Regulation of Vascular Smooth Muscle Cell Proliferation and Migration by Human Sprouty 2
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动脉硬化血栓血管生物学 2005年第3期
From the Departments of Pharmacology and Experimental Therapeutics (D.C., J.M.L., T.B.P.) and Pathology (D.M., J.M.L.), Loyola University, Chicago, Stritch School of Medicine, Maywood, Ill; and the Department of Medicine (C.Z., L.J.), University of Tennessee, Memphis.
Correspondence to Tarun B. Patel, Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, 2160 S First Ave, Maywood, IL 60153. E-mail tpatel7@lumc.edu
Abstract
Objectives— To determine whether the human sprouty 2 (hSPRY2) protein, an inhibitor of receptor tyrosine kinase actions, regulates vascular smooth muscle cell (VSMC) proliferation, migration, and neointima formation in injured carotid artery.
Methods and Results— The hSPRY2 protein or green fluorescent protein (GFP; control) was transduced into VSMCs by placing an N-terminal TAT epitope on the proteins. The transduction of TAT-tagged hSPRY2 (TAT-hSPRY2) but not TAT-GFP inhibited the ability of serum and different growth factors to stimulate migration of VSMCs. Likewise, TAT-hSPRY2 also inhibited VSMC proliferation in response to serum. The hSPRY2 microtubule association (amino acids 123–177) and membrane translocation (amino acids 178–194) domains were necessary for the biological actions of hSPRY2. In the rat carotid artery injury model, exposure of the injured vessel for 1 hour to TAT-hSPRY2, but not TAT-GFP, markedly inhibited growth of the neointima over the 28-day postangioplasty period as well as VSMC proliferation. The exogenously applied TAT-hSPRY2 was retained in the carotid arteries for at least 3 days after injury, and endogenous SPRY2 expression was maximized around day 14 after injury. The latter is perhaps a compensatory mechanism to regulate neointima formation.
Conclusions— We conclude that TAT-tagged proteins are efficiently transduced into VSMCs in vitro and in vivo, that hSPRY2 inhibits growth and migration of VSMCs, and that this protein can decrease neointimal growth after blood vessel injury.
The aim of this study was to determine whether the human sprouty 2 (hSPRY2) protein, an inhibitor of receptor tyrosine kinase actions regulates vascular smooth muscle cell (VSMC) proliferation, migration and neointima formation in injured carotid artery. We conclude that TAT-tagged proteins are efficiently transduced into VSMCs in vitro and in vivo, that hSPRY2 inhibits growth and migration of VSMCs, and that this protein can decrease neointimal growth after blood vessel injury.
Key Words: sprouty ? vascular smooth muscle cells ? proliferation ? migration ? growth factors ? carotid artery injury ? neointima formation
Introduction
The sprouty (SPRY) family of proteins has been shown to inhibit the actions of receptor tyrosine kinases and regulate tracheal branching,1,2 as well as cellular migration, proliferation, and angiogenesis.3–5 To date, 4 isoforms of mammalian SPRY proteins have been cloned.1,2,6,7 These proteins have a highly conserved, cysteine-rich, C terminus, but their N terminus is variable. SPRY1, SPRY2, and SPRY4 are expressed during development of the embryo and in several adult tissues, including kidney, lung, brain, heart, and skeletal muscle.7 On the other hand, SPRY3 mRNA is present in the adult brain and testis.7 As observed initially in the Drosophila,1 in mouse, a decrease in endogenous SPRY2 expression by antisense oligonucleotide approach resulted in increased lung branching morphogenesis.2 These findings suggest that SPRY proteins have conserved function to modulate respiratory morphogenesis. The ability of mouse SPRY4 to inhibit angiogenesis3 also demonstrates that the SPRY proteins play a profound role in regulating tubular morphogenesis.
At the cellular level, we and others have shown that SPRY1,4 SPRY2,5 and SPRY43 inhibit migration and proliferation of cells in response to serum and growth factors. Treatment of cells with epidermal growth factor (EGF) results in translocation of the human SPRY2 (hSPRY2) protein from the vicinity of microtubules to membrane ruffles.5,8 We have shown that abrogating the colocalization of hSPRY2 with microtubules or deletion of the region that is necessary for translocation to membrane ruffles obliterates the ability of the protein to inhibit cell migration and proliferation.5 We have also shown that hSPRY2 expression alters the cellular distribution of protein tyrosine phosphatase 1B and that the increase in amount and activity of the cytosolic form of this protein, at least in part, mediates the antimigratory actions of hSPRY2.9 More recently, we reported that the antimigratory actions of hSPRY2 also involve inhibition of Rac1 activation.10
Angiogenesis plays a critical role during embryonic development, tissue growth, or regeneration, and efficient growth of tumors.11 Whereas the role of endothelial cells in angiogenesis is well established, it is also becoming increasingly evident that proliferation and migration of vascular smooth muscle/perivascular cells play an important role in ensuring endothelial cell survival and vasculogenesis.12–14 In addition, the proliferative response of vascular smooth muscle cells (VSMCs) has an important role in recovery from injury as well as atherosclerosis, hypertension, and postangioplasty restenosis.15 Although SPRY4 has been shown to inhibit the migration and proliferation of human umbilical vascular endothelial cells,3 whether SPRY proteins affect the growth and migration of VSMCs remains to be determined. Likewise, whether or not SPRY protein can modulate neointima formation after injury to the blood vessel has also not been investigated. Therefore, in this report, we examined the effects of hSPRY2 on the growth and migration of rat aortic VSMCs. Our data demonstrate that hSPRY2 inhibits the growth factor–elicited migration and proliferation of VSMCs, and the SPRY translocation domain is necessary for this biological action of hSPRY2. Moreover, using the carotid artery balloon injury model in intact rats, we show that hSPRY2 can decrease the growth of the neointima and decrease cellular proliferation. Interestingly, the amount of endogenous rat homologue of SPRY2 is increased after balloon injury to the carotid arteries. This increase in expression of SPRY2 is probably a compensatory mechanism to modulate actions of growth factors.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
A detailed Materials and Methods section is available online at http://atvb.ahajournals.org.
