Exosomes derived from platelet-rich plasma activate YAP and promote the fibrogenic activity of Müller cells via the PI3K/Akt pathway

doi:10.1016/j.exer.2020.107973

Experimental Eye Research

Volume 193, April 2020, 107973

https://doi.org/10.1016/j.exer.2020.107973 Get rights and content

Highlights

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We firstly studied the role of PRP-Exos in the regulation of fibrogenic activity of Müller cells and its mechanism.
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PRP-Exos from diabetic rats could effectively increase proliferative and migratory activities of Müller cells.
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PRP-Exos may contribute to the activation of YAP and promote the fibrogenic activity via the PI3K/Akt pathway.

Abstract

The purpose of this study was to investigate the role of exosomes derived from platelet-rich plasma (PRP-Exos) in the regulation of the fibrogenic activity of Müller cells and the underlying mechanism. We studied the effects of PRP-Exos on the fibrogenic activity of human retinal Müller cells (hMCs) in vitro. PRP-Exos were isolated from the plasma of diabetic rats (DM-PRP-Exos) and normal control rats (Nor-PRP-Exos) and then observed by transmission electron microscopy. After treatment with DM-PRP-Exos or Nor-PRP-Exos, the proliferation and migration of hMCs were measured in vitro. Western blotting was conducted to assess the levels of fibrogenic molecules and activation of Yes-associated protein (YAP) and the PI3K-Akt signalling pathway. In cultured hMCs, DM-PRP-Exos but not Nor-PRP-Exos effectively increased the proliferative and migratory activities and improved connective tissue growth factor (CTGF) and fibronectin expression. Genetic and pharmacological suppression of YAP could reduce the proliferative and migratory activities of hMCs induced by DM-PRP-Exo. Additionally, YAP knockdown inhibited the DM-PRP-Exo-induced up-regulation of CTGF and fibronectin. Furthermore, DM-PRP-Exo-induced PI3K-Akt signalling mediated YAP activation and the expression of CTGF and fibronectin. In summary, DM-PRP-Exos, through YAP activation, enhance both the proliferation and fibrogenic activity of Müller cells via the PI3K/Akt pathway.

Introduction

Diabetic retinopathy (DR) is one of the most serious complications of diabetes and one of the main causes of vision loss. In the late stage of DR, retinal fibrosis occurs, and fibrovascular contraction leads to haemorrhage and retinal detachment (Mata-Cases et al., 2017). Connective tissue growth factor (CTGF) is a profibrotic factor that induces the production of extracellular matrix (ECM) and angiogenesis (Jadhav et al., 2016). The key role of CTGF in pathological fibrosis has been confirmed, and CTGF has been recognized as an important therapeutic target in ocular fibrosis. In human and rodent DR models, CTGF is upregulated and induced by glucose and advanced glycation end-products (Hu et al., 2014; Jiao et al., 2019). Moreover, CTGF is responsible for fibrosis but not angiogenesis, leading to retinal scarring and blindness. During long-term diabetes, structural and functional changes of the ECM result in several microvascular complications, such as DR, peripheral vascular diseases, cerebrovascular disorder and nephropathy (Kase et al., 2016). All of these studies indicate the therapeutic potential of anti-CTGF treatment in DR.

In 2014, Torreggiani et al. (2014) isolated exosomes from platelet-rich plasma (PRP) and demonstrated their potential effect on the proliferation and migration of mesenchymal stem cells. Exosomes derived from platelet-rich plasma (PRP-Exos) are extracellular vesicles (EVs) derived from activated platelets with diameters of 40–100 nm (Tao et al., 2017b). PRP-Exos carry a variety of proteins, and these components depend on the platelet activation process (Tao et al., 2017a). In recent years, increasing interest has been devoted to PRP-Exos, which act as carriers of mRNAs, microRNAs, and bioactive proteins and play an increasingly important role in cell-cell and platelet-cell communication (Guo et al., 2017). Because platelet-derived growth factors are encapsulated in PRP-Exos, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) carried by exosomes promotes fibrogenesis in recipient cells through the PI3K/Akt signalling pathway (Robbins and Morelli, 2014). However, the mechanism by which PRP-Exos contribute to retinal fibrogenesis in DR is still unclear.

The Hippo pathway regulates a variety of biological processes, including differentiation, proliferation, and survival, and is evolutionarily conserved (Yeh et al., 2018). Activation of the Hippo signalling pathway leads to LATS1-mediated phosphorylation of the downstream effectors WW domain-containing protein (TAZ) and Yes-associated protein (YAP) (Hsu et al., 2018). YAP is activated and then transferred to the nucleus to bind to the TEA domain (TEAD) transcription factor complex, thereby stimulating the expression of target genes, including those involved in cell differentiation, migration and proliferation, such as CTGF, AREG, CYR61, and ANKRD1 (Jin et al., 2018). Notably, the profibrotic factor CTGF is closely related to the development and progression of DR. Therefore, YAP is a key regulator of the proliferation and migration of endothelial cells and promotes their fibrogenic activity (Basu-Roy et al., 2016).

Recently, we observed that PRP-Exos mediate hyperglycaemia-induced retinal endothelial injury by upregulating the TLR4 signalling pathway (Zhang et al., 2019b). In addition, we found that exosomes derived from retinal pigment epithelium exacerbate the harmful oxidation induced by photooxidative blue-light stimulation by upregulating the NLRP3 inflammasome (Zhang et al., 2019c). In addition, we further confirmed that exosomes derived from mesenchymal stem cells reduce retinal inflammation induced by hyperglycaemia by downregulating the HMGB1 signalling pathway (Zhang et al., 2019d). In the current study, we investigated whether PRP-Exos mediate diabetes-induced retinal fibrogenesis via activation of YAP. Our findings revealed the key role of PRP-Exos in DR and may help to identify optimal targets and effective treatments to prevent fibrogenesis in DR.

Section snippets

Induction of experimental diabetes

The study was approved by the Medical Ethics Committee of Tianjin Medical University and carried out in accordance with the Declaration of Helsinki; all experiments were performed in accordance with the approved guidelines. Induction of experimental diabetes was performed according to our previously published protocol (Zhang et al., 2019d). In short, 8-week-old Wistar rats were intraperitoneally injected with streptozotocin (STZ; 45 mg/kg, Sigma). The level of blood glucose was examined 48 h

Identification of MSC-Exos

Electron microscopy results showed that both Nor-PRP-Exos and DM-PRP-Exos were clear, intact, and cup-shaped (Fig. 1A). The NanoSight analysis system revealed that Nor-PRP-Exos and DM-PRP-Exos had peak sizes of 84 nm and 95 nm, respectively (Fig. 1B). However, the concentration of exosomes was increased in the DM-PRP-Exos group compared with that of the Nor-PRP-Exos group (Fig. 1B). Western blot analysis demonstrated that PRP-Exos from both diabetic and normal rats expressed the characteristic

Discussion

Fibrosis is a key pathological feature of DR; however, the molecular mechanism of retinal fibrogenesis in DR is still unclear. As a major fibrotic factor, CTGF is overexpressed in DR models and plays an important role in the thickening of the retinal endothelial membrane (Hu et al., 2014). CTGF promotes the overproduction of extracellular matrix proteins such as fibronectin (Zhang et al., 2016). Thus, CTGF is recognized as a prospective target for the treatment of retinal fibrogenesis in DR. In

Disclosure

The authors state that they do not have any financial interest or other relationship with any product manufacturer or provider of services discussed in this article. The authors also do not discuss the use of off-label products, which include unlabelled, unapproved, or investigative products or devices.

Author contributions

Yichun Kong and Wei Zhang conceived and designed the experiments; Wei Zhang performed the experiments and prepared the figures; Wei Zhang and Hao Jiang collected the sample data; Wei Zhang contributed to writing the manuscript; Yichun Kong and Hao Jiang reviewed the manuscript; all authors contributed to writing the manuscript and discussed the manuscript at various stages.

Declaration of competing interest

The authors declare no competing interests.

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Exosomes derived from platelet-rich plasma activate YAP and promote the fibrogenic activity of Müller cells via the PI3K/Akt pathway☆

Highlights

Abstract

Introduction

Section snippets

Induction of experimental diabetes

Identification of MSC-Exos

Discussion

Disclosure

Author contributions

Declaration of competing interest

Mol. Cell

JACC Basic Transl. Sci.

Cell. Signal.

Exp. Eye Res.

Deregulation of Hippo-TAZ pathway during renal injury confers a fibrotic maladaptive phenotype

Faseb. J.

Effects of mesenchymal stem cell-derived exosomes on experimental autoimmune uveitis

Sci. Rep.

PPARgamma agonists promote differentiation of cancer stem cells by restraining YAP transcriptional activity

Oncotarget

Interaction of the EGF receptor and the Hippo pathway in the diabetic kidney

J. Am. Soc. Nephrol.: JASN (J. Am. Soc. Nephrol.)

YAP/TAZ are essential for TGF-beta2-mediated conjunctival fibrosis

Investig. Ophthalmol. Vis. Sci.

Hepatic sinusoids in liver injury, inflammation, and fibrosis: new pathophysiological insights

J. Gastroenterol.