Ann Thorac Surg 2001;72:129-136
© 2001 The Society of Thoracic Surgeons
Original article: cardiovascular
Nitric oxide system in needle-induced transmyocardial revascularization
Takayuki Saito, MD, PhDa,
Marc P. Pelletier, MDa,
Hani Shennib, MDa,
Adel Giaid, PhDa
a Departments of Pathology and Surgery, The Montreal General Hospital and McGill University, Montreal, Quebec, Canada
Address reprint requests to Dr Giaid, The Montreal General Hospital, McGill University, 1650 Cedar Ave, Suite L3-314, Montreal, Quebec, H3G 1A4, Canada
e-mail: adel.giaid{at}mcgill.ca
Presented at the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.
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Abstract
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Background. Nitric oxide (NO) promotes endothelial proliferation and migration, essential for angiogenesis. The purpose of this study was to determine the cellular expression of inducible and endothelial nitric oxide synthases (iNOS and eNOS) in an ischemic cardiomyopathy animal model of needle-induced transmyocardial revascularization (TMR).
Methods. Myocardial infarction was created in rats by ligating the left coronary artery, and animals were divided into two groups: no-TMR group (served as control) and TMR group (underwent concomitant TMR by the creation of six transmural channels with a 25-gauge needle in the ischemic area). Rats were sacrificed at intervals of 1, 2, 4, and 8 weeks. Immunohistochemistry using specific antisera was performed for iNOS, eNOS, and endothelial cell marker factor VIII. Vascular density and positive staining area with either iNOS or eNOS were assessed in the infarcted myocardium.
Results. Vascular density in the infarcted myocardium was significantly increased in the TMR group (p < 0.001). The positive staining area for iNOS and the intensity of iNOS immunoreactivity in cardiomyocytes, vascular endothelium, and macrophages were significantly greater in the TMR group (p < 0.05). However, these differences were seen only in the first 2 weeks after TMR. There was no significant difference in the expression of eNOS between groups.
Conclusions. A mechanical injury using needle puncture in an ischemic myocardium increased vascular density and is associated with increased expression of myocardial iNOS. Increased production of NO derived from iNOS may contribute to the angiogenic response of TMR.
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Introduction
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The possibility of restoring adequate myocardial blood flow in patients with advanced coronary artery disease by creating intramyocardial channels is being currently investigated by various groups [1]. Initial trials suggest that transmyocardial revascularization (TMR) may be effective in revascularizing the myocardium when conventional therapy is not possible. Many investigators have also shown the efficacies of TMR in experimental animal models [2–4]. However, the mechanisms by which TMR achieves its therapeutic effects and the choice of method for creating transmural channels has not been fully elucidated. It is generally believed that TMR induces angiogenesis and improves myocardial collateral circulation through the processes of tissue injury and wound healing. During the inflammatory and proliferating phases of wound healing, there is significant upregulation of various growth factors that promote angiogenesis and neovascularization. Angiogenesis, which consists of endothelial cell proliferation and migration, remodeling of extracellular matrix, and tubular structure formation, is a pathophysiological event occurring after tissue injury, or in tumor growth and metastasis [5]. This process is tightly regulated by the actions of angiogenic cytokines such as vascular endothelial growth factor (VEGF) [6], basic fibroblast growth factor (bFGF) [7], angiopoietin-1 [8], and transforming growth factor-beta (TGFb). It is well known that these angiogenic cytokines are induced in response to ischemia and contribute to regulate neovascularization. We have previously demonstrated that angiogenesis, after mechanical injury, in an infarcted myocardium is associated with a significant increase in the expression of VEGF, bFGF, and TGFb [2–4].
It has been well recognized that activation of the inducible form of nitric oxide (NO) synthase (iNOS) by various insults including myocardial ischemia [9] releases excessive amounts of NO from macrophages and cardiomyocytes. Diverse effects of NO involve not only inducing vascular smooth muscle relaxation but also inhibiting platelet aggregation [10], leukocyte adherence to the endothelium [11], and inducing endothelial cell proliferation [12] and migration. Guo and associates [13] demonstrated that the exogenous administration of NO donor significantly stimulated endothelial proliferation in vitro. Furthermore, production of NO is regulated by several angiogenic growth factors such as VEGF and TGFb [14, 15].
Therefore, we hypothesized that mechanical injury in the ischemic myocardium may be associated with induced expression of iNOS, which in conjunction with other factors, may contribute to the process of angiogenesis associated with TMR.
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Material and methods
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Animals
Lewis male rats weighing 250 to 300 g (Charles River Laboratory, Wilmington, MA) were used in this study. All animal work was performed in accordance with institutional guidelines, which is in compliance with the "Guide for the Care and Use of Laboratory Animals," published by the National Institutes of Health (NIH publication 85-23, revised 1985). Animals were divided into: no-TMR group (n = 25), underwent ligation of the left coronary artery only; TMR group (n = 28), underwent ligation of the left coronary artery along with TMR at the same operation; sham group (n = 6), underwent left thoracotomy only.
Experimental animal model
Left ventricular free-wall myocardial infarction was induced as described previously [16] with a slight modification. In brief, each rat was anesthetized with enflurane, intubated with a 14-gauge intravenous catheter, and mechanically ventilated with room air by use of a small rodent ventilator (Harvard, South Natick, MA) at a rate of 90 cycles per minute and a tidal volume of 1 mL/100 g body weight. A left thoracotomy was then performed in the fourth intercostal space. After the pericardium was incised, the proximal portion of the left coronary artery was ligated with one suture of 5-0 silk. In the TMR group, transmyocardial punctures were performed with a 25-gauge needle. Six transmural channels were created in the distribution of the left coronary artery, using caution to avoid puncturing any large coronary veins. Hemostasis was achieved with slight pressure in all cases. In both groups, the chest was closed in three layers of 4-0 Vicryl (Ethicon, Somerville, NJ). After surgery, rats were allowed to recover and postoperative pain was controlled with subcutaneous injections of buprinorphine (0.1 to 0.5 mg/kg). Both TMR and no-TMR groups were then divided into four subgroups, which were sacrificed at periods of 1, 2, 4, and 8 weeks postoperatively. Apart from the coronary artery ligation, sham rats underwent an identical procedure and were sacrificed at 1 week postoperatively.
Tissue preparation
At sacrifice, animals were again anesthetized, intubated, and ventilated as described above. After the sternum was removed, isolation and cannulation of the aortic root with a 20-gauge intravenous cannula was employed. Beating hearts were arrested and fixed in diastole with 4% paraformaldehyde, then stored at 4°C. After 24 hours, the hearts were washed and stored in phosphate-buffered saline (PBS) solution containing 15% sucrose, again at 4°C.
Immunohistochemistry
Cryostat sections of tissue were immunostained with antisera to eNOS, and iNOS with a modification of the avidin-biotin-peroxidase methods [17]. Briefly, sections were incubated serially with the following solutions: (1) 2% hydrogen peroxide for 30 minutes to block endogenous peroxide activity; (2) 0.3% Triton-X 100 for 15 minutes to permeabilize the membrane; (3) 10% normal goat serum for 60 minutes to reduce nonspecific binding of the antiserum; (4) primary antisera for 16 hours at 4°C; (5) biotinylated goat anti-mouse or goat anti-rabbit IgG for 45 minutes; and (6) avidin-biotinylated horseradish peroxidase complex (Vectastain; Vector Laboratories, Burlingame, CA) for 45 minutes. Immunoreactive sites were visualized by incubation with 0.025% 3,3-diaminobenzidine and 0.01% hydrogen peroxide for 3 minutes. PBS, pH 7.4, was used to dilute each solution and to wash the sections three times between each step. Antiserum to factor VIII (the endothelial cell marker von Willebrand factor) was also used.
Morphometric studies
Angiogenesis was assessed by counting the number of vessels per high-power field (HPF; x400). Vessels were defined as round structures with a certain lumen that is lined by cells staining positively to factor VIII [2]. Protein expression was quantified by measuring the area of tissue sections positively stained for iNOS or eNOS in each HPF (x400). Measurements were performed using 10 sampling sites per infarcted zone. Results were quantitated as square millimeters per square millimeter of myocardial tissue [2]. To compare the immunoreactivity for iNOS and eNOS in myocytes, macrophages, endocardium, and vascular endothelium of the infarcted myocardium, positive stained dots in HPF (x400) were highlighted and the density of dots was calculated after encircling these cells on the computer screen. The average of 10 samples from each cell type in the infarcted myocardium was quantitated as dots per square micrometer. It is important to mention that although most of the animals used in this study were included in our previous study [2], we have taken different slices of the myocardium and increased the number of fields examined in each section (n = 10). This explains the difference in vascular density in both studies.
Statistical analysis
All data were presented as mean ± SD. Two-way repeated-measures analysis of variance and Student’s t test were used. A correlation between the positive staining area with iNOS and vascular density was assessed by linear regression analysis. Values of p less than 0.05 were considered significant.
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Results
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Vascular density
The number of capillaries per HPF was greater in the infarcted area of both TMR and no-TMR groups when compared with sham group (Fig 1). When compared between experimental groups, vascular density was significantly increased by TMR treatment, particularly at 1 and 2 weeks postoperatively (p < 0.001). After 4 weeks, there was no significant difference in the number of vessels between the two experimental groups. In both TMR and no-TMR groups, the number of vessels was greatest at 1 week and decreased progressively until the eighth week.
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Fig 1. Vascular density was expressed as number of vessels per HPF (x400) in the myocardium of sham and the two experimental groups (no-TMR and TMR groups); *p < 0.001.
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Inducible nitric oxide synthase
In the sham group, weak immunoreactivity with iNOS was seen in the cardiomyocytes, endocardium, and endothelium. In the experimental myocardial infarction model, the localization of iNOS immunoreactivity was seen in the cardiomyocytes, endocardium, and endothelium of intramyocardial vessels in both infarcted and noninfarcted myocardium, and macrophages in the infarcted myocardium. The expression of iNOS was more prominent in the infarcted myocardium of the TMR and the no-TMR groups when compared with the sham group (Figs 2A–F and 3A–D). Intensity of iNOS immunoreactivity in cardiomyocytes, endothelium, and macrophages was significantly higher in the TMR groups at either 1 or 2 weeks postoperatively than that of the no-TMR group (p < 0.05). The positive immunoreactive area for iNOS in the infarcted myocardium was significantly greater in the TMR than in the no-TMR group at 1 and 2 weeks postoperatively (Fig 4A, p < 0.05). However, analysis of variance (ANOVA) revealed no significant difference in the time course of both positive staining area and intensity of immunoreactivity between TMR and no-TMR groups. No iNOS immunoreactivity was seen in the negative control sections.
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Fig 2. Immunostaining for iNOS (A–E) and eNOS (F–H) in the myocardium. (A) Strong expression of iNOS in the infarcted myocardium of TMR group at 1 week after TMR procedure. Arrow indicates endocardial endothelium. (B) Higher magnification of the heart in A showing intense expression of iNOS in the vascular endothelium (arrows) and macrophages (arrowheads). (C) Also a higher magnification of the heart in A and shows abundant expression of iNOS in the cardiomyocytes (arrowheads) and endocardial endothelium (arrow). (D) Little expression of iNOS in the infarcted myocardium of the TMR group at 8 weeks after the procedure. Arrow indicates endocardial endothelium. (E) Little expression of iNOS in the noninfarcted myocardium of the sham group. (F) Immunostaining with eNOS in the infarcted myocardium of the no-TMR group at 1 week after surgery. (G) Higher magnification of the heart in F and shows moderate staining with eNOS in the cardiomyocytes (arrowheads) and endocardial endothelium (arrow). (H) Diffuse immunoreactivity for eNOS in the myocytes of the sham group. Magnifications: A, D, and F (x200); B, C, E, G, and H (x400).
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Fig 3. The intensity of iNOS (A–D) and eNOS immunoreactivity (E–G) in the cardiomyocytes, endocardium, endothelium, and macrophages in the infarcted myocardium of no-TMR and TMR groups and in the noninfarcted myocardium of sham group; *p < 0.05.
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Fig 4. Positive immunoreactive area for iNOS (A) and eNOS (B) in the infarcted myocardium of the TMR and the non-TMR groups and in the noninfarcted myocardium of the sham group; *p < 0.05.
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Endothelial nitric oxide synthase
In the sham group, immunoreactivity with eNOS was seen in the endocardium and endothelium, and to some extent in the cardiomyocytes. In the experimental myocardial infarction model, localization of eNOS immunoreactivity was seen in the cardiomyocytes, endocardium, and endothelium of intramyocardial vessels in both infarcted and noninfarcted myocardium. There was no significant difference in eNOS immunoreactivity in cardiomyocytes, endothelium, and endocardium among all groups (Figs 2G–H and 3E–H). Positive immunoreactive area for eNOS was significantly reduced in the infarcted myocardium of both TMR and no-TMR groups compared with the sham group (Fig 4B). ANOVA revealed there was no significant difference in the time course of both positive staining area and intensity of immunoreactivity between TMR and no-TMR groups. No eNOS immunostaining was seen in the negative control sections.
Correlation studies
Figure 5 shows the relationship between the positive immunoreactive area for iNOS and vascular density in the infarcted myocardium of both TMR and no-TMR groups. Vascular density was lineally correlated with iNOS immunoreactive area in both experimental groups (TMR group: R2 = 0.89, p < 0.0001, no-TMR group; R2 = 0.732, p < 0.0001).