Results and Discussion
Transduction of VSMCs With TAT-Tagged Proteins
Because it is difficult to achieve a high transfection efficiency in VSMCs, we used the strategy of introducing hSPRY2 into cells by tagging the protein with an 11-aa sequence (YGRKKRRQRRR) from the TAT protein of HIV. This permits transduction of the protein of interest into >99% of a variety of cells,5,16–20 including VSMCs (see Figure I in online Materials and Methods section).
Inhibition of VSMC Migration and Proliferation by TAT-HA-hSPRY2
It is well established that the migration and proliferation of VSMCs in response to growth factors play a critical role in angiogenesis12–14 as well the development of atherosclerosis and vascular stenosis.21 To investigate the role of hSPRY2 in modulating VSMC migration in response to various growth factors, cellular motogenesis in response to serum was measured using a monolayer-wounding protocol22 (see Figure II in online Methods and Materials section for details and representative figure of the wound healing assay). After serum starvation, confluent VSMCs were treated for 1 hour with increasing concentrations of TAT protein (0, 1, 2, 5, 10, and 20 μg/mL), and migration in response to 10% serum was monitored as described in the online Materials and Methods section. Controls were performed in which cells were treated with TAT–green fluorescent protein (GFP; 10 μg/mL; "0" TAT-tagged hSPRY2 [TAT-hSPRY2] in Figure 1A). As shown in Figure 1A, for VSMCs treated with 10 μg/mL TAT-GFP as a control ("0" TAT-hSPRY2), the wounds closed an average of 84.7±3.6%. Treatment with TAT-hSPRY2 in a concentration-dependent manner inhibited VSMC migration in response to serum. In all subsequent experiments, TAT-GFP and TAT-hSPRY2 were used at a concentration of 10 μg/mL. Next, we investigated whether the VSMC migration in response to different growth factors was also modulated by hSPRY2. As shown in Figure 1B, serum, EGF, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) stimulated migration of control untransduced cells over basal values (reported in figure legend). The transduction of VSMCs with TAT-GFP did not alter migration in response to either serum or any of the growth factors, demonstrating that the TAT-tag does not by itself modulate cell migration. However, in cells transduced with TAT-hSPRY2, cell migration in response to serum and all the growth factors tested was attenuated (Figure 1B). Similar results were observed using the Boyden Chamber assay (Figure III, available online at http://atvb.ahajournals.org). These findings are consistent with our previous observations that serum and growth factor–stimulated migration of human cancer cell line (HeLa) and rat intestinal epithelial cell line (IEC-6) is also inhibited by hSPRY25,9,10 and that the antimigratory actions of hSPRY2 are mediated at some downstream locus/loci in the promigratory pathway, where signals from a number of different growth factors culminate.
Figure 1. TAT-hSPRY2 inhibits growth factor– and serum-stimulated migration and proliferation of rat aortic VSMCs. A, Confluent monolayers of VSMCs were exposed to different concentrations of TAT-hSPRY2 or 10 μg/mL of TAT-GFP (0 TAT-hSPRY2) for 1 hour before making scratch wounds and monitoring migration in response to serum (10%), as described in the online Materials and Methods section. Closure of the wounds in the absence of serum was 22.5±2.0% (not shown on graph). Data are the mean±SEM of 3 experiments (6 different fields for each condition). *P<0.001 compared with control; Student unpaired t test. B, Same as A, except migration of cells in response to serum (10%) or EGF (100 nmol/L), or FGF (150 ng/mL) or PDGF (10 ng/mL) was studied as detailed in the online Materials and Methods section. In absence of serum or growth factors, the percent closure of wounds under different conditions were: no addition, 19.8±1.5%; TAT-hSPRY2, 20.2±2.3%; TAT-GFP, 21.6±3.0%. Values presented are the mean±SEM of 3 experiments (6 different fields/condition). *P<0.001 compared with the corresponding condition in untreated controls or in cells treated with TAT-GFP; Student unpaired t test. C, VSMCs (105/dish) were grown in serum (10%) containing medium supplemented with 10 μg/mL each of TAT-GFP or TAT-hSPRY2, or hSPRY2123 to 177 or TAT-hSPRY2178 to 194. Cells were counted at different time points. The medium supplemented with TAT-tagged proteins was changed every 24 hours. The mean±SEM of 3 different experiments is shown. *P<0.05 and **P<0.01 compared with TAT-GFP (control) at the same time points; Student unpaired t test. D, Same as B, except that in addition to TAT-hSPRY2 and TAT-GFP, cells were also exposed to 10 μg/mL each of TAT-hSPRY2123 to 177 or TAT-hSPRY2178 to 194. Migration in response to serum (10%) was monitored. The mean±SEM of 3 separate experiments is shown.
Like migration, serum-induced VSMC proliferation was also markedly inhibited in cells that had been transduced with TAT-hSPRY2 compared with TAT-GFP (Figure 1C), indicating that hSPRY2 inhibits the proliferation and migration of VSMCs.
The SPRY2 protein contains 2 domains that have been shown to be necessary for the association of the protein with microtubules (amino acids 123–177) and translocation of the protein to membrane ruffles in response to growth factors (amino acids 178–194).5,8 We showed previously that the deletion of either of these regions in the hSPRY2 protein abolishes its ability to inhibit migration and proliferation of HeLa cells.5 To test the generality of our previous findings, we determined whether transduction of VSMCs with TAT-hSPRY2 harboring either of the 2 deletions altered its ability to inhibit cell migration and proliferation. Figure 1C and 1D show that the deletion of either the microtubule association domain (amino acids 123–177) or the translocation domain (amino acids 178–194) obliterated the ability of hSPRY2 to inhibit proliferation and migration of VSMCs in response to serum. These findings demonstrate that the microtubule association domain and translocation domain on hSPRY2 are necessary for its antimitogenic and antimotogenic actions in more than one cell type.
