- Email: [email protected]
Early inhibition of myointimal proliferation by angiopeptin after balloon catheter injury in the rabbit
Early inhibition of myointimal proliferation by angiopeptin after balloon catheter injury in the rabbit Marie L. Foegh, MD, DSc, Satish Asotra, PhD, John V. Conte, MD, Marcus Howell, MD, Elliott Kagan, MD, Kiran Verma, MS, and Peter W. Rarnwell, PhD, Washington) D.C. Purpose: Coronary artery restenosis after percutaneous transluminal angioplasty occurs in
more than 40% of patients. Angiopeptin, a stable synthetic octapeptide analogue of somatostatin, attenuates accelerated coronary artery myointimal thickening in rabbit cardiac allografts and myointimal thickening after arterial injury. In this study the temporal relationship between the angiopeptin treatment schedule and efficacy was explored. The relationship between inhibition of myointimal thickening by angiopeptin and inhibition of vascular cell proliferation was also examined. Methods: The aorta and the common and external iliac arteries of the rabbit underwent balloon injury. Angiopeptin (2 to 200 JLg/kg/day) was administered for 1 day before injury and for 1, 5, and 21 days after injury. Morphometric studies were performed to determine measurement of intimal thickening. Inhibition of vascular cell proliferation by angiopeptin was evaluated by tritiated thymidine incorporation into the balloon-injured rabbit aorta. Thymidine was either administered intraperitoneally or added ex vivo to aorta segments of rabbits treated with angiopeptin (2, 20, or 200 JLg/kgjday) from 1 day before injury until sacrifice 72 hours later. Results: Administration of angiopeptin (2 to 200 JLg/kgjday) significantly reduced intimal thickening by approximately 50% in all three vessels when evaluated 3 weeks after injury. This inhibitory effect was unrelated to duration of treatment and dose. Treatment initiated at the time of injury was found to be effective, but delaying treatment for 8, 18, or 27 hours abrogated the inhibitory effect of angiopeptin on myointimal thickening. Angiopeptin treatment significantly decreased thymidine-labeled nuclei of smooth muscle cells in vitro. Angiopeptin treatment similarly inhibited thymidine uptake in vitro by balloon-injured aorta segments. Conclusion: Angiopeptin significantly inhibits myointimal thickening by inhibiting vascular cell proliferation. Administration of angiopeptin for 2 days is as efficacious as 3 weeks treatment in inhibiting myointimal thickening. Delaying treatment for as little as 8 hours after injury abrogates the inhibitory effects of angiopeptin. This speaks to the importance of early events immediately after vascular tissue injury, suggesting that angiopeptin inhibits the expression of early genes causally related to the vascular injury response and thereby triggering vascular cell proliferation. (J VAse SURG 1994;19:1084-91.)
Owing to the high incidence of restenosis after percutaneous trans luminal coronary angioplasty,1,2 a From the Departments of Surgery, Division of Cardiovascular Surgery, Physiology and Biophysics, and Pathology, Georgetown University Medical Center, and Henri Beaufour Institute-USA, Washington. Supported by a grant from the Henri Beaufour Institute-USA. Reprint requests: Marie L. Foegh, MD, Department of Surgery, Georgetown University Medical Center, 4000 Reservoir Rd., N.W., Washington DC 20007. Copyright © 1994 by The Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter. 0741-5214/94/$3.00 + 0 24/1/52853
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major effort has been directed to exploring the mechanisms involved in myointimal hyperplasia and developing drugs for clinical trials. 3 Most of the clinical studies have been disappointing, and all of the large-scale trials have been unsuccessful. 4 -6 Because a variety of growth factors have been implicated/ we have been exploring a number of endocrinologic approaches. Somatostatin is a widely distributed, naturally occurring tetradecapeptide that inhibits growth hormone release and release of a wide variety of hormones such as insulin and glucagon,8,9 and its analogues have been shown to decrease plasma insulin-like growth factor-I (IGF-I).lO,1l Further-
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more, somatostatin inhibits proliferation of lymphocytes 12 and several tumor celllines. 13 ,14 However, this peptide has a very short half-life, and therefore a number of long-acting analogues have been synthesized that have both longer plasma half-lives and greater specificity. One such compound synthesized by Coy et al.,I5 angiopeptin, is an octapeptide that inhibits accelerated atherosclerosis in rabbit cardiac allografts. 16 It inhibits myointimal thickening in the rat after carotid artery desiccation injury17 and in the rabbit after balloon injury.18 In this report we describe the effect of different treatment time schedules in the latter model. We also examine the effect of angiopeptin on vascular cell proliferation by studying 3H-thymidine uptake in vitro by rabbit aorta after lllJury. METHODS Male New Zealand white rabbits weighing 2.5 to 2.8 kg (Hazelton Labs, Vienna, Va.) were used for all experiments. Animals were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care at 20° C with scheduled 12-hour light cycles and were fed as desired (Lab Rabbit Chow 5321, Purina Mills Inc., Richmond, Va.). All angiopeptin-treated and control animals described in these studies underwent the balloon injury procedure. The animals were killed either at 21 days after injury for morphometry studies or at 72 hours after balloon injury for studies to determine thymidine incorporation and DNA content in the vascular wall. Balloon injury. Rabbits were anesthetized with 50 mg/kg ofketamine intramuscularly (Mobay Corp. Animal Health Division, Shawnee, Kan.) and 5 mg/kg intramuscularly of xylazine (Quad Pharmaceuticals, Indianapolis, Ind.), with additional doses as needed. Under sterile conditions, the left femoral artery was exposed through a groin incision and isolated. An arteriotomy was made and a 3F Fogarty embolectomy catheter (American Edwards Laboratory, Inc., Santa Ana, Calif.) was passed 20 cm to the level of the diaphragm. There, the balloon was inflated with saline solution to 3 atm pressure by use of a handheld inflator (Indeflator; Advanced Cardiovascular Systems, Inc., Temecula, Calif.). The inflated balloon was passed three times from the diaphragm to the femoral artery. Mter removal of the catheter, the artery was ligated, and the incision was closed with absorbable sutures. All angiopeptintreated and control animals described in these studies underwent the balloon injury procedure. Morphometric studies. Three morphometric
Foegh et al.
