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. Author manuscript; available in PMC: 2012 Jun 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2011 Mar 17;31(6):1300–1308. doi: 10.1161/ATVBAHA.111.223701

SDF-1α Induction in Mature Smooth Muscle Cells by Inactivation of PTEN is a Critical Mediator of Exacerbated Injury-Induced Neointima Formation

Raphael A Nemenoff 1,2, Henrick Horita 1, Allison C Ostriker 1, Seth B Furgeson 1, Peter A Simpson 1, Vicki VanPutten 1, Joseph Crossno 2, Stefan Offermanns 4, Mary CM Weiser-Evans 1,2,3
PMCID: PMC3111081  NIHMSID: NIHMS286081  PMID: 21415388

Abstract

Objective

PTEN inactivation selectively in smooth muscle cells (SMC) initiates multiple downstream events driving neointima formation, including SMC cytokine/chemokine production, in particular SDF-1α. We investigated the effects of SDF-1α on resident SMC and bone marrow-derived cells and in mediating neointima formation.

Methods and Results

Inducible, SMC-specific PTEN knockout mice (PTEN iKO) were bred to floxed-stop ROSA26-βGal mice to fate-map mature SMC in response to injury; mice received wild-type GFP-labeled bone marrow to track recruitment. Following wire-induced femoral artery injury, βGal(+) SMC accumulate in the intima and adventitia. Compared to wild-type, PTEN iKO mice exhibit massive neointima formation, increased replicating intimal and medial βGal(+)SMC, and enhanced vascular recruitment of bone marrow cells following injury. Inhibiting SDF-1α blocks these events and reverses enhanced neointima formation observed in PTEN iKO mice. Most recruited GFP(+) cells stain positive for macrophage markers, but not SMC markers. SMC-macrophage interactions result in a persistent SMC inflammatory phenotype that is dependent on SMC PTEN and SDF-1α expression.

Conclusions

Resident SMC play a multifaceted role in neointima formation by contributing the majority of neointimal cells, regulating recruitment of inflammatory cells, and contributing to adventitial remodeling. The SMC PTEN-SDF-1α axis is a critical regulator of these events.

Keywords: smooth muscle, macrophage, restenosis, PTEN, stromal cell-derived factor-1alpha

Restenosis, a wound healing response characterized by unchecked proliferation of resident SMC and vascular accumulation of inflammatory cells, is a major limitation of percutaneous angioplasty procedures. Resident SMC play a multifaceted role in the progression of this pathology. Under physiological conditions, SMC express a highly quiescent, differentiated phenotype. Activation of SMC in response to injury promotes a transition to a highly proliferative, inflammatory phenotype characterized by downregulation of SM contractile markers and increased production of multiple cytokines and chemokines (ie. activated phenotype)1. Many of these factors participate in the remodeling process through direct effects on SMC and through recruitment of inflammatory cells, which sustains progression of lesion formation2,3. Therefore, existing evidence supports the concept that resident SMC are both initiators and effectors of the injury response.

The origin of SMC in intimal lesions has remained a controversial subject. Elegant studies from over 20 years ago provided strong evidence that large numbers of differentiated, medial SMC enter the cell cycle in response to arterial injury, migrate and proliferate thereby contributing the majority of intimal SMC4-6. This long-held theory recently has been challenged by several studies suggesting transdifferentiation of bone marrow-derived progenitors to SMC during vascular repair contributes a significant percentage of intimal SMC7,8. It is clear that bone marrow-derived cells are recruited to injured vessels, although their ability to transdifferentiate to SMC, typically defined by SM-α-actin expression, is under dispute9-12. In fact, a recent report that fate-mapped the time course of bone marrow cell accumulation on injured vessels showed that differentiation into SMC is a rare event9. In contrast, these cells express a macrophage phenotype, although whether their contribution to neointima formation is good or bad remains unclear. From a clinical standpoint, the origin of intimal SMC, the role of resident, differentiated SMC vis-à-vis bone marrow-derived cells to vessel remodeling, and the underlying molecular signals regulating pathological neointima formation are important issues for the design of more effective therapeutics to control restenosis.

