May 2012
Volume 53, Issue 6
Free
Retina  |   May 2012
Fenofibrate, an Anti-Dyslipidemia Drug, Elicits the Dilation of Isolated Porcine Retinal Arterioles: Role of Nitric Oxide and AMP-Activated Protein Kinase
Author Notes
  • From the Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan. 
  • Corresponding author: Taiji Nagaoka, Department of Ophthalmology, Asahikawa Medical University, 2-1-1-1 Midorigaoka Higashi, Asahikawa, Japan 078-8510; nagaoka@asahikawa-med.ac.jp
Investigative Ophthalmology & Visual Science May 2012, Vol.53, 2880-2886. doi:10.1167/iovs.11-8841
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Tsuneaki Omae, Taiji Nagaoka, Ichiro Tanano, Takayuki Kamiya, Akitoshi Yoshida; Fenofibrate, an Anti-Dyslipidemia Drug, Elicits the Dilation of Isolated Porcine Retinal Arterioles: Role of Nitric Oxide and AMP-Activated Protein Kinase. Invest. Ophthalmol. Vis. Sci. 2012;53(6):2880-2886. doi: 10.1167/iovs.11-8841.

      Download citation file:


      © 2016 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

Purpose.: Although recent clinical trials have demonstrated that fenofibrate is effective for treating diabetic retinopathy, the mechanism of this beneficial effect remains unclear. In the current study, we examined the effect of the vasomotor action of fenofibrate on porcine retinal arterioles.

Methods.: Porcine retinal arterioles (internal diameter, 60–90 μm) were isolated, cannulated, and pressurized (55 cmH2O) without flow in vitro. Video-microscopic techniques recorded the diameter responses to fenofibrate.

Results.: The retinal arterioles dilated in a dose-dependent manner in response to fenofibrate (10 nM to 30 μM). This vasodilation significantly decreased after the endothelium was removed. Nω-nitro-L-arginine methyl ester (a nitric oxide [NO] synthase inhibitor), 1H-(1,2,4)oxadiazole(4,3-alpha)quinoxaline-1-one (a soluble guanylyl cyclase inhibitor), wortmannin (a phosphatidylinositol [PI] 3-kinase inhibitor), and compound C (an AMP-activated protein kinase inhibitor) attenuated the effect of fenofibrate-induced vasodilation to an extent comparable to that produced by denudation. Pretreatment with GW6471, a peroxisome proliferator-activated receptor-α blocker, did not significantly inhibit fenofibrate-induced vasodilation.

Conclusions.: Fenofibrate primarily elicited endothelium-dependent dilation of the retinal arterioles. The current findings suggested that fenofibrate-induced endothelium-dependent vasodilation is mediated by the release of NO, which probably mediates dilation via activation of guanylyl cyclase, the PI3-kinase pathway, and the AMP-activated protein kinase pathway. Understanding the vasodilatory effect of fenofibrate on the retinal microvasculature may improve potential therapy for diabetic retinopathy.

