouse, we previously constructed a hamster model lacking Lcat gene, in which the endogenous CETP expression15 and the distribution of apoB editing enzyme were similar to those observed in humans.17 LCAT-deficient hamster model with a lipoprotein profile similar to patients with FLD exhibited more atherosclerotic lesions upon a high-cholesterol/high-fat diet feeding for 3 months compared with wild-type (WT) hamsters. We speculate that the accelerated atherosclerotic lesions are largely attributable to an atherogenic lipoprotein profile with significantly elevated levels of plasma TC and TG due to a diet intervention, in which we could not provide a direct causal link between atherosclerosis and the markedly reduced HDL caused by LCAT deficiency. It should also be noted that although high-cholesterol diet has been widely used for induction of atherosclerosis, this is in fact due to the ineffectiveness of most other risk factors to directly induce atherosclerotic lesions in 2 December 2020
experimental animal models. A conclusion then only can be drawn on whether it has an effect on atherosclerosis from the application of high-cholesterol diet together with the other factor of interest. Thus, studying atherosclerotic development using animals on a standard laboratory diet, which can exclude the influence of a diet intervention, will provide the direct evidence on whether the gene of interest plays a role in atherogenesis. Rationally, in current study, we investigated the spontaneous atherosclerosis in standard laboratory diet-fed LCAT−/− hamsters at different age together with WT counterparts to further define the influence of LCAT deficiency on atherosclerosis.
MATERIALS AND METHODS The data that support the findings of this study are available from the corresponding author upon reasonable request. Detailed materials and methods are in the Data Supplement.
Animals LCAT−/− hamsters were created with CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9). All animals were housed in a temperature and humidity controlled environment under a 14-hour light/10-hour dark cycle with clean conditions. Hamsters were allowed a free access to food and water. The hamsters were divided into 4 groups consisting of younger than 8 (16)-month-old male and female LCAT−/− hamsters, and sex and age-match WT hamsters were as controls. Blood samples were collected from the retro-orbital plexus of the hamsters after 12-hour fasting under isoflurane anesthesia. The heart and the entire aorta were harvested for morphological studies at the end point of the experiments. All experiments were performed under the principle of experimental animal health (National Institutes of Health released no. 85Y231996 Revision) and approved by the laboratory animal ethics committee of Peking University (LA2010-059; March 15, 2010)
Lipids and (Apo)Lipoproteins Assay Plasma TG and TC were determined by commercially available kits for TG and cholesterol, respectively (Zhongsheng
Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.315265
Guo et al
Pathological Evaluation All animals were euthanized and perfused with 40 mL of 0.01 mol/L cold PBS through the left ventricle. Hearts and whole aorta were fixed with 4% paraformaldehyde solution for 24 hours and transferred to 20% sucrose solution. Afterward, hearts were embedded in optimal cutting temperature compound and cross-sectioned (10 µm per slice). The atherosclerotic plaques in whole aortas (en face) and aortic roots were analyzed by staining with 0.3% oil red O solution (Sigma-Aldrich, St. Louis, MO). For the analysis of the atherosclerotic plaque components, the cross-sectioned slices were stained for VCAM-1 (vascular cell adhesion protein 1) and CD68 in macrophages and α-SMA (α-smooth muscle actin) in smooth muscle cells.
Plasma Malondialdehyde Measurement
Downloaded from http://ahajournals.org by on October 3, 2020
Fresh anticoagulant blood was collected from indicated hamsters, centrifuged at 1000g at 4 °C for 10 minutes. The plasma was collected for the measurement by using commercial malondialdehyde kit based on the reaction of malondialdehyde and thiobarbituric to form a malondialdehyde-thiobarbituric adduct (Applygen, Beijing). Briefly, 100 μL fresh plasma sample or serially diluted standard samples were mixed thoroughly with 300 μL buffer containing sodium dodecyl sulfate and thiobarbituric, respectively. The mixture was incubated at 95 °C for 30 minutes and then placed on ice for 5 minutes, followed by a centrifuge at 10 000g for 10 minutes. Two hundred-microliter supernatant was collected for fluorometric measurement using ex535 nm/em553 nm, which was converted to the concentrations according to the standard curve.
Data Analysis Shapiro-Wilk test was used to determine the normality of the data. Data were represented as mean±SEM. Two-way ANOVA with Bonferroni post test and liner regression were applied to analyze the data. GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA) was used for all statistical analysis. Differences at P
Spontaneous Atherosclerosis in Aged LCATDeficient Hamsters With Enhanced Oxidative Stress Mengmeng Guo,* Zongyu Liu,* Yitong Xu, Ping Ma, Wei Huang , Mingming Gao, Yuhui Wang, George Liu, Xunde Xian OBJECTIVE: LCAT (lecithin cholesterol acyltransferase) deficiency results in severe low HDL (high-density lipoprotein). Although whether LCAT is pro- or antiatherosclerosis was in debate in mouse studies, our previous study clearly shows that LCAT deficiency (LCAT−/−) in hamster accelerates atherosclerotic development on high-fat diet. However, unlike in hypercholesterolemia and hypertriglyceridemia, whether LCAT deficiency could lead to spontaneous atherosclerosis has not been studied yet in animal models. We, therefore, sought to investigate the atherosclerosis in LCAT−/− hamsters on standard laboratory diet and explore the potential underlying mechanisms.
Downloaded from http://ahajournals.org by on October 3, 2020
APPROACH AND RESULTS: Young (16 months) male and female wild-type and LCAT−/− hamsters on standard laboratory diet were used. Compared with age- and sex-matched wild-type hamsters, LCAT−/− hamsters showed a complete loss of plasma HDL and an increase in triglyceride by 2- to 8-folds at different stages of age. In aged LCAT−/− hamsters, the lesion areas at the aortic roots were ≈40×104 μm3 in males and 18×104 μm3 in females, respectively, which were consistent with the en face plaques observed in male (1.2%) and (1.5%) female groups, respectively. The results of plasma malondialdehyde measurement showed that malondialdehyde concentrations were markedly elevated to 54.4 μmol/L in males and 30 μmol/L in females, which are significantly associated with the atherosclerotic lesions. CONCLUSIONS: Our study demonstrates the development of spontaneous atherosclerotic lesions in aged male and female LCAT−/− hamsters with higher plasma oxidative lipid levels independent of plasma total cholesterol levels, further confirming the antiatherosclerotic role of LCAT. Key Words: atherosclerosis ◼ esterification ◼ hypercholesterolemia ◼ lipoprotein lipase ◼ malondialdehyde
L
CAT (lecithin cholesterol acyltransferase) is a key secretory enzyme largely synthesized in liver to catalyze the esterification of free cholesterol (FC) in circulation, thus participating in the reverse cholesterol transport from peripheral tissues to liver.1 Loss-of-function mutations in Lcat gene in human cause familial LCAT deficiency (FLD), which is characterized by complete loss of LCAT activity in plasma and impaired FC esterification, then leading to dyslipidemia-related disease.2,3 Recently, Oldoni et al4 reported that carotid intima-media thickness was differentially affected by the mutations with complete and partial loss of LCAT activity in humans, depending
on the esterification of FC carried on apoB (apolipoprotein B)-containing lipoproteins. However, whether LCAT deficiency contributes to the development of atherosclerosis has not been conclusive in other multiple population studies.5–7 Although more evidence still needs to be collected to clarify the relationship between LCAT and atherosclerosis, rhLCAT (recombinant human LCAT) has been shown to significantly correct lipid metabolism disorders in mouse models and patients with FLD with well tolerance and improve in vitro HDL (high-density lipoprotein)-mediated function of endothelium in acute coronary syndrome, suggesting that LCAT replacement
Correspondence to: Xunde Xian, Institute of Cardiovascular Sciences, Peking University, NO. 38 Xueyuan Rd, Haidian District, Beijing, China 100191. Email [email protected] *These authors contributed equally to this article. The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.120.315265. For Sources of Funding and Disclosures, see page xxx. © 2020 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb
Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.315265
December 2020 1
Brief Report - AL
Guo et al
LCAT Deficiency and Spontaneous Atherosclerosis
Nonstandard Abbreviation and Acronyms
Highlights
ABCA1 ATP-binding cassette transporter ACAT acyl-CoA:cholesterol acyltransferase apoB apolipoprotein B apoE apolipoprotein E FC free cholesterol FLD familial LCAT deficiency GPIHBP1 glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 HDL high-density lipoprotein HDL-C HDL-cholesterol LCAT lecithin cholesterol acyltransferase LDL low-density lipoprotein LPL lipoprotein lipase rhLCAT recombinant human LCAT SR-B1 scavenger receptor class membrane I VCAM-1 vascular cell adhesion protein 1 VLDL very low-density lipoprotein WT wild-type α-SMA α-smooth muscle actin
• LCAT−/− hamsters display complete loss of HDL (high-density lipoprotein) and elevated triglyceride levels on standard laboratory diet. • Spontaneous atherosclerotic lesions are developed in aged male and female LCAT−/− hamsters on standard laboratory diet independent of total cholesterol levels. • Aged male and female LCAT−/− hamsters on standard laboratory diet exhibit increased plasma malondialdehyde levels that are associated with the severity of atherosclerosis.
