Published online before print June 16, 2005, doi: 10.1161/01.RES.0000174286.73200.d4
© 2005 American Heart Association, Inc.
Integrative Physiology |
Overexpression of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Induces Intramyocardial Vasculopathy in Apolipoprotein E–Null Mice
From the Department of Vascular Physiology (K.I., Y.A., T.S.), National Cardiovascular Center Research Institute, Osaka; the Department of Cardiovascular Medicine (K.I., T.K.), Graduate School of Medicine, Kyoto University; the Department of Physiological Chemistry and Metabolism (H.K.), Graduate School of Medicine, University of Tokyo; and the Department of Molecular Pathophysiology (T.S.), Graduate School of Pharmaceutical Sciences, Osaka University, Japan.
Correspondence to Dr Tatsuya Sawamura, Department of Vascular Physiology, National Cardiovascular Center Research Institute, Suita, 565-8565 Osaka, Japan. E-mail t-sawamura{at}umin.ac.jp
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Abstract |
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Endothelial dysfunction induced by oxidized low-density lipoprotein (OxLDL) has been implicated in the pathogenesis of atherosclerosis and vasculopathy. Increased expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the receptor for OxLDL in endothelial cells, has been demonstrated in the atherosclerotic plaques from experimental atherosclerotic animal models and human clinical samples. In vitro, activation of LOX-1 alters the expression of several endothelial cell genes that are involved in endothelial dysfunction. To investigate the role of LOX-1 in terms of both endothelial dysfunction and resultant vascular changes, we generated mice overexpressing LOX-1 (LOXtg) in C57BL/6 and apolipoprotein E–null mice (apoEKO) backgrounds. We found that the expression of the transgene was prominent in coronary vessels and cardiomyocytes. The enhancement of OxLDL uptake in LOXtg mice was consistent with the expression level of LOX-1. Under hyperlipidemic conditions, both OxLDL and 8-hydroxy-2'-deoxyguanosine accumulated in the coronary arteries of LOXtg/apoEKO mice. The expression of ICAM-1 and VCAM-1, as well as the number of macrophages around blood vessels, were significantly increased in LOXtg/apoEKO mice compared with control littermates. There were no differences in either the hemodynamic profile or the plasma lipid profile between the 2 groups of animals. LOXtg/apoEKO mice displayed accelerated intramyocardial vasculopathy, and the atheroma-like lesion area was increased 10-fold in the LOXtg/apoEKO mice compared with nontransgenic littermates after 3-weeks on the high-fat diet. Thus, it is demonstrated that LOX-1 overexpression promotes inflammatory intramyocardial vasculopathy in a hyperlipidemic mouse model, and this effect is probably mediated through the endothelial dysfunction induced by overexpression of LOX-1.
Key Words: endothelial dysfunction • lectin-like oxidized low-density lipoprotein receptor-1 • oxidized low-density lipoprotein
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Introduction |
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Endothelial cells respond to chemical factors, mechanical stress, and blood flow by producing molecules that regulate vascular tonus, coagulation, cell proliferation, and leukocyte trafficking to stabilize blood vessels. A number of molecules that alter the normal function of the endothelium have been identified over the last decade. These molecules have facilitated the understanding of both vascular physiology and the vascular diseases caused by the complication of endothelial dysfunction, including atherosclerosis, hypertension, diabetes, inflammation, and various types of organ failure. The diversity of the diseases related to endothelial dysfunction indicates the pivotal role of the endothelium in normal physiologic homeostasis.1,2
Oxidatively modified low-density lipoprotein (OxLDL) is believed to be one of the major causes of endothelial dysfunction associated with proatherogenic conditions.3 OxLDL induces the expression of adhesion molecules, eg, ICAM-1, VCAM-1, and selectins. It also stimulates the release of chemokines and smooth muscle growth factors and impairs endothelium-dependent vasorelaxation. These changes lead to the recruitment of leukocytes into the subendothelial space, proliferation of smooth muscle cells,4 and an increase in vascular tone. The endothelial dysfunction induced by OxLDL is thought to precede tissue morphological changes and is believed to be one of the initiators of vasculopathy and atherosclerosis.
