Homocysteine Inhibition of Endothelium-Dependent Nitric Oxide-Mediated Dilation of Porcine Retinal Arterioles via Enhanced Superoxide Production

Abstract

Purpose.: Elevated plasma concentration of homocysteine, a sulfur-containing amino acid, is an emerging risk factor for cardiovascular diseases. Recent epidemiologic studies have confirmed that elevated homocysteine levels are associated with ocular vascular diseases; however, the direct effect of homocysteine on ocular microvascular reactivity remains unknown. We investigated whether homocysteine affects endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles and whether oxidative stress and distinct protein kinase signaling pathways are involved in the homocysteine-mediated effect.

Methods.: Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Diameter changes were recorded using videomicroscopy techniques.

Results.: Intraluminal treatment with homocysteine (1 mM, 180 minutes) significantly attenuated arteriolar dilation in response to the endothelium-dependent NO-mediated agonists bradykinin and A23187 but not in response to the endothelium-independent NO donor sodium nitroprusside. In the presence of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), the nicotinamide adenine dinucleotide phosphate-oxidase (NAD(P)H oxidase inhibitor apocynin, p38 kinase inhibitor SB203580, and peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist pioglitazone, the detrimental effect of homocysteine on bradykinin-induced dilation was prevented; however, neither the xanthine oxidase inhibitor allopurinol, the JNK inhibitor SP600125, or pioglitazone with PPAR-γ inhibitor GW9662 had that effect.

Conclusions.: Homocysteine inhibits endothelium-dependent NO-mediated dilation in the retinal arterioles by producing superoxide from NAD(P)H oxidase, which appears to be linked with p38 kinase. By impairing endothelium-dependent NO-mediated vasoreactivity, homocysteine potentially facilitates development of retinal vascular diseases. In addition, pioglitazone can prevent homocysteine-induced endothelial dysfunction possibly by activating PPAR-γ.

Introduction

Although homocysteine is a sulfur-containing amino acid that forms during metabolism of methionine, an essential amino acid, ¹ elevation of plasma homocysteine concentrations could be caused by a variety of factors, including excess dietary methionine, inherited enzyme variants, or nutritional deficiency of folate and vitamin 6, which are required for the normal metabolism of homocysteine. ^2,3 Numerous clinical studies have shown that an elevated serum homocysteine level might be an independent risk factor for retinal vascular diseases, including diabetic retinopathy (DR) ⁴ and retinal vein occlusion. ⁵ Furthermore, epidemiologic studies have reported a relationship between hyperhomocysteinemia, which induces apoptosis of retinal ganglion cells ⁶ and ocular degenerative diseases such as glaucoma ⁷ and age-related macular degeneration. ⁸ These results indicated that elevated serum homocysteine concentrations might play a role in the pathogenesis of ocular disorders. In addition, it has been reported that high homocysteine levels caused endothelial dysfunction via production of reactive oxygen species (ROS) in microvascular endothelial cells ⁹ and that hyperhomocysteinemia induced by oral administration of methionine impaired vascular endothelial function in human brachial arteries, ¹⁰ suggesting that homocysteine per se can impair retinal vascular function. However, the effect of homocysteine on retinal vascular function remains unclear. 

Stimulation of peroxisome proliferator-activated receptor-γ (PPAR-γ), a member of the nuclear hormone receptor superfamily, appears to be vasoprotective, ¹¹ whereas loss-of-function mutations in endothelial PPAR-γ induce oxidative stress and result in endothelial dysfunction. ¹² Furthermore, mRNA levels of PPAR-γ decreased in genetic models of hyperhomocysteinemia, ¹³ and homocysteine-induced intercellular adhesion molecule-1 (ICAM-1) were ameliorated by PPAR-γ agonist in vitro. ¹⁴ We recently reported that the thiazolidinedione derivative pioglitazone, a potent and selective activator of PPAR-γ, elicited nitric oxide (NO)-mediated dilation of retinal arterioles. ¹⁵ Because the thiazolidinediones might prevent onset of proliferative DR in humans ¹⁶ and exert antioxidant effects in vivo ¹⁷ and in vitro, ¹⁸ these results implied that the thiazolidinediones might be a novel therapy for DR, to protect the vasomotor function of the endothelium in the retinal microcirculation from homocysteine-induced oxidative stress. 

