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Stroke. 2001;32:1665-1672

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(Stroke. 2001;32:1665.)
© 2001 American Heart Association, Inc.

Original Contributions

Broad-Spectrum and Selective Serine Protease Inhibitors Prevent Expression of Platelet-Derived Growth Factor–BB and Cerebral Vasospasm After Subarachnoid Hemorrhage

Vasospasm Caused by Cisternal Injection of Recombinant Platelet-Derived Growth Factor–BB

Z. Zhang, MD, PhD; I. Nagata, MD, DMSc; H. Kikuchi, MD, DMSc; J-H. Xue, MD, PhD; N. Sakai, MD, DMSc; H. Sakai, MD, DMSc H. Yanamoto, MD, DMSc

From the Laboratory for Cerebrovascular Disorders, Research Institute of National Cardiovascular Center (Z.Z., J-H.X., H.Y.), Department of Neurosurgery (I.N., N.S., H.S., H.Y.), and National Cardiovascular Center (H.K.), Osaka, Japan.

Correspondence to Hiroji Yanamoto, MD, DMSci, Laboratory for Cerebrovascular Disorders, Research Institute of National Cardiovascular Center, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. E-mail hyanamot{at}res.ncvc.go.jp


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background and Purpose—Plasma serine protease cascade, including the complement system and thrombin, is activated in the subarachnoid space during the acute phase after subarachnoid hemorrhage (SAH). To examine the effect of protease cascade–based inflammation and subsequent vascular repair in the development of cerebral vasospasm, we examined the effect of 2 synthetic serine protease inhibitors—FUT-175, an inhibitor of thrombin and the complement system, and argatroban, a selective inhibitor of thrombin—on the development of cerebral vasospasm in a rabbit SAH model.

Methods—One hundred Japanese White male rabbits were used in the study. The SAH was simulated by a single injection of autologous arterial blood into the cisterna magna. To evaluate the development of cerebral vasospasm, the caliber of the basilar artery was measured on x-ray film before and at 2 days after SAH. Nine groups of rabbits (n=6 each) were treated with continuous intravenous injection of FUT-175 (2.5, 5, 10, or 20 mg/d), argatroban (1.25, 2.5, or 5 mg/d), or the same amount of saline (vehicle) for 48 hours, starting 40 minutes after SAH. Two days after SAH, the expression of homodimer of platelet-derived growth factor–BB (PDGF-BB) in the basilar artery was examined with immunohistochemical techniques. In 20 normal rabbits, 5 µg of recombinant PDGF-BB or vehicle was injected into the cisterna magna, and the basilar arteries were examined on angiograms for 48 hours.

Results—Significant differences were observed in the caliber of the basilar arteries between the vehicle group and the groups with the 3 larger doses of FUT-175 (vehicle, 52±5.0%; 5 mg, 79±5.7%; 10 mg, 80±2.5%; 20 mg, 80±3.7%) and between the vehicle group and the groups with the 2 larger doses of argatroban (vehicle, 52±6.4%; 2.5 mg, 81±9.0%; 5 mg, 85±4.1%) (P<0.05). In the histological examination, administration of effective doses of FUT-175 or argatroban suppressed the expression of PDGF-BB in the endothelial and medial smooth muscle cell layers. Exogenous PDGF-BB caused delayed and prolonged vasoconstriction on normal basilar arteries.

Conclusions—Activation of the serine protease cascade and/or thrombin after SAH was demonstrated to play an essential role in the development of cerebral vasospasm. The expression of PDGF-BB–like protein in the arterial walls correlated with the development of cerebral vasospasm. Elevated PDGF-BB level in the subarachnoid space was found to induce delayed and chronic vasoconstriction.


