1178
Biomacromolecules 2001, 2, 1178-1183
Terminally Alkylated Heparin. 2. Potent Antiproliferative Agent for Vascular Smooth Muscle Cells Masahiro Gohda,†,‡ Tomoko Magoshi,† Shinya Kato,† Teruo Noguchi,‡ Satoshi Yasuda,‡ Hiroshi Nonogi,‡ and Takehisa Matsuda*,† Department of Bioengineering, Research Institute, and Division of Cardiology, Hospital, National Cardiovascular Center, 5-7-1, Fujishirodai, Suita, Osaka 565-8565, Japan Received June 5, 2001; Revised Manuscript Received September 10, 2001
The antiproliferative activity of alkylated heparin, in which the terminal end of heparin is derivatized with an alkyl group (butyl, octyl, lauryl, stearyl), was examined using vascular smooth muscle cells. The proliferation of cells, which were growth-arrested prior to addition of heparin, was inhibited in proportion to both increase in the chain length of the alkyl group of alkylated heparin and alkylated heparin concentration in the serum-containing medium. The antiproliferative activity of stearyl group derivatized heparin was significantly stronger than that of nonmodified heparin. Little proliferation was observed at high dose (500 µg/mL). Confocal laser scanning microscopic observation indicated that alkylated heparin was accumulated on the cell membranes at an early incubation time, followed by homogeneous distribution of intracellular space. The therapeutic potential of alkylated heparin for preventing restenosis after balloon angioplasty is discussed. Introduction Proliferation of vascular smooth muscle cells (SMCs) plays an important role in the pathogenesis of atherosclerosis and is an important contributor to the formation of the fibrocellular lesions often observed following surgical intervention such as percutaneous transluminal coronary angioplasty or bypass grafting. The main therapeutic strategies which have been extensively studied for preventing intimal hyperplasia include (1) minimal thrombus formation at injured or denuded endothelium and (2) growth arrest of SMCs. Many papers reported that the highly sulfated glycosaminoglycan, heparin, which is a potent anticoagulant, also acts as a potent modulator of SMC growth. In fact, heparin inhibits the migration and proliferation of cells in response to vascular damage in vivo1-4 and, further, promotes the induction of a phenotype reversion in cultured SMCs5,6 from synthetic to contractile type. Recently, the coronary angiogenic effect of heparin was examined.7 Since heparin is a highly negatively charged polyelectrolyte, stable and strong adsorption on negatively charged vessel wall as well as tissue transport is not expected and wash-out due to hydrodynamic shear stress of circulating blood may diminish these effects. In this paper, to increase local concentration and the residence time of heparin at lipid-rich atherosclerotic sites and to enhance the tissue permeability and transport and for effective interaction with SMCs in tissue, heparin was hydrophobically modified with minimal loss of its biological activity. The alkylated heparins designed for the study had * To whom correspondence may be addressed. Present address: Department of Biomedical Engineering, Graduate School of Medicine, Kyushu University, 3-1-1, Maidashi, Higashiku, Fukuoka 812-8562, Japan. E-mail:
[email protected]. † Department of Bioengineering, Research Institute, National Cardiovascular Center. ‡ Division of Cardiology, Hospital, National Cardiovascular Center.
Figure 1. The schematics of alkylated heparin.
