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. Author manuscript; available in PMC: 2012 Aug 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2011 Jun 2;31(8):e19–e26. doi: 10.1161/ATVBAHA.111.230706

Response Gene to Complement 32 Promotes Vascular Lesion Formation through Stimulation of Smooth Muscle Cell Proliferation and Migration

Jia-Ning Wang 1,2, Ning Shi 1, Wei-bing Xie 1, Xia Guo 1, Shi-You Chen 1
PMCID: PMC3146015  NIHMSID: NIHMS306920  PMID: 21636805

Abstract

Objective

The objectives of this study are to determine the role of response gene to complement 32 (RGC-32) in vascular lesion formation after experimental angioplasty and to explore the underlying mechanisms.

Methods and Results

Using a rat carotid artery balloon-injury model, we documented for the first time that neointima formation was closely associated with a significantly increased expression of RGC-32 protein. shRNA Knockdown of RGC-32 via adenovirus (Ad)-mediated gene delivery dramatically inhibited the lesion formation by 62% as compared to control groups 14 days after injury. Conversely, RGC-32 overexpression significantly promoted the neointima formation by 33%. Gain and loss of function studies in primary culture of rat aortic smooth muscle cells (RASMCs) indicated that RGC-32 is essential for both the proliferation and migration of RASMCs. RGC-32 induced RASMC proliferation by enhancing p34CDC2 activity. RGC-32 stimulated the migration of RASMC via inducing focal adhesion contact and stress fiber formation. These effects were caused by the enhanced ROKα activity due to RGC-32-induced downregulation of Rad GTPase.

Conclusions

RGC-32 plays an important role in vascular lesion formation following vascular injury. Increased RGC-32 expression in vascular injury appears to be a novel mechanism underlying the migration and proliferation of vascular SMCs. Therefore, targeting RGC-32 is a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.

Keywords: Response gene to complement 32, Restenosis, Vascular smooth muscle cells, Migration, Proliferation

Percutaneous coronary intervention (PCI) has been widely employed in patients with coronary artery disease1. However, restenosis following PCI limits the long-term outcome of PCI, and patients frequently require repeated revascularization procedures.2 Systemic pharmacological approaches to reduce restenosis have not been successful in clinical use. Local treatment with drug-eluting stent (DES) has demonstrated significant reduction in restenosis rate and the subsequent need for revascularization.3 However, DES increases the risk of late stent thrombosis.4, 5 Therefore, restenosis remains to be a medical challenge in cardiovascular field.

A large body of clinical and experimental studies have demonstrated that neointima formation contributing to restenosis is triggered by complex biological responses, including inflammation, thrombosis, cellular proliferation, and extracellular matrix production. Among these multiple factors, media-to-intima migration and proliferation of vascular smooth muscle cells (VSMCs) with subsequent synthesis of extracellular matrix is the most critical step in the pathogenesis of neointima formation.6

Although many factors are found to be involved in VSMC migration or proliferation, the endogenous regulators contributing to neointima formation remain largely unknown.7–11 Emerging data suggest response gene to complement 32 (RGC-32) plays a role in VSMC function.12 RGC-32 can be activated by complement C5b-9, serum and other growth factors.12, 13,14,15 Functionally, RGC-32 has been shown to stimulate or suppress endothelial cell growth depending on the physiological or pathological conditions.15, 16 In VSMCs, RGC-32 plays an important role in cell proliferation. RGC-32 physically associates with cyclin-dependent kinase p34CDC2, which increases the kinase activity to induce quiescent aortic smooth muscle cells to enter the S phase.12 However, it is unknown if RGC-32 plays a role in neointima formation following vascular injury in vivo, and if RGC-32 contributes to VSMC migration.