In the current study, we tested the hypotheses that homocysteine impairs endothelium-dependent vasodilation of the retinal arterioles by increasing oxidative stress and that cotreatment with pioglitazone prevents this endothelial dysfunction via activation of PPAR-γ. Using an isolated vessel preparation, we examined the endothelium-dependent NO-mediated dilation of retinal arterioles with and without homocysteine. We also investigated whether key vascular signaling molecules in oxidative stress (i.e., distinct superoxide-generating enzymes and stress-activated protein kinases) are involved in the homocysteine-mediated effect. 

Materials and Methods

Animal Preparation

The Animal Care Committee of Asahikawa Medical University approved all animal procedures. The study was conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The eyes were enucleated immediately from pigs of either sex (age, 16–24 weeks; weight, 20–35 kg) after they were killed in a local slaughterhouse and transported to the laboratory in a moist chamber on ice. 

Isolation and Cannulation of Microvessels

The techniques used for identification, isolation, cannulation, pressurization, and visualization of the retinal vasculature have been described previously. ^15,19–21 Briefly, the anterior segment and vitreous body were removed. The posterior segment was placed in a cooled dissection chamber (∼8°C) containing physiologic salt solution (PSS; pH 7.4). Isolated retinal arterioles (90–130 μm in situ) dissected with microdissection forceps were transferred for cannulation to a Lucite vessel chamber containing PSS-albumin solution equilibrated with room air at ambient temperature. The outside of the arteriole cannulated with a pair of glass micropipettes was secured with sutures. After cannulation, the vessel was pressurized to 55 cm H₂O intraluminal pressure without flow by two independent pressure reservoirs. ²² The internal diameter of the isolated vessel was recorded continuously using videomicroscopy techniques during the experiment. ¹⁹  

Experimental Protocols

Cannulated arterioles were bathed in PSS at 36° to 37°C to allow development of basal tone. After the vessels developed a stable basal tone (∼30–40 minutes), we assessed the effect of homocysteine on NO-mediated vasodilation. For this purpose, the endothelium-dependent NO-mediated vasodilation in response to bradykinin (1 × 10⁻¹² to 1 × 10⁻⁸ M) ^21,23 was established before and after 180 minutes of intraluminal incubation with either a 100 μM or 1 mM concentration of homocysteine. ²⁴ The vessels were exposed to each concentration of bradykinin for 2 to 3 minutes until a stable diameter was established. In our preliminary study, bradykinin-induced dilation was reproducible and did not deteriorate after repeated applications (n = 5). 

To determine whether the effect of homocysteine is selective toward endothelium-dependent NO-mediated dilation, dose-dependent responses to the receptor-independent but endothelial NO-mediated vasodilator A23187 (1 × 10⁻⁸ to 3 × 10⁻⁶ M) and to endothelium-independent NO donor sodium nitroprusside (1 × 10⁻⁸ to 1 × 10⁻⁴ M) were established before and after 180 minutes of intraluminal incubation with 1 mM of homocysteine in another series of experiments. 

The roles of superoxide and the oxidative enzymes nicotinamide adenine dinucleotide phosphate-oxidase [NAD(P)H oxidase] and xanthine oxidase in mediating the effect of homocysteine were determined in a separate group of vessels by examining bradykinin-induced dilation before and after treating the vessels with homocysteine (1 mM) combined with the cell-permeable superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL; 1 mM), ²⁵ the NAD(P)H oxidase inhibitor apocynin (100 μM), ²⁵ and the xanthine oxidase inhibitor allopurinol (1 μM). ²⁶ The roles of stress-activated protein kinases were examined by treating the vessels with homocysteine combined with the p38 kinase inhibitor SB203580 (0.1 μM; Calbiochem, San Diego, CA) ²⁷ or the c-June N-terminal kinase (JNK) inhibitor SP600125 (5 μM; Calbiochem). ²⁸  

To assess the ability of pioglitazone to mitigate the effect of homocysteine on NO-mediated vasodilation, vessels were treated with homocysteine (1 mM) combined with a clinical dose (0.5 μM) of pioglitazone. ²⁹ In addition, to determine the role of PPAR-γ on the ability of pioglitazone to mediate the effect of homocysteine on the vasodilatory response to bradykinin, another group of vessels was preincubated with the PPAR-γ inhibitor GW9662 (10 μM) ³⁰ and subsequently treated with homocysteine in combination with pioglitazone. 

Superoxide Detection

Superoxide production in isolated retinal arterioles was evaluated with the fluorescent dye dihydroethidium (DHE). Isolated and pressurized retinal arterioles (70–100 μm in diameter and 1.5 mm in length) were incubated intraluminally with PSS-containing vehicle, homocysteine (1 mM), or homocysteine plus TEMPOL (1 mM) at 37°C for 180 minutes and stained with DHE (4 μM) for 30 minutes. After being washed, the arterioles were embedded in OCT compound (Tissue-Tek, Sakura Finetek, Torrance, CA) for cryostat sectioning. Embedded arterioles were cut into 10-μm-thick sections and placed on glass slides. Images were taken with a Fluoview model FV 1000 confocal microscope (Olympus, Tokyo, Japan). Fluorescence was detected with a 610-nm emission filter. Control and experimental tissues were placed on the same slide and processed under the same conditions. The settings for image acquisition were identical for the control and experimental tissues. Fluorescence intensities of DHE staining were analyzed using FV10-ASW 3 software (Tokyo, Japan). Data were normalized to the average fluorescence intensity of the vessels treated with PSS-containing vehicle to permit comparisons across several groups. 

Chemicals

Pioglitazone was obtained from LKT Laboratories, Inc. (St. Paul, MN). SB203580 and SP600125 were obtained from Calbiochem. Pioglitazone and GW9662 were dissolved in dimethyl sulfoxide (DMSO). Other drugs were obtained from Sigma-Aldrich (St. Louis, MO) and dissolved in PSS. Subsequent dilutions of these drugs were prepared in PSS. The final concentration of DMSO in the vessel bath was less than 0.1%. ²⁰ Vehicle control studies indicated that this final concentration of solvent did not affect arteriolar function. 

Data Analysis

At the end of each experiment, the vessel was relaxed in ethylenediaminetetraacetic acid (1 mM)-calcium-free PSS to obtain the maximal diameter at 55 cm H₂O intraluminal pressure. ^19,21 All diameter changes in response to agonists were normalized to the maximal vasodilation and expressed as a percentage of the maximal dilation. ^19,21 Data are means ± SEM; n represents the number of vessels studied. Statistical comparisons of changes in resting tone and average fluorescence intensity by homocysteine or pharmacologic inhibitors were performed using Student's t-test. Two-way analysis of variance, followed by the Bonferroni multiple-range test, was used to determine the significance of the difference between the control and experimental interventions. A P value of <0.05 was considered significant. 

Results

Effect of Homocysteine on NO-Mediated Vasodilation

All vessels (n = 68) developed a similar level of basal tone (constricted to 68% ± 1% of maximal diameter) in a bath in which the temperature was 36°C to 37°C with 55 cm H₂O intraluminal pressure. The average resting and maximal vessel diameters were 76 ± 1 μm and 105 ± 2 μm, respectively. Bradykinin dilated the retinal arterioles in a dose-dependent manner. The vasodilation in response to bradykinin was significantly (P < 0.001) attenuated after incubation with 1 mM but not with 100 μM of homocysteine (Fig. 1A). A higher dose of homocysteine (5 mM, n = 4) did not further reduce the bradykinin-induced dilation (data not shown). Figure 1B shows the representative time course of the response of A23187 to retinal arterioles. We found that A23187 produced slight constriction in rapid phase (initiated within 1 minute) and subsequently dilation of retinal arterioles from the baseline of 64 μm to 92 μm in late phase. Homocysteine at 1 mM also significantly (P = 0.017) reduced the A23187-induced vasodilation (Fig. 1B). However, the dilation of the retinal arterioles in response to the endothelium-independent NO donor sodium nitroprusside was unaffected by homocysteine (1 mM) (data not shown). Homocysteine did not change the resting vascular tone (data not shown). 

Figure 1

 

Effect of homocysteine on retinal reactivity. (A) Dilation of isolated retinal arterioles in response to bradykinin was examined before (control, n = 12) and after intraluminal incubation with 1 mM homocysteine (n = 6) or 100 μM homocysteine (n = 6) for 3 hours. (B) Representative trace shows that A23187 (3 μM) induced slight constriction within 1 minute and subsequent dilation of retinal arterioles within 3 minutes. (C) The retinal arteriolar dilation in response to A23187 (n = 4) was examined before and after intraluminal incubation with 1 mM homocysteine for 3 hours. *P < 0.05 versus control.

Roles of Superoxide, NAD(P)H Oxidase, and Xanthine Oxidase

In the presence of TEMPOL, the inhibition of bradykinin-induced vasodilation by homocysteine (1 mM) was prevented (Fig. 2A). This effect also was found in vessels treated with NAD(P)H oxidase inhibitor apocynin (Fig. 2B). However, the xanthine oxidase inhibitor allopurinol did not influence the homocysteine-mediated effect (Fig. 2B). These agents did not affect the basal tone (data not shown). 

Figure 2

 

Blockade of superoxide production or NAD(P)H oxidase activation. Dilation of the retinal arterioles to bradykinin was examined before (control, n = 5) and after intraluminal incubation with 1 mM homocysteine plus the superoxide anion scavenger TEMPOL (1 mM; n = 5). Dilation of the retinal arterioles in response to bradykinin was examined before (control, n = 9) and after intraluminal incubation with 1 mM homocysteine plus the NAD(P)H oxidase inhibitor apocynin (100 μM; n = 5) or the xanthine oxidase inhibitor allopurinol (10 μM; n = 4). *P < 0.05 versus control.

Roles of p38 Kinase and JNK Kinase

In the presence of the p38 kinase inhibitor SB203580 but not the JNK inhibitor SP600125, the detrimental effect of homocysteine on the vasodilatory response to bradykinin was prevented (Fig. 3). These agents did not affect the basal tone (data not shown). 

Figure 3

 

Blockade of p38 kinase activation. Dilation of the retinal arterioles in response to bradykinin was examined before (control, n = 9) and after intraluminal incubation with 1 mM homocysteine plus the p38 kinase inhibitor SB203580 (0.1 μM; n = 5) or the JNK inhibitor SP600125 (5 μM; n = 4). *P < 0.05 versus control.

Effect of Pioglitazone and Role of PPAR-γ Activation

Coadministration of homocysteine and pioglitazone prevented the detrimental effect of homocysteine on bradykinin-induced vasodilation (Fig. 4). However, the inhibitory effect of homocysteine on the vasodilatory response to bradykinin did not change in the presence of pioglitazone in combination with the PPAR-γ inhibitor GW9662 (Fig. 4). These agents did not significantly alter the basal tone (data not shown). 

Figure 4

 

Effects of coadministration of homocysteine with pioglitazone (Pio) and extraluminal incubation with the PPAR-γ inhibitor GW9662 (10 μM) for 1 hour. Dilation of the retinal arterioles in response to bradykinin was examined before (control, n = 4) and after intraluminal incubation with 1 mM homocysteine plus pioglitazone (0.5 μM; n = 4). Dilation of the retinal arterioles in response to bradykinin was examined before (control, n = 4) and after intraluminal incubation with 1 mM homocysteine plus pioglitazone (0.5 μM; n = 4) in the presence of GW9662. *P < 0.05 versus control.

Effect of Homocysteine on Superoxide Production

Without homocysteine (i.e., the vehicle control), DHE fluorescence showed sparse levels of superoxide in the vessel walls (Fig. 5A). In contrast, intraluminal incubation of vessels with homocysteine (1 mM, 180 minutes) significantly increased the superoxide level in the endothelial layer. Endothelial and smooth muscle layers were identified by setting the scanning threshold to obtain a clear background image of the vessel wall. TEMPOL significantly reduced the homocysteine-induced fluorescence signals for superoxide in the endothelium comparable to that observed in vessels without homocysteine (Fig. 5B). 

Figure 5

 

DHE fluorescence imaging of superoxide in the retinal arterioles. (A) Isolated and pressurized retinal arterioles were incubated intraluminally with vehicle (control), 1 mM homocysteine, or homocysteine and TEMPOL (1 mM) for 180 minutes, followed by addition of the oxidative fluorescent dye DHE and were imaged by confocal microscopy. The arrowheads indicate endothelial cells. Note the increased fluorescence reflecting superoxide levels in the endothelium (determined by the overly bright field image). (B) Quantitative analysis of DHE fluorescence signals for the experimental groups is shown in A. Data were obtained from four separate experiments. *P < 0.05 versus control. ^# P < 0.05 versus homocysteine.

