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【摘要】
Objectives- We have previously reported that vascular injury or treatment of cultured vascular smooth muscle cells with platelet-derived growth factor-BB (PDGF-BB) or fibroblast growth factor-2 (FGF2) increases the levels of protein tyrosine phosphatase (PTP)1B. The current study was designed to test the hypothesis that PTP1B attenuates PDGF- or FGF-induced motility and proliferation of cultured cells, as well as neointima formation in injured rat carotid arteries.
Methods and Results- Treatment of cultured cells with adenovirus expressing PTP1B decreased PDGF-BB- or FGF2-induced cell motility and blocked PDGF-BB- or FGF2-induced proliferation, whereas expression of dominant negative PTP1B (C215S-PTP1B) uncovered the motogenic effect of subthreshold levels of PDGF-BB or FGF2, increased neointimal and medial cell proliferation, and induced neointimal enlargement after balloon injury. The inhibitory effect of PTP1B directed against PDGF in cultured cells was associated with dephosphorylation of the PDGFß receptor.
Conclusions- PTP1B suppresses cell proliferation and motility in cultured smooth muscle cells treated with PDGF-BB or FGF2, and the phosphatase plays a counter-regulatory role in vascular injury-induced cell proliferation and neointima formation. Taken together with previous studies indicating increased PTP1B levels in cells treated with growth factors, the current findings are the first to report the existence of an inhibitory feedback loop involving PDGF or FGF, and PTP1B in blood vessels.
The current study was designed to test the hypothesis that PTP1B attenuates vascular smooth muscle motility and proliferation induced by platelet-derived growth factor or fibroblast growth factor-2 in cultured cells and that PTP1B plays a counter-regulatory role in neointima formation. Our results confirm the aforementioned hypotheses.
【关键词】 PTPB growth factors neointima formation cell motility cell proliferation
Introduction
Migration and proliferation of smooth muscle cells are of critical importance in neointima formation and remodeling occurring in response to vascular injury. 1 Increased release of platelet-derived growth factor (PDGF) and/or fibroblast growth factor-2 (FGF2), followed by activation of PDGF and/or FGF2 receptor tyrosine kinase activities, are thought to be major events contributing to vascular remodeling. 2 Several reports indicate that injury-induced movement of smooth muscle cells from media to intima and the proliferation of smooth muscle cells in intima are significantly reduced by pharmacological antagonists of the function or availability of PDGF or FGF2. 3-7 Conversely, administration of PDGF-BB or FGF2 has been reported to enhance smooth muscle cell movement from media to intima, followed by cell proliferation in vessels with minimal endothelial damage. 7,8 These studies indicate that PDGF and FGF are important mediators of neointima formation in models of vascular injury. Tyrosyl phosphorylation of growth factor receptors via their intrinsic tyrosine kinase activities is a pivotal event for activation of downstream signaling that mediates increased motility and proliferation in cultured cells. Furthermore, balloon injury or treatment in vivo with PDGF also induces PDGF receptor tyrosyl phosphorylation in vascular smooth muscle, 6,9 a finding consistent with experiments in vitro.
Protein tyrosine phosphatases (PTPs) are thought to play an important role as counter-regulatory agents that attenuate or terminate signaling induced by activation of receptor tyrosine kinases. PTP1B is a ubiquitously expressed nonreceptor phosphatase targeted to several intracellular domains, including the endoplasmic reticulum and focal adhesions. 10,11 PTP1B has been most prominently linked with regulation of insulin or insulin-like growth factor-1 (IGF-1) signaling in vitro and in vivo. 12-16 We have recently reported that PTP1B attenuates insulin-induced cultured smooth muscle cell motility by decreasing the levels of phosphotyrosyl in insulin receptors. 17 In a separate recent study, we reported that PDGF and FGF but not IGF-1 significantly increased the levels of PTP1B protein in cultured rat aortic smooth muscle cells. 18
Recent studies indicate that PTP1B may also target the PDGF receptor. One such study reported a biochemical association of the PDGF receptor with PTP1B, 19 whereas another study presented evidence consistent with PDGF receptor dephosphorylation by PTP1B, although this effect paradoxically failed to alter signaling downstream of the receptor. 20 To our knowledge, there are no reports on the capacity of PTP1B to act as inhibitor of PDGF function in cultured smooth muscle cells or neointima formation in injured blood vessels. Moreover, the potential role of PTP1B as modulator of FGF2 activity in vitro or in vivo appears not to have been investigated. The current study was therefore targeted at testing the hypotheses that PTP1B has the capacity to regulate PDGF- or FGF-induced motility and proliferation of cultured rat aortic smooth muscle cells and that the phosphatase plays a role in attenuating neointima formation occurring in response to vascular injury by decreasing smooth muscle cell motility, proliferation, and/or apoptosis.