Key Words: cerebral vasospasm • growth factors • subarachnoid hemorrhage


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cerebral vasospasm after subarachnoid hemorrhage (SAH) remains an unresolved process that causes delayed cerebral ischemia with morbidity or mortality in addition to primary brain damage due to SAH.1 Numerous studies have hypothesized that delayed induction of vasoconstrictive substances, supposedly spasmogen(s) released in the subarachnoid space, affects cerebral arteries surrounded by thick blood clot and causes sustained vasoconstriction.2 3 In clinical treatment, a calcium antagonist was shown to improve overall outcome within 3 months of aneurysmal SAH; however, the intermediate factors by which the calcium antagonist exerts its beneficial effect for vasospasm remain unclear.4 Thus, despite the recent substantial progress in our understanding of cerebral vasospasm, including the alterations of gene expressions, production or depletion of chemical mediators, and structural abnormalities in cerebral arteries after SAH, development of a clinically effective inhibitor targeting a key etiology of cerebral vasospasm remains a challenge for basic researchers.3 5

The intracellular analysis of prolonged narrowing of cerebral vessels has demonstrated that such narrowing accompanies components of vasculopathy, which differs from the physiological vasoconstriction that occurs under normal conditions.6 7 In the pathological characteristics of cerebral arteries, attacks of inflammatory responses on the cerebral arteries after SAH have been implicated in the pathogenesis of cerebral vasospasm.8 9 Plasma protease cascades precede initial noncellular inflammatory responses comprising the coagulation, fibrinolytic, and complement systems in the acute phase in the subarachnoid space after SAH.10 11 12 13 14 These responses are considered to initiate a process intended to repair injured cerebral arteries after SAH. In a rabbit SAH model, we used a specific broad-spectrum, nonselective serine protease inhibitor, FUT-175 (nafamostat mesylate), to study the role of the activated protease cascade in the development of cerebral vasospasm, and we used argatroban, a selective serine protease inhibitor for thrombin, to study the role of thrombin, a component of the protease cascade. In addition, expression of platelet-derived growth factor–BB (PDGF-BB), a growth factor possibly induced by thrombin activation, was studied on the arterial wall after SAH.15 16


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
One hundred male Japanese White rabbits weighing 3.0 to 3.5 kg (Shimizu Experimental Animal, Kyoto, Japan) were used. The experimental protocols were approved by the Animal Research Committee at the Research Institute of the National Cardiovascular Center. All efforts were made to minimize suffering and the number of animals used.

Subarachnoid Hemorrhage
Rabbits were anesthetized with an intramuscular injection of 150 mg (3 mL) of ketamine (Ketalal, Sankyo Co) and 20 mg (1 mL) of xylazine (Seractal, Bayer Co). SAH was simulated by transcutaneous single injection of autologous nonheparinized arterial blood. One milliliter of fresh arterial blood, aspirated via a catheter inserted in the right femoral artery, was injected into the cisterna magna with the animal in a tilted, head-down, prone position. Before the injection, 1.0 mL of cerebrospinal fluid (CSF) was removed. After the injection, the animal was maintained in the same position for 40 minutes to allow the injected blood, following a mixture with CSF, to completely coagulate around the targeted basilar artery.

Angiographic Examination
Rabbits were anesthetized with intramuscular anesthetic (as described above) with additional local subcutaneous anesthesia (0.5% lidocaine) at the right femoral region. An angiographic 4F catheter (designed by H.Y.; Medikit Co) was inserted from the right femoral artery into the left vertebral artery under fluoroscopy. Iopamidol (0.5 mL; 300 mg of iodine per 1.0 mL) was injected manually to take a frontoposterior vertebrobasilar angiogram at a constant exposure (50 kV, 8 mA) and at a constant magnification with medical x-ray film (Konica NIF, new A, No. 20289).8 17 Angiography was performed before SAH (day 0) to determine the baseline caliber of the basilar artery and then repeated 48 hours after SAH (day 2). The caliber of the basilar artery was measured at 3 corresponding cross sections (the midpoint of the basilar artery and 0.5 cm above and below) in a blind manner with the use of a computer-assisted image analysis system (SD-510C, WACOM). The mean value of the 3 measurements was expressed on days 0 (before SAH) and 2.

