Effects of selective cyclooxygenase-2 and nonselective cyclooxygenase inhibition on ischemic myocardium Michael P. Robich, MD, MSPH, Louis M. Chu, MD,

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Effects of selective cyclooxygenase-2 and nonselective cyclooxygenase inhibition on ischemic myocardium Michael P. Robich, MD, MSPH, Louis M. Chu, MD, Jun Feng, MD, PhD, Thomas A. Burgess, MA, Roger J. Laham, MD, Cesario Bianchi, MD, PhD, Frank W. Sellke, MD The Journal of Thoracic and Cardiovascular Surgery Volume 140, Issue 5, Pages 1143-1152 (November 2010) DOI: 10.1016/j.jtcvs.2010.06.057 Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Myocardial functional data. A, Mean arterial blood pressure (MAP) is increased in celecoxib (CBX) group (asterisk indicates P < .001). B, Left ventricular pressure (LVP) is increased in celecoxib group (dagger indicates P = .004). C, Vertical segmental shortening (VSS) is increased in celecoxib group (double dagger indicates P = .02). D, Positive first derivative of developed pressure over time (+dP/dt) is decreased in celecoxib group (section mark indicates P = .04). NAP, Naproxen. The Journal of Thoracic and Cardiovascular Surgery 2010 140, 1143-1152DOI: (10.1016/j.jtcvs.2010.06.057) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Blood flow in collateral-dependent territory. A, Blood flow to ischemic myocardium at rest is increased in naproxen (NAP) group (asterisk indicates P = .03. B, There is significantly increased blood flow to ischemic territory during ventricular pacing in naproxen group (dagger indicates P = .02). The Journal of Thoracic and Cardiovascular Surgery 2010 140, 1143-1152DOI: (10.1016/j.jtcvs.2010.06.057) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Microvascular responses. A, Microvessel responses to endothelin-1 (ET-1) demonstrate significantly increased contractions in naproxen (NAP) group at 10−8 (asterisk indicates P = .001 vs control and celecoxib [CBX]) and 10−9 (dagger indicates P = .001 vs control). Percentage contraction is not different between control and celecoxib groups at any concentration. B, Contraction responses to serotonin demonstrate increases in naproxen group at 10−6 (double dagger indicates P = .001 vs control and celecoxib) and 10−5 (section mark indicates P = .04 vs control). C, Relaxation responses to adenosine diphosphate (ADP), endothelium-dependent vasorelaxation agent, show decreased microvascular responses in celecoxib group (parallel lines indicate P = .01 vs control and naproxen). D, Responses to sodium nitroprusside (SNP) are not different among groups (P = .22). The Journal of Thoracic and Cardiovascular Surgery 2010 140, 1143-1152DOI: (10.1016/j.jtcvs.2010.06.057) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Serum and tissue levels of prostaglandins and thromboxane. A, Tissue levels of prostaglandin F1α(PGF1) show marked decrease in naproxen (NAP) group (asterisk indicates P < .001 vs control and celecoxib) and significant decrease in celecoxib group as well (dagger indicates P < .05 vs control). B, Serum prostaglandin (PGI1a) levels are significantly higher in control group (double dagger indicates P = .004 vs naproxen and celecoxib). C, Tissue levels of thromboxane B2(TXB2) are slightly increased in naproxen and celecoxib groups (P = .25 vs control). The Journal of Thoracic and Cardiovascular Surgery 2010 140, 1143-1152DOI: (10.1016/j.jtcvs.2010.06.057) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Levels of protein and lipid oxidative stress (Ox Stress) in normal and ischemic myocardium. A, Protein peroxidation levels in ischemic territory are increased in celecoxib (CBX) group (asterisk indicates P = .001 vs control and naproxen [NAP]). B, There is trend toward decreased lipid oxidative stress in ischemic territory in naproxen group (P = .07). MDA, Malondialdehyde. The Journal of Thoracic and Cardiovascular Surgery 2010 140, 1143-1152DOI: (10.1016/j.jtcvs.2010.06.057) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 Quantification of dividing and total endothelial cells. Double staining for endothelium (CD-31, green) and dividing cells (Ki-67, red) with 4′,6-diamidino-2-phenylindole (blue) allows visualization of dividing endothelial cells in ischemic territory, which appear pink in combined image. A, In this representative section for control group, there are many dividing endothelial cells. B, Representative myocardial section from ischemic area of naproxen group demonstrates fewer dividing endothelial cells. C, Representative image from celecoxib group. All images taken at 200× magnification. D, Quantification of Ki-67–positive endothelial cells demonstrates significant increases in control group relative to naproxen (NAP) and celecoxib (CBX) groups (asterisk indicates P = .003). There is no difference between naproxen and celecoxib groups. E, Similarly, total number of endothelial cells in ischemic territory is significantly lower in naproxen and celecoxib groups than in control (dagger indicates P = .003). The Journal of Thoracic and Cardiovascular Surgery 2010 140, 1143-1152DOI: (10.1016/j.jtcvs.2010.06.057) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 7 Immunoblotting in ischemic myocardium. A, Levels of vascular endothelial growth factor (VEGF) are highest in control group and decreased in naproxen (NAP) and celecoxib (CBX) groups (asterisk indicates P = .001 vs naproxen and celecoxib). B, Expression of phospho–endothelial nitric oxide synthase (p-eNOS) is highest in control group (dagger indicates P = .002 vs naproxen and celecoxib). C, Vascular endothelial growth factor receptor 2 (VEGFR2) is likewise downregulated in naproxen and celecoxib groups (double dagger indicates P = .04 vs naproxen and celecoxib). D, c-Jun N-terminal kinase (JNK) expression level is increased in celecoxib group (section mark indicates P = .04 vs control). There is no significant difference between control and naproxen groups. E, Endostatin, antiangiogenic protein, is significantly downregulated in naproxen group relative to control and celecoxib groups (parallel lines indicate P = .02). Post hoc analysis revealed no difference between control and celecoxib groups. The Journal of Thoracic and Cardiovascular Surgery 2010 140, 1143-1152DOI: (10.1016/j.jtcvs.2010.06.057) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions