targeted deletion of pten in smooth muscle cells results...

Targeted Deletion of PTEN in Smooth Muscle Cells Resultsin Vascular Remodeling and Recruitment of Progenitor

Cells Through Induction of Stromal Cell–Derived Factor-1�

Raphael A. Nemenoff, Peter A. Simpson, Seth B. Furgeson, Nihal Kaplan-Albuquerque,Joseph Crossno, Pamela J. Garl, James Cooper, Mary C.M. Weiser-Evans

Abstract—We previously showed that changes in vascular smooth muscle cell (SMC) PTEN/Akt signaling followingvascular injury are associated with increased SMC proliferation and neointima formation. In this report, we used agenetic model to deplete PTEN specifically in SMCs by crossing PTENLoxP/LoxP mice to mice expressing Cre recombinaseunder the control of the SM22� promoter. PTEN was downregulated with increases in phosphorylated Akt in majorvessels, hearts, and lungs of mutant mice. SMC PTEN depletion promoted widespread medial SMC hyperplasia,vascular remodeling, and histopathology consistent with pulmonary hypertension. Increased vascular deposition of thechemokine stromal cell–derived factor (SDF)-1� and medial and intimal cells coexpressing SM-�-actin and CXCR4,the SDF-1� receptor, was detected in SMC PTEN-depleted mice. PTEN deficiency in cultured aortic SMCs inducedautocrine growth through increased production of SDF-1�. Blocking SDF-1� attenuated autocrine growth and blockedgrowth of control SMCs induced by conditioned media from PTEN-deficient SMCs. In addition, SMC PTEN deficiencyenhanced progenitor cell migration toward SMCs through increased SDF-1� production. SDF-1� production by othercell types is regulated by the transcription factor hypoxia-inducible factor (HIF)-1�. We found SMC nuclear HIF-1�expression in PTEN-depleted mice and increased nuclear HIF-1� in PTEN-deficient SMCs. Small interferingRNA–mediated downregulation of HIF-1� reversed SDF-1� induction by PTEN depletion and inhibition ofphosphatidylinositol 3-kinase signaling blocked HIF-1� and SDF-1� upregulation induced by PTEN depletion. Our datashow that SMC PTEN inactivation establishes an autocrine growth loop and increases progenitor cell recruitmentthrough a HIF-1�–mediated SDF-1�/CXCR4 axis, thus identifying PTEN as a target for the inhibition of pathologicalvascular remodeling. (Circ Res. 2008;102:1036-1045.)

Key Words: smooth muscle cell � PTEN � neointima � autocrine growth � conditional knockout mouse

Smooth muscle cell (SMC) accumulation in the arterialintima is a key event in the pathogenesis of atheroscle-

rosis, postangioplasty/in-stent restenosis, and graft arterio-sclerosis,1 with changes in the biological function and phe-notype of SMCs contributing to the pathology.2 Theseconditions are characterized, to varying degrees, by dediffer-entiation, migration, and proliferation of medial-derivedSMCs to form the neointima. Recent data suggest that bonemarrow– derived, circulating, and/or resident progenitor/proinflammatory cells are recruited to the injured vessel,differentiate down a SMC lineage, and proliferate, therebycontributing to neointimal lesion formation.3–6 Compellingevidence supports the contribution of both processes tointimal hyperplasia, and major advances have identifiednumerous factors involved in this complex pathobiology.However, the underlying mechanism(s) initiating lesion for-

mation are not clearly defined. Increased SMC production ofchemokines, such as stromal cell–derived factor (SDF)-1�

(CXCL12), has been shown to be centrally involved inprogenitor cell recruitment,7 although their role in inducingan autocrine growth pathway within the artery wall itself isunknown. We focused on the hypothesis that SMCs arecentral mediators of the injury response. Perturbations inSMC signaling, result in the production of soluble factors thatregulate significant SMC hyperplasia and progenitor/proin-flammatory cell recruitment through an autocrine/paracrinemechanism.

Under physiological conditions, the mature blood vessel isa highly quiescent tissue,8,9 suggesting that pathologicalvascular remodeling requires the inactivation of activegrowth inhibitory pathways before rendering SMCs permis-sive to growth stimulation. Several studies support the con-

Original received December 13, 2007; revision received March 3, 2008; accepted March 5, 2008.From the Department of Medicine, Divisions of Renal Diseases and Hypertension (R.A.N., P.A.S., S.B.F., N.K.-A., J. Cooper, M.C.M.W.-E.),

Pulmonary Sciences and Critical Care Medicine (J. Crossno), Cardiovascular and Pulmonary Research (R.A.N., J. Crossno, P.J.G., M.C.M.W.-E.),University of Colorado Denver; and Veterans Affairs Medical Center (J. Crossno), Denver.

Correspondence to Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Denver,Denver, CO 80262. E-mail [email protected]

© 2008 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.107.169896

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http://circres.ahajournals.org/


















Figure 1. Arterial remodeling and histopathology consistent with pulmonary hypertension in SMC-specific PTEN-null mice. A, Immuno-fluorescence for SM-�-actin in aorta and pulmonary artery (MPA) from WT and KO mice (red). Blue indicates 4�,6-diamidino-2-phenylindole (DAPI) (nuclei). Images are oriented lumen side up. Two independent animals per genotype are shown for MPA. Arterialwall thicknesses were measured; means�SD are presented in the graph; n�8; P�0.01. Arrowheads indicate internal elastic laminae;arrows, intimal SM-�-actin–positive cells. B, Left, Immunohistochemistry for BrdUrd on representative aorta and MPA showingincreased vascular cell proliferation in KO mice (brown nuclei; arrows). Line indicates arterial media. The percentage of replicating aorticintimal, medial, and adventitial cells was determined; means�SD are presented in the graph; n�3; P�0.01. Right, Double immunofluo-rescence for BrdUrd (green) and SM-�-actin (red). Arrows indicate double-positive; arrowheads, BrdUrd-positive and SM-�-actin–nega-tive. C, Cardiac hypertrophy in KO mice. Top, Total heart weight per body weight. Bottom, Right ventricular hypertrophy in KO mice(right ventricle/left ventricle�septum). Values are the means�SD; n�8. *P�0.01. D, Left and Middle, Representative hematoxylin/eosinstaining of lung from WT (top) and KO (bottom) mice showing increased wall thickness of small pulmonary arteries (arrowheads) andreduced alveolarization. Right, Double immunofluorescence for SM-�-actin (green) and von Willebrand factor (red). Arrows indicate

