Downregulated Expression of Plasminogen Activator Inhibitor-1 Augments Myocardial Neovascularization and Reduces Cardiomyocyte Apoptosis After Acute Myocardial Infarction
Guosheng Xiang, PhD*,
Michael D. Schuster, MS,
Tetsunori Seki, PhD,
Piotr Witkowski, MD,
Shawdee Eshghi, BS and
Silviu Itescu, MD
Departments of Surgery and Medicine, Columbia University, New York, New York
Manuscript received February 1, 2005;
revised manuscript received March 30, 2005,
accepted April 13, 2005.
* Reprint requests and correspondence: Dr. Guosheng Xiang, Columbia-Presbyterian Medical Center, 630 West 168th Street, P&S 14-402, New York, New York 10032 (Email: gx15{at}columbia.edu).
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Abstract
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OBJECTIVES: The aim of this study was to examine whether selective plasminogen activator inhibitor type 1 (PAI-1) downregulation in the acutely ischemic heart increases the myocardial microvasculature and improves cardiomyocyte (CM) survival.
BACKGROUND: Endogenous myocardial neovascularization is an important process enabling cardiac functional recovery after acute myocardial infarction. Expression of PAI-1, a potent inhibitor of angiogenesis, is induced in ischemic heart tissue.
METHODS: A sequence-specific catalytic deoxyribonucleic acid (DNA) enzyme was used to reduce PAI-1 levels in cultured endothelial cells and in ischemic myocardium. At the time of coronary artery ligation, rats were randomized into three groups, each receiving an intramyocardial injection (IMI) of a single dose at three different sites of the peri-infarct region consisting, respectively, of DNA enzyme E2 targeting rat PAI-1 (E2), scrambled control DNA enzyme (E0), or saline. Cardiomyocyte apoptosis, capillary density, and echocardiography were studied two weeks following infarction.
RESULTS: The E2 DNA enzyme, which efficiently inhibited rat PAI-1 expression in vitro, induced prolonged suppression (>2 weeks) of PAI-1 messenger ribonucleic acid and protein in rat heart tissues after a single IMI. At two weeks, hearts from experimental rats had over five-fold greater capillary density, 70% reduction in apoptotic CMs, and four-fold greater functional recovery compared with controls.
CONCLUSIONS: These results imply a causal relationship between elevated PAI-1 levels in ischemic hearts and adverse outcomes, and they suggest that strategies to reduce cardiac PAI-1 activity may augment neovascularization and improve functional recovery.
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Abbreviations and Acronyms
| | AMI = acute myocardial infarction | | CM = cardiomyocyte | | DNA = deoxyribonucleic acid | | EC = endothelial cell | | E0 = scrambled control deoxyribonucleic acid enzyme E0 | | E2 = deoxyribonucleic acid enzyme E2 targeting rat plasminogen activator inhibitor type 1 | | IHC = immunohistochemical staining | | IMI = intramyocardial injection | | LAD = left anterior descending coronary artery | | mRNA = messenger ribonucleic acid | | PIR = peri-infarct region | | PAI-1 = plasminogen activator inhibitor type 1 |
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Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiological regulator of both tissue-type and urokinase plasminogen activators (1). In addition to increasing coagulation and adversely affecting blood flow in large caliber vessels, PAI-1 plays an important role in inhibiting growth of tissue microvasculature (2,3). This is believed to be due to reduced plasmin-dependent matrix degradation required for angiogenesis and tissue neovascularization (4–6), direct pro-apoptotic effects on pre-formed endothelium (7), and reduced migration of endothelial progenitors by inhibiting the binding of cell surface integrins regulating cellular attachment and migration (8,9).
Despite frequent associations between elevated PAI-1 expression and poor cardiovascular outcomes, a causal relationship has yet to be definitively established. Plasma levels of PAI-1 are increased in patients with myocardial infarction, atherosclerosis, and restenosis (10–13). Moreover, PAI-1 messenger ribonucleic acid (mRNA) and protein expression are elevated in atherosclerotic human arteries and failed vein grafts (14–16), as well as in arterial walls and neointima formation in various animal models of arterial injury (17,18). Recently, urokinase-dependent plasminogen activation and plasmin activity have been shown to be required for both efficient in vitro myogenesis and in vivo skeletal muscle regeneration (19,20). Together, these results suggest there may be a direct causal link between the negative effects of elevated PAI-1 levels on macrovasculature and microvasculature, as well as on progenitor cell migration and tissue repair.
