Skip Navigation

Cardiovascular Research 2003 58(1):231-238; doi:10.1016/S0008-6363(02)00833-7
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Takeuchi, H.
Right arrow Articles by Kohno, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takeuchi, H.
Right arrow Articles by Kohno, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

Potentiation of C-type natriuretic peptide with ultrasound and microbubbles to prevent neointimal formation after vascular injury in rats

Hiroto Takeuchia, Koji Ohmoria,*, Isao Kondoa, Akira Oshitaa, Kaori Shinomiyaa, Yang Yua, Yuichiro Takagia, Katsufumi Mizushigea, Kenji Kangawab and Masakazu Kohnoa

aSecond Department of Internal Medicine, Kagawa Medical University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan
bDepartment of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita, Osaka 565-8565, Japan

* Corresponding author. Tel./fax: +81-87-891-2151. komori{at}kms.ac.jp

Received 6 September 2002; accepted 27 November 2002


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Long-term intravenous infusion of high-dose C-type natriuretic peptide (CNP) is known to prevent neointimal formation after vascular injury. Ultrasound (US) irradiation during microbubbles (MBs) infusion (US/MBs) has been used for local delivery of bioactive agents. We examined whether short-term infusion of CNP could also inhibit neointimal development and whether combined US/MBs treatment at the beginning of the CNP infusion could enhance its effect. Methods: In the rat carotid artery-balloon injury model, the intima/media area (I/M) ratio 14 days after injury was compared among various short-term post-injury treatments. For combined US/MBs, a commercial echocardiograph (1.8 MHz, mechanical index 1.0) and albumin-coated octafluoropropane gas MBs were used. Results: Infusion of high-dose CNP (1.0 µg/kg/min) immediately after injury for only 24 h successfully reduced the I/M ratio (0.18±0.05) to 18% of the ratio in control rats (1.00±0.13) that underwent only balloon injury. Although low-dose CNP (0.1 µg/kg/min for 24 h) alone was not effective in reducing the I/M ratio (0.83±0.18), combined US/MBs treatment for the first 80 min of the infusion markedly reduced the I/M ratio (0.17±0.07), which persisted until 28 days after injury (0.16±0.04). Conclusions: The effects of CNP on the events occurring early after arterial injury may be important in preventing subsequent neointimal development. Thus, intravenous infusion of CNP with US/MBs at its initiation may provide a clinically feasible anti-restenosis therapy applicable immediately after vascular interventions.

KEYWORDS Natriuretic peptide; Receptors; Restenosis; Smooth muscle; Ultrasound


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Neointimal hyperplasia follows injury to the arterial wall as a result of migration and proliferation of smooth muscle cells (SMCs) [1,2] and/or bone marrow-derived smooth-muscle progenitor cells [3]. These processes lead to restenosis, one of the unsolved limitations of intravascular interventions [1–3]. Although a sirolimus-eluting stent that inhibits the proliferation of lymphocytes and SMCs has recently been shown to be promising [4], no effective therapy to prevent restenosis after non-stent angioplasty has yet been established.

C-type natriuretic peptide (CNP), first identified in the porcine brain, is the third member of the family of natriuretic peptides [5]. CNP is a secreted polypeptide consisting of 22 amino acids with a ring structure formed by an intramolecular disulfide linkage and binds to guanylate cyclase-linked natriuretic peptide receptor-B (NPR-B) [5,6]. In addition to their vasorelaxant and natriuretic effects, these peptides, especially CNP, can inhibit SMCs migration and proliferation through the cyclic guanosine 3',5'-monophosphate (cGMP) cascade [7–9]. In fact, it has been shown that CNP increases cGMP concentrations in cultured vascular SMCs expressing NPR-B in a dose dependent manner [8] and that a continuous systemic administration of a relatively high dose of CNP prevents neointimal formation following arterial injury in animals [10,11].

