CALL FOR PAPERS Stem Cell Physiology and Pathophysiology

Type-2 pericytes participate in normal and tumoral angiogenesis

Alexander Birbrair,1,2 Tan Zhang,1 Zhong-Min Wang,1 Maria Laura Messi,1 John D. Olson,3

Akiva Mintz,3,4 and Osvaldo Delbono1,2

1Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, North Carolina;2Neuroscience Program, Wake Forest School of Medicine, Winston-Salem, North Carolina; 3Department of Radiology, WakeForest School of Medicine, Winston-Salem, North Carolina; and 4Department of Neurosurgery, Wake Forest School ofMedicine, Winston-Salem, North Carolina

Submitted 20 March 2014; accepted in final form 23 April 2014

Birbrair A, Zhang T, Wang ZM, Messi ML, Olson JD, MintzA, Delbono O. Type-2 pericytes participate in normal and tumoralangiogenesis. Am J Physiol Cell Physiol 307: C25–C38, 2014. Firstpublished April 30, 2014; doi:10.1152/ajpcell.00084.2014.—Tissuegrowth and function depend on vascularization, and vascular insuffi-ciency or excess exacerbates many human diseases. Identification ofthe biological processes involved in angiogenesis will dictate strate-gies to modulate reduced or excessive vessel formation. We examinethe essential role of pericytes. Their heterogeneous morphology,distribution, origins, and physiology have been described. Usingdouble-transgenic Nestin-GFP/NG2-DsRed mice, we identified twopericyte subsets. We found that Nestin-GFP�/NG2-DsRed� (type-1)and Nestin-GFP�/NG2-DsRed� (type-2) pericytes attach to the wallsof small and large blood vessels in vivo; in vitro, type-2, but nottype-1, pericytes spark endothelial cells to form new vessels. Matrigelassay showed that only type-2 pericytes participate in normal angio-genesis. Moreover, when cancer cells were transplanted into Nestin-GFP/NG2-DsRed mice, type-1 pericytes did not penetrate the tumor,while type-2 pericytes were recruited during its angiogenesis. Asinhibition of angiogenesis is a promising strategy in cancer therapy,type-2 pericytes may provide a cellular target susceptible to signalingand pharmacological manipulation in treating malignancy. This workalso reports the potential of type-2 pericytes to improve blood perfu-sion in ischemic hindlimbs, indicating their potential for treatingischemic illnesses.

angiogenesis; pericytes; stem cells; tumor

ALL TISSUES REQUIRE HEALTHY vasculature to supply nutrients andO2 and remove degradation products. Insufficient vasculariza-tion leads to ischemic conditions that inhibit tissue growth andsurvival, while tumors promote excessive and aberrant vascu-larization (36). Angiogenesis is the process by which newblood vessels form from existing vessels (65). It plays a centralrole in human physiology during fetal development, menstru-ation, wound healing, tissue repair after surgery or trauma,cancer, and various ischemic and inflammatory diseases (3,46). Understanding angiogenesis well enough to manipulate itwould create numerous therapeutic opportunities.

Successful angiogenesis requires the participation of variouscell types and associated molecular signaling. Cells attached tothe walls of microvessels, called pericytes, play a critical rolein stabilizing blood vessels. On the basis of their markers,

morphology, and origin, they are heterogeneous (15, 17–19,34, 72, 75, 77, 90). Some have robust angiogenic potential(30), respond to angiogenic factors, guide newly formed ves-sels, and provide survival signals for endothelial cells. Whetherall pericyte subpopulations have angiogenic potential remainsunknown.

As pericytes participate in stem cell support, self-renewal,and proliferation (50), as well as tissue regeneration and repair(15, 18), targeting only the pericyte subpopulation involved inangiogenesis may provide more efficient control of bloodvessel development and a target for treatments designed todecrease (cancer) or increase (ischemic diseases) vasculariza-tion.

Recently, using Nestin-GFP/NG2-DsRed transgenic mice,we identified two pericyte subtypes, type 1 (Nestin-GFP�/NG2-DsRed�) and type 2 (Nestin-GFP�/NG2-DsRed�). Wefound that they play diverse roles in different microenviron-ments (15, 17, 18): while type-2 pericytes generate new muscletissue after injury, type-1 pericytes are fibrogenic and adipo-genic in old and diseased skeletal muscle, respectively.Whether their angiogenic potential differs is unknown.

Here, using a Matrigel plug assay, we show that angiogen-esis occurs when type-2, but not type-1, pericytes are culturedwith endothelial cells or injected in vivo. Moreover, onlyendogenous type-2 pericytes are recruited during tumor angio-genesis. We also found that type-2 pericytes recover bloodflow in a mouse model of hindlimb ischemia. We proposetargeting type-2 pericytes, rather than all pericytes, for antian-giogenic cancer therapy and tissue revascularization. Thisapproach will not interfere with normal type-1 pericyte func-tions, such as extracellular matrix deposition, needed for nor-mal tissue remodeling.

MATERIALS AND METHODS

Animals. Our colony of Nestin-GFP transgenic mice was main-tained homozygous for the transgene on the C57BL/6 genetic back-ground (54). Our colony of C57BL/6 wild-type mice was used as thecontrol. Male athymic nude (nu/nu) mice (Taconic Farms, German-town, NY) were used in transplantation studies. NG2-DsRed trans-genic mice expressing DsRed-T1 under the control of the NG2promoter (89) and �-actin-DsRed transgenic mice expressing the redfluorescent protein variant DsRed.MST under the control of thechicken �-actin promoter coupled with the cytomegalovirus immedi-ate-early enhancer (84) were purchased from the Jackson Laboratory(Bar Harbor, ME). All tissues of �-actin-DsRed transgenic micefluoresce red (84). Nestin-GFP mice were cross-bred with �-actin-

Address for reprint requests and other correspondence: O. Delbono, Sectionon Gerontology and Geriatric Medicine, Dept. of Internal Medicine, WakeForest School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157(e-mail: odelbono@wakehealth.edu).

Am J Physiol Cell Physiol 307: C25–C38, 2014.First published April 30, 2014; doi:10.1152/ajpcell.00084.2014.

0363-6143/14 Copyright © 2014 the American Physiological Societyhttp://www.ajpcell.org C25

DsRed mice to generate Nestin-GFP/�-actin-DsRed double-trans-genic mice. Nestin-GFP/NG2-DsRed double-transgenic mice are de-scribed elsewhere (15, 17, 18). All colonies were housed in a patho-gen-free facility of the Animal Research Program at Wake ForestSchool of Medicine under a 12:12-h light-dark cycle and fed adlibitum. Male and female homozygous mice were used, and their agesranged from 3 to 5 mo. Animal handling and procedures wereapproved by the Wake Forest School of Medicine Animal Care andUse Committee.

Primary antibodies. Table 1 shows the antibodies and source.Mesenteric vessel isolation. Mesenteric vessels were isolated as

described elsewhere (53). Briefly, the mice were anesthetized andtheir arteries were removed, with all precautions to ensure sterility.The abdominal wall was opened in layers, and the celiac gland wasexposed. The superior mesenteric artery was then easily identified inclose proximity to the gland, stripped of surrounding mesentery, andclamped proximally. Blood was cleared from the artery and itsbranches by injection of 1 ml of cold PBS solution into the artery,distal to the clamp. Tissues were kept moist with PBS solution duringdissection. After surrounding mesentery was teased away, the arteryand its branches were quickly removed and stripped of periarterial fatand fibrous tissue. The superior mesenteric artery and its mainbranches were then dissected out and observed under phase-contrastand fluorescence microscopy.

Skeletal muscle immunohistochemistry. To detect DsRed and greenfluorescent protein (GFP) fluorescence in 3-mo-old Nestin-GFP/NG2-DsRed mice, nondissociated extensor digitorum longus muscles weredissected; fixed in 4% paraformaldehyde (PFA) overnight; immersedin 10%, 20%, and 30% sucrose solutions for 60, 45, and 30 min,respectively; embedded in optimal cutting temperature (OCT) com-pound; and rapidly frozen in liquid nitrogen to prepare 10-�m-thickcryosections. Muscle sections were fixed with 4% PFA for 30 min,permeabilized in 0.5% Triton X-100 (Sigma, St. Louis, MO), andblocked to saturate nonspecific antigen sites using 5% (vol/vol) goatserum-PBS (Jackson ImmunoResearch Laboratories, West Grove,PA) overnight at 4°C. On the next day, the sections were incubatedwith primary antibodies at 1:100 dilution for 4 h at room temperatureand visualized using appropriate species-specific secondary antibodiesconjugated with Alexa Fluor 680 at 1:1,000 dilution (Invitrogen,Carlsbad, CA) (87, 88). Muscle sections were counterstained withHoechst 33342, mounted on slides using fluorescence mounting me-dium (DakoCytomation, Carpinteria, CA), and examined under fluo-rescence microscopy.

Fluorescence-activated cell sorting. Fluorescence-activated cellsorting (FACS) was carried out on a flow cytometer (Aria Sorter, BDBiosciences, San Jose, CA). Data acquisition and analyses wereperformed using FACSDiva 5.0.3 software (BD Biosciences), gatedfor a high level of GFP or allophycocyanin (APC) expression. Theclear separation of GFP� from GFP� cells (14) and APC� fromAPC� cells, as well as the low flow rate, explains the ease andaccuracy of sorting (17). Sorted cells were reanalyzed to confirm theirfluorescence profile (14, 17).

Isolation of type-1 and type-2 DsRed� pericytes. Hindlimb musclecells were freshly isolated from young adult (3- to 5-mo-old) Nestin-GFP/�-actin-DsRed mice as described elsewhere (16). Briefly, mus-cles were carefully dissected away from the surrounding connectivetissue and minced, digested by gentle agitation in 0.2% (wt/vol)

type-2 collagenase in Krebs solution at 37°C for 2 h, and dissociatedby trituration and resuspension in 0.25% trypsin-0.05% EDTA in PBSfor 15 min at 37°C. After centrifugation at 1,500 rpm for 5 min, thesupernatant was removed, and the pellet was resuspended in growthmedium. Aggregates were removed by passage through a 40-�m cellstrainer prior to sorting. After they were counted, the cells werecentrifuged at 1,500 rpm for 5 min and resuspended in 100 �l of 1%FBS in PBS per 106 cells. First, an aliquot was collected for use asunlabeled control (labeled with only the secondary APC anti-rabbitantibody, without the primary rabbit anti-NG2 antibody) to set thegate for APC. The remaining cells were incubated with the primaryrabbit anti-mouse NG2 antibody for 45 min and washed in 1% FBS inPBS. They were then incubated for 30 min with APC anti-rabbitsecondary antibody and washed in PBS with 1% FBS. The gate forGFP was set using cells isolated from skeletal muscle of wild-typemice. Sorting was based on GFP and APC fluorescence. IsolatedNestin-GFP�/NG2-APC�/�-actin-DsRed� and Nestin-GFP�/NG2-APC�/�-actin-DsRed� cells were used in angiogenic in vitro assays,in Matrigel plug in vivo assays, and in cell fate-tracking experimentsto evaluate vessel formation in vivo after hindlimb ischemia. Incontrast to Nestin-GFP/NG2-DsRed, all Nestin-GFP/�-actin-DsRedcells express �-actin-DsRed (21), and their red fluorescence persistseven after their transdifferentiation, allowing us to track cell fate.

Cell culture. Human umbilical vein endothelial cells (HUVECs)were obtained from the American Type Culture Collection. Allexperiments used endothelial cells between passages 2 and 4. HUVECswere maintained in Medium 200 with endothelial low-serum growthsupplement (Invitrogen) at 37°C with 5% CO2. The medium waschanged every other day. All cells were maintained as subconfluentcultures and split 1:3 at 24 h before use.