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studies were undertaken. In the first study, 31 rabbits were assigned to placebo, 2, 20, or 200 J.Lg/kg/day angiopeptin (Henri Beaufour Institute, Washington, D.C.) treatment groups and received two divided daily doses subcutaneously beginning 1 day before balloon injury until the rabbits were killed at 21 days after balloon injury. Each angiopeptin treatment group consisted of eight animals, and the placebo group contained seven animals. In the second study, rabbits were assigned to a control group (n = 5) that received saline solution 1 day before and 2 days 'after balloon injury; another group (n = 7) received 20 J.Lg/kg/day of angiopeptin twice daily 1 day before balloon injury and continuing for only 1 day afterward (2 days total). A third group of animals (n = 7) received the same dose of angiopeptin as the previous group but the treatment was continued for 5 days (6 days total). In all three groups of this study, the animals were maintained until sacrifice 21 days after injury. The third study was an experiment to determine the effect of delaying angiopeptin treatment. Rabbits were divided into five groups (n = 4). The control group received saline solution, whereas the four remaining groups received 10 J.Lg/kg angiopeptin subcutaneously twice daily until sacrifice 21 days after balloon injury. One treatment group received the first dose during balloon injury and thereafter until sacrifice at day 21 after the procedure, whereas the remaining three groups received their first dose 8, 18, and 27 hours after balloon injury, respectively. Intimal hyperplasia was determined by morphometry as described below. Blood vessels were harvested from animals that were killed via the intracardiac injection of potassium or an overdose of ketamine and xylazine. A left thoracotomy was performed, and the descending thoracic aorta was ligated distal to the subclavian artery and cannulated with an 18 gauge catheter. The aorta was perfused with lactated Ringer's solution containing 10,000 units/liter of heparin (Organon Inc., West Orange, N.T.) at a pressure of80 mm Hg for 20 minutes. This was followed by a 30-minute perfusion with 10% buffered formalin at a pressure of 88 mm Hg. The abdominal aorta and iliac arteries were harvested and placed in 10% buffered formalin for histologic sectioning. Three- to four-millimeter sections were taken from the infrarenal aorta, the common iliac artery immediately distal to the aortic bifurcation, and the external iliac artery immediately above the inguinal ligament. Computerized morphometric analysis was performed (The Morphometer, Woods Hole Educational Associates, Woods Hole,
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Table I. Degree of percent intimal thickening in balloon-injured vessels after different doses of angiopeptin (1 day before treatment and 21 days after injury treatment) Angiopeptin (WJ/kg/day) 0 2 20 200
Aorta 22.0 10.6 14.6 13.1
± ± ± ±
6.5 3.7* 1.6* 6.4*
Common iliac
External iliac
25.3 12.0 14.1 16.8
30.8 15.2 24.1 23.4
± ± ± ±
3.2 4.5* 3.3* 8.2*
± ± ± ±
6.5 4.1* 3.7* 5.9*
*p < 0.04, Student's unpaired t test. Vabes are mean ± SD. Intimal thickening was determined by morphometric analysis 21 days after injury. Values (± SD) were calculated from the average of two histologic sections from each level of the arteries studied.
Mass.) on elastin-stained (Van Gieson) sections. Intimal thickening was determined from two crosssections of the infrarenal aorta and common iliac and external iliac arteries and expressed as a percent intimal thickening (intimal area/total vessel area x 100%), as previously described. IS Thymidine uptake studies. In vitro labeling of aorta segments. Five animals from each treatment group (2, 20, 200 J.Lg/kg/day angiopeptin and controls) were killed 3 days after balloon injury procedures. The in vitro incorporation of thymidine into rings of aortic segments from these animals was determined by incubating the aortic rings with 3H-thymidine (l-J.LCi/ml) for 3 hours. Unincorporated radioactive material was removed by washing the rings twice with Krebs-Ringer buffer. The samples were then incubated in the presence of 1 mmol/L unlabeled thymidine (Sigma Chemical Co., St. Louis, Mo.) for 1 hour and digested in 1 N sodium hydroxide at 60° C. The tissue lysates were precipitated with 10% trichloroacetic acid and centrifuged, and the pellets were digested in Protosol (New England Nuclear, Wilmington, Del.). The samples were then counted in a Packard TRICARB liquid scintillation counter (Packard, Downers Grove, Ill.). For the quantitation of tissue DNA, arterial segments were digested in 1 N sodium hydroxide. After neutralization with 0.6 N hydrochloric acid, the pellets obtained were washed with 100% ethanol and digested in 0.5 N perchloric acid at 90° C for 3 hours. Samples were centrifuged, and tissue DNA content was determined by the colorimetric method of Burton. 19 Protein content was determined with the Biorad assay (Biorad, Richmond, Calif.). In vivo 3H-thymidine labelling of injured aorta segments. The animals assigned to the treatment groups received 2, 20 or 200 J.Lg/kg/day subcutaneously of angiopeptin dissolved in saline solution in two divided doses, beginning 1 day before balloon
injury, and continuing twice daily until euthanasia, which was 3 days after balloon injury. Control animals received saline solution on the same schedule. Mter balloon injury and approximately 18 hours before euthanasia, tritium-labelled thymidine (80 Ci/mmol, Amersham Inc., Arlington Heights, Ill.) was injected intraperitoneally 0.5 J.LCi/g body weight into a total of 20 rabbits assigned to control, and the three treatment groups. After euthanasia, segments of the aorta were prepared as described for morphometric analyses. A small segment of the duodenum was also taken to confirm 3H-thymidine labelling. After several washes, the tissue segments were fixed in glutaraldehyde buffer, dehydrated in a series of alcohol grades and embedded in epon resin. One J.Lm-thick sections were obtained, rehydrated and coated with Kodak NTB-2 photographic emulsion (Eastman Kodak Co., Rochester, N.Y.) in the dark. After 7 weeks, the slides were developed according to the manufacturer's instructions, and the grain count was determined on toluidine blue-stained sections. With a calibrated eyepiece reticle, which was aligned with the internal elastic lamina, the number oflabeled nuclei within 100 J.Lm x 100 J.Lm area was enumerated at x 1000 magnification on six sections per aortic segmentP Statistical Analysis. All data are expressed as mean ± SD. Statistical analysis was performed with Student's unpaired t test or the Wilcoxon nonparametric test, as appropriate. Each treatment group was compared with its control group. A value ofp < 0.05 was considered to be significant. RESULTS Effect of time of treatment. With three different doses of angiopeptin (2, 20, and 200 J.Lg/kg/day), we first established that treatment for 1 day before balloon injury and for 21 days after injury significantly inhibited myointimal thickening induced in the aorta and common iliac and external iliac vessels
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Table II. Degree of percent intimal thickening in balloon-injured aorta (angiopeptin pretreatment was for 1 day, followed by 1 and 5 days after injury treatment) Angiopeptin ( I'if/Kg/day)
Days of treatment after injury
Aorta
0 20 20
6 2 6
27.9 ± 9.5 18.5 ± 3.8* 17.4 ± 2.5t
*p < 0.04; Wilcoxon nonparametric test. tp < 0.02; Wilcoxon nonparametric test. Values are mean ± SD; intimal thickening was determined by morphometric analysis 21 days after injury. Values were calculated from the average of two histologic sections.
