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Background <p>Diabetic retinopathy&#xa0;(DR) is the most common microvascular complication of diabetes, severely affecting the health and quality of life of individuals with diabetes. Müller cells undergo glial–mesenchymal transition (GMT) in a diabetic environment, contributing to retinal fibrosis. While complement activation is implicated in DR, the specific role of C3a in regulating Müller cells GMT and its underlying molecular mechanisms remains to be fully elucidated.</p> Methods <p>An in vitro high-glucose (HG) induction model was established using human Müller cells (MIO-M1). Cells were treated with recombinant C3a and the C3a receptor antagonist SB290157. Cell proliferation and migration were assessed via CCK-8, EdU, and Transwell assays. Protein expression levels of GMT markers (GS, GFAP, α-SMA) and Hippo-YAP pathway components (total YAP and TEAD) were measured by Western blotting. Gene expression of Yes-associated protein 1 (YAP) downstream targets was analyzed by Quantitative Real-Time Reverse Transcription PCR (qRT-PCR). Inflammatory cytokines were detected by Enzyme-Linked Immunosorbent Assay (ELISA). The role of YAP signaling was investigated using the inhibitor verteporfin.</p> Results <p>High glucose induced Müller cells proliferation and significantly increased C3a production. In the HG-induced model, exogenous C3a treatment promoted cell proliferation, migration, and the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α). C3a treatment upregulated GFAP and α-SMA while downregulating GS, indicative of GMT; these effects were reversed by SB290157. Mechanistically, C3a stimulation led to a significant upregulation of total YAP and TEAD protein levels, accompanied by a significant upregulation of YAP-dependent target genes CTGF and CYR61. These molecular changes suggest the activation of the Hippo-YAP signaling pathway. Treatment with the YAP inhibitor verteporfin suppressed C3a-induced proliferation, migration, and mesenchymal transition.</p> Conclusion <p>Our findings suggest that C3a may regulate the GMT process and inflammatory response in high glucose-stimulated Müller cells by activating the Hippo-YAP signaling pathway. These data provide insights into the potential molecular mechanisms linking complement activation to retinal fibrosis in DR, warranting further validation in vivo.</p>

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C3a targets the Hippo-YAP signaling pathway to promote Müller cells glial–mesenchymal transition in a high-glucose in vitro model

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月29日

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  • Shuang Liu,
  • Long Suo,
  • Hongliang Chen,
  • Qiankun Wang,
  • Dongyuan Mao,
  • Yuanyuan Cai,
  • Bing Qin

摘要

Background

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes, severely affecting the health and quality of life of individuals with diabetes. Müller cells undergo glial–mesenchymal transition (GMT) in a diabetic environment, contributing to retinal fibrosis. While complement activation is implicated in DR, the specific role of C3a in regulating Müller cells GMT and its underlying molecular mechanisms remains to be fully elucidated.

Methods

An in vitro high-glucose (HG) induction model was established using human Müller cells (MIO-M1). Cells were treated with recombinant C3a and the C3a receptor antagonist SB290157. Cell proliferation and migration were assessed via CCK-8, EdU, and Transwell assays. Protein expression levels of GMT markers (GS, GFAP, α-SMA) and Hippo-YAP pathway components (total YAP and TEAD) were measured by Western blotting. Gene expression of Yes-associated protein 1 (YAP) downstream targets was analyzed by Quantitative Real-Time Reverse Transcription PCR (qRT-PCR). Inflammatory cytokines were detected by Enzyme-Linked Immunosorbent Assay (ELISA). The role of YAP signaling was investigated using the inhibitor verteporfin.

Results

High glucose induced Müller cells proliferation and significantly increased C3a production. In the HG-induced model, exogenous C3a treatment promoted cell proliferation, migration, and the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α). C3a treatment upregulated GFAP and α-SMA while downregulating GS, indicative of GMT; these effects were reversed by SB290157. Mechanistically, C3a stimulation led to a significant upregulation of total YAP and TEAD protein levels, accompanied by a significant upregulation of YAP-dependent target genes CTGF and CYR61. These molecular changes suggest the activation of the Hippo-YAP signaling pathway. Treatment with the YAP inhibitor verteporfin suppressed C3a-induced proliferation, migration, and mesenchymal transition.

Conclusion

Our findings suggest that C3a may regulate the GMT process and inflammatory response in high glucose-stimulated Müller cells by activating the Hippo-YAP signaling pathway. These data provide insights into the potential molecular mechanisms linking complement activation to retinal fibrosis in DR, warranting further validation in vivo.