Research Article | Open Access
Increased Inflammation in Atherosclerotic Lesions of Diabetic Akita-LDLr−/− Mice Compared to Nondiabetic LDLr−/− Mice
Background. Diabetes is associated with increased cardiovascular disease, but the underlying cellular and molecular mechanisms are poorly understood. One proposed mechanism is that diabetes aggravates atherosclerosis by enhancing plaque inflammation. The Akita mouse has recently been adopted as a relevant model for microvascular complications of diabetes. Here we investigate the development of atherosclerosis and inflammation in vessels of Akita mice on LDLr−/− background. Methods and Results. Akita-LDLr−/− and LDLr−/− mice were fed high-fat diet from 6 to 24 weeks of age. Blood glucose levels were higher in both male and female Akita-LDLr−/− mice (137% and 70%, resp.). Male Akita-LDLr−/− mice had markedly increased plasma cholesterol and triglyceride levels, a three-fold increase in atherosclerosis, and enhanced accumulation of macrophages and T-cells in plaques. In contrast, female Akita-LDLr−/− mice demonstrated a modest 29% increase in plasma cholesterol and no significant increase in triglycerides, atherosclerosis, or inflammatory cells in lesions. Male Akita-LDLr−/− mice had increased levels of plasma IL-1β compared to nondiabetic mice, whereas no such difference was seen between female diabetic and nondiabetic mice. Conclusion. Akita-LDLr−/− mice display considerable gender differences in the development of diabetic atherosclerosis. In addition, the increased atherosclerosis in male Akita-LDLr−/− mice is associated with an increase in inflammatory cells in lesions.
Atherosclerosis is a chronic inflammatory disease characterized by formation of lesions in large- and medium-sized arteries. Diabetes is associated with increased atherosclerosis, and diabetic patients have a 2–4-fold increased risk of cardiovascular mortality [1, 2]. Moreover, stroke, coronary heart disease, and peripheral artery disease are more common and occur at an earlier age in diabetic patients than in nondiabetic persons . The proposed culprits responsible for the increased risk of atherosclerosis in diabetic patients include dyslipidemia, hypertension, endothelial dysfunction, oxidative stress, and increased generation of advanced glycation end-products (AGEs) . However, the underlying cellular and molecular mechanisms whereby diabetes accelerates atherosclerosis are still poorly understood, and one of the main reasons for this has been the lack of animal models of diabetes that replicate the disease as seen in humans.
One of the most widely used mouse models for cardiovascular disease in type 1 diabetes is the streptozotocin-induced diabetes model in atherosclerotic apolipoprotein E-deficient () or LDL-receptor-deficient () mice. The streptozotocin induced diabetes model, however, has a drawback in the nonspecific toxic effects of streptozotocin to other organs than pancreas, since streptozotocin is known to be both hepatotoxic and nephrotoxic . In addition, streptozotocin can methylate DNA and has genotoxic effects by damaging DNA . Recently, streptozotocin was shown to have a direct toxic effect on lymphocytes in vitro, particularly on CD8-cells and B-cells . Streptozotocin treatment of mice also resulted in a relative increase in regulatory T-cells, and this effect was independent of hyperglycemia . CD8-cells [8, 9], B-cells [10–13], and regulatory T-cells  have been shown to play important roles in atherosclerosis. Thus, these side effects of streptozotocin could have an impact on the atherosclerotic disease, which is not related to the diabetic disease.
The (Akita) mouse is a model of type 1 diabetes, characterized by a point-mutation causing pro-insulin misfolding with subsequent endoplasmic reticulum-stress leading to beta-cell apoptosis . The Akita mouse has previously been successfully used as a model of diabetic microvascular complications, including retinopathy, neuropathy, and nephropathy . Recently, increased atherosclerosis in mice and mice compared to nondiabetic mice has been reported [17, 18]. Both of these studies primarily focused on the role of altered lipid metabolism. Although the cellular and molecular mechanisms behind why diabetes results in increased atherosclerosis are not known, one proposed mechanism is increased inflammation, due to increased oxidative stress in the atherosclerotic lesions. In atherosclerosis, subendothelial retention and oxidation of LDL induce expression of proinflammatory cytokines and recruitment of inflammatory cells to the vessel wall [19, 20]. In diabetes, increased glucose levels result in formation of AGE, which is believed to increase oxidative stress and inflammation [21, 22]. To see if the increased atherosclerosis reported in mice is accompanied or driven by increased plaque inflammation, we measured the extent of inflammatory cells and cytokines in atherosclerotic vessels of mice. We also asked whether mice display an altered immunological T-cell profile compared to nondiabetic mice.
(B6.Cg- /J) and mice were obtained from Jackson Laboratories and bred at our animal facilities. Akita mice were genotyped according to protocols provided by the Jackson Laboratories. Animals had free access to tap water and were fed a high-fat diet, containing 21% cocoa fat (weight%), 0.15% cholesterol (weight%), from six weeks of age, and were sacrificed at 18 or 24 weeks of age. Blood glucose was measured every three weeks using a One-Touch Glucometer (LifeScan Inc., CA, USA) in nonfasting mice. Plasma triglycerides and cholesterol were measured in nonfasting mice by colorimetric assays as described before . All animal experiments were approved by the Malmö-Lund Animal Care and Use Committee and the investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health.
