Functional analysis of mitogen activated protein kinase activated protein kinase-2 (MK2) in atherogenesis

Functional analysis of mitogen activated protein kinase activated protein kinase-2 (MK2) in atherogenesis

Thesis

Submitted for the degree of a Doctor of Philosophy (Ph.D.) in the subject of Molecular Cardiology

By

Kumaravelu Jagavelu November 2007

Hannover Medical School

International M.D./Ph.D. program “Molecular Medicine”

Department of Cardiology and Angiology

Acknowledged by the MD/PhD committee and head of Hannover Medical School

President: Prof. Dr. Dieter Bitter-Suermann

Supervisors: Dr.med. Udo Bavendiek / Prof. Dr. Bernhard Schieffer Co supervisors: Prof. Dr. Matthias Gaestel

Prof. Dr. Michael Kracht

External expert: Prof. Dr. Nour Eddine EL Mokhtari Internal expert: Prof. Dr. Helmut Holtmann

Day of the final exam/public defense: 9th November 2007

Table of contents Introduction

1.1. Artery 4

2. Atherosclerosis 4

3. The Endothelium 5

3.1. Endothelial dysfunction 5

4. Development of Early lesion 6

4.1. Inflammatory response by Endothelial cells 7

4.2. Recruitment of Inflammatory cells 9

4.2.1. Monocyte/Macrophages 9

4.2.2. Reverse cholesterol transport 10

4.2.3. T cells 11

4.3. Foam cell formation 12

5. Advanced lesion/Plaque rupture 13

6. Animal models in atherosclerosis 14

7. p38-MAPK 15

8. MAPKAPK-2 16

9. Aims of study 17

10. Results part I Systemic deficiency of the MAP kinase activated

protein kinase 2 reduces atherosclerosis in hypercholesterolemic mice 19 Results part II Deficiency of MAPKAPK2 reduces ROS formation and improved endothelium-independent vasorelaxation in

hypercholesterolemic mice. 37

11. Conclusion 54

12. References 57

13. Acknowledgements 62

14. Curriculum Vitae 63

15. Declaration 66

1. Introduction:

1.1. Artery

Arteries are elastic vessels that transport blood away from the heart. The largest artery of the body is the aorta. The aorta originates from the heart and branches out into smaller arteries. The smallest arteries are called arterioles which branch into capillaries. The artery wall consists of three layers 1.Tunica adventitia; 2.Tunica media; 3.Tunica intima.

Tunica adventitia is the strong outer covering of arteries and veins. It is composed of connective tissue as well as collagen and elastic fibers. These fibers allow the arteries and veins to stretch to prevent overexpansion due to the pressure that is exerted on the walls by blood flow. The tunica media is the middle layer of the walls of arteries and veins. It is composed of smooth muscle and elastic fibers. This layer is thicker in arteries than in veins. The tunica intima is the innermost layer of arteries and veins. In arteries this layer is composed of an elastic membrane lining and smooth endothelium.

2. Atherosclerosis

Atherosclerosis is an insidious and complex inflammatory disease of large and medium sized arteries. Atherosclerosis, which originates from the Greek words for "gruel" or "goo" and

"hardening," is defined as the presence of atheromas, or lesions, on the inside walls of arteries. The advanced lesions, also known as plaque, consist of fatty deposits and inflammatory cells. Complications of atherosclerosis are the most common causes of death in the world.

Atherosclerosis gets initiated early in life; many genetic and environmental risk factors (Table 1) aggravate the disease to the fatal extent.

Table 1. Risk factors of atherosclerosis. Adopted from (Glass and Witztum et al., 2001)

3. The Endothelium:

Endothelial cells form a monolayer lining the internal wall of the vessel. Endothelial cells act as active autocrine and paracrine cells. They sense the mechanical stimuli, such as pressure and shear stress, and hormonal stimuli such as vasoactive substances (e.g. NO, angiotensin II). They protect underlying tissue, including the basement membrane from blood borne substances. They trigger the inflammatory processes through the expression of inflammatory modulators such as adhesion molecules (e.g. intracellular adhesion molecule-1 (ICAM1), vascular adhesion molecule-1 (VCAM-1), chemokines (e.g. MCP1) and cytokines (e.g. IL-6). The endothelium plays an important role in vascular biology. Its biochemical entity Nitric Oxide (NO) has been ascribed with various roles like regulation of vascular tone, inhibition of platelet aggregation and inhibition of VSMC proliferation (Libby, Aikawa et al. 2006), Prevention of adhesion and recruitment of leukocytes, adhesion and aggregation of platelets as well as thrombogenesis and fibrinolysis (Kubes, Suzuki et al. 1991; Davignon and Ganz 2004). As a major regulator of local vascular homeostasis, the endothelium maintains the balance between vasodilation and vasoconstriction.

3.1 Endothelial dysfunction

The term endothelial dysfunction was first used in the article which showed an obligatory role of endothelial cells in the relaxation of smooth muscle cells by acetylcholine (Furchgott and Zawadzki 1980) and recently the term also includes altered anticoagulant and anti-inflammatory properties of the endothelium mainly caused by the reduced bioavailability of Nitric oxide (NO) (Kobayashi, Hataishi et al. 2001). NO is a potent vasodilator formed in endothelial cells from L- arginine by endothelial nitric oxide synthase (eNOS). eNOS transfers electrons from NADPH to its hemecenter, where L-arginine is oxidized to L-citrulline and thus NO is produced. NO plays a major role in endothelial function. Low bioactivity of NO upregulates VCAM-1 in the endothelial cell layer via induction of NF-κB expression inducing inflammation (Khan, Harrison et al. 1996). The direct link of endothelial dysfunction to early stages of atherosclerosis was shown in patients suffering from mild and advanced coronary artery disease (CAD) having paradoxical constriction of arteries (Ludmer, Selwyn et al. 1986). ROS are known to quench NO with formation of peroxynitrite (Koppenol, Moreno et al. 1992) which is a cytotoxic oxidant, and through nitration, oxidizing sulfhydryl and hydroxylating aromatic groups including tyrosine, tryptophan and guanine of proteins,affecting protein function and therefore endothelial function.

Furthermore, compelling evidence has shown that hypertension increases vascular production of reactive oxygen species (ROS).