alkyl groups of different chain lengths attached to the terminal end of the heparin molecule (-(CH2)nCH3, n ) 3, 7, 11, and 17, designated as heparin-C4, -C8, -C12, and -C18, respectively). The schematics of alkylated heparin is shown in Figure 1. A series of alkylated heparins was prepared to modify the artificial substrate of an assembled extracorporeal circulatory device in which alkylated heparin acts as a “surfactant”.8 In this paper, the effect of alkylated heparin on proliferation of SMCs in vitro was reported, and its potential for therapeutic use is discussed using an atheroscleolytic model of injured coronaries. Materials and Methods Heparin and Alkylated Heparin. Heparin (sodium salt derived from porcine mucosa with a mean molecular weight of 16 000, Wako Pure Chemical Industries, Osaka, Japan) was used with or without modification by attachment of an alkyl group, -(CH2)nCH3 (n ) 3, 7, 11, and 17), at its terminal end. The detailed preparation method is described in the preceeding paper in this issue.8 Briefly, heparin was treated with a reducing agent to generate a lactone ring at its terminal end and subsequently reacted with the corresponding alkylamine to synthesize alkylated heparin.8 Fluorescence-labeled heparin and alkylated heparin were prepared by condensation reaction with 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF, Sigma Chemical Co., St. Louis,
10.1021/bm010097x CCC: $20.00 © 2001 American Chemical Society Published on Web 11/09/2001
Terminally Alkylated Heparin
Biomacromolecules, Vol. 2, No. 4, 2001 1179
Figure 2. Effect of alkyl chain length of alkylated heparins on proliferation of SMCs. After culture for 3 days in FBS-free medium, the cells were cultured in FBS-containing medium with or without heparin or alkylated heparin at a concentration of 500 µg/mL for 5 days. The cell density (OD at 450 nm) was determined with a Cell Counting Kit-8. Day 0 values show basal cell density measured just before heparin or alkylated heparin was added, and control values show final cell density at day 5 serving as controls for normal cell growth. Data are shown as mean ( standard deviation. Key: †, n ) 6 per group; ††, n ) 12 per group; /, P < 0.01 vs day 0; #, P < 0.01 vs control.
MO) according to our method previously reported.9 Irrespective of alkylation, the number of DTAF incorporated was around 2.7 per molecule. Cell Culture. SMCs, harvested from the media of rabbit aorta by a collagenase and elastase method as described previously,10,11 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Laboratories Inc., Grand Island, NY) supplemented with 15% fetal bovine serum (FBS, Gibco Lab. Inc.), 50 IU/mL penicillin, 50 µg/mL streptomycin (Flow Lab., Irvine, Scotland), and 2.5 µg/mL amphotericin B (ICN Biomedicals Inc., Aurora, OH). The SMCs were indentified by their orphological characteristics, namely, the hills-muscle-actin-specific monoclonal antibody (M851, Dako Corp., Glostrup, Denmark). Cell culture was performed at 37 °C in a humidified atmosphere of 5% CO2 in an incubator. SMCs at passage 2 were used for all the experiments. Cell Growth Assay. Cells were plated sparsely (5 × 103 cells/well) in 24-well cluster plates (Sumitomo Bakelite Co., Tokyo, Japan) in DMEM without FBS. After incubation for 3 days and subsequent washing with phosphate buffer solution (PBS, Nissui Pharmaceutical Co., Tokyo, Japan), the medium was replaced with the culture medium (DMEM + 15% FBS) with or without heparin or alkylated heparin. Cell Counting Kit-8 (Dojindo Lab., Kumamoto, Japan) which employs a sensitive colorimetric assay method for determining the number of viable cells by employing a novel tetrazolium salt, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt),12 was used for cell counting. The absorbance due to water-soluble formazan dye produced upon bioreduction in the presence of an electron carrier, 1-methoxy5-methylphenazinium methosulfate, was measured. The amount of formazan produced, determined by the optical density (OD, in this paper, expressed as cell density) at 450 nm, is directly proportional to the number of living cells.12
The degree of antiproliferative activity of heparin or alkylated heparin was determined from the following relationship: % inhibition ) {1 - (net increase of cell density in the presence of heparin or alkylated heparin)/(net increase of cell density in the absence of one)} × 100. Fluorescent Staining in Vitro and in Vivo. Cells were cultured in DMEM with FBS for the first 12 h, and then the medium was replaced with DMEM supplemented with FBS and 500 µg/mL heparin-C18. After 1 or 24 h, in culture, the cells were washed three times with PBS, fixed in 10% formalin neutral buffer solution (Wako Pure Chemical Industries) for 10 min at room temperature, and subsequently washed again three times with PBS. The cells were imaged by confocal laser scanning microscopy (488 nm excitation; Bio-Rad Lab., Hercules, CA), and then, the fluorescent intensity of individual cells was measured by NIH image (National Institutes of Health, USA). As for in vivo study, monogreal dogs were used. Detailed procedures of endothelial denudation of illiac arteries and balloon inflation and drug infusion are described in a separate paper in detail.9 The concept, fabrication, and performance of the newly designed device was also described in detail.13 The sliced tissue was observed under a fluorescent microscope (Nikon, Tokyo, Japan). Statistical Analysis. All values are expressed as mean ( standard deviation. Analysis of variance with the StudentNewman-Keuls test was employed to determine significance of differences in multiple comparisons. Values of P < 0.05 were considered statistically significant. Results Antiproliferation on SMCs. Sparsely plated SMCs were cultured in serum-free DMEM for 3 days, and the culture was continued in the serum-containing medium supplemented with or without heparin or alkylated heparin. Figure 2 shows
1180
Biomacromolecules, Vol. 2, No. 4, 2001
Gohda et al.
