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. 2015 Dec;35(12):2594-604.
doi: 10.1161/ATVBAHA.115.305857. Epub 2015 Oct 8.

Deletion of Methionine Sulfoxide Reductase A Does Not Affect Atherothrombosis but Promotes Neointimal Hyperplasia and Extracellular Signal-Regulated Kinase 1/2 Signaling

Paula J Klutho  1 , Steven M Pennington  1 , Jason A Scott  1 , Katina M Wilson  1 , Sean X Gu  1 , Prakash Doddapattar  1 , Litao Xie  1 , Ashlee N Venema  1 , Linda J Zhu  1 , Anil K Chauhan  1 , Steven R Lentz  1 , Isabella M Grumbach  2
Affiliations

Deletion of Methionine Sulfoxide Reductase A Does Not Affect Atherothrombosis but Promotes Neointimal Hyperplasia and Extracellular Signal-Regulated Kinase 1/2 Signaling

Paula J Klutho et al. Arterioscler Thromb Vasc Biol. 2015 Dec.

Abstract

Objective: Emerging evidence suggests that methionine oxidation can directly affect protein function and may be linked to cardiovascular disease. The objective of this study was to define the role of the methionine sulfoxide reductase A (MsrA) in models of vascular disease and identify its signaling pathways.

Approach and results: MsrA was readily identified in all layers of the vascular wall in human and murine arteries. Deletion of the MsrA gene did not affect atherosclerotic lesion area in apolipoprotein E-deficient mice and had no significant effect on susceptibility to experimental thrombosis after photochemical injury. In contrast, the neointimal area after vascular injury caused by complete ligation of the common carotid artery was significantly greater in MsrA-deficient than in control mice. In aortic vascular smooth muscle cells lacking MsrA, cell proliferation was significantly increased because of accelerated G1/S transition. In parallel, cyclin D1 protein and cdk4/cyclin D1 complex formation and activity were increased in MsrA-deficient vascular smooth muscle cell, leading to enhanced retinoblastoma protein phosphorylation and transcription of E2F. Finally, MsrA-deficient vascular smooth muscle cell exhibited greater activation of extracellular signal-regulated kinase 1/2 that was caused by increased activity of the Ras/Raf/mitogen-activated protein kinase signaling pathway.

Conclusions: Our findings implicate MsrA as a negative regulator of vascular smooth muscle cell proliferation and neointimal hyperplasia after vascular injury through control of the Ras/Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 signaling pathway.

Keywords: ERK pathway; methionine sulfoxide reductase; neointima; oxidation-reduction; proliferation.

