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Tissue factor induction by aggregated LDL depends on LDL receptor-related protein expression (LRP1) and Rho A translocation in human vascular smooth muscle cells

S. Camino-López , V. Llorente-Cortés , J. Sendra , L. Badimon
DOI: http://dx.doi.org/10.1016/j.cardiores.2006.10.017 208-216 First published online: 1 January 2007

Abstract

Objective: Low density lipoprotein (LDL) internalized in the vascular wall and modified by binding to extracellular matrix-proteoglycans (ECM) becomes aggregated (agLDL). AgLDL induces tissue factor (TF) expression and activity in human vascular smooth muscle cells (VSMC). TF expression in vascular cells promotes the prothrombotic transformation of the vascular wall. However, the mechanisms by which agLDL induces TF are not known. The aim of this study was to investigate the mechanisms involved in TF activation by extracellular matrix-modified LDL in human VSMC.

Methods and results: AgLDL significantly induces TF expression (real time PCR and Western blot analysis) and procoagulant activity (factor Xa generation test) in human VSMC. HMG-CoA reductase inhibition completely prevents agLDL-induced TF expression and partially inhibits agLDL-TF activation. These effects are reverted by geranylgeranyl pyrophosphate (GGPP) but not by farnesyl pyrophosphate (FPP), suggesting the involvement of a geranylated protein in agLDL-TF induction. AgLDL increases Rho A translocation (2-fold) from the cytoplasm to the cell membrane in control but not in simvastatin-treated VSMC. Exoenzyme C3, a specific Rho A inhibitor, completely prevents agLDL-induced TF overexpression and partially agLDL-TF activation. Blocking LRP1, the receptor of agLDL, with anti-LRP1 antibodies or inhibiting LRP1 expression by small interference RNA treatment (siRNA-LRP1) impairs agLDL-induced TF overexpression and activation.

Conclusions: These results demonstrate that TF induction by agLDL depends on LRP1 expression and requires Rho A translocation to the cellular membrane.

Keywords
  • Aggregated LDL
  • Thrombosis
  • Statins
  • LRP1
  • Tissue factor

1. Introduction

Tissue factor (TF) is a transmembrane cell surface glycoprotein that plays a key role in the atherothrombotic process and plaque TF content seems to predict plaque thrombogenicity [1–3]. The prothrombotic transformation of an atherosclerotic plaque strongly depends on lipids, since advanced lipid-enriched atherosclerotic plaques are considered highly susceptible to rupture [2] and the lipid core is one of the most thrombogenic substrates [3,4]. Most of the resident lipid in the arterial intima comes from low density lipoprotein (LDL) that is retained forming aggregates (agLDL) by binding to the extracellular matrix (ECM) proteins of the arterial intima [5,6]. Native and different types of modified LDL have the ability to increase TF expression in different cell models [7–9]. However, only oxLDL and agLDL induce sustained cellular TF activity [8,9]. It has been reported that oxLDL, which is taken up through scavenger receptors, induces TF activation through cellular lipid peroxidation [8]. We have previously demonstrated that agLDL is internalized by Low Density Lipoprotein Receptor-Related Protein (LRP1) and that agLDL up-regulates LRP1 expression [9–13]. Interestingly, agLDL induces a much higher TF expression and activity than non-aggregated native LDL in VSMC [9]. Although lipid loading induces TF through apoptotic mechanism in macrophages [14,15], agLDL-lipid loading does not induce apoptosis of VSMC [12]. Presently, the mechanisms involved in agLDL induced TF activation are completely unknown.

