Articles in PresS. Am J Physiol Heart Circ Physiol (July 11, 2008). doi:10.1152/ajpheart.00079.2008
C-reactive Protein and Vein Graft Disease: Evidence for a Direct Effect on Smooth Muscle Cell Phenotype via modulation of PDGF receptor-
Karen J. Ho, Christopher D. Owens, Thomas Longo, Xin X. Sui, Cristos Ifantides, Michael S. Conte
Division of Vascular and Endovascular Surgery, Brigham & Women’s Hospital, Boston, MA
Running head: CRP and saphenous vein smooth muscle cell phenotype
Address: Michael S. Conte, Division of Vascular and Endovascular Surgery, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115 (email:
[email protected])
Copyright © 2008 by the American Physiological Society.
Abstract
Plasma C-reactive protein (CRP) concentration is a biomarker of systemic atherosclerosis and may also be associated with vein graft disease. It remains unclear if CRP is also an important modulator of biologic events in the vessel wall. We hypothesized that CRP influences vein graft healing by stimulating smooth muscle cells (SMCs) to undergo a phenotypic switch. Distribution of CRP was examined by immunohistochemistry in pre-bypass human saphenous veins (HSVs) (n=21) and failing vein grafts (n=18, 25-4400 days postop). Quiescent HSVSMCs were stimulated with human CRP (5-50 gm/L). SMC migration was assessed in modified Boyden chambers with platelet derived growth factor BB (PDGF-BB; 5-10 ng/mL) as the chemoattractant. SMC viability and proliferation were assessed by trypan blue exclusion and reduction of Alamar Blue substrate, respectively. Expression of PDGF ligand and receptor (PDGFR) genes was examined at RNA and protein levels after 24-72h of CRP exposure. CRP staining was present in 13 of 18 diseased vein grafts where it localized to the deep media and adventitia, but it was minimally detectable in most pre-bypass veins. SMCs pre-treated with CRP demonstrated a dose-dependent increase in migration to PDGF-BB (p=.02), which was inhibited by a PDGF neutralizing antibody. SMCs treated with CRP showed a dose-dependent increase in PDGFR- expression and phosphorylation after 24-48h. Exogenous CRP had no effect on SMC viability or proliferation. These data suggest that CRP is detectable within the wall of most diseased vein grafts, where it may exert local effects. Clinically relevant levels of CRP can stimulate SMC migration by a mechanism that may involve upregulation and activation of PDGFR- .
Keywords: C-reactive protein/chemistry; Muscle, Smooth, Vascular/cytology; Cells/Cultured; Biological Markers/metabolism
Introduction
Vein graft failure remains a formidable challenge among patients with critical limb ischemia or coronary ischemia requiring revascularization. Even with autogenous vein conduit, the most durable small vessel substitute, 30-50% of lower extremity vein bypass grafts fail within 5 years (9). Vein graft failure is most commonly ascribed to myo-intimal hyperplasia, which involves a smooth muscle cell (SMC) switch from a quiescent/contractile phenotype to a migratory, proliferative and synthetic phenotype (11). Chronic baseline inflammation in humans, as measured by the high sensitivity C-reactive protein (hsCRP) assay, has emerged as a novel biomarker that adds incremental predictive ability above traditional cardiovascular risk factors for primary and secondary cardiovascular events. Inflammation has been implicated not only in de novo atheroscleross-related complications, i.e., myocardial infarction and stroke, but also in restenosis of arteries after balloon injury and in stenosis and failure of vein grafts used for arterial reconstructions (10, 21, 31, 44-46). We have recently shown that plasma hsCRP concentrations are associated with primary vein graft failure in individuals undergoing lower extremity bypass procedures (40) and that inflammation, as assessed by hsCRP, influences the normal remodeling pattern of arterialized veins (41). A central question arising from this work is whether CRP is not only a biomarker but a direct biomodulator of biologic events in vascular cells and tissues. CRP may contribute to vascular wall inflammation through multiple posited mechanisms: it is deposited in the wall at sites of endothelial injury, where it opsonizes lipids and activates the classical complement cascade; it induces expression of several cell adhesion molecules as well as tissue factor; mediates LDL uptake by macrophages; induces monocyte recruitment into the arterial wall; enhances production of monocyte chemoattractant protein-1; promotes endothelial dysfunction and impairs endothelial progenitor cell survival and differentiation; and enhances reactive oxygen species generation (7, 16, 18, 27, 48, 54, 55, 58, 65). Specific to the question of a potential role in vein graft
disease, CRP has been immunolocalized to the media and adventitia in a canine vein graft model (19). In the present study, we investigated the hypothesis that CRP plays a direct role in vein graft hyperplasia, and demonstrate an induction of a promigratory SMC phenotype that may be related to alterations in PDGF signaling.