PTEN, a dual-specificity lipid and protein phosphatase, functions to suppress multiple signaling networks involved in cellular proliferation, survival, and inflammation13. Tight regulation of PTEN levels and activity is essential for the maintenance of normal physiological states14. PTEN is susceptible to positive and negative transcriptional regulation, post-transcriptional inhibition through specific micro-RNAs, and post-translational regulation by phosphorylation and oxidation leading to inactivation of its phosphatase activity15,16. PTEN oxidation is particularly important in pathological situations characterized by chronic vascular oxidative stress (e,g. diabetes), which would lead to persistent inactivation of PTEN17. Importantly, while multiple stimuli have been reported to promote an activated, inflammatory SMC phenotype, the underlying molecular programs actively repressing this remain unclear. Our group and others have demonstrated that regulation of PTEN signaling in SMC plays a critical role in pathological vascular remodeling18-22. Notably, PTEN negatively regulates SMC phenotypic modulation with loss of PTEN in SMC associated with multiple downstream events regulating neointima formation, including promotion of an activated SMC phenotype18. In addition, our previous work demonstrated that molecular depletion of PTEN in SMC establishes an autocrine growth loop through induction of the chemokine SDF-1α22. Others have demonstrated a critical role for SDF-1α induction in the progression of neointima formation23-26. However direct in vivo effects of increased SDF-1α produced by SMC as a consequence of SMC PTEN loss on resident smooth muscle cells, and their precise role in injury-induced neointima formation has not been examined. Here we used an innovative in vivo approach to fate-map the contribution of highly differentiated resident SMC compared to bone marrow-derived progenitor cells to wire-induced neointima formation and to define the role of SMC PTEN-induced SDF-1α production in this response.

MATERIALS AND METHODS

PTENflox/flox mice (Dr. Tak Mak, Ontario Cancer Institute, University of Toronto, Toronto, Ontario)27, SMMHC-CreERT2 transgenic mice (Dr. Stephen Offermanns, U. Heidelberg, Heidelberg, Ger)28, and ROSA26 reporter (R26R) mice (Jackson Laboratory) were bred to generate tamoxifen-inducible SMC-specific PTEN knockout mice carrying the R26R allele (PTEN iKO). Controls expressed SMMHC-CreERT2 and R26R, but were wild type for PTEN (WT). Mice received bone marrow transplants using transgenic UBI-EGFP donor mice. Mice received 1 mg I.P. tamoxifen injections for 5 consecutive days one week prior to femoral artery wire-induced injury29. For SDF-1⟨ neutralization, mice received 100 μg I.P. control IgG or neutralizing SDF-1⟨ antibody (clone 79014; R&D Systems) 24 hours before and immediately following injury; mice received additional 50 μg I.P. injections twice weekly for the duration of the experiment. Mice were maintained in the Center for Comparative Medicine and procedures performed under a protocol approved by the Institutional Animal Care and Use Committee at the University of Colorado Denver. Morphometric analysis, Immunohistochemistry, SMC-macrophage co-culture, quantitative RT-PCR, Western analysis, and ELISA were performed as described previously18,22,30. Full Materials and Methods are detailed in the Supplement (http://atvb.ahajournals.org).

RESULTS

Generation of Inducible SMC-Specific PTEN Null Mice

Inducible PTEN iKO mice were generated by crossing PTENflox/flox mice with transgenic mice expressing tamoxifen-inducible Cre recombinase fused to a mutant ligand-binding domain of the estrogen receptor (CreERT2) under the control of the murine SMMHC promoter28. Wild type controls expressed CreERT2, but were wild type for PTEN+/+ (WT). To fate-map differentiated SMC in response to vascular injury, mice were crossed with floxed-stop ROSA26-LacZ reporter mice. Prior to tamoxifen and injury, mice were transplanted with bone marrow from eGFP transgenic mice to track the accumulation of bone marrow-derived cells to neointima formation. Flow cytometry of peripheral blood mononuclear cells 10 weeks after transplantation showed that >90% of circulating mononuclear cells were GFP(+)(not shown). Mice received tamoxifen once daily for 5 days prior to vascular injury, which genetically and permanently marked SMMHC-expressing SMC by tamoxifen-induced Cre-mediated βGal knock-in. Since tamoxifen was administered before injury and then stopped, SMC expressing SMMHC at the time of injections and prior to injury and their progeny were the only cells labeled with βGal throughout the experimental time period. This allowed fate mapping of differentiated SMC in response to vascular injury even if these cells subsequently lost SM markers. Following tamoxifen administration and prior to injury, βGal expression was induced in virtually 100% of arterial medial SMC and was specific to SMC; no labeled cells were detected in the absence of tamoxifen (Supplemental Figure IA-C). Tamoxifen resulted in efficient depletion of PTEN and a corresponding increase in phospho-Akt from major arteries of PTEN iKO mice compared to wild type (Supplemental Figure ID).

