Induction of Angiogenesis After TMR: A Comparison of Holmium:YAG, CO 2, and Excimer Lasers

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1 Induction of Angiogenesis After TMR: A Comparison of Holmium:YAG, CO 2, and Excimer Lasers G. Chad Hughes, MD, Alan P. Kypson, MD, Brian H. Annex, MD, Bangliang Yin, MD, James D. St. Louis, MD, Shankha S. Biswas, MD, R. Edward Coleman, MD, Timothy R. DeGrado, PhD, Carolyn L. Donovan, MD, Kevin P. Landolfo, MD, and James E. Lowe, MD Divisions of Cardiovascular and Thoracic Surgery and Cardiology and the Department of Radiology, Duke University Medical Center, Durham, North Carolina Background. Transmyocardial laser revascularization (TMR) is an emerging treatment for end-stage coronary artery disease. A variety of lasers are currently available to perform the procedure, although their relative efficacy is unknown. The purpose of this study was to compare changes in myocardial blood flow and function 6 months after TMR with holmium:yttrium-aluminum-garnet (holmium:yag), carbon dioxide (CO 2 ), and xenon chloride excimer lasers in a model of chronic ischemia. Methods. Miniswine underwent subtotal (90%) left circumflex coronary stenosis. Baseline positron emission tomography and dobutamine stress echocardiography were performed to document hibernating myocardium in the left circumflex coronary artery distribution. Animals were then randomized to sham redo-thoracotomy (n 5) or TMR using a holmium:yag (n 5), CO 2 (n 5) or excimer (n 5) laser. Six months postoperatively, the positron emission tomography and dobutamine stress echocardiography studies were repeated and the animals sacrificed. Results. In animals undergoing TMR with holmium: YAG and CO 2 lasers, a significant improvement in myocardial blood flow to the lased left circumflex regions was seen. No significant change in myocardial blood flow was seen in sham- or excimer-lased animals. There was a significant improvement in regional stress function of the lased segments 6 months postoperatively in animals undergoing holmium:yag and CO 2 laser TMR that was consistent with a reduction in ischemia. There was no change in wall motion in sham- or excimer-lased animals. Significantly greater neovascularization was observed in the holmium:yag and CO 2 lased regions than with either the sham procedure or excimer TMR. Conclusions. Transmyocardial laser revascularization with either holmium:yag or CO 2 laser improves myocardial blood flow and contractile reserve in lased regions 6 months postoperatively. These changes were not seen following excimer TMR or sham thoracotomy, suggesting that differences in laser energy or wavelength or both may be important in the induction of angiogenesis. (Ann Thorac Surg 2000;70:504 9) 2000 by The Society of Thoracic Surgeons Transmyocardial laser revascularization (TMR) is emerging as a potential treatment option for patients with end-stage coronary artery disease who are not candidates for coronary angioplasty or bypass surgery. The procedure involves using a laser to create transmural channels from the epicardial to endocardial surface of the left ventricle in regions of chronically ischemic yet viable (hibernating) myocardium. While studies have demonstrated the effectiveness of TMR for relieving angina [1, 2], the relative efficacy of the three lasers most commonly used in clinical practice, namely holmium:yttriumaluminum-garnet (holmium:yag) [2], carbon dioxide (CO 2 ) [1], and xenon chloride excimer [3], is unknown. The purpose of the present study was to compare these Accepted for publication Mar 14, Address reprint requests to Dr Lowe, Department of Surgery, Duke University Medical Center, Box 3954, Durham, NC 27710; Lowe0004@mc.duke.edu. three lasers in a recently characterized porcine model of hibernating myocardium [4, 5]. Six months postoperatively, laser-treated and sham-treated animals were examined for changes in regional myocardial blood flow (MBF) using positron emission tomography (PET), myocardial function and contractile reserve using dobutamine stress echocardiography (DSE), and the extent of neovascularization with histologic and histochemical techniques. Material and Methods Twenty adult male miniswine weighing approximately 35 kg each (Harlan-Sinclair, Indianapolis, IN) were used. The Animal Care and Use Committee of Duke University approved all procedures and protocols. Animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and 2000 by The Society of Thoracic Surgeons /00/$20.00 Published by Elsevier Science Inc PII S (00)

2 Ann Thorac Surg HUGHES ET AL 2000;70:504 9 COMPARISON OF THREE LASERS 505 Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (National Institutes of Health publications 85 23, revised 1985). Chronic Ischemia Model All animals underwent placement of a hydraulic occluder and ultrasonic flow probe (Transonic Systems, Ithaca, NY) around the proximal left circumflex coronary artery (LCX), as previously described [4, 5]. Three days postoperatively, the occluder was inflated to reduce resting blood flow immediately distal to the occluder to 10% of baseline. Animals were then kept in this low-flow state with blood flow recordings performed 3 times per week to assure continued occlusion. PET and DSE After 2 weeks in the low-flow state, animals underwent PET and DSE to document the presence of hibernating myocardium in the LCX distribution. Once the animals had undergone an overnight fast, dynamic PET emission imaging of the heart using 13 N-ammonia and 18 F- fluorodeoxyglucose was performed at rest, as previously described [4 7], to obtain regional estimates of MBF (ml g 1 min 1 ) and glucose utilization (nanomol g 1 min 1 ). The PET scans were interpreted as showing hibernating myocardium if reduced absolute values of myocardial blood flow were noted in the lateral and posteroinferior walls of the left ventricle supplied by the LCX accompanied by normal or increased 18 F- fluorodeoxyglucose uptake in these same regions; results were compared with those in the nonischemic septum [8]. Dobutamine stress echocardiography was performed in 3-minute stages with incremental doses of dobutamine beginning with 5 g kg 1 min 1 and increasing to 40 g kg 1 min 1, as previously described [4, 5]. Based on a standard 16-segment model, wall motion was graded as 1 (normal), 2 (hypokinetic), 3 (akinetic), or 4 (dyskinetic). Regional wall motion score index (WMSI) was calculated at rest, at low dobutamine doses, and at peak stress. Echocardiograms were interpreted in a blinded manner by a cardiologist with expertise in stress echocardiography. Using DSE, viability in the LCX region was defined as an improvement in systolic wall thickening with low-dose dobutamine in myocardial regions with severe hypocontractility at rest. Viable segments were considered ischemic if systolic wall motion deteriorated with stress (biphasic response) [9]. TMR and Sham Redo-Thoracotomy Once the presence of hibernating myocardium in the LCX distribution was demonstrated by PET and DSE, animals were randomly assigned to one of the following groups: TMR with a holmium:yag laser (Cardiogenesis, Sunnyvale, CA) (n 5), TMR with a 800-W CO 2 laser (The Heart Laser, PLC Medical Systems, Milford, MA) (n 5), TMR with a xenon chloride excimer laser (Spectranetics CVX 300, United States Surgical Corporation, Norwalk, CT) (n 5), or sham redo-thoracotomy (n 5). All procedures were performed within 3 days of completion of the baseline PET and DSE studies by a single surgeon using previously described techniques [5]. For animals randomized to TMR, 20 channels were created at 1-cm intervals in the hibernating LCX region. This number of channels consistently treats the entire LCX region [5]. Holmium:YAG channels were created using multiple 2-J pulses, with a total energy level of approximately 20 J per channel. Carbon dioxide laser channels were created using a single 40-J pulse. Excimer channels were created using a fluence of 35 mj mm 2 sec 1 and a pulse rate of 30 pulses per second; the total energy level per channel was approximately 20 J. All laser settings were in accordance with manufacturer recommendations. The occluder and flow probe were left intact. The pericardium was left widely open. Those animals randomized to the sham group underwent an identical repeat thoracotomy; the pericardium was opened but TMR was not performed. In all cases, continuous LCX occlusion was confirmed postoperatively by weekly flow monitoring with the flow probe. Follow-up PET and DSE Six months following TMR or sham redo-thoracotomy, animals underwent repeat PET and DSE. This follow-up timepoint was chosen because it corresponds to the period of maximal anginal relief seen in clinical studies of TMR [1]. To allow comparisons between studies performed at baseline and 6 months and to correct for the known interstudy variability of absolute values of MBF by PET [6, 10], normalization of the data were performed using previously described techniques [10]. For each study, sectors representing the anterior septum were used as the normal reference segments. The 13 N- ammonia activity in the sectors representing the LCX distribution were then expressed as a percentage of the activity measured in the reference segments. Vascular Density Analysis Animals were sacrificed 6 months following TMR or sham thoracotomy for histologic and histochemical staining to assess vascular density in the LCX region [5]. At that time, the channels were identified as punctate regions of scar tissue easily visible on the endocardial surface. Of the 20 original channels per animal, six were randomly chosen for histologic analysis. Sections of myocardium measuring 5 5 mm containing the entire channel length from epicardium to endocardium were made. The sections were placed in longitudinal section in OCT (Optimal Cutting Temperature) and snap frozen in liquid nitrogen. Frozen sections (6 m) were made in a cryostat on microscope slides. Vascular density was assessed using endogenous endothelial alkaline phosphatase [11]. Slides were incubated for 1 hour with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Gibco BRL, Galthersburg, MD) and postfixed in 4% paraformaldehyde. The tissue was then stained with eosin, which shows endothelial cells as blue against a red background. Vascular density was quantitated in a blinded fashion by three indepen-

3 506 HUGHES ET AL Ann Thorac Surg COMPARISON OF THREE LASERS 2000;70:504 9 Fig 1. (A) Normalized LCX region myocardial blood flow by PET preoperatively and 6 months postoperatively. Note significant increase in myocardial blood flow to the lased LCX distribution following TMR with both holmium:yag ( before versus after TMR) and CO 2 ( before versus after TMR) lasers. No significant change in myocardial blood flow was seen after excimer TMR ( before versus after TMR, p 0.11) or sham thoracotomy ( before versus after sham procedure, p 0.2). (B) Resting regional WMSI by DSE preoperatively and 6 months postoperatively. See text for details. (C) Peak stress regional WMSI by DSE preoperatively and 6 months postoperatively. Note significant decrease in stress WMSI consistent with a reduction in ischemia following holmium:yag ( before versus after TMR) and CO 2 ( before versus after TMR) laser TMR. No significant change in peak stress WMSI was seen following excimer TMR ( before versus after TMR, p 0.2) or sham thoracotomy ( before versus after sham procedure, p 0.2). (D) Quantitative vascular density analysis. See text for details. (CO 2 carbon dioxide; DSE dobutamine stress echocardiography; LCX left circumflex coronary artery; PET positron emission tomography; TMR transmyocardial laser revascularization; WMSI wall motion score index; YAG yttrium-aluminum-garnet.) dent observers using a modification of previously described techniques [5, 11, 12]. Endothelial alkaline phosphatase staining was measured using an image analysis system (Olympus IX70 inverted microscope, Olympus America, Melville, NY; Optronics DEI-750 imagecapturing hardware, Meyer Instruments, Houston, TX; PowerTower Pro 180 CPU, Power Computing, Round Rock, TX). Images were captured using Adobe Premiere (Adobe Systems Incorporated, San Jose, CA) and quantified using National Institutes of Health image software. Four randomly selected samples, each containing at least one TMR channel remnant, were analyzed per animal for a total of 20 samples per group. Three random highpower (200 ) fields were examined per sample. Vascular density was analyzed for the myocardium within 0.5 cm of the channel remnant but not including the connective tissue channel itself [13]. For the sham-procedure animals, vascular density was analyzed in 20 randomly selected samples (four per animal) from the ischemic LCX distribution. Statistical Analysis Results are presented as the mean standard error. Myocardial blood flow and glucose utilization by pet, as well as WMSI by DSE, were compared within groups using a paired Student s t test. One-way between-groups analysis of variance was used to compare MBF, WMSI, and vascular density between groups. Statistical significance was considered a p value less than Results Positron emission tomography, DSE, and vascular density data are shown for all groups in Figure 1. In all animals, PET demonstrated a significant decrease in LCX region absolute MBF as compared with that in the corresponding nonischemic septal regions ( versus ml g 1 min 1 for LCX region versus nonischemic septum; p 0.001). There was no difference in normalized baseline LCX region MBF between any of the four groups by one-way analysis of variance. 18 F-

4 Ann Thorac Surg HUGHES ET AL 2000;70:504 9 COMPARISON OF THREE LASERS 507 Fig 2. Endogenous endothelial alkaline phosphatase staining (original magnification, 100 ) of representative sections from hibernating myocardium lased with (A) holmium:yag, (B) CO 2, and (C) excimer lasers and from (D) nonlased hibernating myocardium in an animal from the sham group. Note the progressive decrease in blue staining intensity characteristic of endothelial cells from figures 2A to 2D. (Asterisk location of channel remnant.) fluorodeoxyglucose PET revealed a significant increase in glucose utilization in the regions of decreased blood flow ( versus nanomol g 1 min 1 for LCX region versus septum; p 0.001) consistent with myocardial viability and ischemia. Six months following both holmium:yag and CO 2 laser TMR, there was a significant increase in MBF to the lased regions (Fig 1A). No significant change in MBF was seen after either excimer TMR or sham redo-thoracotomy. Baseline DSE in all animals demonstrated severe hypocontractility at rest in the LCX region. There was no difference in baseline rest or stress LCX-region WMSI among any of the four groups by one-way analysis of variance. Six months following TMR (Fig 