Angiogenic assays. Angiogenic assays were performed as de-scribed elsewhere (4). Briefly, after overnight incubation at 4°C, 150�l of precooled Matrigel-reduced growth factor (BD Matrigel356230) were transferred to a 48-well plate on ice. After gel formationat 37°C for 30 min, 4 � 105 HUVECs were diluted in cell culturemedium, and 500 �l of the suspension were added to the 48-well plate.HUVECs were cultured alone as a control or together with 1.5 � 105 type-1DsRed� purified pericytes or 1.5 � 105 type-2 DsRed� purifiedpericytes in the same culture medium. Plating dishes were incubatedat 37°C, and after 3 h the culture medium was gently removed andMatrigel (150 �l) was added to form a sandwich that was finallycovered with 500 �l of culture medium. After 10 days, the formationof endothelial tubular networks was examined under the microscope.

Matrigel plug assays. Matrigel plug assays were performed asdescribed elsewhere (23). Briefly, 4 � 106 HUVECs and 5 � 105

type-1 DsRed� purified pericytes or 5 � 105 type-2 DsRed� purifiedpericytes were suspended in 500 �l of culture medium. The cellsuspensions were mixed with 500 �l of liquid Matrigel-reducedgrowth factor (BD Matrigel 356230) at a ratio of 1:1 at 4°C. Nudemice (2–3 mo old) received a total of 1 ml of this mixture subcuta-neously in the dorsal region, generating Matrigel plugs when warmedto body temperature (49). Plugs were recovered 2 wk later.

Intracerebral transplantation of glioblastoma cells. To evaluatecell recruitment during brain tumor growth, orthotopic glioblastomatumors were established by stereotaxic implantation of 2 � 105

actively growing G26-H2 murine glioma cells in Nestin-GFP/NG2-DsRed mice. Briefly, mice were anesthetized with a mixture of 114

Table 1. Antibodies and source

Antibody Source Location

Rat anti-CD31 (PECAM-1) BD Biosciences San Jose, CARat anti-mouse CD146 BioLegend San Diego, CARabbit anti-PDGFR� Dr. W. Stallcup Sanford-Burnham Medical Research Institute, La Jolla, CARabbit anti-NG2 chondroitin sulfate proteoglycan Chemicon-Millipore Temecula, CA

PECAM, platelet endothelial cell adhesion molecule; PDGFR, platelet-derived growth factor receptor.

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mg/kg ketamine and 17 mg/kg xylazine, and a 0.45-mm burr hole wasmade 2 mm lateral and 0.5 posterior to the bregma in the right cerebralhemisphere through a scalp incision. Stereotaxic injection was per-formed on a Just For Mice stereotaxic apparatus (Harvard Apparatus,Holliston, MA), with insertion of a 10-�l syringe (Hamilton, Reno,NV) with a 30-gauge, 1-in. flat needle through the burr hole to a depthof 3.2 mm. A Nanomite programmable syringe pump (HarvardApparatus) delivered constant infusion at a rate of 0.5 �l/min to a totalvolume of 5 �l (11, 12).

To prevent infection and alleviate pain and/or discomfort, allanimals received an antibiotic (gentamicin, 5.8 mg/kg) and an anal-gesic (buprenorphine, 0.1–0.5 mg/kg). Mice were monitored for bodyweight and ambulatory, feeding, and grooming activities. Animalslosing �20% of their body weight or having trouble ambulating orfeeding were euthanized. Mice with tumors growing outside theirintracranial space were eliminated from the study.

At 5 wk after implantation, mice were anesthetized again andtranscardially perfused with cold PBS and then with 4% PFA solutionin PBS. After decapitation, brains were rapidly dissected out, removedfrom the skull, postfixed for 24 h in the same fixative solution, andcryoprotected with 30% sucrose in PBS for 2 days. The brains werethen placed in embedding cryomolds, covered with tissue embeddingmedium (OCT compound, Tissue-Tek, Sakura Finetek, Tokyo, Ja-pan), snap-frozen in liquid nitrogen, and stored at �80°C.

Brain histological processing. Serial, 20-�m-thick, coronal sec-tions of frozen brains, obtained using a cryostat (Microm HM 500,Zeiss, Oberkochen, Germany) at �20°C, were mounted on Super-Frost Plus microscope slides in series of six (Fisher Scientific) andstored at �20°C. For experiments, the sections were dried at roomtemperature for 1 h, rehydrated in PBS, permeabilized with 0.5%Triton X-100 in PBS solution, and blocked to saturate nonspecificantigen sites using 5% (vol/vol) goat serum-PBS at 4°C overnight. Onthe next day, the sections were incubated, with Hoechst 33342 used asa nuclear marker. The sections were mounted on slides using fluores-cence mounting medium and examined with fluorescence microscopy.

Subcutaneous transplantation of melanoma cells and tumor tissueprocessing. To evaluate cell recruitment during peripheral tumorgrowth, actively growing B16 melanoma cells were implanted sub-cutaneously into Nestin-GFP/NG2-DsRed mice at 3 � 105 cells permouse in 200 �l of PBS-Matrigel (BD Biosciences). When tumorsreached a volume �1,000 mm3, the mice were killed. Tumor tissuesand adjacent muscle were dissected, harvested, and fixed in 4% PFAovernight. Tissues were then immersed in 30% sucrose solutionovernight, embedded in OCT, and rapidly frozen in liquid nitrogen toprepare 10-�m-thick cryosections, which were fixed with 4% PFA for30 min, permeabilized in 0.5% Triton X-100, and blocked to saturatenonspecific antigen sites using 5% (vol/vol) goat serum-PBS over-night at 4°C. On the next day, the sections were incubated with theprimary antibody anti-CD31 (platelet endothelial cell adhesion mol-ecule) at room temperature for 4 h and visualized using appropriatespecies-specific secondary antibody conjugated with Alexa Fluor 680at 1:1,000 dilution (Invitrogen). The sections were counterstainedwith Hoechst 33342, mounted on slides using fluorescence mountingmedium, and examined with fluorescence microscopy.

