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Address correspondence to Silviu Itescu, Columbia-Presbyterian Medical Center, 630 West 168th St., PH 14W, Room 1485, New York, NY 10032. Phone: (212) 305-7176; Fax: (212) 305-8304; email: si5{at}columbia.edu; or Guosheng Xiang, Columbia-Presbyterian Medical Center, 630 West 168th St., P&S 14-402, New York, NY 10032. Phone: (212) 305-1614; Fax: (212) 305-8145; email: gx15{at}columbia.edu
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Abstract |
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Key Words: myocardial infarction angiogenesis bone marrow stem cells myocardial regeneration DNA enzyme
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Introduction |
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Plasmin generation is negatively regulated by local concentrations of the serpin plasminogen activator inhibitor (PAI)-1, which in its native state, is complexed to circulating and tissue vitronectin (9, 10). After reactivity with a proteinase such as u-PA, PAI-1 undergoes a rapid conformational change that causes it to dissociate from vitronectin and increase its affinity for the low density lipoprotein receptor (11), leading to its clearance and degradation. In addition, removal of PAI-1 from vitronectin exposes a cryptic epitope necessary for binding to the cell surface integrin alphavbeta3, which regulates cellular attachment and migration (9, 12).
Plasma levels of PAI-1 are increased in patients with myocardial infarction, atherosclerosis, and restenosis (13-16). Moreover, PAI-1 mRNA and protein expression are elevated in atherosclerotic human arteries and failed vein grafts (1719), as well as in arterial walls and neointima formation in various animal models of arterial injury (20, 21). Despite the frequent associations between elevated PAI-1 expression and poor cardiovascular outcomes, a causal relationship has yet to be definitively established. Because both u-PA surface expression (8) and cellular interactions with tissue vitronectin are important components in new blood vessel growth (9, 22, 23), we hypothesized that increased PAI-1 expression after myocardial infarction might result in poor outcome by directly inhibiting the ability of bone marrowderived angioblasts to induce neovascularization. This study examined the effect of PAI-1 down-regulation on postinfarction neovascularization and myocardial function recovery induced by bone marrowderived angioblasts.
To develop an approach to inhibit PAI-1 expression that might have clinical applicability, we examined various potential strategies for inhibiting specific mRNA activity, including antisense oligonucleotides and ribozymes (2426). Because these approaches are limited by sensitivity to chemical and enzymatic degradation and restricted target site specificity, we focused on the use of a new generation of catalytic nucleic acids containing DNA molecules with catalytic activity for specific RNA sequences (2730). 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. In this study we created DNA enzymes with specificity for target sequences in human and rat PAI-1 mRNA. Our results indicate that inhibition of PAI-1 expression in the heart after a myocardial infarction results in a striking augmentation of human angioblastdependent neovascularization, cardiomyocyte regeneration, and functional cardiac recovery.
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Materials and Methods |
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In Vitro Transcription.
Human PAI-1 cDNA was amplified by RT-PCR from total RNA of cultured human umbilical vein endothelial cells (HUVECs) using the following primer pair: 5'-CCAAGAGCGCTGTCAAGAAGAC-3' (forward primer) and 5'-TCACCGTCTGCTTTGGAGACCT-3' (reverse primer; data are available from GenBank/EMBL/DDBJ under accession no. J03764, position 2510600). Length of the PCR product was 1,598 bases. Rat PAI-1 cDNA was amplified using RT-PCR from total RNA of cultured rat arotic endothelial cells (provided by G. Ceballos-Reyes, Instituto Politecnico Nacional de Mexico, Mexico City, Mexico). The primer pair used was 5'-AGC ACA CAG CCA ACC ACA GCT-3' (forward primer) and 5'-CTT CGA GAG TCT GAG GTC TG-3' (reverse primer; data are available from GenBank/EMBL/DDBJ under accession no. M24067, position 481499) and the length of PCR product was 1,452 bases. Both PAI-1 cDNAs were cloned into pGEM-T vector (Promega) to obtain plasmid constructs pGEM-hPAI and pGEM-rPAI, and cDNA sequences were verified by automatic sequencing. A 32P-labeled nucleotide from the human or rat PAI-1 RNA transcript was prepared by in vitro transcription (SP6 polymerase; Promega) and cut before transcription with NcoI. 20 µl of transcription reaction consisted of 4 µl 5x buffer, 2 µl DTT, 1 µl RNasin inhibitor, 4 µl NTP mixture (1 µl A, G, C, and 1 µl H2O), 100 µM UTP, 2 µl template (0.3 µg/µl), 32P-UTP (10 µci/µl), and 1 µl SP6 polymerase (20 U/µl). Reaction time was 1 h at 32°C. Unincorporated label and short nucleotides (<350 bases) were separated from radiolabeled species by centrifugation on Chromaspin-200 columns (CLONTECH Laboratories, Inc.).