1B), there was a trend towards improved resting wall motion in the holmium:yag and CO 2 treated groups, but no change was seen in resting WMSI following either excimer TMR or sham redothoracotomy (all p 0.2). There was a significant improvement in regional WMSI for the lased segments at peak stress (Fig 1C), consistent with a reduction in ischemia, 6 months following holmium:yag and CO 2 laser TMR. No significant change in peak stress regional WMSI was seen following sham redo-thoracotomy or excimer TMR. The TMR channel remnants were easily identified on histologic staining as hypocellular regions filled with connective tissue. Regions similar in appearance were observed 6 months following TMR with each of the three lasers. In no instance were patent channels seen. Histologic analysis of the ischemic LCX region in animals undergoing sham redo-thoracotomy was unremarkable, showing no areas of increased connective tissue. Endogenous endothelial alkaline phosphatase staining demonstrated numerous blood vessels adjacent to the holmium: YAG and CO 2 laser channel remnants (Fig 2). Fewer numbers of vessels were seen adjacent to excimer channels and in nonlased (sham redo-thoracotamy) LCX myocardium. Quantitative vascular density analysis confirmed these observations (Fig 1D). In addition, quantitative analysis revealed vascular density to be greater in holmium:yag- versus CO 2 - treated myocardium. Interobserver variability for vascular density measurements was less than 10%. Comment Since its description [14], TMR has been increasingly used as a treatment for severe angina pectoris in patients

5 508 HUGHES ET AL Ann Thorac Surg COMPARISON OF THREE LASERS 2000;70:504 9 with otherwise nonreconstructable coronary disease [1, 2, 15, 16]. In addition, TMR may be used to treat nonrevascularizable territories in patients undergoing either percutaneous angioplasty or coronary artery bypass grafting [17]. Despite this wide potential application, little is known about the relative efficacy of the three laser types most commonly used clinically for performing TMR: holmium:yag, CO 2, and xenon chloride excimer. The present study suggests that the type of laser used is important to the functional outcome of the procedure, as improved myocardial perfusion and contractile reserve were observed 6 months following holmium:yag and CO 2 laser TMR, but not following excimer TMR. In addition, this study supports prior clinical [1, 9] and experimental [18, 19] work documenting improved regional perfusion, function, or both following TMR. The results also suggest that the mechanism responsible for the improved perfusion is angiogenesis, since large areas of neovascularization were observed only in myocardium treated with holmium:yag and CO 2 lasers. To our knowledge, this study is the first to compare long-term changes in myocardial perfusion and function following TMR with those lasers currently used in clinical practice. However, the results of the present study apply only to the specific models tested, as different versions of these same lasers may possess differing operating profiles. Both holmium:yag and CO 2 lasers are infrared lasers, which use thermal ablation to create transmyocardial channels. Excimer lasers, on the other hand, are cold lasers that operate deep in the ultraviolet spectrum and produce tissue ablation by dissociation of molecular bonds [3]. Consequently, excimer lasers are more purely ablative and produce less damage of surrounding myocardium than do infrared lasers. Of the infrared lasers, holmium:yag produces greater lateral thermal damage than CO 2 [20]. Interestingly, the present study found these known differences in the degree of tissue damage produced by each laser to be paralleled by the amount of neovascularization observed 6 months following their application to chronically ischemic myocardium. The greatest degree of neovascularization was observed with the holmium:yag and CO 2 lasers, whereas there was no significant difference in vascular density between excimer-treated and nonlased myocardium. These anatomic changes were reflected in the functional data as well: improved perfusion by PET and contractile reserve by DSE were observed only in the holmium:yag and CO 2 groups. Pelletier and colleagues [21] recently reported that levels of the angiogenic growth factors transforming growth factor and basic fibroblast growth factor were significantly higher in TMR-treated than in nontreated ischemic rat myocardium. These growth factor elevations were accompanied by a significant angiogenic response. One potential explanation for the results of the present study is that the infrared lasers, with their more extensive tissue injury, may produce a greater local release of angiogenic growth factors and a subsequent increase in neovascularization. Inflammation is an important potential contributor to angiogenesis, and inflammatory cells such as macrophages and neutrophils infiltrating the region of laser injury may release numerous cytokines capable of stimulating the expression of angiogenic growth factors [22]. In summary, this study demonstrates that TMR with both holmium:yag and CO 2 lasers improves long-term perfusion and stress function in hibernating porcine myocardium and that this functional improvement is associated with a significant neovascularization response. In addition, the study suggests that not all laser energy is equivalent, as functional improvement was not seen following excimer TMR. Finally, a continuum in the amount of neovascularization was observed that paralleled the known degree of thermoacoustic damage associated with each of the three lasers, suggesting that greater injury led to greater increases in angiogenesis. This work was supported in part through a National Research Service Award from the National Institutes of Health and the NHLBI (grant # 1 F32 HL ) (G. Chad Hughes) as well as unrestricted educational grants from Cardiogenesis (Sunnyvale, CA), PLC Medical Systems (Milford, MA), and the United States Surgical Corporation (Norwalk, CT). In addition, the authors gratefully acknowledge the technical assistance of Mr Michael Lowe. References 1. Frazier OH, March RJ, Horvath KA. Transmyocardial revascularization with a carbon dioxide laser in patients with end-stage coronary artery disease. N Engl J Med 1999;341: Allen KB, Dowling RD, Fudge TL, et al. Comparison of transmyocardial revascularization with medical therapy in patients with refractory angina. N Engl J Med 1999;341: Mack CA, Magovern CJ, Hahn RT, et al. Channel patency and neovascularization after transmyocardial revascularization using an excimer laser. Results and comparisons to nonlased channels. Circulation 1997;96 [Suppl II]:II St. Louis JD, Hughes GC, Kypson AP, et al. An experimental model of chronic myocardial hibernation. Ann Thorac Surg 2000;69: Hughes GC, Lowe JE, Kypson AP, et al. Neovascularization after transmyocardial laser revascularization in a model of chronic ischemia. Ann Thorac Surg 1998;66: DeGrado TR, Hanson MW, Turkington TG, et al. Myocardial blood flow estimation for longitudinal studies using 13Nammonia and PET. J Nucl Cardiol 1996;3: Gambhir SS, Schwaiger M, Huang SC, et al. Simple noninvasive quantification method for measuring myocardial glucose utilization in humans employing positron emission tomography and flourine-18 deoxyglucose. J Nucl Med 1989; 30: Camici P, Ferrannini E, Opie LH. Myocardial metabolism in ischemic heart disease: basic principles and application to imaging by positron emission tomography. Prog Cardiovasc Dis 1989;32: Donovan CL, Landolfo KP, Lowe JE, Clements F, Coleman RB, Ryan T. Improvement in inducible ischemia during dobutamine stress echocardiography after transmyocardial laser revascularization in patients with refractory angina pectoris. J Am Coll Cardiol 1997;30: Mélon PG, De Landsheere CM, Degueldre C, Peters J-L, Kulbertus HE, Piérard LA. Relation between contractile reserve and positron emission tomographic patterns of per-

6 Ann Thorac Surg HUGHES ET AL 2000;70:504 9 COMPARISON OF THREE LASERS 509 fusion and glucose utilization in chronic ischemic left ventricular dysfunction. J Am Coll Cardiol 1997;30: Takeshita S, Zheng LP, Brogi E, et al. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest 1994;93: Channon KM, Qian H, Neplioueva V, et al. In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-fed rabbits. Circulation 1998;98: Kohmoto T, DeRosa CM, Yamamoto N, et al. Evidence of vascular growth associated with laser treatment of normal canine myocardium. Ann Thorac Surg 1998;65: Mirhoseini M, Cayton MM. Revascularization of the heart by laser. J Microsurg 1981;2: Schofield PM, Sharples LD, Caine N, et al. Transmyocardial laser revascularization in patients with refractory angina: a randomised controlled trial. Lancet 1999;353: Burkhoff D, Schmidt S, Schulman SP, et al. Transmyocardial laser revascularisation compared with continued medical therapy for treatment of refractory angina pectoris: a prospective randomized trial. Lancet 1999;354: Hughes GC, Abdel-aleem S, Biswas SS, Landolfo KP, Lowe JE. Transmyocardial laser revascularization: experimental and clinical results. Can J Cardiol 1999;15: Yamamoto N, Kohmoto T, Gu A, DeRosa C, Smith CR, Burkhoff D. Angiogenesis is enhanced in ischemic canine myocardium by transmyocardial laser revascularization. J Am Coll Cardiol 1998;31: Horvath KA, Greene R, Belkind N, Kane B, McPherson DD, Fullerton DA. Left ventricular functional improvement after transmyocardial laser revascularization. Ann Thorac Surg 1998;66: Deckelbaum LI. Cardiovascular applications of laser technology. Lasers Surg Med 1994;15: Pelletier MP, Giaid A, Sivaraman S, et al. Angiogenesis and growth factor expression in a model of transmyocardial revascularization. Ann Thorac Surg 1998;66: Ware JA, Simons M. Angiogenesis in ischemic heart disease. Nat Med 1997;3: