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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:194-202

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:194-202.)
© 1997 American Heart Association, Inc.

Articles

Inhibition of Collagen Synthesis, Smooth Muscle Cell Proliferation, and Injury-Induced Intimal Hyperplasia by Halofuginone

Arnon Nagler; Hua-Quan Miao; Helena Aingorn; Mark Pines; Olga Genina; Israel Vlodavsky

the Departments of Bone Marrow Transplantation (A.N.) and Oncology (H.-Q.M., H.E., I.V.), Hadassah-Hebrew University Hospital, Jerusalem, and the Institute of Animal Science (M.P., O.G.), Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel.

Correspondence to Dr Israel Vlodavsky, Department of Oncology, Hadassah Hospital, PO Box 12 000, Jerusalem, 91120, Israel.


*    Abstract
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 *Abstract
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 down arrowResults
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Proliferation of vascular smooth muscle cells (SMCs) and accumulation of extracellular matrix (ECM) components within the arterial wall in response to local injury are important etiologic factors in vascular proliferative disorders such as arteriosclerosis and restenosis after angioplasty. Fibrillar and nonfibrillar collagens are major constituents of the ECM that modulate cell shape and proliferative responses and thereby contribute to the pathogenesis of intimal hyperplasia. Halofuginone, an anticoccidial quinoazolinone derivative, inhibits collagen type I gene expression. We investigated the effect of halofuginone on (1) proliferation of bovine aortic endothelial cells and SMCs derived from the same specimen and maintained in vitro, (2) ECM deposition and collagen type I synthesis and gene expression, and (3) injury-induced intimal hyperplasia in vivo. DNA synthesis and proliferation of vascular SMCs in response to serum or basic fibroblast growth factor were abrogated in the presence of as little as 0.1 µg/mL halofuginone; this inhibition was reversible upon removal of the compound. Under the same conditions, halofuginone exerted a relatively small antiproliferative effect on the respective vascular endothelial cells. Halofuginone also inhibited the synthesis and deposition of ECM components by vascular SMCs as indicated both by a substantial reduction in the amount of sulfated proteoglycans and collagen type I synthesis and gene expression. Local administration of halofuginone in the rabbit ear model of crush injury–induced arterial intimal hyperplasia resulted in a 50% reduction in intimal thickening as measured by a morphometric analysis of the neointima/media ratio. The differential inhibitory effect of halofuginone on vascular SMCs versus endothelial cells, its inhibition of ECM deposition and collagen type I synthesis, and its ability to attenuate injury-induced intimal hyperplasia may place halofuginone alone or in combination with other antiproliferative compounds as a potential candidate for prevention of arterial stenosis and accelerated atherosclerosis.


Key Words: vascular smooth muscle cell • intimal proliferation • extracellular matrix • fibroblast growth factor • collagen type I


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowDiscussion
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Proliferation of arterial SMCs in response to endothelial injury is an important etiologic factor in the pathophysiology of restenosis of coronary arteries after percutaneous transluminal coronary angioplasty.1 2 Coronary bypass surgery or angioplasty are performed to reopen coronary arteries that have been narrowed due to intimal hyperplasia and accelerated atherosclerosis. A major drawback of both procedures is that within 3 to 6 months arteries reclog in {approx}40% of patients undergoing angioplasty and {approx}10% of bypass surgery patients.3 4 Vascular SMCs are ordinarily protected by the smooth luminal lining of the arteries composed of vascular ECs. However, SMCs are often left exposed after angioplasty, and in a futile effort to repair the wound, the cells proliferate and clog the artery.1 2

