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Cardiovascular Research Advance Access first published online on June 7, 2008
This version [Corrected Proof] published online on July 2, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn158
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Rho-GDP dissociation inhibitor alpha downregulated by low shear stress promotes vascular smooth muscle cell migration and apoptosis: a proteomic analysis

Ying-Xin Qi1, Ming-Juan Qu1, Ding-Kun Long1, Bo Liu1, Qing-Ping Yao1, Shu Chien1,2 and Zong-Lai Jiang1,*

1 Institute of Mechanobiology and Medical Engineering, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, PO Box 888, 800 Dongchuan Road, Minhang, Shanghai 200240, P.R. China
2 Departments of Bioengineering and Medicine, University of California, San Diego, La Jolla, CA 92093-0412, USA

* Corresponding author. Tel: +86 21 34204863; fax: +86 21 34204118. E-mail address: zljiang{at}sjtu.edu.cn

Received 13 February 2008; revised 29 May 2008; accepted 5 June 2008

Time for primary review: 22 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Low shear stress (LSS) plays a significant role in vascular remodelling during atherogenesis, which involves migration, proliferation, and apoptosis of vascular smooth muscle cells (VSMCs). The aim of the present study is to elucidate the molecular mechanisms by which LSS induces vascular remodelling.

Methods and results: Using proteomic techniques, two-dimensional electrophoresis, and mass spectrometry, the protein profiles of Sprague–Dawley rat aorta cultured under two levels of shear stress, 5 and 15 dyn/cm2, were determined. The results showed a significantly lower expression of protein-Rho-GDP dissociation inhibitor alpha (Rho-GDI{alpha}) in the LSS vessels. Rho-GDI{alpha} signalling mechanisms and effects on VSMC migration and apoptosis were then studied to understand the role of Rho-GDI{alpha} in the LSS-induced vascular remodelling. A decrease in Rho-GDI{alpha} expression by using target small interfering RNA (siRNA) transfection caused increases in the phosphorylation of Rac1 and Akt and enhancements of VSMC migration and apoptosis. Treatment with the PI3K/Akt-specific inhibitor wortmannin significantly decreased Akt phosphorylation, but had no effect on Rho-GDI{alpha} expression and Rac1 phosphorylation. Wortmannin was able to reverse the Rho-GDI{alpha} siRNA-induced enhancement of VSMC migration, but not VSMC apoptosis.

Conclusion: The results indicate that the LSS-induced VSMC migration and apoptosis are mediated by a downregulation of Rho-GDI{alpha}. The effect of Rho-GDI{alpha} on VSMC migration is mediated by the PI3K/Akt pathway, but its effect on VSMC apoptosis is not.

KEYWORDS Rho-GDI{alpha}; Migration; Apoptosis; Vascular smooth muscle cells; Shear stress


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
An essential pathology of atherosclerosis is vascular remodelling, which involves proliferation, apoptosis, and migration of vascular smooth muscle cells (VSMCs) and disturbance of extracellular matrix homeostasis.1 Pathological studies have shown that atherosclerotic lesions occur preferentially at vessel branch points, bifurcations, and regions of high curvature, suggesting that low and disturbed shear stress is an inducer in atherogenesis, whereas the normal level of laminar shear stress is atheroprotective.1,2

The mechanotransduction in vascular cells induced by shear stress has been investigated both in vivo and in vitro. Transmembrane integrins can sense the shear stress stimulation to undergo binding with focal adhesion kinases and cytoskeletal proteins.3,4 Other mechanical sensors, including K+ and Ca2+ ion channels5 and receptor tyrosine kinase,6 also have been shown to participate in the transduction of shear stress stimulation into vascular cells. Shear stress has been shown to activate signalling pathways that modulate gene expression and cell functions, e.g. Rho family GTPases,7 PI3K/Akt,8 and mitogen-activated protein kinases,9 including C-Jun-N-terminal kinase.10 The molecular mechanism of vascular remodelling due to low shear stress (LSS), however, remains to be elucidated.