Cleavage Reactions.
Synthetic RNA substrate was end-labeled with 32P using T4 polynucletiod kinase. The cleavage reaction system included 60 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 150 mM NaCl, 0.5 µM 32P-labeled RNA oligo, and 0.055 µM DNA enzyme. For cleavage of in vitro transcripts, the reaction system contained 1% of PAI-1 transcripts from transcription reaction system, 25 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 100 mM NaCl, and 0.220 µM DNA enzyme. Reactions were allowed to proceed at 37°C and were quenched by transfer of aliquots to tubes containing 90% formamide, 20 mM EDTA, and loading dye. Samples were separated by electrophoresis on TBE-ureadenaturing polyacrylamide gels (5% gel for in vitro transcripts and 15% for synthetic RNA). Densitometric analysis of band autoradiographs was performed using a Personal Densitometer and ImageQuant software (Molecular Dynamics). The percentage of the transcript mRNA cleaved by DNA enzyme was calculated as density of degraded mRNA bands/density of degraded plus undegraded bands.
DNA Enzyme Stability in Serum.
DNA enzymes were 32P-radiolabeled using T4 polynucleotide kinase. Labeling reaction (20 µl of volume) consisted of 1 µl DNA enzyme (20 uM), 2 µl 10x kinase buffer, 1 µl T4 PNK (10 U/µl), 10 µl H2O, and 6 µl 32P-ATP (3,000 µCi/mmol, 10 µCi/µl). Reaction time was 30 min at 37°C. 32P-labeled DNA enzymes were incubated at 37°C in media containing 2% FCS (EGM; Clonetics) or 20% FCS (DMEM; GIBCO BRL) at a final concentration of 100 nM. Aliquots of the mixture were removed at different time points of 0, 3, 6, 12, and 24 h, quenched with phenol/chloroform, and frozen until use. At the end of each experiment, all samples were phenol extracted and analyzed by 15% denaturing polyacrylamide gels and visualized by autoradiography.
Culture Conditions and DNA Enzyme Transfection.
Primary HUVECs were obtained from Clonetic and grown in EGM medium containing 2% FCS. Primary rat arotic endothelial cells were cultured in DMEM medium, pH 7.4 (Life Technologies), containing 10% FCS, 100 µg/ml streptomycin, and 100 IU/ml penicillin at 37°C in a humidified atmosphere of 5% CO2. For transfection of DNA enzyme, rat endothelial cells were plated into each well of six-well plates (105 cells/well). Subconfluent (
7080%) endothelial cells were washed twice with 1 ml Hepes buffer, pH 7.4, and transfected using 0.5 ml of serum-free DMEM containing 1 µM of test molecule (E2 or E0) and 7 µl/ml Superfect (QIAGEN). 3 h after incubation, 0.5 ml of 5% serum DMEM was added to each well. 3 h later, TGF-ß was added to half of the wells at a final concentration of 1.8 ng/ml. Transfected cells continued to be incubated for 8 h and were lysed to isolate RNA (for RT-PCR) and protein (for Western blot) using Trizol reagent (Life Technologies).
RT-PCR.
Total RNA from each well of six-well plates was isolated using 1 ml Trizol reagent and dissolved in 20 µl DEPC-treated H2O. 2-µl samples were used to perform reverse transcription in 20 µl of reaction system, and 4 µl of products was then used as templates to amplify rat PAI-1 or GAPDH in 50 µl PCR system (5 µl 10x buffer, 1 µl dNTP, 0.25 µl Taq polymerase, 1 µl forward and reverse primer, 38 µl H2O, 2 µl 32P dCTP [3,000 ci/mmol, 10 µci/µl]). The reaction conditions were as follows: 95°C for 0.5 min, 58°C for 1 min, 72°C for 2 min, and 30 cycles at 72°C for 10 min. Rat GAPDH was used as internal control to quantify PAI-1 (PAI-1 primers as noted above). Rat GAPDH forward primer is 5'-CTCTACCCACGGCAAGTTCAA-3' and the reverse primer is 5'-GGGATGACCTTGCCCACAGC-3'. PCR product was 515 bases long.
Western Blot.
Control and DNA enzymetreated cells were lysed and protein pellets were dissolved in 1% SDS solution and then boiled for 4 min after an equal volume of 2x lysis buffer was added. Equal amounts of protein (10 µg/lane) were separated by SDS gel electrophoresis with 10% polyacrylamine separating gel using minigels (Bio-Rad Laboratories). After electrophoresis, proteins were transferred to nitrocellulose membrane (Amersham Biosciences) by electrotransfer and blocked for at least 1 h at room temperature with 5% (wt/vol) nonfat milk in TBS-T buffer (0.1 M Tris-base, pH 7.5, 0.15M NaCl, and 0.1% Tween-20). After this step, membranes were immunoblotted with goat IgG polyclonal anti-PAI antibodies (Santa Cruz Biotechnology, Inc.) and then visualized by the ECL system (Amersham Biosciences) using horseradish peroxidaseconjugated antigoat IgG (Sigma-Aldrich).
Measurement of PAI-1 Activity.
At 7080% confluence, HUVECs seeded in 24-well plates were transfected with 2 µM DNA enzymes (E1 and E3 as well as E0 in the presence or absence of 2 ng/ml TGF-ß1 for 24 h. After washing twice with PBS, cell lysates were collected using 400 µl 0.5% Triton X-100 in PBS. 25 µl of cell lysate was incubated with plasmin substrate in a 96-well microtiter plate (final volume, 230 µl) for 134 min at room temperature. PAI-1 activity was measured by reading the difference of absorbances at 405 and 492 nm and was calculated against a plasmin standard regression line according to the protocol of American Diagnostica, Inc. (catalog no. 101201).
Transendothelial Migration Assay of Human Bone Marrow Stem Cells.
In brief, rat endothelial cell monolayers (2 x 105 cells/well) were grown to subconfluence (
7080%) on PET transwells in the presence or absence of rat vitronectin. To the top chamber of each well in triplicate experiments, 105 human CD34+ cells (use of human cells was approved by the Columbia University Institute for Animal Care and Use Committee) were added from a single donor together with 2% FCS, the presence or absence of 1.8 ng/ml TGF-ß, and either E2 or scrambled DNA enzyme complexed with 20 µg/ml DOTAP. After 24 h in culture at 37°C, the cells in the top and bottom chambers were recovered and counted with a hemocytometer. The proportion of cells migrating across the endothelial monolayer was calculated as the number of cells in the bottom chamber divided by the total number of cells counted, and normalized to the proportion of human CD34+ cells migrating across the membrane in the absence of any endothelial monolayer for each condition tested. Additionally, after supplemental transmigration, an assay was performed to evaluate the effect of exogenous rat PAI-1 or antibody against rat PAI-1 on CD34+ cell transmigration. Rat endothelial cells (3.5 x 104 cells/100 µl) were seeded into the upper chamber of transwell plates (6.5-mm diameter, pore size of 5 µm; Costar, Inc.) and 300 µl DMEM (2% FCS) was placed into the lower chamber. After cultured cells reached full confluence and upper chambers were placed into fresh lower chambers, 2 µg rat PAI-1 (American Diagnostics) in 50 µl DMEM without FCS was added into some upper chambers, and 2 and 3 µg IgG against rat PAI-1 (American Diagnostics) was added into some upper and lower chambers, respectively. 30 min later, 105 CD34+ cells in 100 µl DMEM without FCS were added into each upper chamber. Cells migrating from the upper chamber into the lower chamber were harvested and counted using a hemocytometer 24 h after the addition of CD34+ cells.
