Published online before print March 30, 2001, doi: 10.1161/hh0701.088838
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© 2001 American Heart Association, Inc.
Online First Article |
Adenoviral Delivery of a Leukocyte-Type 12 Lipoxygenase Ribozyme Inhibits Effects of Glucose and Platelet-Derived Growth Factor in Vascular Endothelial and Smooth Muscle Cells
From the Division of Cardiology and Department of Experimental Pathology (M.K.P., F.H., J.A.B.), and Undergraduate Student Research Program (A.N.D.), University of California Los Angeles; Departments of Endocrinology and Metabolism (R.N.) and Molecular Biology (J.J.R.), Beckman Research Institute of City of Hope, Duarte, Calif; Division of Cardiology (R.S.M), University of Texas Southwestern Medical Center, Dallas, Tex; and Division of Endocrinology and Metabolism (J.-L.G., J.L.N, C.C.H), University of Virginia, Charlottesville, Va.
Correspondence to Catherine C. Hedrick, PhD, Division of Endocrinology and Metabolism, University of Virginia, PO Box 801405, Lane Rd/MR4 Bldg, Room 5116, Charlottesville, VA 22908-1405. E-mail cch6n{at}virginia.edu
Abstract
Abstract—The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase–polymerase chain reaction. Reverse-phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an important role for the 12LO pathway in regulating inflammatory changes in endothelial cells and smooth muscle cells.
Key Words: 12-lipoxygenase • ribozyme • adenovirus • endothelium • vascular smooth muscle cells
Lipoxygenase (LO) activation has been implicated in numerous diseases including atherosclerosis and diabetes. Certain pathological changes that occur in diabetic vascular disease, such as adhesion of leukocytes to the endothelium and chemotaxis of smooth muscle cells (SMCs), may share a common pathway through LO. Upregulation of LO activity and enhanced formation of LO products have been reported in vitro and in vivo. We and others have reported increased LO activity and elevated levels of LO products, but not cyclooxgenase products, in porcine aortic endothelial cells (PAECs)1 and porcine SMCs (PSMCs)2 when cultured in high glucose (HG) conditions. Also, enhanced production of HETEs has been observed in patients with diabetic renal disease3 and in vessels from infants of diabetic mothers.4 Exposure to extracellular stimulants such as angiotensin II, platelet-derived growth factor (PDGF), interleukin-4, and interleukin-1 has also led to increased synthesis of LO products in vascular and mononuclear cells.2 5 6 7 8 These results suggest that many of the biological effects of the agents involved in stimulating inflammatory processes may be mediated through LO and its products.
The LOs responsible for regulating specific vascular functions have not been determined. The number of mammalian LO sequences published includes at least 18 different sequences representing 7 isoforms in 7 different species. There are three classifications of 12LO, epidermis-, platelet-, and leukocyte-type. The importance of the leukocyte 12LO in diabetic pathology and atherosclerosis is suggested by studies in which 12LO-null mice show decreased atherosclerosis.9 10 Furthermore, the leukocyte-type 12LO has been detected in human ECs and SMCs.11 This study has addressed the role of the leukocyte 12LO, which is the only 12/15LO cloned in swine,12 in regulating EC and SMC function.
The goal of the present studies was to specifically address the role of 12LO in glucose-induced monocyte-endothelial interactions and in PDGF-mediated SMC migration. Although there are many pharmacological inhibitors that reduce 12LO activity, such as ETYA (eicosatetraynoic acid), NDGA (nordihydroguaiaretic acid), and CDC (cinnamoyl-3,4-dihydroxy-a-cyanocinnamate), many cannot distinguish the different isoforms of LO and may also harbor unwanted side effects.13 A highly selective method to inhibit isoform-specific LO is through the use of ribozyme technology. Ribozymes are catalytically active RNA molecules with the capacity to cleave other RNA substrates in trans or cis,14 and their specificity is derived from the complementary sequences flanking either side of the catalytic core. Among the RNA molecules used to suppress various genes, the hammerhead ribozyme is the best characterized.
We have previously described a catalytically active ribozyme to leukocyte-type 12LO and have shown it to successfully cleave in vitro–transcribed 12LO message.15 In the present study, we have packaged the ribozyme in an adenoviral vector to allow for enhanced transfer of the ribozyme into cells and to achieve longer expression compared with naked oligonucleotides. Previous studies have shown successful use of adenovirus to deliver functionally expressed ribozymes in vitro16 and in vivo.17 We demonstrate the ability of the viral vector system to efficiently deliver the 12LO ribozyme into PAECs and PSMCs and its ability to reduce the target, 12LO mRNA, and its product. In the present study, we document the role of the leukocyte 12LO pathway in glucose-mediated monocyte attachment to the endothelium and in the chemotactic effect of PDGF in PSMCs.
Materials and Methods
Cell Preparation and Culture
PAECs and PSMCs were isolated as previously
described18 19
and used at passages 2 through 6. For HG exposure, cells were passaged
twice in growth medium supplemented with 19.5 mmol/L glucose
(25 mmol/L glucose final concentration). Optimum multiplicity of
infection (MOI) of 100 (pfu/cell) was determined for PAECs and 500
(pfu/cell) for PSMCs. All experiments were conducted 72 hours after
infection. Human monocytes were isolated by using the Recalde method as
described
earlier.20
Construction of 12LORZ Adenovirus
Vector
The structure of the 12LO hammerhead ribozyme has
been previously described by our
group.15 The 36-bp 12LO
ribozyme oligonucleotide,
5'-CGGTAGACTGATGAGTCCGTGAGGACGAAACC- CAT-3' and the corresponding
reverse complementary strand were annealed and
phosphorylated by using T4 polynucleotide
kinase (Promega) before ligation into the pUC19 polylinker of the
adenoviral shuttle vector pACCMVpLpA(+). This plasmid contains the
cytomegalovirus (CMV) immediate-early promoter, pUC19 polylinker, and
small t antigen
splicing/polyadenylation signals from SV40. The ribozyme-containing
plasmid, designated pAC/RZ, and the right 91% fragment of the viral
genome (ClaI cut Ad5 fragment;
derived from digestion of AdLacZ at a unique
ClaI restriction site) were
cotransfected into the replication-permissive 293 cells (E1A
transcomplementing cell line) by using SuperFect Transfection Reagent
(Qiagen) to undergo homologous recombination to produce E1-deleted,
replication-defective recombinant adenovirus. Areas of cytopathic
effect, consistent with viral replication, were picked and
screened by using polymerase chain reaction and Southern blot
analysis, and positive plaques were subjected to 3 rounds of
plaque purification.
Figure 1A
shows the primer set used for PCR that flanks the
ribozyme insertion site on the pACCMVpLpA(+) plasmid: pAC5'
(5'-CGTGTACGG TGGGAGGTCTA-3') and pAC3' (5'-CCTTCACAAAGATCCCA AGC-3'.
The PCR contained 0.5 µmol/L of each primer, 1 uL of intact virus, or
plaque lysate, and the cycling parameters were as follows:
95EC for 10 minutes, followed by 30 cycles at 94EC for 30 seconds, 54EC
for 30 seconds, 72EC for 20 seconds, and a final extension at 72EC for
5 minutes. Nonradioactive Southern blot analysis was performed
by using a Gene Images kit (Amersham Pharmacia) according to the
manufacturer’s instructions, except the hybridization temperature was
42EC. The recombinant adenovirus containing the ribozyme was also
sequenced (with the assistance of the University of California Los
Angeles Sequencing Facility) from the CMV promoter through the entire
SV40 region to determine ribozyme sequence integrity. Large-scale
amplification of recombinant adenovirus in 293 cells was followed by
purification by using a discontinuous CsCl gradient. AdGFP and AdLacZ,
both under the control of the CMV promoter, were obtained from Quantum
Biotechnologies, Inc.
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Analysis of Gene Expression
Total RNA was isolated by Trizol reagent (Gibco
BRL) and treated with RNase-free DNase I (Boehringer) followed
by sample purification with RNEasy mini kit (Qiagen). To detect
expression of the 12LO ribozyme within cells, reverse
transcripterase–polymerase chain reaction (RT-PCR) was performed by
using random hexamers and 2.5 µg of total RNA (Stratagene Pro-Star
first-strand RT-PCR kit). Five microliters of cDNA was subjected to PCR
by using 0.5 µmol/L each of the primers RZ5'
(5'-CGGTAGACTGATGAGTCCGT-3') and SV403' (5'-AAATGAGC-
CTTGGGACTGTG-3'). This primer set produces a 209-bp PCR product as
shown in
Figure 1B
. In separate reactions, GAPDH mRNA was detected by
using GAPDH primers (5'-CCCTCAAGATCGTCA GCAAT-3' and
5'-AGGTCAGATCCACAACCGAC-3'). To quantify levels of 12LO and GAPDH mRNA,
25 ng of total RNA was reverse-transcribed and amplified in triplicate
wells in a multiplex Taqman RT-PCR (Perkin-Elmer Biosystems) by using
the relative standard curve method. The TaqMan EZ-RT-PCR core reagent
kit was used, and each 25-µL reaction contained 300 nmol/L each
porcine leukocyte-type 12LO-specific primers (5'-AACGGCACGAACCCCAT-3'
and 5'-AACTCAGGCGGGCAGG-3') and porcine leukocyte-type 12LO probe
(5'-FAM-TGCTGCGGCACTC-CGTTGAGC-TAMRA-3'), and 40 nmol/L each of
GAPDH primers (5'-TGGAAAGGCCATCACCATCT-3' and 5'-ACCAGCATCGCCCC-
ATTT-3') and GAPDH probe (5'-VIC-CCAGGAGCGAGATCCCGC- CAAC-TAMRA-3').
The 12LO primers and probe are specific for porcine leukocyte-type 12LO
and do not share homology with 15LO or platelet 12LO. The cycling
parameters were 60EC for 30 minutes, 95EC for 5 minutes,
followed by 40 cycles at 94EC for 20 seconds and 62EC for 1 minute.
Primers and probe for TaqMan assays were designed by using Primer
Express software (PE Biosystem), and all TaqMan reactions were
performed on the ABI 7700 sequence detection
system.
Quantitation of 12 HETE by HPLC and RIA
These studies were performed essentially as described
previously.21 PAECs cultured
in normal (5.5 mmol/L) or high (25 mmol/L) glucose in 6-well
plates were infected with either AdLacZ or AdRZ (MOI of 100) and
incubated for 72 hours. After this time, for HPLC analysis,
cells were incubated with 5 µCi/mL
3[H]-arachidonic acid
(3[H]-AA)for 24 hours. Cells were
incubated for 2 hours at 37EC in 0.05% fatty acid–free BSA in DMEM
and then placed in 5% FBS/DMEM containing normal glucose (NG)
or HG for 4 hours and then harvested with PBS containing
Ca2+ and Mg2+.
For HPLC, lipids were hydrolyzed in methanolic NaOH in the presence of
40 mmol/L n-propyl
gallate, acidified, and then fatty acids were isolated by using C18
bond-elution columns and analyzed by using a
reverse-phase C18 column.2
Each sample was spiked with a known amount of cold standard (5-, 12-,
15HETE HPLC mix; Biomol) before extraction and compared against a
standard curve to determine extraction efficiency. The retention time
of the 12HETE peak was confirmed by injecting a known amount of 12HETE
and observing an increase in the 12HETE peak. Radioactive metabolites
were identified by comigration with both authentic cold and tritiated
12(S)-HETE standard. For RIA, cell treatments were as described above.
At 72 hours after infection, cells were incubated for 45 minutes in
DMEM with 0.2% fatty acid–free BSA and placed on ice, the medium was
collected and acidified, and the fatty acid fraction was separated on a
C18 bond-elution column. 12(S)-HETE levels in the fatty acid fraction
were quantitated by using a specific radioimmunoassay (Advanced
Magnetics,
Inc).22
Adhesion Assay and Migration Assays
Adhesion assay was performed as previously
described.23 Migration assay
of PSMCs was performed on a 48-well modified Boyden chamber
(Neuroprobe, Inc) as previously
described.21
Statistics
Data for all experiments were analyzed by
using ANOVA and Fisher’s protected least-significant difference test
by using the Statview 5.0 software program (SAS Institute
Inc).
Results
Construction and Expression of the 12LO
Ribozyme in PAECs and PSMCs
Construction of the recombinant adenovirus containing
the 12LO ribozyme is described in Materials and Methods. Viral plaques
were screened by PCR by using the primer set shown in
Figure 1A.
Figure 1C (top) shows that the positive clone (designated
AdRZ), which contains the 36-bp ribozyme sequence, produced the
expected 160-bp amplified band. Southern blot analysis using
the full ribozyme sequence as the probe confirmed the presence of the
ribozyme in the recombinant virus
(Figure 1C, bottom).
We examined the expression of AdRZ in PAECs and PSMCs.
Initially, optimal MOI was determined by using AdGFP to achieve an
optimal balance of high gene expression and low viral titer to minimize
cytotoxicity. Infection of PAECs and PSMCs with AdRZ revealed positive
expression of the ribozyme by RT-PCR by using the primer set shown in
Figure 1B. As expected, the ribozyme sequence-specific
primers produced a 209-bp PCR fragment only in cells infected with AdRZ
and not in uninfected cells or in cells infected with AdGFP
(Figure 1D).