Previous studies have demonstrated that ultrasound irradiation in the presence of microbubbles (US/MBs) can increase cell membrane permeability, so-called ‘sonoporation’. This has been used to enhance transfection of genes or plasmid DNA [12–14], which subsequently produce proteins, when the transfection is established with the temporary use of US/MBs. However, it remains unclear whether short-term US/MBs treatment can also enhance the effect of CNP, which does not require intracellular delivery but demands a persistent high-dose infusion for 5 days to 2 weeks after arterial injury to inhibit neointimal proliferation [10,11]. In the present study, we investigated whether (1) combined US/MBs treatment can augment the effect of CNP on the cGMP production in cultured vascular SMCs; (2) short-term CNP infusion following balloon injury can also prevent neointimal formation; or (3) combined US/MBs with CNP infusion for a clinically feasible time period potentiates its effect in the rat carotid artery.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 In vitro study
2.1.1 Preparation and treatment of cultured smooth muscle cells
SMCs isolated from young Sprague–Dawley rat thoracic aortas using the standard scraping technique were grown to confluence in RPMI 1640 medium containing 10% fetal calf serum, penicillin (100 µg/ml) and streptomycin (50 µg/ml), and were maintained at 37°C with atmospheric air and 5% CO2 [15]. These cells were identified by their typical morphological and growth characteristics. Monolayers of SMCs subcultured on 24-well cell culture plates between the 4th and 9th passages were washed twice with PBS, and then treated with various combinations of CNP, microbubbles (MBs), and ultrasound (US).

Human CNP-22 (Peptide Institute, Osaka, Japan) was used in this study. The amino acid sequence of CNP is identical in humans, pigs, and rats [5,16,17]. CNP was prepared in Hanks’ balanced salt solution with 20 mM HEPES (pH 7.0) containing 1 mM 3-isobutyl-1-methylxanthine, and was used at a final concentration of 10–7 mol/l. A transpulmonary US contrast agent Optison® (Mallinckrodt Medical, St. Louis, MO, USA) that is a suspension of octafluoropropane-filled albumin microbubbles [18] was added to the solution at a final concentration of 2x107 microbubbles/ml. For US irradiation, an S4 transducer of the Sonos 5500 (Philips Medical Systems, Andover, MA, USA) was placed under the bottom of the culture plate via a 2-cm thick acoustic coupler. US at a transmission frequency of 1.8 MHz and a mechanical index of 1.0 was applied in second harmonic mode at a frame rate of 30 Hz for 60 s immediately after adding CNP and/or MBs to the medium. Ultrasound penetration through the bottom of the culture plate was confirmed by visualizing the suspension of MBs in a well of the plate using the same insonation as the treatment.

Six treatment combinations based on the presence (+) or absence (–) of CNP, MBs and US were compared: (1) CNP(–)MB(–)US(–); (2) CNP(–)MB(+)US(+); (3) CNP(+)MB(–)US(–); (4) CNP(+)MB(+)US(–); (5) CNP(+)MB(–)US(+); and (6) CNP(+)MB(+)US(+). Moreover, we examined the effect of the presence of MBs in the medium on the concentration of free CNP. A mixture of MBs and CNP at the same concentrations applied to cultured SMCs was centrifuged at 1500 rpm for 3 min. After removing the MBs fractions from a sample of the mixture, the CNP concentration in the remaining sample was measured using an enzyme-immunoassay kit (Phoenix Pharmaceuticals, Belmont, CA, USA).

2.1.2 cGMP measurement
The reaction was stopped at 5, 10, 15, and 30 min after its onset by rapid aspiration and the addition of 1 ml of 5% trichloroacetic acid. After centrifugation at 12 000 rpm for 5 min at 4°C, the supernatant was collected and 1 N NaOH 250 µl was added. The cGMP concentrations were then measured using a cGMP enzyme-immunoassay system (Amersham Pharmacia Biotech UK, Buckinghamshire, UK) [8] by a person (KS) who was blinded to the treatment condition of each sample.

2.2 In vivo study
We designed an in vivo study employing the rat carotid artery balloon injury model [19] to determine the effect of short-term CNP infusion and its potentiation by US irradiation combined with MBs infusion to prevent neointimal formation. This study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2.1 Animal preparation
Male Sprague–Dawley rats, weighing 350–400 g, were used in the study. The 36 animals were anesthetized with an intraperitoneal injection of 50 mg/kg sodium pentobarbital. The right jugular and femoral veins were cannulated for infusion of CNP and MBs, respectively. A 2F Fogarty embolectomy balloon catheter (Baxter Healthcare, Irvine, CA, USA) was inserted through the left iliac artery and advanced to the left carotid artery via the aorta. After confirmation of the presence of the catheter in the common carotid artery by echography, mechanical injury in the artery was created by passing an inflated balloon through the lumen five times [19]. After balloon injury, the ultra miniature pressure transducer catheter SPR-671 (Millar Instruments, Houston, TX, USA) was advanced into the left ventricle to examine the hemodynamic effects of CNP infusion in three rats.

2.2.2 Hemodynamic effects of CNP after balloon injury
In the three rats in which a left ventricular catheter was inserted after balloon injury, the left ventricular pressure was digitally recorded at baseline and during CNP infusion at the rate of 0.1 µg/kg/min and 1.0 µg/kg/min for 5 min each.

