High Intraocular Concentration of Fibrinogen Regulates Retinal Function Via the ICAM-1 Pathway

Yasuyuki Sotani; Hisanori Imai; Hiroko Yamada; Akiko Miki; Sentaro Kusuhara; Makoto Nakamura

doi:10.1167/iovs.65.13.34

. 2024 Nov 15;65(13):34. doi: 10.1167/iovs.65.13.34

High Intraocular Concentration of Fibrinogen Regulates Retinal Function Via the ICAM-1 Pathway

Yasuyuki Sotani ¹, Hisanori Imai ^1,^2,^✉, Hiroko Yamada ¹, Akiko Miki ¹, Sentaro Kusuhara ¹, Makoto Nakamura ¹

PMCID: PMC11578147 PMID: 39546292

Abstract

Purpose

Diabetic retinopathy (DR) is a significant complication of diabetes mellitus that can lead to progressive visual impairment. This study aimed to elucidate the role of fibrinogen, a protein whose serum and intraocular concentrations are elevated in patients with diabetes and DR, in the pathogenesis of DR.

Methods

The changes in the protein levels of the neuronal marker tubulin-β3 (TUBB3) and retinal response induced by the intravitreal injections of 1× phosphate-buffered saline, 40 mg/mL of fibrinogen, and 40 mg/mL of fibrinogen in combination with anti–intracellular adhesion molecule-1 (ICAM-1) antibody in normal mice were observed using immunofluorescence, western blotting, and electroretinography.

Results

High concentrations of fibrinogen led to a decrease in the expression of TUBB3 in immunofluorescence and western blotting. The amplitudes of the positive scotopic threshold response and b-wave were notably reduced after the injection of fibrinogen, indicating potential damage to the retinal ganglion cells. The co-administration of anti–ICAM-1 antibody effectively mitigated these fibrinogen-induced changes, indicating that fibrinogen-induced damage is mediated via the ICAM-1 pathway.

Conclusions

The present study underscores the significance of elevated intraocular fibrinogen levels as a pathogenic factor in DR. Involvement of the fibrinogen/ICAM-1 pathway presents new avenues for therapeutic intervention, especially in patients with treatment-resistant conditions.

Keywords: fibrinogen, ICAM-1, diabetic macular edema, diabetic retinopathy

Diabetes mellitus (DM), characterized by the presence of sustained hyperglycemia due to reduced production or effectiveness of insulin in the body, results in various systemic complications.¹ Diabetic retinopathy (DR) is a major complication of DM that can lead to progressive visual impairment due to retinal ischemia and macular edema.² Thus, preventing the development of DR and providing appropriate treatment to patients with DM are crucial for maintaining visual function. Diabetic macular edema (DME) is characterized by the intraretinal accumulation of serum components due to increased permeability of the macular retinal vessels, which can lead to significant visual distortion.³ Various treatment modalities, such as intravitreal injections of anti–vascular endothelial growth factor (VEGF) drugs, retinal photocoagulation, subtenon or intravitreal injections of triamcinolone acetonide, and pars plana vitrectomy (PPV), have been used for the treatment of DME.⁴^–⁷ However, the effectiveness of these conventional treatments in improving visual acuity and resolving DME is limited. Resistance to anti-VEGF therapy has been reported in 18.9% to 25.6% of patients with DME,⁸ and persistent macular edema has been reported in >30% of patients after PPV.⁹ Thus, conventional treatments alone cannot completely cure DME in all cases, indicating the presence of unknown etiologies. In recent years, we have reported that fibrinogen accumulates in the cystoid spaces of cystoid macular edema (CME) in refractory DME and that the en bloc removal of this fibrinogen for the treatment of refractory CME secondary to several vitreoretinal diseases, including DR, can improve both DME and visual function.¹⁰^–¹³ Therefore, we hypothesized that the accumulation of fibrinogen into the retina could be a potential unknown pathogenic factor affecting DME, and research is progressing in this area.

Fibrinogen, the first clotting factor identified in serum, is a glycoprotein with a molecular weight of approximately 340 kDa and is primarily synthesized by the hepatocytes.¹⁴ Approximately 80% of fibrinogen is present in the serum, whereas the remaining 20% is present in tissues, including the eye ball. Serum fibrinogen is involved in wound healing as a substrate for factor XIII and as an acute-phase reactant during inflammation, in addition to its role in clotting and hemostasis. It has been reported that serum fibrinogen levels in patients with DM are significantly higher than those in patients without DM.¹⁵ Moreover, serum fibrinogen levels in patients with DM and DR are higher than in patients with DM but without DR.¹⁵^,¹⁶ Clinically, it has been reported that an increase in serum fibrinogen levels is strongly associated with the pathophysiology of diabetic macroangiopathies, such as ischemic heart disease.¹⁷^–¹⁹ In previous basic research, it was also confirmed that, in diabetic rabbits, the concentration of serum fibrinogen increased, accumulated significantly in the aortic vascular walls, and induced thrombus formation.²⁰ Furthermore, even in patients with diabetic microangiopathy, including diabetic nephropathy, fibrinogen deposits are frequently observed in the glomeruli, indicating that serum fibrinogen levels can be an indicator of the onset of this pathology.²¹ These findings suggest a close association between the overexpression of serum fibrinogen and the pathogenesis of DM and its related major systemic complications.

On the other hand, it has also been reported that the concentration of fibrinogen increases in extravascular tissues in DM. In intraocular tissue, intravitreal fibrinogen levels are increased in patients with DME compared to normal patients, indicating leakage of fibrinogen outside the retinal blood vessels.²²^,²³ Additionally, the fibrinogen deposits have also been observed within the retinas of patients with DR. Kimura et al.²² reported a negative correlation between fibrinogen levels in the vitreous fluid and postoperative visual acuity in patients with DME. As described above, the surgical removal of fibrinogen accumulated in DME has been reported to result in an improvement in visual function.¹⁰^,¹³ It has also been reported that, in central nervous tissue, fibrinogen that accumulates outside the blood vessels increases the activity of astrocytes and glial cells, leading to secondary neuronal damage and the development of inflammatory brain diseases through the production of inflammatory cytokines secondary to the astrocyte activation.²⁴ These results suggest that fibrinogen that accumulates outside the blood vessels can exert cell-damaging effects, indicating that high concentrations of intraretinal fibrinogen may be involved in the pathology of DR and DME. However, no previous study, to our knowledge, has examined the effect of high concentrations of fibrinogen that have extravasated into retinal tissue on retinal function, indicating a potential area for further research. Thus, this study aimed to investigate the involvement of high concentrations of fibrinogen in the pathology of DR and DME by first examining the effects of fibrinogen on various retinal cells through experiments conducted on normal mice.

Materials and Methods

Mice

All animal experiments were reviewed and approved by the Kobe University Animal Care and Use Committee (permission no. P201204). The study was performed in accordance with the Kobe University Animal Experimentation Regulations and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Wild-type 10-week-old mice with a C57BL/6J genetic background were used in this study. All mice were maintained under standard laboratory conditions (12/12-hour light/dark cycle and 20°–24°C, with food and water provided ad libitum). Anesthesia was induced via the intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) for all procedures.

Intravitreal Injection

Human fibrinogen (#FIB3; Enzyme Research Laboratories, South Bend, IN, USA) was dissolved in 1× phosphate-buffered saline (PBS) at a concentration of 120 mg/mL to prepare the drug, or it was dissolved in 1× PBS containing 40 mg/mL of the anti–intercellular adhesion molecule-1 (ICAM-1) antibody to attain the same concentration. The mice were anesthetized, and their heads were immobilized. A conjunctival incision was made using a spring shear to expose the sclera. A 32-gauge needle was used to puncture the anterior chamber and reduce the intraocular pressure. A Hamilton syringe with a 32-gauge needle was filled with the drug solution and inserted slightly posterior to the corneal ring into the vitreous cavity, and 2 µL of the drug was slowly injected. The volume of the mouse vitreous cavity is estimated to be 4 to 6 µL,²⁵ and intravitreal administration of 2 µL of the above drug would result in an intravitreal fibrinogen concentration of approximately 40 mg/mL. Previous reports have indicated that the fibrinogen concentration in mouse plasma is around 2 mg/mL.²⁶ Given that fibrinogen leakage into the vitreous cavity is expected to be minimal under normal conditions, the intravitreal fibrinogen concentration in this experiment is considered to be significantly elevated compared to normal. Hemostasis was confirmed before the administration of levofloxacin eye drops. All mice used in the experiments were maintained under the aforementioned conditions.

Immunofluorescence

The mice were killed, and the retinas were collected 1 week after the intravitreal administration of the three drug solutions. Retinal cryosections of 8-µm thickness were collected on glass slides and fixed with 4% paraformaldehyde (PFA) for 10 minutes. After blocking with 5% bovine serum albumin (BSA), the sections were incubated at 4°C overnight with an appropriate concentration of the following primary antibodies (Table): tubulin-β3 (TUBB3), a protein that serves as a marker for neurons in the mouse retina; glial fibrillary acidic protein (GFAP), a marker for activated Müller cells in the mouse retina whose expression increases when the Müller cells are activated; and ionized calcium-binding adapter molecule 1 (Iba1), a marker for microglia whose expression is increased in activated microglia. The sections were incubated with secondary antibodies (Invitrogen Donkey anti-Rabbit IgG [H+L] Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, #A-21207; Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 hour after rinsing three times with PBS with Tween 20 (PBS-T) for 10 minutes. Thereafter, the sections were washed three times with PBS-T, counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA), and mounted with coverslips. Fluorescent images were acquired and analyzed using a confocal laser scanning microscope (LSM 700; Carl Zeiss Microscopy, Oberkochen, Germany).

Table.

List of Antibodies Used for Immunostaining and Western Blotting

Antibody	Dilution and Purpose	Catalog no.	Company	Host
TUBB3 conjugate A488	1:200, IF	801203	BioLegend	Mouse
GFAP conjugate Cy3	1:200, IF	C9205	Sigma-Aldrich	Mouse
Iba1	1:200, IF	ab178846	Abcam	Rabbit
β-actin	1:40,000, WB	ab115777	Abcam	Rabbit
TUBB3	1:250,000, WB	66375-1	Proteintech	Mouse
GFAP	1:40,000, WB	23935-1-AP	Proteintech	Rabbit
Iba1	1:4000, WB	ab178846	Abcam	Rabbit
ERK1/2	1:2000, WB	9102	Cell Signaling Technology	Rabbit
p-ERK1/2	1:4000, WB	4370	Cell Signaling Technology	Rabbit
AP-1	1:400, WB	A5968	Sigma-Aldrich	Rabbit

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IF, immunofluorescence; WB, western blotting.

Western Blotting

The mice were killed, and the retinas were collected 1 week after intravitreal administration of the three drug solutions. The retinas were lysed in a hypotonic lysis buffer solution containing 10-mM HEPES-KOH (pH 7.9), 10-mM KCl, 1.5-mM MgCl₂, 1-mM dithiothreitol, 0.5-mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail and were then centrifuged at 10,000g at 4°C for 15 minutes. The supernatant was used as the cytoplasmic fraction. After the supernatants were removed, the pellets, including the nuclear fraction, were incubated with a nuclear lysis buffer containing 20-mM HEPES-KOH (pH 7.9), 400-mM NaCl, 1.5-mM MgCl₂, 0.2-mM EDTA, 1-mM dithiothreitol, 5% glycerol, and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and were incubated for 30 minutes on ice. The lysates were centrifuged at 13,000g at 4°C for 5 minutes. The supernatants were used as the nuclear fraction samples. Immunoblotting analysis of β-actin was performed to confirm the absence of contamination between the cytoplasmic and nuclear fractions. Retinal proteins (60 µg) were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; #XP08160BOX; Thermo Fisher Scientific) and transferred onto polyvinylidene difluoride membranes (#10600123; GE Healthcare Life Sciences, Buckinghamshire, UK). The membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature for 1 hour, followed by incubation at 4°C overnight with the primary antibody (Table) in TBST. The membranes were incubated with horseradish peroxidase–conjugated anti-rabbit immunoglobulin G (IgG; 1:10,000) or horseradish peroxidase–conjugated anti-mouse IgG (1:20,000) at room temperature for 1 hour after rinsing three times with PBS-T for 10 minutes. Chemiluminescence was detected using electrochemiluminescence (ECL) reagents (#RPN2232; GE Healthcare Life Sciences) after four 15-minute washes. The signals were quantified using β-actin expression levels as a reference and analyzed using the LAS-3000 Mini Digital Imaging System (Fujifilm, Tokyo, Japan). Six mice were used in each experiment.

Electroretinography Recording

One week after the intravitreal administration of the three drug solutions, electroretinography (ERG) was performed as described previously with some modifications. All animals were dark adapted overnight before the ERG recordings, and all procedures were performed under dim red light. The mice were anesthetized and positioned on a built-in heating pad that maintained the body temperature at 37°C. After the pupils were dilated with 2.5% phenylephrine and 1.0% tropicamide eye drops (Santen Pharmaceutical, Osaka, Japan), a contact lens electrode embedded with a gold wire was placed on the cornea as the active electrode (Mayo Corporation, Aichi, Japan), and a chloride silver plate was placed in the mouth as the reference electrode. ERG was performed using commercially available equipment equipped with a Ganzfeld bowl (Mayo Corporation). Scotopic recordings were obtained from dark-adapted animals under increasing light intensities. The responses were amplified 10,000 times and bandpass filtered from 0.3 to 500 Hz (PuREC PC-100; Mayo Corporation). Serially increasing luminescence intensities of –5.6, –5.1, –4.6, –4.3, –4.1, –3.1, –2.1, –1.1, –0.1, 0.9, and 1.9 log scotopic Troland were used to record scotopic threshold responses (STRs). The responses were amplified differentially and bandpass filtered at 0.125 to 50 Hz, and the responses from 80 repeated stimuli for each intensity were averaged. The responses were amplified differentially and bandpass filtered at 0.3 to 300 Hz, and the responses from 30 (20 for flicker) repeated stimuli for each intensity were averaged.

Statistical Analyses

Data are presented as mean ± standard error of the mean (SEM). All statistical analyses were performed using Microsoft Excel 2013 and MedCalc 19.0.3 (MedCalc Software, Ostend, Belgium). Statistical comparisons were performed using a one-way ANOVA with Bonferroni's test for post hoc analysis when three or more groups were compared. Unpaired t-tests were used when two groups were compared. Statistical significance was set at P < 0.05.

Results

Figure 1 presents the results of immunostaining of retinal cryosections. A reduction in TUBB3 expression was observed in the group that received fibrinogen (Fibrinogen group) compared with that in the group that received PBS (Control group). The reduction in TUBB3 expression due to high-concentration fibrinogen was mitigated in the group that received fibrinogen in combination with the anti–ICAM-1 antibody (Fibrinogen+anti–ICAM-1 antibody group) (Fig. 1A). The expression of GFAP in the retinal Müller cells was increased in the Fibrinogen group. Furthermore, the high-concentration fibrinogen–induced increase in GFAP expression was reduced by co-administration of the anti–ICAM-1 antibody (Fig. 1B). An increase in the Iba1-positive cells and enhanced expression of Iba1 were observed in the Fibrinogen group. Moreover, co-administration of the anti–ICAM-1 antibody mitigated the high-concentration fibrinogen–induced increase in Iba1-positive cells and enhanced expression of Iba1 (Fig. 1C).

The results of the western blotting are presented in Figures 2 through 5. A significant reduction in the retinal expression of TUBB3 was observed in the Fibrinogen group compared with that in the Control group. The fibrinogen-induced reduction in TUBB3 expression was mitigated in the Fibrinogen+anti–ICAM-1 antibody group (Figs. 2A, 2B). The expression of GFAP was increased in the Fibrinogen group. However, the high-concentration fibrinogen–induced increase in GFAP expression was reduced in the Fibrinogen+anti–ICAM-1 antibody group (Figs. 2A, 2C). The expression of Iba1 was increased in the Fibrinogen group, and the high-concentration fibrinogen–induced increase in Iba1 expression was reduced in the Fibrinogen+anti–ICAM-1 antibody group (Figs. 3A, 3B). The expression levels of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 were also examined by western blotting. Regarding ERK1, significant phosphorylation was induced in the fibrinogen group, and the phosphorylation of ERK1 observed in the fibrinogen group was reduced in the Fibrinogen+anti–ICAM-1 antibody group (Figs. 4A, 4B). As for ERK2, the ratio of phosphorylated ERK2 significantly increased in the fibrinogen group, but no significant reduction in phosphorylation was observed in the Fibrinogen+anti–ICAM-1 group (Figs. 4A, 4C). The expression of activator protein 1 (AP-1) was increased in the Fibrinogen group, and the high-concentration fibrinogen–induced increase in AP-1 expression was reduced in the Fibrinogen+anti–ICAM-1 antibody group (Fig. 5).

Figure 2. — Western blot analyses of TUBB3 and GFAP levels in mouse retina treated with intravitreal injections of 1× PBS (Control group), 40 mg/mL of fibrinogen (Fibrinogen group), or 40 mg/mL of fibrinogen in combination with anti–ICAM-1 antibody (Fibrinogen+anti–ICAM-1 group). (A) Representative images of western blot analyses of TUBB3, GFAP, and β-actin. (B) Summary of the western blot results for TUBB3 and GFAP (*P < 0.05, **P < 0.01 for all; n = 6). A significant reduction in the retinal expression of TUBB3 was observed in the Fibrinogen group compared with that in the Control group. The fibrinogen-induced reduction in TUBB3 expression was mitigated in the Fibrinogen+anti–ICAM-1 antibody group. The expression of GFAP was increased in the Fibrinogen group. However, the high-concentration fibrinogen–induced increase in GFAP expression was reduced in the Fibrinogen+anti–ICAM-1 antibody group.

Figure 5. — Western blot analyses of AP-1 levels in mouse retina treated with intravitreal injections of 1× PBS (Control group), 40 mg/mL of fibrinogen (Fibrinogen group), or 40 mg/mL of fibrinogen in combination with ICAM-1 antibody (Fibrinogen+anti–ICAM-1 group). (A) Representative images of western blot analyses of AP-1 and β-actin. (B) Summary of the western blot results for AP-1 (*P < 0.05, **P < 0.01 for all; n = 6). The expression of AP-1 was increased in the Fibrinogen group. However, the high-concentration fibrinogen–induced increase in AP-1 expression was reduced in the Fibrinogen+anti–ICAM-1 antibody group.

Figure 3. — Western blot analyses of Iba1 levels in mouse retina treated with intravitreal injections of 1× PBS (Control group), 40 mg/mL of fibrinogen (Fibrinogen group), or 40 mg/mL of fibrinogen in combination with ICAM-1 antibody (Fibrinogen+anti–ICAM-1 group). (A) Representative images of western blot analyses of Iba1 and β-actin. (B) Summary of the western blot results for Iba1 (*P < 0.05, **P < 0.01 for all; n = 6). The expression of Iba1 was increased in the Fibrinogen group. However, the high-concentration fibrinogen–induced increase in Iba1 expression was reduced in the Fibrinogen+anti–ICAM-1 antibody group.

Figure 4. — Western blot analyses of ERK1/2 levels in mouse retina treated with intravitreal injections of 1× PBS (Control group), 40 mg/mL of fibrinogen (Fibrinogen group), or 40 mg/mL of fibrinogen in combination with ICAM-1 antibody (Fibrinogen+anti–ICAM-1 group). (A) Representative images of western blot analyses of ERK1/2, p-ERK1/2, and β-actin. (B) Summary of the western blot results for ERK1/2 (*P < 0.05, **P < 0.01 for all; n = 6). The ratio of p-ERK1 expression to the total amount of ERK1 expression increased in the Fibrinogen group, and the increase in the p-ERK1 expression ratio induced by high-concentration fibrinogen was significantly suppressed in the Fibrinogen+anti–ICAM-1 antibody group. As for ERK2, although the ratio of p-ERK2 expression to the total amount of ERK2 expression increased in the Fibrinogen group, the suppression of the increase in the p-ERK2 expression ratio induced by high-concentration fibrinogen was not significant in the Fibrinogen+anti–ICAM-1 antibody group.

Figure 6 presents the ERG results. The amplitudes of the positive STR (pSTR) and b-waves were notably reduced in the Fibrinogen group compared with those in the Control group. However, in the Fibrinogen+ICAM-1 group, the reduced amplitudes of both waves were improved compared to the Fibrinogen group.

Discussion

In the present experiment, it was found through immunostaining and western blotting that high-concentration fibrinogen administration resulted in increased activation of Müller cells and microglia and decreased activation of retinal ganglion cells. Furthermore, ERG indicated a subsequent decline in retinal function. These results suggest that high-concentration fibrinogen administration has a detrimental effect on the retina. To date, there have been no reports examining the impact of fibrinogen on retinal function, making these findings highly novel.

There are two potential mechanisms by which fibrinogen exerts its harmful effects on the retina. The first possibility is that intracellular signaling mediated by ICAM-1 receptors may be involved in the ultimate retinal damage. ICAM-1 is a type of receptor expressed on the cell surface of vascular endothelial cells, as well as some lymphocytes and monocytes.²⁷ In the retina, ICAM-1 is known to be expressed on vascular endothelial cells and glial cells such as Müller cells and microglia, but it is minimally expressed on retinal ganglion cells.²⁷ ICAM-1 functions as a cell adhesion molecule during inflammation by binding with various substances and mediating inflammatory responses via intracellular signaling.²⁸ Fibrinogen is one of the ligands for ICAM-1 and is involved in the expression of various inflammatory cytokines through an intracellular signaling pathway mediated by ICAM-1 receptors.²⁹^,³⁰ It has been reported that, in Müller cells and vascular endothelial cells, when ICAM-1 binds to fibrinogen, signaling through ERK1 phosphorylation activates the transcription factor AP-1, leading to an increase in inflammatory cytokines such as IL-1β, IL-6, IL-8, IL-12, TGF-β, and ICAM-1.³¹^,³² In this study, increased phosphorylation of ERK1 and activation in AP-1 were observed in the Fibrinogen group, suggesting that the increase in inflammatory cytokines may be induced via the aforementioned signaling pathway. Furthermore, the addition of anti–ICAM-1 antibody partially mitigated these changes, supporting the notion that the ICAM-1/fibrinogen complex is involved in ERK1-mediated signal transduction. In the central nervous system, it has been reported that fibrinogen binds to ICAM-1 expressed on astrocytes, inducing inflammation through the upregulation of inflammatory cytokines via the fibrinogen/ICAM-1 signaling pathway.³³ Additionally, the activation of astrocytes by fibrinogen binding has been reported to cause inflammatory neurodegenerative diseases.²⁴^,³¹ Moreover, it is also reported that high concentrations of fibrinogen activate astrocytes, leading to neurodegeneration via the activation of tyrosine receptor kinase B.²⁴ Other reports have shown that fibrinogen interacts with ICAM-1 and cellular prion protein (PrP^C)³⁴ on the surface of the astrocytes in cultured mouse astrocytes, and this interaction induces the overexpression of inflammatory cytokines, resulting in neuronal cell damage. In this way, it is suggested that the activation of glial cells and the subsequent inflammation mediated by the fibrinogen/ICAM-1 signaling pathway may also lead to damage to surrounding nerve cells. Although the initiation of glial cell activation differs, prior studies in the retina have shown that TNF-α, IL-1β, and IL-6 are overproduced when the retinal microglia in rats are activated under hypoxic conditions, leading to induction of apoptosis in the ganglion cells.³⁵ It is also reported that TNF-α is produced from activated Müller cells in the eyes of glaucomatous rats, causing damage to the ganglion cells that leads to cell death.³⁶ In addition, the activation of microglia results in a significant increase in the mRNA and protein levels of inflammatory factors, such as TNF-α and IL-6, in the eyes of glaucomatous mice.³⁷ These inflammatory factors upregulate the mRNA expression of inflammatory factors in the Müller cells via positive feedback. Thus, it is speculated that the activated microglia and macroglia may damage nerve cells and induce neurodegeneration even in the retinal tissue via the production of aforementioned inflammatory cytokines. We believe that the retinal ganglion cell damage observed in this study may also be a result of the inflammation caused by the activation of Müller cells and microglia. Further investigation into the specifics of this process is warranted.

The second possibility is that the high concentration of fibrinogen caused direct cytotoxic effects due to high osmotic pressure. For example, indocyanine green (ICG), used during vitrectomy to stain the internal limiting membrane, has been reported to cause retinal toxicity by changing the osmotic pressure of the vitreous cavity.³⁸ The retinal tissue damage observed in this study due to high concentrations of fibrinogen cannot be ruled out as having occurred through a similar mechanism of retinal toxicity caused by osmotic pressure changes induced by ICG injection. However, experiments using rabbit eyes have reported that injecting hyperosmotic solutions into the vitreous cavity results in functional and morphological changes in the retina when the osmotic pressure exceeds 500 mOsm.³⁹ In this study, the osmotic pressure of the solution prepared by dissolving fibrinogen in PBS was approximately 300 mOsm, so the influence of osmotic pressure on the results is considered minimal.

In this study, it was demonstrated that both the changes in the expression of various retinal proteins and the decline in retinal function induced by high concentrations of fibrinogen were almost fully restored to normal levels by co-administration of the anti–ICAM-1 antibody. Previous studies have shown that the activation of astrocytes induced by high concentrations of fibrinogen is mitigated by the administration of an anti–ICAM-1 antibody.²⁴ These results suggest that most of the changes induced by high concentrations of fibrinogen may be regulated by the fibrinogen/ICAM-1 signaling pathway. Furthermore, this study implies that treatments using ICAM-1–neutralizing antibodies could be promising for conditions involving high concentrations of fibrinogen, such as DME. Furthermore, molecules such as ERK1 and AP-1, which are involved in the downstream fibrinogen/ICAM-1 signaling pathway, may also emerge as more practical drug discovery targets for similar conditions in the future. The findings of this study are highly significant, as they clarify the pathogenesis of several vitreoretinal disorders, including DR, and have the potential to offer new treatment options for DR and DME. Further research is needed to explore these possibilities.

Interestingly, in addition to the current analytical results, a decrease in the amplitudes of not only the pSTR and b-wave but also the a-wave and oscillatory potential (OP)-wave in the ERG was observed (data not shown). This finding suggests that high concentrations of fibrinogen may induce cellular damage throughout the neurovascular unit, including photoreceptors, amacrine cells, vascular endothelial cells, Müller cells, and ganglion cells. However, further investigations are necessary to explore these results in greater detail.

A limitation of the present study is that it investigated the retinal toxicity of fibrinogen using normal mice. In humans, the fibrinogen concentration in the vitreous cavity similarly increases in various vitreoretinal diseases, including DR. Future research using animal models of these vitreoretinal diseases may provide more clinically relevant results.

In conclusion, the intravitreal injection of high concentrations of fibrinogen induces retinal damage, which is suggested to be regulated via the fibrinogen/ICAM-1 signaling pathway. Modulation of the fibrinogen/ICAM-1 signaling pathway may serve as a potential therapeutic target for various vitreoretinal diseases, including DR, where intraocular fibrinogen levels are elevated.

Acknowledgments

Supported by grant-in-aid for Japanese scientific research (21K09719).

Disclosure: Y. Sotani, None; H. Imai, None; H. Yamada, None; A. Miki, None; S. Kusuhara, None; M. Nakamura, None

