The search for a novel therapy for endothelial regenerating is an area of intensive investigation. Recent experimental and clinical evidence strongly suggests that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) have several physiological effects independent of low-density lipoprotein cholesterol reduction. We here report that the carotid arterial blood flow after endothelial injury in hamsters treated with simvastatin was restored, in contrast to the situation in nontreated hamsters. Histologic observations showed a prompt recovery of endothelial cells with a much higher DNA synthesis index in repaired endothelium of hamsters treated with simvastatin. The amount of secreted vascular endothelial cell growth factor (VEGF) by cultured vascular smooth muscle cells from hamsters treated with simvastatin was significantly increased. Mevalonate reduced the amount of VEGF secretion by simvastatin in vitro. Finally, an injection of either an anti-VEGF antibody or an anti-VEGF receptor-1 (Flt-1) antibody, but not anti-VEGF receptor-2 (Flk-1), reduced the prompt endothelial healing. Simvastatin regulates endothelial regenerating by an over-release of VEGF and by this may result in prompt endothelial healing after vascular injury. Our results provide new insights into the role of statin and VEGF in the pathogenesis of vascular diseases.
Statins, 3-hydroxy-3-methyl-coenzyme A (HMG-CoA) reductase inhibitors, are widely prescribed to lower cholesterol in hyperlipidemic patients at risk for cardiovascular diseases. 1 It has been recognized that the protective effects of these drugs extend to myocardial infarction patients with average cholesterol concentrations, 2 and that lipid reduction alone cannot entirely account for the benefits of statin therapy. 3 In experiments using normocholesterolemic animals, statin therapy has been shown to protect against stroke, 4 stimulation of the antithrombotic system, 5,6 ischemic heart injury, 7 and vascular inflammatory responses. 8 Moreover, statin therapy in patients has been found to rapidly improve vasomotor response to endothelium-dependent agonists 9 and to increase coronary blood flow. 10 These findings strongly suggested that statins might have various effects independent of cholesterol reduction.
On the other hand, the integrity of endothelial surface is essential for maintaining homeostasis between blood and surrounding tissues. Vascular endothelial growth factor (VEGF) is a potent mitogen displaying high specificity for endothelial cells. 11 It plays a major role in angiogenesis 12 and indeed, mice lacking one or both of the VEGF alleles die before birth and show defects in the development of the cardiovascular system. 13 Moreover, VEGF mediates vascular permeability, 14,15 endothelial chemotaxis, 16 endothelium-derived relaxing factor–dependent vasodilatation 17 and thrombogenicity. 18 Vascular injury is followed by endothelial cell proliferation and migration to repair the vascular surface and vessel wall. Studies show that VEGF is highly expressed in smooth muscle cells (SMCs) after endothelial denudation and that it accelerates reendothelialization. 19,20
The product of HMG-CoA reductase, mevalonate, is an important precursor for many isoprenoids, thereby endowing statins with the ability to directly alter cellular events other than cholesterol synthesis. Indeed, the isoprenoids farnesylpyrophosphate and geranylgeranylpyrophosphate play important roles, and survival via their attachment to critical signaling proteins, such as Ras and Rho. 21,22 Very recently, we reported that simvastatin induced VEGF secretion on A10 cells via p44/42 MAP kinase activation. 23 Therefore, in the present study, we investigate the role of simvastatin in vascular remodeling by using stenosis model of hamsters. Here, we report for the first time a crucial role for simvastatin following endothelial injury.
From the *Department of Pharmacology, Gifu University School of Medicine, Gifu, Japan; and †Division of Cardiology, Internal Medicine III, Hamamatsu University School of Medicine, Hammatsu, Japan.
Received for publication August 8, 2003; accepted October 28, 2003.
Reprints: Dr. H. Matsuno, Department of Pharmacology, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500-8705, Japan (e-mail address: firstname.lastname@example.org).
The study was performed according to the guidelines published in the international guidelines for the care of animal in experimentation.
Male hamsters (Gold, SLC, Sizuoka, Japan) weighing 90–110 g were used. All experiments were performed in accordance with institutional guidelines.
