Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 2007;101:1104-1112
Published online before print September 20, 2007, doi: 10.1161/CIRCRESAHA.107.156075
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
101/11/1104    most recent
CIRCRESAHA.107.156075v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jagavelu, K.
Right arrow Articles by Bavendiek, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jagavelu, K.
Right arrow Articles by Bavendiek, U.
Related Collections
Right arrow Animal models of human disease
Right arrow Pathophysiology
(Circulation Research. 2007;101:1104.)
© 2007 American Heart Association, Inc.

Molecular Medicine

Systemic Deficiency of the MAP Kinase–Activated Protein Kinase 2 Reduces Atherosclerosis in Hypercholesterolemic Mice

Kumaravelu Jagavelu, Uwe J.F. Tietge, Matthias Gaestel, Helmut Drexler, Bernhard Schieffer*, Udo Bavendiek*

From the Department of Cardiology & Angiology (K.J., H.D., B.S., U.B.), Hannover Medical School, Germany; the Center for Liver, Digestive & Metabolic Diseases, Department of Pediatrics (U.J.F.T.), University Medical Center, Groningen, The Netherlands; and the Department of Biochemistry (M.G.), Hannover Medical School, Germany.

Correspondence to Udo Bavendiek, MD, Department of Cardiology & Angiology, Hannover Medical School, Carl-Neuberg-Str. 01, 30625 Hannover, Germany. E-mail Bavendiek.Udo{at}mh-hannover.de


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Atherosclerosis is a chronic inflammatory disease and represents the major cause of cardiovascular morbidity and mortality. A critical regulator of inflammatory processes represents the mitogen-activated protein kinase–activated protein kinase-2 (MK2). Therefore, we investigated the functional role of MK2 in atherogenesis in hypercholesterolemic mice as well as potentially underlying mechanisms in vivo and in vitro. Activation of MK2 (phospho-MK2) was predominantly detected in the endothelium and macrophage-rich plaque areas within aortas of hypercholesterolemic LDL receptor–deficient mice (ldlr–/–). Systemic MK2 deficiency of hypercholesterolemic ldlr–/– mice (ldlr–/–/mk2–/–) significantly decreased the accumulation of lipids and macrophages in the aorta after feeding an atherogenic diet for 8 and 16 weeks despite a significant increase in proatherogenic plasma lipoproteins compared with ldlr–/– mice. Deficiency of MK2 significantly decreased oxLDL-induced foam cell formation in vitro, diet-induced foam cell formation in vivo, and expression of scavenger receptor A in primary macrophages. In addition, systemic MK2 deficiency of hypercholesterolemic ldlr–/– mice significantly decreased the aortic expression of the adhesion molecule VCAM-1 and the chemokine MCP-1, key mediators of macrophage recruitment into the vessel wall. Furthermore, silencing of MK2 in endothelial cells by siRNA reduced the IL-1ß–induced expression of VCAM-1 and MCP-1. MK2 critically promotes atherogenesis by fostering foam cell formation and recruitment of monocytes/macrophages into the vessel wall. Therefore, MK2 might represent an attractive novel target for the treatment of atherosclerotic cardiovascular disease.


Key Words: atherosclerosis • MK2 • hypercholesterolemic mice


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Atherosclerosis is a chronic inflammatory disease.1–5 This process is initiated by trapping and modification of apo-B-containing lipoproteins into the vessel wall resulting in increased expression of adhesion molecules (eg, VCAM-1) and chemokines (eg, MCP-1) by the endothelium. The latter is followed by recruitment of monocytes into the vessel wall, where these cells differentiate into macrophages. Inflammatory macrophages produce a plethora of inflammatory mediators (eg, chemokines, cytokines) sustaining and promoting the inflammatory process. Furthermore, macrophages develop into foam cells by unrestricted uptake of modified lipoproteins (eg, oxLDL) through scavenger receptors (SR-A, CD36).1–8

The mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK-2, MK2) is a direct substrate of the stress activated MAPK p38{alpha} and ß.9 Recent studies performed with MK2-deficient cells in vitro demonstrate a central role of MK2 in the production of proinflammatory mediators such as TNF{alpha}, IL-1ß, MIP-1{alpha}, IL-8, IL-6, and INF{gamma}.10–13 One important mechanism MK2 uses to increase the expression of proinflammatory mediators is the modulation of mRNA-stability by targeting AU-rich elements located in the 3'-untranslated region of the mRNA of proinflammatory mediators (e. g. TNF{alpha}, IL-6) via phosphorylation of RNA-stability regulating proteins (eg, tristetraprolin).11,12,14 Furthermore, MK2 possesses an important functional role in cell migration and chemotaxis by regulating actin remodeling and stress fiber formation via phosphorylation of Hsp27 and activation of the LIM-kinase-cofilin pathway.15–17 Besides these in vitro data the important functional role of MK2 for inflammatory processes was most clearly revealed in MK2-deficient mice used in in vivo models of inflammatory diseases: MK2-deficient mice show a significantly reduced production of TNF{alpha} and IL-6 in response to LPS-challenge resulting in increased survival of LPS-induced septic shock and exhibit significantly reduced severity and incidence of joint inflammation in the collagen induced-arthritis model.10,18 As similar inflammatory pathways are also known to be involved in atherogenesis, MK2 appears to be an attractive and potentially selective target for the treatment of atherosclerosis. Importantly, MK2-deficient mice do not develop a lethal phenotype as well as signs of generalized inflammation per se.10 Therefore, serious side effects should not be expected after blocking MK2 in vivo, which is in contrast to mice deficient for its upstream kinase p38{alpha}/ß-MAPK, that develop an embryonically lethal phenotype.19,20 Therefore, the present study investigated the functional role of MK2 in atherogenesis in hypercholesterolemic mice in vivo as well as potentially underlying molecular mechanisms in MK2-deficient cells in vitro.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Treatment of Mice
LDLR-deficient mice (C57Bl/6, The Jackson Laboratories, Bar Harbor, Me) and MK2-deficient mice (C57Bl/6, backcrossed >10 generations)10 were crossbred to generate ldlr–/–/mk2–/– compound mutant mice. The genotype of each mouse was verified by PCR on genomic DNA (tail tip digest) using the following primers: LDLR: 5'- ACC CCA AGA CGT GCT CCC AGG ATG A -3' (sense), 5' – CGC AGT GCT CCT CAT CTG ACT TGT – 3' (antisense); MK2: 5' – CGT GGG GGT GGG GTG ACA TGC TGG TTG AC –3' (sense), 3' – GGT GTC ACC TTG ACA TCC CGG TGA G –3' (antisense); Neomycin-Cassette: 5' – TGC TCG CTC GAT GCG ATG TTT CGC –3' (sense). 8- to 10-week-old male ldlr–/– and ldlr–/–/mk2–/–compound-mutant mice (each group n=8 to 10) consumed a high-cholesterol diet (product #D12108, Research Diets; 1.25% Cholesterol without added cholate) for 8 and 16 weeks. Subsequently, mice were euthanized and the aortas were removed and analyzed as described below. Aortic arches were frozen in OCT compound (Tissue-Tek) and the thoracic and abdominal parts of the aortas were fixed in 10% buffered formalin, as described previously.21 All mice were housed under specific pathogen-free conditions and all procedures were approved by the Institutional Animal Care and Use Committee at Hannover Medical School and the Bezirksregierung Niedersachsen.

Immunohistochemistry/Immunofluorescence
Serial cryostat sections (6 µm) of mouse aortic arches were fixed in acetone (–20°C, 5 minutes), air dried, and stained by the avidin-biotin-peroxidase method, as previously described.21 Controls for specificity used staining with the respective nonimmune IgG subclass (Pharmingen, DAKO). For immunfluorescence serial cryo-sections (6 µm) of mouse aortic arches were fixed in paraformaldehyde (4°C, 15 minutes) and permeabilized with 0.1% Triton X-100 for 5 minutes. Sections were incubated with primary antibodies overnight at 4°C followed by incubation with a fluorescence dye–conjugated secondary antibody (Molecular Probes/Invitrogen). Nuclei were stained with 4'-6-diamidino-2-phenyl indole-2HCl (DAPI, Santa Cruz Biotechnologies). Control stainings with the respective nonimmune IgG control demonstrated specificity of the anti–phospho-MK2 signal (supplemental Figure I, available online at http://circres.ahajournals.org). Antibodies used were: anti-mouse MOMA-2 (Pharmingen), anti–VCAM-1 (Pharmingen), anti–ICAM-1 (Pharmingen), anti–phopho-MK2 (Cell Signaling Technologies), anti-vWF (DAKO).

Oil Red O Staining for Lipids
Deposition of lipids in en face preparations of the Aorta descendens (fixed with 10% formalin) and cryo-sections of the aortic arch was determined by oil red O staining as described previously.21

Tissue Analysis
To quantify the extent and composition of the aortic lesions longitudinal sections of the aortic arch were analyzed microscopically in all mice, as described previously.21 In the aortic arch, a 2-mm proximal segment of the inner curvature, starting at a perpendicular dropped from the left side of the left subclavian artery origin, was analyzed for the total wall area. The respective areas positively stained for lipids (oil red O), macrophages (MOMA-2), VCAM-1, and ICAM-1 were determined via computer-assisted image quantification (Leica Microscope DM 4000B, Leica Qwin software, Leica). En face analysis of lipid depositions of the pinned Aorta descendens used measurement of percentage of surface area (20 mm from the iliac bifurcation to the thoracic section of the aorta) stained by oil red O using computer-assisted image analysis (Axiovert 200 microscope & Axioversion Rel. 4.4 software, Zeiss).

For other materials and methods, please refer to the supplemental materials and methods section (available online at http://circres.ahajournals.org).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Phosphorylated MK2 Is Localized to the Endothelium and Macrophage-Rich Plaque Areas Within Aortas of Hypercholesterolemic ldlr–/– Mice
The phosphorylation of MK2 by the p38-MAPK is essential for its activation.22 Therefore, we investigated the localization of phosphorylated MK2 in atherosclerotic lesions by performing immunfluorescence-staining in sections of the aortic arch of hypercholesterolemic ldlr–/– mice using specific antibodies against phospho-MK2. Immunfluorescence staining revealed a strong signal of phopho-MK2 in macrophage-rich areas of advanced atherosclerotic lesions (Figure 1A and 1B). Furthermore, strong phospho-MK2 staining could be detected in the endothelial cell layer of early as well as advanced atherosclerotic lesions (Figure 1B through 1E). In contrast, there was no significant phospho-MK2 staining in the smooth muscle cell-rich region of the vascular media (Figure 1B and 1E).


