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Ann Thorac Surg 1996;62:433-434
© 1996 The Society of Thoracic Surgeons
DR CARMELO A. MILANO (Durham, NC): I compliment Dr Magovern and associates on an excellent report, which I had the opportunity to review before this session. This is very novel work, particularly the use of vascular endothelial growth factor, and the potential clinical applications are obvious. We at Duke University Medical Center also have made efforts to transfer genes to the myocardium using an adenovirus vector.
We began, as you did, using a recombinant adenovirus, which was directly injected into the myocardium. We have achieved strong ß-galactosidase expression after direct injection, as you have shown with a different reporter gene.
Our goal in undertaking these studies was to overexpress myocardial ß-adrenergic receptor genes for the purpose of enhancing ventricular function. We have used several different species with direct injection of recombinant, replication-deficient adenovirus containing the ß-adrenergic receptor. Very marked overexpression of the ß-adrenergic receptor can be obtained several days after direct injection of the adenovirus. We are concerned, however, about substantial inflammation and fibrosis, which developed at the site of the injection. We subsequently tried intracoronary administration of the adenovirus (both with reporter genes and with a ß-adrenergic receptor gene) and have been able to achieve good levels of expression. Surprisingly, we have found less inflammatory response with intracoronary administration of the virus.
My first question and comment would be whether you examined the site of injection of the adenovirus and whether there was histologic inflammation or substantial fibrosis; have these characteristics been contrasted with those after intracoronary injection of the adenovirus? Related to that, might it be better to administer a vascular endothelial growth factor intravascularly rather than by direct injection?
My second question relates to the promoter segments used. You used the cytomegalovirus promoter, whereas we have used a number of other promoter sequences that are myocardium specific. We have used the promoter for the 
-myosin heavy chain and found that the level of expression is intense and confined to the heart. Use of such a promoter sequence would further eliminate concerns about systemic expression of the gene product. Have you considered using these other promoter sequences or done any work with promoter sequences besides the cytomegalovirus promoter?
DR MAGOVERN: With respect to your first question, Dr Milano, we evaluated tissues histologically as late as 14 days and did detect a mild inflammatory reaction. It was characterized best as a lymphocyte infiltrate with some patchy fibrosis. We could not detect any substantial inflammation beyond a region of 5 mm from the injection site. Furthermore, our echocardiograms performed postoperatively failed to detect any local wall motion abnormalities at the sites of injection. We therefore believe that we have not compromised myocardial tissue with direct injection.
With respect to the delivery technique, there is clear evidence that coronary delivery of this vector can accomplish gene transfer. We have focused on a direct delivery system for a number of reasons. We believe that if we are ultimately going to be delivering genes that express angiogenic growth factors, we might be able to target the area more specifically using a direct injection technique. Although we think we have demonstrated in this study that a direct myocardial injection has limited amounts of systemic spread, intracoronary delivery has to be considered more of a systemic technique, and we suspect that more of the virus would go into the system. We have tried coronary delivery, and in our experience, using the same dose of vector, we achieved much lower levels of expression.
With respect to the promoter, we did use a cytomegalovirus promoter. We have not tried a cardiac-specific promoter, although that sounds like an excellent idea. I would suspect, however, that it would be more advantageous to use a cardiac-specific promoter for delivery systems that are considered more systemic, such as coronary artery delivery or intravenous delivery. We are confident that we have been able to localize our system with the direct approach alone.
DR JAMES D. FONGER (Baltimore, MD): I too compliment Dr Magovern and associates on a very careful and elegant study and also thank them for the opportunity of reviewing the manuscript before the presentation. I think that this work gives all of us confidence that the dispersion of these angiogenic agents is probably more widespread than we ever imagined, and I think that is an important finding. I have three questions.
First, you saw a difference between the expression endocardially and epicardially, and I note in the manuscript that the depth of penetration of the needle was between 3 and 5 mm. Was that an attempt to put this in the middle of the myocardial wall or near the epicardium or near the endocardium?
Second, you saw that penetration spread outward to about 1
cm from the site of injection. At that margin where you still detected expression, do you think it was a physiologically significant amount of expression or was it only a trace amount that was barely detectable?
And finally, as you might gather from our upcoming presentation, we are curious to know whether you looked at your histologic results for angiogenesis based on the fact that you did have vascular endothelial growth factor reliably expressed.
Once again, this is an excellent piece of work with data that encourage us all.
DR MAGOVERN: With respect to the first question, we focused on a superficial injection technique mainly to provide a consistent delivery system; we did not want to go any deeper for fear of entering the left ventricular cavity. We also recognized that, at least in dogs, a prominent epicardial vasculature is evident, and if we are ultimately targeting these vessels and trying to get them to grow, it might be advantageous to have more growth factor on the surface for this animal model.
With respect to your second question as to whether these are physiologically important levels, we really do not know. With respect to vascular endothelial growth factor in vitro, 1 to 2 ng can be sufficient to stimulate endothelial growth. In larger in vivo animal models, doses on the order of 250 ng have been shown to demonstrate an effect. So we do not know the answer to that question.
With respect to the histologic results, we did not look at them sufficiently to comment on whether there was a quantifiable increase in vascularity. This is an obvious and important question, and these studies are in progress.
Related Article
Ann. Thorac. Surg. 1996 62: 425-433.
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