Regulation of vascular smooth muscle cell expression and function of matrix metalloproteinases is mediated by estrogen and progesterone exposure Oscar.

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Presentation transcript:

Regulation of vascular smooth muscle cell expression and function of matrix metalloproteinases is mediated by estrogen and progesterone exposure Oscar H. Grandas, MD, Deidra H. Mountain, PhD, Stacy S. Kirkpatrick, David C. Cassada, MD, Scott L. Stevens, MD, Michael B. Freeman, MD, Mitchell H. Goldman, MD Journal of Vascular Surgery Volume 49, Issue 1, Pages 185-191 (January 2009) DOI: 10.1016/j.jvs.2008.07.080 Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig 1 Schematic diagram depicting treatment timelines for each study arm. DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSA, bovine serum albumin; Est, estrogen; Prog, progesterone; Doxy, doxycycline. Journal of Vascular Surgery 2009 49, 185-191DOI: (10.1016/j.jvs.2008.07.080) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig 2 Determination of optimal hormone concentration. Vascular smooth muscle cells (VSMCs) were treated with the indicated concentrations of estrogen (Est) for 24 hours. Total cell lysates were analyzed by Western blot analysis using anti-matrix metalloproteinase (MMP)-2 antibodies in order to determine the minimum concentration resulting in upregulation of MMPs. Journal of Vascular Surgery 2009 49, 185-191DOI: (10.1016/j.jvs.2008.07.080) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig 3 Hormone stimulation increases membrane type 1-MMP (MT1-MMP) and MMP-2 protein levels with no corresponding increase in tissue inhibitors of metalloproteinase (TIMP)-2. Vascular smooth muscle cells (VSMCs) were pretreated with estrogen (5 ng/mL; Est) with (Est/Prog) and without progesterone (50 ng/mL; Prog) for 24 hours followed by doxycycline (40 μg/mL; Doxy) for 18 hours. Total cell lysates were analyzed by Western blot using anti-MT1-MMP, anti-MMP-2, and anti-TIMP-2 antibodies. A and B demonstrate representative images from 3 or 4 independent experiments. The graph exhibits the mean data ± SEM for MT1-MMP and MMP-2 protein levels. All values have been normalized with β-actin *P < .05 vs control (C); n = 4. Journal of Vascular Surgery 2009 49, 185-191DOI: (10.1016/j.jvs.2008.07.080) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig 4 Hormone stimulation increases matrix metalloproteinase (MMP)-2 activity. Vascular smooth muscle cells (VSMCs) were pretreated with estrogen (5 ng/mL; Est) with (Est/Prog) and without progesterone (50 ng/mL; Prog) for 24 hours followed by doxycycline (40 μg/mL; Doxy) for 18 hours. Concentrated conditioned media were used to measure matrix metalloproteinase (MMP)-2 activity using gelatin in-gel zymography. The lower panel exhibits the mean data ± SEM. *P < .05 vs control (C); n = 3-4. Journal of Vascular Surgery 2009 49, 185-191DOI: (10.1016/j.jvs.2008.07.080) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions

Fig 5 Hormone stimulation induces migration of vascular smooth muscle cells (VSMCs). The ability of cells to migrate through a collagen type IV lattice was assessed using a modified Boyden chamber assay. VSMCs were pretreated with estrogen with and without progesterone (Est, 5 ng/mL; Prog, 50 ng/mL) for 24 hours. Cells were seeded on porous membranes with or without doxycycline (40 μg/mL; Doxy) for an additional 8 hours. A, demonstrates representative images from three independent experiments. Cell migration was quantified by counting the number of migrated cells present on the lower surface in three random fields per membrane at 40X magnification. B, represents the mean data ± SEM. *P < .05 vs control (C); #P < .05 vs control (C); ‡P < .05 vs Est; n = 3. HASMC, human aortic smooth muscle cells. Journal of Vascular Surgery 2009 49, 185-191DOI: (10.1016/j.jvs.2008.07.080) Copyright © 2009 The Society for Vascular Surgery Terms and Conditions