2.2.3 Treatment of rats
The remaining 33 rats were divided into five groups with various post-injury short-term treatments as follows: (1) control, balloon injury only (n = 6); (2) high-dose CNP, infusion of CNP at 1.0 µg/kg/min for 24 h (n = 6); (3) low-dose CNP, infusion of CNP at 0.1 µg/kg/min for 24 h (n = 6); (4) low-dose CNP+US/MBs, infusion of CNP at 0.1 µg/kg/min for 24 h begun with US/MBs treatment (n = 9); and (5) US/MBs, US/MBs treatment only (n = 6).

The treatment protocol is shown in Fig. 1. Infusion of CNP was initiated immediately after balloon injury. For the US/MBs treatment, the S4 transducer was positioned 4 cm from and perpendicular to the injured carotid artery via an acoustic coupler. US with a transmission frequency of 1.8 MHz and mechanical index of 1.0 was applied at a frame rate of 30 Hz for 10 min, during which MBs (10% Optison®) was infused via the femoral vein at 0.2 ml/min (4x106 microbubbles/min) for the first 5 min of the 10-min insonation. The echograms were recorded at baseline, during, and after MBs infusion to ensure the presence of MBs in the carotid artery. The US/MBs treatment was initiated 10 min after balloon injury and repeated 5 times at 3-min intervals. Thus the US/MBs treatment was completed within the first 80 min of the 24-h infusion of CNP following balloon injury.


Figure 1
View larger version (6K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Treatment protocol after balloon injury. Infusion of CNP was begun immediately after balloon injury and continued for 24 h. The US/MBs treatment shown as a white arrow was begun 10 min after balloon injury and repeated five times at 3-min intervals. The US/MBs treatment consisted of 10 min of US irradiation and MBs infusion for the first 5 min of the 10-min insonation. All US/MBs treatments were completed in the first 80 min of CNP infusion.

 
2.2.4 Morphometric analysis
All the rats excluding three of the nine rats in the low-dose CNP+US/MBs group were euthanized with an overdose of sodium pentobarbital and the carotid arteries were dissected, fixed, and sectioned for analysis 14 days after the balloon injury. The remaining three rats in the low-dose CNP+US/MBs group were kept for an additional 14 days to assess the long-term effect of the treatment following balloon injury and were subjected to the same histopathological analyses at 28 days after treatment. Intimal and medial areas were measured in four consecutive sections in each vessel with a digital analysis system using the NIH Image version 1.62 (National Institutes of Health, Washington, DC, USA), and the intima/media (I/M) ratio was calculated [10,11]. Morphometric analysis was performed by a person (IK) who was blinded to the treatment condition of each rat.

2.3 Statistical analysis
Data are expressed as mean±S.D. For comparison between multiple groups, one-way ANOVA with Bonferroni's correction was used. For comparison of hemodynamic parameters at different infusion rates of CNP, one-way repeated measures of ANOVA was used. Differences were considered significant at P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 In vitro study
The cGMP level in SMCs was rapidly increased and peaked at 10 min after incubation with CNP. Fig. 2 compares the peak cGMP levels among the various conditions. The peak cGMP level was 2-fold higher with CNP plus combined US/MBs than with CNP alone. Neither MBs nor US alone augmented the effect of CNP on cGMP production in SMCs. Treatment with MBs combined with US in the absence of CNP showed no effect on cGMP production. Interestingly, the addition of MBs alone to CNP without US even reduced cellular cGMP as compared with CNP alone. The concentration of free CNP in the medium after the removal of MBs fractions from the mixture was reduced to 0.37x10–7 mol/l (37% of the initial concentration of 1.0x10–7 mol/l), which may suggest that MBs may change the distribution of CNP in the medium, presumably by trapping CNP on their surface.


Figure 2
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Comparison of cellular cGMP concentrations in cultured smooth muscle cells in various conditions. A two-fold increase in the cellular cGMP level was exhibited with CNP(+)MB(+)US(+) compared with CNP(+)MB(–)US(–). Cellular cGMP concentration in CNP(+)MB(+)US(–) was about one-half that in CNP(+)MB(–)US(–). * P<0.01 vs. CNP(–)MB(–)US(–) and CNP(–)MB(+)US(+), # P<0.05 vs. CNP(+)MB(–)US(–), ## P<0.001 vs. CNP(+)MB(–)US(–).