The purpose of the present study was to examine whether selective PAI-1 downregulation in the acutely ischemic heart increases the myocardial microvasculature and improves cardiomyocyte (CM) survival. The PAI-1 gene expression in heart tissue was targeted by a sequence-specific deoxyribonucleic acid (DNA) enzyme, a catalytic DNA molecule that can specifically cleave target mRNA at phosphodiester bonds between unpaired purine and paired pyrimidine residues with similar efficacy to that of ribozymes but with the stability of oligodeoxynucleotides (21,22). Our results strongly imply a causal relationship between elevated PAI-1 levels in ischemic hearts and adverse outcomes, and they suggest that strategies to reduce cardiac PAI-1 activity may augment neovascularization and improve functional recovery.
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Materials and methods
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Please refer to Xiang et al. (23) for the DNA enzymes, in vitro cleavage, DNA enzyme stability in serum, cell transfection, reverse transcription-polymerase chain reaction, Western blots, PAI-1 activity assay, measurement of myocyte apoptosis, quantitation of capillary density, and analyses of myocardial function regarding the methodology in the previous text.
In vitro transcription.
Rat PAI-1 cDNA was amplified using RT-PCR from total RNA of cultured rat aortic endothelial cells (ECs) (gift from Dr. G. Ceballos-Reyes, Instituto Politecnico Nacional de Mexico D.F.). Primer pair used was 5'AGC ACA CAG CCA ACC ACA GCT3' (forward primer) and 5'CTT CGA GAG TCT GAG GTC TG3' (reverse primer) (Genbank accession no. M24067, position 48-1499). For other steps, follow Xiang et al. (23).
Animals, surgical procedures and injection of DNA enzyme.
Rowett (rnu/rnu) athymic nude rats (Harlan Sprague Dawley, Indianapolis, Indiana) were used in studies approved by the Columbia University Institute for Animal Care and Use Committee. After anesthesia, a left thoracotomy was performed, the pericardium was opened, and the left anterior descending coronary artery (LAD) was ligated. At the time of surgery, animals were randomized into three groups, each receiving intramyocardial injection (IMI) of a single dose at three different sites of the peri-infarct region (PIR) consisting, respectively, of DNA enzyme E2 targeting rat PAI-1 (E2), scrambled control DNA enzyme E0 (E0), or saline. A 100-µl injection of solution included 30 µl DNA enzyme (300 µg), 20 µl Superfect, and 50 µl saline. Each group consisted of 6 to 10 rats. Animals were sacrificed at two weeks for various studies (see the following text). An additional group of saline-treated controls was sacrificed at 48 h for immunohistochemical staining (IHC) studies.
Immunohistochemistry.
The IHC staining was performed using the Histostain-SP kit (catalogue no. 95-9551, Zymed Laboratories, California) according to the manufacturer’s recommendations. The slides were incubated with 10 µg/ml of polyclonal rabbit anti-rat PAI-1 antibody (American Diagnostics, Greenwich, Connecticut) containing 0.1% goat serum for 1 h at 37°C. The PAI-1 positively staining cells are visualized as brown and counted in a minimum of five low-power fields (100x).
Statistical analysis.
Data are presented as mean ± SEM. Comparisons between two groups were made by the Student’s t test. Values of p < 0.05 were considered significant.
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Results
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Specific cleavage of rat PAI-1 mRNA by E2 DNA enzyme.