We recently identified the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)5 as the receptor for OxLDL on endothelial cells. LOX-1 is a type II membrane glycoprotein with an apparent molecular weight of 50 kDa. It has a C-terminal extracellular C-type lectin-like domain. The lectin-like domain is essential for binding to OxLDL.6 LOX-1 is highly homologous in the lectin-like domain of NKR-P1, which is essential for activation of natural killer cells and is the NK cell receptor family member in this group of C-type lectin-like receptors.7,8 LOX-1 expression in endothelial cells is relatively low under normal conditions, but can be induced with proinflammatory cytokines and vasoconstrictive peptides in vitro.9–14 The expression of LOX-1 is increased in hypertension, diabetes, and hyperlipidemia, and the highest expression of LOX-1 is found in atherosclerotic lesions.15–19 Interestingly, enhanced expression of LOX-1 has also been observed in the endothelium of the prelesion areas of hyperlipidemic rabbits, suggesting that this expression precedes the changes in vascular function induced by hyperlipidemia.17 In endothelial cells, activation of LOX-1 by OxLDL induces an upregulation of MCP-1, ICAM-1, and VCAM-1 expression20,21 and a reduction in the release of NO,22 all of which are known characteristics of endothelial dysfunction.
LOX-1 has also been demonstrated to bind other ligands in addition to OxLDL, including aged cells, apoptotic cells,23 platelets,24 leukocytes,25 and bacteria,26 suggesting a diversity of functions for LOX-1. The binding of platelets to LOX-1 enhances the release of endothelin-1 (ET-1) and suppresses the release of NO from endothelial cells,24,27 suggesting that a platelet–endothelium interaction via LOX-1 may also have a role in the induction of endothelial dysfunction.
These interesting in vitro data on the effects of activation of the LOX-1 receptor prompted us to investigate the role of LOX-1 and OxLDL in vivo by means of a transgenic mouse. In this study, we generated mice overexpressing LOX-1, and determined the role of LOX-1 in the vasculopathy associated with hyperlipidemia in these animals.
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Materials and Methods |
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Generation of LOXtg
Targeted LOX-1 gene expression in endothelial cells was achieved using the expression vector PEP8, which consists of the murine preproendothelin-1 promoter, a NotI site, and the SV40 intron/polyA signal, as described previously.28 Bovine LOX-1 (bLOX-1; GenBank NM_174132) cDNA tagged with a NotI adaptor was inserted into the NotI site of PEP8 downstream of the preproendothelin-1 promoter. The resultant plasmid was designated PEP8-bLOX-1. PEP8-bLOX-1 was linearized by XhoI digestion and injected into fertilized eggs prepared from superovulated C57BL/6 mice (Japan SLC, Shizuoka, Japan). The eggs were transferred into the oviducts of pseudopregnant ICR foster mothers (Japan SLC, Shizuoka, Japan). Founder mice were identified by Southern blot analysis. Genomic DNA obtained from the tails of the mice was digested by EcoRI, separated by electrophoresis, and transferred to a nylon membrane (Biodine, Pall). The membrane was probed with a [32P]dCTP-labeled EcoRI fragment of PEP8-bLOX-1 and analyzed with a BAS-5000 (Fuji Photo Film). Mice were maintained under controlled temperature and humidity with 12 hours light/dark cycle. They were provided standard chow diet and water ad libitum. All procedures were in accordance with the institutional guidelines for animal research in the National Cardiovascular Center.
Generation of LOXtg/ApoEKO
Heterozygous LOXtg mice lines, carrying 24 and 16 copies of the transgene, were crossbred with homozygous C57BL/6:apoEKO mice. The offspring, which carried the LOX-1 transgene and were obligatorily heterozygous for the apoE gene, were further crossbred with C57BL/6:apoEKO mice to generate LOXtg/apoEKO. Mice were maintained on a standard chow diet and water ad libitum until they were fed the high-fat diet. Homozygous mutation in the apoE gene was confirmed by Southern blot analysis as described.29
Total RNA preparation and Northern blot analysis, RT-PCR, Western blot analysis, immunohistochemistry, and analyses of the uptake of DiI-OxLDL were performed as indicated in the online supplement (available at http://circres.ahajournals.org).
Measurement of Hemodynamic and Plasma Lipid Indexes
Blood pressure and heart rates were measured by tail cuff plethysmography (BP-98A; Softron). Total cholesterol, triglycerides, phospholipids, and nonesterified fatty acids (NEFA) in the sera of the mice were measured using ELISA kits (T-Cho E, TG E, PL C, and NEFA C, respectively; Wako). Serum lipoprotein profiles were analyzed with high-performance liquid chromatography (HPLC) (Tosoh) by the online cholesterol oxidase method. Five µL of serum (diluted 100 times with PBS in the case of the apoEKO mice) was applied to 2 serially connected columns for gel permeation chromatography (TSKgel Lipopropak XL, 
7.8 mmx300 mm, Tosoh), and eluted with the TSK eluent LP-1 (Tosoh) at a flow rate of 0.6 mL/min. The postcolumn effluent was mixed on line with enzyme/chromogen solution consisting of cholesterol esterase, cholesterol oxidase, horseradish peroxidase, and sodium 3,5-dimetoxy-N-ethyl-N-(2-hydroxy-3-sulfopropyl)-aniline, and 4-aminoantipyrine from a kit (Cholesterol E-test, Wako), at a flow rate of 0.3 mL/min in the reaction tube (Teflon, 0.4 mmx7.5 m) at a temperature of 45°C. Then the reaction product was measured on line by the absorbance at 550 nm.30
Evaluation of Intramyocardial Vasculopathy
After weaning for 3 weeks, male LOXtg/apoEKO mice and apoEKO mice were maintained on a regular chow diet (CE-2; Clea Japan). From 8 weeks of age, mice were fed with a high-fat diet (1.25% cholesterol, 0.5% cholate, 20% milk casein, 15% cocoa butter, and reduced 
-tocopherol) for 3 weeks. After 3 weeks of high fat loading, mice were euthanized and used for analyses. The murine heart was sliced perpendicularly on the long axis into 8-µm thickness. To evaluate atheroma-like intramyocardial vasculopathy, sections at every 40 µm from the level of the mitral valve to the level of the aortic valve were subjected to staining with Oil red O (nacalai tesque) followed by counterstaining with Mayer’s hematoxylin. The Oil red O positive area was measured with NIH image software.
Statistical Analysis
Data are expressed as the mean±SEM and processed by Mann–Whitney analysis. A P value of <0.05 was considered statistically significant.
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Results |
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Generation of LOXtg
To obtain transgenic mice with LOX-1 overexpressed in endothelial cells, we used a murine preproendothelin-1 promoter. A LOX-1 expression plasmid was constructed by inserting bovine LOX-1 cDNA into a PEP8 vector, which contains the murine preproendothelin-1 and SV40 intron/polyA signal. The linearized plasmids were microinjected into fertilized eggs from C57BL/6 mice. Four lines of LOXtg in the C57BL/6 background were established, which carry 24, 14, 2, and 2 copies of the transgene, respectively (Figure 1a). Among these, one line that exhibited the highest expression level of the transgene was selected for detailed analysis.
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Tissue Distribution of the Transgenic LOX-1 Expression
Northern blot analysis revealed the transgene was most prominently expressed in the heart. However, almost all tissues exhibited moderate expression levels (Figure 1b). The endogenous murine LOX-1 mRNA was only detected in the heart. Assuming that endogenous murine LOX-1 and transgenic bovine LOX-1 have the same signal intensity for an equal molar level of LOX-1, the expression of the transgenic bovine LOX-1 in terms of mRNA level was 
8-fold greater than that of murine LOX-1 in the heart. Similarly, a marked increase of transgenic bovine LOX-1 protein in the heart was also detected by Western blot analysis. Low levels of the LOX-1 protein were also detected in the kidney (Figure 1c). An analysis of endogenous murine LOX-1 with the same amount of protein revealed a low expression level of endogenous murine LOX-1 in the liver. To identify the cells expressing the transgene in the heart of LOXtg, immunohistochemistry was performed with a monoclonal anti-bovine LOX-1 antibody that does not cross-react with endogenous murine LOX-1 (Figure 1d). Endothelial cells in heart vessels and cardiomyocytes were strongly positive for transgenic bovine LOX-1, whereas heart tissue from wild-type (WT) did not display any positive staining.