 
3.2 In vivo study
3.2.1 Hemodynamic effects of CNP after balloon injury
Table 1 summarizes the hemodynamic effects of low-dose and high-dose CNP obtained with high-fidelity recordings of the left ventricular pressure. Although no significant effects on left ventricular maximum dP/dt, left ventricular end-diastolic pressure, or heart rate were demonstrated, the peak systolic pressure was significantly decreased during high-dose CNP infusion.


View this table:
[in this window]
[in a new window]

  		Table 1 Hemodynamics data

 
3.2.2 Contrast echography of carotid artery
Fig. 3 displays echograms obtained during US/MBs treatment in one rat. Although no appreciable structure for the common carotid artery was demonstrated before MBs infusion (Fig. 3A) because of the low carrier frequency of the ultrasound, a bright linear structure corresponding to the common carotid artery (arrows) was visualized in all rats that received MBs infusion during US/MBs treatment (Fig. 3B).


Figure 3
View larger version (124K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Contrast echograms of carotid artery during US/MBs treatment. Although no appreciable structure for common carotid artery was demonstrated at baseline (A), a bright linear structure was visualized during MBs infusion as indicated by arrows (B).

 
3.2.3 Effects on neointimal formation
Fig. 4 displays representative histological sections of the carotid artery at the injured site at 14 days (Fig. 4A–E) and at 28 days (Fig. 4F) after treatment. Neointimal hyperplasia at the injured site was almost completely inhibited by high-dose CNP (Fig. 4B) compared with the control (Fig. 4A). Although low-dose CNP alone failed to prevent neointimal formation (Fig. 4C), combined US/MBs plus low-dose CNP successfully inhibited intimal thickening (Fig. 4D). The inhibition of intimal thickening persisted until 28 days after treatment in this group (Fig. 4F). US/MBs in the absence of CNP showed no effect on neointimal formation (Fig. 4E).


Figure 4
View larger version (105K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Photomicrographs (hematoxylin–eosin staining, 100x) of the histological sections of carotid arteries 14 days (A–E) and 28 days (F) after balloon injury. Control (A) and those treated with high-dose CNP (B), low-dose CNP (C), low-dose CNP combined with US/MBs (D), and US/MBs alone (E) are shown. Neointimal formation was almost completely inhibited in high-dose CNP (B) and low-dose CNP+US/MBs (D,F). This effect in low-dose CNP+US/MBs persisted until 28 days (F).

 
Fig. 5 compares the I/M ratios on day 14 among the groups. Although high-dose CNP reduced the I/M ratio by 82% compared to control, low-dose CNP produced only a non-significant 17% reduction in the I/M ratio. However, the US/MBs treatment exhibited a 4-fold augmentation of the effect of low-dose CNP, which resulted in an I/M ratio similar to that obtained with high-dose CNP. US/MBs alone resulted in no difference in the I/M ratio compared with control. The I/M ratio of the rats in the low-dose CNP+US/MBs group at 28 days after the short-term treatment was 0.16±0.04, which was similar to the ratio at 14 days obtained in the same treatment group.


Figure 5
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Comparison of the I/M ratios among the groups. US/MBs, combined application of ultrasound and microbubbles; high, 1.0 µg/kg/min; low, 0.1 µg/kg/min. I/M ratios were similarly lower in high-dose CNP and low-dose CNP +US/MBs than balloon injury only (CNP(–)US/MBs(–)). * P<0.0001 vs. CNP(–)US/MBs(–).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study demonstrated that a relatively short-term (24 h) treatment with high-dose CNP (1.0 µg/kg/min) immediately after balloon injury inhibited neointimal hyperplasia for 14 days in the rats, the extent of which was comparable to that achieved in previous studies employing a long-term (5-day [10] or 14-day [10,11]) continuous infusion of the same dose. Moreover, the combination of US/MBs treatment in the first 80 min of the 24-h CNP infusion augmented this effect, which persisted beyond 14 days at least until 28 days after the treatment. Thus, this study suggests a new treatment approach that employs combined US/MBs treatment and CNP infusion for a clinically feasible time period to prevent restenosis following intravascular interventions.

4.1 CNP to prevent vascular remodeling
CNP secreted by endothelial cells has multiple effects, including vasodilation and inhibition of vascular SMCs migration and proliferation through the cGMP cascade [7–9]. In atherosclerotic lesions, endothelial CNP production is decreased [20], whereas the CNP-specific receptor NPR-B of vascular SMCs is overexpressed [6]. Arterial interventions cause endothelial injury resulting in the loss of endothelium-derived anti-proliferative factors including CNP and nitric oxide, both of which regulate the intracellular cGMP levels in the arterial wall [21,22]. Therefore, compensation of CNP at the injured site has been attempted to prevent restenosis.

