Skip Navigation

Cardiovascular Research 2003 58(3):671-678; doi:10.1016/S0008-6363(03)00322-5
© 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 Schmidt, A.
Right arrow Articles by Buddecke, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schmidt, A.
Right arrow Articles by Buddecke, E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

Exogenous nitric oxide causes overexpression of TGF-β1 and overproduction of extracellular matrix in human coronary smooth muscle cells

Annette Schmidta,*, Sven Geigenmuellera, Wolfgang Voelkera, Petra Seilera and Eckhart Buddeckeb

aInstitute of Arteriosclerosis Research, University of Muenster, Department of Molecular Cardiology, Domagkstrasse 3, D-48149 Muenster, Germany
bInstitute of Physiological Chemistry and Pathobiochemistry, University of Muenster, Muenster, Germany

annschm{at}uni-muenster.de

* Corresponding author. Tel.: +49-251-835-8626; fax: +49-251-835-8628.

Received 12 November 2002; accepted 24 January 2003


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Nitric oxide (NO) is a major signalling molecule in the vascular system enhancing vascular smooth muscle cell relaxation and vasodilation. NO donors are the most frequently and repeatedly used drugs for relief from angina pectoris. Methods: We investigated the effects of the synthetic NO donor DETA/NO on cultured human coronary smooth muscle cells. Results: Cells exposed to 100 µM DETA/NO for 48–72 h were channeled into a cell cycle-arrested hypertrophic growth status associated with overexpression of TGF-β1 on both the protein and mRNA levels. Increased TGF-β1 transcription and translation were associated with enhanced synthesis of extracellular matrix components including the collagen types I and III as shown by immunocytochemistry and enhanced incorporation of [3H]proline. Higher incorporation of [35S]sulfate into chondroitin/dermatan sulfate and heparan sulfate containing proteoglycans was observed in DETA/NO treated cells than in controls. The ratio of chondroitin/dermatan sulfate to heparan sulfate did not change significantly. Conclusions: Our results suggest a dual function of the overexpressed TGF-β1. Overexpressed TGF-β1 could stabilize the fibrous cap overlaying atherosclerotic plaques due to the accumulation of extracellular matrix components. However, the findings could also support a proatherogenic role of TGF-β1 resulting from the overexpression of LDL-binding proteoglycans.

KEYWORDS Nitric oxide; Gene expression; Smooth muscle; Connective tissue; Growth factor


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Nitric oxide (NO) is a major signalling molecule of the vascular system. It either acts within the endothelium in which it is produced constitutively by NO synthase, or it penetrates the endothelial layer affecting vascular smooth muscle cells (SMC) which may also generate NO by inducible NO synthase. NO reacts with iron in the active site of guanylyl cyclase stimulating it to produce cGMP that in turn activates cGMP-dependent protein kinases facilitating the extrusion of intracellular calcium resulting in SMC relaxation and vasodilation (for review see Ref. [1]).

In addition to maintaining vascular tone, NO acts as a pleiotropic agent that acts in a variety of cGMP-independent ways, in part by S-nitrosylation of intracellular and extracellular proteins [2–4] or by inhibiting the generation of superoxide anions by oxidative enzymes. It has been found that endogenous NO or synthetic NO donors inhibit endothelial generation of superoxide anions [5], suppress the stimulated exposure of E-selectin [6], attenuate leukocyte adhesion [7], inhibit platelet aggregation [8], inhibit smooth muscle cell proliferation [9–12] and reduce the activity of NF-{kappa}B [13].

Nitrates are commonly used to relieve or prevent episodes of angina pectoris. Since most previous studies have described the short-time actions of endogenous NO or of synthetic NO donors, we addressed the effect of exposure of coronary smooth muscle cells (cSMC) to NO over a period of 48–72 h. We found increased expression of TGF-β1 and enhanced stimulation of extracellular matrix synthesis.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The investigation conforms with the principles outlined in the Declaration of Helsinki.