Critical hindlimb ischemia model. Two- to 3-mo-old athymic nude(nu/nu) male mice were anesthetized with xylazine (20 mg/kg) andketamine (100 mg/kg) by intraperitoneal injection as described else-where (30) and subjected to hindlimb ischemia by femoral arteryligation and transection. Immediately after surgery, mice received 2 �106 type-1 or type-2 DsRed� pericytes or unconditioned DMEM (200�l) by intramuscular injection into the ischemic leg. Limb perfusionwas assessed with in vivo MRI angiography 10 days after treatment,followed by fluorescence microscopy analysis of muscle harvestedfrom the ischemic limb.

In vivo MRI angiography. All in vivo imaging was performed on aBruker 70/30 horizontal-bore, small-animal MRI scanner equippedwith a high-power gradient insert (60-mm inside diameter) capable of

generating a maximal magnetic field gradient of 1,000 mT/m (BrukerBiospin, Ettlingen, Germany). Each animal was placed prone in asliding mouse bed in an induction chamber initially filled with roomair and then with a mixture of isoflurane (2%) and O2 (2 l/min) untilthe animal was anesthetized. A pillow over the abdomen monitoredrespiration rate, and a nose cone supplied isoflurane and O2 (typicallevels: 1.5% and 1 l/min) during scanning. Thermostatically con-trolled warm air was blown into the bore of the magnet to keep theanimal’s skin temperature �35°C.

The animal’s pelvis was placed at the center of the 7-T MRImagnet in a quadrature-volume radio-frequency coil with an insidediameter of 35 mm. A three-plane localizer scout scan was acquiredusing a rapid-acquisition-with-relaxation-enhancement (RARE) pulsesequence with the following parameters: repetition time (TR) � 1,500ms, echo time (TE) � 35 ms, field of view (FOV) � 3 cm, matrix size �128 � 128, slice thickness � 2.0 mm, and number of excitations(NEX) � 1.

The high-resolution, T1-weighted scan and angiography scan wereplanned using the three-plane localizer images. The T1-weighted scanwas acquired using a RARE pulse sequence with the followingparameters: TR � 3,000 ms, TE � 8 ms, matrix � 256 � 256, FOV �2.2 cm, and NEX � 3, for a total acquisition time of 7 min and anin-plane resolution of 86 �m. High-resolution angiography scans wereacquired using a fast low-angle shot two-dimensional gradient echoangiography sequence with the following parameters: TR � 15 ms,TE � 4 ms, FOV � 2.2 cm, matrix � 512 � 360, slice thickness �0.40 mm, slice gap � 0.25 mm, and NEX � 16, for a total acquisitiontime of 1 h 14 min and an in-plane resolution of 43 � 61 �m. Whenscanning was complete, the mouse was removed from the scanner,placed on a warming pad, and allowed to regain consciousness.

Microscopy, cell imaging, and counting. An inverted motorizedfluorescence microscope (model IX81, Olympus, Tokyo, Japan) withan Orca-R2 Hamamatsu charge-coupled device camera was used forimage acquisition. Camera drive and acquisition were controlled by aMetaMorph imaging system (Olympus, Center Valley, PA). Tenarbitrary microscopic fields were counted in each immunostainedplate or tissue section, and values were pooled from parallel duplicatesper time point and individual experiment.

Statistical analysis. Values are means SE. Statistical significancewas assessed by Student’s t-test using GraphPad Prism (GraphPadSoftware, San Diego, CA). P 0.05 was considered significant.

RESULTS

Two pericyte subtypes enwrap blood vessels of variouscalibers. Pericytes have been reported around microvessels (6,76) and possibly larger vessels (1, 7, 27, 31, 56, 83), and theirheterogeneity has been described in various tissues (15, 17–19,38). However, whether distinct classes of pericytes surroundblood vessels of a certain caliber is unknown.

The marker most commonly used to identify pericytes inrecent years is NG2, the neuron-glial 2 chondroitin sulfateproteoglycan (62, 63). We analyzed the small skeletal musclecapillaries and larger mesenteric blood vessels from NG2-DsRed/Nestin-GFP mice in which NG2 and Nestin regulatoryelements control DsRed and GFP expression, respectively. Wefound the two types of pericytes, type 1 and type 2, aroundlarge blood vessel walls (Fig. 1A) and small capillaries (Fig.1B). Type-1 and type-2 pericytes stained positive for platelet-derived growth factor receptor-� (PDGFR�) (15, 18, 43) andCD146 (15, 18, 28, 68) (Fig. 1C). Our results indicate that bothpericyte subpopulations line the outer surface of the vascula-ture, irrespective of its caliber.

Recently, we showed that pericyte subtypes differ in theirdifferentiation capacity. Type-1 pericytes are responsible for

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ectopic adipocyte deposition (15) and fibrous tissue accumu-lation with aging (18), while type-2 pericytes differentiate intothe neural lineage (16, 17) and participate in skeletal muscleregeneration after injury (15). Pericyte participation in engi-neered new blood vessels (30, 42) indicates their therapeuticpotential, but whether the subtypes contribute differently toangiogenesis is unknown.

Only one pericyte subtype has angiogenic potential in vitro.The angiogenic potential of pericyte subpopulations was ini-tially evaluated in vitro by testing their ability to form vessel-like structures when incubated with HUVECs. All type-1 andtype-2 pericytes, sorted from Nestin-GFP/�-actin-DsRed mice,expressed constitutive DsRed. In contrast to Nestin-GFP/NG2-DsRed mice, Nestin-GFP/�-actin-DsRed mice express �-actin-

Fig. 1. Two pericyte subtypes are found in capillaries and larger blood vessels. A and B: pericyte subtypes in mesenteric vessels and skeletal muscle fromNestin-GFP/NG2-DsRed mice. A: pericytes surround mesenteric blood vessels. All panels show the same area for different channels (Nestin-GFP, NG2-DsRed,merged fluorescence, bright-field, and merged fluorescence and bright-field images). B: pericytes surround vessels in skeletal muscle. Muscle longitudinalsections show small blood vessels with CD31� endothelial cells surrounded by NG2-DsRed� pericytes. Red and green arrows indicate Nestin-GFP�/NG2-DsRed� (type-1) and Nestin-GFP�/NG2-DsRed� (type-2) pericytes, respectively. Blood vessels are labeled by the endothelial cell marker CD31. All panelsshow the same area for different channels (Nestin-GFP, NG2-DsRed, Hoechst, CD31 staining, bright-field, merged fluorescence, and merged fluorescence andbright-field images). C: pericyte markers platelet-derived growth factor receptor-� (PDGFR�) and CD146 colocalize with skeletal muscle interstitialNestin-GFP-/NG2-DsRed� and Nestin-GFP�/NG2-DsRed� cells. Panels show identical muscle areas from top to bottom: PDGFR� or CD146 (orange),NG2-DsRed (red), Nestin-GFP� (green), Hoechst (blue), bright-field, and merged images. Red arrow indicates type-1 pericytes (Nestin-GFP�/NG2-DsRed�);green arrow indicates type-2 pericytes (Nestin-GFP�/NG2-DsRed�). Scale bar, 20 �m.

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DsRed in all cells (21), and the red fluorescence persists evenafter their transdifferentiation, allowing us to track them aftertransplantation (Fig. 2, A–D). We confirmed their purity asdescribed previously (18). HUVECs cultured alone in Matrigelusually form an unstable network that disappears after 5 days(57). All DsRed� cells are HUVECs, as they do not derivefrom �-actin-DsRed mice. After 10 days, we did not detect anyvessel-like structures in the culture with type-1 pericytes (Fig.2E). In contrast, type-2 pericytes from Nestin-GFP/�-actin-DsRed mice formed vessel-like networks in the in vitro Matri-gel assay (Fig. 2, E and F). All DsRed� cells were closelyassociated with HUVECs, assuming a clear periendothelialposition (P 0.05; Fig. 2, E and F). These results suggest thatonly type-2 pericytes are angiogenic in vitro.

Only type-2 pericytes form blood vessels in vivo. To examinethe angiogenic potential of type-2 pericytes in vivo, we used

the murine Matrigel plug assay. Matrigel, an extract of theEngelbreth-Holm-Swarm tumor composed of basement mem-brane components, is liquid at 4°C and forms a gel whenwarmed to 37°C (49). Pericyte subpopulations were isolatedfrom Nestin-GFP/�-actin-DsRed mice. In this assay, Matrigelplus HUVECs, Matrigel plus HUVECs and type-1 pericytes,and Matrigel plus HUVECs and type-2 pericytes were injectedseparately and subcutaneously into the dorsal region of nudemice (Fig. 3A). The Matrigel plug solidified after the injection.At 2 wk after implantation, the Matrigel plug containing type-2pericytes formed a large blood vessel network connected withthe host vasculature. These vessels contained blood (n � 3). Incontrast, the Matrigel plug containing only HUVECs (data notshown) or HUVECs plus type-1 pericytes displayed no func-tional vessels (n � 3; Fig. 3B). As the transplanted pericyteswere marked with �-actin-DsRed fluorescence, we detected

Fig. 2. Only type-2 pericytes are angiogenicin vitro. A: procedures for isolation of mono-nucleated cells from skeletal muscle of Nes-tin-GFP/�-actin-DsRed double-transgenic (Tg)mice. B: representative flow cytometry dotplot showing green fluorescent protein(GFP) vs. allophycocyanin (APC) fluores-cence, with the gate set using cells isolatedfrom wild-type mice. C and D: representa-tive dot plots showing GFP vs. APC fluores-cence using mononucleated cells from skel-etal muscle of Nestin-GFP/�-actin-DsRedmice, unlabeled and after labeling with NG2proteoglycan APC antibody. Two cell pop-ulations were isolated by sorting: Nestin-GFP�/NG2-APC�/�-actin-DsRed� (type-1pericytes, yellow) and Nestin-GFP�/NG2-APC�/�-actin-DsRed� (type-2 pericytes,green). All cells are DsRed� and can betracked in vitro after they are mixed withhuman umbilical vein endothelial cells(HUVECs). E: in vitro Matrigel assay con-taining a mixture of HUVECs with type-1 ortype-2 DsRed� pericytes sorted from skele-tal muscle of Nestin-GFP/�-actin-DsRedmice. DsRed fluorescence, bright-field, andmerged images are shown. After 10 days,only HUVECs cultured with type-2 peri-cytes formed stable vessel-like networks. F:percentage of HUVECs forming vessel-likestructures in coculture with type-1 or type-2pericytes. Values are means SE.

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those cells in the Matrigel plugs in vivo participating in thevessel formation (Fig. 3C). These experiments indicate thattype-2 pericytes are angiogenic in vivo.

Brain tumor angiogenesis recruits NG2-DsRed� cells thatexpress Nestin-GFP. Angiogenic activity is enhanced in somepathological conditions, such as tumors, in which pericytescontribute to vessel formation (10, 71, 85). Pericytes stabilizethe endothelium by surrounding the blood vessels and supportangiogenesis by secreting VEGF (41). To examine whetherone pericyte subtype or both are recruited during tumor angio-genesis, we injected actively growing glioblastoma tumor cellsinto the right brain hemisphere of 3-mo-old NG2-DsRed/Nestin-GFP mice (2 � 10 cells/5 �l, n � 3). After 5 wk, weremoved the brains to examine the NG2-DsRed� cells in thetumor margins and adjacent normal brain tissue in coronalsections (Fig. 4A). We detected NG2-DsRed�/Nestin-GFP� andNG2-DsRed�/Nestin-GFP� cells in healthy normal brain tissuesurrounding the tumor tissue (Fig. 4, B and C). Nestin-GFP� cellsrepresented 67.3 7.5% of NG2-DsRed� cells, while 32.7 7.5% were Nestin-GFP�. In contrast, 97.7 0.9% of NG2-DsRed� cells penetrating the tumor expressed Nestin-GFP, while

only 2.3 0.9% were Nestin-GFP� (Fig. 4, B–D). Thus NG2-DsRed�/Nestin-GFP� cells significantly increased, while NG2-DsRed�/Nestin-GFP� cells significantly decreased in the tumor(P � 0.02). These results support the conclusion that type-1pericytes (NG2-DsRed�/Nestin-GFP�) are not recruited duringtumor angiogenesis. Not all brain NG2-DsRed�/Nestin-GFP�

cells correspond to type-2 pericytes, as some are oligodendrocyteprogenitors (33). Whether more type-2 than type-1 pericytesmigrate toward the tumor or the cancer cells stimulate oligoden-drocyte progenitor cell migration is unknown.

