Expression of Nik-related kinase in smooth muscle cells attenuates vascular inflammation and intimal hyperplasia

Yi-Jhu Lu, Yee-Jee Jan, Bor-Sheng Ko, Shu-Man Liang, Lujen Chen, Chih-Cheng Wu, Chih-Hui Chin, Cheng-Chin Kuo, Shaw-Fang Yet, Jun-Yang Liou, Yi-Jhu Lu, Yee-Jee Jan, Bor-Sheng Ko, Shu-Man Liang, Lujen Chen, Chih-Cheng Wu, Chih-Hui Chin, Cheng-Chin Kuo, Shaw-Fang Yet, Jun-Yang Liou

Abstract

Inflammation of the vascular microenvironment modulates distinct types of vascular cells, and plays important roles in promoting atherosclerosis, stenosis/restenosis, and vascular-related diseases. Nik-related kinase (Nrk), a member of the Ste20-type kinase family, has been reported to be selectively expressed in embryonic skeletal muscle. However, whether Nrk is expressed in adult vascular smooth muscle, and if it influences intimal hyperplasia is unclear. Here, we found that Nrk is abundantly expressed in cultured vascular smooth muscle cells (VSMC) and mouse arterial intima. Treatment of mouse VSMCs with lipopolysaccharide (LPS) or platelet-derived growth factor significantly reduced Nrk expression. In addition, expression of Nrk was significantly reduced in regions of neointimal formation caused by guide-wire carotid artery injuries in mice, as well as in human atherosclerotic tissues, when compared to normal vessels. We identified that expression of matrix metalloproteinases (MMP3, MMP8 and MMP12) and inflammatory cytokines/chemokines (CCL6, CCL8, CCL11, CXCL1, CXCL3, CXCL5 and CXCL9) are synergistically induced by Nrk siRNA in LPS-treated mouse VSMCs. Moreover, we found that resveratrol significantly impaired LPS- and Nrk siRNA-induced expression of MMP3, CCL8, CCL11, CXCL3 and CXCL5. These results suggested that Nrk may play important roles in regulating pathological progression of atherosclerosis or neointimal- hyperplasia-related vascular diseases.

Keywords: Nik-related kinase; inflammation; intimal hyperplasia; resveratrol; smooth muscle cell.

Conflict of interest statement

CONFLICTS OF INTEREST: The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Expression of Nrk in VSMCs. (A) Expression of Nrk protein was determined by western blotting analysis in mVSMCs, rVSMCs (A10), hVSMCs, HUVECs, HCAECs, HPAECs, C2C12 and A549 cells. Primary antibodies against mNrk (upper panel) and hNrk (middle panel) were employed for the detection of Nrk. Actin was used as a loading control (lower panel). (B) Expression of mNrk in normal carotid artery of wild-type C57BL/6 mice was examined by immunohistochemical staining with primary antibodies against mNrk, CD31, αSMA, and elastic stain. Bar= 50 μM. (C) Expression and localization of αSMA (green) and mNrk (red) on mouse carotid artery was examined by double staining of immunofluorescence confocal microscopy.
Figure 2
Figure 2
Expression of Nrk was suppressed by PDGF and LPS in mVSMCs. mVSMCs were serum starved (0.5% FBS in DMEM) for 24 h, followed by stimulation with PDGF (10 ng/ml) or LPS (100 ng/ml) for an additional 24 h. Expression of mNrk was determined by (A) western blotting (n=6) and (B) qPCR analysis (n=4). Gene expression of qPCR analysis results were normalized to both control cells as well as to GAPDH. Tubulin was used as a loading control for western blotting analysis. Scale bars: means ± SD. *, p <0.05.
Figure 3
Figure 3
Expression of mNrk, αSMA, and elastic staining in carotid artery of wild-type C57BL/6 mice subjected to guide wire injury for 4 weeks. The expression of mNrk, αSMA, and elastic staining in three set of sections of (A) non-injured and (B) injured carotid arteries at 150-μm intervals was examined by immunohistochemical staining. Bar = 50 μM. (C) Left panel: Quantitation of intima/media (I/M) ratio (left panel, p = 0.00056) and Nrk expression (right panel, p = 1.184 × 10-5) in normal and injured carotid arteries. n = 9 for each group.
Figure 4
Figure 4
Representative immunohistochemical staining of hNrk and calponin 1 in human normal (left panels) and atherosclerotic (right panels) vessels. Bar = 100 μm.
Figure 5
Figure 5
Expression of MMPs and chemokines in LPS- and Nrk-siRNA treated mVSMCs. mVSMCs were serum starved (0.5% FBS in DMEM) for 24 h and then treated with LPS (100 ng/mL) for 24 h. Cells were further transfected with 20 nM of negative control or mNrk siRNA for an additional 48 h. Expression of (A) MMP3 (n=13), MMP8 (n=13) and MMP12 (n=14); (B) CCL6 (n=14), CCL8 (n=14) and CCL11 (n=14); (C) CXCL1 (n=14), CXCL3 (n=11), CXCL5 (n=10) and CXCL9 (n=13) was determined by qPCR. Gene expression results of qPCR analysis were normalized to both control cells as well as GAPDH. Scale bars: means ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001.
Figure 6
Figure 6
Effect of resveratrol on LPS- and Nrk siRNA-stimulated MMPs and chemokines. (A) mVSMCs were serum starved (0.5% FBS in DMEM) for 24 h and then treated with LPS (100 ng/mL) and/or resveratrol (50 μM) for 24 h. Cells were further transfected with 20 nM of negative control or mNrk siRNA for an additional 48 h. Expression of (A) MMP3 (n=8), CCL8 (n=8), CCL11 (n=8), CXCL3 (n=6) and CXCL5 (n=6) was determined by qPCR. Gene expression results of qPCR analysis were normalized to both control cells as well as GAPDH. (B) Protein levels of MMP3 (n=7), CCL8 (n=6) and CCL11 (n=7) in cultured conditioned media were determined by ELISA (normalized to total protein concentration). Scale bars: means ± SD. *, p < 0.05, **, p < 0.01, ***, p<0.001.

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