In the present study, we used a well-defined rat carotid artery balloon-injury model and an adenoviral gene delivery approach to test the hypothesis that RGC-32 contributes to the lesion formation after vascular injury. We found that RGC-32 was activated along with the progression of neointima formation in balloon-injured carotid arteries. Knockdown of RGC-32 markedly inhibited the lesion formation, while RGC-32 overexpression exacerbated the injury-induced vascular remodeling. RGC-32 appeared to stimulate both the migration and proliferation of VSMCs. RGC-32 promoted VSMC migration via enhancing the focal adhesion contact and stress fiber formation, which is due to RGC-32-meidated downregulation of Rad GTPase. Our data indicate that targeting RGC-32 may be a promising therapeutic strategy for the prevention of lesion formation in proliferative vascular diseases.

Methods

Animals

Male Sprague-Dawley rats weighing 450–500 g were purchased from Harlan. All rats were housed under conventional conditions in the animal care facilities. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals. Animal surgical procedures were approved by the Institutional Animal Care and Use Committee of The University of Georgia.

Cell Culture

Rat primary aortic SMCs (RASMCs) were cultured by explant method from rat thoracic aorta as described previously.9, 17 Rat thoracic aortas were removed and washed with DMEM. Aortic media were carefully dissected from the vessels, cut into pieces (<1 mm3), and explanted onto a 0.02% gelatin-coated flask. To obtain a stable attachment of the tissue pieces, the flask was incubated upside down for 1 hour. Then DMEM supplemented with 20% FBS, penicillin and streptomycin was slowly added, and cells were allowed to grow at 37°C in a humidified atmosphere of 5% CO2 for 2 weeks. RASMCs were confirmed by expression of smooth muscle α-actin and SM22α.

Construction of Adenovirus

RGC-32 cDNA was subcloned into the Xho I site of pShuttele-IRES-hrGFP-1 (Agilent) and was confirmed by sequencing. RGC-32 shRNA (ShRGC32) coding sequences were: 5’-CGC GTC GAG CTC GAA GAC TTC ATC GCT GAT CTG GAT TCA AGA GAT CCA GAT CAG CGA TGA AGT CTT CGA GCT CTT TTT TCC AAA-3’ (top strand) and 5’-AGC TTT TGG AAA AAA GAG CTC GAA GAC TTC ATC GCT GAT CTG GAT CTC TTG AAT CCA GAT CAG CGA TGA AGT CTT CGA GCT CGA-3’ (bottom strand). Both strands were annealed and ligated into pRNAT-H1.1/Adeno (Genscript corporation) digested with Mlu I and Hind III. Recombinant adenoviral vector was produced by homologous recombination in AD-1 competent cells following manufacturer’s instruction (Agilent).18–20 The resultant recombinant vector pAd-RGC32 or pAd-shRGC32 digested with Pac I was transfected into AD-293 cells with Lipofectamine LTX and Plus (Invitrogen) in order to package viral particles expressing RGC-32 (Ad-RGC32) or shRGC32 (Ad-shRGC32). The adenovirus was purified with gradient density ultracentrifugation of cesium chloride and dialyzed in dialysis buffer (135 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl, pH 7.5, 10% glycerol). Green fluorescent protein (GFP)-expressing adenovirus (Ad-GFP) was used as a control.

Rat Carotid Artery Injury Model and Adenoviral Gene Transfer

Rat carotid artery balloon injury was performed as described previously.21, 22 Rats were anesthetized by an intraperitoneal injection of Ketamine (80 mg/kg) and Xylazine (5 mg/kg). A 2F Fogarty arterial embolectomy balloon catheter (Baxter Edwards Healthcare) was introduced through the left external carotid artery and advanced 4 cm toward the thoracic aorta. The balloon was inflated with 0.02 mL of 0.9% sodium chloride (saline) and then withdrawn through the common carotid artery to the carotid bifurcation with constant rotation during denudation of the endothelium. This procedure was repeated for two additional times to ensure complete endothelial denudation. Heparin (200 units/kg) was then intraperitoneally injected to prevent thrombus formation. The protocol used for introducing adenovirus into rat balloon-injured carotid artery has been previously described.23 The isolated distal segment of injured artery from the proximal edge of omohyoid muscle to the carotid bifurcation was washed with saline, and incubated with 100 µL saline or adenovirus (5×109 pfu) expressing GFP, RGC-32, or ShRGC32, respectively, for 20 minutes. Fourteen days later, the balloon-injured and adenovirus-dwelled segment was perfused with saline and removed. The vessel segments were then fixed with 4% paraformaldehyde and embedded in paraffin. Subsequent morphometric analyses were performed in a double-blinded manner.