The pathogenesis of restenosis and atherosclerosis involves abnormal migration and proliferation of SMCs infiltrated with macrophages and embedded in ECM.1 2 Under normal physiological conditions, the majority of arterial SMCs remain in the Go phase, and cell growth is controlled by a balance between endogenous growth-promoting factors and proliferation-inhibiting molecules (eg, heparan sulfate proteoglycans).1 2 After EC perturbation due to vascular injury, platelet- and nonplatelet-derived growth factors and cytokines are released and stimulate SMC migration as well as proliferation.1 2 Among these growth factors are platelet-derived growth factor,5 6 bFGF,7 8 interleukin-1,9 and thrombin.10 Blocking the expression and/or activity of each of these growth-promoting factors in experimental models of restenosis has only a limited inhibitory effect.2 5 Intimal hyperplasia is associated with a transition of the vascular SMCs from a contractile to a synthetic phenotype of actively proliferating cells that is similar to the change that occurs when SMCs are adapted to grow in tissue culture.2 11 This phenotypic transformation is associated with an increased production of ECM components such as collagen, which contributes to the regulatory effect of collagen on cell proliferation and differentiation.12 13 14 15 We have shown16 that the ECM upon which cells normally migrate and proliferate may dictate the cell shape and provide SMCs with the necessary growth-promoting signals in the absence of exogenously added growth factors.

We have also shown17 18 that halofuginone, an alkaloid originally isolated from the plant Dichroa febrifuga and commonly used as a coccidiostat in chickens and turkeys, suppresses avian skin collagen synthesis in vivo. Halofuginone attenuates collagen {alpha}1(I) gene expression and collagen production by cultured murine and avian skin fibroblasts17 18 and by human fibroblasts cultured from skin biopsies of patients with chronic graft versus host disease and scleroderma.19 Halofuginone specifically inhibits collagen type {alpha}1(I) but not type II18 or type III20 gene expression. In addition, halofuginone reduces anastomotic intimal hyperplasia20 and abrogates increases in skin collagen and ameliorates dermal manifestations in murine models of chronic graft versus host disease.21

In the present study we evaluated the effect of halofuginone on SMC proliferation and collagen type {alpha}1(I) synthesis by cultured vascular ECs and SMCs. In addition, we applied the rabbit ear model of injury-induced restenosis22 to assess the potential therapeutic applicability of halofuginone in the treatment of intimal hyperplasia and restenosis.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
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Materials
Recombinant human bFGF was kindly provided by Takeda Chemical Industries. DMEM, FCS, CS, penicillin, streptomycin and saline containing 0.05% trypsin, 0.01 mol/L sodium phosphate (pH 7.4), and 0.02% EDTA were obtained from Biological Industries. Tissue-culture dishes were obtained from Falcon Labware Division, Becton Dickinson. Halofuginone was a generous gift of Roussel Uclaf. Rat pro-{alpha}l(I) collagen cDNA was kindly provided by Dr B. Kream, University of Connecticut Health Center. [3H]methylthymidine (5000 mCi/mmol), Na235SO4 (540 to 590 mCi/mmol), and 2,3,4,5[3H]L-proline (122 Ci/mmol) were obtained from New England Nuclear. Protease-free collagenase (type VII), trypsin, Triton X-100, dextran T-40, and all other chemicals were purchased from Sigma.

Cells
SMCs were isolated from bovine aortic media.23 Briefly, the abdominal segment of the aorta was removed, and the fascia was cleaned away under a dissecting microscope. The aorta was cut longitudinally, and small pieces of the media were carefully stripped from the vessel wall. Two or three strips ({approx}2x2 mm) were placed in 60-mm tissue-culture dishes that contained DMEM (4.5 g glucose/L) supplemented with 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Large patches of multilayered cells migrated from the explants within 7 to 14 days. The cells were subcultured into 100-mm tissue-culture plates (4 to 6x105 cells/plate). The cultures obtained exhibited the typical morphological characteristics of vascular SMCs, and the cells were specifically stained with monoclonal antibodies that selectively recognize the muscle form of actin (HHF-35) and do not cross-react with ECs or fibroblasts.24 Clonal populations of BAECs were established and cultured in DMEM (1 g glucose/L) supplemented with 10% CS.25 Cultures of bovine corneal ECs were established from steer eyes.26 Cells were cultured at 37°C in 10% CO2 humidified incubators, and the experiments were performed with early (third through eighth) cell passages.

