| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 22, 2005 as doi:10.1096/fj.05-4114fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* The Seymour Cohn Cardiovascular Surgical Research Laboratory, Department of Cardiothoracic Surgery; and
Department of Cell Biology, New York University School of Medicine, New York, New York, USA
1 Correspondence: Department of Cardiothoracic Surgery, NYU School of Medicine, 530 First Ave., NB-15W16, New York, NY 10016, USA. E-mail: pintug01{at}med.nyu.edu
SPECIFIC AIMS
We tested the hypothesis that incubating veins with the synthetic inhibitor of ERK-1/2 activation UO126 prior to grafting into the arterial circulation effectively blocks arterialization-induced ERK-1/2 activation. We then tested the effects of ERK-1/2 activation blockade on medial cell proliferation and apoptosis, and on myeloperoxidase (MPO) expression, a marker of inflammatory cell infiltration. For this purpose we used a canine model in which the external jugular vein was grafted onto the carotid artery, and vein grafts were harvested at different intervals up to day 4 to characterize MAPK activation, MPO levels, PCNA expression (proliferation), and DNA nick-end labeling (apoptosis).
PRINCIPAL FINDINGS
1. Arterialization-induced ERK-1/2 activation is effectively suppressed in veins preincubated with UO126
Prior to grafting, veins were incubated for 30 min at room temperature with 80 µM UO126 or with dimethyl-sulfoxide (DMSO, vehicle) as a control. Vein grafts and control, femoral veins were harvested 3 h later, and ERK-1/2 activation was analyzed by Western blot using phospho-specific antibodies. This analysis showed that UO126 pretreatment resulted in suppression of ERK-1/2 activation upon vein graft arterialization.
2. Inhibition of ERK-1/2 activation results in decreased medial cell proliferation and increased apoptosis
Vein graft arterialization results in increased medial cell proliferation and apoptosis. To study the involvement of the ERK-1/2 pathway in these early responses to vein graft arterialization, vein grafts pretreated with UO126, or with DMSO were harvested at days 1 and 4 after surgery. Inhibition of the ERK-1/2 pathway resulted in strong reduction of PCNA expression, a marker of cell proliferation, as shown by both Western blot and immunohistochemistry analyses (Fig. 1
A). Conversely, UO126 treatment resulted in high rate of medial cell apoptosis, which was maintained from day 1 through day 4 (Fig. 1B
). Therefore, suppression of the ERK-1/2 pathway resulted in both decreased medial cell proliferation, and increased apoptosis.
|
3. Inhibition of ERK-1/2 activation results in decreased MPO levels associated with arterialized vein graft
Vein graft arterialization is accompanied with a variable degree of inflammation. To study potential effects of MEK-1/2 inhibition on the vein graft inflammatory response, we characterized the levels of MPO associated with arterialized vein grafts. The vein graft levels of MPO, a marker of polymorphonuclear and mononuclear cells, are up-regulated very rapidly after vein graft arterialization. In vein grafts harvested 3 h after arterialization. Inhibition of the ERK-1/2 pathway resulted in a strong decrease of MPO levels, as indicated both by Western blot and immunohistochemistry analyses (Fig. 2
).
|
Vein graft arterialization also resulted in increased levels of circulating C-reactive protein (CRP), an acute phase reactant. High amounts of CRP were also associated with the vein graft but not with control vein, indicating that CRP participates in the local inflammatory response occurring in the vein graft. However, unlike MPO, vein graft levels of CRP were not down-regulated by UO126 pretreatment.
CONCLUSIONS AND SIGNIFICANCE
Vein graft arterialization triggers a local vascular remodeling response characterized by endothelial dysfunction, inflammation, proliferation of medial smooth muscle cells, and deposition of abundant extracellular matrix. These responses lead to the formation of a neointima, with consequent thickening of the grafted vessel and, ultimately, vein graft failure. MAPKs have been identified as key biosensors of vascular injury following arterial interventions or vein graft arterialization. We tested the hypothesis that arterialization-induced activation of the ERK-1/2 pathway could be prevented by simply preincubating the vein with a solution containing a synthetic inhibitor of this pathway, UO126. We found that a short (30 min) ex vivo incubation at room temperature not only prevented arterialization-induced ERK-1/2 activation, but also had effects that could be detected as late as 4 days after intervention. The results presented here show that ERK-1/2 inhibition resulted in: 1) dramatic inhibition of medial cell proliferation; 2) increase of medial cell apoptosis, which is maintained up to day 4; and 3) dramatic down-regulation of vein graft-associated MPO, a marker of inflammatory cell infiltration.
The combined reduction of cell proliferation and increased apoptosis induced by inhibition of ERK-1/2 activation results in decreased number of medial cells that participate in neointima formation. Our results confirm the role of the ERK-1/2 pathway in controlling both proliferation and prosurvival mechanisms. In addition, they show an important role of MAPK signaling in the inflammatory response that follows vein graft arterialization. Inflammatory reactions have been implicated in the generation of intimal hyperplasia and of the thrombogenic potential of injured vessels, a risk factor of vein graft failure. Our finding that UO126 pretreatment reduces MPO levels in arterialized vein grafts points to an additional property of MAPK inhibition that could be beneficial for a more favorable clinical outcome of the vein graft. However, the levels of CRP, elevated in arterialized vein graft, are not affected by MAPK inhibition, indicating that the various components of the inflammatory response of vein graft may be controlled by different mechanisms.
The results presented here show that ERK-1/2 is a major signaling pathway in the control of medial cell proliferation, apoptosis and inflammation in vein grafts, and indicate a pharmacological tool for inhibiting the early pathologic response of vein grafts to arterialization, which ultimately leads to vein graft failure.
These results indicate a safe, non-systemic approach to reduce the early effects of arterialization in vein grafts used for bypass surgery (Fig. 3
).
|
Although previous attempts at reducing MAPK activity in models of arterial injury have shown a certain success either by delivering a first generation MAPK inhibitor to isolated vessels or by transfer of dominant negative genes, reduction of MAPK activation has never before been attempted in arterialized vein grafts. Vein grafts are indeed particularly suitable for ex vivo pharmacological treatments as veins are routinely excised and implanted as bypass conduits after a variable period of incubation in physiological solution for up to 1 h. The approach described here effectively suppresses MAPK activation, decreases medial cell proliferation and local inflammation, and increases apoptosis. In addition, this ex vivo treatment is most likely devoid of toxic effects associated with systemic MAPK inhibition.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4114fje;
This article has been cited by other articles:
|
|
 |
|
  E. J.W. Wallitt, M. Jevon, and P. I. Hornick Therapeutics of Vein Graft Intimal Hyperplasia: 100 Years On Ann. Thorac. Surg., July 1, 2007; 84(1): 317 - 323. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
