Endogenous LXA4 circuits are determinants of pathological angiogenesis in response to chronic injury - PubMed (original) (raw)
Endogenous LXA4 circuits are determinants of pathological angiogenesis in response to chronic injury
Alexander J Leedom et al. Am J Pathol. 2010 Jan.
Abstract
Inflammation and angiogenesis are intimately linked, and their dysregulation leads to pathological angiogenesis in human diseases. 15-lipoxygenase (15-LOX) and lipoxin A(4) receptors (ALX) constitute a LXA(4) circuit that is a key feature of inflammatory resolution. LXA(4) 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 LXA(4) 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 LXA(4), respectively, led to exacerbated inflammatory neovascularization coincident with increased VEGF-A and FLT4 expression. Direct topical treatment with LXA(4), 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 LXA(4) circuit as an endogenous regulator of pathological angiogenesis.
Figures
Figure 1
Inflammatory neovascularization differentially regulates expression of the LXA4 circuit. A: Representative images of an uninjured cornea. B: An 8.0 silk suture was placed intrastromally in female C57/BL6 mice. Image of cornea with neovascularization after 7 days of chronic injury. C: mRNA expression of selected genes was quantified by real-time PCR analyses in corneas after 7 days of chronic injury for heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), soluble VEGF receptor-1 (sFLT1), VEGF receptor-3 (FLT4), 12 and /15 LOX (Alox15), and after 48 hours for ALXR-1 (Fprl1) and ALXR-2 (Fpr-rs2). mRNA expression was compared with matched untreated corneas and is expressed as relative quantity (RQ) compared with a mouse positive mRNA control (n = 4, *P < 0.05) using the ΔΔCT method and β-actin as a reference gene.
Figure 2
Inflammatory neovascularization is associated with the dynamic formation of select lipid autacoids. Endogenous lipid autacoids were quantified in corneas without injury and after 2, 4, and 7 days of chronic injury by MS based lipidomic analyses using triple quadrupole LC/MS/MS system (MDS SCIEX QTRAP 3200). Specific transition ions and multiple reaction monitoring was used to measure levels of >20 eicosanoids in a single mouse corneas. Significant levels of prostaglandin E2 (PGE2), 15-hydroxyeicosatetraenoic acid (15-HETE), thromboxane B2, 5-hydroxyeicosatetraenoic acid (5-HETE), and 12_S_- hydroxyeicosatetraenoic acid (12-HETE) were detected in injured and uninjured corneas (n = 4, *P < 0.05 versus uninjured, **P < 0.05 versus 2 D).
Figure 3
Deletion of a LXA4 biosynthetic pathway or topical treatment with LXA4 regulates PMN recruitment to the injured cornea. A: PMN content of corneas from 12/15-LOX KO mice and matched congenic wild-type (wt) controls was assessed by measuring tissue MPO activity after 7 days of chronic injury (n = 4, P < 0.02). A MPO calibration curve was established with inflammatory exudate peritoneal PMN and used to calculate relative tissue PMN numbers. B: 15-HETE formation, a metabolic precursor for LXA4 formation, was determined by LC/MS/MS-based lipidomic analyses after 4 days of chronic injury (n = 4; *P < 0.05). C: Topical action of LXA4 on PMN recruitment to the injured cornea was determined at 48 hours. Relative PMN numbers in uninjured corneas and injured corneas treated with either saline alone or LXA4 (100 ng, t.i.d.) were assessed by measuring total tissue MPO activity (n = 4, *P < 0.02).
Figure 4
Topical LXA4 attenuates inflammatory neovascularization and inhibition of its biosynthetic pathway exacerbates pathological angiogenesis. A: Heme-angiogenesis in corneas with 7 days of chronic injury was assessed by immunohistochemistry using CD31 as a specific endothelial antigen. Corneas from 12/15-LOX KO or matched congenic wild-type (wt) mice treated with or without topical LXA4 (100 ng, t.i.d., 7 days) were collected and incubated in PBS containing fluorescein isothiocyanate-conjugated CD31/PECAM-1 monoclonal antibody and analyzed using a Zeiss Axiophot laser scanning confocal microscope. Images show neovascularization of representative corneal quadrants. All CD31+ vessels were traced manually and are expressed as total pixels (Image Pro Express 6.0) (n = 3 to 4; *P < 0.05). B: Topical 15-HETE exacerbates inflammatory neovascularization. Injured corneas were treated topically with either 15_S_-HETE (100 ng, t.i.d.) or sterile saline for 7 days. Images show representative whole corneal flat mounts (left panel, saline treatment alone; right panel 15_S_-HETE treatment). All CD31+ vessels were traced manually and are expressed as total pixels (n = 4; *P < 0.05).
Figure 5
5-LOX/LXA4 KO mice have a phenotype of exacerbated neovascularization in response to chronic injury. 5-LOX KO mice, which cannot generate LXA4, and their matched congenic wild-type (wt) controls were subjected to chronic suture injury for 7 days. Corneas were removed and pathological angiogenesis quantified by immunohistochemistry using a Zeiss Axioplan 2 microscope. Mosaic images were taken with a AxioCam MR camera and compiled using MosaiX and Zeiss AxioVision 4.5 software. Inset shows representative whole corneal flatmounts (left panel, wild-type; right panel, 5-LOX KO). Vessel density was documented and expressed as total pixels (Image Pro Express 6.0) (n = 4; *P < 0.05).
Figure 6
Deletion of 12/15 LOX up-regulates key regulators of inflammatory neovascularization. Corneas after 48 hours of chronic injury were collected from 12/15-LOX KO and matched congenic wild-type mice (n = 4). mRNA expression was quantified for HO-1, VEGF-A, sFLT1, and FLT4 using a Step One Plus QPCR system (Applied Biosystems). RNA expression for each gene was calculated using relative quantity (RQ) compared with a mouse positive mRNA control with the ΔΔCT method and β-actin as a reference gene. Data are expressed as fold change compared with the mouse positive mRNA control (n = 4, *P < 0.05).
Figure 7
Direct topical treatment with LXA4 down-regulates expression of key regulators of inflammatory angiogenesis. After suture injury corneas were treated with either saline alone or LXA4 (100 ng, t.i.d.) for 48 hours. mRNA expression was quantified for HO-1, VEGF-A, FLT4, and both LXA4 receptors (ALX-1, ALX-2) using a real-time PCR system. Relative expression was compared with a mouse positive mRNA control (n = 4, *P < 0.05).
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