Discussion

The current study was the first to show that homocysteine significantly reduced the dilation of the retinal arterioles in response to bradykinin and A23187 but not sodium nitroprusside, suggesting that homocysteine impairs retinal endothelial function regarding NO-mediated vasodilation. Similarly, it has been reported that acute exposure (30–180 minutes) of homocysteine at approximately the same concentration used in the current study can induce impaired endothelium-dependent vasodilation in rabbit ²⁴ and rat aortas ^31–34 and guinea-pig pulmonary arteries. ³⁵ Those findings support the current finding that homocysteine impairs the vascular endothelial function in the retinal arterioles. 

In addition to the reduced bradykinin-induced NO-mediated dilation, homocysteine reduced vasodilation of the retinal arterioles in response to the calcium ionophore A23187 (Fig. 1C), which activates endothelial NO synthase (eNOS) by increasing intracellular calcium independently of receptor activation ³⁶ and causing NO-dependent dilation in the retinal arterioles. ³⁷ In cultured human veins and renal microvascular endothelial cells, incubation with homocysteine (50 μM) for 24 hours can decrease release of NO in response to the eNOS stimuli, including bradykinin and A23187, with no change in eNOS protein expression. ³⁸ Moreover, it has been shown that the NO concentration can decrease after acute (4 hours) and chronic (24 hours) exposure of homocysteine (100 μM, 500 μM, or 1 mM) to bovine aortic and human vein endothelial cells without any change in eNOS activity or protein expression. ^34,39,40 Similarly, pretreatment with homocysteine (300 μM) for 2 hours can reduce NO elaboration of the calcium ionophore A23187 in porcine aorta segments in reactivity, ⁴¹ suggesting that homocysteine could damage endothelial cells by decreasing the bioavailability of NO, independent of changes in eNOS activity and protein expression. Taken together, we speculate that homocysteine might decrease NO bioavailability in the endothelium of retinal arterioles, leading to impaired endothelium-dependent dilation. 

Although the underlying mechanisms by which homocysteine reduces the NO bioavailability in retinal arterioles are not fully understood, NO bioavailability could decrease as a result of the reaction of NO with the superoxide anion or the reaction of the thiol groups in the homocysteine molecule with NO. ⁴² Indeed, many previous studies in human umbilical vein endothelial cells (HUVECs) have confirmed that ROS form after short (15 minutes or 1 hour) or long (24 hours) incubation with homocysteine at various concentrations (50 μM, 100 μM, or 1 mM). ^38,43,44 We also found that the inhibitory effect of homocysteine on endothelium-dependent dilation was prevented in the presence of TEMPOL, the membrane-permeable superoxide scavenger (Fig. 2). The salutary effect of TEMPOL seems specific, because this superoxide scavenger did not affect the resting basal tone or vasodilation to bradykinin without homocysteine. Consistent with our results, it has been reported that superoxide dismutase, a scavenger superoxide anion, alone or combined with catalase reverse the inhibitory effect of homocysteine on the endothelium-dependent dilation in rat aortas ^31–33 and guinea pig pulmonary arteries. ³⁵ Furthermore, a previous report has shown that the intracellular O₂ ⁻ scavenger Tiron also improves the impaired endothelium-dependent dilation by homocysteine in rabbit aortas. ²⁴ DHE staining provided further support for superoxide production and showed that homocysteine generates TEMPOL-sensitive superoxide in the endothelial layer of the retinal arterioles (Figs. 5A, 5B). Collectively, we speculate that oxidative stress might be involved in the mechanism of the homocysteine-induced endothelial dysfunction of the retinal arterioles. 