Nemenoff et al Smooth Muscle PTEN Deficiency Induces SDF-1� 1037


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cept that neointimal SMCs exhibit a distinct growth pheno-type similar to fetal-derived SMCs that is characterized bymitogen-independent proliferation and, despite the expressionof functional growth factor receptors (eg, platelet-derivedgrowth factor [PDGF] receptor-�), a blunted mitogenic re-sponse to exogenous growth factors known to stimulatemedial-derived SMCs.10–12 Our previous studies demon-strated that mitogen-independent growth is actively repressedin differentiated SMCs,12 suggesting that loss of growthsuppressors likely leads to enhanced SMC growth potential inpathological conditions such as restenosis. Our subsequentwork strongly implicated the tumor suppressor PTEN as apotent, endogenously produced inhibitor of mitogen-independent SMC proliferation.13,14

PTEN is a dual-specificity lipid and protein phosphatasethat inhibits cell proliferation, survival, and growth predom-inantly through dephosphorylation of phosphatidylinositol3,4,5-trisphosphate, thus antagonizing phosphatidylinositol 3(PI3)-kinase–mediated signaling events.15–17 Regulation ofthe PI3-kinase/Akt/mTOR signaling pathway plays a pivotalrole in SMC proliferation in culture and during the pathogen-esis of restenosis.14,18–20 Important clinically, the use ofrapamycin-eluting stents to inhibit mTOR signaling hassignificantly reduced the incidence of in-stent restenosis.21,22

Previous studies demonstrated that PTEN overexpressionreduces intimal hyperplasia in rat carotid artery injury andsaphenous vein graft models,19,23 and upregulation of SMC-derived PTEN attenuates atherosclerotic lesion formation inhigh-fat-fed rabbits, thus implicating PTEN as an antiathero-genic protein.24 Our studies show that temporally controlledPTEN activity correlated with significant alterations in SMCgrowth rate during vascular development and after experi-mental vascular injury.13,14 Importantly, inactivation of PTENin the setting of vascular injury, leading to constitutive Aktactivation, is an early and critical event involved in neointimaformation. The molecular events mediating the effects ofPTEN inactivation on SMC hyperplasia and on progenitorcell recruitment, however, have yet to be identified. To definethe role of PTEN signaling in this context, we examined theeffects of conditional deletion of SMC PTEN.

By mating PTENLoxP/LoxP mice to transgenic mice express-ing Cre recombinase under the control of the SM22� pro-moter, we show here that SMC-specific PTEN mutant mice(PTEN knockout [KO]) exhibit many features associated withpathological vascular remodeling, including remarkable me-dial and intimal SMC hyperplasia as well as vascular recruit-ment of progenitor cells. In particular, PTEN depletion resultsin hypoxia-inducible factor (HIF)-1�–mediated production ofthe chemokine SDF-1�, which induces an autocrine SMCgrowth loop and increases progenitor cell migration througha paracrine signaling mechanism. Our data thus suggest that

an alteration in SMC PTEN signaling serves as one of theinitiating determinants driving pathological vascularremodeling.

Materials and MethodsAn expanded Materials and Methods section is available in theonline data supplement at http://circres.ahajournals.org.

Animals and Generation of PTEN mutant miceSMC-specific PTEN-null mutant mice were generated by crossingSM22�-Cre transgenic mice to PTENflox/flox mice. PTENflox/flox;SM22�-Cre/�; R26R/� mice were generated by interbreeding PTENflox/flox

mice with R26R mice followed by crossing PTENflox/�;SM22�-Cre/�

to PTENflox/flox; R26R/� mice. Animals were bred and maintainedfollowing guidelines approved by the Institutional Animal Care andUse Committee at the University of Colorado Denver HealthSciences.

Additional MethodsAn expanded Materials and Methods section containing detailsregarding animals and generation of PTEN mutant mice; cell culture;generation of stable short hairpin (sh)RNA-expressing SMCs andsmall interfering (si)RNA transfections; quantitative RT-PCR, West-ern blot analysis, and ELISA; immunohistochemistry, immunofluo-rescence, LacZ staining; peripheral blood mononuclear cell (PBMC)isolation, flow cytometry, and cell labeling; growth and transmigra-tion assays; and statistical analysis is available in the online datasupplement.

ResultsSmooth Muscle–Specific PTEN DepletionPromotes Spontaneous Development of ArterialRemodeling and Histopathology Consistent WithPulmonary HypertensionUsing a rat angioplasty model, we established that PTENinactivation was localized to the developing neointima andexclusively in replicating neointimal SMCs13,14 strongly im-plicating inactivation of PTEN in the pathogenesis of injury-induced intimal hyperplasia. Therefore, to define the role ofPTEN specifically in smooth muscle (SM) tissues using agenetic model, PTENLoxP/LoxP mice25 were mated to transgenicmice expressing Cre recombinase (Cre) under the control ofthe SM22� promoter (Figure IA in the online data supple-ment and Materials and Methods).26 Cre specificity wasverified by crossing SM22�-Cre transgenic mice to ROSA26Reporter (R26R) mice, as described previously (supplementalFigure IB).26 Crossing SM22�-Cre mice with PTENflox/flox

mice generated control mice (PTENflox/flox; �/�, PTENflox/�;�/�, and PTENflox/�; Cre/�) and homozygous mutant mice(PTENflox/flox; Cre/�; PTEN KO). Normal Mendelian ratios ofall genotypes were observed in embryos and at birth (data notshown). PTEN KO mice, however, were smaller (supplemen-tal Figure ID) and weaker, and the majority died by 3 weeksof age (all before 6 weeks); no differences among the other 3groups were observed. All studies reported hereafter com-pared 20-day-old PTEN KO mice with PTENflox/flox; �/�control littermates (WT). PCR for the PTEN� allele (indicat-ing Cre activity) in DNA extracted from aortic, carotid artery,heart, and lung tissues revealed the expected PCR product inPTEN KO but not WT mice (supplemental Figure IC).27

Western blotting showed significant reductions in total PTENprotein in large arteries, hearts, and lungs of PTEN KO mice

Figure 1 (Continued). occluded precapillary arterioles. E, Immu-nohistochemistry for BrdUrd (brown nuclei) on representativelung sections showing increased cell proliferation in KO mice(left and middle). Aw indicates airway; PA, pulmonary artery;arrows, representative BrdUrd-positive. Right, Double immuno-fluorescence for BrdUrd (green) and SM-�-actin (red) on repre-sentative KO lung sections. Arrows indicate double-positive;arrowheads, BrdUrd-positive and SM-�-actin–negative.

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compared with WT, with an accompanying increase inphospho-Akt levels, demonstrating a functional loss of PTENactivity (supplemental Figure ID).