hSPRY2 Actions in Intact Animals
Having established that TAT-hSPRY2 can be conveniently transduced into VSMCs and that hSPRY2 inhibits the migration and proliferation of these cells, we investigated whether the TAT-hSPRY2 can be used to transduce cells in intact animals and whether hSPRY2 can inhibit neointima formation in injured blood vessels. The latter would test the in vivo significance of our in vitro findings with VSMCs. Essentially, using the rat carotid artery injury model (online Materials and Methods section), we investigated whether the transduction of TAT-hSPRY2 or TAT-GFP into cells after injury to the blood vessels decreased growth of the neointima. As shown by the representative cross-sections of carotid arteries in Figure 2Ai, the intima in the uninjured blood vessels is essentially made up of a single endothelial layer. Fourteen days after carotid artery injury, there was significant neointimal growth in the untreated injured artery (Figure 2Aii). The neointima (more granular layer) in these vessels can be separated from the media by the internal elastic lamina (Figure 2A, arrowheads, enlarged sections). The transduction of TAT-GFP did not alter this neointima formation (Figure 2Aiv). However, when the injured arteries had been transduced with TAT-hSPRY2 only once (at the time of the injury), the neointima formation was inhibited (Figure 2Aiii). Quantification of these data from carotid arteries at different times after injury demonstrated that the ability of hSPRY2 to inhibit neointima formation could be observed as early as 7 days after injury, and this inhibitory action was maintained up to 28 days after the intervention (Figure 2B). These findings demonstrate the antiproliferative and antimigratory actions of hSPRY2 in VSMCs that had been documented in the in vitro model (Figure 1) contribute to the attenuation of stenosis observed in the injured carotid artery in vivo. These findings also raise the interesting possibility of using SPRY2 to modulate VSMC migration and proliferation to, for example, attenuate restenosis after angioplasty.
Figure 2. TAT-hSPRY2 inhibits neointima formation in rat carotid arteries after balloon injury. Rat carotid arteries were subjected to balloon injury and then exposed to either vehicle (PBS) or 20 μg/mL each of TAT-GFP, or TAT-hSPRY2 for 1 hour, as described in the online Materials and Methods section. At 7, 14, and 28 days after injury, sections of the carotid arteries were analyzed. A, Representative photomicrographs (magnification x100) of hematoxylin-eosin (HE)–stained normal carotid artery and carotid arteries 14 days after injury. A higher magnification of sections in areas marked by boxes is also shown next to each panel. The methods described previously29 were used for the injury and morphometric analyses. The conditions are: (i) uninjured vessel; (ii) vehicle-treated injured vessel; (iii) injured vessel treated with TAT-hSPRY2 (20 μg/mL); and (iv) injured vessel treated with TAT-GFP (20 μg/mL). The internal elastic lamina (depicted by arrows) separates the intima (I) and media (M). B, Bar graph representing the mean±SEM (n=6) of the ratios of intimal/medial areas in sections of carotid arteries at 7, 14, and 28 days after balloon injury. *P<0.05 vs TAT-GFP and vehicle groups.
TAT-hSPRY2 Retention and Expression of Endogenous SPRY2 After Carotid Artery Injury
Because a single exposure for 1 hour to TAT-hSPRY2 inhibited neointimal formation for up to 28 days after injury, by immunohistochemistry and Western analyses, we investigated how long the TAT-SPRY2 was retained in the blood vessels. As shown in Figure 3B (bottom row), there was a significantly higher amount of SPRY2 staining (brown) in the TAT-hSPRY2–treated arteries compared with vehicle-treated arteries on days 1 and 3 after injury. Moreover, the amount of SPRY2 increased between 7 and 14 days after injury (Figure 3B, bottom row). Western blotting of the arteries that had been treated with a single exposure to TAT-hSPRY2 with anti-hemagglutinin (anti-HA) antibody that recognizes the HA epitope in TAT-hSPRY2 confirmed that the TAT-hSPRY2 was retained in the vessels for 3 days (Figure 3C). Thus, the TAT-SPRY2 is retained in the arteries for at least 3 days after transduction, and between 7 and 14 days after balloon injury, there may be an increase in the expression of endogenous SPRY2 (Figure 3B). The latter notion is confirmed by the findings that in the vehicle-treated arteries, there was increased SPRY2 staining at 14 days after injury; this increase was retained even at 28 days after balloon injury (Figure 3B, top row). That the expression of endogenous SPRY2 is increased at 14 days after injury is also confirmed by Western blotting (Figure 3A). The endogenous SPRY2 is expressed primarily in the neointima, with lesser amounts being present in the medial layer (Figure 3B). In these experiments, we ensured that the anti-SPRY2 antibody was specifically recognizing the SPRY2 protein by neutralizing the antibody with its peptide epitope (NTZ antibody; Figure 3B). An intriguing finding is that although uninjured arteries express SPRY2, their expression is decreased immediately after injury and then increased between 7 and 14 days after carotid artery injury (Figure 3A).
Figure 3. Retention of TAT-SPRY2 and expression of endogenous SPRY2 in injured carotid arteries. Carotid arteries that had been exposed to either vehicle (PBS) or TAT-hSPRY2 after injury were isolated at various indicated times after balloon injury and subjected to Western and immunohistochemical analyses. A, Western analyses with anti-SPRY2 antibody, loading control with anti-actin antibody is also shown in bottom panel. B, Sections of arteries were stained with hematoxylin eosin (violet/blue) for nuclei (depicted by arrows pointing up) and anti-SPRY2 antibody (brown and shown by the black downward-pointing arrows). The black bar on the left of each panel depicts the intima. The medial layer is labeled "M." Representative immunohistochemical photomicrographs (x400) from slices (5-μm thick) of carotid arteries at different time points are shown. Representatives of at least 3 different sections from different animals are shown. C, Western analyses of injured carotid arteries that had been exposed to TAT-hSPRY2. Proteins (30 μg each) were separated on SDS-PAGE gels and analyzed by Western blotting using anti-HA antibody that recognizes TAT-hSPRY2. Note the presence of the TAT-hSPRY2 up to 3 days after injury. NA indicates normal artery; NTZ Ab, neutralized antibody.
To determine whether the expression of SPRY2 in the injured blood vessels was in VSMCs or another cell type, we used sections of carotid arteries that had been injured and treated with vehicle only (PBS). In these arteries, 14 days after injury, the SPRY2 and the vascular smooth muscle–specific -actin were visualized using fluorescent secondary antibodies. Controls in the absence of primary antibodies were also performed. As shown in Figure 4, in the injured blood vessels, hSPRY2 was localized mainly in the neointima and in the medial layer. Most importantly, the SPRY2 was colocalized with the smooth muscle–specific -actin, demonstrating that the increased SPRY2 expression after injury is indeed in VSMCs. The small amount of coexpression of SPRY2 and smooth muscle–specific -actin in the adventitia may be attributable to the presence of myofibroblasts in this region after injury.23
Figure 4. The endogenous SPRY2 after injury to carotid arteries is localized in smooth muscle cells of arteries. Fourteen days after balloon injury, sections of carotid arteries exposed to vehicle (PBS) were stained for smooth muscle–specific -actin and SPRY2 as described in the online Materials and Methods section. Controls shown at x400 magnification for SPRY2, and -actin were performed without the primary antibody. Photomicrographs of the same section at x200 and x400 are shown separately and then merged (bottom panel on right side). The area of the x200 field that is visualized at x400 magnification is shown by rectangles. The lumen of the artery is seen at x200 magnification and marked "L." The neointima and medial layers are depicted as "I" and "M," respectively. A schematic demonstrating how SPRY2 expression may be altered by growth factors is also presented (see text for discussion).
Growth factors have been shown to stimulate the expression of SPRY proteins.1,24,25 Hence, it is possible that in the injured blood vessel when the cells are under the influence of greater concentrations of growth factors (no barrier from endothelium), the expression of SPRY2 may be enhanced, and the increased expression of SPRY2 after injury may serve as a negative feedback to control the biological actions of growth factors. This possibility is presented by the schematic in Figure 4. The schematic also incorporates the possibility that SPRY2, by inhibiting the action of growth factors, may regulate its own expression (indicated by "?" in schematic, Figure 4). Although the increase in SPRY2 may, to some extent, oppose the actions of growth factors in modulating growth and migration of VSMCs, in controls (vehicle- or TAT-GFP–treated) the lack of significant amount of hSPRY2 in the initial days after the injury may be the determining difference in the extent of neointima formation compared with TAT-hSPRY2–treated arteries. In this context, the decrease in SPRY2 expression immediately after injury (Figure 3A) may be deliterious, and the transduction of additional SPRY2 during the initial days after injury to the blood vessel, when endogenous SPRY2 expression is decreased, is clearly beneficial and able to attenuate neointima formation (Figure 2). This latter notion is also supported by the findings of Mori et al26 that the early administration of estrogen protects against neointima formation in injured arteries.
To determine whether the decrease in neointimal growth in TAT-hSPRY2–treated animals was indeed attributable to the decrease in cellular proliferation, sections of injured carotid arteries exposed to either TAT-hSPRY2 or vehicle were stained for proliferative cell nuclear antigen (PCNA) expression. As shown in Figure 5A, 14 days after injury, the proportion of PCNA-positive cells in TAT-hSPRY2–treated arteries was decreased compared with controls. Thus, as observed in vitro with VSMCs (Figure 1), proliferation of cells in vivo is also decreased by hSPRY2 (Figure 5A).
Figure 5. TAT-hSPRY2 decreases cell proliferation in injured carotid arteries but does not modulate leukocyte infiltration. Fourteen days after injury, sections of carotid arteries that had been treated with vehicle (PBS) or TAT-GFP were stained with anti-PCNA antibody (brown) a biomarker for proliferating cells and hematoxylin (A) or anti-CD45 antibody (brown) and hematoxylin (B). The bar graph in A depicts VSMC proliferation index that was calculated by the following formula: [(the number of PCNA-positive cells)/(total cells stained by hematoxylin)x100] for each section. Values are mean±SEM (n=6), and statistical significance is shown. Likewise, the bar graph in B shows the percent area occupied by CD45-positive cells (leukocytes). Mean±SEM from 6 experiments is shown.
Vascular injury is also associated with infiltration of inflammatory cells such as leukocytes.27,28 To determine whether the inflammatory response was also altered by transduction of hSPRY2 into the injured blood vessels, we monitored the infiltration of leukocytes using anti-CD45 antibody. The data in Figure 5B demonstrate that at 14 days after injury to the carotid arteries, leukocyte infiltration was not altered. Please note that we have shown previously, even at 14 days after injury, infiltration of leukocytes in the injured arteries is significantly higher than controls.28 Thus, it would appear that hSPRY2 does not alter the inflammatory response after injury to the carotid arteries. Whether or not SPRY2 also modulates extracellular matrix synthesis during restenosis is not addressed by our studies, and this subject will be investigated in the future.
In summary, we have shown that hSPRY2 inhibits the migration and proliferation of VSMCs in vitro and in vivo, and that the transduction of TAT-hSPRY2 into the injured carotid artery significantly decreases neointima formation. Although the infiltration of inflammatory cells into the injured area is not altered by hSPRY2, these findings suggest that SPRY2 may be useful in preventing neointima formation in pathologic states such as restenosis after angioplasty. Moreover, we show that immediately after injury, when the expression of endogenous SPRY2 is decreased, hSPRY2 transduction is beneficial and attenuates neointima formation. The increased expression of SPRY2 several days after injury to the blood vessel is probably a compensatory mechanism to regulate the actions of growth factors and reduce neointima formation. A formal experimental evaluation of the latter suggestion requires specific knockdown or knockout of SPRY2 in intact arteries and forms the subject of our future investigations.