Homocysteine can induce endothelial toxicity by ROS generated via both enzymatic and nonenzymatic mechanisms. ^9,45 Superoxide can be generated by several enzymatic sources in vascular cells, including NAD(P)H oxidase and xanthine oxidase. The current study showed that homocysteine-induced impaired NO-mediated dilation was prevented by apocynin but not by allopurinol, suggesting that superoxide anions produced by NADPH oxidase are responsible for the inhibitory action of homocysteine (Fig. 2). Indeed, it has been reported that treatment with homocysteine (40 μM) for 6 hours can generate ROS via increasing NADPH oxidase expression in rat heart microvascular endothelial cells ⁹ and that blockade of NADPH oxidase by apocynin can inhibit homocysteine-induced intracellular superoxide anion production in HUVECs, ⁴⁶ which were consistent with the current results. Although a much higher concentration of homocysteine than found in plasma is readily oxidized nonenzymatically to form ROS and subsequently is cytotoxic to cultured endothelial cells, ⁴⁷ the effect of apocynin was comparable to that produced by TEMPOL and combination of apocynin and TEMPOL had no further blockade effect on the inhibitory effect of homocysteine on endothelium-dependent dilation of retinal arterioles (n = 3, data not shown), indicating that ROS generated during auto-oxidation could not be implicated in retinal endothelial dysfunction caused by homocysteine. Taken together, it is likely that homocysteine causes activation of NADPH oxidase and results in the ROS production in the endothelial cells of the retinal arterioles. 

The precise mechanisms leading to homocysteine-induced increase in superoxide production by NADPH oxidase are not fully understood. Although numerous studies have shown that stress-activated kinases, including p38 MAPK and JNK, are important signaling molecules in inflammation and oxidative stress, ^48,49 it is controversial whether stress-activated protein kinases are involved with apoptosis and ROS production in response to homocysteine. Although inhibition of p38 MAPK using the selective inhibitor SB203580 can significantly prevent homocysteine-mediated cell apoptosis in HUVECs, ⁵⁰ another study has reported that homocysteine can enhance apoptosis in HUVECs through a JNK-dependent mechanism, independent of p38 MAPK. ⁵¹ Coincident with the former finding, a function of p38 MAPK in the oxidative response by homocysteine is indicated by our observation that the p38 MAPK inhibitor SB203580 but not the JNK inhibitor SP600125 blocked the inhibitory effect of homocysteine on endothelium-dependent dilation in the retinal arterioles (Fig. 3). Furthermore, results from cultured lung endothelial cell studies suggested that p38 MAPK can contribute to regulation of NADPH oxidase. ⁵² Considering these findings together, we speculate that production of ROS by homocysteine is p38 MAPK-dependently generated from the endothelium of retinal arterioles, probably via activation of NADPH oxidase and subsequently causes retinal endothelial dysfunction. 

Because pioglitazone, a potent and selective activator of PPAR-γ, can improve the vascular endothelial function in human brachial arteries ⁵³ and exert antioxidant effects in the kidneys of obese rats ¹⁷ and human coronary endothelial cells, ¹⁸ the antioxidative properties of pioglitazone might prevent the impaired endothelium-dependent dilation caused by homocysteine in isolated retinal arterioles. Current data show that pioglitazone prevents the detrimental changes caused by homocysteine on endothelium-dependent vasodilation in the retinal arterioles (Fig. 4). Because the protective effect of pioglitazone against homocysteine-induced endothelial dysfunction was identical to the effect of TEMPOL in isolated retinal arterioles (Fig. 4), the current findings indicated that pioglitazone might improve the impaired endothelium-dependent dilation in response to homocysteine by reducing oxidative stress. A previous study also showed that pioglitazone inhibits the superoxide radical generated by stress stimuli such as tumor necrosis factor, oxidized low-density lipoprotein, and angiotensin II in human coronary artery endothelial cells. ¹⁸ Moreover, chronic administration of pioglitazone can normalize the increased superoxide level and significantly decrease the increased NADPH oxidase activity in the streptozotocin-induced diabetic rat aorta. ⁵⁴ Therefore, it is plausible that pioglitazone protects endothelial cells against homocysteine-induced oxidative stress by inhibiting NADPH oxidase in retinal arterioles. 