ROS are typically generated by the tightly regulated NAD(P)H oxidase enzyme-complex, and from less well-regulated sources such as xanthine oxidase, cyclooxygenases, NO synthases, cytochrome P450 monooxygenases and enzymes of the mitochondrial respiratory chain. ROS are mainly produced by activation ofcell surface NAD(P)H oxidase. ROS are then rapidly converted to hydrogen peroxide in the presence of superoxide dismutase (Droge 2002). The NAD(P)H oxidases are membrane-associated enzymes that catalyse the 1-electron reduction of oxygen using NADH or NADPH as the electron donor. NAD(P)H+2O2 →NAD(P)⁺ +H⁺ +2O2

.-

(Griendling, Sorescu et al. 2000).

ROS are thought to promote atherosclerosis through a variety of mechanisms, including enhanced oxidation of lipoproteins (Steinberg 1997), activation of proinflammatory genes (Marui, Offermann et al. 1993), proliferation of smooth-muscle cell growth (Griendling and Ushio-Fukai 1998) and decreasing levels of NO (Koppenol, Moreno et al. 1992), all key events in initiation of atherogenesis.

The endothelium is also exposed to forces of the circulation such as fluid shear stress, circumferential distension, and blood pressure. Shear stress, the tangential hemodynamic force, has been implicated in regulating endothelial function and vascular remodelling. It is also shown that areas of turbulent flow show increased ROS formation. For example shear stress modulates the secretion of nitric oxide and other vasoactive substances, and the expression of genes encoding coagulation molecules and adhesion molecules in endothelial cells. (Chien, Li et al.

1998) as well as stimulating NAD(P)H oxidase activity (Howard, Alexander et al. 1997; De Keulenaer, Chappell et al. 1998). Furthermore, in human endothelial cells NO modulates the expression of NAD(P)H oxidase subunits Nox2/gp91(phox) and p47(phox) by laminar shear stress (Duerrschmidt, Stielow et al. 2006). Thereby an increase in atherosclerotic lesions in regions of disturbed flow might be due to an increased ROS formation causing endothelial dysfunction.

4. Development of early lesion

Atherosclerosis initiates when low density lipoprotein (LDL) gets entrapped and modified in the subendothelial space. Many lines of evidence suggest that oxidative modifications in the lipid and apolipoprotein B (apoB) components of LDL drive the initial formation of fatty streaks and early

atherosclerotic lesions (Steinberg and Witzum, 1999). LDL is protected from oxidation in the plasma compartment but it becomes susceptible to enzymatic and nonenzymatic modifications when retained by extracellular matrix proteins in the artery wall (Schwenke and Carew 1989).

Entrapped LDL is either directly or indirectly oxidized by myeloperoxidase, nitric oxide synthase and 15-lipoxygenase (15-LO) (Heinecke 1998). Modified LDL, such as oxidized LDL (oxLDL), has profound effects on the arterial wall. One such is stimulation of the overlying endothelial cells to produce a number of pro-inflammatory molecules, including adhesion molecules (e.g.VCAM-1, ICAM-1, E-selectins) growth factors (e.g. macrophage colony-stimulating factor (M-CSF)), chemokines (e.g. RANTES (Marfaing-Koka, Devergne et al. 1995)) and cytokines such as IFNγ and TNFα.

4.1. Inflammatory responses by endothelial cells

Inflammatory stimuli induce the expression of surface bound molecules (selectins and adhesins) thereby attracting the inflammatory cells from the circulation and facilitate their migration to the subendothelial cavity called transendothelial migration. Various types of selectins and adhesins play a role in this process. Selectins are a family of glycoproteins sharing an N-terminal C-type lectin domain followed by epidermal growth factor (EGF) conserved domains. There are about three types of selectins namely P, E and L, which are involved in rolling and tethering of leukocytes to the vascular wall. The expression goes by their name: L-selectin is mainly expressed in leukocytes and also on T cells and B cells. P-selectin is majorly expressed in platelets and also on endothelial cells, these are stored in α−granules of platelets and Weibel- Palade bodies of endothelial cells and are recruited only on activation to the surface. Lastly, E- selectin is expressed only on endothelium and is absent in resting endothelium. It is transcriptionaly induced by NF-κB and other inflammatory cytokines. All the above selectins are involved in rolling and tethering of leukocytes (Libby, Aikawa et al. 2006). Studies of mice deficient in P- and E-selectins, revealed an important role of these adhesion molecules in atherosclerosis (Dong, Chapman et al. 1998).

Adhesins belong to the large immunoglobulin (Ig) superfamily and are membrane glycoprotein receptors containing varying extracellular Ig domains. Intercellular adhesion molecules (ICAM) are about ICAM 1-5. Only ICAM 1-3 are important in the context of atherosclerosis. ICAM-1 (CD-54) is widely expressed at the basal level but can be up-regulated by pro-inflammatory cytokines in leukocytes and endothelial cells (Fig.2). ICAM-2 (CD-102) is present on leukocytes,

platelets and endothelium but is down-regulated by inflammatory mediators. ICAM-3 (CD 50) is expressed on endothelial cells, leukocytes and neutrophils. These three molecules are involved in adhesion of leukocytes to activated endothelium inducing firm arrest of inflammatory cells on the vascular surface and mice deficient for ICAM-1 showed reduced atherosclerosis (Collins, Velji et al. 2000; Galkina and Ley 2007).

Vascular cell adhesion molecule-1 (VCAM-1) (CD-106) is not expressed in healthy endothelium.

It is transcriptionally induced on endothelial cells on stimulation (e.g. oxLDL), but also expressed on macrophages, dendritic cells and myoblasts. VCAM-1 interacts with very late antigen 4 (VLA-4) which induces signals in endothelial cells (EC) and allows firm adherence of leukocytes and emigration from the blood into the vessel wall. (Fig.2) (Galkina and Ley 2007). Studies reveal that VCAM-1 plays a major role in the initiation of atherosclerosis (Cybulsky, Iiyama et al. 2001).

Platelet endothelial cellular adhesion molecule 1 (PECAM-1) (CD 31) is expressed by leukocytes, platelets and endothelial cells. The expression is high in the junction between endothelial cells, which mainly supports in homophilic binding between adjacent cells. PECAM- 1 is also involved in endothelial integrity and extravasation of cells from the lumen of the vessel to the underlying tissue (Jackson 2003).

Figure 2. Initation of atherosclerosis. Oxidized/minimally oxidized LDL stimulates the overlying endothelial cells to produce adhesionmolecules (e.g. VCAM-1), chemotactic proteins (e.g. MCP-1), and growth factors (e.g. M- CSF), resulting in the recruitment of monocytes to the vessel wall. Oxidized LDL has other effects, such as inhibiting the production of NO, an important mediator of vasodilation and expression of endothelial leukocyte adhesion molecules (ELAMs). Amongendothelial cell adhesion molecules likely to be important in the recruitment of leukocytes are ICAM-1, P-selectin, E-selectin, PCAM-1 and VCAM-1. Important adhesion molecules on monocytes include β2 integrin, VLA-4, andPCAM-1. Adopted from (Lusis et al., 2000).