ECM Deposition by Cultured ECs and SMCs
For preparation of sulfate-labeled ECM, vascular SMCs were seeded into 24-well plates at a confluent density (2.5x105 cells/well) that within 4 to 6 hours formed a growth-arrested cell layer. Under these conditions the cells remained viable and retained their normal monolayer configuration and morphological appearance up to a concentration of 2 µg/mL halofuginone. Na235SO4 (40 µCi/mL) and increasing concentrations of halofuginone were added 1 and 3 days after seeding, and the cultures were incubated without medium change. Nine days after seeding, the ECM was exposed by dissolving the cell layer with phosphate-buffered saline containing 0.5% Triton X-100 and 20 mmol/L NH4OH for 5 minutes at room temperature followed by four washes in phosphate-buffered saline.27 28 29 The ECM was then digested with 12 U/mL collagenase for 18 hours at 37°C followed by 25 µg/mL trypsin for 24 hours at 37°C, and the solubilized material was counted in a ß-counter.

Cell Proliferation
Cell proliferation was evaluated by either measuring [3H]thymidine incorporation or cell counting. For [3H]thymidine incorporation, SMCs were plated (4x104 cells·16-mm well-1·mL-1) in DMEM supplemented with 10% FCS. Twenty-four hours after seeding, halofuginone and [3H]thymidine (1 µCi/well) were added, and DNA synthesis was assayed 48 hours later by measuring the radioactivity incorporated into TCA-insoluble material.30 Alternatively, the medium was first replaced with medium containing 0.2% FCS, and 48 hours later the cells were exposed to growth stimulants and [3H]thymidine (1 µCi/well), with or without increasing concentrations of halofuginone, for an additional 48 hours. DNA synthesis was then assayed. For cell counting, BAECs or SMCs (1.5x104 cells/well) were seeded into 24-well culture plates and exposed to halofuginone as described above. At various times after seeding, the cells were dissociated with trypsin/EDTA and counted in a Coulter counter (Coulter Electronics Ltd).30 31 Each of the cell proliferation experiments described above was performed at least three times, and the variation between different experiments did not exceed ±20%.

Evaluation of Collagen Synthesis
Cells (5x104 cells/16-mm well) were incubated for 24 hours with varying concentrations of halofuginone in 0.5 mL glutamine-free DMEM containing 5% FCS, 50 µg/mL ascorbic acid, 50 µg/mL ß-aminopropionitrile, and 2 µCi [3H]proline. At the end of the incubation, the medium was decanted and incubated with or without collagenase for 18 hours, followed by TCA precipitation. The amount of radiolabeled collagen was estimated as the difference between total [3H]proline-containing proteins and those left after collagenase digestion.32 33

RNA Isolation and Northern Blot Analysis
Total RNA was isolated by using the guanidinium-thiocyanate-phenol technique.34 RNA was subjected to 1% agarose denaturing gel electrophoresis followed by blotting onto a nylon filter (GeneScreen Plus, New England Nuclear). cDNA probes were labeled by using the random primer procedure35 with a commercial kit (Boehringer). Hybridization was performed overnight at 40°C in a solution containing 10% dextran sulfate, 1% sodium dodecyl sulfate, 1 mol/L NaCl, 40% formamide, and 200 mg/mL denatured herring sperm DNA. Hybridization was followed by two 30-minute washes in 2x SSC (1x SSC contains 0.15 mol/L NaCl and 0.015 mol/L sodium citrate) and 1% sodium dodecyl sulfate and two 20-minute washes in 1x SSC and 0.1% sodium dodecyl sulfate. The filters were exposed to x-ray film (Agfa-Curix) at -70°C by using intensifying screens. The amount of RNA loaded per lane was monitored by methylene blue staining of 18S and 28S RNA.

Rabbit Ear Model of Injury-Induced Arterial SMC Proliferation
Adult New Zealand White rabbits, which were housed in accordance with Animal Welfare Act specifications, were anesthetized with ketamine (50 mg/kg IM). Physical injury was applied for 30 minutes externally to the central artery of each ear.22 Halofuginone (0.2 mL of 0.1 mg/mL SC) was administered around the physical crush area 1 hour after the crush and once every 24 hours during the first 4 days. The rabbits were killed on day 14, and the ears were fixed in 4% buffered formaldehyde for 72 hours. The crush sites were further trimmed at 1-mm intervals, dehydrated in ethanol and xylene, and embedded in paraffin. Each site, {approx}1 cm in length, was cut into 10 cross-sectional segments that were embedded together in paraffin. Five-micrometer serial sections were removed from the top of the block and at two additional points 100 and 200 µm from the first section.22 36 Samples were stained with hematoxylin-eosin and by using the Movat pentachrome method.37

