Reticulon 4B (Nogo-B) is necessary for macrophageinfiltration and tissue repairJun Yua, Carlos Fernandez-Hernandoa, Yajaira Suarezb, Michael Schleichera, Zhengrong Haoa, Paulette L. Wrighta,Annarita DiLorenzoa, Themis R. Kyriakidesc, and William C. Sessaa,1

Departments of aPharmacology, bImmunobiology, and cPathology and Vascular Biology and Therapeutics Program, Amistad Research Building,Yale University School of Medicine, New Haven, CT 06519

Edited by Louis J. Ignarro, University of California, Los Angeles School of Medicine, Los Angeles, CA, and approved August 20, 2009 (received for reviewJuly 6, 2009)

Blood vessel formation during ischemia and wound healing re-quires coordination of the inflammatory response with genes thatregulate blood vessel assembly. Here we show that the reticulonfamily member 4B, aka Nogo-B, is upregulated in response toischemia and is necessary for blood flow recovery secondary toischemia and wound healing. Mice lacking Nogo-B exhibit reducedarteriogenesis and angiogenesis that are linked to a decrease inmacrophage infiltration and inflammatory gene expression in vivo.Bone marrow-derived macrophages isolated from Nogo knock-outmice have reduced spreading and chemotaxis due to impaired Racactivation. Bone marrow reconstitution experiments show thatNogo in myeloid cells is necessary to promote macrophage homingand functional recovery after limb ischemia. Thus, endogenousNogo coordinates macrophage-mediated inflammation with arte-riogenesis, wound healing, and blood flow control.

inflammation � ischemia � vascular

Reticulons (Rtn) are a family of proteins that are localizedprimarily to the endoplasmic reticulon (ER) of most cells by

virtue of an ER targeting motif in the carboxy terminal tail of theirreticulon homology domains (1, 2). In mammals, there are fourfamily members: Rtn 1, 2, 3, and 4, with each gene giving rise tomultiple isoforms. Insights into Rtn functions have been dissectedusing overexpression, knockdown, or knockout strategies, and aclear role for these proteins in tubulogenesis of the peripheral ERand membrane curvature has emerged (3–5). However, despite thesimilarities of these proteins, there is evidence that differentisoforms of each Rtn subclass may exert additional roles in mam-malian cell function other than establishing the ER membranecurvature.

In mammalian cells, the Rtn 4 family has three isoforms, namedNogo-A, -B, and -C. Nogo-A is highly expressed in the nervoussystem and is implicated in controlling axonal regeneration path-ways, Nogo-B is expressed in vascular cells and cardiac myocytes invivo and multiple cell-types in vitro, and Nogo-C is expressed in thenervous system and skeletal muscle cells (1, 6). Previous work hasidentified Nogo-B as a regulator of vascular remodeling in vivo (7)and cardiac function in mice and humans (8, 9). In mice and rabbits,neointimal expansion of injured blood vessels is associated with amarked reduction in endogenous Nogo-B levels suggesting thatNogo-B negatively regulates the extent of vascular injury, and, inhumans, the loss of Nogo-B strongly correlates with stenotic lesionsand plaque rupture (7, 10, 11). This phenotype is most clearlyobserved in mice lacking Nogo-A and -B (Nogo�/� mice), wherethere is no overt developmental vascular phenotype, however, aclear postnatal occlusive vascular remodeling is observed aftervascular injury, a phenotype rescued by local gene transfer ofNogo-B into the vessel wall (7) proving that this phenotype isNogo-B-dependent. However, the endogenous role of Nogo-B ininflammatory tissue repair and scope of Nogo function in non-neural cells is virtually unexplored. In the present study, we show anunanticipated role for Nogo-B in determining the degree of tissue

revascularization by regulating the extent of macrophage recruit-ment to sites of ischemia or wounds.

ResultsNogo-B Levels Are Induced During Tissue Ischemia, and Mice Deficientin Nogo-A/B Exhibit Impaired Responses to Tissue Injury. Since therole of Nogo in regulating the tissue repair are not known, weexamined if the levels of endogenous Nogo are regulated duringischemia-provoked injury in vivo. Hindlimb ischemia was surgicallyinduced as previously described in C57Bl6 mice (12, 13), and thelevels of Nogo-B were examined by Western blotting in tissuesextracts from nonischemic and ischemic tissues 3 days postischemia.As seen in Fig. 1A, Nogo-B1 (�45 kDa on 10% SDS-PAGE gel) isexpressed in nonischemic tissue, and an additional splice-variant,likely Nogo-B2 (�48 kDa), is more clearly observed in ischemictissue. Both isoforms are markedly upregulated in extracts preparedfrom adductor and gastrocnemius muscles, postischemia. The in-crease in Nogo-B levels are associated with an increase in Nogo-B1and -B2 mRNA levels via qRT-PCR (Fig. 1B). Thus, tissue ischemiainduces Nogo-B expression.