Studies in different animal models have demonstrated that HMG-CoA reductase inhibitors or statins have antithrombotic effects in the vascular wall [16,17]. Additionally, statins have been shown to reduce TF expression in aorta [18] and in macrophage-rich atherosclerotic plaques [19,20] in hypercholesterolemic rabbits. We have previously shown that statins significantly inhibited thrombus formation on both eroded and damaged vessel walls in an experimental pig model [21]. These in vivo studies demonstrate a reduction in vascular thrombogenicity due to a reduction on TF expression in the vessel wall which is partially unrelated to a decrease in serum cholesterol (pleiotropic effects) [18–21]. Pleiotropic effects of statins are the result of the ability of HMG-CoA reductase inhibitors to block the synthesis of the two major donors of hydrophobic isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible for the postranslational lipid modification (prenylation) of isoprenylated proteins involved in cell signaling, such as Rho and Ras proteins [22]. The Rho family of small GTP binding proteins cycle between a GDP-bound (inactive) state and a GTP-bound (active state). Activation of Rho GTPases is accompanied by their intracellular translocation and recruitment from the cytoplasm to the plasma membrane.

Our aim was to characterize the mechanism involved in TF induction by LDL retained in the vascular wall (agLDL) in human VSMC. Our results demonstrate that: 1) HMG-CoA reductase inhibition prevents agLDL induced TF overexpression and activation by inhibition of GGPP synthesis; 2) agLDL strongly increases Rho A translocation to the cellular membrane and the inhibition of Rho A translocation prevents TF induction by agLDL; and 3) the inhibition of functional LRP1 with anti-LRP1 antibodies or by small interference RNA treatment (siRNA-LRP1) prevents TF induction by agLDL. These results demonstrate that TF induction by agLDL depends on LRP1 expression and requires Rho A translocation to the cellular membrane.

2. Methods

2.1. VSMC culture and LDL preparation

Primary cultures of human VSMC were obtained from non-atherosclerotic areas of human coronaries of explanted hearts at transplant operations performed at the Hospital de la Santa Creu i Sant Pau, as previously described [10,11]. The study was approved by the Reviewer Institutional Committee on Human Research of the Hospital of Santa Creu i Sant Pau that conforms to the Declaration of Helsinki. To analyze the involvement of statins on TF induction, quiescent VSMC were preincubated with simvastatin (kindly provided by MSD) in absence or presence of several isoprenyl groups (mevalonate, GGPP or FPP) for 4 h prior to the addition of nLDL or agLDL (100 μg/mL, 18 h). To analyze the role of Rho A on agLDL-TF induction, quiescent VSMC were preincubated with exoenzyme C3 (a Rho A inhibitor; Calbiochem, 25 μg/mL, 24 h) prior addition of LDL (18 h). To analyze the requirement of LRP1 for agLDL-TF induction, arrested cells were incubated for 18 h with agLDL (100 μg/mL) in the absence or presence of anti-LRP1 antibodies (RDI-PRO61065, clone 8G1, 30 μg/mL) or non-immune IgG (30 μg/mL). At the end of this period, cells were exhaustively washed and harvested either to analyze TF expression or to analyze TF activity. None of the tested reagents or antibodies produced changes on cell morphology (assessed by staining with Trypan Blue).

Human LDL (d1.019d1.063 g/mL) were obtained from pooled sera of normocholesterolemic volunteers by sequential ultracentrifugation. AgLDL were prepared as previously described [10,11]. TBARS levels (<1.2 mmol malonaldehyde per milligram of protein LDL) remained similar to those in nLDL after LDL aggregation.

2.2. Obtention of LRP1-deficient VSMC

In order to obtain LRP1 deficient cells (siRNA-LRP1-VSMC), human VSMC were transiently transfected with annealed siRNA as previously described [12]. In brief, VSMC were transfected with siRNA-LRP1 (50 nM) using siPORT NeoFx in serum-free DMEM medium (1% glutamine) according to the kit instructions (SilencerTM siRNA Transfection Kit; Ambion no. 4511). This medium with siRNA-LRP1 was maintained for 48 h and it was then replaced by a new medium containing LDL (100 μg/mL). After 18 h, cells were exhaustively washed and harvested to test either for TF expression or TF activity analysis. Extra wells were used in order to test the specificity of siRNA-LRP1 treatment by analyzing LRP1, LDL receptor and CD36 mRNA expression by real time PCR. The cells did not take up Trypan Blue, and their morphology was not altered by the procedure. LRP1 specific sense and antisense oligodeoxynucleotides were synthesized by Ambion (Austin TX, USA) according to our previously published LRP1 target sequences [12]. Fasta analysis (Genetic Computer Group Package) indicated that these sequences would not hybridize to other receptor sequences (including LDL receptors) in the GenBank database.