Proteomic analysis, owing to its high degree of reproducibility and the possibility of assessing the expression profile of a large number of proteins simultaneously, has been widely applied in cancer research, e.g. in detecting biomarkers,11 monitoring therapeutical efficacy,12 and discovering new drugs.13 Some investigators have used proteomic techniques to study cardiac diseases, e.g. myocardial ischaemia/reperfusion injury14 and heart failure,15 but vascular proteomics is still in its infancy. Research on vascular proteomics may provide new insights into the pathogenesis of artherosclerosis and reveal novel therapeutical targets. Therefore, we compared the protein profiles of rat aorta cultured under two levels of shear stress: a LSS at 5 dyn/cm2 and a normal shear stress (NSS) at 15 dyn/cm2. Proteomic analysis was performed using two-dimensional electrophoresis (2-DE) for the separation of a mixture of proteins, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for the detection of differently expressed proteins. We identified a key protein differentially expressed in response to the two shear stress levels, i.e. Rho-GDP dissociation inhibitor alpha (Rho-GDI{alpha}), which could interact with several Rho family proteins, including Rho A, Rho B, and Rac, to regulate their activities. Experiments were then performed to analyse the possible effects of Rho-GDI{alpha} on vascular remodelling and the signalling mechanisms involved.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Vessel culture protocol
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and the protocol was approved by the Animal Research Committee of Shanghai Jiao Tong University.

Adult male Sprague–Dawley rats, 220–260 g in weight, were anaesthetized with intraperitoneal injection of pentobarbital. Following thoracotomy, the thoracic aorta was rapidly dissected with all branches ligated under a dissecting microscope. The vascular segment was mounted in a perfusion culture system (Figure 1). Perfusion pressure was generated by the difference in hydrostatic heights between the medium (DMEM containing 10% heat-inactivated FBS, 100 mmol/L HEPES, 100 U/mL penicillin, 100 µg/mL streptomycin) in the upper reservoir (upstream) and the perfused vessel (downstream), and it was monitored with a pressure transducer (BIOPAC System MP30, Santa Barbara, CA, USA) just upstream to the vascular segment to ensure the maintenance of perfusion pressure at 100 mmHg. A peristaltic pump (7550–30, Cole-Parmer) was used to return the perfusion medium to the upper reservoir. Steady laminar flow was assured using a capacitance in the perfusion circuit (upstream) to eliminate the oscillations due to the peristaltic pump. The culture system was kept in a cell incubator at 37°C, and the perfusion medium was equilibrated with a gas mixture of 95% air and 5% CO2 to keep oxygen tension, pH, and temperature at desired levels.


Figure 1
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  		Figure 1 Schematic drawing of the vessel culture and perfusion system.

 
Under the same perfusion pressure of 100 mmHg, the vascular segments were exposed to wall shear stress at either 15 or 5 dyn/cm2 for 24 h by using different flow rates (Q). The wall shear stress was maintained at the desired level by controlling the flow rate of the peristaltic pump in accordance with the measurement of the vessel radius (r) under the microscope. The wall shear stress ({tau}) was calculated using the formula: {tau} = 4µQ/{pi}r3, where µ is the viscosity of the perfusion medium determined with the use of a Brookfield viscometer (Model LVDV-II+Pro).

2.2 Two-dimensional electrophoresis
Total protein extracts were prepared from the cultured vessels as described previously.16 Briefly, vessel samples were ground and subjected to vortexing in a lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris base, 1% DTT, 0.5% IPG buffer, and protease inhibitor cocktails) at room temperature for 1 h and then centrifuged (12 000 g at 4°C) for 45 min. The total protein in the supernatant was assayed using the Bradford Analysis kit (BioRad).

The first-dimension electrophoresis was carried out on an Ettan IPGphor IEF system (GE Healthcare). The sample solution was applied onto 24 cm IPG dry strips (pH3-10 NL) with a loading of 450 µL per strip. The rehydration and separation programs were automatically processed using the following parameters: 30 V, 12 h; 200 V, 1 h; 500 V, 1 h; 1000 V, 1 h; and 8000 V until the total volt-hours reached 80 000. After the completion of the IEF run, the IPG strips were immediately equilibrated for 2 x 15 min in the SDS equilibration buffer containing 50 mM Tris–HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and traces of bromophenol blue. The first equilibration was performed in the earlier-mentioned equilibration buffer with 1.0% (w/v) DTT, followed by a second equilibration with 2.5% (w/v) iodoacetamide. The strips were subsequently subjected to a second-dimension SDS gel electrophoresis, using an Ettan DALTSixTM electrophoresis unit (GE Healthcare). The SDS–PAGE was performed first at 4 W per gel for 1 h, and then 16 W per gel until the dye front reached the bottom of gels. After fixation in 12% TCA solution, the gels were stained by using the blue-silver method.17 The gels were scanned at 300 d.p.i. resolutions and analysed with an Image Master PlatinumTM software (GE Healthcare).

