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CARDIOVASCULAR
Davis Heart and Lung Research Institute and the Departments of Pharmacology (J.C.H., M.M.B., P.N.K., M.M.M., M.V.M., A.J.C.) and Internal Medicine (H.C., A.R.O., C.A.B., J.L.Z.), The Ohio State University College of Medicine, Columbus, Ohio; and Molecules for Health, Inc., Richmond, Virginia (H.L.E.)
Received for publication
January 26, 2005
Accepted
March 31, 2005.
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Abstract |
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; Elezi et al., 1998; Bennett and O'Sullivan, 2001). During PTCA, the artery walls are expanded by several times their original diameter in an attempt to increase lumen diameter and improve flow. Unfortunately, this technique is plagued by a high incidence of vessel renarrowing or restenosis, occurring in 30 to 40% of patients within 6 months of the procedure (Anderson et al., 1993; Fischman et al., 1994; Elezi et al., 1998; Bennett and O'Sullivan, 2001; Heckenkamp et al., 2002). Prevention of restenosis after successful PTCA remains one of the most challenging tasks in the treatment of obstructive coronary artery disease. Attempts to ameliorate this proliferative response involve the use coronary stents, which have significantly improved both short-term and long-term outcome following interventional coronary revascularization procedures. Despite a reduction in the restenosis rate with stent deployment, restenosis still occurs in 15 to 30% of patients within 6 months (Fischman et al., 1994; Elezi et al., 1998). This incidence of in-stent restenosis is expected to increase as coronary stenting is becoming more frequent and is used in less ideal lesions. Therefore, in addition to mechanical intervention, pharmacological approaches to reduce the incidence and degree of restenosis are needed.
The vascular trauma associated with PTCA involves a cascade of molecular and cellular events occurring within the vessel wall involving the release of a variety of vasoactive, thrombogenic, and mitogenic factors (Bauters and Isner, 1997; Libby and Tanaka, 1997; Goldschmidt-Clermont and Moldovan, 1999). Within this cascade, several mechanisms contribute to restenosis, including elastic recoil, thrombosis, smooth muscle cell migration/proliferation, and matrix formation. The result of these vascular events is intimal hyperplasia, whereby vascular smooth muscle cells (VSMCs) migrate from the media to the intima, proliferate, and consequently, form the neointima. During this proliferative response, SMCs undergo a phenotypic modulation from a contractile to a synthetic phenotype (differentiation) (Epstein et al., 1991; Noda-Heiny and Sobel, 1995; Ueda et al., 1995; Farb et al., 2002; Indolfi et al., 2003). Since, ultimately, the cascade of events following vascular trauma culminates in cell proliferation and neointimal hyperplasia, it would follow then that among targets being pursued would be agents that can impede smooth muscle cell migration and proliferation, as these processes are critical components of restenosis injury.
In this regard, we have tested the ability of two ribonucleotide reductase (RR) inhibitors to limit the degree of restenosis following balloon-mediated dilatation injury in the rat. RR catalyzes the reductive conversion of ribonucleotides to deoxynucleotides. This reductive reaction is a prime target for impeding cellular proliferation, and therefore amenable to inhibiting VSMC replication because it is a rate limiting step in the biochemical pathway, leading to DNA synthesis and thus cell replication (Elford et al., 1970, 1979; Elford, 1972; Takeda and Weber, 1981; Natsumeda et al., 1985; Anderson et al., 1993; Tanaka et al., 2000). DNA synthesis cannot occur without invoking this reaction, since the endogenous pools of dNTP in mammalian cells are inadequate to support new DNA synthesis (Elford et al., 1970, 1979).
Therefore, in the present study we have examined the ability of the polyhydroxy-phenolic compounds Didox (3,4-dihydroxybenzohydraxamic acid) and Imidate (ethyl-3,4,5-hydroxybenzimidate) to limit the degree of restenosis following vascular injury. Treatment with either Didox or Imidate resulted in >60% reduction in neointimal area. In vitro studies demonstrated that these effects are mediated through both a reduction in SMC proliferation and migration. These results suggest that inhibition of RR may serve as a novel therapeutic target in the treatment of vasculoproliferative disorders such as restenosis.
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Materials and Methods |
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Carotid Injury. Male Wistar rats weighing 400 to 450 g (Harlan, Indianapolis, IN) were fed standard pellet feed and given water ad libitum. The experimental protocol was designed in accordance with Institutional Laboratory Animal Care and Use Committee (ILACUC) standards. Animals were anesthetized with isoflurane (1.5–2%) in air. The right carotid artery was exposed, and a 2F Fogarty balloon embolectomy catheter (Baxter, McGaw Park, IL) was inserted via an external carotid arteriotomy incision. The catheter was advanced to the common carotid artery, inflated to a pressure of 2 atm, and rotated in a forward and retrograde direction. The catheter was then deflated, and the process repeated three times. Treatment cohorts were divided into five groups (n = 6–8/group): control (sham-operated), Didox (200 mg · kg-1 · day-1), Imidate (200 mg · kg-1 · day-1), HU (200 mg · kg-1 · day-1), and vehicle (saline). Drugs were administered daily by i.p. injection for a period of 7 days after injury. Two weeks postinjury, rats were euthanized by pentobarbital overdose and perfused with 10% buffered formalin. Carotid arteries were removed and placed in the same fixative. Tissues were then embedded in paraffin, and four to five sections (4 µm) were cut at multiple levels. These sections were then stained with hematoxylin-eosin or elastic-van Gieson stain. Sections were examined microscopically, and the cross-sectional areas of the lumen, neointima (from the internal elastic lamina to the lumen), and media were determined using digital microscopy with SPOT Advanced software (Diagnostic Instruments, Inc., Sterling Heights, MI). The intima-to-media ratio was then calculated from the determined mean. The data represent the mean ± S.D.
Flow Cytometry. SMC were plated on 6-well dishes at a density of 2 x 105 cells per well. Cells were then treated with Didox (0–200 µM), Imidate (0–200 µM), or HU (0–1000 µM) and incubated for 24 h. Following the 24-h incubation period, the cells were trypsinized and collected in 15-ml centrifuge tubes. The cells were then centrifuged 5 min at 800g. The supernatant was discarded, and the pellet was resuspended in 5 ml PBS. The cells were centrifuged for 6 min at 200g. The supernatant was removed, and the pellet was then thoroughly resuspended in 0.5 ml PBS. The cell suspension was transferred into tubes containing 70% ethanol, keeping the cells in fixative >2 h. The ethanol-suspended cells were centrifuged for 5 min at 200g, and the ethanol was decanted. The cell pellet was resuspended in 5 ml PBS, and after 1 min, was centrifuged for 5 min at 200g. The top layer of liquid was again removed, and the cell pellet was resuspended in 1 ml of propidium iodide/Triton X-100 staining solution with RNase A. This staining solution was then incubated at room temperature for 30 min. Flow cytometry was then performed using a FACS Calibur (BD Biosciences, San Jose, CA). The data are presented as the mean.
Intracellular dNTP Quantitation. SMC were plated in T150 flasks and treated with Didox (0–200 µM), Imidate (0–200 µM), or HU (0–1000 µM) and incubated for 24 h. Following the 24-h incubation period, the cells were trypsinized and collected in 50-ml centrifuge tubes along with the incubation media. All the extraction steps were performed on ice. Immediately before processing, cells were counted, and viability was determined using the trypan blue exclusion method. The cells were then centrifuged for 5 min at 800g. The cell pellet was then deproteinized with the same volume of 6% trichloroacetic acid, vortexed for 20 s, and incubated on ice for 10 min. The acid cell extracts were centrifuged for 10 min at 2000g. The supernatant supernatants were then supplemented with an equal volume of distilled water, vortexed for 60 s, and neutralized by the addition of 5 M K2CO3 prior to high-performance liquid chromatography analysis. dNTP detection was carried out using an ESA (Chelmsford, MA) high-performance liquid chromatographic system with UV-visible detection. Chromatographic separations were performed using a TosoHaas (Montgomeryville, PA) C18 reverse phase column (octadecylsilane, 80 tubular maximum, 250 x 4.6 mm, 5-µm pore). The mobile phase was delivered at a rate of 1.0 ml/min during the analysis using the following stepwise gradient elution program: A/B (80:20) at 0 min; (40:60) at 30 min; (40:60) at 40 min, and (80:20) at 45 min. Buffer A contained 10 mM tetrabutylammonium sulfate, 10 mM KH2PO4, and 0.25% MeOH and was adjusted to pH 6.9. Buffer B consisted of 5.6 mM tetrabutylammonium sulfate, 50 mM KH2PO4, and 30% MeOH and was neutralized to pH 7.0. The injection volume for analysis was 50 µl. Detection was carried out at 254 nm.