(Table I). However, no significant quantitative differences were observed between the effect of the different doses. Next, by use of the middle dose (20 /-Lg/kg/day), we determined that 1 day prior treatment and only 1 or 5 days after treatment were sufficient to significantly inhibit myointimal thickening of the aorta (Table II). Finally, in a third experiment, significant inhibition of myointimal thickening in all three vessels was obtained without pretreatment, provided that treatment was initiated at the time of injury. We discovered that delaying treatment for 8, 18, and 27 hours after injury failed to significantly inhibit myointimal thickening (Table III) . As in the first experiment treatment was continued until euthanasia 21 days after injury. Thymidine incorporation by injured aorta segments in vitro and in vivo. 3H-thymidine incorporation in vitro (counts per minute [CPM]/mg protein) was measured in vascular rings prepared from abdominal aorta that was balloon-injured 3 days previously. 3H-thymidine uptake was significantly lower in vascular rings from the groups treated with the medium and highest dose of angiopeptin compared with the control group (p < 0.02, Fig. 1). CPM/mg protein was 199 ± 32.1 X 103 for the control group, values of 154 ± 36.9 X 103, 406.0 ± 9.13 X 103 and 80.7 ± 19.6 X 103 were obtained for the 2, 20, and 200 /-Lg/kg/day angiopeptin-treated groups, respectively. The DNA content per gram of tissue of the vascular rings was significantly inhibited by all three doses of angiopeptin (Fig. 2). Similar data were obtained for the 20 and 200 /-Lg/kg/day of angiopeptin when the DNAspecific activity (CPM per /-Lg DNA) was calculated (47.4 ± 5.9,88.6 ± 3.6,16.8 ± 3.6,17.6 ± 12.1) for control and angiopeptin 2, 20 and 200 /-Lg/kg/ day, respectively. The data reflect mean ± SEM. Where rabbits were given 3H-thymidine in vivo and tissue sections were analyzed by autoradiography, there were significantly fewer (p < 0.005)
3H-thymidine-labeled cell nuclei in sections from rabbits treated with 20 and 200 /-Lg/kg/day angiopeptin than in control animals (0.3 ± 0.3 and 1.0 ± 0.6 versus 4.3 ± 0.3 nuclei within 100 X 100 /-Lm). The vascular sections from rabbits treated with the lowest dose of angiopeptin (2 /-Lg/kg/day) did not exhibit a decrease in labeled cell nuclei (8.0 ± 0.3 versus 4.3 ± 0.3 nuclei within 100 X 100 /-Lm). DISCUSSION The first of the morphometric studies described here established that angiopeptin treatment significantly inhibits myointimal thickening after balloon injury of the rabbit aorta and iliac arteries. Angiopeptin was effective over a 100-fold dose range (2 to 200 /-Lg/kg/day subcutaneously) when the drug was administered, beginning the day before and continuing for 21 days after injury. A dose-dependent response was not observed. In the second study, the duration of treatment was varied, namely 1 day before treatment was followed by treatment for 1, 5, or 21 days after the injury. We found all three treatment schedules equally effective in inhibiting myointimal thickening when measured morphometrically at 21 days after injury. In the third experiment wherein pretreatment was omitted we observed that treatment beginning at the time of injury was effective but not treatment begun after a delay of 8 hours or longer. Thus treatment for the 24 hours before injury·or for 5 and 21 days after injury was unnecessary. Taken together the data indicate that in this model angiopeptin administration can be confined to the peritreatment period. The ineffectiveness of angiopeptin treatment in inhibiting myointimal thickening when delayed for 8 hours or longer after injury is similar to the low efficacy of heparin as shown by others.20 Initially, heparin administration was reported to be effective up to 18 hours after injury to the rat carotid artery.21 However, in a recent study, it is reported that the
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o~~--~----~----~--~~--~----~--~---Control 2 20 200 Angiopeptin
,.,g/Kg
Fig. 1. Effect of three different daily doses of angiopeptin compared with control on
3H-thymidine incorporation in vitro in balloon-injured abdominal aorta of rabbits, as measured 3 days after injury (5 animals/group). Asterisk is p < 0.02.
Table III. Effect of delayed angiopeptide treatment on percent intimal thickening of balloon-injured vessels Treatment delay (hours)
Angiopeptin ( p.gjkgjday)
Control 0 8 18 27
0 20 20 20 20
Aorta 9.7 2.8 6.5 8.6 12.3
± ± ± ± ±
1.8 0.8* 1.8 1.4 1.6
Common iliac artery 17.3 7.0 12.0 14.5 22.3
± ± ± ±
3.1 0.7* 2.0* 1.9 ± 2.4
External iliac artery 24.1 lD.7 15.2 20.7 28.3
± ± ± ±
4.9 2.2* 1.0 4.1 ± 4.5
*p < O.OlD; Student's unpaired t test. Values are mean ± SD. Intimal thickening was determined by morphometric analysis 21 days following injury. Values were calculated from the average of two histologic sections from each level of the arteries studied.
administration of heparin cannot be delayed beyond 6 hours after injury.2o The effectiveness of the short (2 days) angiopeptin treatment in inhibiting myointimal thickening is similar to the morphologic effect of short-term heparin treatment. This acute effect of angiopeptin and heparin suggests that if cells are prevented from entering the growth cycle at the time of injury, then they do not progress further at a later time after withdrawing angiopeptin. The need for only short-term treatment to attenuate myointimal thickening and the loss of efficacy when treatment is delayed speaks to very early events that determine vascular cell proliferation and the subsequent degree of myointimal thickening. Such early events may involve increased expression of immediate early genes, such as c-myb, c-fos and c-myc that encode
transcriptional regulatory factors 22,23 relating to growth factors that promote smooth muscle hyperplasia and perhaps secretion of matrix leading to myointimal thickening. They may also increase growth factor expression by upregulating their receptors. Such protein growth factors include plateletderived growth' factor (PDGF), IGF-I, fibroblast growth factor (FGF), and epidermal growth factors (EGF). Attempts to inhibit proliferation in vivo with antibodies to PDGF and FGF, although illuminating, yielded moderate inhibitory effects. 24,25 Lindner et al. 26 point out that FGF is critical in the medial cell replication that follows during the first 24 to 48 hours after balloon injury, whereas PDGF is critical in the migration from media into intima 3 to 5 days after injury.24 Perhaps the efficacy of strategies directed
JOURNAL OF VASCULAR SURGERY Volume 19, Number 6
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I
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::::I III III
150
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-
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Angiopeptln 119/K9
Fig. 2. Effect of three different daily doses of angiopeptin compared with control on DNA content of balloon-injured abdominal aorta of rabbits 3 days after injury (same animals as in Fig. 1). Asterisk is P < 0.02.
toward antagonizing specific growth factors may be limited because of a redundancy of mechanisms leading to vascular cell proliferation. For example, the concerted expression of multiple growth factors acting through similar signal transduction pathways may be necessary. IGF-I, PGDF, FGF, and EGF all activate tyrosine kinase in the intracellular domain of their respective receptors. 27 One appealing mechanism for angiopeptin inhibition of myointimal thickening is by activation of a membrane-bound phosphatase, which dephosphorylates activated tyrosine kinases. 28,29 As mentioned above, the intracellular domain of the receptors for PDGF, IGF-I, EGF, and FGF are all tyrosine kinases that autophosphorylate when these ligands bind to their receptors. Specific phosphatases dephosphorylate the activated tyrosine kinase and may thereby prevent further signal transduction. Somatostatin and several of its analogues, including angiopeptin, are reported to stimulate such phosphatases in a dose-dependent manner. 27,28 Thus it is possible that phosphatase activation may be one of the mechanisms by which angiopeptin exerts its inhibitory effect. One of the growth factors that may be especially significant is IGF-I because it is not only a potent mitogen that elicits and potentiates the proliferation of cultured arterial smooth muscle cells by PDGF and
FGF, but it may be regulated by growth hormone. 3o Moreover, the rabbit aorta contains high affinityspecific IGF-I receptor binding sites. 31 ,32 Angiopeptin, like other somatostatin analogues, inhibits growth hormone release,33,34 which stimulates IGF-I synthesis in the liver. 34 More recently, it has been demonstrated that superphysiologic doses of growth hormone will also increase IGF-I gene expression, measured as IGF-I messenger RNA and IGF-I in the rat aorta. 35 The growth hormone-induced increase in IGF-I messenger RNA in the aorta is, however, modest compared with the increase seen with IGF-I gene expression after balloon injury. Angiopeptin in the rat has a half-life of 2 hours, and twice daily administration, as used in our experiments, is insufficient to suppress growth hormone. 36 Thus it is unlikely that regulation of growth hormone by angiopeptin has i significant role in the inhibition of myointimal thickening in this study. The remaining two studies reported here show that angiopeptin treatment of rabbits inhibits thymidine uptake by injured vessels excised 72 hours after injury. First, injured aorta segments were excised 72 hours after injury and then incubated in vitro with 3H-thymidine. Both thymidine incorporation and DNA content were significantly reduced byangiopeptin. Second, 3H -thymidine was administered in vivo
