Basal characteristics of HFD-fed WT and MK2^−/− mice.

<p>*p<0.05,</p><p>**p<0.01 vs. WT controls.</p><p>Data are presented as means ± SEM (n = 8 animals per group).</p><p>Basal characteristics of HFD-fed WT and MK2^−/− mice.</p

High-fat diet-fed <i>MK2</i>^−/− mice are insulin resistant.

<p>High-fat diet (HFD) fed wild-type (WT) and <i>MK2^−/−</i> mice were injected intraperitoneally (i.p.) with glucose (1.25 g/kg) and blood glucose levels were measured at the indicated time points (A). Several days later, mice were injected i.p. with insulin (0.6 U/kg) and blood glucose levels were determined at the indicated time points. Blood glucose levels during the insulin tolerance test (ITT) are depicted as percentage of initial blood glucose levels (B). ΔArea under the curve was calculated by subtracting the area under the curve of an individual mouse by the area under a hypothetical curve that remained at 100% throughout the duration of the test (B, insert). Fasting insulin levels were determined using an ultra-sensitive mouse insulin ELISA (C). *p<0.05, **p<0.01 compared to WT mice (n = 8 mice per group).</p

High-fat diet-fed <i>MK2^−/−</i> mice have reduced adipose tissue expression of the insulin-responsive glucose transporter GLUT4.

<p>Adipose tissue <i>Glut4</i> mRNA expression (n = 8 mice per group) was measured by real-time qPCR (A). Western blot was performed (n = 5 mice per group) to determine GLUT4 protein expression in adipose tissue; GAPDH served as loading control (B); band intensities were quantified using ImageJ software and normalized to the mean intensity of the wild-type (WT) control mice (C). *p<0.05 compared to WT control mice.</p

Impact of MK2-deficiency on adipose tissue inflammation.

<p>Abdominal fat was excised from mice that were fed a high-fat diet (HFD) for 24 weeks (n = 8 mice per group). Real-time qPCR was performed to measure mRNA expression of inflammation-related genes in adipose tissue (A). Formalin-fixed paraffin-embedded tissue sections were stained with hematoxyline and eosine to assess infiltration of immune cells in adipose tissue and sections from 5 randomly selected animals of each group were used to quantify the amount of crown-like structures. At least 300 adipocytes were counted per animal (B). Adipocyte size distribution was assessed by measuring the surface of ∼100 adipocytes per animal using freely available ImageJ software (imagej.nih.gov) and sections from 5 randomly selected animals of each group were used for quantification of adipocyte size (C).</p

MAP-Kinase Activated Protein Kinase 2 Links Endothelial Activation and Monocyte/macrophage Recruitment in Arteriogenesis

<div><p>Arteriogenesis, the growth of natural bypass arteries, is triggered by hemodynamic forces within vessels and requires a balanced inflammatory response, involving induction of the chemokine MCP-1 and recruitment of leukocytes. However, little is known how these processes are coordinated. The MAP-kinase-activated-proteinkinase-2 (MK2) is a critical regulator of inflammatory processes and might represent an important link between cytokine production and cell recruitment during postnatal arteriogenesis. Therefore, the present study investigated the functional role of MK2 during postnatal arteriogenesis. In a mouse model of hindlimb ischemia (HLI) MK2-deficiency (MK2KO) significantly impaired ischemic blood flow recovery and growth of collateral arteries as well as perivascular recruitment of mononuclear cells and macrophages. This was accompanied by induction of endothelial MCP-1 expression in wildtype (WT) but not in MK2KO collateral arteries. Following HLI, MK2 activation rapidly occured in the endothelium of growing WT arteries <i>in vivo</i>. <i>In vitro</i>, inflammatory cytokines and cyclic stretch activated MK2 in endothelial cells, which was required for stretch- and cytokine-induced release of MCP-1. In addition, a monocyte cell autonomous function of MK2 was uncovered potentially regulating MCP-1-dependent monocyte recruitment to vessels: MCP-1 stimulation of WT monocytes induced MK2 activation and monocyte migration <i>in vitro</i>. The latter was reduced in MK2KO monocytes, while <i>in vivo</i> MK2 was activated in monocytes recruited to collateral arteries. In conclusion, MK2 regulates postnatal arteriogenesis by controlling vascular recruitment of monocytes/macrophages in a dual manner: regulation of endothelial MCP-1 expression in response to hemodynamic and inflammatory forces as well as MCP-1 dependent monocyte migration.</p></div

MK2 is activated in monocytes recruited to collateral arteries and mediates MCP-1 induced monocyte migration.