Smooth Muscle Cell Migration. A wound scrape assay was performed using rat vascular SMC. The cells were grown to confluence on 60-mm dishes. The cells were then made quiescent by incubating them in media containing 0.1% serum for 24 h followed by treatment with Didox, Imidate (0–100 µM), or HU (0–1000 µM) in media containing 0.1% serum and platelet-derived growth factor (PDGF) at a concentration of 10 ng/ml. Linear wounds were made by scraping each plate with the tip of a 20-µl pipette. The ability of cells to migrate across the wound area was measured using digital microscopy. SPOT advanced software was used to measure the wound immediately, 2, 4, 12, and 24 h following injury. The data are presented as rate of migration and represent the mean ± S.D.
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Results |
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; Vaughan et al., 1989). Furthermore, we and others have demonstrated that these doses are sufficient to inhibit RR activity without causing significant toxicity (Mayhew et al., 1999, 2002; Horvath et al., 2004).
Fourteen days postinjury, the animals were sacrificed, and morphometric analysis was carried out to asses the histopathological changes in the vessel wall (Fig. 1). Didox treatment resulted in a 62% decrease in neointimal area and a 61% decrease in intima/media ratio (Fig. 2). Imidate-treated groups demonstrated a 57% decrease in neointimal area and a 55% decrease in the intima/media ratio (Fig. 2). Because Didox and Imidate possess other chemical attributes in addition to their effects on RR (Fritzer-Szekeres et al., 1997, 2000, 2002; Lee et al., 1997; Inayat et al., 2002), the commercially available RR inhibitor HU (200 mg/kg/day) was also tested. HU afforded similar vascular protective effects to those observed with Didox and Imidate, resulting in a 55% decrease in neointimal area and a 63% decrease in intima/media ratio (Fig. 2). These results suggest that RR inhibition can modulate the remodeling process following vascular injury. However, because the remodeling process occurs over a prolonged period of time, additional studies were performed to determine whether the vascular protective effects observed would be mitigated over time.
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Effects of Didox, Imidate, and HU on SMC Proliferation. Didox, Imidate, and HU are known to be potent RR inhibitors (Elford, 1968; Elford et al., 1970, 1979). RR catalyzes the reductive conversion of ribonucleotides to deoxynucleotides. This reductive reaction is a prime target for impeding cellular proliferation, and therefore amenable to inhibiting VSMC replication, because it is a rate limiting step in the biochemical pathway leading to DNA synthesis (Elford et al., 1970, 1979; Takeda and Weber, 1981; Natsumeda et al., 1985). The ability of Didox and Imidate to inhibit RR activity has been well documented with published reports demonstrating an IC50 of 3 to 30 µM for this class of compounds (Elford et al., 1979). These values represent a >10-fold increased effectiveness over the classical RR inhibitor HU (Elford, 1968). Based on this evidence, experiments were performed to determine whether the ability of these compounds to reduce neointimal formation was due to their ability to inhibit SMC proliferation in vitro. Therefore, we determined the IC50 of each compound on inhibition of smooth muscle cell growth. Cells were incubated in the presence of Didox (0–200 µM), Imidate (0–200 µM), and HU (0–1000 µM) for 24 h. Cell numbers were then counted using flow cytometry, and the concentration at which cell division was inhibited by 50% (IC50) was calculated. Didox yielded an IC50 of 67 µM, and Imidate was slightly less potent, exhibiting an IC50 of 82 µM, whereas HU was the least potent with an IC50 of 266 µM (Fig. 4). These results are consistent with RR activity data and demonstrate that Didox and Imidate are 3 to 4 times more potent than HU at arresting cell division.
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The concentrations at which these drugs afford their in vitro biological effects are well below the range of the peak plasma levels (300–400 µM) measured following Didox and Imidate infusion (200 mg/kg/day) and would be expected to inhibit RR activity based on the published and observed Ki values for these compounds (Elford et al., 1979). These results would suggest that part of the vascular protective effects of these compounds is due to their ability to impede SMC proliferation.