Inhibitory Drugs Treatment Protocol
The synthetic serine protease inhibitor with broad-spectrum FUT-175 (nafamostat mesylate)18 19 (a gift from Torii Pharmaceutical Co Ltd, Tokyo, Japan) competitively and specifically blocks the active sites of multiple serine proteases, including complement components Clr, Cls, B factor, and D factor; a component of the coagulation system, thrombin; and a component of the fibrinolytic system, plasmin.18 19 The serine protease inhibitor argatroban (a gift from Mitsubishi Chemical Co, Yokohama, Japan) competitively and selectively blocks the active site of thrombin.20 21 Fifty-four rabbits were randomly divided into 9 groups (n=6 each) and used for experiments 1 and 2.

Experiment 1
FUT-175 (2.5, 5, 10, 20 mg/d) or the same volume (480 µL) of saline (vehicle) was infused intravenously via an osmotic minipump (Alzet, model 2 ML1) for 48 hours, which was designed to infuse at a speed of 10 µL/h. The minipump was implanted under the femoral skin with the tip of the infusion catheter set in the proximal right femoral vein under a surgical microscope starting 40 minutes after the induction of SAH. No obstruction of the infusion system was confirmed in each rabbit at the time of euthanasia on day 2.

Experiment 2
Argatroban (1.25, 2.5, 5 mg/d) or the same volume (480 µL) of saline (vehicle) was infused with the same intravenous infusion system described above. Argatroban or vehicle was infused continuously for 48 hours, and the absence of catheter obstruction was confirmed at the time of euthanasia on day 2.

In each animal in experiments 1 and 2, the formation of thick SAH around the basilar artery was confirmed at the time of euthanasia. The protocol for FUT-175 or argatroban was designed to inhibit targeted serine proteases in consideration of pharmacological properties and kinetics reported from experimental and human clinical studies.18 19 21 22

Physiological Parameters During Angiographic Examinations
Physiological parameters were monitored to confirm that no parameters were affected by the anesthesia used for the angiographic examinations or by the drug administrations in a separate set of 12 animals. Blood pressure, heart rate, blood pH, blood gases (O2, CO2), and blood glucose levels were monitored during angiographic examinations before and at 48 hours after SAH in groups treated with the highest volumes of FUT-175 (20 mg/d) and argatroban (5 mg/d) and in a group without drug treatment (n=4 each).

Immunohistochemical Analysis
All SAH rabbits used for angiographic examination and another 6 normal rabbits were used for immunohistochemical analysis with the use of the labeled streptavidin-biotin (LASB kit, Dako) method. Forty-eight hours after SAH, rabbits were deeply anesthetized and perfused with 500 mL of PBS at a pressure of 110 mm Hg. The brain was removed with vertebrobasilar arteries in the clot and embedded in methyl Carnoy’s solution (60% methanol, 30% chloroform, 10% acetic acid). Cross sections of brain stem with surrounding arteries were processed for immunohistochemistry by indirect immunoperoxidase with the murine monoclonal antibody PGF-007 (donated by Mochida Pharmaceutical, Tokyo, Japan). The antibody was produced against PDGF-B chain.15 23 The specificity of this antibody for PDGF-BB has been reported elsewhere.24

Direct Effects of PDGF-BB on Normal Basilar Arteries In Vivo
In a separate set of 20 rabbits, the effects of PDGF-BB on the vascular tone of the normal basilar artery were studied. Five micrograms of recombinant rat PDGF-BB (R&D Systems, Inc) or vehicle (1 mL of saline), both in 0.2% rat albumin as a carrier protein for PDGF-BB, was injected into the cisterna magna (n=10 each). Angiography was performed before and 15 minutes, 1 hour, 6 hours, 24 hours, and 48 hours after the injection of PDGF-BB with the use of a mobile digital imaging system (series 9600, OEC Medical Systems Inc). During the angiographic examinations, blood pressure, blood pH, and blood gases were monitored. The caliber of each basilar artery was assessed in a blind manner on the angiographic images with the use of the computer-assisted image analysis system.

Effects of Drug Administrations on Normal Cerebral Arteries
The effects of the chronic drug administrations on the vascular tone of normal basilar arteries were studied in a separate set of 8 animals. The highest volumes of FUT-175 (20 mg/d) or argatroban (5 mg/d) were administered for 48 hours by the method described above, and angiographic examinations were performed with physiological monitoring before and after drug administration for 48 hours in the absence of SAH (n=4 each).

Statistical Analysis
The data were analyzed by 1-way ANOVA. If multiple comparisons were indicated, the Student-Newman-Keuls test was applied. To compare 2 groups at the same time point, the unpaired 2-tailed t test was used. The results are presented as mean±SEM. The difference was considered significant at P<0.05.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Angiographic Examination
Experiment 1
Two days after SAH in the vehicle-treated group, the mean caliber of the basilar artery decreased to 52±5.0% of the baseline value (Figure 1Down, left paired panel, and Figure 2Down). In the groups treated with the larger 3 doses of FUT-175 (5, 10, or 20 mg/d), there were significant differences in the calibers of basilar arteries compared with those of the vehicle-treated groups on day 2 (P<0.05) (Figure 1Down, right 3 paired panels, and Figure 2Down). There were no significant differences between the vehicle-treated group and the group treated with the lowest dose of FUT-175 (2.5 mg/d) (Figure 2Down).



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  		Figure 1. Angiograms of a rabbit basilar artery. Severe vasospasm was demonstrated on day 2 in the vehicle-treated group (Ve). In contrast, reduced vasoconstriction was observed in the FUT-175–treated groups (FUT). There was no significant difference in the development of vasospasm between the 5- and 20-mg/d groups. L indicates low-dose group; M, medium-dose group; and H, high-dose group.



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  		Figure 2. Angiographic evaluation of cerebral vasospasm in rabbit basilar arteries. *There were significant reductions in the development of vasospasm in the groups treated with the larger 3 volumes of FUT-175 but not in the group treated with the lowest volume (2.5 mg/d) compared with the vehicle-treated group.

Experiment 2
Two days after SAH in the vehicle-treated group, the mean caliber of the basilar artery decreased to 52±6.4% of the control value (Figure 3Down, left paired panel, and Figure 4Down). In argatroban-treated groups on day 2, there were significant differences in the calibers of basilar arteries in the medium- to high-dose (2.5 to 5 mg/d) groups but not in the low-dose (1.25 mg/d) group (P<0.05) (Figure 3Down, right 3 paired panels, and Figure 4Down).



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  		Figure 3. Angiograms of a rabbit basilar artery. Severe vasospasm was demonstrated on day 2 in the vehicle-treated group (Ve). Reduced development of vasospasm was observed in the medium-dose (M) (2.5 mg/d) and high-dose (H) (5 mg/d) argatroban-treated groups (Arg) on day 2 but was not apparent in the low-dose group (L) (1.25 mg/d).



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  		Figure 4. Angiographic evaluation of cerebral vasospasm in rabbit basilar artery. *There were significant reductions in the development of vasospasm in the medium-dose and high-dose (2.5 to 5 mg/d) argatroban-treated groups but no significant reduction in the low-dose (1.25 mg/d) group compared with caliber in the vehicle-treated group.

Physiological Parameters During Angiographic Examinations
Physiological parameters monitored during angiographic examinations are shown in Table 1Down. There were no significant differences in any of the parameters between the groups on day 2 and between before and 2 days after SAH.