Figure 1E (bottom) shows positive amplification of GAPDH
from all cells. As a control for DNA carryover, PCR without the RT
reaction was performed for all samples and shown to be negative (data
not shown). These results verify that a functional recombinant
adenovirus capable of expressing the 12LO ribozyme inside cells was
successfully generated.
Ribozymes Block Glucose-Induced 12LO Expression
in PAECs
We examined the ability of the ribozyme to reduce
levels of 12LO mRNA in AdRZ-infected PAECs. Because the expression of
12LO mRNA is relatively low in PAECs and could not be detected by
Northern blot analysis, an RT-PCR approach was used.
Quantitation of 12LO mRNA in PAECs was achieved by real-time TaqMan
RT-PCR by using the relative standard curve method. In this system,
RT-PCR took place in one step, and both 12LO and GAPDH transcripts were
coamplified in the same well. Because of the overwhelming abundance of
GAPDH relative to 12LO mRNA, primer-limiting experiments were performed
to diminish competition. PAECs cultured in glucose showed a 60%
increase in leukocyte 12LO mRNA. Addition of ribozyme to
glucose-cultured PAECs decreased 12LO mRNA levels down to those found
in control cells
(Figure 2). There were no significant differences found in
the level of 12LO mRNA among NG cells infected with AdLacZ or AdRZ or
in uninfected cells. The lack of a difference between infected and
uninfected cells also indicates that infection with the adenovirus
itself did not alter the endogenous 12LO mRNA level. Thus,
the results demonstrate that the ribozyme was highly effective in
blocking the HG-stimulated increase in the 12LO
transcript.
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Ribozymes Block Glucose-Induced 12HETE
Production in PAECs
By using two independent methods, RIA and HPLC, we
evaluated whether there was a corresponding drop in the level of 12HETE
in cells containing the ribozyme. To determine the amount of 12(S)-HETE
released by the cells into the medium, RIA was used. This assay
utilizes an antibody specific for the
S isomer of 12HETE. The
ribozyme decreased by 35% the level of released 12(S)-HETE from PAECs
as measured by RIA
(Figure 3A, left bars). Because problems with variability of
RIA on cell extracts were observed, accumulation of 12HETE within cells
was examined by using HPLC after labeling cells with
3[H]-AA as described
previously.24 In an earlier
study, we have reported that essentially all arachidonate
incorporated into ECs was esterified, and no unesterified 12HETE could
be detected.24 Furthermore,
we found that the majority of 12HETE released from cells into the
medium was unesterified.24
To analyze the cellular content of 12HETE, lipids were
hydrolyzed to release fatty acids, which were then isolated and
examined by using HPLC. As shown in
Figure 3A (right bars), a HG culture of PAECs induced
approximately a 40% increase in 12HETE level as measured by
reverse-phase HPLC. Infection of PAECs with AdRZ completely inhibited
the HG-induced production of 12HETE and reduced 12HETE levels
to those seen in cells incubated with NG. In both assays, there was no
effect of the ribozyme on 12HETE levels in PAECs cultured in
NG.
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Figure 3B shows a typical radioactive HPLC pattern of
3[H]-12HETE standard and a sample tracing
of extracts from PAECs incubated with
3[H]-AA for 24 hours. From this
chromatogram and our previous studies, it is clear that a number of
arachidonate metabolites were formed in cells and
esterified to cellular lipids. We determined using gas
chromatography–mass spectrometry that the peak eluting
with authentic 12HETE on HPLC contained only lipid of the appropriate
mass. By using such chromatograms, levels of cell-associated 12HETE
were compared in infected and uninfected cells.
Adenovirus Delivery of Ribozymes Inhibits
Glucose and PDGF-Induced Effects in Vascular Cells
We have previously shown that both HG and 12LO
products increase monocyte adhesion to human aortic
endothelial
cells.23 Infection of PAECs
with AdRZ resulted in a complete inhibition of glucose-induced monocyte
binding, which confirms the involvement of 12LO in glucose-mediated
adhesion events
(Figure 4). In addition, the similarity in the number of
bound monocytes between cells infected with adenovirus or left
uninfected indicated a lack of an inflammatory response caused by viral
infection alone.
