Upregulation of iNOS Protects Cyclic Mechanical Stretch-Induced Cell Death in Rat Aorta Smooth Muscle Cells

Abstract

Aortic dissection and aneurysm are associated with abnormal hemodynamic loads originating from hypertension. Our previous study demonstrated that cyclic mechanical stretch (CMS, mimicked hypertension) caused the death of rat aortic smooth muscle cells (RASMCs) in a mitogen activated-protein kinases (MAPKs)-dependent manner. The current study investigated the effects of inducible nitric oxide synthase (iNOS) on CMS-induced RASMC death. cDNA microarrays for CMS-treated RASMCs showed that iNOS expression levels were increased in response to CMS. Real-time polymerase chain reaction (PCR) analysis demonstrated that this increase was p38 MAPK (p38)-dependent. NO production was also increased. This increase could be inhibited by p38 and iNOS inhibitors. Thus, CMS-induced iNOS synthesized NO. CMS-induced cell death in RASMCs was increased by the iNOS inhibitor but abrogated by the long-acting NO donor DETA-NONOate. Increased iNOS expression was confirmed in the abdominal aortic constriction mouse model. Signal transducers and activators of transcription 1 (STAT1) was activated in stretched RASMCs, and iNOS expression and NO production were inhibited by the STAT1 inhibitor nifuroxazide. Our findings suggest that RASMCs were protected by iNOS from CMS-stimulated cell death through the STAT1 and p38 signal pathways independently.

Keywords: NOS; cell death; hypertension; stretch; vascular smooth muscle cell.

Figure 1

Molecular networks associated with mitogen activated-protein kinases (MAPKs) (JNK and p38), apoptosis, and aneurysm. The 91 differentially expressed genes (DEGs) dysregulated in rat aortic smooth muscle cells (RASMCs) treated with cyclic mechanical stretch (CMS) were subjected to bioinformatics analyses to identify genes related to both the JNK and p38, apoptosis, and aneurysm. The regulation between the MAPKs, dysregulated genes, apoptosis, and aneurysm is shown in the network.

Figure 2

Expression of candidate genes in RASMCs subjected to CMS. RASMCs were subjected to CMS for 4 h, and the expression levels of iNOS, NR4A1, MMM9, MMP13, and SERPINE1 were evaluated by real-time RT-PCR. The quantity of the transcripts is expressed as a percentage of the control, normalized to GAPDH. Data are presented as the means ± SD (n = 6); * p < 0.05 and ** p < 0.01 versus control; N.S. indicates no statistically significant difference.

Figure 3

Induction of inducible nitric oxide synthase (iNOS) and NO in RASMCs subjected to CMS. RASMCs were incubated with SB203580 (SB, 20 μM, 20 min) (A), SP600125 (SP, 20 μM, 20 min) (B), BAY11-7082 (BAY, 2.5 µM, 20 min) (C), or 1400 W (50 nM, 1 h) (D) followed by CMS for 4 h. Cells were harvested and analyzed by real-time RT-PCR with specific primers for iNOS (A–C) and the medium was analyzed for NO production by ELISA (D). Data are means ± SD (n = 9); * p < 0.05 and ** p < 0.01 versus CMS control; N.S. indicates no significant difference.

Figure 4

Effects of iNOS inhibitor and NO donor on CMS-induced cell death in RASMCs. The RASMCs were pretreated with iNOS inhibitor (1400 W, 1 h) or NO donor (DETA-NONOate, 20 min) at the indicated concentrations and then incubated under normal conditions or subjected to CMS for 4 h. After CMS, the cells were incubated for 24 h, and then cell viability and death were evaluated by the MTT (A,C) and LDH (B,D) assays, respectively. Colorimetric analysis of each value was normalized by arbitrarily setting the absorbance value of the control to 1. Data are presented as the mean ± SD (n = 9); * p < 0.05 and ** p < 0.01 versus the CMS control.

Figure 5

Abdominal aortic constriction (AAC)-induced iNOS expression in the aorta of mice. Immunofluorescence images of iNOS (red) and smooth muscle actin (SMA, green) in the aortas of sham and AAC mice 6 h post-operation. Tissue sections were co-stained with DAPI (A,B, blue), anti-SMA (C,D) and anti-iNOS (E,F) antibodies. The Merge is the overlapping of iNOS and DAPI (G,H). The uncropped pictures of immunohistochemical analysis are shown in Figure S1. Representative images from four individual animals in each group are shown. Original magnification, 400× (scale: 20 µm).

Figure 6

Effects of signal transducers and activators of transcription 1 (STAT1) inhibitor on iNOS protein expression and NO production induced by CMS in RASMCs. RASMCs were incubated with a STAT1 inhibitor (nifuroxazide; Nif) at the indicated concentrations for 1 h and then incubated under normal conditions or subjected to CMS for 4 h. After CMS, the cells and medium were harvested, and iNOS protein expression (ng/mL) and NO production (µmol/L) were evaluated by ELISA (A) and the NO colorimetric assay (B), respectively. Data are presented as the mean ± SD (n = 9); * p < 0.05 and ** p < 0.01 versus the CMS control.

Figure 7

Effects of p38 and STAT1 inhibitors on STAT1 and p38 phosphorylation induced by CMS in RASMCs. RASMCs were pretreated with SB203580 (SB, 20 µM, 20 min), SP600125 (SP, 20 μM, 20 min), or nifuroxazide (Nif, 2 µM, 1 h) and then subjected to CMS for 4 h. Cell lysates were analyzed by immunoblotting using anti-phosphorylated and total STAT1 antibodies (A) and anti-phosphorylated and total p38 antibodies (B). Phosphorylated STAT1 or p38 levels are expressed as a percentage of the control, normalized to total STAT1 or p38, respectively. The uncropped pictures of immunoblotting are shown in Figure S2. Data are presented as the mean ± SD (n = 3); * p < 0.05 and ** p < 0.01 versus the CMS control; N.S. indicates no significant difference.