To examine if the upregulation of Nogo contributes to tissueremodeling postischemia, WT and Nogo�/� mice (14) were ex-posed to limb ischemia, and gastrocnemius blood flow was assessedvia directly measurement in the surgically manipulated left limbcompared to the contralateral right limb, using a deep penetratingLaser Doppler probe. As seen in Fig. 1C, before surgery (BS), theratio of blood flow between the left limb and right limb is 1, andblood flow postsurgery (PS) is reduced to the same extent in WTand Nogo�/� mice. However, the time-dependent recovery of bloodflow over a 4-week period, is reduced in Nogo�/� mice. Identicalresults were obtained in a different source of Nogo�/� mice(Nogo�/�lacZ; Fig. S1) (15) demonstrating that this effect isindependent of the source of Nogo�/� mice (14, 15). The impairedflow recovery in the Nogo�/� mice suggests that perhaps Nogo mayinfluence vascular patterning, thus we examined neonatal vascularpatterning via whole-mount staining and quantification of themouse ear vasculature in 3-week-old WT and Nogo�/� mice. Asseen in Fig. S2 A and B, the loss of Nogo does not influencepatterning of this circulation . It is well accepted that severe limbischemia triggers flow and macrophage-dependent collateral arte-rial remodeling and/or growth (arteriogenesis) in the thigh andincreases capillary density (angiogenesis) in the calf (16–18). Asseen in Fig. 1 D and E, the loss of Nogo reduces arteriogenesis inthe adductor muscle groups (representative angiogram in Fig. 1D

Author contributions: J.Y., C.F.-H., Y.S., Z.H., P.L.W., A.D., T.R.K., and W.C.S. designedresearch; J.Y., C.F.-H., Y.S., M.S., Z.H., P.L.W., and A.D. performed research; J.Y. and T.R.K.contributed new reagents/analytic tools; J.Y., M.S., and Z.H. analyzed data; and J.Y., T.R.K.,and W.C.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed: E-mail: william.sessa@yale.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0907359106/DCSupplemental.

www.pnas.org�cgi�doi�10.1073�pnas.0907359106 PNAS � October 13, 2009 � vol. 106 � no. 41 � 17511–17516

MED

ICA

LSC

IEN

CES

and quantitative angiography in Fig. 1E), and angiogenesis in thegastrocnemius muscle (Fig. 1F, upper panel via PECAM-1 stainingquantified as capillary/muscle fiber ratios), secondary to limbischemia. In addition, the recruitment of stabilizing mural cells tothe angiogenic vessels (Fig. 1F, lower panel, quantified as smoothmuscle �-actin positive/PECAM-1 positive capillaries) is also re-duced in Nogo�/� mice. Thus, mice deficient in Nogo exhibitimpaired recovery after ischemia that may be due to defectivearteriogenesis/angiogenesis.

Nogo�/� Mice Exhibit Defects in Macrophage Recruitment AfterInjury. The recruitment of monocytes/macrophages and associatedmacrophage-derived cytokines are necessary for arteriogenesissecondary to limb ischemia (19), thus, we examined the presence ofF4/80 positive macrophages recruited to the adductor and gastroc-nemius muscle groups after 3 days of ischemia. As seen in Fig. 2A,and quantified in Fig. 2B, the number of F4/80 positive macro-phages were markedly reduced in tissues from Nogo�/� micepostischemia. This difference in F4/80 positive macrophages intissue was not due to differences in circulating monocytes atbaseline or postischemia (side scatter low/CD11b� population inFig. 2C and quantified in Fig. 2D). These findings suggest that thereduction in monocyte/macrophage homing to injured tissue butnot mobilization of monocytes may explain, in part, the reducedarteriogenesis and blood flow recovery in Nogo�/� mice. Next, we