4.2. Recruitment of inflammatory cells 4.2.1. Monocytes/macrophages

Monocytes

Monocytes play an important role in the disease progression. Recent evidences show that there are subsets of monocytes having varying roles. The Ly-6C(hi) monocyte subset increased dramatically in hypercholesterolemic apoE-deficient mice consuming a high-fat diet (Swirski, Libby et al. 2007). Monocytes adhere to the dysfunctional endothelium expressing VCAM-1 through very late antigen 4 (VLA4) and transmigrate into the arterial tissue in response to monocyte chemotactic protein (MCP-1) (Navab, Imes et al. 1991) and respond to local chemokines. Recent work using mice lacking MCP-1 or its receptor CCR2 markedly reduced atherosclerosis in apoE^-/- mice (Boring, Gosling et al. 1998; Gu, Okada et al. 1998). CCR2(+)Ly- 6C(hi) monocytes efficiently get accumulated in lesion, whereas CCR2(-)Ly-6C(lo) monocytes enters less frequently but were more prone to developing into lesion cells having the dendritic cell-associated marker CD11c (Tacke, Alvarez et al. 2007).

Macrophages

Macrophages are the master player in the advancement of atherosclerosis. Transmigrated monocytes in the presence of macrophage colony stimulating factor (M-CSF) differentiate into macrophages and acquire the characteristics of tissue macrophages. M-CSF is produced by numerous cell types such as endothelial cells and fibroblasts. Ox-LDL also induces monocyte-to- macrophage differentiation in vivo (Fuhrman, Partoush et al. 2007). Mice possessing an osteopetrotic (op) mutation in the MCSF gene on an apolipoprotein E (apoE)-deficient mice showed reduced atherosclerosis (Smith, Trogan et al. 1995). Modified lipoproteins stimulate macrophages to increase the expression of scavenger receptors such as the scavenger receptor A (SRA), SR-B1, CD36, CD68, CXCL16 and lectin type oxidized low-density lipoprotein receptor 1 (LOX1).Through these receptors macrophages internalize modified lipoproteins such as oxLDL, accumulating in cytoplasmic droplets. These lipid-loaded macrophages are also known as “foam cells” and characterize the atherosclerotic lesion and plaque.

Once internalized the free cholesterol as well as cholesterol esters are hydrolyzed in lysosomes.

The macrophage has two potential mechanisms for disposing of excess cholesterol: enzymatic modification to more soluble forms and efflux via membrane transporters which is called reverse cholesterol transport (RCT). In addition, the enzyme cholesterol 27 hydroxylase in macrophages

could potentially play a role in cholesterol excretion by converting it to more soluble 27-O- cholesterol (Bjorkhem 1992).

4.2.2. Reverse Cholesterol Transport (RCT) is a pathway by which accumulated cholesterol is transported from the vessel wall to the liver for excretion. Cholesterol efflux, a part of RCT, is a major process by which macrophages secrete cholesterol extracellularly through a variety of molecules like ATP-binding cassette transport protein A1(ABCA1), Scavenger receptor B1 (SR-B1), caveolins and Sterol 27 hydroxylase (CYP27A1) (Ohashi, Mu et al. 2005) and thus preventing atherosclerosis. Major acceptors of RCT are high-density lipoprotein (HDL) and apolipoprotein A-1 (apoA-1) and enzymes such as lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), hepatic lipase (HL) and cholesterol ester transfer protein (CETP). ApoA-1 is produced by the liver and released into the plasma, which interacts with serum phospholipids and forms nascent discoidal HDL (ndHDL), which triggers cholesterol efflux by the macrophages into the subendothelial space. Externalized cholesterol is absorbed by ndHDL and subsequently esterified by LCAT. HDL enriched with cholesteryl ester become larger called HDL3 and HDL2, PLTP fuse the two HDL3 and one HDL2 and make it much larger, and these larger molecules are now processed by HL making it smaller and denser. These are now delivered back to the liver via scavenger receptor B1, converted to bile salts, and eliminated through the gastrointestinal tract (Fig.3).

Intracellular cholesterol pool Macrophage

Extracellular space ^ABCA-1 SR-B1

Passive diffusion

Caveolin Cyp27A1 Effluxed cholesterol

ndHDL Mature HDL Liver

GI tract

Figure 3 Cholesterol efflux. Cholesterol efflux, a part of RCT, is a pathway transporting intracellular cholesterol from macrophages to extracellular acceptors such as apolipoprotein A-1 (apoA-1) of high-density lipoprotein (HDL).

Adopted and modified from (Ohashi et al., 2005).

ABCA1 is a major cholesterol translocating protein translocating cholesterol to the extracellular space (Oram and Vaughan 2000) (Fig 2). Overexpression of ABCA1 on LDLR deficient mice leads to accumulation of pro-atherogenic lipoproteins and enhanced atherosclerosis (Joyce, Wagner et al. 2006).

4.2.3. T cells

T cells are also important players in atherogenesis. The transition from simple fatty streaks to advanced lesions is influenced by interaction between monocyte/macrophage and T cells.

Significant interactions appear to occur among the cellular elements of developing lesions. T cells undergo activation after interacting with antigen presenting cells such as macrophages and dendritic cells and process the oxLDL present in the intima. (Zhou, Robertson et al. 2006) showed that reconstitution of Apoe^−/−/SCID^−/− mice with CD4⁺T cells from atherosclerotic or oxidized low-density lipoprotein (oxLDL)-immunized mice aggravates atherosclerotic lesion development indicating a proatherogenic role for “oxLDL-specific” T cells.

T helper 1 (TH1) cells responds to the local expression of macrophage derived interleukin-12/-18 secrete interferon-γ (IFNγ), tumor-necrosis factor (TNFα) and CD40 ligand (CD40L). These messengers prompt macrophage activation, production of proteases, activate endothelial cells, increase adhesion molecule expression and inhibit smooth muscle proliferation as well as collagen production. IFNγ stimulates the production of proinflammatory cytokines and increased expression of MHC classII molecules. Mice lacking the IFNγ-receptor on an apoE background exhibited significantly reduced atherosclerosis (Gupta, Pablo et al. 1997). Depletion of CD40 signalling using an antibody against mouse CD40L in hypercholesterolemic ldlr^-/- reduced the lesion size and lesion content of macrophages and T lymphocytes (Mach, Schonbeck et al. 1998).

T helper 2 cell (TH2) derived cytokines (IL-4, IL-10) may also have complex effects on lesion formation. IL-4, a prototypic TH2 cytokine, exerts a number of effects that are predicted to be anti-atherogenic like its antagonistic effect on IFNγ and inhibition of Th1 cell function. On the other hand IL-4 is also a potent inducer of 15-LO, which promotes LDL oxidation (Glass and Witztum 2001). IL-4 is also known to stimulate scavenger receptors expression CD36 (Feng, Han et al. 2000), SR-A (Jozefowski, Arredouani et al. 2005) and the induction of the elastin degrading matrix metallo proteinase-12 (MMP12), promoting aneurysm formation (Shimizu, Shichiri et al.

2004). IL-10 has potent anti-inflammatory properties in macrophages and modulates several other cellular processes. IL-10 deficient mice exhibited a 3-fold increase in lipid accumulation

indicating an anti-atherogenic role of IL-10 (Mallat, Besnard et al. 1999). It has been shown that T_H2 mediated immune responses are increased in human abdominal aortic aneurysms (Schonbeck, Sukhova et al. 2002).

4.3. Foam cell formation

Although the uptake of ox-LDL by macrophages in the early stages of atherosclerosis may be considered to be a protective mechanism providing the removal of cytotoxic and pro- inflammatory ox-LDL, the progressive migration of monocytes/macrophages to the intima and the continued uptake of ox-LDL lead to the formation of large numbers of foam cells and thus to the growth of atherosclerotic lesions. Foam cells are characterized by the presence of lipid droplets in the cytoplasm of macrophages. Modified lipoprotein-aggregates are internalized by macrophages through a scavenger receptor dependent pathway involving mainly CD36 and SR- A. Scavenger receptor A (SRA) and CD36 account for 75 to 90% of the uptake and degradation of acetylated and oxidized LDL (Kunjathoor, Febbraio et al. 2002). Disruption of SR-A and CD36 in ApoE deficient mice led to the significant reduction of atherosclerosis (Suzuki, Kurihara et al. 1997; Febbraio, Podrez et al. 2000). A recent report by (Mehta, Sanada et al. 2007) also shows that LOX-1 deletion in LDLR deficient mice reduced atherosclerosis.

Figure 4. Foam-cell formation. Highly oxidized aggregated LDL is formed in the vessel as a result of the action of reactive oxygen species (ROS) and the enzymes secretory phospholipase 2 (sPLA2), other lipases, and myeloperoxidase (MPO). The oxidized aggregated LDL is taken up by macrophages via scavenger receptors such as SR-A, CD36 and CD68. The death of foam cells leaves behind a growing mass of extracellular lipids and other cell debris. Adopted and modified from (Lusis et al., 2000)

5 Advanced lesions/Plaque rupture

Chronic inflammatory responses are characteristics of advanced atherosclerotic lesions, developing finally into atherosclerotic plaques. Atherosclerotic plaques can be divided into two types based on the structure and composition. 1. Stable plaques usually have uniformly dense fibrous caps characterized by a high collagen content, accumulation of smooth muscle cells and having less or no lipid core. (Fig.5D) 2. Vulnerable plaque consists of thin fibrous caps, reduced accumulation of smooth muscle cells, high concentrations of lipid-filled macrophages within the shoulder region, and large necrotic cores. Plaque rupture and thrombosis lead to acute cardiovascular events such as myocardial infarction and stroke (Lee and Libby 1997). Apoptosis of smooth muscle cells and macrophages results from cell-cell interaction and local chemokine/cytokine environment with in the arterial wall. Free, especially crystalline cholesterol also induces apoptosis of macrophage foam cells. Thereby cellular cholesterol esters and free cholesterol are released into the lesion, resulting in lipid or necrotic core formation in advanced atheromata. The functional role of smooth muscle cell in plaque vulnerability was shown in transgenic mice expressing human diphtheria toxin receptor on an apoE background (SM22α- hDTR Apoe^-/-), which resulted in thinning of fibrous cap, loss of collagen, extracellular matrix as well as accumulation of cell debris (Clarke, Figg et al. 2006). Thin fibrous caps are formed as a result of an increased expression

Figure 5 Events in atherosclerosis. A. Normal artery, B. Early atheroma, C. Vulnerable Plaque, D. Stabilized plaque, E. Plaque ruputure. Adopted and modified from (Libby 2002)

of matrix metalloproteinases secreted by macrophages to degrade the extracellular matrix proteins (Carmeliet 2000). Matrix metalloproteinase 9 (MMP9) plays a critical role in plaque disruption as seen in macrophages overexpressing autoactive MMP9 in atherosclerotic lesions of apoE^-/- mice (Gough, Gomez et al. 2006).

Plaque rupture occurs generally at the shoulder region of the plaque owing to the accumulation of macrophages, high activity of matrix metalloproteinases resulting in the degradation of the collagenous matrix rendering the plaque structure weak, friable and susceptible to fracture. The rupture occurs at the weakened region subjected to high haemodynamic stresses. After rupture, the release of the highly thrombogenic gruel into the lumen causes thrombosis and occlusion of arteries leading to acute ischemia and myocardial infarction (Fig.5).

6. Animal Models of Atherosclerosis

To study and to develop preventive treatments for the complex, multifactorial human disease atherosclerosis is dependent on animal models that mimic the human subject metabolically and pathophysiologically and develop lesions comparable to those in humans. The mouse is the most useful, economic, and valid model for studying atherosclerosis and exploring effective therapeutic approaches. Unfortunately, mice are highly resistant to atherosclerosis. However, through induced mutations it has been possible to develop lines of mice that are susceptible to this disease. ApoE is an amphipathic protein that plays a pivotal role inlipoprotein trafficking.

ApoE is a constituent of chylomicrons,VLDL, and HDL and acts as a ligand for the receptor- mediated clearance of these particles. Deletion of this protein in mice leads to high plasma cholesterol levels that are 4 to 5 times higher than the normal mice and these mice develop atheroscleroticlesions spontaneously, even when fed with a normal chow diet, whichis low in fat and cholesterol. The lesions resemble human lesionsand progress over time from an initial fatty streak to a complexlesion with a fibrous cap (Nakashima, Plump et al. 1994). Micelacking the LDLR have less overt disease, with a modest plasma cholesterol level when maintained on a normalchow diet, and they develop atherosclerosis only slowly. However, in response to a high- fat, high-cholesterol diet, LDLR-deficientmice exhibit massive elevations in plasma cholesterol and rapidlydevelop atherosclerotic lesions throughout the arterial tree (Ishibashi, Goldstein et al.

1994). A newly emerging model is the ApoE*3Leiden (E3L) transgenic mouse, in which a mutated form of the human apoE3 gene has been introduced; E3L mice have a hyperlipidemic phenotype, develop atherosclerosis on being fed with a high cholesterol diet (Zadelaar, Kleemann

et al. 2007). Although various mouse models have produced voluminous amount of data and shed light on atherosclerosis disease progression, the end stage of the disease, plaque rupture is not present in these mice and therefore the pathophysiology of plaque rupture, a major reason for cardiovascular death in humans, cannot be studied, which holds a limitation for the model.

7. p38 Mitogen Activated Protein Kinase (p38-MAPK)

Lastly, all the above processes are regulated by several signalling mechanisms for the progressing of the disease; one such is the p38-mitogen activated protein kinase (p38-MAPK). p38-MAPK plays a central role in inflammation by regulating biosynthesis of a variety of cytokines (Han, Lee et al. 1994). It is also known to mediate stress responses and is activated by heat shock, ultraviolet light, lipopolysaccharides and proinflammatory cytokines such as TNFα and IL-1β and endotoxin. p38α-MAPK (p38) was first isolated as a 38-kDa protein rapidly tyrosine phosphorylated in response to LPS stimulation (Han, Lee et al. 1993; Han, Lee et al. 1994). It is one of the four isoforms and is ubiquitously expressed in all cell types. Uptake of oxLDL by macrophages and subsequent foam cell formation requires p38- activation (Zhao, Liu et al. 2002).

It is important for endothelial cell alignment by changing actin dynamics and morphological changes induced by shear stress (Azuma, Akasaka et al. 2001). Endothelial VCAM-1 expression is regulated at the post-transcriptional level by p38- MAPK (Pietersma, Tilly et al. 1997).

Deletion of LOX-1 (receptor for oxLDL on EC) reduced p38-MAPK activity, thereby reducing inflammatory response (Mehta, Sanada et al. 2007). Furthermore in synovial neutrophils of rheumatoid arthritis (RA) patients ROS generation is dependent on p47phox (a subunit of NAD(P)H oxidase) and activation of this subunit occurs via phosphorylation of p47phox-Ser345.

In parallel p38-MAPK activation was shown to be increased and pharmacological inhibition of p38-MAPK decreased ROS formation and phosphorylation of p47phox-Ser345 (Dang, Stensballe et al. 2006). Several factors and processes which seem to be important for atherosclerosis are regulated by p38-MAPK. Unfortunately, to study the role of this molecule in various diseases failed due to the unavailability of a viable mouse model. Complete knockout of p38α revealed, that the mice are not viable at the midgestation due to defects in the placenta and the embryonic vasculature (Adams, Porras et al. 2000). To overcome this problem, several pharmacological inhibitors like SB203580 (pyridinyl imidazole) which specifically inhibits the p38α and p38β were deployed. On activation, p38-MAPK phosphorylates a variety of substrates, primarily

transcription factors and also the mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAP-2, MK2).

1.9.2. MAPKAP-2

MK2 is a serine/threonine kinase and adirect substrate of p38 MAPKα and –β. p38 binds to the C terminus of MK2 and phosphorylates its regulatory sites. Phosphorylation of MK2 serves dual functions; activated MK2 in turn phosphorylates downstream molecules such as small heat shock protein Hsp25/27, tyrosine hydroxylase, and leukocyte-specific protein. Further MK2 cotransports active p38-MAPK to the cytoplasm (Kotlyarov, Yannoni et al. 2002). MK2 is required for the production of several cytokines such as tumor necrosis factor (TNFα) and interleukin-6 (IL-6) in mouse macrophages after LPS challenge (Kotlyarov, Neininger et al.

1999). One important strategy MK2 employs to regulate the expression of proinflammatory mediators is by modulating the mRNA-stability and translation, where stability of mRNA determines the rate of mRNA degradation. mRNA-stability is given by the presence of AU-rich elements (ARE) that are located in the 3´non-coding region of the unstable mRNAs. AREs represent the most common cis-acting elements of mRNA stability in mammalian cells and can be regulated by various extracellular signals. Based on the overlapping AUUUA motifs, AREs are clustered in five groups (I-V) that contain five, four, three, two or one pentameric repeats.

Different ARE- binding proteins have been indentified to stabilize, destabilize or influence mRNA stability. Stabilizing proteins include RNA-binding protein homologous to human A-D or R antigen (HuA-D/R), destabilizing proteins are heterogeneous nuclear ribonucleoprotein (hnRNP)A1, -A2, -C and -D, tristetraprolin (TTP), K homology-type splicing regulatory protein (KSRP) and butyrate - response factor (BRF1) (Gaestel 2006) (Fig.6). MK2, an important regulator of inflammatory processes, controls the expression of proinflammatory mediators by regulating mRNA stability (Winzen, Kracht et al. 1999) via phosphorylating the RNA-binding proteins (e.g. Tristetraprolin) (Brook, Tchen et al. 2006; Hitti, Iakovleva et al. 2006). MK2 also regulates the stability of many other mRNAs such as urokinase plasminogen activator (uPA) (Han, Leng et al. 2002) and cyclooxygenase-2 (COX2) (Lasa, Mahtani et al. 2000).

Apart from the above function MK2 phosphorylates Hsp27 and Hsp25, targeting the LIM- Kinase-cofilin-pathway causing actin remodelling resulting in cell migration and chemotaxis (Kotlyarov and Gaestel 2002; Kobayashi, Nishita et al. 2006). Owing to the tight control on the inflammatory process by MK2, its function in inflammatory processes has been further evidenced

in a mouse model for collagen induced arthritis. In this model MK2 deficient mice showed significantly reduced arthritis and expression of inflammatory markers (e.g. IL-6). (Hegen, Gaestel et al. 2006). However, the functional role of MK2 in inflammatory cardiovascular disease, such as atherosclerosis is not known. Hence, we hypothesise MK2 might play role in atherosclerosis.

Figure 6 Regulation of cytokine gene expression by the p38 MAP-kinase pathway. Adopted from NCB reviews 2000.

Aim of the study

This study aims in dissecting the functional role of

1. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAP-2) in atherosclerosis.

2. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAP-2) in endothelial dysfunction and ROS formation.

Results-Part I

Systemic deficiency of the MAP kinase activated protein kinase 2 reduces atherosclerosis in

hypercholesterolemic mice

Results- II

Deficiency of MAPKAPK-2 reduces ROS formation and improved endothelium-

independent vasorelaxation in

hypercholesterolemic mice.

Deficiency of MAPKAPK-2 reduces ROS formation and improved endothelium- independent vasorelaxation in hypercholesterolemic mice.

