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Cardiovascular Research 2005 66(3):574-582; doi:10.1016/j.cardiores.2005.01.024
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Copyright © 2005, European Society of Cardiology

C-type natriuretic peptide (CNP) suppresses plasminogen activator inhibitor-1 (PAI-1) in vivo

Evette M. Kairuza, Melissa N. Barbera,b, Colin R. Andersonc, Meetali Kanagasundarama, Grant R. Drummonda,b and Robyn L. Woodsa,*

aHoward Florey Institute, University of Melbourne, Victoria, 3010, Australia
bDepartment of Pharmacology, Monash University, Victoria, 3800, Australia
cDepartment of Anatomy and Cell Biology, University of Melbourne, Victoria 3010, Australia

* Corresponding author. Tel.: +61 3 83445264; fax: +61 3 93481707. Email address: r.woods{at}hfi.unimelb.edu.au

Received 7 November 2003; revised 12 January 2005; accepted 30 January 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Elevated vascular plasminogen activator inhibitor-1 (PAI-1) levels are associated with atherosclerosis. In vitro, C-type natriuretic peptide (CNP) has anti-proliferative effects and inhibits the production of PAI-1 in cultured vascular cells. Whether CNP can affect PAI-1 in vivo, particularly in the setting of atherosclerosis, has not been reported.

Methods: Using the rabbit carotid arterial collar model of intimal hyperplasia (collar in place for 7 days), PAI-1 protein was compared in normal, vehicle (saline)-collared, and CNP-treated-collared arteries from the same animal. PAI-1 levels were measured by immunohistochemistry and densitometry and by Western blot. CNP was either infused into the peri-arterial space within one collar (10 fmol/h) or infused directly into the arterial lumen under one collar (100 pmol/h). In some rabbits (n=8), superoxide production in collared and normal artery segments was measured in vitro by chemiluminescence.

Results: PAI-1 was present throughout the vascular wall. Endothelial PAI-1 was elevated in saline-collared arteries (~16%, P<0.05; n=7 rabbits) compared with normal carotid segments. The collar induced both a neointima that contained PAI-1 and the accumulation of macrophages in the adventitia. Peri-arterial CNP reduced PAI-1 (P<0.05) in the endothelium (33%), adventitia (47%) and neointima (39%), compared with levels in the contralateral, saline-collared carotid artery, while macrophage infiltration was reduced. Elevated superoxide production in collared arteries was not altered by chronic in vivo treatment with CNP (n=8). Peri-arterial CNP treatment did not reduce intimal thickening. Intra-luminal CNP (n=6) reduced endothelial, neointimal and total vessel (Western blot) PAI-1, macrophage accumulation, and intimal thickening (all P<0.05).

Conclusions: CNP treatment of collared carotid arteries in vivo for 1 week suppressed endothelial and neointimal PAI-1, independently of intimal thickening. The CNP effects were not via superoxide. This is the first evidence that CNP inhibits activated PAI-1, in vivo.

KEYWORDS Restenosis; Intimal hyperplasia; Neointima; Atherosclerosis; Endothelium; Macrophages; Superoxide; NADPH oxidase


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
C-type natriuretic peptide (CNP) belongs to a family of structurally related cardiovascular hormones which also includes atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP). Unlike ANP and BNP which are made in cardiomyocytes and released into the circulation, CNP is produced by vascular endothelial cells [1,2] and acts as a local regulator of vascular tone and growth [3–6]. CNP also exhibits anti-migratory and anti-proliferative effects on vascular smooth muscle cells (VSMC) in vitro [7–9]. In vivo, CNP inhibits intimal thickening in a number of experimental models of atherosclerosis [10–12]. Recently, it was reported that CNP reduces levels of the atherogenic agent, plasminogen activator inhibitor-1 (PAI-1), in vitro, in VSMC [13] and endothelial cells [14]. Elevated PAI-1 is associated with the development of experimental atherosclerosis [15] and the restenosis-like changes that occur after experimental balloon injury [16]. In normal and mildly atherosclerotic human aorta, a strong positive hybridization signal for PAI-1 mRNA was observed in the endothelium, and a weaker signal was observed in the intima [17]. PAI-1 activity was increased in early human vascular lesions when compared to normal arteries, and PAI-1 immunoreactivity was observed in the endothelium and intima of early lesions [18]. PAI-1 is the major physiological inhibitor of the fibrinolytic system in vivo [19] and contributes to the regulation of the vascular extracellular matrix [20]. PAI-1 is therefore thought to be important in vascular remodelling associated with artery disease.