Recombinant mouse VEGF, anti-mouse VEGF, anti-mouse VEGF receptor-1 (Fit-1) and anti-VEGF receptor-2 (Flk-1) antibodies were purchased from R&D Systems (Minneapolis, MN). The other chemical substances were obtained from Sigma Chemical Co. (St. Louis, MO).
Experimental Endothelial Injury in Hamsters
The experimental procedure to induce an endothelial injury has been described in detail previously. 24,25 Hamsters were divided into 2 groups (n = 8 for each group): a control group and a group treated with simvastatin (2.0 mg/kg/day). Subcutaneous injection of simvastatin was started 2 hours before injury and continued 3 days after the initiation of injury. Animals were anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital. In brief, the right common carotid artery, the left jugular vein, and the right femoral artery were exposed. Catheters (ID = 0.5 mm, OD = 0.8 mm, polyethylene sp3, Natume Co., Tokyo, Japan) were connected to the left jugular vein and to the right femoral artery for the injection of Rose Bengal (20 mg/kg) and for monitoring blood pressure and pulse rate using a pressure transducer (AP601G Nihon Koden, Tokyo, Japan) during experiments on day 0. Blood flow in the carotid artery was continuously monitored using a Doppler flow probe (Model PDV-20, Crystal Biotech Co., Tokyo, Japan) positioned proximally to the injured area of the carotid artery. Irradiation by green light (540 nm) proximal to the flow probe was started, blood flow was monitored for 10 minutes to control its stability, and then a bolus injection of Rose Bengal was performed. The irradiation was continued for 15 minutes after the injection of Rose Bengal. This procedure results in destruction of endothelial cells in the irradiated area by oxygen radicals induced by the photochemical reaction between Rose Bengal and green light. Our previous histologic observations revealed that a platelet-rich thrombus including fibrin was formed when the blood flow was 0. 25 The flow probe was removed after the first observation (day 0) and replaced on each consecutive observation day (days 1, 2, and 3). The presence of an occlusive thrombus was detected when blood flow was 0. After recovery from anesthesia, the animals were kept in individual cages and fed standard chow (RC4, Oriental Yeast Co., Japan).
Electron Microscopic Analysis
Hamsters were killed by injection of an overdose of pentobarbital 24 or 72 hours after the initiation of endothelial injury. At the time of death, mice were exsanguinated and 2 mL of PBS was injected into the right jugular vein to perfuse the whole body. Carotid arteries were removed, incubated in a 4% formaldehyde or a 2% glutaraldehyde solution for 24 hours, and transferred to a solution containing 50 mM sodium phosphate buffer. The samples, fixed by glutaraldehyde, were cut longitudinally to allow visual inspection using scanning electron microscopy (SEM) as described. 23,24
Immunohistochemical Staining of vWF
Staining for vWF was used to detect repairing endothelial cells in the injured area of the murine vessels. Hamster (n = 4 each time point) were killed by injection of an overdose of pentobarbital before and 2 days after the initiation of endothelial injury. At the time of death, mice were exsanguinated and 2 mL of saline was injected into the right jugular vein to perfuse the whole body. Following removal of the carotid artery, frozen cross-sections were prepared. Endothelial cells were stained with a peroxidase-conjugated monoclonal anti-vWF antibody (P 0226, Dako Japan, Kyoto, Japan) and detected with diaminobenzidine (DAB). Sections were also stained for background with hematoxylin.
Index of Newly Synthesized DNA In Vivo
In separate animals, SMCs with newly synthesized DNA were identified by the thymidine analogue 5-bromo-2-deoxy-uridine (BrdU). 24,25 BrdU tests were performed at 1, 2, and 3 days after injury. BrdU (50 mg/kg) was injected subcutaneously 1, 8, 16, and 24 hours prior to removal of the carotid arteries. Following removal of the artery, frozen cross-sections were prepared from these arteries. BrdU-positive cells were stained with a murine monoclonal antibody (Sigma, St. Louis, MO), followed by goat antimouse Ig-antibodies conjugated to peroxidase, and the coloring reaction was performed using DAB and H2O2. Sections were also counterstained with hematoxylin. The number of positive and negative nuclei were counted in the media and newly formed intima. The BrdU labeling index was calculated using the following formula: (the number of positive nuclei stained with DAB)/(the number of total nuclei stained with hematoxylin) × 100.
Spontaneous Secretion of VEGF in Primary Cultured Cells
Vascular SMCs were obtained from the thoracic aorta of hamsters treated with simvastatin at a dose of 2 mg/kg/day or from nontreated hamsters. The cultured cells (1 × 105) were seeded into 35-mm-diameter dishes and maintained in 2 mL of Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum at 37°C in a humidified atmosphere of 5% CO2/95% air. After 6 days, the medium was exchanged for serum-free DMEM. After 48 hours, VEGF expression levels in the culture medium were measured by ELISA (Quantikine, VEGF immunoassay, R&D System). In separate experiments, cells were stimulated by platelet-derived growth factor (PDGF) at a dose of 30 ng/mL for 24 hours, and VEGF levels in the culture medium were measured by ELISA.
Effects of Mevalonate on VEGF Release In Vitro
In the separate experiments, vascular SMCs were obtained from the hamsters. The cultured cells were treated as mentioned above. Various doses of mevalonate were added in the cultured cells, and then cells were stimulated by simvastatin at a dose of 10 μM. The reaction was terminated by collecting the medium, and VEGF in the medium was measured by a VEGF enzyme immunoassay kit.
Treatment of Hamsters with anti-VEGF Antibodies or VEGF
Hamsters treated with simvastatin (3 groups of n = 6) received a bolus injection of either an anti-VEGF antibody (25 μg per body) or antibodies against VEGFR-1 or VEGFR-2, which are expressed almost exclusively on endothelial cells, 26 5 minutes before the initiation of endothelial injury in the hamsters. To define the effect of VEGF after endothelial injury in hamsters not treated with simvastatin, VEGF (8 ng per body, n = 6) was injected as a bolus a few minutes before the initiation of the injury. Vascular patency was observed until 3 days after injury.
All data are expressed as the mean ± SEM. The significance of the effect of each compound (* P < 0.01) was determined by ANOVA followed by the Student-Newman-Keuls test.
Simvastatin Restores the Blood Flow After Endothelial Injury
In hamsters treated with or without simvastatin, cyclic reocclusion and reflow were clearly observed in the artery on day 0 (the day of the initiation of endothelial injury) (Fig. 1). Spontaneous reperfusion was observed in 4 of 8 nontreated hamsters at the end of the observation period on day 0 (120 minutes after the initiation of injury). Twenty-four hours later (day 1), persistent occlusion was observed in 5 hamsters, and cyclic reocclusion and reflow were observed in 3 hamsters. At day 3, persistent patency was observed in 2 hamsters and the others did show cyclic reocclusion and reflow. Following reperfusion at day 0, the mean blood flow remained less than 49.3% of the baseline blood flow (Table 1). However, spontaneous reperfusion was observed in all hamsters treated with simvastatin on day 1. These flow patterns clearly changed at day 2 and cyclic reocclusion/reflow was markedly diminished. Persistent patency was observed in all hamsters at day 3, and reperfused blood flow recovered to 82.7% of baseline flow (Table 2).
Histologic Observation of Reendothelialization
Figure 2 shows reendothelialization of hamsters after the injury in hamsters treated with simvastatin (Fig. 2A, C, and E) or without simvastatin (Fig. 2B, D, and F). vWF antibodies were used for identification of endothelial cells and samples were stained by DAB (Fig. 2A–D). Two hours after injury, a few endothelial cells remained on the vascular surface after injury (Fig. 2A and B). No difference between control (without simvastatin) and hamsters treated with simvastatin could be observed. In contrast, the reendothelialization was markedly difference 48 hours after injury. Indeed, the endothelial cell layer was thickly covering the injured area in hamsters treated with simvastatin (Fig. 2C), whereas this was only partial in wild-type mice (Fig. 2D). As observed with SEM at 24 hours after injury, the injured vascular surface was not completely covered by repairing endothelial cells in control hamsters (Fig. 2F). On the other hand, in hamsters treated with simvastatin, supersaturation of endothelial cells was observed on the injured vascular surface (Fig. 2E).
