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Cardiovascular Research 1997 36(1):92-100; doi:10.1016/S0008-6363(97)00139-9
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Low density lipoprotein enhances the thrombin-induced growth of vascular smooth muscle cells

Stefan Seewald1, Georg Nickenig1, Yon Ko, Hans Vetter and Agapios Sachinidis*

Medizinische Universitäts-Poliklinik, Wilhelmstr. 35–37, 53111 Bonn, Germany

* Corresponding author. Tel.: (+49 228) 2872589 or 2580; fax: (+49 228) 2872674 or 2266.

Received 30 January 1997; accepted 12 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: In the present study we investigated whether low density lipoprotein is able to enhance the growth promoting effects of thrombin in vascular smooth muscle cells. Methods: DNA synthesis was examined by measurement of the [3H]thymidine incorporation into the cell DNA. Cell count was measured with a Neubauer cell box. Thrombin receptor mRNA was determined by Northern blotting. Ca2+ was measured by the fura 2-method. Results: Thrombin (5 nmol/l), thrombin receptor activating protein (3 µmol/l) and low density lipoprotein (33 nmol/l) induce a 652±80%, 593±80% and a 316±60% increase in [3H]thymidine incorporation into DNA (mean±SD, n = 3), respectively. A coincubation of thrombin or thrombin receptor activating protein with low density lipoprotein led to a 1245±160% or 1200±40% increase of DNA synthesis (mean±SD, n = 3). Thus, coincubation of low density lipoprotein and thrombin causes a synergistic rather than an additive mitogenic effect on smooth muscle cells. Thrombin and low density lipoprotein induced a 22±8,4% and a 29%±6% increase in cell number, respectively. Simultaneous treatment of vascular smooth muscle cells with thrombin and low density lipoprotein caused a 63±14% increase in cell number (mean±SD, n = 3). To further elucidate the underlying mechanism, we studied the effect of low density lipoprotein on the expression of thrombin receptor mRNA. Low density lipoprotein caused a 2.5-fold increase of thrombin receptor mRNA within 24 h, as assessed by Northern analysis. Preincubation of cells for 24 h with 33 nmol/l low density lipoprotein resulted in an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration from 538±54 to 923±75 nmol/l (mean±SD, n = 4). Conclusion: In summary, low density lipoprotein may enhance the mitogenic effect of thrombin probably by an up-regulation of thrombin receptor gene expression in vascular smooth muscle cells or by an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration.

KEYWORDS Low density lipoprotein; Smooth muscle cell; Thrombin


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Low density lipoprotein (LDL) is one of the most important risk factors for cardiovascular disease. It is well established that elevated serum levels of LDL are associated with the pathogenesis of atherosclerosis and hypertension [1–3]. The molecular and intracellular events which lead to LDL-induced development of chronic vascular disease are a subject of controversy. Retention of LDL in the artery wall is thought to be a primary step in the atherogenetic process. Since abnormal growth of vascular smooth muscle cells (VSMC) correlates with the pathogenesis of cardiovascular disease, the mitogenic effect of LDL on VSMC has been investigated intensively. It has been described, that LDL causes an elevation of intracellular free calcium concentration ([Ca2+]i), which stimulates the Na+/H+ exchanger in VSMC and induces vasoconstriction on rat aortic rings [4]. Moreover, LDL induces expression of immediate early growth response genes such as c-fos [5]and egr-1 [6]as well as promotes VSMC growth [4, 7].

Numerous other mediators have been investigated in respect to their role in the pathogenesis of chronic vascular disease. Thrombin is a principle factor in acute thrombus formation [8, 9]. Besides versatile biological features, thrombin stimulates platelet aggregation, induces vasoconstriction [10]and activates growth-related signalling pathways in VSMC which causes ultimately proliferation of VSMC. Most of its known effects are mediated by the thrombin receptor, a G protein-coupled receptor [11]. Activation of the thrombin receptor by thrombin causes elevation of [Ca2+]i [11], activation of protein kinase C (PKC), Na+/H+ exchanger and the induction of the immediate early gene c-fos [12–14].