Acknowledgments
This work was supported by grant HL48308 from the National Institutes of Health to T.B.P. and a grant from the American Diabetes Association to C.Z. We are grateful to Dr Steven D. Dowdy, University of California, San Diego, for providing us with the pTAT-HA vector to make the desired constructs and for the pTAT-HA-GFP construct. We also thank Dr K.U. Malik, University of Tennessee, for providing VSMCs used in some of the experiments reported here.
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Correspondence to Tarun B. Patel, Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, 2160 S First Ave, Maywood, IL 60153. E-mail tpatel7@lumc.edu
Abstract
Objectives— To determine whether the human sprouty 2 (hSPRY2) protein, an inhibitor of receptor tyrosine kinase actions, regulates vascular smooth muscle cell (VSMC) proliferation, migration, and neointima formation in injured carotid artery.
Methods and Results— The hSPRY2 protein or green fluorescent protein (GFP; control) was transduced into VSMCs by placing an N-terminal TAT epitope on the proteins. The transduction of TAT-tagged hSPRY2 (TAT-hSPRY2) but not TAT-GFP inhibited the ability of serum and different growth factors to stimulate migration of VSMCs. Likewise, TAT-hSPRY2 also inhibited VSMC proliferation in response to serum. The hSPRY2 microtubule association (amino acids 123–177) and membrane translocation (amino acids 178–194) domains were necessary for the biological actions of hSPRY2. In the rat carotid artery injury model, exposure of the injured vessel for 1 hour to TAT-hSPRY2, but not TAT-GFP, markedly inhibited growth of the neointima over the 28-day postangioplasty period as well as VSMC proliferation. The exogenously applied TAT-hSPRY2 was retained in the carotid arteries for at least 3 days after injury, and endogenous SPRY2 expression was maximized around day 14 after injury. The latter is perhaps a compensatory mechanism to regulate neointima formation.
Conclusions— We conclude that TAT-tagged proteins are efficiently transduced into VSMCs in vitro and in vivo, that hSPRY2 inhibits growth and migration of VSMCs, and that this protein can decrease neointimal growth after blood vessel injury.
The aim of this study was to determine whether the human sprouty 2 (hSPRY2) protein, an inhibitor of receptor tyrosine kinase actions regulates vascular smooth muscle cell (VSMC) proliferation, migration and neointima formation in injured carotid artery. We conclude that TAT-tagged proteins are efficiently transduced into VSMCs in vitro and in vivo, that hSPRY2 inhibits growth and migration of VSMCs, and that this protein can decrease neointimal growth after blood vessel injury.
Key Words: sprouty ? vascular smooth muscle cells ? proliferation ? migration ? growth factors ? carotid artery injury ? neointima formation
Introduction
The sprouty (SPRY) family of proteins has been shown to inhibit the actions of receptor tyrosine kinases and regulate tracheal branching,1,2 as well as cellular migration, proliferation, and angiogenesis.3–5 To date, 4 isoforms of mammalian SPRY proteins have been cloned.1,2,6,7 These proteins have a highly conserved, cysteine-rich, C terminus, but their N terminus is variable. SPRY1, SPRY2, and SPRY4 are expressed during development of the embryo and in several adult tissues, including kidney, lung, brain, heart, and skeletal muscle.7 On the other hand, SPRY3 mRNA is present in the adult brain and testis.7 As observed initially in the Drosophila,1 in mouse, a decrease in endogenous SPRY2 expression by antisense oligonucleotide approach resulted in increased lung branching morphogenesis.2 These findings suggest that SPRY proteins have conserved function to modulate respiratory morphogenesis. The ability of mouse SPRY4 to inhibit angiogenesis3 also demonstrates that the SPRY proteins play a profound role in regulating tubular morphogenesis.
At the cellular level, we and others have shown that SPRY1,4 SPRY2,5 and SPRY43 inhibit migration and proliferation of cells in response to serum and growth factors. Treatment of cells with epidermal growth factor (EGF) results in translocation of the human SPRY2 (hSPRY2) protein from the vicinity of microtubules to membrane ruffles.5,8 We have shown that abrogating the colocalization of hSPRY2 with microtubules or deletion of the region that is necessary for translocation to membrane ruffles obliterates the ability of the protein to inhibit cell migration and proliferation.5 We have also shown that hSPRY2 expression alters the cellular distribution of protein tyrosine phosphatase 1B and that the increase in amount and activity of the cytosolic form of this protein, at least in part, mediates the antimigratory actions of hSPRY2.9 More recently, we reported that the antimigratory actions of hSPRY2 also involve inhibition of Rac1 activation.10
Angiogenesis plays a critical role during embryonic development, tissue growth, or regeneration, and efficient growth of tumors.11 Whereas the role of endothelial cells in angiogenesis is well established, it is also becoming increasingly evident that proliferation and migration of vascular smooth muscle/perivascular cells play an important role in ensuring endothelial cell survival and vasculogenesis.12–14 In addition, the proliferative response of vascular smooth muscle cells (VSMCs) has an important role in recovery from injury as well as atherosclerosis, hypertension, and postangioplasty restenosis.15 Although SPRY4 has been shown to inhibit the migration and proliferation of human umbilical vascular endothelial cells,3 whether SPRY proteins affect the growth and migration of VSMCs remains to be determined. Likewise, whether or not SPRY protein can modulate neointima formation after injury to the blood vessel has also not been investigated. Therefore, in this report, we examined the effects of hSPRY2 on the growth and migration of rat aortic VSMCs. Our data demonstrate that hSPRY2 inhibits the growth factor–elicited migration and proliferation of VSMCs, and the SPRY translocation domain is necessary for this biological action of hSPRY2. Moreover, using the carotid artery balloon injury model in intact rats, we show that hSPRY2 can decrease the growth of the neointima and decrease cellular proliferation. Interestingly, the amount of endogenous rat homologue of SPRY2 is increased after balloon injury to the carotid arteries. This increase in expression of SPRY2 is probably a compensatory mechanism to modulate actions of growth factors.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
A detailed Materials and Methods section is available online at http://atvb.ahajournals.org.