Effects of Didox, Imidate, and HU on SMC Migration. SMC migration, however, is also a critical component of neointimal proliferation. Therefore, additional studies were performed to determine the effects of these compounds on SMC migration. Using a wound scrape assay, SMC migration studies were carried out in the presence of Didox, Imidate, and HU. VSMCs were cultured to confluence on 60-mm dishes. The cells were made quiescent by incubating in media containing 0.1% serum. Following 24 h of serum deprivation, Didox, Imidate, and HU were added to the wells (10–1000 µM) in the presence of PDGF (10 ng/ml), and a linear wound was made across the plate. SMC migration across the wound was monitored by digital microscopy over a 24-h period. Results from these studies demonstrated that Didox (100 µM) and Imidate (100 µM) treatment almost completely inhibited SMC migration, decreasing the migratory rate from 15.8 µM/h in the control to 1.7 and 0.9 µM/h, respectively (Fig. 5). In contrast, HU (100 µM) had little effect on SMC migration, resulting in a migratory rate of 15.1 µM/h (Fig. 4). No further inhibition was seen with HU concentrations up to 1 mM. These results demonstrate that Didox and Imidate significantly impair SMC migration. This would be expected to contribute to the vascular protective effects afforded by these drugs. However, RR inhibition appears to be the principal mechanism as the rat arterial injury data demonstrated similar efficacy with HU.
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Discussion |
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; Fischman et al., 1994; Elezi et al., 1998; Bennett and O'Sullivan, 2001; Heckenkamp et al., 2002). This process of vessel renarrowing is characterized by neointimal hyperplasia resulting in lumen loss and impaired vascular function. The vascular response to injury triggers a migratory and proliferative response within the smooth muscle cells, resulting in intimal thickening (Noda-Heiny and Sobel, 1995; Libby and Tanaka, 1997; Elezi et al., 1998; Ward et al., 2000; Bennett and O'Sullivan, 2001; Heckenkamp et al., 2002; Lanza et al., 2002; Segev et al., 2002; Bhargava et al., 2003; Crook and Akyurek, 2003; Indolfi et al., 2003). In this regard, emphasis has been placed on developing pharmacological therapy aimed at reducing the proliferative response. Currently, two pharmacological agents have been approved for clinical use in the treatment of post-PTCA restenosis (Drachman et al., 2000; Heldman et al., 2001; Suzuki et al., 2001). Paclitaxel (Taxol; Bristol-Myers Squibb, Princeton, NJ) and sirolimus (Rapamycin; Cordis, Miami Lakes, FL) are being delivered using coated coronary stents. Preliminary results suggest that these approaches offer significant protection against the restenosis process and validate the use of antiproliferative agents in the treatment of vascular proliferative disorders such as restenosis (Sousa et al., 2001; Gershlick et al., 2004; Stone et al., 2004).
As such, we have explored the use of RR inhibition as a new therapeutic target in ameliorating balloon mediated restenosis injury. The biochemical attributes of this enzyme make it amenable for the treatment of proliferative disorders, since inhibition of RR blocks DNA synthesis and thus cell replication. Therefore, using as balloon model of arterial injury, we have studied the effects of RR inhibition on the restenosis process.
Our results demonstrated that following balloon injury, 1 week of systemic administration of the RR inhibitors Didox and Imidate largely inhibited neointimal formation resulting in a 60% reduction in the intima/media ratio. Morphometric analysis revealed an 
60% reduction in neointimal area with no significant change in the medial area between treated and untreated groups. However, there was a small but statistically significant decrease in medial area following Imidate dosing when this cohort was compared against HU and Didox. These results suggest that Imidate may have some negative effects on smooth muscle remodeling following medial injury that results in medial wall thinning.
These in vivo studies demonstrate that inhibition of RR limits the extent of intimal hyperplasia following mechanical injury. However, these compounds possess a variety of chemical attributes that may contribute to their protective effects. Didox has been shown to inhibit NF-
B, although both compounds are potent free radical scavengers (Fritzer-Szekeres et al., 1997; Lee et al., 1997; Duilio et al., 2001; Turchan et al., 2003). Because of the myriad of effects elicited by these compounds, further experiments were carried out using the commercially available RR inhibitor HU. Following the same dosing regimen for Didox and Imidate, HU afforded similar antirestenotic efficacy, further supporting our observation regarding the importance of RR in the vascular response to injury.
