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Tyrosine Kinase Inhibitor Markedly Suppresses the Development of Coronary Lesions Induced by Long-Term Treatment with Platelet-Derived Growth Factor in Pigs In Vivo

Kozai, Toshiyuki; Shimokawa, Hiroaki; Fukumoto, Yoshihiro; Kobayashi, Sei*; Owada, M. Koji; Kadokami, Toshiaki; Ito, Akira; Kuwata, Kouichi; Egashira, Kensuke; Shiraishi, Tadayoshi; Kanaide, Hideo*; Takeshita, Akira

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Journal of Cardiovascular Pharmacology: April 1997 - Volume 29 - Issue 4 - p 536-545
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Abstract

Atherosclerosis is now regarded as slowly progressive inflammatory and proliferative responses of the vascular wall to various forms of injury (1-3). Indeed, we previously developed a porcine model of coronary atherosclerosis by a combination of endothelial injury and high-cholesterol feeding, in which coronary vasospasm was repeatedly induced at the atherosclerotic lesions (4-6).

Among the cytokines/growth factors, platelet-derived growth factor (PDGF) (7) has been suggested by in vitro experiments to play an important role in the pathogenesis of atherosclerosis (1,8-10). However, no study has attempted to examine the long-term effect of PDGF on the coronary artery in vivo or to suppress the effects of PDGF by pharmacologic interventions in vivo.

Tyrosine kinases are important transducers of a variety of extracellular signals that regulate proliferation, differentiation, and specific functions of differentiated cells (11). Because the receptors for growth factors (including PDGF) are known to have tyrosine kinase activity (12), tyrosine kinase could be regarded as one of the common and key steps for smooth-muscle proliferation. Indeed, we (13,14) recently showed that the development of coronary proliferative/hyperconstrictive lesions induced by long-term treatment with interleukin 1β (IL-1β) are markedly suppressed by ST 638, a specific tyrosine kinase inhibitor (15), but not by ST 494, a biologically inactive form of ST 638 (16).

Our study was thus designed to examine what morphologic and functional changes of the coronary arteries are induced by long-term treatment with PDGF in vivo and whether or not ST 638 could suppress those changes in vivo.

METHODS

Animal preparation

Fifteen male Yorkshire pigs, 2-4 months old and weighing 23-27 kg, were sedated with intramuscular administration of ketamine hydrochloride (12.5 mg/kg) and anesthetized with intravenous administration of sodium pentobarbital (25 mg/kg). The animals were then intubated and ventilated with room air supplemented with oxygen by a positive-pressure respirator (Shinano Inc., Tokyo, Japan). Under aseptic conditions, a left thoracotomy was performed, and the proximal segments of the left anterior descending (LAD) and the left circumflex (LCX) coronary arteries were carefully dissected. The dissected segments of the coronary artery were gently wrapped with cotton mesh absorbing agents (13,14). The following two protocols were examined.

Protocol 1. The dissected segments of the coronary arteries were wrapped with cotton mesh after absorbing sepharose beads suspension with recombinant human PDGF-AA (2.5 μg) or PDGF-BB (2.5 μg) (n = 6). The sites of the treatment were randomized.

Protocol 2. The dissected segments of the coronary arteries were wrapped with cotton mesh after absorbing one of the following agents (n = 6): (a) sepharose beads suspension with recombinant human PDGF-AA (2.5 μg) plus 1% Tween 80, a vehicle for ST 638, or (b) sepharose beads suspension with recombinant human PDGF-AA (2.5 μg) plus ST 638 (12.5 mg) dissolved in Tween 80. The sites of the treatments were randomized.

We confirmed in a preliminary study that ST 638, but not ST 494, markedly inhibits the PDGF-induced tyrosine phosphorylations in cultured rat aortic smooth-muscle cells in vitro (data not shown). We also previously confirmed that the treatment with Tween 80 alone does not cause any vascular lesion (14).

This experiment was reviewed by the committee of the Ethics on Animal Experiment in Faculty of Medicine, Kyushu University, and carried out under the control of the Guidelines for Animal Experiment in Faculty of Medicine, Kyushu University, and The Law (No. 105) and Notification (No. 6) of the Japanese Government.