In this regard, a systemic high-dose administration that potentially affects systemic hemodynamics [23] was performed to yield a sufficient concentration of CNP at the target site for long periods [10,11]. Other authors used an infiltrator angioplasty balloon catheter to perform adenovirus-mediated transfer of the CNP gene [24]. However, after gene transfer, the reduced local CNP concentration may not be compensated until the expression of CNP in the target site becomes sufficient, which may explain the lack of a difference in the degree of neointimal hyperplasia between the sites with control gene transfection and those with CNP gene transfection in their study [24]. By contrast, in our study, a high-dose CNP infusion for only 24 h immediately after the injury was as effective in preventing neointimal formation as the 2-week continuous infusion at the same rate employed in previous studies [10,11]. Moreover, combined US/MBs treatment in the first 80 min of the 24-h CNP infusion at a much lower infusion rate (0.1 µg/kg/min), one-tenth the previously reported rate [10,11], significantly augmented the anti-proliferative effect of CNP to a level similar to that obtained with high-dose CNP. Furthermore, neointimal formation remained inhibited until 28 days after the treatment, after which the I/M ratio is known to barely increase [25,26]. This suggests that the effect of CNP in preventing neointimal proliferation may be related to the early events that occur in response to vascular injury.

CNP has been shown to inhibit migration of stimulated SMCs [8], which has been thought to lead to their proliferation and resulting in neointimal hyperplasia [27]. More recently, it has been reported that bone marrow-derived smooth muscle progenitor cells mobilized to the injured site can differentiate into SMCs to contribute to subsequent neointimal development [3]. CNP may inhibit the migration of SMCs or their progenitor cells, both of which occur early after vascular injury.

Alternative explanations for the favorable effect of relatively short-term CNP administration may be related to their possible anti-inflammatory actions. Balloon inflation induces an infiltration of inflammatory cells into the vessel wall, the greatest increase being seen at 24 h after injury [28]. Significant accumulation of neutrophils and macrophages occurs in the adventitia in the first 24 h after balloon injury [29], which precedes the onset of cell proliferation beginning between 48 and 72 h [30]. Growth factors or their inhibitors placed in the adventitia can significantly affect intimal development after balloon injury [31,32]. Therefore, the accumulation of inflammatory cells in the adventitia might be an important source for growth factors and cytokines stimulating intimal thickening after angioplasty. In addition, rapid accumulation of adhesion molecules on the luminal surface and neutrophil recruitment occur after endothelial denudation [33]. Thus, regardless of adventitial and luminal sides, infiltration of leukocytes plays a role in restenosis development. In this regard, sirolimus, which has recently been shown to prevent in-stent restenosis, blocks inflammation at the angioplasty site [34]. A monoclonal antibody to ICAM-1 significantly suppresses intimal hyperplasia in the rat model of restenosis [35]. The blockade of the selectin family by an analogue of sialyl-LewisX [36] and the knock-out of P-selectin [37] can also reduce neointimal hyperplasia. Moreover, leukocyte depletion by either mustine or an antibody against the leukocyte common antigen has been shown to inhibit neointimal hyperplasia after balloon angioplasty in a rabbit model [38].

CNP derived from the CNP gene plasmid transfected to the site of balloon angioplasty prevented the adventitial inflammatory/proliferative response [24]. As was expected by the authors who reported the production and secretion of CNP by inflammatory cells [39], our preliminary data have shown that CNP has anti-inflammatory actions including the inhibition of the expression of adhesion molecules on neutrophils, which diminishes reperfusion injury in a renal ischemia/reperfusion model [40]. These data lead us to the hypothesis that CNP may exert anti-leukocyte actions when administered immediately after vascular injury, which may be a mechanism of inhibition of the neointimal proliferation at the early phase of the response to the vascular injury.

