Human retinal Müller cells synthesize collagens of the vitreous and vitreoretinal interface in vitro

Abstract

Purpose: To investigate the capacity of cultured Müller cells to synthesize collagens, since previous studies indicated that Müller cells could be involved in collagen remodeling at the vitreoretinal border in adult human eyes.

Methods: Spontaneously immortalized cultured human Müller cells were analyzed for the presence of mRNA of types I-VII, IX, XI, and XVII collagen by RT-PCR. Furthermore, Müller cells were immunocytochemically stained for light microscopic (LM) evaluation of these collagens and their main characteristics. Finally, cell extracts and culture medium were evaluated by western blot (WB) analysis using anticollagen antibodies.

Results: Cultured Müller cells contained mRNA for types I-VII, IX, and XI collagen, but not for type XVII collagen. LM and WB confirmed the intracellular expression of all the above-mentioned collagens with the exception of type XVII. Collagen secretion into the medium was established for types I-VII, IX, and XI collagen.

Conclusions: Cultured Müller cells can synthesize internal limiting lamina and vitreous collagens. Possible collagen production by Müller cells could explain and expand on previous in vivo morphological findings in the embryonic and postnatal period and in pathologic conditions.

Figure 1

RT–PCR on Müller cell extracts. From left to right, bands indicating the positions of types I, II, III, IV, V, VI, VII, IX, XI, and XVII collagen are depicted. At the left margin, a 100 bp DNA ladder has been added.

Figure 2

Immunocytochemical analyses of cultured Müller cells in medium with fetal bovine serum. A: Type I collagen shows a granular staining with a variable intensity between cells. B: Type II collagen is seen as a faint staining in the cytoplasm. C: Type III collagen is positive in all cells. D: Type IV collagen is visible as a strong granular cytoplasmic staining. E: Type V collagen shows mainly staining in the cytoplasm. F: In the case of type VI collagen, the cells are predominantly stained in the cytoplasm. G: Type VII collagen is faintly positive in the cytoplasm. H: Type IX collagen is also present in the cytoplasm. I: Type XI collagen is primarily seen in the cytoplasm. Bars in all panels equal 50 μm.

Figure 3

Examples of western blot analyses: Müller cell extracts with the addition of type VII collagenase (0, 10, and 30 units/ml). A: The specific band of type II collagen is shown at 150 kDa and disappears without formation of new bands when type VII collagenase is added. B: At 180 kDa, the specific band of type III collagen disappears gradually on the addition of type VII collagenase. C: Addition of type VII collagenase to type VII collagen results in the gradual disappearance of the specific band at 270 kDa and the appearance of breakdown products at 130, 135, and 140 kDa. Specific bands are indicated with arrows.

Figure 4

Immunocytochemical analyses of cultured Müller cells in medium with G5. Types I (A) and V (C) collagen show clear extracellular fibrillar threads and less intracellular staining compared to Figures 2A and 2E, respectively. Types II (B) and XI (D) collagen show some fine extracellular threads and small granules (arrows) and decreased intracellular staining compared to Figures 2B and 2I, respectively. In the inlays of Figures 4B and 4D, the extracellular collagen is magnified two times. Bars in each panel equal 50 μm.