Since LDL as well as thrombin are thought to participate in the pathogenesis of cardiovascular disease and because of their co-localisation in the atherogenic plaque, we investigated the interactions of both factors. In the present study we examined the effects of LDL on thrombin-induced increase of VSMC growth. To elucidate whether LDL enhanced the thrombin-induced mitogenesis via a functional increase of thrombin receptor, we examined whether the effect of LDL is reproducible on thrombin receptor activating protein (TRAP)- induced mitogenesis. We found a significant potentation of thrombin-induced proliferation of VSMC by LDL. A putative mechanism involved in this phenomenon may be the LDL-induced up-regulation of thrombin receptor gene expression.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
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. 8523, revised 1985).

2.1 LDL isolation
LDL (d = 1.019–1.063 g/ml) was isolated from the plasma of normocholesterolaemic subjects (serum cholesterol <6.2 mmol/l) by potassium bromide density-gradient ultracentrifugation according to Redgrave et al. [15]. The LDL fraction was dialysed against 0.15 mol/l NaCl containing 1 mmol/l EDTA. Oxidation of LDL was prevented by addition of 10 µmol/l butylated hydroxytoluene (BHT) to all LDL preparations. Quantification of LDL was performed by determination of protein-component according to the method of Bradford [16]. LDL is a spherical particle with a mass of 3x106 Da consisting of several molecules of cholesterol, cholesteryl ester, phospholipids and one molecule Apoprotein B-100 [1]. The purity of LDL was examined with a commercially available test (Lipidophor System) following agarose electrophoresis as previously described by Wieland and Seidel [17]. The cholesterol:protein ratio of LDL was 7.83. No oxidation of LDL was observed as assessed by measurement of malondialdehyde (MDA) by the thiobarbituric acid (TBA) method after separation of MDA by the HPLC method as described previously [18].

2.2 Isolation of vascular smooth muscle cells
Rat aortic smooth muscle cells were isolated by enzymatic dispersion using a slight modification of the method described by Chamley et al. [19]. The thoracic aorta from Wistar-Kyoto rats (6–8 weeks old, Charles River Wiga GmbH, Sulzfeld, Germany) was removed and transferred on ice in Dulbecco's phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin (w:v). The aorta was freed from connective tissue, transferred into a petri dish containing 3 ml of an enzyme dissociation mixture containing Dulbecco's modified Eagles medium (DMEM) with 400 IU/ml of collagenase type I, 0.5 mg/ml of elastase and 0.5 mg/ml of soybean trypsin inhibitor and was incubated for 30 min at 37°C. The aorta was then transferred into PBS and the adventitia was stripped off with forceps under a binocular microscope. The aorta was then minced and the minced media was transferred into a plastic tube containing 6 ml of the enzyme dissociation mixture. The suspension was incubated for up to 2 h at 37°C under constant agitation and then centrifuged (1500 rpm for 10 min). The pellet was resuspended in DMEM with 10% fetal calf serum (FCS) and plated in a petri dish (diameter: 3 cm). Cells were cultured over several passages according to Ross [20]. VSMC were cultured in DMEM supplemented with 10% FCS, nonessential amino acids, penicillin 100 IU/ml and streptomycin 100 µg/ml in 75 cm2 flasks at 37°C in a humidified atmosphere of 95% air and 5% CO2 (Steri-cult incubator, Forma Scientific, Göttingen, FRG). VSMC in culture showed the characteristic "hills and valleys" growth pattern. The purity of VSMC was confirmed by immunocytochemical localization of smooth muscle specific {alpha}-smooth muscle actin using fluoresceinisothiocyanate (FITC)-conjugated monoclonal anti-{alpha}-smooth muscle actin plus FITC-conjugated F(ab')2 fragment of goat anti-mouse immunoglobulins. Experiments were performed with three different cell lines from passage 6 to 15.

2.3 Determination of DNA synthesis and cell count
The effect of LDL and thrombin on DNA synthesis was measured by a slight modification of a previously described method [21]. VSMC were seeded on 24-well culture plates and cultivated in culture medium to confluence. Then the medium was replaced by serum-free medium consisting of a mixture of DMEM and Ham's F-10 medium (1:1). Following 24 h cultivation in serum-free medium, stimulators were added to the cells. Cultures were then exposed to the stimulating agents for 20 h before 3 µCi/ml [3H]thymidine were added to the quiescent medium. Experiments were terminated four hours later by aspirating the medium and subjecting the cultures to sequential washes with PBS containing 1 mM CaCl2, 1 mM MgCl2, 10% trichloroacetic acid and ethanol/ether (2:1, v/v). Phase-contrast microscopy was used to inspect the dishes for evidence of cell detachment or changes in cell morphology. Acid-insoluble [3H]thymidine was extracted into 250 µl/dish 0.5 mol/l NaOH and 0.1 ml of this solution was mixed with 5 ml scintillant and quantified by using a Packard Instrument liquid scintillation counter Model Beckmann (Düsseldorf, Germany). 50 µl of the residual solution were prepared for the determination of protein using the Bio-Rad protein assay according to the method of Bradford [16].

For cell counting, VSMC were seeded in 24-well culture plates [(5x104 cells/well, well diameter 12 mm)] and cultured in DMEM, supplemented with 10% FCS, non-essential amino acids, penicillin 100 IU/ml and streptomycin 100 µg/ml at 37°C for 48 h. The medium was then replaced by serum-free medium consisting of DMEM and Ham's F-10 (1:1, v/v) and cells were incubated for 24 h in serum-free medium. VSMC were then stimulated with thrombin in the presence and absence of LDL. After 24 h, cells were trypsinized and resuspended in DMEM plus trypan blue. Cell counting was performed with a Neubauer-cell-box by light microscopy.

2.4 RNA isolation and Northern analysis
VSMC were grown in 6 cm diameter dishes to confluency. Following 24 h cultivation period in serum-free medium, cells were exposed to thrombin (5 nmol/l), LDL (33 nmol/l) and thrombin in the presence of LDL for different time periods. Then cells were lysed in situ with 1 ml of TRI reagent and then scraped and processed according to the manufacturer's protocol to obtain total cellular RNA. This was quantified spectrophotometrically by obtaining absorbance values at 260 nm and 280 nm. Ten microgram aliquots were electrophoretically separated in 1.2% agarose–0.67% formaldehyde gels and stained with ethidium bromide to verify the quantity and quality of the RNA. After capillary transfer to Hybond N membranes in 20x saline sodium citrate (SSC; 1x SSC=150 mmol/l sodium chloride, 15 mmol/l sodium citrate) the membranes were exposed to ultraviolet radiation utilizing a Stratalinker 2400 (Stratagene, Inc.; LaJolla, CA). Northern blots were prehybridized for 2 h at 42°C in a buffer containing 50% deionized formamide, 0.5% SDS, 6x SSC, 10 µg/ml denatured salmon sperm DNA, and 5x Denhardt's buffer and then hybridized for 16 h with a random primed, [32P]dCTP-labeled rat thrombin receptor cDNA probe at 42°C in the same buffer except without Denhardt's. The rat thrombin receptor cDNA probe was a 800 bp fragment generated from VSMC RNA by reverse transcriptase-polymerase chain reaction using the primer pair (5'-ATA GTC AGC CTT CCC CTG AAC-3') and (5'-CAC GCT CGT CAC GCA GAC GCA-3'), and Vent polymerase (New England Biolabs; Beverly, MA). The hybridized filters were washed for 15 min at room temperature in 2x SSC and then for 15 min at 50°C in 2x SSC, 0.1% SDS and exposed for 4–12 h to Hyperfilm (Amersham) at –80°C.

2.5 Measurement of intracellular free Ca2+
For the measurement of [Ca2+]i, confluent cells were detached with 0.04% trypsin/0.02% EDTA in PBS after 5 to 10 min at 37°C. The cells were then cultured on round glass microscope slides (diameter 12 mm) under normal tissue culture conditions until confluence. Cells were cultured in serum-free medium for 24 h and then cells were exposed to LDL for 24 h. Then cells were washed three times with PBS and then incubated with 2 µmol/l fura-2 pentaacetoxymethyl ester at 37°C for 20 min in HEPES buffer (20 mmol/l HEPES, 16 mmol/l glucose, 130 mmol/l NaCl, 1 mmol/l MgSO4·7H2O, 0.5 mmol/l CaCl2, Tris-base, pH 7.4) supplemented with 1% bovine serum albumin [(BSA) (w:v)]. Just prior to the measurements, the cell monolayer was rinsed with HEPES buffer, containing 1 mmol/l CaCl2, and the glass slide was positioned diagonally in the cuvette. The Ca2+-fura-2 fluorescence was measured at 37°C in a Perkin-Elmer LS 50B fluorescence spectrofluorometer (Überlingen, Germany) at excitation wavelengths of 340 and 380 nm and at an emission wavelength of 505 nm. Maximum (Rmax) and minimum (Rmin) fluorescence were determined by adding digitonin at a final concentration of 3x10–5 mol/l followed by the addition of Tris-base/EGTA at a final concentration of 0.1 mol/l Tris-base/25 mmol/l EGTA. Fluorescence was corrected for cellular autofluorescence. Fluorescence signals were calibrated according to Grynkiewicz et al. [22]using the following equation [Ca2+]i=Kdx(R–Rmin)/(Rmax–R)x(Sf2/Sb2). Kd for the fura-2/Ca2+ complex at 37°C is assumed to be 224 nmol/l. Sf2 is the 380 nm-exited fluorescence in the absence of Ca2+ (EGTA added) and Sb2 is the 380 nm-excited fluorescence in the presence of saturating Ca2+ (1 mmol/l Ca2+).

2.6 Materials
DMEM, Ham's F-10, antibiotics and PBS were obtained from Gibco BRL (Eggenstein, Germany), {alpha}-thrombin (100 units/mg protein) was obtained from Sigma (Deisenhofen, Germany). TRAP was obtained from Bachem (Heildelberg, Germany). BHT was obtained Fluka Chemika (Buchs, Switzerland). [Methyl-3H]thymidine, [32P]dCTP and Hybond N-nylon membranes were obtained from Amersham (Braunschweig, Germany). TRI-reagent was from Molecular Research Centre (Cincinnati, OH, USA). Oligonucleotides were synthesized using Pharmacia Chemicals, with an automated DNA synthesizer (Pharmacia LKB, gene assembler plus).

2.7 Statistics
Values are expressed as means±SD. Statistical analysis of the data was performed using the Mann-Whitney U-test (Stat View 512+TM, version 1.0, Apple Computer, Inc.). Triplicate wells were analyzed for each [3H]thymidine incorporation experiment and each experiment was performed independently a minimum of three times. Data presented are from representative experiments unless otherwise indicated. Data obtained from individual experiments with triplicate determinations were normalized by calculating the mean±SD of the individual experiments and expressed as percent change from the basal value of the unstimulated cells (=100%). P<0.05 was considered to be statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Results from three individual experiments — each performed in triplicate wells — were normalized by calculation of the mean±SD of individual experiments and were expressed as percent increase above the basal value of unstimulated cells (=100%) (Fig. 1). These results show that thrombin (5 nmol/l=18 units/l) and LDL (100 µg/ml=33 nmol/l) induce a 652±80% and a 316±60% increase in [3H]thymidine incorporation into DNA (mean±SD, n = 3), respectively. A coincubation of thrombin and LDL led to a 1245±160% increase of DNA synthesis (mean±SD, n = 3). TRAP (3 µmol/l) induced a 593±80% increase in [3H]thymidine incorporation into DNA. A coincubation of TRAP and LDL led to a 1200±140% increase in [3H]thymidine incorporation (mean±SD, n = 3).


Figure 1
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  		Fig. 1 Effect of thrombin and LDL on DNA synthesis in VSMC. Confluent cells (24-well plates) were precultured for 24 h in serum-free medium. Cells were stimulated with thrombin (5 nmol/l) and TRAP (3 µmol/l) in the presence and absence of LDL (33 nmol/l). Following another 20 h incubation, cells were exposed to 3 µCi/ml [3H]thymidine. Four hours later the reaction was terminated and cell protein and [3H]thymidine incorporation into cell DNA were quantified. Data from three individual experiments each performed in triplicate wells were standardized by calculating the mean±SD of individual experiment and are expressed as percent increase above the basal value of unstimulated cells. (*P<0.05 for thrombin, TRAP, or LDL vs. control, n = 3; Figure 1<0.05 for the LDL+thrombin or LDL+TRAP effect vs. thrombin or LDL effect, n = 3.

 
To examine whether enhanced DNA synthesis is correlated with cell proliferation we measured the cell count. Fig. 2 shows that thrombin and LDL induced a 22±8.4% and a 29±6% increase in cell number over basal value (=100%) (mean±SD, n = 3, Formula <0.05), respectively. Coincubation of thrombin and LDL causes a marked increase of 63±4% in cell number (mean±SD, n = 3, Formula <0.05).


Figure 2
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  		Fig. 2 Effects of thrombin and LDL on VSMC cell number: Cells (5x104) were seeded on 24-well plates. Cells were serum-deprived for 24 hours and then stimulated with thrombin (5 nmol/l), LDL (33 nmol/l) and thrombin in the presence of LDL. After 24 h cells were trypsinized and counted as described in Section 2. Data represent the means±SD of three experiments. (*P<0.05 for thrombin or LDL vs. control, n = 3, Figure 2<0.05 for LDL+thrombin effect vs. thrombin or LDL effect, n = 3).

 
To elucidate the influence of LDL on thrombin receptor mRNA we incubated confluent and serum deprived VSMC with 33 nmol/l LDL and analyzed VSMC-RNA by Northern Blotting, isolated at the indicated time points. Fig. 3 (blot) illustrates autoradiographic results from a Northern blot showing hybridization of a rat vascular thrombin receptor cDNA probe to 10 µg of electrophoretically separated, total cellular RNA extracted from VSMC at the indicated time points after addition of LDL to the culture medium. The probe hybridizes to an abundant 3.5 kb transcript, as observed previously [23]. This autoradiogram reveals a significant time-dependent elevation of the transcript level. Thrombin receptor mRNA signal appears maximally increased 24 h after exposure to LDL. Fig. 3 (blot) also shows the hybridization of a glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA probe to the same Northern blot. Compared with the thrombin receptor mRNA, GAPDH mRNA appears relatively stable over the time course of the experiment. Autoradiographic data, generated from four separate experiments, using different VSMC cell lines and various LDL preparations, were analyzed by laser densitometry. A 12 h incubation with 33 nmol/l LDL causes an up-regulation of thrombin receptor mRNA levels to 187±15% (mean±SD, n = 4, Formula <0.05 for LDL effect at 12 h vs. control at 0 h=100%). Fig. 3 (diagram) shows the LDL-induced up-regulation of thrombin receptor mRNA hybridization signal, relative to vehicle-treated control levels at 0 h. After 24 h of LDL stimulation, the thrombin receptor mRNA signals are measured at 264±20.6% (mean±SD, Formula <0.05 for LDL or LDL+thrombin effect at 24 h vs. control at 0 h) relative to the control level at 0 h (Fig. 3, diagram). GAPDH mRNA expression is not significantly regulated by LDL. These observations indicate that LDL selectively up-regulates the expression of thrombin receptor mRNA in this system. In an other set of experiments we incubated VSMC with thrombin as well as with LDL for 24 h and analyzed the thrombin receptor mRNA level. Thrombin (5 nmol/l) exerted no significant effect on thrombin receptor mRNA (Fig. 3, diagram). Coincubation of 5 nmol/l thrombin and 33 nmol/l LDL for 24 h led to an up-regulation of the thrombin receptor transcript but this effect was not significantly different from the LDL-induced up-regulation of thrombin receptor mRNA (mean±SD, n = 4).


Figure 3
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  		Fig. 3 Effect of LDL on the thrombin receptor mRNA. Blot, VSMC were grown to confluency. Following incubation of cells for 24 h in serum-free medium, cells were exposed to 33 nmol/l LDL for different time periods. Hybridization of a thrombin receptor cDNA probe to Northern Blots of 10 µg total RNA extracted from VSMC at the indicated time points. Hybridization of a GAPDH cDNA probe to the same blot stripped of the thrombin receptor cDNA probe (representative Northern hybridization autoradiograms for four separate experiments). Diagram, Quantification of Northern hybridization signal intensity of thrombin receptor as well as GAPDH mRNA after VSMC treatment with 33 nmol/l LDL, thrombin (5 nmol/l) or LDL and thrombin for 24 h. Each point represents the relative hybridization signal normalized to the 0 h treatment with vehicle (100%) from four separate experiments(mean±SD, Figure 3<0.05 for LDL or LDL+thrombin effect at 24 h vs. control at 0 h).

 
Finally, we tried to gain insight into the mechanism whereby LDL enhances the thrombin-dependent effect on cell DNA synthesis. Therefore, we preincubated VSMC for 24 h with either vehicle or 33 nmol/l LDL and measured the thrombin-induced increase in [Ca2+]i. As demonstrated in Fig. 4A(a), thrombin at concentrations of 5 nmol/l induced a rapid increase in [Ca2+]i from approximately 100 to 500 nmol/l with a maximum at 15 s (a representative tracing). [Ca2+]i declined after this peak value toward a stable [Ca2+]i value of approximately 250 nmol/l within 2 min. As shown in Fig. 4A(b), treatment of the cells with LDL for 24 h enhanced the thrombin-induced increase in [Ca2+]i from approximately 500 (non LDL-pretreated cells) to 900 nmol/l after 15 s. [Ca2+]i declined after this peak value toward a stable [Ca2+]i value of approximately 300 nmol/l within 2 min. Evaluation of three separate experiments was performed by calculation of the maximal increase in [Ca2+]i at 15 s. Thrombin (5 nmol/l) caused an increase in [Ca2+]i from 86±12 (basal value) to 538±54 nmol/l (Fig. 4B). In LDL-pretreated cells, thrombin induced an increase in [Ca2+]i from 95±10 to 923±75 nmol/l (mean±SD, n = 3, Formula <0.05 for thrombin effect vs. basal value, Formula <0.05 for LDL+thrombin effect vs. thrombin effect).


Figure 4
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  		Fig. 4 Effect of thrombin on [Ca2+]i in LDL treated VSMC. A, Cells were cultured on round glass microscope slides under normal tissue culture conditions until confluence. They were cultured then in serum-free medium for 24 h. Cells were then exposed to 33 nmol/l LDL for 24 h. Measurements were performed in 2 ml HEPES buffer and the glass slide was positioned diagonally in the cuvette. Arrow indicates addition of 20 µl of a 500 nmol/l thrombin solution (HEPES buffer) in the cuvette (final concentration 5 nmol/l). (a), Thrombin (5 nmol/l) was added to fura-2-loaded untreated cells. (b) Thrombin (5 nmol/l) was added to fura-2-loaded LDL-treated cells. After subtraction of autofluorescence, changes in 340/380 nm excitation wavelength ratio by the emission wavelength of 505 nm were converted into corresponding levels of [Ca2+]i. B, Evaluation of three separate experiments was performed by calculating the maximal thrombin-induced increase in [Ca2+]i at 15 s (*P<0.05 for thrombin effect vs. basal value, Figure 4<0.05 for LDL+thrombin effect vs. thrombin effect).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
LDL may influence the onset of cardiovascular disease at several different levels. Many steps of the beginning of the atherosclerosis process such as the recruitment and migration of circulating monocytes are promoted by LDL [24–27]. There is a direct effect of LDL on elevation of [Ca2+]i and vasocontriction of VSMC [4]as well as on the expression of transcriptional factors such as c-fos [5]and egr-1 [6]. These observations may explain the mitogenic effects of LDL. LDL may also influence DNA synthesis by delivering cholesterol for membrane synthesis. In our experiments, we observed changes in [3H]thymidine incorporation as well as changes in cell number as an indicator for cell proliferation following cell stimulation with LDL, thrombin or their combination.

Furthermore, LDL induces an increase in the expression of platelet-derived growth factor (PDGF) alpha and beta-receptors and of PDGF A-chain transcripts in smooth muscle cells [28]. In addition, LDL causes an enhanced expression of PDGF-B chain in endothelial cells [29]. We recently described that LDL enhances the angiotensin II (Ang II)-induced mitogenesis and upregulates the angiotensin II -type1-(AT1) receptor gene expression [30]. Moreover, preincubation of the cells with LDL for 24 h resulted in a 40% elevation of the PDGF-BB-induced DNA synthesis in these cells [31].

The effect of thrombin on VSMCs is mediated by a G protein coupled receptor [11, 32]. As most of the known biological effects of thrombin are mediated by this receptor, its expression level in VSMC may influence the response of VSMC upon thrombin stimulation. It has already been shown that the thrombin receptor expressed on VSMC is regulated by growth factors such as basic fibroblast growth factor [23]. We now provide evidence that LDL upregulates thrombin receptor mRNA in VSMC. This increased level of thrombin receptor mRNA transcript suggests that thrombin receptor gene expression is enhanced. Therefore, it seems reasonable to presume that increased thrombin receptor expression should lead to an elevated functional response of VSMC upon stimulation with thrombin. Indeed, thrombin-induced VSMC mitogenesis is increased when co-incubated with LDL. The same effect is reproducible for the TRAP-induced mitogenesis. Furthermore, cells pretreated with LDL show an enhancement of thrombin-induced increase in [Ca2+]i.

However, it has been proposed that in contrast to classical growth factors such as PDGF-BB or basic fibroblasts growth factor (bFGF) that initiated DNA synthesis after 16–19 h, thymidine incorporation in response to thrombin is delayed by an additional time of 3 to 6 h [33]. Authors suggested that thrombin-stimulated VSMC proliferation is delayed and requires the de novo expression of one or more autocrine mitogens [33]. In this context, PDGF-AA [34]and basic fibroblast growth factor (bFGF) [35]have been discussed to be candidates as autocrine mitogens induced by thrombin in VSMC. However, in a last study both growth factors have been excluded as significant thrombin-induced mitogens in VSMCs [33]. Thus, up to now, it is unclear which paracrine thrombin-induced growth factors are responsible for the delayed mitogenic effect of thrombin.

On the basis of these findings [33]it is not entirely clear whether the increased expression of thrombin receptor mRNA beginning after 12 h of co-incubation of LDL with thrombin is exclusive responsible for the enhanced effects of thrombin on the DNA synthesis of the LDL-treated cells. Therefore, further experiments are required to elucidate which amount of the enhanced DNA synthesis in LDL-treated cells is caused by the enhanced thrombin mRNA expression and which part of this effect can be referred to thrombin-induced autocrine mitogens.

In contrast to our results, it has been reported that incubation of quiescent VSMCs with thrombin (10, 1 and 0.1 nmol/l) induced up-regulation of thrombin receptor (TR) mRNA within 6 h [36]. Experiments of Bachhuber et al. were performed in serum-free medium containing selenium, insulin and transferrin which may promote TR mRNA up-regulation by thrombin [36]. Therefore, the inconsistencies between our results and those of Bachhuber et al. may reflect differences in cultured experimental conditions [36]. However, the question whether thrombin per se is able to up-regulate TR mRNA remains to be elucidated.