2.1 Reagents
Cell culture medium and smooth muscle growth supplement were purchased from TEBU (Offenbach, Germany). Tissue culture flasks were from Falcon Labware Division, Becton Dickinson (Heidelberg, Germany). DETA NONOate (DETA/NO; (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate) was a product of Alexis Biochemicals, QBIOGENE-ALEXIS (Grünberg, Germany). DETA/NO is stable in 0.01 M NaOH [14]. Addition of excess buffer at pH 7.4 initiates release of NO with a half-life of 20–23 h at 37°C in a first order reaction [14,17]. The kinetic law dictates that both dose rate and cumulative dose can be predicted. DETA/NO was shown to be active during a 2–5-day culture period [9]. [Methyl-3H]thymidine (specific activity 1.3 TBq mmol–1), [2,3-3H]proline (specific activity 1.74 TBq mmol–1) and sodium [35S]sulfate (carrier free, 0.8–1.5 TBq mg–1 sulfur) were obtained from Amersham Pharmacia Biotech (Freiburg, Germany) and ICN Biomedicals (Eschwege, Germany). TGF-β1 ELISA-system was obtained from R&D Systems (Wiesbaden, Germany). Antibodies against TGF-β1 were from R&D Systems (MAB240). Antibodies against collagen I (ICN 631701, Clone I-8H5) and collagen III (Sigma C-7805, Clone FH-7A) and against the glycosaminoglycans chondroitin sulfate (Sigma C-8035, Clone CS-56) and heparan sulfate (Seikagaku Epitop 10E4, 370255) were commercially available. All other chemicals were of analytical grade or the best grade available and were purchased from Roche Diagnostics (Mannheim, Germany), Merck (Darmstadt, Germany) or Serva (Heidelberg, Germany).

2.2 Cell culture
Human coronary artery smooth muscle cells from TEBU were cultured in Medium 231 enriched with smooth muscle growth supplement, 2 mM glutamine (Seromed, Berlin, Germany) and 10 µg ml–1 ciprofloxacin (Bayer, Leverkusen, Germany) (standard medium) at 37°C in a humidified atmosphere of 5% CO2. The cells identity was confirmed by immunostaining smooth muscle cells for {alpha}-actin. Cultures of the 3rd to the 5th passage were used in the experiments. An effect of DETA/NO was determined as follows: cells were seeded at a density of 75 000/dish (35 mm diameter). After 40–48 h the medium was replaced by the standard medium (control) or the standard medium containing 50–100 µM DETA/NO and incubation was continued for a further 48–72 h. Then medium and cells were processed for analysis according to the described protocol. Parallel cultures treated with DETA (diethylentriamine) served as additional controls. Cell growth and cell growth inhibition were estimated on the basis of [3H]thymidine incorporation and cell counting by standard methods as previously described [15]. [3H]Thymidine was added 8 h prior to the end of the experiment. Synthesis of collagen and proteoglycans was determined by [3H]proline and [35S]sulfate incorporation into total cell protein and glycosaminoglycans.

2.3 Isolation of total RNA, real-time quantitative RT-PCR and agarose gel electrophoresis
cSMC were grown in the presence or absence of 100 µM DETA/NO for 72 h. After extensive washing, total RNA was isolated by Qiagen RNeasy kit and further purified by DNase digestion. Two µg of total RNA were then reverse transcribed into cDNA using the Superscript II Polymerase (Life Technologies, Karlsruhe, Germany). The sequences of forward and reverse oligonucleotide primers for PCR were designed by R&D Systems (Minneapolis, MN, USA). PCR products (35 cycles) were visualized by agarose gel electrophoresis. For Taqman analysis the mRNA for TGF-β1 was measured by real-time quantitative RT-PCR. The mRNA levels for TGF-β1 were corrected for 18S ribosomal RNA.

2.4 Cell proteins and proteoglycans
The total protein content was determined according to Lowry. Collagen was labelled for 48 h in standard medium containing 370 kBq ml–1 [2,3-3H]proline. Incorporation into medium proteins was determined from the cell-free culture medium by GPC for separation of the protein-bound 3H-radioactivity. Sulfated glycosaminoglycans were labeled for 48 h in standard medium containing 370 kBq ml–1 of [35S]sulfate and processed as described [16].

2.5 TGF-β1, collagen and glycosaminoglycans
TGF-β1 was quantified by a specific enzyme immunoassay technique (Quantikine, R&D Systems). Aliquots of culture medium were taken for quantification of secreted TGF-β1. For quantitative determination of the cellular TGF-β1 the cells were washed three times with phosphate-buffered saline (PBS, 0.138 M sodium chloride, 0.0027 M potassium chloride, 0.01 M phosphate buffer, pH 7.4, at 25°C) and solubilized on ice in 200–300 µl PBS containing 0.5% Brij35 and protease inhibitors for 15 min. The cell lysate was centrifuged at 10 000xg for 10 min at 4°C and the clear supernatant was used for TGF-β1 analysis by ELISA. The matrix-bound TGF-β1 was obtained after lysis of the cells with 300 µl PBS containing 0.5% TX-100 in 25 mM NH4OH for 3 min at 22°C followed immediately by an extensive washing with PBS and extraction of the matrix-bound TGF-β1 by 250 µl PBS containing 0.5% Brij35, protease inhibitors and 0.16 M HCl at 4°C in order to release the active form of TGF-β1. After 1 h the extract was neutralized, centrifuged and the clear supernatant was submitted to ELISA analysis. The assay recognizes recombinant and natural human soluble TGF-β1 in a concentration range of 31.2–2000 pg/ml. Standard data were linearised by plotting the log of the TGF-β1 concentration vs. the log of the optical density at 450 nm (OD450). No significant cross-reactivity or interference were observed with TGF-β2 and TGF-β3. The minimum detected concentration of TGF-β1 was less than 7 pg/ml. The matrix-bound TGF-β1 and the nonlabelled extracellular matrix components were obtained by exposure of the cells to a solution of 0.5% Triton X-100 and 25 mM NH4OH in PBS for 3 min at 22°C as previously described [15]. Thereafter, the extracellular (subcellular) matrix was washed with PBS and exposed to specific antibodies (against TGF-β1, collagen I, III, chondroitin sulfate, heparan sulfate). After 1 h the excess antibody was removed by three washing steps, and the appropriate secondary antibody conjugated with peroxidase (Sigma A-8786, A-2304) was added followed by an extensive wash-out. After addition of horseradish peroxidase substrate the absorption at 405 nm was determined in an ELISA reader. The results are expressed in arbitrary units. In addition, the [2,3-3H]proline or [35S]sulfate-labelled extracellular matrix components were monitored for radioactivity after proteolytic degradation of the matrix.

2.6 Morphometric analysis
The bottom of the flat-embedded cell layer was inverted for light microscopic examination and digital documentation of the cells. Micrographs were taken with a video camera system (Visitron System, Puchheim, Germany), digitized and calculated with IPLab morphometry (Visitron Systems). Areas of the cytosolic compartment of individual cells were outlined with a mouse cursor. Statistical analysis of treated and non-treated cells was performed using Student's t-test.

2.7 Other methods
Apoptosis and necrosis of SMCs were monitored by the cellular DNA fragmentation ELISA and cell death detection ELISA (Roche Diagnostics, #1585045 and #1544675).

2.8 Statistics
Results are expressed as means±standard deviation (S.D.) of the specified number of experiments carried out on different cultures in duplicates or triplicates. Statistical significance was assessed using the Student's paired t-test.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 NO induces a hypertrophic phenotype in coronary smooth muscle cells
Human cSMC were converted to a cell cycle arrested growth status by culturing for 72 h in the presence of 100 µM DETA/NO (Fig. 1). Under cell culture conditions, DETA/NO releases NO with a half-life of 20 h in a strictly first order reaction [14,17]. The growth arrested hypertrophic phenotype of the cells in the NO-treated cultures was indicated by the increased content of total cell protein (P<0.01) and by the lower number of cells at the end of the experiment (P<0.01) (Fig. 2). The alterations are time- and concentration-dependent (Figs. 1 and 2Go). At 50 µM DETA/NO the increased content of cell protein is not significant, but the arrest of cell cycle is evident at this concentration (P<0.05). The DETA/NO effect is reversible. When the culture medium of DETA/NO-treated cells is replaced by a standard medium after 48 h, the [3H]thymidine incorporation rate recovers over the following 48 h (inset of Fig. 1). DETA, the reaction product of DETA/NO had no effect on the examined parameter (not shown).


Figure 1
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Dose–response inhibition of cSMC proliferation by DETA/NO. Cells were seeded at a density of 75 000 cells/dish (35 mm diameter) and cultured in standard medium. DETA/NO in the concentration specified was added only once 24 h after seeding (time zero). Inhibition of proliferation was monitored after an incubation period of 3 days. Values are the mean±S.D. of three experiments performed in duplicate. Inhibition of cell proliferation was measured by [3H]thymidine incorporation into DNA (bullet). Inset, proliferation kinetics of cSMC under the influence of 100 µM DETA/NO (bullet). In parallel cultures the medium was replaced after 48 h by a DETA/NO-free standard medium ({blacksquare}). Other conditions as described.

 

Figure 2
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Cell number and total cell protein content after 48 h treatment with DETA/NO. Means and S.D. of four experiments in duplicate. Other conditions as in Fig. 1. *P<0.05; **P<0.01 vs. control.

 
Morphometric measurements of the cell size confirms the conversion to a hypertrophic growth status (Fig. 3). The values are expressed as area of cytosol compartment, but due to a considerable variation of minimal and maximal values (control 128–798 µm2 vs. NO-treated cells 560–6114 µm2), the error probability of cell size difference was 0.056.


Figure 3
View larger version (159K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Light micrograph of control (a) and DETA/NO-treated (b) cultured cSMC. Culture conditions as in Fig. 1. The cell layer was inverted for light microscopic examination and used for morphometric analysis that revealed a remarkable increase of cell size. Bar 100 µm.

 
3.2 NO promotes TGF-β1 expression and TGF-β1 gene transcription
When we studied the expression of TGF-β1 protein, we found a dose-dependent increase in TGF-β1 protein at proliferation-inhibiting doses determined in a TGF-β1 specific ELISA system. The overexpressed TGF-β1 is delivered to three compartments (Fig. 4). The bulk of TGF-β1 in DETA/NO-treated cells is found in the culture medium where it exceeds that of the control cells 3–4-fold. The remaining proportion of the overexpressed TGF-β1 is deposited in the subcellular matrix where TGF-β1 was detected by specific antibodies after lysis of the cells by a method exposing the cell-free subcellular matrix for further analysis [14]. The values obtained for the cell-free matrix relate predominantly to the active form of TGF-β1 since the monoclonal antibodies used recognize the active form and exhibit an ~15% cross-reactivity only with latent TGF-β1. The results are given in arbitrary units, since no standard values for matrix bound TGF-β1 could be elaborated.


Figure 4
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 DETA/NO-induced changes of TGF-β1 level in the culture medium (a) and in the cellular and extracellular (b) compartment of cSMC after exposure for 3 days. Measurements of the medium and cellular TGF-β1 concentration were performed on aliquots of the culture medium and on cell lysates using a commercial ELISA system. For quantification of extracellular TGF-β1 the cells were lysed by a method that preserved the subcellular matrix (see Methods). The matrix was washed extensively and exposed to a primary and secondary antibody. Values are means of three experiments. The increase in TGF-β1 was significant in the medium (P<0.001) and in the matrix associated material (P<0.01). ECM, extracellular matrix.

 
The data shown in Fig. 4 demonstrate the impact of NO on the expression of TGF-β1 at the protein level. To confirm the effect of DETA/NO on the upregulation of TGF-β1 transcription we isolated mRNA from cSMC pretreated with 100 µM for 72 h. The total RNA was transcribed to cDNA and submitted to PCR using forward and reverse oligonucleotide primers. The PCR products, visualized by agarose gel electrophoresis (Fig. 5), show a pronounced band in NO-treated cells, while only a weak band could be detected in the absence of DETA/NO. Taqman analysis (not shown) assessed a 2.1-fold increase of TGF-β1-specific mRNA.


Figure 5
View larger version (60K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Agarose gel electrophoresis of TGF-β1-specific PCR products. Total RNA was isolated from cultures of confluent cSMC incubated in the presence or absence of 100 µM DETA/NO for 3 days. Two µg of RNA were reverse transcribed into cDNA and submitted to PCR using TGF-β1-specific forward and reverse oligonucleotide primers. PCR products were visualized by agarose gel electrophoresis. C, control; C+, positive control (cDNA of TGF-β1). The bands are representative for three experiments.

 
3.3 Stimulation of extracellular matrix biosynthesis by DETA/NO
The synthesis of extracellular matrix components was monitored by assessing the incorporation of 3H-labelled proline and inorganic [35S]sulfate into the medium and the cell-free subcellular matrix generated by the cells during the culture period (see Methods). Additionally, the matrix was analyzed by immunocytometry using antibodies against collagen types I and III and glycosaminoglycans. The effect of NO on collagen and glycosaminoglycan biosynthesis is seen in Fig. 6. In all compartments investigated a higher amount of protein-bound [3H]proline (and [3H]hydroxyproline, respectively) was observed in NO-treated cultures. Both in the medium and in the cell lysate the incorporated [3H]proline activity exceeded the control values by more than 100%. The matrix associated [3H]proline incorporation was one order of magnitude lower and was probably due to the retarded assembly of soluble collagen molecules to insoluble fibres under culture conditions. However, the antibody-reactive collagen I and III of the matrix (Fig. 8) was found to be 100% more in the NO-treated cells than in control cultures. The artery-specific ratio of collagen I and III (2:1) is maintained after NO stimulation, but due to probable differences in the quality of the antibodies this value is lacking specificity. Cultured cSMC synthesize chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) containing proteoglycans in a ratio of 70:30 and distribute them to an extracellular, cell-associated and an intracellular compartment. Under cell culture conditions the bulk of sulfated proteoglycans (>90%) is found in the medium, both in control and NO-treated cells, but a higher amount of the incorporated [35S]sulfate radioactivity is detectable in all compartments of NO-treated cells than in control cells (Figs. 7 and 8Go). The ratio of chondroitin/dermatan sulfate:heparan sulfate was measured after degradation of the 35S-labelled material by chondroitin sulfate lyase ABC and separation of the undegraded heparan sulfate and the CS/DS split products. The ratio did not change significantly under NO treatment. The antibodies used to monitor chondroitin/dermatan sulfate and heparan sulfate in the cell-free matrix (Fig. 8) have different affinities to their epitopes, so that no conclusions can be drawn about the ratio of CS/DS:HS biosynthesis from the OD405 values.


Figure 6
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Biosynthetic activity of DETA/NO-treated cSMC as monitored by the incorporation of [3H]proline. After a 3-day incubation period the incorporation of [3H]proline into the medium, cellular and extracellular protein was measured. An aliquot of the medium was lyophilized, redissolved in a small volume of distilled water and submitted to Sephadex G-50 column chromatography. The V0 peak was collected for analysis of radioactivity. **P<0.01 vs. control.

 

Figure 8
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Immunocytometric analysis of collagen and glycosaminoglycan content of the extracellular matrix. The extensively washed cells were lysed by a method that preserved the extra(sub)cellular matrix before exposure to a primary and secondary antibody, n = 3; means and S.D. expressed as arbitrary units.

 

Figure 7
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Biosynthesis of sulfated proteoglycans in cSMC as influenced by 100 µM DETA/NO. After a culture period in the presence or absence of DETA/NO the incorporated [35S] radioactivity was determined in the cellular and pericellular (trypsin-removable) compartment and in the medium. The medium [35S]proteoglycans were obtained by adsorption to a DEAE column and elution with 1 M NaCl. The eluate contained the total sulfated glycosaminoglycans. The trypsin-releasable pool represents the cell membrane-integrated proteoglycans. The extra(sub)cellular pool contains proteoglycans interacting with the insoluble extracellular matrix.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Nitric oxide, a highly reactive biological signal transduction molecule plays a key role in vascular homeostasis and is capable of affecting atherogenesis and restenosis after vascular injury. In endothelial cells constitutively produced and/or exogenous NO function as vasodilators. They also limit LDL oxidation by inhibiting the generation of superoxide, inhibit leukocyte and platelet activation [7,8], smooth muscle cell proliferation [9–12] and suppress the stimulated expression of E-selectin, VCAM-1 and MCP-1 [6].

Here, we describe a new function of NO mediated by the NO donor compound DETA/NO on cSMC. At a DETA/NO concentration of 50–100 µM, the continuously released NO in a first order reaction causes cell cycle arrest, but maintains essential metabolic activities. Thus, a hypertrophic cell growth status is initiated that is associated with an overexpression of TGF-β1 both on the protein and mRNA levels and an increased synthesis of extracellular matrix compounds.

The applied dose of DETA/NO (100 µM) used in our study is 2–3-fold higher than physiological NO plasma levels in healthy adults (36–42 µM) [18]. After treatment with a 10 mg dose of isosorbide dinitrate the plasma levels of patients reach values of 345.6±83.3 µMxmin (AUC) [19].

Numerous earlier studies have indicated that TGF-β1 is an active and central participant in vascular processes. It affects the production of extracellular matrix [20] leading to an accumulation of extracellular matrix components such as proteoglycans and collagen [21]. The assumption that the NO-induced overexpression of TGF-β1 effects the observed increased synthesis of extracellular matrix compounds is supported by the findings that exogenous TGF-β1 stimulates the synthesis of collagen types I and III in human [22] and bovine [23] arterial smooth muscle cells and increases versican- and biglycan-specific mRNA in monkey arterial smooth muscle cells [24]. In addition, it has been shown that these two proteoglycans are localized in lipid-rich regions of atherosclerotic plaques along with TGF-β1 [25] and exhibit increased binding to LDL due to longer glycosaminoglycan chains in these proteoglycans [26].

Intimal hyperplasia (neointima formation) in the course of atherogenesis in response to arterial injury (e.g. after percutaneous transluminal coronary angiography) is represented by the expression of a variety of cellular activities involving recruitment of macrophages and other inflammatory cells as well as the secretion of extracellular matrix by resident SMC. While the presence of immigrated mononuclear blood cells enhances the risk of plaque rupture by the production of degradative enzymes, the TGF-β1-triggered synthesis of extracellular matrix strengthens the fibrous cap overlaying the plaque thereby increasing plaque stability. This explains the finding that the application of antisense oligodeoxynucleotides to TGF-β1 significantly retards balloon injury-induced neointima expansion [27]. Moreover, patients with decreased levels of TGF-β1 circulating in the active form have more advanced coronary diseases [28]. This is in accordance with our finding that the TGF-β1 released from DETA/NO-treated cells is found in large amounts in the culture medium.

Studies on experimental animals suggest that NO inhibits atherogenesis and the neointimal response to vascular injury. It is therefore reasonable to anticipate that augmentation of NO synthesis or the systemic/local delivery of NO donor compounds may be beneficial. Augmentation of NO synthesis or systemic delivery of NO compounds have been found to be beneficial in experimental animals for the prevention and/or treatment of atherosclerosis or for attenuation of neointima formation after vascular injury [1]. However, none of these drugs has been reported to limit the progression of atherosclerosis in humans. The limitation of the most commonly used NO donor compounds, nitrates, is the requirement for biotransformation by nitrosothiol formation and nitrate tolerance. Tolerance with loss of efficiency develops within 12–24 h of continuous exposure due to depletion of sulfhydryl groups, but useful response is restored after 8 h freedom from the drug [1].

Other nitric oxide donors such as SNAP (S-nitroso-N-acetylpenicillamine) or the molsidomine derivative SIN-1 (3-morpholino-sydonimine) could reinforce the role of NO for the modulation of vascular SMC metabolism. The use of these compounds, however, is complicated by the need for metabolic activation and therefore they were not used in this study. The biological actions of FK-409 [29], a NO donor which does not require transformation, are thought to be similar to those of endogenous NO [30].