Enhanced Neointima Formation in PTEN iKO Mice and Role of Resident Mature SMC

To assess the contribution of differentiated SMC to vessel remodeling, appearance of βGal(+) cells was examined in the intima, media, and adventitia of injured arteries. βGal(+) cells populated the media and accumulated in the intima and adventitia at 7-days post-injury in both WT and PTEN iKO mice (Figure 1A and Supplemental Figure II), establishing that migration of differentiated medial SMC is a major contributor to neointima formation and furthermore that these cells are surprisingly found in the adventitia. Double staining for βGal and α-SMA revealed that most βGal(+) intimal SMC co-expressed α-SMA, although some βGal(+);α-SMA(−) SMC were detected (Figure 1A). Levels of α-SMA were generally lower in the PTEN iKO mice. In addition, we consistently detected βGal(+) cells in the adventitia after injury; all βGal(+) adventitial cells were α-SMA(−)(Figure 1A). To test whether loss of PTEN selectively in mature SMC enhances neointima formation, WT and PTEN iKO mice were subjected to wire-induced femoral artery injury following tamoxifen (Figure 1B). Injury to PTEN iKO mice resulted in the development of a larger neointima at 3 weeks post-injury compared to WT mice (Figure 1B). Morphometric measurements confirmed 5-fold and 3-fold increases in intima-to-media ratio and percent stenosis, respectively, in PTEN iKO mice compared to controls (Figure 1B). The increase in neointima size in PTEN iKO mice was associated with increased numbers of replicating cells in the arterial intima and media (Figure 1C and Supplemental Figure IIIA). Double staining for BrdU and βGal showed that the majority of replicating intimal and medial cells in WT and iKO mice were βGal(+) (Figure 1C and Supplemental Figure IIIB) indicating that mature SMC contribute to the bulk of replication in these arterial compartments. Replication of mature SMC accounted for only a percentage of replicating adventitial cells (Figure 1C). These data thus confirm that mature SMC contribute to intimal, medial, and adventitial remodeling and that loss of PTEN exacerbates their proliferation.

Figure 1. Highly Differentiated SMC Contribute to Injury-Induced Vessel Remodeling and Enhanced Neointima Formation in PTEN iKO Mice.

Figure 1

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(A) Immunofluorescence staining for βGal and α-SMA on 7-d injured femoral arteries from WT and PTEN iKO mice. Arrows = representative intimal βGal(+);α-SMA(+) SMC; arrowheads = representative adventitial βGal(+) SMC; open arrowheads = representative intimal βGal(+);α-SMA(−) SMC; lines delineate the arterial media. (B) (Top) Time course of experimental protocol. H&E staining of representative arterial lesions in WT and PTEN iKO mice. Medial and intimal areas were measured using SPOT software. Intima-to-media ratios (left) and percent stenotic areas (right) are presented in the graphs. (C) Total numbers of BrdU(+) compared to βGal(+);BrdU(+) SMC in the arterial intima, media, and adventitia at 3-w post-injury were counted separately and the data reported in the graphs. *Different from WT; n=8.

