Introduction: Vascular cells communicate through gap junctions, which are formed by connexin (Cx) proteins. Cx43 is expressed in both endothelial and smooth muscle cells. Studies have demonstrated alterations in gap junctions with atherosclerosis and hypertension, diseases that involve changes in mechanical forces. However, regulation of arterial gap junctions by mechanical forces has not been well understood. Methods: In the present study, ex vivo perfusion culture of rabbit thoracic aortas was used to investigate the regulation of Cx43 by pressure magnitude and pulsatility. After culturing for 6 or 24 h, the Cx43 protein and mRNA levels were detected by Western blot and real-time PCR, respectively. The Src inhibitor PP1 or NADPH oxidase inhibitor apocynin was added to the culture medium to study the molecular mechanisms in some experiments. Results: (1) An increase in the steady pressure level (from 80 to 150 mmHg) significantly increased both mRNA and protein levels of Cx43 at 6 h, which were blocked by PP1. High steady pressure also upregulated Cx43 mRNA at 24 h, although the Cx43 protein levels were similar. This pattern of steady pressure-induced regulation of Cx43 was not altered by the presence of pressure pulsatility or flow levels. (2) Cyclic stretch, elicited by pulsatile perfusion (mean: 80 mmHg, pulse: 30 mmHg, 192 cycles/min), decreased Cx43 protein for both 6 and 24 h, compared with steady stretch controls (mean: 80 mmHg, pulse: 0 mmHg). Concomitantly, levels of active and total Src were reduced by cyclic stretch at 24 h. PP1 in steady perfusion culture or apocynin in pulsatile perfusion culture eliminated the observed differences in Cx43 protein between cyclic and steady stretch. In addition, apocynin elevated active and total Src in aortas under cyclic stretch at 24 h. The ratio of active to total Src was not significantly altered in any case. Conclusions: Both pressure magnitude and pulsatility regulates Cx43 expression. High pressure upregulates Cx43 mRNA and is time-independent. High pressure upregulates Cx43 protein and is time-dependent. Cyclic stretch downregulates Cx43 protein and is time-independent. Src and NADPH oxidase may be involved in the signaling pathway.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-01232008-222025 |
| Date | 09 June 2008 |
| Creators | He, Yong |
| Contributors | David A. Vorp, Harvey S. Borovetz, Kirk P Konrad, James H-C. Wang, Sanjeev G. Shroff, Sandra A. Murray |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-01232008-222025/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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