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Ocular instillation of vitamin A–coupled liposomes containing HSP47 siRNA ameliorates dry eye syndrome in chronic GVHD

Hiroyuki Ohigashi, Daigo Hashimoto, Eiko Hayase, Shuichiro Takahashi, Takahide Ara, Tomohiro Yamakawa, Junichi Sugita, Masahiro Onozawa, Masao Nakagawa and Takanori Teshima

Article Figures & Data

Figures

  • Figure 1.

    HSP47+myofibroblasts accumulated in fibrotic lesions of the lacrimal gland after allogeneic BMT. BALB/c mice were transplanted with 8 × 106 BM cells plus 2 × 107 splenocytes from allogeneic (Allo) B10.D2 or syngeneic (Syn) BALB/c donors on day 0, following 6 Gy TBI. Lacrimal glands were harvested on day +35 after BMT. H&E-stained (A) and MT-stained (B) images of the lacrimal glands. Scale bars, 100 μm. (C) MT staining (upper panels) and immunofluorescent images (lower panels) for HSP47 (red), with DAPI nuclear staining (blue) on the pairs of consecutive serial sections. Areas in the rectangles (middle panels) were magnified (right panels). Scale bars, 50 μm. Crosses indicate glandular parenchyma. Arrows indicate HSP47+ fibroblasts. (D) Immunofluorescent staining of HSP47 (green) and α-SMA (red) with DAPI nuclear staining (blue). Arrows indicate HSP47+ myofibroblasts. Scale bars, 50 μm. (E) Proportion of fibrotic area stained with MT compared with the total area of the sections of lacrimal glands from Syn (n = 6) and Allo (n = 9) mice. (F) The amount of collagen deposition in the lacrimal glands from Syn (n = 7) and Allo (n = 7) mice was normalized with tissue weight. Data from 2 independent experiments were combined and are shown as mean ± SEM. (G) The volume of tear secretion was measured as the length of wet cotton threads from Syn (n = 8) and Allo (n = 9) mice on day +35 posttransplant. Data from 2 independent experiments were combined and are shown as mean ± SEM. Dashed lines indicate the borders between the glandular parenchyma and interstitium. *P < .05, **P < .01, ***P < .005. Du, ductal lumen.

  • Figure 2.

    VA-lip HSP47 ameliorates fibrosis of the lacrimal glands in chronic GVHD. Mice were transplanted as in Figure 1. (A) VA-lip Dy647 eye drops were administered daily in 1 eye from day +29 to day +33 after allogeneic BMT. One hour later, the lacrimal glands were harvested from the treated and control sides. Images of VA-lip Dy647 particles (red) with DAPI (blue) counterstaining. The area in the dashed rectangle (middle panel) is magnified (lower panel). Scale bars, 20 μm. (B-F) A group of allogeneic recipients (Allo) was treated with VA-lip HSP47 eye drops from day +2 to day +34, and the lacrimal glands were harvested on day +35 after BMT. VA-lip Scr was administered to Allo control mice. (B) MT staining (upper panels) and immunofluorescent (lower panels) images for HSP47 (red) with DAPI nuclear staining (blue) on pairs of consecutive serial sections. Scale bars, 50 μm. Dashed lines indicate the borders between the glandular parenchyma and interstitium, crosses represent glandular parenchyma; and arrows point to HSP47+ fibroblasts. (C) H&E staining (upper panels) and MT staining (lower panels) of pairs of consecutive serial sections. Scale bars, 100 µm. (D) Proportion of fibrotic area to whole area of the sections of lacrimal glands stained with MT. Data from syngeneic mice (Syn; n = 3), allogeneic controls treated VA-lip Scr (Allo+Scr; n = 6), and allogeneic recipients treated with VA-lip HSP47 (Allo+VA-lip HSP47, n = 9) are shown as mean ± SEM. (E) The amount of collagen deposition in the lacrimal glands from Syn (n = 8), Allo+Scr (n = 9), and Allo+VA-lip HSP47 (n = 8) mice was normalized with the weight of tissues. (F) The volume of tear secretion was measured as the length of wet cotton thread inserted under the lower eyelids on day +35 posttransplant. Data from Syn (n = 3), Allo+Scr (n = 6), and Allo+VA-lip HSP47 (n = 9) mice are shown as mean ± SEM. (G-H) Allogeneic recipient mice (n = 13) were treated with VA-lip HSP47 eye drops in the right eye and VA-lip Scr eye drops in the left eye from day +2 to day +34 after BMT. (I-J) Allogeneic recipient mice were treated with VA-lip HSP47 eye drops once daily (n = 8) or twice daily (n = 8) from day +2 to day +34 after BMT. The amount of collagen deposition in the lacrimal glands (G,I) and the volume of tear secretion (H,J) on day +35 are shown as mean ± SEM. (D-J) Data from 2 independent experiments were combined. *P < .05; **P < .01.

  • Figure 3.

    VA-lip HSP47 eye drops resolve established lacrimal gland fibrosis in chronic GVHD. Mice were transplanted as in Figure 1. A group of allogeneic recipients (Allo) was treated with VA-lip HSP47 eye drops or VA-lip Scr eye drops daily from day +21 to day +34, and the lacrimal glands were harvested on day +35 post-BMT. (A) Representative images of MT staining of lacrimal glands harvested from syngeneic (Syn; upper panel) or Allo (lower panel) recipients on day +21 posttransplant. Scale bars, 50 µm. (B) Immunofluorescent images for HSP47 (red) with DAPI (blue) counterstaining on day +35. Arrows point to HSP47+ fibroblasts. Scale bars, 50 µm. (C) MT staining of lacrimal glands harvested from Allo mice given VA-lip Scr (Allo + Scr; upper panel) and Allo mice given VA-lip HSP47 (Allo + VA-lip HSP47; lower panel) on day +35 post-BMT. Scale bars, 50 µm. (D) The amount of collagen deposition in the lacrimal glands was evaluated before (Allo Day 21, n = 8) and after ocular instillation with VA-lip Scr (Allo + Scr Day 35, n = 8) or VA-lip HSP47 (Allo + VA-lip HSP47 Day 35, n = 7). Syn controls were evaluated on day +21 (n = 8). (E) Tear secretion was assessed before initiation of ocular instillation (Allo Day 21, n = 18) and after ocular instillation with VA-lip Scr (Allo + Scr Day 35, n = 8) or VA-lip HSP47 (Allo + VA-lip HSP47 Day 35, n = 10). Syn controls were evaluated on day +21 (n = 8). (D-E) Data from 2 similar experiments were combined. *P < .05; **P < .01; ***P < .005.