Materials and Methods
Materials
Detailed materials information is available in the data supplement at http://atvb.ahajournals.org.
Cell Culture
Rats were purchased from Charles River Laboratories (Wilmington, Mass) or they were bred in the University of Tennessee vivarium. Smooth muscle cells were isolated from thoracic aortas of 6- to 9-day-old Sprague-Dawley rats and cultured as described in a previous report from our laboratory. 21 The choice of cells was made on the basis of studies indicating that primary cultured cells from rat pups have characteristics similar to those of the neointima. 22,23 In vitro experiments were carried out using primary cultures to minimize culture-induced phenotypic dedifferentiation of cells. Each experiment was performed using cells isolated from different litters of pups.
Measurement of Cell Motility
Cell motility in cultured cells was measured via a protocol described in a recent publication from our laboratory. 24 Details of the procedure are provided in the Data Supplement.
Measurement of Cell Proliferation
Cell proliferation in cultured cells was measured by using the In Situ Cell Proliferation Kit (purchased from Roche). The detailed procedure is provided in the Data Supplement.
Measurement of PDGFß Receptor Phosphotyrosyl Levels
PDGF receptor tyrosyl phosphorylation was determined by immunoprecipitation of PDGFß receptor with antibody directed against PDGFß receptor and blotting with antibody directed against PDGFß receptor phosphotyrosyl residue 770. The detailed procedure is described in the Data Supplement.
Rat Carotid Artery Injury Model
Rat carotid artery injury was generated via a standard procedure. 25 Details are provided in the data supplement.
Morphometric Measurement of Neointima Formation
Rat carotid arteries were collected 3, 7, or 14 days after balloon injury. Carotid arteries were fixed by in situ perfusion through the left ventricle with 10% formalin followed by embedding in paraffin. Cross-sections were stained with hematoxylin and eosin for morphometric analysis. Images were collected by using Spot 3.3.2 software. The cross sectional surface areas of neointima and media were measured by using a computerized image analysis system (NIH v.1.62).
Immunohistochemistry
Paraffin-embedded carotid arteries were sectioned (5 µm), dewaxed, rehydrated, and irradiated in a microwave oven at 94°C in 0.1 mol/L citrate buffer, pH 6.0. Levels of proliferating cell nuclear antigen (PCNA) in tissue sections were measured by incubation with anti-PCNA at a dilution of 1:200. Detection was carried out by using the ABC (Avidin-Biotin Complex) method with DAB (3, 3'-diaminobenzidine) as substrate. Total and PCNA-positive cell numbers were determined from analysis of four microscopic fields from each of the tissue sections, at magnification of x 400. The fraction of proliferating cells was calculated as the mean ratio of the number of positive cells to total cell number in each set of tissue sections.
Measurement of Apoptosis
Paraffin-embedded carotid arteries were sectioned (5 µm), dewaxed, rehydrated, and irradiated in a microwave oven at 94°C in 0.1 mol/L citrate buffer, pH 6.0. Apoptosis was measured by following the manufacturer?s instructions, using In Situ Cell Death Detection kit. Detection was carried out by using alkaline phosphatase substrate kit. Total cell number and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cell number were determined in 4 microscopic fields from each of the tissue sections at magnification of x 400. The fraction of apoptotic cells was calculated as the ratio of the number of positive cells to total cell number in each set of tissue sections.
Statistical Analysis
Results are expressed as mean±SEM and were analyzed by using 2-way ANOVA followed by Fisher?s least significant difference test or unpaired t test. P <0.05 is considered statistically significant.