Bone Marrow-derived Progenitors are Recruited to Injured Vessels, but do not Differentiate into SMC

To determine the contribution of bone marrow-derived progenitors to neointima formation, all mice received bone marrow from eGFP transgenic mice 6-wk prior to tamoxifen injections and vascular injury. At 3-wk post-injury, we detected abundant numbers of GFP(+) cells in all arterial compartments of WT and PTEN iKO mice. Consistent with a previous report9, double staining for GFP and α-SMA revealed few, if any, GFP(+);α-SMA(+) cells (Figure 2A); no GFP(+);SMMHC(+) cells were detected (not shown). Spatially distinct regions of either GFP(+) or α-SMA(+) cells were detected, indicating that recruited bone marrow cells do not differentiate into SMC. In addition, although an occasional GFP(+) bone marrow cell was found in the media of uninjured arteries, none of these medial GFP(+) cells co-expressed βGal following tamoxifen administration (Figure 2B), confirming that bone marrow cells do not serve as SMC progenitors. In contrast to α-SMA, the large majority of GFP(+) cells co-expressed the macrophage marker, Mac3 (Figure 2C and Supplemental Figure IV). GFP(+) macrophage recruitment to injured vessels was increased in the intima and media of injured vessels from PTEN iKO mice compared to WT (Figure 3A); however macrophage replication accounted for only a small number of replicating cells in these vessel areas (Figure 3B). In contrast, increased numbers of replicating macrophages were detected in the adventitia of PTEN iKO mice compared to WT (Figure 3B). Therefore, bone marrow-derived macrophages contribute to injury-induced vascular remodeling likely through crosstalk with SMC rather than through differentiation into SMC. PTEN loss in mature SMC augments macrophage recruitment and retention in injured vessels.

Figure 2. Bone Marrow-Derived Cells are Recruited to Injured Vessels, but do not Differentiate into SMC.

Figure 2

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(A) Immunofluorescence staining for GFP and α-SMA on 3-w injured femoral arteries from WT and PTEN iKO mice. N=Neointima; M=Media; A=Adventitia. (B) Immunofluorescence staining for βGal and GFP on an uninjured femoral artery. Right panel shows merged image. Arrows = GFP(+);βGal(−) cell in the arterial media; arrowheads = internal elastic lamina. (C) Immunofluorescence staining for GFP and Mac3 on 3-w injured femoral arteries from WT (top panels) and PTEN iKO (bottom panels) mice. Right panels show merged images. Scale bars A&C = 20 μm.

Figure 3. Increased Accumulation of Bone Marrow-Derived Macrophages in Injured Vessels from PTEN iKO Mice.

Figure 3

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(A) Immunofluorescence staining for GFP on 3-w injured femoral arteries from WT and PTEN iKO mice. Total numbers of GFP(+) cells in the arterial intima, media, and adventitia were counted separately and the data reported in the graphs. (B) Immunofluorescence staining for GFP and BrdU on 3-w injured femoral arteries from WT and PTEN iKO mice. GFP(+);BrdU(+) cells in the arterial intima, media, and adventitia were counted separately and the data reported in the graphs. Arrows = representative GFP(+);BrdU(+) cells; arrowheads = representative GFP(−);BrdU(+) cells. *Different from WT; n=8. N=Neointima; M=Media; A=Adventitia.

Enhanced Neointima Formation in PTEN iKO Mice is Dependent on SDF-1α

We previously showed that loss of PTEN in SMC results in induction of the chemokine, SDF-1α18,22 and others have implicated SDF-1α in the mobilization and recruitment of bone marrow-derived smooth muscle progenitor cells following vascular injury25,26. Compared to WT, SDF-1α mRNA and protein were increased in injured arteries (Figure 4A) as well as in serum (Figure 4B) from PTEN iKO mice. Mice were treated with neutralizing antibodies against SDF-1α to determine its role in mediating increased neointima formation. Blocking SDF-1α reversed increases in I/M ratio and percent stenosis observed in PTEN iKO mice compared to WT mice (Figure 4C). Inhibition of neointima formation was associated with attenuated proliferation of resident βGal(+) SMC in the intima, media, and adventitia of PTEN iKO mice (Figure 5A). These in vivo findings are consistent with our earlier cell culture studies showing that SDF-1α induction establishes an autocrine growth loop driving SMC proliferation22. In addition to effects on SMC, SDF-1α neutralization also blocked accumulation of GFP(+) bone marrow-derived cells in the intima and media and proliferation of adventitial GFP(+) cells (Figure 5B&C and Supplemental Figure V). These findings support the concept that induction of SDF-1α is a major regulator of exacerbated neointima formation mediated by loss of PTEN signaling through profound effects on resident SMC proliferation combined with inflammatory cell accumulation.