4.2 Mechanisms of enhancement of CNP by US/MB
Previous investigators successfully utilized ultrasound-mediated MBs destruction to induce ‘cavitation’ for ‘sonoporation’ [12] of the cell membrane to deliver genes or plasmid DNA into cells and tissues [13,14]. The ‘cavitation’ may allow a direct ‘micro-injection’ [41] of CNP into the arterial wall from the lumen side or a microvascular disruption [42] at the level of the vasa vasorum, both of which may increase CNP concentration in the arterial wall. In addition, the occurrence of a ‘micro-stream’ near the cell surface may increase the opportunities for ligand–receptor contacts. In fact, US/MBs augmented cGMP production in cultured SMCs that were not in the tissue but were exposed in the medium. Moreover, our in vitro experiments demonstrated that CNP concentration in the medium was reduced after removal of MBs fractions from the mixture and that production of cGMP in SMCs combined with both CNP and MBs in the absence of US was less compared with CNP alone. These results may suggest another mechanism by which MBs trap CNP molecules on their surface to limit the action of CNP until the MBs are disrupted by exposure to US. This mechanism may be beneficial in in vivo settings, because diffusion of CNP or its degradation by neutral endopeptidase [43] can be minimized in the systemic circulation and local release of CNP only at the insonated site may be provided.

4.3 Clinical implications
Although the precise mechanisms remain unknown, the local enhancement of the bioeffects of CNP shown in this study has several advantages. The use of CNP, an endogenous molecule, eliminates possible hazards arising from the use of viral vectors or the gene itself in gene delivery. Both the MBs and US systems employed in the present study are approved for clinical use and safety. Moreover, when US and MBs were combined, one-tenth of the effective dosage of intravenous CNP achieved nearly complete prevention of neointimal formation. In contrast to the reported high-dose of CNP that decreased systolic blood pressure, low-dose CNP infusion exerted almost no significant hemodynamic effects in this study. This may be an advantage for the application of this proposed treatment in patients undergoing intravascular interventions, in whom an unfavorable blood pressure decline should be avoided. Of importance, a relatively short-term treatment with CNP and US/MBs early after the vessel injury has been found to be effective. This not only suggests that the effect of CNP may be related to the early events in response to the vascular injury, but also implies its future clinical application as an immediate adjunct to intravascular interventions.

4.4 Study limitations
Several limitations exist in this study. First, the long-term effects of the proposed treatment beyond 28 days post-procedure remain unknown. However, previous studies employing the carotid artery injury model also evaluated the neointimal formation 2 weeks after injury [10,11,44–46]. It is known that the mitogenesis of the intimal smooth muscle cells reaches its maximum level within 7 days after the injury [26] and that the I/M ratio peaks by 4 weeks and plateaus thereafter at least until 16 weeks after the injury [25]. Therefore, we also evaluated the outcome of the treatment at 14 days post injury and confirmed the persistence of the effect of the proposed treatment up to 28 days after treatment. Second, the precise mechanisms for the effectiveness of the present treatment are unknown. Finally, future studies are necessary to determine the minimally required dose or duration of CNP administration, and optimal MBs concentration and US parameters specific to variable acoustic properties of the tissues surrounding the target vessels.

4.5 Conclusions
A relatively short-term intravenous infusion of CNP immediately after vascular injury can inhibit neointimal formation. Short-term ultrasound irradiation with microbubble infusion at the beginning of the CNP infusion potentiates the effect. Thus, CNP infusion combined with ultrasound and microbubbles may provide a practical and safe approach for the prevention of restenosis following intravascular interventions.