For assessment of atherosclerosis, plaque characteristics, myocardial fibrosis, and inflammation mice were sacrificed at 24 weeks of age. En face preparations of the aorta, Oil red O-staining, and quantification were performed as described before . Staining of monocytes/macrophages (MOMA-2, Biomedicals AG, Switzerland, detecting a glycoprotein located in the cytoplasm and on the cell surface on monocytes and macrophages), T-cells (anti-CD3) , collagen (Masson’s trichrome), and AbcA1 (abcam) were performed as described before . Subvalvular plaque area was determined in haematoxylin stained sections of the aortic root.
2.3. Plasma Cytokines
Cytokines in plasma from 24-week-old mice were measured using a Th1/Th2 9-plex assay (Meso Scale Discovery, USA) according to manufacturer’s instructions.
2.4. Gene Expression Analysis
A separate subset of mice was sacrificed at 18 weeks of age to measure mRNA levels of inflammatory markers in brachiocephalic arteries. Mice were perfused with RNAlater (Applied Biosystems). Brachiocephalic arteries were isolated and snap-frozen in Trizol (Invitrogen). Total RNA was extracted as described previously  and cDNA was synthesized with RevertAid First Strand cDNA Synthesis Kit (Fermentas Life Sciences). mRNA levels were analyzed by quantitative real-time PCR using Taqman assays (Applied Biosystems): Mm00436767_m1 for osteopontin (OPN), Mm00446190_m1 for interleukin-6 (IL-6), Mm01336189_m1 for interleukin 1β (IL-1β), Mm01320970_m1 for vascular cell adhesion molecule (VCAM), Mm00442991_m1 for matrix metalloproteinase-9 (MMP9), Mm00436450_m1 for macrophage inflammatory protein-2 (MIP2), and Mm00441242_m1 for monocyte chemotactic protein-1 (MCP-1). Expression levels of target genes were normalized to the expression of cyclophilin B (PPIb), Mm00478295_m1, as the housekeeping gene.
2.5. Splenocyte Preparation and Culture
Splenocytes were isolated as previously described . Briefly, splenocytes from 24-week-old mice were isolated, washed, and stimulated with Concanavalin A (2.5 μg/ml) or left unstimulated. After 72 hours, [methyl-3H]-thymidine was added to wells. To quantify DNA synthesis, cells were harvested 16 hours after [methyl-3H]-thymidine addition and measured using a liquid scintillation counter.
2.6. Flow Cytometry
Splenocytes were washed and stained with fluorochrome-conjugated antibodies and analyzed with a CyAn ADP flow cytometer (Beckman Coulter). The antibodies used were AF488-CD69, PE-Cy7-CD3ε, PB-CD4, and APC-CD25 (all from Biolegend).
2.7. Statistical Analysis
Values are presented as mean ± SD unless otherwise specified. Statistical analyses were performed with Graph-Pad 5 (Prism) or PASW Statistics 18 software. Statistical significance was determined using two-way ANOVA followed by Bonferroni post hoc tests unless otherwise specified. Correlation analyses were performed using Pearson (normally distributed variables) or Spearman (skewed variables). Linear regression analysis was performed with PASW Statistics 18.
3.1. Akita-LDL−/− Mice Display Gender-Specific Metabolic Profiles
male mice displayed severe hyperglycemia with an average glucose level of mM, whereas female mice had a milder phenotype with an average glucose of mM (Figure 1(a) and see Figure 1(a) in Supplementary Material available online at http://dx.doi.org/10.1155/2012/176162). mice were normoglycemic ( mM and mM; male and female, resp.). As it has been described for Akita mice , males failed to gain as much weight as nondiabetic males, and therefore had a lower body weight at 24 weeks of age ( g versus g, ). No differences in body weight were observed between females ( g and g; diabetic and nondiabetic, resp.). Cholesterol levels in male mice were increased two-fold compared to male controls ( mM versus mM, ; Figure 1(b)). Female mice displayed a more modest increase in plasma cholesterol compared to female controls ( mM versus mM, ). Nondiabetic female mice had elevated cholesterol levels when compared to nondiabetic male mice ( mM versus mM, ; Figure 1(b)). Moreover, males, but not females, exhibited elevated triglyceride levels compared to their nondiabetic counterparts (Figure 1(c)).
3.2. Male Akita-LDLr−/− Mice Display Increased Atherosclerosis Compared to LDLr−/− Controls
En face Oil red O staining of aortas of 24 weeks of age mice was performed in order to quantify atherosclerotic burden. Male mice had a five-fold increase in lesion area compared to male control mice ( versus , ; Figure 2(a)). Nondiabetic female were found to have a significantly larger lesion area than nondiabetic male ( versus , ; Figure 2(a)). However, diabetes had no significant effect on lesion area in the aorta of female mice ( versus ; Figure 2(a)). In an analysis of the plaque area in the aortic arch separately, male mice had a 5-fold increase in lesion area compared to male mice ( versus , ), whereas female mice only displayed a trend toward a 1.4-fold increase in lesion area compared to female ( versus , ; unpaired t-test). When atherosclerosis was studied in the aortic root, the subvalvular lesion area was significantly increased in male mice compared to nondiabetic male mice ( μm2 versus μm2, , Figure 2(b)). As we found in the aorta, nondiabetic female mice had significantly larger subvalvular plaque area than male mice; however, diabetes had no further impact on plaque size in female mice ( versus , Figure 2(b)).