2.3 Matrix-assisted laser desorption/ionization-time of flight mass spectrometry
Protein spots that showed differential expressions between vessels exposed to the two levels of shear stress were subjected to automatic MS analysis in an Ettan Spot Handling Workstation. The dried peptide fragments were resuspended with 3 µL of matrix solution, which consists of 50% acetonitrile, 0.03% trifluoroacetic acid, and semi-saturated {alpha}-cyano-4-hydroxycinnamic acid. The samples were analysed in a time-of-flight mass spectrometer (Ettan MALDI-TOF/Pro, GE Healthcare). Peptide matching and protein searching were performed by using the Mascot research software. The peptide masses were compared with the theoretical peptide masses of all available proteins from Rattus sub-database in the NCBInr database. Mono-isotopic masses were used and 100 p.p.m. peptide mass tolerance was allowed.

2.4 Culture of vascular smooth muscle cells
VSMCs culture was prepared from the rat aorta by the explant technique.18 Briefly, the media layer of the rat aorta was isolated surgically and minced into small pieces, which were plated onto 25 cm2 culture flasks for culture in DMEM with 10% heat-inactivated FBS, 2 mmol/L glutamine, 100 mmol/L HEPES, 100 U/mL penicillin, 100 µg/mL streptomycin, and incubated at 37°C with humidified 5% CO2. VSMCs displayed the typical spindle-shaped morphology and ‘‘hill-and-valley’’ pattern of growth and were characterized by immunohistochemical staining for smooth muscle-specific {alpha}-actin (Sigma). Cells between passages 5–7 were used.

2.5 RNA interference and inhibitor treatment
The mRNA sequences of rat Rho-GDI{alpha} (NM_001007005) and Rac1 (NM_134366 [GenBank] ) were acquired from NCBI GenBank. Small interfering RNAs (siRNAs) against rat Rho-GDI{alpha} and Rac1 were designed and synthesized by GenePharma Biological Company (Shanghai, P.R. China). The double strands of siRNA for Rho-GDI{alpha} were 5'-AGCA CUCU GUGA ACUA CAAdT dT-3' and 5'- UUGU AGUU CACA GAGU GCUC dG-3', and those for Rac1 were 5'-CAAA CAGA CGUG UUCU UAAT T-3' and 5'-UUAA GAAC ACGU CUGU UUGC G-3'. Non-silencing siRNA that does not recognize any known homology to rat genes was synthesized as a negative control. The cells were seeded at a density of 2.0 x 105 cells per well in six-well plates and grown in DMEM with 10% heat-inactivated FBS. After seeding for 24 h, the cells were transfected with 100 nmol siRNA and 5 µL LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instruction. Briefly, LipofectamineTM 2000 was diluted in 250 µL opti-MEM (Invitrogen) and incubated for 5 min at room temperature; then 100 nmol siRNA in 250 µL opti-MEM was added. After mixing for 20 min at room temperature, the mixture was added dropwise onto the cells. In some experiments, 6 h after the treatment with Rho-GDI{alpha} siRNA, the mixture (5 µL LipofectamineTM 2000 and 100 nmol siRNA in 500 µL opti-MEM) was replaced with DMEM containing 5% heat-inactivated FBS. The cells were then cultured for 48 h for the next analysis. Control cells were exposed to the transfectant with non-silencing siRNA instead of target siRNA.

To assess the role of PI3K/Akt on Rho-GDI{alpha}-induced VSMC migration and apoptosis, the VSMCs were treated with wortmannin or vehicle in 5% FBS/DMEM after 6 h incubation of transfection mixture (5 µL LipofectamineTM 2000 and 100 nmol siRNA in 500 µL opti-MEM). Dimethyl sulphoxide (DMSO) was present at equal concentration (0.2%) in all groups.

2.6 Real-time polymerase chain reaction and western blotting
The mRNA expression of Rho-GDI{alpha} in cultured vessels subjected to the two levels of shear stress was detected by real-time polymerase chain reaction (PCR) (Bio-Rad). Specific primers for rat Rho-GDI{alpha} were synthesized by Sangon Biological Company (Shanghai, P.R. China) (sense 5'-TGTG CTGC TGTT GCTT CC-3'; antisense 5'-GCTC GGCT GGCT TTGT-3'; product length 230 bp). Amplification was performed in parallel samples using rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (sense 5'-GGCA GCCC AGAA CATC ATCC-3'; antisense 5'-GCCA GCCC AAGC ATCA AAG-3'; product length 298 bp) as a control.

For the detection of protein expression levels, tissue/VSMC lysates were subjected to electrophoretic separation with 10% SDS–PAGE and transferred to nitrocellulose membranes (Hybond, Amersham). Western blots were performed using antibodies directed against Rho-GDI{alpha} (1:500, Santa Cruz Technologies), phospho-Rac1 (1:500, Cell Signaling Technologies), Rac1/2/3 (1:500, Cell Signaling Technologies), phosphor-Akt (1:500, Cell Signaling Technologies), total-Akt (1:500, Cell Signaling), and GAPDH (1:500, Santa Cruz Technologies).

2.7 Cell migration and flow cytometry
Studies on VSMC migration were performed with the Transwell system (Costar), which allows cells to migrate through a polycarbonate membrane with a pore size of 8 µm in six-well plates, as described previously.19 Briefly, VSMCs were plated at a concentration of 1.0 x 106 cells/mL on the upper chamber, and the lower chamber was filled with 600 µL DMEM with 50% heat-inactivated FBS to serve as a chemoattractant. After 6 h of incubation, the number of migrated cells was counted under a microscope (magnification x200, Olympus). Six randomly chosen fields were evaluated per transwell membrane.

After siRNA transfection, the VSMCs were fixed with cold 4% paraformaldehyde, stained with PI, and analysed by flow cytometry (FACScan, Becton Dickinson) to determine cell cycle and ratio of apoptotic cells.

2.8 Statistical analysis
Each experiment was performed at least in triplicate, and all values are expressed as mean ± SD. The one-way ANOVA was used to compare the results between the two groups followed by Fisher's t-test for multiple comparisons. Values of P < 0.05 were accepted as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Proteomic analysis
To search for variations in protein profiles between the vessels cultured under shear stresses of 15 and 5 dyn/cm2, 2-DE combined with blue silver staining and computer-assisted Image Mater PlatinumTM software analysis were employed. This technique allowed screening of an average of 1900 protein spots per gel with loading of 800 µg of total proteins. After analysing the 2-DE gel maps of vessels cultured under 15 and 5 dyn/cm2, we found a protein spot that was highly expressed in vessels cultured under 15 vs. 5 dyn/cm2, with isoelectric point and molecular weight of 4.65 and 25 kDa, respectively (Figure 2A). The peptide mass of this spot was identified by MALDI-TOF MS and proved to be Rho-GDI{alpha} (Figure 2B). The identification of Rho-GDI{alpha} peptide mass was evaluated automatically by the Mascot and ProFound software using NCBInr protein data bank.


Figure 2
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  		Figure 2 Different expressions of Rho-GDP dissociation inhibitor alpha between vessels cultured under 15 (the normal shear stress) and 5 dyn/cm2 (the low shear stress) revealed by proteomic analysis. (A) A typical example of two-dimensional electrophoresis gel maps. Filled triangle marks the protein spot, with a higher expression in vessels cultured under normal shear stress. Three gels for normal shear stress and three gels for low shear stress, each gel was run with a protein sample from an independent rat aorta. (B) The matrix-assisted laser desorption/ionization-time of flight mass spectrometry result of the marked spot showed it to be Rho-GDP dissociation inhibitor alpha. (C) Western blotting revealed that the expression of Rho-GDP dissociation inhibitor alpha was suppressed in the low shear stress-cultured vessels compared with the normal shear stress, whereas the phosphorylations of Rac1 and Akt were increased. The left panel shows the gels from a typical study; the right histograms show densitometric quantification (compared with glyceraldehyde-3-phosphate dehydrogenase). Values shown are mean ± SD for each condition from three independent experiments. *P < 0.05 for low shear stress vs. normal shear stress.

 
3.2 Differential gene and protein expressions of Rho-GDP dissociation inhibitor alpha between vessels cultured under 15 and 5 dyn/cm2
The differential expression of Rho-GDI{alpha} under the two levels of shear stress found by 2-DE and MALDI-TOF MS analysis was verified by real-time PCR analysis and western blotting, at the gene and protein levels, respectively. These measurements showed that the mRNA (data not shown) and protein expression levels of Rho-GDI{alpha} in the vessels cultured under 5 dyn/cm2 were significantly lower than those under 15 dyn/cm2 (P < 0.05, Figure 2C). The immunohistochemistry analysis of the rat aorta showed that Rho-GDI{alpha} was mainly expressed in the cytoplasm of VSMCs (data not shown). Therefore, we focused our attention on Rho-GDI{alpha} in terms of its downstream signalling and effects on VSMC migration and apoptosis.

