DOI: 10.1126/science.1203165, 238 (2011);333 Science

, et al.Christian GöritzA Pericyte Origin of Spinal Cord Scar Tissue

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and one malignant germ cell tumor). Six out ofthe 12 patients were designated as cachectic ac-cording to the definition of Evans et al. (31).Total lipase, ATGL, and HSL activities weresignificantly higher in visceral WAT of cancerpatients compared with individuals without can-cer and significantly higher in cancer patientswith cachexia compared with cancer patientswithout cachexia (Fig. 4, A to C). Lipase ac-tivities in cancer patients without cachexia weresimilar to those of noncancer patients. A signif-icant inverse correlation was found betweentotal lipase, ATGL, and HSL activities in WATof cancer patients and their body mass index(BMI) (Fig. 4, D to F). In contrast, lipolytic ac-tivities in WAT of noncancer patients showedno correlation with their BMI (fig. S15). Thus,our study provides compelling evidence thatthe previously observed increase in FA and glyc-erol release fromWATof patientswithCAC (9, 10)is due to up-regulation of ATGL and HSL acti-vities and that increased lipase activities strong-ly correlate with cachexia.

In summary, our data are consistent with theview that lipolysis plays an instrumental role inthe pathogenesis of CAC. The increased catabo-lism of adipose lipid stores leads to the completeloss of WAT followed by a reduction in musclemass. The absence of ATGL and, to a lesser de-gree, HSL reduces FAmobilization, retains WATand muscle mass, and prevents CAC. Whetherthe protection of adipose and muscle loss inlipase-deficient mice is a consequence of defec-tive tissue autonomous lipolysis or due to endo-

crine signaling from the tumor or WAT remainsto be elucidated. However, pharmacologicalinhibition of lipases may represent a powerfulstrategy to avoid the devastating condition ofcachexia in response to cancer or other chronicdiseases.

References and Notes1. M. J. Tisdale, Nat. Rev. Cancer 2, 862 (2002).2. J. E. Morley, D. R. Thomas, M. M. Wilson, Am. J. Clin.

Nutr. 83, 735 (2006).3. W. D. Dewys et al., Am. J. Med. 69, 491 (1980).4. C. Deans, S. J. Wigmore, Curr. Opin. Clin. Nutr.

Metab. Care 8, 265 (2005).5. K. C. Fearon, A. G. Moses, Int. J. Cardiol. 85, 73

(2002).6. M. Fouladiun et al., Cancer 103, 2189 (2005).7. K. C. Fearon, Proc. Nutr. Soc. 51, 251 (1992).8. M. Lainscak, G. S. Filippatos, M. Gheorghiade,

G. C. Fonarow, S. D. Anker, Am. J. Cardiol. 101, 8E(2008).

9. M. J. Tisdale, Physiol. Rev. 89, 381 (2009).10. T. Agustsson et al., Cancer Res. 67, 5531 (2007).11. A. Hyltander, P. Daneryd, R. Sandström, U. Körner,

K. Lundholm, Eur. J. Cancer 36, 330 (2000).12. S. Klein, R. R. Wolfe, J. Clin. Invest. 86, 1403

(1990).13. A. Legaspi, M. Jeevanandam, H. F. Starnes Jr.,

M. F. Brennan, Metabolism 36, 958 (1987).14. M. Rydén et al., Cancer 113, 1695 (2008).15. J. H. Shaw, R. R. Wolfe, Ann. Surg. 205, 368

(1987).16. M. Jeevanandam, G. D. Horowitz, S. F. Lowry,

M. F. Brennan, Metabolism 35, 304 (1986).17. J. M. Argilés, S. Busquets, M. Toledo, F. J. López-Soriano,

Curr. Opin. Support. Palliat. Care 3, 263 (2009).18. C. Bing et al., Proc. Natl. Acad. Sci. U.S.A. 101,

2500 (2004).19. R. Zechner, P. C. Kienesberger, G. Haemmerle,

R. Zimmermann, A. Lass, J. Lipid Res. 50, 3 (2009).

20. G. Haemmerle et al., Science 312, 734 (2006).21. G. Haemmerle et al., J. Biol. Chem. 277, 4806

(2002).22. M. van Royen et al., Biochem. Biophys. Res. Commun.

