Expression of the oxygen-regulated protein ORP150 accelerates wound healing by modulating intracellular VEGF transport

Abstract

Expression of angiogenic factors such as VEGF under conditions of hypoxia or other kinds of cell stress contributes to neovascularization during wound healing. The inducible endoplasmic reticulum chaperone oxygen-regulated protein 150 (ORP150) is expressed in human wounds along with VEGF. Colocalization of these two molecules was observed in macrophages in the neovasculature, suggesting a role of ORP150 in the promotion of angiogenesis. Local administration of ORP150 sense adenovirus to wounds of diabetic mice, a treatment that efficiently targeted this gene product to the macrophages of wound beds, increased VEGF antigen in wounds and accelerated repair and neovascularization. In cultured human macrophages, inhibition of ORP150 expression caused retention of VEGF antigen within the endoplasmic reticulum (ER), while overexpression of ORP150 promoted the secretion of VEGF into hypoxic culture supernatants. Taken together, these data suggest an important role for ORP150 in the setting of impaired wound repair and identify a key, inducible chaperone-like molecule in the ER. This novel facet of the angiogenic response may be amenable to therapeutic manipulation.

Figure 1

ORP150 expression in healing human wound. (a–e) A human wound (31-year-old male) was analyzed either by H&E staining (×2) (the area indicated by open box was further studied in b–k) or immunohistochemically using anti-ORP150 (×40) (b), anti-CD68 Ab (×40) (c), anti-VEGF Ab (×40) (d), or anti-CD31 Ab (×40) (e), followed by visualization with the peroxidase method. (f–h) Sections adjacent to b (areas indicated by filled arrowheads) were double-stained with anti-ORP150 Ab (f) and anti-CD68 Ab (g). Both images were digitally overlapped (×100) (h). (i–k) Sections adjacent to f were double-stained with anti-ORP150 Ab (i) and anti-VEGF Ab (j). Both images were digitally overlapped (×100) (k). Open arrowheads in a denote the edge of the granulation tissue. See text for the description of filled arrowheads in b–e.

Figure 2

Expression of VEGF and ORP150 in human macrophages exposed to hypoxia. (a) Macrophages were either exposed to hypoxia (filled bars; 0–24 hours) or maintained under normoxic conditions (open bars). VEGF content in the culture supernatant was measured by ELISA as described in the text (n = 8; mean ± SD). *P < 0.01 by multiple contrast analysis. (b–d) Macrophages were exposed to hypoxia (0–24 hours). (b) RNA was prepared at the indicated times and subjected to RT-PCR analysis using primers for either VEGF (upper panel) or β-actin (lower panel). Protein extracts were prepared and subjected to Western blot analysis using anti-human ORP150 Ab (c). Cell lysates were incubated with either anti-human ORP150 Ab or anti-VEGF Ab. (d) The immunoprecipitant was separated in SDS-PAGE (8–12%; nonreducing condition for VEGF and reducing condition for ORP150) and subjected to Western blot analysis using either anti-VEGF Ab (left panel) or anti-ORP150 Ab (right panel). Migrations of molecular-weight markers are shown in the middle. The filled circles denote the signals derived from IgG used as primary Ab. (e–g) Human macrophages were double-stained with anti-ORP150 Ab (e) and anti-VEGF Ab (f). VEGF and ORP150 signals were digitally overlapped (×400) (g).

Figure 3

Dependence of VEGF processing on ORP150 in hypoxic macrophages. (a and b) Macrophages were exposed to hypoxia in the presence of Ad/S-ORP150, Ad/AS-ORP150, AxCALacZ, or AxGFP. Cell lysates were subjected to Western blot analysis using either anti-ORP150 Ab (a) (upper panel) or anti-KDEL mAb’s (lower panels). RNA was also prepared from cells incubated in the presence of adenovirus. (b) RT-PCR analysis was performed using primers specific for either VEGF (upper panel), β-actin (middle panel), or GAPDH (lower panel). (c) Cell lysates were fractionated, as described, and fractions corresponding to ER (lanes 1 and 2), Golgi apparatus (lane 3), plasma membrane (lane 4), and cytosol (lane 5) were subjected to immunoblot analysis using cellular organelle-specific Ab’s. (d–i) Cultured macrophages were infected with AxCALacZ (100 moi), Ad/S-ORP150 (0–100 moi), or Ad/AS-ORP150 (0–100 moi), and further incubated under hypoxic conditions for 24 hours, either in the absence (d, e, g, and h) or presence (f and i) of SNP. The content of VEGF antigen in the ER fraction (d–f) and culture supernatant (panels g–i) was measured by ELISA as described (n = 6; mean ± SD). *P < 0.01 compared with AxCALacZ-treated culture by either multiple comparison analysis (d, g, h, and f) or multiple contrast analysis, following two-way ANOVA (i). (j–o) Macrophages were infected with either AxCALacZ (j–l) or Ad/AS-ORP150 (m–o) and immunostained with either anti-KDEL mAb (j and m) or anti-VEGF mAb. (l and o) Both images were digitally overlapped. ×400.