Index of Newly Synthesized DNA In Vivo
Figure 3 shows the percentage of BrdU-positive cells in repairing endothelial cells or in the media on day 1, 2, or 3 after the initiation of the vascular injury. The treatment with simvastatin caused a significant increase in BrdU-positive cells. Typical observations of BrdU-positive cells in cross sections of arteries obtained 2 days after injury are shown in Figure 3C and D).
Spontaneous Secretion of VEGF in Cultured Cells Obtained from the Hamster Thoracic Aorta
Spontaneous release of VEGF was observed in vascular SMCs isolated from the thoracic aorta from both groups of hamsters. VEGF in the secreted culture medium was measured by ELISA at different time points (Fig. 4). The level of VEGF released from vascular SMCs of hamsters treated with simvastatin was significantly higher than that of cells from control hamsters. Moreover, when cells were stimulated with PDGF, the amount of VEGF released from vascular SMCs of hamsters treated with simvastatin was 2.4 times higher than that of cells from control hamsters.
Effects of Mevalonate In Vitro
Pretreatment with mevalonate reduced secretion of VEGF in cultured cells obtained from hamsters (Fig. 5).
Effect of Injection of Anti-VEGF Antibodies in Hamsters
In hamsters treated with simvastatin, intravenous injection of an anti-VEGF antibody did not improve mean arterial blood flow (Table 3) nor did it result in vascular patency after spontaneous reperfusion (Table 4). The repaired endothelial surface in hamsters treated with simvastatin was almost the same as the one in control hamsters (data not shown). Moreover, the action of VEGF is mediated by a particular family of receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (KDR), which are expressed almost exclusively on endothelial cells. 27 When an anti-VEGFR-1 antibody was administered to hamsters treated with simvastatin, vascular patency was not different as compared with that of control hamsters, whereas all hamsters treated with simvastatin after an injection of an anti-VEGFR-2 antibody showed a significant change in blood flow and vascular patency (Tables 3 and 4). On the other hand, vascular patency was not improved when VEGF was injected in hamsters before the initiation of endothelial injury. Mean arterial blood flows and vascular patency after spontaneous reperfusion are shown in Table 5.
The present study demonstrates that treatment with simvastatin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins), improves vascular patency after experimental endothelial injury and induces prompt healing of endothelial cells. These effects could be mainly explained by the over-release of VEGF in the injured vascular surface when hamsters are treated with simvastatin.
In our first experiment, we could demonstrate that the arterial blood flow after spontaneous reperfusion was restored in hamsters treated with simvastatin at a dose of 2.0 mg/kg/day, even though the time to occlusion was not significantly changed in both groups of hamsters. However, the status of spontaneous reperfusion in hamsters treated with simvastatin was improved on days 1, 2, and 3. Indeed, following reperfusion on day 0, the mean blood flow of hamsters remained less than 49.3% of the baseline blood flow. The blood flow measured after reperfusion, in hamsters treated with simvastatin, recovered to 82.7% of baseline flow on day 3 in contrast to the mean arterial blood flow in control hamsters, which only recovered to 61.3% at day 3. In both groups, levels of plasma lipids were not significantly changed (data not sown). These results indicate that simvastatin could affect vascular remodeling independent of plasma lipid reduction by statin.
Our histologic observations confirm the above-mentioned differences in both groups of hamsters. Time-dependent profiles of the BrdU index showed that cells with newly synthesized DNA were densely located in the repaired endothelial cells when studied 24 hours after the arterial injury in the hamsters treated with simvastatin. Moreover, immunohistochemical and electron microscopic observations clearly showed that reendothelialization occurred after vascular injury. The recovered vascular surface of hamsters treated with simvastatin was markedly different from that of nontreated hamsters, where repairing endothelial cells did not yet completely cover the injured area 72 hours after the injury. These results indicate that a significant regeneration of endothelial cells is rapidly induced by the treatment with simvastatin following experimental vascular injury.
