Effect of tissue plasminogen activator on vascular smooth muscle cells

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Effect of tissue plasminogen activator on vascular smooth muscle cells Zhe Yang, MD, PhD, Darwin Eton, MD, Feng Zheng, MD, Alan S. Livingstone, MD, Hong Yu, PhD Journal of Vascular Surgery Volume 42, Issue 3, Pages 532-538 (September 2005) DOI: 10.1016/j.jvs.2005.05.035 Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 1 Cell proliferation. A, DNA synthesis assay. SMCs (black), SMCs/lacZ (grey), and SMCs/tPA (white) were cultured in the medium with 2% fetal bovine serum after being treated for 24 hours in serum-free medium, either in the presence or the absence of plasminogen. The cells were pulse labeled with 3[H]thymidine for 6 hours. The incorporated 3[H]thymidine was expressed as count per minute (cpm) per cell. B, Cell proliferation in conditioned media. Confluent cells were cultured for 48 hours with serum-free media either without plasminogen (SMCs, white; SMCs/tPA, hatched) or with plasminogen (SMCs, diamonds; SMCs/tPA, black). These supernatants were used to culture SMCs on a 24-well plate. After being cultured for 24 and 48 hours, the cell numbers in the wells were counted. Cell proliferation was expressed as cell number increase compared with that at time = 0 (1 × 104 cell/well). *P < .01 compared with others (one-way analysis of variance). Similar results as SMCs were obtained for SMCs/lacZ (data not shown). SMCs, Smooth muscle cells; tPA, tissue plasminogen activator. Journal of Vascular Surgery 2005 42, 532-538DOI: (10.1016/j.jvs.2005.05.035) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 2 Effect of tPA/plasminogen on cell morphology. SMCs (a, d, g), SMCs/lacZ(b, e, h), and SMCs/tPA (c, f, i) were cultured in growth medium until confluent. They were then cultured for 48 hours with serum-free media containing no plasminogen (upper panel) and plasminogen (10 μg/mL) (middle panel) and stained with H&E. SMCs/tPA (×10) shrinkage with no cell loss was observed (f). DAPI staining (lower panel) shows a similar number of apoptotic cells (arrow points at white, condensed pyknotic nuclei) in shrunk SMCs/tPA (i) compared with SMCs (g) and SMCs/LacZ (h). Scale bar = 100 μm. SMCs, Smooth muscle cells; tPA, tissue plasminogen activator; DAPI, 4’,6-diamidino-2-phenylindole. Journal of Vascular Surgery 2005 42, 532-538DOI: (10.1016/j.jvs.2005.05.035) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 3 Inhibition of smooth muscle cell/tissue plasminogen activator (SMC/tPA) shrinking. A, Confluent SMCs/tPA were cultured for 48 hours with serum-free media containing plasminogen plus (a) none, (b) aprotinin, (c) matrix metalloproteinase-2 (MMP-2) inhibitor, (d) MMP inhibitor II, (e) MMP inhibitor III, and (f) MMP inhibitor IV. Cells were stained with 0.1% crystal violet. B, Assessment of inhibitor effect. The area of cell coverage in panel A was measured digitally. Inhibition of cell shrinking by aprotinin (b) is defined as 100%. Inhibition by MMP inhibitors was normalized to the aprotinin effect. Each column represents 36 data points from three experiments, with triplicates at the specified condition. Each well was photographed at four microscopic views at ×100 magnification for digital analysis. Journal of Vascular Surgery 2005 42, 532-538DOI: (10.1016/j.jvs.2005.05.035) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 4 Migration of smooth muscle cells (SMCs). SMCs (white), SMCs/LacZ (hatched), and SMCs/tissue plasminogen activator (black) were subconfluently plated on top of the transwell filter inserts inside a 24-well plate. After being cultured for 48 hours in serum-free medium, with or without plasminogen, 5% fetal bovine serum was added to the medium in the lower chamber. After overnight incubation, the total cells in the inserts and the cells that had migrated to the lower surface were counted. Percent migration is the ratio of migrated cells to the total number of cells. Each column represents data from three experiments, with triplicates at the specified condition. *P < .01 compared with others (one-way analysis of variance). Journal of Vascular Surgery 2005 42, 532-538DOI: (10.1016/j.jvs.2005.05.035) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 5 Collagen degradation. A, Smooth muscle cells/tissue plasminogen activator (SMCs/tPA) were cultured in the absence (gray) or presence (black) of plasminogen for 48 hours. Type I and IV collagens in the supernatant were measured using an enzyme-linked immunosorbent assay. Data were from two experiments of triplicate assays. B, SMCs (white) and SMCs/tPA (black) were embedded within fluorescein isothiocyanate-conjugated type I collagen gel and cultured in the presence or absence of plasminogen for 48 hours. Fluorescence in the conditioned medium was measured. Journal of Vascular Surgery 2005 42, 532-538DOI: (10.1016/j.jvs.2005.05.035) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 6 Effect of tissue plasminogen activator (tPA) on matrix metalloproteinase-2 (MMP-2) activity. The zymography shows the MMP-2 and MMP-9 activity in the conditioned medium cultured with smooth muscle cells (SMCs) (lanes 1 and 2) and SMCs/tPA (lanes 3 to 5). In the absence of plasminogen, no active MMP-2 was detected either in SMCs (lane 1) or in SMCs/tPA (lane 3). In the presence of plasminogen, an active MMP-2 band was detected in SMCs/tPA (lane 4), but not in SMCs (lane 2). Lane 5: same as lane 4 but with the addition of the plasmin inhibitor aprotinin showing the faint active MMP-2 band. The MMP-2 and MMP-9 activity in the neointima on the polytetrafluoroethylene grafts that were recovered 100 days after implantation in the rabbit abdominal aorta were similarly analyzed. Lane 6: neointima on the graft seeded with no cells; lane 7: seeded with SMCs/LacZ; and lane 8: seeded with SMCs/tPA. Lane M: MMP markers. Journal of Vascular Surgery 2005 42, 532-538DOI: (10.1016/j.jvs.2005.05.035) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions