Allosteric MAPKAPK2 inhibitors improve plaque stability in advanced atherosclerosis

Lale Ozcan; Canan Kasikara; Arif Yurdagul Jr; George Kuriakose; Brian Hubbard; Michael H. Serrano-Wu; Ira Tabas

doi:10.1371/journal.pone.0246600

Abstract

Atherosclerotic vascular disease resulting from unstable plaques is the leading cause of morbidity and mortality in subjects with type 2 diabetes (T2D), and thus a major therapeutic goal is to discover T2D drugs that can also promote atherosclerotic plaque stability. Genetic or pharmacologic inhibition of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2 or MK2) in obese mice improves glucose homeostasis and enhances insulin sensitivity. We developed two novel orally active small-molecule inhibitors of MK2, TBX-1 and TBX-2, and tested their effects on metabolism and atherosclerosis in high-fat Western diet (WD)-fed Ldlr^-/- mice. Ldlr^-/- mice were first fed the WD to allow atherosclerotic lesions to become established, and the mice were then treated with TBX-1 or TBX-2. Both compounds improved glucose metabolism and lowered plasma cholesterol and triglyceride, without an effect on body weight. Most importantly, the compounds decreased lesion area, lessened plaque necrosis, and increased fibrous cap thickness in the aortic root lesions of the mice. Thus, in a preclinical model of high-fat feeding and established atherosclerosis, MK2 inhibitors improved metabolism and also enhanced atherosclerotic plaque stability, suggesting potential for further clinical development to address the epidemic of T2D associated with atherosclerotic vascular disease.

Citation: Ozcan L, Kasikara C, Yurdagul A Jr, Kuriakose G, Hubbard B, Serrano-Wu MH, et al. (2021) Allosteric MAPKAPK2 inhibitors improve plaque stability in advanced atherosclerosis. PLoS ONE 16(5): e0246600. https://doi.org/10.1371/journal.pone.0246600

Editor: Christian Schulz, Ludwig-Maximilians-Universitat Munchen, GERMANY

Received: January 31, 2021; Accepted: April 20, 2021; Published: May 13, 2021

Copyright: © 2021 Ozcan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: This work was supported by National Institutes of Health grants HL087123 (to I.T. and L.O.), DK124457 (to L.O), K99 HL145131 (to A.Y), and an American Heart Association postdoctoral fellowship (20POST35210962) to CK. Tabomedex provided support in the form of consultancy payments for authors [B.H. and M.H.S-W.], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

Competing interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: L.O., B.H., M.H.S.-W., and I.T. are members of Tabomedex Biosciences, Inc., which is developing allosteric MK2 inhibitors for the treatment of type 2 diabetes. All the authors confirm that this commercial affiliation does not alter our adherence to all PLOS ONE policies on sharing data and materials.

Introduction

The prevalence of type 2 diabetes (T2D) is becoming a significant public health problem owing to the worldwide obesity epidemic [1]. Patients with T2D have a two- to four-fold increased risk of developing atherosclerotic cardiovascular disease (CVD), which accounts for ~40% of all T2D mortality [2, 3]. Reducing the incidence and severity of CVD is a key unmet medical need in T2D patients. Over the past decade, many anti-diabetic agents have been tested for cardiovascular safety [4]. Despite good glycemic control, most of these studies failed to show a significant CVD benefit [5]. Therefore, identification of common signaling pathways and molecular targets in distinct cell types that contribute to both T2D and CVD pathogenesis may lead to the development of novel diabetes therapies with proven CVD benefits.

We have previously shown that dysregulated calcium signaling in hepatocytes in obesity activates a calcium/calmodulin-dependent protein kinase II (CaMKII)‒MK2 pathway, which then promotes hyperglycemia and insulin resistance via increasing hepatic glucose production and disrupting insulin receptor signaling [6–8]. Genetic inhibition of hepatic CaMKII or MK2 in obese insulin-resistant mouse models markedly lowers blood glucose and insulin resistance [7]. Further, treatment with an allosteric MK2 inhibitor called compound (cmpd) 28 was shown to improve glucose homeostasis and insulin sensitivity in obese mice [9]. The metabolic benefit of cmpd 28 treatment in this study was mechanistically "on-target" and, importantly, was additive with the current leading T2D drug, metformin. Consistent with these results, MK2 deletion has recently been shown to lower hyperglycemia and improve insulin tolerance in the low-dose streptozotocin-induced diabetic mouse model [10]. Interestingly, a different CaMKII-MK2‒driven downstream pathway in atherosclerotic lesional macrophages promotes advanced atherosclerosis progression by impairing clearance of apoptotic cells (efferocytosis) and suppressing resolution of inflammation [11, 12].

The mechanisms linking T2D to CVD include insulin resistance, hyperglycemia, dyslipoproteinemia, and inflammation [13]. Our pre-clinical studies have shown that inhibition of the CaMKII‒MK2 pathway ameliorates all of these processes [7, 9], and germline deletion of MK2 in Ldlr^-/- mice was shown to suppress atherosclerosis, but effects on plaque stability and glucose metabolism was not reported in that study [14]. Using two new orally available MK2 inhibitors, TBX-1 and TBX-2, we now report that MK2 inhibitor treatment of Ldlr^-/- mice with established atherosclerosis improves metabolism and features of unstable plaques.

Materials and methods

Synthesis of MK2 inhibitors

TBX-1 and TBX-2 were synthesized as described in the patent literature [15]. All solvents and reagents were obtained from commercial sources and used without further purification. NMR spectra were obtained on a Bruker Neo 400M spectrometer operating at 400 MHz. Chemical shifts are reported in parts per million (δ) from the tetramethysilane resonance in the indicated solvent. LC-Mass spectra were taken with Agilent 1260-6125B single quadrupole mass spectrometer using a Welch Biomate column (C18, 2.7 um, 4.6*50 mm) eluting with a mixture of solvents A (ACN with 0.05% trifluoroacetic acid) and B (Water with 0.05% trifluoroacetic acid) using a gradient elution. Detection was by DAD (254 nm and 210 nm). Ionization was by ESI. Detailed description of TBX-1 and TBX-2 synthesis is included in the S1 File.