Figure 4. Enhanced Neointima Formation in PTEN iKO mice is Dependent on SDF-1α.

Figure 4

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(A) qPCR analysis for SDF-1α mRNA in injured femoral arteries from WT and PTEN iKO mice. β-Actin was used for normalization of cDNA. (B) Serum from 7-d and 3-w post-injured WT and PTEN iKO mice was analyzed by ELISA for SDF-1α levels. (C) (Top) Timeline of experimental protocol. H&E staining on 3-w injured femoral arteries from WT and PTEN iKO mice treated with control or SDF-1α neutralizing antibodies. Intima-to-media ratios (left) and percent stenotic areas (right) were calculated and are presented in the graphs. *Different from WT; **PTEN iKO + αSDF-1α different from PTEN iKO + control IgG; n=6.

Figure 5. Increased SMC Proliferation and Accumulation of Bone Marrow-Derived Macrophages in PTEN iKO mice is Dependent on SDF-1α.

Figure 5

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Mice were subjected to the experimental protocol outlined in Figure 4. (A) Total numbers of BrdU(+) cells compared to βGal(+);BrdU(+) SMC in the arterial intima, media, and adventitia at 3-w post-injury were counted separately and the data reported in the graphs. (B) Total numbers of GFP(+) cells in the arterial intima, media, and adventitia were counted separately and the data reported in the graphs. (C) GFP(+);BrdU(+) cells in the arterial intima, media, and adventitia were counted separately and the data reported in the graphs. *Different from WT; **PTEN iKO + αSDF-1α different from PTEN iKO + control IgG; n=6.

Persistent Inflammatory Environment is Promoted Through Crosstalk Between SMC and Macrophages and is Dependent on SDF-1α

Our data suggest that SMC-macrophage crosstalk contributes to enhanced vascular remodeling. To examine this in vitro, bone marrow–derived cells isolated from wild type mice were cultured in the presence of M-CSF to promote macrophage maturation as previously described30. These cells have the morphology of macrophages and are >95% F4/80 positive (not shown). Co-cultures of WT or PTEN-depleted SMC with WT macrophages were used to analyze the effects of interactions on macrophage adhesion to SMC, cytokine production by SMC, and macrophage-mediated SMC proliferation. As shown previously18, PTEN-depleted SMC exhibit an activated, inflammatory phenotype, characterized by increased Akt phosphorylation and cytokine production (Supplemental Figure VI). We examined if this activated phenotype enhances macrophage adhesion and found increased numbers of WT macrophages adhered to PTEN-depleted SMC compared to WT SMC (Figure 6A). To determine if SMC-macrophage interactions perpetuate an inflammatory response, macrophages were co-cultured with WT or PTEN–depleted SMC using Transwells, which allows diffusible mediators to act on each cell type. Co-culture of macrophages with WT SMC resulted in induction of SDF-1α, MCP-1, and KC mRNAs, which correlated with decreased PTEN mRNA expression by SMC (Figure 6B); cytokine production was further enhanced in co-cultures of macrophages with PTEN-depleted cells (Figure 6B). Co-culture of macrophages with WT SMC increased SMC proliferation under basal conditions (Figure 6C). While PTEN-depleted SMC cultured under basal conditions exhibited higher rates of proliferation compared to WT, co-culture with macrophages significantly enhanced their proliferation (Figure 8C).

Figure 6. Persistent Inflammatory Environment is Promoted Through Crosstalk Between SMC and Macrophages and is Dependent on SDF-1α.