Results and Discussion
Transduction of VSMCs With TAT-Tagged Proteins
Because it is difficult to achieve a high transfection efficiency in VSMCs, we used the strategy of introducing hSPRY2 into cells by tagging the protein with an 11-aa sequence (YGRKKRRQRRR) from the TAT protein of HIV. This permits transduction of the protein of interest into >99% of a variety of cells,5,16–20 including VSMCs (see Figure I in online Materials and Methods section).
Inhibition of VSMC Migration and Proliferation by TAT-HA-hSPRY2
It is well established that the migration and proliferation of VSMCs in response to growth factors play a critical role in angiogenesis12–14 as well the development of atherosclerosis and vascular stenosis.21 To investigate the role of hSPRY2 in modulating VSMC migration in response to various growth factors, cellular motogenesis in response to serum was measured using a monolayer-wounding protocol22 (see Figure II in online Methods and Materials section for details and representative figure of the wound healing assay). After serum starvation, confluent VSMCs were treated for 1 hour with increasing concentrations of TAT protein (0, 1, 2, 5, 10, and 20 μg/mL), and migration in response to 10% serum was monitored as described in the online Materials and Methods section. Controls were performed in which cells were treated with TAT–green fluorescent protein (GFP; 10 μg/mL; "0" TAT-tagged hSPRY2 [TAT-hSPRY2] in Figure 1A). As shown in Figure 1A, for VSMCs treated with 10 μg/mL TAT-GFP as a control ("0" TAT-hSPRY2), the wounds closed an average of 84.7±3.6%. Treatment with TAT-hSPRY2 in a concentration-dependent manner inhibited VSMC migration in response to serum. In all subsequent experiments, TAT-GFP and TAT-hSPRY2 were used at a concentration of 10 μg/mL. Next, we investigated whether the VSMC migration in response to different growth factors was also modulated by hSPRY2. As shown in Figure 1B, serum, EGF, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) stimulated migration of control untransduced cells over basal values (reported in figure legend). The transduction of VSMCs with TAT-GFP did not alter migration in response to either serum or any of the growth factors, demonstrating that the TAT-tag does not by itself modulate cell migration. However, in cells transduced with TAT-hSPRY2, cell migration in response to serum and all the growth factors tested was attenuated (Figure 1B). Similar results were observed using the Boyden Chamber assay (Figure III, available online at http://atvb.ahajournals.org). These findings are consistent with our previous observations that serum and growth factor–stimulated migration of human cancer cell line (HeLa) and rat intestinal epithelial cell line (IEC-6) is also inhibited by hSPRY25,9,10 and that the antimigratory actions of hSPRY2 are mediated at some downstream locus/loci in the promigratory pathway, where signals from a number of different growth factors culminate.
Figure 1. TAT-hSPRY2 inhibits growth factor– and serum-stimulated migration and proliferation of rat aortic VSMCs. A, Confluent monolayers of VSMCs were exposed to different concentrations of TAT-hSPRY2 or 10 μg/mL of TAT-GFP (0 TAT-hSPRY2) for 1 hour before making scratch wounds and monitoring migration in response to serum (10%), as described in the online Materials and Methods section. Closure of the wounds in the absence of serum was 22.5±2.0% (not shown on graph). Data are the mean±SEM of 3 experiments (6 different fields for each condition). *P<0.001 compared with control; Student unpaired t test. B, Same as A, except migration of cells in response to serum (10%) or EGF (100 nmol/L), or FGF (150 ng/mL) or PDGF (10 ng/mL) was studied as detailed in the online Materials and Methods section. In absence of serum or growth factors, the percent closure of wounds under different conditions were: no addition, 19.8±1.5%; TAT-hSPRY2, 20.2±2.3%; TAT-GFP, 21.6±3.0%. Values presented are the mean±SEM of 3 experiments (6 different fields/condition). *P<0.001 compared with the corresponding condition in untreated controls or in cells treated with TAT-GFP; Student unpaired t test. C, VSMCs (105/dish) were grown in serum (10%) containing medium supplemented with 10 μg/mL each of TAT-GFP or TAT-hSPRY2, or hSPRY2123 to 177 or TAT-hSPRY2178 to 194. Cells were counted at different time points. The medium supplemented with TAT-tagged proteins was changed every 24 hours. The mean±SEM of 3 different experiments is shown. *P<0.05 and **P<0.01 compared with TAT-GFP (control) at the same time points; Student unpaired t test. D, Same as B, except that in addition to TAT-hSPRY2 and TAT-GFP, cells were also exposed to 10 μg/mL each of TAT-hSPRY2123 to 177 or TAT-hSPRY2178 to 194. Migration in response to serum (10%) was monitored. The mean±SEM of 3 separate experiments is shown.
Like migration, serum-induced VSMC proliferation was also markedly inhibited in cells that had been transduced with TAT-hSPRY2 compared with TAT-GFP (Figure 1C), indicating that hSPRY2 inhibits the proliferation and migration of VSMCs.
The SPRY2 protein contains 2 domains that have been shown to be necessary for the association of the protein with microtubules (amino acids 123–177) and translocation of the protein to membrane ruffles in response to growth factors (amino acids 178–194).5,8 We showed previously that the deletion of either of these regions in the hSPRY2 protein abolishes its ability to inhibit migration and proliferation of HeLa cells.5 To test the generality of our previous findings, we determined whether transduction of VSMCs with TAT-hSPRY2 harboring either of the 2 deletions altered its ability to inhibit cell migration and proliferation. Figure 1C and 1D show that the deletion of either the microtubule association domain (amino acids 123–177) or the translocation domain (amino acids 178–194) obliterated the ability of hSPRY2 to inhibit proliferation and migration of VSMCs in response to serum. These findings demonstrate that the microtubule association domain and translocation domain on hSPRY2 are necessary for its antimitogenic and antimotogenic actions in more than one cell type.
hSPRY2 Actions in Intact Animals
Having established that TAT-hSPRY2 can be conveniently transduced into VSMCs and that hSPRY2 inhibits the migration and proliferation of these cells, we investigated whether the TAT-hSPRY2 can be used to transduce cells in intact animals and whether hSPRY2 can inhibit neointima formation in injured blood vessels. The latter would test the in vivo significance of our in vitro findings with VSMCs. Essentially, using the rat carotid artery injury model (online Materials and Methods section), we investigated whether the transduction of TAT-hSPRY2 or TAT-GFP into cells after injury to the blood vessels decreased growth of the neointima. As shown by the representative cross-sections of carotid arteries in Figure 2Ai, the intima in the uninjured blood vessels is essentially made up of a single endothelial layer. Fourteen days after carotid artery injury, there was significant neointimal growth in the untreated injured artery (Figure 2Aii). The neointima (more granular layer) in these vessels can be separated from the media by the internal elastic lamina (Figure 2A, arrowheads, enlarged sections). The transduction of TAT-GFP did not alter this neointima formation (Figure 2Aiv). However, when the injured arteries had been transduced with TAT-hSPRY2 only once (at the time of the injury), the neointima formation was inhibited (Figure 2Aiii). Quantification of these data from carotid arteries at different times after injury demonstrated that the ability of hSPRY2 to inhibit neointima formation could be observed as early as 7 days after injury, and this inhibitory action was maintained up to 28 days after the intervention (Figure 2B). These findings demonstrate the antiproliferative and antimigratory actions of hSPRY2 in VSMCs that had been documented in the in vitro model (Figure 1) contribute to the attenuation of stenosis observed in the injured carotid artery in vivo. These findings also raise the interesting possibility of using SPRY2 to modulate VSMC migration and proliferation to, for example, attenuate restenosis after angioplasty.
Figure 2. TAT-hSPRY2 inhibits neointima formation in rat carotid arteries after balloon injury. Rat carotid arteries were subjected to balloon injury and then exposed to either vehicle (PBS) or 20 μg/mL each of TAT-GFP, or TAT-hSPRY2 for 1 hour, as described in the online Materials and Methods section. At 7, 14, and 28 days after injury, sections of the carotid arteries were analyzed. A, Representative photomicrographs (magnification x100) of hematoxylin-eosin (HE)–stained normal carotid artery and carotid arteries 14 days after injury. A higher magnification of sections in areas marked by boxes is also shown next to each panel. The methods described previously29 were used for the injury and morphometric analyses. The conditions are: (i) uninjured vessel; (ii) vehicle-treated injured vessel; (iii) injured vessel treated with TAT-hSPRY2 (20 μg/mL); and (iv) injured vessel treated with TAT-GFP (20 μg/mL). The internal elastic lamina (depicted by arrows) separates the intima (I) and media (M). B, Bar graph representing the mean±SEM (n=6) of the ratios of intimal/medial areas in sections of carotid arteries at 7, 14, and 28 days after balloon injury. *P<0.05 vs TAT-GFP and vehicle groups.
TAT-hSPRY2 Retention and Expression of Endogenous SPRY2 After Carotid Artery Injury
Because a single exposure for 1 hour to TAT-hSPRY2 inhibited neointimal formation for up to 28 days after injury, by immunohistochemistry and Western analyses, we investigated how long the TAT-SPRY2 was retained in the blood vessels. As shown in Figure 3B (bottom row), there was a significantly higher amount of SPRY2 staining (brown) in the TAT-hSPRY2–treated arteries compared with vehicle-treated arteries on days 1 and 3 after injury. Moreover, the amount of SPRY2 increased between 7 and 14 days after injury (Figure 3B, bottom row). Western blotting of the arteries that had been treated with a single exposure to TAT-hSPRY2 with anti-hemagglutinin (anti-HA) antibody that recognizes the HA epitope in TAT-hSPRY2 confirmed that the TAT-hSPRY2 was retained in the vessels for 3 days (Figure 3C). Thus, the TAT-SPRY2 is retained in the arteries for at least 3 days after transduction, and between 7 and 14 days after balloon injury, there may be an increase in the expression of endogenous SPRY2 (Figure 3B). The latter notion is confirmed by the findings that in the vehicle-treated arteries, there was increased SPRY2 staining at 14 days after injury; this increase was retained even at 28 days after balloon injury (Figure 3B, top row). That the expression of endogenous SPRY2 is increased at 14 days after injury is also confirmed by Western blotting (Figure 3A). The endogenous SPRY2 is expressed primarily in the neointima, with lesser amounts being present in the medial layer (Figure 3B). In these experiments, we ensured that the anti-SPRY2 antibody was specifically recognizing the SPRY2 protein by neutralizing the antibody with its peptide epitope (NTZ antibody; Figure 3B). An intriguing finding is that although uninjured arteries express SPRY2, their expression is decreased immediately after injury and then increased between 7 and 14 days after carotid artery injury (Figure 3A).
Figure 3. Retention of TAT-SPRY2 and expression of endogenous SPRY2 in injured carotid arteries. Carotid arteries that had been exposed to either vehicle (PBS) or TAT-hSPRY2 after injury were isolated at various indicated times after balloon injury and subjected to Western and immunohistochemical analyses. A, Western analyses with anti-SPRY2 antibody, loading control with anti-actin antibody is also shown in bottom panel. B, Sections of arteries were stained with hematoxylin eosin (violet/blue) for nuclei (depicted by arrows pointing up) and anti-SPRY2 antibody (brown and shown by the black downward-pointing arrows). The black bar on the left of each panel depicts the intima. The medial layer is labeled "M." Representative immunohistochemical photomicrographs (x400) from slices (5-μm thick) of carotid arteries at different time points are shown. Representatives of at least 3 different sections from different animals are shown. C, Western analyses of injured carotid arteries that had been exposed to TAT-hSPRY2. Proteins (30 μg each) were separated on SDS-PAGE gels and analyzed by Western blotting using anti-HA antibody that recognizes TAT-hSPRY2. Note the presence of the TAT-hSPRY2 up to 3 days after injury. NA indicates normal artery; NTZ Ab, neutralized antibody.
