Elsevier

Experimental Eye Research

Volume 222, September 2022, 109166
Experimental Eye Research

Research article
LncRNA Meg3 knockdown reduces corneal neovascularization and VEGF-induced vascular endothelial angiogenesis via SDF-1/CXCR4 and Smad2/3 pathway

https://doi.org/10.1016/j.exer.2022.109166Get rights and content

Highlights

  • Meg3 has a time-dependent relationship with corneal neovascularization(CNV).

  • Knockdown of Meg3 alleviated pathological changes and angiogenic responses in CNV.

  • SiMeg3 reduced VEGF-induced proliferation, migration and angiogenesis in HUVEC.

  • SiMeg3 inhibited expression of SDF-1/CXCR4 and Smad2/3 in VEGF-induced HUVEC.

Abstract

The crucial effect of vascular endothelial growth factor (VEGF)-induced vascular angiogenesis has been well known in corneal neovascularization (CNV). This research aimed to determine the underlying value and mechanism of Meg3 on CNV in vivo and in vitro. In an alkali-burned mouse model, length and area of new vessels were increased along with thinning of corneal epithelium, accompanied by the overexpression of Meg3. Notably, subconjunctival injection of shMeg3 suppressed the degree of injury in cornea, causing expression of the angiogenesis markers--VEGF-A and CD31 decreased. In VEGF-induced human umbilical vein endothelial cells (HUVECs), knockdown of Meg3 antagonized the enhancement of viability, proliferation, wound healing ability and angiogenesis by VEGF. The proteins expression of VEGF-A, CD31, SDF-1/CXCR4 as well as phosphoraylation-Smad2/3 pathways, which were related to angiogenesis, were reduced with Meg3 deficiency. Overall, knockdown of Meg3 alleviated formation of neovascularization in alkali-burned corneas and reduced VEGF-induced angiogenesis by inhibiting SDF-1/CXCR4 and Smad2/3 signaling in vitro.

Introduction

Corneal avascularity, which is known as “angiogenic privilege”, plays an essential role in corneal transparency and refraction (Mukwaya et al., 2019). Under certain conditions, including trauma, inflammation, infections, autoimmunity and allergies, capillaries invade from the limbal vascular plexus, leading to corneal neovascularization (CNV) Barbariga et al. (2019) Hsu et al. (2015); (Zhang et al., 2018). As the second common cause of blindness worldwide after cataract, corneal diseases are characterized by complex pathogenesis, while always associated with the formation of CNV (Whitcher et al., 2001). When the balance between angiogenic (e.g. VEGF and bFGF); and anti-angiogenic is out of control, abnormal neovascularization occurs (Chang et al., 2001). As CNV is a high-risk factor for corneal allograft rejection, the therapy of CNV is mainly aimed at anti-inflammation and antiangiogenesis, such as steroids and anti-VEGF drugs (Nicholas and Mysore, 2021). However, due to the systemic side effects of steroids (Gupta and Illingworth, 2011) and the study of anti-VEGF drugs are still under the animal research stage (Al-Debasi et al., 2017), it is urgent to discover a novel method to treat CNV.

Long non-coding RNAs (lncRNAs) consist of over 200 nucleotides, pervasively take part in fluctuation of gene expression from DNA to RNA and posttranscriptional stage (Bayarmaa et al., 2019). LncRNAs play a crucial role in proliferation, apoptosis and metastasis (Shi et al., 2013). Human maternally expressed gene 3 (Meg3) is one of the lncRNAs located on the human chromosome 14q32 region (Sherpa et al., 2018). Meg3 is mostly considered as a tumor-related tag, which is connected with P53, miRNAs and so on (Zhou et al., 2007). Currently, evidence has shown a relationship between Meg3 and cardiovascular disease. Research proved that decreasing Meg3 protects brain lesions by the notch pathway and results in a proangiogenic effect (Liu et al., 2017). Lan et al. demonstrated that Meg3 upregulated Girdin, a protein related with vascular remodeling, which reduced the length of telomeres and enhanced the platelet phagocytosis, thus protecting vascular endothelial cells against senescence (Lan et al., 2019). Moreover, Meg3 expression was associated with P53 activation in promoting viability and reducing DNA damage of endothelial cells (Shihabudeen Haider Ali et al., 2019).

However, whether Meg3 contributes to CNV and its possible mechanisms remains unknown. In this report, we explored the effect of Meg3 in CNV and pathways of VEGF-triggered sprouting angiogenesis in HUVECs.

Section snippets

Animals

We used six-week-old male BALB/c mice in animal research. The environment of mice were kept out of pathogen with consistent (24 ± 2 °C, 50 ± 5% humidity) and regular conditions (12-h diurnal cycle). Water and standard laboratory chow were unrestrained provided to the mice. This research was authorized and conducted by the animal care and use committee of Shanghai Tenth People's Hospital. All animal experiments were performed under the guidance of ARVO Statement for the Use of Animals in

Relative expression of Meg3 in alkali-burned cornea in vitro

First, the CNV was monitored at 3,7 and 14 days after alkali-burning, as previously described, and the lengths and areas of new vessels were measured by Image J. Seven days after injury, normal corneal structure was disturbed along with thinning of corneal epithelium (Fig. 1A and D). PAS staining did not show obvious goblet cells until 14 days after injury (Fig. S2A). The areas and lengths of new vessels increased significantly at 7 days compared to those of normal corneas as well as 3 days and

Discussion

CNV is a major reason of corneal blindness disease worldwide. There is consensus that disequilibrium between angiogenesis and antiangiogenesis causes vascularization in the cornea (Chang et al., 2012). Alkali substances activate pathologic angiogenic and inflammatory processes, leading the cumulation of proangiogenic and inflammatory factors (such as VEGF, IL-1β, TNF-a) (Ferrara, 2010). VEGF is known as a promoter of mitogenesis, angiogenesis and survival factors in endothelial cells derived

Conclusion

Knockdown of Meg3 alleviated formation of neovascularization in alkali-burned BALB/c corneas, and reduced VEGF-induced angiogenesis by inhibiting SDF-1/CXCR4 and Smad2/3 signaling in HUVECs. Our finding provides new possibility into CNV and siMeg3 could be potential targets for the therapeutic intervention.

Author contributions

TY Shen, Y Jing and W Zhu designed the research; TY Shen, Y Wu, WT Cai, HZ Jin, DH Yu and Q Yang performed the experiments; TY Shen and Y Wu conducted data analysis and wrote the initial draft of the manuscript; and all authors verified the final version of the manuscript.

Declaration of competing interest

The authors declared no competing or financial interests.

Acknowledgments

This work was supported by the Youth Program of National Natural Science Foundation of China (No. 81700804, 82000903) and Natural Science Foundation of Shanghai (No. 19ZR1439500). This work was also supported by Fundamental Research Funds for the Central Universities (No. 22120180509) and Medical and Health Research Project of Zhejiang (No. 2019KY643).

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