We designed two DNA enzymes, termed E2 and E3, to target specific pyrimidine-purine junctions at or near the translational start site AUG of rat and human PAI-1 messenger RNA, a region that is conserved between species and has low relative free energy. Evidence for the specificity of the DNA enzymatic reactions can be seen in Figure 1. The E2 cleaved the 24-base oligonucleotide S2, synthesized from the sequence of rat PAI-1 mRNA, in a dose- and time-dependent manner (Fig. 1a). The E2 also cleaved a rat PAI-1 transcript in a dose-dependent manner by 2 to 4 h to give the 156 nucleotide cleavage product (Fig. 1b). In contrast, neither the scrambled control DNA enzyme E0 nor E3 cleaved the rat PAI-1 transcript. Since the rat PAI-1 transcript differs by only one nucleotide from the human PAI-1 transcript, which can be cleaved by E3 (Fig. 1c), these results demonstrate the exquisite target specificity of these DNA enzymes.
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Figure 1 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.
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DNA enzymes inhibit induction of endogenous PAI-1 mRNA and protein.
To determine the effect of DNA enzymes on endogenous PAI-1 production, rat ECs were grown to subconfluence and transfected with E2 or E0. Transfected cells were then activated with transforming growth factor (TGF)-beta for 8 h to induce maximal expression of PAI-1. Densitometric analysis of polymerase chain reaction products following reverse transcription of cellular mRNA showed that E2 inhibited the induction of PAI-1 mRNA by TGF-beta in cultured rat ECs by 52%, relative to E0 (1.62 ± 0.10 for E0 vs. 0.85 ± 0.03 for E2; p < 0.01) (Fig. 2a). Similar inhibition of TGF-beta-induced PAI-1 protein expression was observed following transfection of ECs with E2, but not E0 (0.37 ± 0.05 for E0 vs. 0.26 ± 0.06 for E2; p < 0.05) (Fig. 2b). Further experiments indicate that an inverted thymidine at the 3' end can protect the PAI-1 DNA enzyme against nucleolytic degradation at physiologic serum concentrations likely to be encountered in vivo (Fig. 2c), enabling inhibition of cellular PAI-1 activity by  25%. Additionally, we also used two human DNA enzymes E1 and E3 targeting human PAI-1 mRNA to transfect human umbilical vein ECs and observed marked reduction of cellular human PAI-1 activity (6.43 ± 0.29 U/ml for E1 or 5.58 ± 1.33 U/ml for E3 vs. 8.09 ± 1.19 U/ml for E0; p < 0.01).
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Figure 2 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) 32P-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).
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IMI of E2 at the time of acute myocardial infarction (AMI) reduces early PAI-1 mRNA and protein expression in the ischemic heart.
Next, we investigated the in vivo biological activities of the PAI-1 DNA enzyme in a rat model of AMI. As shown in Figure 3a, at 48 h after LAD ligation and induction of AMI in rats, direct IMI of E2 at the PIR at the time of LAD ligation resulted in significantly reduced PAI-1 mRNA levels in the rat heart left ventricle, as measured by RT-PCR (0.86 ± 0.12 vs. 0.35 ± 0.11; p < 0.01). The IHC studies in consecutive sections showed that PAI-1 protein expression was dramatically increased in rat CMs at the PIR, but not at sites distal to the infarct zone in animals treated with E0 (Fig. 3b, left panel). Increased PAI-1 expressions are also observed in some ECs at the PIR (data not shown). In contrast, animals treated with E2 did not show such an increase in PAI-1 expression at 48 h. Moreover, as shown in Figure 3b, right panel, at 48 h, there was a >2.5-fold reduction in the number of PAI-1 positive cells at the PIR of E2-treated animals than E0-treated controls (67.2 ± 13.3/low-power field for E0 vs. 26.6 ± 9.4/low- power field for E2; p < 0.01).
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Figure 3 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).
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IMI of E2 results in prolonged suppression of PAI-1 mRNA and enhanced myocardial neovascularization.
