Lipidomic Analysis Reveals Drug-Induced Lipoxins in Glaucoma Treatment

Abstract

Synthetic prostaglandin analogues, such as latanoprost, are first-line treatments to reduce intraocular pressure (IOP) in the management of glaucoma, treating millions of patients daily. Glaucoma is a leading cause of blindness, characterized by progressive optic neuropathy, with elevated IOP being the sole modifiable risk factor. Despite this importance, the underlying latanoprost mechanism is still not well defined, being associated with both acute and long term activities, and ocular side effects. Prostaglandins are eicosanoid lipid mediators. Yet, there has not been a comprehensive assessment of small lipid mediators in glaucomatous eyes. Here we performed a lipidomic screen of aqueous humour sampled from glaucoma patients or healthy control eyes. The resulting signature was surprisingly focused on significantly elevated levels of arachidonic acid (AA) and the potent proresolving mediator, lipoxin A₄ (LXA₄) in glaucoma eyes. Subsequent experiments revealed that this response is due to latanoprost actions, rather than a consequence of elevated IOP. We demonstrated that increased LXA₄ inhibits pro-inflammatory cues and promotes TGF-β₃ mediated tissue remodeling in the anterior chamber. In concert, an autocrine prostaglandin circuit mediates rapid IOP-lowering. This work reveals parallel mechanisms underlying acute and long-term latanoprost activities during the treatment of glaucoma.

Keywords: arachidonic acid; glaucoma; intraocular pressure; latanoprost; lipoxin; prostaglandin.

Figure 1:. The arachidonic acid-lipoxin pathway is specifically elevated in glaucomatous aqueous humor.

(A) Representative OCT scans from a control patient showing healthy RNFL and optic nerve head in both eyes. (B) Representative OCT scans from a glaucomatous patient showing significant superior and inferior RNFL thinning in the right eye and superior RNFL thinning in the left eye. (C-F) Lipidomic analysis of mediators and metabolites from glaucomatous and healthy aqueous humor showed significantly elevated concentrations of (C) AA and (D) LXA₄. (E) In comparison13-HODE was detected at significantly lower levels in glaucomatous aqueous humor. (F) 12-HEPE levels were significantly elevated in the glaucoma group, though statistically driven by only four samples (p values are indicated, bars are SE). (G-H) Concentrations of additional analytes detected in human aqueous humor samples included (G) PGD2, (H) PGE2, (I) DHA, and (J) EPA. However, none of these differences reached statistical significance. (For all charts p values are indicated, bars are SE). (OD; right eye, OS; left eye, RNFL; retinal nerve fiber layer).

Figure 2:. The AA-lipoxin circuit is induced by latanoprost and timolol treatment in human trabecular meshwork cells.

(A) Graph representing the percentage of glaucoma patients taking topical glaucoma eye drops, including prostaglandin analogues, beta blockers, carbonic anhydrase inhibitors and alpha-2 adrenergic agonists. (B) Human trabecular meshwork cells were treated with latanoprost (prostaglandin analogue), timolol (beta blocker), dorzolamide (carbonic anhydrase inhibitor), brimonidine (alpha-2 adrenergic agonist), or vehicle for one hour before collecting RNA for qPCR and conditioned media for lipidomic analyses. (C) Quantification of qPCR results showed that treatment with latanoprost caused a significant, dose-dependent upregulation of ALOX5, ALOX15, PLA2G2A and PTGDS expression. (D) Lipidomic analyses of the culture media showed a significant increase in arachidonic acid and LXA₄ levels with increasing latanoprost treatment. In addition, EPA and DHA substrate levels were also significantly elevated by treatment compared to vehicle. (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns; not significant, bars are SE) (AA; arachidonic acid, ALOX5; arachidonate 5-lipoxygenase, ALOX15; arachidonate 15-lipoxygenase, DHA; docosahexaenoic acid, EPA; eicosapentaenoic acid, LXA₄; lipoxin A₄, PLA2G2A; phospholipase A₂ group IIA, PTGDS; prostaglandin D₂ synthase, PTGS2; prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2), RQ; relative quantification).

Figure 3:. The AA-lipoxin circuit is not induced by ocular hypertension alone.