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In a different biological assay, we sought to determine
whether PDGF-induced PSMC migration could be abrogated with the 12LO
ribozyme. We and others have previously shown that the PDGF-induced SMC
migration could be blocked by pharmacological LO
inhibitors.21 25
PSMCs were infected with AdRZ, and the response to PDGF was evaluated
in a migration assay by using a modified Boyden’s chamber.
Figure 5 shows a 50% inhibition in PDGF-induced migration
by AdRZ in PSMCs. These results show the efficacy of the ribozyme in
two different biological settings and the utility of expressing the
ribozyme in an adenoviral vector.
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Discussion
Our studies document the ability of 12LO ribozyme
packaged within adenovirus to reduce 12LO activity and function in
activated ECs and SMCs. We obtained high levels of transfection
and 12LO ribozyme expression in PAECs
(Figure 1D) without an adenoviral vector–related toxicity.
We observed an almost complete inhibition of a glucose-induced increase
in 12LO mRNA by the ribozyme
(Figure 2). No reduction in 12LO mRNA compared with basal
levels was observed when the ribozyme was introduced into ECs cultured
in NG (5.5 mmol/L). Although ribozymes are catalytically active
molecules and therefore are theoretically able to achieve multiturnover
reactions, the rate-limiting step is usually the association of the
ribozyme arms with its complementary mRNA target. The ribozyme and
target mRNA transcript must be physically colocalized and the ribozyme
must also contend with competition with mismatched
substrates.26 There are
several potential causes for the lack of inhibition in cells cultured
in NG: (1) 12LO levels are considerably lower in cells cultured in
normal compared with HG; thus, RNA/ribozyme interactions could be less
frequent; (2) 12 LO mRNA in NG-cultured cells may be associated with
different proteins or may be localized in different compartments than
cells cultured in HG, causing a difference in ribozyme accessibility;
(3) the ribozyme may target only newly synthesized mRNA; or (4) glucose
increases ribozyme expression. With regard to these points, 12LO mRNA
is 50% lower in NG cells and can be detected under normal conditions
by using real-time quantitative PCR technologies or conventional RT-PCR
followed by Southern blot
analysis.11 Thus, in
NG cells, the LO mRNA is present in low levels. It is
possible that we cannot detect a decrease in LO mRNA in the NG cells
because of the limits of detection of our assay. It is also possible
that, as a result of the low LO mRNA abundance in NG cells, that
RNA/ribozyme interactions are less frequent. Another possibility is
that the ribozyme may target only newly synthesized mRNA. This would
certainly explain our results, although there is little evidence for
such specific mRNA targeting in other ribozyme studies. Finally, there
is the possibility that glucose increases expression of ribozyme in our
model. We have not observed an increase in ribozyme expression (or
control LacZ expression) in HG cells using conventional RT-PCR
techniques, and we have had some difficulty using Taqman real-time
quantitative PCR technology to detect the ribozyme. A modest increase
in ribozyme expression in HG cells could lead to a dramatic reduction
in LO mRNA in those cells. We cannot rule out this possibility. There
is also the possibility that the transcriptional start site for LO is
different in NG cells and thus the ribozyme site would be eliminated.
This could occur if a different promoter is used by the cells in NG
versus HG conditions. Although this is unlikely, we cannot yet rule out
this possibility, and we are currently examining this hypothesis.
Despite these unanswered questions in NG cells, our findings clearly
demonstrate the ability of the 12LO ribozyme to reduce 12LO mRNA levels
in glucose-cultured PAECs and imply also a reduction in 12LO mRNA
levels in PDGF-treated PSMCs.
The 12LO metabolite of arachidonic acid,
12HETE, was also measured to determine whether the inhibition of 12LO
mRNA by the ribozyme led to a corresponding decrease in its
product. The HPLC studies revealed, as have our previous studies,
that a number of different arachidonate metabolites are
formed in ECs, but for the present studies, we focused on 12HETE,
the major primary product of 12LO. We demonstrate that
ribozyme-treated cells released less 12(S)-HETE into the medium and
contained less esterified 12HETE
(Figure 3).
Hyperglycemia as a result of diabetes has been shown to alter eicosanoid metabolism. Elevated levels of glucose have been shown to upregulate LO products in ECs,1 SMCs,19 and islets,27 and elevated levels of HETEs have been detected in patients with diabetes3 and in animals induced to develop diabetes.28 29 30 This study provides new evidence that glucose increases 12LO mRNA in PAECs similar to earlier results found in PSMCs.2 Furthermore, in the present studies, the level of 12LO products is directly linked to the levels of 12LO mRNA. Another potential mechanism of augmenting 12LO products is by increasing substrate availability, and studies have shown that many agents that enhance production of 12HETE can also activate phospholipase A2 to liberate arachidonic acid.31 32 However, taken together, the findings in this study suggest that glucose regulates the level of 12LO product by modulating the level of 12LO enzyme.
