Effect of myeloid-derived suppressor cells on retinal epithelial cells in retinopathy of prematurity model

Introduction

Retinopathy of prematurity (ROP) is a proliferative vascular disorder that occurs in the retinas of premature infants with incomplete retinal vascularization. ROP is a significant cause of blindness and low vision in infants and young children. With the remarkable advancements in obstetric techniques and the care of premature infants, the number of infants with a birth weight of less than 1,500 g has increased annually, with a notable rise in survival rates. Consequently, the high incidence of ROP has emerged as a serious concern. Research in China has indicated that the incidence of ROP is 60% in infants with a birth weight of less than 1,000 g and 23.6% in those weighing less than 1,500 g. Approximately 20,000 newborns experience blindness or severe visual impairment due to this condition each year (1). Despite the increased awareness of the need for strict oxygen control and proactive measures to prevent and treat ROP, the incidence of ROP remains at a high level. This indicates that the country’s preventive and therapeutic strategies for ROP still require significant improvement. Current treatment options for ROP include laser therapy, vitrectomy, anti-vascular endothelial growth factor (VEGF) drug therapy, and direct ablation of abnormal blood vessels (2). These traditional treatment options exhibit several limitations, including the risks associated with general anesthesia, the potential disruption of normal retinal development leading to visual field defects, the induction of new vascular abnormalities, the systemic effects of anti-VEGF drug therapy in neonates, and the possibility of disease recurrence (3,4). Regenerative medicine approaches utilizing cell therapy have shown promising results in treating retinal diseases and other conditions with similar inflammatory and pathophysiological bases (5). The stem cells employed in animal studies of ROP include mouse bone marrow-derived mesenchymal stem cells (MSCs), human progenitor cell combinations (bone marrow-derived and vascular wall-derived endothelial colony-forming cells), human endothelial progenitor cells, and human peripheral blood stem cells (6,7). These studies indicate that stem cells migrate to the avascular regions of the retina, primarily inhibiting angiogenesis through the stimulation of the hypoxia-inducible factor (HIF)-α pathway. This mechanism allows the retina to adapt more flexibly to fluctuations in oxygen concentration, thereby promoting vascular repair. These cells can differentiate into microglia, where they play an immunomodulatory role during ischemia and in the phagocytosis of cellular debris, thus aiding retinal regeneration (8). Recent research has demonstrated that myeloid-derived suppressor cells (MDSCs), a type of stem cell, possess significant regenerative and reparative capabilities. They have been shown to have angiogenic properties and can release various growth factors and cytokines that promote the formation and repair of new blood vessels (9). Additionally, MDSCs exhibit immunomodulatory and anti-inflammatory effects, which enable them to suppress inflammatory responses and immune-mediated damage (10). However, the role of MDSCs as a stem cell therapy in treating ROP remains unclear. Therefore, elucidating the potential mechanisms of MDSCs in ROP is essential for identifying new therapeutic targets. We present this article in accordance with the MDAR reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2024-578/rc).

Methods

Cell culture and treatment

Adult retinal pigment epithelial cell line-19 (ARPE-19; No. SCSP-5277; National Collection of Authenticated Cell Cultures, Shanghai, China) were cultured and treated for the experiments. MDSCs were extracted from an 8-week-old male C57BL/6J mouse (Shanghai Laboratory Animal Research Center, Shanghai, China), the cell donor, for obtaining bone marrow-derived MDSCs. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Guangzhou First People’s Hospital (No. K-2022-103-01). Informed consent is waived as this is a zoological in vitro study. Experiments were performed under a project license (No. Scutlac2023289) granted by the South China University of Technology Animal Experiment Ethics Committee, in compliance with the institutional guidelines of South China University of Technology for the care and use of animals.