Introduction
Recently, the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study found that treatment with fenofibrate, which is clinically used to treat dyslipidemia, 1 was associated with a reduction in the need for laser therapy for diabetic retinopathy (DR) in patients with type 2 diabetes and good glycemic control (HbA1c < 7.0%). 2 In addition, the Action to Control Cardiovascular Risk in Diabetes (ACCORD) Eye study showed that adding fenofibrate to statin therapy significantly reduced the risk of DR progression in patients with type 2 diabetes, compared with statin monotherapy. 3 These two large-scale trials indicated that fenofibrate, which has an anti-inflammatory and anti-atherosclerotic effect on the artery wall in addition to a lipid-modifying effect, 4 may slow the progression of DR and be a novel therapy for DR in patients with type 2 diabetes mellitus. Although this effect does not seem to be related to the plasma concentrations of lipids, 2 the exact mechanisms underlying the beneficial effect of fenofibrate on DR in patients with type 2 diabetes remains uncertain. 
Although DR is a leading cause of blindness in adults worldwide, the exact pathogenesis of DR remains unclear. We recently reported that retinal blood flow (RBF) decreases in patients with type 2 diabetes mellitus with no retinopathy and mild DR. 5 This observation implies that improvements in impaired retinal microcirculation after intervention during the early stage of diabetic mellitus may prevent the development and progression of DR. Fenofibrate has been reported to stimulate endothelial cells to increase production of nitric oxide (NO). 69 Because NO is a powerful vasodilator in the retinal arterioles, fenofibrate may be effective against DR by improving RBF. However, the effect of fenofibrate on retinal microcirculation has not been previously examined. We investigated the effect of fenofibrate on the retinal microvascular diameter and the signaling mechanisms involved in this vasomotor activity, using an isolated vessel technique. 
Methods
Animal Preparation
All animal procedures were approved by the Animal Care Committee of Asahikawa Medical University and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Porcine eyes were enucleated immediately from pigs of either sex (age range, 16–24 weeks; weight range, 10–20 kg) after they were killed at a local abattoir and transported to the laboratory in a moist chamber on ice. The techniques to identify and isolate the retinal microvessels have been described previously. 1012 Briefly, the anterior segment and vitreous body were removed carefully under a dissecting microscope. The posterior segment was placed in a cooled dissection chamber (∼8°C) containing a physiologic salt solution (PSS—NaCl, 145.0 mM; KCl, 4.7 mM; CaCl2, 2.0 mM; MgSO4, 1.17 mM; NaH2PO4, 1.2 mM; glucose, 5.0 mM; pyruvate, 2.0 mM; EDTA, 0.02 mM; and MOPS, 3.0 mM) with 1% albumin. Single second-order retinal arterioles (0.6–1.0 mm in length) were carefully dissected with the aid of a stereomicroscope (Model SZX12; Olympus, Tokyo, Japan). After careful removal of any remaining neural/connective tissues, the arterioles then were transferred for cannulation to a polymethylmethacrylate vessel chamber containing PSS-albumin solution equilibrated with room air at ambient temperature. One end of the arteriole was cannulated with a glass micropipette filled with PSS-albumin solution, and the outside of the arteriole was tied securely to the pipette with an 11-0 ophthalmic suture (MANI, Tochigi, Japan). The other end of the vessel was cannulated with a second micropipette and secured with a suture. After cannulation, the vessel and pipettes were transferred to the stage of an inverted microscope (Model CKX41; Olympus) coupled to a video camera (WAT-902B; Watec Co., Ltd., Yamagata, Japan), a video micrometer (V-94; Living Systems Instrumentation, Burlington, VT), and a data acquisition system (PowerLab; ADInstruments, Colorado Springs, CO) for continuous measurement and recording of the internal diameter throughout the experiment. 10 The vessel was pressurized to an intraluminal pressure of 55 cmH2O (∼40 mm Hg) without flow, using two independent pressure reservoirs. This level of pressure was used based on previously documented pressure ranges in the retinal arterioles in vivo. 13 To examine the role of the endothelium, we intraluminally administered a pharmacologic inhibitor as described previously. 14 Briefly, a tube filled with the drug was inserted into the sidearm of the micropipette holder, and the micropipette was replaced with this drug. Flow was started into the vessel from the drug-filled micropipette by creating a pressure gradient (∼5 cmH2O) across two pressure reservoirs. After perfusing the vessels with the drug-containing solution for 10 minutes, the flow was stopped and the vessels were incubated with an inhibitor for at least 30 minutes before agonist stimulation. 
Experimental Protocols
Cannulated arterioles were bathed in PSS at a temperature of 36°C to 37°C to allow development of basal tone. After the vessels had developed a stable basal tone (approximately 30–40 minutes), the dose-dependent vasodilation in response to fenofibrate (10 nM to 30 μM) was evaluated based on evidence that the plasma level of fenofibrate can reach 10 nM to 30 μM in patients treated with therapeutic doses of fenofibrate. 15 After the control responses were completed, the vessels were washed with PSS to allow for redevelopment of basal tone. The vessels then were exposed to each concentration of agonist for 3 to 5 minutes until a stable diameter was established. The vasodilation elicited by fenofibrate was reexamined after 30 minutes to confirm the reproducibility of the response (n = 6). 
Endothelial Denudation