The suppressive effects of CNP on PAI-1 have, to date, only been demonstrated in vitro [13,14]. CNP inhibits angiotensin II- and platelet-derived growth factor stimulated PAI-1 mRNA and protein expression by 50% in cultures of rat and human aortic VSMC [13] and PAI-1 mRNA and activity in rat aortic endothelial cells [14]. A direct link between CNP and PAI-1 in the pathological remodelling associated with atherosclerosis or restenosis has not yet been investigated. The major aim of the present study was to determine whether CNP attenuates PAI-1 in an in vivo model of intimal hyperplasia. Placement of a peri-arterial collar around rabbit carotid arteries induces sub-endothelial intimal hyperplasia [21–23], functional changes in the endothelium [24] and increases superoxide production from NADPH oxidase provided endogenous superoxide dismutase is inhibited [25]. Morphological changes with this rabbit model are similar to the characteristic early changes in the human artery wall that predispose the site to atherosclerosis or that occur during restenosis after angioplasty [26]. A feature of this model is that treatments (CNP in the present study) can be applied locally to the arterial wall, within the confines of the collar. In the present study, we investigated whether PAI-1 protein is elevated in the collared carotid artery and whether local administration of CNP suppresses this response. Furthermore, given our previous demonstrations that elevated NADPH oxidase activity appears to underlie the impairment of vasorelaxation responses to ACh in this model [25], and that CNP prevented this endothelial dysfunction [27], we also examined the effects of CNP on superoxide production.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animal model of atherosclerosis
Male New Zealand White rabbits, weighing between 3.0 and 4.0 kg, were fed a standard laboratory diet (zero cholesterol). Rabbits were anesthetized with Propofol (i.v. 0.5 mg/kg bolus; Abbott Australasia) followed by intramuscular injection of Xylazine (20 mg/kg; Xylaze 100, Parnell Laboratories, Australia) and Ketamine (100 mg/kg; Ketapex, Apex Laboratories, Australia). A non-occlusive, soft, silastic collar (length 2.5 cm, external diameter 1 cm tapering to 2 mm at each end) was placed bilaterally around each carotid artery as previously described [21,22]. All procedures were approved by the Howard Florey Institute Animal Ethics Committee. The investigation conforms with 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).

2.1.1. Peri-arterial administration of CNP
The peri-arterial space of one collar was filled with 0.9% saline. A catheter was inserted into the contralateral collar and attached to an osmotic mini-pump (Alzet, Model 2001, Alzet Pharmaceuticals, rate of 1 µl/h) that infused 10 fmol/h CNP (rat-CNP-22, Bachem, Switzerland, dissolved in 0.9% saline) onto the outside of the artery for 7 days. The peri-arterial space was filled with 10 µmol/L CNP. In the first group of these rabbits (n=7), central segments of each arterial region were fixed, as described below for immunohistochemistry and morphology. The remaining tissue was cut into rings for functional analysis in the organ bath (results described elsewhere [27]).

In a second group of rabbits (n=8) treated with peri-arterial CNP (10 fmol/h) for 7 days, as described above, tissues from both carotid arteries from within the collar and proximal to the collar were harvested for immediate in vitro measurement of superoxide activity (described below) [25].

2.1.2. Intra-luminal infusion of CNP
In these rabbits (n=6), a collar was placed around the carotid artery and a catheter was inserted approximately 1 cm into the lumen of one carotid artery via an arterial side branch. The collar was placed distal to the side branch. The catheter was attached to an osmotic mini-pump that infused 100 pmol/h CNP (in 1 µl/h) into the arterial lumen for 7 days. The peri-arterial space was filled with 10 µmol/L CNP at the time of surgery. The collar placed around the contralateral artery was filled with saline as described above. Central segments of each arterial region were fixed, as described below for immunohistochemistry and morphology. The remaining tissue was snap frozen for subsequent analysis of PAI-1 protein by Western blot.

2.2. Tissue retrieval and processing
In all cases, after 7 days the rabbits were heparinized (1500U, i.v.) and killed with an overdose of sodium pentobarbitone (Nembutal, 60 mg/kg i.v.; Merial Australia Pty Ltd). Carotid arteries were excised and placed in ice-cold Krebs-HEPES buffer (pH 7.4). Collars were removed and the arteries were cleared of connective tissue.

For immunohistochemistry and morphology, arterial rings (~2–3 mm) were taken from the mid-region of artery within each collar and from a section of ‘normal’, uncollared artery that was always proximal to each collar and, in the case of group 3 rabbits, proximal to the catheter. These ‘normal’ sections of artery display the same vascular responsiveness and histological appearance as naïve carotid arteries [21,22]. The artery rings were fixed in 4% paraformaldehyde overnight and processed for paraffin embedding. Transverse sections (4 µm) of each of the four carotid artery segments from each rabbit were mounted onto a single slide to ensure that subsequent processing of all sections from an individual rabbit was identical. Sections distal to the collared region were also taken from the third group of rabbits for comparison with the normal artery proximal to the collar and catheter. No differences between these non-collared regions were observed so only results from proximal sections are included here.

For superoxide activity measurements, 3 ring segments (~3 mm) were cut from each unfixed collared artery and its proximal, uncollared region immediately after dissection and placed in a sterile multi-well plate containing ice-cold Krebs-HEPES buffer.

2.3. Morphology
Transverse sections (4 µm) from the normal and collared regions of each artery were stained with haematoxylin and eosin. The cross-sectional areas of the media of all vessels and the luminal areas and neointima of collared arteries were measured three times using the image analysis program MCID-M2 (Imaging Research Inc, Canada) and the average of these measurements was used for further analysis. The neointimal and medial areas were used to calculate the intima/media ratio (IMR) for each collared artery.

2.4. Immunohistochemistry
Rabbit liver was used as a positive control for all immunohistochemistry and negative controls consisted of omitting the primary antibody.