Uptake of DiI-OxLDL in Heart
To verify whether the transgenic bovine LOX-1 protein was functional in the heart, OxLDL uptake was analyzed in the cultured tissue of the heart by incubating each slice of the heart tissue with Dil-oxLDL. Dil-OxLDL uptake was enhanced in both endothelial cells and cardiomyocytes in LOXtg, probably reflecting the expression of the LOX-1 transgene in both endothelial cells and cardiomyocytes. The number of DiI-OxLDL–positive vessels was greatly increased in the heart sections from LOXtg compared with those from WT (Figure 2). Inhibition of DiI-OxLDL uptake by simultaneous incubation of an excess amount of unlabeled OxLDL or anti-bovine LOX-1 antibody with DiI-OxLDL further confirmed the specificity of the LOX-1 activity.
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Baseline Characteristics of LOXtg
At 16 weeks, there were no significant differences between WT and LOXtg fed a normal chow diet in terms of body weight, mean blood pressure, heart rate, total cholesterol, phospholipid, triglyceride, or NEFA (Table). HPLC analyses further showed that the subfraction profiles of lipoproteins yielded no significant differences in the WT and LOXtg mice.
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Generation of LOXtg/ApoEKO
To further investigate the effect of LOX-1 overexpression on the heart of LOXtg, LOXtg were cross-bred with apoEKO in a C57BL/6 background. In LOXtg/apoEKO mice, LOX-1 transgene was carried in the same copy number and was expressed at significantly high levels in both endothelial cells in the heart and cardiomyocytes, as in the case with LOXtg (data not shown). We then determined the effect of a high-fat diet on the transgenic mice. ApoEKO and LOXtg/apoEKO were fed a high-fat diet (1.25% cholesterol, 0.5% cholic acid) for 3 weeks starting at the age of 8 weeks. After exposure to the high-fat diet, the total cholesterol, phospholipids, and NEFA exhibited significantly higher levels compared with the levels before the diet. However, no significant differences were observed between apoEKO and LOXtg/apoEKO in terms of body weight, mean blood pressure, heart rate, total cholesterol, phospholipids, triglycerides, NEFA, or subfraction profile of lipoproteins (Table).
Accumulation of OxLDL and the Products of Oxidative Stress
To examine whether the overexpression of LOX-1 accelerates accumulation of OxLDL in the heart under hypercholesterolemia conditions, heart tissue sections were probed with a polyclonal antibody against OxLDL. This antibody does not cross-react with unmodified LDL. In the LOXtg/apoEKO heart, endothelial cells were stained positively, whereas little positive staining was observed in apoEKO (Figure 3a). To determine the oxidative stress in heart overexpressing LOX-1, we measured 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidation product of 2'-deoxyguanosine hydroxylated at the C-8 position. We found that 8-OHdG was detected in endothelial cells of LOXtg/apoEKO, but not in apoEKO under the same conditions (Figure 3b).
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Leukocyte Adhesion Molecule Expression and Macrophage Infiltration
To determine the effects of LOX-1 overexpression on the regulation of adhesion molecules and the consequences of this effect, we measured the expression levels of ICAM-1 and VCAM-1 mRNA in the hearts of apoEKO and LOXtg/apoEKO. We found that both genes were expressed at significantly higher levels in the LOXtg/apoEKO (n=7) than apoEKO mice (n=7) (Figure 4a). Immunohistochemical analysis similarly revealed high expression levels of ICAM-1 and VCAM-1 in the endothelial cells of LOXtg/apoEKO. The results are consistent with the mRNA levels of the molecules (Figure 4b and 4c). The expression was not limited to the lesion but also extended to the normal blood vessels in the heart. We further examined the number of macrophages that had infiltrated into the heart tissue. We found that the number of the cells that stained positively with an antimacrophage antibody were increased 6-fold in the LOXtg/apoEKO mice compared with macrophages found in the coronary arteries of the apoEKO mice (LOXtg/apoEKO, 5.60±0.71 cells/mm2, n=13; apoEKO, 0.88±0.36 cells/mm2, n=14) (Figure 5). This data suggests that LOX-1 overexpression induces a chronic inflammatory response.
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LOX-1 Overexpression Induces Intramyocardial Vasculopathy
To evaluate the physiological consequence of LOX-1 overexpression, we examined the intramyocardial vasculopathy to determine any pathological or histological differences between the 2 male mice lines. The heart tissues were cut into 8-µm thick sections every 40 µm along the long axis from the mitral valve to the aortic valve and stained with Oil red O. LOXtg/apoEKO displayed significant lipid deposition in the blood vessels of the sections (5.8 slices of 10 per mouse) from the coronary artery, whereas apoEKO displayed only occasional deposition in a small number of sections (1.6 slices of 10 per mouse) (Figure 6a). Quantitative analysis of the Oil red O–positive area showed that LOXtg/apoEKO developed an 10-fold larger area of lesions than apoEKO (34.31±15.41, n=7; 3.01±1.83, n=8; respectively [x10–6 Oil red O staining area/heart area]; Figure 6b).
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Discussion |
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Oxidized LDL (OxLDL) is implicated in the pathogenesis of atherosclerosis, including both the formation of foam cells and the induction of endothelial dysfunction. These changes that occur in the vascular wall might be mediated by several receptors for oxidized LDL such as SR-A, CD36, SR-BI, and LOX-1, which together are collectively termed the "scavenger receptor." Macrophages that have migrated into the subendothelial space take up OxLDL, and eventually become lipid-laden foam cells. The role of SR-A and CD36 in this process has been demonstrated with knockout mice deprived of those genes.31,32 However, the in vivo function of LOX-1 has yet to be determined.
LOX-1, which is expressed mainly in endothelial cells, has been suggested to initiate endothelial dysfunction. A number of in vitro studies have shown that the generation of superoxide by NADPH oxidase in endothelial cells induced by OxLDL is mediated by LOX-1.33 Furthermore, in vitro studies indicate that activation of the LOX-1 also initiates a reduction in NO release22 and an upregulation of gene expression, including ET-1, AT1 receptor, MCP-1,20 and cell-adhesion molecules.21
It is clearly demonstrated that in vivo LOX-1 overexpression results in pathological changes of blood vessels under hyperlipidemia. In the transgenic mice, overexpression of the LOX-1 transgene occurs in the blood vessels in the heart and the myocardium at both the mRNA and protein level. In addition, the overexpressed LOX-1 is shown to be functional, because an enhanced uptake of OxLDL in these same tissues was demonstrated. Cross-breeding the LOXtg mice with apoEKO mice established a double transgenic mouse (LOXtg/apoEKO), which was subsequently used to study the pathological consequences of LOX-1 overexpression. Despite the fact that in this mouse model of hyperlipidemia no significant differences were found in the plasma lipoprotein profiles between the LOXtg/apoEKO and apoEKO mice. OxLDL was found to accumulate more in the hearts of LOXtg/apoEKO than apoEKO mice, especially around the coronary blood vessels, where the LOX-1 transgene expression was shown to be particularly high. These results indicate that the pathology associated with the overexpression of LOX-1 is not caused by changes in the amount of VLDL/LDL per se, but rather, to the accumulation of OxLDL in tissues, which accumulation is mediated by the overexpression of functional LOX-1.