It is widely thought that [Ca2+]i plays a substantial role in the regulation of cell growth [37, 38]. Since pretreatment of the cells with LDL led to an elevation of the thrombin-dependent effect on [Ca2+]i it is possible that the LDL-induced elevation of the thrombin effect on DNA synthesis may be attributed to its effect on [Ca2+]i.

Although these observations are derived from cell culture experiments, it is attractive to speculate that LDL-induced enhancement of thrombin-mitogenesis may be a putative mechanism by which LDL may influence the pathogenesis of atherosclerosis and hypertension. This is especially relevant since thrombin receptor activation by thrombin mediates vasoconstriction and proliferation of VSMC [22, 23]. Moreover, thrombin receptor-specific antisense sequences inhibit growth-related effects of thrombin on smooth muscle cells [39].

Several studies found that LDL particles promote VSMC and endothelial cells growth but it is controversially discussed whether oxidatively modified LDL [40]or native LDL [4, 41, 42, 18]accounted for the mitogenic effect of LDL. It has been proposed that endothelial cells [43]VSMC [44]and fibroblasts [44]are able to modify LDL oxidatively so it becomes mitogenic for VSMC. Several observations contradict the speculation that oxidized LDL (ox-LDL) is responsible for the proliferative effects of LDL: (1) It is known that BHT is a potent antioxidant which was used in all LDL preparations and completely prevents oxidation of LDL [45]. LDL oxidation by endothelial cells and VSMC is a time dependent process and occurs by endothelial cells [43]within 12 to 18 h and by VSMC over three days of contact between LDL and VSMC [46], (2) Native LDL induces immediate putative mitogenic signals such as increase in [Ca2+]i and intracellular pHi [4]as well as expression of the transcription factors egr-1 in VSMC [6]and endothelial cells [39]between 20 s and 30 min, (3) ox-LDL may be cytotoxic for endothelial cells [24, 45]and VSMC [46]. Since it has been reported that a possible mitogenic or toxic effect of ox-LDL is dependent on the extent of oxidation [47], it can not be excluded that the observed effect of LDL on cell DNA synthesis are derived from middle oxidized LDL (also known as minimally modified LDL) which may be produced during incubation with VSMC.

One has to question whether experiments with cultured cells have any physiological relevance. However, it is difficult to study cell proliferation in intact multicellular animal. Nevertheless, cultured VSMCs are a useful model for understanding the mechanisms causing VSMCs proliferation induced by growth factors.

In the formation of atherosclerotic plaques, deposition of LDL in the artery wall as well as platelet aggregation are important steps. According to the "response to injury hypothesis" from Ross et al., VSMC proliferation participate in the formation of atheromatous plaques [48]. Thus, it is conceivable that in addition to other classical growth factors such as PDGF, thrombin alone or in combination with LDL may also contribute to the formation of an atheromatous plaque. In patients with abnormal plasma lipid levels abnormal platelet function has been demonstrated [49]. In addition, platelets show increased tendency to aggregate and express an increased number of fibrinogen binding sites on its surface when exposed to hypercholesterolaemic serum. Moreover, it has been shown that platelets exposed to LDL show a reduced sensitivity to prostacyclin, an antiaggregatory agent [50]. It is of special interest to further elucidate the links between LDL, platelet aggregation and thrombin which are thought to be involved in the onset of atherosclerosis and hypertension. Therefore, further investigations will be necessary in order to determine whether thrombin receptor gene expression is also increased in human with elevated LDL serum levels.

Time for primary review 27 days.


   Acknowledgements
 
This work was supported by a grant of Deutsche Forschungsgemeinschaft (Sa 568/2-1).

The excellent technical assistance of Claudia Seul und Petra Epping is greatly appreciated.


   Notes
 
1 Equal contributor to this work. Back


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

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