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.

Full text

Page 1

The Journal of Experimental Medicine
J. Exp. Med.
Volume 200, Number 12, December 20, 2004 1657–1666
http://www.jem.org/cgi/doi/10.1084/jem.20040221
©
The Rockefeller University Press • 0022-1007/2004/12/1657/10 $8.00
1657
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
Abstract
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
Key words:
myocardial regeneration • DNA enzyme
myocardial infarction • angiogenesis • bone marrow stem cells •
Introduction
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: si5@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@columbia.edu
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

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PAI-1 and Myocardial Neovascularization by CD34
?
Cells
1658
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.
3
?
-3
?
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:
5
?
-CCAAGAGCGCTGTCAAGAAGAC-3
and 5
?
-TCACCGTCTGCTTTGGAGACCT-3
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
?
(forward primer)
?
(reverse primer;
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
sequencing. A
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
NcoI. 20
?
l of transcription reaction consisted of 4
2
?
l DTT, 1
?
l RNasin inhibitor, 4
C, and 1
?
l H
2
O), 100
?
M UTP, 2
?
– P-UTP (10
?
ci/
?
l), and 1
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
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-
degraded bands.
DNA Enzyme Stability in Serum.
32
P-radiolabeled using T4 polynucleotide kinase. Labeling reac-
tion (20
?
l of volume) consisted of 1
2
?
l 10
?
kinase buffer, 1
?
l T4 PNK (10 U/
6
?
l P-ATP (3,000
?
Ci/mmol, 10
30 min at 37
?
C. P-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.
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-
?
-AGC
?
(forward primer) and 5
?
(reverse primer; data
?
-
32
?
l 5
?
?
buffer,
l A, G,
?
g/
?
l NTP mixture (1
?
l template (0.3
l SP6 polymerase (20 U/
?
?
l),
l).
32
?
32
2
,
32
?
M
2
, 100 mM
DNA enzymes were
?
l DNA enzyme (20 uM),
?
l), 10
Ci/
?
l). Reaction time was
?
l H
2
O, and
32
?
32
Primary
2
. For
?
10
5
cells/well). Subconfluent
?
l/
?
was added

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Xiang et al.
1659
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).
RT-PCR.
Total RNA from each well of six-well plates was
isolated using 1 ml Trizol reagent and dissolved in 20
treated H
2
O. 2-
?
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
system (5
?
l 10
?
buffer, 1
?
l dNTP, 0.25
?
l forward and reverse primer, 38
ci/mmol, 10
?
ci/
?
l]). The reaction conditions were as follows:
95
?
C for 0.5 min, 58
?
C for 1 min, 72
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 for-
ward primer is 5
?
-CTCTACCCACGGCAAGTTCAA-3
the reverse primer is 5
?
-GGGATGACCTTGCCCACAGC-3
PCR product was 515 bases long.
Western Blot.
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
Cells.
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
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-
?
, 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 DEPC-
?
l of products was then
?
l PCR
?
l Taq polymerase, 1
O, 2
?
l P dCTP [3,000
?
l H
2
32
?
C for 2 min, and 30 cycles
?
and
?
.
?
lysis buffer was
?
g/lane) were separated by
At 70–80% confluence,
?
M
?
l 0.5% Triton X-100
?
l) for 134 min at
?
2
?
10
5
cells/
?
70–80%) on PET trans-
5
human
?
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
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 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
400).
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-
To
Cardiomyocyte

Page 4

PAI-1 and Myocardial Neovascularization by CD34? Cells
1660
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.
Statistical Analysis.
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
Results
Construction of DNA Enzymes Targeting Human and Rat
PAI-1 mRNA.
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
Figure
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
1.
Construction of
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.

Page 5

Xiang et al.
1661
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
DNA Enzymes.
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-
lial Monolayers.
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
Figure 2.
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
separate experiments.
DNA enzymes inhibit induction of endoge-