Histomorphometry and Statistical Analysis
Histopathologic analysis was performed by observers blinded to the experimental groups. Sections were video-digitized and analyzed by using a Power Macintosh 7200/90 computer in association with a ZEISS Axioskop and a Scion Image 1.60b7 program (National Institutes of Health). The lumen area (circumscribed by the lining of the apical surface of ECs), the area circumscribed by the IEL, and the area circumscribed by the external elastic lamina were traced. The ratio between neointimal area (area bounded by IEL-luminal area) and medial area (area bounded by external elastic lamina-area bounded by IEL) was calculated. The percent cross-sectional area narrowed by neointima was evaluated as neointimal areax100/area bounded by IEL. All sections ({approx}10 per ear) with neointimal formation were subjected to planimetry. Data are presented as mean±SD. The histological differences were analyzed by using a two-sample (two-tailed) Student's t test for differences in means. A value of P<=.05 was significant. The *StatWorks statistical package (Cricket Software, Inc) was used to perform these calculations.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Inhibition of Vascular SMC Proliferation by Halofuginone
Migration and proliferation of vascular SMCs play an important role in the pathogenesis of restenosis after angioplasty. We investigated the ability of halofuginone to inhibit the proliferation of actively growing bovine aortic SMCs. Subconfluent vascular SMCs maintained in medium containing 10% FCS were exposed for 48 hours at 37°C to [3H]thymidine in the absence and presence of increasing concentrations of halofuginone (Fig 1ADown). Complete inhibition of [3H]thymidine incorporation was observed at 150 ng/mL halofuginone, while 65% inhibition was obtained at a concentration as low as 50 ng/mL. In a subsequent experiment inhibition of cell proliferation in response to halofuginone was evaluated by actual measurements of the number of SMCs maintained under optimal growth conditions. Sparsely seeded vascular SMCs were exposed to 10% FCS in the absence and presence of increasing concentrations of halofuginone added 24 hours after seeding the cells (Fig 1BDown). Halofuginone at 50 ng/mL caused 50% inhibition of SMC proliferation (not shown), while an almost complete inhibition was observed with 125 ng/mL halofuginone.



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  		Figure 1. Line graphs. A, Bovine aortic SMCs (4x104 cells/well) were seeded into 24-well plates in medium containing 10% FCS. Halofuginone (25 to 150 ng/mL) and [3H]thymidine (1 µCi/well) were added 24 hours after seeding. DNA synthesis was assayed 48 hours later by measuring the amount of radioactivity incorporated into TCA-insoluble material. B, SMCs were seeded in complete medium into 24-well plates (1.5x104 cells/well), and after 18 hours the cells were exposed to halofuginone at 75 ({bullet}), 100 ({blacksquare}), or 125 ({blacktriangleup}) ng/mL. Control cells ({square}) were maintained in the absence of halofuginone. At intervals after seeding, the cells were dissociated and counted in a Coulter counter. Each point represents mean±SD from triplicate wells. All experiments were performed at least three times; variations between different experiments did not exceed ±20%.

To evaluate the time needed for the cells to recover after drug removal, SMCs were exposed to halofuginone for 72 hours followed by replacement of the medium with halofuginone-free medium (Fig 2Down). Removal of the drug resulted in an accelerated growth rate that approached that of the untreated SMCs. This result indicates that the antiproliferative effect of halofuginone is reversible and is not due to a toxic effect, which was also indicated by the lack of lactic dehydrogenase release above the basal level detected in untreated SMC cultures (data not shown).



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  		Figure 2. Line graph. SMCs were seeded into 24-well plates (1.5x104 cells/well) and exposed to halofuginone at 75 ({bullet}), 100 ({blacksquare}), or 125 ({blacktriangleup}) ng/mL. Control cells ({square}) were maintained in the absence of halofuginone. The cultures were washed 72 hours after seeding (arrow), and fresh medium was added. At intervals after seeding, the cells were dissociated and counted in a Coulter counter. Each point represents mean±SD from triplicate wells.

bFGF is involved in intimal hyperplasia in response to vascular injury and is a potent stimulant of SMC proliferation.3 4 5 6 7 8 9 10 SMCs were seeded at a subconfluent density (4x104 cells/16-mm well), arrested at the Go/G1 phase by serum deprivation (0.2% FCS for 48 hours), and incubated for 48 hours at 37°C with varying concentrations of halofuginone in the presence of 1 ng/mL recombinant bFGF (Fig 3Down). bFGF caused a four- to fivefold increase in [3H]thymidine incorporation into DNA. Exposure of SMCs to halofuginone 200 ng/mL resulted in an almost complete inhibition of bFGF-stimulated thymidine incorporation in growth-arrested SMCs. Similar results were obtained with cells that were stimulated with 10% FCS, 1 ng/mL platelet-derived growth factor, or 1 ng/mL bFGF plus 5x10-6 mol/L thrombin (not shown). Halofuginone had no effect on the cell counts and basal level of [3H]thymidine incorporation by growth-arrested SMCs maintained in DMEM containing 0.2% FCS (not shown).



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  		Figure 3. Line graph. SMCs were seeded into 24-well plates (4x104 cells/well) in medium containing 10% FCS. Twenty-four hours after seeding the cells were arrested by 48 hours' incubation in medium containing 0.2% FCS. SMCs were then incubated with 1 ng/mL bFGF and increasing concentrations of halofuginone for an additional 48 hours in the presence of [3H]thymidine. Thymidine incorporation into TCA-insoluble material was determined as described in "Methods." The background level of thymidine incorporation by growth-arrested SMCs ranged from 12 600 to 16 400 cpm in different plates. Each point represents mean±SD from triplicate wells.

Effect of Halofuginone on Vascular EC Proliferation
Because an intact EC layer is held responsible for the integrity of the blood vessel wall, contributing to the quiescent state of the underlying SMCs, we assessed the effect of halofuginone on BAEC proliferation. Sparsely seeded BAECs were cultured in medium containing 10% CS in the absence and presence of increasing concentrations of halofuginone. The cells were dissociated and counted daily. Inhibition of EC proliferation was observed primarily during the first 4 days of treatment with 100 to 125 ng/mL halofuginone (Fig 4Down). Unlike SMCs, starting on day 5 the ECs regained an almost normal growth rate, indicating that vascular ECs are less susceptible to the inhibitory effect of halofuginone than vascular SMCs.



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  		Figure 4. Line graph. BAECs were seeded into 24-well plates (1.5x104 cells/well) in medium containing 10% CS . Twenty-four hours after seeding, the cells were exposed to halofuginone at 75 ({bullet}), 100 ({blacksquare}), and 125 ({blacktriangleup}) ng/mL, and at later intervals the cells were dissociated from the plastic and counted with a Coulter counter. Control cells ({square}) were maintained in the absence of halofuginone. Each point represents mean±SD from triplicate wells.

Effect of Halofuginone on Deposition of ECM by Cultured SMCs
The ECM promotes the proliferation of vascular SMCs, an activity that is attributed to both macromolecular constituents of the ECM and to heparin-binding growth factors (eg, bFGF) that are associated with heparan sulfate proteoglycans in the ECM.16 38 39 40 To assess the effect of halofuginone on ECM deposition, labeled sulfate (Na235SO4) or amino acids (ie, [3H]proline, [14C]glycine, or [14C]lysine) and increasing concentrations of halofuginone were added to confluent, resting vascular SMCs 24 hours after seeding. Eight days later, the cell layer was dissolved to expose the underlying ECM, which was then digested with collagenase to evaluate the effect of halofuginone on the amount of proteins released from the ECM and/or digested by collagenase. The remaining material was trypsinized, and the solubilized radioactivity was counted in a ß-scintillation counter. Deposition of ECM was inhibited by 60% to 70% in the presence of 50 ng/mL halofuginone regardless of whether the amounts of labeled sulfate (Fig 5Down) or amino acids were determined. A profound inhibition of ECM deposition was also observed with confluent, contact-inhibited vascular and corneal ECs (not shown). This inhibition was also shown by a microscopic examination of the denuded culture dishes, which revealed a very thin or no ECM produced in the presence of halofuginone.