Kumaravelu Jagavelu¹, Carola Doerries¹, Ulf Landmesser¹, Matthias Gaestel², Helmut Drexler¹, Bernhard Schieffer¹, Udo Bavendiek¹

1Department of Cardiology & Angiology, Hannover Medical School, Germany (K. J., C.D., U.L., H. D., B. S., U. B.)

2 Department of Biochemistry, Hannover Medical School, Germany (M. G.)

Address of Correspondence:

Udo Bavendiek, MD

Department of Cardiology & Angiology Hannover Medical School

Carl-Neuberg-Str. 01 30625 Hannover, Germany Tel: (0)511-532-9597 Fax: (0)511-532-3357

Email: Bavendiek.Udo@mh-hannover.de

Journal Subject Heads: endothelial dysfunction, ROS formation, hypercholesterolemic mice Total word count: (3606)

Abstract

Atherosclerosis is a multifactorial chronic inflammatory disease and represents the major cause of cardiovascular morbidity and mortality. Endothelial dysfunction and reactive oxygen species (ROS) are important mediators of atherogenesis. We investigated the role of the mitogen - activated protein kinase activated protein kinase-2 (MK2) in endothelial dysfunction and ROS production in hypercholesterolemic ldlr^-/-- deficient mice. Deficiency of MK2 leads to reduced blood pressure in ldlr^-/--deficient mice before and after feeding a high cholesterol diet. MK2 deficiency did not affect endothelium-dependent vasorelaxation induced by acetylcholine, but improved endothelium-independent vasorelaxation induced by nitroglycerine and reduced total ROS formation as well as NAD(P)H oxidase dependent ROS formation in aortas isolated from hypercholesterololemic ldlr^-/- deficient mice. These data suggest that MK2 may be critically involved in atherogenesis by fostering ROS formation. In addition MK2 seems to impair endothelial-independent vasorelaxation, probably by desensitizing the vascular response to NO.

Keywords: Atherosclerosis, MK2, Endothelial dysfunction, hypercholesterolemic mice, ROS

Introduction

Atherosclerosis is a chronic inflammatory disease characterised by the recruitment of inflammatory cells to the inflamed vessel wall and deposition of lipids^1-3. The integrity and function of endothelial cells are fundamental for normal homeostasis of the vessel wall. In health, the endothelium has an anti-inflammatory phenotype that maintains the uninterrupted circulation of leukocytes and platelets, whereas endothelial dysfunction and injury results in the increased adhesion and recruitment of leukocyte and platelets to the vascular wall, initial stages of atherosclerosis. Also, clinical and experimental studies have shown that a marked reduction in endothelial-dependent vasodilation is a characteristic of an early stage of atherosclerosis^{4, 5}. Therefore, endothelial dysfunction seems to be a key factor in the initiation and progression of atherosclerosis⁶. Endothelial dysfunction is mainly caused by the reduced bioavailability of Nitric oxide (NO), a potent vasodilator formed in endothelial cells from L-arginine by eNOS. NO released towards the vascular lumen is a potent inhibitor of platelet aggregation and adhesion to the vascular wall^{7, 8}. It also inhibits leukocyte adhesion to the vessel wall by interfering with the leukocyte adhesion molecules CD11 and CD18^{9, 10}. As leukocyte adherence is an early event in the development of atherosclerosis NO may protect against the onset of atherogenesis. A reduction in the bioavailability of NO has been implicated in various diseases and also in atherosclerosis and hypertension. Studies on hypercholesterolemic primates have demonstrated a loss of endothelium-dependent vasodilatation in response to acetylcholine and bradykinin⁴ because of a reduced availability of NO and an increased formation of reactive oxygen species (ROS)¹¹. Reactive oxygen species (ROS) are mainly derived from the vascular NAD(P)H oxidase system. ROS are thought to promote atherosclerosis through multiple mechanisms, including enhanced oxidation of lipoproteins¹², activation of proinflammatory mediators¹³, reduction in the bioavailability of NO and proliferation of smooth-muscle cell growth¹⁴ which are all known as key events in atherogenesis.

The mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAP-2, MK2) is a direct physiological substrate of MAPK p38α/β. MK2 regulates the expression of several proinflammatory genes by regulating the mRNA-stability through targeting AU-rich elements¹⁵. In smooth muscle cells angiotensin II induced ROS-dependent p38-activation through the angiotensin type1 (AT1) receptor resulting in activation of MAPKAP kinase-2 and phosphorylation of HSP27¹⁶.In synovial neutrophils of rheumatoid arthritis (RA) patients ROS

generation is dependent on p47phox (a subunit of NAD(P)H oxidase). The activation of this subunit occurs via phosphorylation of p47phox-Ser345. The activation of NAD(P)H oxidase is probably dependent on p38-MAPK as pharmacological inhibition of p38-MAPK decreased TNF- α induced phosphorylation of p47phox-Ser345 and subsequently reduced ROS formation¹⁷. In addition, long term treatment with a selective p38 MAPK inhibitor suppresses both NAD(P)H oxidase expression and ROS generation and protects against Ang II-induced target-organ damage in rats ¹⁸. Furthermore, inhibition of p38 MAP kinase in rats with chronic heart failure reduced MK-2 phosphorylation, preserved acetylcholine-induced vasorelaxation and reduced vascular superoxide formation ¹⁹. In this regard, Ju et.al (2003) has shown that hypertension induced endothelial dysfunction depends on p38-MAPK activation.

Therefore, in the present study we investigated the functional role of MK2 in endothelial dysfunction and ROS formation in hypercholesterolemic mice deficient for MK2 compared to ldlr^-/- deficient control mice.

Materials and Methods

Materials

All chemicals were purchased from Sigma-Aldrich unless otherwise stated.

Mouse model:

All procedures followed were in accordance with approved guidelines set by the Institutional Animal Care and Use Committee at Hannover Medical School and the Bezirksregierung Niedersachsen. To understand the pathophysiological alteration of atherosclerosis and to assess the potential role of MK2 in atherosclerosis, low-density-lipoprotein-receptor-deficient mice (ldlr^-/- C57Bl/6, Jackson Laboratories) and MK2-deficient mice (mk2^-/- C57Bl/6, backcrossed

>10 generations) ²¹were crossbred to generate ldlr^-/-/mk2^-/-compound mutant mice. The genotype of each mouse was confirmed by PCR after isolating 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 GGGGTG 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). 12 week old male ldlr^-/-and ldlr^-/-/mk2^-/- compound-mutant mice (each group n=8-14)

consumed a high cholesterol diet (product #D12108, Research Diets; 1.25% Cholesterol without added cholate) for 7 weeks. Subsequently, mice were sacrificed and the aortas were removed and analyzed as described below. All mice were housed under specific pathogen-free conditions.