examined a different model of macrophage-dependent tissue re-modeling after full-thickness wounding of the skin. As seen in Fig.S3 A and B, Nogo�/� mice exhibited a delayed wound healingresponse compared to WT mice. Macrophage infiltration was alsoimpaired in Nogo�/� mice during wound healing compared to WTmice (Fig. S3C and quantified in Fig. S3D). Next, we examined theexpression of 92 proinflammatory and angiogenic genes by qPCRarrays in total RNA extracted from the gastrocnemius muscle groupfrom WT and Nogo�/� mice after 3 days of limb ischemia (Fig. S4).Nogo�/� mice showed a marked decrease in the expression of genesimplicated in inflammation [CCL2, CCL11, CCR5, CSF-1, IL-1�,TNF�, TNFRSF1B (TNF-R1)], macrophage homing (CCR2,MSR-1), and angiogenesis/vascular remodeling (angiopoetin 2)consistent with lower numbers of macrophages detected immuno-chemically and reduced recovery of blood flow postischemia.Analysis of blood chemistry demonstrated no differences in totalblood cell populations or leukocyte differentials between the strains(Table S1). To examine if Nogo regulates the pool of circulatingprogenitor cells released into the circulation secondary to ischemia,true CD34� cells (Fig. S5 A and B) and early outgrowth endothelialprogenitor cells (EPC) isolated from blood (Fig. S5 C and D) wereexamined in WT and Nogo�/� mice before and 7 days afterischemia. Ischemia increased the number of CD34�, and earlyoutgrowth EPCs in WT and Nogo�/� mice showing that ischemiainduced mobilization of endothelium progenitor cells is essentiallynormal in Nogo�/� mice.

Nog

o-/-

WT

BS PS 1 wk 2 wks 4 wks0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2WT

Nogo-/-

** *

Per

fusi

on u

nit r

atio

(Lef

t/Rig

ht)

Nogo-B1

Hsp 90

Hsp 90

Adductors

Gastrocnemius

Non-ischemic Ischemic

Nogo-B2

Nogo-B1Nogo-B2

WT Nogo-/-0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

*

Ves

sel l

engt

h ra

tio(L

eft/R

ight

)

WT Nogo-/-0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Ves

sel a

rea

ratio

(Lef

t/Rig

ht) *

0

1

2

3

4

5

6

7

*

WT Nogo-/-

Cap

illar

y de

nsity

(per

mus

cle

fiber

)

0

5

1 0

1 5

2 0

2 5

3 0

3 5

*

WT Nogo-/-

% S

MA

/cap

illar

y

0

5

10

15

20

25

Nogo-B1 Nogo-B2

Fol

d in

duct

ion

(Lef

t/Rig

ht)

A B C

D E F

Fig. 1. Nogo-B is induced with ischemia and is necessary for arteriogenesis and angiogenesis, thus functional recovery after ischemia. (A) Nogo-B1 and -B2proteins were induced in both adductor and gastrocnemius muscles by ischemia, and Hsp 90 was used as a loading control. (B) qPCR of RNA isolated from WTgastrocnemius muscle (n � 3) showed induction of Nogo expression 3 days after ischemia. Fold changes of ischemic (Left) versus nonischemic (Right) are shown.Ribosomal RNA (18s) was used as internal control. (C) Gastrocnemius blood flow in WT (n � 9) and Nogo�/� (n � 10) mice BS, PS, 1 week, 2 weeks, and 4 weeksafter arteriectomy. (D and E) Representative arteriograms (D) and quantification (E) of arteriogenesis after 2 weeks of ischemia in WT and Nogo�/� mice (n �7). (F) Quantification of capillary density (PECAM-1) and pericyte recruitment (smooth muscle �-actin) in gastrocnemius muscles before and 2 weeks after ischemia(n � 5). Data are expressed as mean � SEM. Two-way ANOVA; *, P � 0.05.

17512 � www.pnas.org�cgi�doi�10.1073�pnas.0907359106 Yu et al.

Bone Marrow Transfer Experiments Document that Nogo in Circulat-ing and in Resident Tissue Monocytes/Macrophages Contributes toImpaired Tissue Recovery in Nogo�/� Mice. To delineate if Nogo incirculating monocytes influences functional recovery of blood flowpostischemia, bone marrow transplantation (BMT) experimentswere performed. In this experiment, Nogo�/� mice were lethallyirradiated, and reconstituted with WT or Nogo�/� bone marrow(BM) cells for 6 weeks, followed by hindlimb ischemia. BMreconstitution was confirmed by complete blood counts (Table S1)and PCR from whole blood. Reconstitution of WT BM intoNogo�/� mice improves blood flow recovery postischemia close tothat seen in WT mice, suggesting that Nogo-B in circulating cells issufficient for functional recovery after ischemia (Fig. 3A). Next,lethally irradiated WT mice were reconstituted with Nogo�/� orWT BM, and blood flow recovery was assessed after hindlimbischemia. As seen in Fig. 3B, transfer of Nogo�/� BM into WT micesignificantly reduces blood flow recovery, albeit to a lesser extentthen WT marrow correcting the Nogo�/� defect. Examination ofmacrophage infiltration (via F4/80) postischemia documents thattransfer of WT BM into Nogo�/� mice rescues the defect inmacrophage homing as quantified in the adductor and gastrocne-mius muscles, respectively (Fig. 3 C and D). Nogo�/� BM failed tofully home in Nogo�/� and WT mice. Next, we studied whetherresident tissue macrophages in lethally irradiated WT mice werepresent and if they could produce cytokines to partially explain whythe Nogo�/� BM only partially promoted ischemic disease. As seenin Fig. 3F, resident macrophages in WT mice transplanted withNogo�/� BM can generate proangiogenic cytokine/chemokines(i.e., MCP-1, CSF-1, and CCR2, but not TNF� and IL-1�), that maycontribute to partial functional recovery in WT mice transplanted