2.3. Real time PCR

Total RNA and protein were isolated by using the Tripure™ isolation Reagent (Roche Molecular Biochemicals) according to the manufacturer. LRP1 and TF mRNA levels were analyzed by real time PCR. TaqMan fluorescent real time PCR primers and probes (6′FAM-MGB) for LRP1, LDL receptor (LDLR) and TF were designed by use of Primer Express software from PE biosystems and were as follows: LRP1 forward: 5′-gagctgaaccacgcctttg-3′; LRP1 reverse: ggtagacactgccactccgatac-3′; LRP1 probe: 5′-ttgccatggtgacacag-3′; LDLR forward: 5′-tgacaatgtctcaccaagctctg-3′; LDLR reverse: 5′-ctcacgctactgggcttcttct-3′: LDLR probe: 5′-ctgccagcaacgtcg-3′; TF forward: 5′-ttcacaccttacctggagacaaac-3′; TF reverse: 5′-aacatcccggaggcttagga-3′; TF probe: 5′-caaaagtgaatgtgaccgtag-3′. Assays on demand (Applied Biosystems) were used for RhoA (Hs 00357608 m1), Rac1 (Hs 00251654 m1) and CD36 (Hs00169627 m1). Human gapdh (4326317E) was used as endogenous control. Taqman real time PCR was performed as previously described [11,12].

2.4. Western blot analysis

Proteins were analyzed by Western blot analysis as previously described [11,12]. Blots were incubated with monoclonal antibodies against human LRP1 (β-chain; Research Diagnostics; clone 8B8 RDI 61067) or human TF (American Diagnostica 4501). Equal loading of protein in each lane was verified staining filters with Pounceau and also by incubating blots with monoclonal antibodies against human α-actinin (MAB 1682, Chemicon International). Western blot bands were quantified with a Chemi-doc (BioRad) using the Quantity One 1-D Analysis Software. Results are expressed as arbitrary units that refer to units of intensity×millimeters. TF and LRP1 protein band intensities were normalized by the respective α-actinine band intensities.

2.5. Determination of TF activity in VSMC

VSMC were collected in TBS-EDTA buffer (0.1 M NaCl, 0.05 M Tris, EDTA 0.1 M, pH 7.5) and tissue factor procoagulant activity (TF-PCA) was measured in extracted cellular membranes by using a factor Xa generation test as previously described [9,23,24]. The sample (containing approximately between 10 and 15 μg of cellular membranes) was added to a solution containing liposomes of phosphatidylserine 30%-phosphatidylcholine 70% (PS 30:PC 70) 100 μM, 4 nM factor VIIa and 5 mM CaCl2. The mixture was incubated for 15 min and then factor X was added at 300 nM. After 15 min of incubation, EDTA buffer (TBS, 0.1% BSA, 300 mM EDTA, pH 7.5) was added to stop the production of activated factor X (factor Xa). A chromogenic substrate, factor Xa chromogenic substrate (Sigma) (25 μL, 0.5 mM), was added and the OD was measured at 405 nm for 30 min using a kinetic ELISA reader at 37 °C (SpectraMax). TF-PCA values were obtained from a standard curve performed with factor Xa. The variance between the samples was controlled by protein determination at the end of the extraction procedure.

2.6. Membrane translocation of Rho A and Rac1

Human VSMC were cultured and stimulated with lipoproteins as indicated above in the absence or presence of simvastatin. Cell monolayers were washed with PBS and lysed with Subcellular Proteome Extraction kit (Calbiochem) to obtain membrane and cytoplasmic protein. The protein fractions were cleaned using clean-up kit (Amersham) according to manufacturer's instructions. Proteins were analyzed by Western blot; blots were incubated with monoclonal antibodies against human Rho A (26C4, Santa Cruz Biotechnology) or Rac1 (610650, BD Transduction Laboratories).