Methods and Materials
Lower extremity vein and vein graft specimens. Specimens of human lower extremity veins and vein grafts were collected from discarded conduits at the time of coronary or lower extremity bypass grafting and lower extremity bypass graft revision, respectively. Specimens were fixed in 10% neutral buffered formalin (Sigma-Aldrich), embedded in paraffin, sectioned (6 m) and collected on Superfrost/Plus slides (Fisher Scientific). A total of 21 human greater saphenous veins and 18 saphenous vein grafts were available for this study. Serial sections were cut and used for hematoxylin and eosin staining, elastin staining and immunohistochemistry. IRB approval was obtained for the collection of all human specimens and relevant clinical data as part of a tissue repository. Immunohistochemistry, elastin staining and image analysis. The mouse monoclonal antibody (clone CRP-8) directed against denatured and native human CRP, used at 1:100 and 1:250 dilution, was purchased from SigmaAldrich. It has no cross-reactivity with human serum amyloid P, haptoglobin, alpha-1-acid glycoprotein, IgG or CRP from Limulus (54). A mouse monoclonal anti-human -smooth muscle actin antibody was obtained from Biogenex (used at 1:200), a mouse monoclonal anti-human CD31 antibody was from DAKO (1:30), and a rabbit polyclonal anti-human PDGFR- antibody was from Santa Cruz Biotechnology (1:1000). Alpha actin , CD31, PDGFR- or CRP immunolocalization was achieved with an indirect immunoperoxidase technique and enhanced diaminobenzidine (DAB+; DAKO) as the peroxidase substrate. Hematoxylin was the counterstain. The intensity of CRP staining was graded in a blinded manner as none, weak or strong by 2 investigators on 2 separate occasions; if staining was detected, the vessel layer in which the staining was present was noted. For morphometric analysis, elastin was stained using the Verhoeff-Van Gieson technique. All sections were dehydrated and mounted permanently with Cytoseal (Richard-Allan Scientific). Microscopy was performed with a Nikon
Eclipse 80i microscope and Spot Flex digital camera (Micro Video Instruments, MA). Neointima, media, total wall area and thickness were measured from 20x and 40x images of sections using Image J (NIH). Cell culture. HSVSMCs were cultured from explants of veins obtained at the time of bypass surgery and maintained in high glucose DMEM (Gibco) containing 10% fetal calf serum, glutamine and penicillin/streptomycin as described (60). Immunophenotyping after passage 3 using smooth muscle specific alpha-actin staining revealed that cultured cells were completely smooth muscle cells. Cells were used between passage 4 to 8 in all experiments. All cell culture experiments were performed at least 3 different times. CRP preparation. Commercial purified human CRP (Polysciences) was supplied in 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM CaCl2 and 0.1% NaN3. To remove NaN3, CRP was dialyzed against 2 1L-changes of the same buffer without NaN3 using a slide-a-lyzer dialysis cassette (10K MWCO, Pierce). This technique has been previously described to be sufficient for the removal of NaN3 (26, 57). The concentration of dialyzed CRP was quantified using a modified Lowry assay (DC Assay, Bio-Rad) and purity was grossly confirmed by separating the CRP preparation by SDS-PAGE and confirming the presence of a single band at approximately 25 kDa on a Coomassie-stained gel (data not shown). The level of endotoxin in CRP preparations after dialysis was determined using Limulus Amebocyte Lysate pyrogent (Cambrex, sensitivity 0.06 EU/mL) following manufacturer’s instructions. All experiments were performed with 2 different lots of CRP preparation. Semi-quantitative and quantitative real time RT-PCR. To determine the effect of CRP on gene expression, HSVSMCs were serum starved for 48h before the addition of increasing doses of dialyzed CRP (0, 5, 50 g/mL) for 1h and 24h. Cells were lysed with Trizol reagent (Invitrogen) and total RNA was purified according to manufacturer’s instructions and quantified by spectrophotometry. Reverse transcription was performed for semi-quantitative RT-PCR using 1 g total RNA and random hexamer primers (First-Strand cDNA Synthesis Kit, Amersham Biosciences) and for real-time quantitative RT-PCR using the High
Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer instructions. Gene-specific primers for PDGFR- , PDGFR- , PDGF-A, PDGF-B and GAPDH and PCR amplification conditions for semiquantitative RT-PCR have been described previously (60). A 474-bp product from PDGF-D was amplified using Accuprime Taq Polymerase (Invitrogen) with the forward primer 5’- CCATCCAGGTGAAAGGAAACG-3’ and reverse primer 5’TTTTTGTCCAGAGCATCCGC-3’ under the following conditions: 94 x 2min for 1 cycle; 94 x 30sec, 56 x 30sec, 68 x 1min for 35 cycles; 68 x 3min for 1 cycle. Inventoried Taqman gene expression assays (Applied Biosystems) were used for quantitative real-time PCR (PDGFR- – Hs00182163_m1, PDGFR- – Hs00183486_m1, PDGF-A – Hs00234994_m1, PDGF-B – Hs00234042_m1). Samples were run in triplicate in 25 L reactions using 2x TaqMan Universal PCR Master Mix (Applied Biosystems) on an ABI 7900HT sequence detector (Applied Biosystems). Target gene expression levels are expressed relative to GAPDH expression levels using the comparative CT method (34). Transwell migration assay. HSVSMC migration was determined using gelatin-coated modified Boyden chambers (Costar) with 8 M pores in 24-well plates. SMCs were serum starved for 48h, treated with vehicle or dialyzed CRP (20 g/mL) for an additional 48h, trypsinized lightly with 0.05% trypsin, and seeded in transwells at a density of 20,000 cells/well. PDGF-BB (20ng/mL; R&D Systems) in serum free-media was added as a chemoattractant in the bottom wells. In some cases, neutralizing antibody to PDGF-BB (5 g/mL; R&D Systems) was added to both the top and bottom wells. All treatments were done in duplicate or triplicate. After 6-9 hours, unmigrated cells were scrubbed and rinsed twice from the top of the membranes with cotton swabs and PBS. Membranes were fixed in methanol, stained with DAPI (Invitrogen) and carefully cut from the inserts. Membranes were mounted on slides in PBS and photographed under high power (400x) with a fluorescent microscope. Three to five high power fields were photographed per membrane. Nuclei were counted and migration is expressed relative to that of the control group (no chemoattractant).
Cell viability and proliferation. HSVSMC viability and proliferation were assessed as functions of trypan blue exclusion, morphology of fluorescentstained nuclei, and reduction of Alamar Blue substrate (60). For trypan blue exclusion, HSVSMCs were plated in 96-well plates and allowed to adhere overnight. The following day, the wells were rinsed with PBS and vehicle, 5 g/mL CRP or 50 g/mL CRP were added in low serum conditions (0.1%) in triplicate wells. Forty-eight hours later, cells were simultaneously trypsinized and stained with trypan blue (Sigma-Aldrich). Viable cells (which exclude dye) were counted and viability was expressed as percent difference in the absolute number of viable cells from vehicle-treated wells. To detect morphological changes in nuclei that accompanies apoptosis (such as chromatiin condensation), HSVSMCs were plated in chamber slides and treated with vehicle or CRP in triplicate as before. After 48 hours, cells were rinsed, permeabilized with triton X-100 (SigmaAldrich), and stained with DAPI (Invitrogen). Slides were coverslipped and analyzed by fluorescent microscopy. Three high-powered fields (400x) of each well were photographed. Vehicle and CRP-treated nuclei were compared qualitatively. For the Alamar Blue assay, HSVSMCs were plated in 24-well plates and allowed to adhere overnight in normal serum conditions. The following day (day 0), the wells were rinsed with PBS and vehicle or CRP were added in low serum conditions (0.1%) as above. The positive control group consisted of cells that continued to grow in normal serum conditions. All treatment groups were done in triplicate wells. Reduction of Alamar Blue was measured on days 0, 2, 4 and 8, converted to cell number based on a standard curve, and expressed as percent increase over vehicle-treated wells. Media was changed and CRP was replenished every 48 hours. Immunoprecipitation and Western blot analysis. To determine the effect of CRP on PDGFR- protein levels, HSVSMCs were serum starved for 48h and treated with increasing doses of dialyzed CRP (0, 5, 50 g/mL) for an additional 24h or 48h. Cells were lysed in buffer (25mM HEPES, pH 7.5, 150mM NaCl, 1% Triton X-100, 0.1% beta-mercaptoethanol, 10% glycerol, 5mM EDTA, 5mM EGTA) containing protease inihibitor (Roche) and phosphatase inhibitor (Pierce)
cocktails. Lysates were cleared by centrifugation and protein concentration was determined using a modified Lowry assay (RC DC assay, Bio-Rad). Thirty micrograms of total protein was boiled briefly before being separated on 7.5% SDS-PAGE gels and transferred to nitrocellulose membranes using a semi-dry technique. Membranes were blocked in 5% milk and incubated with a rabbit polyclonal antibody to PDGFR- (1:200; Santa Cruz Biotechnology) overnight. Bound antibody was detected using a HRP-conjugated goat anti-rabbit antibody (1:10,000; Jackson Immuno) with enhanced chemiluminescent substrate (West Pico; Pierce). Beta actin (1:10,000; Sigma-Aldrich) served as the loading control. To determine the effect of CRP on phosphorylation levels of PDGFR- , total receptor was immunoprecipitated from 250 g cleared total protein lysates with 1 g of anti- PDGFR- overnight at 4 deg. Protein A/Gbeads (Pierce) were added and after 1 hour, protein A/G-antibody conjugates were pelleted, washed, and disrupted by boiling. Western blotting was performed as described above using mouse monoclonal antibody to phospho-tyrosine (1:200; Santa Cruz Biotechnology). Total PDGFR- served as the loading control. All blots were scanned. Bands were quantified by densitometry (GelDoq, Bio-Rad) and normalized relative to bands in control groups (no CRP treatment). Statistical analysis. Values are expressed as mean
standard error
unless otherwise specified. Student’s t-test and ANOVA were used to assess differences between two and more than two groups, respectively. Differences were considered significant at P