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  		Figure 5. Bar graph. Bovine SMCs were seeded at a confluent cell density (2.5x105 cells/well) into a 24-well plate. One and 3 days after seeding, the cells were exposed to halofuginone (0 to 200 ng/mL) and Na235SO4 (40 µCi/well). Control cells were maintained in the absence of halofuginone. On day 9, the cell layer was solubilized to expose the underlying ECM, which was washed three times and subjected to sequential digestions with collagenase (hatched bar) and trypsin (solid bar). The released radioactivity was counted in a ß-scintillation counter as described in "Methods." Results are mean±SD of triplicate wells.

Effect of Halofuginone on Collagen Synthesis
To evaluate the effect of halofuginone on collagen synthesis, SMCs and BAECs were incubated with [3H]proline and halofuginone (10-7 mol/L, 41.5 ng/mL) for 24 hours. At the end of the incubation, collagen type {alpha}1(I) gene expression (Fig 6ADown) and incorporation of radiolabeled proline into collagenase-digestible proteins (Fig 6BDown) were determined. Lower levels of collagen type {alpha}1(I) gene expression were observed in BAECs than SMCs. Halofuginone inhibited collagen type {alpha}1(I) gene expression in SMCs without affecting the expression of the gene in BAECs (Fig 6ADown). The effect of halofuginone on collagen type {alpha}1(I) gene expression resulted in an inhibition of incorporation of radiolabeled proline to collagenase-digestible proteins by SMCs but not by BAECs (Fig 6BDown).



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  		Figure 6. A, Autoradiography. Vascular SMCs and ECs were seeded at a subconfluent density (5x104 cells/well) and treated for 24 hours at 37°C with halofuginone 10-7 mol/L (41.5 ng/mL). Total RNA was then extracted, subjected to 1% agarose electrophoresis, blotted onto Nytran membrane, and hybridized with a labeled cDNA probe for collagen type {alpha}1(I). The amount of RNA loaded per lane was monitored by methylene blue staining of 18S and 28S RNA. B, Bar graph. Vascular SMCs and ECs were seeded at a subconfluent density and treated for 24 hours at 37°C with (+) or without (-) halofuginone 10-7 mol/L in glutamine-free DMEM in the presence of [3H]proline. Incorporation of proline to collagenase-digestible proteins (CDP) was determined as described in "Methods." Results are mean±SD of triplicate wells.

Effect of Halofuginone on Injury-Induced Arterial Stenosis
In view of the differential effects of halofuginone on collagen type {alpha}1(I) gene expression and proliferation of vascular SMCs and BAECs in vitro, we investigated its effect in an in vivo model of injury-induced intimal hyperplasia. We employed the rabbit ear model, in which a single external crush is applied under controlled conditions to the central artery of the rabbit ear, causing a marked SMC proliferation with neointimal formation and significant luminal encroachment in virtually 100% of the treated arteries.22 Halofuginone (20 µg/0.2 mL SC) or saline (0.2 mL SC) was administered around the crush area 1 hour after the arterial crush and once every 24 hours during the first 4 days. On day 14 the animals were killed, and the ears were fixed, subjected to serial sectioning, and processed for histological evaluation.36 A marked neointimal formation was evident in all the untreated controls, and the crush injury to the central ear artery resulted in an extensive SMC proliferation, formation of neointima, and a significant decrease in the luminal area (Fig 7A and 7BDownDown). SMCs migrated from the media into the neointima through the disrupted IEL (Fig 7BDown). Local administration of halofuginone around the crush area resulted in a profound reduction of intimal hyperplasia (Fig 7C and 7DDownDown). Quantification of the results by image analysis showed that halofuginone reduced both the percent cross-sectional stenosis (30.75±11.19% versus 62.42±17.09%, P<.001) (Fig 8ADown) and the neointima/media ratio (0.24±0.18 versus 0.40±0.15, P=.001) (Fig 8BDown) in the halofuginone-treated animals (n=10) compared with untreated control rabbits (n=6), respectively.