2.4.1. PAI-1
Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min and sections were blocked with 1.5% rabbit serum for 30 min at room temperature. The sections were stained with a sheep anti-human PAI-1 IgG (1:200; Chemicon International, Temecula, CA, USA) at room temperature overnight. This antibody recognises PAI-1 that is complexed to either of the plasminogen activators. Sections were washed with 0.01 M phosphate buffered saline (PBS) and incubated at room temperature with a biotinylated anti-sheep IgG (1:200; Vector Laboratories, Burlingame, CA, USA). The avidin–biotin horseradish peroxidase complex (1:100; Vector Laboratories) was added for 1 h. Antibody binding was detected by soaking the sections in diaminobenzidine tetrachloride (DAB) (0.5 mg/ml in PBS) for 5 min, then by adding hydrogen peroxide to a final concentration of 0.009% for 1 further minute. The sections were then placed in PBS to stop the reaction. Sections were rinsed in tap water, dehydrated in alcohol and cleared in histolene. Slides were mounted in DPX mounting media and coverslipped and the sections were examined by light microscopy.

2.4.2. Macrophages
A mouse anti-rabbit alveolar macrophage (RAM 11) antibody (1:100, a gift from Rod Dilley, Baker Institute, Prahran, Australia) and a biotinylated anti-mouse IgG (1:200, Vector Laboratories) secondary antibody were applied using the methods described above, except the sections were not blocked for endogenous peroxidase activity.

2.5. Quantification of immunohistochemistry
Using image analysis software (MCID-M2), PAI-1 immunostaining was measured in logarithmic greyscale units of relative optical density (ROD) as compared to an internally calibrated system. For each individual transverse section, four regions 90 ° apart were photographed at high power (60 x objective). The outline of each of these regions was traced in the MCID software and the average ROD calculated from PAI-1 staining in each layer of the vascular wall: endothelium, neointima (collared vessels only), media and adventitia. A background reading was taken from a clear region of the slide, usually within the lumen of the vessel, and subtracted from all individual measurements. Results were expressed as raw ROD values and then normalised against the ROD of the normal section (saline-collared artery).

Light microscopy revealed that RAM-11 immunopositive cells were present as individual cells or as large clusters of immunopositive cells. The size of the clusters was variable and in each case it was not possible to count the cells within a cluster. Individual cells were counted within an individual transverse section, in a blinded study of macrophage immunostaining. Sections used for macrophage detection were cut immediately adjacent to the sections used for PAI-1 measurements.

2.6. PAI-1 protein measured by Western blot
Carotid arteries were homogenized on ice in buffer containing 0.1 M Pipes (pH 6.9), 5 mM MgCl2, 5 mM EGTA, 0.5% Triton X-100, 20% glycerol, 1 mM NaF, 1 mM PMSF and protease inhibitor cocktail used according to manufacturers instructions (Roche, Mannheim, Germany). The homogenates were centrifuged at 12,500 rpm for 15 min and the protein content of the supernatant was measured against bovine serum albumin standards using the Bradford method (Biorad, Hercules, CA, USA). Fifteen micrograms of protein was boiled for 5 min in sodium dodecyl sulphate (SDS) sample buffer and separated using 10%SDS-PAGE. Protein was transferred onto a nitrocellulose membrane (Amersham, Buckinghamshire, UK) and blocked for non-specific binding in 5% non-fat-milk and TBS-T (20 mM Tris–HCl (pH 7.6), 137 mM NaCl, 0.1% Tween-20) for 1 h at room temperature, agitating. The membranes were then incubated with sheep anti-human PAI-1 IgG (1:5000; Chemicon International) in 5% non-fat milk-TBS-T overnight at 4 °C, agitating. Membranes were washed three times for 5 min in TBS-T and then horse radish peroxidase-conjugated secondary anti-sheep IgG (1:6000; Dako, Carpinteria, CA, USA) was added for 1 h at room temperature in 5% non-fat milk-TBS-T, agitating. Following four 5-min washes in TBS-T, the immune complexes were detected by enhanced chemiluminescence according to the manufacturer's instructions (Amersham) and exposed to X-ray film (Amersham). Bands on developed film were quantitated by image analysis (Quantity One, Biorad). PAI-1 levels were measured in units of optical density adjusted for area and the values expressed relative to the normal (uncollared) segments of saline-collared carotid in each animal.

2.7. Superoxide activity measured by lucigenin chemiluminescence assay
Superoxide production by normal (proximal to the collar) and collared (± CNP treatment for 7 days) carotid artery ring segments from each rabbit was measured using lucigenin-enhanced chemiluminescence, as previously described [25], during the following treatments, in vitro (all wells contained 3 mM diethyldithiocarbamate (DETCA) to inactivate endogenous Cu+2/Zn+2 superoxide dismutase): no additions; NADPH (100 µM); NADPH+diphenyleneiodonium (DPI, 5 µM).