Figure 6

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Bone marrow–derived cells were cultured as described30. (A) Time course analysis of macrophage adhesion to WT or PTEN-depleted SMC. Representative images showing adhesion of macrophage to WT (a) or PTEN-depleted (b) SMC. Quantification of macrophage adhesion by total fluorescence (left) or total cell number (right). Shown are the means±SD from three independent experiments; p<0.05. (B&C) WT or PTEN-depleted SMC were co-cultured with WT macrophages. (B) qRT-PCR analysis for the indicated mRNAs. βActin was used for normalization of cDNA. Shown are fold changes in mRNA copy number from WT SMC alone; PTEN mRNA: means from three independent experiments; SDF-1α, MCP-1, and KC mRNAs: shown are one of three independent experiments. (C) BrdU immunocytochemistry for SMC proliferation under basal conditions (alone) or in co-culture with macrophages. Shown are the percent means±SE of BrdU-positive SMC of triplicates from one of three representative experiments. * = p<0.05; different from WT alone; ** = p<0.05; different from WT SMC + macrophages. (D) qRT-PCR analysis for MCP-1, IL-6, and KC mRNAs in WT SMC under basal conditions (Vehicle) or after 48 hr stimulation with 100 ng/ml rSDF-1α. βActin was used for normalization of cDNA. Shown are fold changes in mRNA copy number from vehicle; means±SE from three independent experiments; p<0.05.

SMC PTEN inactivation promotes induction of several cytokines (18 and Supplemental Figure VI). However, despite this, the present in vivo data suggests SDF-1α is a critical mediator of both the proliferative and inflammatory events associated with SMC PTEN loss. To determine if SDF-1α regulates SMC cytokine production and macrophage adhesion, WT SMC were stimulated with recombinant SDF-1α. MCP-1, IL-6, and KC mRNAs were induced in WT SMC stimulated with SDF-1α (Figure 6D), and this was associated with protein release into culture media (Supplemental Figure VIIA). Consistent with these in vitro findings, serum levels of MCP-1, IL-6, and KC were reduced in PTEN iKO mice treated with neutralizing anti-SDF-1α antibodies compared to control IgG-treated mice (Supplemental Figure VIIB). No change in macrophage adhesion was observed in SDF-1α-treated SMC compared to controls (data not shown). Collectively, these data suggest that SMC-macrophage crosstalk creates a persistent inflammatory SMC phenotype that is dependent on SDF-1α induction. Chronic SMC PTEN inactivation enhances these responses, which likely contribute to the exacerbated neointima formation observed in PTEN iKO mice.

DISCUSSION

Molecular changes in SMC initiate multiple events associated with restenosis. While numerous stimuli activate SMC, restenosis reflects an underlying failure in the regression of wound repair due to defects in anti-proliferative and anti-inflammatory processes. The central importance of PTEN in SMC in restricting neointima formation is supported by our findings using the inducible system described here. We show that genetic depletion of PTEN in SMC, which physiologically recapitulates events associated with vascular injury, was sufficient to promote massive neointima formation in a genetic strain of mice that are normally low-to-moderate responders to vascular injury. Enhanced neointima formation was associated with increased resident SMC proliferation combined with increased accumulation of inflammatory cells. Importantly, inhibition of SDF-1α, induced in SMC by PTEN loss, reversed resident SMC proliferation and blocked the accumulation of inflammatory cells, supporting previous studies suggesting SDF-1α could be an important pharmacological target to limit restenosis.

Local production of SDF-1α has been shown to be an important factor in mediating injury-induced neointima formation. Previous studies using apolipoprotein E-null mice showed that HIF-1α-mediated induction of SDF-1α in SMC after injury promotes progenitor cell recruitment and neointima formation23. Using culture systems, we demonstrated that PTEN inactivation in SMC promotes an autocrine growth loop through HIF-1α-dependent induction of SDF-1α, thus establishing that SDF-1α directly effects SMC function22. Consistent with our in vitro data, enhanced neointima formation in PTEN iKO mice was accompanied by increased local production of SDF-1α. Surprisingly, despite induction of many cytokines/chemokines in the setting of PTEN loss18, blocking SDF-1α in vivo resulted in complete reversal of neointima formation. Our findings provide the first evidence that SDF-1α has significant direct effects in vivo on resident SMC proliferation. Thus, PTEN operates as an upstream regulator of SDF-1α, which functions as a final common mediator of both the proliferative and inflammatory events that are fundamentally important to restenosis. It should be noted, however, that at this time, we cannot rule out the possibility that the pronounced sensitivity observed in this study to anti-SDF-1α treatment is unique to SMC PTEN null background.

Through our fate-mapping approach, we definitively showed that the large majority of replicating neointimal cells are derived from SMMHC-expressing, mature SMC originally residing in the medial wall, consistent with concepts proposed more than 20 years ago4-6. In addition and previously not reported, we consistently detected resident SMC βGal(+)-derived cells in the adventitia of injured vessels. Adventitial βGal(+) SMC-derived cells did not express SM marker proteins, demonstrating a previously unidentified role for SMC in vessel repair through their contribution to the cellular component of the adventitia. The precise fate and subsequent role of adventitial βGal(+);α-SMA(−) SMC-derived cells is a topic of our current ongoing research. Importantly, however, most studies have used α-SMA to define the role of SMC to injury-induced vessel remodeling. The use of α-SMA to identify SMC is somewhat unreliable, as it has been demonstrated that infiltrating cells and myofibroblasts often transiently express α-SMA10. Additionally, our data suggest that sole use of α-SMA as a marker for SMC will lead to underestimating the numbers of mature SMC contributing to vessel remodeling and therefore underestimating the overall importance of SMC in vessel repair.

While large numbers of bone marrow cells accumulated in injured vessels, we detected very few that co-expressed SM markers supporting a growing number of reports that suggest these cells do not serve as definitive SMC progenitors. In further support, an occasional GFP(+) cell was detected in the arterial media of uninjured vessels. However, none of these medial GFP(+) cells co-expressed βGal following tamoxifen treatment, which would be anticipated if bone marrow progenitors differentiated into SMMHC-expressing SMC. Therefore, in agreement with recent reports9,11,12, our data suggest that while recruitment of these cells is an important contributor to neointima formation, this is not through differentiation into SMC, but rather through crosstalk with SMC and persistence of the inflammatory microenvironment. We propose, consistent with a well-accepted cascade model31, that interactions of these circulating cells with vascular cells leads to sustained neointima formation in part through continual production of various growth factors and cytokines.

Macrophage infiltration, adhesion, and retention have been shown to predict the severity of restenosis32. Our co-culture studies demonstrate that macrophage-SMC crosstalk results in inactivation of PTEN, which is associated with SMC production of several cytokines, thus indicating that the inflammatory environment can mediate sustained PTEN loss. Consistent with this, PTEN-depleted SMC, which exist in an activated, inflammatory state, promote enhanced macrophage adhesion and the synergistic induction of cytokines and enhanced SMC proliferation observed in co-cultures with macrophages. While SDF-1α did not enhance macrophage adhesion, it did mediate production of MCP-1, IL-6, and KC thus contributing to the inflammatory environment. Consistent with the in vitro data, blocking SDF-1α in vivo reduced circulating levels of MCP-1, IL-6, and KC, which likely underlies some of the efficacy on neointima formation. Importantly, the functional changes mediated by SMC PTEN depletion phenocopies the behavior of SMC isolated from experimental diabetic animal models33. Therefore, our findings have potential clinical importance, especially related to high-risk patient populations (eg. type 2 diabetics) who exhibit augmented inflammation and accelerated rates of restenosis. Therapeutically, it appears equally important to prevent SMC activation and macrophage infiltration to successfully limit lesion formation. Our data indicate that inhibiting SDF-1α̣ will impact both of these processes. Howe ver, additional signaling pathways are likely to contribute, and further studies will be required to define the molecular signals underlying the functional effects observed in response to SMC-macrophage interactions.

Neointimal hyperplasia continues to be a major obstacle to the long-term success of percutaneous interventions as well as surgical procedures (eg. bypass grafting). While existing drug-eluting stents have reduced the rates of in-stent restenosis, they have proven less effective in high-risk patient populations. Defining the origin of neointimal SMC, the functional implications of interactions between resident SMC and infiltrating macrophages and other circulating cells, and the molecular signals that regulate pathological neointima formation remain important issues for the design of more effective therapeutics to control restenosis. Our data implicate mature SMC as critical participants in this process, by contributing the vast majority of neointimal cells, regulating the recruitment of inflammatory cells, and contributing to adventitial remodeling. Targeting the PTEN pathway represents a powerful approach to affect the complex biology associated with restenosis.

Supplementary Material

1

Acknowledgements

This work was supported by grants from the NIH to M.C.M.W-E (1RO1 HL88643 and 2PO1 HL014985) and R.A.N (2PO1 HL014985).

Footnotes

Disclosures: None.

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