Time for primary review 31 days.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Austin G.E., Ratliff N.B., Hollman J., Tabei S., Phillips D.F. Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary artery stenosis after percutaneous transluminal coronary angioplasty. J Am Coll Cardiol (1985) 6:369–375.[Abstract]
  2. Libby P., Schwartz D., Brogi H., Tanaka H., Clinton S.K. A cascade model for restenosis: A special case of atherosclerosis progression. Circulation (1992) 86:III47, III52.
  3. Sata M., Saiura A., Kunisato A., et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nature Med (2002) 4:403–409.
  4. Morice M.C., Serruys P.W., Sousa J.E., et al. A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. New Engl J Med (2002) 346:1773–1780.[Abstract/Free Full Text]
  5. Sudoh T., Minamino N., Kangawa K., Matsuo H. C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Biochem Biophys Res Commun (1990) 168:863–870.[CrossRef][ISI][Medline]
  6. Koller K.J., Lowe D.G., Bennett G.L., et al. Selective activation of B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science (1991) 252:120–123.[Abstract/Free Full Text]
  7. Ikeda M., Kohno M., Yasunari K., et al. Natriuretic peptide family as a novel antimigration factor of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol (1997) 17:731–736.[Abstract/Free Full Text]
  8. Kohno M., Yokokawa K., Yasunari K., et al. Effect of natriuretic peptide family on the oxidized LDL-induced migration of human coronary artery smooth muscle cells. Circ Res (1997) 81:585–590.[Abstract/Free Full Text]
  9. Hutchinson H.G., Trindade P.T., Cunanan D.B., Wu C.F., Pratt R.E. Mechanisms of natriuretic-peptide-induced growth inhibition of vascular smooth muscle cells. Cardiovasc Res (1997) 35:158–167.[Abstract/Free Full Text]
  10. Furuya M., Aisaka K., Miyazaki T., et al. C-type natriuretic peptide inhibits intimal thickening after vascular injury. Biochem Biophys Res Commun (1993) 193:248–253.[CrossRef][ISI][Medline]
  11. Shinomiya M., Tashiro J., Saito Y., et al. C-type natriuretic peptide inhibits intimal thickening of rabbit carotid artery after balloon catheter injury. Biochem Biophys Res Commun (1994) 205:1051–1056.[CrossRef][ISI][Medline]
  12. Bao S., Thrall B.D., Miller D.L. Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound Med Biol (1997) 23:953–959.[CrossRef][ISI][Medline]
  13. Anwer K., Kao G., Proctor B., et al. Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration. Gene Ther (2000) 7:1833–1839.[CrossRef][ISI][Medline]
  14. Shohet R.V., Chen S., Zhou Y.T., et al. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation (2000) 101:2554–2556.[Abstract/Free Full Text]
  15. Fukunaga M., Makita N., Roberts II L.J., et al. Evidence for the existence of F2-isoprostane receptors on rat vascular smooth muscle cells. Am J Physiol (1993) 264:C1619–1624.[ISI][Medline]
  16. Kojima M., Minamino N., Kangawa K., Matsuo H. Cloning and sequence analysis of a cDNA encoding a precursor for rat C-type natriuretic peptide (CNP). FEBS Lett (1990) 276:209–213.[CrossRef][ISI][Medline]
  17. Tawaragi Y., Fuchimura K., Tanaka S., et al. Gene and precursor structures of human C-type natriuretic peptide. Biochem Biophys Res Commun (1991) 175:645–651.[CrossRef][ISI][Medline]
  18. Clark L.N., Dittrich H.C. Cardiac imaging using Optison. Am J Cardiol (2000) 86:14G–18G.[ISI][Medline]
  19. Inoue Y., Toga K., Sudo T., et al. Suppression of arterial intimal hyperplasia by cilostamide, a cyclic nucleotide phosphodiesterase 3 inhibitor, in a rat balloon double-injury model. Br J Pharmacol (2000) 130:231–241.[CrossRef][ISI][Medline]
  20. Naruko T., Ueda M., van der Wal A.C., et al. C-type natriuretic peptide in human coronary atherosclerotic lesions. Circulation (1996) 94:3103–3108.[Abstract/Free Full Text]
  21. Garg U.C., Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest (1989) 83:1774–1777.[ISI][Medline]
  22. Marumo T., Nakaki T., Hishikawa K., et al. Natriuretic peptide-augmented induction of nitric oxide synthase through cyclic guanosine 3',5'-monophosphate elevation in vascular smooth muscle cells. Endocrinology (1995) 136:2135–2142.[Abstract]
  23. Stingo A.J., Clavell A.L., Aarhus L.L., Burnett J.C. Jr. Cardiovascular and renal actions of C-type natriuretic peptide. Am J Physiol (1992) 262:H308–312.[ISI][Medline]
  24. Morishige K., Shimokawa H., Yamawaki T., et al. Local adenovirus-mediated transfer of C-type natriuretic peptide suppresses vascular remodeling in porcine coronary arteries in vivo. J Am Coll Cardiol (2000) 35:1040–1047.[Abstract/Free Full Text]
  25. Gabeler E.E.E., van Hillegersberg R., Statius van Eps R.G., et al. A comparison of balloon injury models of endovascular lesions in rat arteries. BMC Cardiovasc Disord (2002) 2:16.[CrossRef][Medline]