Mouse experiments

Eight-week-old male Ldlr^-/- mice on the C57BL/6J background were purchased from Jackson Laboratory (stock # 002207) and fed a Western-type Diet (WD) for 16 weeks (Envigo, TD.88137; 21.2% fat, 0.2% cholesterol, 48.5% carbohydrate, and 17.3% protein by weight). Between weeks 10–16 of feeding, the WD of the experimental group was supplemented with TBX-1 or TBX-2 (600 mg/kg diet) to achieve a dose of ∼30 mg/kg body weight per day, whereas control mice remained on regular WD. The study included four separate cohorts, each comprised of 5 to 10 mice per group. The mice were randomly assigned to the different cohorts. Fasting blood glucose was measured in mice that were fasted for 5 h, with free access to water, using a glucose meter. Insulin tolerance test was performed in 5 h-fasted mice by assaying blood glucose at various times after injection of insulin (0.7 IU/kg body wt i.p.). Plasma insulin levels were measured in mice that were fasted for 5 h using an ultra-sensitive mouse insulin ELISA kit (Crystal Chem). At the time of harvest, mice were euthanized using isoflurane, and blood was then removed by left ventricular puncture. Animals were provided with food and water ad libitum and housed in standard cages at 22°C in a 12-12h light-dark cycle in a barrier facility. The Columbia University IACUC provided ethical approval for these studies, and procedures were conducted in accordance with approved animal protocols.

Pharmacokinetic studies

The pharmacokinetics of the compounds were evaluated in CD-1 mice by single oral and intravenous routes of drug administration. The mice were fasted overnight before oral-gavage dosing of the compounds. The solutions used for PK studies were prepared by dissolving the compounds in 5% Solutol HS 15 in water. Following oral or intravenous administration of compounds, blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after dosing. Blood samples were centrifuged to separate plasma, and compound concentrations were determined by LC-MS. Dosing was well tolerated following oral and intravenous administration of both compounds, and no abnormal behavior was observed.

Plasma cholesterol and triglyceride measurements

Total plasma cholesterol and triglyceride levels were measured using commercially available kits (Wako Diagnostics). Plasma lipoproteins were analyzed by running 200 μl of pooled plasma onto a fast protein liquid chromatography system consisting of 2 Superose 6 columns connected in series (Amersham Pharmacia Biotech) as described previously [16].

Murine atherosclerotic lesion analysis

Total lesion area and necrotic area analysis were carried out on hematoxylin and eosin (H&E)-stained aortic root lesional cross sections as previously described [12]. Briefly, total lesion area (from internal elastic lamina to the lumen) and acellular/anuclear areas (negative for hematoxylin-positive nuclei) per cross section were quantified by taking the average of 6 sections spaced 30 μm apart beginning at the base of the aortic root. Necrotic areas were defined as anuclear areas based on absence of hematoxylin staining. Collagen staining was performed using picrosirius red as per the manufacturer’s instructions (Polysciences Inc.), and fibrous cap thickness was quantified from 3 distinct regions of the plaque and scored per lesion size as previously described [11]. For immunofluorescence staining, frozen sections were fixed in cold acetone for 10 minutes. After blocking for 1 hour in serum-free protein blocking buffer (DAKO, catalog X0909), sections were incubated overnight at 4°C with anti–phospho-hsp25 (CST, 1:200), anti-Mac2 (1:10,000), and smooth-muscle α actin (SMαA, 1:150) antibodies, rinsed with PBS three times, incubated with fluorescently-labeled secondary antibodies for 2 hours, and counterstained with DAPI. Three slides, each with 2 sections, were assessed for each mouse.

In situ efferocytosis assay

Experimentation was carried out as previously described [12]. In brief, acetone-fixed aortic root sections were incubated with TUNEL (Roche) followed by staining with anti-Mac2 (1:10,000) to label macrophages. Apoptotic cells were then determined to be either macrophage-associated (colocalizing or juxtaposed with macrophages) or free (not associated with macrophages). Data were plotted as a ratio of associated-to free cells, which is a measure of efferocytosis [17].

Primary hepatocytes

Primary mouse hepatocytes were isolated from 8- to 12-week-old mice as described previously [9] and cultured in media containing Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning, #10-013-CV), 10% (vol/vol) heat-inactivated FBS, and 1X penicillin-streptomycin. After overnight incubation, the cells were washed twice with PBS, treated with the indicated concentrations of TBX-1 for 1 h, followed by treatment with TBX-1 and forskolin for 4 h in FBS-free media.

Reagents and antibodies

Forskolin was from Sigma. Anti–phospho-hsp25 antibody was from Cell Signaling, anti–Mac2 antibody was from Cedarlane, anti–smooth-muscle α actin was from Sigma and anti–β-actin antibody was from Abcam.

Immunoblotting and quantitative RT-PCR

Immunoblot and quantitative RT-PCR (RT-qPCR) assays were conducted as previously described [9]. Total RNA was extracted from primary hepatocytes using the RNeasy kit (Qiagen). cDNA was synthesized from 2 mg total RNA using oligo (dT) and Superscript II (Invitrogen).

Statistics

All results are presented as means ± SEM. Statistics were performed using SigmaPlot 14.0 and Graphpad Prism 8 software. Normality was determined using D’Agostino-Pearson and/or Shapiro-Wilk normality testing. P values for normally distributed data were calculated by either Student’s t test (two-tailed) or one-way analysis of variance (ANOVA) with Student–Newman–Keuls post hoc analysis when three or more groups were tested. P values for non–normally distributed data were calculated using the Mann-Whitney U test.

