Lack of mitogen-activated protein kinase phosphatase-1 protects ApoE-null mice against atherosclerosis

Abstract

Rationale: Multiple protein kinases have been implicated in cardiovascular disease; however, little is known about the role of their counterparts: the protein phosphatases.

Objective: To test the hypothesis that mitogen-activated protein kinase phosphatase (MKP)-1 is actively involved in atherogenesis.

Methods and results: Mice with homozygous deficiency in MKP-1 (MKP-1(-/-)) were bred with apolipoprotein (Apo)E-deficient mice (ApoE(-/-)) and the 3 MKP-1 genotypes (MKP-1(+/+)/ApoE(-/-) ; MKP-1(+/-)/ApoE(-/-) and MKP-1(-/-)/ApoE(-/-)) were maintained on a normal chow diet for 16 weeks. The 3 groups of mice exhibited similar body weight and serum lipid profiles; however, both MKP-1(+/-) and MKP-1(-/-) mice had significantly less aortic root atherosclerotic lesion formation than MKP-1(+/+) mice. Less en face lesion was observed in 8-month-old MKP-1(-/-) mice. The reduction in atherosclerosis was accompanied by decreased plasma levels of interleukin-1alpha and tumor necrosis factor alpha, and preceded by increased antiinflammatory cytokine interleukin-10. In addition, MKP-1-null mice had higher levels of plasma stromal cell-derived factor-1a, which negatively correlated with atherosclerotic lesion size. Immunohistochemical analysis revealed that MKP-1 expression was enriched in macrophage-rich areas versus smooth muscle cell regions of the atheroma. Furthermore, macrophages isolated from MKP-1-null mice showed dramatic defects in their spreading/migration and impairment in extracellular signal-regulated kinase, but not c-Jun N-terminal kinase and p38, pathway activation. In line with this, MKP-1-null atheroma exhibited less macrophage content. Finally, transplantation of MKP-1-intact bone marrow into MKP-1-null mice fully rescued the wild-type atherosclerotic phenotype.

Conclusion: These findings demonstrate that chronic deficiency of MKP-1 leads to decreased atherosclerosis via mechanisms involving impaired macrophage migration and defective extracellular signal-regulated kinase signaling.

Figure 1. Effect of MKP-1 deficiency on metabolic characteristics of ApoE-null mice

Body (A, B) and heart (C, D) weights in male and female mice fed a normal chow diet for 16-week. Serum level of total cholesterol and its different components were determined as described in “METHODS” for male (E) and female (F) mice fed a normal diet for 16-week. Numbers in columns indicate the No. of mice in that group.

Figure 2. Effect of MKP-1 deficiency on aortic atherosclerosis formation in ApoE-null mice

Representative Oil red O staining of aortic sinus sections from 16-week-old female mice with indicated genotypes (A). Quantification of atherosclerotic lesion areas in aortic sinus from the 16-week-old male (B) and female (C) mice on the ApoE-null background with indicated MKP-1 genotypes. D, aortic sinus lesion of 8-month-old male mice. E, en face lesion area of entire aorta from 8-month-old male mice, n=7 mice in each group.

Figure 3. MKP-1 deficiency alters plasma levels of chemokines/cytokines in ApoE-null mice

The relative levels of 40 chemokines/cytokines were tested by a micro-array approach using plasma samples pooled from 3 female mice of each genotype (A). Plasma levels of SDF-1α were determined in male (B) and female (C) mice by ELISA assay. The relationship between plasma SDF-1α level and atherosclerotic lesion area was determined by a correlation analysis (D). The plasma levels of IL-1α (E) and TNFα (F) were determined by a Luminex bead-based multiplexing assay. Numbers in columns indicate the No. of mice in that group. Plasma levels of IL-10 (G) in ApoE-null mice of indicated ages were determined by ELISA, n=6 mice in each group. *, p<0.05 to respective controls.

Figure 3. MKP-1 deficiency alters plasma levels of chemokines/cytokines in ApoE-null mice

The relative levels of 40 chemokines/cytokines were tested by a micro-array approach using plasma samples pooled from 3 female mice of each genotype (A). Plasma levels of SDF-1α were determined in male (B) and female (C) mice by ELISA assay. The relationship between plasma SDF-1α level and atherosclerotic lesion area was determined by a correlation analysis (D). The plasma levels of IL-1α (E) and TNFα (F) were determined by a Luminex bead-based multiplexing assay. Numbers in columns indicate the No. of mice in that group. Plasma levels of IL-10 (G) in ApoE-null mice of indicated ages were determined by ELISA, n=6 mice in each group. *, p<0.05 to respective controls.

Figure 4. MKP-1 expression in atherosclerotic plaque and macrophages

Immuno-histochemical staining of MKP-1 protein in aortic sinus sections of representative 16-week-old ApoE-null mice. Macrophage-rich area was indicated by Mac-3 staining and SMC actin staining revealed smooth muscle-rich areas. The green arrows point to the localization of atheroma, MKP-1 protein and macrophage-rich versus smooth muscle-rich areas, respectively.

Figure 5. MKP-1 deficiency affects macrophage spreading and migration

Macrophage spreading capacity was observed by cell morphology after 2 d in culture (A). Macrophage migration in vitro in response to 10% FBS was determined using a Boyden chamber system (B); left panel are representative pictures of Boyden chamber membranes containing migrated cells (B). The number of thioglycollate-elicited macrophages in the peritoneum was counted and compared between the indicated groups of mice (C). Numbers in columns indicate the No. of mice in that group. *, p<0.05 to respective controls.

Figure 6. Macrophage content in atheroma of MKP-1-intact versus MKP-1-null mice

Macrophage accumulation in the aortic sinus of indicated genotype was examined by Immunohistochemical staining of Mac-3. Brown color in representative images (A) stands for Mac-3 positive macrophages. Relative macrophage numbers in the plaque areas of indicated mice (n=5 in each group) were quantified (B). *, p<0.05 vs. control.

Figure 7. MAPKs activation in macrophage of MKP-1-intact versus MKP-1-null mice with ApoE-null background

The time courses of ERK, JNK and p38 phosphorylation in response to serum were determined by western blotting in macrophages of female mice fed normal chow diet for 16-week. Data are expressed as fold increase over control after normalization to the respective controls. The experiment was repeated 3 times (3 mice in each genotype) with similar results (A). The effect of U0126 on migration capacity of MKP-1-intact versus MKP-1-null macrophages was determined using a Boyden chamber system (B). *, p<0.05 vs. respective controls.

Figure 8. Contribution of MKP-1 in bone marrow-derived cells to atherosclerotic lesion formation

Female MKP-1^+/+ /ApoE^−/− and MKP-1^−/− /ApoE^−/− mice were lethally irradiated at 6-week, and transplanted with donor bone marrow (MKP-1^+/+ /ApoE^−/−) as indicated. Mice were continuously fed with normal chow diet for additional 16 weeks after bone marrow transplantation, after which they were sacrificed for evaluation of atherosclerotic lesion size in the aortic sinus. Age-matched (22 weeks) female mice served as non-transplanted controls.