There are 3 reasonable interpretations of the present results. First, the modified LDL present in hyperlipidemic plasma may be more effectively bound by the LOX-1 overexpressed on the surface of endothelial cells, which would confirm that LOX-1 is an OxLDL receptor that facilitates OxLDL uptake. Second, LDL that has permeated through the endothelium and deposited in vascular wall can be oxidized and bound by LOX-1, leading to superoxide generation and increased LDL oxidation. There is evidence that burden of oxidative stress is enhanced by ligands for LOX-1 such as OxLDL33 and activated platelets.24 The detection of 8-OHdG, a marker of oxidative stress, in the blood vessels of LOXtg/apoEKO supports the hypothesis that LOX-1 might actually contribute to the increase in oxidative stress under hyperlipidemia. Third, a change in the adhesive capacity of endothelial cells may contribute to the accumulation of OxLDL. Specifically, LOX-1–mediated activation of endothelial cells results in the enhanced expression of chemokines and adhesion molecules.20,21 The activity of LOX-1 as a leukocyte-adhesion molecule would further enhance the adhesiveness of endothelial cells.25 These events in turn would further facilitate the infiltration of macrophages into the vascular wall, which infiltrated cells would then scavenge and accumulate OxLDL. In fact, we did show that the expression of ICAM-1 and VCAM-1 is indeed enhanced, notably even in apparently normal vessels, in the heart of LOXtg/apoEKO, indicating LOX-1 activates endothelial cells under the condition of hyperlipidemia. The increased infiltration of macrophages in LOXtg/apoEKO further supports the effect of LOX-1 on the vascular adhesiveness of leukocytes.
Atheroma formation can be understood as a downstream event of endothelial dysfunction. The formation of atheroma-like lesions was dramatically accelerated in LOXtg/apoEKO compared with apoEKO. This suggests the overall impact of the LOX-1 functions described above on oxidative stress, induction of adhesion molecules, and the binding of OxLDL and leukocytes is to promote atherosclerosis-like vasculopathic changes. In the analyses of the aortic atherosclerotic lesion, where the expression of the transgene was very limited both before and after high-fat diet, in the mouse lines used the size of the lesion did not reach a statistically significant difference between the apoEKO and LOXtg/apoEKO strains. This is in good agreement with the low expression level of the LOX-1 transgene and further supports a role for LOX-1 in the current model of atherogenesis. Interestingly, most atheromatous lesions were found in intramyocardial vessels, although the epicardial coronary arteries are the most common region of atherogenesis in humans. This might be related to the expression profile of the LOX-1 transgene, or the higher oxidative stress in the region. It is reported that the cholate-containing diets are associated with toxicity and inflammation. This needs to be considered in interpreting the data in the present study. Such toxic and proinflammtory effects might cooperatively act, in the present study, with the proinflammatory nature of LOX-1, hence promoting lipoprotein-mediated localized arteritis.
Based on the data from experimental animal models including the present study, it is possible that LOX-1 promotes endothelial dysfunction and atherosclerosis in humans. Recently, other researchers along with our laboratory have reported that LOX-1 gene polymorphisms are associated with both the progression of atherosclerosis and the incidence of ischemic heart diseases.34–36 Although these studies were not able to specify whether the disease-related polymorphism of the LOX-1 gene specifically enhances the activity of LOX-1, together with the data reported here, there is support for a proatherogenic effect of LOX-1 in humans. LOX-1 appears to enhance vasculopathic and atherogenic changes through its proinflammatory and prooxidative properties.
In summary, it is demonstrated that LOX-1 enhanced both the inflammatory response and lipid deposition in heart vessels when overexpressed in apoEKO mice. LOXtg/apoEKO should prove to be a useful model for further investigation into the initiation and progression of atheromatous changes in the mouse heart, and in time lead to the development of a mouse model of ischemic heart disease.
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Acknowledgments |
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This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Ministry of Health, Labor, and Welfare of Japan, the Organization for Pharmaceutical Safety and Research, Senri Life Science Foundation, Mitsubishi Pharma Foundation, and Suzuken Memorial Foundation. We thank Drs Antony Johns and Jiing-Huey Lin for critical reading of the manuscript, and PacificEdit for a review before submission.
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Footnotes |
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Original received May 11, 2004; resubmission received April 5, 2005; revised resubmission received June 3, 2005; accepted June 8, 2005.
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