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  		Figure 7. Light photomicrographs. Rabbit ear central arteries were subjected to a crush injury, and 20 µg halofuginone in 0.2 mL saline or 0.2 mL saline alone was injected locally 1 hour later and once every 24 hours for 4 consecutive days. On day 14 the rabbits were killed, and the ear tissue was processed for histology. A, Untreated control shows striking neointimal formation resulting in substantial obliteration of the arterial lumen (original magnification x100). B, Higher magnification (x200) of A showing SMCs migrating from the media toward the neointima, crossing the disrupted IEL. Disruption of the IEL was seen in only a small number of sections, both in untreated and halofuginone-treated animals. C and D, Halofuginone-treated arteries in which neointimal formation is significantly inhibited (original magnification x100).



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  		Figure 8. Bar graphs show effect of halofuginone on (A) percent cross-sectional area narrowing and (B) neointima/media ratio of rabbit ear central arteries. Rabbit ears were subjected to crush injury and treated with saline alone (Control, n=6) or halofuginone (Treated, n=10) as described in the legend to Fig 7Up. Digital planimetry of multiple tissue sections was performed as described in "Methods." Data are mean±SD. P<.001 for A and B.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
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We have demonstrated21 that halofuginone efficiently inhibits the fibrosis associated with chronic graft versus host disease and scleroderma. This inhibition is attributed to a specific inhibition of collagen type {alpha}1(I) synthesis and gene expression.17 18 This type of collagen is a major constituent of the ECM upon which cells migrate, proliferate, and differentiate in vivo. Type I collagen promotes the transition of vascular SMCs from a contractile to a synthetic phenotype that characterizes intimal hyperplasia, restenosis, and accelerated atherosclerosis.11 Significant increases in collagen synthesis and tissue content coincide with the increases in intimal area and SMC proliferation observed in restenotic lesions of rabbits and humans after balloon angioplasty.13 14 15 41 Likewise, inhibition of collagen synthesis and assembly inhibit SMC proliferation and response to various mitogenic factors.42 43 Halofuginone at relatively low concentrations (50 to 150 ng/mL) almost completely inhibited SMC proliferation but had only a limited antiproliferative effect on the respective vascular ECs. Halofuginone effectively inhibited collagen type {alpha}1(I) synthesis and gene expression by vascular SMCs but not by vascular ECs, which unlike the SMCs exhibited low levels of collagen type I synthesis. This differential effect is beneficial since the integrity of the luminal EC lining is of prime importance in preventing the SMC migration and proliferation associated with restenosis and atherosclerosis.11 44 45 The antiproliferative effect of halofuginone was not due to a toxic effect, as indicated by the lack of LDH release above the basal level detected in untreated SMCs. Likewise, halofuginone did not affect the viability of the vascular ECs and SMCs, and its effect was reversible, indicating that its inhibition of cell proliferation was not due to cell toxicity. It is therefore conceivable that exposure to halofuginone will not interfere with the subsequent EC relining of the perturbed vessel, allowing the underlying SMCs to regain their normal differentiated nonproliferative properties.