We then investigated whether a single IMI of E2 at the time of AMI resulted in short-term or prolonged downregulation of myocardial PAI-1 mRNA expression. Two weeks after LAD ligation, myocardium from saline-treated animals demonstrated two-fold higher levels of PAI-1 mRNA than sham operated rats after normalization for glyceraldehyde-3-phosphate dehydrogenase control (1.96 ± 0.32 vs. 1.00 ± 0.05; p < 0.01) (Fig. 3c). Animals who received E2 at the time of infarction continued to demonstrate reduced myocardial PAI-1 mRNA at two weeks, with mean levels being 60% lower than in LAD-ligated controls (1.33 ± 0.25 vs. 1.96 ± 0.32; p < 0.01) (Fig. 3c). Because these results indicated a surprisingly prolonged effect of the early, single-dose administration of the DNA enzyme, we next examined whether prolonged PAI-1 downregulation influenced myocardial neovascularization after AMI. Histologic examination revealed that a single injection with E2 at the time of infarction was followed at two weeks by a significant increase in infarct zone microvascularity, cellularity, and numbers of factor VIII-positive capillaries, and by reduction in matrix deposition and fibrosis in comparison to controls. Hearts from animals injected with E2 had five-fold greater capillary density at the PIR compared with hearts injected with E0 (13.3 ± 2.5 for E2 vs. 2.5 ± 1.4 for E0; p < 0.01) (Fig. 4a). No increase in capillary numbers was observed at sites distal to the infarct zone.
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Figure 4 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.
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Myocardial PAI-1 downregulation reduces CM apoptosis and improves functional cardiac recovery after AMI.
Finally, we sought to determine the effects of myocardial PAI-1 downregulation on CM apoptosis and functional cardiac recovery after AMI. By concomitant staining for the CM-specific marker troponin I and DNA end-labeling, 70% lower numbers of apoptotic CMs were detected in infarcted left ventricles of rats injected with E2 compared with controls receiving E0 (18.0 ± 2.5 for E0 vs. 5.6 ± 1.1 for E2; p < 0.01) (Fig. 4b). These differences were particularly evident within the peri-infarct rim, where the small, irregularly shaped CM in the controls treated with either saline or E0 had the highest index of apoptotic nuclei. By two-dimensional echocardiography, rats receiving E2 demonstrated a nearly four-fold greater recovery in global cardiac function at two weeks than animals receiving E0 (mean left ventricular ejection fraction recovery 11 ± 2% vs. 3 ± 1%; p < 0.01) (Fig. 4c). Together, these results indicate that downregulation of PAI-1 by E2 enhanced peri-infarct neovascularization, increased CM survival, and thereby improved myocardial function.
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Discussion
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Endogenous myocardial neovascularization is an important process enabling cardiac functional recovery after AMI (24,25). Approaches that enhance myocardial neovascularization after acute ischemia, such as bone marrow mobilization and/or direct injection of endothelial progenitor cells, result in protection of "at-risk" CMs at the PIR against apoptosis and prevention of adverse remodeling (26). In this study, expression of PAI-1 markedly increased in the border zone of the infarct compared with sites distal to the infarct zone, and such an elevation may be affected more significantly by PAI-1 DNA enzymes, therefore leading to more obvious effects. Because targeted inhibition of PAI-1 expression in cardiac tissue including CMs and ECs was associated with enhanced myocardial neovascularization and reduced CM apoptosis, our data suggest that the complex effects of PAI-1 on CMs and vascular endothelium directly result in reduced viability and survival of CMs in vivo. Furthermore, our results strongly imply a direct causal relationship between elevated serum/tissue PAI-1 levels and adverse cardiovascular outcomes, including myocardial infarction, atherosclerosis, and restenosis (13–16).
To develop an approach to inhibit PAI-1 expression that might have clinical applicability, we focused on the use of a new generation of catalytic nucleic acids containing DNA molecules with catalytic activity for specific RNA sequences (21,22). These DNA enzymes exhibit greater catalytic efficiency than hammerhead ribozymes, offer greater substrate specificity, are more resistant to chemical and enzymatic degradation, and are far cheaper to synthesize.
Together, our results indicate that inhibition of cardiac PAI-1 mRNA by a sequence-specific catalytic DNA enzyme is a feasible approach for enhancing cardiac neovascularization after AMI. More importantly, our data demonstrate the general concept that strategies to reduce cardiac PAI-1 activity may result in protection against CM apoptosis and in improved functional recovery after acute ischemia.
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Footnotes
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This study was partially funded by National Institutes of Health Grants RFA-HL-02-017 and RFA-AG-01-006.
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Abstract
Materials and methods