Blood Pressure Measurement:

Systolic blood pressure and heart rate were recorded by tail cuff measurement with the aid of a computerized system (BP2000 Blood Pressure Analysis System, Visitech System, Apex, NC, USA) in conscious animals. Animals were pretrained for blood pressure measurements 3 consecutive days prior to the real measurements. On each day blood pressure determination was measured at the same time, 30 measurements were taken and averaged for each individual animal every day. Blood pressure reading was taken before starting the high cholesterol diet and after high cholesterol diet and mice were pretrained in both cases.

Organ Chamber Experiments

After excision of the descending aorta, the vessel was placed in ice cold buffer I (mmol/L, NaCl 118.0, CaCl2 2.5, KCL 4.73, MgCl2 1.2, KH2PO4 1.2, NaHCO3 25.0, C8H17N2NaO4S 1.355, D(+)glucose 5.5) ²². The aorta was cut into 3 mm rings, after the removal of surrounding fat, the rings were placed in the organ baths filled with the buffer I (37^° C continuously aerated with 95%

O₂ and 5% CO₂). After inserting the force transducers into the rings while maintaining the endothelial integrity, the rings were stretched gradually using a passive tension of 1 g, which was maintained throughout the experiment. Rings were precontracted with KCl (80 mmol/L) and again precontracted to 50% of the maximal tension (KCL) with phenylepherine. To assess endothelium-dependent and -independent relaxation,acetylcholine (ACH 10^-9 to 10^-5 µmol/L) or nitroglycerine, (NTG 10^-9 to 10^-5 µmol/L) was cumulatively added to the organ chambers respectively. The isometric tension was recorded using Biopac systems and analysis using Acqknowledge 3.7.2 software.

Measurement of Vascular Reactive oxygen species (ROS) and NAD(P)H-Oxidase dependent ROS formation by Electron Paramagnetic Resonance Spectroscopy/ESR Detection of Superoxide Radicals.

After feeding the high cholesterol diet as mentioned above mice were sacrificed by CO2

inhalation and the aorta from the thoracic region was dissected. Care was taken to excise off the

fat and extra tissue without destroying the endothelium and aortas were cut into 4 mm rings from ldlr^-/-and ldlr^-/-/mk2^-/-(each group n = 10-12 mice) The dissected rings were placed in ice cold buffer 1containingdeferoxamine (25 µmol/litre, metal chelator). The rings were placed in 50 µl capillary tube (Corning). ESR spectra were recorded as mentioned previously ²³ by using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) (Noxygen GmBH) to measure the total superoxide anions. To measure the NAD(P)H oxidase dependent superoxide anion production, NADH was used as a substrate and NAD(P)H dependent ROS formation was measured using CMH using a MiniScope ESR spectrometer (Magnettech, Berlin, Germany). The ESR setting was the following: field center: 3356.53G; field sweep width: 50G; microwave frequency: 9.82GHz; microwave power: 32mW (5dB); magnetic field modulation frequency: 100 kHz; modulation amplitude: 2.5G. The intensity of ESR spectra was quantified after subtraction of the ESR signal of samples and without the sample. Data from each spectrum were quantified as sum of the total 3-peak intensities and two rings were analysed for each mouse and averaged.

Statistical analysis

All values are given as mean ± SEM. Results were analyzed using Student's t-test or analysis of variance followed by Bonferroni/Tukey for multiple comparisons using Instat software (Graph pad). A value of P<0.05 was considered statistically significant.

Reduced Systolic Blood Pressure in hypercholesterolemic ldlr^-/- and ldlr^-/-/mk2^-/-mice.

Systolic blood pressure was monitored by tail cuff plethysmography in ldlr^-/- and ldlr^-/- /mk2^-/- mice before and after feeding a high cholesterol diet for 7 weeks. Ldlr^-/-/mk2^-/- mice (n=20) showed a significantly reduced blood pressure before the start of the high cholesterol diet (ldlr^-/- vs ldlr^-/- /mk2^-/-: 127±2.8 mmHg vs. 110±3.4 mmHg n=20, P<0,002) and after feeding the high cholesterol diet compared to age-matched ldlr^-/- mice. (ldlr^-/- vs ldlr^-/- /mk2^-/-: 116±2.6 mmHg vs. 106±2.2 mmHg, n=20, P<0,007) (Fig. 1).

MK2 deficiency improved endothelial independent vasorelaxation, but not endothelium- dependent vasorelaxation in hypercholesterolemic mice.

To examine the role of MK2 in vascular function of hypercholesterolemic mice, we fed ldlr^-/- /mk2^-/- mice and ldlr^-/- control mice with a high cholesterol diet for 7 weeks and the endothelium-dependant and -independent vasorelaxation was studied. Endothelium - dependent vasorelaxation of aortic rings was not different between ldlr^-/- mice and ldlr^-/- /mk2^-

/- mice elicited by acetylcholine concentrations ranging from 10^-9 to 10^-4.5 mol/L. (n=14 in both groups) (Fig.2a). In contrast, endothelium-independent vasorelaxation in response to nitroglycerine was substantially improved at a concentration range of (10^-8 to 10^-5 mol/L, n=

14, P<0, 01) in ldlr^-/- /mk2^-/- compared to ldlr^-/- mice.(Fig. 2b).

MK2-deficiency reduced ROS formation in hypercholesterolemic ldlr^-/-mice.

To elucidate the underlying mechanism of the observed impact of MK2 deficiency in the endothelium-dependent and -independent vasorelaxation, we investigated if deficiency of MK2 affects the formation of ROS in the aortas of hypercholesterolemic ldlr^-/- by electron spin resonance (ESR). Total ROS formation in ldlr^-/-/mk2^-/- mice was significantly reduced (1.17 pmol O₂^.- / min, n=12, P<0,009) compared to ldlr^-/- mice (2.55 pmol O₂^.- / min) (Fig. 3a).

Furthermore, to determine if the NADPH dependent ROS formation is affected by MK2- deficiency we also analysed the NAD(P)H oxidase/activity in these hypercholesterolemic mice. We found a significantly reduced NAD(P)H oxidase dependent ROS formation in ldlr^-/-

/mk2^-/- mice (0.977 pmol O₂^.- / min, n= 12, P<0,01) compared to ldlr^-/- mice (1.817 pmol O₂^.-

/ min) (Fig. 3b).