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  		Table 1. Physiological Parameters During Angiographic Examinations With or Without Treatment With FUT-175 or Argatroban After SAH

Immunohistochemical Analysis
Figure 5Down shows the results of immunostaining for PDGF-BB on the cross sections of basilar arteries 2 days after or without SAH (normal control). Two days after SAH, strong immunoreactivity for PDGF-BB was observed in all layers: the endothelial cell, smooth muscle cell, and adventitial cell layer in the vehicle-treated SAH group (Figure 5Down, top panel). In contrast, in normal basilar artery, there was faint or no immunoreactivity detected for PDGF-BB (Figure 5Down, bottom panel). The enhanced immunoreactivity was observed mainly in the nuclei of endothelial and smooth muscle cells. In contrast, in groups treated with 3 different doses of FUT-175 (Figure 6Down), the immunoreactivity in the endothelial and smooth muscle cells was weakly positive compared with that of vehicle-treated vessels (Figure 5Down, top panel, and Figure 6Down). Furthermore, the level of immunoreactivity in the smooth muscle cells correlated well with the degree of vascular constriction indicated by corrugation of the internal elastic lamina (Figure 6Down).



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  		Figure 5. Immunostaining with the monoclonal antibody to PDGF-BB on cross sections of rabbit basilar artery. On day 2 after SAH, the basilar artery expressed vasoconstriction by winding of the internal elastic lamina (top). The nuclei of endothelial cells, smooth muscle cells, and fibroblasts in the adventitial layer were immunopositive for PDGF-BB. In contrast, there was no immunostaining in the normal basilar artery in any layer without SAH (bottom) (magnification x600).



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  		Figure 6. In the groups treated with FUT-175 (FUT) 5 mg/d (top), 10 mg/d (middle), or 20 mg/d (bottom) on day 2, the immunoreactivity for PDGF-BB was positive but weak compared with that of the vehicle-treated SAH group (Figure 5Up, top panel) on cross sections of rabbit basilar artery. The immunoreactivity for PDGF-BB was detected in the nuclei of smooth muscle cells. When the vasoconstriction was moderate (top, with winding internal elastic lamina and thickened vascular wall), the endothelial cell expressed immunoreactivity. In contrast, when the vasoconstriction was mild (bottom, with less winding internal elastic lamina), endothelial cells were immunolucent (magnification x600).

Figure 7Down shows the immunostaining in the argatroban-treated groups. The level of immunoreactivity for PDGF-BB was highest in the low-dose (1.25 mg/d) and lowest in the high-dose (5 mg/d) argatroban-treated group. The immunoreactivity for PDGF-BB in the endothelial and smooth muscle cells correlated well with the degree of vascular constriction and also correlated well with the degree of vasoconstriction demonstrated on angiographic examinations (Figure 7Down). Replacing the primary antibody with nonimmune murine IgG completely abolished the positive immunostaining (data not shown).



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  		Figure 7. In the groups treated with argatroban (Arg) 1.25 mg/d (top), 2.5 mg/d (middle), or 5 mg/d (bottom) on day 2, the immunoreactivity for PDGF-BB was positive but weak compared with that of the vehicle-treated SAH group (Figure 5Up, top panel) on cross sections of rabbit basilar artery. Immunoreactivity for PDGF-BB was detected in the nuclei of smooth muscle cells. When vasoconstriction was moderate (top, with winding internal elastic lamina and thickened vascular wall), the endothelial cell expressed immunoreactivity. In contrast, when the vasoconstriction was mild (bottom, with less winding internal elastic lamina), endothelial cells were immunolucent (magnification x600).

Direct Effects of PDGF-BB on Normal Basilar Arteries In Vivo
The caliber of the basilar arteries slowly decreased after the injection of recombinant PDGF-BB (Figure 8Down). There were significant differences in calibers between the vehicle and PDGF-BB–injected groups at 1 hour, 6 hours, and 24 hours but not at 15 minutes or 48 hours after the injection. The development of vasoconstriction was delayed and was gradually enhanced. The peak of the chronic and long-lasting vasoconstriction was observed 24 hours after the cisternal injection of recombinant PDGF-BB. Regarding the physiological parameters, there were no significant differences between PDGF-BB or vehicle injection (Table 2Down).