Histomorphometric Analysis and Immunohistochemistry (IHC) Staining

Vessel segments were cut by serial sectioning (5µm). Ten sections that were evenly distributed in the vessel segment were collected for analysis.24 The sections were stained with modified hematoxylin and eosin or Elastica van Gieson staining. Cross-sectional images were captured with a Nikon microscope (Nikon America Inc). The areas of the lumen, internal elastic lamina, and external elastic lamina were measured using Image-pro Plus Software. For immunohistochemistry, sections were rehydrated, blocked with 5% goat serum and permeabilized with 0.01% Triton X-100 in PBS, and incubated with rabbit anti-RGC-32 25, 26 or proliferating cell nuclear antigen (PCNA) antibody (Santa CruZ) overnight at 4°C followed by incubation with HRP-conjugated secondary antibody. The sections were counterstained with hematoxylin.

Immunofluorescent Staining

Exponentially growing cells were seeded on glass coverslips in 6-well cell culture plates and incubated overnight at normal cell growth conditions. The immunofluorescent staining was performed as previously described.15

Western Blot Analysis

RASMCs or rat carotid arteries were homogenized in homogenization buffer (50 mmol/L Tris.HCl, pH 7.5/150 mmol/L NaCl/1% SDS/protease inhibitor cocktail (Sigma-Aldrich)). After removal of cell or tissue debris by centrifugation, 20 µg of proteins were separated on 10% SDS-PAGE and were transferred to PVDF membrane (Bio-Rad). Antibodies against RGC-32, Rad GTPase, α-Tubulin were used for immunoblotting. Western blot was performed as described previously. 27, 28

Wound Healing Assay

RASMC migration was evaluated by wound healing assay using CytoSelect Wound Healing Assay Kit (Cell Biolabs).9, 29 Wound healing inserts were put into 24-well cell culture plates coated with fibronectin. Cell suspension (250 µL) was added to either side of the insert and incubated overnight to form a monolayer. The inserts were then removed to allow the cells to migrate. Images of wound healing were captured using a dissection microscope at a magnification of 40 ×. Cell migration was quantified by blind measuring the migration distances.

Cell Proliferation Assay

RASMC cell proliferation was evaluated with MTT assay using TACS MTT Cell Proliferation Assay Kit (Trivegen).30 The optical density at 570 nm was measured.

Immunoprecipitation and ROK Kinase Assay

RASMCs was transfected with myc-tagged ROCK II (ROKα) expression plasmid31 using Lipofectamine LTX plus (Invitrogen), and infected with adenovirus expressing GFP, dominant negative Rad GTPase (Ad-Rad DN)11, and/or RGC-32 shRNA. 48 hours after the transduction, the cells were scraped off and lysed in an immunoprecipitation buffer (Pierce). The cell lysates were subjected to immunoprecipitation with anti-myc monoclonal antibody (clone 4A6, Upstate). Immunoprecipitates were washed five times with kinase-assay buffer and resuspended in 50 µl of kinase-assay buffer. ROKα Kinase activity was measured by using a ROCK Assay Kit following the manufacturer’s recommended protocols (Cell Biolabs, Inc). Phosphorylated MYPT-1 (ROKα substrate) was detected by western blot using p-MYPT-1 antibody provided in the kit.

