Fig 1.
MK2 essentially regulates blood flow recovery after hind limb ischemia and growth of collateral arteries during postnatal arteriogenesis.
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 (*p< 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), #p< 0.05 control vs. HLI, *p< 0.01 WT vs. MK2KO, n = 6).
Fig 2.
MK2 is essential for the vascular recruitment of macrophages and expression of MCP-1 during postnatal arteriogenesis.
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).
Fig 3.
MK2 is activated in the endothelium of collateral arteries and mediates the expression of MCP-1 induced by pro-inflammatory cytokines as well as cyclic stretch in endothelial cells.
A, after hind limb ischemia (6 h after arterial ligation) sections of collateral arteries from the ischemic (HLI) and the contralateral non-ischemic limb (control) of wildtype mice (WT) and MK2-deficient mice (MK2KO) were stained for activated phospho-MK2 and analyzed by fluorescence laser scanning confocal microscopy (phopho-MK2, green), endothelial cells (red, CD31), nuclear staining (DAPI, blue). Representative stainings of at least 3 different experiments are shown. B-E, cultured primary murine endothelial cells isolated from WT and MK2KO mice were stimulated with IL-1β (10 ng/ml), TNF-α (50 ng/ml) or cyclic stretch (0.5 Hz, 10% elongation) for 0–60 min. (B-C) or 6 h (D-E). Activation of MK2 was determined by immunoblotting for phospho-MK2 (B-C) or release of MCP-1 protein was quantified by ELISA in cell culture supernatants (D-E). Blots of experiments employing endothelial cell isolations from 5 different mice of each genotype are shown (B-C). #p< 0.05 control vs. cytokine or cyclic stretch, *p< 0.05 WT vs. MK2KO, n = 4–6).
Fig 4.
MK2 is activated in monocytes recruited to collateral arteries and mediates MCP-1 induced monocyte migration.
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). #p< 0.05 control vs. MCP-1, *p< 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.
Fig 5.
Model of MK2-regulated postnatal arteriogenesis.
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.