Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival Johan.

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Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival Johan Ekstrand, MD, Anton Razuvaev, MD, PhD, Lasse Folkersen, MSc, Joy Roy, MD, PhD, Ulf Hedin, MD, PhD Journal of Vascular Surgery Volume 52, Issue 1, Pages 167-175 (July 2010) DOI: 10.1016/j.jvs.2010.02.282 Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 1 DNA synthesis of SMCs under static conditions compared with SMCs exposed to FSS (14 dynes/cm2) for 24 hours. DNA synthesis as measured by BrdU uptake was 22.2% to that of controls in the FSS exposed cells. Data presented as median (vertical line) ± 10th to 90th percentile (box). Min and max represented by whiskers and outliers are denoted by dots. ***P < .001. Journal of Vascular Surgery 2010 52, 167-175DOI: (10.1016/j.jvs.2010.02.282) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 2 Real-time PCR (A) demonstrating a 437.8-fold increase in TFPI-2 mRNA (mean with standard error of the mean), and (B) Western blot demonstrating an upregulation of TFPI-2 protein (at 32 kDa) in cells exposed to FSS (14 dynes/cm2) for 24 hours compared with static controls. ***P < .001. Journal of Vascular Surgery 2010 52, 167-175DOI: (10.1016/j.jvs.2010.02.282) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 3 (A) BrdU incorporation in rat SMCs following treatment with rhTFPI-2. Treatment with rhTFPI-2 at 50 and 100 ng/mL in 10% fetal calf serum significantly inhibited SMC DNA synthesis. (B) BrdU incorporation in human SMCs following treatment with rhTFPI-2. Treatment with rhTFPI-2 at 100 ng/mL in 15% fetal calf serum significantly inhibited SMC DNA synthesis. Data expressed as median (vertical line) ± 10th to 90th percentile (box) with min and max represented by whiskers. Outliers are denoted by dots. ns, not significant. ***P < .001. Journal of Vascular Surgery 2010 52, 167-175DOI: (10.1016/j.jvs.2010.02.282) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 4 Apoptosis as measured by caspase 3/7 activity. Treatment with rhTFPI-2 significantly increased rSMC apoptosis at 0.5 and 1.0 ug/mL. Data expressed as mean ± SEM. ns, not significant. **P < .01. Journal of Vascular Surgery 2010 52, 167-175DOI: (10.1016/j.jvs.2010.02.282) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 5 TFPI-2 mRNA signal as determined by microarray analysis at 0 hours (n = 3), 5 hours (n = 3), 10 hours (n = 3), and 20 hours (n = 3) after balloon injury to the rat carotid. Data expressed as mean ± SEM. **P < .01. Journal of Vascular Surgery 2010 52, 167-175DOI: (10.1016/j.jvs.2010.02.282) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 6 Immunohistochemistry of the rat carotid artery. Vessel lumen at top of each picture. Uninjured (A, C) and at 10 days after balloon injury (B, D), with visible neointima formation. In the injured artery, no positive luminal von Willebrand Factor (vWF) staining was seen (B) as compared with the uninjured control (A). Perivascular capillaries (*) served as positive control for vWF. Medial and neointimal cells were identified as SMCs by positive SM α-actin staining (C; control, D; 10 days after balloon injury). Arrowheads: Internal elastic lamina. Journal of Vascular Surgery 2010 52, 167-175DOI: (10.1016/j.jvs.2010.02.282) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 7 Immunohistochemistry of the rat carotid artery. (A) Uninjured control vessel revealed no positive TFPI-2 protein staining in the vessel wall. At 2 days after injury (B), TFPI-2 was diffusely expressed in the cytoplasm of adluminal SMCs. At days 5 (C) and 10 (D), increased TFPI-2 protein was visible in adluminal, FSS-exposed SMCs. These cells also expressed activated caspase-3 (E) at day 10. Double staining for TFPI-2 (red) and caspase-3 (blue) at 10 days after injury (F) showed colocalization (purple) of these two proteins adluminally in the neointima. Arrowheads: Internal elastic lamina. Journal of Vascular Surgery 2010 52, 167-175DOI: (10.1016/j.jvs.2010.02.282) Copyright © 2010 Society for Vascular Surgery Terms and Conditions