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. 2026 Apr 27;9(5):1099–1108. doi: 10.1021/acsptsci.5c00314

Bemarituzumab Suppresses Choroidal Neovascularization in Mice by Downregulating Melanoma Cell Adhesion Molecule and Yes-Associated Protein 1

Liujun Ding †, Bintao Xie †, Jineng Lv †, Zhonglou Zhou †, Xin Zhou †, Kunchao Wu †,‡, Qin Zhang †, Fan Lu †, Cong Wang §, Jia Qu †,*, Lue Xiang †,*, Qi Chen †,*
PMCID: PMC13162049  PMID: 42130720

Abstract

In recent years, some fibroblast growth factors (FGFs) have been reported to be promising therapeutic targets for neovascular age-related macular degeneration (nAMD). Interestingly, we found that the FGFR2b inhibitor bemarituzumab (FPA144) exerts a beneficial effect in a mouse choroidal neovascularization (CNV) model. Our current study aims to uncover the potential molecular mechanism by which FPA144 alleviates CNV. The effect of FPA144 was evaluated in a laser-induced CNV mouse model. Fundus fluorescein angiography (FFA), optical coherence tomography (OCT), and choroidal flat mounts were carried out for the quantitative assessment of CNV. Label-free quantitative proteomics analysis was performed to assess changes in molecular pathways. Primary cultured human umbilical vein endothelial cells (HUVECs) were used for further confirmation of the target pathway in vitro. FFA, OCT, and choroidal flat mounts demonstrate the protective effect of FPA144 in a mouse CNV model. Compared to the control group, the treated group has smaller vascular leakage areas or smaller CNV lesions. Proteomic analyses indicate that the melanoma cell adhesion molecule (MCAM, CD146)–Yes-associated protein 1 (Yap1) pathway may be involved in the effect of FPA144. Immunostaining of choroidal flat mounts and cryosections reveals decreased expression of CD146 and Yap1 in the vascular endothelial cells of the treated animals. Experiments on HUVECs further verify the inhibitory effect of FPA144 on vascular endothelial cells. Our findings demonstrate that FPA144 can efficiently inhibit the development of choroidal neovascularization in mice by downregulating CD146 and Yap1.

Keywords: Neovascular age-related macular degeneration, Choroidal neovascularization, FGFR2, CD146, Yap1

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Age-related macular degeneration (AMD) is a disease involving fundus degeneration that impairs central vision and has a major negative impact on quality of life. The prevalence of AMD is estimated to continue to increase due to the aging of the population worldwide. Over 90% of legally blind AMD patients have neovascular AMD (nAMD), the primary clinical characteristic of which is choroidal neovascularization (CNV). A consensus has been reached on the role of vascular endothelial growth factor (VEGF) and its receptors in promoting neovascularization. − Currently, anti-VEGF therapy has become the mainstream treatment for nAMD. , However, most patients receiving this therapy experience intolerable side effects or significant financial difficulties due to the need for repeated intravitreal injections over a long period of time. , Moreover, not every patient responds well to this treatment. Thus, alternative or adjuvant therapies for nAMD are desperately needed.

FGF and its receptor (FGFR) are essential for embryonic development, angiogenesis, wound healing, and the proliferation and differentiation of various cells. − Recent studies have indicated that multiple FGF family members, including FGF2, FGF5, and FGF21, are involved in the development of CNV, − suggesting that they are promising therapeutic targets for nAMD. Furthermore, conditional knockout of FGFR, a receptor tyrosine kinase similar to VEGFR, significantly affects the development of CNV in mouse endothelial cells. Thus, we are interested in studying FGFR and its potential as a target for the development of drugs for nAMD.

As antitumor medications, pan-FGFR inhibitors have been shown to cause retinal damage in phase I studies. Therefore, the use of pan-FGFR inhibitors for treating nAMD is not practical because of these safety concerns. A humanized IgG monoclonal antibody called FPA144 is thought to prevent FGFR2b from binding and activating FGF. According to published papers, FPA144 can potentially avoid some side effects of pan-FGFR inhibitors. Recently, a global phase III study on FPA144 was performed, which inspired clinical and basic research scientists to further study the effects of FPA144. However, little is known about the effect of this drug on ocular neovascular diseases. Here, we found that the FGFR2b inhibitor FPA144 exerts a protective effect against choroidal neovascularization in mice. Our data revealed that FPA144 can efficiently suppress choroidal neovascularization in a laser-induced CNV mouse model and in vascular endothelial cells by inhibiting CD146 and Yap1.

Results

FPA144 Reduces Vascular Leakage in the CNV Mouse Model

A laser-induced CNV mouse model was used to assess the development of CNV after FPA144 treatment. This model mimics the key characteristics of human nAMD, and is most frequently used model in the field of choroidal neovascularization. The CNV model mice were randomly divided into two groups and treated with FPA144 or PBS on days 1 and 4. The experimental design is shown in Figure A. FP and FFA images were captured 3, 7, and 14 days after laser injury (Figure B). FP was performed to ensure that all of the experimental mice had a normal fundus. The FFA images taken on day 3 were used as baseline images, and laser spots with similar leakage areas were used for comparison with images taken on days 7 and 14 (Figure B). Statistical analysis confirmed no significant difference in CNV area among the groups at this baseline (Supporting Figure S1). The FFA results showed that the FPA144-treated group had a significantly decreased vascular leakage area. Statistically, compared with that in the control group, the vascular leakage areas in the FPA144-treated group on days 7 and 14 were 48% smaller (Figure C) and 34% smaller (Figure D), respectively.

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FPA144 decreased vascular leakage in the CNV mouse model. (A) Schematic diagram of the experimental design, including model establishment, treatment, and data acquisition. (B) Representative fundus bright field images and FFA images of the PBS (left) and FPA144 (right) groups. (C, D) Quantitative analysis of CNV vascular leakage in FFA images. The data were collected on days 7 (C) (n = 16 for PBS group; n = 12 for FPA144 group) and 14 (D) (n = 10 for PBS group; n = 9 for FPA144 group). The data are presented as the mean ± SEM **P < 0.01, and ***P < 0.001.

FPA144 Reduces the Extent of CNV

To further confirm the inhibitory effect of FPA144 on laser-induced CNV in mice, OCT was used to measure the thickness and length of CNV lesions in the mouse fundus on day 7 (Figure A). The results showed that the average CNV lesion thickness and length in the PBS group were 104.8 and 2201.0 μm, respectively. However, those in the FPA144 group were decreased to 91.9 and 1751.5 μm (Figure B and C), and these differences were statistically significant. Subsequently, choroidal flat mounts were prepared on the same day for IB4 immunostaining (Figure D). Quantitative analysis of the cells revealed that the CNV surface area in the drug-treated group was 79.3% smaller than that in the PBS group (Figure E and F). These results collectively demonstrate that FPA144 can effectively suppress the development of CNV in a laser-induced CNV mouse model.

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Effects of FPA144 on the volume of CNV lesions on day 7. (A) Representative OCT images of the PBS (top) and FPA144 (bottom) groups. The CNV lesions are circled in red. Measurements of CNV lesion thickness (B) and length (C) on OCT images (n = 21). (D) Immunofluorescence images of choroidal flat mounts stained with IB4; the CNV lesions in the PBS (top) and FPA144 (bottom) groups are circled in white. (E) Statistical analysis of the surface area of CNV lesions in the immunofluorescence images. The data are presented as the mean ± SEM **P < 0.01, and ****P < 0.0001.

Differential Protein Expression Analysis: Identification of Proteins Related to the Antiangiogenic Effect of FPA144

To understand how FPA144 regulates protein expression in RPE-choroid complexes, label-free quantitative proteomics analysis of CNV model mice after injection of PBS or FPA144 was carried out with an Orbitrap Fusion Lumos mass spectrometer. A total of 4220 protein groups were identified, including 4051 in the FPA144 group and 4201 in the PBS group (Figure A). The PBS group was used as the baseline for protein expression levels. Among the 4032 proteins identified in both groups, there were 17 significantly upregulated and 185 significantly downregulated proteins (considered differentially expressed proteins (DEPs)) according to criteria fold change > 1.5 or < 0.8 and P value < 0.05 (Figure B). Gene Ontology enrichment analysis revealed that these DEPs were enriched mainly in biological processes such as antigen processing and presentation, wound healing and actin filament organization (Figure C). KEGG pathway analysis suggested that the DEPs were mainly enriched in the pathways phagosome, protein processing in the endoplasmic reticulum, antigen processing and presentation, and cell adhesion molecules (Figure D). FGF plays major roles in the wound healing process. Thus, we generated a heatmap of the key DEPs in the FPA144-treated groups, such as Yap1, Thbs1, Axl, Pecam1 and Ccn2. We noticed that most of these proteins were downregulated in the FPA144-treated groups (Figure E). Interestingly, the protein MCAM, also known as CD146, was significantly downregulated, which is known to be associated with wound healing (Figure F).

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Proteomic analysis of RPE-choroid complex from CNV model mice treated with PBS or FPA144. (A) The protein groups identified in RPE-choroid complex. (B) Volcano plot of differentially expressed proteins in RPE-choroid complex from CNV model mice treated with PBS or FPA144. The cutoffs for significant changes was as follows: fold change > 1.5 or < 0.8 and p value < 0.05. (C) The biological processes (BPs), cellular components (CCs) and molecular functions (MFs) in which the differentially expressed proteins were enriched according to Gene Ontology enrichment analysis. (D) The top 20 pathways in which the differentially expressed proteins were enriched based on KEGG pathway analysis. (E) Heatmap showing the differentially expressed proteins involved in the wound healing pathway. (F) Normalized abundance of MCAM (CD146) in RPE-choroid complex from CNV model mice treated with PBS (red) or FPA144 (blue).

CD146 and Yap1 Mediate the Suppression of CNV by FPA144

CD146 and Yap are regarded as key proteins in mediating the suppression of neovascularization. − To verify the results of the comparative proteomics analysis, the protein expression of CD146 and Yap1 was measured by immunostaining of choroidal neovascularization tissues obtained on day 7. Immunostaining of choroidal flat mounts and cryosections revealed decreased expression of CD146 (Figure A) and Yap1 (Figure B) in the vascular endothelial cells of the treated animals. We found that the percentage of CD146 and Yap1 fluorescent signals was decreased by 37.32% and 42.31%, respectively, in the FPA144-treated group.

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Expression levels of CD146 and Yap1 were decreased in FPA144-treated CNV model mice. (A) Representative immunofluorescence staining images of choroidal flat mounts. The FPA144 group exhibited less CD146 (green) fluorescence in CD31 (red)-positive endothelial cells than the PBS group. Nuclei were stained with DAPI (blue). Statistical analysis of the relative fluorescence intensity of CD146 was depicted at right (n = 6). (B) Coimmunostaining of Yap1 (green) and CD31 (red) in ocular posterior segment cryosections. Statistical analysis of the relative fluorescence intensity of Yap1 was depicted at right (n = 4). The data are presented as the mean ± SEM **P < 0.01, and ***P < 0.001.

FPA144 Inhibits the Angiogenic Activity of HUVECs via the CD146/YAP Pathway

We used the tube formation (Figure A), CCK-8 (Figure B), and wound healing (Figure C and D) assays to determine the impact of FPA144 on vascular endothelial cell proliferation, migration, and morphology formation, all of which are important for angiogenesis. According to the results, the proliferative, migratory, and tube-forming abilities of the HUVECs were all decreased, further validating the effect of FPA144 in inhibiting angiogenesis in vitro. Notably, the protein expression levels of CD146 and YAP were decreased to 74.57% and 79.01% in the drug-treated groups (Figure E–G). This result was consistent with that observed in the CNV animal model. Taken together, these findings demonstrate that FPA144 can effectively suppress the development of angiogenesis by downregulating vascular endothelial cell CD146 and Yap in vitro and in vivo.

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FPA144 can inhibit the angiogenic activity of HUVECs by downregulating CD146 and YAP. (A) Representative images of the tubular structures formed by HUVECs. (B) The proliferation of HUVECs was measured by the CCK-8 assay. (C) Representative images of HUVEC wound healing were captured at 0, 6, and 12 h after scratching. (D) Statistical analysis of the migration area of HUVECs (n = 3). (E) Representative confocal images of HUVECs stained with antibodies against CD146 and YAP. (F, G) Statistical analysis of the relative fluorescence intensity of CD146 (n = 3) (F) and YAP (n = 3) (G). The above-mentioned data were also compared between the FPA144 (80 μg/mL)-treated group and untreated group. The data are presented as the mean ± SEM *P < 0.05.

Discussion

While VEGF and VEGFR are the most common foci of research on ophthalmic neovascularization, FGF and FGFR have attracted the attention of researchers in recent years. Although it has been reported that several FGFs play a therapeutic role in choroidal neovascularization, − little is known about the role of their receptors in ophthalmic neovascularization. Moreover, unlike the mechanism downstream of VEGF/VEGFR signaling, − the detailed molecular mechanism downstream of FGF/FGFR signaling remains largely unclear. Without question, FGF/FGFR signaling plays a role in angiogenesis and angiodysplasia. In the present study, we identified it as a promising target for the treatment of not only a number of cancers but also nAMD. The FGFR2b inhibitor FPA144 was found to effectively inhibit the development of choroidal neovascularization by downregulating CD146 and Yap1 in vitro and in vivo.

As mentioned in the introduction section, FGF2, FGF5, and FGF21 are thought to be potential novel therapeutic targets for nAMD. − These FGF ligands have the highest affinity for FGFR1. Our results showed that FGFR2 has an impact on choroidal neovascularization, which is consistent with the findings from FGFR2 knockout mice. Based on research on the role of FGFRs in cancer, we speculate that FGFR3 and FGFR4 may also play a role in choroidal neovascularization. A previous study showed that FGFR activation increases the invasion of murine brain capillary endothelial cells (MBECs) via the MAPK signaling pathway. FGF/FGFR causes matrix metalloproteinase-2 (MMP2) and MMP9, which are essential for HUVEC invasion and tube formation, to be released from the surface of HUVECs. Additionally, the FGFR pathway has complementary effects on the VEGFR and PDGFR pathways, indirectly promoting tumor neovascularization. Moreover, FGFR has been demonstrated to mediate resistance to anti-VEGF therapy. These studies all point to the potential of targeting FGFRs in treating angiogenesis.

Both CD146 and Yap have long been hot topics in the fields of malignancy and neovascularization. ,− A clinical study revealed that nAMD patients had increased serum soluble CD146 (sCD146) levels, suggesting that sCD146 might be a biomarker of nAMD. Yap is the main downstream effector molecule of the Hippo pathway. A previous study revealed that the expression of Yap was upregulated in choroid and retinal tissues in a CNV mouse model and that CNV progression was inhibited by Yap siRNA, suggesting that Yap is a therapeutic target for nAMD. Interestingly, we found that FPA144 can downregulate both CD146 and Yap1 simultaneously. The interaction between CD146 and Yap has been reported previously. According to a study by Liang, Yap is activated by CD146 via the Hippo pathway. In addition, FGF has been verified to upregulate Yap nuclear localization, and it could be reversed by an FGFR inhibitor. All of these suggests a potential connection between the treatment of FPA144 and these two factors in CNV mouse. FPA144 may exert its inhibitory effect on pathological angiogenesis by suppressing the FGFR2b–CD146–YAP1 axis, or alternatively, by directly regulating each of these two factors independently. Further investigation is warranted to clarify the precise mechanism(s) through which FPA144 influences CD146 and YAP1 expression.

Our research has several limitations. First, while we verified that CD146 and Yap1, which are thought to be downstream targets of FGF/FGFR2, are play key roles in wound healing through proteomics analysis, we cannot rule out that they play important roles in CNV processes, such as antigen processing and presentation and actin filament organization. Second, we did not perform FPA144 concentration gradient experiments in the CNV mouse model. It is possible that FPA144 may inhibit neovascularization more strongly at higher dosages. The original of FPA144 used in this study was 2.13 mg/mL. Due to the small vitreous volume in mice, we were unable to investigate the effects of higher FPA144 dosages in this mouse model. Testing higher concentrations of FPA144 may provide additional information about the effect of the drug on CNV.

Overall, our study found that FPA144, which has already been exploited as an anticancer drug, could be repurposed to treat nAMD. We believe that this drug may also have further application in treatment of other ocular neovascular diseases. Through proteomics analysis of FPA144-treated mice, we first identified CD146 and Yap1 as key downstream targets through which FPA144 exerts its pharmacological effects. This study provides important information for the further application of FPA144 to target FGFR2.

Methods

Animals

Male wild-type C57BL/6J mice aged between 6 and 8 weeks were used in this study. All mice were housed in a mouse facility at a constant temperature of 22 °C on a 12 h light–dark cycle at Wenzhou Medical University. All animal experiments were conducted according to the ARVO statement for the use of animals in ophthalmic and vision research, and the experiments protocols involving animals (NO. YSG23102718) were approved by the Experimental Animal Ethics and Management Committee of Eye Hospital of Wenzhou Medical University.

Laser-Induced CNV and Drug Treatment

CNV was induced in mice with a Micron IV image-guided laser system (Phoenix Research Laboratories, Pleasanton, CA, USA) according to a previous protocol. Six- to eight-week-old C57BL/6J mice were anesthetized intraperitoneally with ketamine (80 mg/kg) and xylazine (16 mg/kg), and 0.5% tropicamide was applied to dilate the pupils. Laser photocoagulation (240 mW intensity, 50 μm in size, 100 ms duration) was performed at 4 locations in the fundus, which were located 1–2 disc diameters away from the optic disc. The production of bubbles was the criterion for successful establishment of the model, as it indicated the disruption of Bruch’s membrane. The mice were treated with 2 μL of 2.13 mg/mL FPA144 or an equivalent volume of PBS by intravitreal injection on day 1 (D1) and day 4 (D4) after laser injury.

Fundus Photography and Fundus Fluorescein Angiography

At 3, 7, and 14 days after laser photocoagulation, fundus photography (FP) and fundus fluorescein angiography (FFA) were applied to analyze the CNV area with a Micron IV retinal imaging system. Bright field images were captured after the mice were anesthetized and the pupils were dilated. Then, the mice were intraperitoneally injected with AK-FLUOR (Akorn, Lake Forest, IL) (fluorescein injection, USP), and 5 min later, FFA images were collected. To ensure comparability of the FFA findings at days 7 and 14, the day-3 FFA was used as an inclusion criterion to confirm that a consistent level of neovascularization had been initially achieved in all animals included in the study.

Optical Coherence Tomography

On the seventh day after CNV modeling, high-resolution spectral domain-optical coherence tomography (SD-OCT) (Spectralis HRA + OCT; Heidelberg Engineering, Heidelberg, Germany) was used to assess the morphologic characteristics of CNV lesions. After the mice were anesthetized and the pupils were dilated, a corneal contact lens was applied to the eyes of the mice, and OCT images were captured at the maximum transverse diameter of each spot. The thickness and length of CNV lesions were analyzed by using ImageJ software (version 1.53; National Institutes of Health, Bethesda, MD, USA).

Immunostaining of Choroidal Flat Mounts, Cryosections, and Cultured Cells

To prepare choroidal flat mounts, the eyeballs of the mice were removed and fixed in 4% paraformaldehyde for 1 h. After the removal of the cornea, lens, and retina, the eyecups, composed of the RPE, choroid, and sclera, were obtained and trimmed in a clover shape. The method used to prepare cryosections was similar to that used to prepare choroidal flat mounts. In brief, eyeballs were fixed for 20 min both before and the cornea, lens were removed. The eyeballs were embedded in embedding agent (Neg-50, Thermo) after dehydration with 30% sucrose. Subsequently, 20 μm thick cryosections were prepared. A total of 1 × 105 cells were seeded in each well of a 24-well plate. The cells were treated with FPA144 or left untreated for 12 h after being starved overnight. The tissues and cells were washed with PBS, blocked and permeabilized for 2 h, after which they were incubated with primary antibodies overnight at 4 °C. The next day, the sections were incubated with secondary antibody at room temperature for 2 h after being washed. The following antibodies were used for this study: rabbit anti-CD146 (1:200; Abcam); rabbit anti-YAP (1:200; Cell Signaling Technology); mouse anti-CD146 (1:200; Cell Signaling Technology); mouse anti-CD31 (1:200; Thermo Fisher); Alexa Fluor 594-conjugated isolectin B4 (1:500; Thermo Fisher); Alexa Fluor 488-conjugated donkey antirabbit (1:200; Jackson Laboratories); and Alexa Fluor 594-conjugated donkey antimouse (1:200; Jackson Laboratories). DAPI (1:5000; Sigma-Aldrich Corp) was also used in this study.

Protein Extraction and Digestion for Proteomics Analysis

Mouse RPE-choroid complexes collected 7 days after modeling were lysed in 60 μL of T-PER buffer containing 1× EDTA-free protease inhibitor cocktail (Thermo Fisher), followed by 2 min of homogenization and 20 min of incubation at 4 °C with rotation. The lysate was centrifuged twice at 10000 × g for 10 min, after which the supernatant was collected as the protein extract. The protein concentration was determined by BCA assay (Thermo Fisher), and 4 μg of protein was taken from each sample for subsequent digestion. Protein digestion was performed according to the manual of the EasyPep Mini MS Sample Prep Kit (Thermo Scientific). Briefly, 50 μL of reduction solution and 50 μL of alkylation solution were added to the extracted proteins and gently mixed. Then, the proteins were reduced and alkylated by incubating at 95 °C using a heat block for 10 min. After the sample was cooled to room temperature, 50 μL of Trypsin/Lys-C Protease Mix was added. Incubate the sample in an Eppendorf Thermomixer C at 37 °C and 300 rpm for 3 h to digest the proteins. Digestion was stopped by adding 50 μL of Digestion Stop Solution, and the resulting peptides were cleaned up in a Peptide Clean-Up column and resuspended in 0.1% formic acid solution. Then, the peptide concentration was quantified using the Pierce Quantitative Colorimetric Peptide Assay. A total of 200 ng of peptide was loaded onto the column for LC–MS/MS analysis.

LC–MS/MS for Proteomics Analysis

The peptides were separated and analyzed with the Easy-nLC 1200 system (Thermo Scientific) coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer using a commercial trap column (Acclaim PepMap 100 C18, 75 μm × 2 cm, Thermo Scientific) and an analytical column (Acclaim PepMap RSLC, 75 μm × 25 cm, Thermo Scientific). Mobile phase A was H2O with 0.1% formic acid, and mobile phase B was 80% acetonitrile with 0.1% formic acid. The flow rate was 300 nL/min, and the elution gradient was as follows: 0∼2 min, 3%∼8% B; 2∼12 min, 8%∼10% B; 12∼22 min, 10%∼15% B; 22∼37 min, 15%∼25% B; 37∼47 min, 25%∼32% B; 47∼57 min, 32%∼100% B; hold at 100% B for 10 min. The spray voltage, ion transfer tube temperature and RF lens (%) were set at 2100 V, 320 °C and 50, respectively. Both full MS and MS/MS scans were acquired by Orbitrap. The resolution of the full MS scans was set at 60000, and the AGC target of the full MS scans was 100%. The mass range of the full MS scans was 350 to 1500 m/z. For MS/MS scans, data-dependent acquisition (DDA) mode was applied. The resolution and AGC target for the MS/MS scans were 15000 and 100%, respectively.

Data Analysis

The raw data were analyzed via Proteome Discoverer 2.5.0.400 using the common label-free quantitative workflow. All the data were searched against mouse UniProt Reference Proteomes (36,404 entries) downloaded at 08/2020 via the Sequest HT node. For the identification of peptides, precursor and fragment mass tolerances of 10 ppm and 0.2 Da, respectively, were used. Trypsin was specified as the enzyme, and the maximum number of missed cleavage sites was 2. Carbamidomethylation was set as the static modification. The FDR for peptide and protein levels was 1%. The quantification of peptide levels was based on precursor abundance. Protein abundance was calculated as the average abundance of the top 3 peptides. Proteins with more than 50% missing values were filtered out. The fold change was calculated by normalizing to the average abundance of each protein, and the p value was calculated by unpaired Student’s t test. The cutoff for identifying significant differentially expressed proteins was a fold change > 1.5 or < 0.8 and a p value < 0.05. Hierarchical clustering analysis, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed with R 4.2.3 using the clusterProfiler package.

Cell Culture

Primary cultured human umbilical vein endothelial cells (HUVECs) were maintained in an incubator at a constant temperature of 37 °C in 5% CO2. The culture medium consisted of 5% fetal bovine serum (FBS) (ScienCell Research Laboratories).

Cell Counting Kit-8 (CCK-8) Assay

The proliferation and viability of HUVECs were measured by the Cell Counting Kit-8 (CCK-8) (MedChem Express) assay. A total of 8 × 103 or 1 × 104 cells were seeded in each well of a 96-well plate. The adherent cells were starved with 1% FBS medium overnight. After that, the medium was changed to fresh 1% FBS medium supplemented with or without FPA144, and the cells were cultured for 24 h. Then, 10 μL of CCK-8 solution was added to each well, the mixture was incubated at 37 °C for 2 h, and the absorbance at 450 nm was measured and recorded.

Endothelial Cell Migration Assay

A total of 5 × 105 cells were seeded in each well of a 6-well plate. After the cells adhered to the plate, they were starved by culture in ECM containing 1% FBS for 12 h and then scratched with a 200 μL pipet tip to create a wound. PBS was used to remove the floating cells. The culture medium was changed, and one group of cells was treated with FPA144. The cells were photographed at 0, 6, and 12 h after scratching. ImageJ software was used to quantify the wound area.

Endothelial Cell Tube Formation Assay

Cells were starved as described for the CCK-8 assay, and 48-well plates were coated with 140 μL of Matrigel (Corning) per well before being incubated for 30 min at 37 °C. Suspended cells were seeded onto the solidified Matrigel and treated with or without FPA144. After incubation at 37 °C for 6 h, the formed tubes were imaged with an inverted phase-contrast microscope (Axio Observer3, Zeiss, Germany).

Statistical Analysis

The data are presented as the mean ± SEM and were tested using GraphPad Prism. Statistical significance was calculated using a two-tailed Student’s t test. P values less than 0.05 were considered to indicate a significant difference between the FPA144-treated group and the control group.

Supplementary Material

pt5c00314_si_001.pdf (72KB, pdf)

Acknowledgments

This research was funded by the Zhejiang Provincial Natural Science Foundation of China (LTGD23H120001, LTGC23H120001, LGD22H120001), the Project of the State Key Laboratory of Ophthalmology, Optometry and Vision Science (No. K03-20220205), and the Guizhou Science and Technology Support Project (Qiankehezhicheng [2020]4Y146).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.5c00314.

  • Baseline PBS vs FPA144 CNV vascular leakage area quantitative analysis in FFA images; changes in CNV leakage area from Day 3 to Day 7 in PBS vs FPA144 groups (PDF)

∥.

L.D. and B.X. contributed equally to this work and should be regarded as first authors. L.J.D.: Writingoriginal draft, Visualization, Investigation, Data curation, Formal analysis. B.T.X.: Writingoriginal draft, Visualization, Investigation, Data curation, Formal analysis, Funding acquisition. J.N.L.: Resources, Methodology. Z.L.Z.: Resources, Methodology. X.Z.: Investigation, Data curation. K.C.W.: Resources, Funding acquisition. Q.Z.: Investigation. F.L.: Methodology. C.W.: Methodology. J.Q.: Writingreview and editing, Conceptualization, Supervision. L.X.: Writingreview and editing, Methodology, Project administration, Funding acquisition. Q.C.: Writingreview and editing, Visualization, Supervision, Funding acquisition.

The authors declare no competing financial interest.

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