Geniposide alleviates choroidal neovascularization by downregulating HB-EGF release from RPE cells by downregulating the miR-145-5p/NF-κB axis

Highlights

• GEN inhibited the transcription and expression of HB-EGF, a proangiogenic factor, in hypoxic RPE cells and a mouse laser-induced CNV model.

• Inhibition of GLP-1R, a GEN receptor blocker, eliminated the protective effect of GEN.

• GEN decreased the transcription and expression of HB-EGF in hypoxia-exposed RPE cells by downregulating the miR-145-5p/NF-κB axis.

Abstract

Age-related macular degeneration (AMD), mainly wet AMD, is the major reason for nonreversible vision loss worldwide. Choroidal neovascularization (CNV) is a characteristic pathological manifestation of wet AMD. Stress or injury to the retinal pigment epithelium (RPE) induces proangiogenic factors that drive CNV. An iridoid glycoside extracted from the fruit of gardenia, geniposide (GEN) plays an antiangiogenic role. In this study, GEN inhibited the transcription and expression of heparin-binding epidermal growth factor (HB-EGF), a proangiogenic factor, in hypoxic RPE cells and a mouse laser-induced CNV model. Inhibition of glucagon-like peptide-1 receptor (GLP-1R), a GEN receptor blocker, eliminated the protective effect of GEN. Additionally, GEN decreased the transcription and expression of HB-EGF in hypoxia-exposed RPE cells by downregulating the miR-145-5p/NF-κB axis. Therefore, our research provides a promising novel strategy for wet AMD therapy.

Introduction

Age-related macular degeneration (AMD) is the major cause of irreversible vision loss worldwide and is the third leading cause of blindness globally (Flaxman et al., 2017; Taylor, 2019). Clinically, AMD is mainly classified into dry and wet types. Among the cases of AMD with an acute loss of visual acuity, wet AMD accounts for approximately 90% (Ferris et al., 1984). Choroidal neovascularization (CNV) is a characteristic pathological manifestation of wet AMD that is caused by retinal pigment epithelial (RPE) dysfunction, extraretinal hypoxia, and Bruch's membrane abnormalities (Jager et al., 2008; Roth et al., 2004). It is characterized by the abnormal invasion of choroidal vessels into the retinal epithelium or subretinal tissues, causing dysfunction and vascular leakage, leading to blood accumulation in the macula and ultimately fibrosis (Chirco et al., 2017). Vascular endothelial growth factor (VEGF) is a critical proangiogenic component of wet AMD. Currently, the main strategy for wet AMD therapy consists of repeated injections of anti-VEGF drugs into the vitreous. Although this treatment can obviously improve vision, the therapeutic effect of anti-VEGF drugs declines over time and may also cause ocular side effects, such as endophthalmitis (Sadda et al., 2020; Wolff et al., 2020). Consequently, there is an urgent need to explore more effective targets for wet AMD therapy.

Heparin-binding epidermal growth factor (HB-EGF), a member of the EGF superfamily with a high affinity to heparin, is synthesized as a membrane-bound growth factor and is hydrolyzed by several specific metalloproteinases. Released soluble HB-EGF (sHB-EGF) activates EGF receptor (EGFR) to play an extensive role in processes such as tissue injury, wound healing and angiogenesis (Scuderi and Failla, 2008; Taylor et al., 2014). Additionally, HB-EGF is upregulated in proliferative vitreoretinopathy, proliferative diabetic retinopathy (PDR) and neovascular glaucoma, suggesting the involvement of HB-EGF in ocular neovascularization (Hollborn et al., 2005; Kovacs et al., 2015). Additionally, HB-EGF promotes the development of CNV. Therefore, the inhibition of HB-EGF is a possible therapeutic target for ocular neovascularization in wet AMD, PDR and retinopathy of prematurity (ROP) (Inoue et al., 2018).

Geniposide (GEN) is a kind of iridoid glycoside extracted from the fruit of gardenia. Past research has indicated that GEN plays antiangiogenic, anti-inflammatory and neuroprotective roles (Habtemariam and Lentini, 2018; Li et al., 2019a; Liu et al., 2015; Zhou et al., 2019). It has been shown that GEN acts as an agonist of glucagon-like peptide-1 receptor (GLP-1R) (Li et al., 2019b; Song et al., 2020), which is extensively expressed in human tissues, including the retina (Hebsgaard et al., 2018). The latest studies have demonstrated that functional GLP-1R exists in RPE cells and has potential beneficial effects on RPE apoptosis and DR (Kim et al., 2015; Puddu et al., 2013). However, the specific effect and mechanism of GEN on the regulation of CNV are still unclear.

MicroRNAs (miRNAs) are a category of noncoding single-stranded RNA molecules approximately 22 nucleotides long that can regulate various stages of angiogenesis (Kir et al., 2018; Rupaimoole and Slack, 2017). MiR-145-5p acts primarily as a tumor suppressor that can inhibit NF-κB signaling pathways in multiple cancers, such as esophageal squamous cell carcinoma and malignant melanoma (Jin et al., 2019; Mei et al., 2017). The transcription factor NF-κB is a key regulator of the inflammatory response. The NF-κB/Rel family comprises five members: p50, p52, p65 (Rel-A), c-Rel and Rel-B (Giridharan and Srinivasan, 2018). The most abundant form of NF-κB activated by pathological stimulation via the canonical pathway is the p65:p50 heterodimer, and activated p65 then transactivates target genes involved in diverse physiological activities, such as cellular proliferation, inflammatory cytokines and mediators of apoptosis (Baldwin, 2012; Yde et al., 2011). Furthermore, the expression of miR-145-5p was found to be lower in patients with wet AMD than in the control group, suggesting that miR-145-5p dysregulation is associated with the pathogenesis of wet AMD (Blasiak et al., 2019). GEN protects PC12 cells (rat neuronal cell line) from lipopolysaccharide (LPS)-induced inflammatory injury through up-regulating miR-145-5p, showing that miR-145-5p is a target of GEN (Ma et al., 2019). However, there is still a lack of sufficient evidence on the role and mechanism of miR-145-5p in the pathogenesis of CNV. The objective of this study was to investigate the pharmacological effects of GEN on CNV and the underlying mechanism.