Animals, Surgical Procedures, and Injection of DNA Enzyme and Human Cells.
Rowett (rnu/rnu) athymic nude rats (Harlan Sprague Dawley) 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 (LAD) coronary artery was ligated. At the time of surgery, animals were randomized into three groups, each receiving three intracardiac injections at the peri-infarct region consisting, respectively, of E2 DNA enzyme, E0 scrambled control, or saline. 100 µl of injection solution included 30 µl DNA oligonucleotide (300 µg), 20 µl Superfect, and 50 µl saline. For studies on neovascular rization and effects on myocardial viability and function, 2.0 x 106 human cells obtained from a single donor after G-CSF mobilization were reconstituted with 2.0 x 105 immunopurified CD34+ CD117bright cells (1) and injected into the rat tail vein 48 h after LAD ligation. Each group consisted of 610 rats.
Immunohistochemistry and Quantitation of Capillary Density.
To quantitate and characterize PAI-1expressing cells at 48 h after LAD ligation, sections from the hearts of control animals killed at this time point were freshly stained with goat IgG polyclonal anti-PAI antibodies (Santa Cruz Biotechnology, Inc.), mouse mAbs directed against rat CD68, factor VIIIrelated antigen, and cardiac troponin I (DakoCytomation), and then visualized by immunoperoxidase technique using an Avidin/Biotin Blocking Kit, a rat-absorbed biotinylated antigoat IgG, and a peroxidase conjugate (all from Vector Laboratories). Capillary density and species origin of the capillaries were determined in sections from the hearts of animals killed at 2 wk. Sections were freshly stained with mAbs directed against rat CD31 (Serotec and Research Diagnostics, respectively), factor VIIIrelated antigen (DakoCytomation), and rat MHC class I (Accurate Chemicals). Arterioles were differentiated from large capillaries by the presence of a smooth muscle layer, identified by staining sections with an mAb against myocyte-specific desmin (DakoCytomation). Staining was performed by immunoperoxidase technique using an Avidin/Biotin Blocking Kit, a rat-absorbed biotinylated antimouse IgG, and a peroxidase conjugate (all from Vector Laboratories). Capillary density was determined at 2 wk after infarction from sections labeled with anti-CD31 mAb and confirmed with mAb against factor VIIIrelated antigen and compared with the capillary density of the unimpaired myocardium. Values are expressed as the number of CD31+ capillaries per high power field (a magnification of 400).
Quantitation of Cardiomyocyte Proliferation.
Cardiomyocyte DNA synthesis and cell cycling was determined by dual staining of rat myocardial tissue sections obtained from LAD-ligated rats 2 wk after injection of either saline or CD34+ human cells, and from healthy rats as negative controls, for cardiomyocyte-specific troponin I and human- or rat-specific Ki-67. In brief, paraffin-embedded sections were microwaved in 0.1 M EDTA buffer and stained with either a polyclonal rabbit antibody with specificity against rat Ki-67 at a 1:3,000 dilution (provided by G. Cattoretti, Columbia University, New York, NY), or a mouse mAb recognizing both human and rat Ki-67 and MIB-1 at a 1:300 dilution (DakoCytomation), and incubated overnight at 4°C. After washes, sections were incubated with a species-specific secondary antibody conjugated with alkaline phosphatase at a 1:200 dilution (Vector Laboratories) for 30 min, and positively staining nuclei were visualized as blue with a BCIP/NBT substrate kit (DakoCytomation). Sections were then incubated overnight at 4°C with an mAb against cardiomyocyte-specific troponin I (Accurate Chemicals), and positively staining cells were visualized as brown through the Avidin/Biotin system described above. Cardiomyocytes progressing through cell cycle in the infarct zone, peri-infarct region, and areas distal to the infarct were calculated as the proportion of troponin I+ cells per high power field coexpressing Ki-67. For confocal microscopy, FITC-conjugated rabbit antimouse IgG was used as secondary antibody to detect Ki67 in nuclei. A Cy5-conjugated mouse mAb against -sarcomeric actin (clone 5C5; Sigma-Aldrich) was used to detect cardiomyocytes, and propidium iodide was used to identify all nuclei. In separate experiments, animals receiving saline or CD34+ cells after LAD ligation were fed BrdU ad libitum daily in drinking water. After death, paraffin-embedded tissue was incubated with a mouse anti-BrdUrd antibody (Roche Molecular Biochemicals) followed by a biotinylated rabbit antimouse IgG antibody (Jackson ImmunoResearch Laboratories), diluted at 1:3,000 with D-PBS. The biotin is detected by using the Avidin/Biotin Complex Kit (Vector Laboratories) as described above.
Analyses of Myocardial Function.
Echocardiographic studies were performed using a high frequency liner array transducer (SONOS 5500; Hewlett Packard). Two-dimensional images were obtained at mid-papillary and apical levels. End-diastolic (EDV) and end-systolic (ESV) left ventricular volumes were obtained by bi-plane area length method, and the percent of left ventricular ejection fraction was calculated as [(EDV-ESV)/EDV] x 100. All echocardiographic studies were performed by a blinded investigator.
Statistical Analysis.
Data are presented as mean ± SD. Comparisons between groups were made by Student's t test. Values of P < 0.05 were considered significant.
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Results |
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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, endothelial monolayers of human and rat origin were grown to confluence and transfected with species-specific DNA enzymes or scrambled control. Transfected cells were then activated with TGF-ß for 8 h to maximally induce expression of PAI-1. Densitometric analysis of RT-PCR products after reverse transcription of cellular mRNA showed that E2 inhibited TGF-ßinducible steady-state mRNA levels in cultured rat endothelium by 52%, relative to the E0 scrambled DNA enzyme (Fig. 2 A). Basal level of PAI-1 mRNA revealed a small decrease after transfection with the DNA enzymes (ratio of PAI1 mRNA to GAPDH, 27 ± 1% for E0 vs. 19 ± 2% for E2). Fig. 2 B shows the effect of endothelial cell transfection with E2 DNA enzyme on TGF-ßmediated induction of PAI-1 protein. Endothelial cells transfected with scrambled DNA enzyme demonstrated an 50% increase in cytoplasmic PAI-1 protein as detected by Western blot. In contrast, this effect was almost completely abrogated by transfection with the PAI-1 DNA enzyme E2.
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PAI-1 DNA Enzymes Increase Transendothelial Migration of Human Bone Marrow Stem Cells.
To investigate the physiologic significance of inhibiting PAI-1 activity in endothelial cells, we examined migration of human CD34+ cells across rat endothelial monolayers transfected with either E2 or the scrambled DNA enzyme E0. As shown in Fig. 3 A, treatment of rat endothelial monolayers with E2 increased human CD34+ migration by approximately threefold relative to medium alone in the presence of vitronectin, but not in the absence of vitronectin (P < 0.05). In contrast, treatment with the scrambled DNA enzyme did not significantly increase transendothelial migration relative to medium alone. The addition of exogenous recombinant rat PAI-1 or antibody against rat PAI-1 caused, respectively, either a marked reduction or an increase in transmigrating CD34+ cells compared with untreated controls (53.1 ± 18.7% or 168.7 ± 33% vs. 100 ± 20.4%, P < 0.05 for both; Fig. 3 B). These results demonstrate that inhibition of PAI-1 mRNA and protein expression augments the ability of human CD34+ cells to migrate across endothelial monolayers via interactions involving plasmin generation and vitronectin binding.