Results

Our previous MK2 inhibitor study in obese mice used cmpd 28 [9]. However, the low oral bioavailability of cmpd 28 (BAV < 3%) precluded use of this tool compound in longer term animal experiments. Following a series of scaffold re-designs (to be described elsewhere), two MK2 inhibitors with greatly improved oral bioavailability were identified: TBX-1 (hMK2 IC₅₀ = 67 nM) and TBX-2 (hMK2 IC₅₀ = 19 nM). Similar to cmpd 28 [9], TBX-1 inhibited forskolin-induced G6pc mRNA in a dose-dependent manner (S1A Fig in S1 File). The improved pharmacokinetic profiles of TBX-1 and TBX-2 were affirmed by intravenous and oral administration routes in normal chow-fed mice (Table 1). The plasma half-lives of TBX-1 and TBX-2 were 2.7 and 3.1 hours, respectively. Both compounds displayed clearance and absorption profiles that were suitable for oral delivery, making them attractive small molecule inhibitors to study the role of MK2 in advanced atherosclerotic lesion formation in mice.

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Table 1. In vivo pharmacokinetic evaluation of compounds TBX-1 and TBX-2.

https://doi.org/10.1371/journal.pone.0246600.t001

To determine the therapeutic potential of MK2 inhibitors and provide clinically relevant findings, we tested whether they could improve features of plaque stability when administered after plaques were established. For this purpose, Ldlr^-/- mice were first fed a Western-type diet (WD) for 10 weeks to induce atherosclerotic plaque development. Based on the pharmacokinetic profile of the compounds, we supplemented the WD with 600 mg/kg TBX-1 or TBX-2 to achieve a dose of ∼30 mg/kg body weight per day. Half of the mice were continued for 6 additional weeks on the MK2 inhibitor supplemented diets, while the other half continued to receive WD alone (control). Neither treatment was associated with overt signs of toxicity, e.g., body weights, food intake, and activity levels were similar to those of control mice. As predicted, inhibitor treatment efficiently reduced the phosphorylation of the MK2 substrate, heat-shock protein (hsp) 25 in liver, as demonstrated with TBX-1 (S1B Fig in S1 File), indicating successful targeting of MK2 activity in liver. As with cmpd 28, both inhibitors improved glucose homeostasis and enhanced insulin sensitivity (Fig 1). Both compounds also lowered plasma levels of total cholesterol (TC) and triglyceride (TG) (Fig 2A, 2B, 2D and 2E). Fast protein liquid chromatography analysis of the plasma showed that very-low-density lipoprotein (VLDL)-cholesterol and low-density lipoprotein (LDL)-cholesterol were reduced after treatment with TBX-1 and TBX-2 (Fig 2C and 2F). Analysis of white blood cells in the blood revealed no differences in the number of circulating leukocytes or monocytes (S2 Fig in S1 File).

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Fig 1. MK2 inhibitor treatment improves glucose homeostasis in WD-fed Ldlr^-/- mice.

8-week-old Ldlr^-/- mice were placed on WD for 10 weeks. The mice were continued for 6 additional weeks on WD alone (control) or on WD containing TBX-1 or TBX-2. (A-B) 5-hour-fasting plasma insulin levels were measured, and an insulin tolerance test was conducted in a randomly selected subset of Ldlr^-/- mice treated withTBX-1 (n = 6 mice/group; mean ± SEM, p < 0.001, p = 0.02 and p = 0.03, respectively). (C-D) 5-hour fasting blood glucose and plasma insulin levels were measured in a randomly selected subset of Ldlr^-/- mice treated with TBX-2 (n = 7–8 mice/group; mean ± SEM, p = 0.006 and p = 0.009, respectively).

https://doi.org/10.1371/journal.pone.0246600.g001

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Fig 2. MK2 inhibitor treatment lowers plasma lipids in WD-fed Ldlr^-/- mice.

Plasma total cholesterol (TC) and triglyceride (TG) levels were measured, and fast protein liquid chromatography-separated lipoproteins were assayed for cholesterol, in Ldlr^-/- mice treated with TBX-1 (A-C) or TBX-2 (D-F) as in Fig 1 (n = 29 mice/group for TBX-1 and n = 28 mice/group for TBX-2; mean ± SEM, p < 0.001, p < 0.001, p < 0.001 and p = 0.002, respectively).

https://doi.org/10.1371/journal.pone.0246600.g002

Turning to atherosclerotic lesion analysis, we first documented that MK2 activity was reduced in the atherosclerotic plaques of inhibitor-treated mice, as phospho-hsp25 levels were significantly reduced in the lesions of Ldlr^-/- mice treated with TBX-1 (Fig 3A). We then quantified the overall lesion area and necrotic area of the aortic root plaques and found that both treatments lowered total lesion area compared with untreated control mice (Fig 3B and 3C). Moreover, total necrotic area was significantly reduced in mice treated with TBX-1 and TBX-2 (Fig 3B and 3C).

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Fig 3. MK2 inhibitor treatment lowers p-hsp25, lesion size, and plaque necrosis in aortic root lesions of WD-fed Ldlr^-/- mice.

