REGULATION OF ARTERIAL GAP JUNCTIONS BY MECHANICAL FACTORS: AN EX VIVO STUDY
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.
Advisor:David A. Vorp; Harvey S. Borovetz; Kirk P Konrad; James H-C. Wang; Sanjeev G. Shroff; Sandra A. Murray
School:University of Pittsburgh
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Date of Publication:06/09/2008