Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis

Abstract

Aims: Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR.

Materials & methods: We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC-MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression.

Results: Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR.

Conclusion: Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.

Keywords: 12-HETE; 12/15-Lipoxygenase; 15-HETE; Bioactive lipids; Blood retinal barrier; Diabetic retinopathy; Eicosanoids; ICAM-1; Leukostasis; Permeability.

Figure 1. Clustered heat map of circulating bioactive lipid metabolites in the plasma of diabetic mice

The metabolites were initially clustered into four major groups according to their polyunsaturated fatty acid (PUFA) origin; linoleic acid (LA), arachidonic acid (AA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA). This primary clustering is followed by secondary subclustering bioactive lipid metabolites within each PUFA group according to their putative enzymatic biosynthesis pathways and hence chemical structure similarity. The LA group includes the following subclusters: hydroxyoctadecadienoic acids (HODEs), oxooctadecadienoic acids (oxoODEs), epoxyoctadecamonoenoic acids (EpOME), dihydroxyoctadecamonoenoic acids (DiHOME), and LA-derived 1-series prostanoids. The AA group includes: hydroxyeicosatetraenoic acids (HETEs), oxoeicosatetraenoic acids (oxoETEs), epoxyeicosatrienoic acids (EpETrEs), Dihydroxyeicosatrienoic acids (DiHETrEs), AA-derived 4-series leukotrienes (LTs), AA-derived 4-series lipoxins (LXs), and AA-derived 2-series prostanoids and thromboxane (TXs). The EPA group includes: hydroxyeicosapentaenoic acids (HEPEs), epoxyeicosatetraenoic acids (EpETEs), dihydroxyeicosatetraenoic acids (DiHETEs), EPA-derived 5-series LTs, EPA-derived 5-series LXs, EPA-derived 3-series prostanoids, and E-series Resolvins (Rvs). The DHA group includes: hydroxydecosahexaenoic acids (HDoHEs), epoxydecosapentaenoic acids (EpDOPEs), Dihydoxydecosapentaenoic acids (DIHDOPEs), and D-series Rvs. The significant (P<0.05) increased metabolites (9,10-DiHOME, 5,6-DiHETrE, 11,12-DiHETrE, 14,15-DiHETrE, 15d-D12,14-PGJ3, and RvD2) were indicated by * while decreased metabolites were indicated by #. Data shown for the comparison are the fold change of 6 diabetic and 8 nondiabetic mice relative to the average of nondiabetic mice ± SD. The highest fold change is indicated by the red color, the lowest fold change is indicated by the green color, and the undetected metabolite is represented by the black color.

Figure 2. Circulating lipidomic profile in diabetes is dominated by cytochrome P 450 (CYP) /sEH pathway derived metabolites rather than 12/15-LO metabolites

A) The activity of circulating 12/15-LO was not altered during diabetes as indicated by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including LA (9- and 13-HODEs), AA (12- and 15- HETEs), EPA (12- and 15- HEPEs), or DHA (14- and 17-HDoHE). B) In contrast to 12/15-LO metabolites, levels of CYP/sEH metabolites derived from different PUFAs showed an increasing trend in diabetes. This trend was significantly evident among 9,10-DiHOME, 5,6-DiHETrE, 11,12-DiHETrE, 14,15-DiHETrE. Data shown for the comparison are from the same experimental groups used in figure 1 and represented here by the mean fold change of 6 diabetic mice relative to the average of 8 nondiabetic mice ± SD.