1090 Foegh et al.
to angiopeptin-treated rabbits 48 hours after injury and segments of these aorta were used for autoradiography. Counting of thymidine-labeled cells confirmed that angiopeptin treatment decreased cell proliferation. Inhibition of myointimal thickening can relate to decreased extracellular matrix formation and inhibition of both migration and smooth muscle cell proliferation. The thymidine incorporation studies evaluated the events 72 hours after balloon injury and may not include the period of smooth muscle cell migration from the media to the intima, because in studies by others this migration is reported to occur in rats at days 3 to 4. 37,38 The thymidine uptake studies show that angiopeptin inhibits mitogenesis, because the number of smooth muscle cells as shown by counting of labeled cells and DNA content, and thymidine incorporation in the aorta are reduced in angiopeptin-treated animals. In conclusion, angiopeptin, a stable octapeptide analogue of somatostatin inhibits myointimal thickening and smooth muscle cell proliferation induced by balloon injury. The inhibition is independent of dose over a wide dose range. The need for only short-term treatment and the lack of efficacy of delayed treatment suggests that angiopeptin inhibits expression of early events such as induction of early response genes, triggering smooth muscle cell proliferation. We thank Dominique Y. Pifat, PhD, and Denise Cook for their invaluable assistance. REFERENCES 1. Kent KM. Restenosis after percutaneous transluminal coronary angioplasry. Am J CardioI1988;61:67G-70G. 2. Klein LW, Rosenblum J. Restenosis after successful percutaneous transluminal coronary angioplasty. Prog Cardiovasc Dis 1990;32:365-82. 3. Hermans WR, Rensing BJ, Strauss BH, Serruys PW. Prevention of restenosis after percutaneous transluminal coronary angioplasty: the search for a "magic bullet." Am Heart J 1991;122:171-87. 4. Knudtson ML, Flintoft VF, Roth DL, Hansen JL, DuffHJ. Effect of short-term prostacyclin administration on restenosis after percutaneous transluminal coronary angioplasty. JACC 1990;15:691-7. 5. Pepine CJ, Hirshfeld JW, Macdonald RG, et al. A controlled trial of corticosteroids to prevent restenosis after coronary angioplasty. Circulation 1990;81: 1753-61. 6. Popma JJ, Califf RM, Topol EJ. Clinical trials of restenosis following coronary angioplasty. Circulation 1991;84:142636. 7. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature 1993;362:101-9. 8. Alberti KG, Christensen NT, Christensen SE, et al. Inhibition of insulin secretion by somatostatin. Lancet 1973;2:1299301.
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9. Bloom SR, Polak. JM. Somatostatin. Brit Med J 1987;295: 288-90. 10. Flyvbjerg A, Jorgensen KD, Marshall SM, Orskov H. Inhibitory effect of octreotide on growth hormone-induced IGF-I generation and organ growth in hypophysectomized rats. Am J PhysioI1991;260:E568-74. 11. Parmar H, Bogden A, Mollard M, de Rouge B, Phillips RH, Lightman SL. Somatostatin and somatostatin analogues in oncology. Cancer Treat Rev 1989;16:95-115. 12. Payan DG, Hess CA, Goetzl EJ. Inhibition by somatostatin of the proliferation ofT-lymphocytes and molt-4Iymphoblasts. Cell Immunol 1984;84:433-8. 13. Hierowski MT, Liebow C, du Sapin K, Schally AV. Stimulation by somatostatin of dephosphorylation of membrane proteins in pancreatic Gancer MIA PaCa-2 cell lines. FEBS Lett 1985;179:252-6. 14. Bogden A, Taylor J, Moreau JP, Coy DH, LePage DJ. Response of human lung tumor xenografts to treatment with a somatostatin analogue (somatuline). Cancer Res 1990;50: 4350-65. 15. Coy DH, Heiman ML, Rossowski J, et al. Receptor selective somatostatin (SRIF) analogues. In: Marshall GR, ed. Peptides, chemistry and biology. Leiden, ESOM, 1988:46264. 16. FoeghML, Khirabadi SS, Chambers E, Amamoo S, Ramwell PW. Peptide inhibition of coronary artery transplant atherosclerosis in rabbits with angiopeptin, an octapeptide. Atherosclerosis 1989;78:229-36. 17. Lundergan C, Foegh ML, Vargas R, et al. Inhibition of myointimal proliferation of the rat carotid attery by the peptides angiopeptin and BIM 23034. Atherosclerosis 1989; 80:49-55. 18. Conte JV, Foegh ML, Calcagno D. Peptide inhibition of myointimal proliferation following angioplasty in rabbits. Transpl Proc 1989;21:3686-8. 19. Burton K. A Study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:315-23. 20. Lindner V, Olson NE, Clowes AW, Reidy MA. Inhibition of smooth muscle cell proliferation in injured rat arteries. J Clin Invest 1992;90:2044-9. 21. Clowes AW, Clowes MM. Kinetics of cellular proliferation after atterial injury: heparin inhibits rat smooth muscle mitogenesis and migration. Circ Res 1986;58:839-45. 22. Banskota NK, Taub R, Zellner K, Olsen P, King GL. Characterization of induction of protooncogene c-myc and cellular growth in human vascular smooth muscle cells by insulin and IGF-I. Diabetes 1989;38:123-9. 23. Pukac LA, Ottlinger ME, Kamovsky MJ. Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells. J Bioi Chern 1992;267:37{}7-11. 24. Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science 1991;253: 1129-32. 25. Olsen NE, Chao S, Lindner V, Reidy MA. Rapid communication: intimal smooth muscle cell proliferation after balloon catheter injury. Am J PathoI1992;140:1017-23. 26. Lindner V, Majack RA, Reidy MA. Role of basic fibroblast growth factor in vascular lesion formation. Circ Res 1991; 68:106-13. 27. Wilks AF: Structure and function of the protein tyrosine kinases. Prog Growth Factor Res 1990;2:97-111.
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28. Lee MT, Liebow C, Kamer AR, Schally AV. Effects of epidermal growth factor and analogues of luteinizing hormone-releasing hormone and somatostatin on phosphorylation and dephosphorylation of tyrosine residues of specific protein substrates in various tumors. Proc Nat! Acad Sci USA 1991;88:1656-60. 29. Colas B, Cambillau C, Buscail L, et al. Membrane tyrosine phosphatase activity is regulated by somatostatin analogues in rat pancreatic acinar cells. Eur J Biochem 1992;207:1017-24. 30. Pfeifle B, Horst B, Ditschunseit H. Interaction of receptors for insulin-like growth factor I, platelet-derived growth factor, and fibroblast growth factor in rat aortic cells. Endocrinology 1987;120:2251-8. 31. Pfeifle B, Ditschuneit HH, Ditschuneit H. Binding and biological actions of insulin-like growth factors on human smooth muscle cells. Horm Metab Res 1982;14:409-14. 32. Sidawy AN, Termanini B, Nardi RV, Harmon JW, Korman LY. Insulin-like growth factor I receptors in the arteries of the rabbit: autoradiographic mapping and receptor characterization. Surgery 1990;108:165-71. 33. Moreau SC, Murphy W A, Coy DH. Comparison of Lanreotide (BIM-23014) and somatostatin on endocrine and exocrine activities in the rat. Drug Dev Res 1991;22:79-93.
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34. Magnan E, Cataldi M, Guillaume V, et al. Acute changes in growth hormone releasing hormone secretion after injection ofBIM 23014, a long acting somatostatin analogue, in rams. Life Sci 1992;51:831-8. 35. Cercek B, Fishbein MC, Fortester JS, Helfant RH, Fagin JA. Induction of insulin-like growth factor I messenger RNA in rat aorta after balloon denudation. Circ Res 1990;66:175560. 36. Cathapermal S, Foegh ML, Rau CS, et al. Disposition and tissue distribution of Angiopeptin in the rat. J Drug Metab Dispos 1991;19:735-9. 37. Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res 1985;56:139-45. 38. Goldberg ID, Stemerman MB, Schnipper LE, Ransil BJ, Crooks GW, Fuhro RL. Vascular smooth muscle cell kinetics: a new assay for studying patterns of cellular proliferation in vivo. Science 1979;205:920-2.