Despite the beneficial effects of PPAR-γ activation on the vasculature, the relationship between oxidative stress, which plays a critical role in the pathogenesis of cardiovascular disease, ⁵⁵ and PPAR-γ activation has not yet been elucidated. Indeed, genetic disruption of endothelial PPAR-γ can accelerate ROS production and impair endothelial-dependent vasodilation in rat aorta, ¹² indicating that PPAR-γ may play a potential role in the regulation of oxidative stress. Furthermore, since mRNA levels of PPAR-γ can be decreased and mRNA levels of NADPH oxidase, which catalyzes the production of ROS, simultaneously can be increased in a genetic model of hyperhomocysteinemia, ¹³ stimulation of PPAR-γ may reduce oxidative stress in response to homocysteine and subsequently protect retinal endothelial cells. In the current study, blockade of PPAR-γ attenuated the protective effect of pioglitazone on homocysteine-impaired endothelium-dependent dilation (Fig. 4), suggesting that pioglitazone reversed the inhibitory effect of homocysteine with a PPAR-γ-dependent mechanism. It has been shown that pioglitazone can inhibit hydrogen peroxide-induced activation of nuclear factor-κB, which has been implicated in the cellular response to oxidative stress, ⁵⁶ through PPAR-γ-dependent mechanisms in rat cardiac myocytes. ⁵⁷ Therefore, we postulated that pioglitazone mitigated oxidative stress induced by homocysteine by inhibiting activation of NAD(P)H oxidase with a PPAR-γ-dependent mechanism. Although the direct action of pioglitazone on the NOS activity in the endothelium of the retinal arterioles cannot be excluded, the current study did not support this because intraluminal treatment with pioglitazone alone for 180 minutes failed to enhance bradykinin-induced vasodilation (data not shown). 

It was reported that A23187 can induce calcium release from intracellular calcium stores in lung fibroblast cells within 1 minute. ⁵⁸ Furthermore, elevated intracellular calcium concentrations are thought to be a major mechanism of vasoconstriction. ⁵⁹ In addition to A23187-induced endothelium dilation, we found that A23187 rapidly produced slight constriction of retinal arterioles within 1 minute (Fig. 1B). These results indicated that A23187 reached smooth muscle in rapid phase with slight vasoconstriction and subsequently penetrated to endothelium of retinal arterioles with marked vasodilation. 

There is one limitation to this study that should be noted. We used a relatively higher concentration of homocysteine in the present study. Indeed, normal plasma homocysteine concentrations range from 5 to 15 μM in the fasting state. ⁶⁰ Moreover, mild or moderate hyperhomocysteinemia (10–100 μM), which is highly prevalent in the general population, ⁶¹ has been reported to be an independent risk factor for cardiovascular diseases. ⁶² In the current study, homocysteine at a dose of 1 mM but not at the dose of 100 μM, for 180 minutes inhibited endothelial-dependent dilation of the retinal arterioles (Fig. 1A). Although similar results have been reported in other vascular beds, ^24,31,32,35 the concentrations of homocysteine used in the current study seem to be higher compared with those experienced in hyperhomocysteinemia in humans. Therefore, a limitation of our in vitro study was that the use of a higher concentration of homocysteine may be required in vitro to reproduce the in vivo condition as closely as possible. 

In conclusion, we reported for the first time that homocysteine inhibits the endothelium-dependent NO-mediated dilation of isolated porcine retinal arterioles. The mechanism underlying the acute effect of homocysteine involves p38 kinase and production of superoxide by vascular NAD(P)H oxidase. Furthermore, pioglitazone protects against homocysteine-induced endothelial dysfunction in retinal arterioles, possibly through activation of PPAR-γ and reduction of oxidative stress. The current results showed that homocysteine impairs endothelial dilation of the retinal arterioles, indicating that hyperhomocysteinemia is implicated in the pathogenesis of ocular vascular disorders. A better understanding of the mechanisms by which homocysteine causes endothelial dysfunction of the retinal arterioles will help to develop strategies to prevent and treat retinal vascular diseases. 

Footnotes

 Supported by Grant-in-Aid for Scientific Research (C) 18591904 (TN) and Young Scientists (B) Grant 24791828 (TO) from the Ministry of Education, Science, and Culture, Tokyo, Japan, Uehara Memorial Foundation, Takeda Foundation, Science Foundation (TN), and Akiyama Life. The authors alone are responsible for the content and writing of the paper.

Footnotes

 Disclosure: T. Omae, None; T. Nagaoka, None; I. Tanano, None; A. Yoshida, None