Down-regulation of plasminogen activator inhibitor 1 expression promotes myocardial neovascularization by bone marrow progenitors.
ABSTRACT Human adult bone marrow-derived endothelial progenitors, or angioblasts, induce neovascularization of infarcted myocardium via mechanisms involving both cell surface urokinase-type plasminogen activator, and interactions between beta integrins and tissue vitronectin. Because each of these processes is regulated by plasminogen activator inhibitor (PAI)-1, we selectively down-regulated PAI-1 mRNA in the adult heart to examine the effects on postinfarct neovascularization and myocardial function. Sequence-specific catalytic DNA enzymes inhibited rat PAI-1 mRNA and protein expression in peri-infarct endothelium within 48 h of administration, and maintained down-regulation for at least 2 wk. PAI-1 inhibition enhanced vitronectin-dependent transendothelial migration of human bone marrow-derived CD34+ cells, and resulted in a striking augmentation of angioblast-dependent neovascularization. Development of large, thin-walled vessels at the peri-infarct region was accompanied by induction of proliferation and regeneration of endogenous cardiomyocytes and functional cardiac recovery. These results identify a causal relationship between elevated PAI-1 levels and poor outcome in patients with myocardial infarction through mechanisms that directly inhibit bone marrow-dependent neovascularization. Strategies that reduce myocardial PAI-1 expression appear capable of enhancing cardiac neovascularization, regeneration, and functional recovery after ischemic insult.
- [show abstract] [hide abstract]
ABSTRACT: Left ventricular remodeling is a major cause of progressive heart failure and death after myocardial infarction. Although neoangiogenesis within the infarcted tissue is an integral component of the remodeling process, the capillary network is unable to support the greater demands of the hypertrophied myocardium, resulting in progressive loss of viable tissue, infarct extension and fibrous replacement. Here we show that bone marrow from adult humans contains endothelial precursors with phenotypic and functional characteristics of embryonic hemangioblasts, and that these can be used to directly induce new blood vessel formation in the infarct-bed (vasculogenesis) and proliferation of preexisting vasculature (angiogenesis) after experimental myocardial infarction. The neoangiogenesis resulted in decreased apoptosis of hypertrophied myocytes in the peri-infarct region, long-term salvage and survival of viable myocardium, reduction in collagen deposition and sustained improvement in cardiac function. The use of cytokine-mobilized autologous human bone-marrow-derived angioblasts for revascularization of infarcted myocardium (alone or in conjunction with currently used therapies) has the potential to significantly reduce morbidity and mortality associated with left ventricular remodeling.Nature Medicine 05/2001; 7(4):430-6. · 22.86 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The generation of blood cells, haematopoiesis, in the mouse embryo begins with the development of primitive nucleated erythroid cells in the yolk sac followed by the appearance of precursors for multiple definitive haematopoietic lineages. The later developing lineages arise from multipotential stem cells, but the relationship of primitive erythroid cells to these other haematopoietic populations is unknown. Using an in vitro embryonic stem (ES) cell differentiation system, we show that primitive erythrocytes and other haematopoietic lineages arise from a common multipotential precursor that develops within embryoid bodies generated from differentiated ES cells. In response to vascular endothelial growth factor and c-kit ligand these precursors give rise to colonies containing immature cells (blasts) expressing marker genes characteristic of haematopoietic precursors. Many blast colonies also expressed betaH1 and beta major globins but not Brachyury, a mesodermal marker. Kinetic analysis demonstrated that the blast colony-forming cells represent a transient population, preceding the establishment of the primitive erythroid and other lineage-restricted precursors. This precursor population may represent the earliest stage of embryonic haematopoietic commitment.Nature 05/1997; 386(6624):488-93. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Embryonic stem cell-derived embryoid bodies contain a unique precursor population which, in response to vascular endothelial growth factor, gives rise to blast colonies in semi-solid medium. Upon transfer to liquid culture with appropriate cytokines, these blast colonies generate both hematopoietic and adherent, stromal-type cells. Cells within the adherent population display characteristics of endothelial lineage including the expression of CD31, flk-1, flt-1, tie-2, the capacity to take up acetylated LDL and the presence of cytoplasmic Weibel-Palade bodies. Mixing studies demonstrated that the hematopoietic and endothelial precursors within the blast colonies develop from the same cell, the blast colony-forming cell. Kinetic analysis showed that the blast colony-forming cell represents a transient cell population that develops early and is lost quickly during embryoid body development. These findings provide strong evidence that the blast colony-forming cell represents the long-hypothesized hemangioblast, the common precursor of the hematopoietic and endothelial lineages.Development 03/1998; 125(4):725-32. · 6.21 Impact Factor