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Intracardiac Injection of E2 Together with Angioblasts Results in Enhanced Neovascularization at the Peri-Infarct Region.
When CD34+ cells containing angioblasts were administered intravenously with intramyocardial E2 DNA enzyme, as shown in Fig. 3 (F and G), a further 3.5-fold increase in neovascularization was seen over that induced by the CD34+ cells alone (P < 0.01). Moreover, thin-walled capillaries with very large lumens (more than six nuclei) and diameters in excess of 30 µm were frequently seen at the peri-infarct region when E2 and CD34+ cells were coadministered, but not when either was injected alone (Fig. 3 F). Notably, the large lumen capillaries overlapped in size with arterioles, but differed in that they only contained a thin endothelial layer, whereas the arterioles could be distinguished by an outer layer containing two to three smooth muscle cells of rat origin, as determined by positive staining with desmin and rat MHC class I mAbs. Although many of the newly formed vessels were of rat origin (angiogenesis), at least 25% of the vessels at the peri-infarct region were of human origin (vasculogenesis), as defined by a human anti-CD31 mAb. Fig. 3 H shows the effect of E2 on coalescence and vascular incorporation of human CD34+ cells in infarcted rat myocardium. In the presence of E2, the number of unincorporated, discrete, interstitial human CD34+ cells in the infarct zone and peri-infarct region, as determined by staining with a human anti-CD31 mAb, was reduced by 5.2-fold compared with E0 control enzyme (P < 0.01). Conversely, the number of large lumen capillaries (more than six nuclei) at the peri-infarct region was increased by 14-fold after E2 injection as compared with E0. We conclude that by inhibiting PAI-1 expression at the peri-infarct region, E2 enables human CD34+ cells to more effectively migrate through cardiac tissue, coalesce, and participate in new vessel formation.
Angioblast-dependent Neovascularization Induces Cardiomyocyte Regeneration.
Although myocyte hypertrophy and increase in nuclear ploidy have generally been considered the primary mammalian cardiac responses to ischemia, damage, and overload (32, 33), recent observations have suggested that human cardiomyocytes have the capacity to proliferate and regenerate in response to injury (34, 35). Because organogenesis in the prenatal period is preceded by signals derived from neovasculature (36), and fetal cardiomyocytes have the capacity to enter the cell cycle, we next examined whether neovascularization of the adult heart by human angioblasts might induce proliferation/regeneration of endogenous cardiomyocytes. As shown in Fig. 4 A, at 2 wk after LAD ligation, rats receiving human CD34+ cells demonstrated numerous "fingers" of cardiomyocytes of rat origin, as determined by expression of rat MHC class I molecules, extending from the peri-infarct region into the infarct zone. Similar extensions were seen very rarely in animals receiving saline. The islands of cardiomyocytes at the peri-infarct rim in animals receiving human CD34+ cells contained a high frequency of rat myocytes with DNA activity, as determined by dual staining with an mAb reactive against cardiomyocyte-specific troponin I and a polyclonal rabbit antiserum with reactivity against rat, but not human, Ki67. Triple immunofluorescence using confocal microscopy confirmed the presence of cycling rat cardiomyocytes and demonstrated a speckled pattern of Ki67 reactivity within cycling nuclei (Fig. 4 B). In contrast, in animals receiving saline, there was a high frequency of cells with fibroblast morphology and reactivity with rat Ki67, but not troponin I, within the infarct zone. The number of cardiomyocytes progressing through cell cycle at the peri-infarct region of rats receiving human CD34+ cells was 40-fold higher than that at sites distal to the infarct, where myocyte DNA activity was no different than in sham-operated rats.
