To the Editor:
I read with interest the article by Indolfi et al in the December 13, 2002, issue of Circulation Research entitled “Physical Training Increases eNOS Vascular Expression and Activity and Reduces Restenosis After Balloon Angioplasty or Arterial Stenting in Rats” in which the authors show that regular exercise reduces neointima formation after stenting and balloon angioplasty and also inhibits negative remodeling after balloon angioplasty.1 They concluded that the benefits of exercise were attributable to enhanced eNOS activity. I have some doubts on this conclusion.
First, the differences in neointima formation, remodeling, and reendothelialization could at least in part be explained by the effects of exercise on these vessels before the injury. Exercise training started 14 days before the balloon or stent injury and if this significantly increased vessel size prior to the injury, then the amount of injury created by standard techniques would be significantly reduced in the larger vessel. Data from the operated vessels 2 days after injury showed that there was little if any increase in external elastic lamina (EEL) area in the uninjured vessel, which is reassuring that the injured vessel was unlikely to be bigger 2 days prior. In studies of physiological remodeling, permanent change in arterial size is apparent 7 days after changes in flow,2 so any measurements of carotid blood flow (CBF) at varying time points after exercise training would be useful in discerning whether 14 days of training is required to result in sustained increases in CBF.
Second, the site of arteriotomy for the carotid balloon injury is also important as an external carotid site and with subsequent ligation of the external carotid would substantially reduce carotid blood flow in the sedentary animals as shown in online Figure 2. This in turn could also explain the negative remodeling and enhanced intima formation in this group.3
Finally, the assay used to detect endothelial nitric oxide synthase (eNOS) activity is not specific for the eNOS and would also reflect activity of inducible NOS (iNOS), which is also significantly affected by vessel injury. While the changes in eNOS activity were mirrored by changes in detectable eNOS by immunoblotting, it would be useful to know the specificity of the antibodies used to eNOS relative to iNOS and why different antibodies were used for immunoblotting versus immunohistochemistry for eNOS.
References
Response
We thank Dr Ward for his interest in our study focused on the effects of physical exercise on restenosis after angioplasty or stenting.1
However, Dr Ward was wondering whether the beneficial effects of training are related to (a) endothelial nitric oxide synthase (eNOS) upregulation; (b) the effects of flow induced by exercise prior to the angioplasty/stenting; or (c) the ligation of carotid collateral vessel (that could reduce absolute flow in the common carotid artery).
In our manuscript, we documented for the first time that regular exercise reduces neointimal formation after angioplasty and, more interestingly, after stenting.1
How could exercise affect the neointimal formation? One possibility is that exercise prior to the angioplasty increased the vessel diameter (by eNOS upregulation). As stated in the manuscript, the histological findings at 2 days after balloon angioplasty did not show any difference in lumen diameter between groups, making it unlikely that this effect could explain the beneficial effect of exercise in preventing neointimal hyperplasia. Furthermore, regarding the stent protocol, it has to be pointed out that in order to minimize the role of arterial diameter before the stent deployment, we oversized the carotid arteries by implanting the stents with a balloon/artery ratio of 1.5:1.2 In addition, since the neointimal formation is proportional to the inflation pressure,3 we kept the atmosphere constant at 1.5.2,3 Therefore, as described in our study protocol, the ligation of the external carotid artery cannot explain the different results of neointimal formation between sedentary and exercised animals, since this artery was ligated in both groups. Nevertheless, as we stated in the limitation section of our manuscript, we cannot exclude that changes in blood flow and shear stress after exercise could have contributed to our results by increasing eNOS expression and activity.1
Finally, in the kit used for NOS activity, incubations were performed in either the presence or absence of EGTA (2 mmol/L) to determine the Ca2+-dependent NOS activities as previously described.4 Because of this, we strongly believe that the measured activity reflects mainly constitutive NOS activity. Furthermore, it should be noted that the role of inducible NOS (iNOS) on neointimal hyperplasia after vascular injury has been a matter of debate because of its inflammatory property, this of course being at odds with the beneficial effect described in our study.1 We used different eNOS antibodies for immunoblotting versus immunohistochemistry (both of them specific for eNOS and not cross-reacting with iNOS), because these antibodies were more efficient given the methods used.
In conclusion, we strongly believe that the evidence provided in our study is a striking demonstration that exercise is beneficial in terms of neointimal proliferation reduction after vascular injury, and that upregulation of eNOS expression and activity is linked to these effects.
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