Endogenous LXA4 Circuits Are Determinants of Pathological Angiogenesis in Response to Chronic Injury

Inflammation and angiogenesis are intimately linked, and their dysregulation leads to pathological angiogenesis in human diseases. 15-lipoxygenase (15-LOX) and lipoxin A₄ receptors (ALX) constitute a LXA₄ circuit that is a key feature of inflammatory resolution. LXA₄ analogs have been shown to regulate vascular endothelial growth factor (VEGF)-A-induced angiogenic response in vitro. 15-LOX and ALX are highly expressed in the avascular and immune-privileged cornea. However, the role of this endogenous LXA₄ circuit in pathological neovascularization has not been determined. We report that suture-induced chronic injury in the cornea triggered polymorphonuclear leukocytes (PMN) infiltration, pathological neovascularization, and up-regulation of mediators of inflammatory angiogenesis, namely VEGF-A and the VEGF-3 receptor (FLT4). Up-regulation of the VEGF circuit and neovascularization correlated with selective changes in both 15-LOX (Alox15) and ALX (Fpr-rs2) expression and a temporally defined increase in basal 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key and obligatory enzymes in the formation of LXA₄, respectively, led to exacerbated inflammatory neovascularization coincident with increased VEGF-A and FLT4 expression. Direct topical treatment with LXA₄, but not its metabolic precursor 15-hydroxyeicosatetraenoic acid, reduced expression of VEGF-A and FLT4 and inflammatory angiogenesis and rescued 15-LOX knockout mice from exacerbated angiogenesis. In summary, our findings and the prominent expression of 15-LOX and ALX in epithelial cells and macrophages place the LXA₄ circuit as an endogenous regulator of pathological angiogenesis.Formation of a new functional microvasculature, neovascularization, is a fundamental response to ischemia and a salient feature of wound healing. The primary function of newly formed blood vessels is to increase tissue oxygen tension and delivery of essential nutrients and effector cells to restore normal function. However, aberrant neovascularization is a hallmark feature of chronic inflammation and is associated with numerous pathological conditions that include diabetic retinopathy, Crohn''s Disease, atherosclerosis, and cancer.^1–3The growth of microvessels from existing vessels, angiogenesis, is tightly controlled by a range of angiogenic factors and inhibitors; a circuit that is highly evolved in avascular tissues such as the cornea. The vascular endothelial growth factor (VEGF) family of angiogenic factors and their receptors are key mediators of this process, which has led to the recent development and clinical use of anti-VEGF strategies for the treatment of pathological neovascularization in the retina, colon cancer, and lung cancer.^2–5 Many insights into the endogenous role of the VEGF network have been gained by using the cornea,^5,6 which maintains an immune-privileged and avascular state despite expression of VEGF-A and its immediate proximity to the vasculature.⁷ Specifically, recent findings have demonstrated that avascularity of the cornea requires expression of a soluble VEGF receptor-1 (sFLT1), which traps VEGF-A.⁸ In addition, the receptor for VEGF-C/VEGF-D, namely VEGF receptor-3 (VEGFR-3, FLT4), is a critical regulator of inflammatory neovascularization.^9–11 FLT4 is of special of interest because its essential expression during development becomes restricted primarily to lymph vessels and activation of this endothelial receptor is a critical step in initiating lymphangiogenesis. However, FLT4 expression is also up-regulated in microvessels of tumors and wounds, and in macrophages and in addition is constitutively expressed in corneal epithelial cells.^3,10–13 A recent report demonstrates that FLT4 is highly expressed in angiogenic sprouts and is a critical regulator of sustained heme-angiogenesis,¹² which underscores the potential key role of this receptor in pathological neovascularization.Inflammation is intimately associated with neovascularization especially during wound healing and ischemic injury. Lipid autacoids are some of the earliest signals that are released in response to injury or insult. In this regard, the 15-lipoxygenase pathway^14,15 is of interest as it is one of the most inducible genes in macrophages and highly expressed in mucosal and corneal epithelial cells. Macrophages and epithelial cells are important regulators of angiogenesis, especially in avascular tissue such as the cornea.^3,10,16 Macrophages have a central and well-documented role in angiogenesis, especially in tumors and inflammatory neovascularization. In the cornea, a well-established model tissue for studying inflammatory neovascularization, VEGF-A recruits macrophages, the major cell type to generate VEGF, which drives inflammatory heme and lymphangiogenesis. Corneal epithelial cells constitutively express the receptor for VEGF-C/VEGF-D (ie, FLT4), which has been proposed as a critical pathway for regulating inflammatory neovascularization.¹⁰Human 15-LOX (ALOX15 and ALOX15B) generate 15S-hydroxyeicosatetraenoic acid (HETE) and mouse 12/15-LOX (Alox15) generates 15S-HETE and 12S-HETE from arachidonic acid. 15-HETE and 12-HETE have been shown to induce proliferation, migration, and tube formation in endothelial cells.¹⁷ More importantly, 15-HETE is a key intermediate in the formation of the well-studied anti-inflammatory mediator lipoxin A₄ (LXA₄) that is generated via the rate-limiting enzyme 5-lipoxygenase (5-LOX). A body of work^18–24 has established that the anti-inflammatory actions, which are associated with the up-regulation of 15-LOX and/or 15S-HETE formation are mediated by LXA₄ and its G-protein coupled receptor ALX. Recent reports have demonstrated that stable analogs of LXA₄ inhibit VEGF induced angiogenic responses in endothelial cells.^25–27 These metabolically stable analogs are mimetics of aspirin-triggered LXA₄, an endogenous isomer whose synthesis can be triggered by aspirin-acetylated cyclooxygenase-2 rather than 15-LOX. This 15-epi-isomer of LXA₄ resists metabolic inactivation and mediates it bioactions, like LXA₄, via the ALX receptor. The intimate link between inflammation and angiogenesis and the ability of LXA₄²⁸ and analogs of 15-epi LXA₄^25–27 to inhibit VEGF-A induced angiogenic responses in vitro points toward a potential role of endogenous LXA₄ circuits in pathological angiogenesis. However, the endogenous role of 15-LOX in the regulation of angiogenesis remains controversial. Reports have demonstrated that the enzyme or its products promote or inhibit angiogenic responses in in vitro studies.^29–32 More importantly, the in vivo role of the 15-LOX pathway or the LXA₄ circuit in pathological neovascularization remains to be clearly defined. To this end, we assessed the role the 15-LOX pathway and LXA₄ circuit in chronic injury-induced inflammatory neovascularization.Here, we report that inflammatory neovascularization and up-regulation of the VEGF circuit correlate with changes in both 15-LOX (Alox15) and LXA₄ receptor (ALX) expression and temporally defined 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key enzymes in the formation of LXA₄, led to amplified neovascularization and expression of VEGF-A and FLT4 in the avascular cornea during chronic injury. LXA₄, but not 15S-HETE, attenuated expression of VEGF-A and FLT4 and the angiogenic response, which provides evidence that selective autacoids from the prominent 15-LOX pathway have an endogenous role in limiting pathological neovascularization.