  26. Fishman J.A., Ryan G.B., Karnovsky M.J. Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening. Lab Invest (1975) 32:339–351.[ISI][Medline]
  27. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (1993) 362:801–809.[CrossRef][Medline]
  28. Kennedy S., McPhaden A.R., Wadsworth R.M., Wainwright C.L. Correlation of leukocyte adhesiveness, adhesion molecule expression and leukocyte-induced contraction following balloon angioplasty. Br J Pharmacol (2000) 130:95–103.[CrossRef][ISI][Medline]
  29. Okamoto E., Couse T., De Leon H., et al. Perivascular inflammation after balloon angioplasty of porcine coronary arteries. Circulation (2001) 104:2228–2235.[Abstract/Free Full Text]
  30. Scott N.A., Cipolla G.D., Ross C.E., et al. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation (1996) 93:2178–2187.[Abstract/Free Full Text]
  31. Edelman E.R., Nugent M.A., Smith L.T., Karnovsky M.J. Basic fibroblast growth factor enhances the coupling of intimal hyperplasia and proliferation of vasa vasorum in injured rat arteries. J Clin Invest (1992) 89:465–473.[ISI][Medline]
  32. Prescott M.F., McBride C.K., Court M. Development of intimal lesions after leukocyte migration into the vascular wall. Am J Pathol (1989) 135:835–846.[Abstract]
  33. Roque M., Fallon J.T., Badimon J.J., et al. Mouse model of femoral artery denudation injury associated with the rapid accumulation of adhesion molecules on the luminal surface and recruitment of neutrophils. Arterioscler Thromb Vasc Biol (2000) 20:335–342.[Abstract/Free Full Text]
  34. Suzuki T., Kopia G., Hayashi S., et al. Stent-based delivery of sirolimus reduces neointimal formation in a porcine coronary model. Circulation (2001) 104:1188–1193.[Abstract/Free Full Text]
  35. Yasukawa H., Imaizumi T., Matsuoka H., Nakashima A., Morimatsu M. Inhibition of intimal hyperplasia after balloon injury by antibodies to intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1. Circulation (1997) 95:1515–1522.[Abstract/Free Full Text]
  36. Barron M.K., Lake R.S., Buda A.J., Tenaglia A.N. Intimal hyperplasia after balloon injury is attenuated by blocking selectins. Circulation (1997) 96:3587–3592.[Abstract/Free Full Text]
  37. Kumar A., Hoover J.L., Simmons C.A., Lindner V., Shebuski R.J. Remodeling and neointimal formation in the carotid artery of normal and P-selectin-deficient mice. Circulation (1997) 96:4333–4342.[Abstract/Free Full Text]
  38. Miller A.M., McPhaden A.R., Wadsworth R.M., Wainwright C.L. Inhibition by leukocyte depletion of neointima formation after balloon angioplasty in a rabbit model of restenosis. Cardiovasc Res (2001) 49:838–850.[Abstract/Free Full Text]
  39. Kubo A., Isumi Y., Ishizaka Y., et al. C-type natriuretic peptide is synthesized and secreted from leukemia cell lines, peripheral blood cells, and peritoneal macrophages. Exp Hematol (2001) 29:609–615.[CrossRef][ISI][Medline]
  40. Shinomiya K., Ohmori K., Kondo I., et al. C-type natriuretic peptide limits renal ischemia/reperfusion injury by inhibiting migration of activated leukocytes [abstract]. Circ J (2002) 66:692.
  41. Mukherjee D., Wong J., Griffin B., et al. Ten-fold augmentation of endothelial uptake of vascular endothelial growth factor with ultrasound after systemic administration. J Am Coll Cardiol (2000) 35:1678–1686.[Abstract/Free Full Text]
  42. Skyba D.M., Price R.J., Linka A.Z., Skalak T.C., Kaul S. Direct in vivo visualization of intravascular destruction of microbubbles by ultrasound and its local effects on tissue. Circulation (1998) 98:290–293.[Abstract/Free Full Text]
  43. Brandt R.R., Mattingly M.T., Clavell A.L., Barclay P.L., Burnett J.C. Jr. Neutral endopeptidase regulates C-type natriuretic peptide metabolism but does not potentiate its bioactivity in vivo. Hypertension (1997) 30:184–190.[Abstract/Free Full Text]
  44. Indolfi C., Esposito G., Lorenzo E.D., et al. Smooth muscle cell proliferation is proportional to the degree of balloon injury in a rat model of angioplasty. Circulation (1995) 92:1230–1235.[Abstract/Free Full Text]
  45. Indolfi C., Cioppa A., Stabile E., et al. Effects of hydroxymethylglutaryl coenzyme A reductase inhibitor simvastatin on smooth muscle cell proliferation in vitro and neointimal formation in vivo after vascular injury. J Am Coll Cardiol (2000) 35:214–221.[Abstract/Free Full Text]
  46. Ohlstein E.H., Romanic A.M., Clark L.V., et al. Application of in vivo and ex vivo magnetic resonance imaging for evaluation of tranilast on neointima formation following balloon angioplasty of the rat carotid artery. Cardiovasc Res (2000) 47:759–768.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Takeuchi, H.
Right arrow Articles by Kohno, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takeuchi, H.
Right arrow Articles by Kohno, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?