To determine whether the expression of SPRY2 in the injured blood vessels was in VSMCs or another cell type, we used sections of carotid arteries that had been injured and treated with vehicle only (PBS). In these arteries, 14 days after injury, the SPRY2 and the vascular smooth muscle–specific -actin were visualized using fluorescent secondary antibodies. Controls in the absence of primary antibodies were also performed. As shown in Figure 4, in the injured blood vessels, hSPRY2 was localized mainly in the neointima and in the medial layer. Most importantly, the SPRY2 was colocalized with the smooth muscle–specific -actin, demonstrating that the increased SPRY2 expression after injury is indeed in VSMCs. The small amount of coexpression of SPRY2 and smooth muscle–specific -actin in the adventitia may be attributable to the presence of myofibroblasts in this region after injury.23
Figure 4. The endogenous SPRY2 after injury to carotid arteries is localized in smooth muscle cells of arteries. Fourteen days after balloon injury, sections of carotid arteries exposed to vehicle (PBS) were stained for smooth muscle–specific -actin and SPRY2 as described in the online Materials and Methods section. Controls shown at x400 magnification for SPRY2, and -actin were performed without the primary antibody. Photomicrographs of the same section at x200 and x400 are shown separately and then merged (bottom panel on right side). The area of the x200 field that is visualized at x400 magnification is shown by rectangles. The lumen of the artery is seen at x200 magnification and marked "L." The neointima and medial layers are depicted as "I" and "M," respectively. A schematic demonstrating how SPRY2 expression may be altered by growth factors is also presented (see text for discussion).
Growth factors have been shown to stimulate the expression of SPRY proteins.1,24,25 Hence, it is possible that in the injured blood vessel when the cells are under the influence of greater concentrations of growth factors (no barrier from endothelium), the expression of SPRY2 may be enhanced, and the increased expression of SPRY2 after injury may serve as a negative feedback to control the biological actions of growth factors. This possibility is presented by the schematic in Figure 4. The schematic also incorporates the possibility that SPRY2, by inhibiting the action of growth factors, may regulate its own expression (indicated by "?" in schematic, Figure 4). Although the increase in SPRY2 may, to some extent, oppose the actions of growth factors in modulating growth and migration of VSMCs, in controls (vehicle- or TAT-GFP–treated) the lack of significant amount of hSPRY2 in the initial days after the injury may be the determining difference in the extent of neointima formation compared with TAT-hSPRY2–treated arteries. In this context, the decrease in SPRY2 expression immediately after injury (Figure 3A) may be deliterious, and the transduction of additional SPRY2 during the initial days after injury to the blood vessel, when endogenous SPRY2 expression is decreased, is clearly beneficial and able to attenuate neointima formation (Figure 2). This latter notion is also supported by the findings of Mori et al26 that the early administration of estrogen protects against neointima formation in injured arteries.
To determine whether the decrease in neointimal growth in TAT-hSPRY2–treated animals was indeed attributable to the decrease in cellular proliferation, sections of injured carotid arteries exposed to either TAT-hSPRY2 or vehicle were stained for proliferative cell nuclear antigen (PCNA) expression. As shown in Figure 5A, 14 days after injury, the proportion of PCNA-positive cells in TAT-hSPRY2–treated arteries was decreased compared with controls. Thus, as observed in vitro with VSMCs (Figure 1), proliferation of cells in vivo is also decreased by hSPRY2 (Figure 5A).
Figure 5. TAT-hSPRY2 decreases cell proliferation in injured carotid arteries but does not modulate leukocyte infiltration. Fourteen days after injury, sections of carotid arteries that had been treated with vehicle (PBS) or TAT-GFP were stained with anti-PCNA antibody (brown) a biomarker for proliferating cells and hematoxylin (A) or anti-CD45 antibody (brown) and hematoxylin (B). The bar graph in A depicts VSMC proliferation index that was calculated by the following formula: [(the number of PCNA-positive cells)/(total cells stained by hematoxylin)x100] for each section. Values are mean±SEM (n=6), and statistical significance is shown. Likewise, the bar graph in B shows the percent area occupied by CD45-positive cells (leukocytes). Mean±SEM from 6 experiments is shown.
Vascular injury is also associated with infiltration of inflammatory cells such as leukocytes.27,28 To determine whether the inflammatory response was also altered by transduction of hSPRY2 into the injured blood vessels, we monitored the infiltration of leukocytes using anti-CD45 antibody. The data in Figure 5B demonstrate that at 14 days after injury to the carotid arteries, leukocyte infiltration was not altered. Please note that we have shown previously, even at 14 days after injury, infiltration of leukocytes in the injured arteries is significantly higher than controls.28 Thus, it would appear that hSPRY2 does not alter the inflammatory response after injury to the carotid arteries. Whether or not SPRY2 also modulates extracellular matrix synthesis during restenosis is not addressed by our studies, and this subject will be investigated in the future.
In summary, we have shown that hSPRY2 inhibits the migration and proliferation of VSMCs in vitro and in vivo, and that the transduction of TAT-hSPRY2 into the injured carotid artery significantly decreases neointima formation. Although the infiltration of inflammatory cells into the injured area is not altered by hSPRY2, these findings suggest that SPRY2 may be useful in preventing neointima formation in pathologic states such as restenosis after angioplasty. Moreover, we show that immediately after injury, when the expression of endogenous SPRY2 is decreased, hSPRY2 transduction is beneficial and attenuates neointima formation. The increased expression of SPRY2 several days after injury to the blood vessel is probably a compensatory mechanism to regulate the actions of growth factors and reduce neointima formation. A formal experimental evaluation of the latter suggestion requires specific knockdown or knockout of SPRY2 in intact arteries and forms the subject of our future investigations.
Acknowledgments
This work was supported by grant HL48308 from the National Institutes of Health to T.B.P. and a grant from the American Diabetes Association to C.Z. We are grateful to Dr Steven D. Dowdy, University of California, San Diego, for providing us with the pTAT-HA vector to make the desired constructs and for the pTAT-HA-GFP construct. We also thank Dr K.U. Malik, University of Tennessee, for providing VSMCs used in some of the experiments reported here.
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