Zinc finger protein 191 deficiency attenuates vascular smooth muscle cell proliferation, migration, and intimal hyperplasia after endovascular arterial.

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Zinc finger protein 191 deficiency attenuates vascular smooth muscle cell proliferation, migration, and intimal hyperplasia after endovascular arterial injury Lei Lv, MD, Jiwei Zhang, MD, Peng Wang, MD, Qiurong Meng, MD, Wei Liang, MD, PhD, Lan Zhang, MD, PhD Journal of Vascular Surgery Volume 59, Issue 2, Pages 500-509 (February 2014) DOI: 10.1016/j.jvs.2013.03.049 Copyright © 2014 Terms and Conditions

Fig 1 Zinc finger protein 191 (ZFP191) is activated in guidewire-injured mouse femoral arteries. Artery proteins were extracted from sham and guidewire-injured mouse femoral arteries on day 7. Samples were probed with anti-ZFP191 polyclonal antibody. Tubulin served as an internal control. A, Representative Western blots for heterozygous ZPD191 knockout mice (KO) and wild-type (WT) mice. B, Bar graph shows relative densitometric values of the Western blots. Values are expressed as mean ± standard deviation from three independent experiments. *P < .05 vs sham. §P < .05 vs WT. Representative photomicrographs (original magnification, ×200) show immunohistochemistry analysis of ZFP191 in (C) uninjured femoral arteries and at (D) 7 days after guidewire injury. The brown staining represents cellular ZFP191 expression. Results shown are representative of three independent experiments. Journal of Vascular Surgery 2014 59, 500-509DOI: (10.1016/j.jvs.2013.03.049) Copyright © 2014 Terms and Conditions

Fig 2 Transduction of lentiviral short-hairpin zinc finger protein 191 (ZFP191) vector suppresses the ZFP191 protein level in vascular smooth muscle cells. The subconfluent cells were infected with control (empty lentivirus vector) or short-hairpin ZFP191 lentiviruses. Cells were collected and lysed for analysis at 7 days after infection. Tubulin served as an internal control. A, Representative Western blots. B, Bar graph shows relative mean densitometric values of Western blots, with error bars indicating the standard deviation. Results shown are representative of three independent experiments. ∗P < .05 vs CON/NC. CON, Control (vascular smooth muscle cells uninfected with lentivirus); KD, vascular smooth muscle cells infected with lentivirus carrying shZFP191; NC, vascular smooth muscle cells infected with control lentivirus. Journal of Vascular Surgery 2014 59, 500-509DOI: (10.1016/j.jvs.2013.03.049) Copyright © 2014 Terms and Conditions

Fig 3 Silencing zinc finger protein 191 (ZFP191) inhibits vascular smooth muscle cell (VSMC) growth. VSMCs were first transfected with control (CON, VSMCs uninfected with lentivirus) or short-hairpin ZFP191 lentiviruses. Cell proliferation was assessed by the (A) MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (B) and [3H]-thymidine incorporation analyses. Values are expressed as mean ± standard deviation (range bars) from six independent experiments. *P < .05 vs CON/NC. KD, VSMCs infected with lentivirus carrying short-hairpin ZFP191; NC, VSMCs infected with control lentivirus. Journal of Vascular Surgery 2014 59, 500-509DOI: (10.1016/j.jvs.2013.03.049) Copyright © 2014 Terms and Conditions

Fig 4 Silencing zinc finger protein 191 (ZFP191) suppresses vascular smooth muscle cell (VSMC) migration. VSMCs were first transfected with control (CON, VSMCs uninfected with lentivirus) or short-hairpin ZFP191 lentiviruses. The confluent VSMC monolayers were wounded by scraping. A, Cell migration to the wound surface was monitored from 0 to 24 hours. B, The number of migrated cells was counted and averaged. C, Migration assay was performed with transwell culture chambers (original magnification, ×200). D, Five different areas of migrated cells were counted. Values are expressed as mean ± standard deviation (error bars) from six independent experiments. *P < .05 vs CON/NC. KD, VSMCs infected with lentivirus carrying short-hairpin ZFP191; NC, VSMCs infected with control lentivirus. Journal of Vascular Surgery 2014 59, 500-509DOI: (10.1016/j.jvs.2013.03.049) Copyright © 2014 Terms and Conditions