Results
Overexpression of PTP1B Attenuates PDGF-BB- or FGF2-Induced Motility in Cultured Smooth Muscle Cells
These experiments were performed to test the hypothesis that overexpression of PTP1B is sufficient to decrease cell motility induced by PDGF-BB or FGF2. As shown in Figure I (available online at http://atvb.ahajournals.org), treatment of cells with recombinant adenovirus encoding for PTP1B induced a significant increase of PTP1B protein levels. Moreover, as shown in Figure 1 A, overexpression of PTP1B decreased PDGF-BB-induced cell motility by 10% to 90%. PTP1B was more effective in opposing the motogenic response directed against relatively low than high concentrations of PDGF-BB, and indeed the phosphatase essentially blocked the motogenic effect of the lowest concentration of PDGF-BB (0.5 ng/mL) used in our study. As shown in Figure 1 B, overexpression of PTP1B also attenuated FGF2-induced cell motility; however, unlike PDGF-BB-induced motility, PTP1B decreased FGF2-induced motility by at least 50%, even at a relatively high concentration of FGF2. These results indicate that an increase of PTP1B levels is sufficient to attenuate PDGF-BB- as well as FGF2-induced motility in primary cultured rat aortic smooth muscle cells.
Figure 1. Overexpression of PTP1B attenuates PDGF-BB- or FGF2-induced cell motility in cultured rat aortic smooth muscle cells. Cells were infected for 24 hours with adenovirus expressing EGFP (enhanced green fluorescent protein, as control virus) or PTP1B at multiplicity-of-infection (MOI) values of 10 to 15. Virus-containing media were removed and cells were cultured for an additional 24 hours to allow for expression of transgenic PTP1B. Cell motility was determined via a wounded culture assay in response to treatment with PDGF-BB (0, 0.5, 1 or 10 ng/mL, A) or FGF2 (0, 2, or 20 ng/mL, B) for 24 hours as described in Materials and Methods. Results are the mean+SEM of 3 independent experiments. A, * P <0.01 compared with treatment category lacking PTP1B. B, Open bars indicate cells infected with control virus; striped bars, cells infected with virus expressing PTP1B. Numerals shown inside bars indicate FGF2 concentration in ng/mL. * P <0.01 compared with control virus; ** P <0.01 compared with control virus + FGF2.
Overexpression of PTP1B Blocks PDGF-BB- or FGF2-Induced Proliferation in Cultured Smooth Muscle Cells
Smooth muscle cell proliferation is an independent pivotal event in vascular injury-induced neointima formation. The present study was also designed to test the hypothesis that overexpression of PTP1B is sufficient to decrease cell proliferation induced by PDGF-BB or FGF2. As shown in Figure 2, treatment of cultured cells with adenovirus expressing PTP1B blocked PDGF-BB- or FGF2-induced proliferation, indicating that upregulation of PTP1B is sufficient to decrease PDGF-BB- or FGF2-induced proliferation in primary cultured rat aortic smooth muscle cells.
Figure 2. Overexpression of PTP1B blocks PDGF-BB- or FGF2-induced proliferation of cultured rat aortic smooth muscle cells. Cells were infected for 24 hours with "empty" adenovirus (as control, containing no inserted protein) or PTP1B at MOI values of 10 to 15. Virus-containing culture media were removed and cells were cultured for an additional 24 hours to allow for expression of transgenic PTP1B. Cell proliferation was determined by counting the number of BrdU-labeled cells in the microscope view field in response to treatment with PDGF-BB (0.25 ng/mL, A) or FGF2 (1 ng/mL, B) for 24 hours as described in Materials and Methods. Results are mean+SE of 3 independent experiments. * P <0.01 compared with control (empty virus); ** P <0.01 compared with PDGF+ control virus or FGF2 + control virus (ANOVA and Fisher?s PLSD test).