Figure 4

Characterization of wounds infected with adenovirus. (a–h) Two days after the introduction of a wound (6 mm in diameter) in db/db mice, a mixture of adenovirus (AxGFP and Ad/S-ORP150; 5 × 10⁷ pfu each) was administered at each wound, as described. Two days after the infection, the tissue was sampled and subjected to H&E staining (×4) (a), immunostaining with anti-F4/80 Ab (×40) (b), or visualization of GFP signal by fluorescent microscopy (×40) (c). Signals derived from F4/80 and GFP were digitally overlapped (×40) (d). (a) Filled arrowheads indicate the initial wound area, and the open arrowheads indicate the edge of the granulation tissue. (e–h) Granulation tissue was stained with anti-ORP150 Ab and signals derived from (e) GFP (×40) and (f) ORP150 (×40) were visualized by fluorescent microscopy. (g) Signals of GFP and ORP150 were digitally overlapped. (×40.) (h) The adjacent section was immunostained with anti-F4/80 Ab. (×40.) (i–k) Either the indicated amount of AxGFP and Ad/S-ORP150 (open bars) or (i and j) AxCALacZ and Ad/S-ORP150 (10⁸ pfu each; filled bars), or (k) AxCALacZ and AxGFP (10⁸ pfu each; filled bar) was administered, as described. Three days after the infection, either GFP-positive cells (i) or the percentage of GFP-positive cells in F4/80-positive cells (j) was counted in the granulation tissue as described in the text. The content of tissue ORP150 antigen was assessed by ELISA as described. (k). For i–k, n = 6, mean ± SD. *P < 0.01 by multiple comparison analysis.

Figure 5

Effect of ORP150 on wound closure. A secondary intention wound (12 mm in diameter) was introduced in either C57BL/6J-m^+/+ Lepr^db (db/db) mice or C57BL/6J (wild-type) mice. Photographs of the wounds are shown on days 3 and 9 after induction of the wounds (a and d). The db/db mice were treated with either AxCALacZ (LacZ) or Ad/S-ORP150 (sense), and wild-type mice were treated with either Ad/AS-ORP150 (antisense) or AxCALacZ. In each case, 2 × 10⁸ pfu of adenovirus was subcutaneously injected at each octal angle of the wound 2 mm from the edge. Wound closure, shown as percentage of area of the initial wound (b and e) and histologic score (c and f) were determined on the indicated day after wounding: db/db mice were treated with AxCALacZ (db/db + LacZ) or Ad/S-ORP150 (db/db + sense), and wild-type were treated with AxCALacZ (wild-type + LacZ) or Ad/AS-ORP150 (wild-type + antisense). n = 4,; mean ± SD. *P < 0.01 by multiple contrast analysis.

Figure 6

ORP150 content in wounds after infection with recombinant adenoviruses. A secondary intention wound model (6 mm in diameter) was introduced in C57BL/6J-m^+/+ Lepr^db homozygous mice (db/db) (a–e) and C57BL/6J mice (wild-type) (f–j). Two days after the injury, recombinant adenovirus (5 × 10⁷ pfu each) was administered as described above. Photographs of the wounds are shown on days 3 and 9 after induction of the wounds (a and f). At the indicated time points, wounds were sampled (10 mm diameter), and protein extracts were prepared from the wound tissue and subjected to Western blot analysis with anti-ORP150 Ab. A typical example of four repeated experiments is shown (b and g). Densitometric analysis of multiple Western blots was performed in (c) (db/db mice) and (h) (wild-type mice). *P < 0.01 by multiple contrast analysis. (d and i) Angiogenesis was estimated semiquantitatively 7 days after the injury based on the area occupied by PECAM-1 immunoreactive tissue. n = 6; mean ± SD. **P < 0.01 by nonpaired t test. (e and j) Tissue VEGF content was measured in wound tissue. n = 6; mean ± SD. *P < 0.01 by multiple contrast analysis. Statistics were performed in comparison with animals infected with AxCALacZ (open bars) in each panel.