Rac1 and Akt activations were assessed by western blotting with the phospho-specific Rac1 and Akt antibodies that recognize the phosphorylated Ser71 residue on Rac1 and Thr308 residue on Akt, which are the critical sites required for the activation of Rac1 and Akt, respectively. As shown in Figure 2C, the expressions of phospho-Rac1 and phospho-Akt were increased in vessels cultured under 5 dyn/cm2 (P < 0.05).

3.3 Silencing of Rho-GDP dissociation inhibitor alpha and its effect on vascular smooth muscle cell migration and apoptosis
After siRNA treatments for 24, 48, and 72 h, determination of the Rho-GDI{alpha} protein expression by analysing the mean grey scale of the specific protein bands showed that the specific Rho-GDI{alpha} siRNA caused significant suppression of the protein level at all three time points when compared with the control cells transfected with non-silencing siRNA (P < 0.05; for reference, see Supplementary material online, Figure S3A). The most effective silencing of Rho-GDI{alpha} was detected at 48 h, and hence this time period was used in subsequent studies.

After siRNA treatment for 48 h, the decreased expression of Rho-GDI{alpha} was associated with significantly enhanced Rac1 and Akt phosphorylations (P < 0.05, RNA interference vs. control, Figure 3A). The migration rate of VSMCs treated with specific Rho-GDI{alpha} siRNA was significantly higher than that of control cells treated with non-silencing siRNA (P < 0.05, Figure 3B). Flow cytometry analysis revealed that the VSMC apoptosis was also increased after 48 h of transfection with the specific Rho-GDI{alpha} siRNA (P < 0.05, Figure 3C; for reference, see Supplementary material online, Figure S3C). These results suggest that the suppressed expression of Rho-GDI{alpha} enhanced the migration ability and induced apoptosis of VSMCs.


Figure 3
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  		Figure 3 The effect of 48 h transfection with non-silencing small interfering RNA (siRNA) (left) or specific Rho-GDP dissociation inhibitor alpha small interfering RNA (right) on the protein expression levels of Rho-GDP dissociation inhibitor alpha, phospho-Rac1, and phospho-Akt in vascular smooth muscle cells, and the migration and apoptosis of vascular smooth muscle cells. (A) After 48 h transfection, the expression of Rho-GDP dissociation inhibitor alpha was significantly suppressed, and the phosphorylations of Rac1 and Akt were increased. The top panel represents the blots from a typical study; the bottom histogram shows the relative intensities of Rho-GDP dissociation inhibitor alpha, phospho-Akt, and phospho-Rac1 (densitometric quantification relative to glyceraldehyde-3-phosphate dehydrogenase). Values shown are the mean ± SD for each condition from four independent experiments. *P < 0.05 vs. the control. (B) The role of Rho-GDP dissociation inhibitor alpha in vascular smooth muscle cell migration, which was determined by transwell assays. The number of cells illustrates the migration ability of vascular smooth muscle cells. Bar 100 µm. Histogram shows fold changes in vascular smooth muscle cell migration relative to the control. Values shown are the mean ± SD for each condition from four independent experiments, and six images were randomly acquired in each independent experiment. *P < 0.05 vs. the control. (C) The role of Rho-GDP dissociation inhibitor alpha in vascular smooth muscle cell apoptosis. Histogram shows fold changes in vascular smooth muscle cell apoptosis relative to the control (the original data collected by FAC are available in the Supplementary material online, Figure S3C). Values shown are the mean ± SD for each condition from three independent experiments. *P < 0.05 vs. the control.

 
3.4 Silencing of Rac1 and its effect on vascular smooth muscle cell migration and apoptosis
When treated with specific Rac1 siRNA, the expression of both total-Rac (Rac1/2/3) and phospho-Rac1 were decreased (P < 0.05, Figure 4A). The downexpression of phospho- Rac1 suppressed the activation (phosphorylation) of Akt (P < 0.05, Figure 4A), but had no effect on the Rho-GDI{alpha} expression (P > 0.05, Figure 4A).


Figure 4
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  		Figure 4 The effect of 48 h transfection with non-silencing small interfering RNA (siRNA) (left) or specific Rac1 small interfering RNA (right) on the protein expression levels of total-Rac, phospho-Rac1, Rho-GDP dissociation inhibitor alpha, total-Akt, and phospho-Akt in vascular smooth muscle cells, and the migration and apoptosis of vascular smooth muscle cells. (A) After specific Rac1 small interfering RNA transfection, the expressions of total-Rac and phospho-Rac1 were significantly suppressed, the expression of Rho-GDP dissociation inhibitor alpha was similar to the control, and the phosphorylation of Akt was decreased. The top panel represents the blots from a typical study; the bottom histogram shows the relative intensities of total-Rac, phospho-Rac1, Rho-GDP dissociation inhibitor alpha, and phospho-Akt (densitometric quantification relative to glyceraldehyde-3-phosphate dehydrogenase). Values shown are the mean ± SD for each condition from four independent experiments. *P < 0.05 vs. the control. (B) Vascular smooth muscle cell migration analysis determined by transwell assays. The silencing of Rac1 expression induced by RNA interference suppressed vascular smooth muscle cell migration. Bar 100 µm. Histogram shows fold changes of vascular smooth muscle cell migration relative to the control. Values shown are mean ± SD for each condition from four independent experiments, and six images were randomly acquired in each independent experiment. *P < 0.05 vs. the control. (C) Transfection with specific Rac1 small interfering RNA decreased the mean value of vascular smooth muscle cell apoptosis, but this effect had no statistical significance. Histogram shows fold changes in vascular smooth muscle cell apoptosis relative to the control (the original data collected by FAC are available in the Supplementary material online, Figure S4C). Values shown are the mean ± SD for each condition from three independent experiments. *P < 0.05 vs. the control.

 
The migration rate of VSMCs treated with specific Rac1 siRNA was significantly reduced when compared with that of control cells treated with non-silencing siRNA (P < 0.05, Figure 4B). The apparent decrease in VSMC apoptosis analysed by flow cytometry after transfection with the specific Rac1 siRNA was not statistically significant compared with the control (P > 0.05, Figure 4C; for reference see Supplementary material online, Figure S4C).

3.5 The effect of Akt activation on the modulations of vascular smooth muscle cell migration and apoptosis by Rho-GDP dissociation inhibitor alpha
Wortmannin is a specific inhibitor of the PI3K/Akt signal pathway. As shown in Figure 5A, the phosphorylation of Akt was significantly suppressed after wortmannin incubation (P < 0.05), whereas the expressions of Rho-GDI{alpha} and phospho-Rac1 were not significantly changed from their respective controls (P > 0.05).


Figure 5
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  		Figure 5 The effect of Akt activation on the modulations of vascular smooth muscle cell migration and apoptosis by Rho-GDP dissociation inhibitor alpha. (A) The protein expression levels of total-Akt, phospho-Akt, Rho-GDP dissociation inhibitor alpha, total-Rac, and phospho-Rac1 in vascular smooth muscle cells after 48 h treatment with vehicle (left) and wortmannin (right). The top panel represents the blots from a typical study; the bottom histogram shows the relative intensities of phospho-Akt, Rho-GDP dissociation inhibitor alpha, and phospho-Rac1 (densitometric quantification relative to glyceraldehyde-3-phosphate dehydrogenase). Values shown are the mean ± SD for each condition from four independent experiments. *P < 0.05 vs. the control. (B) The protein expression levels of phospho-Akt and Rho-GDP dissociation inhibitor alpha in vascular smooth muscle cells after 48 h treatment with wortmannin and 48 h transfection with specific Rho-GDP dissociation inhibitor alpha small interfering RNA, respectively. The top panel represents the blots from a typical study; the bottom histogram shows the relative intensities of phospho-Akt and Rho-GDP dissociation inhibitor alpha (densitometric quantification relative to glyceraldehyde-3-phosphate dehydrogenase). Values shown are the mean ± SD for each condition from four independent experiments. *P < 0.05 vs. dimethyl sulphoxide. #P < 0.05 for Rho-GDP dissociation inhibitor alpha small interfering RNA + wortmannin vs. Rho-GDP dissociation inhibitor alpha small interfering RNA. (C) Vascular smooth muscle cell migration analysis determined by transwell assays. The silencing of Rho-GDP dissociation inhibitor alpha expression induced by RNA interference increased vascular smooth muscle cell migration, and this increase was reversed by wortmannin. Bar 100 µm. Histogram shows fold changes of vascular smooth muscle cell migration relative to the controls treated with dimethyl sulphoxide. Values shown are mean ± SD for each condition from four independent experiments, and six images were randomly acquired in each independent experiment. *P < 0.05 vs. dimethyl sulphoxide. #P < 0.05 for Rho-GDP dissociation inhibitor alpha small interfering RNA + wortmannin vs. Rho-GDP dissociation inhibitor alpha small interfering RNA. (D) When transfected with specific Rho-GDP dissociation inhibitor alpha small interfering RNA, vascular smooth muscle cell apoptosis was increased, and wortmannin did not decrease this effect (the apparent increase was not statistically significant). Histogram shows fold changes in vascular smooth muscle cell apoptosis relative to the controls treated with dimethyl sulphoxide (the original data collected by FAC are available in the Supplementary material online, Figure S5D). Values shown are the mean ± SD for each condition from four independent experiments. *P < 0.05 vs. dimethyl sulphoxide.

 
As mentioned earlier, the decrease of Rho-GDI{alpha} expression by its siRNA transfection increased the migration ability and apoptosis of VSMCs (Figures 3B and C). The solvent DMSO did not cause such changes (P < 0.05, Figures 5C and D). After the transfection with specific Rho-GDI{alpha} siRNA for 6 h, addition of wortmannin to the growth medium caused a marked decrease in the expression of phospho-Akt (P < 0.05, Figure 5B), but had no effect on the reduced level of Rho-GDI{alpha}. Wortmannin was able to reverse the increase of VSMC migration caused by Rho-GDI{alpha} siRNA transfection (P < 0.05, Figure 5C), but it had no statistically significant effect on the enhancement of VSMC apoptosis caused by Rho-GDI{alpha} siRNA transfection, though the mean value was actually higher (P > 0.05, Figure 5D; for reference, see Supplementary material online, Figure S5D).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
Mechanical stress has been recognized as an important factor in the regulation of vascular remodelling, and LSS has been shown to be an inducer for atherogenesis. The proteomic data set of rat aorta and its response to oxidative stress has been studied.20 However, the proteomic variation of aorta induced by LSS remains to be established. In our current study, the protein profiles of the rat aorta cultured under NSS (15 dyn/cm2) and LSS (5 dyn/cm2) were compared using proteomic techniques of 2-DE and MALDI-TOF MS. The results revealed that a modulator of Rho family signal transduction, Rho-GDI{alpha}, was differentially expressed between these two levels of shear stress: the expression of Rho-GDI{alpha} was significantly lower when the aorta was exposed to LSS for 24 h. Our previous studies demonstrated that Rho-GDI{alpha} was differentially expressed in aorta between spontaneously hypertensive rats and their normotensive counterpart Wistar–Kyoto rats.21 All these results suggest that Rho-GDI{alpha} plays a significant role in the vascular remodelling caused by mechanical stimuli.

Rho-GDI{alpha}, the molecule of interest that was found by proteomic analysis, is a member of Rho-GDP dissociation inhibitors (Rho-GDIs), which have been proved to negatively regulate the activities of small G proteins of the Rho family by shutting off their GDP/GTP cycling and cytosol/membrane translocation.22,23 Rho family proteins cycle between an active GTP-bound form and an inactive GDP-bound form to function as molecular switch to regulate the downstream signal transduction processes.24 The essential role of Rho-GDIs is to form the Rho/Rho-GDIs complex, thus inhibiting guanine nucleotide exchange factors that stimulate GDP/GTP exchange.22 Rho-GDIs also could shut down the activity of Rho proteins by keeping them in the cytosol, where these proteins are attached by an isoprenoid moiety located at their C terminus.23 There are three isoforms of Rho-GDIs: Rho-GDI{alpha} (also named Rho-GDI 1), β (also named Rho-GDI 2 or D4/LyGDI), and {gamma} (also named Rho-GDI 3). Among these Rho-GDIs, Rho-GDI{alpha} is ubiquitously expressed and has a broad range of activities towards several Rho family proteins in vivo, including RhoA, RhoB, Rac1, Rac2, and Cdc42.25

It has been reported that Rho family proteins participate in the regulation of polarity,26 proliferation,26,27 adhesion, spreading,28 migration,29,30 and cytoskeleton organization31 of VSMCs. Studies in vivo revealed that the activation of Rho family proteins are significantly increased in atherosclerotic plaque.32 Xu et al.33 reported that the application of mechanical stress to VSMCs could modulate the activation of Rac and Ras, which participate in the vascular remodelling in response to haemodynamic stimulation. All these results suggest that the active/inactive cycles of Rho family proteins are crucial molecular switches to control mechanical stress-induced VSMC signalling processes. However, because of the complexity of intracellular machinery and signalling pathways, the exact molecular regulation mechanism of the activity of Rho family proteins has not been clarified.

Our study revealed that the expression of Rho-GDI{alpha} was significantly decreased when the aorta was subjected to LSS for 24 h. Immunohistochemistry analysis of the rat aorta showed that Rho-GDI{alpha} was mainly expressed in the cytoplasm of VSMCs (data not shown). Since Rho-GDI{alpha} is the negative regulator of Rho family proteins, we hypothesize that the decreased expression of Rho-GDI{alpha} may lead to the activation of Rho family proteins during vascular remodelling. To test this hypothesis, the expression of Rho-GDI{alpha} in the cultured VSMCs was silenced by target siRNA transfection. Because migration and apoptosis of VSMCs are key processes during vascular remodelling, the effects of Rho-GDI{alpha} silencing on these two processes were studied. Our results showed that the suppression of Rho-GDI{alpha} expression in VSMCs led to marked enhancements of both migration and apoptosis of VSMCs. These results are in agreement with the hypothesis that LSS induces the VSMC apoptosis and migration by downregulating the expression of Rho-GDI{alpha}.

To investigate the possible intracellular signalling involved in the downregulation of Rho-GDI{alpha} in the LSS-induced vascular remodelling, the relationships between Rho-GDI{alpha}, Rac1, and PI3K/Akt were studied by targeted siRNA silencing for Rho-GDI{alpha} and Rac1, as well as specific inhibitor treatment for PI3K/Akt pathway, and then VSMC migration and apoptosis were analysed. With the decreased expression of Rho-GDI{alpha} induced by siRNA transfection, the activations of Rac1 and Akt were significantly increased, as reflected by the phosphorylation levels of the Ser71 residue on Rac1 (GTP-bound form) and the Thr308 residue on Akt (GTP-bound form). When the expression of phospho-Rac1 was suppressed by specific Rac1 siRNA transfection, the activation (phosphorylation) of Akt was decreased, but the expression of Rho-GDI{alpha} was not significantly changed. The VSMC migration was suppressed by specific Rac1 siRNA transfection, suggesting that Rho-GDI{alpha} might regulate the migration of VSMCs via the modulation of Rho family protein-Rac1. Our results showed that the VSMC apoptosis was not significantly repressed by the specific Rac1 siRNA. This might be because that the basal apoptosis ratios of VSMCs in control groups were low (1.02–1.65%), and it would be difficult to detect a decrease caused by Rac1 siRNA transfection.

Treatment with the PI3K/Akt-specific inhibitor wortmannin significantly reduced the phosphorylation of Akt, but had no effect on the expression of Rho-GDI{alpha} and phosphorylation of Rac1. The increase of VSMC migration caused by Rho-GDI{alpha} siRNA transfection could be reversed by wortmannin treatment. But wortmannin had no statistically significant effect on VSMC apoptosis induced by Rho-GDI{alpha} inhibition. These results suggest that the LSS probably enhances the apoptosis and migration of VSMCs by divergent pathways after downregulating the expression of Rho-GDI{alpha} to modulate the activity of Rho family protein-Rac1. The effect of Rho-GDI{alpha} on VSMC migration is dependent on the PI3K/Akt signal transduction pathway (Figure 6), whereas its effect on VSMC apoptosis is not.


Figure 6
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  		Figure 6 Schematic drawing outlining the pathway Rho-GDP dissociation inhibitor alpha, phospho-Rac, and phospho-Akt that mediates the enhancement of migration by low shear stress. The inhibition of each of the molecules in the pathway blocks the effect of low shear stress on the member(s) downstream, but not upstream. These studies allow the determination of the hierarchical relationships among the signalling molecules shown in this diagram.

 
In summary, our results suggest that the LSS-induced downregulation of Rho-GDI{alpha} may play a role in the enhancements of VSMC migration and apoptosis by LSS. These results suggest that the normal expression of Rho-GDI{alpha} might be a beneficial factor in vascular biology. Shimokawa and Takeshita,34 in reviewing animal and clinical studies, found that Rho-kinase inhibitors, which negatively regulate the activity of small G proteins like Rho-GDI{alpha}, have broad pharmacological effects for arteriosclerosis and many other cardiovascular diseases. Although LSS enhances both VSMC migration and apoptosis via the downregulation of Rho-GDI{alpha}, the signalling pathways for these two cellular functions are not the same; the effect on migration is mediated by the PI3K/Akt pathway, but that on apoptosis is not. Further research on Rho-GDI{alpha} can elucidate these signalling pathways and might contribute to the discovery of a new target in the prevention and treatment of vascular remodelling during atherosclerosis.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
National Natural Science Foundation of China (No.10732070 to Z.-L.J., No.10702043 to Y.-X.Q., and No. 30472034 to Z.-L.J.).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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