Figure 3

Monocytic/macrophagic 12/15-LO does not play a role in high glucose-induced leukocyte adhesion to retinal endothelial cells. A) Real-time (RT)-PCR expression of 12-LO and 15-LO mRNAs in mouse leukocytes. Delta Rn versus cycle number plots showing amplification curves from wild-type (WT) mouse peripheral blood mononuclear cells (PBMCs) versus knockout (KO) PBMCs. Rn represents the normalized fluorescent signal of the reporter divided by the passive reference dye, ROX. B) and C) Leukocyte adhesion assay was performed on high glucose, HG- (B), or LPS- (C) activated mouse retinal endothelial cells (mRECs) using leukocytes isolated from either 12/15-LO knockout (KO) or wild type (WT) mice. mRECs activated either by 72-hours HG treatment (B) or LPS (C) significantly increased the number of adherent leukocytes isolated from KO mice relatively to the same extent as those derived from WT mice. The data are presented as the fold change in Myeloperoxidase (MPO) activity (an indicator for leukocyte adhesion) ± SD relative to the corresponding control from non-activated mRECs, n =4–6 for each group.

Figure 4. Activation of human retinal endothelial cells (HRECs) for leukocyte adhesion followed by hyperpermeability under different hyperglycemic conditions

A) HRECs were treated, as described in material and method, with HG, glycated albumin (AGA), or LPS as a positive control, then subjected to leukocyte adhesion using either U937 monocyte-like cells or purified CD14⁺ monocytes. The number of adherent leukocytes to HRECs was significantly increased by HG-treatment compared to normo-osmotic control and the effect of HG was more robust compared with that obtained with the AGA. Additionally, CD14⁺ monocytes showed a better response towards activated HRECs than U937. The data presented are the fold changes in Myeloperoxidase (MPO) activity (an indicator for leukocyte adhesion) ± SD relative to the corresponding control from non-activated HRECs, n =4–6 in each group. B & C) Normalized TER resistance ± SD over time for HRECs incubated with HG or LG before and after CD14⁺ monocytes addition, respectively. The experiment was carried out after HRECs formed confluent mature monolayers as indicated by plateau of electronic resistance under (i) full media, (ii) serum free media (SFM). Thereafter, HG or LG in SFM was added (iii) to confluent mature monolayers before adding CD14⁺ monocytes in C), n=4–6 in each experimental group.

Figure 5. Contribution of endothelial 12/15-LO to high glucose (HG)-induced ICAM-1 expression in HRECs

A) Expression of 12-LO and 15-LO in HRECs and human monocyte-like U937 cells. B) Inhibition of HG-induced ICAM-1 expression, as assessed by Western blot analysis, in HRECs pre-treated with the broad 12/15-LO pharmacological inhibitor, baicalein (10 μM). Cells were pretreated with baicalein for 30 min, and then incubated with (HG; D-Glucose, 30 mM) or normo-osmotic control (LG, 5mM D-glucose+25 mM L-glucose) for 5-days. C&D) Inhibition of HG-induced ICAM-1 expression, as assessed by Western blot analysis, in HRECs transfected with 15-LO siRNA. Cells were transfected with siRNA or mock siRNA for 24 hours and then treated with (HG; D-Glucose, 30 mM) or normo-osmotic control (LG, 5mM D-glucose+25 mM L-glucose) for 5-days. C) Measurement of 15-LO expression relative to actin by Western blot after transfection with 15-LO siRNA. D) Relative ICAM-1 expression in HRECs incubated with HG or LG in the presence or absence of 15-LO siRNA normalized to that of LG and mocked siRNA-treated cells, which was arbitrarily set at 1, n=4–5. E) HRECs were incubated in (HG; D-Glucose, 30 mM) or normo-osmotic control (LG, 5mM D-glucose+25 mM L-glucose) for 5-days in presence of baicalein (10 μM) or its vehicle (DMSO), then subjected to leukocyte adhesion using CD14⁺ monocytes. The data presented are the fold changes in Myeloperoxidase (MPO) activity (an indicator for leukocyte adhesion) ± SD relative to the corresponding control LG-treated HRECs, n =4 in each group.

Figure 6. Proposed cascade of events in the pathogenesis of diabetic retinopathy

Hyperglycemia activates 12/15-LO to release 12- and 15-hydroxyeicosatetraeanoic acids (HETEs) that in turn activate retinal endothelial cells through various inflammatory signaling system leading to increases in ICAM-1 expression, leukocyte adhesion, followed by hyperpermeability, the cardinal signs of diabetic retinopathy.