Drugs, main reagents, and instruments

A 3% hydrogen peroxide (H₂O₂) solution was obtained from Fuzhou Weibokan Biotechnology Co., Ltd. (Fuzhou, China). Protein markers, agarose, tris, anhydrous ethanol, and phosphate-buffered saline (PBS) were sourced from Zhejiang Liansuo Biotechnology (Hangzhou, China). DNA markers were acquired from Guangzhou Dongsheng Biotechnology (Guangzhou, China). Radioimmunoprecipitation assay (RIPA) protein lysis buffer and protein quantification analysis kits were supplied by Thermo Fisher Scientific (Waltham, MA, USA). Goat anti-mouse secondary antibodies, enhanced chemiluminescence detection kits, and reverse transcription kits were provided by Promega (Madison, WI, USA). The NanoDrop 2000 instrument was sourced from NanoDrop Technologies (Wilmington, DE, USA), while the Agilent Bioanalyzer 2100 was obtained from Agilent Technologies Co., Ltd. (Beijing, China). Hunan Xiangyi Laboratory Instrument Development Co., Ltd. (Changsha, China) supplied the TCL-16M desktop high-speed refrigerated centrifuge. An ultraviolet (UV)-visible spectrophotometer was acquired from Shanghai Yidian Analytical Instrument Co., Ltd. (Shanghai, China). Horizontal and vertical electrophoresis tanks and electrophoresis equipment were sourced from Jinan Ailai Bao Instrument Equipment Co., Ltd. (Jinan, China).

Modeling and grouping

An oxidative stress environment was simulated in ARPE-19 cell cultures by adding H₂O₂, thereby inducing pathological changes similar to those associated with ROP. A co-culture system was established involving MDSCs and ARPE-19 cells, with three experimental groups defined: the ROP model group, which consisted of ARPE-19 cells cultured alone with 300 µM H₂O₂; the ROP model + MDSCs group, which included ARPE-19 cells co-cultured with MDSCs in the presence of 300 µM H₂O₂; and the model + medium control group, which contained ARPE-19 cells co-cultured with the medium, also in the presence of 300 μM H₂O₂.

Detection of gene expression levels of glial fibrillary acidic protein (GFAP) and VEGF in different cell groups using quantitative real-time polymerase chain reaction (qRT-PCR)

The gene expression levels in different cell groups were assessed by extracting total RNA from the three groups of cells using the Trizol method. Complementary DNA (cDNA) was synthesized through reverse transcription from RNA with reverse transcription kit (TAKARA, Kusatsu, Shiga Prefecture, Japan). PCR amplification was performed using cDNA on the CFX96 real-time fluorescence polymerase chain reaction system (Bio-Rad, Hercules, CA, USA), and the reaction procedure was set as follows: pre-denature at 95 ℃ for 30 s; denaturation at 95 ℃ for 5 s; annealing at 60 ℃ and extending for 30 s; 40 cycles. The relative expression levels of selected microRNAs (miRNAs), identified through gene chip screening, were measured with U6 as the internal reference gene. The relative expression of each gene was calculated using the 2^−∆∆CT method.

The protein expression of GFAP and VEGF in different groups of cells detected by western blot

The protein expression levels of VEGF and GFAP were assessed using Western blotting. Total protein was extracted from each group using cell lysis buffer, and protein concentrations were determined with a bicinchoninic acid (BCA) protein quantification kit. Equal amounts of protein were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a polyvinylidene fluoride (PVDF) membrane and blocking of non-specific sites. Corresponding monoclonal antibodies were incubated overnight at 4 ℃. Subsequently, horseradish peroxidase-conjugated secondary antibodies were applied for 2 h of hybridization. Enhanced chemiluminescence was used for visualization. A digital imaging analysis system was employed to analyze the results, and Image Pro Plus 6.0 software was utilized for densitometric analysis of the protein bands. The experiments were performed in triplicate.

The protein expression and localization of GFAP and VEGF in different groups of cells detected by immunofluorescence

Cells from each group were washed in PBS and collected onto slides. They were fixed in 4% paraformaldehyde for 20 min and washed with PBS. The cells were then permeabilized with 0.3% Triton X-100 at room temperature for 20 min and washed with PBS. Goat serum was applied to block non-specific binding for 30 min. The cells were incubated with the primary antibody at 37 ℃ for 60 min. After washing with PBS, the cells were treated with the secondary antibody and incubated at 37 ℃ for 30 min, followed by additional PBS washes. 4'-6-diamidino-2-phenylindole (DAPI) was then added to stain the nuclei, and the samples were protected from light during a 5-min incubation. After washing with PBS, an anti-fade mounting medium was applied. The samples were observed and photographed under a fluorescence microscope. Under UV excitation, the nuclei appeared blue, while the fluorescein-labeled positive expression was observed as green fluorescence.

Statistical analysis

The experimental data were processed using Statistical Package for the Social Sciences (SPSS) 26.0 statistical analysis software. There were three biological replicates for all experiments. Measurement data were expressed as mean ± standard deviation (SD). The Student’s t-test was used to compare two groups, while one-way analysis of variance (ANOVA) was employed to compare multiple groups. A P<0.05 was considered statistically significant.

Results

The gene expression levels of GFAP and VEGF in different groups of cells were detected by qRT-PCR

GFAP and VEGF expression levels in the model + medium group were comparable to those in the model group (GFAP: 0.955 vs. 1.021; VEGF: 0.986 vs. 0.992; P>0.05). In contrast, the expression levels of GFAP and VEGF in the model + MDSCs group were significantly reduced compared to the model group (GFAP: 0.476 vs. 1.021; VEGF: 0.512 vs. 0.992; P<0.05), as shown in Figure 1.

Figure 1 The gene expression levels of GFAP (A) and VEGF (B) in different groups of cells were detected by qRT-PCR. *, P <0.05; **, P<0.01. GFAP, glial fibrillary acidic protein; MDSC, myeloid-derived suppressor cell; MED, medium; qRT-PCR, quantitative real-time polymerase chain reaction; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth factor.

The protein expression levels of GFAP and VEGF in different groups of cells were detected by western blot

GFAP and VEGF expression levels in the model + medium group were comparable to those in the model group (GFAP: 0.946 vs. 1.21; VEGF: 0.979 vs. 1.19; P>0.05). In contrast, the expression levels of GFAP and VEGF in the model + MDSCs co-culture group were significantly reduced compared to the model group (GFAP: 0.256 vs. 1.21; VEGF: 0.497 vs. 1.19; P<0.05), as shown in Figure 2.

Figure 2 The protein expression of GFAP (A,C) and VEGF (A,B) in different groups of cells was detected by western blot. 1, RPE group; 2, RPE + MED group; 3, RPE + MDSCs group. *, P<0.05. GFAP, glial fibrillary acidic protein; MDSC, myeloid-derived suppressor cell; MED, medium; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth factor.

The protein expression and localization of GFAP and VEGF in different groups of cells were detected by Immunofluorescence

In the model + medium group, the expression levels of GFAP and VEGF were significantly elevated, whereas in the model + MDSCs group, the expression levels of GFAP and VEGF were markedly reduced, as illustrated in Figures 3,4.

Figure 3 Immunofluorescence detected the protein expression and localization of GFAP in different groups of cells. The expression of GFAP was detected by immunofluorescence staining (×200), with nuclei stained with DAPI. The samples were observed and photographed under a fluorescence microscope. Under UV excitation, the nuclei appeared blue, while the fluorescein-labeled positive expression was observed as green fluorescence. ***, P<0.001; ns, no statistical significance. DAPI, 4'-6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; MDSC, myeloid-derived suppressor cell; MED, medium; RPE, retinal pigment epithelium; UV, ultraviolet.

Figure 4 Immunofluorescence detected the protein expression and localization of VEGF in different groups of cells. The expression of VEGF was detected by immunofluorescence staining (×200), with nuclei stained with DAPI. The samples were observed and photographed under a fluorescence microscope. Under UV excitation, the nuclei appeared blue, while the fluorescein-labeled positive expression was observed as green fluorescence. ***, P<0.001; ns, no statistical significance. DAPI, 4'-6-diamidino-2-phenylindole; MDSC, myeloid-derived suppressor cell; MED, medium; RPE, retinal pigment epithelium; UV, ultraviolet; VEGF, vascular endothelial growth factor.

Discussion

This study systematically evaluated the regulatory effect of MDSCs on GFAP and VEGF expression through q-PCR, Western blot, and immunofluorescence techniques, untangling the potential mechanisms of MDSCs in inhibiting glial cell activation and angiogenesis-related molecular pathways.

GFAP is a cytoskeletal protein expressed in astrocytes and is commonly used as a marker for astrocyte activation. The role of GFAP in ROP remains not fully understood. However, several studies have provided relevant insights. In the context of ROP, GFAP may be associated with cell proliferation, apoptosis, and responses to injury (11). First, in ROP, the expression of GFAP may relate to local immune responses, influencing the inflammatory process. Second, GFAP plays a role in the repair process following retinal damage, potentially participating in cell proliferation and scar formation. The levels of GFAP could serve as a biomarker for assessing the severity and prognosis of ROP, with elevated concentrations in serum being associated with certain pathological changes in ROP.

Our experimental results demonstrate that MDSCs co-culture significantly reduces both gene and protein expression levels of GFAP. This observation is highly consistent with the role of GFAP as a marker of astrocyte activation (12-14). Previous studies have indicated that upregulation of GFAP is closely associated with reactive gliosis, and its expression intensity can reflect the severity of central nervous system injury or inflammation (12,15). For instance, in epilepsy models, high GFAP expression is correlated with neuronal loss and enhanced neuroinflammation (16). In spinal cord injury, suppression of GFAP can reduce glial scar formation and improve functional recovery (13,17). In this study, MDSCs may downregulate GFAP expression by secreting anti-inflammatory factors [such as interleukin-10 (IL-10) or transforming growth factor-β (TGF-β)] or through direct cell-to-cell contact to inhibit glial cell activation. This mechanism is similar to previously reported findings where MSCs inhibited astrocyte activation via exosomes (18).

VEGF is a growth factor that specifically stimulates the proliferation of vascular endothelial cells and the formation of new blood vessels. In the pathological process of ROP, local ischemia and hypoxic conditions in the retina lead to a compensatory increase in intraocular VEGF expression, which subsequently induces pathological retinal vascular growth. VEGF plays a central role in the pathological mechanisms of ROP. The pathogenesis of ROP primarily involves two stages: the vascular obstructive phase and the vascular proliferative phase (19,20). (I) Vascular obstructive phase: following birth, the influence of the hyperoxic environment outside the womb results in reduced VEGF levels, inhibiting normal retinal vascular growth. This phase typically lasts from birth until at least 32 weeks of corrected gestational age. (II) The vascular proliferative phase occurs between 32 and 42 weeks of corrected gestational age. As the metabolic demands of the developing neuroretina increase, the retina enters an ischemic and hypoxic state, leading to an upregulation of VEGF levels that promotes pathological retinal vascular proliferation. This may subsequently result in tractional retinal detachment. Therefore, anti-VEGF drug therapy is important in treating ROP, as it can lower intraocular VEGF levels, thereby inhibiting pathological angiogenesis while promoting the continued development of the non-vascularized retina.

Our results demonstrate that the regulatory effects of VEGF and its expression levels related to angiogenesis were significantly reduced in the MDSCs co-culture group, suggesting that MDSCs may exert therapeutic effects by inhibiting VEGF-mediated angiogenic pathways. VEGF serves as a critical regulator of endothelial cell differentiation and vascular permeability, and its overexpression is often associated with pathological angiogenesis (21,22). For instance, in osteosarcoma, fibroblast activation protein (FAP) promotes the phosphorylation of the a kinase transforming/protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) pathway by upregulating VEGF-A, thereby enhancing the proliferation of vascular endothelial cells (22). Our findings align with these mechanisms, indicating that MDSCs may block abnormal angiogenesis by interfering with the FAP-VEGF axis or directly inhibiting VEGF secretion. Additionally, immunofluorescence revealed a decrease in VEGF localization within the cytoplasm, which may be related to MDSCs regulating VEGF secretion or degradation pathways (23,24). However, the specific molecular mechanisms underlying these observations require further validation.