The Journal of Experimental Medicine
J. Exp. Med.
Volume 200, Number 12, December 20, 2004 1657–1666
The Rockefeller University Press • 0022-1007/2004/12/1657/10 $8.00
Down-regulation of Plasminogen Activator Inhibitor 1
Expression Promotes Myocardial Neovascularization
by Bone Marrow Progenitors
Guosheng Xiang, Michael D. Schuster, Tetsunori Seki,
Alfred A. Kocher, Shawdee Eshghi, Andrew Boyle, and Silviu Itescu
Department of Surgery and Department of Medicine, Columbia University, New York, NY 10032
Human adult bone marrow–derived endothelial progenitors, or angioblasts, induce neovascular-
ization of infarcted myocardium via mechanisms involving both cell surface urokinase-type plas-
minogen activator, and interactions between
integrins and tissue vitronectin. Because each of
these processes is regulated by plasminogen activator inhibitor (PAI)-1, we selectively down-
regulated PAI-1 mRNA in the adult heart to examine the effects on postinfarct neovasculariza-
tion and myocardial function. Sequence-specific catalytic DNA enzymes inhibited rat PAI-1
mRNA and protein expression in peri-infarct endothelium within 48 h of administration, and
maintained down-regulation for at least 2 wk. PAI-1 inhibition enhanced vitronectin-dependent
transendothelial migration of human bone marrow–derived CD34
augmentation of angioblast-dependent neovascularization. Development of large, thin-walled
vessels at the peri-infarct region was accompanied by induction of proliferation and regeneration
of endogenous cardiomyocytes and functional cardiac recovery. These results identify a causal
relationship between elevated PAI-1 levels and poor outcome in patients with myocardial in-
farction through mechanisms that directly inhibit bone marrow–dependent neovascularization.
Strategies that reduce myocardial PAI-1 expression appear capable of enhancing cardiac neovascu-
larization, regeneration, and functional recovery after ischemic insult.
cells, and resulted in a striking
myocardial regeneration • DNA enzyme
myocardial infarction • angiogenesis • bone marrow stem cells •
We have recently isolated endothelial progenitor cells, or
angioblasts, in human adult bone marrow that have pheno-
typic and functional characteristics of embryonic angio-
blasts (1), which are cells that are derived from the human
ventral aorta and give rise to the definitive vascular net-
work during early development (2–5). Intravenous admin-
istration of these cells to athymic rats who have undergone
experimental myocardial infarction results in selective hom-
ing to ischemic myocardium, induction of infarct bed
neovascularization, and significant improvement in myo-
cardial function (1). Although the precise chemotactic factors
regulating migration of bone marrow–derived angioblasts
to the site of acute ischemia remain to be identified, trans-
endothelial egress, extracellular matrix degradation, and
neovascularization are thought to require activation of la-
tent metalloproteinases by plasmin, which is derived from
plasminogen through activation by urokinase-type plas-
minogen activator (u-PA) expressed on the surface of the
infiltrating bone marrow–derived cells (6-8).
Plasmin generation is negatively regulated by local con-
centrations 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 in-
crease 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 epi-
tope necessary for binding to the cell surface integrin
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: email@example.com;
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: firstname.lastname@example.org
Abbreviations used in this paper:
cell; LAD, left anterior descending; PAI, plasminogen activator inhibitor;
u-PA, urokinase-type plasminogen activator.
HUVEC, human umbilical vein endothelial
PAI-1 and Myocardial Neovascularization by CD34
alphavbeta3, which regulates cellular attachment and mi-
gration (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 ele-
vated in atherosclerotic human arteries and failed vein grafts
(17–19), 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 vitronec-
tin are important components in new blood vessel growth
(9, 22, 23), we hypothesized that increased PAI-1 expres-
sion after myocardial infarction might result in poor out-
come by directly inhibiting the ability of bone marrow–
derived angioblasts to induce neovascularization. This study
examined the effect of PAI-1 down-regulation on post-
infarction neovascularization and myocardial function re-
covery induced by bone marrow–derived angioblasts.
To develop an approach to inhibit PAI-1 expression that
might have clinical applicability, we examined various po-
tential strategies for inhibiting specific mRNA activity, in-
cluding antisense oligonucleotides and ribozymes (24–26).
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 (27–30). These
DNA enzymes exhibit greater catalytic efficiency than ham-
merhead 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 angioblast–
dependent neovascularization, cardiomyocyte regenera-
tion, and functional cardiac recovery.
Materials and Methods
DNA Enzymes and RNA Substrates.
inverted thymidine were synthesized by Integrated DNA
Technologies and purified by RNase-free ion exchange HPLC
or reverse phase HPLC. The short RNA substrates correspond-
ing to target DNA enzyme sequences were chemically synthe-
sized followed by RNase-free PAGE purification and also made
by in vitro transcription from a DNA template.
In Vitro Transcription.