Figure 3. Dose-dependent antiproliferative effect of heparin-C18 on SMCs. Culture conditions were the same as those for Figure 2. The cell density (OD at 450 nm) was determined with a Cell Counting Kit-8. Day 0 values show basal cell density measured just before heparin or heparin-C18 was added and control values show final cell density at day 5 serving as controls for normal cell growth. Data are shown as mean ( standard deviation, n ) 6 for each group. Key: /, P < 0.01 vs day 0; #, P < 0.01 vs control.
the cell density at 5 days of culture after replacement with culture medium containing 500 µg/mL heparin or alkylated heparin. Upon culture in the serum-containing medium, the cell density almost doubled at 5 day of culture. Addition of heparin reduced the cell density significantly as compared with that without heparin. In the case of alkylated heparin, with increased chain length of the alkyl group, the cell density decreased. The percent inhibition was almost 70% for heparin, 17% for heparin-C4, 56% for heparin-C8, 76% for heparin-C12, and 120% for heparin-C18. The cell density in the presence of heparin-C18 on day 5, which was almost 75% of that on day 0, was significantly low as compared with that of heparin. Figure 3 shows the dose-dependent inhibitory effect of heparin-C18 on SMC growth. The cells were starved in serum-free medium for the first 3 days and then subjected to culture in serum-containing culture medium for 5 days at different doses of heparin-C18. At low concentration (100 µg/mL), little antiproliferative effect was observed, whereas above 300 µg/mL, a strong inhibitory effect was observed. That is, the percent inhibition was almost zero for 100 µg/ mL, 19% for 200 µg/mL, 79% for 300 µg/mL, 102% for 400 µg/mL, and 105% for 500 µg/mL heparin-C18. Figure 4 shows cell growth profiles at a concentration of 500 µg/mL heparin or heparin-C18. Heparin induced moderately cell growth at 3 and 5 days, whereas heparinC18 did not induce cell proliferation during the culture period and, on the contrary, induced cell loss to some extent. On day 5, the cell density in the presence of heparin-C18 was almost 87% of that on day 0. Cellular Uptake. Confocal laer scanning microscope (CLSM) microscopy revealed that morphologically cells
Figure 4. SMC growth profiles in the presence or absence of heparin or heparin-C18. After culture for 3 days in FBS-free medium, the cells were cultured in FBS-containing medium with or without heparin or heparin-C18 for 1, 3, and 5 days. The cell density (OD at 450 nm) was determined with a Cell Counting Kit-8 (white, control; diagonally hatched, 500 µg/ mL heparin; black, 500 µg/mL heparinC18). Data are shown as mean ( standard deviation, n ) 6 per group. Key: /, P < 0.05; //, P < 0.01 vs day 0.
treated in the absense of heparin were elongated shapes on day 5, whereas cells cultured in the presence of heparin or heparin-C18 (500 µg/mL) appeared to be less spread and tended to be round on day 5. When fluorescence-labeled (DTAF-conjugated) heparins, regardless of alkylation, were added into the culture medium, cells were fluorescencestained with DTAF (Figure 5). The fluorescence intensity of cells cultured in the presence of heparin-C18 derivatized with DTAF irrespective of alkylation and concentration was
Terminally Alkylated Heparin
Biomacromolecules, Vol. 2, No. 4, 2001 1181
Figure 5. Laser scanning confocal micrographs of cultured SMCs treated with fluorescence-labeled heparin or heparin-C18 after 1 or 24 h. The images were reconstructed from top view: A, 500 µg/mL heparin derivatized with DTAF; B, 100 µg/mL heparin-C18 derivatized with DTAF; C, 500 µg/mL heparin-C18 derivatized with DTAF. Original magnification 600×.