PTEN KO mice exhibited significant SMC hyperplasia inmajor vessels, leading to increased medial thickness alongwith aortic intimal accumulation of SM-�-actin–positive cells(Figure 1A). Increased numbers of SM-�-actin–positive,

5-bromodeoxyuridine (BrdUrd)-positive replicating cellswere observed throughout the aortic and pulmonary arterialwalls in PTEN KO mice (Figure 1B). In addition, PTEN KOmice spontaneously developed right ventricular hypertrophyand pulmonary vascular remodeling at Denver, Colo, ambientair (1609 meters). RV/LV�S ratios, a measure of rightventricular hypertrophy, were significantly higher in PTEN

Figure 2. Circulating PBMCs traffic to the vasculature in SMC-specific PTEN-null mice. A, PBMCs were isolated, stained for indicatedcell surface markers, and sorted by flow cytometry. Left, Representative fluorescence-activated cell sorting profiles. Right, Percentagesof gated progenitor marker–positive cells; data are from 1 of 3 representative experiments. B, Donor PBMCs from WT or KO mice werelabeled with fluorescing nanocrystals and reinjected into WT or KO recipients. Tissues from recipient mice were analyzed for labeledPBMC accumulation (positive indicates cytoplasmic nanocrystal [green] plus a nucleus [DAPI, blue]). Top, Representative aorta fromKO-to-KO mice. Arrows indicate labeled PBMCs on the intimal and adventitial surfaces. Bottom, Representative lung from controls(WT-to-WT, WT-to-KO, KO-to-WT) and from KO-to-KO mice. Arrows indicate labeled PBMCs. Labeled cells in lung tissue were quanti-fied by counting 20 high-power fields per section (graph). C, Immunofluorescence for SM-�-actin (red) on lung sections from KO-to-KOmice. Arrows indicate SM-�-actin–positive labeled PBMCs; arrowhead, SM-�-actin–negative labeled PBMCs. D, Immunohistochemistryfor CD45 (brown) on representative aorta (left) and lung (right) showing increased vascular and perivascular accumulation in KO mice.Arrows indicate CD45-positive cells; PA, pulmonary artery.



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KO compared with WT mice, suggesting the development ofpulmonary hypertension (Figure 1C). Compared with WT,PTEN KO mice exhibited increased wall thickness of smallpulmonary arteries, occluded precapillary arterioles, and re-duced alveolarization (Figure 1D). Occluded precapillaryvessels stained positive for von Willebrand factor (an endo-thelial marker) and SM-�-actin, characteristic of plexiformlesions observed in human primary pulmonary hyperten-sion.28 Increased numbers of replicating SM-�-actin–positiveand –negative cells were observed throughout the lungs ofPTEN KO mice, including small, muscularized pulmonaryarteries (Figure 1E). In addition, increased numbers of repli-cating coronary artery SMCs, coronary perivascular cells,cardiomyocytes, and interstitial fibroblasts were observed inhearts of PTEN KO mice (supplemental Figure II). Cardio-myocyte proliferation was consistent with early developmen-tal cardiac expression of SM22� and, therefore, inactivationof PTEN. Enhanced extracellular matrix deposition wasdetected in major arteries and in perivascular locations inlungs and coronary vessels of mutants (supplemental FigureIII), demonstrating vascular, lung, and cardiac interstitialfibrotic changes. The observed myocardial fibrosis is a keypathological feature of heart failure, the likely cause of deathof mutants.

Recruitment of Circulating Progenitor Cells andIncreased SDF-1� Expression in PTEN KO MiceIn addition to the above, PTEN KO mice consistentlyexhibited significant splenomegaly (supplemental FigureIVA). Increased bone marrow hyperplasia and hyperplasia ofsplenic red pulp, consistent with increased extramedullaryhematopoiesis, were detected in PTEN KO mice (supplemen-tal Figure IVB through IVD); no changes were observed inlivers or kidneys (not shown). Because enhanced bonemarrow progenitor/hematopoietic cell mobilization can resultin extramedullary hematopoiesis, we hypothesized that SMC-

specific PTEN deletion results in production of systemicfactors involved in mobilization and trafficking of bonemarrow–derived progenitors. To determine whether histolog-ical changes were accompanied by increases in peripheralblood progenitor cells, PBMCs were analyzed by 2-colorflow cytometry for the hematopoietic leukocyte markerCD45; the progenitor markers CD34, c-Kit, Sca-1, andCXCR4; the endothelial cell progenitor marker FLK-1; or themonocyte progenitor marker CD14. Higher numbers of cir-culating CD34�, c-Kit�, CD14�, and CXCR4� hematopoieticcells were detected in PTEN KO mice compared with WT(Figure 2A); no changes in FLK-1� or Sca-1� cells weredetected (supplemental Figure VA). In PTEN KO miceexpressing the ROSA26 reporter allele, where LacZ is ex-pressed in cells lacking PTEN, no Cre activity was detected inbone marrow, therefore direct inactivation of PTEN in bonemarrow cells did not mediate progenitor cell mobilization(supplemental Figure IVE).

To determine whether mutant-derived PBMCs traffic to thevasculature, PBMCs isolated from 3-week-old WT or PTENKO donor mice were labeled with fluorescent quantum dotnanocrystals, and reinjected retroorbitally to either 3-week-old WT or PTEN KO recipient mice. Target tissues wereanalyzed for fluorescence 24-hour postinjection; cells werescored positive if they contained significant amounts ofcytoplasmic, granular fluorescent nanocrystals plus a nucleus.No labeled cells were detected on the aorta of control animals(WT donor-to-WT recipient; WT donor-to-KO recipient; KOdonor-to-WT recipient). Only single positive cells weredetected in the lungs (Figure 2B), and these were notassociated with recipient lung alveolar or vascular surfaces.In contrast, labeled cells were identified on aortic intimal andadventitial surfaces and in lungs of KO donor-to-KO recipi-ent mice; large numbers of labeled progenitors were identi-fied in the lungs (Figure 2B). SM-�-actin–positive and

Figure 3. SDF-1� deposition and vascu-lar accumulation of CXCR4-positive cellsin SMC-specific PTEN-null mice. A,Serum ELISA measurements for SDF-1�levels. Shown are the means�SD; n�6.B, Immunofluorescence for SDF-1�(green, aorta; red, lung) and SM-�-actin(red, aorta; green, lung) on aortic (left)and lung (right) sections from WT andKO mice. Blue indicates DAPI (nuclei).Aorta: merged images (top); SDF-1�staining (bottom). Merged images areshown only for lung sections. C, Immu-nofluorescence for CXCR4 (green) andSM-�-actin (red) on representative aorticsections showing CXCR4 expression inSM-�-actin–positive medial SMCs in WTand KO mice and in SM-�-actin–positiveintimal cells (arrows) in KO mice. Arrow-heads indicate internal elastic lamina.Right images are nonspecific rat IgGcontrol.



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–negative PBMCs were detected often in a perivascularlocation (Figure 2C), suggesting some of the recruitedPBMCs differentiate toward SMCs. Increased numbers oflabeled cells were also found in spleens (supplemental FigureVIA), but not livers (not shown), of KO donor-to-KOrecipient mice. Consistent with increased trafficking ofPBMCs, accumulation of CD45� and c-Kit� cells wasobserved on major arteries and in perivascular locations inlungs of PTEN KO mice (Figure 2C and supplementalFigure VB). Taken together, these results support theconcept that SMC-specific PTEN depletion produces alocal microenvironment favorable for trafficking and ac-cumulation of circulating progenitor cells.