Submitted Aug. 26, 1993; accepted Nov. 8, 1993.
BOUND VOLUMES AVAILABLE TO SUBSCRIBERS Bound volumes of the JOURNAL OF VASCULAR SURGERY for 1994 are available to subscribers only. They may be purchased from the publisher at a cost of $76.00 for domestic, $97.32 for Canadian, and $92.00 for international subscribers for Vol. 19 (January to June) and Vol. 20 (July to December). Price includes shipping charges. Each bound volume contains a subject and author index, and all advertising is removed. Copies are shipped within 60 days after publication of the last issue in the volume. The binding is durable buckram with the journal name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact Subscription Services, Mosby-Year Book, Inc., 11830 Westline Industrial Dr., St. Louis, MO 63146-3318, USA. In the United States call toll free (800)325-4177, ext. 435l. In Missouri or foreign countries call (314)453-435l. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular JOURNAL subscription.
more than 40% of patients. Angiopeptin, a stable synthetic octapeptide analogue of somatostatin, attenuates accelerated coronary artery myointimal thickening in rabbit cardiac allografts and myointimal thickening after arterial injury. In this study the temporal relationship between the angiopeptin treatment schedule and efficacy was explored. The relationship between inhibition of myointimal thickening by angiopeptin and inhibition of vascular cell proliferation was also examined. Methods: The aorta and the common and external iliac arteries of the rabbit underwent balloon injury. Angiopeptin (2 to 200 JLg/kg/day) was administered for 1 day before injury and for 1, 5, and 21 days after injury. Morphometric studies were performed to determine measurement of intimal thickening. Inhibition of vascular cell proliferation by angiopeptin was evaluated by tritiated thymidine incorporation into the balloon-injured rabbit aorta. Thymidine was either administered intraperitoneally or added ex vivo to aorta segments of rabbits treated with angiopeptin (2, 20, or 200 JLg/kgjday) from 1 day before injury until sacrifice 72 hours later. Results: Administration of angiopeptin (2 to 200 JLg/kgjday) significantly reduced intimal thickening by approximately 50% in all three vessels when evaluated 3 weeks after injury. This inhibitory effect was unrelated to duration of treatment and dose. Treatment initiated at the time of injury was found to be effective, but delaying treatment for 8, 18, or 27 hours abrogated the inhibitory effect of angiopeptin on myointimal thickening. Angiopeptin treatment significantly decreased thymidine-labeled nuclei of smooth muscle cells in vitro. Angiopeptin treatment similarly inhibited thymidine uptake in vitro by balloon-injured aorta segments. Conclusion: Angiopeptin significantly inhibits myointimal thickening by inhibiting vascular cell proliferation. Administration of angiopeptin for 2 days is as efficacious as 3 weeks treatment in inhibiting myointimal thickening. Delaying treatment for as little as 8 hours after injury abrogates the inhibitory effects of angiopeptin. This speaks to the importance of early events immediately after vascular tissue injury, suggesting that angiopeptin inhibits the expression of early genes causally related to the vascular injury response and thereby triggering vascular cell proliferation. (J VAse SURG 1994;19:1084-91.)
Owing to the high incidence of restenosis after percutaneous trans luminal coronary angioplasty,1,2 a From the Departments of Surgery, Division of Cardiovascular Surgery, Physiology and Biophysics, and Pathology, Georgetown University Medical Center, and Henri Beaufour Institute-USA, Washington. Supported by a grant from the Henri Beaufour Institute-USA. Reprint requests: Marie L. Foegh, MD, Department of Surgery, Georgetown University Medical Center, 4000 Reservoir Rd., N.W., Washington DC 20007. Copyright © 1994 by The Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter. 0741-5214/94/$3.00 + 0 24/1/52853
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major effort has been directed to exploring the mechanisms involved in myointimal hyperplasia and developing drugs for clinical trials. 3 Most of the clinical studies have been disappointing, and all of the large-scale trials have been unsuccessful. 4 -6 Because a variety of growth factors have been implicated/ we have been exploring a number of endocrinologic approaches. Somatostatin is a widely distributed, naturally occurring tetradecapeptide that inhibits growth hormone release and release of a wide variety of hormones such as insulin and glucagon,8,9 and its analogues have been shown to decrease plasma insulin-like growth factor-I (IGF-I).lO,1l Further-
JOURNAL OF VASCULAR SURGERY Volume 19, Number 6
more, somatostatin inhibits proliferation of lymphocytes 12 and several tumor celllines. 13 ,14 However, this peptide has a very short half-life, and therefore a number of long-acting analogues have been synthesized that have both longer plasma half-lives and greater specificity. One such compound synthesized by Coy et al.,I5 angiopeptin, is an octapeptide that inhibits accelerated atherosclerosis in rabbit cardiac allografts. 16 It inhibits myointimal thickening in the rat after carotid artery desiccation injury17 and in the rabbit after balloon injury.18 In this report we describe the effect of different treatment time schedules in the latter model. We also examine the effect of angiopeptin on vascular cell proliferation by studying 3H-thymidine uptake in vitro by rabbit aorta after lllJury. METHODS Male New Zealand white rabbits weighing 2.5 to 2.8 kg (Hazelton Labs, Vienna, Va.) were used for all experiments. Animals were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care at 20° C with scheduled 12-hour light cycles and were fed as desired (Lab Rabbit Chow 5321, Purina Mills Inc., Richmond, Va.). All angiopeptin-treated and control animals described in these studies underwent the balloon injury procedure. The animals were killed either at 21 days after injury for morphometry studies or at 72 hours after balloon injury for studies to determine thymidine incorporation and DNA content in the vascular wall. Balloon injury. Rabbits were anesthetized with 50 mg/kg ofketamine intramuscularly (Mobay Corp. Animal Health Division, Shawnee, Kan.) and 5 mg/kg intramuscularly of xylazine (Quad Pharmaceuticals, Indianapolis, Ind.), with additional doses as needed. Under sterile conditions, the left femoral artery was exposed through a groin incision and isolated. An arteriotomy was made and a 3F Fogarty embolectomy catheter (American Edwards Laboratory, Inc., Santa Ana, Calif.) was passed 20 cm to the level of the diaphragm. There, the balloon was inflated with saline solution to 3 atm pressure by use of a handheld inflator (Indeflator; Advanced Cardiovascular Systems, Inc., Temecula, Calif.). The inflated balloon was passed three times from the diaphragm to the femoral artery. Mter removal of the catheter, the artery was ligated, and the incision was closed with absorbable sutures. All angiopeptintreated and control animals described in these studies underwent the balloon injury procedure. Morphometric studies. Three morphometric
Foegh et al.
