The autologous saphenous vein graft is currently the most suitable conduit for small-caliber arterial reconstruction of the lower limbs. However, the patency is not always complete, and graft failure may occur (1-3). Intimal hyperplasia of the vein graft appears to be one of the main causes of graft failure (4,5).
OPC-29030, (S)-(+)-6-[3-(1-o-tolylimidazol-2-yl)sulfinylpropoxy]-3,4-dihydro-2(1H)-quinolinone, is a newly developed inhibitor of 12(S)-hydroxyeicosatetraenoic acid (12-HETE) released from platelets. OPC-29030 prevented platelet adhesion to the vascular subendothelial matrix (6) and thrombus formation under rheologic conditions (7). In platelets, thromboxane A2 (TXA2) and 12-HETE are major arachidonic acid metabolites. It is known that platelets play a significant role in the early phase of arteriosclerosis through the release of 12-HETE, which is one of the most potent inducers of platelet adhesion (8-10), and platelet adhesion plays an important role in thrombus formation in the threat of a stenosis (11,12). In addition, 12-HETE stimulates the migration of smooth muscle cells from the media to intima (11).
In our present experiment, the poor-runoff model in the rabbit femoral artery is considered to be similar to that in human patients with peripheral vascular disease, because under such poor-runoff conditions, intimal thickening of the autologous vein graft was significantly thicker than that in the control group (13,14). Conversely, hypercholesterolemia is well known as one of the most important risk factors for the development of atherosclerosis, (15,16) and is also related to failure of vein grafts in both aortocoronary (17,18) and peripheral arterial bypasses (19). The effects of hypercholesterolemia on intimal hyperplasia in vein grafts have been reported (14,20). However, the precise mechanisms of intimal hyperplasia caused by poor runoff or hyperlipidemia are still unknown.
In this study, we examined whether there is a relation between 12-HETE and intimal hyperplasia induced by a poor-runoff model and a hyperlipidemic model in rabbit autologous vein grafts.
MATERIALS AND METHODS
Effect of OPC-29030 on intimal hyperplasia
Material and methods. Male New Zealand White rabbits, weighing approximately 2.5 kg, were divided into two groups, poor distal runoff group (PR group: n = 18) and hyperlipidemic diet group (HL group: n = 16). In the poor distal runoff group, 18 rabbits received autologous vein grafts under abnormal flow conditions, according to the experimental poor-runoff model (14), as described later. In the hyperlipidemic diet group, 16 rabbits were given a 1% cholesterol diet for 4 weeks and received the autologous vein grafts under normal-flow conditions. Nine rabbits in the PR group and eight rabbits in the HL group were given a diet containing 0.1% OPC-29030 (dry powder) until they were killed, and the other nine rabbits in the PR group and eight rabbits in the HL group were given a diet without OPC-29030. Thus, the PR group and the HL group were divided into two groups, OPC-29030 group and control group. Among them, eight vein grafts in the PR group and six vein grafts in the HL group were used for the examination of bromodeoxyuridine (BrdU) incorporation, as described later.
Animal care complied with the "Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals" (National Institutes of Health, publication no. 86-23, revised 1985).
Surgical procedures. Anesthesia was induced with an intra-muscular injection of xylazine (10 mg/kg) and ketamine hydrochloride (25 mg/kg).
We developed a PR model according to the Itoh's method (14). In the poor distal runoff group, a longitudinal medial incision was made in the right lower thigh, and the popliteal, saphenous, and distal femoral arteries were exposed. The distal femoral artery, the saphenous artery, and the caudal femoral artery were ligated and divided, and only the middle muscle branch was preserved. Four weeks later, we deemed that the hemodynamics were stabilized with collateralization and that the PR models were completed, and a second procedure was initiated. Anesthesia was induced with xylazine (10 mg/kg) and ketamine hydrochloride (40 mg/kg), and was maintained with an additional intramuscular injection of ketamine hydrochloride (15 mg/kg) every 30 min. A skin incision was made on the right thigh, proximal to the previous incision, the femoral artery and vein were exposed, and a ∼1.5-cm segment of the femoral vein was taken for the autologous vein graft. Harvesting of the vein graft was completed with meticulous care to avoid injury to the vein wall. The harvested graft was preserved in heparinized saline (5 U/ml) for 20 min, before grafting, at room temperature. The femoral artery, 1 cm in length, distal to the orifice of the lateral circumflex femoral artery, was resected and replaced with the harvested autologous femoral vein. The vein graft was anastomosed in a reversed end-to-end fashion with interrupted 10-0 monofilament sutures under a surgical microscope. Throughout these procedures, neither anticoagulant drug nor antibiotics were administered (14).