Recent data support a role for the chemokine SDF-1� andits receptor expressed by hematopoietic progenitor cells,CXCR4, in promoting vascular remodeling.29–31 Changes inthe SDF-1� gradient from bone marrow to blood result in

movement of precursor cells into the circulation.7 Increasedplasma levels and local vascular accumulation of SDF-1� inresponse to vascular injury recruit CXCR4� bone marrowcells to remodeling vessels.7,32,33 Because circulating CXCR4�

PBMCs were detected in PTEN KO mice, SDF-1� expres-sion in tissues from WT and PTEN KO mice was examined.There was a nonsignificant trend toward increased serumSDF-1� levels (Figure 3A) and increased vascular andperivascular deposition of SDF-1� in aortae and lungs ofPTEN KO mice (Figure 3B). This was associated with intimalaccumulation of CXCR4� cells in the vasculature (Figure 3C)and in spleens of PTEN KO mice (supplemental Figure VIB).CXCR4 expression was also detected on medial SMCs of WTand KO mice (Figure 3C), suggesting SMCs express func-tional SDF-1� receptors. These results support a model inwhich SMC-specific PTEN inactivation indirectly promotesvascular recruitment of progenitor cells through induction of

Figure 4. PTEN depletion induces autocrine growth of SMCs through increased SDF-1� production. A, Cultured aortic SMCs stablyexpressing CTRL or PTEN-specific shRNA were analyzed for total PTEN and phospho-Akt expression under basal and PDGF-stimulated conditions in the presence or absence of LY294002 to inhibit PI3-kinase activity (LY). B, CTRL and PTEN shRNA-expressingSMCs were analyzed by BrdUrd immunohistochemistry for SMC proliferation under basal and PDGF- or serum-stimulated conditions.Left, Means�SD percentage of BrdUrd-positive SMCs of triplicates from 1 of 3 representative experiments. Right, Fold changes inBrdUrd-positive SMCs from basal. *Different from CTRL in the same condition (P�0.01). C, CTRL and PTEN-deficient SMCs were ana-lyzed by BrdUrd immunohistochemistry for SMC proliferation in response to media conditioned by CTRL or PTEN-depleted SMCs(source). Graph shows means�SD percentage of BrdUrd-positive SMCs of triplicates from 1 of 3 representative experiments. Note thatPTEN-depleted SMC conditioned media drives proliferation of CTRL SMCs, whereas CTRL SMC conditioned media attenuatesmitogen-independent proliferation of PTEN-deficient SMCs. D, CTRL and PTEN-deficient SMCs were serum-restricted for 24 hours.Left, Quantitative RT-PCR for SDF-1� mRNA. �-Actin was used for normalization of cDNA. Shown are fold changes in mRNA copynumber�SD from control SMCs from 3 independent experiments. *P�0.05. Right, Conditioned media were analyzed by ELISA, andSDF-1� levels were normalized to total cell number. Shown are fold changes�SD from CTRL SMCs from 3 independent experiments.*P�0.05. E, SMCs were maintained in serum-free media (SFM) in the presence or absence of recombinant SDF-1�, recombinantPDGF-BB, neutralizing anti–SDF-1� or anti–PDGF-BB, or PTEN-deficient SMC conditioned media with or without anti–SDF-1� or anti–PDGF-BB and analyzed for proliferation. Shown are means�SD percentage of BrdUrd-positive SMCs of triplicates from 1 of 3 repre-sentative experiments. Inset, Western blot analysis for CXCR4 and total PTEN on CTRL and PTEN-deficient SMCs maintained underbasal conditions.



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SDF-1�. Because CXCR4 was detected on medial SMCs,increased SMC-derived SDF-1� could also directly affect thebiological function of SMCs.

PTEN Depletion Induces SMC Autocrine Growthand Enhances Progenitor Cell Migration ThroughIncreased SDF-1� ProductionTo verify the direct effect of PTEN depletion on SMCfunction in vitro, aortic SMCs stably expressing PTEN-specific shRNA were generated. PTEN depletion resulted inenhanced Akt activity under basal conditions and in responseto PDGF stimulation compared with SMCs transfected withempty vector (CTRL). Increased Akt phosphorylation wasblocked by treatment with LY294002 to inhibit PI3-kinaseactivity, demonstrating a functional loss of PTEN (Figure4A). Primary aortic SMCs transfected with control scrambledor PTEN-specific siRNA exhibited similar responses (supple-mental Figure VII). PTEN depletion increased cell prolifer-ation under basal conditions and reduced responsiveness toPDGF-BB, a known mitogen for medial-derived SMCs (Fig-ure 4B), similar to what is observed in fetal and neointimalSMCs.10–12 As with fetal and neointimal SMCs, reducedmitogenic responsiveness to PDGF was not attributable to aloss of functional PDGF receptors (data not shown). Condi-tioned media from PTEN-depleted SMCs stimulated CTRLSMC proliferation, indicating that PTEN depletion results insecretion of a mitogen, which may act in an autocrine fashion(Figure 4C). Consistent with results in PTEN KO mice,PTEN deficiency in vitro increased SDF-1� mRNA andsecretion into the cell media (Figure 4D). RecombinantSDF-1� stimulated proliferation of CTRL SMCs that wasblocked with an anti–SDF-1� neutralizing antibody (Figure4E). A neutralizing anti–SDF-1� antibody also attenuatedautocrine growth of PTEN-depleted SMCs and blockedgrowth of CTRL SMCs induced by conditioned media fromPTEN-depleted SMCs (Figure 4E). This effect was specificfor SDF-1�, because neutralization of PDGF had no effect ongrowth responses to conditioned media from PTEN-depletedSMCs (Figure 4E). Consistent with in vivo data, CXCR4 wasexpressed by CTRL and PTEN-depleted SMCs (Figure 4E,inset), thus supporting the concept that SDF-1� upregulationvis a vis PTEN loss induces an autocrine growth loopinvolving an SDF-1�/CXCR4 axis.

To determine whether increased production of SDF-1� byPTEN-deficient SMCs results in enhanced migration ofcirculating progenitor cells in vitro, CTRL and PTEN-depleted SMCs were cocultured with PBMCs from WT orPTEN KO mice using a Transwell system (Figure 5A).PBMCs from both WT and PTEN KO mice showed increasedmigration toward PTEN-depleted SMCs compared withCTRL SMCs, consistent with enhanced SDF-1� levels me-diating chemotaxis of CXCR4� progenitor cells (Figure 5B).Higher numbers of migrating PBMCs were observed fromPTEN KO mice compared with WT mice, consistent withhigher numbers of circulating CXCR4� cells in PTEN KOmice. Migration toward PTEN-depleted SMCs was attenu-ated by the addition of neutralizing anti–SDF-1� and in-creased toward CTRL SMCs in response to recombinantSDF-1� (Figure 5B), suggesting that enhanced secretion of

SDF-1� induced by PTEN depletion drives progenitor cellmigration through a paracrine signaling mechanism.