with Nogo�/� BM. Collectively, these results suggest that Nogo-Bin BM-derived monocytes (BMM) or in resident tissue macro-phages is critical for functional recovery after ischemia.

Nogo-B Is Highly Expressed in Monocytes/Macrophages and Nogo�/�

Monocytes Are Defective in Cell Migration and Spreading. Westernblot analysis of human blood-borne monocytes and murine BMMdemonstrates that Nogo-B, but not Nogo-A (not shown), is highlyexpressed (Fig. S6A), consistent with a previous report (20). Toexamine if endogenous Nogo-B directly influences monocyte/macrophage function, BMM were isolated from WT and Nogo�/�

mice, and their migration and spreading were assessed. As seen inFig. 6B, using a modified Boyden chamber, the loss of Nogo did notaffect BMM chemokinesis, but reduced migration in response to agradient of the murine chemokines, CSF-1 and MCP-1. The loss ofNogo-B reduced BMM spreading onto glass or fibronectin-coatedslides and decreased the adhesive area of BMM to glass andfibronectin (Fig. S6 C and D), suggesting that endogenous Nogo-Bregulates chemotaxis and spreading, which may explain, in part, thereduced number of macrophages trafficking into the ischemic limbsor wounds from Nogo�/� mice.

BMM Isolated from Nogo�/� Mice Show Defects in Rac Activity,Morphology, and Chemokine Production. The reduction in chemo-kine-mediated migration in Nogo�/� BMM is reminiscent of cellsexhibiting impaired Rac-mediated cytoskeletal changes (21–23).Thus, we examined Rac localization, activation, and cytoskeletaldynamics in WT and Nogo�/� BMM. As seen in Fig. 4A, innonstimulated BMM, Rac (red) localizes mainly in a diffuse patternin the cytosol (upper panel) and stimulation with CSF-1 promotes

Add

ucto

rsG

astr

ocne

miu

s

ogoN -/-WT

100μm

0

5

1 0

1 5

2 0 WTNogo-/-

*

*

Adductors Gastrocnemius

% F

4/80

pos

itive

are

a

WT Nogo-/-

SS

C

CD11b PE

Non-ischemic Ischemic

% C

ircul

atin

g m

onoc

yte

0

1

2

3 WT

Nogo-/-

A B

C D

Fig. 2. Loss of Nogo impairs macrophage homing but not activation. (A) Representative images of macrophage (F4/80) staining of adductor and gastrocnemiusmuscles 3 days after ischemia and (B) quantification of F4/80 staining indicating impaired macrophage recruitment in Nogo�/� compared to WT mice (n � 5).(C and D) FACS analysis of circulating monocytes before and 3 days after ischemia (n � 3). Blood monocytes (green population) were defined by CD11bhigh/sidescatterlow (SSClow) in CD45� leukocytes. Data are expressed as mean � SEM. One-way ANOVA analysis is used; *, P � 0.05.

Yu et al. PNAS � October 13, 2009 � vol. 106 � no. 41 � 17513

MED

ICA

LSC

IEN

CES

Rac localization to the plasma membrane (lower panel). Endoge-nous Nogo-B (green) also partially colocalizes with Rac undercontrol conditions and colocalizes with Rac after stimulation.CSF-1 increases Rac activity, an effect significantly reduced inBMM isolated from Nogo�/� mice, (Fig. 4B). We also exploredother signaling pathways activated by CSF-1, such as Akt andmitogen-activated protein kinases (p42/44 ERK and p38 MAPK),which are known pathways that influence BMM motility andmorphology. As seen in Fig. S7, these pathways are not different inWT versus Nogo�/� BMM.

Primary BMM isolated from mice can exhibit multiple morphol-ogies when studied in vitro. For example, BMM can exhibit stellate,elongated, or migratory phenotypes as previously described (23).Next, we quantified these morphological end points in BMMisolated from WT and Nogo�/� mice. As seen in Fig. 5C, BMMisolated from WT mice exhibited a prominent migratory pheno-type, with less elongated and stellate-like cells. In contrast, BMM

cells isolated from Nogo�/� mice were less migratory and moreelongated and stellate shape consistent with the reduced spreadingand chemotaxis in these cells. F-actin levels upon stimulation ofCSF-1 were also accessed by phalloidin staining. As seen in Fig. 5D,the amount of F-actin was significantly decreased in Nogo�/�

BMM.To further examine the effect of Nogo-B on BMM gene expres-

sion function, we WT and Nogo�/� BMM with LPS (10 ng/mL).LPS induction of most genes were not different between the strains,however BMM from Nogo�/� mice showed a marked decrease inthe expression of genes implicated in inflammation (TNF, MMP12,IL-1�, IL-6ra) and the macrophage chemokine CCL2 (aka MCP-1)as shown in Fig. S8. Collectively, these data show that the loss ofNogo impairs several mononcyte/macrophages functions includingmigration, spreading, Rac activation, actin reorganization, andcytokine/chemokine gene expression, all of which may explain thedefective tissue repair processes in the Nogo�/� mice.

BS PS 1wk 2wk 3wk 4wk0.0

0.2

0.4

0.6

0.8

1.0

1.2 WT BM Nogo-/-

Nogo-/- BM Nogo-/-

Per

fusi

on u

nit r

atio

(Lef

t/Rig

ht)

BS PS 1wk 2wk 3wk 4wk0.0

0.2

0.4

0.6

0.8

1.2

1.0 Nogo-/- BM WTWT BM WT

Per

fusi

on u

nit r

atio

(Lef

t/Rig

ht)

WT BM Nogo-/-Nogo-/- BM Nogo-/- Nogo-/- BM WTWT BM WT

100μm

Add

ucto

rsG

astr

ocne

miu

s

Adductors

0

5

10

15

20

* *

% F

4/8

0 p

osi

tive

are

a

Gastrocnemius

0

10

20

30

%F

4/8

0 p

osi

tive

are

a

*

*

WT

BMW

TNog

o-/- B

MNog

o-/-

WT

BMN

ogo

-/-N

ogo

-/- B

MW

T

TNFα MCP-1 CSF-1 IL-1β CCR20

25

50

75

100

125

150

175

Fo

ld i

nd

uc

tio

n

(Is

ch

em

ic/n

on

-is

ch

em

ic)

* #

* *

*

*

WT BM WT

Nogo-/- BM Nogo-/-

WT BM Nogo-/-

Nogo-/- BM WT

#

WT BM Nogo-/- WT BM Nogo-/-Nogo-/- BM WT Nogo-/- BM WT

cimehcsIcimehcsi-noN

5050μm

*

*

**

**

A B

C D

E F

Fig. 3. WT BM can rescue the impairment of blood flow recovery and macrophage homing in Nogo�/� mice. (A) Hindlimb ischemia was performed on Nogo�/�

mice reconstituted with BM from WT or Nogo�/� mice (n � 6 of each group), and gastrocnemius blood flow was measured. (B) Hindlimb ischemia was performedon WT mice reconsituted with BM from WT or Nogo�/� mice (n � 6 of each group), and gastrocnemius blood flow was measured. (C) Representative images ofmacrophage (F4/80) staining of adductor and gastrocnemius muscles 3 days after ischemia. (D) Quantification of F4/80 staining indicating WT but not Nogo�/�

BM (n � 3 of each group) rescued the defect of macrophage recruitment in Nogo�/�. (E) Representative merged IF images of macrophage (F4/80 in green) andNogo (in red) staining in nonischemic and ischemic gastrocnemius muscles after BM transplantation. Arrow heads indicate resident macrophage, and arrowsindicate macrophage from circulation. (F) qRT-PCR analysis of cytokine/chemokine gene expression in gastrocnemius muscles 3 days after ischemia in each BMtransplantation groups (n � 3 of each group). Data are expressed as mean � SEM. One-way ANOVA; *, P � 0.05 compare to WT mice reconstituted with WT BM;#, P � 0.05 compare to WT mice reconstituted with Nogo�/� BM.

17514 � www.pnas.org�cgi�doi�10.1073�pnas.0907359106 Yu et al.

DiscussionThis paper documents an unanticipated role of Rtn 4 in inflam-mation and tissue repair. Here we show that Rtn 4, aka Nogo, is anendogenous regulator of inflammatory tissue remodeling andwound healing that is mediated, in part, via impaired macrophagehoming to ischemic tissue and wounds in vivo. Since the Nogo-deficient mice used in this study lack Nogo isoforms -A and -B (14),and only Nogo-B is detectable in vasculature and macrophages, weinterpret these results supporting a critical role of Nogo-B. Therelative importance of host versus inflammatory cell Nogo-B isskewed toward the role of Nogo in circulating inflammatory cells,since transfer of WT BM into irradiated Nogo-deficient micerescues the impaired ischemic vascular response and homing ofmacrophages to ischemic tissue. Mechanistically, the loss of Nogo-Bin BMM reduces the migratory phenotypes of isolated cells in vitro.Previous work has shown that Nogo-B in macrophages is a substratefor the protein kinase MAPKAP-K2, however, the loss of Nogo inBMM did not influence their chemotactic response to C5a (24).

Since the specific functional roles of the Rtn family members in vivoare still virtually unexplored data showing a marked reduction inmacrophage infiltration and macrophage-mediated tissue remod-eling in Nogo�/� mice combined with defects in macrophagefunction in vitro are striking and suggests an important Nogo-Bfunction during inflammation in vivo.

Little is known regarding the expression of Nogo-B in vivo andin vitro. In vivo, using mice that express LacZ in the Nogo-A/Blocus, the expression of Nogo is found in neurons (15, 25), atrialmyocytes, arteries, and veins (7), whereas in vitro, neural cellsexpress Nogo-A and primary cultures of EC and VSM and cancercell lines express Nogo-B (26, 27). In the model of hindlimbischemia, there was a marked increase in Nogo-B protein levels intissue extracts and gene expression in the ischemic limb. Theincrease in Nogo-B expression in tissue is likely due to increasedgene expression in vascular cells as well as the recruitment ofmonocytes/macrophages into tissue. In this model of severe limbischemia, it is believed that ischemia triggers the redistribution of

Stellate Elongated Migratory WT Nogo-/-

0

5

10

15

20

WT Nogo-/-

F-a

ctin

/bac

kgro

und

inte

nsity

*

CSF-1: 0 2 5 10 0 2 5 10 min

mar

ker

WT Nogo-/-

Rac-GTP

Nogo-B

Total Rac

Hsp 90

0.0

0.5

1.0

1.5

2.0

2.5

Rel

ativ

e R

ac-G

TP

ogoN -/-

0 2 5 10 min

*

TW

0

25

50

75

100

*

*

*

WTNogo-/-

Stellate Elongated Migratory

% o

f pop

ulat

ion

Rac Nogo Merged

10μm

Con

trol

CS

F-1

A B

C D

Fig. 4. Nogo colocalizes with Rac; loss of Nogo impairs Rac activation and F-actin assembly in BMM. (A) Representative confocal images showing Rac localization inWT BMM under quiescent (Upper) and CSF-1-stimulated (5 min, Lower) condition. Rac conpartially colocalizes with Nogo-B in plasma membrane upon stimulation inWT BMM. (B) Western blotting for active Rac indicates impaired kinetics of Rac activation upon CSF-1 stimulation. Lower panel show densitometric analysis from fourindividual Rac activity assays. (C) Confocal images illustrating stellate, elongated, and migratory morphology of BMM in vitro (Upper); quantification of BMMmorphology in WT and Nogo�/�. More than 100 cells from three individual experiments in each group were quantified (Lower). (D) Confocal images of phalloidinstaining of BMM stimulated with CSF-1 (Upper); quantification of F-actin intensity of the basal plane of BMM. Data are expressed as mean � SEM; *, P � 0.05.

Yu et al. PNAS � October 13, 2009 � vol. 106 � no. 41 � 17515

MED

ICA

LSC

IEN

CES

blood flow into preformed collaterals, and the increase in shearstress concomitant with the recruitment of macrophages and mac-rophage-derived cytokines such as MCP-1, TNF�, or VEGF par-ticipate in arteriogenesis in the adductor muscles, blood flowrecovery, and angiogenesis in the gastrocnemius muscle (16, 17, 28).Our data support this model since Nogo�/� mice had defects inmacrophage recruitment and arteriogenesis, blood flow recovery,and angiogenesis. Similar results were obtained in a model offull-thickness wounds, where Nogo�/� mice exhibit delayed mac-rophage infiltration and wound healing. A causal role for defectivemacrophages in the hindlimb ischemia model is suggested via theBMT experiments since WT BMM almost completely rescued thedefective macrophage homing and impaired function in Nogo�/�

recipients. However, since transfer of Nogo�/� BM in WT recipi-ents did not fully recapitulate the same degree of impaired limbfunction as in Nogo�/� mice, we cannot unequivocally exclude arole for host Nogo in this response until tissue-specific knockoutmice are used in similar experiments. Tissue macrophages arenotoriously difficult to completely eliminate from tissues, havedifferent kinetics of repopulating (29) and local macrophages mayproliferate in tissue after ischemia (30), thus, the presence ofresidual Nogo-positive macrophages after irradiation in WT micemay contribute to the recovery of function postischemia in micetransplanted with Nogo�/� BMM. Indeed our data suggests thatskeletal muscle resident macrophages exist in irradiated micebefore and after ischemia, and they contribute to cytokine geneexpression.

During inflammation, monocytes are actively recruited to sitesof inflammation where they differentiate into tissue macro-phages, processes critically dependent on cytoskeletal remodel-ing. Since our in vivo data suggested that the impaired responsesto ischemia and wounding may be due, in part, to defectivemacrophage recruitment, we focused on aspects of macrophagefunction in vitro. Indeed, BMM lacking Nogo did not spread welland exhibited attenuated migratory responses to the chemoat-tractants, CSF-1 and MCP-1. Since Rac is necessary for the

responses to these chemokines, we examined Rac localizationand activation in WT and Nogo-deficient cells. We found thatRac partially colocolizes with Nogo in the peripheral ER andplasma membrane and loss of Nogo significantly reduced Racactivation. Nogo�/� BMM has altered cell morphology andreduced F-actin clustering, consistent with reduced activation ofRac. Thus, we surmise that Nogo deficiency influences theactivation of Rac, which in turn reduces F-actin polarization andcell migration. Precisely how Nogo-B regulates Rac activation isnot known, however, there is precedence showing that other Rtnfamily members can modulate ER function and protein traffick-ing. Rtns are critical for assembly of the tubulated ER in yeastand mammalian cells (3, 31). In addition, Rtn 2B regulates thetrafficking of the glutamate transporter (32), Rtn 1B negativelyregulates the localization of the ER associated GTP activationprotein, TBC1D20, a GAP for the small GTPase Rab1 (33), andRtn 3 overexpression blocks ER-Golgi trafficking (34). Our datashowing an impairment of Rac activation can explain the spread-ing/migratiory and actin defects in Nogo-deficient BMM. Thiseffect, in turn, limits the number of macrophages infiltrating intotissue, thereby reducing arteriogenesis and delaying healing.Thus, understanding the endogenous roles of Rtns and Nogomay provide insights into pathways that regulate vascular in-f lammation associated with atherosclerosis, wound healing, andtumor progression.

Experimental ProceduresAll animal studies were approved by the institutional animal care and use com-mittees of Yale University. Two strains of Nogo�/� mice used were from MarcTessier-Lavigne (MTL) (14) and Steven Strittmatter (SS) (15) mice.

ACKNOWLEDGMENTS. We thank Stephen Strittmatter and Marc Tessier-Lavigne for Nogo�/� mice used throughout the studies; Dan Wu, AnthonyKoleske, and Martin Schwartz for helpful discussions; and Zhenwu Zhuang fortechnical assistance. This work was supported in part by National Institutes ofHealth Grants R01 HL 064793, RO1 HL 061371, R01 HL 081190, and PO1 HL 70295;Yale Proteomics Contract N01-HV-28186 (to W.C.S.); awards from the AmericanHeartAssociation(toJ.Y.andY.S.);andProgram3�3FellowshipfromtheCentroNacional de Investigaciones Cardiovasculares.

1. Oertle T, Schwab ME (2003) Nogo and its paRTNers. Trends Cell Biol 13:187–194.2. Teng FY, Tang BL (2008) Cell autonomous function of Nogo and reticulons: The

emerging story at the endoplasmic reticulum. J Cell Physiol 216:303–308.3. Voeltz GK, Prinz WA, Shibata Y, Rist JM, Rapoport TA (2006) A class of membrane

proteins shaping the tubular endoplasmic reticulum. Cell 124:573–586.4. Shibata Y, et al. (2008) The reticulon and DP1/Yop1p proteins form immobile oligomers

in the tubular endoplasmic reticulum. J Biol Chem 283:18892–18904.5. Shnyrova A, Frolov VA, Zimmerberg J (2008) ER biogenesis: Self-assembly of tubular

topology by protein hairpins. Curr Biol 18:R474–R476.6. Yang YS, Strittmatter SM (2007) The reticulons: A family of proteins with diverse

functions. Genome Biol 8:234.7. Acevedo L, et al. (2004) A new role for Nogo as a regulator of vascular remodeling. Nat

Med 10:382–388.8. Gramolini AO, et al. (2008) Comparative proteomics profiling of a phospholamban

mutant mouse model of dilated cardiomyopathy reveals progressive intracellular stressresponses. Mol Cell Proteomics 7:519–533.

9. Bullard TA, et al. (2008) Identification of Nogo as a novel indicator of heart failure.Physiol Genomics 32:182–189.

10. Paszkowiak JJ, et al. (2007) Evidence supporting changes in Nogo-B levels as a markerof neointimal expansion but not adaptive arterial remodeling. Vascul Pharmacol46:293–301.

11. Rodriguez-Feo JA, et al. (2007) Low levels of Nogo-B in human carotid atheroscleroticplaques are associated with an atheromatous phenotype, restenosis, and stenosisseverity. Arterioscler Thromb Vasc Biol 27:1354–1360.

12. Ackah E, et al. (2005) Akt1/protein kinase Balpha is critical for ischemic and VEGF-mediated angiogenesis. J Clin Invest 115:2119–2127.

13. Yu J, et al. (2005) Endothelial nitric oxide synthase is critical for ischemic remodeling,mural cell recruitment, and blood flow reserve. Proc Natl Acad Sci USA 102:10999–11004.

14. Zheng B, et al. (2003) Lack of enhanced spinal regeneration in Nogo-deficient mice.Neuron 38:213–224.

15. Kim JE, Li S, GrandPre T, Qiu D, Strittmatter SM (2003) Axon regeneration in youngadult mice lacking Nogo-A/B. Neuron 38:187–199.

16. Heil M, et al. (2004) Collateral artery growth (arteriogenesis) after experimentalarterial occlusion is impaired in mice lacking CC-chemokine receptor-2. Circ Res 94:671–677.

17. Weihrauch D, Arras M, Zimmermann R, Schaper J (1995) Importance of monocytes/macrophages and fibroblasts for healing of micronecroses in porcine myocardium. MolCell Biochem 147:13–19.

18. Heil M, Eitenmuller I, Schmitz-Rixen T, Schaper W (2006) Arteriogenesis versus angio-genesis: Similarities and differences. J Cell Mol Med 10:45–55.

19. Helisch A, Schaper W (2003) Arteriogenesis: The development and growth of collateralarteries. Microcirculation 10:83–97.

20. Rousseau S, Peggie M, Campbell DG, Nebreda AR, Cohen P (2005) Nogo-B is a newphysiological substrate for MAPKAP-K2. Biochem J 391:433–440.

21. Allen WE, Jones GE, Pollard JW, Ridley AJ (1997) Rho, Rac and Cdc42 regulate actinorganization and cell adhesion in macrophages. J Cell Sci 110:707–720.

22. Wells CM, Walmsley M, Ooi S, Tybulewicz V, Ridley AJ (2004) Rac1-deficient macro-phages exhibit defects in cell spreading and membrane ruffling but not migration.J Cell Sci 117:1259–1268.

23. Wheeler AP, et al. (2006) Rac1 and Rac2 regulate macrophage morphology but are notessential for migration. J Cell Sci 119:2749–2757.

24. Rousseau S, et al. (2006) CXCL12 and C5a trigger cell migration via a PAK1/2-p38alphaMAPK-MAPKAP-K2-HSP27 pathway. Cell Signal 18:1897–1905.

25. Huber AB, Schwab ME (2000) Nogo-A, a potent inhibitor of neurite outgrowth andregeneration. Biol Chem 381:407–419.

26. Li Q, et al. (2001) Link of a new type of apoptosis-inducing gene ASY/Nogo-B to humancancer. Oncogene 20:3929–3936.

27. Oertle T, Merkler D, Schwab ME (2003) Do cancer cells die because of Nogo-B?Oncogene 22:1390–1399.

28. Arras M, et al. (1998) Monocyte activation in angiogenesis and collateral growth in therabbit hindlimb. J Clin Invest 101:40–50.

29. Kennedy DW, Abkowitz JL (1997) Kinetics of central nervous system microglial andmacrophage engraftment: Analysis using a transgenic bone marrow transplantationmodel. Blood 90:986–993.

30. Khmelewski E, Becker A, Meinertz T, Ito WD (2004) Tissue resident cells play a dominantrole in arteriogenesis and concomitant macrophage accumulation. Circ Res 95:E56–E64.

31. De Craene JO, et al. (2006) Rtn1p is involved in structuring the cortical endoplasmicreticulum. Mol Biol Cell 17:3009–3020.

32. Liu Y, et al. (2008) Reticulon RTN2B regulates trafficking and function of neuronalglutamate transporter EAAC1. J Biol Chem 283:6561–6571.

33. Haas AK, et al. (2007) Analysis of GTPase-activating proteins: Rab1 and Rab43 are keyRabs required to maintain a functional Golgi complex in human cells. J Cell Sci120:2997–3010.

34. Wakana Y, et al. (2005) Reticulon 3 is involved in membrane trafficking between theendoplasmic reticulum and Golgi. Biochem Biophys Res Commun 334:1198–1205.

17516 � www.pnas.org�cgi�doi�10.1073�pnas.0907359106 Yu et al.