In the hyperlipidemic diet group, after a 1% cholesterol diet for 4 weeks, the vein graft was implanted in the same manner. Ten minutes after reestablishment of blood flow, a flow probe connected to a ultrasonic transit-time flowmeter (Transonic T201; Transonic Systems, Inc., Ithaca, NY, U.S.A.) was applied on the midportion of the implanted graft, and the rate of blood flow and flow waveforms were recorded (14). The rate of blood flow was measured at the implantation of the vein graft and the harvest of the implanted graft in both groups.
Blood sample analysis. Blood was obtained from a cannula placed in the middle ear artery at the time of graft implantation and killing, and collected into plastic tubes containing ethylenediaminetetraacetic acid (EDTA; final concentration, 0.1%). Platelet-rich plasma was obtained by centrifuging the blood at 1,500 g for 10 min. Eicosanoid extraction was performed by the method described by Raghunath et al. (21). The concentration of 12-HETE was measured using a TiterZyme 12-HETE Enzyme Immunoassay Kit (PerSeptive Diagnostics, Inc., Framingham, MA, U.S.A.) (6, 6-keto-PGF1α (6-keto-PGF1α KIT; Cayman Chemical Co., Ann Arbor, MI, U.S.A.) and thromboxane B2 (TXB2 EIA KIT; PerSeptive Biosystems, Framingham, MA, U.S.A.), metabolites of prostacycline and thromboxane A2, were also measured at the time of graft implantation and killing. The total plasma cholesterol level was measured with a cholesterol kit (Monotest Cholesterol; Boehringer Mannheim GmbH, Mannheim, West Germany).
Harvest of implanted grafts. At 4 weeks after implantation of the autologous vein grafts, the rabbits were anesthetized, and the vein grafts were harvested. A laparotomy was performed, and a cannula was inserted into the artery. The graft was perfused and fixed with 4% paraformaldehyde at a pressure of 100 mm Hg for 30 min. The perfused autologous vein graft, including anastomoses, was totally removed and immersed in the same fixative overnight at 4°C.
Sample processing. The midportion of the harvested graft was used for histologic studies. Each sample was paraffin-embedded and cut with a microtome into 4-μm-thick sections, which were mounted on glass slides.
BrdU staining and measurement of the labeling index. At 2 weeks after implantation of the autologous vein grafts, the rabbits were anesthetized, and the vein grafts were harvested. One hour before killing, 40 mg/kg of BrdU (Sigma Chemical Co., St. Louis, MO, U.S.A.) was administered intravenously, and the vein graft was exposed. Then a laparotomy was performed, and a cannula was inserted into the artery. The graft was perfused and fixed with 4% paraformaldehyde at a pressure of 100 mm Hg for 30 min. The perfused autologous vein graft, including anastomoses, was totally removed and immersed in the same fixative overnight at 4°C.
Each section was deparaffinized in a xylene/ethanol series. For DNA denaturation, the sections were first incubated in 2N HCl for 1 h and neutralized with 0.1 M sodium tetraborate, and then digested with 0.05% protease (Type XXV; Sigma Chemical Co.) for 5 min at 37°C, and rinsed with phosphate-buffered saline (PBS). After inhibition of endogenous peroxidase and incubation of normal rabbit serum, sections were incubated with anti-BrdU monoclonal mouse antibody (1:50; Becton Dickinson Immunocytometry Systems, Mountain View, CA, U.S.A.), overnight at 4°C. After rinsing in PBS, the ABC technique was completed using the Histofine SAB-PO mouse immunoglobulin kit (Nichirei, Tokyo, Japan). Peroxidase visualization was achieved by the DAB method, and counterstaining was performed with methyl green (22,23). Cells that had incorporated BrdU were identified by the presence of brown pigment over the nuclei. BrdU-labeled cells were counted in the intima of each cross section. BrdU labeling index was expressed as a percentage of the total number of cells scored in the intima (14,23).
Assessment of intimal hyperplasia. The sections were also stained either with hematoxylin-eosin or the elastic van Gieson's method. Intimal hyperplasia was measured with an ocular cytometer placed on the ocular lens of a light microscope at a magnification of ×400. The average intimal hyperplasia of more than eight randomly selected points from each sample was taken as the degree of intimal hyperplasia (13,14,20).