Preparation of sepharose beads suspension

The method of preparing the beads suspension has been previously reported in detail (13,14). In brief, 1 g of sepharose microbeads (CNBr-activated sepharose 4B, 45-165 μm in diameter; Pharmacia, Uppsala, Sweden), which bind amino-residues of proteins, were suspended in 20 ml of NaHCO3/NaCl solution with 1 mg of PDGF-AA or PDGF-BB. The beads were allowed to bind with PDGF-AA or PDGF-BB at room temperature for 1 h and then at 4°C overnight. After centrifugation at 1,200 rev/min for 5 min, the supernatant was measured by enzyme-linked immunosorbent assay (17). The PDGF-AA-bound and PDGF-BB-bound beads were washed and resuspended so that the final concentration of PDGF-AA and PDGF-BB was 50 μg/ml. The number of beads in the suspension was ∼70/μl. All preparations mentioned were performed under sterile conditions.

Because in our bead preparations, most of the PDGF molecules are bound inside the beads by a covalent bond at the amino-residues of the proteins, ≤1.2% of the PDGF molecules are actually bound to the surface of the beads and biologically active (13). Thus when 2.5 μg of PDGF that is bound to the beads is applied to the coronary artery, ≤30 ng of PDGF is biologically active (13).

We also confirmed in the previous study that the treatment with sepharose beads alone caused only minimal thickening and no hyperconstrictive responses to intracoronary administration of serotonin or histamine (13).

Pharmacokinetics and bioavailability of ST 638

In the previous study, we measured the local concentration of ST 638 in the porcine carotid arteries treated with ST 638 (12.5 mg) to elucidate its pharmacokinetics, by using high-performance liquid chromatography (14). The average amount of ST 638 per 1 g wet weight of the vessel 1, 3, 7, and 14 days after the treatment was 602, 651, 288, and 121 μg/g, respectively (14). This means that the estimated local concentration of ST 638 was 1.7, 1.8, 0.81, and 0.34 mM, respectively, while regarding 1 g of wet weight of the vessel as 1 ml of fluid. Thus throughout the experimental period, the local concentrations of ST 638 were higher enough than those (25-50 μM) that inhibit the intracellular tyrosine kinases, such as tyrosine kinase of epidermal growth factor (EGF) receptor (15).

Coronary arteriography

Two weeks after the operation, animals were again anesthetized and ventilated as described, and coronary angiography was performed. A preshaped Kifa or Judkins catheter was inserted into the carotid or femoral artery, respectively, and coronary arteriography in a left anterior oblique view was performed. Electrocardiograms (I, II, III, V1, and V6 leads) and blood pressure were continuously recorded during the experiments (13,14).

Coronary vascular responses were examined in response to intracoronary administration of nitroglycerin (10 μg/kg), serotonin (1, 3, and 10 μg/kg), histamine (1, 3, and 10 μg/kg), and prostaglandin F (5 and 50 μg/kg) (13,14). Coronary angiography was performed 2 min after intracoronary administration of nitroglycerin and serotonin, 1 min after that of histamine, and 5 min after that of prostaglandin F, when hemodynamic variables returned to basal levels (13,14).

Coronary-diameter measurement

Cineangiograms at end diastole were chosen and printed, and the coronary luminal diameters were measured with calipers (13,14). With this technique, excellent correlations between repeated measurements (r = 0.99) and between different observers (r = 0.98) were confirmed in the range of the coronary diameter from 0.98-5.58 mm (13,14). Coronary stenosis of the segment treated with drugs was expressed as the percentage decrease in the diameter compared with the luminal diameter of adjacent proximal and distal nonstenotic coronary segments after intracoronary administration of nitroglycerin (10 μg/kg). The coronary responses to serotonin, histamine, and prostaglandin F were expressed as the percentage luminal narrowing compared with the coronary luminal diameter after intracoronary administration of nitroglycerin (10 μg/kg). The extent of relaxation induced by nitroglycerin was calculated by the following equation; [(DNTG − DCONT)/DNTG − 1] × 100 (%). In this equation, DCONT and DNTG express the diameter before and after intracoronary administration of nitroglycerin, respectively.