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		Figure 1. Phosphorylated MK2 is localized to the endothelium and macrophage-rich plaque areas within aortas of hypercholesterolemic ldlr–/– mice. Frozen sections of aortic arches of hypercholesterolemic ldlr–/– mice were immunfluorescence-stained in situ with a specific anti–phospho-MK2 antibody and surface markers specific for endothelial cells (vWF) and macrophages (MOMA-2). Positive staining for phopho-MK2 (B and E, green) can be detected in macrophage-rich areas (A, green, MOMA-2) of advanced atherosclerotic lesions (A and B) as well as in the endothelial cell layer (C and D, red, vWF) of early (D through F) and advanced (B and C) atherosclerotic lesions of hypercholesterolemic mice. E, double-immunfluorescence-staining for phospho-MK2 and vWF (yellow). A through E, nuclear staining (blue, DAPI). Magnifications of images are indicated (x20, x40). Analysis of sections obtained from 4 different mice showed similar results.

MK2 Deficiency Inhibits Atherosclerosis by Reducing Vascular Lipid Deposition and Macrophage Content in Hypercholesterolemic ldlr–/– Mice
The functional role of MK2 in atherosclerosis was investigated by comparing the development of atherosclerosis in MK2-deficient (ldlr–/–/mk2–/–) and not MK2-deficient (ldlr–/–) hyperercholesterolemic LDL receptor–deficient mice. Atherosclerosis was markedly reduced in ldlr–/–/mk2–/– mice compared with ldlr–/– mice after feeding an atherogenic diet for 8 or 16 weeks as indicated by a significantly reduced lipid-positive area (oil red O) in the aorta descendens (en face, Figure 2A and Table 1) and the aortic arch (longitudinal sections, Figure 2B and Table 1). In addition, ldlr–/–/mk2–/– mice had a significantly decreased macrophage content (MOMA-2) in longitudinal sections of the aortic arch (Figure 2C and Table 1). Strikingly, the reduced lipid deposition and macrophage content observed in the aortas of hypercholesterolemic ldlr–/–/mk2–/– mice occurred despite significantly increased plasma levels of total cholesterol and non–HDL-cholesterol compared with ldlr–/– mice (Table 2 and supplemental Figure II).


Figure 2
View larger version (38K):
[in this window]
[in a new window]

  		Figure 2. Systemic deficiency of MK2 significantly reduces atherosclerosis in hypercholesterolemic ldlr–/– mice. A, ldlr–/– and ldlr–/–/mk2–/– were fed a high-cholesterol diet for 8 and 16 weeks (8 to 10 animals per group), and deposition of lipids in the aorta descendens was analyzed en face by oil red O staining. Quantitative analysis of lipid positive–stained area given as the percentage of the total analyzed area of the aorta descendens is displayed as mean±SEM on the left and a representative staining of aortas (oil red O, 16 weeks atherogenic diet) is shown on the right. *P<0.05 and #P<0.01 ldlr–/– vs ldlr–/–/mk2–/–. B and C, ldlr–/– and ldlr–/–/ mk2–/– were fed a high-cholesterol diet for 8 and 16 weeks (8 to 10 animals per group) and the content of lipids (B, oil red O) and macrophages (C, MOMA-2) was analyzed in longitudinal sections of the aortic arch. Quantitative analysis of the positive stained area given as the percentage of the total analyzed area of the aortic arch is displayed as mean±SEM on the left and a representative staining of longitudinal sections of the aortic arch (oil red O, MOMA-2) is shown on the right. *P<0.05, #P<0.01, and §P<0.001 ldlr–/– vs ldlr–/–/mk2–/–.


View this table:
[in this window]
[in a new window]

  		Table 1. Table 1. Aortic Lipid Deposition and Macrophage Content in Hypercholesterolemic ldlr–/– and ldlr–/–/mk2–/– Mice


View this table:
[in this window]
[in a new window]

  		Table 2. Table 2. Plasma Lipids and Lipoproteins in Hypercholesterolemic ldlr–/– and ldlr–/–/mk2–/– Mice

MK2-Deficiency Reduces Macrophage Foam Cell Formation and Scavenger Receptor A Expression
One possibility to explain reduced atherosclerosis observed in ldlr–/–/mk2–/– mice in the face of a proatherogenic lipid profile could be a functional role of MK2 in macrophage foam cell formation. Therefore, we investigated the oxLDL-induced formation of foam cells in murine peritoneal macrophages isolated from MK2-deficient mice (mk2–/–) or wild-type control mice (wt). Deficiency of MK2 in murine macrophages caused a significantly reduced formation of oil red O–positive foam cells (Figure 3A and 3B, wt versus mk2–/–: 64.14±2.09% versus 31.92±1.78%, P<0.05). This was furthermore supported by a significantly reduced uptake of Dil-labeled oxLDL by MK2-deficient macrophages compared with wild-type controls (Figure 3D, relative Dil-oxLDL-uptake wt versus mk2–/–: 1.00±0.03 versus 0.57±0.02, P<0.01). Additionally, we used an in vivo model of foam cell formation as described by Li et al23 to verify a functional role of MK2 in foam cell formation in vivo. In line with the impact of MK2-deficiency on foam cell formation in vitro, MK2 deficiency caused a significant reduction of oil red O–positive foam cells in preparations of peritoneal macrophages isolated from hypercholesterolemic ldlr–/– mice fed an atherogenic diet for 10 weeks compared with wild-type controls (Figure 3C, ldlr–/– versus ldlr–/–/mk2–/–: 52.92±8.28% versus 20.44±3.09%, P<0.05). Furthermore, the functional role of MK2 in cholesterol efflux from foam cells was investigated in oxLDL-loaded peritoneal macrophages isolated from wild-type control or MK2-deficient mice. Cholesterol efflux assays revealed no significant difference in cholesterol efflux towards HDL between oxLDL-loaded wild-type control and MK2-deficient macrophages (percentage of released 3H-cholesterol to total 3H-cholesterol: wt versus mk2–/–: 4.43±0.52% versus 5.67±0.45%, P>0.05, n=3). As the impaired foam cell formation in MK2-deficient macrophages could be caused by reduced expression of oxLDL scavenging receptors (CD36, SR-A), we determined mRNA and protein expression of SR-A and CD36 by quantitative real-time PCR and Western blotting, respectively. Analysis of the expression of SR-A and CD36 revealed a markedly reduced mRNA and protein expression of the scavenger receptor SR-A in MK2-deficient macrophages compared with wild-type macrophages, whereas no difference in the protein expression levels for CD36 was detected between wild-type and MK2-deficient macrophages (Figure 3E and 3F; relative mRNA expression wt versus mk2–/–: SR-A 1.00±0.08 versus 0.31±0.11, P<0.001; CD36 1.00±0.13 versus 0.41±0.10, P<0.05).