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Discussion |
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Transendothelial egress, extracellular matrix degradation, and neovascularization are all related processes thought to require intravascular activation of latent metalloproteinases by plasmin, which is derived from plasminogen through activation by u-PA on the surface of the infiltrating bone marrowderived cells and inhibited by PAI-1 (68). Recent studies have suggested that u-PA also binds to PAI-1 in tissues, where it is complexed to vitronectin (9, 10). The u-PAPAI-1 interaction results in a rapid conformational change in PAI-1 that causes it to dissociate from vitronectin and increase its affinity for the low density lipoprotein receptor (11), leading to its clearance and degradation. Removal of PAI-1 from vitronectin exposes a cryptic epitope necessary for binding to the cell surface integrin alphavbeta3, which regulates cellular attachment and migration (9, 12). Together, these data indicate that angioblast-dependent neovascularization of the adult heart is inhibited in situations of excess circulating and tissue PAI-1 levels by overlapping mechanisms, including interference with u-PAdependent plasmin generation and integrin binding to tissue vitronectin (8, 9, 22, 23).
In addition to augmenting cardiac neovascularization, inhibition of myocardial PAI-1 mRNA expression resulted in striking induction of cardiomyocyte regeneration and functional cardiac recovery in the presence of intravenously administered angioblasts. Although myocyte hypertrophy and increase in nuclear ploidy have generally been considered to be the primary mammalian cardiac responses to ischemia, damage, and overload (32, 33), recent observations have suggested that human cardiomyocytes have the capacity to proliferate and regenerate in response to injury (34, 35), although the signals required to induce such regeneration in meaningful numbers have not been identified. Common precursors giving rise to both cells of smooth muscle and cardiomyocyte lineage have been identified in the adult murine bone marrow (37), and it is possible that these cells might be mobilized coincident with acute ischemia or may seed the developing heart early in ontogeny and be locally resident in the adult. Whether the regenerative process described herein indeed involves bone marrowderived or resident cardiomyocyte progenitors remains to be determined; however, it bears noting the striking similarity to the spontaneous myocardial regeneration seen to accompany prominent neovascularization after myocardial injury in MRL mice, a strain with abnormal stem cell development (38).
To develop an approach to inhibit PAI-1 expression that might have clinical applicability, various potential strategies for inhibiting specific PAI-1 mRNA activity were examined. Antisense oligonucleotides hybridize with their complementary target site in mRNA, blocking translation to protein by sterically inhibiting ribosome movement or by triggering cleavage by endogenous RNase H (24). Although current constructs are made more resistant to degradation by serum through phosphorothioate linkages, nonspecific biological effects due to "irrelevant cleavage" of nontargeted mRNA remains a major concern (25). Ribozymes are naturally occurring RNA molecules that contain catalytic sites, making them more potent agents than antisense oligonucleotides. However, wider use of ribozymes has been hampered by their susceptibility to chemical and enzymatic degradation and restricted target site specificity (26). Consequently, we focused on the use of a new generation of catalytic nucleic acids containing DNA molecules with catalytic activity for specific RNA sequences (2730). These DNA enzymes exhibit greater catalytic efficiency than hammerhead ribozymes, producing a rate enhancement of 10 million-fold over the spontaneous rate of RNA cleavage, offer greater substrate specificity, are more resistant to chemical and enzymatic degradation, and are far cheaper to synthesize. Our results demonstrate that inhibition of myocardial PAI-1 mRNA by a sequence-specific catalytic DNA enzyme is a feasible approach for enhancing cardiac neovascularization, regeneration, and functional recovery after an acute ischemic insult.
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Acknowledgments |
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The authors have no conflicting financial interests.
Submitted: 3 February 2004
Accepted: 16 November 2004
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References |
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