
==== Front
Invest Ophthalmol Vis Sci
Invest Ophthalmol Vis Sci
IOVS
Investigative Ophthalmology & Visual Science
0146-0404
1552-5783
The Association for Research in Vision and Ophthalmology

39287590
10.1167/iovs.65.11.25
IOVS-24-40179
Lens
Lens
Prospective Cytokine Profiling in Aqueous Humor Reveals a Proinflammatory Microenvironment in Highly Dense Nuclear Cataracts
Inflammatory Cytokines in Dense Nuclear Cataracts
Latz Catharina 1
Licht Annika 1
Mirshahi Alireza 1
Latz Eicke 2
Zimmer Kai 3
1 Dardenne Eye Clinic, Bonn, Germany
2 Deutsches Rheumaforschungszentrum, DRFZ, Berlin, Germany
3 Department of Internal Medicine V, Hematology and Oncology, Comprehensive Cancer Center Innsbruck (CCCI), Medical University of Innsbruck, Innsbruck, Austria
* Correspondence: Catharina Latz, Dardenne Eye Clinic, Friedrich-Ebert-Str. 23-25, Bonn D-53177, Germany; c_latz@hotmail.com.
17 9 2024
9 2024
65 11 2505 8 2024
11 4 2024
Copyright 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Purpose

To investigate if the cytokine profile in the aqueous humor (AH) of cataract patients varies according to cataract type and severity.

Methods

This prospective study included 397 eyes of 397 patients (median age, 76 years; range, 30–94 years) who underwent standard small-incision phacoemulsification surgery. Cataracts were graded using the LOCS III system: mild (≤3), moderate (3.5–5), and severe (≥5). Biometric measurements from the IOL master 700 (Zeiss, Oberkochen, Germany) were used to differentiate between thick (>4.5 mm) and thin lenses. Information about age, gender, and self-reported diseases was obtained from patient records. Eleven different proteins were measured in AH using a multiplex cytokine assay (AYOXXA Biosystems, Cologne, Germany), including IL- 6, IL-8, angiopoietin 2, C-reactive protein (CRP), vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor BB, placental growth factor, CXCL12, CXCL13, and CXCL10. Statistical analysis was performed using R and included nonparametrical testing, linear regression, and k-means clustering.

Results

Higher nuclear LOCS grades correlated with increased levels of CCL2 (360 vs. 387 vs. 517 pg/mL, P < 0.001), VEGF-A (270 vs. 292 vs. 390 pg/mL, P = 0.012), IL-8 (3.1 vs. 4.2 vs. 5.7 pg/mL, P = 0.018), and CXCL10 (52 vs. 61 vs. 90 pg/mL, P = 0.003). No associations were observed for cortical and subcapsular cataracts. Thicker lenses were associated with significantly increased levels of CRP, CXCL10, CXCL12, IL-6, IL-8, and VEGF-A.

Conclusions

The cytokine profile of AH varies based on cataract grading and lens thickness. In highly dense nuclear cataracts, CCL2, VEGF-A, IL-8, and CXCL10 were elevated.

aqueous humor
LOCS
cataract
cytokine
proinflammatory
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pmcCataract surgery is one of the most successful and standardized surgeries of our time. In the past decades, surgical technique and indication have further evolved. Clear lens extraction for refractive purposes brings younger and less dense cataracts to the operating room, while advances in technology have enabled phacoemulsification surgery on highly dense cataracts. Except for developing countries, extracapsular cataract extraction is virtually extinguished. Highly dense cataracts yield a longer postoperative recovery time. So far, this has been explained by a more traumatic surgical burden.

Aqueous humor (AH) supplies nutrients and removes waste from the lens.1 Three mechanisms are involved in AH formation: diffusion, ultrafiltration, and active secretion. Diffusion and ultrafiltration are responsible for the accumulation of plasma ultrafitrate in the stroma, behind tight junctions of the nonpigmented epithelium, from which the posterior chamber AH is derived. Active secretion is thought to be the major contributor to aqueous formation, responsible for approximately 80% to 90% of the total AH formation.1 The protein content of AH has both quantitative and qualitative differences compared to plasma. Most AH proteins are intrinsic glycoproteins of the vitreous, which are secretory products of the inner epithelial layer of the ciliary body.2 Because of the necessity for clear visual structures, normal AH contains a hundredfold less protein than plasma.3 This is established through the blood–aqueous barrier.4,5

Similarly, Mitrovic et al.6 observed that mediators in AH were generally not associated with the corresponding values in plasma, suggesting that these proteins were produced intraocularly while the blood–AH barrier was intact.