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  		Figure 8. Time course of the angiographic caliber of the basilar arteries after a cisternal injection of recombinant PDGF-BB or vehicle. There was no vasoconstriction soon after the injection of PDGF-BB; however, the caliber was significantly decreased beginning at 1 hour after the injection. The significant difference lasted for approximately 24 hours, with a peak at 24 hours after the injection.


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  		Table 2. Physiological Parameters During Angiographic Examinations After Injection of PDGF-BB or Vehicle

Effects of Chronic Drug Administrations on Normal Cerebral Arteries
After 48 hours of continuous intravenous administration with 20 mg/d FUT-175 or 5 mg/d argatroban, the calibers of the basilar arteries were 100±1.8% and 101±1.7%, respectively, compared with the baseline calibers. Calibers of the basilar arteries were not affected and remained consistent after the drug administrations of FUT-175 or argatroban in normal rabbits. Physiological parameters were within the normal range and were not significantly different from the values monitored before drug administration (data not shown).

Side Effects of Drug Treatment
There were no functional impairments or mortalities during treatment after SAH. In human clinical studies, the side effects of these compounds have been fully clarified in the clinical phase I trials and were within the acceptable range for clinical use.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
An inflammatory defense reaction against a coagulated clot (non-self) in the subarachnoid space is differentiated into protein (protease)-based and subsequent infiltrated cells–based inflammation.8 After SAH, the coagulated clot around the cerebral arteries triggers the initial protease-based noncellular inflammation in the subarachnoid space.8 The protease-based inflammation proceeds in spiral and dynamic activations of interrelated multiple cascades of the complement, coagulation, fibrinolytic, and kallikrein-kinin systems.25 26 These closely linked systems that comprise multiple proteases have cascades with which to stimulate and activate each other to exert local inflammatory responses. The levels of activated proteases in these systems reportedly elevate in the CSF during the acute phase after SAH, which is considered to be an etiologic factor of cerebral vasospasm.10 12 13 14 27 FUT-175 inhibits complement factors thrombin and plasmin and suppresses the protease-based inflammation.18 19 FUT-175 has been used for disseminated intravascular coagulation in clinical practice.22 Before the present study, it was reported that intermittent intravenous administration of FUT-175 prevented the development of experimental and clinical vasospasm after SAH17 28 29 ; however, an overdose phenomenon (>4 mg/d) was suggested by the intermittent administration in rabbits.17 The present study examined the effect of FUT-175 using continuous (slow) intravenous administration and demonstrated that larger doses of 5 to 20 mg/d were similarly effective in preventing cerebral vasospasm.

Argatroban, a selective thrombin inhibitor, effectively inhibits thrombin-induced cleavage of fibrinogen, factor V, factor VIII, and protein C, as well as the initiation of platelet aggregation, and it has been used in the treatment of acute cerebroarterial thrombosis and other chronic vascular occlusive diseases in clinical practice in Japan (the US Food and Drug Administration approved this drug for the treatment of heparin-induced thrombocytopenia in October 2000).30 Thrombin had been considered an enzyme belonging solely to the coagulation system; however, it is now recognized as a multifunctional organizer leading to a healing process from inflammation, and it stimulates vascular endothelial cells, smooth muscle cells, fibroblasts, neutrophils, and macrophages.31 32 33 After SAH, it has been reported that levels of thrombin-antithrombin III complex and F1+2, molecular markers of thrombin activation in the CSF, were elevated and correlated well with the clinical severity of SAH at the onset and occurrence of cerebral vasospasm.11 12 34 35 Recently, it has been reported that intrathecal placement of collagen pellets releasing thrombin inhibitor prevented the development of canine vasospasm.36 Although vascular endothelial and smooth muscle cells are capable of expressing thrombin receptor,37 and thrombin causes the immediate contraction of smooth muscle cells via activated thrombin receptor,38 39 the peak thrombin-antithrombin III complex concentration in the CSF of SAH patients was apparently earlier (on days 2 to 5) than the development of human cerebral vasospasm, which indicated that thrombin activation and its vasoconstrictive effect were not a direct cause of cerebral vasospasm.34 39

