Departments of 1Physiology and 2Cardiology, Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania
Submitted 7 July 2004 ; accepted in final form 15 September 2004
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ABSTRACT |
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Immune products, including granulocyte-monocyte colony-stimulating factor (GM-CSF), IFN, IL-1, IL-6, and TNF
, influence the VSMC phenotype and mediate the vascular response to injury (27, 38). Although it has been shown that cytokine-activated but not quiescent VSMC secrete both GM-CSF and M-CSF (8), little has been reported on the expression of granulocyte colony-stimulating factor (G-CSF) cytokine in injured arteries or activated VSMC or on the effect of G-CSF on VSMC pathophysiology. G-CSF is considered to be a lineage-restricted hematopoietic growth factor which stimulates terminal mitotic divisions and the final cellular maturation of hematopoietic progenitors, particularly granulocytes (18). G-CSF has multiple effects on its target cells, including promotion of cell survival, proliferation, chemotaxis, and maturation (20). All of these pathophysiological responses are involved in VSMC activation and development of intimal hyperplasia. Indeed, recently it was observed that infusion of G-CSF into patients undergoing coronary interventional procedures unexpectedly increased the rate of in-stent restenosis (13).
Using cDNA microarray analysis to investigate the VSMC transcriptional response to inflammatory stimuli, we observed an unexpected expression of G-CSF mRNA. Very little has been reported regarding the expression and effects of G-CSF in VSMC. In this report, we describe how we verified the expression of G-CSF by examining its expression in cytokine-stimulated VSMC and balloon angioplasty-injured rat carotid arteries. We also show that G-CSF is chemotactic to human VSMC and activates the GTPase Rac1, which is responsible for regulation of migration. Inhibition of Rac1 by dominant-negative or pharmacological inhibition abrogates G-CSF-initiated migration. G-CSF also initiates signal transduction pathways in VSMC by activating several cellular protein kinases, including Akt, p44/42 MAPK, and pS6. Together, this suggests an important, previously unrecognized role for this cytokine in vascular immune cell communication.
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MATERIALS AND METHODS |
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Rat left common carotid artery balloon angioplasty. Left common carotid artery balloon angioplasty was performed in 350-g male Sprague-Dawley rats (Charles River Breeding Laboratory, Wilmington, MA) that were under pentobarbital sodium anesthesia (65 mg/kg delivered by intraperitoneal injection; Steris Laboratories, Phoenix, AZ) as described previously (1). Briefly, the left external carotid artery was cleared of adherent tissue, allowing the insertion of a 2-French Fogarty arterial embolectomy catheter (model 12 060 2F; Baxter Healthcare, Santa Ana, CA). The catheter was guided a fixed distance down the common carotid artery to the aortic arch, inflated with a fixed volume of fluid, and withdrawn back to the site of insertion a total of three times. Once this step was completed, the catheter was removed and the wound was closed (9-mm Autoclips; Clay Adams, Parsippany, NJ) and swabbed with Povadyne surgical scrub (7.5% povidone-iodine; Chaston, Danville, CT). Animals were housed in Plexiglas cages, maintained under a 12:12-h light-dark cycle, and allowed access to standard laboratory chow and drinking water ad libitum until tissue collection. All surgical procedures were performed in accordance with the guidelines of the Animal Care and Use Committee of Temple University and the American Association for Laboratory Animal Care.
Tissue processing and immunohistochemistry. Primary antibodies G-CSF (Santa Cruz Biotechnology, Santa Cruz, CA) and proliferating cell nuclear antigen (PCNA; Transduction Labs, Lexington, KY) were used at 2 µg/ml in 1% BSA-PBS and applied for 1 h, followed by biotinylated secondary antibody (1:200 dilution) and then avidin-biotin-peroxidase complex, each for 30 min. Nonspecific isotype antibodies were used as negative controls. Staining was visualized with the substrate diaminobenzidine (Vector Laboratories, Burlingame, CA), producing a reddish brown color, and then counterstaining with hematoxylin was performed as described previously (2).
RNA isolation and cDNA microarray analysis.
The complete detailed protocol for microarray analysis can be found on the Gene Logic web site (http://www.genelogic.com/docs/pdfs/backgrounder_DataGeneration.pdf). Briefly, total cellular RNA was extracted using TriReagent (Molecular Research Center, Cincinnati, OH), which allowed simultaneous extraction of RNA and protein as described previously (2). cDNA was synthesized using T7-oligo(dT)24 and SuperScript II reverse transcriptase and labeled using biotinylated CTP in an in vitro transcription reaction. cRNA (10 µg) was fragmented and analyzed using the Affymetrix U-133 chip (Affymetrix, Santa Clara, CA) and hybridized and washed according to the manufacturer's protocol. GeneChips were scanned and data were normalized to several housekeeping genes as well as spike-in cDNA to construct a standard curve using the Affymetrix MAS 5.0 algorithm electronic Northern blotting based on relative change values (E Northern) were computed from the percentile and median values of expression from the test sample over the control sample. Relative change and E Northern blot analysis were performed using the GeneExpress algorithm.
Western blotting. Human VSMC extracts were prepared as described elsewhere (2). Membranes were incubated with a 1:2,000 dilution of primary antibody and a 1:2,000 dilution of secondary antibody. G-CSF antibody was obtained from Santa Cruz Biotechnology. Equal protein concentrations of cell extracts were determined using a Bradford assay, and equal loading on gels was verified by performing Ponceau red S staining of the membrane. Reactive proteins were visualized using enhanced chemiluminescence. Multiplex phospho-p90 ribosomal S6 kinase (p90RSK), Akt, p44/42, and S6 were purchased from Cell Signaling (Beverly, MA) and used according to the manufacturer's instructions. Statistics based on densitometric analysis of at least three independent experiments were obtained using a paired sample t-test.
Rac1 activation.
Rac1 activation was determined using the p21-activated kinase 1 protein (PAK) pull-down assay as described previously (3). Human VSMC were serum starved in 0.5% FCS overnight, challenged with 200 pg/ml G-CSF for various time periods, and lysed in sample buffer (25 nM HEPES, 150 mM NaCl, 5 mM MgCl, 0.5 mM EGTA, 20 mM -glycerophosphate, 0.5% Triton X-100, 5% glycerol, 10 mM NaF, and 2 mM Na-vanadate, plus protease inhibitors). The volume of lysate was adjusted to normalize for equal concentrations of proteins. Cell suspensions were incubated with glutathione S-transferase (GST)-PAK Sepharose (Cytoskeleton, Denver, CO) for 1 h at 4°C. Only activated Rac1 binds the PAK protein (9). Beads were washed three times, bound proteins were detected using Western blotting with Rac1 antibody, and bands were quantitated by performing densitometry.
Migration and chemotaxis. Transwell Boyden chamber plates (6.5-mm diameter; Costar, Cambridge, MA) with 8-µm polycarbonate membrane pore size were seeded with 40,000 VSMC/membrane in medium containing 0.5% FCS as previously described (3). G-CSF at 1,000, 100, or 10 pg/ml were placed in the lower chamber, and cells were incubated for varying times as indicated at 37°C, and then cells were fixed and stained in Dif-Quick cell stain (American Hospital Supply, Deerfield, IL). The upper layer was scraped free of cells. VSMC that had migrated to the lower surface of the membrane were quantitated by counting four high-powered fields in each membrane. Experiments were performed in triplicate with three independently transduced groups of VSMC. Dominant-negative Rac1 (Rac1N17) adenovirus, described previously (40), was a gift from Dr. Satoru Eguchi (Dept. of Physiology, Temple University, Philadelphia, PA). VSMC were infected with 30 multiplicities of infection (MOI) of AdRac1N17 48 h before the migration assay. Sch-51344, a Rac1 pathway inhibitor (15), a gift from Dr. Satya Kunapuli (Dept. of Physiology, Temple University), was used at 10 µM concentration and added directly to the lower chamber. Statistical analysis, based on three experiments, was performed using ANOVA.
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RESULTS |
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G-CSF is expressed in injured, not naive, rat carotid artery. Cytokine-activated VSMC are the most abundant neointimal cell type in the injured artery, and it was important to determine whether G-CSF was expressed by VSMC in response to a pathophysiological process. Rat carotid arteries were injured by performing balloon angioplasty, and after 3 and 15 days, serial sections were examined for G-CSF and PCNA expression using immunohistochemistry. Figure 2 shows that G-CSF protein was not detectable in uninjured arteries but was detectible in medial VSMC 3 days postinjury and in neointimal cells 15 days postinjury. At both time points, antibody recognition was particularly concentrated in an area closest to the lumen of the vessel. This finding is in contrast to that for PCNA, which was predominately expressed in adventitial cells 3 days postinjury and was undetectable 15 days postinjury.
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DISCUSSION |
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Because activated VSMC constitute the major cell type in restenotic lesions, we investigated the expression of G-CSF in balloon angioplasty-injured rat carotid arteries using immunohistochemistry. No detectable G-CSF was observed in naive, uninjured arteries, but G-CSF was identified in medial VSMC in injured arteries after 3 days or in neointimal cells in injured arteries after 15 days. This is not entirely unexpected, because the restenotic injury is a complex lesion consisting of a multiple-cytokine milieu (28). This corroborates the Western blot analysis data indicating cytokine-dependent induction of G-CSF expression, and this is the first report of G-CSF expression in response to mechanical arterial injury. Also important is the contrast to PCNA staining in terms of both expression and location, suggesting that G-CSF-expressing cells, while activated, do not necessarily proliferate. Although no studies have reported G-CSF expression in injured arteries, G-CSF expression could represent an important prorestenotic event in the initiation and development of the restenotic lesion by several means. First, G-CSF is a potent leukocyte chemoattractant (24), and its expression in the arterial wall could act as a homing mechanism to promote leukocyte infiltration. Second, G-CSF is proliferative for hematopoietic cells, and the ED50 for mouse myeloblastic NFS-60 cell proliferation is 100 pg/ml (32). In this way, once present in the restenotic lesion, immune cells are stimulated to divide and mature. Third, we recently showed that G-CSF is proliferative to human VSMC (6). Consequently, expression of G-CSF by activated VSMC could act as an autocrine regulator of VSMC proliferation.
G-CSF is chemotactic for granulocytes (24). An early report also shows that G-CSF is chemotactic for endothelial cells (5). In a myeloid cell line, G-CSF induced Egr-1 upregulation through interaction of serum response element-binding proteins (19). Because Egr-1 plays a key role in vascular cell pathophysiology (14), we hypothesized that G-CSF would also promote VSMC migration as well. Indeed, we noted a dose- and time-dependent effect of G-CSF on VSMC migration. The present study is the first to establish a chemotactic effect of G-CSF on VSMC. G-CSF exerts its proliferative effects through activation of the Ras family of GTPases, which are intimately involved in the regulation of migration as well as proliferation (25, 26). These proteins act as molecular switches and cycle between inactive GDP-bound and active GTP-bound molecules to modify upstream signals to downstream effectors as required. Rac1 is a Ras family member that also plays an obligate role in cell migration. The Rac proteins in particular play important roles in VSMC pathophysiology, including the regulation of oxidative processes, proliferation, and migration (22). The experiments presented in Figs. 5 and 6 are the first to determine that G-CSF activates Rac1 in VSMC. Elimination of Rac1 activity results in significant reduction of G-CSF-induced VSMC migration. Accordingly, Rac1 activation is an obligatory molecular event that mediates G-CSF-induced migration of VSMC.
Activated Rac1 can transduce signals from cell surface receptors to corresponding cytoplasmic targets (10, 22). Although the G-CSF receptor does not contain a cytoplasmic kinase domain, binding of G-CSF to its receptor results in a rapid tyrosine autophosphorylation of its receptor as well as the phosphorylation and activation of numerous intracellular protein tyrosine kinases (7). These include the Ras-MAPK, JNK/SAPK, and p38 pathway activation (4, 24, 25). In our experiments, we show that G-CSF induces rapid activation of p90RSK and p44/42 MAPK, and Akt and more gradual, less robust activation of pS6 kinase.
Several reports have demonstrated a MAPK- and NF-B-mediated autocrine growth loop for G-CSF in nonhematopoietic tumor cells in which serum deprivation stimulated constitutive production of G-CSF (21, 36). Although known for some time to be proliferative for granulocytes and human endothelial cells (5, 26), there is only one other report in the literature describing expression of G-CSF in human VSMC (34). In this report, G-CSF mRNA was expressed in response to the inflammatory cytokine IL-1
. The authors of that study suggested that because G-CSF is proliferative for granulocytes, it might be involved in the progression of vascular inflammatory diseases. Our study extends that hypothesis and is the first to report that G-CSF has activating effects on human VSMC and confirms that this cytokine may exert pleiotropic effects outside the hematopoietic system.
Numerous recent studies have indicated that bone marrow-derived and circulating vascular cell precursors, or stem cells, contribute to the cellular component of the neointimal lesion in both mechanical and allograft-injured arteries by homing to the site of the lesion (29, 31, 33). These reports suggested that migration and transdifferentiation of multipotent cells can be an important source of neointimal occlusion in several vascular pathologies. G-CSF is a known powerful chemoattractant and mobilizer of bone marrow-derived stem cells (15, 24). It has been demonstrated that bone marrow stem cell transplantation improves myocardial perfusion but is limited by the invasiveness of stem cell collection. A recent report (13) described the MAGIC cell randomized clinical trial, in which endogenous stem cells were mobilized by infusion of G-CSF in patients with coronary stents. In this study, an unexpectedly high rate of in-stent restenosis was noted in patients who were administered G-CSF, so much so that enrollment in that arm of the study was terminated. It was suggested that the stem cells contributed to arterial occlusion within the lesion. Consequently, G-CSF secretion by activated arterial VSMC may contribute to recruitment and homing of vascular precursor cells to the site of the arterial lesion.
There are several novel points in this study. First, G-CSF can be expressed by human VSMC, and this expression can be increased by several cytokines. Second, G-CSF is not detectable in normal rat carotid arteries but can be induced in medial and neointimal VSMC by injury. Third, G-CSF is chemotactic for VSMC, likely through Rac1 activation. Fourth, G-CSF can increase activation of signal transduction pathways in VSMC. Taken together, these points indicate that G-CSF expression by activated VSMC has important implications for vascular inflammatory cell communication, resulting in the progressive nature of several vascular pathologies.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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