Connective tissue growth factor tends to increase in the AH as the severity of age-related cataracts increases,7 while ascorbic acid concentration and AH total antioxidant capacity correlate negatively with cataract severity.8 In addition, the cytokine profile of AH changes with ocular pathology such as diabetes, uveitis, or branch retinal vein occlusion.9–14

We hypothesized that the cytokine profile in AH might become proinflammatory as the severity of cataracts increases. While previous studies have investigated the cytokine profile in AH in relation to ocular pathology, to the best of our knowledge, there has not been a correlation to cataract severity.9–14

The LUNARIS Human 11-Plex Ophthalmology Kit (AYOXXA Biosystems, Cologne, Germany) offers multiplex technology for low-volume samples as small as 2 µL. It allows accurate and precise quantitation of 11 cytokines15: C-reactive protein (CRP) is a commonly used marker for both acute and chronic inflammation. CCL2, CXCL10, CXCL12, and CXCL13 are involved in the recruitment of various immune cells. CCL2 is found in active inflammation such as endophthalmitis, while CXCL10 is associated with Th1 responses and CXCL13 in B-cell recruitment. IL-6 is involved in the acute phase response of inflammation, while IL-8 attracts neutrophil migration. Both IL-6 and IL-8 are elevated in pseudoexfoliation syndrome. Angiopoetin (ANG) 2, vascular endothelial growth factor (VEGF) A, placental growth factor (PLGF), and platelet-derived growth factor (PDGF) BB are involved in angiogenesis, vascular stability or maturation, and ocular pathology such as diabetic retinopathy or age-related macular disease.16

This study measured the AH cytokine profile in various cataract grades and correlated it to patient characteristics.

Methods

Patients

Aqueous humor samples were obtained from 397 patients (median age, 76 years; range, 30–94 years). All patients underwent ophthalmologic examinations, including measurements of best-corrected visual acuity, IOP measured by noncontact tonometry, and slit-lamp and funduscopic examinations under pupillary dilation. Cataract severity was graded at the slit lamp using the LOCS III system. LOCS III is a standardized system for grading and comparing cataract severity and type, and it has 95% tolerance limits for within-grader and between-grader reproducibility.16

For statistical analysis, we organized cataract grades as mild (≤3), moderate (3.5–5), and severe (≥5). We measured axial length, lens thickness, and anterior chamber depth using the IOL master 700 (Zeiss, Oberkochen, Germany). In addition, we included information from patient records on comorbidities such as glaucoma, pseudoexfoliation, type 2 diabetes, arterial hypertension, hypercholesteremia, and use of eye drops. The demographic data of the patients, including age, sex, cataract grades, optical biometry, and comorbidities, are summarized in Table 1. All patients provided written informed consent for AH collection in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of the North Rhine Medical Chamber.

Table 1. Baseline Characteristics of the Cohort Stratified by Sex

Characteristic	Men, n = 169	Women, n = 228	P Value*	q Value†	
Age, median (IQR), y	76 (70–82)	76 (70–80)	0.16	0.53	
Nuclear color, n (%)			0.77	>0.99	
 ≤3	40 (24)	61 (27)			
 3–<5	111 (66)	145 (64)			
 ≥5	18 (11)	22 (9.6)			
Nuclear color, n (%)					
 2	3 (1.8)	4 (1.8)			
 2.5	2 (1.2)	1 (0.4)			
 3	35 (21)	56 (25)			
 3.5	25 (15)	49 (21)			
 4	64 (38)	68 (30)			
 4.5	22 (13)	28 (12)			
 5	11 (6.5)	19 (8.3)			
 5.5	5 (3.0)	3 (1.3)			
 6	2 (1.2)	0 (0)			
Nuclear opalescence, n (%)			>0.99	>0.99	
 ≤3	48 (28)	66 (29)			
 3–<5	108 (64)	145 (64)			
 ≥5	13 (7.7)	17 (7.5)			
Posterior cataract, n (%)			0.48	0.96	
 ≤3	138 (86)	188 (86)			
 3–<5	10 (6.2)	18 (8.3)			
 ≥5	13 (8.1)	12 (5.5)			
Cortical cataract, n (%)			0.087	0.52	
 ≤3	131 (80)	159 (71)			
 3–<5	24 (15)	53 (24)			
 ≥5	8 (4.9)	11 (4.9)			
Side, n (%)			0.56	0.96	
 Left	88 (52)	112 (49)			
 Right	81 (48)	116 (51)			
Glaucoma, n (%)	18 (11)	17 (7.5)	0.27	0.64	
PEX, n (%)	10 (5.9)	16 (7.0)	0.66	>0.99	
T2D, n (%)	49 (29)	35 (15)	<0.001	0.012	
Arterial hypertension, n (%)	112 (66)	136 (60)	0.18	0.53	
Hypercholesterolemia, n (%)	8 (4.7)	11 (4.8)	0.97	>0.99	
Eye drops, n (%)	38 (22)	53 (23)	0.86	>0.99	
IQR, interquartile range; PEX, pseudoexfoliation syndrome.

* Wilcoxon rank sum test; Pearson's χ2 test.

† False discovery rate correction for multiple testing.

Table 2. Cytokine Levels and Statistics for the LOCS III Grading Category Nuclear Opalescence

	Nuclear Opalescence			
Cytokine	≤3, n = 114, Median (IQR)	3–5, n = 253, Median (IQR)	≥5, n = 30, Median (IQR)	P Value*	q Value†	
ANG-2	105 (42–378)	147 (37–352)	171 (83–529)	0.27	0.40	
CCL2	360 (293–454)	387 (312–498)	517 (394–658)	<0.001	0.004	
CRP	1,309 (726–2,994)	1,864 (777–4,269)	2,320 (1,227–5,000)	0.086	0.19	
CXCL10	52 (30–78)	61 (36–92)	90 (65–112)	0.003	0.014	
CXCL12	271 (166–501)	316 (153–472)	332 (181–740)	0.73	0.80	
CXCL13	19 (7–60)	23 (6–55)	27 (10–81)	0.46	0.57	
IL-6	4 (2–9)	5 (2–13)	7 (2–18)	0.29	0.40	
IL-8	3.1 (2.1–6.7)	4.2 (2.3–7.3)	5.7 (3.5–10.4)	0.018	0.049	
PDGF-BB	6 (2–16)	6 (2–17)	12 (5–22)	0.12	0.21	
PLGF	17 (5–54)	17 (3–51)	24 (5–72)	0.80	0.80	
VEGF-A	270 (191–404)	292 (223–405)	390 (283–510)	0.012	0.044	
* Kruskal–Wallis rank-sum test.

† False discovery rate correction for multiple testing

Aqueous Humor Collection

Patients received a peribulbar block (4 mL bupivacaine 0.75%) with 2 mL mepivacaine 2% and 75 IE hyaluronidase (ESTEVE Pharmaceuticals GmbH, Berlin, Germany) in the preoperative area and dilating eye drops (phenylephrine hydrochloride 5%, URSAPHARM Arzneimittel GmbH, Saarbrücken, Germany; tropicamide 0.5%, Pharma Stulln GmbH, Stulln, Germany). Patients who were taking warfarin with a high international normalized ratio received topical anesthesia (proparacaine hydrochlorid 0.5%, Ursapharm Arzneimittel GmbH, 1×; bupivacaine hydrochlorid 0.75%, Puren Pharma GmbH & Co. KG, München, Germany). Nonsteroidal anti-inflammatory drug eye drops were not given routinely. Eyes were disinfected with Betadine 5%. We obtained AH samples (100–200 µL) at the beginning of cataract surgery through a limbal paracentesis using a 1-mL syringe with a 27-gauge Sauter cannula avoiding blood and intraocular tissue contamination. After retrieving the AH, the anterior chamber was reformed with balanced salt solution, and the cataract surgery was performed. All samples were snap-frozen on dry ice and stored at –80°C until analyses were performed. Prior to analysis, samples were centrifuged. The cell-free supernatant was diluted in the assay buffer (AYOXXA Biosystems) 1:2.

We measured levels of IL-6, IL-8, CXCL10, CXCL12, CXCL13, CRP, PDGF-BB, VEGF-A, PLGF, ANG-2, and CCL2 in AH using a bead-based multiplex assay (AYOXXA Biosystems) according to the manufacturer's instructions measuring fluorescence on the Lunaris plate reader (AYOXXA Biosystems).

Statistical Analysis

Statistical analysis was performed using R (V.4.0.1; R Project for Statistical Computing, Vienna, Austria). For statistical analysis, we organized cataract grades as mild (≤3), moderate (3.5–<5), and severe (≥5). Nonparametric Wilcoxon rank-sum test for pairwise comparisons of two groups and Kruskal–Wallis rank-sum test for comparison of more than two groups were used. A two-sided P value <0.05 was considered statistically significant. If appropriate, P values were corrected for multiple testing using the Benjamini–Hochberg method and were reported as q values.17 For k-means clustering of the cohort by cytokine profile, cytokine data were z-score normalized, and the optimal number of clusters was determined as k = 2 by the sum of squared errors using the R package factoextra (V1.0.7).18 Simple and multiple linear regression was performed using the R package stats (V4.2.1).

Results

Cohort

Sixty percent of study participants were women (n = 228). Median age was 75 years (interquartile range, 70–80). When assessed by LOCS III criteria, median nuclear opalescence and color grade were LOCS 4, while median cortical cataract grading was 2 and median posterior cataract grading was 1 (Table 1). Regarding baseline characteristics, we identified rates of self-reported type 2 diabetes to be significantly higher in men (Table 1).

LOCS Classification

LOCS category gradings were highly intercorrelated, with the highest correlation for nuclear opalescence and nuclear color (R = 0.86) and cortical cataract and posterior cataract gradings (R = 0.45, Supplementary Table S1).

The distribution of LOCS gradings in the patient cohort regarding cataract type (nuclear opalescence, cortical or posterior subcapsular) is shown in Figure 1A.

Figure 1. Association of cytokines to LOCS III grading. (A) Histograms displaying the distribution of the LOCS III grading categories in our cohort. (B) Boxplot showing the significantly dysregulated cytokines and their respective levels for the LOCS III category nuclear opalescence. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. (C) Correlation matrix displaying the level of positive (shades of blue) or negative (shades of red) correlations for the indicated parameters. Size of the circle indicates higher levels of correlation.

Cytokines

We identified CCL2 (q = 0.04), CXCL 10 (q = 0.014), IL-8 (q = 0.049), and VEGF-A (q = 0.044) levels to be positively correlated with nuclear opalescence grading as well as significantly elevated in pairwise comparisons (Kruskal–Wallis rank-sum test, P < 0.001 for CCL2, P = 0.003 for CXCL10, P = 0.018 for IL-8, and P = 0.012 for VEGF-A) (Fig. 1B, Table 2, Supplementary Table S3). For cortical and posterior cataract, we did not observe any differentially regulated cytokines with higher gradings (Supplementary Table S2).

Association to Demographic Characteristics

Regarding sex, men had significantly higher levels of CRP (2271 vs. 1469 pg/mL, P = 0.008, q = 0.018), CXCL10 (69 vs. 56 pg/mL, P = 0.005, q = 0.004), and CXCL12 (346 vs. 274 pg/mL, P = 0.004, q = 0.018) compared to women (Fig. 2A, Supplementary Table S4).

Figure 2. Association of aqueous humor cytokine levels to baseline characteristics. (A) Boxplot showing the significantly dysregulated cytokines and their respective levels stratified by sex. (B) Boxplot showing the significantly dysregulated cytokines and their respective levels stratified by self-reported T2D. (C) Boxplot showing the significantly dysregulated cytokines and their respective levels stratified by the presence of arterial hypertension. (D) Boxplot showing the significantly dysregulated cytokines and their respective levels stratified by the presence of glaucoma. (E) Boxplot showing the significantly dysregulated cytokines and their respective levels stratified by lens thickness with a cutoff of 4.5 mm. (F) Boxplot showing the significantly dysregulated cytokines and their respective levels stratified by cardiovascular risk factors. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Patients with self-reported type 2 diabetes (T2D) showed higher levels of CRP (2273 vs. 1759 pg/mL, P = 0.005), CXCL10 (77 vs. 58 pg/mL, P < 0.001), CXCL12 (346 vs. 301 pg/mL, P = 0.009), IL-8 (4.5 vs. 3.6 pg/mL, P = 0.029), and VEGF-A (316 vs. 283 pg/mL, P = 0.01) (Fig. 2B, Supplementary Table S4). After controlling these values for repeated testing, none of them reached statistical significance.

Patients with hypertension presented with higher levels of CRP (1286 vs. 2031 pg/mL, P < 0.001, q = 0.003), CXCL10 (49 vs. 66 pg/mL, P < 0.001, q = 0.004), and VEGF-A (277 vs. 307 pg/mL, P = 0.006, q = 0.022) (Fig. 2C). Further, patients with glaucoma had higher levels of CXCL10 (77 vs. 58 pg/mL, P = 0.002, q = 0.024) (Fig. 2D, Supplementary Table S4).

We stratified the cohort regarding cardiovascular risk factors by selecting patients with self-reported hypertension, T2D, cholesterol-lowering medications, or inhibitors of thrombocyte aggregation. This group of patients showed increased AH levels of CRP, VEGF-A, and CXCL10 (Fig. 2F).

In correlation analysis, age was positively correlated to lens thickness (Spearman R = 0.26, P < 0.001). In a simple linear regression model, age explained only 8.4% of the variance in lens thickness (adjusted R² = 0.0844, F(1, 394) = 37.39, P < 0.001) (Table 3). Also, age showed a positive correlation to several cytokines (Fig. 1C), and we evaluated its explanatory value in simple linear regression for significantly correlated cytokines, with VEGF-A having the highest adjusted R² of 0.0381 (F(1, 395) = 16.7, P < 0.001). This indicates that age as a single factor only mildly influences cytokine levels in the anterior chamber.

Table 3. Statistics of Simple and Multiple Linear Regression Analysis to Predict Lens Thickness

Dependent Variable	Explanatory Variable(s)	F	df	P Value	R2 Adjusted	
Lens Thickness	Age	37.390	1,394	<0.001	0.0844	
	CCL2	3.926	1,394	0.048	0.0074	
	ANG2	1.338	1,394	0.24	0.0009	
	CRP	16.790	1,394	<0.001	0.0384	
	CXCL10	1.463	1,394	0.22	0.0012	
	CXCL12	4.390	1,394	0.036	0.0085	
	CXCL13	1.221	1,394	0.27	0.0006	
	IL6	2.063	1,394	0.15	0.0027	
	IL8	5.054	1,394	0.021	0.0102	
	PDGF-BB	2.924	1,394	0.088	0.0048	
	PLGF	0.857	1,394	0.35	−0.0004	
	VEGFA	5.630	1,394	0.018	0.0116	
	Multiple linear regression of all	4.980	12,383	<0.001	0.1079	
Bold indicate statistical significance.

To evaluate the influence of lens-derived factors on the cytokine profile, patients were stratified according to the lens thickness as measured by the IOL Master: lenses thicker than 4.5 mm had significantly elevated cytokine levels of CRP, CXCL10, CXCL12, IL-6, IL-8, and VEGF-A (Fig. 2E, Table 3). Adding lens thickness to age in multiple linear regression cytokine led to a mild increase in the predictive value of the respective models (Table 4).

Table 4. Statistics of Simple and Multiple Linear Regression Analysis to Predict Cytokine Values by Age and or Lens Thickness

Dependent Variable	Explanatory Variable(s)	F	df	P Value	R2 Adjusted	
CRP	Age	5.605	1395	0.018	0.0115	
	LT	16.79	1394	<0.001	0.0383	
	Age + LT	9.172	2393	<0.001	0.0397	
CCL2	Age	10.14	1395	<0.001	0.0225	
	LT	3.926	1394	0.048	0.0073	
	Age + LT	5.672	2393	0.004	0.0231	
VEGF-A	Age	16.7	1395	<0.001	0.0381	
	LT	5.63	1394	0.018	0.0116	
	Age + LT	9.118	2393	<0.001	0.0395	
CXCL10	Age	9.464	1395	<0.001	0.0209	
	LT	1.463	1394	0.23	0.0012	
	Age + LT	4.751	2393	0.009	0.0186	
IL-6	Age	11.63	1395	<0.001	0.0261	
	LT	2.063	1394	0.15	0.0027	
	Age + LT	5.898	2393	0.003	0.0242	
IL-8	Age	13.47	1395	<0.001	0.0305	
	LT	5.054	1394	0.025	0.0102	
	Age + LT	7.492	2393	<0.001	0.0367	
LT, lens thickness.

Bold indicate statistical significance.

No differences in cytokine levels were observed in cytokine levels for pseudo-exfoliation, hypercholesterinemia, or use of eye drops.

To explore the data set in a more unbiased approach, we used k-means cluster analysis to identify subgroups of individuals with similar cytokine patterns. With an a priori determined optimal k = 2, n = 336 individuals were assigned to cluster 1, and n = 60 individuals were assigned to cluster 2 (Fig. 3A). Pairwise comparison of cytokine levels between clusters revealed significantly higher levels in all measured cytokines (all P < 0.001, Figs. 3B, 3C). Analysis of baseline characteristics to identify epidemiologic factors that contribute to the observed differences revealed a modest but significantly elevated age in cluster 2 (median 78 vs. 76 years, P = 0.012) and higher rates of self-reported T2D (33% vs. 20%, P = 0.013). No differences in LOCS grading categories were observed (Fig. 3B).

Figure 3. K-means cluster analysis. (A) Visual representation of k-means cluster analysis dividing the cohort into two cohorts. Each dot represents one individual, and lines connect dots to the cluster centroid. (B) Characteristics of cohort stratified by clusters as identified by k-means clustering. (C) Boxplots of cytokines stratified by clusters. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Discussion

In this study, we analyzed AH coming from a large real-world cohort of n = 397 eyes of n = 397 patients and showed that hyper-mature cataracts are associated with higher cytokine levels in AH. Importantly, our study cohort represents a typical cohort of patients undergoing cataract surgery in the developed world with a median age of 75 years and slightly higher percentage of women than men. Only visually significant nuclear cataracts underwent surgery, represented by a median LOCS III grading of 4.0 in terms of nuclear color and nuclear opalescence.

We found that higher dense nuclear cataracts showed statistically significant higher levels of the proinflammatory proteins CCL2, CXCL10, IL-8, and the proangiogenic VEGF-A. The other measured cytokines showed at least trends in higher levels in more advanced cataracts. This suggests higher inflammatory tonus in the direct surroundings of the lens probably reflects lens-induced inflammation. As sampling was performed upfront phacoemulsification directly after incision, we are confident that the measured cytokine levels reflect the status quo of inflammation in AH before surgery. Under the assumption that the lens itself causes the inflammation, our data suggest that in hyper-mature nuclear cataracts, either the leakage or the autoantigenicity of lens proteins such as crystallin increases, possibly activating an inflammatory signaling cascade. Retained lens material or nuclear fragments after cataract surgery were shown to induce inflammation, which may result in uveitis.19 In support of this, it has been shown that levels of lens proteins are higher in eyes with cataract and that the solubility of crystallins decreases with age.20 Rosenbaum et al.21 demonstrated chemotactic properties in gamma crystallin lens proteins or substances that coeluted with these low molecular weight proteins. All the cytokines that were elevated in our study were low molecular weight proteins ranging from 8.4 to 13 kDa, with VEGF-A being the biggest with 45 kDA. Also, lens-induced uveitis is associated with advanced cataracts22 by leaking of lens proteins through the semipermeable lens capsule into the anterior chamber. Correlation of crystallin levels, as a surrogate for lens protein leakage, as well as exact subtyping of lens proteins in AH would have supported this hypothesis but was out of the scope of this project. One could also speculate that the measured cytokines in the AH reflect the systemic inflammatory burden in these patients.

Unfortunately, no paired sampling of peripheral blood and AH samples was performed. However, in a small published analysis comparing the levels of VEGF-A, FasL, and IL-10 between serum and AH, no significant correlation could be identified supporting the hypothesis of lens-induced inflammation.6

To have a surrogate for systemic inflammatory burden, we stratified patients based on their self-reported cardiovascular risk profile, as it is well accepted that cardiovascular diseases promote systemic inflammatory burden. We found that aqueous CRP levels, as well as CXCL10 and VEGF-A levels, were significantly higher compared to patients with no reported risk factors. However, this study was not a priori designed to accurately determine the cardiovascular risk profile, and thus only self-reported T2D or self-reported hypertension, hypercholesteremia, or use of antiplatelet drugs could be used for stratification, making us very cautious in interpreting these analyses. Still, a large-scale study on how subtle systemic inflammation influences inflammation in immune-privileged sites such as the eye would be interesting.

To identify patterns of cytokine dysregulation and clinical phenotypes in this large cohort, we performed k-means clustering to stratify patients based on cytokine expression. Cluster 2 with n = 60 individuals showed significant upregulation across all measured cytokines. Analysis of baseline characteristics showed significant differences in age and rates of self-reported T2D. Interestingly, no differences in rates between LOCS grading categories or lens metrics were observed between the two clusters. This suggests that other factors, which were not depicted in our data set, contribute to cytokine expression in cataract patients. As stated earlier, deep clinical and inflammatory phenotyping could help to better understand these mechanisms.

Huang et al.23 obtained in a similar study AH cytokines from cataract patients who served as a control and patients shortly after acute primary angle closure (APAC). They found significantly elevated levels of VEGF and IL-8 in the APAC eyes.24 This sparked our interest in the relationship between VEGF-A and risk factors for acute angle closure, such as short axial length and increased lens thickness, both of which were available from biometric measurements. Indeed, we were able to demonstrate a mild positive correlation of VEGF-A and lens thickness paired with a mild negative correlation of VEGF-A and axial length. In our opinion, this demonstrates the complexity of cytokine analysis in vivo: different factors such as lens morphology, systemic diseases such as T2D, and sex play a role in addition to yet unknown factors.

A limitation of this study is the small number of patients with highly dense nuclear cataracts in comparison to mild and moderate subgroups. Moreover, we did not follow up on patients after surgery for practical reasons, so we cannot conclude about the influence or correlation of baseline inflammation on postsurgery recovery time or complications. We cannot explain the origin of the studied cytokines, nor do we have serum samples of our patients. In this regard, there is no evidence in the literature that increased serum levels implicate increased AH levels. To truly understand serum and AH interaction, a much larger study would be needed.

Regardless, it is worth knowing that surgery on highly dense cataracts is conducted in a proinflammatory AH microenvironment and might warrant further investigation of an anti-inflammatory treatment strategy. Further comprehensive studies using standardized surgical procedures and follow-up would allow for correlation of elevated cytokine levels with postoperative recovery and complications.

Conclusions

In summary, we could demonstrate that with increasing density of nuclear cataract, there is a significant increase in proinflammatory cytokines such as CCL2, CXCL10, IL-8, and VEGF-A. The change in cytokine profile causes a proinflammatory burden. Additional factors such as systemic disease, age, gender, and yet unknown factors also have a significant influence on the AH cytokine profile. Better understanding of these mechanisms could help tailor appropriate pre- and postoperative anti-inflammatory treatment.

Supplementary Material

Supplement 1

Acknowledgments

AYOXXA Biosystems (Cologne, Germany) collaborated with EL and provided testing kits and the plate reader at no cost. The company filed for bankruptcy in 2022.

Disclosure: C. Latz, None; A. Licht, None; A. Mirshahi, None; E. Latz, None; K. Zimmer, None
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