Statistical analysis. Statistical evaluation of the data was performed using Student's t test for unpaired observations. When more than two means were compared, an analysis of variance was used. If the value was statistically significant, Scheffé's test for multiple comparisons was used to identify differences among the groups. A value of p < 0.05 was considered to have statistical significance.
Effect of OPC-29030 on rat aortic smooth muscle cells in culture
Rat aortic smooth muscle cells (A10 cells) were purchased from Dai-Nippon Pharmaceutical Co., Ltd. The OPC-29030 used in this study was obtained from the Second Tokushima Factory of Otsuka Pharmaceutical Co., Ltd., and dissolved in 100% dimethyl sulfoxide (DMSO), and the solution was added to the samples to a final concentration of 0.1% DMSO.
Effect of OPC-29030 on the proliferation of rat aortic smooth muscle cells in culture(24).
Proliferation of A10 cells with 1, 3, and 10% fetal bovine serum (FBS). A10 cells (40,000 cells/dish) were cultured with 1, 3, and 10% FBS, and then harvested and counted using a hemocytometer at 1, 2, 3, 4, or 5 days after seeding to determine the proliferation index for A10 cells.
Effect of OPC-29030 on the proliferation of A10 cells with 10% FBS. A10 cells (40,000 cells/dish) were cultured with 10% FBS. OPC-29030 (0.1, 0.1, 1, or 10 μM) was added to each dish. The cultured A10 cells were then harvested and counted using a hemocytometer at 4 days after seeding to determine the proliferation index for A10 cells.
Effect of OPC-29030 on the migration of rat aortic smooth muscle cells in culture(25).
(a) Effect on migration of A10 cells when OPC-29030 was added to human platelets.
(b) Effect on migration of A10 Cells when OPC-29030 was added to A10 cells...
Migration of A10 cells was measured in a Boyden chamber (Neuro Probe Co., Ltd). A10 cells (∼2 × 104M cells/well) were placed into the upper chamber, and washed human platelets (3 × 107 platelets/well) were placed into the lower chamber, with a polycarbonate filter membrane (pore size, 8 μm) positioned between the two chambers. For case (a), OPC-29030 was added to the human platelets. For case (b), OPC-29030 was added to the A10 cells. After incubation in CO2 for 5 h at 37°C, the A10 cells that had passed through the membrane were fixed with 10% neutral formalin buffer solution and stained with 0.5% gentian violet R.
The number of migrated A10 cells at each concentration of OPC-29030 was expressed as a percentage of the number of migrating cells without OPC-29030 (control).
Statistical analysis. The difference in the number of proliferating or migrating A10 cells between the control and OPC-29030 groups was analyzed using the Statistical Analysis System (SAS).
All animals survived, and the 34 vein grafts were patent at harvest.
Effect of OPC-29030 on intimal hyperplasia
Hemodynamic data. At graft implantation, the mean flow rates in the poor-runoff limbs (n = 10: 1.6 ± 0.5 ml/min) were significantly lower than those of the normal runoff in hypercholesterolemic rabbits (n = 10: 4.5 ± 1.4 ml/min, p < 0.01).
At harvesting, the mean flow rates in the poor-runoff limbs (n = 10: 1.7 ± 0.6 ml/min) were also significantly lower than those of the normal runoff in hypercholesterolemic rabbits (n = 10: 4.6 ± 1.2 ml/min, p < 0.01). There was no significant difference in mean flow rates at the time of implantation of the vein graft and at the time of harvesting the implanted graft in the PR and HL groups.
Blood sample analysis. Plasma cholesterol levels at the time of graft implantation in the HL group were significantly higher than those of the PR group. Plasma cholesterol levels at the time of graft harvest was maintained in both groups, and were significantly higher in the HL group than in the PR group. OPC-29030 did not affect the plasma cholesterol levels in the PR and HL groups (Table 1).
In the PR group, the plasma 12-HETE levels in the OPC-29030 group were significantly lower than those of the control group. However, plasma 6-keto-PGF1α levels and plasma TXB2 levels were not significantly different between the OPC-29030 group and the control group (Fig. 1).
In the HL group, the plasma 12-HETE, 6-keto-PGF1α, and TXB2 levels were not significantly different between the OPC-29030 group and the control group (Fig. 2).
Progression of intimal hyperplasia. In the PR group at 4 weeks after grafting, the intimal hyperplasia in the OPC-29030 group (n = 5: 56.4 ± 17.5 μm) was significantly reduced compared with that of the control group (n = 5: 124.0 ± 18.8 μm; Figs. 3 and 4).