Figure 3
View larger version (35K):
[in this window]
[in a new window]

  		Figure 3. MK2-deficiency in macrophages reduces foam cell formation in vitro and in vivo and expression of scavenger receptor A. A through C, Cultured primary murine peritoneal macrophages were harvested from wt and mk2–/– mice and incubated with oxLDL (25 µg/mL, 4 hour, 37°C) or harvested from ldlr–/– and ldlr–/–/mk2–/– mice after feeding an atherogenic diet (1.25% Cholesterol) for 10 weeks and foam cell formation in vitro (A) and in vivo (C), respectively, was analyzed after staining cellular lipids with oil red O. Quantitative analysis of lipid-positive cells given as percentage of the total number of analyzed cells is displayed as mean ± SEM (A, C) and representative images of oxLDL-treated macrophages stained with oil red O are shown (B). n=3, *P<0.05 wt vs mk2–/– and ldlr–/– versus ldlr–/–/mk2–/–. D, Cultured primary murine peritoneal macrophages harvested from wild-type (wt) and mk2–/– mice were incubated with Dil-oxLDL (10 µg/mL, 4 hours, 37°C) and oxLDL-uptake was analyzed with a fluorescence-photometer at excitation/emission wavelength of 520/580 nm and normalized to total cellular protein content. OxLDL-uptake is displayed as mean±SEM given relative to wild-type. n=4, #P<0.01 wt vs mk2–/–. E and F, Primary murine peritoneal macrophages isolated from wt and mk2–/– mice were cultured and used for isolation of total RNA or protein lysates. mRNA and protein expression of scavenger receptors (SR-A, CD36) and housekeeping genes (18S, GAPDH) were determined by quantitative RT-PCR (E) and Western blotting (F) using specific primers and antibodies for SR-A, CD36, 18S, and GAPDH. Relative gene expression of SR-A and CD36 as determined by qRT-PCR is displayed as mean±SEM, n=4, *P<0.05, §P<0.001 (E). Representative Western blots (F) of 4 different preparations of peritoneal murine macrophages are shown.

MK2-Deficiency Decreases the Expression of VCAM-1 and MCP-1 in Aortas of Hypercholesterolemic Mice and Endothelial Cells
Based on the strong reduction of the macrophage content in atherosclerotic vessels of ldlr–/–/mk2–/– mice, especially in early stages of atherosclerosis (8 weeks high-cholesterol diet), MK2 could conceivably play a causal role in the recruitment of monocytes/macrophages into the atherosclerotic vessel wall. As adhesion molecules such as E-selectin, VCAM-1, ICAM-1, and the chemokine MCP-1 are key mediators in this process,1,2,4–6 we investigated the vascular expression of these mediatores in aortas isolated from ldlr–/– and ldlr–/–/mk2–/– mice fed an atherogenic diet for 8 weeks using quantitative RT-PCR. mRNA expression of E-selectin, VCAM-1, ICAM-1, and MCP-1 was significantly reduced in ldlr–/–/mk2–/– mice compared with ldlr–/– mice (Figure 4A, relative expression ldlr–/– versus ldlr–/–/mk2–/–; E-selectin: 1.00±0.12 versus 0.57±0.07, VCAM-1: 1.00±0.06 versus 0.43±0.06, ICAM-1: 1.00±0.04 versus 0.59±0.05, MCP-1: 1.00±0.17 versus 0.47±0.10, all P<0.05). Furthermore, we investigated the vascular expression of E-selectin, VCAM-1, ICAM-1, and MCP-1 in sections of the aortic arch of these mice by immunhistochemistry using specific antibodies. Whereas specific staining could be obtained for VCAM-1 and ICAM-1, specific staining could not be obtained in aortic sections of hypercholesterolemic ldlr–/– mice using different antibodies available for MCP-1 and E-selectin. In line, a previous report demonstrated lack of detection of E-selectin expression by immunhistochemistry in atherosclerotic murine aortas, although E-selectin expression was detectable by RT-PCR.24 Aortic arches of hypercholesterolemic ldlr–/–/mk2–/– mice revealed a significant reduced area positively stained for VCAM-1 compared with aortic arches of hypercholesterolemic ldlr–/– mice, whereas the area positively stained for ICAM-1 was not significantly different between these genotypes (Figure 4B, % positive stained area ldlr–/– versus ldlr–/–/mk2–/–, VCAM-1: 8.76±1.63% versus 4.46±0.58%, P<0.05, ICAM-1: 7.24±0.49% versus 6.22± 0.53%, P>0.05.


