1. Date of download: 11/11/2016 Copyright © The American College of Cardiology. All rights reserved. From: Downregulated Expression of Plasminogen Activator Inhibitor-1 Augments Myocardial Neovascularization and Reduces Cardiomyocyte Apoptosis After Acute Myocardial Infarction J Am Coll Cardiol. 2005;46(3):536-541. doi:10.1016/j.jacc.2005.04.047 Figure Legend: Deoxyribonucleic acid (DNA) enzymes inhibit induction of endogenous plasminogen activator inhibitor type 1 (PAI-1) messenger ribonucleic acid (mRNA) and protein, and demonstrate serum resistance. (a) Results of reverse transcription-polymerase chain reaction demonstrating that transfection of rat endothelial cells (ECs) with E2 results in significant inhibition of transforming growth factor-beta-induced expression of PAI-1 mRNA, relative to E0 (p < 0.01, n = 4). (b) Results of Western blot using PAI-1 specific antisera, demonstrating that transfection of rat ECs with E2 results in significant inhibition of TGF-beta induced expression of PAI-1 protein, relative to the E0 (p < 0.01, n = 3). (c) 32 P-labeled PAI-1 DNA enzyme with inverted thymidine at the 3′ end demonstrates resistance to degradation in medium with 2%, but not 20%, serum (representative of two separate experiments).

2. Date of download: 11/11/2016 Copyright © The American College of Cardiology. All rights reserved. From: Downregulated Expression of Plasminogen Activator Inhibitor-1 Augments Myocardial Neovascularization and Reduces Cardiomyocyte Apoptosis After Acute Myocardial Infarction J Am Coll Cardiol. 2005;46(3):536-541. doi:10.1016/j.jacc.2005.04.047 Figure Legend: Effects of intramyocardial plasminogen activator inhibitor type 1 (PAI-1) deoxyribonucleic acid (DNA) enzyme injection on myocardial PAI-1 messenger ribonucleic acid (mRNA) and protein expression. (a) Results of reverse transcription-polymerase chain reaction (RT-PCR) 48 h after left anterior descending coronary artery (LAD) ligation and direct injection of DNA enzymes at the peri-infarct region (PIR), showing reduced myocardial PAI-1 mRNA expression after E2 injection relative to E0 (p < 0.01, n = 6 to 10). (b) Immunohistochemical staining (IHC) analysis of normal rat myocardium and rat PIR 48 h after LAD ligation. (Left) The PAI-1 expression in rat cardiomyocytes of animals treated with E0, but not with E2. PAI-1 positively stained cells were visualized as brown with immunoperoxidase technique and 3,3′-diaminobenzidine tetrahydrochloride substrate. (Right) quantitative IHC analyses showing reduced numbers of PAI-1 expressing cells at the PIR after E2 injection relative to E0 (p < 0.01, n = 6 to 10). (c) The RT-PCR analyses 2 weeks after LAD ligation and direct injection of E2 at the PIR, showing reduced level of PAI-1 mRNA in infarcted left ventricles after E2 injection relative to saline (p < 0.01, n = 6 to 10).

3. Date of download: 11/11/2016 Copyright © The American College of Cardiology. All rights reserved. From: Downregulated Expression of Plasminogen Activator Inhibitor-1 Augments Myocardial Neovascularization and Reduces Cardiomyocyte Apoptosis After Acute Myocardial Infarction J Am Coll Cardiol. 2005;46(3):536-541. doi:10.1016/j.jacc.2005.04.047 Figure Legend: Effects of intramyocardial plasminogen activator inhibitor type 1 (PAI-1) deoxyribonucleic acid (DNA) enzyme injection on myocardial neovascularization, cardiomyocyte (CM) apoptosis, and functional recovery after acute myocardial infarction. (a) E2 injection induced significantly greater neovascularization at the peri-infarct region (PIR) 2 weeks later in comparison to E0 (p < 0.01, n = 6 to 10). Capillaries are characterized as small lumen vessels lacking smooth muscle layers and positively staining for CD31. (b) E2 injection at the time of left anterior descending coronary artery ligation resulted in 70% reduction in CM apoptosis at the PIR 2 weeks later compared with infarct controls (p < 0.01, n = 6 to 10), as determined by concomitantly TUNEL staining (blue) and troponin I staining (brown). This was not observed with E0 injection. (c) E2 injection induced significantly greater improvement in left ventricular ejection fraction 2 weeks later in comparison to E0 (p < 0.01, n = 6 to 10), as measured by echocardiographic studies.

4. Date of download: 11/11/2016 Copyright © The American College of Cardiology. All rights reserved. From: Downregulated Expression of Plasminogen Activator Inhibitor-1 Augments Myocardial Neovascularization and Reduces Cardiomyocyte Apoptosis After Acute Myocardial Infarction J Am Coll Cardiol. 2005;46(3):536-541. doi:10.1016/j.jacc.2005.04.047 Figure Legend: Specificity of cleavage reaction for rat plasminogen activator inhibitor type 1 (PAI-1) messenger ribonucleic acid (mRNA) by E2 deoxyribonucleic acid (DNA) enzyme. (a) E2 cleaved the 24-base oligonucleotide S2, derived from the sequence of rat PAI-1 mRNA, in a dose- and time-dependent manner. (b) E2 also cleaved a rat PAI-1 mRNA transcript in a dose-dependent manner to give the 156 nucleotide cleavage product. Neither the scrambled control E0 nor E3 cleaved the rat PAI-1 mRNA transcript. (c) Single nucleotide substitution between rat and human PAI-1 mRNA target sequence prevents cleavage of rat PAI-1 mRNA transcript by E3.