In vivo effect of 8-epi-PGF2α on retinal circulation in diabetic and non-diabetic rats

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(6), 349-355 @1998 HarcourtBrace& Co. Ltd

In v i v o e f f e c t of 8-epi-PGF2 on r e t i n a l c i r c u l a t i o n in d i a b e t i c a n d non. diabetic rats E. Michoud, M. Lecomte, M. Lagarde, N. Wiernsperger ~Diabetic Microangiopathy Research Unit, LIPHA-INSERM U352, INSA-Lyon, 69621 Villeurbanne Cedex, France

Summary Retinal hemodynamic responses to a F2-isoprostane, 8-epi-PGF2~, were quantitated in vivo in non-diabetic and diabetic rats using a video fluorescein angiography system. Vascular diameters and retinal mean circulation time were determined before and after 5 gl intra-vitreous injection of 8-epi-PGF2~ (10-s to 104 M), 1 0 -4 M 8-epi-PGF2~ + 1 0 -3 M SQ29,548 or 10-3 M LCB2853 (two inhibitors of TXA2 receptor), 104 M 913-PGF2~, or the carrier in non-diabetic animals. Diabetic rats received either 8-epi-PGF2~ 10-4 M, or the carrier. Compared to control animals, diabetic rats presented in the basal state a venous vasodilation (P<0.01), without modification of retinal mean circulation time or blood flow. After intravitreous injection of 8-epi-PGF2~, a significant arterial vasoconstriction was observed in control but not in diabetic animals. This vasoconstriction was concomitant with increased retinal mean circulation time in control but not in diabetic rats, inducing an impaired reduction of blood flow. No vasoconstriction was observed after injection of either the carrier, 913-PGF2~or the isoprostane associated to the inhibitors of TXA2 receptors. This is the first direct observation that the isoprostane 8-iso-PGF2~, is a potent vasoconstricting agent in the retina. It occurs at the arterial but not venous level, and is likely mediated through a TXA2-1ike receptor. Differences observed between control and diabetic animals suggest altered adaptative mechanisms toward vasoconstrictor substances (such as isoprostanes) in diabetic rats.

INTRODUCTION

Isoprostanes are a unique series of prostaglandin-like compounds formed in vivo non-enzymatically from freeradical-catalysed peroxidation of arachidonic acid, cyclooxygenase (COX-2) catalysing only minor amounts of these substances2 '2 The discovery of these unique products of lipid peroxidation was reported in 1990 by Morrow et al.,3 explaining old data concerning particular PG-like compounds formed by auto-oxidation in vitro. 4 They provide a reliable measure of oxidant injury not only in vitro but, more importantly, in vivo, for example after brain injury, 5 after ischemia-reperfusion episodes, 6 or in atherosclerotic lesions. 7 Among these isoprostanes, 8-epi-PGF2~ has received considerable attention because Received 20 August 1998 Accepted 9 October 1998 Correspondence to: E. Michoud, Diabetic Microangiopathy Research Unit, LIPHAqNSERM U352, tNSA-Lyon B406, 69621 Villeurbanne cedex, France. Tel: +33 4 72 43 81 12; Fax: +33 4 72 43 81 13; e-mail: [email protected]

of its potential biological activity, having been found to be a vasoconstrictor in kidney, lungs, brain, heart and lymphatics.8-~z The renal vasoconstriction induced by isoprostanes can be abrogated by the thromboxane/ endoperoxide (TXA2/PGH2)receptor antagonist SQ29,548, suggesting that they exert these effects by interacting through a TXA2-1ike receptor in the vasculature. 3 NonetheIess, it has been suggested that isoprostanes induce vasoconstriction by interaction with a unique receptor similar to, but distinct from the TXA2/PGH2 receptor. ~3'14 In the newborn pig retina, 8-epi-PGF2~ levels have been shown to increase after in vitro oxidative stress, or after in vivo ischemia reperfusion episode induced by asphyxia (5 min) and reoxygenation (45 rain). 15 A vasoconstrictor effect of 8-epi-PGF2~ has also been observed in the retina ex vivo with this animal model. ~S Oxidative stress has been proposed as one of the mechanisms explaining the development and evolution of diabetic vascular complications. 1¢t7 Elevated glucose and growth factors increase the formation of Fa-isoprostanes in vascular smooth muscle cells in vitro. ~s Furthermore 349