Statistical analysis

All data were evaluated with a 2-tailed, unpaired Student t test or compared by 1-way ANOVA followed by Fisher t test and are expressed as mean±SD. A value of P<0.05 was considered statistically significant.

Results

RGC-32 was activated in carotid artery following balloon withdrawal injury

As an initial step to define the role of RGC-32 in vasculature, we established a highly characterized rat balloon withdrawal injury model. Normal and balloon-injured rat carotid arteries at 1, 3, 7, 14 days after surgery were harvested and paraffin-embedded. Vessel sections were stained with hematoxylin & eosin (Figure 1A). Neointima was first observed at 3 days, and progressively increased at 7 and 14 days after the injury, as reported previously.21, 22

Figure 1. RGC-32 expression is activated in balloon-injured carotid artery.

Figure 1

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A, Paraffin-embedded sections prepared from control (uninjured) and balloon-injured carotid arteries at 1, 3, 7 and 14 days after surgery were stained with hematoxylin and eosin. B, RGC-32 was induced in balloon-injured rat carotid artery. The artery sections were incubated with rabbit anti-RGC-32 polyclonal antibody followed by HRP-conjugated goat anti-rabbit secondary antibody and DAB staining. C, Protein was extracted from control and balloon-injured rat carotid arteries at different time points as indicated. Western blots were performed using RGC-32 antibody. Data shown are a representative result of three independent experiments. D, RGC-32 expression level was quantified by normalization to α-tubulin. *P<0.01 compared to control group; #P<0.05 compared to 1, 3 or 14 day group after injury, n=3.

To determine the role of RGC-32 in neointima formation, RGC-32 expression in normal and injured vessels was examined by IHC staining (Figure 1B). RGC-32 expression was negligible in the uninjured arteries and the injured vessels at 1 day after surgery. In contrast, strong expression of RGC-32 was observed in the developing neointima and media at 3, 7, 14 days after the injury. To quantify RGC-32 expression following vascular injury, we performed western blot analysis using proteins extracted from balloon-injured rat carotid arteries at different time points. RGC-32 protein expression was upregulated at 3 days, and reached its peak at 7 days, and remained at the elevated level at 14 days after the injury (Figure 1C). Quantitative analysis showed that RGC-32 protein expression was up-regulated by 2.4 fold at one day, 4.9 fold at 3 days, 16 fold at 7 days, and 8.1 fold at 14 days after the injury compared to uninjured vessels, respectively (Figure 1D). These data suggest that RGC-32 is involved in vascular lesion formation.

RGC-32 promoted neointima formation in vivo

To investigate the role of RGC-32 in neointimal formation in vivo, recombinant adenoviruses Ad-GFP, Ad-RGC32, and Ad-shRGC32 were generated. We knocked down or overexpressed RGC-32 in the injured vessels via adenovirus-mediated gene transfer. The distal segment of injured left carotid arteries were incubated with saline, Ad-GFP, Ad-RGC32, or Ad-shRGC32 for 20 min immediately after the balloon injury, and was harvested, fixed, paraffin-embedded, and sectioned 14 days later. Immunohistochemistry staining showed no significant change in RGC-32 expression between saline-treated and Ad-GFP-transduced vessels. However, RGC-32 expression was markedly increased in Ad-RGC32-transduced balloon-injured vessels but was greatly inhibited in Ad-shRGC32-transduced vessels, as compared to the saline or Ad-GFP-incubated vessels (Figure 2A). These results indicate that exogenously-introduced Ad-RGC32 was effectively expressed in the distally injured carotid artery, and Ad-shRGC32 successfully blocked RGC-32 expression in the neointima in vivo (Figure 2A).

Figure 2. Effect of RGC-32 on neointima formation in rat balloon-injured carotid artery.

Figure 2

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A, RGC-32 expression in rat balloon-injured carotid arteries. Immediately after balloon injury, arteries were treated with 0.9% saline solution, Ad-GFP, Ad-RGC32 or Ad-shRGC32 as indicated. 14 days later, carotid arteries were isolated, perfused with saline, fixed with 4% paraformaldehyde, paraffin-embedded, and sectioned. The cross sections were stained with RGC-32 antibody. RGC-32 expression was visualized by DAB staining. B, Representative photomicrographs of elastic fiber-stained carotid arteries 14 days after balloon injury. The artery cross sections were stained with elastic fiber van Gieson’s solution. Arrows indicate internal elastic lamina. C and D, Quantitative analysis of intima area and intima/media ratio, respectively. Results were expressed as mean±SD; n=6. *P<0.05, #P<0.01, compared to Saline- or Ad-GFP-treated arteries.

Importantly, balloon-injured carotid arteries transduced with Ad-RGC32 exhibited a marked increase in neointima formation compared with arteries transduced with Ad-GFP (Figure 2B). Morphometric analysis of elastic-stained sections showed that the expression of Ad-RGC32 increased neointima area by 33% compared to Ad-GFP-transduced vessels (0.180±0.005 versus 0.135±0.003 mm2; p<0.05, n=6, Figure 2C). Conversely, arteries transduced with Ad-shRGC32 exhibited a significantly reduced neointima compared with those treated with Ad-GFP or saline (Figure 2B). Quantitative analysis showed that RGC-32 knockdown inhibited neointimal formation by 62% compared with Ad-GFP-transduced vessels (0.051±0.002 versus 0.135±0.003 mm2; p<0.01, n=6, Figure 2C). Similar results was obtained when Intima/Media area ratios were compared (Figure 2D). No significant difference in neointima formation was observed between Ad-GFP- and saline-treated groups (Figure 2C), consistent with the RGC-32 expression (Figure 2A).

RGC-32 promoted VSMC proliferation

VSMC proliferation plays an important role in neointima formation after vascular injury. To delineate the mechanism by which RGC-32 promoted neointimal formation, we first evaluated RGC-32 function in VSMC proliferation in vitro using the adenoviral vectors that were used in the in vivo studies. Quiescent RASMCs did not express RGC-32. Ad-RGC32 transduction, however, caused robust expression of RGC-32 in the RASMCs (Figure 3A, left panel). RGC-32 expression is induced in RASMCs by serum. Serum-induced RGC-32 was effectively blocked by Ad-shRGC32 transduction, but not by Ad-GFP transduction (Figure 3A, right panel). To determine the effect of RGC-32 on RASMC proliferation, MTT proliferation assay was performed. As shown in Figure 3B, RGC-32 overexpression by Ad-RGC32 transduction significantly promoted the proliferation of RASMCs as compared with control or Ad-GFP-transduced RASMCs (p<0.01, n=6). On the other hand, shRNA knockdown of serum-induced RGC-32 by Ad-ShRGC32 significantly inhibited the proliferation of RASMCs as compared with that of Ad-GFP or control (p<0.01, n=6, Figure 3C). These data demonstrate that RGC-32 plays an important role in VSMC proliferation. RGC-32 appeared to stimulate VSMC proliferation by regulating p34CDC2 (Cdk1) activity because RGC-32 overexpression by Ad-RGC32 significantly increased Cdk1 activation (phosphorylation at Thr161) (Figure 3D), consistent with previous study showing that RGC-32 regulates cell cycle via modulating Cdk1 activity. 12 Since RGC-32 also regulates Akt signaling in endothelial cells, whether or not RGC-32-mediated Cdk1 activity directly regulates VSMC cell cycle requires further investigation.16

Figure 3. Effect of RGC-32 on aortic VSMC proliferation.

Figure 3

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A, RGC-32 overexpression and knockdown efficiency. Ad-RGC32 transduction efficiently expressed RGC-32 in RAMCs (Left panel), while FBS-induced RGC-32 was effectively blocked by Ad-shRGC32 (Right panel). B, RGC-32 overexpression promoted RASMC proliferation. Data were expressed as mean±SD. *P<0.01 compared to control or Ad-GFP group, n=6. MTT assay was performed 48h after Ad-GFP or Ad-RGC32 infection. C, RGC-32 knockdown inhibited RASMC proliferation. MTT assay was performed 48h and 72h after Ad-GFP or Ad-shRGC32 infection. Data were expressed as mean±SD. *P<0.01 compared to control or Ad-GFP, n=6. D, RGC-32 promoted p34CDC2 (Thr161) phosphorylation (p-Cdk1). Data shown are representatives of three independent experiments. E, RGC-32 promoted PCNA expression in VSMCs of rat balloon-injured carotid arteries. Ad-GFP, Ad-RGC32, or Ad-shRGC32 was transduced into balloon-injured arteries. The artery sections were stained with PCNA antibody, and PCNA expression was visualized by DAB staining. F, Calculation of percentage of PCNA expressing cells by Image-Pro Plus 6.0 software (Media Cybernetics). *, #P<0.01 compared to Saline- or Ad-GFP-treated arteries.

To determine if RGC-32 promotes VSMC proliferation in neointima formation in vivo, we examined the expression of a cell proliferation marker proliferating cell nuclear antigen (PCNA) in the artery sections treated with or without Ad-RGC32 or Ad-shRGC32. We found that vascular injury caused PCNA expression in VSMCs (Figure 3E). RGC-32 overexpression increased the number of VSMCs expressing PCNA. RGC-32 Knockdown, however, markedly decreased the number of PCNA-positive VSMCs (Figure 3E–3F). These data suggest that RGC-32 may play a role in VSMC proliferation in vivo, which contributes to the neointima formation following vascular injury.

RGC-32 promoted VSMC migration

VSMC migration from the media into the intimal surface of blood vessels is an important step during neointima formation after vascular injury. To further explore the mechanisms by which RGC-32 promotes intimal hyperplasia, we used wound healing assay to evaluate the effect of RGC-32 on VSMC migration. RASMCs transduced with Ad-RGC32 were cultured in FBS-free DMEM, while those transduced with Ad-shRGC32 were cultured in medium containing 10% FBS. Immunostaining showed that RGC-32 was highly expressed in Ad-RGC32-transduced RASMCs whereas it was effectively suppressed in Ad-shRGC32-transdced serum-treated RASMCs (Figure 4A, FBS groups). Wound healing assay showed that RGC-32 overexpression promoted migration of quiescent RASMCs as compared with Ad-GFP-treated cells (273±31 versus 92±28 µm; p<0.01, n=6; Figure 4A and 4B, left panel). RGC-32 knockdown, however, inhibited serum-induced RASMC migration as compared with Ad-GFP-transduction (212±19 versus 315±20 µm; p<0.01, n=6, Figure 4A and 4B, right panel). These data demonstrate that RGC-32 stimulates VSMCs migration in vitro.

Figure 4. RGC-32 stimulates VSMC migration.

Figure 4

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A, Representative microphotographs of RGC-32 expression (upper panel) and wound healing assay (lower panel) in RASMCs transduced with Ad-GFP, Ad-RGC32 or Ad-shRGC32. B, Quantitative analysis of the migration distance. *P<0.01 compared to Ad-GFP, n=6. RGC-32 overexpression promoted while RGC-32 knockdown inhibited RASMC migration. C, RGC-32 was induced in RASMCs by PDGF-BB (10 ng/ml) in a time-dependent manner. D, PDGF-BB-induced RGC-32 was efficiently blocked by Ad-shRGC32. E, RGC-32 knockdown inhibited PDGF-BB-induced VSMC migration. Shown are the representative wound healing assays with VSMCs induced by PDGF-BB. F, Quantitative analysis of migration distances demonstrated that PDGF-BB-induced migration was inhibited by Ad-shRGC32. *P<0.01 compared to Ad-GFP group, n=6.

Platelet derived growth factor (PDGF) is a potent regulator for VSMC migration. 32 In order to determine whether RGC-32-induced VSMC migration is related to PDGF activity or if RGC-32 is a downstream target of PDGF, we treated RASMC with PDGF-BB and detected RGC-32 expression. We found that RGC-32 was induced by PDGF-BB in a time-dependent manner (Figure 4C). Importantly, shRNA knockdown of RGC-32 significantly inhibited PDGF-BB-induced RASMC migration (Figure 4D–4F). These data suggest that PDGF-BB is a specific regulator of RGC-32 expression in VSMC, and that RGC-32 mediates, at least in part, the VSMC migration induced by PDGF.

RGC-32 enhanced focal adhesions and stress fiber formation

To investigate the mechanism by which RGC-32 promotes VSMC migration, we examined whether RGC-32 affects cytoskeletal organization. Ad-GFP-, Ad-RGC32- or Ad-shRGC32-transduced RASMC cells were cultured on fibronectin-coated glass coverslip. The focal adhesion contacts and actin stress fibers were then labeled by anti-vinculin and anti-α-actin antibodies, respectively. As shown in Figure 5, overexpression of RGC-32 in RASMCs enhanced while knockdown of RGC-32 inhibited both focal adhesion contacts (Figure 5A) and stress fiber formation (Figure 5B). These data document that RGC-32 stimulates VSMC migration via enhancing focal adhesion and stress fiber organization.

Figure 5. RGC-32 affects focal adhesion contacts and stress fiber formation.

Figure 5

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A, Effect of RGC-32 overexpression or knockdown on focal adhesion contacts. The focal adhesion contacts were labeled with vinculin antibody followed by Tritc-conjugated secondary antibody. Shown are the representative results of three independent experiments. B, Effect of RGC-32 overexpression or knockdown on the formation of actin stress fibers. The actin stress fibers were labeled with smooth muscle α-actin (α-SMA) followed by Tritc-conjugated secondary antibody. Shown are the representative results of three independent experiments. RGC-32 overexpression stimulates while RGC-32 knockdown blocks both focal adhesion contact and stress fiber formation.

RGC-32 suppressed Rad GTPase expression in VSMCs

Rad (Ras associated with diabetes) is the prototypic member of Ras-related GTPase family.33 It has been reported that Rad inhibits VSMC migration through inhibition of focal adhesion and stress fiber formation, leading to suppression of neointima formation following vascular injury.11 Since RGC-32 promoted focal adhesion and stress fiber organization, we hypothesized that RGC-32 regulates Rad GTPase expression. To test this hypothesis, we examined the effect of RGC-32 on the expression of Rad GTPase in RASMCs. Western blot analysis showed that overexpression of RGC-32 completely blocked whereas RGC-32 knockdown by shRNA significantly increased Rad GTPase expression as compared with the expression in Ad-GFP-transduced RASMCs (Fig 6A and 6B). These data indicate that RGC-32 may promote VSMC migration and neointima formation, at least partially, through inhibition of Rad GTPase expression.

Figure 6. RGC-32 regulates ROKα activity by controlling Rad GTPase expression.

Figure 6

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A, RGC-32 regulated Rad GTPase expression. RASMCs were transduced with Ad-GFP, Ad-shRGC32, or Ad-RGC32, respectively at a MOI of 100. 48 hours later, cells were lysed for western blotting to detect the expression of RGC-32 and Rad GTPase. α-Tubulin was an internal control. Three independent experiments were performed. B, Relative RGC-32 or Rad GTPase was quantified by normalized to α-Tubulin. *P<0.01 compared to Ad-GFP group, n=3. C, RASMCs were transduced with c-myc-tagged ROKα followed by transduction with Ad-GFP, Ad-shRGC32, or both Ad-shRGC32 and Ad-Rad DN (S105N) as indicated. Cell lysates were subjected to immunoprecipitation with c-Myc antibody followed by ROKα kinase assay. Phosphorylation of ROKα-specific substrate MYPT-1 was detected by western blot. D, Relative activity of ROKα was calculated by comparing to ROKα activity in Ad-GFP group (set as 1). Values were expressed as mean±SD (n=3, *P<0.01 compared to Ad-GFP group, #P<0.05 compared to Ad-shRGC32 group).

Rad GTPase inhibits VSMC migration by inhibiting the ROKα singling pathway.11 To determine if down-regulation of Rad GTPase by RGC-32 affects ROKα activity, we performed ROKα kinase assays. We found that RGC-32 knockdown by shRNA significantly inhibited ROKα kinase activity (Figure 6C–6D), suggesting that RGC-32 may enhance ROK signaling to induce VSMC migration. Importantly, RGC-32 shRNA attenuated ROKα activity can be restored by dominant negative Rad GTPase (Figure 6C–6D), suggesting that Rad GTPase mediated RGC-32-induced ROKα activity.

Discussion

RGC-32 has pleiotropic effects in diverse cellular processes such as cell proliferation12, differentiation14, and immunity.34 RGC-32 overexpression causes quiescent human aortic smooth muscle cells to enter S-phase and G2/M in vitro.12 However, the role of RGC-32 in proliferative vascular diseases has not been reported. Our studies demonstrate for the first time that RGC-32 plays a critical role in vascular lesion formation after experimental angioplasty. RGC-32 is progressively expressed in the neointima following vascular injury. Importantly, RGC-32 silencing dramatically inhibits the neointima formation, whereas RGC-32 overexpression markedly increases the size of the vascular lesion compared to the controls.

Accumulation of VSMCs in the vascular lesion is attributed to their proliferation and migration. RGC-32 appears to stimulate both VSMC proliferation and migration. Although RGC-32 function in endothelial cell proliferation could be stimulatory or inhibitory depending on physiological conditions15,16, our data strongly support that RGC-32 is a positive regulator for VSMC proliferation. RGC-32 stimulates VSMC proliferation by enhancing the activation of cycle cell regulator p34CDC2, consistent with previous report. 12, 13 RGC-32 not only induces VSMC proliferation in vitro, but also appears to play a role in VSMC proliferation in vivo. Vascular injury causes marked expression of PCNA, which is blocked by RGC-32 shRNA, suggesting that RGC-32 is involved in stimulating PCNA expression, leading to VSMC proliferation in the neointima formation.

In addition to its role in VSMC proliferation, our data demonstrate for the first time that RGC-32 also stimulates VSMC migration. Focal adhesion formation and cytoskeletal organization are key processes in cell locomotion and migration. RGC-32 appears to induce VSMC migration by enhancing focal adhesion and stress fiber organization. Mechanistically, RGC-32 regulates VSMC migration by altering the expression of Rad GTPase. Rad GTPase has been shown to inhibit VSMC migration, resulting in an attenuation of vascular lesion formation.11 RGC-32 overexpression inhibits while RGC-32 knockdown markedly increases Rad GTPase expression in RASMCs. Downregulation of Rad GTPase by RGC-32 causes an enhanced ROKα kinase activity, which ultimately induces VSMC focal adhesion and stress fiber formation, resulting in VSMC migration.

Taken together, the present study has identified RGC-32 as an essential mediator for the progression of vascular lesion formation in the experimental angioplasty. RGC-32 expression associated with vascular injury provides a novel mechanism underlying the VSMC proliferation and migration in proliferative vascular diseases. Therefore, targeting RGC-32 may be a potential therapeutic strategy for the prevention of vascular remodeling in proliferative vascular diseases.

Supplementary Material

1

Acknowledgment

The authors wish to thank Dr Shuh Narumiya for generously providing the ROKα (ROCK-II) plasmid.

Source of funding: This work was supported by grants from National Institutes of Health (HL093429 and HL107526 to Dr. Chen).

Footnotes

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