Human PAI-1 cDNA was amplified by
RT-PCR from total RNA of cultured human umbilical vein en-
dothelial cells (HUVECs) using the following primer pair:
data are available from GenBank/EMBL/DDBJ under accession
no. J03764, position 25–10600). 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 Mex-
DNA enzymes with
ico, Mexico City, Mexico). The primer pair used was 5
ACA CAG CCA ACC ACA GCT-3
CTT CGA GAG TCT GAG GTC TG-3
are available from GenBank/EMBL/DDBJ under accession no.
M24067, position 48–1499) 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
P-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
l of transcription reaction consisted of 4
l DTT, 1
l RNasin inhibitor, 4
C, and 1
M UTP, 2
– P-UTP (10
l), and 1
Reaction time was 1 h at 32
C. Unincorporated label and short
350 bases) were separated from radiolabeled species
by centrifugation on Chromaspin-200 columns (CLONTECH
Synthetic RNA substrate was end-labeled
P using T4 polynucletiod kinase. The cleavage reac-
tion system included 60 mM Tris-HCl, pH 7.5, 10 mM MgCl
150 mM NaCl, 0.5
M P-labeled RNA oligo, and 0.05–5
DNA enzyme. For cleavage of in vitro transcripts, the reaction
system contained 1% of PAI-1 transcripts from transcription reac-
tion system, 25 mM Tris-HCl, pH 7.5, 5 mM MgCl
NaCl, and 0.2–20
M 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-urea–
denaturing polyacrylamide gels (5% gel for in vitro transcripts and
15% for synthetic RNA). Densitometric analysis of band autora-
diographs was performed using a Personal Densitometer and Im-
ageQuant 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 un-
DNA Enzyme Stability in Serum.
P-radiolabeled using T4 polynucleotide kinase. Labeling reac-
l of volume) consisted of 1
kinase buffer, 1
l T4 PNK (10 U/
l P-ATP (3,000
30 min at 37
C. P-labeled DNA enzymes were incubated at
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.
HUVECs were obtained from Clonetic and grown in EGM me-
dium containing 2% FCS. Primary rat arotic endothelial cells
were cultured in DMEM medium, pH 7.4 (Life Technologies),
containing 10% FCS, 100
g/ml streptomycin, and 100 IU/ml
penicillin at 37
C in a humidified atmosphere of 5% CO
transfection of DNA enzyme, rat endothelial cells were plated
into each well of six-well plates (
70–80%) 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
M of test molecule (E2 or E0) and 7
ml Superfect (QIAGEN). 3 h after incubation, 0.5 ml of 5% se-
rum DMEM was added to each well. 3 h later, TGF-
(forward primer) and 5
(reverse primer; data
l A, G,
l NTP mixture (1
l template (0.3
l SP6 polymerase (20 U/
, 100 mM
DNA enzymes were
l DNA enzyme (20 uM),
l). Reaction time was
Xiang et al.
to half of the wells at a final concentration of 1.8 ng/ml. Trans-
fected 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).
Total RNA from each well of six-well plates was
isolated using 1 ml Trizol reagent and dissolved in 20
l samples were used to perform reverse transcrip-
tion in 20
l of reaction system, and 4
used as templates to amplify rat PAI-1 or GAPDH in 50
l dNTP, 0.25
l forward and reverse primer, 38
l]). The reaction conditions were as follows:
C for 0.5 min, 58
C for 1 min, 72
C for 10 min. Rat GAPDH was used as internal control to
quantify PAI-1 (PAI-1 primers as noted above). Rat GAPDH for-
ward primer is 5
the reverse primer is 5
PCR product was 515 bases long.
Control and DNA enzyme–treated cells were
lysed and protein pellets were dissolved in 1% SDS solution and
then boiled for 4 min after an equal volume of 2
added. Equal amounts of protein (10
SDS gel electrophoresis with 10% polyacrylamine separating gel
using minigels (Bio-Rad Laboratories). After electrophoresis, pro-
teins were transferred to nitrocellulose membrane (Amersham Bio-
sciences) 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 poly-
clonal anti-PAI antibodies (Santa Cruz Biotechnology, Inc.) and
then visualized by the ECL system (Amersham Biosciences) using
horseradish peroxidase–conjugated anti–goat IgG (Sigma-Aldrich).
Measurement of PAI-1 Activity.
HUVECs seeded in 24-well plates were transfected with 2
DNA enzymes (E1 and E3 as well as E0 in the presence or ab-
sence of 2 ng/ml TGF-
1 for 24 h. After washing twice with
PBS, cell lysates were collected using 400
in PBS. 25
l of cell lysate was incubated with plasmin substrate
in a 96-well microtiter plate (final volume, 230
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 proto-
col of American Diagnostica, Inc. (catalog no. 101201).