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Fig. 5. Correlation between iNOS immunoreactive area and vascular density in the infarcted myocardium of the TMR (A) and no-TMR groups (B).
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Comment
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In this study, we have demonstrated that myocardial ischemia promotes neovascularization and this is enhanced by the additional mechanical injury in this area. These results suggest that some component of the needle-induced TMR procedure leads to the formation of new microvessels. However, the effect of TMR on neovascularization was seen only within the first 2 weeks postprocedure. Our current observations are somewhat consistent with recent clinical studies showing no improvement in myocardial blood perfusion at intermediate term after TMR [18, 19]. Decreasing vascular density over time may be explained by the well-known process of wound healing. With time, the inflammatory process may lead to increased scar tissue formation via fibroblast proliferation and collagen deposition. Dense scar tissue may allow fewer vessels to survive. Moreover, because the surface area of surviving cardiomyocytes is less than 10% over total infarcted myocardium of the same animal model (our unpublished data), fewer numbers of vessels may be required in this area to supply blood to the remaining cardiomyocytes.
Induction of NOS in cells of the cardiovascular system in response to a variety of stimuli, and its possible pathologic role, have been well documented. NO has specifically been shown to have angiogenic actions in vitro [20]. In the present study, we have demonstrated that myocardial ischemia significantly induced iNOS expression particularly in cardiomyocytes, endocardial and vascular endothelium, and macrophages. Interestingly, both positive immunoreactive area for iNOS and intensity of the immunostaining were significantly increased in the infarcted myocardium of the TMR group. The most significant difference in the expression of iNOS was seen in only first 2 weeks after the TMR procedure. Also, the number of vessels in the infarcted myocardium was lineally correlated with the positive immunoreactive area for iNOS in the infarcted myocardium. This phenomenon was true for both TMR and no-TMR groups. Because we show that myocardial ischemia induced iNOS expression, and we know from previous literature that the resultant increase in NO production can have angiogenic effects, it is reasonable to speculate that NO contributes to the increased vascular density after TMR.
Another isoform of NOS, eNOS, is constitutively produced in endothelial cells as well as other cell types, and plays an important role in maintaining normal vascular tone. Murohara and associates have recently demonstrated deteriorated angiogenic response to hindlimb ischemic model in eNOS gene knockout mice [21]. Therefore, eNOS has been considered to be critical for angiogenesis. However, we did not detect an increase in eNOS expression in the infarcted myocardium. In other words, neither myocardial ischemia nor additional mechanical injury could influence the pattern of eNOS expression. On the contrary, we observed a reduction in the area of eNOS immunostaining in the TMR and no-TMR groups. This might be explained by loss of immunoreactive myocytes in the infarcted myocardium. Our results are also supported by the findings of Wildhirt and associates, who showed that iNOS but not eNOS activity is significantly increased in the infarcted myocardium [22]. Taken into consideration that iNOS produces high levels of NO, which may have serious cytotoxic and negative inotropic effects on the myocardium [23], and that eNOS produces physiological levels of NO, future studies should include the use of selective iNOS inhibitor, and the use of combined TMR and eNOS gene transfer therapy.
We have previously reported that neovessel formation after TMR is associated with the increased expression of several growth factors such as VEGF, bFGF, and TGFb [2–4]. Among these cytokines, VEGF is an important regulator of endothelial cell proliferation, migration, and permeability, and is secreted from tumor and hypoxic cells [24]. Myocardial ischemia has previously been shown to induce expression of VEGF mRNA in cardiac myocytes and vascular smooth muscle cells [25]. VEGF binds to high-affinity tyrosine kinase receptors [26] on vascular endothelial cells, which leads to the release of NO through the activation of both eNOS and iNOS [14]. Interestingly, a recent study has shown that nonselective inhibition of NOS prevents VEGF-mediated angiogenesis [27]. Moreover, we have recently shown that expression of VEGF was highest at 1 week after coronary artery ligation, which is consistent with the current findings of increased vascular density and iNOS expression at the same time. In addition to VEGF, TGFb also promotes proliferation of endothelial cells in vitro, and induces the formation of capillary-like tubes of bovine microvascular endothelial cells [28]. TGFb has a potent chemoattractant effect for monocytes and fibroblasts [29, 30]. These cells can then secret angiogenic molecules such as VEGF and bFGF, which act directly on endothelial cells. Consistent with our present finding, it has recently been shown that myocardial mRNA and protein levels for iNOS and TGFb were increased in the postischemic myocardium [31]. TGFb has been shown to inhibit iNOS expression in vitro and to be upregulated as a negative feedback response to elevated levels of NO [15, 32, 33]. Therefore, an increase of NO may lead to an abundant production of TGFb and may consequently accelerate vessel formation. Interestingly, the highest expression of both iNOS and TGFb was also seen during the first week after ligation, particularly in the TMR group [2]. These results suggest a possible interactive pathway between iNOS, VEGF, and TGFß in the ischemic myocardium.
