Chengpei Xu, MD, PhDa, Sheila Lee, BSa, Tej M

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Chengpei Xu, MD, PhD Hemodynamics and Vascular Remodeling Symposium In Honor of Dr. Seymour Glagov Thursday February 20, 2003.

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Presentation transcript:

Molecular mechanisms of aortic wall remodeling in response to hypertension Chengpei Xu, MD, PhDa, Sheila Lee, BSa, Tej M. Singh, MDa, Eiketsu Sho, MD, PhDb, Xiangqi Li, MDa, Mien Sho, MDb, Hirotake Masuda, MD, PhDb, Christopher K. Zarins, MDa Journal of Vascular Surgery Volume 33, Issue 3, Pages 570-578 (March 2001) DOI: 10.1067/mva.2001.112231 Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 1 Left panels, top : Representative film resulted from an RPA procedure showing major bands, which correspond to TGF-β1 at the 256 base level and TGF-β3 at 226 base level as well as GAPDG at 97 base level according to manufacturer's design. Far left lane shows multiple probes without ribonuclease digestion. Far right lane shows molecular markers. Bottom: Relative mRNA levels of TGF-β1 and β3. Data are expressed as density ratio of TGF-β over GAPDH from three separate experiments. TGF-β1 and TGF-β3 were highly expressed with a peak at 1 day for each. The mRNA levels were declined after 1 day and returned to control levels by 2 weeks. *P <.0033 compared with controls (Ctrl ). Right panels, top: Representative bands of Western blot analysis for TGF-β1 and TGF-β3. Bottom: Densitometry values show similar trend to mRNA levels for both TGF-β1 and β3. *P <.0030 compared with controls (Ctrl ). Data are means from three separate experiments. D, Day; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; L32, ribosomal protein L32; mRNA, messenger RNA; TNF, tumor necrosis factor; W, week. Journal of Vascular Surgery 2001 33, 570-578DOI: (10.1067/mva.2001.112231) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 2 Left panels, top: Representative film resulted from RPA procedure showing major bands, which correspond to Bcl-xS at 325 base level and FasL at 286 base level as well as GAPDG at 97 base level according to manufacturer's design. Far left lane shows multiple probes without ribonuclease digestion. Far right lane shows molecular markers. Bottom: Relative mRNA levels of Bcl-xS and FasL. Data are expressed as density ratio over GAPDH from three separate experiments. Bcl-xS and FasL were decreased immediately after coarctation and remained so until 2 weeks when Bcl-xS returned to control level and 4 weeks, when FasL returned to higher than control level. The remarkable change is that FasL was highly expressed after 2 weeks. *P <.0033 compared with controls (Ctrl ). Right panels, top: Representative bands of Western blot analysis for Bcl-xS and FasL. Bottom: Densitometry values show similar trend to mRNA levels for Bcl-xS and FasL. *P <.0030 compared with controls (Ctrl ). Data are means from three separate experiments. Bax, bel-2 associated χ protein; Caspase, aspartate-specific cysteine proteinase; D, day; FasL, Fas ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; L32, ribosomol protein L32; W, week. Journal of Vascular Surgery 2001 33, 570-578DOI: (10.1067/mva.2001.112231) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 3 Cellularity of ECs and SMCs was within normal population (Ctrl ). Starting from 1 day, cellularity of ECs and SMCs increased, and they appeared crowded (1D-4W). Ctrl, Controls; D, day; W, week. Journal of Vascular Surgery 2001 33, 570-578DOI: (10.1067/mva.2001.112231) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 4 BrdU-incorporated nuclei were labeled dark blue by the immunodetection mediated by alkaline phosphatase. All nuclei were counterstained pink with fast red. ECs and SMCs showed BrdU incorporation. Higher power view of the framed zone on 1-W panel is shown on far right side. Ctrl, Control; D, day; W, week. Journal of Vascular Surgery 2001 33, 570-578DOI: (10.1067/mva.2001.112231) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 5 BrdU incorporation rate (mean ± SD) was expressed as number of labeled nuclei of ECs and SMCs per section obtained from three animals of each time interval. Incorporation rate increased at 3 days (52 ± 11) after coarctation. It peaked at 1 week (73 ± 7), dropped to 31 ± 4 at 2 weeks, and returned to control level by 4 weeks (4 ± 1). *P <.001 compared with controls (4 ± 2). Journal of Vascular Surgery 2001 33, 570-578DOI: (10.1067/mva.2001.112231) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 6 Cell apoptosis detection with TUNEL. Sections were observed under a laser-scanning confocal microscope. All nuclei were counterstained red with propidium iodide, and nuclei of apoptotic cells were labeled green with fluorescein. ECs and SMCs showed positive labeling. Elastic lamellae appeared light green because of their auto fluorescence. By 4 weeks, number of labeled nuclei seemed to be increased. Ctrl, Control; D, day; NC, negative control; PC, positive control; W , week. Journal of Vascular Surgery 2001 33, 570-578DOI: (10.1067/mva.2001.112231) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 7 Apoptosis rate (mean ± SD) was expressed as number of TUNEL positive nuclei per section obtained from three animals of each interval. Apoptosis rate was not remarkably changed until 4 weeks (25 ± 6). *P <.0033 compared with controls (10 ± 6). D, Day; W, week. Journal of Vascular Surgery 2001 33, 570-578DOI: (10.1067/mva.2001.112231) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 8 Wall thickness was increased by 1 week, and such thickness was maintained at 2 and 4 weeks. *P <.002 as compared with age-matched controls. D, Day; W, week. Journal of Vascular Surgery 2001 33, 570-578DOI: (10.1067/mva.2001.112231) Copyright © 2001 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions