Molecular Vision 2024; 30:268-277 <http://www.molvis.org/molvis/v30/268>
Received 03 June 2023 | Accepted 01 July 2024 | Published 02 July 2024

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Complement C3 is downregulated following ranibizumab intervention in experimental central retinal vein occlusion

Lasse Jørgensen Cehofski,1,2,3 Anders Kruse,4 Mads Odgaard Mæng,4 Benedict Kjaergaard,2 Benn Falch Sejergaard,4 Anders Schlosser,5 Grith Lykke Sorensen,5 Bent Honoré,6,7 Henrik Vorum4

1Department of Ophthalmology, Odense University Hospital, Odense, Denmark; 2Biomedical Research Laboratory, Aalborg University Hospital, Aalborg, Denmark; 3Department of Clinical Research, University of Southern Denmark, Odense, Denmark; 4Department of Ophthalmology, Aalborg University Hospital, Aalborg, Denmark; 5Department of Cancer and Inflammation Research, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark; 6Department of Biomedicine, Aarhus University, Aarhus, Denmark; 7Department of Clinical Medicine, Aalborg University, Aalborg University Hospital

Correspondence to: Lasse Jørgensen Cehofski, 19 Department of Ophthalmology, Odense University Hospital Sdr. Boulevard 29, 5000 Odense, Denmark; Phone +45 53558878 24; email: lassecehofski@hotmail.com

Abstract

Purpose: Ranibizumab (Novartis, Copenhagen, Denmark) is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the treatment of macular edema following central retinal vein occlusion (CRVO). Studying proteins that mediate the beneficial effects of ranibizumab in CRVO can potentially lead to the improved management of macular edema.

Methods: In 14 Danish Landrace pigs, experimental CRVO was induced in the right eyes and treated with either intravitreal ranibizumab (n=6) or an intravitreal sodium chloride 9 mg/mL solution as a sham injection (n=8). Successful CRVO was confirmed by fluorescein angiography. Retinal samples were collected 15 days after induced CRVO and analyzed with label-free, quantification, nano-liquid chromatography–tandem mass spectrometry. Validation was performed with western blotting and immunohistochemistry.

Results: CRVO was successfully induced and confirmed by fluorescein angiography. A total of 28 proteins were upregulated, and 31 proteins were downregulated following ranibizumab treatment. A high concentration of the ranibizumab component immunoglobulin kappa chain C region was observed in retinas treated with ranibizumab. Complement C3, the Ig lambda chain C region, and nucleobindin-2 were downregulated following ranibizumab intervention. The downregulation of complement C3 was confirmed by western blotting. The Ig lambda chain C region and nucleobindin-2 were downregulated following ranibizumab intervention. Modest changes were observed in the remaining significantly regulated proteins.

Conclusions: Retinal complement C3 was downregulated following ranibizumab intervention in CRVO. The decrease in complement C3 may potentially downregulate the inflammatory response in CRVO. A high retinal concentration of ranibizumab was reached 15 days after injection of the compound.

Introduction

Central retinal vein occlusion (CRVO) is a visually disabling condition resulting from impaired outflow of the central retinal vein, the major outflow vessel of the eye [1,2]. The visual outcome in CRVO can be devastating, and blindness may be the ultimate result. Visual impairment in CRVO is predominantly caused by macular edemaand visual acuity rarely improves above 20/40 if treatment is not initiated [6].

Macular edema in CRVO arises through a complex multifactorial mechanism. Occlusion of the central retinal vein increases the resistance to blood flow in retinal arterioles with closure of retinal capillaries and small arterioles, leading to retinal hypoxia. Retinal hypoxia results in increased production of vascular endothelial growth factor A (VEGF-A) and a complex inflammatory response mediated by interleukin (IL)-6, IL-8, IL-18, S100A12, fibrinogen, fibronectin, galectin-3, and monocyte chemotactic protein-1 [7-11], which increases vascular permeability and leads to the accumulation of fluid in the macula [8]. In retinal vein occlusion, intraocular complement factors have recently been found to correlate with visual acuity and the severity of macular edema, indicating involvement in pathogenic processing and leading to visual loss [10,11].

Macular edema is effectively treated with intravitreal anti-VEGF injections as a first-line therapy, while dexamethasone intravitreal implants are considered second-line treatments [12-15]. Despite significant therapeutic advances, CRVO remains a burden to patients and continues to challenge clinicians, as well as the healthcare system in general. Macular edema is recurrent in most cases, and approximately 50% of patients need anti-VEGF injections four years after being diagnosed with either CRVO or branch retinal vein occlusion (BRVO) [16]. In a Danish population of patients with retinal vein occlusion (RVO), the yearly number of injections for macular edema in doubled from 2014 to 2022 [17].

Ranibizumab is a monoclonal antibody fragment (Fab) that neutralizes all isoforms of VEGF-A [18]. Studying protein changes downstream of VEGF-A neutralization by ranibizumab may elucidate novel mechanisms that mediate the beneficial response to anti-VEGF treatment. The retinal proteome in CRVO following ranibizumab treatment has not previously been explored. Here, we report on retinal proteome changes following ranibizumab intervention in an experimental model of CRVO.

Methods

Animal preparation

The study was approved by the Danish Animal Experiments Inspectorate, permission no. 2019–15–0201- 01,651, and the experiments were conducted in accordance with the guidelines published by the Institute for Laboratory Animal Research. A total of 14 Danish Landrace pigs were used for the experiments and housed under a 12-h light/dark cycle. General anesthesia, topical anesthesia with eye drops, and dilation of the pupils were performed as previously described [19].

Experimental central retinal vein occlusion (CRVO)

Experimental CRVO was induced in proximity to the optic nerve head with a standard argon laser (532 nm), given by indirect ophthalmoscopy using a 20D lens. The laser energy was set to 400 mW, with an exposure time of 550 ms. A total of 30–40 laser applications were used per occlusion. By applying the laser directly to the retinal veins close to the optic nerve head, the thrombotic material was directed toward the optic nerve head and lamina cribrosa. Experimental CRVO was considered successful when stagnation of venous blood and the development of flame-shaped hemorrhages were observed by indirect ophthalmoscopy. In the intervention group, the right eye of the animal (n=6) received an intravitreal injection of 0.05 ml ranibizumab 10 mg/ml (Novartis A/S, Copenhagen, Denmark). In the control group, the right eye of each animal (n=8) received a vehicle injection with 0.05 ml of sodium chloride 9 mg/mL (NaCl; B. Braun, Denmark).

Fifteen days after induced CRVO, the eyes were enucleated and dissected on ice under a microscope. The animals were euthanized immediately after enucleation. The anterior segment was removed, and the vitreous body was removed by aspiration into a 5 ml syringe. In eyes used for proteomic analysis, the neurosensory retina was peeled from the retinal pigment epithelium/choroid complex with tweezers and stored at −80 °C. In eyes collected for immunohistochemistry, complexes consisting of the neurosensory retina, RPE/choroid complex, and sclera were excised.

Sample preparation for mass spectrometry

Label-free, quantification, nano-liquid chromatography–tandem mass spectrometry (LFQ nLC-MS/MS) was performed to compare CRVO + ranibizumab (n=5) and CRVO + NaCl (n=7). Sample preparation was undertaken, as previously described [20]. Briefly, lysis with a buffer of 5% sodium deoxycholate and 20 mM triethyl ammonium bicarbonate was performed, as previously described [21]. The protein concentration was measured by infrared spectrometry with a Direct Detect Spectrometer (Merck KGaA, Darmstadt, Germany), and alkylation with iodoacetamide was performed, as previously described [20]. The resulting lysates were added to filter units (Microcon Centrifugal Filter Devices 30 K, MilliporeSigma, Sigma Aldrich, Søborg, Denmark), and digestion with trypsin was performed using S-Trap micro spin columns from Protifi (Hintington, NY), as described previously [10]. Finally, the samples were dried in a vacuum centrifuge and resuspended in 100 mM etraethylammonium bromide, followed by measurement of the peptide concentration by fluorescence, as previously described [21]. Peptides were dried and dissolved in 1% formic acid, and 1 µg was injected into the mass spectrometer.

Label-free, quantification, nano-liquid chromatography–tandem mass spectrometry (LFQ nLC-MS/MS)

LFQ nLC-MS/MS was performed on an Orbitrap Fusion Tribrid mass spectrometer equipped with an EasySpray ion source coupled to a Dionex UltimateTM 3000 RSLC nanosystem (Thermo Fisher Scientific Instruments, Waltham, MA) using the Universal Method for label-free quantification, as previously described [10] [15]. Peptide separation was performed, as previously described [20]. Mass spectrometry was performed with the Universal Method for label-free quantification, and full scans in the Orbitrap were obtained, as previously described [20]. Isolation of precursor ions in the quadrupole, collision-induced dissociation, and detection of MS2 scans in the ion trap were performed, as previously described [20]. MaxQuant software version 1.6.6.0 [22] (Max Planck Institute of Biochemistry, Martinsried, Germany) was used to search raw data files against the Uniprot Sus scrofa and Homo sapiens databases.

