α-Galactosidase A Deficiency Accelerates Atherosclerosis in Mice With Apolipoprotein E Deficiency
Background— α-Galactosidase A (Gla) deficiency leads to widespread tissue accumulation of neutral glycosphingolipids and is associated with premature vascular complications such as myocardial infarction and stroke. Glycosphingolipids have been shown to accumulate in human atherosclerotic lesions, although their role in atherogenesis is unclear.
Methods and Results— To determine whether Gla affects the progression of atherosclerosis, mice were generated with combined deficiencies of apolipoprotein E and Gla. At 45 weeks of age, Gla-deficient mice had developed more atherosclerosis than mice with normal Gla expression (25.1±14.0 versus 12.3±9.3 mm2 of total lesion area, P<0.02). This increase in atherosclerosis was associated with the presence of increased Gb3, enhanced inducible nitric oxide synthase expression, and increased nitrotyrosine staining.
Conclusions— These findings suggest that deficiency of Gla leads to increased inducible nitric oxide synthase expression and accelerated atherosclerosis.
Received June 7, 2004; revision received October 6, 2004; accepted October 12, 2004.
Alpha-Galactosidase A (Gla) deficiency (Fabry disease) is an X-linked disorder that leads to widespread tissue accumulation of neutral glycosphingolipids with α-galactosyl linkages consisting primarily of globotriaosylceramide (Gb3).1 Clinical manifestations of Fabry disease include renal failure, painful neuropathies, angiokeratoma, myocardial infarction, and stroke, which lead to premature mortality.1 Although premature vascular complications are more common in subjects with Fabry disease,2–4 the effect of Gla deficiency on atherogenesis is unknown. Glycosphingolipids have been shown to accumulate in atherosclerotic plaques even in subjects without Fabry disease, which suggests they may play a role in atherogenesis.5 A mouse model of Gla deficiency has been generated by targeted disruption of the Gla gene.6 These mice accumulate glycosphingolipids in multiple organs with age, including the large blood vessels7,8; thus, they provide a useful model to study the vascular consequences of Gla deficiency. The present study was designed to test the consequences of Gla deficiency in vascular disease with a mouse model of atherosclerosis.
To test the consequences of Gla deficiency on vascular disease, we examined the development of lipid lesions between apolipoprotein E-deficient (ApoE−/−) littermate mice with genetic variation in Gla. ApoE−/− mice on the C57BL6/J background were purchased from Jackson Laboratory (Bar Harbor, Me). Gla-deficient (Gla−/0 or Gla−/−) mice were bred from mice provided by Drs Ashok Kulkarni and Roscoe Brady (National Institutes of Health, Bethesda, Md). The “0” in Gla−/0 denotes the absence of the second X chromosome of the male mice in this X-linked disease. These mice were backcrossed at least 6 generations to the C57BL6/J strain before being bred to ApoE−/− mice. Gla−/0, ApoE+/− mice were intercrossed to produce Gla−/0, ApoE−/− and Gla+/0, ApoE−/− mice. To generate groups of female mice, Gla−/0, ApoE−/− mice were then crossed to Gla+/−, ApoE−/− mice to generate Gla+/−, ApoE−/− and Gla−/−, ApoE−/− mice. Mice were genotyped by polymerase chain reaction analysis of tail DNA specimens with primers as described previously8 and grouped according to gender (3 to 5 mice/cage) at weaning. All mice were maintained in specific pathogen-free facilities and fed a normal chow (LabDiet #5001) from weaning until 41 weeks, followed by 4 weeks of Western chow (TD88137, Harlan Teklad) to enhance atherogenesis. All animal care and experimental procedures complied with the Principals of Laboratory and Animal Care established by the National Society for Medical Research and were approved by the University of Michigan Committee on Use and Care of Animals.
Analysis of Atherosclerosis
At 45 weeks of age, mice were euthanized via exsanguination while under intraperitoneal pentobarbital anesthesia (100 mg/kg). The mice were perfused with phosphate-buffered saline and fixed with formalin with a 25-gauge needle, inserted into the left ventricle, at a rate of 1 mL/min. For quantification of surface area occupied by atherosclerosis, the thoracic and abdominal aorta and its major branches (including brachiocephalic trunk, right and left subclavian arteries to first major bifurcation, right and left carotid arteries distal to internal/external bifurcation, and right and left common iliac arteries distal to first major bifurcation) were dissected and stained with oil red O and then subjected to quantitative morphometry as described previously.9 The lesion area was calculated for each genotype and expressed as the total lesion area (mm2) and the percent lesion area (expressed as %), which was total lesion area (mm2) divided by the total surface area examined (mm2).
