1. Levels of angiopoietin-like 4 (ANGPTL4), a protein involved in new blood vessel production (angiogenesis), were highly upregulated in various models of retinopathy and in the eyes of patients with proliferative diabetic retinopathy (PDR).
2. Treatment with an ANGPTL4-blocking antibody reduced the ability of ocular aqueous fluid from PDR patients to foster endothelial cell tubule growth, an in vitro measurement of PDR angiogenic potential.
Evidence Rating Level: 3 (Average)
Study Rundown: Nearly one-third of diabetic adults in the United States over 40 years of age have diabetic retinopathy (DR). The most severe stage of DR, PDR is characterized by formation of new, fragile blood vessels in the eye which can severely impact vision upon bursting. Recent research has suggested that therapy targeting vascular endothelial growth factor (VEGF), a molecule that promotes angiogenesis, can prevent progression of early-stage DR to PDR. However, treatment is not always successful and may cause serious side effects.
The authors of this study initially found that VEGF levels in aqueous fluid from PDR patients did not correlate with the fluid’s ability to promote endothelial cell tubule formation in vitro. Further, treatment with the anti-VEGF drug bevacizumab did not reduce tubule formation, indicating that other factors may be involved in PDR angiogenesis. In searching for other factors, the authors found that ANGPTL4 gene expression levels were equal to or greater than VEGF levels in both in vitro and in vivo models of PDR. ANGPTL4 levels were also highly upregulated in the aqueous fluid and vitreous of PDR patients as compared to controls, even when PDR patients had recently received anti-VEGF therapy. Treatment with an ANGPTL4- targeting antibody reduced the stimulation of tubule formation by aqueous fluid from PDR patients.
This work importantly identifies ANGPTL4 as an angiogenic factor which can serve as a novel therapeutic target in treating PDR, possibly in conjunction with anti-VEGF treatment. Future work should further elucidate the mechanisms of ANGPTL4’s involvement in PDR in order to enable the development of drugs targeting this pathway.
Relevant Reading: Microvascular modifications in diabetic retinopathy
In-Depth [in vitro and animal study]: Initial experiments showed that aqueous fluid from PDR patients significantly increased endothelial cell tubule formation as compared to fluid from control patients (p<0.001, n=4-15 per group) and diabetic patients without DR. Tubule formation was not correlated with fluid VEGF levels or affected by the addition of bevacizumab to the fluid.
In an in vitro model of PDR, retinopathy was triggered in retinal Müller cells by exposure to hypoxia. The in vivo PDR model used was the oxygen-induced retinopathy mouse, previously characterized to have neovascularization that promotes retinopathy. Both models have been shown to result in the stabilization of hypoxia-inducible factor 1α, which is a critical starting point in promoting PDR angiogenesis. Compared to 21 other potential cytokines and angiogenic factors studied, ANGPTL4 mRNA expression was notably upregulated in both Müller cells (p<0.0001 at 48 hours post-hypoxia) and mice (p<0.01 at 1 and 2 days post-hypoxia). The addition of interfering RNA (RNAi) against either VEGF or ANGPTL4 mRNA reduced the stimulation of tubule formation by hypoxic Müller cells by ~30% (p<0.01 for each), while combining both treatments resulted in a 50% reduction (p<0.05 vs. VEGF RNAi alone; p<0.01 vs. ANTPTL4 RNAi alone).
ANGPTL4 levels were increased in aqueous fluid and vitreous of PDR patients as compared to control patients (for vitreous, p<0.0001 and n=10 per group). Finally, treatment with an antibody that targeted ANGPTL4 reduced the stimulation of tubule formation by aqueous fluid from PDR patients (p<0.01 compared to untreated), even when using fluid from patients who had recently received anti-VEGF treatment (p<0.05 compared to untreated).
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