Only type-2 pericytes are recruited during tumor vesselformation. As oligodendrocyte progenitors are restricted to thecentral nervous system (60), we implanted growing B16 mela-noma cells hypodermically into 3-mo-old Nestin-GFP/NG2-DsRed double-transgenic mice to evaluate cell recruitment duringtumor growth outside the central nervous system (3 � 105

cells/200 �l, n � 3). After 2 wk, the tumors were surgicallyremoved with a good margin of normal surrounding tissue (Fig.5A). Consistent with a previous report (17), we found two pericytesubpopulations on the abluminal surface of vessels in the normalskeletal muscle surrounding the tumor (Fig. 5, B and C). Endo-

Fig. 3. Matrigel plug assay shows that only type-2pericytes are angiogenic in vivo. A: transplantationscheme of an in vivo Matrigel plug assay. A mixture ofHUVECs and either type-1 or type-2 pericytes sortedfrom skeletal muscle are embedded in Matrigel andimplanted subcutaneously into nude mice. Matrigel plugis recovered after 2 wk for analysis. B: gross anatomy offreshly removed Matrigel plug with type-1 or type-2pericytes 2 wk after implantation. Type-2 pericytes,together with endothelial cells, form blood vessels invivo. C: �-actin-DsRed fluorescence around blood ves-sels in a Matrigel plug tracks type-2 pericytes in vivo.

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Fig. 4. Nestin-GFP�/NG2-DsRed�, but not Nestin-GFP�/NG2-DsRed�, cells, invade brain tumor mass. A: intracranial injection of allograft tumor cells and brainpreparation for histology. Growing G26-H2 murine glioblastoma cells were injected into the brain of a Nestin-GFP/NG2-DsRed double-transgenic mouse.B: representative image of a Nestin-GFP/NG2-DsRed mouse brain coronal section showing margin of a G26-H2 murine brain tumor. Dashed line indicatesseparation between tumor and normal surrounding tissue. Nestin-GFP�/NG2-DsRed� cells are shown invading the tumor; Nestin-GFP-/NG2-DsRed� cells arepresent only in adjacent healthy tissue. Left: identical areas in the brain section: NG2-DsRed (red), Nestin-GFP� (green), Hoechst (blue), bright-field,NG2-DsRed (red) and Nestin-GFP� (green) merged, and all images merged. Right: NG2-DsRed (red), Nestin-GFP� (green), and Hoechst (blue) merged. Regionsin red, yellow, and white boxes show Nestin-GFP�/NG2-DsRed� and Nestin-GFP�/NG2-DsRed� in normal tissue and Nestin-GFP�/NG2-DsRed� cellsinvading the tumor tissue, respectively; these areas are magnified in C. White arrows indicate Nestin-GFP�/NG2-DsRed� cells in the tumor. C: magnificationof NG2-DsRed� cells in normal and tumor cells in B. Identical brain tissue areas are shown for different channels: NG2-DsRed (red); Nestin-GFP� (green);Hoechst (blue); bright-field (BF); merged NG2-DsRed (red) and Nestin-GFP� (green) (N/N); all images merged; and merged NG2-DsRed (red), Nestin-GFP�

(green), and Hoechst (blue) (N/N/H). In normal tissue, NG2-DsRed� cells express Nestin-GFP transgene or not, while only NG2-DsRed�/Nestin-GFP� cellsare present in the tumor. D: percentage of Nestin-GFP�/NG2-DsRed� and Nestin-GFP�/NG2-DsRed� cells in normal brain or tumor 35 days after cellimplantation (n � 3 preparations). Predominantly type-2 pericytes invade the tumor.

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thelial cells forming capillary tubes are located near pericytes(86). In the walls of small vessels, we found both pericytesubtypes associated with endothelial cells marked with CD31(Fig. 5, B and C).

A fraction of NG2-DsRed� cells (56.7 5.0%) expressedNestin-GFP, while 43.3 5.0% were Nestin-GFP�. In con-

trast, we only detected NG2-DsRed�/Nestin-GFP� cells (99.7 0.3% of NG2-DsRed� intratumoral cells), which showed asignificant increase in the tumor (P � 0.0001), surroundingCD31� microvessels (Fig. 5D). Our results indicate that onlyendogenous type-2 pericytes (NG2-DsRed�/Nestin-GFP�) areangiogenic.

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Type-2 pericytes improve blood flow in a mouse model ofcritical hindlimb ischemia. Stimulation of angiogenesis canimprove perfusion and function in ischemic tissues (81). Crit-ical limb ischemia, a leading cause of nontraumatic amputation(61), is a condition of severe arterial obstruction in whichblood flow to the legs and feet is not sufficient to maintaintissue viability. We used a well-established mouse model withunilateral hindlimb ischemia (30) to test whether pericytesubtypes can be used to promote angiogenesis. We trans-planted type-1 �-actin-DsRed� pericytes, type-2 �-actin-DsRed� pericytes, or DMEM as a control into the ischemic legof a nude mouse with unilateral hindlimb ischemia (Fig. 6A).After 10 days, we assessed limb perfusion using in vivo MRIangiography. We found that while type-1 pericytes did notrecover femoral artery blood flow in the ischemic leg, type-2pericytes induced partial recovery (Fig. 6, A–D) (the imagesare representative of 2 experiments). To test whether type-2pericytes remain in the tissue, we analyzed muscles from thesemice by fluorescence microscopy. We found that �-actin-DsRed�/Nestin-GFP� type-2 pericytes formed the walls ofnewly formed blood vessels (Fig. 6E). These results indicatethat type-2 pericytes can be used for vascular therapy.

DISCUSSION

We propose that type-1 and type-2 pericytes in small capil-laries and larger blood vessels vary in their angiogenic capac-ity. Only type-2 pericytes are angiogenic in vitro and in vivoand can be used in cell therapy to improve perfusion inischemic tissues. In cancer, only type-2 pericytes from thesurrounding normal tissue contribute to angiogenesis and canbe a target for antiangiogenic therapy.

Pericytes are heterogeneous and differ in their regenerativecapacity. We propose that pericytes are multipotent, but theirsubtypes are oligopotent (18). They can behave like stem cells,but their potential in health and disease remains unknown (22).Despite their putative common identity, pericytes are a heter-ogeneous cell population and differ in their developmentalorigins. Their ontogeny indicates that those in the head derivefrom the neurectoderm, while those in other organs derive fromthe mesoderm (34). Pericytes from different anatomic locationsdiffer morphologically, biochemically, and physiologically (6,72), and different pericyte subsets occupy the periendothelialcompartment (9).

Their multiplicity raises a question: Do pericyte subpopu-lations vary in function like stem cells do? Our previouswork shows that pericyte subpopulations are oligopotent,differentially committed to specific lineages. Pericytes in-volved in repairing skeletal muscle differ from those that

contribute to scar and fat formation. Our transplantationstudies indicate that type-2 pericytes are myogenic (15),while type-1 pericytes do not form muscle but contribute tofat deposition in diseased skeletal muscle (15) and fibroustissue deposition with aging (18).

Angiogenesis is the process of blood vessel assembly, be-ginning with cell clustering and ending with a vascular net-work. Angiogenic potential has been reported in pericytes (2,30, 59, 79). They rapidly form neovasculature, which readilyanastomoses with the host vasculature and significantly ame-liorates hindlimb ischemia in a femoral artery ligation model(30). We show for the first time that only type-2 pericytes haveangiogenic potential.

Blood vessels consist of endothelial and mural cells (32, 64),the latter of which refers to pericytes or smooth muscle cells(8). Pericytes’ molecular markers can be down- or upregulatedin various culture conditions, pathologies, and developmentalstates (9, 31, 32). Identification of pericytes in tissue sectionsrelies on the combination of their anatomic location and spe-cific markers such as the NG2 proteogylcan (39, 50, 55, 78).We distinguished two populations of pericytes in the skeletalmuscle on the basis of Nestin-GFP expression (17); however,we do not know whether pericyte subtypes can interconvert.Two strategies, a pericyte subtype ablation in vivo and thediscovery of additional specific markers, will address thisquestion. We have shown that both pericyte subtypes candifferentiate into smooth muscle cells in vitro (17). Whetherpericytes differentiate into smooth muscle cells under specificconditions in vivo remains to be studied. Interestingly, wefound that only Nestin-expressing (type-2) pericytes, which donot express neural markers, form neural progenitor cells (14,16, 17).

The type-2 pericyte may be a cellular target for inhibitingtumor angiogenesis. Cancer is characterized by excessive an-giogenesis (24). Traditional antiangiogenic therapy aimed toinhibit as much as possible and to prune existing tumor vessels.The cellular targets of antiangiogenic drugs are normal hostcells, such as pericytes or endothelial cells, which circumventsthe acquired drug resistance when tumor cells are the target.Pericytes play an important role in stabilizing blood vessels inthe microvasculature (58, 76) through cross talk with endothe-lial cells. Pericytes deposit matrix or releasing factors that canpromote endothelial cell differentiation or quiescence (8). Tu-mor blood vessels associated intimately with pericytes aremore functional and stable than those lacking the support ofpericytes (25).

The combination of VEGF receptor and PDGFR� inhibitorsaffects pericyte-mediated endothelial cell survival, resulting in

Fig. 5. Type-2 pericytes are recruited during tumor angiogenesis. A: protocol for subcutaneous allograft tumor growth. B16 melanoma cells are injected intoNestin-GFP/NG2-DsRed double-transgenic mice, and tumor plus normal adjacent healthy tissue is surgically removed 2 wk later. B: representativeimmunofluorescence image of a Nestin-GFP/NG2-DsRed mouse tumor section stained with anti-CD31 antibody illustrating margin of the melanoma tumor andsurrounding normal tissue. Vertical dashed line indicates separation between tumor and normal skeletal muscle. The same tissue area is shown for differentchannels: NG2-DsRed (red), Nestin-GFP� (green), CD31 (orange or blue), Hoechst (blue), and merged images. Regions selected in red, yellow, and white boxesshow type-1 pericytes in normal tissue, type-2 pericytes in normal tissue, and type-2 pericytes associated with tumor blood vessels, respectively. These areas aremagnified in C. Only type-2 pericytes are associated with CD31� blood vessel endothelial cells in the tumor, while type-1 and type-2 pericytes associate withthese cells in the normal skeletal muscle. Type-1 pericytes are present only in adjacent healthy tissue. C: magnification of pericytes in normal and tumor tissuesin B. Identical tissue areas are shown for different channels: NG2-DsRed (red), Nestin-GFP� (green), CD31 (orange), Hoechst (blue), bright-field, and allfluorescent merged images. Type-1 and type-2 pericytes are associated with vessels (CD31�) in the normal tissue, while only type-2 pericytes associate withtumor vessels. D: percentage of Nestin-GFP�/NG2-DsRed� (type-1) and Nestin-GFP�/NG2-DsRed� (type-2) pericytes in normal and cancer tissues 15 daysafter tumor implantation (n � 3 preparations). Only type-2 pericytes are recruited during tumor blood vessel formation.

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Fig. 6. Type-2 pericytes recover blood flow in a mouse model of hindlimb ischemia. A: hindlimb ischemia protocol in nude mice. Femoral artery is ligated andtransected, type-1 or type-2 pericytes are intramuscularly injected into the ischemic leg, and limb perfusion is assessed with in vivo MRI angiography. B:high-resolution in vivo MRI. Angiography from mouse hindlimbs right after ligation of the femoral artery (time 0) and after 10 days of treatment. Red circlesindicate femoral artery location. C: MRI angiography of hindlimbs below the knee 10 days after treatment. D: new, partially developed femoral artery in ischemiclegs of mice injected with type-2 pericytes. Red rectangles indicate location of femoral arteries. E: DsRed and GFP fluorescence around vessels in whole muscles10 days after type-2 pericyte transplantation. Scale bar, 100 �m. O, occlusion of femoral artery; P1, type-1 pericytes; P2, type-2 pericytes.

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regression of tumor blood vessels and inhibiting tumor growth(13). In parallel, the genetic depletion of pericytes using viralthymidine kinase slows primary tumor growth (26). However,this strategy can also increase invasiveness, possibly by in-creasing normal blood vessel leakage and reducing the barrierthat tumor cells intravasate. Thus distinguishing tumor peri-cytes from normal pericytes could provide a specific cellulartarget for more efficient therapy. We found that only type-2pericytes participate in new blood vessel formation duringtumor angiogenesis (Fig. 7, A and B). Future work will analyzewhether and how tumor type-2 pericytes differ from type-2pericytes in normal vasculature. As pericytes are heteroge-neous and subsets have different functions, targeting only thepericyte subpopulation involved in angiogenesis may be moreefficient. Since antiangiogenic drugs are the leading therapy toarrest tumor growth, type-2 pericytes may provide a centralcellular target susceptible to signaling and pharmacologicalmanipulation.

Role of type-1 pericytes in tumor growth remains unclear.Our results indicate that endogenous type-1 pericytes do notparticipate in tumor angiogenesis, but they do not exclude arole in tumor growth. In cancer, stromal cells may acquire aphenotype of activated fibroblasts (69). The signals that medi-ate the transition of normal cells into cancer-associated fibro-blasts are not fully understood. Cancer-associated fibroblastsare commonly identified by their expression of �-smoothmuscle actin (37, 67, 82), which pericytes express in culture(48). Phenotypic features of cancer-associated fibroblasts canbe induced by transforming growth factor-�, which mediatesfibroblast activation in organ fibrosis (51). Similar pathways

may also be responsible for the emergence of cancer-associatedfibroblasts in tumors (66). These cells produce an extracellularmatrix rich in type I collagen, which is conducive to initiatingtumor angiogenesis (20).

Recently, we showed that type-1 pericytes are fibrogenicand, when exposed to transforming growth factor-�, maydifferentiate into fibroblasts, which produce type I collagen(18). Additional studies are needed to determine whethertype-1 pericytes can contribute to cancer-associated fibroblasts.However, using the Nestin-GFP/NG2-DsRed double-trans-genic mice, we cannot track the fate of type-1 pericytes thatchange or lose their marker expression. We used NG2 andNestin expression to distinguish type-1 pericytes (NG2�/Nes-tin�) in tissues, but we do not have a positive marker for them.As NG2 is expressed in type-1 and type-2 pericytes (15), wemust explore the complete type-1 and type-2 pericyte transcrip-tome to find a specific marker for type-1 pericytes.

Type-2 pericytes and therapeutic angiogenesis. Pathologicalchanges in the vascular system, such as constriction andobstruction, may lead to ischemia and limb amputation. Thegoal of therapeutic angiogenesis is to treat ischemia by stim-ulating new blood vessel growth from existing vessels (35, 36,44). Several cell types have been used to induce neovascular-ization. Of the available cell therapy approaches, endothelialprogenitor cells are lineage-committed and grow slowly (47).By contrast, induced pluripotent stem cells exhibit high repli-cative capacity (80), but they have a tendency to lead to cancer.

Transplantion of mesenchymal stem cells (MSCs) inducesneovascularization and improves blood flow to ischemichindlimbs in animal models (40, 45, 74). The presence of

Fig. 7. Schematic representation of type-2 pericyte in-volvement in angiogenesis. A: type-1 (yellow) andtype-2 (green) pericytes are associated with blood ves-sels. We propose that only type-2 pericytes are angio-genic. B: type-2 pericytes participate in angiogenesisassociated with ischemic conditions and tumor progression.

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MSCs in many adult tissues suggests a common origin. Somehave proposed that MSCs are pericytes on the basis of sharedmarkers in vivo and in vitro (22), and attention has turned topericytes as stem cells with broad organ distribution (5, 28, 29,52, 58, 70, 73). Consistently, pericytes have been proposed forangiogenic therapy on the basis of their role in forming andstabilizing engineered blood vessels (30, 42).

Because of their pivotal role in angiogenesis, pericytesrepresent a promising target for treatments designed to increasevascularization in ischemic diseases. Specific manipulation oftype-2 pericytes, instead of the whole pericyte population, canpreclude complications associated with excluding the benefitsof type-1 functions. Future research should compare the an-giogenic potency of various cell types: endothelial progenitorcells, embryonic stem cells, induced pluripotent stem cells, andMSCs. Our long-term studies will investigate the mechanismsunderlying the angiogenic potential of type-2 pericytes andwhether their ablation affects normal vascular function.

ACKNOWLEDGMENTS

We thank Dr. G. N. Enikolopov (Cold Spring Harbor Laboratory) and Dr.W. Stallcup (Sanford-Burnham Medical Research Institute) for sharing with usthe Nestin-GFP mouse and the rabbit anti-PDGFR� antibody, respectively. Dr.James Wood (Wake Forest School of Medicine Comprehensive Cancer Cen-ter) contributed expertise on flow cytometry to our project.

GRANTS

The present study was supported by a Wake Forest Pepper Center PilotProject and PUSH grant from the Wake Forest Comprehensive Cancer Center(to O. Delbono and A. Mintz), National Institute on Aging Grants AG-13934and AG-15820 (to O. Delbono) and P30-AG-21332 (to the Wake ForestClaude D. Pepper Older Americans Independence Center), and a Glenn/American Federation for Aging Research Scholarship for Research in theBiology of Aging (to A. Birbrair).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.B. and O.D. are responsible for conception and design of the research;A.B., T.Z., Z.-M.W., M.L.M., and J.D.O. performed the experiments; A.B. andO.D. analyzed the data; A.B., A.M., and O.D. interpreted the results of theexperiments; A.B. and O.D. prepared the figures; A.B. and O.D. drafted themanuscript; A.B., A.M., and O.D. edited and revised the manuscript; O.D.approved the final version of the manuscript.

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