Figure 4
View larger version (31K):
[in this window]
[in a new window]

  		Figure 4. Vascular expression of E-selectin, VCAM-1, ICAM-1, and MCP-1 in hypercholesterolemic ldlr–/– and ldlr–/–/mk2–/– mice. A, Aortas harvested from ldlr–/– and ldlr–/–/mk2–/– mice after feeding an atherogenic diet (1.25% Cholesterol) for 8 weeks were used for isolation of total RNA. mRNA expression of E-selectin, VCAM-1, ICAM-1, MCP-1, and housekeeping genes (18S, GAPDH) was determined by quantitative RT-PCR using specific primers. Relative gene expression is displayed as mean±SEM. n=5, *P<0.05. B, ldlr–/– and ldlr–/–/mk2–/– were fed a high-cholesterol diet for 8 weeks (9 to 10 animals per group) and the expression of VCAM-1 and ICAM-1 was analyzed by immunhistochemistry in longitudinal sections of the aortic arch using specific antibodies. Quantitative analysis of the positive stained area given as the percentage of the totally analyzed area of the aortic arch is displayed as mean±SEM on the left and representative stainings of longitudinal sections of the aortic arch (VCAM-1, ICAM-1) are shown on the right. n=9 to 10, *P<0.05 ldlr–/– vs ldlr–/–/mk2–/–.

In addition, we investigated, whether the endothelial protein expression of VCAM-1 and MCP-1 is decreased by inhibition of MK2 expression in vitro. Using siRNA, protein expression of MK2 in human endothelial cells (HUVECs) was substantially reduced as determined by Western blotting (Figure 5A). In line, the IL-1ß–induced phosphorylation of Hsp27, a major downstream target of MK2, was markedly reduced after silencing the expression of MK2 in HUVECs (Figure 5B). After successful inhibition of the expression and function of MK2 in HUVECs, the IL-1ß–induced expression of VCAM-1 (Figure 5C) and MCP-1 (Figure 5D, control-siRNA versus MK2-siRNA, IL-1ß: 108.09±6.56 versus 67.74±6.91 ng/mL, P<0.01) was markedly and significantly reduced in endothelial cells, demonstrating a functional role of MK2 for the expression of VCAM-1 and MCP-1 in endothelial cells. These results indicate that the reduced accumulation of macrophages in atherosclerotic vessels of MK2-deficient mice might be caused by reduced expression of endothelial VCAM-1 and MCP-1.


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		Figure 5. MK2 knockdown decreased expression of VCAM-1 and MCP-1 by endothelial cells. A, Cultured endothelial cells (HUVECs) were transfected with control-siRNA (C) and MK2-specific siRNA (MK2). Silencing of protein expression of MK2 was confirmed in cell lysates harvested at day 3 post transfectionem by Western blotting using a MK2-specific antibody. B, HUVECs transfected with control- and MK2-specific siRNA were incubated 3 days post transfectionem with IL-1ß (1 ng/mL) for the indicated times and phophorylation of the MK2-downstream target Hsp27 was analyzed in cell lysates by Western blotting using a phospo-Hsp27 specific antibody. Representative Western blots of 3 independent experiments using 3 different donors (HUVECs) are shown. C and D, HUVECs transfected with control- and MK2-specific siRNA were incubated 3 days post transfectionem with IL-1ß (1 ng/mL, 24 hours). Then protein expression of VCAM-1 was analyzed in cell lysates by Western blotting (C), and release of MCP-1 into cell culture supernatants was determined by ELISA (D). Representative Western blots (C) of 3 independent experiments using 3 different donors (HUVECs) are shown. Release of MCP-1 (D) is displayed as mean±SEM. n=5, *P<0.01 control-siRNA vs MK2-siRNA.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
MK2 is an important regulator of inflammatory processes.15,16 Because atherosclerosis is considered a chronic inflammatory disease,1–5 we hypothesized that MK2 might also play a central role in atherogenesis. The present study provides evidence for a causal involvement of MK2 in atherosclerosis on different levels of the disease process, namely increased macrophage foam cell formation and increased expression of mediators that are crucial for the recruitment of monocytes/macrophages into the vascular wall.

Initially performed experiments detected phosphorylated MK2 (indicating the active form of the enzyme22) within the endothelial cell layer as well as advanced macrophage-rich atherosclerotic lesions, however, not in the smooth muscle cell-rich media of hypercholesterolemic mice. These data pointed to a role of MK2 in atherosclerosis, a process primarily initiated in endothelial cells and monocytes/macrophages. To further elucidate the involvement of MK2 in atherogenesis, the impact of MK2 deficiency was investigated in hypercholesterolemic ldlr–/– mice. ldlr–/–/mk2–/– mice fed an atherogenic diet developed—for yet unknown reasons—a significant increase in apoB-containing lipoproteins without changes in HDL-cholesterol compared with controls. Strikingly, despite this clearly proatherogenic lipid profile ldlr–/–/mk2–/– mice had a substantial decrease in early (8 weeks) as well as late (16 weeks) aortic atherosclerosis. MK2 deficiency apparently had a dual effect causing a significant reduction ({approx}50% to 70%) in aortic lipid-depositions as well as macrophage infiltration.