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plasma 8-epi-PGF2~ levels, reflecting an increased plasma lipid peroxidation, have been shown to be elevated in patients with non-insulin dependent diabetes mellitus. 19 Urinary excretion of 8-epi-PGF2~ has also been described to be higher in rats made diabetic with streptozotocin (4 weeks before) than in control rats. a° The retina is particularly rich in polyunsaturated fatty acids. 21 Retina is, therefore, an ideal target for oxidative damage leading to lipid peroxidation products such as isoprostanes, which could be implicated in the development of diabetic retinopathy. In order to test the reactivity properties of 8-epi-PGF2~ in the retina in vivo, and their potential consequences in diabetic conditions, the retinal hemodynamic changes in response to intravitreal injections of 8-epi-PGF2~ were quantitated in diabetic and non-diabetic rats using a video fluorescein angiography system.

MATERIALS AND METHODS

Before intravitreal injections, three angiographic recomings were performed to provide a baseline measurement of the retinal circulatory parameters. The intravitreal injection was performed with a 30 gauge needle connected to a 10 gl Hamilton Luer Lock syringe, inserted carefully into the vitreous cavity under direct visualization and positioned directly above the optic disk region. The entire 5 gl volume of agent was slowly injected into the vitreous cavity and time recorded.

Instrumentation

The video fluorescein angiography system used for the real time recording of retinal fluorescein angiograms is built around a fluorescence microscope (Zeiss Orthoplan, Oberchen, Germany). The retina was observed by a closecircuit video system, including a black and white CCD camera (4912, Cohu, San Diego, California), a S-VI-ISvideo cassette recorder (BR-S605E, WC, Tokyo, Japan) and a black and white monitor.

Animals

Male Wistar rats (Iffa Credo, L'Arbresle, France), weighing 190-210g at the beginning of the protocols, were used for these experiments. Streptozotocin (Sigma, France, 60 mg/kg in 10 mM citrate buffer), or the carrier were injected intravenously to obtain diabetic or control rats, respectively. Experiments were performed 4 to 5 weeks after this treatment, before any cataract formation and when an increased appearance of 8-epi-PGF2~ in urine has been found in streptozotocin rats. 2° Each rat was anesthetized (pentobarbital, 60 mg/kg, i.p.) before hemodynamic measurements. The body temperature of animals was kept constant at 37 + I°C with a heating pad. The trachea was intubated with a polyethylene tubing (PE-250; Guerbet Biomedical, Louvres, France) to facilitate spontaneous breathing. Polyethylene catheters (PE-50) were inserted into the right jugular vein for fluorescein injection, and into the right carotid artery for blood pressure measurement and blood sampling. Arterial blood sample was collected at the end of the experiment for glycemia determination. Blood glucose concentrations were measured by the glucose oxidase method. The pupil of the left eye was dilated using atropine 0.3%. A piano-concave contact lens was placed on the cornea, which permitted a high quality view of the fundusY The rat eye was positioned under the microscope. A syringe was filled with sodium fluorescein 0.1 M and connected to the venous catheter. The optic disk of the retina was centered and focused in the field of view. For angiographic recording, a 40 gl bolus of fluorescein dye was rapidly injected into the catheter, and the time of injection was recorded.