Currently, anti-VEGF drug therapy for ROP has gradually gained acceptance in clinical practice; however, some studies have raised concerns regarding the potential effects of anti-VEGF drugs on the neurodevelopment of premature infants. ROP patients frequently experience damage to the blood-retinal barrier, leading to an imbalance in the retinal microenvironment. Anti-VEGF drugs can easily cross the blood-retinal and blood-brain barriers into systemic circulation, potentially impacting neurodevelopment (25,26). Therefore, the use of anti-VEGF drugs requires careful consideration of their therapeutic efficacy for ROP against potential systemic effects. MDSCs play a unique role in promoting neovascularization, regulating immune responses, and suppressing inflammation. This may mitigate the adverse effects of anti-VEGF drugs on the neurodevelopment of premature infants and even on systemic circulation, presenting a promising new avenue for the treatment of ROP.

This study still has the following limitations. Firstly, the specific signaling pathway through which MDSCs regulate GFAP/VEGF remains elusive. Additionally, the experiment did not include multi-time point detections, making it difficult to untangle the dynamic changes of GFAP/VEGF. Secondly, the current data are derived from an in vitro co-culture system, which cannot fully reflect the actual effects of MDSCs in complex microenvironments. Future research should focus on constructing animal models (such as cerebral ischemia or glioma models) to evaluate the long-term impact of MDSCs on gliosis and angiogenesis.

Conclusions

In conclusion, treatment strategies for ROP are continually evolving, and cell therapies such as MDSCs offer new therapeutic avenues. However, further research is needed to optimize these treatment methods to enhance their efficacy and minimize potential side effects. As regenerative medicine continues to advance, it is anticipated that more innovative treatment options will be developed, providing safer and more effective solutions for ROP patients.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2024-578/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2024-578/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2024-578/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2024-578/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of Guangzhou First People’s Hospital (No. K-2022-103-01). Informed consent is waived as this is a zoological in vitro study. Experiments were performed under a project license (No. Scutlac2023289) granted by the South China University of Technology Animal Experiment Ethics Committee, in compliance with the institutional guidelines of South China University of Technology for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Blencowe H, Lawn JE, Vazquez T, et al. Preterm-associated visual impairment and estimates of retinopathy of prematurity at regional and global levels for 2010. Pediatr Res 2013;74:35-49. [Crossref] [PubMed]
2. Mutlu FM, Sarici SU. Treatment of retinopathy of prematurity: a review of conventional and promising new therapeutic options. Int J Ophthalmol 2013;6:228-36. [PubMed]
3. Quinn GE, Darlow BA. Concerns for Development After Bevacizumab Treatment of ROP. Pediatrics 2016;137:e20160057. [Crossref] [PubMed]
4. Gotz-Więckowska A, Chmielarz-Czarnocińska A, Pawlak M, et al. Ranibizumab after laser photocoagulation failure in retinopathy of prematurity (ROP) treatment. Sci Rep 2017;7:11894. [Crossref] [PubMed]
5. Adak S, Magdalene D, Deshmukh S, et al. A Review on Mesenchymal Stem Cells for Treatment of Retinal Diseases. Stem Cell Rev Rep 2021;17:1154-73. [Crossref] [PubMed]
6. Medina RJ, O'Neill CL, Humphreys MW, et al. Outgrowth endothelial cells: characterization and their potential for reversing ischemic retinopathy. Invest Ophthalmol Vis Sci 2010;51:5906-13. [Crossref] [PubMed]
7. Li Calzi S, Shaw LC, Moldovan L, et al. Progenitor cell combination normalizes retinal vascular development in the oxygen-induced retinopathy (OIR) model. JCI Insight 2019;4:e129224. [Crossref] [PubMed]
8. Ritter MR, Banin E, Moreno SK, et al. Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy. J Clin Invest 2006;116:3266-76. [Crossref] [PubMed]
9. Boelte KC, Gordy LE, Joyce S, et al. Rgs2 mediates pro-angiogenic function of myeloid derived suppressor cells in the tumor microenvironment via upregulation of MCP-1. PLoS One 2011;6:e18534. [Crossref] [PubMed]
10. Vetsika EK, Koukos A, Kotsakis A. Myeloid-Derived Suppressor Cells: Major Figures that Shape the Immunosuppressive and Angiogenic Network in Cancer. Cells 2019;8:1647. [Crossref] [PubMed]
11. Huang Q, Zhao P. Anti-vascular endothelial growth factor treatment for retinopathy of prematurity. Ann Eye Sci 2017;2:47. [Crossref]
12. Brenner M, Messing A. Regulation of GFAP Expression. ASN Neuro 2021;13:1759091420981206. [Crossref] [PubMed]
13. Cai J, Yan Y, Zhang D, et al. Silencing of lncRNA Gm14461 alleviates pain in trigeminal neuralgia through inhibiting astrocyte activation. IUBMB Life 2020;72:2663-71. [Crossref] [PubMed]
14. Akhoundzadeh K, Shafia S. Association between GFAP-positive astrocytes with clinically important parameters including neurological deficits and/or infarct volume in stroke-induced animals. Brain Res 2021;1769:147566. [Crossref] [PubMed]
15. Yang XL, Wang X, Shao L, et al. TRPV1 mediates astrocyte activation and interleukin-1β release induced by hypoxic ischemia (HI). J Neuroinflammation 2019;16:114. [Crossref] [PubMed]
16. Hu QP, Yan HX, Peng F, et al. Genistein protects epilepsy-induced brain injury through regulating the JAK2/STAT3 and Keap1/Nrf2 signaling pathways in the developing rats. Eur J Pharmacol 2021;912:174620. [Crossref] [PubMed]
17. Guo Y, Chen Y, Zhang H, et al. Emodin attenuates hypoxic-ischemic brain damage by inhibiting neuronal apoptosis in neonatal mice. Neuroscience 2024;554:83-95. [Crossref] [PubMed]
18. He GH, Zhang W, Ma YX, et al. Mesenchymal stem cells-derived exosomes ameliorate blue light stimulation in retinal pigment epithelium cells and retinal laser injury by VEGF-dependent mechanism. Int J Ophthalmol 2018;11:559-66. [PubMed]
19. Sjöbom U, Hellström W, Löfqvist C, et al. Analysis of Brain Injury Biomarker Neurofilament Light and Neurodevelopmental Outcomes and Retinopathy of Prematurity Among Preterm Infants. JAMA Netw Open 2021;4:e214138. [Crossref] [PubMed]
20. Arima M, Fujii Y, Sonoda KH. Translational Research in Retinopathy of Prematurity: From Bedside to Bench and Back Again. J Clin Med 2021;10:331. [Crossref] [PubMed]
21. Zhang S, Zhang W, Li Y, et al. Human Umbilical Cord Mesenchymal Stem Cell Differentiation Into Odontoblast-Like Cells and Endothelial Cells: A Potential Cell Source for Dental Pulp Tissue Engineering. Front Physiol 2020;11:593. [Crossref] [PubMed]
22. Zeng C, Wen M, Liu X. Fibroblast activation protein in osteosarcoma cells promotes angiogenesis via AKT and ERK signaling pathways. Oncol Lett 2018;15:6029-35. [Crossref] [PubMed]
23. Cao F, Wang S, Wang H, et al. Fibroblast activation protein-α in tumor cells promotes colorectal cancer angiogenesis via the Akt and ERK signaling pathways. Mol Med Rep 2018;17:2593-9. [PubMed]
24. Mi C, Ma J, Wang KS, et al. Imperatorin suppresses proliferation and angiogenesis of human colon cancer cell by targeting HIF-1α via the mTOR/p70S6K/4E-BP1 and MAPK pathways. J Ethnopharmacol 2017;203:27-38. [Crossref] [PubMed]
25. Bayramoglu SE, Sayin N. The Effect of Intravitreal Bevacizumab Dose on Retinal Vascular Progression in Retinopathy of Prematurity. Ophthalmologica 2022;245:161-72. [Crossref] [PubMed]
26. Tian Y, Zhang F, Zhang G. Effect of anti-VEGF intravitreal injections on the development of nervous system in retinopathy of prematurity. Chinese Journal of Experimental Ophthalmology 2022;40:271-5.