higher at 24 h than that at 1 h after incubation, the average fluorescent intensity of individual cell (n ) 10 for each group) was 72.4 ( 5.8 for cells treated with heparin derivatized with DTAF (500 µg/mL), 151.2 ( 17.6 for ones with heparin-C18 derivatized with DTAF (100 µg/mL), and 190.9 ( 16.4 for ones with heparin-C18 derivatized with DTAF (500 µg/mL), respectively. In addition, of interest is the fact that the fluorescent intensity was very strong at the peripheral region of cell membranes at an early incubation time, which can be easily detected by cross-sectional vertical sections. Gradually the intracellular fluorescent intensity became stronger with time, but at 24 h, the entire intracellular space appeared to be homogeneously stained (Figure 5, a detailed study will be reported in a forthcoming paper). Residency in Tissue. After the intraluminal surfaces of iliac arteries of a canine were mechanically injured to remove the endothelium and break the elastic laminae, fluorescently stained alkylated or nonalkylated heparin was infused for 20 min through the specially designed catheter after balloon inflation. Irrespective of nonalkylated or alkylated (HepC18) heparin, high fluorescence intensity was observed at the outer layer of inimal tissues 6 h after infusion. Twentyfour hours later, the fluorescent intensity of a cross section of sliced vessel segment up to 100 µm from the luminal surface of vessel was determined (Figure 6), and the intensity was measured by a NIH image analyzer. Alkylated heparin resided mainly on and even at the deeper tissue region of the injured arteries at much higher concentration and broader regions than nonalkylated heparin, which was slightly recognized on the outermost layer of the luminal region. The overall NIH image up to 100 µm of depth from the luminal
surface indicated that an almost 15 times higher intensity was noticed for alkylated heparin (Figure 7). In the macroscopic appearance of luminal surfaces where vessel segments were removed 24 h after infusion, a marked difference in morphological change between alkylated and nonalkylated and heparin was observed. At 48 h, a very faint fluorescence intensity was observed for nonalkylated heparin, whereas still a high intensity at the luminal surface of artery was noticed for the Hep-C18 infused artery. For nonalkylated heparin, massive thrombus formation with a fibrin-platelet complex was macroscopically observed, whereas for alkylated heparin, minimal platelet adhesion without fibrin formation was observed (Figure 8). Some platelets adhered on exposed cells, probably SMCs, at denudated surfaces. Discussion The development of catheters suitable for drug infusion, while a balloon was inflated and partial blood perfusion was allowed, has made it possible to infuse drugs which minimize thrombus formation and induce rapid endothelialization and rapid phenotypic alteration (redifferentiation) of SMCs at the site of balloon inflation. We devised such a triple functional catheter system for the first time.9 We anticipate that heparin may be the best-suited drug for preventing intimal hyperplasia after balloon angioplasty since heparin functions as an antiproliferative for SMCs as well as a potent anticoagulant agent. Although the antiproliferative activity of heparin on SMCs has been acknowledged over the last 2 decades, the mechanism of action of the drug is not well understood. For example, in the late 1970s, Clowes et al.1 showed that heparin markedly inhibited SMC proliferation in vivo following endothelial denudation of the rat carotid artery. Since
1182
Biomacromolecules, Vol. 2, No. 4, 2001
Gohda et al.
Figure 6. Cross-sectional fluorescent images of a luminal surface 24 h after a 20 min infusion of fluorescent dye conjugated heparin: (A) fluorescent dye derivatized heparin; (B) fluorescent dye derivatized heparin-C18.
