Inhibition of 20-HETE attenuates diabetes-induced decreases in retinal hemodynamics

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Abstract

The mechanisms of early diabetes-induced decreases in retinal blood flow have yet to be fully determined. The aim of this study was to explore the hypothesis that 20-hydroxyeicosatetraenoic acid (20-HETE) plays a role in the early decrease of retinal hemodynamics in diabetic mice. 20-HETE has been implicated previously in the diabetes-enhanced vasoconstriction of mesenteric and renal vessels; however, its role in the diabetic retinal microcirculation has not been investigated. Diabetes was induced by multiple low-dose injections of streptozotocin (STZ; 50 mg/kg for 5 consecutive days), then ∼2 weeks later the mice were administered daily intraperitoneal injections with or without the 20-HETE inhibitor HET0016 (2.5 mg/kg/day) for the following 2 weeks. Non-diabetic age-matched mice were included as controls. Intravital microscopy was used to obtain measurements of retinal vascular diameters and red blood cell (RBC) velocities for the feed arterioles and draining venules extending out of and into the optic disk. From these values, wall shear rates and blood flow rates were calculated. Diabetes induced approximately 30–40% decreases in RBC velocity, wall shear rate, and blood flow rate. These decreases were attenuated to 5–10% in the mice given HET0016. In summary, the 20-HETE inhibitor HET0016 is able to attenuate the retinal hemodynamic changes induced by diabetes.

Highlights

► This is the first study to investigate 20-HETE in diabetic retinal blood flow. ► Diabetes reduces retinal blood velocity, vessel shear rate, and blood flow rate in mice. ► Diabetes-induced reductions in retinal hemodynamics are attenuated by a 20-HETE inhibitor.

Introduction

Diabetic retinopathy is classified as a microvascular disease, and is characterized by microaneurysms, hemorrhages, pericyte loss, increased microvascular permeability, exudates, capillary basement membrane thickening, capillary occlusion, shunts, venous beading, edema, and neovascularization (Blair et al., 2009, Kador et al., 2009). Diabetes also induces changes in vascular reactivity (induction of altered vasodilation or vasoconstriction) in various tissues throughout the body, including the retina (Justesen et al., 2010, Lorenzi et al., 2010, Pemp et al., 2009). Animal experiments, including those from our own lab (Lee and Harris, 2008, Wright and Harris, 2008, Wright et al., 2009, Wang et al., 2010), have demonstrated that diabetes induces significant decreases in retinal blood flow; however, the complete mechanisms of these decreases have yet to be determined.

The cytochrome P450-dependent metabolism of arachidonic acid can result in the production of 20-HETE, which has been found to be a potent vasoconstrictor. The mechanisms of 20-HETE-induced vasoconstriction have been attributed to increased Ca++ sensitivity, increased Ca++ entry via the depolarization of KCa channels, and activation of L-type Ca++ channels via protein kinase C (Roman et al., 2006). Additionally, the vasoactive actions of 20-HETE have been determined to be associated with a number of other constrictor pathways, including those of angiotensin II, vasopressin, endothelin, serotonin, and thromboxane (Miyata and Roman, 2005, Roman et al., 2006). 20-HETE has been implicated previously in the diabetes-enhanced mesenteric and renal responses to vasoconstrictors (Yousif et al., 2009); however, to date, its role in the diabetic retinal microcirculation has not been investigated.

HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)Formamidine) is a potent and selective inhibitor of 20-HETE formation, and demonstrates three orders of magnitude higher inhibition of 20-HETE than of epoxyeicosatrienoic acids (EETs) (Miyata and Roman, 2005). In the current study, we investigate the possibility that daily administration of HET0016 can attenuate the retinal hemodynamic changes induced in mice injected with streptozotocin. Measurements included arteriolar and venular diameters, red blood cell (RBC) velocities, wall shear rates, and blood flow rates.

Section snippets

Animals

Nine-week old C57BL/6 male mice (Jackson Laboratories) were randomly assigned to intraperitoneal (i.p.) injection of STZ (Sigma, St. Louis, MO; 50 mg/kg dissolved in pH 4.5 sodium citrate buffer), or sodium citrate buffer alone, for 5 consecutive days, with the STZ injection performed within 15 min of mixing into solution. Changes in blood glucose levels were detected with a tail vein puncture after the 5th day with an AlphaTRAK glucometer (Abbott; Abbott Park, IL); diabetic mice were included

Results

The study included four groups of animals, i.e., control and diabetic mice with or without daily treatment with HET0016. However, the dose of 2.5 mg/kg/day HET0016 was not tolerated by the non-diabetic controls: of the eight mice initially in this group, one died prior to the day of the experiment, and another five died following anesthesia, prior to the collection of retinal hemodynamic data. However, the same dose of HET0016 had no such lethal effects on the diabetic mice.

Table 1 provides the

Discussion

To our knowledge, this is the first study to investigate the influence of 20-HETE in the diabetic retinal vasculature, with a unique feature of the study being the direct in vivo microscopic measurement of retinal blood flow and hemodynamics. The results indicate that 20-HETE inhibition attenuates the significant decrease in retinal RBC velocity, shear rate, and blood flow rate induced by diabetes. This finding could have clinical relevance, since decreases in retinal blood flow and shear force

Acknowledgments

This study was funded by NIH EY017599 (NRH).

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