Because the vascular response to injury is a chronic process, additional studies were performed to assess whether the protective effects elicited by early RR inhibition are maintained throughout the remodeling period. The RR inhibitors (Didox, Imidate, and HU) were administered daily for 1 week, and the extent of injury was assessed 6 weeks after balloon dilatation. This additional recovery time resulted in a >50% increase in the intima/media ratio, demonstrating the progression of the lesion over time. Interestingly, Didox and HU treatment reduced the intima/media ratio by >70%, although slightly less efficacy was observed with Imidate. Moreover, the degree of protection afforded by these compounds was significantly increased when compared with the results of the 2-week study. This suggests that RR is an early target in the vascular response to injury and that inhibition of this enzyme affects the long-term vascular remodeling associated with restenosis.
These results demonstrate that RR inhibition limits the degree of restenosis following arterial dilation injury. We believe these effects are mediated through an inhibition of SMC proliferation, since this process precedes neointimal formation. Therefore, studies were performed using a cell proliferation assay. Didox, Imidate, and HU treatment resulted in the arrest of cell division. Analysis of dNTP pools demonstrated a greater than 50% reduction in dATP levels, further supporting that the observed effects of these drugs are mediated at least in part through the inhibition of ribonucleotide reductase. These effects were independent of any cytotoxic action that these compounds may possess, since flow cytometry revealed <3% apoptotic cells following the dosing regimes tested. In addition, prior to dNTP analysis, cells were counted and viability assessed using trypan blue exclusion. Cell viability was greater than 95% among all groups.
As previously stated, Didox and Imidate possess various chemical attributes in addition to RR inhibition, many of which may confer protection against restenosis (Fritzer-Szekeres et al., 1997; Lee et al., 1997; Duilio et al., 2001; Shet et al., 2003; Turchan et al., 2003). Among these is their ability to scavenge free radicals, which may modulate SMC migration, a critical component of neointimal proliferation (Duilio et al., 2001; Turchan et al., 2003). Therefore, we tested the effects of Didox and Imidate on SMC proliferation using a wound scrape assay. Results demonstrated that both compounds almost completely inhibited SMC migration. This would suggest that inhibition of SMC proliferation may contribute to the protection afforded by these compounds. Similarly, a number of studies have demonstrated that HU, when oxidized, can release NO (Stolze and Nohl, 1990; Pacelli et al., 1996). Since NO has been shown to inhibit cell migration, we tested whether HU possesses antimigratory properties that could be involved in its antirestenotic effects (Sarkar et al., 1997; Sarkar and Webb, 1998). HU at concentrations up to 1 mM had no effect on SMC migration. This is an important observation and would suggest that inhibition of RR is the principal mechanism through which these compounds afford their protection, as similar efficacy was seen with all compounds. However, if the in vivo results are interpreted on a molar basis, HU (2.6 mmol/kg/day) doses are 2- to 3-fold higher than that of Didox (1.2 mmol/kg/day) and Imidate (0.9 mmol/kg/day) and suggests that the effects of Didox and Imidate on SMC migration may contribute to the vascular protective effects observed with these two compounds.
Together, the in vivo and in vitro data demonstrate that activation of RR is a critical, early component in the proliferative response associated with vascular injury and that inhibition of this enzyme may reduce the vascular pathology associated with restenosis injury. Although the incidence of restenosis has markedly decreased with the advent of drug-coated stents, restenosis still occurs in up to 20% of patients within the first year, whereas results on late lumen loss are still being gathered (Drachman et al., 2000; Heldman et al., 2001; Grube et al., 2003; Kastrati et al., 2005; Kim et al., 2005). In addition, because the use of coated stents may increase the risk of thrombosis, there is a need for agents that can be administered systemically in patients at high risk for thrombotic events (Morice, 2005). We believe that inhibition of RR may be a pathway that can be therapeutically targeted through either local or systemic delivery based on the low toxicity associated with current RR inhibitor therapy using HU. Additionally, our data suggests that RR is an early target in the restenosis process, and as such, early pharmacological intervention may preclude chronic therapy and its associated adverse side effects. We believe that these observations have important therapeutic potential and implicate RR as a promising therapeutic target in the treatment of vascular proliferative disorders.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PTCA, percutaneous transluminal coronary angioplasty; VSMC, vascular smooth muscle cell(s); SMC, smooth muscle cell(s); RR, ribonucleotide reductase; HU, hydroxyurea; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; NF-B, nuclear factor B; NO, nitric oxide.
1 These authors contributed equally to this work. 
Address correspondence to: Arturo J. Cardounel, Davis Heart and Lung Research Institute, 473 W. 12th Ave., Columbus, OH 43210. E-mail: cardounel-1{at}medctr.osu.edu
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, significantly different at p < 0.05 among treated groups.