To elucidate the signaling mechanisms involved in the retinal arteriolar dilation induced by fenofibrate, the following series of experiments was performed. The role of the endothelium in fenofibrate-induced dilation was evaluated by comparing the responses before and after removal of the endothelium by luminal perfusion with the nonionic detergent CHAPS (0.4%) as described previously. 12,14,16 To ensure that the vascular smooth muscle function was not compromised by the CHAPS treatment, the dose-dependent dilation of the vessel in response to the endothelium-independent vasodilator sodium nitroprusside (SNP) (0.1–100 μM) was examined before and after denudation. Only vessels with normal basal tone with no vasodilation in response to the endothelium-dependent vasodilator bradykinin (10 nM) 17 and those with unaltered vasodilation in response to SNP after removal of the endothelium were used for further study with fenofibrate. 
Mechanistic Study of Fenofibrate-Induced Dilation
Role of Endothelium-Derived Factors.
The involvement of endothelium-derived vasodilators (i.e., prostaglandins, NO, and cytochrome-P450 metabolites) in mediating the vascular response was assessed in the presence of known effective concentrations of the specific enzyme inhibitors indomethacin (10 μM), 11,18 NG-nitro-L-arginine methyl ester (L-NAME) (10 μM), 10,11 and sulfaphenazole (10 μM), 19 respectively. The role of guanylyl cyclase/cyclic guanosine monophosphate (cGMP) signaling was assessed by treating the vessels with 0.1 μM soluble guanylyl cyclase inhibitor, 1H-1,2,4-oxadia- zolo[4,3-a]quinoxalin-1-one (ODQ). 11,12 To probe for the involvement of phosphatidylinositol (PI) 3-kinase and AMP-activated protein kinase (AMPK) in the endothelium, we studied the fenofibrate-induced response after incubating the vessels intraluminally with the PI3-kinase inhibitor wortmannin (0.1 μM) 20 and the AMPK inhibitor compound C (10 μM). 21 Moreover, because AMPK is expressed in both endothelial and smooth muscle cells, 22,23 we also administered extraluminal compound C (10 μM). 21 Since peroxisome proliferator–activated receptor α (PPARα) activated by fenofibrate is expressed in endothelial 24 and smooth muscle cells, 25 we examined the role of PPARα on the effect of fenofibrate using isolated vessels pretreated intraluminally and extraluminally with the PPARα antagonist GW6471 (5 μM) 26 before we examined the fenofibrate-induced response. Moreover, we also treated the vessels with the PPARα agonist WY14643 (10 nM to 30 μM) 27 to assess the direct effect of PPARα on this dilation. SNP (1 nM to 10 μM) was used to probe endothelium-independent NO-mediated vasodilation. All drugs were administered extraluminally unless otherwise stated. In addition to the extraluminal administration that may involve both the endothelium and smooth muscles, we also added the AMPK inhibitor compound C and PPARα inhibitor GW6471 intraluminally to confirm that drugs can reach the endothelium without permeation through smooth muscle extraluminally, because AMPK and PPARα are expressed in both the endothelium and smooth muscle. 2225 Each pharmacologic inhibitor was incubated with the vessels for at least 30 minutes. 
Chemicals
Compound C was obtained from Calbiochem (San Diego, CA). The other drugs were obtained from Sigma-Aldrich (St. Louis, MO). L-NAME and SNP were dissolved in PSS. Indomethacin, sulfaphenazole, and ODQ were dissolved in ethanol. Fenofibrate, wortmannin, GW6471, compound C, and WY14643 were dissolved in dimethyl sulfoxide (DMSO). Subsequent dilutions of these drugs were prepared in PSS. The final concentration of ethanol and DMSO in the vessel bath was less than 0.1%. 11 Vehicle control studies indicated that these final concentrations of solvents did not affect arteriolar function. 
Data Analysis
At the end of each experiment, the vessel was relaxed in EDTA (1 mM) calcium-free PSS, and the maximal vessel diameter at an intraluminal pressure of 55 cmH2O was measured. 10,12 All diameter changes in response to agonists were normalized according to this maximal vasodilation value and expressed as a percentage of the maximal dilation. 10,12 Data are reported as the mean ± SEM, and n represents the number of vessels studied. All variables were normally distributed (Kolmogorov–Smirnov test). Statistical comparisons of the changes in resting tone caused by the antagonists were performed using the Student's t-test. A two-way ANOVA, followed by the Bonferroni multiple-range test, was used to determine the significance of the difference between control and experimental interventions. A one-way AVOVA followed by a Dunnett post-hoc comparison was used to determine the significance of changes in the baseline diameter using different concentrations of agonists. P < 0.05 was considered significant. 
Results
Dilation of Retinal Arterioles Induced by Fenofibrate
All vessels (n = 72) had a basal tone in the range of 55% to 75% (average, approximately 68% ± 1%) of their maximal diameter when placed in a bath at a temperature of 36°C to 37°C and with an intraluminal pressure of 55 cmH2O. The average resting and maximal diameters of the vessels were 76 ± 2 and 112 ± 2 μm, respectively. Fenofibrate produced consistent dose-dependent dilation of the retinal arterioles, and the vasodilatory response reached its maximum within 2 to 3 minutes. The threshold concentration for vasodilation was 3 nM, and the highest concentration (30 μM) elicited a vasodilation of approximately 50% of the maximal dilation (Fig. 1). To avoid confounding effects produced by a high concentration of solvent (i.e., DMSO), concentrations of fenofibrate higher than 30 μM were not examined. Further study showed that fenofibrate-induced dilation was reproducible and did not deteriorate after repeated applications (Fig. 1). 
Figure 1.
 
Response of isolated retinal arterioles to fenofibrate. The dose-dependent vasodilatory effect of fenofibrate was examined (first trial, resting diameter: 81 ± 4 μm; maximum diameter: 113 ± 5 μm; n = 7) and then repeated after a 30-minute washout period (second trial, resting diameter: 81 ± 4 μm; maximum diameter 113 ± 5 μm; n = 7).
Figure 1.
 
Response of isolated retinal arterioles to fenofibrate. The dose-dependent vasodilatory effect of fenofibrate was examined (first trial, resting diameter: 81 ± 4 μm; maximum diameter: 113 ± 5 μm; n = 7) and then repeated after a 30-minute washout period (second trial, resting diameter: 81 ± 4 μm; maximum diameter 113 ± 5 μm; n = 7).
Role of the Endothelium
In this series of studies, 12 vessels were subjected to the denudation protocol. After perfusion with CHAPS, 3 of the 12 vessels lost their basal tone, and 3 showed partial inhibition by the endothelium-dependent vasodilator bradykinin. These apparently damaged or partially denuded vessels were excluded from further study. The remaining six vessels maintained their basal tone (control, 72% ± 2% vs. denudation, 72% ± 2%; P = 0.37), and the vasodilation induced by bradykinin (10 nM) was abolished (control, 85% ± 5% vs. denudation, 2% ± 1%). In addition, these vessels exhibited normal vasodilation in response to SNP (Table 1). In these accepted denuded vessels, the dilation induced by fenofibrate was greatly attenuated (P < 0.001) (Fig. 2). 
Table.
 
Diameter Responses of Retinal Arterioles to SNP
Table.
 
Diameter Responses of Retinal Arterioles to SNP
n SNP (μM)
0.1 1 10 100
Control 19 5.9 ± 0.9 21.6 ± 2.1 50.5 ± 0.7 83.4 ± 1.7
Denudation 5 9.4 ± 3.1 31.5 ± 5.4 52.7 ± 5.0 82.0 ± 1.2
L-NAME 4 7.2 ± 2.7 30.3 ± 6.7 52.2 ± 6.4 82.8 ± 1.1
Wortmannin 4 4.5 ± 1.1 18.1 ± 3.1 50.6 ± 5.2 83.6 ± 3.7
Compound C
Intraluminal 5 6.3 ± 1.7 17.4 ± 3.4 42.1 ± 6.2 80.9 ± 5.1
Extraluminal 4 8.6 ± 2.5 30.0 ± 10.2 55.2 ± 10.5 87.3 ± 5.4
WY-14643 6 5.3 ± 1.2 25.1 ± 7.0 62.6 ± 3.8 89.3 ± 0.6
Figure 2.
 
Role of endothelium in retinal arteriolar dilation in response to fenofibrate. The dose-dependent vasodilation in response to fenofibrate was examined before (control) and after the removal of the endothelium by perfusion with 0.4% CHAPS. *P < 0.05 versus control.
Figure 2.
 
Role of endothelium in retinal arteriolar dilation in response to fenofibrate. The dose-dependent vasodilation in response to fenofibrate was examined before (control) and after the removal of the endothelium by perfusion with 0.4% CHAPS. *P < 0.05 versus control.
Role of Endothelium-Derived Factors
The inhibition of cytochrome P450 epoxygenase and prostaglandins by sulfaphenazole and indomethacin, respectively, did not affect the vasodilatory response to fenofibrate (Fig. 3). Fenofibrate-induced vasodilation was significantly reduced by the nitric oxide synthase (NOS) inhibitor L-NAME (P < 0.001; Fig. 3) and was inhibited to the same extent by denudation (L-NAME versus denudation, P > 0.05; Figs. 2, 3). The basal tone was not significantly altered by sulfaphenazole (control, 68% ± 3% vs. sulfaphenazole, 68% ± 3%; P = 0.43), indomethacin (control, 65% ± 2% vs. indomethacin, 65% ± 2%; P = 0.62), or L-NAME (control, 69% ± 2% vs. L-NAME, 69% ± 2%; P = 0.77). ODQ significantly reduced the vasodilatory response to fenofibrate in a manner similar to L-NAME (Fig. 4). Similar to the effect of L-NAME, intraluminal wortmannin and compound C significantly (P < 0.001) reduced the fenofibrate-induced vasodilation (Fig. 5). Furthermore, extraluminal and intraluminal compound C significantly (P < 0.001) reduced fenofibrate-induced dilation to the same extent (Fig. 5), suggesting that fenofibrate elicited vasodilation of the retinal arterioles through activation of AMPK in the endothelial cells. These agents did not affect the basal tone (data not shown). 
Figure 3.
 
Role of endothelium-derived factor in the dilation of retinal arterioles in response to fenofibrate. The dose-dependent vasodilation induced by fenofibrate was examined before (control) and after incubation with the NOS inhibitor L-NAME (10 μM), the cyclooxygenase inhibitor indomethacin (10 μM), or the cytochrome-P450 epoxygenase inhibitor sulfaphenazole (10 μM). *P < 0.05 versus control.
Figure 3.
 
Role of endothelium-derived factor in the dilation of retinal arterioles in response to fenofibrate. The dose-dependent vasodilation induced by fenofibrate was examined before (control) and after incubation with the NOS inhibitor L-NAME (10 μM), the cyclooxygenase inhibitor indomethacin (10 μM), or the cytochrome-P450 epoxygenase inhibitor sulfaphenazole (10 μM). *P < 0.05 versus control.
Figure 4.
 
Role of guanylyl cyclase in the vasodilatory response to fenofibrate in isolated retinal arterioles. The dose-dependent vasodilatory response to fenofibrate was examined before (control) and after incubation with the soluble guanylyl cyclase inhibitor ODQ (0.1 μM). *P < 0.05 versus control.
Figure 4.
 
Role of guanylyl cyclase in the vasodilatory response to fenofibrate in isolated retinal arterioles. The dose-dependent vasodilatory response to fenofibrate was examined before (control) and after incubation with the soluble guanylyl cyclase inhibitor ODQ (0.1 μM). *P < 0.05 versus control.
Figure 5.
 
Role of the PI3-kinase and AMPK pathways in the vasodilatory response to fenofibrate in the endothelium of isolated retinal arterioles. The dose-dependent vasodilatory response was examined before (control) and after intraluminal incubation with the PI3-kinase inhibitor wortmannin and the AMPK inhibitor compound C. In addition, to investigate the involvement of AMPK in retinal smooth muscle arterioles, the dose-dependent vasodilatory response was examined before and after extraluminal pretreatment with compound C. *P < 0.05 versus control.
Figure 5.
 
Role of the PI3-kinase and AMPK pathways in the vasodilatory response to fenofibrate in the endothelium of isolated retinal arterioles. The dose-dependent vasodilatory response was examined before (control) and after intraluminal incubation with the PI3-kinase inhibitor wortmannin and the AMPK inhibitor compound C. In addition, to investigate the involvement of AMPK in retinal smooth muscle arterioles, the dose-dependent vasodilatory response was examined before and after extraluminal pretreatment with compound C. *P < 0.05 versus control.
Role of the PPARα Pathway
Intraluminal and extraluminal GW6471 did not significantly affect vasodilation in response to fenofibrate (Fig. 6A), indicating that the fenofibrate-induced dilation of the retinal arterioles was not involved in PPARα in both the endothelial cells and smooth muscle cells. In addition, extraluminal and intraluminal administration of the PPARα inhibitor did not affect the basal tone of the retinal arterioles (data not shown). Moreover, lower concentrations of WY14643 (<10 μM), a specific PPARα agonist, 27 did not significantly elicit dilation of the retinal arterioles, while a high concentration of WY14643 significantly dilated the retinal arterioles (Fig. 6B). 
Figure 6.
 
Role of PPARα pathway in the vasodilatory response to fenofibrate in isolated retinal arterioles. (A) The dose-dependent vasodilatory response to fenofibrate was examined before (control) and after extraluminal incubation with the PPARα inhibitor GW6471 (5 μM) and intraluminal incubation with GW6471 (5 μM). (B) To examine the dose-response effect of fenofibrate and WY14643, the retinal arterioles were treated with fenofibrate and WY14643, respectively (10 nM to 30 μM). *P < 0.05 versus control. # P < 0.05 versus baseline.
Figure 6.
 
Role of PPARα pathway in the vasodilatory response to fenofibrate in isolated retinal arterioles. (A) The dose-dependent vasodilatory response to fenofibrate was examined before (control) and after extraluminal incubation with the PPARα inhibitor GW6471 (5 μM) and intraluminal incubation with GW6471 (5 μM). (B) To examine the dose-response effect of fenofibrate and WY14643, the retinal arterioles were treated with fenofibrate and WY14643, respectively (10 nM to 30 μM). *P < 0.05 versus control. # P < 0.05 versus baseline.
Response to SNP
Various interventions did not affect the SNP-induced dilation of the retinal arterioles (Table 1), suggesting that the vascular smooth muscle function was unaltered by these interventions. 
Discussion
Fenofibrate has attracted attention as a novel therapeutic agent because of its beneficial effect in patients with DR in two clinical studies, the FIELD study 2 and the ACCORD Eye study. 3 However, the underlying mechanism responsible for the beneficial effect of fenofibrate is poorly understood. In the current study, we clarified, for the first time, that fenofibrate has a vasodilatory effect on isolated retinal arterioles. Because the concentration of serum fenofibric acid, the active metabolite of fenofibrate, reportedly reaches a concentration of about 30 μM for 3.5 hours when oral fenofibrate is administered to human subjects at a therapeutic dose, 15 our findings suggested that fenofibrate elicited a dose-dependent vasodilation of the retinal arterioles of 30% to 40% at a therapeutic concentration (Fig. 1). Therefore, the concentrations of fenofibrate used in the current study are considered clinically relevant. Because we recently reported that RBF may be impaired in patients with type 2 diabetes with no or early-stage retinopathy, 5 we believe that the vasodilatory actions of fenofibrate observed in the retinal arterioles in the current study may increase the RBF, possibly protecting against the onset or progression of DR. 
The current data showed that endothelial disruption by CHAPS 14 almost completely inhibited fenofibrate-induced vasodilation (Fig. 2). Although no study has examined whether the effect of fenofibrate on the vessels is endothelium dependent or independent, bezafibrate, another fibrate, did not significantly induce vasodilation in guinea-pig mesenteric artery without the endothelium. 28 The current findings suggested that the endothelium, but not the smooth muscle, is mainly involved in the fenofibrate-induced vasodilation of retinal arterioles. 
In the current study, we investigated the possible role of endothelium-derived vasodilator factors such as NO, prostaglandin, and endothelium-derived hyperpolarizing factor in fenofibrate-induced vasodilation and found that fenofibrate-induced dilation decreased significantly with blockade of NOS and denudation to the same extent (Fig. 3). Because fenofibrate reportedly phosphorylates eNOS in human umbilical vein endothelial cells (HUVEC) 7,8 and in human glomerular microvascular 9 and upregulates endothelial NOS (eNOS) activity in human glomerular microvascular 9 and bovine aortic endothelial cells, 6 the current results suggested that NO may be highly involved in the endothelial-dependent vasodilation induced by fenofibrate in retinal arterioles. However, the vasodilatory response to fenofibrate was not affected by inhibition of the synthesis of prostacyclin and cytochrome P450 metabolites, which are generated by the endothelium as vasodilators (Fig. 3), although no reports have investigated the acute vasodilatory effects of this agent in relation to prostacyclin and cytochrome P450 metabolites. Taken together, we concluded that NO from the endothelium was the main contributor to the fenofibrate-induced vasodilation, independent of prostaglandin and cytochrome P450 metabolites. 
In the current experiment, fenofibrate elicited dilation of the retinal arterioles within 5 minutes (data not shown). Although no studies have examined whether fenofibrate rapidly phosphorylates eNOS in the retinal vascular endothelial cells, one study reported that the phosphorylation of eNOS increased from 2.5 to 10 minutes after fenofibrate treatment in HUVECs. 7 Although the species differed, their results support our hypothesis that fenofibrate phosphorylates eNOS in retinal endothelial cells within 5 minutes. 
In the current study, similar to the inhibitory effect of L-NAME and denudation, pretreatment with ODQ significantly inhibited the fenofibrate-induced vasodilation (Fig. 4). Since the NO/cGMP pathway is generally considered to be a major vasodilatory mechanism, the fenofibrate-induced vasodilation of the retinal arterioles may occur by NO release in the endothelium and consequent cGMP production in the smooth muscle cells. 
Inhibition of the PI3-kinase/Akt pathway by wortmannin decreased fenofibrate-induced vasodilation to an extent comparable to that produced by L-NAME (Fig. 5). Fenofibrate rapidly phosphorylates Akt, which plays an important role in the phosphorylation of eNOS, 29,30 in HUVECs. 8 Although no study has examined whether fenofibrate can cause rapid phosphorylation of Akt in retinal endothelial cells, fenofibrate may elicit vasodilation of the retinal arterioles via the Akt/NO pathway. Further studies of the time course are needed. 
AMPK, which is expressed in both endothelial 23 and smooth muscle cells, 22 is reportedly phosphorylated by fenofibrate in HUVECs within 2.5 to 10 minutes. 7 Consistent with this previous report, we found that the fenofibrate-induced dilation of retinal arterioles was significantly inhibited by the AMPK inhibitor compound C intraluminally in a manner comparable to the inhibitory effect of wortmannin and L-NAME (Fig. 5). In addition, not only intraluminal but also extraluminal administration of compound C inhibited fenofibrate-induced vasodilation to the same extent, suggesting that AMPK in endothelial cells might be involved in this vasodilation. Therefore, fenofibrate might elicit dilation of the retinal arterioles via the AMPK/NO and the PI3-kinase/Akt/NO pathways in endothelial cells. Because previous reports have shown that activation of AMPK by fenofibrate occurs upstream from that of Akt 8 and that PI3-kinase is located upstream of AMPK, 31 we assumed that activation of the PI3-kinase/AMPK/Akt/NO pathway in the endothelial cells may play an important role in the fenofibrate-induced vasodilation of retinal arterioles. 
Although fenofibrate, an agonist of PPARα, is expressed in endothelial 24 and smooth muscle cells, 25 both intraluminal and extraluminal administration of the PPARα antagonist GW6471 was unaffected by fenofibrate-induced vasodilation, indicating that PPARα may not be associated with fenofibrate-induced vasodilation. Moreover, because only a high concentration of WY14643, a specific PPARα agonist, slightly dilated the retinal arterioles (Fig. 6B), the activation of PPARα may have only a weak vasodilatory effect on the retinal arterioles. Because numerous reports have shown that the fenofibrate-induced phosphorylation of AMPK is independent of PPARα, 7,32 the current results imply that fenofibrate-induced vasodilation via activation of AMPK was mainly regulated by a PPARα-independent pathway. 
Our study had some limitations. One is the inability to determine whether fenofibrate may have a beneficial effect on retinal vessels in a diseased state, especially those affected by DR. Previous reports have indicated that fibrates improve retinal hard exudates in patients with DR. 33 Moreover, recent reports have demonstrated that pharmacologic agents including fenofibrate prevent apoptosis in bovine retinal pericytes 34 and human retinal endothelial cells, 35 which deteriorate in DR, 36 through activation of AMPK. Therefore, we speculated that activation of AMPK in response to fenofibrate in the retinal endothelial cells may be responsible for the fenofibrate-induced vasodilation of retinal arterioles. Second, because we examined only the acute effect of fenofibrate on the retinal arterioles in the current study, the effects of chronic administration of fenofibrate on the retinal circulation are unknown. Because chronic administration of fenofibrate impairs endothelium-dependent vasodilation in reaction to acetylcholine by increased vasoconstrictor prostanoid release in rat aorta, 37 another clinical study is needed to examine the effect of chronic administration of fenofibrate on the retinal circulation in in-vivo and human studies. 
In summary, we showed that fenofibrate elicited potent dilation of the retinal arterioles via an endothelial-dependent pathway. The current findings suggested that the endothelial-dependent dilation induced by fenofibrate may be mediated via activation of the AMPK and PI3-kinase/Akt/eNOS signal pathways in endothelial cells, resulting in NO release and consequent activation of the soluble guanylyl cyclase/cGMP pathway (Fig. 7). Because the RBF decreases during the early stage of DR in patients with type 2 diabetic mellitus, 5 fenofibrate-induced vasodilation may be associated with beneficial effects on DR, which have been demonstrated in recent large-scale clinical trials. 2,3 Further clinical study is required to determine whether fenofibrate can improve ocular blood flow in patients with type 2 diabetes. 
Figure 7.
 