(A) Gradual ocular hypertension (gOHT) was induced by a circumlimbal suture in six-week-old Long Evans rats and maintained for 8–10 weeks before eyes were collected for lipidomic analyses. (B) As expected, circumlimbal suturing induced a gradual increase in IOP, consistently exceeding 20 mmHg from weeks 3–5 post-suturing (*p<0.0001, bars are SE). (C) Lipidomic analyses detected AA levels that were not significantly altered in the OHT group compared to control. (D-F) Similarly, endogenous prostaglandins D₂ and E₂, and 6-keto-prostaglandin F_1α were detected, but were not significantly altered by ocular hypertension alone (bars are SE). (PGD₂, prostaglandin D₂; PGE₂, prostaglandin E₂).

Figure 4:. The LXA₄ pathway and cox pathways are induced by latanoprost treatment in vivo.

(A) Six-week-old Long Evans rats were administered topical latanoprost for 7 days, followed by the analyses of angle tissues. (B) Concentrations of AA and DHA were slightly reduced in latanoprost treated samples compared to vehicle (bars are composites of 8 samples). (C) Elevated levels of LXA₄ were detected, along with several pathway intermediates and products, including 12-HETE, 15-HETE, 17-HDHA, and 13-HODE (bars are composites of 8 samples). (C) Elevated cox pathway products were also detected, including PGE₂, PGD₂, PGF_2α, 6Keto-PGF1α, and TBX2 (bars are composites of 8 samples). (D) Levels of LTB4 were sharply reduced in latanoprost treated samples compared to vehicle (bars are composites of 8 samples).

Figure 5:. LXA₄ does not cause acute IOP-lowering, but inhibits proinflammatory cytokines and induces production of TGF-β3.

(A) Six-week-old Long Evans rats were treated with LXA₄ once daily for one week, and the eyes were collected for cytokine analyses of the angle tissues. (B) Daily IOP measurements indicate that LXA₄ did not cause significant IOP changes compared to vehicle treated controls (bars are SE). (C-F) At the end of the study angle tissue samples were subjected to a panel of 30 cytokines, with those showing significant difference presented here. There was a significant decrease in the levels of pro-inflammatory cytokines (C) IL-12, (D) MIP-1α and (E) TNF-α, and (F) a significant increase in TGF-β₃ levels (p values indicated, bars are SE). (IL-12p70; interleukin-12p70, MIP-1α; macrophage inflammatory protein-1 alpha, TGF-β₃, transforming growth factor-beta₃, TNF-α; tumor necrosis factor-alpha).

Figure 6:. Prostaglandin synthesis is required for latanoprost acute IOP-lowering activity.

(A) Six-week-old Long Evans rats were treated with the COX inhibitor, bromfenac, or vehicle for 48 hours prior to starting concomitant latanoprost treatment over the next seven days. Both treatments were administered once daily, with daily IOP monitoring. (B) Analyses of rat angle tissues following administration of bromfenac (B) or vehicle (V) showed strong inhibition of levels of the prostaglandins PGD₂, PGE₂, PGF_2α, and 6-keto-PGF_1α (bars are composites of 6 samples). (C) Bromfenac treatment alone had no effect on IOP, while latanoprost treatment caused a rapid and sustained IOP reduction. When administered together, bromfenac treatment attenuated the IOP-lowering activity of latanoprost (***p<0.001, *p<0.05 between latanoprost and latanoprost + bromfenac, bars are SE). (D) Comparison of the average IOP from days 2 to 7 demonstrated significant attenuation of the IOP-lowering effect of latanoprost by bromfenac (****p<0.0001, bars are SE). (COX; cyclooxygenase).

Figure 7:. Flowchart depicting a proposed parallel AA-dependent drug mechanism.

(A) The upregulation of PLA_2, 5-LOX and 15-LOX by latanoprost induces synthesis of LXA₄, resulting in tissue remodeling and inflammation resolution to exert sustained IOP lowering effects. (B) In concert, endogenous prostaglandins are generated via COX activity, resulting in acute IOP lowering. (PGD₂; prostaglandin D₂, PGE₂; prostaglandin E₂, PGF_2α; prostaglandin F_2α).