It is well known that VEGF is a major regulator of endothelial cell production under both physiological and pathologic conditions. 28 Previous studies carried out in a variety of animal species have repeatedly shown that extensive endothelial denudation of the arterial wall leads to neointimal thicking. 29,30 Local delivery of VEGF to the site of vascular injury resulted in expeditious reendothlialization. 30 In the present study, the plasma concentrations of VEGF in both groups were almost the same before and after the initiation of endothelial injury (data not shown). However, we confirmed that spontaneous release of VEGF by cultured vascular SMCs from hamsters treated with simvastatin was significantly higher than that from cells from nontreated hamsters. Indeed, we previously showed that simvastatin induced secretion of VEGF via p44/42 MAP kinase activation 23 and, in the present experiment, treatment with mevalonate in cultured cells reduced the amount of secreted VEGF by simvastatin in a dose-dependent manner. These findings indicated that inhibition of HMG-CoA would directly affect VEGF secretion. Moreover, the amount of VEGF in the culture medium was much higher when SMCs were stimulated by PDGF. PDGF is secreted from activated platelets and plays a role in the region of the injured vascular wall. Therefore, we speculated that local platelet activation due to endothelial injury could induce conditions where PDGF might stimulate vascular SMCs. Under these conditions, PDGF will stimulate the secretion of VEGF and an effect of simvastatin can be expected. However, a recent study reported that statins interfere with angiogenesis and reduce proliferation of endothelial cells induced by VEGF. 31 More recently, it was demonstrated that statins show biphasic (namely, proangiogenic) effects at low concentrations and angiostatic effects at high concentrations. 32 Indeed, in our experiments, when a high dose of simvastatin of 20 mg/kg/day was administered to hamsters, vascular patency after spontaneous reperfusion was not improved and a prompt healing of endothelial cells after the vascular injury was not observed (data not shown). Therefore, a therapeutic dose of statins might lead to regeneration of endothelial cells after vascular injury.
To further define the physiological relation between simvastatin and VEGF in endothelial healing, we performed several additional experiments. When an anti-VEGF-antibody or an anti-VEGF receptor-1 antibody was administered to hamsters treated with simvastatin, no difference in the repair process of the vascular surface was observed when compared with the situation in nontreated hamsters. However, when an anti-VEGF receptor-2 antibody was administered, a significant difference in vascular patency was observed when simvastatin-treated hamsters were compared with nontreated hamsters. This result supports the previous observation that the effect of VEGF is mainly mediated via VEGFR-1. Previous reports showed that proliferation of endothelial cells in vivo is mainly regulated by VEGFR-1, but not by VEGFR-2. 32,33
A bolus injection of VEGF (8 ng per body) before the initiation of injury in hamsters not treated with simvastatin resulted in a low number of repaired endothelial cells in the injured area. The half-life of recombinant VEGF in the circulation is only minutes. Indeed, administration of recombinant VEGF has recently been shown to be ineffective also in man. 34,35 These findings clearly indicated that only local release of a high concentration of VEGF could promote a prompt and dense endothelial healing after vascular injury.
To the best of our knowledge, the present report is the first to describe an essential role of simvastatin in vivo within the framework of an event after endothelial injury. The physiological scenario of such a response might be as follows: first, the event of vascular injury locally induces acute thrombus formation containing many activated platelets. Under this condition, PDGF can play a role in vascular remodeling. Treatment with simvastatin then stimulates the secretion of VEGF and PDGF further stimulates the release of VEGF. VEGF is mainly released from vascular SMCs after vascular injury and physiologically regulates the repairing of endothelial cells. During this process, over-release of VEGF by the treatment with simvastatin induces prompt healing of endothelial cells. In the injured vascular surface in vivo, which greatly changes the rate of endothelial healing mainly via activation of VEGFR-1. VEGF is also known as a vascular permeability factor to induce vascular leakage. 36 Therefore, local highly elevated levels of VEGF may in addition induce vascular permeability, followed by VEGF over-secretion from SMCs stimulated by simvastatin. Indeed, our previous findings showed that over-secretion of VEGF in experimental acute myocardial infarction in mice increased the vascular permeability. 37
In conclusion, simvastatin improves the vascular patency after endothelial injury, which is mainly due to the enhancement of regeneration of endothelial cell via an over-release of VEGF. We therefore conclude that simvastatin has an important local physiological role in vascular remodeling. The findings in this report have identified a new target for the development of new therapeutics for the clinical therapy for cardiovascular diseases.
The work was supported by a Grant for Scientific Research (13670085) from the Ministry of Education, Science, Sports and Culture of Japan and by the grant provided by the Ichiro Kanehara Foundation.
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