Substrate stiffness and pressure alter retinal Müller glia response and extracellular matrix production

Abstract

Background: The retina is highly influenced by its mechanical environment, with Müller glia (MG) acting as key mechanosensors and extracellular matrix (ECM) producers. This study examined MG responses to substrate stiffness and high pressure (HP), and whether TGF-β1 modulation could mitigate these effects.

Methods: Primary MG from adult rat retinas were cultured on glass (Young's modulus E'=∼1 gigapascal (GPa)) and polyacrylamide gels (10 kPa and 100 kPa). MG were exposed to atmospheric and 70 mmHg (HP) conditions, with TGF-β1 pharmacologically blocked.

Results: On glass and 100 kPa gels, MG survival, cell area, and ECM deposition (collagen I, IV, and fibronectin) increased, with cells adopting a fusiform shape and more dedifferentiated state. Under HP, survival decreased on stiffer substrates, though cell area and morphology remained unchanged. HP increased ECM deposition, which was reduced by TGF-β1 inhibition.

Conclusions: Our findings suggest that MG response to mechanical stress alter their survival and cell area, and increases ECM secretion, highlighting TGF-β1 as a potential therapeutic target.

Keywords: Extracellular matrix; Müller glia; Polyacrylamide gels; Pressure; Stiffness; TGF-β1.

Graphical abstract

Fig. 1

MG behavior on substrates of different stiffnesses. (A) Survival, cell area and morphology of MG cultured on PAA gels of 10 kPa and 100 kPa, and glass. The same number of MG were seeded on all substrates. (B) Immunolabelling of α-SMA in MG cultured on substrates of different stiffnesses. (C, D) Histograms of the percentage of MG and their cell area in the cultures on PAA gels relative to glass. (E) The percentage of MG positive to α-SMA in each substrate is shown. MG were labeled with antibodies against vimentin (green) and α-SMA (red). Nuclei were stained with DAPI (blue). Data is represented as mean ± SEM. *p-value < 0.05. Scale bar: 20 μm.

Fig. 2

HP conditions reduce MG survival but has no effect on cell area and morphology. Survival and morphology of MG cultured on PAA gels of 10 kPa and 100 kPa, and glass (A) in atmospheric conditions and (B) after being subjected to HP for 72 h. The same number of MG were seeded in all substrates. (C) Histogram of the percentage of MG in the cultures on PAA gels relative to glass. MG were labeled with antibody against vimentin (green). Nuclei were stained with DAPI (blue). Data is represented as mean ± SEM. *p-value < 0.05. Scale bar: 50 μm.

Fig. 3

HP increases MG expression of mechanoreceptor TRPV4 on stiffer substrates. Expression of the mechanosensitive channel TRPV4 by MG cultured on PAA gels of 10 kPa and 100 kPa, and glass (A) in atmospheric conditions and (B) after being subjected to HP for 72 h. The same number of MG were seeded in all substrates. (C) The percentage of TRPV4 expressed by MG relative to glass under atmospheric pressure condition. MG were labeled with antibody against vimentin (green) and TRPV4 (red). Nuclei were stained with DAPI (blue). Data is represented as mean ± SEM. *p-value < 0.05. Scale bar: 50 μm.

Fig. 4

HP and stiffer substrates promote MG deposition of ECM components. Deposition of collagen I, collagen IV and fibronectin by MG under atmospheric conditions (A, C and E, respectively) and after being subjected to HP for 72 h (B, D and F, respectively). The same number of MG were seeded in all substrates. Prior to immunolabelling, MG from the glass control condition were removed to ensure that only deposited ECM was taken into account. Histogram of the percentage of (G) collagen I, (H) collagen IV and (I) fibronectin deposited by MG relative to glass under atmospheric pressure. MG were labeled with antibody against vimentin (green), collagen I (red, A,B), collagen IV (red, C, D) and fibronectin (red, E, F). Nuclei were stained with DAPI (blue). Data is represented as mean ± SEM. *p-value < 0.05. Scale bar: 50 μm.

Fig. 5

TGF-β1 inhibition reduces MG survival and cell area under atmospheric conditions, but only cell area under HP. Survival, morphology and expression of TGF-β1 of MG cultured on PAA gels of 10 kPa and 100 kPa, and glass (A) in atmospheric conditions, (B) after being subjected to HP for 72 h, and after treatment with 10μg/mL of the TGF-β1 inhibitor SB-431542 (C) under control and (D) HP conditions. The same number of MG were seeded in all substrates. (E, F) Histograms of the percentage of MG and their cell area in the cultures on PAA gels relative to glass. (G) The percentage of TGF-β1 expressed by MG relative to untreated glass under atmospheric pressure condition. MG were labeled with antibodies against vimentin (green) and TGF-β1 (red). Nuclei were stained with DAPI (blue). Data is represented as mean ± SEM. *p-value < 0.05. Scale bar: 50 μm.

Fig. 6

TGF-β1 inhibition reduces MG deposition of ECM components under HP on stiffer substrates. Deposition of (A-D) collagen I, (E-H) collagen IV and (I-L) fibronectin by MG at (A,E,I) atmospheric pressure and (B,F,J) after being subjected to HP for 72 h, and after treatment with 10μg/mL of the TGF-β1 inhibitor SB-431542 (C,G,K) under control and (D,H,L) HP conditions. The same cell number was seeded in all substrates. Prior to immunolabelling, MG from the glass control condition were removed to ensure that only deposited ECM was taken into account. Histogram of the percentage of (M) collagen I, (N) collagen IV and (O) fibronectin deposited by MG relative to untreated glass under atmospheric pressure. Only deposition is shown for simplifying purposes. MG were labeled with antibody against collagen I (red, A-D), collagen IV (red, E-H) and fibronectin (red, I-L). Data is represented as mean ± SEM. *p-value < 0.05. Scale bar: 50 μm.