PEER REVIEWED

Cytokines in RVO: Exploring New Horizons

A scientific overview and possible treatment paradigm

ADITYA MODI, MBBS, DNB, FVRS • P. NEHA SUDHAKAR, MBBS, MS • NARESH KUMAR YADAV, MBBS, DOMS, FMRF, FRCS (GLAS)

Retinal vein occlusion is the most common retinal vascular disease after diabetic retinopathy.¹ According to population-based studies, the incidence ranges from 0.5% to 1.8% and the prevalence from 0.7% to 1.6% in middle-aged and elderly adults.²

Pooled data from population studies have shown a lower prevalence of central RVO (Figure 1) than branch RVO, with an estimated 16.4 million adults affected by RVO, 2.5 million of which are affected by CRVO and 13.9 million by BRVO.³

Figure 1. A 60-year-old male patient with CRVO in the left eye (A), who previously received three injections of IVB, showed initial response (B) but later demonstrated recurrence (C) and worsening after a third dose of IVB (D) Subsequently, cytokine levels were analyzed (VEGF, IL-6, IL-8 levels of 29.94, 1.31, 82.12 pg/mL, respectively); correspondingly, IVTA was administered with complete resolution of ME and no recurrenceatn two consecutive monthly follow-ups. CFT – Central foveal thickness, IVB – Intravitreal bevacizumab, IVTA – Intravitreal triamcinolone acetonide.

RVO is of multifactorial causation owing to a complex interplay between local and systemic factors in which vascular and inflammatory mediators play a very crucial role.⁴

Macular edema (ME), macular nonperfusion, retinal neovascularization, vitreous or intraretinal hemorrhage, tractional retinal detachment, and a combination of these conditions are responsible for vision loss in eyes with RVO, of which ME is the cause, is of prime importance because it can affect the quality of life of patients if not treated promptly and precisely.⁵

CYTOKINES IN RVO PATHOGENESIS

The pathogenesis of ME is a perplexing enigma, with the key role being played by cytokines, which are important to the functioning of endothelial cells and leukocytes, which in turn accentuate their secretions.

However, long-term exposure of endothelial cells to proinflammatory cytokines results in leukocyte extravasation into the perivascular space, promoting interactions with vascular endothelial cells by means of adhesion molecules, which induce breakdown of the blood-retinal barrier and subsequent increases in vascular permeability and fluid leakage into the retina.⁶ Interestingly, a particular cytokine can function in different ways depending on its cellular context and microenvironment.

Drastic upregulation of the cytokines that enhance retinal vascular permeability and fluid leakage is correlated significantly with the severity of ME in RVO.⁷ Of all inflammatory mediators, VEGF, interleukin (IL)-6, and IL-8 are the primary molecules involved in the pathogenesis of ME in RVO.^7-9

VEGF is a multifunctional cytokine produced in response to ischemia by RPE cells, pericytes, and Müller cells. By nature, it is a double-edged sword, with a central role in angiogenesis in normal retina¹⁰ and neuroprotective effects in the central nervous system¹¹ and conversely a proinflammatory nature in diseased retina by permitting leukocyte infiltration, increasing vasopermeability by disrupting the blood-retina barrier, and acting as a chemoattractant for endothelial cell precursors.¹²

IL-6 is the main stimulator of most acute-phase proteins, and it plays a significant role in facilitating the transition from the acute to chronic phase of inflammation, directly increasing capillary permeability by rearranging actin filaments in gap junctions between adjacent cells and upregulating VEGF, thereby linking the inflammatory process with angiogenesis.¹³ IL-8 is a chemokine produced by endothelial and glial cells that attracts and activates neutrophils and T lymphocytes, with both angiogenic and vasopermeability effects.¹⁴

The complex chain of events among VEGF, cytokines, chemokines, angiotensin II, prostaglandins, matrix metalloproteinases, interleukins, selectins, adhesion molecules, and inflammatory cells has yet to be fully defined.^15-16

EVIDENCE-BASED TREATMENT STRATEGY

The management of RVO centers around two main objectives: modifying the risk factors and managing sight-threatening complications, such as ME. New treatment modalities and combination therapies continue to emerge with positive results. According to randomized, controlled trials, the standard treatment for many years for ME was macular grid laser photocoagulation in BRVO¹⁷ and observation in CRVO.¹⁸

The multifactorial pathophysiology of ME in RVO is further supported by the multifaceted treatment approach that has evolved in the form of intravitreal therapy. The rationale for using steroids to treat ME lies in their diverse actions, including reducing the activity of inflammatory cells that release cytokines, stabilizing cell membranes and tight junctions, acting upstream of pigment epithelium-derived factor expression, and inhibiting the expression of the VEGF gene and the metabolic pathway of VEGF.¹⁹ The SCORE study demonstrated significant improvement in VA with intravitreal triamcinolone acetonide in BRVO,²⁰ compared to observation in CRVO.²¹

The GENEVA trials were two phase 3 trials that tested the dexamethasone implants (in the 0.35-mg and 0.7-mg dosages) vs sham injections in patients with BRVO and CRVO. There was a statistically significant improvement in VA in the dexamethasone implant group in both BRVO and CRVO.²²

Anti-VEGF drugs, such as bevacizumab (Avastin, Genentech, South San Francisco, CA) or ranibizumab (Lucentis, Genentech), have emerged as mainstays of treatment, revolutionizing the outcomes significantly in patients with ME secondary to RVO. In both the BRAVO²³ and CRUISE²⁴ trials, ranibizumab groups had significantly higher average gains in VA and significantly lower mean foveal thickness, compared to the sham injection group.

Another intravitreal agent is aflibercept (Eylea, Regeneron, Tarrytown, NY), which acts as a VEGF trap and inhibits VEGF along with placental growth factor. The VIBRANT²⁵ and COPERNICUS²⁶ trials demonstrated significant gains in VA, along with a reduction in ME in eyes receiving aflibercept compared to sham groups in both BRVO and CRVO.

A NEW PARADIGM

The risk-benefit ratio is best for intraocular injections of anti-VEGF agents, the first-line therapy for most patients, while intraocular steroids are likely to play adjunctive roles.²⁷

Few patients fail to respond to treatment with a particular pharmacotherapeutic agent, suggesting the role of different inflammatory pathways, in turn making the choice of intravitreal drug to be administered an arduous task and creating a therapeutic dilemma in the mind of treating specialist.

Hence, we introduce a whole new concept of a tailor-made or “bespoke” approach to therapy (Figure 2), which constitutes determining the level of responsibility of primary inflammatory mediators by obtaining a sample of ocular fluids, such as tears, aqueous, or vitreous humor, and modulation of therapy in the form of anti-VEGF agents or steroids, depending upon the predominant inflammatory mediator in question.

Figure 2. Treatment algorithm: “Bespoke” approach to treatment of macular edema in retina vein occlusions. Concept of customization. IL-6 – Interleukin 6, IL-8 – Interleukin 8, CFT – Central foveal thickness.

It has already been documented that no correlation exists between cytokine levels in the aqueous humor and plasma,²⁸ while favorable correlations have been found in aqueous and vitreous, providing evidence for intraocular sources and clinical benefits in indicating the severity of ME secondary to RVO.^7,8

However, surgical harvesting of vitreous fluid is associated with the risk of vitreous hemorrhage, retinal tears, and retinal detachment. In contrast, obtaining aqueous humor samples is a far easier and less risky procedure.

With this knowledge, we propose our approach of customization of intravitreal therapy, depending on the aqueous levels of primary cytokines (VEGF, IL-6, IL-8) in ME secondary to RVO. We call it a “three-step bespoke approach.”

Step 1 involves a comprehensive baseline examination, including best-corrected LogMAR VA, using charts, slit-lamp biomicroscopy with a 90 D lens and indirect ophthalmoscopy, widefield fundus fluorescein angiography to identify peripheral capillary nonperfusion areas and neovascularization, and spectral-domain OCT for grading the severity of edema by measuring central foveal and subretinal fluid thickness if present.

If ME is the predominant pathology, and there is a primary need to administer intravitreal therapy, we proceed to Step 2, where we collect and process aqueous from the affected eye for cytokine level determination. Normative data of primary cytokine levels were derived by analyzing a control group as follows: 32.55 pg/mL of VEGF; 16.40 pg/mL for IL-6; and 6.69 pg/mL for IL-8.

Under aseptic precautions, aqueous humor is withdrawn with a 30-gauge needle attached to a 1-mL tuberculin syringe after insertion of a speculum. Undiluted aqueous samples (50 to 100 µL) are obtained at presentation and on follow-up before an intravitreal injection of steroid or anti-VEGF. Aqueous cytokine concentrations are determined using BD Cytometric Bead Array (BD Biosciences, San Jose, CA), which consists of capture beads, detection reagent, and their respective standards.

The assay is performed and analyzed according to the manufacturer’s instructions. Briefly, 50 µL of capture beads are mixed with 50 µL of the standards or test sample and incubated for one hour in the dark at room temperature. After the addition of detection beads, the mixture is further incubated for two hours in the dark at room temperature.

The mixture is then washed with 1 mL of wash buffer (300 revolutions per minute for five minutes). The pellet is then resuspended in 300 µL of wash buffer. The flow cytometer (BD FACSCalibur, BD Biosciences) is calibrated with setup beads, and the samples are analyzed using BD Cell Quest Pro Software (version 6.0; BD Biosciences). Individual analyte concentrations are indicated by their fluorescence intensities and are computed using the standard reference curves with the help of BD FCAP Array Software (version 3; BD Biosciences). Once we have the cytokine analysis in our hands, we proceed to third and final step of our therapy.

Step 3 involves the delivery of targeted therapy depending on the level of cytokines. Customization involves classifying patients into four groups based on the levels of primary cytokines: a “VEGF” group, in which VEGF levels are high and the patient receives anti-VEGF therapy; an “interleukin” group, in which IL-6 and IL-8 are high and the patient receives steroid injection; and a “mixed” group, in which patients have either low levels of all three cytokines and receive steroids, or all three cytokine levels are high, and the patient receives anti-VEGF.

The rationale behind this decision making in the “mixed” group with low levels is speculation about the predominant involvement of other inflammatory cytokines, such as monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor α (TNF-α), and intercellular adhesion molecule 1 (ICAM-1), which respond well to steroids.²⁹

In the category of patients with elevated cytokine levels, anti-VEGF drugs are administered because VEGF is the predominant vasomediator,¹² and in terms of risk-benefit ratio, anti-VEGF drugs are more effective than steroids.

Follow-up examination includes monitoring of central foveal thickness on SD-OCT and collecting aqueous samples for cytokine analysis every month to modulate therapy accordingly. We cease treating cases with central foveal thickness <250 µm and normal cytokine levels on two consecutive monthly follow-ups.

Treatment-naïve, recurrent, and refractory cases are approached in the similar fashion. Fundus fluorescein angiography is repeated every three months and OCT every month to monitor the disease on the whole. Laser photocoagulation or surgical intervention is advised when indicated.

CONCLUSION

The future direction of tailor-made treatment modalities for venous occlusions offers varied advantages, such as quicker resolution and fewer injections, thereby reducing economic burden, reducing the incidence of tachyphylaxis and thromboembolic events, and providing a whole new insight into the treatment of RVO. Although it has not been tested in a clinical trial, we believe that our approach has merit. RP

REFERENCES