Mice were treated as in Fig 1. (A) Aortic root sections from a randomly selected subset of Ldlr^-/- mice treated with TBX-1 were co-immunostained for phospho-hsp25 (p-hsp25, green) and Mac2 (red). Scale bar: 50 μm. Phospho-hsp25 staining was quantified as mean fluorescence intensity (MFI) within Mac2⁺ area. Data are presented relative to the average value obtained from the control group (n = 10 mice/group; mean ± SEM, p = 0.001). (B-C) Total lesion area and necrotic area were quantified from the aortic root sections of Ldlr^-/- mice treated with TBX-1 (B) or TBX-2 (C). Representative images with necrotic cores outlined in black are shown. Scale bar: 150 μm (n = 29 mice/group for TBX-1 and n = 28 mice/group for TBX-2; mean ± SEM, p = 0.006, p < 0.001, p = 0.008 and p = 0.002, respectively).

https://doi.org/10.1371/journal.pone.0246600.g003

Impaired efferocytosis is a key driver of necrotic core formation in advanced atherosclerosis [18]. To determine whether MK2 inhibition improved lesional efferocytosis, we used an in-situ measure of efferocytosis in the lesions in which the ratio of macrophage-associated to free apoptotic cells is quantified. We found that both compounds significantly enhanced lesional efferocytosis, without affecting macrophage number, which could explain the decrease in plaque necrosis (Fig 4A). Fibrous-cap thickness and collagen content of the lesions, which promote plaque stability [19], were significantly increased in TBX-1‒ and TBX-2‒treated mice (Fig 4B and S3A Fig in S1 File). Of note, neither treatment affected lesional macrophage or smooth muscle cell content (S3B and S3C Fig in S1 File). Collectively, these data indicate that MK2 inhibition induces a more stable plaque phenotype with less plaque necrosis and thicker fibrous caps.

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Fig 4. MK2 inhibitor treatment enhances fibrous cap formation and lesion efferocytosis in WD-fed Ldlr^-/- mice.

Mice were treated as in Fig 1. (A) Lesional efferocytosis was quantified from aortic sections as the ratio of TUNEL⁺ cells associated with Mac2+ macrophages to free TUNEL⁺ cells in a randomly selected subset of TBX-1– or TBX-2–treated Ldlr^-/- mice (n = 7–10 mice/group; mean ± SEM, p < 0.001 and p = 0.04, respectively). Representative images of TUNEL+ apoptotic cells (green) associated with lesional macrophages (red) versus apoptotic cells not associated with macrophages (‘free’) are shown. Scale bar: 100 μm. (B) Aortic root sections from a randomly selected subset of TBX-1– or TBX-2–treated Ldlr^-/- mice were stained with picrosirius red, and fibrous cap was measured at the midpoint and shoulder regions of each lesion and quantified as the ratio of cap thickness to lesion size. Data are presented relative to the average value obtained from the control group. Representative images are shown on the left. Scale bar: 100 μm (n = 7–10 mice/group; mean ± SEM, p = 0.02 and p < 0.001, respectively).

https://doi.org/10.1371/journal.pone.0246600.g004

Discussion

In response to diverse stimuli, MK2 is activated via p38 MAPK-mediated phosphorylation, and activated MK2 plays a key role in a variety of inflammatory and metabolic diseases, including heart disease and T2D [20]. Inhibition of MK2 activity causes a partial decrease in p38 via p38 destabilization and offers a safe therapeutic advantage over p38 inhibitors [21]. We recently reported that treatment of obese insulin-resistant mice with a small-molecule inhibitor of MK2 improves metabolism and lowers blood glucose [9]. As insulin resistance and hyperglycemia are linked to the development of clinically significant unstable atherosclerotic plaques [22], and activated MK2 in lesional macrophages can impair efferocytosis and resolution [11, 12], we hypothesized that MK2 could represent a promising therapeutic target to prevent unstable plaque formation in the setting of insulin resistance. In this study, we have identified two new orally active small-molecule MK2 inhibitors, TBX-1 and TBX-2, and tested their effect on advanced atherosclerosis progression of established plaques in high fat-fed Ldlr^-/- mice. We found that both compounds promoted features of plaque stability as evidenced by lower lesion area and plaque necrosis and increased fibrous cap thickness. The consistent biological effects elicited by two chemically distinct MK2 inhibitors provides strong support for the therapeutic benefit of MK2 inhibition to ameliorate chronic inflammation in CVD.

The ability of the compounds to lower circulating levels of atherogenic apolipoprotein B-containing lipoproteins undoubtedly contributes to their atheroprotective effect. Our previous work revealed that cmpd 28 treatment of DIO mice also resulted in a trend towards lowering of plasma lipids [9]. However, it is possible that other mechanisms could have contributed to the overall lesional phenotype. Chronic unresolved inflammation is a key feature of advanced atherosclerosis. In this regard, MK2 has been shown to stimulate many proinflammatory cytokines, and MK2-deficient mice are protected from LPS-induced endotoxic shock and inflammation-driven tumorigenesis [23, 24]. Further, MK2 directly phosphorylates and regulates the nuclear localization of 5-lipoxygenase (5-LOX), which limits the synthesis of proresolving molecules and favors proinflammatory mediator formation [25, 26]. Proresolving mediators enhance lesional efferocytosis and increase fibrous cap thickness in athero-prone mice [27], and so it is possible that a direct effect of MK2 inhibition on macrophage inflammatory/proresolving phenotype could have contributed to the improved plaque stability in the mice in the current study.