2.7. Immunocytochemistry

Cells were seeded in glass coverslips, grown to confluence, arrested for 24 h in absence of foetal calf serum, and then incubated in absence or presence of agLDL (100 μg/mL) for 18 h. They were then fixed at room temperature for 10 min in PBS containing 4% paraformaldehyde and washed twice with PBS.

To analyze Rho A staining, fixed VSMC were incubated with primary antibodies against Rho A (26C4, Santa Cruz Biotechnology, dilution 1:5). Coverslips were then washed and incubated with FITC-conjugated goat anti-mouse IgG (dilution 1:100). Images of immunostained cells were analyzed on a Leica inverted fluorescence confocal microscope (Leica TCS SP2-AOBS, Wetzlar, Germany) and processed with the Leica Standard Software TCS-AOBS.

2.8. Data analysis

Data were expressed as mean±SEM. A statview (Abacus Concepts) statistical package for the Macintosh computer system was used for all analysis. Multiple groups were compared by ANOVA or Wilcoxon test as needed. Statistical significance was considered when P<0.05.

3. Results

3.1. Effects of HMG-CoA reductase inhibition and isoprenoids on agLDL induced TF overexpression and activation

Simvastatin (at 2.5 μM) strongly decreased agLDL-induced TF protein overexpression (by 36±1.5%) (Fig. 1A) and TF activation (by 40±0.13%) (Fig. 1B).

Fig. 1

HMG-CoA reductase inhibitors decrease agLDL-TF protein overexpression and agLDL-TF activation though inhibition of GGPP synthesis. A and B, quiescent VSMC preincubated for 4 h with simvastatin (0, 2.5, 5 and 10 μM) and then incubated in absence of LDL (control VSMC, romboid) or in presence of nLDL (squares) or agLDL (triangles) (100 μg/mL) for 18 h. C and D, quiescent VSMC were preincubated for 4 h with buffer (black bars), simvastatin (SIMV) (5 μM) (white bars) and in the presence of GGPP (10 μM) (scatched bars) or FPP (10 μM) (pointed bars). WSMC were then incubated with nLDL or agLDL (100 μg/mL) for 18 h. (A and C) Western blot analysis of cellular membrane TF antigen. Results are normalized by the respective α-actinin band intensities. (B and D) Factor Xa generation test of TF activity in VSMC treated with simvastatin. TF activity is expressed as mU/mg protein. Results are shown as mean±SEM of three experiments performed in duplicate. P<0.05: # vs no LDL exposed VSMC; ‡ vs nLDL exposed VSMC; * vs simvastatin untreated VSMC.

Simvastatin reduced TF mRNA expression (more than 50%) in control, nLDL or agLDL-exposed VSMC (data not shown). To characterize the main prenyl groups involved in the effect of simvastatin on agLDL induced TF overexpression and activation, we added either GGPP or FPP during statin treatment. GGPP but not FPP completely prevented the effect of simvastatin on agLDL-induced TF protein overexpression (Fig. 1C) or TF activation (Fig. 1D). These prenyl intermediates by themselves did not alter TF protein expression or activity in control or LDL-exposed VSMC (data not shown).

3.2. Effect of agLDL on Rho A translocation and effect of exoenzyme C3, specific inhibitor of Rho A, on agLDL induced TF overexpression and activation

As shown in Fig. 2A, agLDL increased Rho A mRNA expression levels over those induced by nLDL (agLDL: by 2-fold vs nLDL: 1.22-fold) but agLDL did not exert any significant effect on Rac1 mRNA expression (data not shown). Simvastatin strongly increased Rho A mRNA expression (Fig. 2A) and the cytoplasmic (inactive) form of Rho A protein (Fig. 2B) in control and LDL-exposed cells. On the contrary, simvastatin did not exert any effect in the cytoplasmic form of Rac1 (Fig. 2B). Interestingly, agLDL strongly increased the membrane (active) form of RhoA (agLDL: by 2.67-fold vs nLDL: by 1.50-fold) and simvastatin completely prevented agLDL induced RhoA activation (Fig. 2B). In contrast, agLDL did not exert any significant effect on Rac1 protein translocation; however, simvastatin was able to decrease Rac1 translocation by 63±0.37% in control cells, by 69±0.76% in nLDL-exposed VSMC and by 34±0.32% in agLDL-exposed VSMC (Fig. 2B). Confocal laser scanning microscopy (Fig. 2C) showed a strong increase on Rho A staining and suggested that agLDL increased Rho A membrane translocation, in agreement with the results obtained by western blot analysis after cell subfractionation.