Histologic examination

On completion of these experiments, animals were anesthetized with a lethal dose of sodium pentobarbital, and hearts were removed. Left coronary arteries were perfused via a constant-pressure perfusion system (120 cm H2O) with saline (500 ml) and subsequently with 6% formaldehyde (1,000 ml) (13,14). After the fixation, both LAD and LCX were cut transversely into segments at 5-mm intervals along their main trunk with small surrounded tissues. These segments were stained with hematoxyline-eosin and van Gieson's elastic staining for photomicroscopy. With a photomicroscopic photograph system (MICROPHOT-FXA; Nikon Co., Tokyo, Japan), pictures of coronary arteries were taken at a ×40 magnification, and the degree of the intimal thickening was analyzed quantitatively by using a computer-assisted picture system (Genlocker System; Sony, Tokyo, Japan) (13,14). This system consists of a high-resolution television monitor, an image processing and calculation unit with a microprocessor, a light pen controller with a microprocessor, and a printer. The inner border of the intimal layer and the internal elastic lamina (IEL) were traced by the light pen, and the areas encircled by the tracings were calculated automatically.

Three areas [luminal area, IEL area and external elastic lamina (EEL) area] were measured. The intimal area (Ai) was calculated by the following formula; Ai = Ae − Al, where Ae and Al are the areas within the IEL area and the luminal area, respectively. The degree of the neointimal formation was expressed by the following three parameters; intimal area = Ai (mm2), maximal intimal thickness (mm) measured with a caliper, and % intima, calculated by the following equation: Ai/Ae × 100 (%) (13,14). The degree of the vascular remodeling was expressed by the change in the three vessel areas (lumen area, IEL area, and EEL area), which were calculated by the formula (ATx − ACONT)/ACONT × 100 (%), where ATx and ACONT are the vessel areas of the coronary segments at the treated site and at the adjacent control site, respectively. The geometric remodeling was defined as the reduction of the IEL and EEL areas (18-20).

Antiphosphotyrosine immunoblotting

Three pigs were killed 3 days after the operation with the PDGF-AA treatment for antiphosphotyrosine immunoblotting. In these pigs, the hearts were removed, and the coronary segments treated with PDGF-AA (2.5 μg), PDGF-AA (2.5 μg), plus ST 638 (12.5 mg), and the untreated segments were dissected. Each segment was then homogenized in the extraction buffer [containing 62 mM TRIS-HCl, 2.5 mM MgCl2, 1.0 mM ethylene glycol-bistetraacetic acid (EGTA), 1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 100 kallikrein inhibitor unit/ml aprotinine, 12 μM pAPMSF, 25 μM leupeptin, 5 μM E-64, and 200 μM orthovanadate). The cell lysate was centrifuged at 12,000 r/min for 30 min at 4°C. The supernatant was treated with SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Then immunoblotting was performed by using monoclonal antiphosphotyrosine antibody (PY20; ICN Biomedicals, Irvine, CA, U.S.A.) and detected by horseradish peroxidase-coupled goat anti-mouse immunoglobulin G (IgG) and chemiluminescence (21).

Drugs

The following drugs were used: recombinant human PDGF-AA and -BB (Genzyme Co., Cambridge, MA, U.S.A.), ST 638 and ST 494 (Kanegafuchi Chemical Co., Osaka, Japan), 5-hydroxytryptamine (serotonin) and histamine (Sigma Chemical Co., St. Louis, MO, U.S.A.), nitroglycerin (Nihon-kayaku Pharmaceutical Co., Tokyo, Japan), and prostaglandin F (Ono Pharmaceutical Co., Osaka, Japan). ST 638 was dissolved in 1% Tween 80. Serotonin, histamine, and prostaglandin F were dissolved in physiologic saline.

Statistical analysis

The results were expressed as the mean ± SEM. Throughout the text and in the figures, n represents the number of animals examined. Multiple comparisons were made by analysis of variance (ANOVA) for repeated examinations followed by a Scheffé's test. Paired data were analyzed by Student's t test. A p value of <0.05 was considered to be statistically significant.