In the HL group at 4 weeks after grafting, both the OPC-29030 group (n = 5: 165.0 ± 23.1 μm) and the control group (n = 5: 196.3 ± 34.3 μm) showed a remarkable degree of intimal thickness, and there was no significant difference between the two groups (Figs. 3 and 4).
BrdU incorporation. In the PR group, the BrdU labeling index at 2 weeks after surgery was significantly lower in the OPC-29030 group (n = 4) compared with that in the control group (n = 4) (Fig. 5). However, in the HL group, there was no significant difference between the OPC-29030 group (n = 3) and the control group (n = 3).
Effect of OPC-29030 on rat aortic smooth muscle cells in culture
Effect of OPC-29030 on the proliferation of rat aortic smooth muscle cells in culture. A logarithmic relationship was observed between A10 cell proliferation and FBS concentration ranging from 1% to 10% in the culture medium for 2-5 days after cell seeding (Fig. 6). Adhesion of A10 cells to the dish improved with increasing FBS concentration. OPC-29030 did not affect the proliferation of A10 cells in 10% FBS medium at concentrations from 0.01 to 10 μM(Table 2).
Effect of OPC-29030 on the migration of rat aortic smooth muscle cells in culture. Coincubation with washed platelets and OPC-29030 inhibited the migration of A10 cells in a dose-dependent manner, and was statistically significant at concentrations of 1 and 10 μM. Coincubation of A10 cells with OPC-29030 did not show any inhibitory effect on migration up to a concentration of 10 μM(Fig. 7).
Late graft failure caused by progressive intimal hyperplasia is a well-known persistent complication in arterial reconstruction, particularly in cases with poor-runoff vessels (1-3,13,14). The antiplatelet agents have been widely used in an attempt to improve vascular graft patency, and the antiplatelet therapy has been tested repeatedly. However, the effects of the antiplatelet therapy are still controversial (26-28).
In our previous studies, we have demonstrated that the development of intimal hyperplasia was more progressive in autologous vein grafts under abnormal blood-flow conditions. Hemodynamic factors such as a low flow velocity and low shear stress result in progression of late graft failure because of intimal thickening (13,14,29-31). In our previous studies, we also classified the electromagnetically measured blood flow waveforms at reconstructive surgery into five types. We reported a close relationship between the ultimate results of the arterial reconstruction and intraoperative blood flow wave-forms (14). Grafts with a type 0 or I flow wave pattern (normal flow group, characterized by steep acceleration and deceleration) had a long-term patency. In grafts with a type II, III, or IV flow waveform pattern (abnormal flow group, characterized by a gentle sloping), graft failure was more frequent than in the normal flow group. In our present experiment, we used a poor-runoff model in the rabbit femoral artery (14), which is similar to a human patient with peripheral vascular disease, because under this poor-runoff condition, the intimal thickening of the autogenous vein graft was significantly thicker than that in the control group (14), and this intimal hyperplasia also correlated well with the enhanced platelet adherence on the intima of the vein grafts during an early period after implantation (13,14,23,32).
In addition, hypercholesterolemia accelerated the intimal hyperplasia in experimental vein grafts (14,20). However, the precise mechanism of the intimal thickening under poor-runoff conditions or hypercholesterolemia was still unknown.
Intimal hyperplasia is the universal response of a vein graft to insertion into the arterial circulation and is considered to result from both the migration of smooth muscle cells out of the media into the intima and proliferation of these smooth muscle cells (33), although a recent report suggested that adventitial myofibroblasts may contribute to the intimal thickening (34). 12-HETE is released from platelets by activating lipooxygenase, and accelerated platelet adhesion to the endothelial cells (35). 12-HETE is one of the most potent inducers of platelet adhesion (7-9). In addition, 12-HETE stimulates the migration of smooth muscle cells from the media to intima (11).
Uno et al. (6,7) found 2(1H)-quinolinone derivatives having an azole group in the side chain showed inhibitory activity on 12-HETE released from platelets. They synthesized and tested many 2(1H)-quinolinone derivatives, and then found that OPC-29030, (S)-(+)-6-[3-(1-o-tolylimidazol-2-yl)sulfinylpropoxy]-3,4-dihydro-2(1H)-quinolinone was the most potent inhibitor of 12-HETE release, and it has inhibitory effects on platelet adhesion in vivo (6) and thrombus formation under rheologic conditions (7). OPC-29030 suppressed 12-HETE production by inhibiting a certain step of the 12-lipoxygenase translocation (36). Although OPC-29030 did not inhibit cyclooxygenase activity, it decreased prostaglandin biosynthesis, especially thromboxane A2 synthesis. The mechanisms of OPC-29030 differed from those of other antiplatelet agents.