2.8. Statistics
A two-way analysis of variance (ANOVA) was performed on the raw data for all PAI-1 and superoxide measurements, based on within animal comparisons, with the Bonferroni post-hoc test for multiple comparisons. Macrophage studies were analyzed using a non-parametric two-way ANOVA and all multiple comparisons were evaluated using the Student–Newman–Keuls method. Values are expressed as the mean ± S.E. from the ANOVA. Significance level of P<0.05 was accepted.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Localization of PAI-1 immunohistochemistry
The most intense PAI-1 staining in the normal and collared carotid artery walls was located in the endothelium (Fig. 1). PAI-1 was also present in the extracellular matrix of the media and adventitia, however the staining was less intense. In collared arteries, cellular components of the neointima and adventitia exhibited positive immunoreactivity for PAI-1 (Fig. 1F and G). Quantitatively, the ROD of PAI-1 immunostaining in the endothelium was 16 ± 6% higher in saline-collared arteries compared to the normal uncollared region from the same vessel (Fig. 2A).


Figure 1
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  		Fig. 1 Panels A, B and D–G are representative photomicrographs of transverse sections of carotid arteries immunostained for PAI-1. Panel C is a photomicrograph of macrophages in the adventitia stained with RAM-11 (scale bar represents 10 µm). Panels A (uncollared, normal section of saline-treated artery) and B (saline-collared section) are identically processed sections from the same rabbit treated with CNP (10 fmol/h) within the collar for 7 days. Low power magnification (scale bar represents 50 µm) in panels A and B illustrates PAI-1 immunostaining across all layers. High power magnification (scale bar represents 20 µm) in panels D–G illustrates PAI-1 immunostaining in endothelium and neointima. Panels D–G are sections that were processed under identical conditions from the same rabbit infused for 7 days intra-luminally under one collar with CNP (100 pmol/h). Panels D and E are uncollared, normal sections proximal to the collars while panels F and G are collared sections from saline- and CNP-treated carotids, respectively. Note the increase in PAI-1 staining, particularly in the endothelium (B and F) and neointima (F), of the saline-collared artery sections and the general reduction in staining in the collared artery section (G) infused directly with CNP. The arrow heads indicate the internal elastic lamina, the arrows in (A) and (B) indicate the boundary of the media and adventitia and the arrows in (C) point to macrophages.

 

Figure 2
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  		Fig. 2 Relative optical density (ROD) of PAI-1 immunostaining in carotid arteries from rabbits treated peri-arterially with CNP (n=7). Four types of tissue were collected from each rabbit: saline-collared artery (striped bars) and normal artery proximal to this collar (open bars); CNP-treated collared artery (hatched bars) and normal artery proximal to this collar (black bars). Due to the differences in PAI-1 immunostaining observed throughout the vascular wall, PAI-1 levels were measured in each layer of the vascular wall separately: (A) endothelium, (B) neointima, (C) media and (D) adventitia. Bars represent the mean ± S.E. and the percentages are the within animal mean for each tissue normalised to the uncollared (saline side) section of normal artery (100%). For neointima, saline-collared artery was deemed 100%. The subendothelial layer of normal carotid arteries could not be measured accurately at the magnification (20 x) used for morphometric analysis, thus neointimal areas were only measured in saline-collared (striped bars) and CNP-treated collared (hatched bars) arteries. *P<0.05 versus normal (saline) artery, {dagger}P<0.05 versus saline-collared artery.

 
Rabbit liver, used as a positive control, was positive for PAI-1. PAI-1 immunostaining was not detected in any tissue when the primary antibody was omitted. PAI-1 immunostaining in one femoral artery, one renal artery, one non-collared sham operated carotid artery and one untouched normal carotid artery shared a similar pattern of PAI-1 immunostaining with the sections of normal carotid arteries taken from regions proximal to the collars (data not shown). There were no differences in PAI-1 immunostaining between normal sections taken from the region proximal to each collar (Fig. 2).

3.2. Effects of peri-arterial administration of CNP on PAI-1
Staining intensity for PAI-1 appeared to be reduced throughout the vascular wall of collared arteries treated peri-arterially with CNP, compared with the contralateral saline-collared arteries. CNP treatment significantly reduced endothelial PAI-1 immunostaining by 33 ± 7%, compared to saline-collared arteries, which is lower than the levels seen in sections from normal carotids (Fig. 2A). In the neointima of collared arteries, peri-arterial CNP treatment reduced PAI-1 immunostaining by 39 ± 10% compared with saline-collared arteries (Fig. 2B). CNP treatment significantly reduced adventitial PAI-1 levels by 47 ± 14% compared to saline-collared arteries (Fig. 2D).

3.3. Effects of intra-luminal administration of CNP on PAI-1
Although the four segments of carotid arteries from rabbits treated intra-luminally with CNP displayed similar PAI-1 immunostaining (Fig. 1D–G) to those described above (peri-arterial CNP), the saline collars in these rabbits did not significantly increase endothelial PAI-1 (Fig. 3A). Adventitial PAI-1 tended to be elevated (by 35 ± 14%; P=0.08) by the collar (Fig. 3D). Western blot analysis indicated that total PAI-1 protein in saline-collared segments from these rabbits was not significantly increased (Fig. 3E; P=0.29).


Figure 3
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  		Fig. 3 Panels A–D depict the relative optical density (ROD) of PAI-1 immunostaining in carotid arteries from rabbits infused intra-luminally with CNP (n=6). The four types of tissue (panels A–D) collected from each rabbit, the symbols and abbreviations are the same as depicted in Fig. 2. Panel E depicts the total PAI-1 protein levels across the whole vessel segments (measured by Western blot), expressed as a percentage of the uncollared section (saline) of normal artery (100%). *P<0.05 versus normal (saline) artery, {dagger}P<0.05 versus saline-collared artery, {ddagger}P<0.05 versus normal (CNP) artery.

 
Collared arteries infused intra-luminally with CNP for 7 days displayed significantly reduced PAI-1 levels in the endothelium (by 17 ± 5%; Fig. 4A), the neointima (by 30 ± 3%; Fig. 3B) and the media (by 28 ± 7%; Fig. 3C) compared with the same regions in saline-collared arteries. CNP also tended to return adventitial PAI-1 levels to normal (reduced by 34 ± 7%, P=0.10 compared with contralateral saline-collar, Fig. 3D). CNP significantly lowered total PAI-1 protein (Western blot) in collared segments by 18.4 ± 7.5% (Fig. 3E).


Figure 4
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  		Fig. 4 Effect of NADPH (grey bars) and NADPH+DPI (black bars) on superoxide levels in all four carotid arterial segments (n=8 rabbits). White bars on the left of each group represent basal superoxide activity (no NADPH added). Ring segments are the same as described in Fig. 2 (from rabbits treated with peri-arterial CNP infusion into one collar). All rings were incubated with lucigenin (5 µmol/L), DETCA (3 mmol/L) and the appropriate drug treatment for 60 min before assay for superoxide. Values are mean ± S.E.M. *P<0.05 effect of NADPH treatment.

 
3.4. Macrophages
RAM-11 immunoreactive macrophages were observed as individual cells or as large clusters of cells, predominantly in collared arteries (Table 1 and Fig. 1C). The majority of these immunopositive cells were located in the adventitia. Macrophages were not present in the neointima of collared carotid arteries. There were significantly more individual macrophages, and clusters (data not shown), in saline-collared arteries compared to normal arteries (Table 1) in both groups of rabbits. Peri-arterial treatment with CNP tended to reduce the number of RAM-11-positive cells (P=0.3; Table 1). Intra-luminal CNP treatment significantly reduced macrophage cell numbers in collared arteries compared to saline-collared arteries (Table 1). Clusters of macrophages were never observed with CNP treatment, administered via either route.


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  		Table 1 Rabbit alveolar macrophage 11 (RAM-11) positive cell counts in rabbit carotid arteries

 
3.5. Morphometric analyses
A substantial neointima formed in the collared arterial regions of all rabbits, as reported previously [21–23,27]. Intimal areas and IMR data from all except one rabbit included in the present manuscript were reported recently [27]. Briefly, peri-arterial CNP did not reduce the intimal areas in collared arteries, but intra-luminal CNP infusion significantly (P<0.05) reduced intimal areas by 16 ± 5%. In addition, the present study showed that neither peri-arterial infusion of CNP nor intra-luminal CNP affected the luminal areas of collared arteries (data not shown), indicating that the peptide did not have a major vasoactive action on collared arteries.

3.6. Superoxide production
Basal superoxide production was minimal in all carotid segments but the addition of NADPH increased the production of superoxide in all segments (Fig. 4). Collared artery segments produced ~3-times more (P<0.05) superoxide than uncollared segments. The NADPH-oxidase inhibitor DPI reduced superoxide to a level not significantly different from basal production in all vessels. CNP treatment of collared arteries did not change the superoxide production in the presence of NADPH or with NADPH plus DPI (Fig. 4).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The major finding of the present study is that CNP reduces PAI-1 in vivo in rabbit carotid arteries with intimal hyperplasia. PAI-1 immunostaining in all sections of normal rabbit carotid arteries was most intense in the endothelium. The increased endothelial PAI-1 and the presence of PAI-1 in the neointima 7 days after collar placement are similar to findings in early human atherosclerosis [17,18]. Levels of PAI-1 were markedly reduced by CNP in the vessel layers that are critically involved in processes leading to restenosis [16] and longer-term atherosclerosis [17,18], namely the endothelium and sub-endothelial neointima. Although previous studies have shown that CNP reduces PAI-1 in vitro, this study is the first in vivo evidence, to our knowledge, supporting a role for CNP's suppression of PAI-1. The localization and changes in levels of PAI-1 measured immunohistochemistically were supported by the changes in total PAI-1 protein levels measured by Western blot analysis of whole vessel segments.

Intra-luminal CNP reduced intimal thickening [27] and suppressed endothelial and neointimal PAI-1. Although peri-arterial CNP had no effect on intimal thickening (present results and [27]), peptide administered to the outside of the vessel reduced PAI-1 immunostaining in both the endothelium and neointima. These findings indicate that reducing PAI-1 is not the critical step in controlling intimal hyperplasia in this model. A recent study investigating the time course of effects of PAI-1 on balloon injured rat carotid arteries showed that the intimal area of PAI-1 transfected arteries (compared to control arteries from different animals) was smaller at 4 days, unchanged at 8 days and significantly increased 14 days after balloon injury [15]. The role of PAI-1 may depend on the time after injury and also on the type of injury induced. It is difficult to extrapolate the findings from a model of acute vessel injury to those of our collar-induced inflammatory model of intimal thickening. Nevertheless, it is possible that if the collars were in place for a longer period, the role of PAI-1 in extracellular matrix formation and intimal hyperplasia may become more important. Elevated levels of PAI-1 have also been linked with decreases in neointimal formation in LDL receptor deficient and apolipoprotein E deficient mice [28] and balloon injured rat carotid arteries [29]. Clearly, further investigation of the role of PAI-1 in intimal hyperplasia, particularly at different stages of development, is required.