For analysis of Gb3 within atherosclerotic lesions, we stained cross sections of the brachiocephalic artery of 52-week-old male ApoE−/− mice with or without Gla. This site was chosen because a particularly robust, consistent lesion forms at this location. After PBS perfusion and excision of these sections, the samples were frozen for subsequent staining with verotoxin B as described previously.8
Inducible Nitric Oxide Synthase, Endothelial Nitric Oxide Synthase, and Nitrotyrosine Quantification
Immunostaining for endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and nitrotyrosine was performed on formalin-fixed, paraffin-embedded 5-μm sections obtained from the atherosclerotic, brachiocephalic artery. For these analyses, sections with similar plaque size were chosen for comparison between groups of mice with and without Gla. The following antibodies were used for antigen detection: iNOS, rabbit polyclonal antibody to mouse iNOS, 1:100 (Laboratory Vision-NeoMarkers); eNOS, rabbit polyclonal antibody to human eNOS, 1:100 (Laboratory Vision-NeoMarkers); nitrotyrosine, rabbit polyclonal antibody to synthetic nitrated protein, 1:100 (Upstate Cell Signaling Solutions). Staining was performed according to manufacturer’s instructions and was quantified with automated color-detection software (Image Pro-Plus) after digitization of the slides with a Spot Insight color camera system (Diagnostic Instruments, Inc). The observer was blinded to sample genotype for these analyses. The data are expressed as percentage of the lesion stained with antibody.
Real-Time Polymerase Chain Reaction
For real-time polymerase chain reaction (RT-PCR) studies, atherosclerotic and nonatherosclerotic carotid and aortic arch samples were excised and stored immediately after PBS perfusion with an RNA stabilization reagent (QIAGAN Inc). Isolation and purification of total RNA was performed with the RNeasy kit and RNase free-DNase (QIAGEN) according to the manufacturer’s instructions. cDNA was generated from total RNA with TaqMan Reverse Transcription reagents (Applied Biosystems) with RNA (800 ng) as a template and oligo(dT)16 as primers. The resulting cDNA was then used for RT-PCR. The Sequence Detection System 7100 (Applied Biosystems) was used for amplification and specific sequence detection. Primers for iNOS and probe mix solution were purchased from Applied Biosystems (assay ID MM00440485). Equal amounts of cDNA were used in duplicate with the TaqMan Master Mix provided by ABI. Amplification efficiencies were validated against the housekeeping gene, β-actin. The data were normalized to β-actin mRNA level and expressed in arbitrary units. The formula 2−ΔΔCT was used for each run according to the manufacturer’s instructions and published methods for this system.10
Values are expressed as mean±SD. The statistical significance of differences in the quantification of atherosclerosis and immunostaining between mice with and without Gla was determined with the Student’s t test. P<0.05 was considered significant.
Effect of Gla on Atherosclerosis
To determine the role of Gla on the development of atherosclerosis, 45-week-old ApoE−/− mice with or without Gla were examined. There were no significant differences in atherosclerosis between the male and female mice, which were equally represented between groups of mice expressing normal Gla and mice with Gla deficiency (5 females and 5 males for a total of 10 in each group). Gla-deficient mice (Gla−/0, ApoE−/− and Gla−/−, ApoE−/−) had significantly greater total lesion area (25.1±14.0 mm2) than normal Gla mice (Gla+/0, ApoE−/− and Gla+/+, ApoE−/−; 12.3±9.3 mm2). The percent lesion area of the dissected arterial tree was also significantly increased by ≈2-fold (Figures 1A and 1B). Gla heterozygotes displayed an intermediate phenotype (15.2±6.0 mm2), although this was not statistically different from the normal Gla or Gla-deficient groups. Immunostaining of the brachiocephalic artery revealed intense Gb3 staining within the atherosclerotic plaque of Gla-deficient mice (Figures 2A and 2B). No atherosclerosis was detected by surface oil red O staining of arterial trees from 3 male Gla-deficient mice with wild-type ApoE expression (ApoE+/+) euthanized at 12 months of age.