The substantial reduction of lipid depositions we observed in the aorta of hypercholesterolemic MK2-deficient mice—especially in advanced atherosclerotic disease (16 weeks atherogenic diet)—is most likely attributable to an impaired uptake of modified atherogenic lipoproteins25,26 by MK2-deficient macrophages in vivo based on our data: MK2 deficiency reduced oxLDL-induced as well as diet-induced foam cell formation of macrophages in vitro and in vivo, respectively, whereas cholesterol efflux towards HDL of oxLDL-loaded macrophages seems not to be affected by MK2 deficiency. The underlying mechanistic basis of decreased macrophage oxLDL uptake by MK2-deficient macrophages is probably a markedly decreased expression of scavenger receptor A. This mechanism apparently even overrides the impact of the proatherogenic plasma lipoprotein profile observed in hypercholesterolemic MK2-deficient mice.

Interestingly, the phenotype of hypercholesterolemic MK2-deficient mice remarkably resembles the phenotype of hypercholesterolemic SR-A–deficient mice. Similarly to ldlr–/–/mk2–/– mice apoE–/–/sra–/– mice developed {approx}40% higher plasma cholesterol concentrations compared with hypercholesterolemic control mice.27,28 Despite this increased plasma concentrations of proatherogenic lipoproteins, which have been shown to positively correlate with atherosclerotic lesion formation in murine models of atherosclerosis,29,30 apoE–/–/sra–/– mice like ldlr–/–/mk2–/– mice did not show a therefore expected increase in atherosclerosis but a decrease or no change in atherosclerosis compared with hypercholesterolemic control mice.27,28 Furthermore, SR-A–deficient macrophages accumulated {approx}50% less cholesterol after exposition to modified LDL-cholesterol in vitro or in vivo compared with control macrophages27,28 similarly to the experiments performed in this study employing MK2-deficient peritoneal macrophages. Therefore, the resembling phenotype of MK2-deficient and SR-A–deficient mice/macrophages observed in our experiments and previous studies27,28 supports our data indicating a functional role of MK2 for vascular foam cell formation via regulation of SR-A expression.

Recent work has shown that TLR2/4-dependent regulation of SR-A expression is abolished after inhibition of p38-MAPK,31 the upstream kinase of MK2, suggesting a MK2-dependent regulation of SR-A. However, although MK2 is known to regulate the expression of inflammatory mediators (e. g. TNF{alpha}, IL-6) by increasing mRNA stability via targeting AU-rich elements of 3'-untranslated regions,11,12,14 expression of SR-A seems not to be primarily regulated by this mechanism as transcript stability was only modestly increased in murine macrophages after increasing mRNA-expression by stimulation with LPS.32 Therefore, further studies are mandatory to identify the molecular mechanism of MK2-dependent regulation of SR-A expression.

In addition to the reduced vascular deposition of lipids, infiltration of macrophages was strongly reduced ({approx}70%) in ldlr–/–/mk2–/– mice especially in early atherosclerotic lesions, indicating that MK2 regulates processes important for the recruitment of monocytes/macrophages into the diseased vessel wall. Therefore, our study also aimed at identifying potentially underlying processes regulated by MK2 that are crucial for macrophage recruitment into the vessel wall. We demonstrate that MK2 deficiency in hypercholesterolemic ldlr–/– mice in vivo and MK2 knockdown in endothelial cells by siRNA in vitro significantly decreased the respective aortic and endothelial cell expression of VCAM-1 and MCP-1, key mediators of macrophage recruitment into the vessel wall.33,34 In addition, our data are supported by a recent study demonstrating a reduced pulmonal expression of VCAM-1 and MCP-1 in MK2-deficient mice in an airway inflammation model.35 As recent studies have shown that MK2 regulates expression of inflammatory mediators (eg, TNF{alpha}, IL-6) by increasing transcript stability via targeting AU-rich elements located in the 3'-untranslated region,11,12,14 the presence of AU-rich elements located in the 3'-untranslated region of the VCAM-1 and MCP-1 coding genes36 would be consistent with a direct impact of MK2 on VCAM-1 and MCP-1 expression in endothelial cells. Conceivably, reduced recruitment of monocytes/macrophages into the diseased vessel wall would also result in a reduction of the number of macrophages prone to develop into foam cells. This mechanism would be additive to the reduced modified lipid uptake per se observed in this study resulting as a net effect in a significantly decreased vascular deposition of lipids in MK2-deficient mice.

Based on the present results we conclude that MK2 is importantly involved in atherogenesis by promoting the recruitment of inflammatory monocytes/macrophages through increasing endothelial expression of VCAM-1 and MCP-1. In addition, our experiments show that MK2 promotes the formation of foam cells by affecting the uptake of oxLDL via regulating SR-A expression (Figure 6). Importantly, MK2-deficient mice do not develop a lethal phenotype as well as generalized inflammatory disease per se and are even protected against LPS-induced endotoxic shock.10 In contrast, deficiency of its upstream mediator p38{alpha}/ß-MAPK by genetic disruption causes an embryonically lethal phenotype.19,20 Consequently, MK2 seems to be a more selective target for the inhibition of processes important for atherogenesis compared with p38-MAPK and hence the risk for serious side effects after pharmacological inhibition of MK2 in vivo by specific inhibitors to be developed might be low. Therefore, based on our results, MK2 might represent an attractive novel target for the treatment of atherosclerotic cardiovascular disease.


Figure 6
View larger version (13K):
[in this window]
[in a new window]

  		Figure 6. Proposed model of the functional role of MK2 in atherogenesis. MK2 promotes expression of VCAM-1 and MCP-1 in endothelial cells and uptake of modified lipoproteins (eg, oxLDL) by enhancing expression of scavenger receptor A in macrophages fostering vascular recruitment of monocytes/ macrophages, foam cell formation, and consequently atherosclerosis.


*    Acknowledgments
 
We thank Sandra Witting and Tatjana Yakowlava for skillful technical assistance. We also express our thanks to the MD/PhD program "Molecular Medicine" of the Hannover Medical School.

Sources of Funding

This work was supported by the Hannover Medical School grant HILF-I (to U.B.), by the Deutsche Forschungsgemeinschaft grant SFB 566/B9 (to H.D. and B.S.) and grant BA 1997/3-1 (to U.B.) and by the Netherlands Organization for Scientific Research grant (NOW) VIDI grant 917-56-358 (to U.J.F.T.).

Disclosures

None.


*    Footnotes
 
*Both authors share senior authorship. Back

Original received May 13, 2007; revision received August 20, 2007; accepted September 12, 2007.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999; 340: 115–126.[Free Full Text]
  2. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.[CrossRef][Medline] [Order article via Infotrieve]
  3. Steinberg D. Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime. Nat Med. 2002; 8: 1211–1217.[CrossRef][Medline] [Order article via Infotrieve]
  4. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685–1695.[Free Full Text]
  5. Tedgui A, Mallat Z. Cytokines in atherosclerosis: pathogenic and regulatory pathways. Physiol Rev. 2006; 86: 515–581.[Abstract/Free Full Text]
  6. Glass CK, Witztum JL. Atherosclerosis. the road ahead. Cell. 2001; 104: 503–516.[CrossRef][Medline] [Order article via Infotrieve]
  7. Williams KJ, Tabas I. The response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol. 1995; 15: 551–561.[Free Full Text]
  8. Lusis AJ. Atherosclerosis Nature. 2000; 407: 233–241.
  9. Stokoe D, Campbell DG, Nakielny S, Hidaka H, Leevers SJ, Marshall C, Cohen P. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase. Embo J. 1992; 11: 3985–3994.[Medline] [Order article via Infotrieve]
  10. Kotlyarov A, Neininger A, Schubert C, Eckert R, Birchmeier C, Volk HD, Gaestel M. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat Cell Biol. 1999; 1: 94–97.[CrossRef][Medline] [Order article via Infotrieve]
  11. Neininger A, Kontoyiannis D, Kotlyarov A, Winzen R, Eckert R, Volk HD, Holtmann H, Kollias G, Gaestel M. MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post-transcriptional levels. J Biol Chem. 2002; 277: 3065–3068.[Abstract/Free Full Text]
  12. Hitti E, Iakovleva T, Brook M, Deppenmeier S, Gruber AD, Radzioch D, Clark AR, Blackshear PJ, Kotlyarov A, Gaestel M. Mitogen-activated protein kinase-activated protein kinase 2 regulates tumor necrosis factor mRNA stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/uridine-rich element. Mol Cell Biol. 2006; 26: 2399–2407.[Abstract/Free Full Text]
  13. Culbert AA, Skaper SD, Howlett DR, Evans NA, Facci L, Soden PE, Seymour ZM, Guillot F, Gaestel M, Richardson JC. MAPK-activated protein kinase 2 deficiency in microglia inhibits pro-inflammatory mediator release and resultant neurotoxicity. Relevance to neuroinflammation in a transgenic mouse model of Alzheimer disease. J Biol Chem. 2006; 281: 23658–23667.[Abstract/Free Full Text]
  14. Winzen R, Kracht M, Ritter B, Wilhelm A, Chen CY, Shyu AB, Muller M, Gaestel M, Resch K, Holtmann H. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. Embo J. 1999; 18: 4969–4980.[CrossRef][Medline] [Order article via Infotrieve]
  15. Kotlyarov A, Gaestel M. Is MK2 (mitogen-activated protein kinase-activated protein kinase 2) the key for understanding post-transcriptional regulation of gene expression? Biochem Soc Trans. 2002; 30: 959–963.[CrossRef][Medline] [Order article via Infotrieve]
  16. Gaestel M. MAPKAP kinases - MKs - two’s company, three’s a crowd. Nat Rev Mol Cell Biol. 2006; 7: 120–130.[CrossRef][Medline] [Order article via Infotrieve]
  17. Kobayashi M, Nishita M, Mishima T, Ohashi K, Mizuno K. MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration. Embo J. 2006; 25: 713–726.[CrossRef][Medline] [Order article via Infotrieve]
  18. Hegen M, Gaestel M, Nickerson-Nutter CL, Lin LL, Telliez JB. MAPKAP kinase 2-deficient mice are resistant to collagen-induced arthritis. J Immunol. 2006; 177: 1913–1917.[Abstract/Free Full Text]
  19. Adams RH, Porras A, Alonso G, Jones M, Vintersten K, Panelli S, Valladares A, Perez L, Klein R, Nebreda AR. Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development. Mol Cell. 2000; 6: 109–116.[CrossRef][Medline] [Order article via Infotrieve]