How does CNP reduce PAI-1 in collared vessels? Based on in vitro evidence, a direct inhibitory action of CNP on synthesis and release of endothelial [13] and VSMC [14] PAI-1 is likely, via guanylate cyclase natriuretic peptide receptor activation of cyclic GMP. Additionally, CNP's effects on PAI-1 may occur through actions on an intermediary such as macrophages, superoxide or iNOS, each of which has been shown to have an influence on PAI-1 levels and to be influenced by natriuretic peptides. Macrophages produce PAI-1, and soluble products from macrophages have been shown to upregulate PAI-1 production in endothelial and VSMC [30]. The inhibitory effect of CNP on macrophage accumulation that we observed in collared vessels may be part of an anti-inflammatory action of the peptide [31], via guanylate cyclase natriuretic peptide receptors (B-type) known to exist on macrophages [32]. Therefore, at least some of the CNP-induced reduction in PAI-1 may result from its action on macrophages. Increased superoxide production has been linked to a decrease in PAI-1 activity and mRNA expression in human endothelial cells in culture [33]. Thrombin-stimulated PAI-1 gene expression in VSMC can be suppressed by antioxidant treatment and is inhibited when a critical subunit for NADPH oxidase (p22phox) is disabled [34], adding support to a role for NADPH-driven superoxide in PAI-1 release. Although CNP's action on superoxide has not been studied previously, the sister hormone ANP has been reported to ‘prime’ neutrophils to secrete superoxide [35]. Our present results confirm that NADPH-driven superoxide is elevated in 7-day collared vessels but we could find no evidence that in vivo treatment with CNP-modulated superoxide production in collared vessels. A final possible intermediary is iNOS. It was recently shown that virally vectored CNP reduced intimal hyperplasia and macrophage infiltration in balloon injured vessels via iNOS [31]. A more recent report that iNOS can inhibit PAI-1 production [36] suggests a tantalising link between CNP, iNOS and PAI-1. Although further studies are needed to determine a role for iNOS, our present study can confirm that superoxide production via NADPH oxidase does not play a critical role in the in vivo actions of CNP on PAI-1.

The presence of a perivascular collar was an effective in vivo stimulus for enhancing vascular PAI-1, except in the rabbits where CNP was administered intra-luminally under the collar. In those intra-luminal rabbits, the ipsilateral saline-collared artery did not show the same increases in PAI-1 as when CNP was confined to the peri-arterial space of the contralateral collar (Fig. 2 compared with Fig. 3). CNP infused directly into the carotid artery under the collar would have spilled over to the ipsilateral collared artery, albeit at a much reduced concentration, and this may have been sufficient to prevent the collar-induced increase in endothelial PAI-1. Previous studies by others investigating the effects of CNP on neointimal formation in vivo used systemic infusions of CNP at the quite high dose of 1 µg/kg/min (or ~30 nmol/kg/h) [10,37,38]. This was ~1000-fold higher than the dose of 100 pmol/rabbit/h (or ~30 pmol/kg/h) employed in our present study. Our aim was to infuse a dose of CNP that would achieve an effective arterial concentration locally under the collar, but that would have minimal potentially confounding effects on the rest of the circulation. The increase in circulating CNP levels with this dose should have been <6 fmol/ml (~2–3 times an estimated baseline), calculated from the CNP infusion rate and an estimate of the rabbit's cardiac output. Therefore, it is possible that the presence of even small increments in circulating CNP may constrain increases in PAI-1 caused by a sustained inflammatory stimulus.

In summary, for the first time CNP has been shown to reduce PAI-1 levels in an in vivo model of intimal hyperplasia. This effect was observed independently of CNP's ability to reduce intimal thickening. The current study supports the body of evidence describing CNP as an important anti-atherogenic agent. At the early stages of atherosclerosis or restenosis, which the collar model mimics, factors other than PAI-1 appear to be responsible for intimal hyperplasia. Nevertheless, the successful inhibition of chronically elevated PAI-1 may be an action of CNP that could be exploited to therapeutic advantage for prevention or treatment of atherosclerotic disease and restenosis.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the National Health and Medical Research Council of Australia (Institute Block Grant No. 983001) and a Dora Lush (Biomedical) Postgraduate Research Scholarship (No. 008101 for Melissa Barber). We are grateful for the assistance of Yanan (Nancy) Guo and Tony Dornom with the care of the rabbits.