Video angiogram analysis

The analysis of the video fluorescein angiograms was realized by a playback analysis of the video recording using a self-made image processing system. The image was coded as 768 by 572 pixels, with 256 gray levels. All angiograms were analyzed densitometfically for changes in fluorescence intensity over time. Sample sites were chosen in retinal vessels crossing a circle, centered on the optic disc, that had a radius equal to 1.2 fold the radius of the optic disc. Resultant fluorescence data were then fit to a lognormal distribution 23-2s using a least-squares fit algorithm. The log-normal distribution function used here is as follows: [ = I0 + Ip exp (-[3 ln2(t/tp)) where I0 represents the background vessel intensity, Ip represents the peak fluorescence intensity, ~ represents the coefficient of the log-normal exponent, and describes the shape of the curve, tp represents the time at which the peak fluorescence intensity occurred, and In represents the natural logarithm. The mean retinal vessel circulation time, t~, is defined as: tm = t~ exp(3/(413)) and the difference in retinal artery and vein mean circulation times defines the retinal mean circulation time (MCT). The vessel diameters at the same sites were determined using the maximum contrast obtained during the fluorescein bolus injection. The isotropic orientation of the

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vessel was used to increase the signal/noise ratio, by integration of the segments perpendicular to the user's defined segment. The diameters were computed at 50% of the gray level amplitude of the integrated signal. Retinal blood flow was calculated from the MCT and the vessel diameter determinations. It has been shown empirically that retinal blood flow is proportional to the sum of the square of the arterial and venous diameters derided by the retinal MCT. 26 The computed value integrates the diameter o f the feeding vessels, and the hemodynamics of all the microvessels of the retinal microcirculation.

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Mann-Whimey U-test. Values of P < 0.05 were considered to be statistically significant. RESULTS 1. Control rats Effects of intravitreal 8-epi-PGF2~ on retinal circulation

The intravitreal injection of 5 ul 8-epi-PGF2~ induced a marked vasoconstriction of the retinal arteries (P< 0.05) (Fig. 1A). This vasoconstriction was dose-dependent and

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Experimental protocol

In a first series of experiments, the retinal circulato13r dose-response characteristics to different concentrations of 8-epi-PGF2~, (Cayman Chemical, Ann Arbor, MI) injected intravitreally in non-diabetic rats was investigated (from 10-5 to 10-3M). 0.9% NaC1 and 10-4M 913-PGF2~ (a stereo-isomer of 8-epi-PGF2~ without known biological activity to our knowledge, Cayman Chemical, Ann Arbor, MI) were used as control. The injection of solutions containing either 10 4 M 8-epi-PGF2¢ and 10 -3 M SQ29,548 (Cayman Chemical, Ann Arbor, MI), or 10-4 M 8-epi-PGF2~ and 10-aM LCB285327 (Lipha S.A., Lyon, France), two different antagonists of the thromboxane/endoperoxide (TXA2/PGH2) receptor, were also performed. In a second series of experiments, intravitreal injections of 10-4M 8-epi-PGF2~ were performed in control and in streptozotocin-injected rats after one month of diabetes. A minimum of five rats was tested for each group in the experimental protocol.

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Statistical analysis

Results are expressed as mean+SEM. Both diameters and retinal mean circulation time was computed before (mean value of 3 measurements) and after injections (mean value of 3 to 6 measurements made between 3 and 8 min after the injection) for each rat. Between group comparisons were performed using the Kruskall Wallis and © 1998 Harcourt Brace & Co. Ltd

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Retinal and vitreal isoprostane content

In order to estimate the 8-cpi-PGF2~ concentration reaching the retina and remaining in the vitreous 5 min after injection, three rats were injected intravitreally with 5gl of lO-4M 8-epi-PGF2~ (177ng injected) and killed 5 min later. The eyes were sampled and vitreous and retinas dissected and frozen in liquid nitrogen. Tissues were homogenized and isoprostane content was quantified with a 'total 8-epi-PGF2~ correlated EIA kit' (Assay Designs Inc., Ann Arbor, MI).