270, 533 (2000).23. I. Kawamura et al., Anticancer Res. 19, 341

(1999).24. Materials and methods are available as supporting

material on Science Online.25. J. Laurencikiene et al., Cancer Res. 68, 9247

(2008).26. X. Zhou et al., Cell 142, 531 (2010).27. S. Busquets et al., Clin. Nutr. 26, 239 (2007).28. M. J. Rennie et al., Clin. Physiol. 3, 387 (1983).29. J. Khal, A. V. Hine, K. C. Fearon, C. H. Dejong,

M. J. Tisdale, Int. J. Biochem. Cell Biol. 37, 2196 (2005).30. J. E. Belizário, M. J. Lorite, M. J. Tisdale, Br. J. Cancer 84,

1135 (2001).31. W. J. Evans et al., Clin. Nutr. 27, 793 (2008).Acknowledgments: We thank E. Zechner and C. Schober-

Trummler for reviewing the manuscript. The researchwas supported by the doctoral program MolecularMedicine of the Medical University of Graz (S.D.);GOLD, Genomics of Lipid-Associated Disorders aspart of the Austrian Genome Project GEN-AU funded byForschungsförderungsgesellschaft and Bundesministeriumfür Wissenschaft und Forschung (Ru.Ze.); SFB LIPOTOXgrant no. F30 (Ru.Ze., G.H.), the Wittgenstein Award 2007grant no. Z136 funded by the Austrian Fonds zur Förderungder Wissenschaftlichen Forschung (Ru.Ze.). S.K.D., Ro.Zi.,G.H., and Ru.Ze. hold a patent related to the modulationof ATGL for prevention and treatment of cachexia.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/science.1198973/DC1Materials and MethodsFigs. S1 to S15References

12 October 2010; accepted 27 May 2011Published online 16 June 2011;10.1126/science.1198973

A Pericyte Origin of SpinalCord Scar TissueChristian Göritz,1 David O. Dias,1 Nikolay Tomilin,2 Mariano Barbacid,3

Oleg Shupliakov,2 Jonas Frisén1*

There is limited regeneration of lost tissue after central nervous system injury, and the lesion issealed with a scar. The role of the scar, which often is referred to as the glial scar because ofits abundance of astrocytes, is complex and has been discussed for more than a century. Here weshow that a specific pericyte subtype gives rise to scar-forming stromal cells, which outnumberastrocytes, in the injured spinal cord. Blocking the generation of progeny by this pericyte subtyperesults in failure to seal the injured tissue. The formation of connective tissue is common tomany injuries and pathologies, and here we demonstrate a cellular origin of fibrosis.

Most studies on the scar tissue that formsat injuries in the central nervous sys-tem (CNS) have focused on astrocytes,

and it is often referred to as the glial scar (1–5).

There is also a connective tissue or stromal, non-glial, component of the scar (6–10), but it hasreceived much less attention. The generation ofconnective tissue, with large numbers of fibroblastsdepositing extracellular matrix (ECM) proteins, isa general feature of scarring and fibrosis in allorgans and in diverse types of pathology (11). Inspite of being a major clinical problem that hasbeen extensively studied, the origin of scar-formingfibroblasts has been difficult to establish. Moststudies have suggested that they may derive from

circulating cells, proliferating resident fibroblasts,endothelial cells, or epithelial cells (12–14). Thereare also data indicating that pericytes, perivascu-lar cells enwrapping the endothelial cells of cap-illaries, may differentiate into collagen-producingcells in models of dermal scarring and in kidneyfibrosis (15–17).