   Notes
 
Time for primary review 27 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Okahara K., Kambayashi J., Ohnishi T., Fujiwara Y., Kawasaki T., Monden M. Shear stress induces expression of CNP gene in human endothelial cells. FEBS Lett (1995) 373:108–110.[CrossRef][Web of Science][Medline]
  2. Suga S., Itoh H., Komatsu Y., Ogawa Y., Hama N., Yoshimasa T., et al. Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells-evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells. Endocrinology (1993) 133:3038–3041.[Abstract/Free Full Text]
  3. Drewett J.G., Fendly B.M., Garbers D.L., Lowe D.G. Natriuretic peptide receptor-B (guanylyl cyclase-B) mediates C-type natriuretic peptide relaxation of precontracted rat aorta. J Biol Chem (1995) 270:4668–4674.[Abstract/Free Full Text]
  4. Furuya M., Takehisa M., Minamitake Y., Kitajima Y., Hayashi Y., Ohnuma N., et al. Novel natriuretic peptide, CNP, potently stimulates cyclic GMP production in rat cultured vascular smooth muscle cells. Biochem Biophys Res Commun (1990) 170:201–208.[CrossRef][Web of Science][Medline]
  5. Ikeda M., Kohno M., Yasunari K., Yokokawa K., Horio T., Ueda M., 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]
  6. Furuya M., Yoshida M., Hayashi Y., Ohnuma N., Minamino N., Kangawa K., et al. C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells. Biochem Biophys Res Commun (1991) 177:927–931.[CrossRef][Web of Science][Medline]
  7. Doi K., Itoh H., Ikeda T., Hosoda K., Ogawa Y., Igaki T., et al. Adenovirus-mediated gene transfer of the C-type natriuretic peptide causes G1 growth inhibition of cultured vascular smooth muscle cells. Biochem Biophys Res Commun (1997) 239:889–894.[CrossRef][Web of Science][Medline]
  8. Kohno M., Horio T., Yokokawa K., Kurihara N., Takeda T. C-type natriuretic peptide inhibits thrombin- and angiotensin II-stimulated endothelin release via cyclic guanosine 3',5'-monophosphate. Hypertension (1992) 19:320–325.[Abstract/Free Full Text]
  9. Kohno M., Yokokawa K., Yasunari K., Kano H., Minami M., Ueda M., 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]
  10. Shinomiya M., Tashiro J., Saito Y., Yoshida S., Furuya M., Oka N., 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][Web of Science][Medline]
  11. Ueno H., Haruno A., Morisaki N., Furuya M., Kangawa K., Takeshita A., et al. Local expression of C-type natriuretic peptide markedly suppresses neointimal formation in rat injured arteries through an autocrine/paracrine loop. Circulation (1997) 96:2272–2279.[Abstract/Free Full Text]
  12. Doi K., Ikeda T., Itoh H., Ueyama K., Hosoda K., Ogawa Y., et al. C-type natriuretic peptide induces redifferentiation of vascular smooth muscle cells with accelerated reendothelialization. Arterioscler Thromb Vasc Biol (2001) 21:930–936.[Abstract/Free Full Text]
  13. Bouchie J.L., Hansen H., Feener E.P. Natriuretic factors and nitric oxide suppress plasminogen activator inhibitor-1 expression in vascular smooth muscle cells–role of cGMP in the regulation of the plasminogen system. Arterioscler Thromb Vasc Biol (1998) 18:1771–1779.[Abstract/Free Full Text]
  14. Yoshizumi M., Tsuji H., Nishimura H., Masuda H., Kunieda Y., Kawano H., et al. Natriuretic peptides regulate the expression of tissue factor and PAI-1 in endothelial cells. Thromb Haemost (1999) 82:1497–1503.[Web of Science][Medline]
  15. DeYoung M.B., Tom C., Dichek D.A. Plasminogen activator inhibitor type 1 increases neointima formation in balloon-injured rat carotid arteries. Circulation (2001) 104:1972–1981.[Abstract/Free Full Text]
  16. Sawa H., Lundgren C., Sobel B.E., Fujii S. Increased intramural expression of plasminogen activator inhibitor type 1 after balloon injury: a potential progenitor of restenosis. J Am Coll Cardiol (1994) 24:1742–1748.[Abstract]
  17. Schneiderman J., Sawdey M.S., Keeton M.R., Bordin G.M., Bernstein E.F., Dilley, et al. Increased type 1 plasminogen activator inhibitor gene expression in atherosclerotic human arteries. Proc Natl Acad Sci U.S.A. (1992) 89:6998–7002.[Abstract/Free Full Text]
  18. Padro T., Emeis J.J., Steins M., Schmid K.W., Kienast J. Quantification of plasminogen activators and their inhibitors in the aortic vessel wall in relation to the presence and severity of atherosclerotic disease. Arterioscler Thromb Vasc Biol (1995) 15:893–902.[Abstract/Free Full Text]
  19. Irigoyen J.P., Munoz-Canoves P., Montero L., Koziczak M., Nagamine Y. The plasminogen activator system: biology and regulation. Cell Mol Life Sci (1999) 56:104–132.[CrossRef][Web of Science][Medline]
  20. Drew A.F., Tucker H.L., Kombrinck K.W., Simon D.I., Bugge T.H., Degen J.L. Plasminogen is a critical determinant of vascular remodeling in mice. Circ Res (2000) 87:133–139.[Abstract/Free Full Text]
