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.

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.

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