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studies were undertaken. In the first study, 31 rabbits were assigned to placebo, 2, 20, or 200 J.Lg/kg/day angiopeptin (Henri Beaufour Institute, Washington, D.C.) treatment groups and received two divided daily doses subcutaneously beginning 1 day before balloon injury until the rabbits were killed at 21 days after balloon injury. Each angiopeptin treatment group consisted of eight animals, and the placebo group contained seven animals. In the second study, rabbits were assigned to a control group (n = 5) that received saline solution 1 day before and 2 days 'after balloon injury; another group (n = 7) received 20 J.Lg/kg/day of angiopeptin twice daily 1 day before balloon injury and continuing for only 1 day afterward (2 days total). A third group of animals (n = 7) received the same dose of angiopeptin as the previous group but the treatment was continued for 5 days (6 days total). In all three groups of this study, the animals were maintained until sacrifice 21 days after injury. The third study was an experiment to determine the effect of delaying angiopeptin treatment. Rabbits were divided into five groups (n = 4). The control group received saline solution, whereas the four remaining groups received 10 J.Lg/kg angiopeptin subcutaneously twice daily until sacrifice 21 days after balloon injury. One treatment group received the first dose during balloon injury and thereafter until sacrifice at day 21 after the procedure, whereas the remaining three groups received their first dose 8, 18, and 27 hours after balloon injury, respectively. Intimal hyperplasia was determined by morphometry as described below. Blood vessels were harvested from animals that were killed via the intracardiac injection of potassium or an overdose of ketamine and xylazine. A left thoracotomy was performed, and the descending thoracic aorta was ligated distal to the subclavian artery and cannulated with an 18 gauge catheter. The aorta was perfused with lactated Ringer's solution containing 10,000 units/liter of heparin (Organon Inc., West Orange, N.T.) at a pressure of80 mm Hg for 20 minutes. This was followed by a 30-minute perfusion with 10% buffered formalin at a pressure of 88 mm Hg. The abdominal aorta and iliac arteries were harvested and placed in 10% buffered formalin for histologic sectioning. Three- to four-millimeter sections were taken from the infrarenal aorta, the common iliac artery immediately distal to the aortic bifurcation, and the external iliac artery immediately above the inguinal ligament. Computerized morphometric analysis was performed (The Morphometer, Woods Hole Educational Associates, Woods Hole,
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Table I. Degree of percent intimal thickening in balloon-injured vessels after different doses of angiopeptin (1 day before treatment and 21 days after injury treatment) Angiopeptin (WJ/kg/day) 0 2 20 200
Aorta 22.0 10.6 14.6 13.1
± ± ± ±
6.5 3.7* 1.6* 6.4*
Common iliac
External iliac
25.3 12.0 14.1 16.8
30.8 15.2 24.1 23.4
± ± ± ±
3.2 4.5* 3.3* 8.2*
± ± ± ±
6.5 4.1* 3.7* 5.9*
*p < 0.04, Student's unpaired t test. Vabes are mean ± SD. Intimal thickening was determined by morphometric analysis 21 days after injury. Values (± SD) were calculated from the average of two histologic sections from each level of the arteries studied.
Mass.) on elastin-stained (Van Gieson) sections. Intimal thickening was determined from two crosssections of the infrarenal aorta and common iliac and external iliac arteries and expressed as a percent intimal thickening (intimal area/total vessel area x 100%), as previously described. IS Thymidine uptake studies. In vitro labeling of aorta segments. Five animals from each treatment group (2, 20, 200 J.Lg/kg/day angiopeptin and controls) were killed 3 days after balloon injury procedures. The in vitro incorporation of thymidine into rings of aortic segments from these animals was determined by incubating the aortic rings with 3H-thymidine (l-J.LCi/ml) for 3 hours. Unincorporated radioactive material was removed by washing the rings twice with Krebs-Ringer buffer. The samples were then incubated in the presence of 1 mmol/L unlabeled thymidine (Sigma Chemical Co., St. Louis, Mo.) for 1 hour and digested in 1 N sodium hydroxide at 60° C. The tissue lysates were precipitated with 10% trichloroacetic acid and centrifuged, and the pellets were digested in Protosol (New England Nuclear, Wilmington, Del.). The samples were then counted in a Packard TRICARB liquid scintillation counter (Packard, Downers Grove, Ill.). For the quantitation of tissue DNA, arterial segments were digested in 1 N sodium hydroxide. After neutralization with 0.6 N hydrochloric acid, the pellets obtained were washed with 100% ethanol and digested in 0.5 N perchloric acid at 90° C for 3 hours. Samples were centrifuged, and tissue DNA content was determined by the colorimetric method of Burton. 19 Protein content was determined with the Biorad assay (Biorad, Richmond, Calif.). In vivo 3H-thymidine labelling of injured aorta segments. The animals assigned to the treatment groups received 2, 20 or 200 J.Lg/kg/day subcutaneously of angiopeptin dissolved in saline solution in two divided doses, beginning 1 day before balloon
injury, and continuing twice daily until euthanasia, which was 3 days after balloon injury. Control animals received saline solution on the same schedule. Mter balloon injury and approximately 18 hours before euthanasia, tritium-labelled thymidine (80 Ci/mmol, Amersham Inc., Arlington Heights, Ill.) was injected intraperitoneally 0.5 J.LCi/g body weight into a total of 20 rabbits assigned to control, and the three treatment groups. After euthanasia, segments of the aorta were prepared as described for morphometric analyses. A small segment of the duodenum was also taken to confirm 3H-thymidine labelling. After several washes, the tissue segments were fixed in glutaraldehyde buffer, dehydrated in a series of alcohol grades and embedded in epon resin. One J.Lm-thick sections were obtained, rehydrated and coated with Kodak NTB-2 photographic emulsion (Eastman Kodak Co., Rochester, N.Y.) in the dark. After 7 weeks, the slides were developed according to the manufacturer's instructions, and the grain count was determined on toluidine blue-stained sections. With a calibrated eyepiece reticle, which was aligned with the internal elastic lamina, the number oflabeled nuclei within 100 J.Lm x 100 J.Lm area was enumerated at x 1000 magnification on six sections per aortic segmentP Statistical Analysis. All data are expressed as mean ± SD. Statistical analysis was performed with Student's unpaired t test or the Wilcoxon nonparametric test, as appropriate. Each treatment group was compared with its control group. A value ofp < 0.05 was considered to be significant. RESULTS Effect of time of treatment. With three different doses of angiopeptin (2, 20, and 200 J.Lg/kg/day), we first established that treatment for 1 day before balloon injury and for 21 days after injury significantly inhibited myointimal thickening induced in the aorta and common iliac and external iliac vessels
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Foegh et al.
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Table II. Degree of percent intimal thickening in balloon-injured aorta (angiopeptin pretreatment was for 1 day, followed by 1 and 5 days after injury treatment) Angiopeptin ( I'if/Kg/day)
Days of treatment after injury
Aorta
0 20 20
6 2 6
27.9 ± 9.5 18.5 ± 3.8* 17.4 ± 2.5t
*p < 0.04; Wilcoxon nonparametric test. tp < 0.02; Wilcoxon nonparametric test. Values are mean ± SD; intimal thickening was determined by morphometric analysis 21 days after injury. Values were calculated from the average of two histologic sections.