Fig. 2

AgLDL induces Rho A expression and Rho A translocation from the cytoplasm to the membrane. Control VSMC (black bars) and simvastatin-treated VSMC (5 μM, 4 h) (gray bars) were incubated for a further 18 h in the absence or presence of nLDL or agLDL (100 μg/mL). (A) Real time PCR quantification of Rho A mRNA expression levels. Data were processed with a specially designed software program based on Ct values of each sample and normalized to gapdh mRNA (n=3) (B) Representative Western blot of cytoplasmic and membrane Rho A and Rac1 and bar graphs showing the densitometric quantification of blots. Results are expressed as arbitrary units. Results are shown as mean±SEM of three experiments performed in duplicate. P<0.05: # vs no LDL exposed VSMC; ‡ vs nLDL exposed VSMC; * vs statin untreated VSMC. (C) Confocal laser microscopy microphotographs of VSMC incubated with antibodies anti-Rho A. VSMC were incubated in absence or presence of agLDL (100 μg/mL) for 18 h. Cells were then exhaustively washed, fixed and incubated with anti-Rho A antibodies. Photomicrographs are representative of two experiments and show 16 consecutive images obtained by optical sectioning of cells.

As shown in Table 1, exoenzyme C3, a specific Rho A inhibitor, completely prevented the strong agLDL induced TF protein overexpression. However, exoenzyme C3 only partially inhibited agLDL induced TF activation. Exoenzyme C3 also induced a slight decrease on basal VSMC-TF expression.

View this table:
Table 1

Rho A inhibitor (exoenzyme C3) prevent agLDL-TF overexpression and activation

TF proteinTF activity
Exoenzyme C3Exoenzyme C3
++
No LDL2.68±0.162.24±0.10*0.9±0.0290.84±0.054
AgLDL3.80±0.22#2.37±0.23*2.75±0.015#1.61±0.140#*
  • Untreated VSMC or exoenzyme C3 treated VSMC were incubated in absence or presence of agLDL (100 μg/mL) for further 18 h. TF protein expression was determined by Western blot analysis and TF activity by factor Xa generation test. Results are expressed as mean±SEM of three experiments performed in duplicate.

    p<0.05: * vs exoenzyme C3 untreated VSMC; # vs cells incubated in absence of LDL.

3.3. TF induction by agLDL depends on LRP1 expression

To analyze whether LRP1 is required for the induction of TF by agLDL, we assessed the effect of monoclonal antibodies against the α chain of LRP1. Fig. 3A shows microphotographs of representative cells after incubation of untreated, anti-LRP1 or non-immune IgG-treated cells with agLDL. As shown, VSMC had many aggregates of LDL bound (arrows) on the cell surface, whereas anti-LRP1 treated VSMC did not. In anti-LRP1 treated VSMC, agLDL was unable to up-regulate TF protein expression (Fig. 3B) or TF activity (Fig. 3C).

Fig. 3

Effect of anti-LRP1 antibodies on agLDL induced TF overexpression in human VSMC. VSMC were arrested and incubated with agLDL (100 μg/mL) in the absence or presence of anti-LRP1 antibodies (30 nM) or non-immune IgG (30 nM) for 18 h. (A) VSMC were then washed and photographed (magnification × 10). Arrows indicate agLDL bound to the cell surface (B) Western blot analysis of cellular membrane TF antigen. Results are normalized by the respective α-actinin band intensities. (C) Factor Xa generation test of TF activity. TF activity is expressed as mU/mg protein. Results are shown as mean±SEM of three experiments performed in duplicate. P<0.05: # vs no LDL exposed VSMC; * vs VSMC incubated in absence of antibodies.

To further evidence the role of LRP1 on the induction of TF by agLDL, we analyzed the effects of agLDL in siRNA-LRP1-treated VSMC. Silencing LRP1 mRNA showed efficacy and specificity to inhibit LRP1 expression. siRNA-LRP1-VSMC showed a significant decrease in LRP1 mRNA expression in control (24±1.5%) in nLDL- (29±0.7%) and agLDL-exposed VSMC (42±1.1%) (Fig. 4A), and siRNA-LRP1 treatment did not show any significant decrease in LDLR (Fig. 4B) or CD36 mRNA expression (data not shown). In agreement, reduction in LRP1 mRNA expression was associated to a significant decrease in LRP1 protein expression in control (34±1.2%), nLDL- (33±1.6%) and agLDL-exposed VSMC (35±2.2%) (Fig. 4C). In siRNA-LRP1-treated VSMC, agLDL was unable to induce Rho A mRNA overexpression (Fig. 5A) or Rho A translocation (Fig. 5B). siRNA-LRP1 treatment did not show any effect on nLDL induced Rho A translocation and it slightly reduced Rho A mRNA expression and membrane Rho A protein in control VSMC.

Fig. 5

AgLDL fails to induce Rho A mRNA expression and Rho A translocation in LRP1-deficient VSMC. Untreated VSMC (black bars) or siRNA-LRP1-VSMC (gray bars) were incubated in absence or presence of nLDL or agLDL (100 μg/mL) for further 18 h. (A) Real time PCR quantification of Rho A mRNA expression levels. (B) Representative Western blot of cytoplasmic and membrane Rho A and bar graphs showing the densitometric quantification of blots. Results are expressed as arbitrary units. Results are shown as mean±SEM of three experiments performed in duplicate. P<0.05: # vs no LDL exposed VSMC; ‡ vs nLDL exposed VSMC; * vs siRNA-LRP1 untreated VSMC.

Fig. 4

siRNA-LRP1 treatment decreases LRP1 mRNA and LRP1 protein expression. Untreated VSMC (black bars) or siRNA-LRP1-VSMC (gray bars) were incubated in absence or presence of nLDL or agLDL (100 μg/mL) for further 18 h. (A) Real time PCR quantification of LRP1 mRNA expression levels. (B) Real time PCR quantification of LDLR mRNA expression levels. Data were processed with a specially designed software program based on Ct values of each sample and normalized to gapdh mRNA. (C) Western blot analysis of membrane LRP1 protein expression. Results are normalized by the respective α-actinin band intensities. Results are shown as mean±SEM of three experiments performed in duplicate. P<0.05: # vs no LDL exposed VSMC; ‡ vs nLDL exposed VSMC; * vs siRNA-LRP1 untreated VSMC.

As shown in Table 2, siRNA-LRP1 treatment led to the prevention of agLDL induced TF protein overexpression, and agLDL induced TF activation.

View this table:
Table 2

AgLDL fails to induce TF protein overexpression and TF activation in siRNA-LRP1 treated VSMC

TF protein expressionTF activity
siRNA-LRP1siRNA-LRP1
++
No LDL3.82±0.224.2±0.41.72±0.181.44±0.22
AgLDL5.15±0.5#3.46±0.23*3.17±0.36#2.21±0.15*
  • Untreated VSMC or siRNA-LRP1-VSMC were incubated in absence or presence of agLDL (100 μg/mL) for further 18 h. TF protein expression was determined by Western blot analysis and TF activity by factor Xa generation test. Results are expressed as mean±SEM of three experiments performed in duplicate. p<0.05:* vs siRNA-LRP1 untreated VSMC; # vs cells incubated in absence of LDL.

4. Discussion

Previous studies in our laboratory showed that agLDL, one of the main LDL modifications in the arterial intima [5,6], increased TF expression and activity in human VSMC [12,13]. Several studies have shown that statins influence TF expression and activity in different in vitro and in vivo models [16–19]. Our results demonstrate for the first time that TF induction by agLDL depends on LRP1 engagement, and requires Rho A translocation to the VSMC-membrane. According to our results, the effect of simvastatin preventing agLDL-TF induction is associated to inhibition of GGPP synthesis, suggesting the involvement of a geranylgeranylated protein on agLDL-TF induction. Western blot and immunocytochemistry demonstrate that agLDL strongly and specifically induce Rho A but not Rac1 translocation to the membrane, an effect that is abrogated by simvastatin, in agreement with the ability of statins to interfere Rho A membrane translocation [25–27]. Simvastatin also prevents the translocation of Rac1 to the membrane but with higher efficiency in control and nLDL-exposed cells than in agLDL-cells. These results suggest that agLDL might up-regulate the translocation of Rac1 to the membrane in a situation of limited protein isoprenylation. Since agLDL is a ligand for LRP1 [10,12] and LRP1 seems to be a key modulator of Rac1 activation [28], agLDL might decrease the modulatory capacity of LRP1 on Rac1 activation. These effects will require further analysis. While there is no effect of simvastatin on Rac1 cytoplasmic protein levels, simvastatin strongly increased Rho A cytoplasmic protein in all tested conditions. This effect seems to be related to the capacity of mevalonate depletion to induce the up-regulation of Ras, Rap1, Rho A and Rho B [29,30]. Mechanisms underlying this up-regulation are increased mRNA synthesis, increased protein synthesis and decreased protein degradation. The relative contribution of these mechanisms to the up-regulation differs among Ras-related proteins.

Like simvastatin, exoenzyme C3, a specific Rho A inhibitor, completely prevents agLDL-induced TF expression and it partially prevents agLDL-induced TF activation. These results suggest that while agLDL induces TF overexpression mainly through a mechanism that involves Rho A translocation, agLDL might induce TF activation by other mechanisms independent of Rho A translocation. Several previous studies suggest that TF expression and TF procoagulant activation are independent processes [31,32]. TF activation has been shown to be regulated by mechanisms that alter TF quaternary structure [33]. We have recently demonstrated that agLDL has the ability to alter the sphyngomyelin (SM) content of VSMC cellular membrane [9]. SM is one of the main phospholipids structuring caveolae, structures that are highly enriched in TF in encrypted form [34]. Thus, by altering the SM content of the caveolae, agLDL might influence TF topology and consequently its activation.

According to our results, agLDL show a much stronger capacity than nLDL to induce Rho A translocation to the cellular membrane in human VSMC. Rho A induction by agLDL is specifically prevented by siRNA-LRP1 inhibition, in agreement with the pivotal role of LRP1 on VSMC-agLDL uptake previously demonstrated in our group [9–11]. The stronger capacity of agLDL to induce Rho A activation might thus be related to the ability of agLDL to up-regulate its own receptor LRP1 [11]. In agreement, the inhibition of LRP1 expression by siRNA-LRP1 treatment or by anti-LRP1 (α chain) antibodies leads to the complete prevention of agLDL induced TF overexpression and TF activation. These results demonstrate that both agLDL-TF overexpression and agLDL-TF activation specifically depend on LRP1 expression. The strong increase of Rho A protein translocation by LRP1-mediated agLDL internalization, one of the main mechanisms contributing to VSMC-foam cell formation [9–13], might have relevance for the synthesis of TF in the vasculature. Our results suggest that the maintenance of regulated levels of LRP1 expression during atherosclerotic lesion progression might be a chance to prevent the proatherothrombotic changes induced by agLDL, one of the main LDL modifications contributing to lipid deposition during atherosclerotic plaque progression.

Acknowledgements

This work was possible thanks to funding from FIS C03-01 (RECAVA), SAF2003-03187, MSD-unrestricted grant, FIS PI051717, Sociedad Española de Arteriosclerosis grant 2001 and Fundación Investigación Cardiovascular (FIC) Catalana-Occidente. SCL is a pre-doctoral fellow at ICCC. The authors thank Cardiology and Cardiac Surgery Service Heart Transplant Team at Hospital Santa Creu i Sant Pau and the Blood Bank at Hospital Clinic, Barcelona, for their collaboration. The authors also thank Dr. E. Peña by her help with confocal microscopy images. Thanks also to Laura Nasarre and Vanessa Martín for their technical support.

Footnotes

  • Time for primary review 27 days

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View Abstract