NaCI

Fig. 1 Effects of 5 gl intravitreous injection of NaCi; 8-epi-PGF2~ (104 to 10-3 M); a solution mixing 8-epi-PGF2,~ (10-~ M) + SQ29,548 (10 -3 M); a solution mixing 8-epi-PGF2~ (104 M) + LCB2853 (10 -a M); and 913-PGF2~ (104-3M). (A) On retinal arterial diameter. (B) On retinal mean circulation time. (C) On blood flow. Results are expressed as percentage compared to control parameter before intravitreal injection. *P < 0.05 vs NaCI. **P < 0.01 vs NaCI.

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diameter variations reached statistical significance with 10-4M of the injected solution (-9% at 10-4M, -19% at 10-3 M). On the other hand, the intravitreal injection of the carrier, or of a diastereoisomer of this isoprostane, 9[~-PGF2~ at the same concentration (10 -4 M), did not produce any significant variation of diameter (Fig. 1A). None of the products tested were able to induce diameter change on the venous side of the retinal vessels (data not shown). The retinal mean circulation time, was significantly increased in control rats after the intravitreal injection of 8-epi-PGF2~ but not with the carrier (Fig. 1B). The response to 8-epi-PGF2~ was also dose-dependent (Fig. 1B), with a maximal effect reached at 10-4 M. The retinal blood flow, computed as the sum of the square of the arterial and venous diameter divided by the mean circulation time (MCT), was decreased for all concentrations of 8-epi-PGF2~ (Fig. 1C).

by 7 % over controls (P=O.06) and the venous diameter was also increased by 7% over control (P< 0.01). In these animals, both the mean circulation time and the blood flow were not statistically different in the diabetic group as compared to the control one (data not shown). Effects of intravitreous 8-epi-PGF2~ on retina/circulation

Our results show that an intravitreal injection of 8-epiPGF2~ induced a vascular response which was blunted in diabetic animals. The vasoconstriction observed on the arterial side of the retinal vascular network with 8-epiPGF2~ was lower in diabetic rats as compared to control

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The intravitreous injection of 5 gl of a solution containing both 8-epi-PGF2~ (lO-4M) and a thromboxane A2/PGH2 receptor antagonist (10-3M SQ29,548 or LCB2853) did not produce a vasoconstriction of retinal arteries (Fig. 1A), as compared to 8-epi-PGF2~ alone (see above). The MCT was non significantly increased (Fig. 1B) and blood flow was also decreased non significantly (Fig. 1C) with these solutions. 2. Diabetic rats

One month after the streptozotocin injection, all diabetic animals presented a marked non-fasting hyperglycemia (glycemia > 20 mM) and a loss of weight compared to control rats (230+9 g vs 330+ 17 g in control rats). Basal vessel diameter was increased in rats after one month diabetes (Fig. 2). The arterial diameter, measured during the maximum fluorescent intensity, was increased

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Fig. 2 Basal retinal arteries and veins diameter in one month streptozotocin-induced diabetic rats (1~) compared to control rats ( I ) . * P < 0.05 vs control animals.

Fig. 3 Effects of 10-4 M 8-epi-PGF2~, ([2) and NaCI ( I ) in control and diabetic rats. (A) On retinal arterial diameter. (B) On retinal retinal mean circulation time. (C) On retinal blood flow. Results are expressed as percentage compared to control parameter before intravitreal injection.*P < 0.05 vs NaCl.

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(6), 349-355

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In vivo effect of an isoprostane in the retina

animals. This effect was statistically non significant when compared to NaC1 carrier in diabetic animals (Fig. 3A). The MCT was not increased when compared to NaC1 injection (Fig. 3B). On the other hand, the blood flow, computed with both diameters and MCT, was statistically decreased after the injection of 8-epi-PGF2~, in diabetic rats (Fig. 3C). Compared to an intravitreous injection of saline, the blood flow was then reduced only by around 20% in diabetic rats, vs approximately 40% in control rats. 3. Isoprostane content of the retina and the vitreous