<p>A, Primary murine monocytes isolated from WT mice were stimulated with MCP-1 (10 ng/ml) or LPS (100ng/ml) for the indicated times and activation of MK2 was determined by immunoblotting for phospho-MK2. Representative blots of two independent experiments each employing monocyte isolations from 3 different mice are shown. B, Primary murine monocytes isolated from WT and MK2KO were given into the upper chamber of a transwell cell culture dish and number of cells migrated towards MCP-1 (10 ng/ml) in the lower chamber was quantified after 24 h and normalized to the total number of cells given into the upper chamber (migration-index). #<i>p</i>< 0.05 control vs. MCP-1, *<i>p</i>< 0.05 WT vs. MK2KO, n = 5). C, after hind limb ischemia (6 h after arterial ligation) sections of collateral arteries from the ischemic limb of WT mice were stained for activated phospho-MK2 and analyzed by fluorescence laser scanning confocal microscopy (phospho-MK2, green; endothelial cells, red, CD31; nuclear staining, blue, DAPI). Representative stainings of at least 3 different experiments are shown.</p

MK2 essentially regulates blood flow recovery after hind limb ischemia and growth of collateral arteries during postnatal arteriogenesis.

<p>A, blood flow was determined by laser-doppler-blood flow analysis in wildtype (WT) and MK2-deficient (MK2KO) mice after HLI at the indicated times (day 0 to 21 after arterial ligation). Perfusion is given as relative ischemic perfusion compared to blood flow before arterial ligation (*<i>p</i>< 0.01 WT vs. MK2KO, n = 11). B-E, after HLI (day 21 post arterial ligation) sections (B, H & E staining) of collateral arteries of the ischemic (HLI) and contralateral non-ischemic limb (CTRL) from WT and MK2KO were analyzed by histomorphometry for diameter (C), wall area (D) and number of perivascular mononuclear cells (E). (C-E), #<i>p</i>< 0.05 control vs. HLI, *<i>p</i>< 0.01 WT vs. MK2KO, n = 6).</p

MK2 is essential for the vascular recruitment of macrophages and expression of MCP-1 during postnatal arteriogenesis.

<p>A-B, after hind limb ischemia sections of collateral arteries from the ischemic limb from WT and MK2KO were stained for macrophages (A) or MCP-1 (B) (red, F4/80 or MCP-1), α-smooth muscle cell actin (green, SMA) and cell nuclei (blue, DAPI) and analyzed by fluorescence laser scanning confocal microscopy. Representative staining of at least 3 different experiments (day 3 post arterial ligation) are shown. C, mRNA-expression of MCP-1 was determined in hind limb muscle from wildtype (WT) mice at baseline (bsl) and 6 h, day 1 (d1), days 3 (d3), and day 7 (d7) after HLI. Relative mRNA-expression determined by qRT-PCR is shown (*p< 0.05 baseline vs. day 1, n = 4–5). D, mRNA-expression of MCP-1 was determined in hind limb tissue from wildtype (WT) and MK2-deficient mice (MK2KO) at day 1 (d1) after HLI (*p< 0.01 WT vs. MK2KO, n = 6).</p

Model of MK2-regulated postnatal arteriogenesis.

<p>MK2 controls vascular recruitment of monocytes/macrophages during postnatal arteriogenesis in a dual manner: regulation of endothelial MCP-1 expression in response to hemodynamic and inflammatory forces as well as MCP-1-dependent monocyte migration.</p