Effect of Gla on iNOS and eNOS
Because altered regulation of nitric oxide (NO) has been hypothesized to account for vascular dysfunction in humans with Fabry disease,11 we analyzed diseased vessels for eNOS, iNOS, and nitrotyrosine by immunohistochemistry. Greater immunostaining within the atherosclerotic plaque was apparent for iNOS in Gla-deficient mice (15.8±9.8%, n=4) than in mice expressing normal Gla (0.7±0.5%, n=4, P<0.002; Figure 2), whereas no significant difference (P=0.20) was observed between the groups for eNOS staining (Gla-deficient 4.3±0.88%, n=4, versus normal Gla 5.0±3.8%, n=4). To determine whether elevated iNOS staining was due to increased iNOS transcription, RT-PCR was performed on RNA isolated from the aortic arch. This analysis revealed an 8.6±3.4-fold increase in iNOS expression from tissue obtained from Gla-deficient mice compared with mice with normal Gla expression. Consistent with elevated iNOS expression, more nitrotyrosine was detected in the plaque from Gla-deficient mice (17.3±9.1%, n=4) than in mice with normal Gla expression (2.2±1.1%, n=4, P<0.001; Figure 2). iNOS expression was also elevated more than 2-fold (2.8±1.0 versus 1.3±0.2 arbitrary units) from aortic samples obtained from 12-month-old Gla-deficient mice (n=4) with wild-type ApoE expression, which have no atherosclerosis, compared with age-matched normal Gla mice (n=5).
Premature vascular complications such as stroke and myocardial infarction are devastating complications in patients with Fabry disease.1 Although endothelial accumulation of glycolipids with ischemic pathology appears to play a role in the nephropathy and neuropathy seen in Fabry patients,1 the mechanisms related to premature macrovascular events are unclear. To test the hypothesis that atherosclerosis may be affected, we generated ApoE/Gla double-knockout mice. This model is particularly suitable for the study of alterations in glycosphingolipid metabolism because increased levels of glycosphingolipids have been shown to be elevated in the serum and aorta of ApoE−/− mice.12 At 45 weeks of age, Gla deficiency was associated with a significant increase in total atherosclerotic burden. This is the first study to demonstrate that Gla deficiency promotes atherosclerosis.
Previous clinical studies have demonstrated that Gla deficiency is associated with abnormalities of cerebral blood flow with positron emission tomography.11 The authors hypothesized that dysregulation of NO may affect blood flow and contribute to the risk of vascular complications. Nitrotyrosine staining was found to be increased in brain vessels from specimens with Gla deficiency compared with control vessels, which suggests excessive NO production with peroxynitrite formation. To examine this hypothesis in our mouse model, we performed immunohistochemical analyses of iNOS and eNOS. Although eNOS staining of the plaque was similar between mice with and without Gla, iNOS staining was increased 20-fold in the plaques from mice with Gla deficiency. This enhanced iNOS was confirmed by RT-PCR, which demonstrated an 8-fold increase in expression. Consistent with toxic NO formation by iNOS, nitrotyrosine staining was increased in atherosclerotic plaques from Gla-deficient mice. Therefore, the present findings suggest the presence of NO dysregulation in the atherosclerotic vessels of Gla-deficient mice. Elevated iNOS expression was also observed in aged nonatherosclerotic Gla-deficient mice, which suggests alterations in iNOS are not an epiphenomenon of differences in atherosclerotic burden.
The effect of NO in vascular disease is complex and controversial. iNOS is upregulated in inflammatory conditions, including atherosclerotic lesions, and may be proatherogenic.13 In this setting, NO may react with superoxide to form peroxynitrite, a strong oxidant.14 Consistent with these potentially vasculopathic events, iNOS deficiency has been shown to reduce aortic lesion area by 40% after 24 weeks of high-fat feeding in ApoE−/− mice.13 It is possible that glycosphingolipids within the plaque may promote local cytokine expression and enhance the inflammatory state. Shiga toxin for example, which binds to Gb3, has been shown to increase nuclear factor-κB expression and tumor necrosis factor-α secretion in a human monocytic cell line,15 conditions that would lead to increased iNOS expression. This mouse model of accelerated atherosclerosis in Gla deficiency will provide an avenue for further studies of this complex interaction of glycosphingolipids with the vascular wall. Further studies designed to analyze various stages of atherosclerosis and test potential therapies aimed at attenuating atherogenesis in these mice may be highly informative.
In conclusion, deficiency of Gla is associated with dysregulation of NO production and enhanced atherosclerosis in ApoE−/− mice. Increased atherogenesis may account for some of the premature vascular complications in patients with Gla deficiency. In addition, it is possible that more subtle alterations in glycosphingolipid metabolism could play a role in the regulation of NO and atherosclerosis in the population at large.
This work was supported by grant funding from NIH grant P01 HL57346 (Dr Eitzman) and NIH grant RO1 DK-055823 (Dr Shayman).
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