The 8-epi-PGF2~ content in the retina and the vitreous after its intravitreal injection was also quantitated. Five minutes after injection, the amount of 8-epi-PGF2~ was increased in the injected vitreous (218_+49 vs 10+2 pg/gg protein in the contralateral non-injected control vitreous, P
F2-isoprostanes have been shown to be increased in different pathologies, including diabetes mellitus. 19,2° Diabetes is a chronic metabolic disease associated with microvascular complications, such as nephropathy and retinopathy. In these complications, microvascular hemodynamics are altered before any visible morphometric changes, a8,29 After one month streptozotocin diabetes, the diabetic rats in the present work presented a basal dilation of the retinal vessels (mainly the veins), without variations in the retinal mean circulation time (Fig. 2). This dilation has already been described in the same animal model = or in humans. 3°,3~ For the basal retinal blood flow in diabetes, there is still a controversy in the literature, depending on the subjects or animal model, the diabetes duration, and the method used to quantitate blood flow? 2 Some authors find a decreased blood flow, 23'33-35 but others find the reverse. =,3°,3~,36 Despite this controversy, most authors describe an altered response of the vessels to v a s o a c t i v e agents 3r-39 or systemic s t i m u l i 4°-44 in diabetic conditions. Endothelial dysfunction is a key feature of diabetes mellitus and is thought to be the major cause of vascular complications associated with this disease. The pathways © 1998 Harcourt Brace & Co. Ltd

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mediating endothelinm cell layer dysfunction are unknown, although many candidates have been proposed, including oxidative stress. 45 F2-isoprostanes are formed in vivo from free-radical-catalysed peroxidation of arachidonic acid contained in phospholipids. ~2 They could participate to the altered reactivity observed in the retina of diabetic animals, but their in vivo impact on vascular properties in the retina were not known until now. Our results confirm the vasoconstrictor effect of 8-epi-PGF2~ observed in various organs, ex vivo 3,8,9,11,~2,I5 or in vivo 1° in normal animals. Our study is the first microscopic evaluation of this vasoconstrictor effect in vivo. The vasoconstriction, observed at the level of a whole organ until now, has been differentiated in this study between the arterial and the venous side of the circulation. Indeed, the vasoconstriction was not observed at the level of the veins, suggesting the presence of receptors to 8-epi-PGF2~ only on the arterial side of the vascular network. The vasoconstriction observed is dose-dependent. Using our visualization technique, we could not find out any variations of arterial diameter following an intravitreous injection of 8-epi-PGF2~ at a concentration of l O-SM. However the retinal MCT, which takes into account not only the feeding vessels of the retina but also the whole microcirculation of the retina, was increased, revealing a reduction of the retinal blood flow. At higher concentration, both the arterial diameter and retinal mean circulation time were modified, and indicate a decreased blood flow in the retina. The computation of the effective dose reaching the vessels has to be taken into consideration. One must consider that a few minutes after the injection, only 1% of the injected compound is found in the retina and 5% in the vitreous, respectively. Several hypotheses can explain this phenomenon. Firstly, the injected product can exit the vitreous via the hole made by the needle, in response to the increased hydrostatic pressure in the eye after injection, since each product has the tendency to go where the pressure is minimum. Secondly, 8-epi-PGF2~ can be metabolized rapidly, and the vasculature may have evacuated most of the compound by diffusion. Thirdly and more importantly, ff one assumes a total diffusion in the eye after injection (with a vitreal volume estimated by different authors from 55 g146 to 250 gps), the estimated concentration resulting from a 5 ~tl amount of lO-4M solution would be divided by a factor 10-50. Therefore, our results suggest that the effective concentration observed in the vessels was around 100 fold lower than the one we injected. Then, the intravitreal injection of 10-5 to 10-3M 8-iso-PGF2~ would correspond to an effective concentration of 100 nM to 10 ~tM, respectively, at the level of the retinal vasculature, with a good reproducibility.