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18. Kannel WB, D'Agostino RB, Wilson PW, Belanger AJ, Gagnon DR. Diabetes, fibrinogen, and risk of cardiovascular disease: the Framingham experience. Am Heart J . 1990; 120(3): 672–676. [DOI] [PubMed] [Google Scholar]
19. Vinik A, Flemmer M.. Diabetes and macrovascular disease. J Diabetes Complications . 2002; 16(3): 235–245. [DOI] [PubMed] [Google Scholar]
20. Witmer MR, Hadcock SJ, Peltier SL, Winocour PD, Richardson M, Hatton MW.. Altered levels of antithrombin III and fibrinogen in the aortic wall of the alloxan-induced diabetic rabbit: evidence of a prothrombotic state. J Lab Clin Med . 1992; 119: 221–230. [PubMed] [Google Scholar]
21. Zhang J, Wang Y, Zhang R, et al.. Serum fibrinogen predicts diabetic ESRD in patients with type 2 diabetes mellitus. Diabetes Res Clin Pract . 2018; 141: 1–9. [DOI] [PubMed] [Google Scholar]
22. Kimura K, Orita T, Kobayashi Y, Matsuyama S, Fujimoto K, Yamauchi K.. Concentration of acute phase factors in vitreous fluid in diabetic macular edema. Jpn J Ophthalmol . 2017; 61(6): 479–483. [DOI] [PubMed] [Google Scholar]
23. Murata T, Ishibashi T, Inomata H.. Immunohistochemical detection of extravasated fibrinogen (fibrin) in human diabetic retina. Graefes Arch Clin Exp Ophthalmol . 1992; 230(5): 428–431. [DOI] [PubMed] [Google Scholar]
24. Clark VD, Layson A, Charkviani M, Muradashvili N, Lominadze D.. Hyperfibrinogenemia-mediated astrocyte activation. Brain Res . 2018; 1699: 158–165. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Kaplan HJ, Chiang CW, Chen J, Song SK.. Vitreous volume of the mouse measured by quantitative high-resolution MRI. Invest Ophthalmol Vis Sci . 2010; 51: 4414. [Google Scholar]
26. Emeis JJ, Jirouskova M, Muchitsch EM, Shet AS, Smyth SS, Johnson GJ.. A guide to murine coagulation factor structure, function, assays, and genetic alterations. J Thromb Haemost . 2007; 5(4): 670–679. [DOI] [PubMed] [Google Scholar]
27. Bui TM, Wiesolek HL, Sumagin R.. ICAM-1: a master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis. J Leukoc Biol. 2020; 108(3): 787–799. [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Hubbard AK, Rothlein R.. Intercellular adhesion molecule-1 (ICAM-1) expression and cell signaling cascades. Free Radic Biol Med . 2000; 28(9): 1379–1386. [DOI] [PubMed] [Google Scholar]
29. Davalos D, Akassoglou K.. Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol . 2012; 34(1): 43–62. [DOI] [PubMed] [Google Scholar]
30. Languino LR, Plescia J, Duperray A, et al.. Fibrinogen mediates leukocyte adhesion to vascular endothelium through an ICAM-1-dependent pathway. Cell . 1993; 73(7): 1423–1434. [DOI] [PubMed] [Google Scholar]
31. Lebedeva T, Dustin ML, Sykulev Y.. ICAM-1 co-stimulates target cells to facilitate antigen presentation. Curr Opin Immunol . 2005; 17(3): 251–258. [DOI] [PubMed] [Google Scholar]
32. Gu D, Beltran WA, Li Z, Acland GM, Aguirre GD.. Clinical light exposure, photoreceptor degeneration, and AP-1 activation: a cell death or cell survival signal in the rhodopsin mutant retina? Invest Ophthalmol Vis Sci . 2007; 48(11): 4907–4918. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Etienne-Manneville S, Chaverot N, Strosberg AD, Couraud PO. ICAM-1-coupled signaling pathways in astrocytes converge to cyclic AMP response element-binding protein phosphorylation and TNF-alpha secretion. J Immunol . 1999; 163(2): 668–674. [PubMed] [Google Scholar]
34. Sulimai N, Brown J, Lominadze D.. Fibrinogen interaction with astrocyte ICAM-1 and PrPC results in the generation of ROS and neuronal death. Int J Mol Sci . 2021; 22(5): 2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Li Q, Cheng Y, Zhang S, Sun X, Wu J.. TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway. J Neuroinflammation . 2021; 18(1): 271. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Bai Y, Shi Z, Zhuo Y, et al.. In glaucoma the upregulated truncated TrkC.T1 receptor isoform in glia causes increased TNF-α production, leading to retinal ganglion cell death. Invest Ophthalmol Vis Sci . 2010; 51(12): 6639–6651. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Hu X, Zhao GL, Xu MX, et al.. Interplay between Müller cells and microglia aggravates retinal inflammatory response in experimental glaucoma. J Neuroinflammation . 2021; 18(1): 303. [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Rodrigues EB, Meyer CH, Mennel S, Farah ME.. Mechanisms of intravitreal toxicity of indocyanine green dye: implications for chromovitrectomy. Retina . 2007; 27(7): 958–970. [DOI] [PubMed] [Google Scholar]
39. Marmor MF. Retinal detachment from hyperosmotic intravitreal injection. Invest Ophthalmol Vis Sci . 1979; 18(12): 1237–1244. [PubMed] [Google Scholar]

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[bib34] 34. Sulimai N, Brown J, Lominadze D.. Fibrinogen interaction with astrocyte ICAM-1 and PrPC results in the generation of ROS and neuronal death. Int J Mol Sci . 2021; 22(5): 2391. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib36] 36. Bai Y, Shi Z, Zhuo Y, et al.. In glaucoma the upregulated truncated TrkC.T1 receptor isoform in glia causes increased TNF-α production, leading to retinal ganglion cell death. Invest Ophthalmol Vis Sci . 2010; 51(12): 6639–6651. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib37] 37. Hu X, Zhao GL, Xu MX, et al.. Interplay between Müller cells and microglia aggravates retinal inflammatory response in experimental glaucoma. J Neuroinflammation . 2021; 18(1): 303. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib38] 38. Rodrigues EB, Meyer CH, Mennel S, Farah ME.. Mechanisms of intravitreal toxicity of indocyanine green dye: implications for chromovitrectomy. Retina . 2007; 27(7): 958–970. [DOI] [PubMed] [Google Scholar]

[bib39] 39. Marmor MF. Retinal detachment from hyperosmotic intravitreal injection. Invest Ophthalmol Vis Sci . 1979; 18(12): 1237–1244. [PubMed] [Google Scholar]

PERMALINK

High Intraocular Concentration of Fibrinogen Regulates Retinal Function Via the ICAM-1 Pathway

Yasuyuki Sotani

Hisanori Imai

Hiroko Yamada

Akiko Miki

Sentaro Kusuhara

Makoto Nakamura