3.3. Male Akita-LDLr−/− Mice Display Increased Accumulation of Inflammatory Cells in Atherosclerotic Lesions Compared to LDLr−/− Controls
Monocyte and T-cell recruitment to the plaque plays a central role in atherogenesis. To investigate the effect of diabetes on monocyte/macrophage infiltration, we stained sections of the aortic root with MOMA-2. Subvalvular lesions of male mice had larger areas infiltrated by macrophages as assessed by MOMA-2 immunoreactivity, compared to control mice ( μm2 versus μm2, ; Figure 3(a), see Figure 2 in Supplementary Material available online at doi: 10.1155/2012/176162). However, although having larger macrophage areas, the percentage of plaque area stained with MOMA-2 was lower in male mice than in controls ( versus , ). No statistically significant differences were observed regarding the female mice versus control mice. To address whether diabetes resulted in increased accumulation of T-cells in subvalvular atherosclerotic lesions, sections from the aortic root were stained with anti-CD3 and quantified. Male mice had larger areas infiltrated by T-cells compared to control male mice (m2 versus μm2, ; Figure 3(c)). Since ABCA1-deficient macrophages display enhanced inflammatory responses , we stained and quantified subvalvular lesions for ABCA1 to see if the increased inflammation seen in male mice could be explained by differences in ABCA1. male mice had increased amount of ABCA1 in lesions compared to nondiabetic mice, whereas no such differences were observed in female mice (see Figure 3 in Supplementary Material available online at doi: 10.1155/2012/176162). Plaque from female mice contained increased amount of ABCA1 compared to male mice. The percentage of plaque area stained with anti-ABCA1 was decreased in male mice ( versus , ) compared to nondiabetic mice, but the same trend was seen in female mice ( versus , ). Plaques from male mice had significantly larger areas of collagen compared to male mice ( μm2 versus μm2; ), whereas there was no difference between female mice and nondiabetic female mice (Figure 3(b)). Again, gender differences were observed in nondiabetic mice, with female mice having significantly larger collagen areas than male mice (Figure 3(b)).
3.4. Effect of Diabetes on the Expression of Inflammatory Genes in the Brachiocephalic Artery
To determine if the increase in inflammatory cells in lesions of male mice was preceded by inflammatory cytokines, we measured the expression of IL-1β, IL-6, VCAM, OPN, MMP-9, MCP-1, and MIP-2 in the brachiocephalic artery of 18 weeks of age or mice (Table 1). MIP-2 levels were increased in female mice compared to female mice, whereas there was no difference between diabetic and nondiabetic male mice. There was a tendency to increased MMP-9 levels in mice compared to mice including both genders ( versus , ), though this increase was more evident in female mice than in male mice. Moreover, in diabetic mice there were associations between plasma glucose levels and osteopontin expression (, ), MCP-1 expression (, ), or IL-6 expression (, ), which were not conserved in nondiabetic mice.
versus females; Mann-Whitney.|
Levels of mRNA were measured by real-time RT-PCR and were normalized to the expression of the housekeeping control gene cyclophilin B. values represent mean ± SD.
3.5. Effect of Diabetes on Myocardial Fibrosis
To determine if the increased atherosclerosis in mice was associated with increased myocardial fibrosis, we measured collagen content in the muscular tissue of the heart, but we were only able to detect minor amounts of collagen in the tissue. Moreover, we did not find any differences between the groups (data not shown). In addition, we analyzed macrophage content in the heart muscle tissue, but did not find any signs of inflammation.
3.6. Male Akita-LDLr−/− Mice Display Increased Levels of IL-1β
Plasma levels of the proinflammatory cytokine IL-1β were significantly elevated in 24-week-old males compared to males of the same age (Figure 4(a)). Moreover, both TNF-α (; Figure 4(b)) and the neutrophil activating chemokine KC (CXCL1; ; Figure 4(c)) displayed trends towards being increased in male mice compared to male control mice. In a linear regression analysis model, IL-1β was associated with average glucose levels in diabetic male and female mice, but not with cholesterol levels. There were no significant changes in plasma levels of IFNγ, IL-2, IL-4, IL-5, or IL-10 between the groups (Table 2). Total IL-12, including both anti-inflammatory IL-12p40 and proinflammatory IL-12p70, was decreased in male mice compared to male control mice (Table 2).
versus males; Bonferroni post-hoc test.|
Two-way ANOVA revealed interactions between diabetes and gender for IL-12 (), and significant effect of gender for IL-5 ().
Values represent mean ± SD.
T-cell subsets have been shown to greatly influence the development of experimental atherosclerosis in mice . To assess whether the increased plaque development reflected a change in the balance between activated conventional T-cells and anti-inflammatory regulatory T-cells, we performed flow cytometry on isolated splenocytes from 24-week-old mice. No differences in percentages of regulatory T-cells (CD4+CD25+FoxP3+, Figure 5(a)) or activated T-cells (CD4+CD69+, Figure 5(b)) were observed between the groups. To further determine the T-cell activation status, we measured basal and Concanavalin A- (ConA-) induced proliferation. Male mice displayed higher basal proliferation compared to male mice, whereas there was no difference in ConA stimulated proliferation (Figures 5(c) and 5(d)). No significant difference was observed between female mice.
In this study, we characterize the mouse as a model of diabetic atherosclerosis. We also investigate if the atherosclerotic disease in mice is accompanied by increased inflammation in atherosclerotic lesions. We show that the mouse has considerable gender differences with regard to metabolic profile, atherosclerotic disease, and inflammatory cells in atherosclerotic lesions. While male mice exhibit severe hyperglycemia, hypercholesterolemia, and hypertriglyceridemia compared to nondiabetic mice, female mice show marked hyperglycemia, but only a modest increase in cholesterol levels and no changes in plasma triglycerides. Male mice have a five-fold increase in aortic lesion area, a three-fold increase in subvalvular lesion area, and increased areas of macrophage and T-cell infiltration in the lesions compared to male mice, but no such differences were seen in female mice.
Estrogen and its receptors are regulators of glucose and lipid metabolism and rodent studies link estrogen to anti diabetic effects. Estrogen is also known to exert antiinflammatory effects in both humans and rodents . It is possible that the gender differences reported in our study at least partly are due to the effects of estrogen, for example, plasma IL-1β levels are increased in male mice compared to female mice (Figure 4(a)). The reason why female mice, despite induction of hyperglycemia, have no or only a minor increase in atherosclerosis, macrophage, and T-cell infiltration compared to female mice could be due to the anti-inflammatory effects of estrogen.In this respect, it is important to note that hyperglycemia is less pronounced in females than in males ( mM versus mM). It is also important to note that nondiabetic female mice have a 1.5-fold increase in cholesterol levels and 3-fold increase in subvalvular lesion area compared to nondiabetic male mice. In fact, female nondiabetic mice have the same subvalvular lesion area as male diabetic mice, but the lesion area in female mice is not further increased in response to hyperglycemia. It is possible that the already high cholesterol levels in female nondiabetic mice mask the effect of hyperglycemia on lesion formation in female diabetic mice. Such observations have been made in other mouse models of diabetic atherosclerosis. For example, Reaven et al. studied male mice on a high fat diet, which were made diabetic using streptozotocin . In that study, diabetic and nondiabetic mice had similar plasma cholesterol levels (25.1 versus 25.9 mM); however, diabetic mice had increased glucose levels, increased triglyceride levels, and increased formation of AGE epitopes in the artery wall. Despite this, there were no differences in atherosclerosis between diabetic and nondiabetic mice. The authors suggest that under conditions of marked hypercholesterolemia, there is no effect of hyperglycemia and/or of enhanced AGE formation on atherogenesis in mice. Thus the observed gender differences in atherosclerotic lesions and inflammation in our study may have two possible explanations: (1) the already high cholesterol levels in female nondiabetic mice could mask the effect of hyperglycemia on lesion formation in female diabetic mice, and (2) the less pronounced hyperglycemia in female mice compared to male mice may result in less atherosclerotic disease and less inflammation.
Our results partly confirm a recent study of LDLr-deficient Akita mice published by Zhou et al. . Zhou et al. reported hyperglycemia and hyperlipidemia accompanied by increased atherosclerotic disease in male and female mice. In that study, however, they found no difference in blood glucose levels between male and female mice ( mM versus mM), whereas we found that male mice had higher glucose levels than females ( mM versus mM, ). High-fat diet induces insulin resistance and diabetes in C57/Bl6 mice  and there is gender difference [32, 33]. However, it has also been reported that there is a large difference in glucose levels in male versus female Akita mice, even on a low-fat diet ( mM in males versus mM in females) , and the authors speculate that estrogen and prolactin play a protective role in the females. The levels reported by Yoshioka et al. are similar to the levels we report ( mM in males versus mM in females). In addition, whereas we found no significant difference in lesion area in female mice, neither in en face preparations of the aorta nor in subvalvular lesions, Zhou et al. reported a significant increase in subvalvular lesions of female mice compared to female mice. However, in agreement with our study, the increase in lesion area in diabetic mice compared to nondiabetic mice were larger in males (224%) than in females (30%). The mice in our study were fed a high fat diet containing 0.15% cholesterol, whereas Zhou et al. fed the mice a low-fat diet with 0.02% cholesterol, thus there was a considerable difference in the size of the subvalvular lesion area in our study compared to Zhou et al. (630000 μm2 versus approx. 350000 μm2, resp., for female diabetic mice). The difference in size of subvalvular lesions may also explain why Zhou et al. report significant differences in diabetic female mice compared to nondiabetic mice. The subvalvular lesions in our study, which is almost the double size compared to the ones reported by Zhou et al., may be at a later atherosclerotic stage, at which the differences in female mice may be evened. Thus, since the subvalvular lesions are one of the earliest atherosclerotic lesions in mice, there may be differences in lesion size at other locations. This is supported by our finding that female mice displayed a trend towards larger lesion area in the aortic arch compared to female mice.
Further, Zhou et al. determined liver specific expression of genes involved in lipid metabolism and inflammation by quantitative PCR . In a recent report Jun et al. showed increased atherosclerosis in male apoE-deficient Akita mice, but did not analyze female mice . In the latter study, the authors analyzed expression of lipoprotein receptors in the liver, lipid secretion from the liver and, plasma lipid profile. Thus, both these recently published studies have focused on lipid metabolism and thus mainly studied differences in the liver. To assess whether the increased atherosclerosis seen in diabetic mice is accompanied by increased inflammation in atherosclerotic vessels, we characterized and quantified inflammatory cells in subvalvular lesions by immunohistochemistry. In addition, we used quantitative PCR to analyze the expression of inflammatory genes in the brachiocephalic artery, which is one of the most plaque prominent locations in the arterial tree in mice.
Studies of plaques derived from both type 1 and 2 diabetes patients have shown increased accumulation of T-cells and macrophages . Similar to humans, subvalvular lesions of male mice were enriched in both T-cells and macrophages compared to controls. Surprisingly, mice have a higher percentage of macrophages in the lesions than diabetic mice, which probably reflects that the lesions are at an earlier stage, whereas more advanced lesions display increased infiltration of smooth muscle cells and more fibrous tissue. This is supported by increased collagen content in male mice compared to controls.
It was previously shown that mice displayed decreased mRNA levels in the liver of several genes involved in lipid metabolism (Srebp1a, Srebp1c, AbcA1, Lxrα, and Cyp7b1) . Some of these genes have been shown to modulate atherosclerotic disease. For example, since LXRs promote cholesterol efflux via upregulation of the ABC family , one would expect that LXRs would have antiatherogenic properties due to increased reverse cholesterol transport in the aorta. Indeed, LXRαβ-deficient macrophages displayed increased accumulation of cholesterol , and apoE- and LXRα-deficient mice show increased atherosclerotic disease [37, 38]. In agreement with this, treatment with LXR agonist reduces atherosclerosis in mice [39–42]. However, LXRs do not only affect atherosclerosis due to increased reverse cholesterol transport. It has been shown that LXRs are negative regulators of macrophage inflammatory gene expression. In vitro studies show that LXR ligands inhibit the expression of inflammatory genes such as IL-6, IL-β, MCP-1, MMP-9, and osteopontin [43–45]. Moreover, in vivo treatment of mice with LXR agonists resulted in substantially reduced MMP-9 gene expression in the aortas . In our study, quantitative gene expression analysis of either IL-6, IL-1β, MCP-1, or osteopontin in the brachiocephalic artery did not reveal any difference between or control mice. There was a tendency to increased MMP-9 gene expression in mice compared to mice including both genders, though this increase was more evident in female mice than in male mice. Also ABCA1 has been reported to have anti-inflammatory properties shown by ABCA1-deficient macrophages, which displayed increased TNFα expression upon LPS stimulation . Moreover, bone marrow transplantations demonstrated an antiatherogenic function of ABCA1 in macrophages independently of changes in plasma lipids . In our study, total ABCA1 levels, analyzed by immunohistochemistry, were increased in subvalvular lesions of male mice. Although the percentage of plaque area stained with anti-ABCA1 was decreased in male mice, the same difference was present in female mice. In conclusion, in our study we find no evidence suggesting that downregulation of AbcA1 or Lxrα gene in lesions could explain the gender difference in inflammatory cells observed in subvalvular lesions of mice. On the other hand, Zhou et al. reported increased TNFα, MCP-1, and IL-1 staining in lesions of mice compared to mice. Differences in our study compared to the study by Zhou et al., for example, the diet, location of the lesions, or protein versus mRNA analysis, may explain the reported differences in cytokines in atherosclerotic lesions.
Diabetes is a strong independent risk factor for heart failure [47, 48]. In a previous paper Basu et al. found that normolipidemic Akita C57Bl6 mice were characterized by diastolic dysfunction at three and six months of age, but preserved systolic function . Moreover, there was no evidence of myocardial hypertrophy or fibrosis in the diabetic mice. The latter is in agreement with our data, showing no or only minor collagen content in cardiac tissue and no differences between diabetic and nondiabetic mice.
Several papers have shown that Akita male mice (on normolipidemic background C57bl6 background) develop elevated systolic blood pressure [50–52], which is not present in female mice . Basu et al. measured heart rate in Akita C57BL/6J mice, but did not find any differences between diabetic and nondiabetic mice . In humans, increased blood pressure is a risk factor for the development of atherosclerotic disease; however, in mice the relationship between blood pressure and atherosclerosis is less clear. While several reports have demonstrated reduced atherosclerosis in mice with decreased blood pressure, other studies have shown that changes in blood pressure do not affect atherosclerotic disease (reviewed in ). Thus, it is difficult to predict whether differences in blood pressure could underlie the gender differences in atherosclerosis observed in mice.
It is widely recognized that T-cells influence atherosclerosis in mice . For example, regulatory T-cells have in several studies been shown to protect against atherosclerosis . Since we did not find any differences in percentages in either the protective regulatory (CD4+CD25+FoxP3+) T-cells or the putatively harmful activated (CD4+CD69+) T-cells, we suggest that diabetic atherosclerosis in these mice is not induced by an overall immune activation.
In conclusion, both male and female mice are hyperglycemic compared to control mice. However, whereas male mice have a 2-fold increase in plasma cholesterol and 3-fold increase in triglyceride levels, female mice have only a modest diabetes-induced increase in cholesterol and no increase in triglyceride levels. This is accompanied by a dramatic increase in atherosclerosis as well as increased plaque inflammation in male mice, but no significant changes in plaque size or inflammatory cells in lesions in female mice compared to nondiabetic mice. We propose that the mouse is a promising tool for studying development of cardiovascular disease both in a setting of severe as well as a more moderate increase in cholesterol levels.
Conflict of Interests
The authors declare that they have no conflict of interests.
This paper was supported by the Innovative Medicines Initiative Joint Undertaking under Grant Agreement no. 115006 (the SUMMIT Consortium), with funding from the European Union's Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in-kind contribution. The work was also supported by grants from the Crafoord, Åke Wiberg foundation, Magnus Bergvall foundation, Albert Påhlsson, Knut & Alice Wallenberg and Swedish Heart and Lung Foundations, and the Swedish Society for Medical Research, as well as from the Malmö Hospital Research Funds, Skåne County Council, the Foundation for Strategic Research (SSF), and Swedish Research Council.
Supplementary fig.1: Blood glucose (a) and plasma cholesterol (b) levels in Akita-LDLr-/- and LDLr-/- male and female mice. Values represent mean ± SEM.
Supplementary fig.2: Macrophage staining of subvalvular lesions (high magnification). The positive staining (brown) reflects both the presence of macrophages in the plaque and the presence of cell debris from necrotic macrophages in the core of the lesion. Scale bar=100 μm.
Supplementary fig.3: Male Akita-LDLr-/- mice have increased ABCA1 levels in subvalvular lesions compared to male LDLr-/- mice. Subvalvular lesions from 24-week old mice were stained and quantified for ABCA1. Values are presented as individual mice and as mean ± SEM. Two-way ANOVA revealed interactions between diabetes and gender (***), and significant effect of diabetes (***) and of gender (*). Bonferroni post hoc test yielded * P<0.05, *** P<0.001.
- S. P. Laing, A. J. Swerdlow, S. D. Slater et al., “Mortality from heart disease in a cohort of 23,000 patients with insulin-treated diabetes,” Diabetologia, vol. 46, no. 6, pp. 760–765, 2003.
- D. M. Nathan, P. A. Cleary, J. Y. C. Backlund et al., “Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes,” New England Journal of Medicine, vol. 353, no. 25, pp. 2643–2653, 2005.
- N. B. Ruderman and C. Haudenschild, “Diabetes as an atherogenic factor,” Progress in Cardiovascular Diseases, vol. 26, no. 5, pp. 373–412, 1984.
- M. Brownlee, “Biochemistry and molecular cell biology of diabetic complications,” Nature, vol. 414, no. 6865, pp. 813–820, 2001.
- S. Lenzen, “The mechanisms of alloxan- and streptozotocin-induced diabetes,” Diabetologia, vol. 51, no. 2, pp. 216–226, 2008.
- A. D. Bolzán and M. S. Bianchi, “Genotoxicity of streptozotocin,” Mutation Research, vol. 512, no. 2-3, pp. 121–134, 2002.
- Y. D. Muller, D. Golshayan, D. Ehirchiou et al., “Immunosuppressive effects of streptozotocin-induced diabetes result in absolute lymphopenia and a relative increase of T regulatory cells,” Diabetes, vol. 60, no. 9, pp. 2331–2340, 2011.
- P. C. Dimayuga, K. Y. Chyu, J. Kirzner et al., “Enhanced neointima formation following arterial injury in immune deficient rag-1-/- mice is attenuated by adoptive transfer of CD8+ T cells,” PLoS ONE, vol. 6, no. 5, Article ID e20214, 2011.
- D. Kolbus, O. H. Ramos, K. E. Berg et al., “CD8+ T cell activation predominate early immune responses to hypercholesterolemia in Apoe−/− mice,” BMC Immunology, vol. 11, article 58, 2010.
- H. Ait-Oufella, O. Herbin, J. D. Bouaziz et al., “B cell depletion reduces the development of atherosclerosis in mice,” Journal of Experimental Medicine, vol. 207, no. 8, pp. 1579–1587, 2010.
- A. C. Doran, M. J. Lipinski, S. N. Oldham et al., “B-cell aortic homing and atheroprotection depend on Id3,” Circulation Research, vol. 110, no. 1, pp. e1–e12, 2012.
- T. Kyaw, C. Tay, A. Khan et al., “Conventional B2 B cell depletion ameliorates whereas its adoptive transfer aggravates atherosclerosis,” Journal of Immunology, vol. 185, no. 7, pp. 4410–4419, 2010.
- T. Kyaw, C. Tay, S. Krishnamurthi et al., “B1a B lymphocytes are atheroprotective by secreting natural IgM that increases IgM deposits and reduces necrotic cores in atherosclerotic lesions,” Circulation Research, vol. 109, no. 8, pp. 830–840, 2011.
- H. Ait-Oufella, B. L. Salomon, S. Potteaux et al., “Natural regulatory T cells control the development of atherosclerosis in mice,” Nature Medicine, vol. 12, no. 2, pp. 178–180, 2006.
- J. Wang, T. Takeuchi, S. Tanaka et al., “A mutation in the insulin 2 gene induces diabetes with severe pancreatic β-cell dysfunction in the Mody mouse,” Journal of Clinical Investigation, vol. 103, no. 1, pp. 27–37, 1999.
- W. Hsueh, E. D. Abel, J. L. Breslow et al., “Recipes for creating animal models of diabetic cardiovascular disease,” Circulation Research, vol. 100, no. 10, pp. 1415–1427, 2007.
- J. Y. Jun, Z. Ma, and L. Segar, “Spontaneously diabetic Ins2+/Akita: apoe-deficient mice exhibit exaggerated hypercholesterolemia and atherosclerosis,” American Journal of Physiology, vol. 301, no. 1, pp. E145–E154, 2011.
- C. Zhou, B. Pridgen, N. King, J. Xu, and J. L. Breslow, “Hyperglycemic Ins2AkitaLdlr−/− mice show severely elevated lipid levels and increased atherosclerosis: a model of type 1 diabetic macrovascular disease,” Journal of Lipid Research, vol. 52, no. 8, pp. 1483–1493, 2011.
- K. Hartvigsen, M. Y. Chou, L. F. Hansen et al., “The role of innate immunity in atherogenesis,” Journal of Lipid Research, vol. 50, pp. S388–S393, 2009.
- I. Tabas, K. J. Williams, and J. Borén, “Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications,” Circulation, vol. 116, no. 16, pp. 1832–1844, 2007.
- G. Basta, A. M. Schmidt, and R. De Caterina, “Advanced glycation end products and vascular inflammation: implications for accelerated atherosclerosis in diabetes,” Cardiovascular Research, vol. 63, no. 4, pp. 582–592, 2004.
- S. F. Yan, R. Ramasamy, and A. M. Schmidt, “Mechanisms of Disease: advanced glycation end-products and their receptor in inflammation and diabetes complications,” Nature Clinical Practice Endocrinology and Metabolism, vol. 4, no. 5, pp. 285–293, 2008.
- G. N. Fredrikson, I. Söderberg, M. Lindholm et al., “Inhibition of atherosclerosis in apoE-null mice by immunization with apoB-100 peptide sequences,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 23, no. 5, pp. 879–884, 2003.
- M. Wigren, D. Bengtsson, P. Dunér et al., “Atheroprotective effects of alum are associated with capture of oxidized LDL antigens and activation of regulatory T cells,” Circulation Research, vol. 104, no. 12, pp. e62–e70, 2009.
- C. Gustavsson, C. D. Agardh, A. V. Zetterqvist, J. Nilsson, E. Agardh, and M. F. Gomez, “Vascular cellular adhesion molecule-1 (VCAM-1) expression in mice retinal vessels is affected by both hyperglycemia and hyperlipidemia,” PloS ONE, vol. 5, no. 9, Article ID e12699, 2010.
- M. Yoshioka, T. Kayo, T. Ikeda, and A. Koizumi, “A novel locus, Mody4, distal to D7Mit189 on chromosome 7 determines early-onset NIDDM in nonobese C57BL/6 (Akita) mutant mice,” Diabetes, vol. 46, no. 5, pp. 887–894, 1997.
- M. Koseki, K. I. Hirano, D. Masuda et al., “Increased lipid rafts and accelerated lipopolysaccharide-induced tumor necrosis factor-α secretion in Abca1-deficient macrophages,” Journal of Lipid Research, vol. 48, no. 2, pp. 299–306, 2007.
- G. K. Hansson and P. Libby, “The immune response in atherosclerosis: a double-edged sword,” Nature Reviews Immunology, vol. 6, no. 7, pp. 508–519, 2006.
- M. H. Faulds, C. Zhao, K. Dahlman-Wright, and J. Gustafsson, “The diversity of sex steroid action: regulation of metabolism by estrogen signaling,” Journal of Endocrinology, vol. 212, no. 1, pp. 3–12, 2012.
- P. Reaven, S. Merat, F. Casanada, M. Sutphin, and W. Palinski, “Effect of streptozotocin-induced hyperglycemia on lipid profiles, formation of advanced glycation endproducts in lesions, and extent of atherosclerosis in LDL receptor-deficient mice,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 17, no. 10, pp. 2250–2256, 1997.
- R. Burcelin, V. Crivelli, A. Dacosta, A. Roy-Tirelli, and B. Thorens, “Heterogeneous metabolic adaptation of C57BL/6J mice to high-fat diet,” American Journal of Physiology, vol. 282, no. 4, pp. E834–E842, 2002.
- N. Garg, S. Thakur, C. A. McMahan, and M. L. Adamo, “High fat diet induced insulin resistance and glucose intolerance are gender-specific in IGF-1R heterozygous mice,” Biochemical and Biophysical Research Communications, vol. 413, no. 3, pp. 476–480, 2011.
- Y. Macotela, J. Boucher, T. T. Tran, and C. R. Kahn, “Sex and depot differences in adipocyte insulin sensitivity and glucose,” Diabetes, vol. 58, no. 4, pp. 803–812, 2009.
- A. P. Burke, F. D. Kolodgie, A. Zieske et al., “Morphologic findings of coronary atherosclerotic plaques in diabetics: a postmortem study,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 24, no. 7, pp. 1266–1271, 2004.
- A. Venkateswaran, B. A. Laffitte, S. B. Joseph et al., “Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXRα,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 22, pp. 12097–12102, 2000.
- R. K. Tangirala, E. D. Bischoff, S. B. Joseph et al., “Identification of macrophage liver X receptors as inhibitors of atherosclerosis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 18, pp. 11896–11901, 2002.
- M. N. Bradley, C. Hong, M. Chen et al., “Ligand activation of LXR beta reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR alpha and apoE,” Journal of Clinical Investigation, vol. 117, no. 8, pp. 2337–2346, 2007.
- C. Hong, M. N. Bradley, X. Rong et al., “LXRalpha is uniquely required for maximal reverse cholesterol transport and atheroprotection in ApoE-deficient mice,” The Journal of Lipid Research, vol. 53, no. 6, pp. 1126–1133, 2012.
- X. Y. Dai, X. Ou, X. R. Hao et al., “The effect of T0901317 on ATP-binding cassette transporter A1 and niemann-pick type C1 in ApoE-/-mice,” Journal of Cardiovascular Pharmacology, vol. 51, no. 5, pp. 467–475, 2008.
- S. B. Joseph, E. McKilligin, L. Pei et al., “Synthetic LXR ligand inhibits the development of atherosclerosis in mice,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 11, pp. 7604–7609, 2002.
- N. Levin, E. D. Bischoff, C. L. Daige et al., “Macrophage liver X receptor is required for antiatherogenic activity of LXR agonists,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 25, no. 1, pp. 135–142, 2005.
- L. Verschuren, J. de Vries-van der Weij, S. Zadelaar, R. Kleemann, and T. Kooistra, “LXR agonist suppresses atherosclerotic lesion growth and promotes lesion regression in apoE*3Leiden mice: time course and mechanisms,” Journal of Lipid Research, vol. 50, no. 2, pp. 301–311, 2009.
- A. Castrillo, S. B. Joseph, C. Marathe, D. J. Mangelsdorf, and P. Tontonoz, “Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages,” Journal of Biological Chemistry, vol. 278, no. 12, pp. 10443–10449, 2003.
- S. B. Joseph, A. Castrillo, B. A. Laffitte, D. J. Mangelsdorf, and P. Tontonoz, “Reciprocal regulation of inflammation and lipid metabolism by liver X receptors,” Nature Medicine, vol. 9, no. 2, pp. 213–219, 2003.
- D. Ogawa, J. F. Stone, Y. Takata et al., “Liver x receptor agonists inhibit cytokine-induced osteopontin expression in macrophages through interference with activator protein-1 signaling pathways,” Circulation Research, vol. 96, no. 7, pp. e59–e67, 2005.
- R. J. Aiello, D. Brees, P. A. Bourassa et al., “Increased atherosclerosis in hyperlipidemic mice with inactivation of ABCA1 in macrophages,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 22, no. 4, pp. 630–637, 2002.
- S. Boudina and E. D. Abel, “Diabetic cardiomyopathy revisited,” Circulation, vol. 115, no. 25, pp. 3213–3223, 2007.
- I. G. Poornima, P. Parikh, and R. P. Shannon, “Diabetic cardiomyopathy: the search for a unifying hypothesis,” Circulation Research, vol. 98, no. 5, pp. 596–605, 2006.
- R. Basu, G. Y. Oudit, X. Wang et al., “Type 1 diabetic cardiomyopathy in the Akita (Ins2WT/C96Y) mouse model is characterized by lipotoxicity and diastolic dysfunction with preserved systolic function,” American Journal of Physiology, vol. 297, no. 6, pp. H2096–H2108, 2009.
- S. B. Gurley, S. E. Clare, K. P. Snow, A. Hu, T. W. Meyer, and T. M. Coffman, “Impact of genetic background on nephropathy in diabetic mice,” American Journal of Physiology, vol. 290, no. 1, pp. F214–F222, 2006.
- S. B. Gurley, C. L. Mach, J. Stegbauer et al., “Influence of genetic background on albuminuria and kidney injury in Ins2+/C96Y(Akita) mice,” American Journal of Physiology, vol. 298, no. 3, pp. F788–F795, 2010.
- C. S. Lo, F. Liu, Y. Shi, H. Maachi et al., “Dual RAS blockade normalizes angiotensin-converting enzyme-2 expression and prevents hypertension and tubular apoptosis in Akita angiotensinogen-transgenic mice,” American Journal of Physiology, vol. 302, no. 7, pp. F840–F852, 2012.
- H. Lu, L. A. Cassis, and A. Daugherty, “Atherosclerosis and arterial blood pressure in mice,” Current Drug Targets, vol. 8, no. 11, pp. 1181–1189, 2007.
- I. Gotsman, R. Gupta, and A. H. Lichtman, “The influence of the regulatory T lymphocytes on atherosclerosis,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 27, no. 12, pp. 2493–2495, 2007.
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