Arachidonic acid metabolites and elevated
glucose levels have been implicated to play a role in the interaction
of monocytes and endothelium. Both ECs cultured in high
glucose33 and rabbits with
alloxan-induced diabetes show increased accumulation of leukocytes to
the
endothelium.34
Honda et al24 have shown
that treatment of human aortic endothelial cells with
LO inhibitors blocked monocyte adhesion stimulated by
minimally oxidized LDL. Other studies have shown that 12(S)-HETE can
induce transendothelial cell migration of
monocytic-like cells.35 In
addition, we have shown that direct treatment of ECs with 12(S)-HETE
can induce monocyte attachment in a dose-dependent
manner.23 In this study, we
wanted to determine whether glucose-mediated stimulation of monocyte
adhesion to ECs was mediated by the 12LO pathway. The ribozyme
completely blocked the increase in adherent monocytes to PAECs induced
by HG
(Figure 4). The number of monocytes bound in HG-cultured
PAECs infected with AdRZ was similar to background levels, which is
consistent with data in which the 12LO mRNA level in
ribozyme-infected PAECs was similar to basal levels
(Figure 2). These results support our previous finding that
indicates the involvement of 12LO in the hyperglycemic effect because
only the stereospecific product of 12LO, 12(S)-HETE, but not
12(R)-HETE, stimulated increased adhesion of monocytes to
ECs.23 Although there is
evidence that infection with adenovirus itself may elicit an
inflammatory response,36 we
did not observe any differences in the number of adherent monocytes
between ECs infected with adenovirus and uninfected cells. This
demonstrates that, in our assay system, the increase in monocyte
attachment in PAECs was caused by the HG culture and that the
inhibition of this increase was the result of the activity of the
ribozyme.
Chemotaxis of SMCs is one of the steps involved in the development of the atherosclerotic lesion. Because we have shown that PDGF, a potent chemotactic agent for SMCs, can upregulate 12LO activity,21 we sought to test the functionality of the ribozyme in this setting. In this study, we demonstrated that inhibition of leukocyte 12LO in PSMC significantly blocked the PDGF-induced migration compared with cells infected with the control virus. The inhibition was partial but consistent with findings by others21 25 who also show partial but significant decreases in SMC migration using pharmacological LO inhibitors. Our results indicate the particular role of the leukoctye-type 12LO in mediating, at least in part, the PDGF effect in PSMCs.
An important question with respect to LO products is whether their cellular effect is mediated by a secreted product binding to a receptor or whether they act as second messengers within the cell. Studies have shown that HETEs may participate as second messengers or may act as direct ligands to receptors.8 37 Although our studies do not directly address this issue, we document that 12HETE levels increase both intracellulary and extracellularly. Previous studies from our group in which dose-response curves were generated show that 1x10–9 mol/L 12(S)-HETE (which we now document is produced by incubation with high glucose) is sufficient to stimulate monocyte binding. However, it is also possible that 12(S)-HETE may function intracellularly as a signal transduction molecule because most HETE is retained in the cells (at one order of magnitude higher than extracellular HETE).
In summary, we demonstrated the derivation of a ribozyme-expressing recombinant adenovirus and verified the utility and effectiveness of the ribozyme in studying the mechanism by which hyperglycemia and PDGF induce their effects on vascular wall cells. Although it will require the combined efforts of many approaches such as the use of chemical inhibitors and the generation of transgenic/knockout mice to fully understand the biological roles of LO, the use of the ribozyme may be key in delineating the individual significance of the various LO isozymes.
Acknowledgments
This study was supported by National Institutes of Health Grants HL55798 (C.C.H., J.A.B., R.N., and J.L.N.), HL-30568 (J.A.B.), and by the Juvenile Diabetes Foundation (C.C.H., J.A.B., R.N., and J.L.N.). The authors thank Dr Kym F. Faull and Dr Ganesamoorthy Subbanagounder for their valuable advice on lipid analysis. We also thank Lia Langi for her dedicated work in tissue culture preparations.
Footnotes
Original received September 28, 2000; revision received February 19, 2001; accepted February 19, 2001.
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