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(Circulation Research. 2001;88:659.)
© 2001 American Heart Association, Inc.

Molecular Medicine

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

Mary Kim Patricia, Rama Natarajan, Alek N. Dooley, Felicia Hernandez, Jia-Li Gu, Judith A. Berliner, John J. Rossi, Jerry L. Nadler, Robert S. Meidell, Catherine C. Hedrick

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Preparation and Culture
PAECs and PSMCs were isolated as previously described^{18 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.

View larger version (26K): [in this window] [in a new window]   Figure 1. Detection of 12LO ribozyme in recombinant adenovirus, PAECs, and PSMCs. A, Schematic representation of the primer set (pAC5' and pAC3') used to determine incorporation of the ribozyme (RZ) into the viral genome. B, Schematic drawing of the primer set (RZ5' and SV403') used to verify expression of the RZ in PAECs and PSMCs. C, PCR screening to determine whether RZ is incorporated into the viral genome. Primers flanking the ribozyme insertion site (pAC5' and pAC3') were used to directly PCR 1 µL of AdRZ virus and run on a 2% agarose gel and stained with ethidium bromide (top). Lane 1, no DNA; lane 2, AdRZ virus; lane 3, pACCMVpLpA; and lane 4, pAC/RZ. Southern blot analysis of the above using full-length RZ oligonucleotide as probe (bottom). D and E, Confirmation of expression of RZ in PAECs and PSMCs at 72 hours after infection. Total RNA was subjected to RT-PCR by using ribozyme sequence-specific primers, RZ5' and SV403' (D), or GAPDH primers (E). Lane 1, AdGFP-infected PAECs; lane 2, AdRZ- infected PAECs; lane 3, uninfected PAECs; lane 4, AdGFP- infected PSMCs; lane 5, AdRZ-infected PSMCs; lane 6, no DNA; lane 7, pAC/RZ as positive control; and lane 8, pACCMVpLpA as negative control.

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

View larger version (10K): [in this window] [in a new window]   Figure 2. Inhibition of HG-induced 12LO expression in PAECs by the 12LO ribozyme. A one-step, multiplex RT-PCR was performed on 25 ng of DNase I–treated total RNA from PAECs grown in normal (NG, 5.5 mmol/L) or high (HG, 25 mmol/L) glucose and infected with AdRZ or AdGFP or left uninfected for 72 hours. 12LO-specific and GAPDH primers and probe were used, and fluorescence of amplification at threshold was measured and compared against a relative standard curve. Each experiment was performed in triplicate. 12LO was normalized to GAPDH. Data are expressed as a percent of control (NGGFP) of 4 experiments±SE. *P<0.005 NGGFP control vs HGGFP; **P<0.02 HGGFP vs HGRZ.

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.

View larger version (22K): [in this window] [in a new window]   Figure 3. Inhibition of HG-stimulated 12LO product formation in PAECs by the 12LO ribozyme. A, Levels of released 12HETE analyzed by RIA (left bars) and of cell-associated 12(S)HETE levels by reverse-phase HPLC (right bars). These results show RZ inhibition of released and cell-associated 12HETE levels in PAECs cultured in HG (25 mmol/L). For HPLC analysis, PAECs were incubated for 24 hours with 3[H]-AA, and the cells were scraped and lipids were hydrolyzed and extracted before HPLC. Twelve-second fractions were collected and the radioactivity measured in a scintillation counter. The level of 12HETE formation was determined after normalization of the measured 12HETE radioactivity to efficiency of lipid extraction, HPLC column recovery, and total milligrams of cellular protein. Data are the mean as a percent of control (NGLacZ) of 3 experiments±SE; each experiment was performed in duplicate. *P<0.02 NGLacZ vs HGLacZ; **P<0.006 HGLacZ vs HGRZ. For the RIA, the data were normalized against total cellular protein and are the mean±SE of 2 experiments as a percent of control (NGLacZ). *P<0.006 NGLacZ vs HGLacZ; **P<0.03 HGLacZ vs HGRZ. B, Radioactive reverse-phase HPLC chromatogram. PAECs were incubated for 24 hours with 3[H]-AA, and the cells were scraped and lipids were hydrolyzed and extracted before HPLC. Twelve-second fractions were collected and the radioactivity measured in a scintillation counter. Shown are a typical radioactive HPLC chromatogram (top) and a chromatogram of the 3[H]-12HETE standard alone (bottom). The position of 12HETE, 15HETE, and arachidonic acid (AA) standard peaks are indicated.

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.

View larger version (24K): [in this window] [in a new window]   Figure 4. Inhibition of HG-mediated monocyte adhesion to PAECs by the 12LO ribozyme. Monocyte binding to PAECs cultured in NG (5.5 mmol/L) or HG (25 mmol/L) were performed at 72 hours after infection with AdRZ (RZ) or AdLacZ (LacZ) or left uninfected (NG). Monocytes (75 000) were added for 15 minutes at 37°C and bound monocytes fixed with 1% gluteraldehyde and 8 fields/well counted. Data are the mean±SE of bound monocytes as a percent of control (NGLacZ) and are representative of 5 experiments. *P<0.001 NGLacZ vs HGLacZ; **P<0.01 HGLacZ vs HGRZ.

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.

View larger version (8K): [in this window] [in a new window]   Figure 5. Inhibition of PSMC migration in response to PDGF by 12LO ribozyme. PSMCs (65 000) infected with AdRZ (+RZ) or AdLacZ (+LacZ) were placed in the upper well of a modified Boyden’s chamber. The lower well contained either medium alone (as a control) or medium plus 1x10–10 mol/L PDGF and the cells from the upper chamber were allowed to migrate down through the collagen-coated polycarbonate filter for 5 hours at 37°C. The migrated cells were fixed and stained, and the densitometry was measured. Data are the mean of 3 experiments as a percent of medium control±SE; each experiment was performed in triplicate. *P<0.0001 medium control vs PDGF+LacZ; **P<0.001 PDGF+LacZ vs PDGF+RZ.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 others^{21 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.

	References

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