Discussion

Hypercholesterolemia and modified lipoproteins (oxLDL) are known to cause endothelial dysfunction and ROS formation and therefore seems to be key factors in the initiation of atherosclerosis⁶. Earlier studies by Widder et.al (2004) showed that pharmacological inhibition of p38-MAPkinase with SB239063 reduced MAPKAPK-2 phosphorylation, preserved acetylcholine-induced vasorelaxation and reduced vascular superoxide formation in CHF rats, revealing that the p38-MAPK downstream target MK2 may increase endothelial dysfunction. The unimpaired endothelium-dependent vasodilator response in MK2-deficient hypercholesterolemic mice (this study) could be due to the presence of high concentrations of proatherogenic lipoproteins, known to decrease the expression of endothelial nitric oxide synthase (eNOS) in endothelial cells and thereby impaired formation of NO and endothelial- dependent vasorelaxation²⁴, masking a potentially increased endothelium-dependent vasorelaxation caused by MK2- deficiency in hypercholesterolemic ldlr^-/- mice. The increased endothelial-independent relaxation caused by MK2-deficiency in hypercholesterolemic ldlr^-/- mice may be due to an increased sensitivity of the sGC-cGMP signalling cascade to NO in smooth muscle cells. This might result in the observed increased endothelium-independent vasorelaxation caused by the potentially reduced NO formation in hypercholesterolemic mk2^-/- mice, similarly as demonstrated in the eNOS^-/- mice²⁵. In line, overexpressing of bovine eNOS was followed by a reduced NO-elicited vasorelaxation^{26, 27}.

Furthermore, contraction and relaxation of smooth muscle cells (SMC) are dependent on cytoskeletal changes. The increased endothelium independent vasorelaxation in MK2 deficient mice could also be due to the cytoskeletal changes observed in endothelial cells due to a casual link between MK2 activation, HSP27 phosphorylation and actin redistribution²⁸ or may be due to the improper actin reorganization mediated by MK2 phosphorylation/activation of LIMK1 as shown in endothelial cells²⁹. Cytoskeletal changes are also mediated via the cGMP signalling cascade. Cyclic GMP-dependent protein kinase I (PKGI) interacts with formin homology (FH) domain-containing protein (FHOD1)³⁰. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics and cell migration. Recently it has been shown that p38 MAPK also interact with FHOD1³¹, which may result in increased vasorelaxation.

Previous studies^18,19 show that the endothelial-dependent vasorelaxation response is dependent on ROS production. To further elucidate the unimpaired endothelial-dependent

vasorelaxation in MK2-defecient hypercholesterolemic mice, we hypothesised that this could be due to reduced level of ROS formation. As expected hyperchosterolemic mice lacking MK2 showed less aortic ROS level. The reduced ROS formation could be attributed to a reduced NAD(P)H oxidase/activity. Previous studies have shown that the ROS production is regulated through NAD(P)H oxidase, constituting a major contributor for ROS production, and is also shown to be increased in early stages of atherosclerosis through renin-angiotensin system (RAS) activation ³². We hypothesised that in MK2-defecient mice NAD(P)H oxidase- dependent ROS formation may be less. As expected MK2 deficient mice showed less production of NADH-dependent ROS formation. It is shown that in bovine leukocytes p38MAP kinase regulates the activation of the NAD(P)H-oxidase and that pharmacological inhibition of p38MAPK reduces phosphorylation of p47phox leading to reduced production of ROS ³³. The phosphorylation of p47phox could be directly mediated by MK2, a direct substrate of p38, thereby regulating the production of ROS. A recent study by Dang et.al., 2006 showed that TNFα primes p38-dependent ROS production through phosphorylation of Ser345 of the p47phox subunit of NAD(P)H oxidase. However, further studies are essential to prove that the MK2 is actually regulating NAD(P)H oxidase /activity.

Furthermore, we have previously shown (Jagavelu et al., 2007 in revision) that mice deficient for MK2 have significantly less atherosclerosis. This could be potentially due to the observed reduced ROS production in these mice with subsequently reduced lipid oxidation. Other studies have shown that ROS are known to be important for monocyte migration³⁴ and oxidation of lipids³⁵. Thereby the initiation and progression of the disease may be attenuated or delayed. In addition, ROS are known to quench NO with the formation of peroxynitrite ³⁶. MK2 deficient mice showed reduced ROS formation thereby potentially an increased bioavailability of NO, eventually recovering the potentially reduced NO caused by increased proatherogenic lipoproteins compared to the hypercholesterolemic ldlr^-/- mice. This could be one reason for the observed unimpaired endothelium-dependent vasodilation.

Taken together, these data indicate a potential role for MK2 in atherogenesis by regulating the vascular production of ROS. In addition, MK2 seems to impair the endothelial independent vasorelaxation, probably by desensitizing the vascular response to NO. Therefore, MK2 may provide a feasible target for a therapeutical regime to treat patients suffering from atherosclerotic cardiovascular disease.

Figure Legends

Fig. 1: MK2-deficiency reduced systolic blood pressure in hypercholesterolemic ldlr^-/- mice.

Systolic blood pressure was measured using tail cuff plethysmography before and after feeding a high cholesterol diet for 7 weeks (Pre and Post HCD n=20, ldlr^-/- vs ldlr^-/- /mk2^-/- * p<0.05).

Fig. 2: MK2-deficiency improved endothelial-independent vasorelaxation but did not affect endothelium-dependent vasorelaxation in hypercholesterolemic ldlr^-/- mice.

Vasorelaxation was determined in aortas isolated from ldlr^-/- and ldlr^-/-/mk2^-/- in response to acetylcholine (Ach) and nitroglycerine (NTG). Vascular reactivity was determined as changes in isometric tension of aortic rings submaximally precontracted with phenylepherine.

Endothelium-dependent relaxation of the aortas from ldlr^-/- (squares) and ldlr^-/- /mk2^-/- (triangles) induced by cumulative additions of ACh (10^-8 to 10^-4.5 mol/L, A) and endothelium- independent relaxation by NTG (10^-8.5 to 10^-5 mol/L, B) were demonstrated. (ldlr^-/- vs ldlr^-/- /mk2^-/- , n= 14, *P < 0.05).

Fig. 3: MK2-deficiency reduced ROS formation in hypercholesterolemic ldlr^-/-/mk2^-/-mice.

Vascular superoxide production was measured by electron spin resonance (ESR). Total ROS formation (A) and NAD(P)H oxidase dependent ROS formation (B) was determined (ldlr^-/- vs.

ldlr^-/- /mk2^-/-, n=12, *P < 0.05).

Fig. 1

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Fig. 2 A

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