  21. Booth R.F., Martin J.F., Honey A.C., Hassall D.G., Beesley J.E., Moncada S. Rapid development of atherosclerotic lesions in the rabbit carotid artery induced by perivascular manipulation. Atherosclerosis (1989) 76:257–268.[CrossRef][Web of Science][Medline]
  22. Dusting G.J., Curcio A., Harris P.J., Lima B., Zambetis M., Martin J.F. Supersensitivity to vasoconstrictor action of serotonin precedes the development of atheroma-like lesions in the rabbit. J Cardiovasc Pharmacol (1990) 16:667–674.[CrossRef][Web of Science][Medline]
  23. De Meyer G.R., Bult H., Van Hoydonck A.E., Jordaens F.H., Buyssens N., Herman A.G. Neointima formation impairs endothelial muscarinic receptors while enhancing prostacyclin-mediated responses in the rabbit carotid artery. Circ Res (1991) 68:1669–1680.[Abstract/Free Full Text]
  24. Arthur J., Dusting G.J. Selective endothelial dysfunction in early atheroma-like lesions in the rabbit. Coron Artery Dis (1992) 3:623–629.[Web of Science]
  25. Paravicini T.M., Gulluyan L.M., Dusting G.J., Drummond G.R. Increased NADPH oxidase activity, gp91phox expression, and endothelium-dependent vasorelaxation during neointima formation in rabbits. Circ Res (2002) 91:54–61.[Abstract/Free Full Text]
  26. De Meyer G.R., Bult H. Mechanisms of neointima formation-lessons from experimental models. Vasc Med (1997) 2:179–189.[Medline]
  27. Barber M.N., Gaspari T.A., Kairuz E.M., Dusting G.J., Woods R.L. Atrial natriuretic peptide preserves endothelial function during intimal hyperplasia. J Vasc Res (2005) 42(2):101–110. [Epub ahead of print].[CrossRef][Web of Science][Medline]
  28. Sjoland H., Eitzman D., Gordon D., Westrick R., Nabel E.G., Ginsburg D. Atherosclerosis progression in LDL-receptor-deficient and apolipoprotein E-deficient mice is independent of genetic alterations in plasminogen activator inhibitor-1. Arterioscler Thromb Vasc Biol (2000) 20:846–852.[Abstract/Free Full Text]
  29. Hasenstab D., Lea H., Clowes AW. Local plasminogen activator inhibitor type 1 overexpression in rat carotid artery enhances thrombosis and endothelial regeneration while inhibiting intimal thickening. Arterioscler Thromb Vasc Biol (2000) 20:853–859.[Abstract/Free Full Text]
  30. Tipping P.G., Davenport P., Gallicchio M., Filonzi E.L., Apostolopoulos J., Wojta J. Atheromatous plaque macrophages produce plasminogen activator inhibitor type-1 and stimulate its production by endothelial cells and vascular smooth muscle cells. Am J Pathol (1993) 143:875–885.[Abstract]
  31. Qian J.Y., Haruno A., Asada Y., Nishida T., Saito Y., Matsuda T., et al. Local expression of C-type natriuretic peptide suppresses inflammation, eliminates shear stress-induced thrombosis, and prevents neointima formation through enhanced nitric oxide production in rabbit injured carotid arteries. Circ Res (2002) 91:1063–1069.[Abstract/Free Full Text]
  32. Kiemer A.K., Vollmar A.M. Effects of different natriuretic peptides on nitric oxide synthesis in macrophages. Endocrinology (1997) 138:4282–4290.[Abstract/Free Full Text]
  33. Shatos M.A., Doherty J.M., Stump D.C., Thompson E.A., Collen D. Oxygen radicals generated during anoxia followed by reoxygenation reduce the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cell culture. J Biol Chem (1990) 265:20443–20448.[Abstract/Free Full Text]
  34. Gorlach A., Diebold I., Schini-Kerth V.B., Berchner-Pfannschmidt U., Roth U., Brandes R.P., et al. Thrombin activated the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells. Role of the p22phox-containing NADPH oxidase. Circ Res (2001) 89:47–54.[Abstract/Free Full Text]
  35. Wiedermann C.J., Niedermuhlbichler M., Braunsteiner H., Widermann C.J. Priming of polymorphonuclear neutrophils by atrial natriuretic peptide in vitro. J Clin Invest (1992) 89:1580–1586.[Web of Science][Medline]
  36. Davila H.H., Magee T.R., Vernet D., Rajfer J., Gonzalez-Cadavid N.F. Gene transfer of inducible nitric oxide synthase complementary DNA regresses the fibrotic plaque in an animal model of Peyronie's disease. Biol Reprod (2004) 71:1568–1577.[Abstract/Free Full Text]
  37. Furuya M., Miyazaki T., Honbou N., Kawashima K., Ohno T., Tanaka S., et al. C-type natriuretic peptide inhibits intimal thickening after vascular injury. Ann N Y Acad Sci (1995) 748:517–523.[Web of Science][Medline]
  38. Takeuchi H., Ohmori K., Kondo I., Oshita A., Shinomiya K., Yu Y., et al. Potentiation of C-type natriuretic peptide with ultrasound and microbubbles to prevent neointimal formation after vascular injury in rats. Cardiovasc Res (2003) 58:231–238.[Abstract/Free Full Text]

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