RESULTS

Protocol 1

Two weeks after the operation, mild to moderate stenotic lesions were noted angiographically at the sites treated with PDGF-AA and -BB, and the extent of the coronary stenosis was comparable between the two sites (Figs. 1 and 2). The extent of the nitroglycerin-induced coronary dilation was comparable among the sites treated with PDGF-AA, -BB, and the untreated site (13 ± 8, 15 ± 4, and 17 ± 3%, respectively; n = 5). Intracoronary administration of serotonin and histamine caused coronary hyperconstriction at the site treated with PDGF-AA and -BB, whereas the autacoids caused only mild vasoconstriction at the untreated site (Figs. 1 and 2). In contrast, intracoronary administration of prostaglandin F caused a comparable degree of vasoconstriction at the sites treated with PDGF-AA and -BB and at the untreated site (Figs. 1 and 2).

FIG. 1
FIG. 1:
Coronary angiograms of the left coronary artery after the intracoronary administration of nitroglycerin (NG, 10 μg/kg; left upper), serotonin (S, 10 μg/kg; left lower), histamine (H, 10 μg/kg; right upper), prostaglandin F (PF, 50 μg/kg; right lower). Black and translucent arrows indicate the coronary artery segments treated with PDGF-AA and PDGF-BB, respectively.
FIG. 2
FIG. 2:
Coronary stenosis and coronary vasoconstricting responses to intracoronary administration of serotonin, histamine, and prostaglandin F induced by long-term treatment with PDGF-AA and -BB.

Histologic examination showed that a comparable degree of neointimal formation was induced at the sites treated with PDGF-AA and -BB (Figs. 3 and 4). Compared with vessel area in the adjacent segment, all three vessel areas (luminal, IEL, and EEL areas) were significantly reduced at the sites treated with PDGF-AA and -BB (geometric remodeling; Fig. 4).

FIG. 3
FIG. 3:
Histology of the coronary segment with long-term treatment with PDGF-AA (left) and -BB (middle) and of the untreated coronary segment (right).
FIG. 4
FIG. 4:
Neointimal formation (A) and geometric remodeling (B) in the coronary artery with long-term treatment with PDGF-AA and -BB.

Protocol 2

The development of coronary stenosis induced by PDGF was also significantly suppressed by cotreatment with ST 638 (Figs. 5 and 6). Coronary hyperconstrictive responses to intracoronary serotonin and histamine induced at the PDGF-treated site were also significantly suppressed by the cotreatment with ST 638 (Figs. 5 and 6).

FIG. 5
FIG. 5:
Coronary angiograms of the left coronary artery after the intracoronary administration of nitroglycerin (10 μg/kg; left upper), serotonin (10 μg/kg; left lower), histamine (10 μ/kg; right upper), and prostaglandin F (50 μg/kg; right lower). Black and translucent arrows indicate the coronary artery segments treated with PDGF-AA and PDGF-AA plus ST 638 (12.5 mg), respectively.
FIG. 6
FIG. 6:
Inhibitory effects of ST 638 (12.5 mg) on coronary stenosis and vasoconstricting responses to serotonin, histamine, and prostaglandin F induced by long-term treatment with PDGF-AA.

The neointimal formation induced by the treatment with PDGF was significantly suppressed by the cotreatment with ST 638 (Figs. 7 and 8). The geometric remodeling induced by the long-term treatment with PDGF was abolished by the cotreatment with ST 638 (Figs. 7 and 8).

FIG. 7
FIG. 7:
Histology of the coronary segment chronically treated with PDGF-AA alone and with PDGF-AA plus ST 638 (12.5 mg).
FIG. 8
FIG. 8:
Inhibitory effects of ST 638 (12.5 mg) on the neointimal formation (A) and the geometric remodeling (B) induced by long-term treatment with PDGF-AA.

Antiphosphotyrosine immunoblotting showed that the levels of phosphotyrosines were elevated at the coronary segment treated with PDGF at 75, 130, 150, and 180 kDa, whereas those elevations were markedly suppressed by cotreatment with ST 638 (Fig. 9).

