Results of the present study show that in the aqueous humor of POAG patients, cytokines involved in vascular endothelial function, including sVEGFR-1, vWF, and angiopoietin-2, as well as in inflammatory reactions, including TNF-α, are significantly greater compared with control patients. Therefore, there is a potential association between POAG pathogenesis and vascular endothelial dysfunction as well as inflammation.
Since vWF is synthesized by endothelial cells and released when endothelial cell are injured, it is a good marker of endothelial dysfunction [13,14]. In ocular diseases, vWF is highly expressed in vitreous and serum of diabetes retinopathy . In glaucoma patients, vWF levels are shown to be high in plasma (by ELISA)  and iris vasculature (by immunohistochemistry)  compared to control groups. However, considering that endothelial cells are distributed over the whole body, the plasma level could be easily influenced by systemic conditions. Additionally, vWF is a kind of secreted cytokines; therefore, it is hard to reflect its true level using immunohistochemistry in real time. By investigating vWF levels in the aqueous humor, our study showed high levels in POAG patients, which is in accordance with previous results and supports the hypothesis that endothelium dysfunction is involved in POAG pathogenesis.
VEGF is a vasoactive cytokine produced by endothelial cells, epithelial cells, pericytes, astrocytes, Mϋller cells and tumor cells . Higher levels of VEGF are thought to be a response to active angiogenesis in some diseases and could be observed in physiological repair processes [19,20]. Animal model studies as well as clinical patient research show that VEGF levels increased in certain intraocular diseases with active intraocular neovascularization including retinopathy of prematurity, central retinal vein occlusion, branch retinal vein occlusion, proliferative diabetic retinopathy, and neovascular glaucoma [7,20-22]. A previous study showed the VEGF levels in the plasma were significantly elevated both in POAG and normal tension glaucoma patients when compared to cataract patients . Our present array study reveals no significant difference in VEGF levels (VEGF-A, VEGF-C and VEGF-D) between POAG aqueous humor and control patients, which is consistent with a previous study , though the result is in opposition to the conclusions from other studies . Using a sandwich ELISA kit, another study demonstrated a statistically significant increase in aqueous levels of VEGF in both POAG and primary angle closure glaucoma patients when compared to cataract patients . The difference may be influenced by the assessment methods used in our research and in the previous study, namely using an array-based multiplex sandwich ELISA system and not the traditional ELISA method.
Splicing the mRNA that can be translated into VEGF receptor-1 produces sVEGFR-1, a soluble form of VEGFR-1. VEGFR-1 is expressed mainly on the surface of vascular endothelial cells and is one of the main VEGF receptors; it regulates and combines with VEGF to recruit monocytes and macrophages and influences vascular permeability involved in angiogenesis and inflammation. Unlike VEGFR-1, soluble receptors act as endogenous antagonists that bind to VEGF-A to exert an anti-hemangiogenic effect and impair the vasodilatory response . In some ocular diseases related to ischemia, sVEGFR-1 was reported to increased, such as in cases of diabetic retinopathy  and retinal vein occlusion [7,22]. Until now, changes in sVEGFR-1 levels in glaucomatous eyes have not been reported. Only one previous study reported that in the plasma of POAG patients, sVEGFR-1 expression levels were lower than that in the control group , which was not in accordance with our present result. There may be several reasons for this discrepancy. First, the test methods were different: the previous study used a traditional ELISA while our group utilized an antibody-based cytokine array; different principles, sensitivity and analytical methods may result in variance. Furthermore, quite different from aqueous humor, plasma is exposed to systemic circumstances and do not reflect the ocular condition with the same accuracy. Moreover, the blood ocular barrier is a special physiological structure that can intensify this variation.
Angiopoietin-2 is a crucial regulator of angiogenesis and vascular homeostasis, exerting an antagonistic effect for angiopoietin-Tie2 signaling . In patients with age-related macular degeneration, angiopoietin-2 is upregulated and correlates with central macular thickness . Moreover, angiopoietin-2 is reported to be essential for the formation and integrity of Schlemm’s canal and in turn stabilizing the IOP: a mice experiment showed that the loss of angiopoietin-Tie2 signaling severely impaired Schlemm’s canal integrity, resulting in elevated IOP and retinal neuron damage . In our present results, the expression of angiopoietin-2 was remarkably higher in POAG patients, also suggesting that there is an impairment of angiopoietin-Tie2 signaling damaging Schlemn’s canal which may underly POAG pathogenesis.
Additionally, TNF-α as a cytokine causing cell death and involving in inflammation, has proven to be increased in glaucomatous aqueous humor [8,28] and was also elevated in POAG patients in our study. Other inflammatory cytokines in this study, including CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, IL-1b, IL-6, IL-8, IL-12 showed no difference between control and glaucoma groups, in accordance with previous study [8,29].
This study has a few limitations. First, it is a case-control study with a relatively small number of participants. Consequently, our study cannot determine causality. In addition, POAG patients had accepted treatments before surgery, which is another difference from control group patients. Nonetheless, patients with age-related cataracts may have other unaccounted differences from healthy people. We implemented our inclusion and exclusion criteria to select the most appropriate non-age-related cataract patients for our control group.
In conclusion, the present study profiled vascular and inflammatory cytokines in the aqueous humor of POAG patients and showed the associations between POAG and cytokine changes. Furthermore, we first found the elevated concentrations of vWF, sVEGFR-1, and angiopoietin-2 in glaucomatous aqueous humor. The elevated cytokines are consistent with the hypothesis that vascular and inflammatory factors are related to POAG pathogenesis. Further research is needed to clarify the role of cytokines and to understand the altered micro-environment in the anterior chamber.