Transendothelial Migration Assay of Human Bone Marrow Stem
In brief, rat endothelial cell monolayers (
well) were grown to subconfluence (
wells in the presence or absence of rat vitronectin. To the top
chamber of each well in triplicate experiments, 10
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-
, and either E2 or scrambled DNA
enzyme complexed with 20
g/ml DOTAP. After 24 h in cul-
ture at 37
?C, the cells in the top and bottom chambers were re-
covered 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 ab-
sence of any endothelial monolayer for each condition tested.
Additionally, after supplemental transmigration, an assay was per-
formed to evaluate the effect of exogenous rat PAI-1 or antibody
against rat PAI-1 on CD34? cell transmigration. Rat endothelial
l of products was then
l Taq polymerase, 1
l P dCTP [3,000
C for 2 min, and 30 cycles
lysis buffer was
g/lane) were separated by
At 70–80% confluence,
l 0.5% Triton X-100
l) for 134 min at
70–80%) on PET trans-
cells (3.5 ? 104 cells/100 ?l) were seeded into the upper chamber
of transwell plates (6.5-mm diameter, pore size of 5 ?m; Costar,
Inc.) and 300 ?l 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 ?g rat PAI-1
(American Diagnostics) in 50 ?l DMEM without FCS was added
into some upper chambers, and 2 and 3 ?g IgG against rat PAI-1
(American Diagnostics) was added into some upper and lower
chambers, respectively. 30 min later, 105 CD34? cells in 100 ?l
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 ad-
dition of CD34? cells.
Animals, Surgical Procedures, and Injection of DNA Enzyme and
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 ar-
tery 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 en-
zyme, E0 scrambled control, or saline. 100 ?l of injection solu-
tion included 30 ?l DNA oligonucleotide (300 ?g), 20 ?l Super-
fect, and 50 ?l saline. For studies on neovascular rization and
effects on myocardial viability and function, 2.0 ? 106 human
cells obtained from a single donor after G-CSF mobilization were
reconstituted with 2.0 ? 105 immunopurified CD34? CD117bright
cells (1) and injected into the rat tail vein 48 h after LAD ligation.
Each group consisted of 6–10 rats.
Immunohistochemistry and Quantitation of Capillary Density.
quantitate and characterize PAI-1–expressing 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 VIII–related antigen, and
cardiac troponin I (DakoCytomation), and then visualized by im-
munoperoxidase technique using an Avidin/Biotin Blocking Kit,
a rat-absorbed biotinylated anti–goat IgG, and a peroxidase con-
jugate (all from Vector Laboratories). Capillary density and spe-
cies 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 Diagnos-
tics, respectively), factor VIII–related antigen (DakoCytomation),
and rat MHC class I (Accurate Chemicals). Arterioles were differ-
entiated from large capillaries by the presence of a smooth muscle
layer, identified by staining sections with an mAb against myo-
cyte-specific desmin (DakoCytomation). Staining was performed
by immunoperoxidase technique using an Avidin/Biotin Block-
ing Kit, a rat-absorbed biotinylated anti–mouse IgG, and a perox-
idase 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
VIII–related 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
Quantitation of Cardiomyocyte Proliferation.
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-
PAI-1 and Myocardial Neovascularization by CD34? Cells
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 recog-
nizing 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 (Dako-
Cytomation). 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. Cardiomyo-
cytes 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 anti–
mouse IgG was used as secondary antibody to detect Ki67 in nu-
clei. 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 anti–mouse IgG antibody (Jackson Im-
munoResearch 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.
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 cal-
culated as [(EDV-ESV)/EDV] ? 100. All echocardiographic studies
were performed by a blinded investigator.
Data are presented as mean ? SD. Com-
parisons between groups were made by Student’s t test. Values of
P ? 0.05 were considered significant.
Echocardiographic studies were
Construction of DNA Enzymes Targeting Human and Rat
We designed three DNA enzymes to spe-
cifically target pyrimidine–purine junctions at or near the
translational start site AUG of human and rat PAI-1 mes-
senger RNA, a region that is conserved between species
and has low relative free energy (31). As shown in Fig. 1 A,
the three DNA enzymes, termed E1, E2, and E3, con-
tained identical 15-nucleotide catalytic domains that were
flanked by two arms of eight nucleotides (E1 and E2) or
DNA enzymes and specific cleav-
age of human PAI-1 mRNA by
E1 and E3. (A) Three DNA en-
zymes were constructed, termed
E1, E2, and E3, containing iden-
tical 15-nucleotide catalytic do-
mains that were flanked by two
arms of eight nucleotides (E1
and E2) or nine nucleotides (E3)
with complementarity to human
(E1 and E3) or rat (E2) PAI-1
mRNA. To produce the control
DNA enzyme E0, the nucleotide
sequence in the two flanking
arms of E2 was scrambled with-
out altering the catalytic domain.
The 3? terminus of each mole-
cule was capped with an inverted
3?-3?–linked thymidine for resis-
tance to 3? to 5? exonuclease di-
gestion. (B) 32P-labeled 21-base
oligomer S1, synthesized from hu-
man PAI-1 mRNA, was cleaved
in a time-dependent manner by
E1 at a 10:1 substrate/enzyme
excess. (C) Sequence-specific
cleavage of human PAI-1 mRNA
in vitro generated transcript by
DNA enzyme E1 compared
with control DNA enzyme E0.
In lanes 2 and 4, incubation with
E1, but not E0, after preheating
transcript to 72?C for 10 min
demonstrates further increase in
cleavage. (D) Concentration-dependent cleavage of 32P-labeled human PAI-1 mRNA in vitro generated transcripts by E3. The 520 nucleotide transcript
was cleaved to products of 320 and 200 nucleotides. (E) Quantitation of representative transcript cleavage by PAI-1 DNA enzymes from two separate
experiments, showing time and dose dependence of the cleavage by DNA enzyme.
Xiang et al.
nine nucleotides (E3) with complementarity to human (E1
and E3) or rat (E2) PAI-1 mRNA. To produce the control
DNA enzyme E0, the nucleotide sequence in the two
flanking arms of E2 was scrambled without altering the cat-
alytic domain (Fig. 1 A). The 3? terminus of each molecule
was capped with an inverted 3?-3?–linked thymidine for
resistance to 3? to 5? exonuclease digestion (see below).
Specific Cleavage of Human PAI-1 mRNA by E1 and E3
As shown in Fig. 1 B, the 21-base oligo-
nucleotide S1, synthesized from human PAI-1 mRNA and
labeled at the 5? end with 32P, was cleaved within 2 min
when cultured with E1 at a 10:1 substrate/enzyme excess,
with maximal cleavage occurring by 2 h. The 10-nucleotide
cleavage product is consistent with the size of the 32P-labeled
fragment at the 5? end. E1 also cleaved larger 32P-labeled
fragments of human PAI-1 mRNA prepared by in vitro
transcription (Fig. 1 C). The 520-nucleotide transcript was
cleaved by 4 h to expected cleavage products of 320 and 200
nucleotides. The sequence-specific nature of the DNA en-
zymatic cleavage is also shown in Fig. 1 C, where the con-
trol DNA enzyme E0, containing an identical catalytic do-
main to E1 and E3, but scrambled sequences in the flanking
arms, caused no cleavage of human PAI-1 mRNA tran-
scripts. Preheating the transcript to 72?C for 10 min (Fig. 1
C, lane 4) before incubation with E1, but not E0, further in-
creased cleavage (percent of cleaved mRNA increased from
65 to 76%). Fig. 1 D shows similar cleavage of human PAI-1
mRNA transcripts by DNA enzyme E3 for 2 h. As shown
in Fig. 1 E, the cleavage of mRNA transcripts by DNA en-
zymes occurred in a time- and concentration-dependent
manner, with 65% cleavage by E1 for 4 h and 74% cleavage
by E3 for 2 h, both at a concentration of 20 ?M.
DNA Enzymes Inhibit Induction of Endogenous PAI-1
mRNA and Protein.
To determine the effect of DNA en-
zymes on endogenous PAI-1 production, endothelial mono-
layers 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-? 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 in-
hibited TGF-?–inducible steady-state mRNA levels in cul-
tured rat endothelium by 52%, relative to the E0 scrambled
DNA enzyme (Fig. 2 A). Basal level of PAI-1 mRNA re-
vealed a small decrease after transfection with the DNA en-
zymes (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-?–medi-
ated induction of PAI-1 protein. Endothelial cells trans-
fected with scrambled DNA enzyme demonstrated an ?50%
increase in cytoplasmic PAI-1 protein as detected by West-
ern blot. In contrast, this effect was almost completely abro-
gated by transfection with the PAI-1 DNA enzyme E2.
PAI-1 DNA Enzyme Is Resistant to Serum-dependent Deg-
radation and Inhibits PAI-1 Activity in Serum-cultured Endothe-
Because DNA enzymes with a thymidine
in the correct orientation at the 3? end are significantly de-
graded by factors in serum (1–5% concentration) within 24 h
(25), we investigated the protective effect of an inverted
thymidine at the 3? end on serum-dependent nucleolytic
degradation of PAI-1 DNA enzymes. The DNA enzyme
E2 was labeled with 32P and incubated at 37?C for 3–24 h
in medium containing 2 or 20% serum concentrations.
Although the DNA enzyme was significantly degraded
within 6 h in medium containing 20% serum, it remained
intact during the entire 24-h incubation with medium con-
taining 2% serum (Fig. 2 C). To evaluate whether PAI-1
was functionally inhibited by DNA enzymes after pro-
longed serum exposure, the conversion of plasminogen to
plasmin corresponding to residual tissue type plasminogen
nous PAI-1 mRNA and protein, demonstrate serum resis-
tance, and reduce PAI-1 activity. (A and B) Results of
RT-PCR and immunoblots demonstrating that transfec-
tion of rat endothelial monolayers with DNA enzyme E2
results in significant inhibition of TGF-?–induced expression
of PAI-1 mRNA and protein, relative to the E0 scrambled
DNA (P ? 0.01 for both). (C) 32P-labeled PAI-1 DNA
enzyme with inverted thymidine at the 3? end demon-
strates resistance to degradation in culture with 2%, but not
20%, serum. (D) Transfection of HUVEC monolayers
with the human PAI-1–specific DNA enzymes E1 or E3
reduces functional PAI-1 activity, as measured by chro-
mogenic reaction of plasmin substrate (P ? 0.01). (A, B,
and D) Results are expressed as the mean ? SEM of three
DNA enzymes inhibit induction of endoge-
PAI-1 and Myocardial Neovascularization by CD34? Cells
activator 1 was measured in endothelial monolayers trans-
fected with PAI-1–specific or –scrambled DNA enzymes
and cultured for 12 h in 2% serum. Under these conditions,
the E1 and E3 DNA enzyme inhibited TGF-?–dependent
PAI-1 activity in endothelial monolayers by a mean of 25%
(P ? 0.01; Fig. 2 D). Because the residual tissue type plas-
minogen activator activity in the sample catalyzed the con-
version of plasminogen to plasmin, which in turn hy-
drolyzed the chromogenic substrate, reduction in PAI-1
activity by DNA enzymes meanwhile indicated increase in
plasmin activity. Together, these results indicate that an in-
verted thymidine at the 3? end can protect the PAI-1 DNA
enzyme against nucleolytic degradation at 2% serum con-
centrations likely to be encountered in vivo, enabling inhi-
bition of cellular PAI-1 activity.
PAI-1 DNA Enzymes Increase Transendothelial Migration of
Human Bone Marrow Stem Cells.
ologic 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, treat-
ment of rat endothelial monolayers with E2 increased hu-
man CD34? migration by approximately threefold relative
to medium alone in the presence of vitronectin, but not in
To investigate the physi-
myocardial neovascularization. (A) Treatment of rat endothelial cell
monolayers with the rat PAI-1 DNA enzyme E2, but not with the
scrambled DNA enzyme E0, increased transendothelial migration of hu-
man CD34? cells by approximately threefold relative to medium alone in
the presence, but not in the absence, of vitronectin (P ? 0.05). (B) The
addition of rat recombinant PAI-1 or antibody against rat PAI-1 caused,
respectively, either a significant reduction or an increase in CD34? cells
transmigrating across endothelial cell monolayers (P ? 0.05 for both). (C)
Immunohistochemical analyses using polyclonal PAI antiserum, showing
reactivity in endothelial cells lining capillaries at the peri-infarct region
(arrows). (D) Results of RT-PCR showing that intramyocardial injection with E2 results in reduced myocardial PAI-1 mRNA levels at 48 h and 2 wk
after LAD ligation (P ? 0.01 for both, relative to E0- and saline-treated controls). (E) Results of quantitative immunohistochemical analyses, showing reduced
numbers of PAI-1–expressing cells at the peri-infarct region after E2 injection relative to the E0-treated controls (P ? 0.01). (F) Immunohistochemical
studies using anti-CD31 mAb demonstrating thin-walled capillaries with very large lumens (more than six nuclei), diameters in excess of 30 ?m, and no
smooth muscle outer layers, at the peri-infarct region after coadministration of E2 and human CD34? cells (right). (G) Coadministration of E2 DNA enzyme
intramyocardially induced significantly greater neovascularization at the peri-infarct region in comparison to E0 when human CD34? cells were injected in-
travenously (P ? 0.01). (H) Coadministration of E2 intramyocardially with human CD34? cells intravenously resulted in reduced numbers of unincor-
porated, discrete, interstitial human CD34? cells and increased numbers of large lumen capillaries (more than six nuclei) at the peri-infarct region, as de-
termined by staining with a human anti-CD31 mAb, compared with E0 scrambled control enzyme (P ? 0.01). (A, B, D, E, G, and H) Results are
expressed as the mean ? SEM of three or four separate experiments.
Effects of intramyocardial PAI-1 DNA enzyme injection on
Xiang et al.
the absence of vitronectin (P ? 0.05). In contrast, treatment
with the scrambled DNA enzyme did not significantly in-
crease transendothelial migration relative to medium alone.
The addition of exogenous recombinant rat PAI-1 or anti-
body against rat PAI-1 caused, respectively, either a marked
reduction or an increase in transmigrating CD34? cells com-
pared 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 interac-
tions involving plasmin generation and vitronectin binding.
Intracardiac Injection of E2 Reduces PAI-1 mRNA and Pro-
tein Expression after Acute Infarction.
the in vivo biological activities of the PAI-1 DNA enzyme.
As shown in Fig. 3 C, at 48 h after ligation of the LAD ar-
tery and induction of myocardial infarction in rats, immuno-
histochemical studies showed that PAI-1 expression oc-
curred predominantly in vascular endothelial cells at the
peri-infarct region and in infiltrating mononuclear cells.
PAI-1 expression was not detected in cardiomyocytes either
within or distal to the infarct zone. As shown in Fig. 3 D,
direct intramyocardial injection of E2 at the peri-infarct re-
gion at the time of LAD ligation resulted in 60% lower lev-
els of PAI-1 mRNA in the heart 48 h later, whereas the
scrambled E0 control did not significantly reduce PAI-1 lev-
els as measured by RT-PCR (P ? 0.01). This effect was
maintained for at least 2 wk, with cardiac PAI-1 mRNA
levels remaining 39% lower in LAD-ligated animals treated
with E2 compared with saline-treated animals at this time
point (P ? 0.01). As shown in Fig. 3 E, quantitative immu-
nohistochemical analyses showed that at 48 h after LAD
ligation and DNA enzyme injection, there was an over
threefold reduction in the number of PA-1? cells at the peri-
infarct region of PAI-1 DNA enzyme-treated animals com-
pared with scrambled DNA enzyme-treated controls (17 ?
5 low power field vs. 5 ? 1 low power field, P ? 0.01).
Intracardiac Injection of E2 Together with Angioblasts Results
in Enhanced Neovascularization at the Peri-Infarct Region.
When CD34? cells containing angioblasts were adminis-
tered 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 cap-
illaries with very large lumens (more than six nuclei) and
diameters in excess of 30 ?m were frequently seen at the
peri-infarct region when E2 and CD34? cells were coad-
ministered, 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 dis-
tinguished 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 (an-
giogenesis), at least 25% of the vessels at the peri-infarct re-
gion were of human origin (vasculogenesis), as defined by a
human anti-CD31 mAb. Fig. 3 H shows the effect of E2
Next, we investigated
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 re-
gion, 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 lu-
men capillaries (more than six nuclei) at the peri-infarct re-
gion was increased by 14-fold after E2 injection as com-
pared 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 Cardiomyo-
Although myocyte hypertrophy and in-
crease 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 orga-
nogenesis in the prenatal period is preceded by signals de-
rived 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 an-
gioblasts might induce proliferation/regeneration of endog-
enous cardiomyocytes. As shown in Fig. 4 A, at 2 wk after
LAD ligation, rats receiving human CD34? cells demon-
strated 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 deter-
mined by dual staining with an mAb reactive against cardio-
myocyte-specific troponin I and a polyclonal rabbit anti-
serum with reactivity against rat, but not human, Ki67.
Triple immunofluorescence using confocal microscopy con-
firmed the presence of cycling rat cardiomyocytes and dem-
onstrated a speckled pattern of Ki67 reactivity within cy-
cling 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 activ-
ity was no different than in sham-operated rats.
E2 Augments Human CD34? Cell–dependent Cardio-
myocyte Regeneration and Improvement in Cardiac Function.
shown in Fig. 4 C, animals receiving human CD34? cells
intravenously had a fourfold higher number of cell-cycling
cardiomyocytes at the peri-infarct region than those found
in LAD-ligated controls receiving saline (P ? 0.01). Com-
bining intramyocardial E2 injection with intravenously
delivered human CD34? cells resulted in a striking in-
crease in the degree of cardiomyocyte regeneration, to
levels 7.5-fold higher than in saline controls (P ? 0.01).
PAI-1 and Myocardial Neovascularization by CD34? Cells
Moreover, as shown in Fig. 4 D, combining intramyocar-
dial injection of E2 with human CD34? cells delivered in-
travenously resulted in an almost doubling of the positive
effect of CD34? cells alone on cardiac function, as deter-
mined by recovery in left ventricular ejection fraction at 2
wk (P ? 0.01). The scrambled DNA enzyme E0 had no
such effect on either cardiomyocyte regeneration or car-
diac functional recovery.
Numerous clinical studies have documented direct cor-
relations between elevated PAI-1 levels and poor cardio-
vascular outcomes, including myocardial infarction, athero-
sclerosis, and restenosis (13–16). Moreover, PAI-1 mRNA
and protein expression are elevated in atherosclerotic hu-
man arteries and failed vein grafts (17–19), as well as in
arterial walls and neointima formation in various animal
tion of endogenous cardiomyocytes and on functional
cardiac recovery. (A) Section from infarct of represen-
tative animal receiving CD34? cells intravenously
showing “finger” of cardiomyocytes of rat origin, as
determined by expression of rat MHC class I mole-
cules, extending from the peri-infarct region into the
infarct zone is shown. These cellular islands contain a
high frequency of myocytes staining positively for
both cardiac-specific troponin I (brown) and rat-specific
Ki-67 (dark blue; arrows). Sections from infarcts of
representative animals receiving saline do not show
same frequency of dual staining myocytes. (B) Confo-
cal microscopy of peri-infarct tissue from representa-
tive animal receiving human CD34? cells demon-
strates cardiomyocytes whose nuclei (labeled blue by
Cy5) concomitantly expressed Ki67 antigen (labeled
green by FITC-conjugated secondary antibody react-
ing with polyclonal rabbit anti–rat primary antibody)
and whose cytoplasm concomitantly stained for troponin
I (labeled red by propidium iodide–conjugated anti-
troponin mAb). (C) Animals receiving human CD34?
cells intravenously had a fourfold higher number of
cycling cardiomyocytes at the peri-infarct region than
that found in LAD-ligated controls receiving saline (P ?
0.01). Combining intramyocardial E2 injection with
intravenously delivered human CD34? cells increased
cardiomyocyte regeneration to levels 7.5-fold higher
than in saline controls (P ? 0.01). The scrambled
DNA enzyme E0 had no such effect. (D) Combining
intramyocardial injection of E2 with intravenous human
CD34? cells increased functional recovery of left ven-
tricular ejection fraction at 2 wk from a mean of 22–
50% (P ? 0.01). The scrambled DNA enzyme E0 had
no such effect. (C and D) Results are expressed as the
mean ? SEM of three separate experiments.
Effects of PAI-1 inhibition on regenera-
Xiang et al.
models of arterial injury (20, 21). However, despite these
associations, no study has to date established a causal link
between elevated PAI-1 levels and cardiovascular disease.
The results in this study suggest that elevated PAI-1 levels
in patients with myocardial infarction might be causally re-
lated to poor outcome through mechanisms that result in
direct inhibition of bone marrow–dependent neovascular-
ization. Indeed, we demonstrated that reducing PAI-1 mRNA
expression in situations of endogenous PAI-1 excess re-
sulted in augmented transendothelial migration of human
CD34? cells in a vitronectin-dependent manner, increased
fusogenicity and vascular formation by discrete CD34? cells
within ischemic myocardial tissue, and improved cardiac
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
marrow–derived cells and inhibited by PAI-1 (6–8). Re-
cent studies have suggested that u-PA also binds to PAI-1
in tissues, where it is complexed to vitronectin (9, 10). The
u-PA–PAI-1 interaction results in a rapid conformational
change in PAI-1 that causes it to dissociate from vitronec-
tin 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 mi-
gration (9, 12). Together, these data indicate that angio-
blast-dependent neovascularization of the adult heart is in-
hibited in situations of excess circulating and tissue PAI-1
levels by overlapping mechanisms, including interference
with u-PA–dependent plasmin generation and integrin bind-
ing 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 consid-
ered to be the primary mammalian cardiac responses to
ischemia, damage, and overload (32, 33), recent observa-
tions 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 re-
generation in meaningful numbers have not been identi-
fied. Common precursors giving rise to both cells of
smooth muscle and cardiomyocyte lineage have been iden-
tified in the adult murine bone marrow (37), and it is possi-
ble 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
marrow–derived or resident cardiomyocyte progenitors re-
mains to be determined; however, it bears noting the strik-
ing 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 complemen-
tary 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 ef-
fects due to “irrelevant cleavage” of nontargeted mRNA re-
mains a major concern (25). Ribozymes are naturally occur-
ring RNA molecules that contain catalytic sites, making them
more potent agents than antisense oligonucleotides. How-
ever, wider use of ribozymes has been hampered by their sus-
ceptibility to chemical and enzymatic degradation and re-
stricted target site specificity (26). Consequently, we focused
on the use of a new generation of catalytic nucleic acids con-
taining DNA molecules with catalytic activity for specific
RNA sequences (27–30). These DNA enzymes exhibit
greater catalytic efficiency than hammerhead ribozymes, pro-
ducing 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 deg-
radation, and are far cheaper to synthesize. Our results dem-
onstrate that inhibition of myocardial PAI-1 mRNA by a se-
quence-specific catalytic DNA enzyme is a feasible approach
for enhancing cardiac neovascularization, regeneration, and
functional recovery after an acute ischemic insult.
This research was supported in part by National Institutes of Health
grants RFA-HL-02-017 and RFA-AG-01-006.
The authors have no conflicting financial interests.
Submitted: 3 February 2004
Accepted: 16 November 2004
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