FIG. 9
FIG. 9:
Inhibitory effects of ST 638 (12.5 mg) on the elevated levels of phosphotyrosines 3 days after the treatment with PDGF-AA (antiphosphotyrosine immunoblotting).

DISCUSSION

The novel findings of our study were that (a) long-term treatment with PDGF, either PDGF-AA or -BB, induced neointimal formation, geometric remodeling, and the hyperconstrictive responses to autacoids of the coronary artery in vivo, and (b) ST 638 markedly suppressed those functional and histologic changes of the coronary artery in vivo. These results thus suggest that PDGF plays an important role in coronary arteriosclerosis-like changes and vasospastic responses in vivo and that those responses are substantially mediated by tyrosine kinases in vivo.

Coronary arteriosclerosis and PDGF

Previous studies in vitro suggested that PDGF plays an important role in the pathogenesis of atherosclerosis (1). Three types of PDGF isoforms (PDGF-AA, -AB, and -BB) are known to exist, and these isoforms consist of two types of PDGF chains (PDGF-A and -B) (12). In addition, two types of PDGF-receptor isoforms (α- and β-receptor) are known to exist (12). The α-receptor binds all PDGF isoforms, whereas the β-receptor binds PDGF-BB with high affinty and PDGF-AB with lower affinity, but does not bind PDGF-AA with any appreciable affinity (12). Previous in vitro studies demonstrated that the mitogenic activity of PDGF-BB is more potent than that of PDGF-AA in some cell lines (12,22,23). In this study, however, long-term treatment with PDGF-AA and -BB induced a comparable extent of proliferative lesions and of coronary hyperconstrictions in vivo.

Recent studies demonstrated that a long-term reduction in the total vessel area (geometric remodeling) substantially contributes, in addition to the neointimal formation, to the process of restenosis after angioplasty of the coronary (18,19) and femoral arteries (20). Scott et al. (24) recently demonstrated that after balloon injury of the porcine coronary artery, cell proliferation is first noted at the adventitial site 3 days after the procedure and threreafter shifts to the intima 7 days after the procedure; PDGF is substantially involved in all these processes. They also demonstrated that the migration of adventitial cells activated by PDGF into the intima largely accounts for the subsequent neointimal formation (24). Our study provided the first and direct experimental evidence that adventitial treatment with PDGF causes subsequent neointimal formation and geometric remodeling of the coronary artery in vivo.

Coronary vasospastic responses induced by long-term treatment with PDGF

In our study, long-term treatment with PDGF caused proliferative coronary lesions where hyperconstrictive responses to autacoids were induced. PDGF itself is known to cause vasoconstriction in the rat aorta in vitro (25). Thus PDGF, when released into the coronary circulation after coronary vasospasm, may further exacerbate the vasospasm (26). However, because no significant increase in the coronary artery tone was noted at the PDGF-treated site in our study, PDGF may not directly induce coronary hyperconstriction in this model. Instead, the development of coronary lesions by long-term treatment with PDGF appears to be responsible for the coronary hyperconstriction to the autacoids (serotonin and histamine).

We have previously demonstrated that the intracellular pathway mediated by protein kinase C (PKC) plays an important role in the pathogenesis of coronary spasm at the atherosclerotic lesion induced by a combination of balloon endothelial denudation, x-ray irradiation and high-cholesterol feeding (27). Recently we also demonstrated that the PKC-mediated pathway plays an important role in the pathogenesis of coronary hyperconstrictive responses in our model with IL-1β (28). Thus an important link appears to exist between the proliferative changes (probably induced primarily by PDGF) and the altered PKC-mediated pathway in the pathogenesis of coronary hyperconstriction (28).