In the rabbit distal poor-runoff model, the present results demonstrated that OPC-29030 inhibited the degree of intimal hyperplasia with decreased plasma 12-HETE levels, although the plasma 6-keto-PGF1α and plasma TXB2 levels were not influenced. The present study also demonstrated that the BrdU labeling index was significantly lower in the OPC-29030 group compared with that in the control group in the poor distal runoff model, although OPC-29030 itself did not prevent the smooth muscle cell proliferation in vitro. In addition, coincubation with platelets and OPC-29030 inhibited migration of the cultured smooth muscle cells, although coincubation of A10 cells with OPC-29030 did not effect migration. These results suggest that platelet-derived factors, such as platelet-derived growth factor (PDGF) or other factors, may play an important role in migration (37), and OPC-29030 inhibited the platelet-derived product, which resulted in inhibiting the migration. These findings support the hypothesis that OPC-29030 decreases plasma 12-HETE levels and prevents platelet adhesion and smooth muscle cell migration from the media to intima under poor-runoff conditions, which result in inhibiting intimal thickening.
By contrast, in the presence of hyperlipidemia, we demonstrated that OPC-29030 did not inhibit the degree of intimal hyperplasia without changing plasma 12-HETE levels, plasma 6-keto-PGF1α levels, and plasma TXB2 levels. Itoh et al. (14) showed that hyperlipidemia may accelerate intimal hyperplasia as a result of acceleration of the proliferation of intimal cells and that the enhancement of cell proliferation by hyperlipidemia is augmented in case of a poor distal runoff. Accelerated intimal cell proliferation of vein grafts by hyperlipidemia may be due to secretion of growth factor from infiltrated macrophages in the thickened intima. In addition, the enhancement of intimal cell proliferation by hyperlipidemia may be accelerated by blood-flow stagnation, as under such conditions, oxidized macrophages readily adhere to the surface of the vein graft, infiltrate beneath the neoendothelium, and then secrete growth factors (14). Thus, the mechanisms for intimal hyperplasia may be different between the intimal hyperplasia caused by poor-runoff conditions and hyperlipidemia. However, the reason that the plasma levels of 12-HETE in hyperlipidemic rabbits was significantly higher than that of poor-runoff model is unknown. One possibility may be that OPC-29030 just inhibited platelet aggregation more in the poor-runoff model than in the hypercholesterolemia model and consequently had a greater effect on 12-HETE levels.
Probucol is used to treat hypercholesterolemia and also has antiatherogenic effects. These later properties are considered to be due to inhibition of oxidative modification of low-density lipoprotein (LDL), such that macrophages neither take up modified LDL, nor convert into foam cells. A recent report (28) demonstrated that probucol suppressed intimal hyperplasia in rabbit vein grafts under hyperlipidemic conditions, although in vein grafts under poor-runoff conditions, the intimal hyperplasia was not inhibited by probucol. These results also supported the possibility that the mechanisms of intimal hyperplasia enhancement due to poor-runoff conditions are different from those induced by hyperlipidemia. Detailed experiments to elucidate the precise mechanism must be carried out.
In summary, we investigated the efficacy of OPC-29030 in modulating the progression of vein graft intimal hyperplasia using a rabbit poor-runoff model and a hyperlipidemic model. OPC-29030 reduced the degree of intimal hyperplasia by inhibiting 12-HETE release under poor-runoff condition. In addition, OPC-29030 did not reduce the degree of intimal hyperplasia and 12-HETE release in hyperlipidemic rabbits. These results suggested the possible involvement of 12-HETE for the intimal hyperplasia induced by the poor-runoff model. There is a difference in the process of intimal hyperplasia between poor distal runoff and hyperlipidemic models.
Acknowledgment: This work was supported in part by a Grant-in-aid for General Scientific Research from the ministry of Education, Science and Culture of Japan. We are grateful to Dr. Timothy D. Keeley, Ph.D., for reading the manuscript and providing helpful comments.
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Keywords:© 2000 Lippincott Williams & Wilkins, Inc.
12-HETE; Vein graft; Hypercholesterolemia; Poor runoff