The possibility of a direct anti-atherosclerosis effect of the MK2 inhibitors, i.e., independent of lipid-lowering, is supported by a study in which genetic targeting of MK2 led to decreased lesion area in fat-fed Ldlr^-/- mice, as, curiously, plasma lipids were actually higher, not lower, in the Mapkapk2^-/- Ldlr^-/- mice used in this study [14]. The authors showed that MK2 activity is enriched in endothelial cells and macrophages in atherosclerotic lesions, and they provided evidence that the atheroprotective effects of MK2 targeting might be due to suppression of macrophage foam cell formation and decreased endothelium-mediated recruitment of monocytes into lesions. In that study, parameters of glucose metabolism and plaque stability were not reported. Although the difference in lipoprotein effects between that study and ours could be due to an MK2-independent mechanism of lipoprotein lowering by the class of compounds used here, another explanation could be the difference in study design. In the previous study, MK2 was completely absent before the high fat diet was begun. Moreover, germline gene targeting can sometimes lead to unexpected compensatory changes in the expression of other genes, leading to phenotypic changes not directly related to the targeted gene [28]. In our study, MK2 was inhibited, not completely deleted, 10 weeks after the high-fat diet was begun in mice with genetically intact MK2. Furthermore, given the high sequence identity (75%) between MK2 and MK3, it is reasonable to expect that both TBX-1 and TBX-2 inhibit MK2 and MK3 with similar efficiency, as the two molecules share a common pharmacophore [29, 30]. Although a previous study reported that MK3 expression was not increased in MK2-deficient cells [31], residual MK3 activity could have effects in Mapkapk2^-/- mice. Indeed, phenotypic differences between Mapkapk2^-/- and Mapkapk2^-/-Mapkapk3^-/- cells have been documented [32].

A growing body of evidence, including causation studies in mice and correlative studies in human disease tissues, supports a role for MK2 activation in the pathogenesis of chronic inflammatory diseases, including CVD [20]. In addition to the current study and the aforementioned atherosclerosis study using Mapkapk2^-/- mice [14], other studies have shown that MK2 inhibition can protect against ischemia/reperfusion injury and cardiac hypertrophy [33, 34], including in a diabetic setting [10]. These studies have used genetic targeting of MK2, and the cardiac benefits of MK2 inhibition in these studies were not linked to benefits in metabolism. We have shown that a previously developed non-ATP-competitive allosteric MK2 inhibitor with high specificity, cmpd 28, can improve glucose metabolism in obese mice [9], and now we show that two new inhibitors have benefits for both metabolism and atherosclerosis. This demonstration that allosteric MK2 inhibitors can have combined metabolic and vascular benefits in preclinical models should help advance their testing in cardiometabolic disease in humans.

Supporting information

S1 Raw images.

https://doi.org/10.1371/journal.pone.0246600.s001

(PDF)

S1 File.

https://doi.org/10.1371/journal.pone.0246600.s002

(PDF)

References

1. Barnes AS. The epidemic of obesity and diabetes: trends and treatments. Tex Heart Inst J. 2011;38(2):142–4. pmid:21494521
- View Article
- PubMed/NCBI
- Google Scholar
2. Martin-Timon I, Sevillano-Collantes C, Segura-Galindo A, Del Canizo-Gomez FJ. Type 2 diabetes and cardiovascular disease: Have all risk factors the same strength? World J Diabetes. 2014;5(4):444–70. pmid:25126392
- View Article
- PubMed/NCBI
- Google Scholar
3. Low Wang CC, Hess CN, Hiatt WR, Goldfine AB. Clinical Update: Cardiovascular Disease in Diabetes Mellitus: Atherosclerotic Cardiovascular Disease and Heart Failure in Type 2 Diabetes Mellitus—Mechanisms, Management, and Clinical Considerations. Circulation. 2016;133(24):2459–502. pmid:27297342
- View Article
- PubMed/NCBI
- Google Scholar
4. Azimova K, San Juan Z, Mukherjee D. Cardiovascular safety profile of currently available diabetic drugs. Ochsner J. 2014;14(4):616–32. pmid:25598727
- View Article
- PubMed/NCBI
- Google Scholar
5. Secrest MH, Udell JA, Filion KB. The cardiovascular safety trials of DPP-4 inhibitors, GLP-1 agonists, and SGLT2 inhibitors. Trends Cardiovasc Med. 2017;27(3):194–202. pmid:28291655
- View Article
- PubMed/NCBI
- Google Scholar
6. Ozcan L, Wong CC, Li G, Xu T, Pajvani U, Park SK, et al. Calcium signaling through CaMKII regulates hepatic glucose production in fasting and obesity. Cell Metab. 2012;15(5):739–51. pmid:22503562
- View Article
- PubMed/NCBI
- Google Scholar
7. Ozcan L, Cristina de Souza J, Harari AA, Backs J, Olson EN, Tabas I. Activation of calcium/calmodulin-dependent protein kinase II in obesity mediates suppression of hepatic insulin signaling. Cell Metab. 2013;18(6):803–15. pmid:24268736
- View Article
- PubMed/NCBI
- Google Scholar
8. Ozcan L, Ghorpade DS, Zheng Z, de Souza JC, Chen K, Bessler M, et al. Hepatocyte DACH1 Is Increased in Obesity via Nuclear Exclusion of HDAC4 and Promotes Hepatic Insulin Resistance. Cell Rep. 2016;15(10):2214–25. pmid:27239042
- View Article
- PubMed/NCBI
- Google Scholar
9. Ozcan L, Xu X, Deng SX, Ghorpade DS, Thomas T, Cremers S, et al. Treatment of Obese Insulin-Resistant Mice With an Allosteric MAPKAPK2/3 Inhibitor Lowers Blood Glucose and Improves Insulin Sensitivity. Diabetes. 2015;64(10):3396–405. pmid:26068544
- View Article
- PubMed/NCBI
- Google Scholar
10. Ruiz M, Coderre L, Lachance D, Houde V, Martel C, Thompson Legault J, et al. MK2 Deletion in Mice Prevents Diabetes-Induced Perturbations in Lipid Metabolism and Cardiac Dysfunction. Diabetes. 2016;65(2):381–92. pmid:26558681
- View Article
- PubMed/NCBI
- Google Scholar
11. Doran AC, Ozcan L, Cai B, Zheng Z, Fredman G, Rymond CC, et al. CAMKIIgamma suppresses an efferocytosis pathway in macrophages and promotes atherosclerotic plaque necrosis. J Clin Invest. 2017;127(11):4075–89. pmid:28972541
- View Article
- PubMed/NCBI
- Google Scholar
12. Tao W, Yurdagul A Jr., Kong N, Li W, Wang X, Doran AC, et al. siRNA nanoparticles targeting CaMKIIgamma in lesional macrophages improve atherosclerotic plaque stability in mice. Sci Transl Med. 2020;12(553). pmid:32718990
- View Article
- PubMed/NCBI
- Google Scholar
13. Razani B, Chakravarthy MV, Semenkovich CF. Insulin resistance and atherosclerosis. Endocrinol Metab Clin North Am. 2008;37(3):603–21, viii. pmid:18775354
- View Article
- PubMed/NCBI
- Google Scholar
14. Jagavelu K, Tietge UJ, Gaestel M, Drexler H, Schieffer B, Bavendiek U. Systemic deficiency of the MAP kinase-activated protein kinase 2 reduces atherosclerosis in hypercholesterolemic mice. Circ Res. 2007;101(11):1104–12. pmid:17885219
- View Article
- PubMed/NCBI
- Google Scholar
15. McComas CC S-W, MH, Vacca JP, inventor; Tabomedex Biosciences LLC, assignee. Fused quadracyclic compounds, compositions and uses thereof. United States 2020.
16. Spolitu S, Okamoto H, Dai W, Zadroga JA, Wittchen ES, Gromada J, et al. Hepatic Glucagon Signaling Regulates PCSK9 and Low-Density Lipoprotein Cholesterol. Circ Res. 2019;124(1):38–51. pmid:30582457
- View Article
- PubMed/NCBI
- Google Scholar
17. Thorp E, Cui D, Schrijvers DM, Kuriakose G, Tabas I. Mertk receptor mutation reduces efferocytosis efficiency and promotes apoptotic cell accumulation and plaque necrosis in atherosclerotic lesions of apoe-/- mice. Arterioscler Thromb Vasc Biol. 2008;28(8):1421–8. pmid:18451332
- View Article
- PubMed/NCBI
- Google Scholar
18. Doran AC, Yurdagul A Jr., Tabas I. Efferocytosis in health and disease. Nat Rev Immunol. 2020;20(4):254–67. pmid:31822793
- View Article
- PubMed/NCBI
- Google Scholar
19. Finn AV, Nakano M, Narula J, Kolodgie FD, Virmani R. Concept of vulnerable/unstable plaque. Arterioscler Thromb Vasc Biol. 2010;30(7):1282–92. pmid:20554950
- View Article
- PubMed/NCBI
- Google Scholar
20. Ruiz M, Coderre L, Allen BG, Des Rosiers C. Protecting the heart through MK2 modulation, toward a role in diabetic cardiomyopathy and lipid metabolism. Biochim Biophys Acta Mol Basis Dis. 2018;1864(5 Pt B):1914–22. pmid:28735097
- View Article
- PubMed/NCBI
- Google Scholar
21. Fiore M, Forli S, Manetti F. Targeting Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MAPKAPK2, MK2): Medicinal Chemistry Efforts To Lead Small Molecule Inhibitors to Clinical Trials. J Med Chem. 2016;59(8):3609–34. pmid:26502061
- View Article
- PubMed/NCBI
- Google Scholar
22. Bornfeldt KE, Tabas I. Insulin resistance, hyperglycemia, and atherosclerosis. Cell Metab. 2011;14(5):575–85. pmid:22055501
- View Article
- PubMed/NCBI
- Google Scholar
23. Kotlyarov A, Neininger A, Schubert C, Eckert R, Birchmeier C, Volk HD, et al. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat Cell Biol. 1999;1(2):94–7. pmid:10559880
- View Article
- PubMed/NCBI
- Google Scholar
24. Suarez-Lopez L, Sriram G, Kong YW, Morandell S, Merrick KA, Hernandez Y, et al. MK2 contributes to tumor progression by promoting M2 macrophage polarization and tumor angiogenesis. Proc Natl Acad Sci U S A. 2018;115(18):E4236–E44. pmid:29666270
- View Article
- PubMed/NCBI
- Google Scholar
25. Werz O, Szellas D, Steinhilber D, Radmark O. Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2). J Biol Chem. 2002;277(17):14793–800. pmid:11844797
- View Article
- PubMed/NCBI
- Google Scholar
