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. 2009 Oct 13;106(41):17511-6.
doi: 10.1073/pnas.0907359106. Epub 2009 Sep 25.

Reticulon 4B (Nogo-B) is necessary for macrophage infiltration and tissue repair

Jun Yu  1 , Carlos Fernández-Hernando, Yajaira Suarez, Michael Schleicher, Zhengrong Hao, Paulette L Wright, Annarita DiLorenzo, Themis R Kyriakides, William C Sessa
Affiliations

Reticulon 4B (Nogo-B) is necessary for macrophage infiltration and tissue repair

Jun Yu et al. Proc Natl Acad Sci U S A. .

Abstract

Blood vessel formation during ischemia and wound healing requires coordination of the inflammatory response with genes that regulate blood vessel assembly. Here we show that the reticulon family member 4B, aka Nogo-B, is upregulated in response to ischemia and is necessary for blood flow recovery secondary to ischemia and wound healing. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. Bone marrow-derived macrophages isolated from Nogo knock-out mice have reduced spreading and chemotaxis due to impaired Rac activation. Bone marrow reconstitution experiments show that Nogo in myeloid cells is necessary to promote macrophage homing and functional recovery after limb ischemia. Thus, endogenous Nogo coordinates macrophage-mediated inflammation with arteriogenesis, wound healing, and blood flow control.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Nogo-B is induced with ischemia and is necessary for arteriogenesis and angiogenesis, thus functional recovery after ischemia. (A) Nogo-B1 and -B2 proteins 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 WT gastrocnemius 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 weeks after 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 α-actin) in gastrocnemius muscles before and 2 weeks after ischemia (n = 5). Data are expressed as mean ± SEM. Two-way ANOVA; *, P < 0.05.
Fig. 2.
Fig. 2.
Loss of Nogo impairs macrophage homing but not activation. (A) Representative images of macrophage (F4/80) staining of adductor and gastrocnemius muscles 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/side scatterlow (SSClow) in CD45+ leukocytes. Data are expressed as mean ± SEM. One-way ANOVA analysis is used; *, P < 0.05.
Fig. 3.
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 performed on WT mice reconsituted with BM from WT or Nogo−/− mice (n = 6 of each group), and gastrocnemius blood flow was measured. (C) Representative images of macrophage (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) and Nogo (in red) staining in nonischemic and ischemic gastrocnemius muscles after BM transplantation. Arrow heads indicate resident macrophage, and arrows indicate macrophage from circulation. (F) qRT-PCR analysis of cytokine/chemokine gene expression in gastrocnemius muscles 3 days after ischemia in each BM transplantation 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.
Fig. 4.
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 in WT BMM under quiescent (Upper) and CSF-1-stimulated (5 min, Lower) condition. Rac conpartially colocalizes with Nogo-B in plasma membrane upon stimulation in WT BMM. (B) Western blotting for active Rac indicates impaired kinetics of Rac activation upon CSF-1 stimulation. Lower panel show densitometric analysis from four individual Rac activity assays. (C) Confocal images illustrating stellate, elongated, and migratory morphology of BMM in vitro (Upper); quantification of BMM morphology in WT and Nogo−/−. More than 100 cells from three individual experiments in each group were quantified (Lower). (D) Confocal images of phalloidin staining 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.
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