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(Circulation Research. 2004;94:e87.)
© 2004 American Heart Association, Inc.

UltraRapid Communications

Proteomic and Metabolomic Analysis of Vascular Smooth Muscle Cells

Role of PKC

Manuel Mayr, Richard Siow, Yuen-Li Chung, Ursula Mayr, John R. Griffiths, Qingbo Xu

From the Department of Cardiac and Vascular Sciences (M.M., U.M., Q.X.) and Department of Basic Medical Sciences (Y.-L.C., J.R.G.), St George’s Hospital Medical School, London, UK; and Centre for Cardiovascular Biology and Medicine (R.S.), King’s College, London, UK.

Correspondence to Prof Qingbo Xu, Department of Cardiac and Vascular Sciences, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. E-mail q.xu{at}sghms.ac.uk

	Abstract

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Recent developments of proteomic and metabolomic techniques provide powerful tools for studying molecular mechanisms of cell function. Previously, we demonstrated that neointima formation was markedly increased in vein grafts of PKC-deficient mice compared with wild-type controls. To clarify the underlying mechanism, we performed a proteomic and metabolomic analysis of cultured vascular smooth muscle cells (SMCs) derived from PKC^+/+ and PKC^–/– mice. Using 2-dimensional electrophoresis and mass spectrometry, we identified >30 protein species that were altered in PKC^–/– SMCs, including enzymes related to glucose and lipid metabolism, glutathione recycling, chaperones, and cytoskeletal proteins. Interestingly, nuclear magnetic resonance spectroscopy confirmed marked changes in glucose metabolism in PKC^–/– SMCs, which were associated with a significant increase in cellular glutathione levels resulting in resistance to cell death induced by oxidative stress. Furthermore, PKC^–/– SMCs overexpressed RhoGDI, an endogenous inhibitor of Rho signaling pathways. Inhibition of Rho signaling was associated with a loss of stress fiber formation and decreased expression of SMC differentiation markers. Thus, we performed the first combined proteomic and metabolomic study in vascular SMCs and demonstrate that PKC is crucial in regulating glucose and lipid metabolism, controlling the cellular redox state, and maintaining SMC differentiation.

Key Words: proteomics • metabolomics • smooth muscle cells • PKC • signal transduction

	Introduction

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Proteomic and metabolomic techniques are ideal for clarifying quantitative protein and metabolite changes in physiological and diseased conditions, respectively.^1–5 In vascular research, however, proteomics and metabolomics are still in their infancies^6–8 and no studies have been performed so far comparing proteomic and metabolomic profiles in vascular smooth muscle cells (SMCs).

PKC represents a novel PKC isoform as characterized on the basis of its structure and maximal activation by diacylglycerol in the absence of calcium.^9,10 We recently developed knockout mice lacking PKC and studied its effect on neointima formation in vein grafts.¹¹ We demonstrated that loss of PKC markedly accelerated neointima formation, resulting in complete occlusion of the vessel lumen in one-third of the vein grafts. As with p53-deficient mice,¹² neointimal lesions in PKC^–/– vein grafts contained twice as many SMCs as wild-type controls and showed significantly lower numbers of apoptotic SMCs.¹¹ In vitro experiments revealed that SMCs derived from PKC^–/– mice were less sensitive to various apoptotic stimuli, including cytokine treatment. Their apoptotic resistance appeared to involve a loss of free radical generation as evidenced by redox-sensitive fluorescent dyes.¹¹ Besides modulating apoptosis, PKC was found to be important for cytoskeleton rearrangement and cell migration.¹³ However, the molecular mechanisms of resistance to apoptosis and cytoskeletal abnormalities in PKC^–/– SMCs are unknown. In the present study, we performed a thorough analysis of the proteome and metabolome of vascular SMCs derived from PKC^+/+ and PKC^–/– mice. We demonstrate that PKC is crucial for SMC homeostasis by regulating the balance between glucose and lipid metabolism and maintaining SMC differentiation.

	Materials and Methods

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All procedures were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals. PKC-deficient mice were generated by targeted disruption of an endogenous PKC gene.¹¹ Vascular SMCs from PKC^–/– and PKC^+/+ mice were cultivated from aortas of 5 different animals, SMCs from PKC^+/– mice were cultivated from aortas of 3 different animals, as described elsewhere.¹⁴

Proteomic Analysis
Protein extracts of PKC^–/– and PKC^+/+ SMCs were separated by 2-dimensional gel electrophoresis (2-DE) as described by McGregor et al.¹⁵ Spot patterns were analyzed using Proteomweaver 2.0 (Definiens) and PDQuest Software 7.1 (Biorad). Spots showing a statistically significant difference in intensity were excised for identification by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or tandem mass spectrometry (MS/MS). A detailed methodology is provided in the online data supplement available at http://circres.ahajournals.org.

Proton Magnetic Resonance Spectroscopy
SMC monolayers were washed twice with chilled saline and SMC metabolites were extracted in 6% perchloric acid.¹⁶ Neutralized extracts were freeze-dried and reconstituted in D₂O; 0.5 mL of the extracts were placed in 5 mm proton nuclear magnetic resonance (NMR) tubes. ¹H NMR spectra were obtained using a Bruker 500 MHz spectrometer. The water resonance was suppressed by using gated irradiation centered on the water frequency. Sodium 3-trimethylsilyl-2,2,3,3-tetradeuteropropionate (TSP) was added to the samples for chemical shift calibration and quantification. Immediately before the NMR analysis, the pH was readjusted to 7 with perchloric acid or KOH.

Standard Biochemical Methods
The methodology for reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting, Rho-activation assays, cell viability, and spreading assays is provided in the online data supplement (http://www.circresaha.org).

Statistical Analysis
Statistical analysis was performed using the analysis of variance and Student t test, respectively. The association of SMC metabolites with PKC genotypes was assessed using generalized linear models. Results were given as means±SE. A P<0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Proteomic Analysis
To analyze changes in the proteome, we created a protein profile of SMCs by 2-DE. Average gels for PKC^+/+ and PKC^–/– SMCs were obtained from cultures obtained from 4 different animals per group (mean passage 25±3 and 26±4 for PKC^+/+ and PKC^–/– SMCs, respectively). A direct overlay is presented in Figure 1. Using a broad range pH gradient (pH 3 to 10 NL), 2-DE gels compromised 1200 protein features. Differentially expressed spots are highlighted in color (blue and orange indicate an increase in PKC^+/+ and PKC^–/– SMCs, respectively). Enlarged silver-stained gels highlight quantitative differences in images (Figure 2). Numbered spots were excised and subject to in-gel tryptic digestion. Protein identifications as obtained by MALDI-MS are listed in Table 1. For proteins marked with a dagger in Table 1, further proof of identification was obtained by tandem mass spectrometry (Table 2). A representative MALDI-MS spectrum is shown in Figure 3.

View larger version (135K): [in this window] [in a new window]   Figure 1. 2-DE map of SMC proteins. Protein extracts were separated on a pH 3 to 10 NL IPG strip, followed by a 12% SDS polyacrylamide gel. Spots were detected by silver staining. Figures represent a direct overlay of average gels from PKC+/+ and PKC–/– SMCs. Each average gel was created from 4 single gels (total n=8). Differentially expressed spots are highlighted in color (blue and orange for PKC+/+ and PKC–/– SMCs, respectively). Proteins identified by MALDI-MS are marked with numbers and listed in Table 1.

View larger version (83K): [in this window] [in a new window]   Figure 2. Enlargements of silver stained gels. Representative areas of 2-dimensional gels from wild-type and PKC–/– SMCs highlight quantitative differences in images. Numbers correspond to proteins listed in Table 1.

View this table: [in this window] [in a new window]   Table 1. Differences in Protein Profiles Between Vascular SMCs of PKC+/+ and PKC–/– Mice

View this table: [in this window] [in a new window]   Table 2. Protein Identification by Tandem MS

View larger version (18K): [in this window] [in a new window]   Figure 3. Mass spectrometry spectrum. Peptide mass profiling of a silver-stained spot from a 2-DE separation of murine SMC proteins. The protein of spot 3 (Figure 1) was digested in situ within the gel with trypsin. The resulting tryptic peptides were analyzed using MALDI-MS in reflectron mode. The protein was identified as glucose 6-phosphate dehydrogenase (Table 1).

Strikingly, many changes observed in PKC^–/– SMCs were related to energy metabolism, including enzymes involved in glucose and lipid metabolism: triose phosphate isomerase and phosphoglycerate kinase represent glycolytic enzymes, whereas glucose 6-phosphate dehydrogenase and aldose reductase are the rate-limiting enzymes in the pentose phosphate and sorbitol pathways, respectively. Concomitantly, 3 isoforms of aldehyde dehydrogenase 3A1 and a highly acidic isoform of acyl-CoA dehydrogenases were found only in PKC^–/–, but not in PKC^+/+ SMCs (Table 1). Additionally, the soluble form of the isocitrate dehydrogenase, which has recently been implicated in glutathione (GSH) recycling,¹⁷ appeared to be upregulated in PKC^–/– SMCs.

Besides enzymatic alterations, we observed profound changes in cytoskeletal proteins in PKC^–/– SMCs, including actin and myosin light chain, which were associated with a compensatory increase in intermediate filaments, eg, vimentin and lamin, and alterations in calcium binding proteins, eg, calmodulin and caldesmon 1 (Table 1). Moreover, PKC deficiency resulted in marked changes of cellular chaperones, including heat shock protein 4 (Hsp4), the tubulin binding-subunit of the T-complex polypeptide 1 (CCT-1),¹⁸ and the redox sensitive chaperone protein disulfide isomerase.^19,20 Further alterations were observed for proteins involved in cell division, eg, septin and immunoregulation, eg, Fkbp9 and annexin 1. Taken together, our proteomic data suggest that PKC deficiency is associated with altered energy generation and cytoskeletal dysregulation in vascular SMCs.

Metabolomic Analysis
To prove the functional relevance of the described enzymatic changes, we applied high-resolution NMR spectroscopy to analyze cellular metabolites (Figure 4). In PKC^+/– and PKC^–/– SMCs levels of alanine, a surrogate marker for the activity of the glycolytic pathway in metabolomic analysis were significantly decreased (Table 3, Figure 5A), whereas lactate tended to accumulate, indicating impaired glucose metabolism. Notably, carnitine, required for the mitochondrial import of long chain fatty acids, was markedly elevated in PKC^–/– SMCs and associated with higher levels of phosphocholine, an essential phospholipid for the synthesis of cell membranes. The metabolic changes in PKC^–/– SMCs resulted in an accumulation of amino acids, such as glutamate, valine, isoleucine, and a diminished creatine pool, a major energy reserve in muscle tissue. ATP levels were similar to PKC^+/+ SMCs when cells were grown in high glucose medium (25 mmol/L) but significantly decreased under normal glucose concentrations (5 mmol/L) (Figure 5B; 90 versus 65 µmol ATP/g protein, P<0.05). Thus, higher levels of glucose are required to maintain cellular energy production in the absence of PKC.

View larger version (21K): [in this window] [in a new window]   Figure 4. NMR spectra of PKC+/+ (A) and PKC–/– SMCs (B). Resonances have been assigned to alanine (Ala), creatine 2 (Cr), phosphocreatine (PCr), carnitine (Car), phosphocholine (PC), glutamate (Glu), lactate (Lac), acetate (Acet), succinate (Suc), glycine (Gly), myoinositol (Myo), valine (Val), and isoleucine (Iso-Leu). Sodium 3-trimethylsilyl-2,2,3,3-tetradeuteropropionate (TSP) was added to the samples for calibration.

View this table: [in this window] [in a new window]   Table 3. Metabolic Effects of PKC Deficiency in Vascular SMCs

View larger version (31K): [in this window] [in a new window]   Figure 5. Comparison of SMC metabolites. Relative changes of metabolites in PKC+/– (gray bars) and PKC–/– SMCs (white bars) compared with PKC+/+ SMCs (black line) (A). Abbreviations for metabolites are explained in the legend to Figure 2. +Near significant difference from PKC+/+ SMCs P<0.1. *Significant difference from PKC+/+ SMCs, P<0.05. **P<0.01. Differences in ATP (B) and GSH levels (C) between PKC+/+ SMCs (black bars) and PKC–/– SMCs (white bars) under high and low glucose conditions. *Significant difference from high glucose conditions, P<0.05. **P<0.01

Elevated Glutathione Levels Protect PKC^–/– SMCs
One of the most prominent enzymatic changes in PKC^–/– SMCs were observed for the soluble form of isocitrate dehydrogenase and glucose 6-phosphate dehydrogenase, 2 enzymes related to GSH metabolism. This prompted us to measure GSH concentrations (Figure 5C): PKC deficiency was associated with a significant increase in GSH levels (25 versus 70 µmol/g protein, P<0.001). The difference to PKC^+/+ SMCs was less pronounced under high glucose conditions (15 versus 22 µmol/g protein, P<0.01), which represents a considerable oxidative stress leading to GSH consumption.^21,22

GSH is a tripeptide with a free sulfhydryl group and is of paramount importance in maintaining the reducing intracellular environment.^21,23 Consequently, increased GSH protected PKC^–/– SMCs against oxidative stress-induced cell death: treatment with 100 µmol/L diethylmaleate (DEM), a sulfhydryl-reactive agent, resulted in rapid depletion of GSH (Figure 6A), followed by a drop in ATP levels (Figure 6B) and cell death in PKC^+/+ SMCs (Figure 6C). In contrast, PKC^–/- SMCs were less sensitive to DEM-induced cell death (Figure 6A to C), tolerating up to 20-times higher concentrations of DEM than PKC^+/+ SMCs (data not shown). Corresponding to GSH depletion, the antioxidant protein heme oxygenase 1 (HO-1) was rapidly induced in PKC^+/+, but not in PKC^–/– SMCs (Figure 6D). Differences in HO-1 expression were restricted to oxidative stress, because HO-1 expression after exposure to heavy metals, eg, cadmium chloride (CdCl₂), was similar in PKC^+/+ and PKC^–/– SMCs (Figure 6E). Taken together, our data clearly demonstrate that loss of PKC alters the cellular redox state by elevating GSH levels, providing protection against oxidative stress-induced cell death.

View larger version (22K): [in this window] [in a new window]   Figure 6. Increased resistance to oxidative stress-induced cell death in PKC–/– SMCs. PKC+/+ SMCs (black bars) and PKC–/– SMCs (white bars) were treated with 100 µmol/L diethylmaleate (DEM). GSH (A) and ATP (B) concentrations were measured at the indicated time-points and SMC survival was quantified after 24 hours using a proliferation/cell death kit (Promega). Black, gray, and white bars represent absorbance values for PKC+/+ SMCs, PKC+/–, and PKC–/– SMCs, respectively (C). *Significant difference from controls P<0.05, **P<0.01. DEM-induced expression of heme oxygenase-1 (HO-1) in PKC+/+ and PKC–/– SMCs (n=5) (D). Note that the decrease in actin in PKC+/+ SMCs is a consequence of increased cell death. Comparison of HO-1 induction in PKC+/+ and PKC–/– SMCs after treatment with DEM (50 µmol/L) and cadmium chloride (CdCl2, 10 µmol/L) (n=3) (E).

Impaired Rho Signaling in PKC^–/– SMCs
In addition to using a wide-range pH gradient (pH 3 to 10 NL), we separated proteins on a pH 4 to 7 gradient (data not shown). Because the same amount of protein was used for all analytical gels, only the spatial resolution was superior compared with the pH3–10 NL gradient. Using this gradient, we observed differential expression for Rho guanine dissociation inhibitor alpha (RhoGDI) (Figure 7A), an endogenous inhibitor of RhoGTPases including Rho, Rac, and Cdc42,²⁴ which orchestrate the regulation of actin polymerization.²⁵ We explored its functional relevance by Western blotting and immunoprecipitation of activated Rho (RhoGTP): increased expression of RhoGDI in PKC^–/– SMCs (Figure 7B) attenuated Rho activation in response to mechanical stress (Figure 7C, D).

View larger version (19K): [in this window] [in a new window]   Figure 7. Impaired Rho signaling in PKC–/– SMCs. Protein extracts of PKC+/+ and PKC–/– SMCs were separated on a pH 4 to 7 IPG strip, followed by a 12% SDS polyacrylamide gel. The spots corresponding to RhoGDI are marked with an arrow (A). Results of Western blot analysis are shown for expression differences of RhoGDI (n=3) (B) and mechanical stress-induced Rho activation in quiescent SMCs as determined by RhoGTP pull-down assays (C). Rho activation after mechanical stress (10 minutes, 15% elongation, 1 Hz) was quantified by densitometry (n=4) (D). Black and white bars represent absorbance values for PKC+/+ SMCs and PKC–/– SMCs, respectively. *Significant difference from unstressed controls and PKC–/– SMCs, P<0.05.

Loss of PKC Causes SMC Dedifferentiation
The small GTPases of the Rho/Rac family orchestrate the regulation of p38MAPK pathways and actin polymerization.^25–28 Cytoskeletal dynamics²⁹ and organization play a crucial role in maintaining SMC differentiation.^30,31 Impaired Rho signaling in PKC deficient SMCs was associated with a disassembly of stress fibers (Figure 8A). Additionally, decreased abundance of the differentiation marker SM22 in the proteomic profile suggested a phenotypic modulation (spot 16, Table 1). This was further investigated by use of RT-PCR analysis: loss of PKC was associated with transcriptional downregulation of SM22 (Figure 8B). Similarly, lower expression levels were observed for SM myosin heavy chain (SMMHC) and calponin (Figure 8C), but not -SM actin (Figure 8B). Thus, inhibition of Rho signaling in PKC^–/– SMCs is associated with a loss of cytoskeletal organization resulting in SMC dedifferentiation.

View larger version (24K): [in this window] [in a new window]   Figure 8. Loss of PKC results in SMC dedifferentiation. Actin fiber formation during cell spreading as visualized by rhodamine phalloidin staining (A). Absence of PKC in cultivated SMCs as confirmed by PCR (B, upper panel). RT-PCR data showing decreased expression of SMC differentiation markers in PKC–/– SMCs (n=5 for PKC+/+ and PKC–/– SMCs, n=2 for PKC+/– SMCs) (B, C).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study provides the first proteomic profile of murine vascular SMCs that was markedly influenced by mutational ablation of the PKC gene. Importantly, proteomic findings were translated into a functional context by combining proteomic techniques with NMR spectroscopy. This new research strategy allows us to decipher the effects of specific genes, drugs, or other treatments on global alterations of cellular proteins, metabolism and function.

Most of our knowledge about the role of PKC is derived from studies using rottlerin, a putative PKC inhibitor.^13,32,33 However, its specificity has recently been questioned as it appears to block PKC activity indirectly in vivo by uncoupling mitochondria.³⁴ In the present study, we delineate the effects of PKC on vascular SMCs by using PKC^–/– mice. Our proteomic and metabolomic data suggest that loss of PKC interferes with glucose metabolism, affecting energy reserves and promoting an antioxidant state of cells reflected by decreased levels of intracellular reactive oxygen species¹¹ and increased GSH concentrations. GSH turnover was more efficient in PKC^–/– SMCs after DEM treatment and provided protection against oxidative stress-induced cell death.

Our metabolomic findings are in line with a recent study by Caruso et al³³ demonstrating that PKC is required for stimulation of the pyruvate dehydrogenase complex. Pyruvate dehydrogenase catalyzes the oxidation of pyruvate to acetyl-CoA, which represents the irreversible step from glycolysis to the citric acid cycle. SMC metabolism, when viewed in terms of ATP synthesis, is primarily oxidative, with glucose being the main source of energy for contractile energy requirements, whereas aerobic lactate production appears to be specifically coupled to sodium and potassium transport processes.^35,36 Hence, decreased activity of the pyruvate dehydrogenase complex in the absence of PKC provides a likely explanation for the diminished creatine pool and reduced ATP levels at 5 mmol/L glucose. Impaired glucose metabolism in PKC^–/– SMCs was reflected as a decrease in alanine, accumulation of lactate, decreased oxidation of certain amino acids, and compensatory upregulation of alternative metabolic pathways. First, lipid metabolism was increased as evidenced by proteomic changes in acyl-CoA dehydrogenase and aldehyde dehydrogenase 3A1 and a corresponding elevation of carnitine and phosphocholine, the precursor for phosphatidylcholine. The biosynthesis of phosphatidylcholine is driven by the availability of free fatty acids, which are preferentially converted to phospholipids if they escape mitochondrial oxidation. Aldehyde dehydrogenases catalyze the oxidation of medium and long-chain fatty aldehydes to their corresponding carboxylic acids. Acyl-CoA dehydrogenases are responsible for ß-oxidation of short chain fatty acids. Second, the pentose phosphate pathway can account for complete oxidation of glucose, the main products being NADPH and CO₂. All tissues in which this pathway is active use NADPH in reductive synthesis including synthesis of GSH.³⁷ Glucose 6-phosphate dehydrogenase is the first and rate-limiting enzyme in the pentose phosphate pathway. Two other NADP⁺-linked dehydrogenases contribute to the generation of cytosolic NADPH, malic enzyme, and cytoplasmic isocitrate dehydrogenase.^17,38 Both glucose 6-phosphate dehydrogenase and cytoplasmic isocitrate dehydrogenase were altered in our proteomic analysis of PKC^–/– SMCs. Thus, PKC-associated changes in glucose metabolism appear to contribute to an increase in GSH, which plays an essential role in maintaining cellular redox balance.

Another important observation of this study is the upregulation of RhoGDI, an endogenous inhibitor of Rho signaling pathways, which was associated with cytoskeletal abnormalities and a phenotypic modulation in PKC^–/– SMCs. Rho signaling is a key regulator of SMC differentiation.³⁰ SMC-specific markers are regulated at a transcriptional level. Except for -SM actin, transcription of these genes is downregulated in dedifferentiated SMCs. Loss of PKC coincided with decreased expression of SMC differentiation markers, including SM22, SMMHC, and calponin, suggesting that PKC is required for maintaining SMC differentiation.

Hemodynamic forces are known to be instrumental in the pathogenesis of vein graft stenosis.³⁹ We have demonstrated previously that mechanical stress can induce SMC apoptosis in vivo and in vitro.^26,27,40,41 Two signaling pathways appear to be involved in initiating SMC apoptosis after mechanical stress: Rac/p38 MAPK activation and oxidative DNA damage.^26,27 These findings were subsequently confirmed by others.^42–44 Importantly, enhanced apoptosis after mechanical injury is associated with a decrease in GSH levels,⁴⁵ and the response of SMCs to mechanical strain is modulated by glucose 6-phosphate dehydrogenase activity.²³ Therefore, our mechanistic data provide a better explanation of why PKC^–/– SMCs are resistant to apoptosis and contribute to accelerated neointima formation in PKC^–/– vein grafts.

In summary, the present study provides new insights into PKC isoform specific effects, which could not have been obtained by studying individual signaling pathways. Our integrated approach highlights the intimate connections between glucose metabolism and susceptibility to cell death, and identifies PKC as one of the key kinases in vascular SMCs, ideally positioned to serve as a "sentinel" responding to abnormalities in glucose metabolism, oxidative stress, and cytoskeleton rearrangement. Our findings highlight potential targets for gene or drug therapy, because enhanced PKC induction in the vessel wall could reduce neointima formation by promoting SMC apoptosis and maintaining SMC differentiation after mechanical injury.

	Acknowledgments

 
This work was supported by grants from British Heart Foundation (PG/02/234/13592) and the Oak Foundation. The use of the facilities of the Medical Biomics Centre at St George’s Hospital Medical School and the help of Dr Robin Wait (Imperial College, London, UK) are gratefully acknowledged.

	Footnotes

 
Received December 22, 2003; revision received April 23, 2004; accepted April 27, 2004.

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