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In t h e n e w b o r n pig r e t i n a ex vivo, a p o t e n t vasoconstriction to 8-epi-PGF2¢ h a s b e e n s h o w n w i t h a n ECs0 of 6 nM. is Differences o b s e r v e d m a y b e d u e to t h e fact t h a t o u r w o r k is p e r f o r m e d in a d u l t rats in vivo. The a n i m a l age c o u l d explain t h e differences o b s e r v e d b e t w e e n rats a n d piglets m e a s u r e s b e c a u s e 8-epi-PGF2~ is m o r e efficient in n e w b o r n animals t h a n in a d u l t ones. 4r Fa-isoprostanes exhibit t h e i r v a s o c o n s t r i c t o r effects b y b i n d i n g to a r e c e p t o r r e l a t e d to, b u t distinct from, t h a t of t h r o m b o x a n e A2. Following this h y p o t h e s i s , t h e conc o m i t a n t injection of SQ29,548 (or LCB2853) a n d 8-epiPGFR~, w i t h a 10 fold h i g h e r c o n c e n t r a t i o n of t h e former, d i d n o t i n d u c e a n y v a s o c o n s t r i c t i o n of retinal arteries. O u r results s h o w a n i n c r e a s e d retinal m e a n circulation time a n d a d e c r e a s e d b l o o d flow s i t u a t e d b e t w e e n t h e effect of NaC1 a n d of 8-epi-PGF2~, a l t h o u g h b e i n g n o n significant. One i n t e r p r e t a t i o n c o u l d be that, at t h e microcirculation level, a n arteriolar v a s o c o n s t r i c t i o n m a y still exist. Alternatively, t h e possibility exists t h a t t h e i n h i b i t o r injected c o n c o m i t a n t l y with t h e a g o n i s t c o u l d n o t c o m p l e t e l y b l o c k t h e receptor. The m e c h a n i s m of a c t i o n of t h e vasoconstrictive effect of 8-epi-PGF2~ h a s b e e n s h o w n to b e m e d i a t e d b y cycloo x y g e n a s e - g e n e r a t e d f o r m a t i o n of t h r o m b o x a n e and, to a lesser extent, b y e n d o t h e l i n , following c a l c i u m e n t r y into cells p o s s i b l y via r e c e p t o r - o p e r a t e d channels. 15 A n i n c r e a s e d p r o d u c t i o n of F2-isoprostanes h a s a s t i m u l a t o r y effect on endothelin-1 release. 4s W i t h t h e s e v a s o a c t i v e peptides, w h i c h c a n m o d u l a t e retinal pericyte contractility in vitro, a significant b l u n t i n g of t h e v a s c u l a r r e s p o n s e has also b e e n o b s e r v e d in diabetic h u m a n s 4s a n d animals. 3s In conclusion, t h e s e results s h o w t h a t a n i n t r a v i t r e o u s injection of 8-epi-PGF2~ i n d u c e s a d o s e - d e p e n d e n t vasoconstriction of retinal arteries m e d i a t e d , at least partially b y a r e c e p t o r to 8-epi-PGFa~. Taking into a c c o u n t t h e effective dose r e a c h i n g t h e vessels, this effect c o u l d be o b s e r v e d at a n e s t i m a t e d tissular c o n c e n t r a t i o n of 100 nM. The a b s e n c e of v a s o c o n s t r i c t i o n of a n o t h e r PGF2~ epimer, 913PGF2~, shows t h a t this effect is specific. The vasoconstrictor effect of 8-epi-PGF2~ o b s e r v e d in m a n y organs can be e x t e n d e d to a n o t h e r tissue, t h e retina, w h i c h is a reservoir of p o l y u n s a t u r a t e d fatty acids for i s o p r o s t a n e f o r m a t i o n in oxidative stress c o n d i t i o n s s u c h as d e s c r i b e d in diabetes. Moreover, t h e results we o b t a i n e d s h o w t h a t t h e v a s c u l a r r e s p o n s e to 8-epi-PGF2~ is altered in diabetic rats.

ACKNOWLEDGEMENTS The authors thank Dr Dao-Yi Yu for useful information on the

contact lenses. They also thank Dr Daniel Ruggiero-Lopez for helpful discussion and revision of the manuscript, and Mrs Patricia Leveque for technical help.

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