Accumulation of ECM components contributes to the pathogenesis of restenosis and atherosclerosis13 14 15 via stimulation of SMC proliferation42 45 and migration as well as activation of platelets and thrombus formation.1 2 46 It is now well recognized that the ECM is not simply an inert scaffolding that stabilizes the physical structure of tissues, but rather that it plays an important role in actively modulating the proliferative responses of the cells that contact it.38 40 47 The ability of halofuginone to inhibit the deposition of ECM by vascular SMCs, in addition to its inhibition of both smooth muscle and fibroblast cell proliferation,17 18 19 20 21 may potentiate its efficacy in reducing the progression of restenosis and accelerated atherosclerosis. Studies on the effect of halofuginone on the amounts of Na235SO4 and labeled amino acids (ie, [3H]proline, [14C]glycine, or [14C]lysine) in the ECM revealed a profound inhibition of the synthesis and/or assembly of ECM constituents. Our results suggest that the effect of halofuginone on ECM deposition can be attributed both to its known inhibitory effect on collagen synthesis17 18 19 20 21 and to an effect on the deposition and/or assembly of other macromolecular constituents into intact ECM. A possible effect on the synthesis of noncollagenous components is being investigated. It is conceivable that by inhibiting collagen type I synthesis, halofuginone interferes with the assembly of other ECM macromolecules (ie, heparan sulfate proteoglycans) that are known to specifically interact with collagen (ie, through its heparin binding domain)48 and form the supramolecular structure comprising the ECM. This hypothesis is supported by the profound reduction in the amount of sulfate-labeled material, since inorganic sulfate is incorporated primarily into sulfated glycosaminoglycans and not collagen. It should be noted that the inhibition of ECM deposition by halofuginone was not due to its antiproliferative activity, since the drug was effective when added to highly confluent SMCs. Likewise, halofuginone inhibited deposition of ECM by contact-inhibited nondividing vascular and corneal ECs (not shown). Although the antiproliferative activity of halofuginone is attributed to its inhibition of collagen type {alpha}l(I) gene expression, the exact mechanism of action (ie, binding to cell surface receptors, internalization, interaction with intracellular mediators) has not been elucidated.

Based on the in vitro data described above, we evaluated the potential use of halofuginone in the inhibition of intimal hyperplasia in the rabbit ear central artery model of external injury–induced stenosis.22 The vascular lesions produced in this manner are indistinguishable from those formed after experimental endovascular injury.22 Halofuginone effectively inhibited intimal hyperplasia in this model. An attempt was made to investigate whether this inhibition was associated with a reduction in collagen type {alpha}1(I) gene expression. In situ hybridization analysis performed on a few representative histological sections of the rabbit ear central artery revealed a significant decrease in collagen type I gene expression in arterial sections derived from halofuginone-treated rabbits compared with those from untreated animals. This reduction was detected in both the neointima and adventitia, but the results were somewhat weakened due to variation between sections. Likewise, an effect of halofuginone on the ECM content and cell density at the site of injury could not be readily detected and quantified. Our animal studies are in accordance with those of Choi et al,20 who have shown that orally administered halofuginone inhibited anastomotic intimal thickening in the common carotid artery of rabbits. It appears that the effect of halofuginone is not species specific, since the drug inhibits collagen type I synthesis in chickens, mice, rats, rabbits, and human cells.17 18 19 20 21 49 50 Taking into account our in vitro and in vivo results, it appears that halofuginone effectively interferes with multiple components of the restenotic process (ie, SMC and fibroblast proliferation, collagen type I synthesis, and ECM deposition) while only minimally inhibiting the EC proliferation required for restoration of a normal vascular EC lining. This, together with the observation that halofuginone is not toxic and can be administered both locally and orally, makes halofuginone a potentially useful compound for preventing intimal hyperplasia after vascular injury. Caution must be exercised, however, when extrapolating results obtained in animal models to the human clinical setting. Therapeutic approaches for prevention of restenosis have used antibodies against growth factors,5 51 growth factor receptor antagonists,52 inhibitors of signal transduction,36 antiproliferative species of heparin and heparan sulfate,53 54 antisense oligonucleotides,36 55 and antithrombotic agents,46 56 but little attention has been paid to the role of ECM and in particular to collagen type {alpha}1(I). Since intimal hyperplasia after angioplasty is multifactorial, one would expect that a combination of several therapeutic modalities would yield an optimal result. Our studies indicate that halofuginone should be included in such a combined approach.


*    Selected Abbreviations and Acronyms
 
BAEC = bovine aortic endothelial cell
bFGF = basic fibroblast growth factor
CS = calf serum
DMEM = Dulbecco's modified Eagle's medium
EC = endothelial cell
ECM = extracellular matrix
FCS = fetal calf serum
IEL = internal elastic lamina
SMC = smooth muscle cell
TCA = trichloroacetic acid


*    Acknowledgments
 
This work was supported by grants from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities and by Collgard/IPC, Medica, Israel.


*    Footnotes
 
The first two authors contributed equally to this work.

Received January 17, 1996; revision received September 10, 1996;
*    References
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