Figure 7. Relative fluorescent intensity of cross-section vessel segments between fluorescent dye derivatized alkylated (C18) and nonalkylated heparin (up to 100 µm thickness, NIH Image Analysis).
then, there have been many in vitro studies on the antiproliferative effects of heparin.5,6 Studies on antiproliferative effects of heparin on SMCs in conjunction with antiatherosclerosis therapy have recently become popular.14-24 As for possible mechanisms underlying antiproliferative activity of heparin, it probably inhibits induction or transcription of several genes associated with cell cycle progression, including c-myb, c-myc, and c-fos protooncogenes, the mitochon-
drial ATP/ADP carrier protein 2F1, and histone H3.17 It could selectively act on a PKC-dependent pathway, which is thought to be involved in cell proliferation, and modulate kinase activity.17 In addition to the direct interaction of heparin with SMCs, heparin appears to reduce proliferation also by forming a complex with the potent mitogenic growth factor, basic fibroblast growth factor (bFGF), thus antagonistically inhibiting the binding of bFGF to the heparan sulfate proteoglycans present on the cell surface or in the extracellular matrix.25 Several recent studies have demonstrated successful, local delivery of heparin via a catheter to balloon angioplasty sites with beneficial impact on both platelet deposition and smooth muscle cell proliferation.20,22 Autoradiography demonstrated homogeneous distribution of heparin throughout the intima, media, and adventitia, with localization in the nucleus, cytoplasm, and extracellular space.20 Wash-out of heparin from the arterial wall is initially rapid, although the drug is detectable for up to 1 week following delivery.20 Our strategy using modified heparin is to increase the residence time of infused heparin on and in the arterial wall and to facilitate interaction with the cell membrane. To this end, heparin was hydrophobically modified without loss of biological activity. A hydrophobic moiety, which has high
Terminally Alkylated Heparin
Biomacromolecules, Vol. 2, No. 4, 2001 1183
Figure 8. Luminal surface appearances of heparin infusion into mechanically injured iliac arteries (a-1 and b-1, macroscopic tissue appearance; a-2 and b-2, scanning electron microscopic photos of proximal site; a-3 and b-3, scanning electron microscopic photos of distal site): left, nonalkylated heparin; right, alkylated heparin (C18).
affinity to lipid, was attached to the terminal end of heparin. Such an alkylated heparin is expected to be adsorbed on and be transported through lipid-rich extracellular matrixes. Our results are summarized as follows. (1) Increase in the chain length of the alkyl group reduced the proliferation rate of SMCs in culture. Among the alkylated heparins, the heparin with the longest alkyl chain length exhibited the largest antiproliferative potential, even larger than that of unmodified heparin. (2) At high dose (500 µg/mL), heparinC18 totally inhibited cell proliferation shortly after release from growth arrest and appeared to exhibit some cytotoxic effects, since the number of living cells gradually decreased with time. (3) The fluorescence staining of cells upon incubation with fluorescence-labeled alkylated heparin may indicate that alkylated heparin directly interacts with cellular membranes. (4) Much higher residential time of alkylated heparin was noted on injured arteries as compared with nonalkylated heparin as shown in Figure 6, indicating that alkylated heparin has higher affinity to a lipid-like tissue, which may facilitate prolonged residential time as well as tissue permeability. In conclusion, alkylated heparin, in addition to a surface modifier, appears to act as a cellular membrane as