We have explored the role of pericytes inscar formation after spinal cord injury. We foundthat Glast-CreER transgenic mice (18) enabledrecombination of the R26R-yellow f luorescentprotein (R26R-YFP) reporter allele (19) in a sub-set of pericytes lining blood vessels in the spinalcord parenchyma, which allowed us to stably andheritably label these cells (20) (Fig. 1 and figs. S1to S5). The recombined cells had the typical ul-trastructural features of pericytes (21), includingbeing encased in the vascular basal lamina, whichseparates them from endothelial cells and astro-cytes (Fig. 1, A to D). The recombined cellsrepresent a distinct pericyte subpopulation thatconstitutes ~10% of all pericytes in the adultspinal cord [assessed by electron microscopy(EM)]. At positions where processes intersect,the Glast-CreER–expressing pericytes were in-variably located abluminal to the other pericytesubtype (Fig. 1A and fig. S6). We refer to thepericyte subclass that is recombined in Glast-CreER mice as type A pericytes and the other

1Department of Cell and Molecular Biology, Karolinska Insti-tute, SE-171 77 Stockholm, Sweden. 2Department of Neuro-science, Karolinska Institute, SE-171 77 Stockholm, Sweden.3Molecular Oncology Programme, Centro Nacional de Inves-tigaciones Oncológicas, 28029 Madrid, Spain.

*To whom correspondence should be addressed. E-mail:jonas.frisen@ki.se

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subclass as type B pericytes. Most or all CNSpericytes express platelet-derived growth factorreceptor (PDGFR) a and b; and CD13 (Fig. 1, Eand F, and fig. S3) (22–24), but the expression of

some other markers is heterogeneous (25). TypeB pericytes can be distinguished by the expres-sion of desmin and/or alpha smooth muscle actin(Fig. 1G and fig. S4).

To address whether pericytes participate inscar formation after spinal cord injury, we gen-etically labeled type A pericytes before a dorsalfuniculus incision or a dorsal hemisection andassayed the fate of recombined cells for up to7 months after the lesion (fig. S1B). The injurywas made following a 7-day clearing periodwithout tamoxifen, which ensures that all re-combination occurs before the insult, so thateven if cells other than type A pericytes wouldstart to express the Glast-CreER transgene in re-sponse to the injury, it would not result in re-combination (26). The injury induced an increasein the number of recombined cells (Fig. 2, A toG).This reaction was restricted to the injured seg-ment. After 9 days, the number of recombinedcells had increased more than 25-fold in the in-jured segment. The number of pericyte-derivedcells peaked at 2 weeks and then, as the scarcondensed (9), decreased after 4 months to a lev-el at which it remained for at least 7 months afterthe injury (Fig. 2G). This can be compared withthe dynamics of the astrocyte population, whichundergoes an approximate doubling in the first2 weeks in the same injury paradigm and thendecreases thereafter with similar kinetics (27).There are about 10 times as many astrocytes astype A pericytes in an uninjured spinal cord seg-ment (480 T 22 and 49 T 3 cells in a 20-mmcoronal section, respectively) (27), but 2 weeksafter a lesion, there are about two times as manypericyte-derived cells as newly generatedastrocytes in an injured spinal cord segment(Fig. 2H). The scar is compartmentalized, withpericyte-derived cells located in the center

Fig. 1. Genetic labeling of type A pericytes. (A to C) Electron micrographs showing a recombined type Apericyte (arrow) on a blood vessel. (A) Pseudocolors indicate a recombined pericyte (green), nonre-combined type B pericytes (blue), endothelial cells (red), and astrocytes (cyan). The inset shows the lightmicroscopic image of the section with the same recombined pericyte revealed by DAB reaction (arrow)before cutting ultrathin sections. Higher magnifications of the recombined pericyte from (A) show (B) thatit is surrounded by basal lamina (bl, arrowheads) and (C) the plasma membrane (pm, arrowheads) of theastrocyte. (D) Two recombined pericytes encapsulating two endothelial tubes (stained for von Willebrandfactor, vWF), surrounded by glial fibrillary acidic protein (GFAP)+ astrocyte processes. Type A pericytesexpress CD13 (E) and PDGFRb (F) but not desmin (G). Cell nuclei are visualized with 4′,6′-diamidino-2-phenylindole (DAPI) in (D) to (G) and appear blue. Scale bars: 2 mm in (A), 0.5 mm in (C), and 10 mm in (G).