26. Fredman G, Ozcan L, Spolitu S, Hellmann J, Spite M, Backs J, et al. Resolvin D1 limits 5-lipoxygenase nuclear localization and leukotriene B4 synthesis by inhibiting a calcium-activated kinase pathway. Proc Natl Acad Sci U S A. 2014;111(40):14530–5. pmid:25246560
- View Article
- PubMed/NCBI
- Google Scholar
27. Fredman G, Hellmann J, Proto JD, Kuriakose G, Colas RA, Dorweiler B, et al. An imbalance between specialized pro-resolving lipid mediators and pro-inflammatory leukotrienes promotes instability of atherosclerotic plaques. Nat Commun. 2016;7:12859. pmid:27659679
- View Article
- PubMed/NCBI
- Google Scholar
28. El-Brolosy MA, Stainier DYR. Genetic compensation: A phenomenon in search of mechanisms. PLoS Genet. 2017;13(7):e1006780. pmid:28704371
- View Article
- PubMed/NCBI
- Google Scholar
29. Gaestel M. MAPKAP kinases—MKs—two’s company, three’s a crowd. Nat Rev Mol Cell Biol. 2006;7(2):120–30. pmid:16421520
- View Article
- PubMed/NCBI
- Google Scholar
30. Lindin I, Wuxiuer Y, Kufareva I, Abagyan R, Moens U, Sylte I, et al. Homology modeling and ligand docking of Mitogen-activated protein kinase-activated protein kinase 5 (MK5). Theor Biol Med Model. 2013;10:56. pmid:24034446
- View Article
- PubMed/NCBI
- Google Scholar
31. Ronkina N, Kotlyarov A, Dittrich-Breiholz O, Kracht M, Hitti E, Milarski K, et al. The mitogen-activated protein kinase (MAPK)-activated protein kinases MK2 and MK3 cooperate in stimulation of tumor necrosis factor biosynthesis and stabilization of p38 MAPK. Mol Cell Biol. 2007;27(1):170–81. pmid:17030606
- View Article
- PubMed/NCBI
- Google Scholar
32. Ehlting C, Ronkina N, Bohmer O, Albrecht U, Bode KA, Lang KS, et al. Distinct functions of the mitogen-activated protein kinase-activated protein (MAPKAP) kinases MK2 and MK3: MK2 mediates lipopolysaccharide-induced signal transducers and activators of transcription 3 (STAT3) activation by preventing negative regulatory effects of MK3. J Biol Chem. 2011;286(27):24113–24. pmid:21586572
- View Article
- PubMed/NCBI
- Google Scholar
33. Ashraf MI, Ebner M, Wallner C, Haller M, Khalid S, Schwelberger H, et al. A p38MAPK/MK2 signaling pathway leading to redox stress, cell death and ischemia/reperfusion injury. Cell Commun Signal. 2014;12:6. pmid:24423080
- View Article
- PubMed/NCBI
- Google Scholar
34. Streicher JM, Ren S, Herschman H, Wang Y. MAPK-activated protein kinase-2 in cardiac hypertrophy and cyclooxygenase-2 regulation in heart. Circ Res. 2010;106(8):1434–43. pmid:20339119
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Barnes AS. The epidemic of obesity and diabetes: trends and treatments. Tex Heart Inst J. 2011;38(2):142–4. pmid:21494521
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Martin-Timon I, Sevillano-Collantes C, Segura-Galindo A, Del Canizo-Gomez FJ. Type 2 diabetes and cardiovascular disease: Have all risk factors the same strength? World J Diabetes. 2014;5(4):444–70. pmid:25126392
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Low Wang CC, Hess CN, Hiatt WR, Goldfine AB. Clinical Update: Cardiovascular Disease in Diabetes Mellitus: Atherosclerotic Cardiovascular Disease and Heart Failure in Type 2 Diabetes Mellitus—Mechanisms, Management, and Clinical Considerations. Circulation. 2016;133(24):2459–502. pmid:27297342
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Azimova K, San Juan Z, Mukherjee D. Cardiovascular safety profile of currently available diabetic drugs. Ochsner J. 2014;14(4):616–32. pmid:25598727
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Secrest MH, Udell JA, Filion KB. The cardiovascular safety trials of DPP-4 inhibitors, GLP-1 agonists, and SGLT2 inhibitors. Trends Cardiovasc Med. 2017;27(3):194–202. pmid:28291655
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Ozcan L, Wong CC, Li G, Xu T, Pajvani U, Park SK, et al. Calcium signaling through CaMKII regulates hepatic glucose production in fasting and obesity. Cell Metab. 2012;15(5):739–51. pmid:22503562
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Ozcan L, Cristina de Souza J, Harari AA, Backs J, Olson EN, Tabas I. Activation of calcium/calmodulin-dependent protein kinase II in obesity mediates suppression of hepatic insulin signaling. Cell Metab. 2013;18(6):803–15. pmid:24268736
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Ozcan L, Ghorpade DS, Zheng Z, de Souza JC, Chen K, Bessler M, et al. Hepatocyte DACH1 Is Increased in Obesity via Nuclear Exclusion of HDAC4 and Promotes Hepatic Insulin Resistance. Cell Rep. 2016;15(10):2214–25. pmid:27239042
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Ozcan L, Xu X, Deng SX, Ghorpade DS, Thomas T, Cremers S, et al. Treatment of Obese Insulin-Resistant Mice With an Allosteric MAPKAPK2/3 Inhibitor Lowers Blood Glucose and Improves Insulin Sensitivity. Diabetes. 2015;64(10):3396–405. pmid:26068544
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Ruiz M, Coderre L, Lachance D, Houde V, Martel C, Thompson Legault J, et al. MK2 Deletion in Mice Prevents Diabetes-Induced Perturbations in Lipid Metabolism and Cardiac Dysfunction. Diabetes. 2016;65(2):381–92. pmid:26558681
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Doran AC, Ozcan L, Cai B, Zheng Z, Fredman G, Rymond CC, et al. CAMKIIgamma suppresses an efferocytosis pathway in macrophages and promotes atherosclerotic plaque necrosis. J Clin Invest. 2017;127(11):4075–89. pmid:28972541
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Tao W, Yurdagul A Jr., Kong N, Li W, Wang X, Doran AC, et al. siRNA nanoparticles targeting CaMKIIgamma in lesional macrophages improve atherosclerotic plaque stability in mice. Sci Transl Med. 2020;12(553). pmid:32718990
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Razani B, Chakravarthy MV, Semenkovich CF. Insulin resistance and atherosclerosis. Endocrinol Metab Clin North Am. 2008;37(3):603–21, viii. pmid:18775354
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Jagavelu K, Tietge UJ, Gaestel M, Drexler H, Schieffer B, Bavendiek U. Systemic deficiency of the MAP kinase-activated protein kinase 2 reduces atherosclerosis in hypercholesterolemic mice. Circ Res. 2007;101(11):1104–12. pmid:17885219
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. McComas CC S-W, MH, Vacca JP, inventor; Tabomedex Biosciences LLC, assignee. Fused quadracyclic compounds, compositions and uses thereof. United States 2020.