Inhibitory effects of a specific tyrosine kinase inhibitor on the development of PDGF-induced coronary lesions

ST 638 inhibits tyrosine-specific protein kinase activity of the EGF receptor with IC50 values of 0.37 μM(15). It has no inhibitory effect on the enzyme activities of serine- or threonine-specific protein kinases (or both) such as cyclic adenosine monophosphate (cAMP)-dependent protein kinase, Ca2+/phospholipid-dependent PKC, casein kinase I and casein kinase II, Na+/K+-ATPase, or 5′-nucleotidase (15). Its inhibitory effect is mediated by competing with the substrate protein of the tyrosine kinase binding site (16). We previously demonstrated that ST 638 markedly suppressed the neointimal formation induced by IL-1β (14) and by balloon injury (29) in vivo. We confirmed in a preliminary study that ST 638 inhibits the PDGF-induced tyrosine phosphorylations of cultured rat smooth-muscle cells in vitro, whereas ST 494 has no such inhibitory effect (unpublished observations). Furthermore, in our study, ST 638 markedly suppressed the PDGF-induced tyrosine phophorylations of the coronary artery in vivo. These results indicate that the inhibitory effects of ST 638 on the development of PDGF-induced coronary proliferative/vasospastic lesions were indeed the result of the inhibition of tyrosine kinases.

ST 638 also abolished the geometric remodeling induced by PDGF. We recently demonstrated that ST 638 also suppresses the geometric remodeling of the porcine coronary artery after balloon injury (28). ST 638 inhibited the increase in the phosphotyrosines at various levels, including at 180 kDa, the mobility of which corresponds to that of phosphorylated receptors for growth factors, including PDGF (12) and EGF (30). These results suggest that activation of tyrosine kinases at the receptor levels for PDGF (14,23) and other growth factors (31) is substantially involved in the pathogenesis of the geometric remodeling and that a specific tyrosine kinase inhibitor may be useful to suppress the pathologic process. However, the detailed molecular mechanisms for the coronary geometric remodeling and the identification of the specific tyrosine kinase responsible for the geometric remodeling remain to be examined.

Inhibitory effects of a specific tyrosine kinase inhibitor on the PDGF-induced coronary vasospastic responses

In our study, the development of coronary vasospastic responses to the autacoids induced by long-term treatment with PDGF also were suppressed by cotreatment with ST 638. It has been reported that tyrosine kinase inhibitor itself attenuates smooth-muscle contraction to several agonists in vitro (32). However, in our previous study, coronary vasomotion was not altered by the long-term treatment with ST 638 alone (14). Thus the inhibitory effects of ST 638 appear to be the result of a suppression of the formation of the inflammatory/proliferative lesions of the coronary artery but not caused by a direct inhibition of coronary vasoconstriction.

Clinical implications

Both neointimal formation and geometric remodeling play important roles in the pathogenesis of restenosis after coronary angioplasty (18-20,23). Neointimal formation after the angioplasty has been shown to be suppressed by treatment with a neutralizing antibody against several growth factors, such as PDGF (9), FGF-2 (33), and insulin-like growth factor-1 (34). Our study also demonstrated that geometric remodeling could be directly induced by growth factors. However, the strategy of using an antibody against each growth factor may have limited effectiveness in vivo because many growth factors/cytokines are sequentially or simultaneously (or both) induced at the inflammatory/proliferative lesions in the cytokine network in vivo (1,14,29). In contrast, tyrosine kinases could be regarded as one of the common and key steps for smooth-muscle proliferation. This study, together with our previous findings (14,29), demonstrated that a specific tyrosine kinase inhibitor can suppress the neointimal formation, geometric remodeling, and hyperconstrictive responses induced by cytokines/growth factors. Thus the inhibition of tyrosine kinases may be one of the potentially important strategies for suppressing coronary atherosclerosis in general and the restenosis after coronary angioplasty in particular in humans.

Acknowledgment: We thank Drs. K. Sueishi, H. Yasutake, and N. Katsumata for their cooperation, and T. Takebe and S. Tomita for their excellent technical assistance.

This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture, Tokyo, Japan, and the Ministry of Health and Welfare, Tokyo, Japan, and grants-in-aid from the Sandoz Foundation for Gerontological Research, Basel, Switzerland, the Japan Research Foundation for Clinical Pharmacology, Tokyo, Japan, Kimura Memorial Heart Foundation, Kurume, Japan, and the Japanese Medical Association, Tokyo, Japan.

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Keywords:

Platelet-derived growth factor; Tyrosine kinases; Restenosis; Vascular remodeling; Coronary vasospasm

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