Fig. 2. Pericytes form the core of the scar in the injured spinal cord. (A)Distribution of recombined type A pericytes (YFP) and astrocytes (GFAP) in anuninjured thoracic spinal cord segment. (B to F) Type A pericyte progenyoccupy the core of the scar and are surrounded by astrocytes after a dorsalfuniculus incision. (G) Number of type A pericyte–derived cells at the lesion

site. (H) Net addition of type A pericyte–derived cells compared with astro-cytes [data from (27)] 14 days and 4 months after injury. Cell nuclei arevisualized with DAPI in (A) to (F). (A) to (E) show coronal sections and (F) asagittal section. The quantifications show the average number of recombinedcells per 20-mm coronal section. Error bars represent SD. Scale bar, 200 mm.

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surrounded first by a layer of astrocytes originat-ing from ependymal cells and then by a layer ofastrocytes originating by self-duplication of resi-dent astrocytes (fig. S7) (27).

To gain insight into the dynamics of thepericyte injury response, we analyzed the earlyevents of pericyte recruitment. The lesion centerwas nearly devoid of blood vessels on days1 and 2 after the injury, but on days 3 to 5 afterthe injury, blood vessel sprouts, with an increaseddensity of associated pericytes, appeared at thelesion (Fig. 3A). All recombined pericytes weretightly associated with endothelial cells outside

the injury site, but many recombined cells hadlost contact with blood vessels at the lesion (Fig.3, B and C). Ultrastructural analysis showed anincrease in the number of type A pericytes anda change in their morphology. Five days afterinjury, they had detached from the basal laminaencasement and developed thin processes, someof which penetrated through the basal lamina toinvade the surrounding tissue (Fig. 3, D to F, andfig. S8). Type A, but not type B, pericytes de-posited abundant ECM within their basal laminaencasement. Type A pericyte progeny that hadinvaded the tissue were also surrounded by ECM

(Fig. 3, D, F, and I, and fig. S8 and S9). Five daysafter injury, the number of pericytes associatedwith blood vessels had significantly increasedin number (0.016 T 0.001 pericyte nuclear planesper 10-mm capillary surface by EM in the un-injured situation; n = 94 versus 0.151 T 0.045after injury; n = 82 (mean T SEM); P < 0.001Student’s t test). Type A pericytes increasedthree times more in number as compared withtype B pericytes, and most important, only typeA pericytes showed signs of leaving the bloodvessel wall (Fig. 3, C to G, and fig. S6). Therecombined cells that no longer had contact with

Fig. 3. Pericytes giverise to stromal cells anddeposit ECM in the in-jured spinal cord. (A) Ac-cumulation of type Apericyte–derived cells(YFP) and their detach-ment from the vascularwall (arrowheads) in thelesion area, 5 days afterinjury. (B) A blood ves-sel crossing the border(dashed line) betweenintact and injured (upperright) tissue, 5 days afterinjury. Type A pericytes(YFP) densely cover en-dothelial cells (vWF) with-in the intact tissue andtheirprogenydetach fromthe blood vessel wall andinvade the surroundingdamaged tissue (arrow-heads). (C) A blood ves-sel within the lesion withtype B pericytes (visual-ized with antibodiesagainst desmin) and typeA pericytes (YFP), 5 daysafter injury, showing theexpansion and detach-ment of the YFP-labeledcells. (D) Pseudocoloredelectron micrographshowing a blood vesselwith three recombinedtype A pericytes (green)5 days after injury. TypeA pericytes detach fromthe surroundingbasal lam-ina (bl), form thin pro-cesses, and deposit ECM.A type B pericyte (blue)remains tightly attachedto the basal lamina (seealso fig. S8). Its ultrastructure is retained, similar to that in uninjured tissues(Fig. 1A). An endothelial cell is colored red and astrocytes cyan. Boxedarea shows the fibrous ECM deposited around type A pericytes. (E) Three-dimensional reconstruction of a series of electron micrographs showing aleading process (lp) of a recombined type A pericyte breaking through itsbasal lamina (bl) encasement (gray). (F) Electron micrograph of the lesionarea 14 days after injury. Several type A pericyte–derived cells (green)have left the vascular wall and show abundant fibrous ECM (arrows) in