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Figures

Figure 1
Figure 1. MsrA deletion does not affect atherosclerotic lesion size or susceptibility to experimental thrombosis
A) Immunofluorescence for MsrA in human non-atherosclerotic and atherosclerotic coronary arteries showing expression in all layers. I=Intima, M=Media. Inset shows 63x magnification. MsrA=green, SMA (Smooth muscle actin)=red, ToPro-3 (Nucleus)=Blue B) Total cholesterol levels in MsrA+/+ or MsrA−/− mice in ApoE−/− background following 15-17 weeks of control or Western diet (n=12-24). C) and D) Quantification and representative images of percent lesion area (Oil red O area/total area; n=8-11 per group). Scale bar = 500 μm. E) and F) Representative images and quantification of atherosclerotic lesion area in aortic sinus. G) Time to first occlusion and H) stable occlusion of the carotid artery following photochemical injury (n=7-13). I) Activated protein C levels following thrombin infusion (n=6-9). *p<0.05 compared to control diet.
Figure 2
Figure 2. Deletion of MsrA significantly increases neointimal formation
A) Expression of MsrA in MsrA+/+ mouse carotid arteries at Day 0 (Non-ligated) and Day 14 after ligation (Ligated). MsrA is detectable in the endothelium, vessel wall, adventitia and neointima. MsrA=green, SMA (Smooth muscle actin) = red, ToPro-3 (Nucleus) = blue. B) Quantification of MsrA signal intensity in the media at Day 0 and in the media and neointimal at Day 14. C) H&E staining and (D) quantification of neointimal area in arteries from MsrA+/+ or MsrA−/− mice. Scale bar = 200μm *p<0.05 vs. MsrA+/+ (n=6-9).
Figure 3
Figure 3. Deletion of MsrA accelerates proliferation of VSMC
A) Left panels, representative images of BrdU staining of carotid artery sections from MsrA+/+ and MsrA−/− mice 14 days after carotid ligation. Right panels, quantification of number and percentage of BrdU positive cells within the neointima (n=6-10). B) Quantification of the number of BrdU positive cells in the medial layer 14 days after carotid ligation (n=5-9). C) MsrA expression by immunofluorescence in MsrA+/+ VSMC. MsrA= green, Nuclei (ToPro-3) = blue. D) 3H-Thymidine uptake in MsrA+/+ and MsrA−/− VSMC (n=12). E) Cell counts of MsrA+/+ and MsrA−/− VSMC (n=3). F) Cell cycle analysis of MsrA+/+ and MsrA−/− VSMC when growth arrested (0hr) and 6 hr and 24hr after release from growth arrest. Left panels, representative flow cytometry images; right panel, distribution of cells in phases of the cell cycle (n=6-11). * p<0.05 vs. MsrA+/+; ** p <0.01 vs. MsrA+/+. # = p<0.05 vs. growth arrested (0 hr).
Figure 4
Figure 4. MsrA controls expression of cell cycle regulators in proliferating VSMC
A) Western blot and quantification of cell cycle regulators in proliferating MsrA+/+ and MsrA−/− VSMC. (n=7-12). B) mRNA levels of cyclin D isoforms in MsrA+/+ and MsrA−/− VSMC by qRT-PCR. (n=3-4) C) Western blot and densitometry of cyclin D1 protein in MsrA−/− and MsrA+/+ VSMC at growth arrest (left panel) and 24 h after release from growth arrest (right panel). *p < 0.05 **p<0.001 vs. MsrA+/+.
Figure 5
Figure 5. Cyclin D activity is increased in MsrA−/− VSMC
A) Analysis of cyclin D1/CDK4 complex formation by immunoprecipitation with anti-CDK4 and Western blot with anti-cyclin D1. Left panel, representative blots; right panel, quantification of cyclin D1/CDK4 complex formation in MsrA−/− VSMC relative to MsrA+/+(normalized to total CDK4 levels; n=3). B) Western blot and quantification of Rb phosphorylation at S780 (pRb) in proliferating MsrA+/+ and MsrA−/− VSMC (n=9). C) In vitro kinase assay for CDK4 activity as determined by Rb phosphorylation (n=4). D) mRNA levels of E2F1, E2F2, and E2F3 in MsrA+/+ and MsrA−/− VSMC (n=6-15).
Figure 6
Figure 6. ERK activity is upregulated in MsrA−/− VSMC
A) Activation of ERK1/2 pathway as determined by Western blotting (n=5-6). B) Activation of ERK1/2 as determined by ELISA for phospho-ERK (n=3). C) ERK1/2 activation following treatment of serum-starved VSMC (ctl) with 10% FBS for 15 min (n=6). D) Activation of Ras as determined by Raf-1 IP and Western blot for Ras. E) Inhibition of ERK1/2 activity decreases cyclin D1 protein levels in MsrA−/− VSMC. Cells were treated with 10 μM U0126 for 16hr to inhibit ERK1/2 (n=3). * p < 0.05 vs. MsrA+/+. F) Cyclin D1 protein levels and G) ERK1/2 activation 72 hr after adenoviral overexpression of MsrA (Ad MsrA) or control (Ad control) in MsrA−/− and MsrA+/+ VSMC
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