Filtration of proteins and statistics

Unfiltered MaxQuant output files (Appendix 1) were uploaded to Perseus software version 1.6.2.3 [23] (Max Planck Institute of Biochemistry, Martinsried, Germany). Proteins were filtrated by removal of proteins only identified by the modification site, followed by removal of proteins identified from a peptide that was found to be part of a protein derived from the reversed part of the decoy database. Proteins identified as contaminants were also removed from the data set. Quantitative values were log2 transformed, and technical replicates were averaged. Successful identification of a protein requires at least two unique peptides. Successful identification and quantification were required in 100% of the samples. A Student t test was performed in Perseus to compare CRVO + ranibizumab (n=5) versus CRVO + NaCl (n=7). Proteins were considered significantly regulated if p<0.05 by Student t test.

Immunohistochemistry

Eyes from one animal were used to compare CRVO + ranibizumab (n=1) versus CRVO + NaCl (n=1). Complexes consisting of the retina, choroid, and sclera were fixated in formalin for 24 h, followed by removal of the formalin solution. The tissue was then stored in a PBS solution at 4 °C until further use. Immunohistochemistry was performed as previously described [24] with a rabbit anti-pig complement component 3 antibody (MBS2028490, MyBiosource, San Diego, CA), diluted 1:100.

Western blotting

Western blotting was performed as previously described [24] using a polyclonal rabbit anti-pig complement component 3 antibody (MBS2028490, MyBiosource, San Diego, CA), diluted 1:1,000. Western blotting was performed to compare CRVO + ranibizumab (n=5) versus CRVO + NaCl (n=7). Statistical analysis of densitometric data was performed with the Mann–Whitney U test.

Results

Retinal proteome changes following aflibercept intervention in experimental CRVO

Experimental CRVO was successfully induced in all eyes. Venous tortuosity and flame-shaped hemorrhages were observed in all animals within 30 min after CRVO was induced (Figure 1). Venous tortuosity, leakage, and areas with retinal capillary nonperfusion were observed on angiography in all quadrants, consistent with successfully induced CRVO (Figure 2).

In total, 2,342 proteins were successfully identified. All successfully identified proteins are provided in Appendix 2. A total of 1,518 proteins were successfully identified and quantified in 100% of the samples (Appendix 3). Statistical analysis was performed on the proteins present in 100% of the samples. A total of 59 proteins were significantly regulated in the ranibizumab intervention in CRVO (Figure 3; Table 1). A total of 28 proteins were upregulated, and 31 proteins were downregulated. The largest upregulation was observed in the Ig kappa chain C region, a component of ranibizumab. Other upregulated proteins included the tubulin beta-6 chain (fold change=2.2; p=0.031) and the very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase 3 (fold change=1.66; p=0.018; Table 1). The remaining upregulated proteins had fold changes between 1.07 and 1.36. Downregulated proteins included complement C3 (fold change=0.44; p=0.039), the Ig lambda chain C region (fold change=0.47; p=0.049), nucleobindin-2 (fold change=0.64; p=0.008), transthyretin (fold change=0.67; p=0.037), prolow-density lipoprotein receptor-related protein 1 (fold change=0.68; p=0.048), and 14 kDa phosphohistidine phosphatase (fold change=0.68; p=0.020; Table 1). All other downregulated proteins had fold changes between 0.68 and 0.95; western blotting confirmed a decreased level of complement C3 following ranibizumab intervention (p=0.048; Figure 4). Complement C3 was predominantly expressed in the retinal nerve fiber layer, inner plexiform layer, outer plexiform layer, and outer nuclear layer (Figure 5).

Discussion

Our study reported on proteome changes in an experimental model of CRVO. As the majority of CRVO cases are nonischemic [2], a CRVO model corresponding to nonischemic CRVO was chosen for the study. Leakage of fluorescein along retinal veins, as well as areas with retinal capillary nonperfusion, were observed on fluorescein angiography consistent with angiographic findings following CRVO in humans. Alternatively, an ischemic CRVO can be created by occluding four branch retinal veins in the porcine eye, but stages with severe ischemia may be more resistant to anti-VEGF therapy [25]. The experimental CRVO model was discussed in detail in a recent paper [26]. The model has several advantages that make it suitable for proteome studies of retinal vascular diseases. The porcine retina is fully vascularized and similar to the human retina in size and photoreceptor distribution [26,27]. Significant drawbacks associated with the use of porcine eyes include the high expenses related to acquisition and housing, which generally make it necessary to conduct experiments with small sample sizes.