Vascular injury–induced SDF-1� expression in SMCs wasrecently shown to be regulated by the transcription factorHIF-1� in a hypoxia-independent manner,34 and previousstudies showed HIF-1� upregulation in other cell systems ismediated by PI3-kinase/Akt signaling.35 We thus examinedthe expression of HIF-1� in PTEN KO mice and SMCs.Compared with WT mice, strong, nuclear HIF-1� stainingwas observed in aortic SMCs of PTEN KO mice (Figure 6A).Consistent with in vivo results, PTEN-depleted SMCs ex-pressed increased HIF-1� mRNA and HIF-1� protein innuclear protein extracts compared with CTRL SMCs (Figure6B and 6C). siRNA-mediated depletion of HIF-1� reversedSDF-1� induction by PTEN depletion (Figure 6D), andinhibition of PI3-kinase signaling blocked HIF-1� andSDF-1� upregulation induced by PTEN depletion (Figure6E), suggesting that SMC PTEN inactivation directly in-creases HIF-1� expression, leading to SDF-1� upregulation.

DiscussionWe previously demonstrated that vascular injury leads toinactivation of PTEN in SMCs.13,14 The goal of the presentstudy was to examine the consequences of PTEN inactivationusing a genetic model in which PTEN is specifically deletedin SMCs. Because changes in SMC PTEN signaling areobserved in the early stages following vascular injury, wepostulated that PTEN loss would initiate key events involvedin pathological vascular remodeling. Our data indicate that

Figure 5. Enhanced SDF-1� production increases PBMC migra-tion to PTEN-null SMCs. A, Schematic for migration experiment.B, CTRL and PTEN-deficient SMCs were maintained in serum-free media (SFM) in the presence or absence of a neutralizinganti–SDF-1� or recombinant SDF-1� or stimulated with serum(10% CS). Equal numbers of PBMCs from WT or PTEN KOmice were isolated and added to 8-�m Transwell filters, andmigrated PBMCs were determined 24 hours later. Data shownumbers of migrated PBMCs from WT mice (left graph) or PTENKO mice (right graph) to CTRL or PTEN-deficient SMCs andrepresent fold changes�SD from WT PBMCs-to-CTRL SMCs(set at 1.0) from 3 independent experiments. *Different fromCTRL SMCs, same condition (P�0.01); ** and ***, different fromserum-free media, same cell types (P�0.05).



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deletion of PTEN is sufficient to induce proliferation ofmedial SMCs and accumulation of SMCs in the vascularintima. Our findings are consistent with a previous study thatused a similar genetic inactivation strategy and showed SMCPTEN loss resulted in widespread SMC hyperplasia associ-ated with the initiation of leiomyosarcoma development by 2months of age.36 We did not detect leiomyosarcomas becauseour mutants rarely survived past 21 days, considerably shorterthan what was reported in this study (mean, 70.3 days).Differences are likely attributable to genetic background,because our mice were fully backcrossed to the C57BL/6background. Nevertheless, in both instances, PTEN depletionspecifically in SMCs induced significant SMC hyperplasia inthe absence of other stimuli. In addition, we report that SMCPTEN depletion is sufficient to recruit hematopoietic progen-itor cells to the vasculature. Based on both our in vivo and invitro data, a critical mediator of PTEN inactivation is in-creased HIF-1�–mediated production of SDF-1� by SMCs.

Several studies have demonstrated that vascular remodel-ing is associated with increased chemokine production bySMCs, as well as other cell types.7,37 Increased SMC produc-tion of SDF-1� has been implicated in the recruitment ofbone marrow– derived progenitor cells expressing theSDF-1� receptor CXCR4, which contribute to neointimaformation. Vascular accumulation of SDF-1� is critical for

targeting CXCR4-positive cells to the site of injury. Consis-tent with these studies, our data show that in PTEN KO mice,CXCR4-expressing intimal cells and PBMCs homing to thevasculature express SM-�-actin, suggesting recruitment ofSMC progenitors. The role of SDF-1� on SMC proliferation,however, has been less studied. Consistent with previousstudies,38 our data indicate that SMCs express CXCR4, andtherefore production and release of SDF-1� would be antic-ipated to establish an autocrine growth loop. Our in vitrostudies demonstrate that PTEN silencing is sufficient toinduce SDF-1� expression and increase autonomous growthof SMCs. In fact, blocking SDF-1� reduced autonomousproliferation of PTEN-depleted cells to control levels, sug-gesting that, in addition to its effects on progenitor cellrecruitment, increased SMC-derived SDF-1� may be a majorregulator of enhanced SMC proliferation.

Although we have not defined the downstream effectorsleading to SDF-1� induction, our data suggest that Akt-dependent upregulation of HIF-1� is likely to be important.Hypoxia-induced SDF-1� upregulation in endothelial cellswas shown to be HIF-1�–dependent,39 and forced overex-pression of PTEN in glioma cells was shown to significantlyreduce HIF-1� expression.40 Karshovska et al34 demonstratedinduction of HIF-1� in SMCs following vascular injury;HIF-1� inhibition resulted in reduced neointimal area and

Figure 6. SMC PTEN inactivation increases HIF-1� expression, leading to SDF-1� upregulation. A, Immunohistochemistry for HIF-1�(brown) on representative aorta from WT (top) and PTEN KO (middle) mice showing increased SMC nuclear staining in PTEN KO mice.Bottom, Nonspecific IgG control. Arrows indicate representative HIF-1�–positive cells; lines, arterial media. B, Quantitative RT-PCR forHIF-1� mRNA on CTRL and PTEN-deficient SMCs. �-Actin was used for normalization of cDNA. Shown are fold changes in mRNAcopy number�SD from CTRL SMCs. *P�0.05. C, Cytoplasmic and nuclear proteins from CTRL and PTEN-deficient SMCs were ana-lyzed for HIF-1� and total PTEN expression under basal conditions. D, CTRL and PTEN-deficient SMCs were transfected with HIF-1�–specific siRNA oligonucleotides and analyzed by quantitative RT-PCR for HIF-1� (left) and SDF-1� (right) mRNA. �-Actin was used fornormalization of cDNA. E, CTRL and PTEN-deficient SMCs were analyzed by quantitative RT-PCR for HIF-1� (left) and SDF-1� (right)mRNA under basal conditions in the presence or absence of LY294002 to inhibit PI3-kinase activity. �-Actin was used for normalizationof cDNA.



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decreased SDF-1� expression. Our studies show that inacti-vation of PTEN in SMCs is sufficient to upregulate HIF-1� inan Akt-dependent manner; blocking HIF-1� upregulationreverses the induction of SDF-1�. Studies are ongoing todetermine the molecular pathways responsible for the regu-lation of HIF-1� and SDF-1� production mediated by PTENinactivation.