In the search for factors downstream to thrombin activation due to SAH, elevated immunoreactivity for PDGF-BB was first demonstrated in the endothelial and smooth muscle cells of the basilar artery after SAH. Regulations of PDGF production in smooth muscle cells by thrombin have been reported.16 40 Furthermore, it has been reported that levels of PDGF-BB concentration in the CSF in the acute phase of SAH patients were significantly higher in patients with rather than without symptomatic vasospasm.41 Direct vasoconstrictive effects on arteries by exogenous PDGF are controversial in the literature. Recombinant PDGF has caused potent concentration-dependent contraction on rat aortic strips within 2 minutes after the application; however, it did not constrict rat intracerebral arterioles (perforating arteries) in vitro.42 43 44 In the present study it was first demonstrated that exogenous PDGF-BB caused delayed and prolonged but not rapid vasoconstriction on cerebral arteries in rabbits. The peak of the vasoconstriction (24 hours) was earlier than that of vasospasm in rabbits (48 hours) caused by blood injection17 ; however, it can be speculated that a time delay of approximately 1 day is due to a phase for the active production of PDGF-BB after thrombin activation in the subarachnoid space or within the vascular wall.

The preventive effects on the developing cerebral vasospasm by intravenous administration of the 2 serine protease inhibitors reduced the immunoreactivity for PDGF-BB in vessel walls as well as cerebral vasospasm. These results were in agreement with the previous study in that an effective inhibition of neointimal formation was achieved by administration of FUT-175, and the preventive effects correlated well with the reduced expression of PDGF-BB in the neointimal as well as smooth muscle cells after balloon (stretching) injury to rat carotid arteries.45 Although the pathophysiology of the proliferative or constricting abnormalities, after injury or bleeding-derived inflammation, is considered different, both of these vascular responses may have a common cascade through PDGF-BB expression linked to a healing process. It was striking that cerebral vasospasm, which normally does not accompany significant proliferation of smooth muscle cells at the peak of vasospasm, expressed strong PDGF-BB–like immunoreactivity in the arterial wall after SAH. In general, growth factors are produced after inflammation to initiate repair injury.31 It is postulated that SAH triggered the activation of a protease cascade, which in turn triggered the production of the growth factor PDGF-BB, and the accumulated PDGF-BB in the smooth muscle cells, via the autocrine or paracrine mechanism,46 47 48 caused delayed and prolonged vasoconstriction. PDGF-BB or its metabolite in smooth muscle cells is a new candidate for spasmogen or inducer of pathological vascular remodeling. This could explain why vascular narrowing is delayed and prolonged. In this context, a significant stretching injury on cerebral arteries, possibly caused by SAH or surgery, may enhance cerebral vasospasm through PDGF-BB production. Cerebral vasospasm was suggested to occur during the healing process in cerebral arteries after SAH.


*    Acknowledgments
 
This study was supported by Japan Health Sciences Foundation, Special Coordination Funds for Promoting Science and Technology, and by a grant-in-aid for scientific research from the Ministry of Education, Culture, Science, and Technology of Japan. Dr Zhang was supported in part by Japan-China Sasakawa Medical Fellowship. We acknowledge the valuable assistance of Atsuko Okuno, Tomoko Yasuda, and Mihoko Kurimoto.

Received May 24, 2000; revision received February 27, 2001; accepted March 20, 2001.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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