[ref16] 16. Spolitu S, Okamoto H, Dai W, Zadroga JA, Wittchen ES, Gromada J, et al. Hepatic Glucagon Signaling Regulates PCSK9 and Low-Density Lipoprotein Cholesterol. Circ Res. 2019;124(1):38–51. pmid:30582457
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref17] 17. Thorp E, Cui D, Schrijvers DM, Kuriakose G, Tabas I. Mertk receptor mutation reduces efferocytosis efficiency and promotes apoptotic cell accumulation and plaque necrosis in atherosclerotic lesions of apoe-/- mice. Arterioscler Thromb Vasc Biol. 2008;28(8):1421–8. pmid:18451332
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref18] 18. Doran AC, Yurdagul A Jr., Tabas I. Efferocytosis in health and disease. Nat Rev Immunol. 2020;20(4):254–67. pmid:31822793
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref19] 19. Finn AV, Nakano M, Narula J, Kolodgie FD, Virmani R. Concept of vulnerable/unstable plaque. Arterioscler Thromb Vasc Biol. 2010;30(7):1282–92. pmid:20554950
View Article
PubMed/NCBI
Google Scholar

[71] View Article

[72] PubMed/NCBI

[73] Google Scholar

[ref20] 20. Ruiz M, Coderre L, Allen BG, Des Rosiers C. Protecting the heart through MK2 modulation, toward a role in diabetic cardiomyopathy and lipid metabolism. Biochim Biophys Acta Mol Basis Dis. 2018;1864(5 Pt B):1914–22. pmid:28735097
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref21] 21. Fiore M, Forli S, Manetti F. Targeting Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MAPKAPK2, MK2): Medicinal Chemistry Efforts To Lead Small Molecule Inhibitors to Clinical Trials. J Med Chem. 2016;59(8):3609–34. pmid:26502061
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref22] 22. Bornfeldt KE, Tabas I. Insulin resistance, hyperglycemia, and atherosclerosis. Cell Metab. 2011;14(5):575–85. pmid:22055501
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref23] 23. Kotlyarov A, Neininger A, Schubert C, Eckert R, Birchmeier C, Volk HD, et al. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat Cell Biol. 1999;1(2):94–7. pmid:10559880
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref24] 24. Suarez-Lopez L, Sriram G, Kong YW, Morandell S, Merrick KA, Hernandez Y, et al. MK2 contributes to tumor progression by promoting M2 macrophage polarization and tumor angiogenesis. Proc Natl Acad Sci U S A. 2018;115(18):E4236–E44. pmid:29666270
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref25] 25. Werz O, Szellas D, Steinhilber D, Radmark O. Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2). J Biol Chem. 2002;277(17):14793–800. pmid:11844797
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref26] 26. Fredman G, Ozcan L, Spolitu S, Hellmann J, Spite M, Backs J, et al. Resolvin D1 limits 5-lipoxygenase nuclear localization and leukotriene B4 synthesis by inhibiting a calcium-activated kinase pathway. Proc Natl Acad Sci U S A. 2014;111(40):14530–5. pmid:25246560
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref27] 27. Fredman G, Hellmann J, Proto JD, Kuriakose G, Colas RA, Dorweiler B, et al. An imbalance between specialized pro-resolving lipid mediators and pro-inflammatory leukotrienes promotes instability of atherosclerotic plaques. Nat Commun. 2016;7:12859. pmid:27659679
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref28] 28. El-Brolosy MA, Stainier DYR. Genetic compensation: A phenomenon in search of mechanisms. PLoS Genet. 2017;13(7):e1006780. pmid:28704371
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref29] 29. Gaestel M. MAPKAP kinases—MKs—two’s company, three’s a crowd. Nat Rev Mol Cell Biol. 2006;7(2):120–30. pmid:16421520
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref30] 30. Lindin I, Wuxiuer Y, Kufareva I, Abagyan R, Moens U, Sylte I, et al. Homology modeling and ligand docking of Mitogen-activated protein kinase-activated protein kinase 5 (MK5). Theor Biol Med Model. 2013;10:56. pmid:24034446
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref31] 31. Ronkina N, Kotlyarov A, Dittrich-Breiholz O, Kracht M, Hitti E, Milarski K, et al. The mitogen-activated protein kinase (MAPK)-activated protein kinases MK2 and MK3 cooperate in stimulation of tumor necrosis factor biosynthesis and stabilization of p38 MAPK. Mol Cell Biol. 2007;27(1):170–81. pmid:17030606
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref32] 32. Ehlting C, Ronkina N, Bohmer O, Albrecht U, Bode KA, Lang KS, et al. Distinct functions of the mitogen-activated protein kinase-activated protein (MAPKAP) kinases MK2 and MK3: MK2 mediates lipopolysaccharide-induced signal transducers and activators of transcription 3 (STAT3) activation by preventing negative regulatory effects of MK3. J Biol Chem. 2011;286(27):24113–24. pmid:21586572
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref33] 33. Ashraf MI, Ebner M, Wallner C, Haller M, Khalid S, Schwelberger H, et al. A p38MAPK/MK2 signaling pathway leading to redox stress, cell death and ischemia/reperfusion injury. Cell Commun Signal. 2014;12:6. pmid:24423080
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref34] 34. Streicher JM, Ren S, Herschman H, Wang Y. MAPK-activated protein kinase-2 in cardiac hypertrophy and cyclooxygenase-2 regulation in heart. Circ Res. 2010;106(8):1434–43. pmid:20339119
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Synthesis of MK2 inhibitors

Mouse experiments

Pharmacokinetic studies

Plasma cholesterol and triglyceride measurements

Murine atherosclerotic lesion analysis

In situ efferocytosis assay

Primary hepatocytes

Reagents and antibodies

Immunoblotting and quantitative RT-PCR

Statistics

Results

Discussion

Supporting information

S1 Raw images.

S1 File.

References