In summary, we report that SMC PTEN-deficient micespontaneously develop features in both the systemic andpulmonary vasculature associated with pathological vascularremodeling that are mediated, at least in part, through theinduction of the chemokine, SDF-1�. It should be noted that,although SDF-1�/CXCR4 signaling has been shown to pro-mote pathological vascular remodeling, recent data alsosuggest SDF-1�/CXCR4 signaling exerts protective effects inprimary atherosclerosis.41 Unfortunately, early lethality in thepresent model precludes the ability to fully examine molec-ular events associated with the pathogenesis of remodeling onPTEN inactivation. Although our data suggest PTEN inacti-vation promotes medial SMC proliferation and the potentialrecruitment of SMC progenitors, these mice cannot be used asrecipients for bone marrow transplant studies. It is, therefore,difficult to determine whether recruited cells are pathologicalor protective in the present system. We are in the process ofdeveloping an inducible SMC-specific system to more clearlyanswer these questions. Nevertheless, our data are consistentwith the proposal that sustained SMC PTEN signaling servesas a central regulator of the vascular injury response and,therefore, have important clinical implications in pathologiesassociated with vascular remodeling, including restenosis andpulmonary hypertension.

Sources of FundingSupported by NIH grants EB003999 (to M.C.M.W.-E.) andDK19928 (to R.A.N.).

DisclosuresNone.

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Crossno, Pamela J. Garl, James Cooper and Mary C.M. Weiser-EvansRaphael A. Nemenoff, Peter A. Simpson, Seth B. Furgeson, Nihal Kaplan-Albuquerque, Joseph

αDerived Factor-1−Recruitment of Progenitor Cells Through Induction of Stromal Cell Targeted Deletion of PTEN in Smooth Muscle Cells Results in Vascular Remodeling and

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2008 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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1

METHODS

Animals and Generation of PTEN mutant mice

PTENflox/flox mice, generated as previously described and generously provided to us by Dr. Tak Mak

(Ontario Cancer Institute, University of Toronto, Toronto, Ontario)1, were fully backcrossed (>10

generations) to the C57BL6/J background. SM22α-Cre transgenic mice were generated as previously

described and generously provided to us by Dr. J. Miano (U. Rochester, Rochester, NY)2. SM22α-Cre

activity was verified by crossing SM22α-Cre transgenic mice with the ROSA26 reporter (R26R) line, as

described2. PTENflox/flox mice were mated to SM22α-Cre transgenic mice to generate control mice that

do not express Cre, but have both (PTENflox/flox;+/+) or only one (PTENflox/+;+/+) PTEN floxed alleles,

heterozygous mice that express Cre and have only one PTEN floxed allele (PTENflox/+;Cre/+), and

homozygous mutant mice that express Cre and have both PTEN alleles floxed (PTENflox/flox;Cre/+;

PTEN KO). PTENflox/flox; SM22α-Cre/+; R26R/+ mice were generated by interbreeding PTENflox/flox

mice with R26R mice followed by crossing PTENflox/+;SM22α-Cre/+ to PTENflox/flox;R26R/+ mice.

Animals were bred and maintained following guidelines approved by the Institutional Animal Care and

Use Committee at the University of Colorado Denver and Health Sciences Center. For genotyping,

genomic DNA from mouse tails was isolated and amplified using the REDExtract-N-Amp tissue PCR

kit (Sigma) and primer sequences, as previously described1, 3.

Cell culture, generation of stable shRNA-expressing SMC, and siRNA transfections

Primary rat aortic SMC were isolated and cultured as previously described4. A short hairpin RNA

(shRNA) sequence based on the mouse PTEN gene (NCBI accession #NM008960) was designed and

cloned into a retroviral expression vector driven by the U6 promoter (Open Biosystems, Huntsville,

AL). To generate stable SMC clones, control or PTEN shRNA vectors were packaged into replication-

2

defective retrovirus, as described previously5. SMC were incubated with secreted virus for 48 hrs and

cells expressing control or PTEN shRNA were selected by culturing in medium containing puromycin.

Individual clones were screened by immunoblotting with total PTEN and phosphoAkt antibodies.

Transient transfections were performed on primary rat aortic SMC (passage 3) using Oligofectamine and

0.2 µg of PTEN-specific or control mismatched siRNA according to protocols provided. To deplete

HIF-1α from control or PTEN shRNA-expressing SMC, transient transfections were performed using

0.2 µg of HIF-1α-specific or control mismatched siRNA. siRNA oligoneucleotides were obtained

through Dharmacon (Lafayette, CO).

Quantitative RT-PCR

To assay for SDF-1α mRNA expression, total RNA was isolated from control or PTEN shRNA-

expressing SMC using the QIAshredder and RNeasy Plus kits (Qiagen) and first strand cDNA was made

using the iScript cDNA synthesis kit (BioRad). Sequence-specific primers were designed using the

MacVector program: SDF-1α: sense (5’-TTTGAGAGCCATG TCGCCAG-3’), antisense (5’-

TGTTGTTGCTTTTCAGCCTTGC-3’); β-Actin: sense (5’-AGGGTGTGATGGTGGGTATGG-3’),

antisense (5’-AATGCCGTGTTCAATGGGG-3’); HIF-1α: sense (5’-TAGACTTGGAAAT

GCTGGCTCCCT-3’), antisense (5’-TGGCAGTGACAGTGATGGTAGGTT). Quantitative real-time

PCR was performed as previously described5, 6 and β-Actin was used for normalization.

Western analysis and ELISA

Whole tissues were harvested, snap frozen in liquid nitrogen, and crushed into a fine powder under

liquid nitrogen. Tissues or SMC were lysed with ice-cold RIPA buffer, pH 7.4, equal amounts of

solubilized proteins were separated by SDS-PAGE and transferred for Western analysis, as described

3

previously4, 7, 8. The NE-PER nuclear and cytoplasmic extraction kit (Pierce Biotechnology) was used

for separation and preparation of nuclear and cytoplasmic proteins from cultured SMC. Total PTEN,

total Akt, phosphoSer473Akt (Cell Signaling), and CXCR4 (Capralogics) antibodies were used at 1:1000

dilutions. A HIF-1α antibody (Novus Biologicals) was used at 1:500 and β-actin (Sigma) was used at

1:5000; β-actin served as a loading control. To detect secreted SDF-1α, conditioned media from control

or PTEN-expressing shRNA SMC was assayed using a mouse SDF-1α-specific solid-phase ELISA kit

according to the protocols provided (R&D Systems). Values were normalized to total cell protein from

individual cell cultures.

Immunohistochemistry/immunofluorescence/LacZ staining

Formalin-fixed, paraffin-embedded tissues were analyzed by H&E staining for morphology.