well as tissue modifier, both of which have antithrombogenic potential. Our next step, as a continuation of this study, is focused on studies at the tissue level as well as cellular level. As described here, a tissue level study verified a larger residence time of alkylated heparin, which is infused through a newly developed multifunctional balloon catheter which allows perfusion even during inflation and drug infusion at balloon sites. This may eventually result in reduced thrombus formation and intimal hyperplasia. A more in-depth study on antiproliferative effects at the cellular level will be primarily important. Such studies are now in progress, and the results will be reported soon. Acknowledgment. One of the authors (M.G.) thanks Professor Ichiro Nishio, Division of Cardiology, Department of Medicine, Wakayama Medical College, for allowing a leave of absence. This study was financially supported by the Promotion of Fundamental Studies in Health Science of
the Organization for Pharmaceutical Safety and Research (OPSR) under Grant No. 97-15. This study was partially performed at the Collaborative Center, Kyushu University. References and Notes (1) Clowes, A. W.; Karnowsky, M. J. Nature 1977, 265, 625-626. (2) Guyton, J. R.; Rosenberg, R. D.; Clowes, A. W.; Karnovsky, M. J. Circ. Res. 1980, 46, 625-634. (3) Clowes, A. W.; Clowes, M. M. Lab. InVest. 1985, 52, 611-616. (4) Clowes, A. W.; Clowes, M. M. Circ. Res. 1986, 58, 839-845. (5) Hoover, R. L.; Rosenberg, R.; Haering, W.; Karnovsky, M. J. Circ. Res. 1980, 47, 578-583. (6) Castellot, J. J.; Jr.; Addonizio, M. L.; Rosenberg, R.; Karnovsky, M. J. J. Cell. Biol. 1981, 90, 372-379. (7) Bombardini, T.; Picano, E. Angiology 1997, 48, 969-976. (8) Matsuda, T.; Magoshi, T. Biomacromolecules 2001, 2, 1169-1177. (9) Itou, T.; Noguchi, T.; Nonogi, H.; Matsuda, T.; Sugawara, T.; Arai, T.; Kanda, K.; Tsutsui, N. Jpn. J. Artif. Organs 1998, 27, 641-646. (10) Campbell, J. H.; Campbell, G. R. J. Cell. Biol. 1981, 89, 379-383. (11) Campbell, J. H.; Campbell, G. R. In Vascular smooth muscle in culture; Campbell, J. H., Campbell, G. R., Eds.; CRC Press: Boca Raton, FL, 1987; p 15. (12) Ishiyama, M.; Miyazone, Y.; Sasamoto, K.; Ohkura, Y.; Ueno, K. Talanta 1997, 44, 1299-1305. (13) Noguchi, T.; Yasuda, S.; Itoh, T.; Arai, T.; Kanda, N.; Nonogi, H.; Matsuda, T. J. Cardiol. 2000, 35, 41-45. (14) Tina, Au, Y. P.; Kenagy, R. D.; Clowes, M. M.; Clowes, A. W. Haemostasis 1993, 23 (suppl 1), 177-182. (15) Sto¨hr, S.; Meyer, T.; Smolenski, A.; Kreuzer, H.; Buchwald, A. B. J. CardioVasc. Pharm. 1995, 25, 782-788. (16) Geary, R. L.; Koyama, N.; Wang, T. W.; Vergel, S.; Clowes, A. W. Circulation 1995, 91, 2972-2981. (17) Herbert, J. M.; Clowes, M.; Lea, H. J.; Pascal, M.; Clowes, A. W. J. Biol. Chem. 1996, 271, 25928-25935. (18) Herbert, J. M.; Bono, F.; Lamarche, I.; Carmeliet, P. Thromb. Res. 1997, 86, 317-324. (19) Vadiveloo, P. K.; Filonzi, E. L.; Stanton, H. R.; Hamilton, J. A. Atherosclerosis 1997, 133, 61-69. (20) Fram, D. B.; Mitchel, J. F.; Azrin, M. A.; Chow, M. S. S.; Waters, D. D.; Mckay, R. G. Catheter Cardio. Diag. 1997, 41, 275-286. (21) Oberhoff, M.; Herdeg, C.; Baumbach, A.; Shamet, K.; Kranzho¨fer, A.; Weinga¨rtner, O.; Ru¨bsamen, K.; Kluge, M.; Karsch, K. R. Catheter Cardio. Diag. 1997, 41, 268-274. (22) Oberhoff, M.; Novac, S.; Herdeg, C.; Baumbach, A.; Kranzho¨fer, A.; Bohnet, A.; Horch, B.; Hanke, H.; Haase, K. K.; Karsch, K. R. CardioVasc. Res. 1998, 38, 751-762. (23) Kohno, M.; Yokokawa, K.; Yasunari, K.; Minami, M.; Kano, H.; Mandai, A. K.; Yoshikawa, J. Metabolism 1998, 47, 1065-1069. (24) Delafontaine, P. Eur. Heart J. 1998, 19 (suppl G), G18-G22. (25) Bono, F.; Rigon, P.; Lamarche, I.; Savi, P.; Salel, V.; Herbert, J. M. Biochem. J. 1997, 326, 661-668.
BM010097X