Downloaded from http://ahajournals.org by on October 3, 2020
could be a potential therapeutic approach for human disease caused by LCAT deficiency.8–10 Given that the heterogeneity of samples in human studies and the difficulty in obtaining the tissues with atherosclerotic lesions, animal models have been widely used to study the relationship between LCAT and atherosclerosis. However, both human LCAT transgenic or LCAT-deficient mouse models showed contradictory results.11–14 In view of the lack of CETP in mice15 and the presence of apoB48 in VLDL (very low-density lipoprotein) caused by the same apoB editing enzyme in small intestine as well as in the liver,16 which lead to an overt difference in metabolic profiles between human and mouse, we previously constructed a hamster model lacking Lcat gene, in which the endogenous CETP expression15 and the distribution of apoB editing enzyme were similar to those observed in humans.17 LCAT-deficient hamster model with a lipoprotein profile similar to patients with FLD exhibited more atherosclerotic lesions upon a high-cholesterol/high-fat diet feeding for 3 months compared with wild-type (WT) hamsters. We speculate that the accelerated atherosclerotic lesions are largely attributable to an atherogenic lipoprotein profile with significantly elevated levels of plasma TC and TG due to a diet intervention, in which we could not provide a direct causal link between atherosclerosis and the markedly reduced HDL caused by LCAT deficiency. It should also be noted that although high-cholesterol diet has been widely used for induction of atherosclerosis, this is in fact due to the ineffectiveness of most other risk factors to directly induce atherosclerotic lesions in 2 December 2020
experimental animal models. A conclusion then only can be drawn on whether it has an effect on atherosclerosis from the application of high-cholesterol diet together with the other factor of interest. Thus, studying atherosclerotic development using animals on a standard laboratory diet, which can exclude the influence of a diet intervention, will provide the direct evidence on whether the gene of interest plays a role in atherogenesis. Rationally, in current study, we investigated the spontaneous atherosclerosis in standard laboratory diet-fed LCAT−/− hamsters at different age together with WT counterparts to further define the influence of LCAT deficiency on atherosclerosis.
MATERIALS AND METHODS The data that support the findings of this study are available from the corresponding author upon reasonable request. Detailed materials and methods are in the Data Supplement.
Animals LCAT−/− hamsters were created with CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9). All animals were housed in a temperature and humidity controlled environment under a 14-hour light/10-hour dark cycle with clean conditions. Hamsters were allowed a free access to food and water. The hamsters were divided into 4 groups consisting of younger than 8 (16)-month-old male and female LCAT−/− hamsters, and sex and age-match WT hamsters were as controls. Blood samples were collected from the retro-orbital plexus of the hamsters after 12-hour fasting under isoflurane anesthesia. The heart and the entire aorta were harvested for morphological studies at the end point of the experiments. All experiments were performed under the principle of experimental animal health (National Institutes of Health released no. 85Y231996 Revision) and approved by the laboratory animal ethics committee of Peking University (LA2010-059; March 15, 2010)
Lipids and (Apo)Lipoproteins Assay Plasma TG and TC were determined by commercially available kits for TG and cholesterol, respectively (Zhongsheng
Arterioscler Thromb Vasc Biol. 2020;40:00–00. DOI: 10.1161/ATVBAHA.120.315265
Guo et al
Pathological Evaluation All animals were euthanized and perfused with 40 mL of 0.01 mol/L cold PBS through the left ventricle. Hearts and whole aorta were fixed with 4% paraformaldehyde solution for 24 hours and transferred to 20% sucrose solution. Afterward, hearts were embedded in optimal cutting temperature compound and cross-sectioned (10 µm per slice). The atherosclerotic plaques in whole aortas (en face) and aortic roots were analyzed by staining with 0.3% oil red O solution (Sigma-Aldrich, St. Louis, MO). For the analysis of the atherosclerotic plaque components, the cross-sectioned slices were stained for VCAM-1 (vascular cell adhesion protein 1) and CD68 in macrophages and α-SMA (α-smooth muscle actin) in smooth muscle cells.
Plasma Malondialdehyde Measurement
Downloaded from http://ahajournals.org by on October 3, 2020
Fresh anticoagulant blood was collected from indicated hamsters, centrifuged at 1000g at 4 °C for 10 minutes. The plasma was collected for the measurement by using commercial malondialdehyde kit based on the reaction of malondialdehyde and thiobarbituric to form a malondialdehyde-thiobarbituric adduct (Applygen, Beijing). Briefly, 100 μL fresh plasma sample or serially diluted standard samples were mixed thoroughly with 300 μL buffer containing sodium dodecyl sulfate and thiobarbituric, respectively. The mixture was incubated at 95 °C for 30 minutes and then placed on ice for 5 minutes, followed by a centrifuge at 10 000g for 10 minutes. Two hundred-microliter supernatant was collected for fluorometric measurement using ex535 nm/em553 nm, which was converted to the concentrations according to the standard curve.
Data Analysis Shapiro-Wilk test was used to determine the normality of the data. Data were represented as mean±SEM. Two-way ANOVA with Bonferroni post test and liner regression were applied to analyze the data. GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA) was used for all statistical analysis. Differences at P
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