Proteomic analysis identified downregulation of retinal complement C3 in eyes treated with ranibizumab. The quantitative validation of the mass spectrometry data was performed by western blotting. Complement C3 was found to be increased in aqueous humor samples from patients with CRVO [11] and BRVO [10]. In aqueous humor from patients with BRVO, a correlation between complement C3 and the severity of macular edema has previously been identified [10]. At the retinal level, complement C3 is increased in experimental CRVO, where it increases with the level of retinal ischemia [9,24]. Interestingly, our study observed a reversal of complement C3 in retinas treated with ranibizumab.

The complement system is part of the innate immune response, the fast, unspecific arm of the immune system, and it is involved in the early response against pathogens [28,29]. The complement system can be activated through three different pathways: the classical pathway, the lectin pathway, and the alternative pathway [28].

The alternative pathway is constitutively active at low levels due to the spontaneous hydrolysis of complement component C3 to C3(H2O). In the alternative pathway, factor B can bind C3(H20) and is cleaved by factor D to form a distinct C3 convertase termed C3(H20), which produces C3b and provides an amplification loop for the activation of the complement system [29]. There is mounting evidence that the complement system plays an important role in the pathogenesis of age-related macular degeneration [30]. Interestingly, a bispecific fusion protein that neutralizes VEGF along with complement C3b and complement C4b has shown promising results in animal models of neovascular age related macular degeneration and has been reported to be well tolerated in a phase 1 trial [30]. Genotyping of patients with neovascular AMD has identified an association between these complements.

Furthermore, the C3 gene has been associated with favorable outcomes of anti-VEGF therapy [31]. There is still limited knowledge of the role of complement activation in CRVO, but our data suggest that ranibizumab may downregulate the inflammatory response in CRVO by decreasing the expression of complement C3.

Ranibizumab intervention was associated with a decreased level of nucleobindin-2, a protein with two Ca2+ binding EF-hand domains separated by an acidic amino acid-rich region and a leucine zipper [32]. The heterogenic structure of nucleobindin-2 is consistent with its multitude of functions. In a variety of cancers, nucleobindin-2 promotes cell proliferation, migration, and invasion, with high nucleobindin-2 levels associated with poor outcomes [32], but its retinal function remains to be elucidated. A downregulation of transthyretin was observed following ranibizumab intervention. Ocular transthyretin is synthesized by RPE cells and has a wide distribution within the retinal layers [33,34], but the role of transthyretin in the RPE remains to be elucidated [35]. The immunoglobulin kappa chain C region of ranibizumab was identified in samples treated with ranibizumab, reflecting high retinal concentrations of the compound 15 days after the intervention. The porcine protein Ig lambda chain C region was downregulated following ranibizumab intervention. The downregulation of the porcine Ig lambda chain C region is likely to result from a restored blood–retinal barrier after anti-VEGF therapy [27,36].

While proteomics allows for the identification and quantification of a large set of proteins, the approach has several limitations. In particular, the quantification of low-abundance proteins is a significant challenge in proteomics. VEGF, a well known, low-abundance protein, was not identified in the proteomic analysis in our study. Several factors contribute to difficulties in detecting low-abundance proteins [10,11]. These include sample complexity, technical variation, and fragmentation efficiency [37,38]. Furthermore, the complexity of the retina, with its multilayered structure and multiple cell types, contributes to the complexity of the tissue, which can make it difficult to detect VEGF [25].

Conclusion

Our study identified downregulation of complement C3 in CRVO treated with ranibizumab. Ranibizumab intervention may potentially contribute to downregulation of the inflammatory response in CRVO by decreasing the retinal level of complement C3. Ranibizumab intervention was also associated with downregulation of the Ig lambda chain C region, which may reflect the reestablishment of the blood–retinal barrier with decreased leakage of plasma proteins. A high concentration of the ranibizumab component Ig kappa chain C region was detected, reflecting high levels of ranibizumab reached in the retina 15 days after injection.

Appendix 1. Supplementary Table 1.

Appendix 2. Supplementary Table 2.

Appendix 3. Supplementary Table 3.

Financial support

The study was funded by Fight for Sight Denmark, Helene og Viggo Bruuns Fond, the Svend Andersen Foundation, the Bagger-Sørensen Foundation, the Obel Family Foundation, the Herta Christensen Foundation, the North Denmark Region (2013–0076797), Overlægerådets Forskningsfond at Odense University Hospital, and Speciallæge Heinrich Kopps Legat. The mass spectrometers used for the present study were funded by A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til almene Formaal.

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