Fig 5 Effect of lentivirus-mediated short-hairpin zinc finger protein 191 (shZFP191) on related proteins of ZFP191 in vascular smooth muscle cells (VSMCs). VSMCs were first transfected with control (CON, VSMCs uninfected with lentivirus) or shZFP191 lentiviruses. The cells were lysed, and proteins from cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and blotted with (A) anti-β-catenin, anti-c-myc, anti-cyclin D1, and (C) anti-matrix metalloproteinase (MMP) 9, anti-MMP2, anti-MMP7, or anti-tubulin antibodies to analyze protein expression. B and D, Bands were quantified by densitometric analysis, and the results are shown as relative density compared with control. Values are expressed as mean ± standard deviation from three independent experiments; ∗P < .05 vs CON/NC. KD, VSMCs infected with lentivirus carrying shZFP191; NC, VSMCs infected with control lentivirus. Journal of Vascular Surgery 2014 59, 500-509DOI: (10.1016/j.jvs.2013.03.049) Copyright © 2014 Terms and Conditions

Fig 6 Alterations of related proteins of zinc finger protein 191 (ZFP191) in uninjured or injured femoral arteries from heterozygous ZFP191 knockout (KO) mice compared with wild-type (WT) mice. The samples were sonicated and vortexed at 10,000g for 30 minutes at 4°C. Equivalent amounts of total protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and blotted with anti-β-catenin, anti-c-myc, anti-cyclin D1, anti-matrix metalloproteinase (MMP) 9, anti-MMP2, anti-MMP7, or anti-tubulin antibodies to analyze protein expression. Representative blots were shown (A and C). Bands were quantified by densitometric analysis, and the results were shown as relative density compared with control (B and D). Values are expressed as mean ± standard deviation from three independent experiments. *P < .05 compared with sham; §P < .05 compared with WT. Journal of Vascular Surgery 2014 59, 500-509DOI: (10.1016/j.jvs.2013.03.049) Copyright © 2014 Terms and Conditions

Fig 7 Zinc finger protein 191 (ZFP191) knockdown reduced the secretion and activation of matrix metalloproteinase (MMP) 9, MMP2, and MMP7. Vascular smooth muscle cells (VSMCs) were cultured at a concentration of 4 × 104 cells/mL on 12-well plates. The conditioned medium was prepared and used for zymography. Representative zymography gels are shown for (A) MMP9, MMP2, and MMP7. B, Bar graphs show relative densitometric values of zymography. Results are mean values representative of three independent experiments with the standard deviation (error bars). *P < .05 vs CON/NC. CON, Control; VSMCs uninfected with lentivirus; KD, VSMCs infected with lentivirus carrying shZFP191; NC, VSMCs infected with control lentivirus. Journal of Vascular Surgery 2014 59, 500-509DOI: (10.1016/j.jvs.2013.03.049) Copyright © 2014 Terms and Conditions

Fig 8 Lesion formation in wild-type (WT) and heterozygous zinc finger protein 191 (ZFP191) knockout (KO) mice after injury of the femoral artery. Wire-mediated vascular injury was produced in WT (n = 8) and heterozygous ZFP191 KO (n = 8) mice. The femoral arteries were excised at 7 days after injury. A, Representative photomicrographs show sample sections that were stained with hematoxylin and eosin, and intimal hyperplasia formation was evaluated (original magnification, ×100). B, Bar graphs showed the intima (I)/media (M) ratio quantified by Image-Pro Plus 6.0 software (Media Cybernetics, Inc, Rockville, Md). Values represent means ± standard deviation of 10-12 independent experiments. *P < .05 vs sham. Journal of Vascular Surgery 2014 59, 500-509DOI: (10.1016/j.jvs.2013.03.049) Copyright © 2014 Terms and Conditions