their immediate surrounding. Boxed area shows abundant fibrous ECM.(G) Large numbers of type A pericyte–derived cells (YFP) distant to bloodvessels (platelet endothelial cell adhesion molecule, PECAM) 14 days afterinjury. Dashed line outlines the ependymal layer. (H) Type A pericyte–derivedcells express smooth muscle actin (SMA) 5 days after injury. (I) Thedistribution of recombined cells overlaps with that of fibronectin 14 daysafter injury. Scale bars: 20 mm in (A) to (C) and (G) to (I), 2 mm in (D) and(E), and 5 mm in (F).

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blood vessels lost their expression of CD13 andPDGFRa, but remained positive for PDGFRband became positive for the fibroblast marker

fibronectin and transiently expressed the myofi-broblast marker smooth muscle actin (28) up to9 days after the injury (Fig. 3, H and I). They

were never positive for astrocyte or oligodendro-cyte lineage markers (figs. S10 and S11). Thevast majority of cells expressing markers of

Fig. 4. Pericyte-derived cells are essential for regaining tissue integrity. (Aand B) Many type A pericytes are Ki67+ 5 days after the injury and incorporateBrdU during the first 5 days after injury. (C) Schematic depiction of the strategyto block the generation of progeny by type A pericytes. (D and E) The gen-eration of type A pericyte progeny is abrogated in Glast-Rasless mice 5 daysafter spinal cord injury. (F) Comparison of the scar core volume within the glialborders that is occupied by PDGFRb+ stromal cells in vehicle- and tamoxifen-treated animals (Student’s two-tailed t test). (G) The percentage of the scar corevolume occupied by PDGFRb+ stromal cells correlates with the recombinationefficacy in Glast-Rasless mice (Pearson’s correlation coefficient). (H) Correla-

tion of the tissue defect volume to the recombination efficacy in Glast-Raslessmice. Individual animals are indicated with the same color in (G) and (H). (Iand J) The injury site (indicated by dashed line) of dissected spinal cords from avehicle-treated (I) and a tamoxifen-treated animal (J) 18 weeks after injury.Arrows point to the tissue defect in (J). (K to P) Sections of the spinal cords from(I) and (J) showing a scar with PDGFRb+ stromal cells and fibronectin in thevehicle animal and the absence of a corresponding stroma in the tamoxifenanimal, which has an open tissue defect lined by GFAP+ astrocytes. The animalin (J) is represented by a green dot in (G) and (H). Scale bars: 20 mm in (B),50 mm in (D), 0.5 mm in (I), and 0.1 mm in (O).

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stromal cells at the lesion, such as fibronectin,PDGFRb, and smooth muscle actin, were re-combined, which established type A pericytesas the main source of the scar connective tissue.Thus, pericytes enter the lesion area with bloodvessel sprouts, and type A pericytes give riseto cells that leave the blood vessel wall andform the stromal component of the scar tissue(fig. S12).

Analysis of bromodeoxyuridine (BrdU) in-corporation and the mitotic marker Ki67 revealedabundant proliferation of type A pericytes duringthe first days after the spinal cord injury. By day 5after the injury, 36.4 T 3.9% of YFP+ cells wereKi67+, and 95.5 T 1.9% had incorporated BrdU,which indicated that the large increase in thenumber of recombined cells is the result of pro-liferation of type A pericytes (Fig. 4, A and B).The absence of recombined cells in bone mar-row or blood excluded a circulating source of re-combined cells to the injured spinal cord (fig.S13). Furthermore, the Glast-CreER line did notrecombinemicroglia or macrophages, and type Apericyte–derived cells were distinct from thesecell types (fig. S14).

To assess the role of type A pericyte–derivedcells in the injured spinal cord, we devised agenetic strategy to inhibit their generation. Weestablished mice that, in addition to carrying theGlast-CreER and R26R-YFP alleles, were homo-zygous for H-ras and N-ras null alleles and forfloxed K-ras alleles, in which type A pericyteswould lack all ras genes after induction of re-combination (Fig. 4C, we refer to these mice asGlast-Rasless). ras genes are necessary for cellcycle progression and mitosis (29), and inducingrecombination before spinal cord injury drasti-cally reduced the appearance of recombined cellsat the lesion site (Fig. 4, D and E). Deleting allras genes did not result in any apparent alterationof the morphology or number of type A pericytesoutside the lesion nor did it alter the number ordistribution of blood vessels (fig. S15).

Adult Glast-Rasless mice in which recom-bination had been induced by five daily injec-tions of tamoxifen, followed by a 7-day clearingperiod, were subjected to a dorsal spinal cordhemisection and analyzed 18 weeks after theinjury. Mice of the identical genotype that re-ceived vehicle without tamoxifen served as con-trols and were indistinguishable from wild-typemice with regard to spinal cord scar formation.The scar of vehicle control animals was com-

posed of a core of PDGFRb-expressing cells en-cased in fibronectin and surrounded by astrocytes,similar to mice wild type for ras genes (Fig. 4).The tamoxifen group had significantly lessPDGFRb-positive stromal cells in the scar corecompared with the vehicle group (P = 0.001,Student’s t test) (Fig. 4F). Tamoxifen-inducedgenetic recombination with CreER is seldomcomplete, and we asked if the variation in thegeneration of stromal cells within the tamoxi-fen group was related to variation in recombina-tion efficacy. The size of the stromal componentin individual animals did indeed negatively cor-relate to the recombination efficacy (P = 0.0015,r = –0.8857, Pearson’s correlation coefficient)(Fig. 4G).

It became obviouswhen analyzing the injuredspinal cords that the generation of progeny bypericytes is important for sealing the injury, as33% of the tamoxifen-treated Glast-Rasless ani-mals had failed to close the lesion and had anopen tissue defect at the site of the injury (com-pared with none of the vehicle controls) (Fig. 4).We found a correlation between the recombina-tion efficacy and the failure to regain tissue in-tegrity, with the animals showing the highestrecombination efficacy having open tissue defectsat the lesion site (Fig. 4H). The tamoxifen-treatedGlast-Rasless mice with the highest recombina-tion efficacy were largely devoid of a stromal celland fibronectin scar core, which demonstrated thattype A pericyte–derived cells are required to sealspinal cord lesions (Fig. 4, I to P).

We have identified pericytes as a source ofscar-forming cells in the adult spinal cord. Pre-vious studies have demonstrated altered pericytemorphology in response to traumatic brain injuryand suggested that they may leave the vessel wall(30, 31), which in the light of our data indicatesthat scar formation by pericytes may be a generalresponse to injuries in the CNS and potentially inother organs. It is well known that pericytes areheterogeneous on the basis of the expressionof markers and morphology (25, 32). Here wedemonstrate functional heterogeneity of pericytepopulations, with scar formation restricted to adistinct subclass. Although the presence of stromalcells in CNS scar tissue has been long recognized(6–9), their role has been difficult to establish inthe absence of knowledge on their origin. We con-clude that the generation of progeny by pericytesis essential to regain tissue integrity after spinalcord injury.

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(2007).29. M. Drosten et al., EMBO J. 29, 1091 (2010).30. P. Dore-Duffy et al., Microvasc. Res. 60, 55 (2000).31. P. Dore-Duffy, Curr. Pharm. Des. 14, 1581 (2008).Acknowledgments: We thank A. Simon and the Frisén and

Shupliakov groups for valuable discussions. This studywas supported by the Swedish Research Council, theSwedish Cancer Society, Tobias Stiftelsen, Hjärnfonden,Knut och Alice Wallenbergs Stiftelse, the Swedish Agencyfor Innovation Systems, and the European ResearchCouncil (ERC-AG/250297-RAS AHEAD). D.D. wassupported by the Foundation for Science and Technologyfrom the Portuguese government (SFRH/BD/63164/2009).

Supporting Online Materialwww.sciencemag.org/cgi/content/full/333/6039/238/DC1Materials and MethodsFigs. S1 to S15References

21 January 2011; accepted 13 May 201110.1126/science.1203165

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