(Table I). However, no significant quantitative differences were observed between the effect of the different doses. Next, by use of the middle dose (20 /-Lg/kg/day), we determined that 1 day prior treatment and only 1 or 5 days after treatment were sufficient to significantly inhibit myointimal thickening of the aorta (Table II). Finally, in a third experiment, significant inhibition of myointimal thickening in all three vessels was obtained without pretreatment, provided that treatment was initiated at the time of injury. We discovered that delaying treatment for 8, 18, and 27 hours after injury failed to significantly inhibit myointimal thickening (Table III) . As in the first experiment treatment was continued until euthanasia 21 days after injury. Thymidine incorporation by injured aorta segments in vitro and in vivo. 3H-thymidine incorporation in vitro (counts per minute [CPM]/mg protein) was measured in vascular rings prepared from abdominal aorta that was balloon-injured 3 days previously. 3H-thymidine uptake was significantly lower in vascular rings from the groups treated with the medium and highest dose of angiopeptin compared with the control group (p < 0.02, Fig. 1). CPM/mg protein was 199 ± 32.1 X 103 for the control group, values of 154 ± 36.9 X 103, 406.0 ± 9.13 X 103 and 80.7 ± 19.6 X 103 were obtained for the 2, 20, and 200 /-Lg/kg/day angiopeptin-treated groups, respectively. The DNA content per gram of tissue of the vascular rings was significantly inhibited by all three doses of angiopeptin (Fig. 2). Similar data were obtained for the 20 and 200 /-Lg/kg/day of angiopeptin when the DNAspecific activity (CPM per /-Lg DNA) was calculated (47.4 ± 5.9,88.6 ± 3.6,16.8 ± 3.6,17.6 ± 12.1) for control and angiopeptin 2, 20 and 200 /-Lg/kg/ day, respectively. The data reflect mean ± SEM. Where rabbits were given 3H-thymidine in vivo and tissue sections were analyzed by autoradiography, there were significantly fewer (p < 0.005)
3H-thymidine-labeled cell nuclei in sections from rabbits treated with 20 and 200 /-Lg/kg/day angiopeptin than in control animals (0.3 ± 0.3 and 1.0 ± 0.6 versus 4.3 ± 0.3 nuclei within 100 X 100 /-Lm). The vascular sections from rabbits treated with the lowest dose of angiopeptin (2 /-Lg/kg/day) did not exhibit a decrease in labeled cell nuclei (8.0 ± 0.3 versus 4.3 ± 0.3 nuclei within 100 X 100 /-Lm). DISCUSSION The first of the morphometric studies described here established that angiopeptin treatment significantly inhibits myointimal thickening after balloon injury of the rabbit aorta and iliac arteries. Angiopeptin was effective over a 100-fold dose range (2 to 200 /-Lg/kg/day subcutaneously) when the drug was administered, beginning the day before and continuing for 21 days after injury. A dose-dependent response was not observed. In the second study, the duration of treatment was varied, namely 1 day before treatment was followed by treatment for 1, 5, or 21 days after the injury. We found all three treatment schedules equally effective in inhibiting myointimal thickening when measured morphometrically at 21 days after injury. In the third experiment wherein pretreatment was omitted we observed that treatment beginning at the time of injury was effective but not treatment begun after a delay of 8 hours or longer. Thus treatment for the 24 hours before injury·or for 5 and 21 days after injury was unnecessary. Taken together the data indicate that in this model angiopeptin administration can be confined to the peritreatment period. The ineffectiveness of angiopeptin treatment in inhibiting myointimal thickening when delayed for 8 hours or longer after injury is similar to the low efficacy of heparin as shown by others.20 Initially, heparin administration was reported to be effective up to 18 hours after injury to the rat carotid artery.21 However, in a recent study, it is reported that the
JOURNAL OF VASCULAR SURGERY
1088 Foegh et at.
June 1994
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o~~--~----~----~--~~--~----~--~---Control 2 20 200 Angiopeptin
,.,g/Kg
Fig. 1. Effect of three different daily doses of angiopeptin compared with control on
3H-thymidine incorporation in vitro in balloon-injured abdominal aorta of rabbits, as measured 3 days after injury (5 animals/group). Asterisk is p < 0.02.
Table III. Effect of delayed angiopeptide treatment on percent intimal thickening of balloon-injured vessels Treatment delay (hours)
Angiopeptin ( p.gjkgjday)
Control 0 8 18 27
0 20 20 20 20
Aorta 9.7 2.8 6.5 8.6 12.3
± ± ± ± ±
1.8 0.8* 1.8 1.4 1.6
Common iliac artery 17.3 7.0 12.0 14.5 22.3
± ± ± ±
3.1 0.7* 2.0* 1.9 ± 2.4
External iliac artery 24.1 lD.7 15.2 20.7 28.3
± ± ± ±
4.9 2.2* 1.0 4.1 ± 4.5
*p < O.OlD; Student's unpaired t test. Values are mean ± SD. Intimal thickening was determined by morphometric analysis 21 days following injury. Values were calculated from the average of two histologic sections from each level of the arteries studied.
administration of heparin cannot be delayed beyond 6 hours after injury.2o The effectiveness of the short (2 days) angiopeptin treatment in inhibiting myointimal thickening is similar to the morphologic effect of short-term heparin treatment. This acute effect of angiopeptin and heparin suggests that if cells are prevented from entering the growth cycle at the time of injury, then they do not progress further at a later time after withdrawing angiopeptin. The need for only short-term treatment to attenuate myointimal thickening and the loss of efficacy when treatment is delayed speaks to very early events that determine vascular cell proliferation and the subsequent degree of myointimal thickening. Such early events may involve increased expression of immediate early genes, such as c-myb, c-fos and c-myc that encode
transcriptional regulatory factors 22,23 relating to growth factors that promote smooth muscle hyperplasia and perhaps secretion of matrix leading to myointimal thickening. They may also increase growth factor expression by upregulating their receptors. Such protein growth factors include plateletderived growth' factor (PDGF), IGF-I, fibroblast growth factor (FGF), and epidermal growth factors (EGF). Attempts to inhibit proliferation in vivo with antibodies to PDGF and FGF, although illuminating, yielded moderate inhibitory effects. 24,25 Lindner et al. 26 point out that FGF is critical in the medial cell replication that follows during the first 24 to 48 hours after balloon injury, whereas PDGF is critical in the migration from media into intima 3 to 5 days after injury.24 Perhaps the efficacy of strategies directed
JOURNAL OF VASCULAR SURGERY Volume 19, Number 6
250
Foegh et at.
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-
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I
QI
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150
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-
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Angiopeptln 119/K9
Fig. 2. Effect of three different daily doses of angiopeptin compared with control on DNA content of balloon-injured abdominal aorta of rabbits 3 days after injury (same animals as in Fig. 1). Asterisk is P < 0.02.
toward antagonizing specific growth factors may be limited because of a redundancy of mechanisms leading to vascular cell proliferation. For example, the concerted expression of multiple growth factors acting through similar signal transduction pathways may be necessary. IGF-I, PGDF, FGF, and EGF all activate tyrosine kinase in the intracellular domain of their respective receptors. 27 One appealing mechanism for angiopeptin inhibition of myointimal thickening is by activation of a membrane-bound phosphatase, which dephosphorylates activated tyrosine kinases. 28,29 As mentioned above, the intracellular domain of the receptors for PDGF, IGF-I, EGF, and FGF are all tyrosine kinases that autophosphorylate when these ligands bind to their receptors. Specific phosphatases dephosphorylate the activated tyrosine kinase and may thereby prevent further signal transduction. Somatostatin and several of its analogues, including angiopeptin, are reported to stimulate such phosphatases in a dose-dependent manner. 27,28 Thus it is possible that phosphatase activation may be one of the mechanisms by which angiopeptin exerts its inhibitory effect. One of the growth factors that may be especially significant is IGF-I because it is not only a potent mitogen that elicits and potentiates the proliferation of cultured arterial smooth muscle cells by PDGF and
FGF, but it may be regulated by growth hormone. 3o Moreover, the rabbit aorta contains high affinityspecific IGF-I receptor binding sites. 31 ,32 Angiopeptin, like other somatostatin analogues, inhibits growth hormone release,33,34 which stimulates IGF-I synthesis in the liver. 34 More recently, it has been demonstrated that superphysiologic doses of growth hormone will also increase IGF-I gene expression, measured as IGF-I messenger RNA and IGF-I in the rat aorta. 35 The growth hormone-induced increase in IGF-I messenger RNA in the aorta is, however, modest compared with the increase seen with IGF-I gene expression after balloon injury. Angiopeptin in the rat has a half-life of 2 hours, and twice daily administration, as used in our experiments, is insufficient to suppress growth hormone. 36 Thus it is unlikely that regulation of growth hormone by angiopeptin has i significant role in the inhibition of myointimal thickening in this study. The remaining two studies reported here show that angiopeptin treatment of rabbits inhibits thymidine uptake by injured vessels excised 72 hours after injury. First, injured aorta segments were excised 72 hours after injury and then incubated in vitro with 3H-thymidine. Both thymidine incorporation and DNA content were significantly reduced byangiopeptin. Second, 3H -thymidine was administered in vivo