Metamorph software was used to measure vessel wall thickness; six measurements per vessel were

obtained from 5 animals per genotype. For immunohistochemistry, formalin-fixed, paraffin-embedded

tissues were deparaffinized, rehydrated and underwent antigen retrieval by heating for 20 min at 115oC

in a decloaking chamber (Biocare). Sections were then exposed to specific antibodies overnight at 4oC.

Antigen:antibody complexes were visualized using kits from Vector Laboratories and sections lightly

counterstained with hematoxylin. Negative controls included the use of mouse or rabbit IgG. For BrdU

analysis, mice were injected with 100 mg of BrdU (Sigma) per kg body weight 12 and 2 hours before

death. Sections stained for BrdU incorporation were pretreated with 2N HCl prior to antibody steps.

Sections were visualized using an Olympus light microscope equipped with SPOT software. To

quantify in vivo replication rates, intimal, medial, and adventitial cells were analyzed independently for

BrdU-positive nuclei; the percentages of BrdU-positive nuclei were determined by counting a minimum

of 200 intimal, medial, or adventitial cells per tissue section from three animals per genotype4, 9. For

4

immunofluorescence, tissue sections were treated as above. Following incubations with primary

antibodies, antigen:antibody complexes were visualized using rhodamine (Alexa Fluor-568)-coupled or

fluorescein isothiocyanate (Alexa Fluor-488)-coupled secondary antibodies (Molecular Probes). For

double labeling, sections were sequentially incubated with specific primary and secondary antibodies.

Coverslips were mounted with VectaShield medium containing DAPI to detect all cell nuclei (Vector

Laboratories), and sections were visualized using a Nikon inverted fluorescence microscope equipped

with Metamorph software. Antibodies used include BrdU (1:100; BD Pharmingen), SM-α-actin-Cy3-

conjugated (1:1000; Sigma), SM-α-actin (1:1000; Abcam), vWF (1:1000; Dako), CD45 (1:50; BD

Pharmingen), CXCR4 (1:50; R&D Systems), c-Kit (1:100; Dako), SDF-1α (1:200; Abcam), and HIF-1α

(1:25; Novus Biologicals). To stain for LacZ activity, tissues were fixed in glutaraldehyde and whole

mount staining was performed at 37oC overnight using a kit from GTS, Inc according to the protocols

provided. Tissues were then paraffin-embedded for histological analysis.

Peripheral blood mononuclear cell (PBMC) isolation, flow cytometry, and cell labeling

PBMCs from WT or PTEN KO mice were isolated from equal volumes of peripheral blood by

differential centrifugation with Histopaque (Sigma) and resuspended in PBS containing 5% FBS.

Hemolyzed PBMCs were stained with fluorescein isothiocyanate- (FITC), phycoerythrin (PE), or APC-

conjugated antibodies (BD Pharmingen): CD45-PE (30-F11), CD45-FITC (30-F11), CXCR4-FITC

(2B11), cKit-FITC (2B8), Sca-1-FITC (D7), CD34-PE (581), Flk-1-APC (12α1), or CD14-PE (rmC5-3).

Cells were analyzed by two-color flow cytometry using a Beckman Coulter FC500 with CXP software.

For cell trafficking experiments, purified PBMCs from donor WT or PTEN KO mice were aseptically

labeled with 5nM green fluorescent Qdot 525 nanocrystals using the Qtracker 525 cell labeling kit

(Invitrogen) according to the protocols provided. Labeled cells were washed extensively and equal

5

numbers re-injected retro-orbitally to WT or PTEN KO recipient mice. Tissues from recipient mice

were harvested 24 hr post-injection, fixed, stained with DAPI, and analyzed for fluorescence as

described above.

Growth and transmigration assays

Cell replication was analyzed by BrdU immunocytochemistry, as previously described4. Briefly, SMC

were plated in triplicate, maintained in serum-free conditions for 72 hrs, and then stimulated with 10

ng/ml PDGF-BB or 10% CS or left in serum-free medium for 24 hrs in the presence of 100 µM BrdU.

Some wells received neutralizing antibodies against SDF-1α or PDGF (R&D Systems), 100 ng/ml

recombinant SDF-1α (R&D Systems), or 20 ng/ml recombinant PDGF-BB (R&D Systems). Cells were

fixed in methanol and stained as described above. To determine the effect of SMC-specific PTEN

depletion on progenitor cell recruitment, PBMCs from WT or PTEN KO mice were isolated as

described above. Control or PTEN-shRNA expressing SMC were plated in the bottom chamber of a

Transwell chamber and maintained under serum-free conditions or in the presence of 10% CS. Some

wells received a neutralizing antibody against SDF-1α or 100 ng/ml recombinant SDF-1α. Equal

numbers of PBMCs, suspended in serum-free DMEM, were added to the top chamber and migration

measured 24 hrs later. Cells that had migrated to the bottom of the Transwell membrane were fixed,

stained with DAPI, and quantified by counting five high power fields (400x) per well.

Statistical Analysis

Data are expressed as means+/-SD and were determined using either two-tailed t-test analyses or 1-way

ANOVA followed by Fisher’s exact test analyses. P values less than 0.05 were considered statistically

significant.

6

SUPPLEMENTAL FIGURE LEGENDS

Supplemental Figure S1. Targeted deletion of PTEN in smooth muscle. (A). Schematic of the strategy

used to generate SMC-specific PTEN null mice (PTEN KO). (B). LacZ activity in smooth muscle tissue

in SM22α-Cre transgenic mice carrying a ROSA reporter (R26R) allele (blue reaction color due to Cre-

mediated excision of a floxed neo cassette upstream of the lacZ gene within the ROSA26 locus). (C).

DNA was extracted from whole aortae, carotid arteries, hearts, and lungs from WT (PTENfl/fl; +/+) and

PTEN KO (PTENfl/fl; SM22α-Cre/+) mice and analyzed by RT-PCR for the PTENΔ allele; positive band

= Cre-mediated excision of floxed PTEN alleles. (D). Right: Western analysis of whole aorta, carotid

artery, heart, and lung showing reduced total PTEN levels with accompanying increased phosphoAkt in

tissues from SMC-specific PTEN KO mice. Left: SMC-specific PTEN KO mice exhibit reduced body

size (graph); all die before 6 weeks of age. Values are presented as the means+/-SD; n=8; p<0.01.

Supplemental Figure S2. Increased cell replication in hearts of PTEN KO mice.

Immunohistochemical staining for BrdU on representative heart sections from WT (top panel) and

PTEN KO (bottom panel) mice showing increased cell proliferation in PTEN KO mice (brown nuclei).

CoA = coronary artery; arrows = representative BrdU-positive cells.

Supplemental Figure S3. Increased perivascular extracellular matrix accumulation in PTEN KO

mice. Representative Masson’s Trichrome staining of aortic (top left), heart (bottom left), and lung

(right) tissues from WT and PTEN KO mice. Note increased vascular ECM deposition in PTEN KO

tissues (blue reaction color). Arrowheads = internal elastic laminae; Arrows in aortic sections = ECM-

rich intimal cell accumulation in PTEN KO mice; Arrows in heart sections = peri-vascular ECM

accumulation; * = coronary artery; PA = pulmonary arteries; Aw = airway.

7

Supplemental Figure S4. Bone marrow hypercellularity and splenomegaly in PTEN KO mice. (A).

Macroscopic image showing splenomegaly in PTEN KO mice. Spleen weights were obtained and

shown in the graph as spleen size per body weight. * = different from WT; p<0.01. (B). Representative

H&E staining of tibiae (left; cross sections shown) and spleen (middle and right) sections from WT (top

panels) and PTEN KO (bottom panels) mice. Note increased bone marrow cellularity in PTEN KO and

expansion of red pulp in PTEN KO spleen tissue. (C). Immunohistochemical staining for BrdU on

representative spleen sections showing increased cell proliferation in PTEN KO mice (brown nuclei).

(D). Immunohistochemical staining for BrdU on representative sections of tibiae showing increased

bone marrow cell proliferation in PTEN KO mice (brown nuclei; longitudinal sections shown). Arrows

= trabecular bone. (E). LacZ activity in PTEN KO mice carrying a ROSA reporter (R26R) allele (blue

reaction color due to SM22α-Cre-mediated excision of a floxed neo cassette upstream of the lacZ gene

within the ROSA26 locus). PA = pulmonary artery; CA = carotid artery; CoA = coronary artery; M =

bone marrow; B = bone. Note uniform SM22α-Cre activity in SMC of major arteries and the

myocardium of PTEN KO mice, but no SM22α-Cre activity in bone marrow of PTEN KO mice.

Supplemental Figure S5. Circulating FLK-1+ or Sca-1+ cells in SMC-specific and vascular

accumulation of c-Kit+ cells in PTEN null mice. (A). PBMCs were isolated, stained with antibodies

against the indicated cell surface markers and sorted by flow cytometry. Left: Representative FACS

profiles. Right: Percents of gated progenitor marker-positive cells; data from one of three representative

experiments. (B). Immunohistochemistry for c-Kit on representative aortic sections; brown reaction

color. Arrows = c-Kit-positive cells. Arrowheads = internal elastic laminae.

8

Supplemental Figure S6. Trafficking of labeled PBMCs to the spleen. (A). Donor PBMCs from WT or

PTEN KO mice were labeled with fluorescing nanocrystals and re-injected into WT or PTEN KO

recipients. Spleen tissues from recipient mice were analyzed for labeled PBMC accumulation (positive

score = significant quantities of cytoplasmic nanocrystal [green] plus a visible nucleus [DAPI, blue]).

Arrows = representative labeled PBMCs; * = background fluorescence from red blood cells. (B).

Immunofluorescence staining for CXCR4 (red) in spleen tissues from WT and PTEN KO mice. Blue =

DAPI (nuclei).

Supplemental Figure S7. Transient siRNA-mediated depletion of PTEN in primary cultured SMC.

Cultured aortic SMC were transfected with control (CTRL) or PTEN-specific siRNA oligoneucleotides

and were analyzed by Western for total PTEN and phosphoAkt expression under basal and PDGF- or

serum-stimulated conditions. Note that PTEN depletion results in enhanced phosphorylation of Akt

under basal conditions and increased and sustained activation of Akt following stimulation.

REFERENCES

1. Suzuki A, Yamaguchi MT, Ohteki T, Sasaki T, Kaisho T, Kimura Y, Yoshida R, Wakeham A, Higuchi T, Fukumoto M, Tsubata T, Ohashi PS, Koyasu S, Penninger JM, Nakano T, Mak TW. T cell-specific loss of Pten leads to defects in central and peripheral tolerance. Immunity. 2001;14:523-534.

2. Miano JM, Ramanan N, Georger MA, de Mesy Bentley KL, Emerson RL, Balza RO, Jr., Xiao Q, Weiler H, Ginty DD, Misra RP. Restricted inactivation of serum response factor to the cardiovascular system. Proc Natl Acad Sci U S A. 2004;101:17132-17137.

3. Suzuki A, Itami S, Ohishi M, Hamada K, Inoue T, Komazawa N, Senoo H, Sasaki T, Takeda J, Manabe M, Mak TW, Nakano T. Keratinocyte-specific Pten deficiency results in epidermal hyperplasia, accelerated hair follicle morphogenesis and tumor formation. Cancer Res. 2003;63:674-681.

4. Weiser-Evans MC, Quinn BE, Burkard MR, Stenmark KR. Transient reexpression of an embryonic autonomous growth phenotype by adult carotid artery smooth muscle cells after vascular injury. J Cell Physiol. 2000;182:12-23.

9

5. Kaplan-Albuquerque N, Van Putten V, Weiser-Evans MC, Nemenoff RA. Depletion of serum response factor by RNA interference mimics the mitogenic effects of platelet derived growth factor-BB in vascular smooth muscle cells. Circ Res. 2005;97:427-433.

6. Kaplan-Albuquerque N, Bogaert YE, Van Putten V, Weiser-Evans MC, Nemenoff RA. Patterns of gene expression differentially regulated by platelet-derived growth factor and hypertrophic stimuli in vascular smooth muscle cells: markers for phenotypic modulation and response to injury. J Biol Chem. 2005;280:19966-19976.

7. Mourani PM, Garl PJ, Wenzlau JM, Carpenter TC, Stenmark KR, Weiser-Evans MC. Unique, highly proliferative growth phenotype expressed by embryonic and neointimal smooth muscle cells is driven by constitutive Akt, mTOR, and p70S6K signaling and is actively repressed by PTEN. Circulation. 2004;109:1299-1306.

8. Walker HA, Whitelock JM, Garl PJ, Nemenoff RA, Stenmark KR, Weiser-Evans MC. Perlecan up-regulation of FRNK suppresses smooth muscle cell proliferation via inhibition of FAK signaling. Mol Biol Cell. 2003;14:1941-1952.

9. Cook CL, Weiser MC, Schwartz PE, Jones CL, Majack RA. Developmentally timed expression of an embryonic growth phenotype in vascular smooth muscle cells. Circ Res. 1994;74:189-196.

SM22α-Cre activity in the R26R mouse

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bronchial SMC pulmonary artery

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Supplemental Figure 1

A.LoxP LoxP

E3 E6E5E4PTEN Flox

LoxPE6PTEN Δ4/5

SM22α: embryonic expression in primitive heart tube, dorsal aortae, cephalic mesenchyme, and yolk sac vasculature; postnatal expression limited to arterial, venous, and visceral SMC

SM22α-Cre transgenic

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CXCR4 DAPI

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Total PTEN

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PDGF15 min

PDGF6 hrs

targeted deletion of pten in smooth muscle cells results...

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