The present study has several methodological limitations. First, our experimental TMR procedure involved needle punctures for an acute ischemic myocardium, which is no relation to the clinical scenario. In addition, although we have previously compared the efficacy of neovascularization by a needle and a laser [3], in this study, we did not use a laser TMR. To mimic clinical situations, therefore, mechanical punctures by either a needle or a laser have to be carried out in the nonscar area adjacent to the infarction area. Second, in our present study, we measured only immunoreactivity for both iNOS and eNOS in the infarcted myocardium. Because availability of substrates for these enzymes and microenvironment of the ischemic myocardium such as calcium concentration and pH could influence the enzymatic activity and NO production, direct measurements of NOS activity and NO concentration might be significantly important to support our present results. Third, although we compared the number of vessels in the infarcted myocardium and showed an augmented angiogenesis by needle punctures, we have not measured myocardial blood flow, which might be more important and relevant to clinical outcome. Finally, we have not shown direct interaction between iNOS and angiogenesis. Therefore, it is important to use a selective iNOS inhibitor to elucidate the role of this enzyme on angiogenesis in vivo. If our hypothesis is pathophysiologicaly correct, the selective iNOS inhibitor may theoretically diminish the efficacy of the mechanical puncture on angiogenesis. To overcome these limitations may provide us with a more detailed mechanism of this procedure on angiogenesis.
In conclusion, a mechanical injury in the ischemic myocardium, such as that induced by needle puncture, promotes transient vessel formation. Associated increased production of NO derived from iNOS, shown in this study, may significantly contribute to the process of neovessel formation seen after needle-induced TMR.
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Acknowledgments
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This work was supported in part by a grant from Medical Research Council of Canada. Dr Giaid is a recipient of an FRSQ scholarship. The animal model used in this study was done in collaboration with Dr R.C.J. Chiu.
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Discussion
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DR. RAINALD SEITELBERGER (Vienna, Austria): Congratulations for your nice paper. Although I had some trouble understanding, you prove that you really did induce angiogenesis. You did show some increase in vascular density after 1 week; however, there was no vascular density increase at all in comparison to the infarction group after 2 weeks, and then again, after 4 weeks, there was a little increase in vascular density. So, my question is, is this just something that is induced by the method of measuring vascular density, because theoretically you should have a consistent increase in vascular density over time? You may have a consistent decrease again over time, but you have some sort of jumping changes over time. Have you an explanation for that?
DR SAITO: Yes. As you suggested, at the first week, it might have been due to no specific reaction to the tissue injury that enhanced neovascularization. We found significant difference not only at the first week but also at 4 weeks. At that time, it might be due to a significantly different level of expression of angiogenic markers, including NO and VGF. However, there are several articles in which vascular density has been progressively decreased after transmyocardial needle revascularization.
DR. SEITELBERGER: But that is not logical, is it? I mean, if you have an increase in vascular density, it at least should be stable over time, because why should it vanish again under stable conditions?
DR LOUIS P. PERRAULT (Montreal, Quebec, Canada): A very interesting study, however, you have not shown really the exact role of iNOS in the exact process here, because your staining studies alone do not substantiate it is one good thing but function studies would be even better, so I am sure you are planning experiments with L-NAME or aminoguanidine to substantiate the interesting hypothesis that you bring out with this work. Do you have any comments? Thank you.
DR. SAITO: I did not understand.
DR. FULLERTON: Have you given any agents to block nitric oxide?
DR SAITO: We did not use any drugs; for example, no selective inhibitor of iNOS. But it has been reported that no selective iNOS inhibitor prevents VGF-mediated angiogenesis.
DR GIAID: If I could answer the first and second question, too. I am the senior author on this paper. What we are actually doing now is we are using the specific inhibitor for iNOS in the same animal model and we are also measuring the left ventricular end-diastolic pressure in the same animal model.
As to the question related to the increased vascularity during the first week, as we all know, when you cause injury, the first phase of it involves angiogenesis, which explains the increased number of vessels seen in the first week, however, this process is followed by the healing and the scar formation, which explained the reduction of the number of vessels during the 2-week and the 4-week period.
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Abstract
Introduction