1090 Foegh et al.
to angiopeptin-treated rabbits 48 hours after injury and segments of these aorta were used for autoradiography. Counting of thymidine-labeled cells confirmed that angiopeptin treatment decreased cell proliferation. Inhibition of myointimal thickening can relate to decreased extracellular matrix formation and inhibition of both migration and smooth muscle cell proliferation. The thymidine incorporation studies evaluated the events 72 hours after balloon injury and may not include the period of smooth muscle cell migration from the media to the intima, because in studies by others this migration is reported to occur in rats at days 3 to 4. 37,38 The thymidine uptake studies show that angiopeptin inhibits mitogenesis, because the number of smooth muscle cells as shown by counting of labeled cells and DNA content, and thymidine incorporation in the aorta are reduced in angiopeptin-treated animals. In conclusion, angiopeptin, a stable octapeptide analogue of somatostatin inhibits myointimal thickening and smooth muscle cell proliferation induced by balloon injury. The inhibition is independent of dose over a wide dose range. The need for only short-term treatment and the lack of efficacy of delayed treatment suggests that angiopeptin inhibits expression of early events such as induction of early response genes, triggering smooth muscle cell proliferation. We thank Dominique Y. Pifat, PhD, and Denise Cook for their invaluable assistance. REFERENCES 1. Kent KM. Restenosis after percutaneous transluminal coronary angioplasry. Am J CardioI1988;61:67G-70G. 2. Klein LW, Rosenblum J. Restenosis after successful percutaneous transluminal coronary angioplasty. Prog Cardiovasc Dis 1990;32:365-82. 3. Hermans WR, Rensing BJ, Strauss BH, Serruys PW. Prevention of restenosis after percutaneous transluminal coronary angioplasty: the search for a "magic bullet." Am Heart J 1991;122:171-87. 4. Knudtson ML, Flintoft VF, Roth DL, Hansen JL, DuffHJ. Effect of short-term prostacyclin administration on restenosis after percutaneous transluminal coronary angioplasty. JACC 1990;15:691-7. 5. Pepine CJ, Hirshfeld JW, Macdonald RG, et al. A controlled trial of corticosteroids to prevent restenosis after coronary angioplasty. Circulation 1990;81: 1753-61. 6. Popma JJ, Califf RM, Topol EJ. Clinical trials of restenosis following coronary angioplasty. Circulation 1991;84:142636. 7. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature 1993;362:101-9. 8. Alberti KG, Christensen NT, Christensen SE, et al. Inhibition of insulin secretion by somatostatin. Lancet 1973;2:1299301.
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9. Bloom SR, Polak. JM. Somatostatin. Brit Med J 1987;295: 288-90. 10. Flyvbjerg A, Jorgensen KD, Marshall SM, Orskov H. Inhibitory effect of octreotide on growth hormone-induced IGF-I generation and organ growth in hypophysectomized rats. Am J PhysioI1991;260:E568-74. 11. Parmar H, Bogden A, Mollard M, de Rouge B, Phillips RH, Lightman SL. Somatostatin and somatostatin analogues in oncology. Cancer Treat Rev 1989;16:95-115. 12. Payan DG, Hess CA, Goetzl EJ. Inhibition by somatostatin of the proliferation ofT-lymphocytes and molt-4Iymphoblasts. Cell Immunol 1984;84:433-8. 13. Hierowski MT, Liebow C, du Sapin K, Schally AV. Stimulation by somatostatin of dephosphorylation of membrane proteins in pancreatic Gancer MIA PaCa-2 cell lines. FEBS Lett 1985;179:252-6. 14. Bogden A, Taylor J, Moreau JP, Coy DH, LePage DJ. Response of human lung tumor xenografts to treatment with a somatostatin analogue (somatuline). Cancer Res 1990;50: 4350-65. 15. Coy DH, Heiman ML, Rossowski J, et al. Receptor selective somatostatin (SRIF) analogues. In: Marshall GR, ed. Peptides, chemistry and biology. Leiden, ESOM, 1988:46264. 16. FoeghML, Khirabadi SS, Chambers E, Amamoo S, Ramwell PW. Peptide inhibition of coronary artery transplant atherosclerosis in rabbits with angiopeptin, an octapeptide. Atherosclerosis 1989;78:229-36. 17. Lundergan C, Foegh ML, Vargas R, et al. Inhibition of myointimal proliferation of the rat carotid attery by the peptides angiopeptin and BIM 23034. Atherosclerosis 1989; 80:49-55. 18. Conte JV, Foegh ML, Calcagno D. Peptide inhibition of myointimal proliferation following angioplasty in rabbits. Transpl Proc 1989;21:3686-8. 19. Burton K. A Study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:315-23. 20. Lindner V, Olson NE, Clowes AW, Reidy MA. Inhibition of smooth muscle cell proliferation in injured rat arteries. J Clin Invest 1992;90:2044-9. 21. Clowes AW, Clowes MM. Kinetics of cellular proliferation after atterial injury: heparin inhibits rat smooth muscle mitogenesis and migration. Circ Res 1986;58:839-45. 22. Banskota NK, Taub R, Zellner K, Olsen P, King GL. Characterization of induction of protooncogene c-myc and cellular growth in human vascular smooth muscle cells by insulin and IGF-I. Diabetes 1989;38:123-9. 23. Pukac LA, Ottlinger ME, Kamovsky MJ. Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells. J Bioi Chern 1992;267:37{}7-11. 24. Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science 1991;253: 1129-32. 25. Olsen NE, Chao S, Lindner V, Reidy MA. Rapid communication: intimal smooth muscle cell proliferation after balloon catheter injury. Am J PathoI1992;140:1017-23. 26. Lindner V, Majack RA, Reidy MA. Role of basic fibroblast growth factor in vascular lesion formation. Circ Res 1991; 68:106-13. 27. Wilks AF: Structure and function of the protein tyrosine kinases. Prog Growth Factor Res 1990;2:97-111.
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28. Lee MT, Liebow C, Kamer AR, Schally AV. Effects of epidermal growth factor and analogues of luteinizing hormone-releasing hormone and somatostatin on phosphorylation and dephosphorylation of tyrosine residues of specific protein substrates in various tumors. Proc Nat! Acad Sci USA 1991;88:1656-60. 29. Colas B, Cambillau C, Buscail L, et al. Membrane tyrosine phosphatase activity is regulated by somatostatin analogues in rat pancreatic acinar cells. Eur J Biochem 1992;207:1017-24. 30. Pfeifle B, Horst B, Ditschunseit H. Interaction of receptors for insulin-like growth factor I, platelet-derived growth factor, and fibroblast growth factor in rat aortic cells. Endocrinology 1987;120:2251-8. 31. Pfeifle B, Ditschuneit HH, Ditschuneit H. Binding and biological actions of insulin-like growth factors on human smooth muscle cells. Horm Metab Res 1982;14:409-14. 32. Sidawy AN, Termanini B, Nardi RV, Harmon JW, Korman LY. Insulin-like growth factor I receptors in the arteries of the rabbit: autoradiographic mapping and receptor characterization. Surgery 1990;108:165-71. 33. Moreau SC, Murphy W A, Coy DH. Comparison of Lanreotide (BIM-23014) and somatostatin on endocrine and exocrine activities in the rat. Drug Dev Res 1991;22:79-93.
Foegh et at.
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Submitted Aug. 26, 1993; accepted Nov. 8, 1993.
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