Overexpression of PTP1B Blocks PDGF-BB-Induced PDGFß Receptor Phosphorylation in Cultured Smooth Muscle Cells
Tyrosyl phosphorylation of growth factor receptors is of critical importance for activation of downstream signaling that mediates increased motility and proliferation in cultured cells. 26 Moreover, it has been recently reported that dephosphorylation of PDGF receptor by PTP1B fails to alter signaling downstream of the receptor in fibroblasts. 20 These findings prompted us to test the hypothesis that PTP1B-induced inhibition of cell motility and proliferation are associated with phosphotyrosyl dephosphorylation of PDGFß receptor. In the present experiments, we targeted tyrosyl residue 770 in the PDGFß receptor for investigation because phosphorylation of this residue has been reported to mediate signaling relevant to cell motility and proliferation, involving the small GTP-binding protein Ras. 26 As shown in Figure II (available online at http://atvb.ahajournals.org), treatment of cells with adenovirus expressing PTP1B blocked PDGF-BB-induced phosphorylation of Y770 in PDGFß receptor, consistent with the view that the antimotogenic and antimitogenic effects of the phosphatase can be attributed to dephosphorylation of residue Y770.
Expression of Dominant Negative PTP1B (C215S-PTP1B) Uncovers the Motogenic Effect of a Subthreshold Level of PDGF-BB or FGF2 in Cultured Smooth Muscle Cells
The next experiments were performed to test the hypothesis that expression of a catalytically-inactive PTP1B allele, previously shown by us to function in dominant-negative manner against the insulin receptor in cultured vascular smooth muscle cells, 17 would enhance PDGF-BB- or FGF2-induced cell motility. The data shown in Figure III (available online at http://atvb.ahajournals.org) demonstrate effective expression of C215S-PTP1B. It should also be noted that expression of dominant negative PTP1B can, by itself, induce cell motility if it occurs at sufficiently high levels, as demonstrated in our previous studies. 17,27 Therefore, for the present experiments, we titrated dominant negative PTP1B expression down to the level at which it produced no significant increase in motility to avoid the potential confounding effect of altered baseline motility. As shown in Figure 3 A, treatment of cells with dominant negative PTP1B uncovered a motogenic effect of PDGF-BB at a low concentration of PDGF-BB, which, when used alone, failed to induce significant cell motility. Similarly, dominant negative PTP1B uncovered the motility stimulatory effect of a subthreshold level of FGF2 ( Figure 3 B). Taken together, the results support the hypothesis that PTP1B plays a counter-regulatory role against both PDGF-BB and FGF2-induced cell motility.
Figure 3. Expression of dominant negative (DN) PTP1B (C215S-PTP1B) uncovers motility induced by subthreshold levels of PDGF-BB or FGF2 in cultured rat aortic smooth muscle cells. Cells were infected for 24 hours with adenovirus expressing EGFP (as control virus) or dominant negative PTP1B (C215S-PTP1B) at MOI values of 10 to 15. Virus-containing media were removed and cells were cultured for an additional 24 hours to allow for expression of dominant negative PTP1B. Images were taken before and after treatment for 24 hours with PDGF-BB (0.1 ng/mL, A) or FGF2 (20 pg/mL, B), and cell motility was determined via a wounded culture assay as described in Materials and Methods. Results are the mean+SE of 3 independent experiments. * P <0.05 compared with control virus, control virus+PDGF, control virus+FGF2, or dominant negative PTP1B.
Expression of Dominant Negative PTP1B Increases Balloon Injury-Induced Neointima Formation in Rat Carotid Arteries
PDGF and FGF are considered to be the principal growth factors mediating neointima formation in blood vessels injured in relatively robust manner via balloon catheter. 2 On the basis of this finding and the aforementioned observations in cultured cells, we next tested the hypothesis that PTP1B plays a counter-regulatory role in neointima formation. The strategy we implemented for these experiments was the use of dominant negative rather than wild-type PTP1B, based on our earlier experiments indicating that vascular injury increases the levels of endogenous PTP1B, 18 theoretically making it more difficult to reveal an effect of overexpressed wild-type PTP1B. Moreover, based on our expectation that dominant negative PTP1B would enhance neointima formation, we subjected rat carotid arteries to a relatively mild degree of injury. As shown in Figure IV (available online at http://atvb.ahajournals.org), treatment with adenovirus expressing dominant negative PTP1B induced significant expression of the mutant protein in medial layers of carotid arteries for at least 14 days, but much less so, if at all, in neointimal or adventitial cells, as determined by Western blot analysis of hemagglutinin (HA)-tagged dominant negative PTP1B. As shown in Figure 4A and 4 B, no neointima was evident 3 days after injury; moreover, treatment with dominant negative PTP1B induced a significant increase in neointima formation, both 7 and 14 days after injury.
Figure 4. Expression of dominant negative (DN) PTP1B (C215S-PTP1B) increases neointima formation in injured rat carotid arteries. A, Stained cross sections from injured representative carotid arteries treated with control virus (expressing lac Z) or with virus expressing DN-PTP1B at a concentration of 10 10 pfu/mL. Carotid arteries were fixed, removed, embedded in paraffin, cross-sectioned, and stained with hematoxylin-eosin 3, 7, or 14 days after injury. The surface areas of intima (I) and media (M) were measured by using NIH 1.62 software at magnification of x 100. B, Summary of vascular remodeling experiments. Results are the mean+SE of 4 experiments, each representing an artery from a single rat. * P <0.05, ** P <0.01 compared with lac Z, via unpaired t test.
Expression of Dominant Negative PTP1B Increases Balloon Injury-Induced Cell Proliferation but Fails to Alter Apoptosis
Vascular injury-induced neointima formation is determined in part by the balance between cell proliferation and cell death. It has been reported that vascular injury induces a rapid increase of apoptosis, followed by increased cell proliferation. 28,29 To determine whether increased neointima formation induced by dominant negative PTP1B occurred via altered cell proliferation and/or apoptosis, we next performed experiments to measure the expression of a protein specifically associated with cell proliferation, namely PCNA, via immunohistochemistry, and apoptosis via the TUNEL method involving staining for DNA fragmentation in injured carotid arteries. As shown in Figure 5, expression of DN-PTP1B markedly increased cell proliferation in neointima and media 7 days after injury and, to a lesser extent in neointima only, 14 days after injury. Although there was a tendency toward increased proliferation 3 days after injury, the difference was not statistically significant. In addition to increased cell proliferation, total cell number in intima but not in media was also increased by expression of DN-PTP1B at 7 and 14 days after injury as shown in Table I (available online at http://atvb.ahajournals.org). However, the levels of apoptosis were not significantly altered at any time point in injured carotid arteries treated with adenovirus expressing DN-PTP1B, compared with arteries treated with adenovirus expressing lac Z (data not shown).
Figure 5. Expression of dominant negative (DN) PTP1B (C215S-PTP1B) increases cell proliferation in injured rat carotid arteries. A, Stained cross sections from injured representative carotid arteries treated with control virus (lac Z) or with virus expressing DN-PTP1B at a concentration of 10 10 pfu/mL. Carotid arteries were fixed, removed, embedded in paraffin, cross sectioned, and immunostained with PCNA and counter-stained with hematoxylin 3, 7, or 14 days after injury. White arrows indicate negative staining; black arrows, positive staining; I, intima; and M, media. B, Summary of in vivo proliferation experiments. Cells were counted by using NIH1.62 software. Open bars indicate neointima; striped bars, media. Results are the mean+SE of 4 experiments, each representing a carotid artery section from a single rat. ** P <0.01 compared with lac Z via unpaired t test.
Discussion
In contrast to receptor protein tyrosine kinases and associated downstream signaling events in the cardiovascular system, the protein tyrosine phosphatases that potentially function as counter-regulatory agents have been less extensively investigated. Thus, the present study addressed the functional role of the ubiquitous phosphatase PTP1B in cultured rat aortic smooth muscle cells and in carotid arteries. In addition to targeting the PDGF receptor, PTP1B has been reported to induce dephosphorylation of several other receptor tyrosine kinases, including the insulin receptor, 17 epidermal growth factor receptor, 30 and IGF-1 receptor. 15
We report for the first time that PTP1B targets PDGF- or FGF-induced cell motility and proliferation in vitro and neointima formation in vivo. It is interesting to note a recent study indicating that signaling events downstream of the PDGF receptor were not significantly altered in fibroblasts genetically lacking PTP1B, although receptor tyrosyl phosphorylation levels were increased significantly. 20 Because the above-mentioned study did not investigate functional end points, it did not establish whether PTP1B failed to play an important physiological role in cultured fibroblasts or if other mechanisms compensated for the inhibitory effect of PTP1B. In contrast, our data indicate that PTP1B is effective in antagonizing the motogenic and mitogenic effects of PDGF or FGF in cultured rat aortic smooth muscle cells. The effect of PTP1B expression on PDGF-induced cell proliferation and migration is associated with tyrosyl dephosphorylation of PDGFß receptors. Of note, the antimotogenic activity of PTP1B is more effective at lower concentrations of PDGF or FGF, suggesting a competitive antagonism of growth factor versus phosphatase activity. Alternatively, the finding that PDGF and FGF2 both increase the levels of endogenous PTP1B in cultured vascular smooth muscle 18 may also explain the relative ineffectiveness of ectopic PTP1B expression, occurring on top of elevated endogenous expression induced by growth factors.
In addition to the above-mentioned findings, indicating that wild-type PTP1B attenuated whereas dominant negative PTP1B increased cultured cell motility and/or proliferation, we observed results consistent with similar effects of PTP1B in injured arteries. Thus, we found that treatment of injured arteries with adenovirus expressing dominant negative PTP1B but not lac Z increased cell proliferation, intimal cell number, and neointima formation without affecting apoptosis. These results support the view that PTP1B decreases cell proliferation and neointima formation in injured carotid arteries. On the basis of our experiments using cultured cells, it is likely that migration of cells from media to neointima may also have been increased by DN-PTP1B, although our in vivo experiments did not directly provide information on this issue. It is interesting to note that adenovirus-mediated expression of PTP1B was mostly confined to the medial layer of arteries, presumably because of dilution of adenovirus after cell division and/or clearance of adenovirus via mechanisms involving the immune system. Given the sequence of events thought to induce neointima formation, which involves migration of cells to the neointima followed by intimal proliferation, 2 the present results are consistent with the view that expression of dominant negative PTP1B in the vascular media is sufficient to affect relatively late events, such as cell proliferation leading to neointimal enlargement.
Our observations in cultured cells support the notion that PTP1B plays an important role in attenuating growth factor-induced vascular remodeling by inducing dephosphorylation of the PDGF receptor, leading to inhibition of smooth muscle cell proliferation and/or migration. Because of the established enzymatic promiscuity of PTP1B, we do not yet know the identity of all tyrosine kinase receptors that may be targeted in vascular injury. On the basis of the notion that both PDGF and FGF play an important role in mediating neointimal expansion, 2 however, it is reasonable to speculate that both PDGF and FGF receptors are prime targets of PTP1B in vivo, although epidermal growth factor receptor, 31 IGF-1 receptor, 32 and insulin receptor, 33 which were documented to play a role in vascular remodeling, may also be targeted.
We have reported that treatment of cultured cells with PDGF-BB or FGF2, but not IGF-1 or epidermal growth factor, increases the protein levels of PTP1B. 18 In addition, vascular injury is associated with increased levels of PTP1B mRNA 34 and protein. 18 Our previous results, taken together with the current findings, indicate the existence of a feedback mechanism whereby PDGF and FGF2 function is not only mediated by the intrinsic activity of cognate receptor tyrosine kinases but also modulated in negative feedback fashion by PTP1B. Moreover, because PTP1B targets IGF-1, insulin, and PDGF receptors, the present studies raise the possibility of PTP1B-mediated negative cross-talk between PDGF or FGF and IGF-1 or insulin activity. On the basis of these results, we believe that further studies into the details of the vascular effects of PTP1B are warranted.
Acknowledgments
This work was funded by United States Public Health Service grants HL63886 and HL72902, American Heart Association-Southeast Affiliate grant 0465176B, American Heart Association grant 0530106N, and American Diabetes Association grant 105JF60. We acknowledge the technical assistance of Leena Desai, Ruirui Ji, and Jian Yang.
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作者单位：Department of Physiology (Y.C., B.C., D.Z., Q.P., A.C.C., A.H.), Vascular Biology Center (C.Z., A.H.), and Department of Surgery (C.Z.), University of Tennessee Health Science Center, Memphis.