Pavan Atluri, MD, Corinna M. Panlilio, BA, George P. Liao, MB, Eric E

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Transmyocardial revascularization to enhance myocardial vasculogenesis and hemodynamic function Pavan Atluri, MD, Corinna M. Panlilio, BA, George P. Liao, MB, Eric E. Suarez, MD, Ryan C. McCormick, BS, William Hiesinger, MD, Jeffrey E. Cohen, BA, Maximilian J. Smith, Abha B. Patel, BS, Wei Feng, MD, Y. Joseph Woo, MD The Journal of Thoracic and Cardiovascular Surgery Volume 135, Issue 2, Pages 283-291.e1 (February 2008) DOI: 10.1016/j.jtcvs.2007.09.043 Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Therapy with transmyocardial laser revascularization upregulates circulating CD31− CD133+VEGFR2+ endothelial progenitor cells. Representative flow cytometric plots of circulating blood 48 hours after either sham treatment (thoracotomy alone) or transmyocardial laser revascularization. A, Dot plots of forward and side scatter determination of lymphocyte subpopulation; representative gate for lymphocytes is visualized. B, Histogram of 7-amino-actinomycin D (7AAD) labeling of lymphocyte cell viability. C, Histogram representing a CD31− population. Representative, gated viable, CD31− cell population is delineated. D and E, EPC density in representative animals from sham (D) and transmyocardial laser revascularization (E) groups after gating for CD31− population, as determined by colabeling of CD133 and VEGFR2. Quantification of endothelial progenitor cell density demonstrates significant increase relative to sham control in circulating CD31− CD133+VEGFR2+ endothelial progenitor cells after transmyocardial laser revascularization. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 283-291.e1DOI: (10.1016/j.jtcvs.2007.09.043) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 A, Western blot analysis of nuclear factor κB (NFk-B) myocardial protein levels in sham and transmyocardial laser revascularization (TMR) groups reveals dramatic upregulation after TMR in hibernating myocardium. Nuclear factor κB was detected at 116 kDa. B, Western blot analysis of angiopoietin 1 (Ang-1) expression in hibernating myocardium in sham and transmyocardial laser revascularization groups demonstrates marked increase in angiopoietin 1 after transmyocardial laser revascularization. Expression at 57 kDa corresponds to native angiopoietin 1, whereas 75-kDa band demonstrates expression of highly glycosylated form of angiopoietin 1. Glycosylated angiopoietin 1 protein plays vital role in vascular maturation. C, Western blot analysis of actin in sham and transmyocardial laser revascularization groups confirms equivalent protein loading. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 283-291.e1DOI: (10.1016/j.jtcvs.2007.09.043) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Three-dimensional microvascular lectin angiogram demonstrates enhanced myocardial perfusion after therapy with transmyocardial laser revascularization (TMR). Representative angiograms from remote myocardium and ischemic myocardium from animals in sham and transmyocardial laser revascularization groups are presented (z-series, 25× oil magnification, Zeiss LSM-510 Meta Confocal Microscope; Carl Zeiss, Inc, Thornwood, NY). There was no difference in remote myocardial perfusion between sham and transmyocardial laser revascularization groups. Perfusion of the ischemic myocardium was significantly increased after transmyocardial laser revascularization. Orientation is presented in lower left corners. Red indicates y-axis; green indicates x-axis; blue indicates z-axis. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 283-291.e1DOI: (10.1016/j.jtcvs.2007.09.043) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Representative Masson trichrome—stained images of myofilament density within an ischemic territory of the left circumflex coronary artery of myocardium from sham and transmyocardial laser revascularization (TMR) Groups demonstrating enhanced myofilament density in latter (original magnification 20×). Red indicates muscle; blue indicates collagen; black indicates nuclei. The Journal of Thoracic and Cardiovascular Surgery 2008 135, 283-291.e1DOI: (10.1016/j.jtcvs.2007.09.043) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Therapy with transmyocardial laser revascularization (TMR) enhances myocardial vasculature. Representative immunofluorescent images of myocardium from ischemic left circumflex coronary myocardium of animals in sham and transmyocardial laser revascularization groups labeled for vasculature (green for CD31) and nuclei (blue for 4',6-diamidino-2-phenylindole) are demonstrated (original magnification 20×; scale bar represents 50 μm). The Journal of Thoracic and Cardiovascular Surgery 2008 135, 283-291.e1DOI: (10.1016/j.jtcvs.2007.09.043) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions