The clinical effects and mechanism of action of ranibizumab in treating myopic choroidal neovascularization

The clinical effects and mechanism of action of ranibizumab in treating myopic choroidal neovascularization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The clinical effects and mechanism of action of ranibizumab in treating myopic choroidal neovascularization Wenting Gu, Zhen Wang, Duo Peng, Qinghuai Liu, Yonghui Gu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4281961/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To investigate the clinical effects and factors influencing repeated intravitreal injections of ranibizumab among patients with choroidal neovascularization (CNV) secondary to pathologic myopia. Methods There were 39 patients with myopic CNV and a control group of 10 cataract patients. Logarithm of the minimum angle of resolution units (logMAR) was adopted to evaluate best-corrected visual acuity (BCVA), while the measurement of central macular thickness was made via optical coherence tomography. Luminex xMAP suspension array was adopted to collect and analyze plasma and aqueous humor samples to compare cytokine concentrations between the two groups and assess changes after intravitreal ranibizumab injections. Additionally, BCVA values and CMT were monitored. Results A great diversity in the BCVA values and CMT was found after intravitreal ranibizumab injections (p<0.05 for both). The study group showed greatly higher plasma concentration of vascular endothelial growth factor-A than the cataractgroup (p<0.05). The research group (p<0.05 for both) showed greatly lower epidermal growth factor (EGF) and angiopoietin-2 concentrations. In the aqueous humor, the study group showed greatly higher concentration of fibroblast growth factor (FGF)-2 than the cataract group (p<0.05), and the research group (p<0.05 for both) displayed greatly lower concentrations of EGF and VEGF-A. The decrease in average VEGF-A contents was significant by comparing with baseline at both 1 month (p<0.05) and 2 months (p<0.05), and the reduction in mean VEGF-D and endoglin contents compared to baseline at 2 months were significant (p<0.05 for both), and were significant when compared at 1 month to 2 months (p<0.05 for both). The average EGF contents were significantly higher at 2 months than at baseline (p<0.05). Conclusion Ranibizumab had obvious effects on myopic CNV. For different individuals, different treatment projects should be chosen. Myopic horoidal neovascularization Ranibizumab Cytokine Plasma Aqueous humor Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In many countries, myopic choroidal neovascularization (CNV) secondary to pathologic myopia represents a prevalent source of visual impairment, notably among individuals < 50 years of age [ 1 – 3 ]. Complications associated with pathologic myopia encompass posterior staphyloma, retinal pigment epithelium (RPE) changes, choroidal atrophy, retinal detachment and CNV [ 4 ]. Of these pathologic myopic complications, myopic CNV is one of the most significant threats to vision [ 5 ]. This disease decreases productivity and quality of life. Based on previous research, vascular endothelial growth factor has been proposed as the most significant mechanism of new blood vessel growth and vascular leakage during the formation of CNV [ 6 – 7 ]. Ranibizumab (Lucentis; Novartis Pharma AG, Switzerland) is specifically adopted to stop the active forms of VEGF-A [ 8 ] as a humanized, recombinant, monoclonal antibody fragment. Intravitreal injections of ranibizumab have demonstrated a curative effect on visual acuity improvement among patients with myopic CNV [ 9 – 10 ]. Further investigation is required to elucidate the clinical effects and mechanism of action of ranibizumab in the treatment of myopic CNV. In addition to VEGF, the potential influence of other cytokines on the pathogenesis of myopic CNV warrants further investigation. In this study, we explored the differences in plasma and aqueous humor concentrations of cytokines between patients with myopic CNV and those with cataracts. In addition, we investigated the variations in the concentrations of aqueous humor cytokines after consecutive intravitreal injections of ranibizumab. Combining this with the best modified visual acuity and the central macular thickness (CMT), we assessed the efficacy of ranibizumab in treating myopic CNV. Materials and Methods This research utilized a single-center non-randomized clinical cohort research design. Participants receiving consecutive intravitreal injections of ranibizumab for myopic CNV due to pathologic myopia were enrolled from the ophthalmology clinic at the First Affiliated Hospital of Nanjing Medical University. The Institutional Review Ethics Committee of the hospital approved the protocol. From July 2022 through December 2023, we enrolled 39 patients (8 males and 31 females) who had active myopic CNV. The inclusion standards were shown below: ≥18 years of age; -6.00 diopters of refractive error or greater; CNV secondary to pathologic myopia and involving the foveal center; and recent disease progression. The exclusion standards were shown below: CNV secondary to age-associated macular degradation, polypoidal choroidal vasculopathy, inflammatory disease, angioid streaks, or trauma; subfoveal fibrosis or atrophy; any prior treatment such as photodynamic therapy or retinal laser photocoagulation for CNV secondary to pathologic myopia; and systemic diseases. Before the first intravitreal injection, all patients received a systematic ophthalmic examination, such as BCVA, slit-lamp examination, fundus examination, optical coherence tomography (OCT; OCT spectral domain, Humphrey Field Analyzer 3 from Zeiss), fundus fluorescein angiography, and indocyanine green angiography. The logarithm of the minimum angle of resolution (logMAR) was used as the BCVA for statistical analysis. The central macular thickness (CMT) was assessed by OCT centering on the fovea, and the scan length was 6.0 mm, measuring the neural epithelium thickness. The average thickness of the central foveal (1 mm in diameter) was adopted for comparison. The examinations of BCVA and OCT were repeated every month after intravitreal injections of ranibizumab. The time of the follow-up visit was three months. Another injection would be carried out based on the examination results. Blood and aqueous humor samples were gathered from the study group before intravitreal injections of ranibizumab and from the control group immediately before cataract operation. Blood samples (5 mL) were taken from the antecubital vein into EDTA routine blood tubes, next to 15-minute centrifugal at 4,500 ×g to isolate the plasma, then aliquoted and kept at − 80°C until use. Undiluted aqueous humor samples (100 µL) were also acquired using a standard sterilization procedure. Anterior chamber paracentesis was performed before each intravitreal injection to avoid a spike in intraocular pressure after ranibizumab (0.05 mg/0.05 mL) was injected intravitreally. After disinfecting the periorbital skin and conjunctiva with a povidone-iodine solution, sterile draping of the patient, and insertion of a lid speculum, aqueous humor was aspirated with a 30-gauge needle linked to a tuberculin syringe at the temporal limbus. Intravitreal injections and cataract surgery were then performed. Aqueous humor samples were gathered in sterile tubes and kept at − 80°C until use. Samples were measured with the Luminex xMAP suspension array. A Milliplex Kit was used for the detection of epidermal growth factor (EGF) [ 11 ], angiopoietin-2 [ 12 ], granulocyte colony-stimulating factor (G-CSF) [ 13 ], endoglin [ 14 ], endothelin-1 [ 15 ], fibroblast growth factor (FGF)-1 [ 16 ], FGF-2 [ 17 ], interleukin-8 (IL-8) [ 18 ], hepatocyte growth factor (HGF) [ 19 ], heparin-binding epidermal growth factor (HB-EGF) [ 20 ], placental growth factor (PLGF) [ 21 ], vascular endothelial growth factor (VEGF)-C [ 22 ], VEGF-D [ 23 ], and VEGF-A [ 24 ]. It is necessary to conduct intensive studies, as many previous investigations have demonstrated that these cytokines may take part in the formation and development of myopic CNV. Before commencing the assay, all reagents were warmed to room 20–25°C. The first step involved adding 25 µL of assay buffer, 25 µL of appropriate matrix solution, 25 µL of plasma sample (1:3 dilution) or undiluted aqueous humor sample, and 25 µL of beads to every well. The plate capacity was 96-well.Wrapped with foil, the plate was incubated on a plate shaker overnight at 4°C or 2 h at 20–25°C. Then, after putting 25 µL of detection antibodies and 25 µL streptavidin-phycoerythrin to every well, the plate was covered with foil, next to 30-minute incubation on a plate shaker at 20–25°C. All incubation procedures were made in the dark to prevent light exposure to the beads. Finally, the plate was run on a Luminex 200™ with xPONENT software. SPSS (version 27.0; SPSS Inc., Chicago, IL, USA) was adopted to analyze data. An independent sample t -test was adopted to compare the diversities in plasma and aqueous humor cytokine concentrations between the two groups. The differences in BCVA values, CMT, and cytokine concentrations among various groups of aqueous humor samples collected at baseline, one month and two months were compared with a one-way analysis of variance (ANOVA). Statistical significance was set at p < 0.05. Results There were 49 plasma samples collected from 39 patients with myopic CNV and 10 patients with cataracts. A total of 15 myopic CNV patients and 10 cataract patients received aqueous sampling upon intravitreal ranibizumab injections at baseline. Subsequently, 14 and 9 myopic CNV patients’ aqueous humor samples were collected at 1 month and 2 months. The average patient age was 54.3 ± 11.7 years (scope 24–75 years). The average patient diopter was − 11.85 ± 3.61 D (range − 7 to − 20 D). As controls, 10 senile cataract patients (5 males and 5 females) without other ocular or systemic diseases were gathered. The average patient age in the control group was 70.7 ± 8.0 years (range 52–80 years). According to our study, the mean patient BCVA (logMAR) value was 1.00 ± 0.49 at baseline, 0.74 ± 0.58 at 1 month, and 0.42 ± 0.23 at 2 months (Table 1 ). However, a great diversity between the BCVA values was observed at baseline and at 2 months (p < 0.05) (Fig. 1 ). The CMT of the patients with myopic CNV was 311.03 ± 54.32 µm at baseline, then reduced to 255.57 ± 50.97 µm at 1 month and 266.42 ± 37.64 µm at 2 months after consecutive intravitreal injections of ranibizumab (Table 1 ). The CMT at 1 and 2 months was greatly lower than that at baseline (p < 0.05 and p < 0.05) (Fig. 1 ). Table 1 Changes in BCVA and CMT in the myopic choroidal neovascularization (CNV) group after ranibizumab injections At baseline At 1 month P-value a At 2 months P-value b P-value c BCVA (logMAR) 1.00 ± 0.49 0.74 ± 0.58 0.411 0.42 ± 0.23 0.001 0.234 CMT (µm) 311.03 ± 54.32 255.57 ± 50.97 0.013 266.42 ± 37.64 0.021 0.990 BCVA, best-corrected visual acuity; logMAR, logarithm of the minimum angle of resolution; CMT, central macular thickness. a One-way analysis of variance between baseline and 1 month for BCVA and CMT In plasma, the concentrations of VEGF-A, EGF, and angiopoietin-2 in the myopic CNV group were 42.61 ± 34.36, 0.74 ± 0.35, and 897.71 ± 366.29 pg/mL, respectively. In the cataract group, the concentrations of these cytokines were 14.94 ± 11.57, 1.13 ± 0.60, and 1242.91 ± 347.91 pg/mL, respectively (Table 2 ). The concentration of VEGF-A in the myopic CNV group was greatly higher than that in the cataract group (p < 0.05). In contrast, EGF and angiopoietin-2 concentrations were greatly lower in the myopic CNV group (p < 0.05 for both) (Fig. 2 ). Table 2 Plasma contents of cytokines in myopic CNV and control groups Control CNV p-value d VEGF-A (pg/mL) 14.94 ± 11.57l 42.61 ± 34.36 0.000 EGF (pg/mL) 1.13 ± 0.60 0.74 ± 0.35 0.010 Angiopoietin-2 (pg/mL) 1242.91 ± 347.91 897.71 ± 366.29 0.010 VEGF-A, vascular endothelial growth factor A; HB-EGF, heparin-binding epidermal growth factor; EGF, epidermal growth factor. d T -test between control and myopic CNV groups for plasma contentss of cytokines. In aqueous humor, the concentrations of FGF-2, EGF, and VEGF-A were 34.11 ± 23.30, 0.64 ± 0.08, and 135.09 ± 70.29 pg/mL, respectively, in the myopic CNV group, and 9.49 ± 1.26, 0.74 ± 0.08, and 272.71 ± 138.91 pg/mL, respectively, in the cataract group (Table 3 ). The myopic CNV group showed greatly higher concentration of FGF-2 than the cataract group (p < 0). In contrast, the concentrations of EGF and VEGF-A were greatly lower in the myopic CNV group (p < 0.05 for both) (Fig. 3 ). Table 3 Aqueous humor contents of cytokines in myopic CNV and control groups Control CNV p-value e VEGF-A (pg/mL) 272.71 ± 138.91 135.09 ± 70.29 0.003 EGF (pg/mL) 0.74 ± 0.08 0.64 ± 0.08 0.007 FGF-2 (pg/mL) 9.49 ± 1.26 34.11 ± 23.30 0.002 VEGF-A, vascular endothelial growth factor A; EGF; FGF, fibroblast growth factor. e T -test between control and myopic CNV groups for aqueous humor contents of cytokines. Changes in the contents of VEGF-A, VEGF-D, EGF, and endoglin among the aqueous humor of patients with myopic CNV were observed after consecutive intravitreal injections of ranibizumab. Baseline VEGF-A concentration in the aqueous humor was 135.09 ± 70.29 pg/mL, but decreased to 5.41 ± 0.58 and 8.33 ± 4.70 pg/mL at 1 and 2 months, after intravitreal injections of ranibizumab. Baseline VEGF-D concentration in the aqueous humor was 2.99 ± 0.39 pg/mL but decreased to 2.84 ± 0.41 and 2.32 ± 0.16 pg/mL at 1 and 2 months, after injections. Baseline endoglin concentration in the aqueous humor was 15.41 ± 1.35 pg/mL and decreased to 14.76 ± 1.83l and 11.69 ± 1.19 pg/mL at 1 and 2 months, after injections. Baseline EGF concentration in the aqueous humor was 0.64 ± 0.08 pg/mL and increased to 0.71 ± 0.17 and 0.78 ± 0.09 pg/mL at 1 and 2 months, after injections (Table 4 ). The decrease in average VEGF-A contents by comparing with baseline at both 1 month (p < 0.05) and 2 months (p < 0.05) was great, and the decrease in average VEGF-D and endoglin contents by comparing with baseline at 2 months was significant (p < 0.05 for both) and were also significant when compared at 1 month to 2 months (p < 0.05 for both). The average EGF contents were significantly higher at 2 months than at baseline (p < 0.05) (Fig. 4 ). Table 4 Changes in aqueous humor contents of cytokines in the myopic CNV group after ranibizumab injections At baseline At 1 month p-value f At 2 months p-value g p-value h VEGF-A (pg/mL) 135.09 ± 70.29 5.41 ± 0.58 0.000 8.33 ± 4.70 0.000 0.389 VEGF-D (pg/mL) 2.99 ± 0.39 2.84 ± 0.41 0.264 2.32 ± 0.16 0.000 0.002 EGF (pg/mL) 0.64 ± 0.08 0.71 ± 0.17 0.150 0.78 ± 0.09 0.010 0.171 Endoglin (pg/mL) 15.41 ± 1.35 14.76 ± 1.83 0.257 11.69 ± 1.19 0.000 0.000 f One-way analysis of variance (ANOVA) between baseline and 1 month for changes in aqueous humor contents of cytokines. g ANOVA between baseline and 2 months for changes in aqueous humor cytokine contents. h ANOVA between 1 month and 2 months for changes in aqueous humor contents of cytokines. Discussion The current research investigated the therapeutic effects of ranibizumab in patients with myopic CNV. A great improvement in BCVA was found, along with a significantly decreased CMT measured by OCT and aqueous humor contents of VEGF-A, VEGF-D, and endoglin, but significantly decreased content of EGF after consecutive intravitreal injections of ranibizumab. The results were similar to those from another study on the effects of ranibizumab in AMD cases [ 25 ]. The concentrations of cytokines may be quantitative indicators of the effects of ranibizumab in patients with myopic CNV. Ranibizumab demonstrated significant effects on CNV secondary to pathologic myopia by improving BCVA values, reducing CMT, and modulating cytokines associated with angiogenesis. For different individuals, different treatment projects should be chosen. The myopic CNV group showed higher plasma concentration of VEGF-A than the control group, whereas EGF and angiopoietin-2 contents were decreased in the myopic CNV group by comparing with the control group. Moreover, compared to controls, aqueous humor content of FGF-2 increased, whereas those of EGF and VEGF-A decreased in the eyes of patients with myopic CNV. VEGF concentration in the plasma is greater among patients with active CNV secondary to AMD than in healthy individuals [ 26 ]. However, there is no research on the plasma VEGF concentration among patients with myopic CNV. It is well known that VEGF is the most significant signaling protein taking part in both vasculogenesis and angiogenesis, and the rise of plasma VEGF is a prerequisite for CNV generation. Therefore, in the present research, it is reasonable to detect a higher plasma concentration of VEGF-A in the myopic CNV group. Based on past research, it is controversial whether the VEGF concentration in the aqueous humor is high in myopic CNV. Tong et al. disclosed that VEGF concentration in the aqueous humor grew significantly among patients with CNV secondary to AMD, polypoidal choroidal vasculopathy, and CNV secondary to pathologic myopia, compared to healthy controls [ 27 ]. In contrast, in research by Sawada et al., VEGF concentrations in the aqueous humor of the myopic CNV group were greatly lower than those in the control group, which is consistent with our results [ 28 ]. Vascular endothelial growth factor receptor VEGFR-2 is mainly located in the retinal blood vessel cells of the leakage area, which is related to vascular leakage, whereas VEGFR-3 is mainly located in deep retinal capillaries and is relevant to VEGFR-2. VEGF-A induces VEGFR-2 and VEGFR-3 expression in the ischemic area and leads to retinal leakage, thereby promoting the formation of new blood vessels [ 6 ]. We hypothesize that VEGF-A may be localized in a small subfoveal area in combination with VEGFR-2 and VEGFR-3, resulting in decreased levels in the aqueous humor during CNV formation. EGF contents are greatly higher in the aqueous humor of patients with AMD [ 11 ]. However, in the current research, the result was different when patients with myopic CNV were the research focus. The EGFR primarily resides in the cell membrane, where it binds with a significant quantity of EGF. It transduces biological signals by invagination pinocytosis, leading to its internalization into the cytoplasm. Once internalized, EGF is degraded by lysosomes, and EGFR is subsequently recycled back to the membrane. The expression of transforming growth factor-α (TGF-α) will increase while CNV forms [ 29 ]. TGF-α and EGF have 33–44% homology in structure, and TGF-α may competitively inhibit the expression of EGF. This leads to a decrease in EGF level in the plasma and aqueous humor of patients with myopic CNV. Angiopoietin-2 exerts a key effect on vessel maturation, angiogenesis, and vessel regression and is expressed most heavily in tumor tissue (especially in tumor neovascularization) but not in normal tissue [ 30 ]. Myopic CNV expresses a large quantity of angiopoietin-2 and angiopoietin receptor (TEK) restricted to CNV lesions; thus, we found lower contents of angiopoietin-2 in both the plasma and aqueous humor among patients with myopic CNV by comparing with the control group. In the normal retina, the mRNA of FGF-2 and its receptor were discovered in the ganglion cell layer and kernel, and transient retinal ischemia induced the synthesis of FGF-2 mRNA [ 31 ]. In vitro studies have found that the complex of anti-VEGF antibody/anti-FGF-2 antibody could completely inactivate VEGF and FGF-2 in the retinal pigment epithelium containing CNV, better inhibiting the growth of vascular endothelial cells than simply adding anti-VEGF antibody [ 32 ]. Unlike VEGF-A, EGF, and angiopoietin-2, FGF-2 is not expressed in retinal capillary cells and is limited to CNV lesions, which may result in higher aqueous humor level of FGF-2 in patients with myopic CNV than in controls. The present research showed lower aqueous humor concentrations of VEGF-A, VEGF-D, and endoglin after consecutive intravitreal injections of ranibizumab, which are anti-VEGF-A monoclonal antibodies [ 8 ]. There is little doubt that the concentration of VEGF-A decreases after intravitreal injection [ 33 ]. As a result, the atrophy of CNV will lower the aqueous humor concentrations of the other two cytokines. VEGF-D works in the processes of angiogenesis and lymphangiogenesis. As there are no lymphatic vessels in the eyes, the VEGF-D expressed by RPE is related to ocular angiogenesis [ 34 ]. VEGF-D can also upregulate VEGF-A expression and promote angiogenesis [ 35 ]. Previous studies have shown that endoglin has strong immunogenicity in the endothelial cells of CNV membranes [ 14 ]. In contrast, the aqueous humor contents of EGF increased after consecutive intravitreal injections of ranibizumab. The atrophy of CNV led to a decrease in TGF-α contents and weakened the inhibition of EGF. As a result, the aqueous humor content of EGF increased. This study had some limitations. First, there was relatively low number of participants recruited into our research. Although the outcomes revealed statistical significance for some of the detected cytokines, more molecular mechanisms should be explored further. Second, we did not gather three aqueous humor samples from each patient, mainly due to the high price of ranibizumab and the economic burden. Some of the patients could not afford three consecutive intravitreal injections of ranibizumab. Third, caused by the restricted volume of the samples, all cytokines that may be related to myopic CNV was not detected. There are still a few molecules potentially associated with the disease that were not examined, such as TGF-β [ 36 ], matrix metalloproteinase [ 37 ], tumor necrosis factor [ 38 ], and pigment epithelium-derived factor [ 27 ]. Finally, we selected patients with cataracts as the control group, so the diopter did not match between the two groups. Conclusion This study will be beneficial to further explain the pathogenesis of myopic CNV, as we identified additional cytokines associated with myopic CNV apart from those already known. We hope that this promotes new drug research and development and that patients with myopic CNV may receive individualized and targeted treatment. Declarations Acknowledgements We thank the participants of this study. Author Contributions All authors made contributions to the preparation and design of this study. Concept and design: Liu QH and Gu YH. Acquisition, analysis, or interpretation of data: Gu WT, Wang Z and Peng D. Drafting of the manuscript: Gu WT and Wang Z. Statistical analysis: Gu WT. Technical. Material support: Liu QH and Gu YH. All authors reviewed the manuscript. Funding No funding or sponsorship was obtained for this research. The authors funded the journal’s Rapid Service Fee. Availability of data and materials The datasets generated and/or explored in the present research are available from the corresponding author upon proper request. Ethics approval and consent to participate The Ethics Committee of the First Affiliated Hospital of Nanjing Medical University reviewed and approved this study. This research insisted on the tenets of the 1964 Declaration of Helsinki and its subsequent modifications. Consent for publication Not applicable. Competing interests No competing interests were declared. References Noble KG, Carr RE. Pathologic myopia. 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Vascular endothelial growth factor(VEGF)-D stimulates VEGF-A,stanniocalcin-1,and neuropilin-2 and has potent angiogenic effects. Arterioscler Thromb Vasc Biol. 2011;31(7):1617–24. Zarranz-Ventura J, Fernández-Robredo P, Recalde S, et al. Transforming Growth Factor-Beta Inhibition Reduces Progression of Early Choroidal Neovascularization Lesions in Rats: P17 and P144 Peptides. PLoS ONE. 2013;8(5):e65434. Lecomte J, Louis K, Detry B, et al. Bone marrow-derived mesenchymal cells and MMP13 contribute to experimental choroidal neovascularization. Cell Mol Life Sci. 2011;68(4):677–86. Lichtlen P, Lam TT, Nork TM et al. Relative contribution of VEGF and TNF-α in the cynomolgus laser-induced CNV model: comparing the efficacy of bevacizumab,adalimumab and ESBA105. Investigative Ophthalmology & Visual Science. 2010;51(9):4738–4745. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4281961","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":293833601,"identity":"7cdb5a82-18dc-4123-8c62-201aba97eb4a","order_by":0,"name":"Wenting Gu","email":"","orcid":"","institution":"Nanjing Medical University, Suzhou Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenting","middleName":"","lastName":"Gu","suffix":""},{"id":293833604,"identity":"699a5e42-7625-4a6d-952b-55995d8f348c","order_by":1,"name":"Zhen Wang","email":"","orcid":"","institution":"Nanjing Medical University, Suzhou Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Wang","suffix":""},{"id":293833607,"identity":"ab7a0029-87ad-427b-8634-bf10f0ae45aa","order_by":2,"name":"Duo Peng","email":"","orcid":"","institution":"Nanjing Medical University, Suzhou Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Duo","middleName":"","lastName":"Peng","suffix":""},{"id":293833610,"identity":"c15a7315-12d1-4052-9fe7-1d7fb84b2b2d","order_by":3,"name":"Qinghuai Liu","email":"","orcid":"","institution":"The First Affiliated Hospital with Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qinghuai","middleName":"","lastName":"Liu","suffix":""},{"id":293833613,"identity":"a7e9298a-72b6-4584-b979-de024b56f18e","order_by":4,"name":"Yonghui Gu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYPCCAwxsQJIZiOXY2NsPkKbFmI/nTAJxWhigWhLnSTgY4FUrH5Fj+Ljg153EPv4zxp8LKg6nt0kwJDD8qNiGU4vhjRxj45l9zxLbJHIMjGecOZzbJt14gLHnzG3cWmbkmEnz9hwGauExSOZtA2qROZDAzNhGjBb+MwaHgVrS2SQSDPBqkZcAauH5AdTCkGPYDNSSQFCLAc+zYmPehsPGbRJpxcw8Z9IN24CBfBCfX+Tbkzc+5vlzWHZ+/+HNn3kqrOXl29sPPvhRgceWAxwGDIxtaKIHcKoH2dLA/oCB4Q8+JaNgFIyCUTDiAQCRZ1jS31W4nwAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing Medical University, Suzhou Municipal Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yonghui","middleName":"","lastName":"Gu","suffix":""}],"badges":[],"createdAt":"2024-04-17 12:42:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4281961/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4281961/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55405018,"identity":"1182e136-b171-4a4b-92a2-71104f8adfaf","added_by":"auto","created_at":"2024-04-26 20:34:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":208740,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of BCVA and CMT in the myopic CNV group after ranibizumab injection.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4281961/v1/18fc8b5e972c5d5a84ca8413.jpg"},{"id":55405459,"identity":"8dd63407-822b-49e3-af87-a1ae502f4dd8","added_by":"auto","created_at":"2024-04-26 20:42:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162572,"visible":true,"origin":"","legend":"\u003cp\u003ePlasma cytokine contents in myopic CNV and control groups.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4281961/v1/2aa336526f3663cb90cc2634.jpg"},{"id":55404667,"identity":"ca561f05-ac14-4790-9472-542153e587a0","added_by":"auto","created_at":"2024-04-26 20:26:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":159130,"visible":true,"origin":"","legend":"\u003cp\u003eAqueous humor contents of cytokines in myopic CNV and control groups.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4281961/v1/39df2cebd43017bb33ffc7f8.jpg"},{"id":55404663,"identity":"54d143f6-a83c-4c9e-b500-33b9a2b30caf","added_by":"auto","created_at":"2024-04-26 20:26:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":262842,"visible":true,"origin":"","legend":"\u003cp\u003eVariations in aqueous humor cytokine contents in the myopic CNV group after ranibizumab injection.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4281961/v1/75c964b0089dbf3258a3b9d4.jpg"},{"id":59888967,"identity":"8a2f0f7a-1c02-4ec1-bbf5-e2a998fca1d4","added_by":"auto","created_at":"2024-07-09 01:55:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1199239,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4281961/v1/f5fdee40-4555-4af8-83ab-4bec8735ac3d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The clinical effects and mechanism of action of ranibizumab in treating myopic choroidal neovascularization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn many countries, myopic choroidal neovascularization (CNV) secondary to pathologic myopia represents a prevalent source of visual impairment, notably among individuals\u0026thinsp;\u0026lt;\u0026thinsp;50 years of age [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Complications associated with pathologic myopia encompass posterior staphyloma, retinal pigment epithelium (RPE) changes, choroidal atrophy, retinal detachment and CNV [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Of these pathologic myopic complications, myopic CNV is one of the most significant threats to vision [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This disease decreases productivity and quality of life. Based on previous research, vascular endothelial growth factor has been proposed as the most significant mechanism of new blood vessel growth and vascular leakage during the formation of CNV [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Ranibizumab (Lucentis; Novartis Pharma AG, Switzerland) is specifically adopted to stop the active forms of VEGF-A [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] as a humanized, recombinant, monoclonal antibody fragment. Intravitreal injections of ranibizumab have demonstrated a curative effect on visual acuity improvement among patients with myopic CNV [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Further investigation is required to elucidate the clinical effects and mechanism of action of ranibizumab in the treatment of myopic CNV. In addition to VEGF, the potential influence of other cytokines on the pathogenesis of myopic CNV warrants further investigation.\u003c/p\u003e \u003cp\u003eIn this study, we explored the differences in plasma and aqueous humor concentrations of cytokines between patients with myopic CNV and those with cataracts. In addition, we investigated the variations in the concentrations of aqueous humor cytokines after consecutive intravitreal injections of ranibizumab. Combining this with the best modified visual acuity and the central macular thickness (CMT), we assessed the efficacy of ranibizumab in treating myopic CNV.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThis research utilized a single-center non-randomized clinical cohort research design. Participants receiving consecutive intravitreal injections of ranibizumab for myopic CNV due to pathologic myopia were enrolled from the ophthalmology clinic at the First Affiliated Hospital of Nanjing Medical University. The Institutional Review Ethics Committee of the hospital approved the protocol. From July 2022 through December 2023, we enrolled 39 patients (8 males and 31 females) who had active myopic CNV. The inclusion standards were shown below: \u0026ge;18 years of age; -6.00 diopters of refractive error or greater; CNV secondary to pathologic myopia and involving the foveal center; and recent disease progression. The exclusion standards were shown below: CNV secondary to age-associated macular degradation, polypoidal choroidal vasculopathy, inflammatory disease, angioid streaks, or trauma; subfoveal fibrosis or atrophy; any prior treatment such as photodynamic therapy or retinal laser photocoagulation for CNV secondary to pathologic myopia; and systemic diseases.\u003c/p\u003e \u003cp\u003eBefore the first intravitreal injection, all patients received a systematic ophthalmic examination, such as BCVA, slit-lamp examination, fundus examination, optical coherence tomography (OCT; OCT spectral domain, Humphrey Field Analyzer 3 from Zeiss), fundus fluorescein angiography, and indocyanine green angiography. The logarithm of the minimum angle of resolution (logMAR) was used as the BCVA for statistical analysis. The central macular thickness (CMT) was assessed by OCT centering on the fovea, and the scan length was 6.0 mm, measuring the neural epithelium thickness. The average thickness of the central foveal (1 mm in diameter) was adopted for comparison. The examinations of BCVA and OCT were repeated every month after intravitreal injections of ranibizumab. The time of the follow-up visit was three months. Another injection would be carried out based on the examination results.\u003c/p\u003e \u003cp\u003eBlood and aqueous humor samples were gathered from the study group before intravitreal injections of ranibizumab and from the control group immediately before cataract operation. Blood samples (5 mL) were taken from the antecubital vein into EDTA routine blood tubes, next to 15-minute centrifugal at 4,500 \u0026times;g to isolate the plasma, then aliquoted and kept at \u0026minus;\u0026thinsp;80\u0026deg;C until use. Undiluted aqueous humor samples (100 \u0026micro;L) were also acquired using a standard sterilization procedure. Anterior chamber paracentesis was performed before each intravitreal injection to avoid a spike in intraocular pressure after ranibizumab (0.05 mg/0.05 mL) was injected intravitreally. After disinfecting the periorbital skin and conjunctiva with a povidone-iodine solution, sterile draping of the patient, and insertion of a lid speculum, aqueous humor was aspirated with a 30-gauge needle linked to a tuberculin syringe at the temporal limbus. Intravitreal injections and cataract surgery were then performed. Aqueous humor samples were gathered in sterile tubes and kept at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eSamples were measured with the Luminex xMAP suspension array. A Milliplex Kit was used for the detection of epidermal growth factor (EGF) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], angiopoietin-2 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], granulocyte colony-stimulating factor (G-CSF) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], endoglin [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], endothelin-1 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], fibroblast growth factor (FGF)-1 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], FGF-2 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], interleukin-8 (IL-8) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], hepatocyte growth factor (HGF) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], heparin-binding epidermal growth factor (HB-EGF) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], placental growth factor (PLGF) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], vascular endothelial growth factor (VEGF)-C [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], VEGF-D [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and VEGF-A [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It is necessary to conduct intensive studies, as many previous investigations have demonstrated that these cytokines may take part in the formation and development of myopic CNV.\u003c/p\u003e \u003cp\u003eBefore commencing the assay, all reagents were warmed to room 20\u0026ndash;25\u0026deg;C. The first step involved adding 25 \u0026micro;L of assay buffer, 25 \u0026micro;L of appropriate matrix solution, 25 \u0026micro;L of plasma sample (1:3 dilution) or undiluted aqueous humor sample, and 25 \u0026micro;L of beads to every well. The plate capacity was 96-well.Wrapped with foil, the plate was incubated on a plate shaker overnight at 4\u0026deg;C or 2 h at 20\u0026ndash;25\u0026deg;C. Then, after putting 25 \u0026micro;L of detection antibodies and 25 \u0026micro;L streptavidin-phycoerythrin to every well, the plate was covered with foil, next to 30-minute incubation on a plate shaker at 20\u0026ndash;25\u0026deg;C. All incubation procedures were made in the dark to prevent light exposure to the beads. Finally, the plate was run on a Luminex 200\u0026trade; with xPONENT software.\u003c/p\u003e \u003cp\u003eSPSS (version 27.0; SPSS Inc., Chicago, IL, USA) was adopted to analyze data. An independent sample \u003cem\u003et\u003c/em\u003e-test was adopted to compare the diversities in plasma and aqueous humor cytokine concentrations between the two groups. The differences in BCVA values, CMT, and cytokine concentrations among various groups of aqueous humor samples collected at baseline, one month and two months were compared with a one-way analysis of variance (ANOVA). Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThere were 49 plasma samples collected from 39 patients with myopic CNV and 10 patients with cataracts. A total of 15 myopic CNV patients and 10 cataract patients received aqueous sampling upon intravitreal ranibizumab injections at baseline. Subsequently, 14 and 9 myopic CNV patients\u0026rsquo; aqueous humor samples were collected at 1 month and 2 months.\u003c/p\u003e \u003cp\u003eThe average patient age was 54.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11.7 years (scope 24\u0026ndash;75 years). The average patient diopter was \u0026minus;\u0026thinsp;11.85\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61 D (range \u0026minus;\u0026thinsp;7 to \u0026minus;\u0026thinsp;20 D). As controls, 10 senile cataract patients (5 males and 5 females) without other ocular or systemic diseases were gathered. The average patient age in the control group was 70.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.0 years (range 52\u0026ndash;80 years). According to our study, the mean patient BCVA (logMAR) value was 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 at baseline, 0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 at 1 month, and 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 at 2 months (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, a great diversity between the BCVA values was observed at baseline and at 2 months (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The CMT of the patients with myopic CNV was 311.03\u0026thinsp;\u0026plusmn;\u0026thinsp;54.32 \u0026micro;m at baseline, then reduced to 255.57\u0026thinsp;\u0026plusmn;\u0026thinsp;50.97 \u0026micro;m at 1 month and 266.42\u0026thinsp;\u0026plusmn;\u0026thinsp;37.64 \u0026micro;m at 2 months after consecutive intravitreal injections of ranibizumab (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The CMT at 1 and 2 months was greatly lower than that at baseline (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in BCVA and CMT in the myopic choroidal neovascularization (CNV) group after ranibizumab injections\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAt baseline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAt 1 month\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAt 2 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP-value\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP-value\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCVA (logMAR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.234\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCMT (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e311.03\u0026thinsp;\u0026plusmn;\u0026thinsp;54.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e255.57\u0026thinsp;\u0026plusmn;\u0026thinsp;50.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e266.42\u0026thinsp;\u0026plusmn;\u0026thinsp;37.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.990\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBCVA, best-corrected visual acuity; logMAR, logarithm of the minimum angle of resolution; CMT, central macular thickness.\u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eOne-way analysis of variance between baseline and 1 month for BCVA and CMT\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn plasma, the concentrations of VEGF-A, EGF, and angiopoietin-2 in the myopic CNV group were 42.61\u0026thinsp;\u0026plusmn;\u0026thinsp;34.36, 0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35, and 897.71\u0026thinsp;\u0026plusmn;\u0026thinsp;366.29 pg/mL, respectively. In the cataract group, the concentrations of these cytokines were 14.94\u0026thinsp;\u0026plusmn;\u0026thinsp;11.57, 1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60, and 1242.91\u0026thinsp;\u0026plusmn;\u0026thinsp;347.91 pg/mL, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The concentration of VEGF-A in the myopic CNV group was greatly higher than that in the cataract group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, EGF and angiopoietin-2 concentrations were greatly lower in the myopic CNV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePlasma contents of cytokines in myopic CNV and control groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCNV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF-A (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.94\u0026thinsp;\u0026plusmn;\u0026thinsp;11.57l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e42.61\u0026thinsp;\u0026plusmn;\u0026thinsp;34.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEGF (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAngiopoietin-2 (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1242.91\u0026thinsp;\u0026plusmn;\u0026thinsp;347.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e897.71\u0026thinsp;\u0026plusmn;\u0026thinsp;366.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eVEGF-A, vascular endothelial growth factor A; HB-EGF, heparin-binding epidermal growth factor; EGF, epidermal growth factor.\u003c/p\u003e \u003cp\u003e \u003csup\u003ed\u003c/sup\u003e \u003cem\u003eT\u003c/em\u003e-test between control and myopic CNV groups for plasma contentss of cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn aqueous humor, the concentrations of FGF-2, EGF, and VEGF-A were 34.11\u0026thinsp;\u0026plusmn;\u0026thinsp;23.30, 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, and 135.09\u0026thinsp;\u0026plusmn;\u0026thinsp;70.29 pg/mL, respectively, in the myopic CNV group, and 9.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26, 0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, and 272.71\u0026thinsp;\u0026plusmn;\u0026thinsp;138.91 pg/mL, respectively, in the cataract group (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The myopic CNV group showed greatly higher concentration of FGF-2 than the cataract group (p\u0026thinsp;\u0026lt;\u0026thinsp;0). In contrast, the concentrations of EGF and VEGF-A were greatly lower in the myopic CNV group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAqueous humor contents of cytokines in myopic CNV and control groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCNV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF-A (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e272.71\u0026thinsp;\u0026plusmn;\u0026thinsp;138.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e135.09\u0026thinsp;\u0026plusmn;\u0026thinsp;70.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEGF (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFGF-2 (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e34.11\u0026thinsp;\u0026plusmn;\u0026thinsp;23.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eVEGF-A, vascular endothelial growth factor A; EGF; FGF, fibroblast growth factor.\u003c/p\u003e \u003cp\u003e \u003csup\u003ee\u003c/sup\u003e \u003cem\u003eT\u003c/em\u003e-test between control and myopic CNV groups for aqueous humor contents of cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChanges in the contents of VEGF-A, VEGF-D, EGF, and endoglin among the aqueous humor of patients with myopic CNV were observed after consecutive intravitreal injections of ranibizumab. Baseline VEGF-A concentration in the aqueous humor was 135.09\u0026thinsp;\u0026plusmn;\u0026thinsp;70.29 pg/mL, but decreased to 5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 and 8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.70 pg/mL at 1 and 2 months, after intravitreal injections of ranibizumab. Baseline VEGF-D concentration in the aqueous humor was 2.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 pg/mL but decreased to 2.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 and 2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 pg/mL at 1 and 2 months, after injections. Baseline endoglin concentration in the aqueous humor was 15.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35 pg/mL and decreased to 14.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83l and 11.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 pg/mL at 1 and 2 months, after injections. Baseline EGF concentration in the aqueous humor was 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 pg/mL and increased to 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 and 0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 pg/mL at 1 and 2 months, after injections (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The decrease in average VEGF-A contents by comparing with baseline at both 1 month (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 2 months (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was great, and the decrease in average VEGF-D and endoglin contents by comparing with baseline at 2 months was significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both) and were also significant when compared at 1 month to 2 months (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both). The average EGF contents were significantly higher at 2 months than at baseline (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in aqueous humor contents of cytokines in the myopic CNV group after ranibizumab injections\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAt baseline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAt 1 month\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAt 2 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep-value\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF-A (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e135.09\u0026thinsp;\u0026plusmn;\u0026thinsp;70.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.389\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF-D (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.264\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEGF (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.171\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndoglin (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ef\u003c/sup\u003eOne-way analysis of variance (ANOVA) between baseline and 1 month for changes in aqueous humor contents of cytokines.\u003c/p\u003e \u003cp\u003e \u003csup\u003eg\u003c/sup\u003eANOVA between baseline and 2 months for changes in aqueous humor cytokine contents.\u003c/p\u003e \u003cp\u003e \u003csup\u003eh\u003c/sup\u003eANOVA between 1 month and 2 months for changes in aqueous humor contents of cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current research investigated the therapeutic effects of ranibizumab in patients with myopic CNV. A great improvement in BCVA was found, along with a significantly decreased CMT measured by OCT and aqueous humor contents of VEGF-A, VEGF-D, and endoglin, but significantly decreased content of EGF after consecutive intravitreal injections of ranibizumab. The results were similar to those from another study on the effects of ranibizumab in AMD cases [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The concentrations of cytokines may be quantitative indicators of the effects of ranibizumab in patients with myopic CNV. Ranibizumab demonstrated significant effects on CNV secondary to pathologic myopia by improving BCVA values, reducing CMT, and modulating cytokines associated with angiogenesis. For different individuals, different treatment projects should be chosen.\u003c/p\u003e \u003cp\u003eThe myopic CNV group showed higher plasma concentration of VEGF-A than the control group, whereas EGF and angiopoietin-2 contents were decreased in the myopic CNV group by comparing with the control group. Moreover, compared to controls, aqueous humor content of FGF-2 increased, whereas those of EGF and VEGF-A decreased in the eyes of patients with myopic CNV.\u003c/p\u003e \u003cp\u003eVEGF concentration in the plasma is greater among patients with active CNV secondary to AMD than in healthy individuals [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, there is no research on the plasma VEGF concentration among patients with myopic CNV. It is well known that VEGF is the most significant signaling protein taking part in both vasculogenesis and angiogenesis, and the rise of plasma VEGF is a prerequisite for CNV generation. Therefore, in the present research, it is reasonable to detect a higher plasma concentration of VEGF-A in the myopic CNV group. Based on past research, it is controversial whether the VEGF concentration in the aqueous humor is high in myopic CNV. Tong et al. disclosed that VEGF concentration in the aqueous humor grew significantly among patients with CNV secondary to AMD, polypoidal choroidal vasculopathy, and CNV secondary to pathologic myopia, compared to healthy controls [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In contrast, in research by Sawada et al., VEGF concentrations in the aqueous humor of the myopic CNV group were greatly lower than those in the control group, which is consistent with our results [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Vascular endothelial growth factor receptor VEGFR-2 is mainly located in the retinal blood vessel cells of the leakage area, which is related to vascular leakage, whereas VEGFR-3 is mainly located in deep retinal capillaries and is relevant to VEGFR-2. VEGF-A induces VEGFR-2 and VEGFR-3 expression in the ischemic area and leads to retinal leakage, thereby promoting the formation of new blood vessels [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. We hypothesize that VEGF-A may be localized in a small subfoveal area in combination with VEGFR-2 and VEGFR-3, resulting in decreased levels in the aqueous humor during CNV formation.\u003c/p\u003e \u003cp\u003eEGF contents are greatly higher in the aqueous humor of patients with AMD [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, in the current research, the result was different when patients with myopic CNV were the research focus. The EGFR primarily resides in the cell membrane, where it binds with a significant quantity of EGF. It transduces biological signals by invagination pinocytosis, leading to its internalization into the cytoplasm. Once internalized, EGF is degraded by lysosomes, and EGFR is subsequently recycled back to the membrane. The expression of transforming growth factor-α (TGF-α) will increase while CNV forms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. TGF-α and EGF have 33\u0026ndash;44% homology in structure, and TGF-α may competitively inhibit the expression of EGF. This leads to a decrease in EGF level in the plasma and aqueous humor of patients with myopic CNV.\u003c/p\u003e \u003cp\u003eAngiopoietin-2 exerts a key effect on vessel maturation, angiogenesis, and vessel regression and is expressed most heavily in tumor tissue (especially in tumor neovascularization) but not in normal tissue [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Myopic CNV expresses a large quantity of angiopoietin-2 and angiopoietin receptor (TEK) restricted to CNV lesions; thus, we found lower contents of angiopoietin-2 in both the plasma and aqueous humor among patients with myopic CNV by comparing with the control group.\u003c/p\u003e \u003cp\u003eIn the normal retina, the mRNA of FGF-2 and its receptor were discovered in the ganglion cell layer and kernel, and transient retinal ischemia induced the synthesis of FGF-2 mRNA [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In vitro studies have found that the complex of anti-VEGF antibody/anti-FGF-2 antibody could completely inactivate VEGF and FGF-2 in the retinal pigment epithelium containing CNV, better inhibiting the growth of vascular endothelial cells than simply adding anti-VEGF antibody [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Unlike VEGF-A, EGF, and angiopoietin-2, FGF-2 is not expressed in retinal capillary cells and is limited to CNV lesions, which may result in higher aqueous humor level of FGF-2 in patients with myopic CNV than in controls.\u003c/p\u003e \u003cp\u003eThe present research showed lower aqueous humor concentrations of VEGF-A, VEGF-D, and endoglin after consecutive intravitreal injections of ranibizumab, which are anti-VEGF-A monoclonal antibodies [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. There is little doubt that the concentration of VEGF-A decreases after intravitreal injection [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. As a result, the atrophy of CNV will lower the aqueous humor concentrations of the other two cytokines. VEGF-D works in the processes of angiogenesis and lymphangiogenesis. As there are no lymphatic vessels in the eyes, the VEGF-D expressed by RPE is related to ocular angiogenesis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. VEGF-D can also upregulate VEGF-A expression and promote angiogenesis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Previous studies have shown that endoglin has strong immunogenicity in the endothelial cells of CNV membranes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In contrast, the aqueous humor contents of EGF increased after consecutive intravitreal injections of ranibizumab. The atrophy of CNV led to a decrease in TGF-α contents and weakened the inhibition of EGF. As a result, the aqueous humor content of EGF increased.\u003c/p\u003e \u003cp\u003eThis study had some limitations. First, there was relatively low number of participants recruited into our research. Although the outcomes revealed statistical significance for some of the detected cytokines, more molecular mechanisms should be explored further. Second, we did not gather three aqueous humor samples from each patient, mainly due to the high price of ranibizumab and the economic burden. Some of the patients could not afford three consecutive intravitreal injections of ranibizumab. Third, caused by the restricted volume of the samples, all cytokines that may be related to myopic CNV was not detected. There are still a few molecules potentially associated with the disease that were not examined, such as TGF-β [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], matrix metalloproteinase [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], tumor necrosis factor [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and pigment epithelium-derived factor [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Finally, we selected patients with cataracts as the control group, so the diopter did not match between the two groups.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study will be beneficial to further explain the pathogenesis of myopic CNV, as we identified additional cytokines associated with myopic CNV apart from those already known. We hope that this promotes new drug research and development and that patients with myopic CNV may receive individualized and targeted treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the participants of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors\u0026nbsp;made\u0026nbsp;contributions\u0026nbsp;to the\u0026nbsp;preparation\u0026nbsp;and design of this study. Concept and design: Liu QH and Gu YH. Acquisition, analysis, or interpretation of data: Gu WT, Wang Z and Peng D. Drafting of the manuscript: Gu WT and Wang Z. Statistical analysis: Gu WT. Technical. Material support: Liu QH and Gu YH. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo funding or sponsorship was obtained for this research. The authors funded the journal\u0026rsquo;s Rapid Service Fee.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or explored in the present research are available from the corresponding author upon proper request. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ethics Committee of the First Affiliated Hospital of Nanjing Medical University reviewed and approved this study. This research insisted on the tenets of the 1964 Declaration of Helsinki and its subsequent modifications.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo competing interests were declared.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNoble KG, Carr RE. Pathologic myopia. Ophthalmology. 1982;89:1099\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhno-Matsui K, Yoshida T, Futagami S, et al. Patchy atrophy and lacquer cracks predispose to the development of choroidal neovascularisation in pathological myopia. Br J Ophthalmol. 2003;87(5):570\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong TY, Ferreire A, Hughes R, et al. 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The genetic variant rs4073 A\u0026rarr;T of the Interleukin-8 promoter region is associated with the earlier onset of exudative age-related macular degeneration. Acta Ophthalmol. 2015;93(8):726\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu WL, Criswell MH, Fong SL, et al. Differences in the temporal expression of regulatory growth factors during choroidal neovascular development. Exp Eye Res. 2009;88(1):79\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInoue Y, Shimazawa M, Nakamura S, et al. Both Autocrine Signaling and Paracrine Signaling of HB-EGF Enhance Ocular Neovascularization. Arterioscler Thromb Vasc Biol. 2018;38(1):174\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrespo-Garcia S, Corkhill C, Roubeix C, et al. Inhibition of Placenta Growth Factor Reduces Subretinal Mononuclear Phagocyte Accumulation in Choroidal Neovascularization. Invest Ophthalmol Vis Sci. 2017;58(12):4997\u0026ndash;5006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao JF, Hua HR, Chen QB, et al. Impact of fenofibrate on choroidal neovascularization formation and VEGF-C plus VEGFR-3 in Brown Norway rats. Exp Eye Res. 2018;174:152\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJauhiainen S, H\u0026auml;kkinen SK, Toivanen PI, et al. Vascular endothelial growth factor (VEGF)-D stimulates VEGF-A,stanniocalcin-1 and neuropilin-2 and has potent angiogenic effects. Arterioscler Thromb Vasc Biol. 2011;31(7):1617\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrisanti S, Zhu Q, Tatar O, et al. Differential expression of vascular endothelial growth factor-a isoforms in neovascular age-related macular degeneration. Retina. 2015;35(4):764\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGungel H, Osmanbasoglu OA, Altan C, et al. The effects of ranibizumab injections on fluorescein angiographic findings and visual acuity recovery in age-related macular degeneration. Clin Ophthalmol. 2014;19(8):981\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarneiro AM, Costa R, Falcao MS, et al. Vascular endothelial growth factor plasma contents before and after treatment of neovascular age-related macular degeneration with bevacizumab or ranibizumab. Acta Ophthalmol. 2012;90(1):e25\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTong JP, Chan WM, Liu DT, et al. Aqueous humor contents of vascular endothelial growth factor and pigment epitheliumderived factor in polypoidal choroidal vasculopathy and choroidal neovascularization. Am J Ophthalmol. 2006;141(3):456\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSawada O, Kawamura H, Kakinoki M, et al. 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Exp Eye Res. 2006;83(5):1031\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJauhiainen S, H\u0026auml;kkinen SK, Toivanen PI, et al. Vascular endothelial growth factor(VEGF)-D stimulates VEGF-A,stanniocalcin-1,and neuropilin-2 and has potent angiogenic effects. Arterioscler Thromb Vasc Biol. 2011;31(7):1617\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZarranz-Ventura J, Fern\u0026aacute;ndez-Robredo P, Recalde S, et al. Transforming Growth Factor-Beta Inhibition Reduces Progression of Early Choroidal Neovascularization Lesions in Rats: P17 and P144 Peptides. PLoS ONE. 2013;8(5):e65434.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLecomte J, Louis K, Detry B, et al. Bone marrow-derived mesenchymal cells and MMP13 contribute to experimental choroidal neovascularization. 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Investigative Ophthalmology \u0026amp; Visual Science. 2010;51(9):4738\u0026ndash;4745.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Myopic horoidal neovascularization, Ranibizumab, Cytokine, Plasma, Aqueous humor","lastPublishedDoi":"10.21203/rs.3.rs-4281961/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4281961/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the clinical effects and factors influencing repeated intravitreal injections of ranibizumab among patients with choroidal neovascularization (CNV) secondary to pathologic myopia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were 39 patients with myopic CNV and a control group of 10 cataract patients. Logarithm of the minimum angle of resolution units (logMAR) was adopted to evaluate best-corrected visual acuity (BCVA), while the measurement of central macular thickness was made via optical coherence tomography. Luminex xMAP suspension array was adopted to collect and analyze plasma and aqueous humor samples to compare cytokine concentrations between the two groups and assess changes after intravitreal ranibizumab injections. Additionally, BCVA values and CMT were monitored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA great diversity in the BCVA values and CMT was found after intravitreal ranibizumab injections (p\u0026lt;0.05 for both). The study group showed greatly higher plasma concentration of vascular endothelial growth factor-A than the cataractgroup (p\u0026lt;0.05). The research group (p\u0026lt;0.05 for both) showed greatly lower epidermal growth factor (EGF) and angiopoietin-2 concentrations. In the aqueous humor, the study group showed greatly higher concentration of fibroblast growth factor (FGF)-2 than the cataract group (p\u0026lt;0.05), and the research group (p\u0026lt;0.05 for both) displayed greatly lower concentrations of EGF and VEGF-A. The decrease in average VEGF-A contents was significant by comparing with baseline at both 1 month (p\u0026lt;0.05) and 2 months (p\u0026lt;0.05), and the reduction in mean VEGF-D and endoglin contents compared to baseline at 2 months were significant (p\u0026lt;0.05 for both), and were significant when compared at 1 month to 2 months (p\u0026lt;0.05 for both). The average EGF contents were significantly higher at 2 months than at baseline (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRanibizumab had obvious effects on myopic CNV. For different individuals, different treatment projects should be chosen.\u003c/p\u003e","manuscriptTitle":"The clinical effects and mechanism of action of ranibizumab in treating myopic choroidal neovascularization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-26 20:26:13","doi":"10.21203/rs.3.rs-4281961/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ada2c8eb-60b2-4a25-942c-557de9d5c10f","owner":[],"postedDate":"April 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-09T01:47:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-26 20:26:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4281961","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4281961","identity":"rs-4281961","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}