  20. Tamura K, Sudo T, Senftleben U, Dadak AM, Johnson R, Karin M. Requirement for p38alpha in erythropoietin expression: a role for stress kinases in erythropoiesis. Cell. 2000; 102: 221–231.[CrossRef][Medline] [Order article via Infotrieve]
  21. Bavendiek U, Zirlik A, LaClair S, MacFarlane L, Libby P, Schonbeck U. Atherogenesis in mice does not require CD40 ligand from bone marrow-derived cells. Arterioscler Thromb Vasc Biol. 2005; 25: 1244–1249.[Abstract/Free Full Text]
  22. Ben-Levy R, Leighton IA, Doza YN, Attwood P, Morrice N, Marshall CJ, Cohen P. Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. Embo J. 1995; 14: 5920–5930.[Medline] [Order article via Infotrieve]
  23. Li AC, Binder CJ, Gutierrez A, Brown KK, Plotkin CR, Pattison JW, Valledor AF, Davis RA, Willson TM, Witztum JL, Palinski W, Glass CK. Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARalpha, beta/delta, and gamma. J Clin Invest. 2004; 114: 1564–1576.[CrossRef][Medline] [Order article via Infotrieve]
  24. Everts M, Asgeirsdottir SA, Kok RJ, Twisk J, de Vries B, Lubberts E, Bos EJ, Werner N, Mejer DK, Molema G. Comparison of E-selectin expression at mRNA and protein levels in murine models of inflammation. Inflamm Res. 2003; 52: 512–518.[CrossRef][Medline] [Order article via Infotrieve]
  25. van Berkel TJ, Out R, Hoekstra M, Kuiper J, Biessen E, van Eck M. Scavenger receptors: friend or foe in atherosclerosis? Curr Opin Lipidol. 2005; 16: 525–535.[Medline] [Order article via Infotrieve]
  26. Moore KJ, Freeman MW. Scavenger receptors in atherosclerosis: beyond lipid uptake. Arterioscler Thromb Vasc Biol. 2006; 26: 1702–1711.[Abstract/Free Full Text]
  27. Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka M, Jishage K, Ueda O, Sakaguchi H, Higashi T, Suzuki T, Takashima Y, Kawabe Y, Cynshi O, Wada Y, Honda M, Kurihara H, Aburatani H, Doi T, Matsumoto A, Azuma S, Noda T, Toyoda Y, Itakura H, Yazaki Y, Kodama T, et al. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature. 1997; 386: 292–296.[CrossRef][Medline] [Order article via Infotrieve]
  28. Moore KJ, Kunjathoor VV, Koehn SL, Manning JJ, Tseng AA, Silver JM, McKee M, Freeman MW. Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice. J Clin Invest. 2005; 115: 2192–2201.[CrossRef][Medline] [Order article via Infotrieve]
  29. Freigang S, Horkko S, Miller E, Witztum JL, Palinski W. Immunization of LDL receptor-deficient mice with homologous malondialdehyde-modified and native LDL reduces progression of atherosclerosis by mechanisms other than induction of high titers of antibodies to oxidative neoepitopes. Arterioscler Thromb Vasc Biol. 1998; 18: 1972–1982.[Abstract/Free Full Text]
  30. Skalen K, Gustafsson M, Rydberg EK, Hulten LM, Wiklund O, Innerarity TL, Boren J. Subendothelial retention of atherogenic lipoproteins in early atherosclerosis. Nature. 2002; 417: 750–754.[CrossRef][Medline] [Order article via Infotrieve]
  31. Xu WY, Wang L, Wang HM, Wang YQ, Liang YF, Zhao TT, Wu YZ. TLR2 and TLR4 agonists synergistically up-regulate SR-A in RAW264.7 through p38. Mol Immunol. 2007; 44: 2315–2323.[CrossRef][Medline] [Order article via Infotrieve]
  32. Fitzgerald ML, Moore KJ, Freeman MW, Reed GL. Lipopolysaccharide induces scavenger receptor A expression in mouse macrophages: a divergent response relative to human THP-1 monocyte/macrophages. J Immunol. 2000; 164: 2692–2700.[Abstract/Free Full Text]
  33. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, Davis V, Gutierrez-Ramos JC, Connelly PW, Milstone DS. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest. 2001; 107: 1255–1262.[Medline] [Order article via Infotrieve]
  34. Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell. 1998; 2: 275–281.[CrossRef][Medline] [Order article via Infotrieve]
  35. Gorska MM, Liang Q, Stafford SJ, Goplen N, Dharajiya N, Guo L, Sur S, Gaestel M, Alam R. MK2 controls the level of negative feedback in the NF-kappaB pathway and is essential for vascular permeability and airway inflammation. J Exp Med. 2007; 204: 1637–1652.[Abstract/Free Full Text]
  36. Clark AR, Dean JL, Saklatvala J. Post-transcriptional regulation of gene expression by mitogen-activated protein kinase p38. FEBS Lett. 2003; 546: 37–44.[CrossRef][Medline] [Order article via Infotrieve]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
101/11/1104    most recent
CIRCRESAHA.107.156075v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jagavelu, K.
Right arrow Articles by Bavendiek, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jagavelu, K.
Right arrow Articles by Bavendiek, U.
Related Collections
Right arrow Animal models of human disease
Right arrow Pathophysiology

Circulation Research Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2007 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.