Taking the results together, we suggest that NO-induced overexpressed TGF-β1 has a dual function. On the one hand TGF-β1 may contribute to the appearance of a dense fibrous cap of extracellular matrix that covers and stabilizes atherosclerotic plaques possibly converting unstable into stable angina pectoris. On the other hand the TGF-β1-initiated increase in proteoglycans with enhanced binding to native LDL would support a proatherogenic role for TGF-β1.

Time for primary review 27 days.


   Acknowledgements
 
We thank Dr. K. Yoshida, Tokyo, for a generous gift of antibodies and Marc Lewejohann for skillful technical assistance.


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

  1. Lloyd-Jones I.M, Block K.D. The vascular biology of nitric oxide and its role in atherogenesis. Annu Rev Med (1996) 47:365–375.[CrossRef][ISI][Medline]
  2. Mallis R.J, Buss J.E, Thomas J.A. Oxidative modification of H-ras: S-thiolation and S-nitrosylation of reactive cysteines. Biochem J (2001) 355:145–153.[CrossRef][ISI][Medline]
  3. Castel H, Vaudry H. Nitric oxide directly activates GABA(A) receptor function through a cGMP/protein kinase-independent pathway in frog pituitary melanotrophs. J Neuroendocrinol (2001) 13:695–705.[CrossRef][ISI][Medline]
  4. Sun J, Xin C, Eu J.P, Stamler J.S, Meissner G. Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO. Proc Natl Acad Sci USA (2001) 98:11158–11162.[Abstract/Free Full Text]
  5. Clancy R.M, Leszczynska P, Piziak J, Abramson S.B. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on NADPH oxidase. J Clin Invest (1992) 90:1116–1121.[ISI][Medline]
  6. Kosonen O, Kankaanranta H, Uotila J, Moilanen E. Inhibition by nitric oxide-releasing compounds of E-selectin expression in and neutrophil adhesion to human endothelial cells. Eur J Pharmacol (2000) 394:149–156.[CrossRef][ISI][Medline]
  7. Villarreal D, Grisham M.B, Granger D.N. Nitric oxide donors improve gut function after prolonged hypothermic ischemia. Transplantation (1995) 59:685–689.[ISI][Medline]
  8. Mellion B.T, Ignarro L.J, Ohlstein E.H, et al. Evidence for the inhibitory role of guanosine 3',5'-monophosphate in ADP induced human platelet aggregation in the presence of nitric oxide and related vasodilator. Blood (1981) 57:946–955.[Free Full Text]
  9. Mooradian D.L, Hutsell T.C, Keefer L.K. Nitric oxide (NO) donor molecules: effect of NO release rate on vascular smooth muscle cell proliferation in vitro. J Cardiovasc Pharmacol (1995) 25:674–678.[ISI][Medline]
  10. Young D.V, Serebryanik D, Janero D.R, Tam S.W. Suppression of proliferation of human coronary artery smooth muscle cells by the nitric oxide donor, S-nitrosoglutathione, is cGMP-independent. Mol Cell Biol Res Commun (2000) 4:32–36.[CrossRef][Medline]
  11. Sarkar R, Gordon D, Stanley J.C, Webb R.C. Cell cycle effects of nitric oxide on vascular smooth muscle cells. Am J Physiol (1997) 272:1810–1818.
  12. 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]
  13. Coke J.P, Tsao P.S. Arginine: a new therapy for atherosclerosis. Circulation (1997) 311–312.
  14. Hrabie J.A, Klose J.R, Wink D.A, Keefer L.K. New nitric oxide-releasing zwitterions derived from polyamines. J Org Chem (1993) 58:1472–1476.[CrossRef][ISI]
  15. Schmidt A, Vlodavsky I, Völker W, Buddecke E. Induction of a hypertrophic growth status of coronary smooth muscle cells is associated with an overexpression of TGF-β. Eur J Cell Biol (2002) 81:138–144.[CrossRef][ISI][Medline]
  16. Schmidt A, Vlodavsky I, Völker W, Buddecke E. Differentiation of coronary smooth muscle cells to a cell cycle-arrested hypertrophic growth status by a synthetic non-toxic heparin-mimicking compound. Atherosclerosis (1999) 147:387–397.[CrossRef][ISI][Medline]
  17. Keefer L.K, Nims R.W, Davies K.M, Wink D.A. ‘NONOates’ (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms. Methods Enzymol (1996) 268:281–293.[ISI][Medline]
  18. Schulz R, Schmidt D, Blum A, et al. Decreased plasma levels of nitric oxide derivatives in obstructive sleep apnoea: response to CPAP therapy. Thorax (2000) 55:1046–1051.[Abstract/Free Full Text]
  19. Vogt D, Trenk D, Bonn R, Jähnchen E. Pharmacokinetics and haemodynamic effects of ISDN following different dosage forms and routes of administration. Eur J Clin Pharmacol (1994) 46:319–324.[ISI][Medline]
  20. McCaffrey T.A. TGF-betas and TGF-beta receptors in atherosclerosis. Cytokine Growth Factor Rev (2000) 11:103–114.[CrossRef][ISI][Medline]
  21. Wight T.N. Cell biology of arterial proteoglycans. Arteriosclerosis (1989) 9:1–20.[Abstract/Free Full Text]
  22. Amento E.P, Ehsani N, Palmer H, Libby P. Cytokines and growth factors positively and negatively regulate interstitial collagen gene expression in human vascular smooth muscle cells. Arterioscler Thromb (1991) 11:1223–1230.[Abstract/Free Full Text]
  23. Lawrence R, Hartmann D.J, Sonenshein G.E. Transforming growth factor β1 stimulates type V collagen expression in bovine vascular smooth muscle cells. J Biol Chem (1994) 269:9603–9609.[Abstract/Free Full Text]
  24. Schönherr E, Järveläinen H.T, Kinsella M.G, Sandell L.J, Wight T.N. Platelet-derived growth factor and transforming growth factor-β1 differentially affect the synthesis of biglycan and decorin by monkey arterial smooth muscle cells. Arterioscler Thromb (1993) 13:1026–1036.[Abstract/Free Full Text]
  25. Evanko S.P, Raines E.W, Ross R, Gold L.I, Wight T.N. Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics, and the proximity of platelet-derived growth factor and transforming growth factor-beta. Am J Pathol (1998) 152:533–546.[Abstract]
  26. Little P.J, Tannock L, Olin K.L, Chait A, Wight T.N. Proteoglycans synthesized by arterial smooth muscle cells in the presence of transforming growth factor-beta1 exhibit increased binding to LDLs. Arterioscler Thromb Vasc Biol (2002) 22:55–60.[Abstract/Free Full Text]
  27. Merrilees M.J, Scott L. Antisense S-oligonucleotide against transforming growth factor-β1 inhibits proteoglycan synthesis in arterial wall. J Vasc Res (1994) 31:322–329.[CrossRef][ISI][Medline]
  28. Grainger D.J, Kemp P.R, Metcalfe J.C, et al. The serum concentration of active transforming growth factor-β is severely depressed in advanced atherosclerosis. Nat Med (1995) 1:74–79.[CrossRef][ISI][Medline]
  29. Aiba M, Takeyoshi I, Ohwada S, et al. Novel nitric oxide donor (FK409) ameliorates liver damage during extended liver resection with warm ischemia in dogs. J Am Coll Surg (2001) 193:264–271.[CrossRef][ISI][Medline]
  30. Hirasawa Y, Nishio M, Maeda K, Yoshida K, Kita Y. Comparison of antiplatelet effects of FK409, a spontaneous nitric oxide releaser, with those of TRK-100, a prostacyclin analogue. Eur J Pharmacol (1995) 272:39–43.[CrossRef][ISI][Medline]

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 Schmidt, A.
Right arrow Articles by Buddecke, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schmidt, A.
Right arrow Articles by Buddecke, E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?