Schematic illustration of proposed signaling mechanisms involved in retinal arteriolar dilation in response to fenofibrate. Inhibition of these signaling pathways by their respective inhibitors is indicated by the vertical lines in reference to the direction of the straight line.
Figure 7.
 
Schematic illustration of proposed signaling mechanisms involved in retinal arteriolar dilation in response to fenofibrate. Inhibition of these signaling pathways by their respective inhibitors is indicated by the vertical lines in reference to the direction of the straight line.
References
Staels B Dallongeville J Auwerx J Schoonjans K Leitersdorf E Fruchart JC . Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation . 1998;98:2088–2093. [CrossRef] [PubMed]
Keech AC Mitchell P Summanen PA Effect of fenofibrate on the need for laser treatment for diabetic retinopathy (FIELD study): a randomised controlled trial. Lancet . 2007;370:1687–1697. [CrossRef] [PubMed]
Chew EY Ambrosius WT Davis MD Effects of medical therapies on retinopathy progression in type 2 diabetes. N Engl J Med . 2010;363:233–244. [CrossRef] [PubMed]
Touyz RM Schiffrin EL . Peroxisome proliferator-activated receptors in vascular biology-molecular mechanisms and clinical implications. Vascul Pharmacol . 2006;45:19–28. [CrossRef] [PubMed]
Nagaoka T Sato E Takahashi A Yokota H Sogawa K Yoshida A . Impaired retinal circulation in patients with type 2 diabetes mellitus: retinal laser Doppler velocimetry study. Invest Ophthalmol Vis Sci . 2010;51:6729–6734. [CrossRef] [PubMed]
Goya K Sumitani S Xu X Peroxisome proliferator-activated receptor alpha agonists increase nitric oxide synthase expression in vascular endothelial cells. Arterioscler Thromb Vasc Biol . 2004;24:658–663. [CrossRef] [PubMed]
Murakami H Murakami R Kambe F Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC. Biochem Biophys Res Commun . 2006;341:973–978. [CrossRef] [PubMed]
Okayasu T Tomizawa A Suzuki K Manaka K Hattori Y . PPARalpha activators upregulate eNOS activity and inhibit cytokine-induced NF-kappaB activation through AMP-activated protein kinase activation. Life Sci . 2008;82:884–891. [CrossRef] [PubMed]
Tomizawa A Hattori Y Inoue T Hattori S Kasai K . Fenofibrate suppresses microvascular inflammation and apoptosis through adenosine monophosphate-activated protein kinase activation. Metabolism . 2011;60:513–522. [CrossRef] [PubMed]
Hein TW Yuan Z Rosa RHJr, Kuo L . Requisite roles of A2A receptors, nitric oxide, and KATP channels in retinal arteriolar dilation in response to adenosine. Invest Ophthalmol Vis Sci . 2005;46:2113–2119. [CrossRef] [PubMed]
Hein TW Xu W Kuo L . Dilation of retinal arterioles in response to lactate: role of nitric oxide, guanylyl cyclase, and ATP-sensitive potassium channels. Invest Ophthalmol Vis Sci . 2006;47:693–699. [CrossRef] [PubMed]
Nagaoka T Hein TW Yoshida A Kuo L . Simvastatin elicits dilation of isolated porcine retinal arterioles: role of nitric oxide and mevalonate-rho kinase pathways. Invest Ophthalmol Vis Sci . 2007;48:825–832. [CrossRef] [PubMed]
Glucksberg MR Dunn R . Direct measurement of retinal microvascular pressures in the live, anesthetized cat. Microvasc Res . 1993;45:158–165. [CrossRef] [PubMed]
Omae T Nagaoka T Tanano I Yoshida A . Pioglitazone, a peroxisome proliferator-activated receptor-{gamma} agonist, induces dilation of isolated porcine retinal arterioles: role of nitric oxide and potassium channels. Invest Ophthalmol Vis Sci . 2011;52:6749–6756. [CrossRef] [PubMed]
Keating GM Croom KF . Fenofibrate: a review of its use in primary dyslipidaemia, the metabolic syndrome and type 2 diabetes mellitus. Drugs . 2007;67:121–153. [CrossRef] [PubMed]
Hein TW Kuo L . cAMP-independent dilation of coronary arterioles to adenosine: role of nitric oxide, G proteins, and K(ATP) channels. Circ Res . 1999;85:634–642. [CrossRef] [PubMed]
Haefliger IO Flammer J Luscher TF . Heterogeneity of endothelium-dependent regulation in ophthalmic and ciliary arteries. Invest Ophthalmol Vis Sci . 1993;34:1722–1730. [PubMed]
Moncada S Vane JR . Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A2, and prostacyclin. Pharmacol Rev . 1978;30:293–331. [PubMed]
Earley S Pastuszyn A Walker BR . Cytochrome p-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia. Am J Physiol Heart Circ Physiol . 2003;285:H127–136. [CrossRef] [PubMed]
Arcaro A Wymann MP . Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J . 1993;296 (pt 2);297–301. [PubMed]
Thors B Halldorsson H Jonsdottir G Thorgeirsson G . Mechanism of thrombin mediated eNOS phosphorylation in endothelial cells is dependent on ATP levels after stimulation. Biochim Biophys Acta . 2008;1783:1893–1902. [CrossRef] [PubMed]
Rubin LJ Magliola L Feng X Jones AW Hale CC . Metabolic activation of AMP kinase in vascular smooth muscle. J Appl Physiol . 2005;98:296–306. [CrossRef] [PubMed]
Davis BJ Xie Z Viollet B Zou MH . Activation of the AMP-activated kinase by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo by promoting the association of heat shock protein 90 and endothelial nitric oxide synthase. Diabetes . 2006;55:496–505. [CrossRef] [PubMed]
Inoue I Shino K Noji S Awata T Katayama S . Expression of peroxisome proliferator-activated receptor alpha (PPAR alpha) in primary cultures of human vascular endothelial cells. Biochem Biophys Res Commun . 1998;246:370–374. [CrossRef] [PubMed]
Staels B Koenig W Habib A Activation of human aortic smooth-muscle cells is inhibited by PPARalpha but not by PPARgamma activators. Nature . 1998;393:790–793. [CrossRef] [PubMed]
Xu HE Stanley TB Montana VG Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARalpha. Nature . 2002;415:813–817. [CrossRef] [PubMed]
Forman BM Chen J Evans RM . Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc Natl Acad Sci U S A . 1997;94:4312–4317. [CrossRef] [PubMed]
Nakamura Y Ohya Y Onaka U Fujii K Abe I Fujishima M . Inhibitory action of insulin-sensitizing agents on calcium channels in smooth muscle cells from resistance arteries of guinea-pig. Br J Pharmacol . 1998;123:675–682. [CrossRef] [PubMed]
Dimmeler S Fleming I Fisslthaler B Hermann C Busse R Zeiher AM . Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature . 1999;399:601–605. [CrossRef] [PubMed]
Fulton D Gratton JP McCabe TJ Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature . 1999;399:597–601. [CrossRef] [PubMed]
Zou MH Kirkpatrick SS Davis BJ Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo: role of mitochondrial reactive nitrogen species. J Biol Chem . 2004;279:43940–43951. [CrossRef] [PubMed]
Araki H Tamada Y Imoto S Analysis of PPARalpha-dependent and PPARalpha-independent transcript regulation following fenofibrate treatment of human endothelial cells. Angiogenesis . 2009;12:221–229. [CrossRef] [PubMed]
Harrold BP Marmion VJ Gough KR . A double-blind controlled trial of clofibrate in the treatment of diabetic retinopathy. Diabetes . 1969;18:285–291. [CrossRef] [PubMed]
Cacicedo JM Benjachareonwong S Chou E Yagihashi N Ruderman NB Ido Y . Activation of AMP-activated protein kinase prevents lipotoxicity in retinal pericytes. Invest Ophthalmol Vis Sci . 2011;52:3630–3639. [CrossRef] [PubMed]
Kim J Ahn JH Kim JH Fenofibrate regulates retinal endothelial cell survival through the AMPK signal transduction pathway. Exp Eye Res . 2007;84:886–893. [CrossRef] [PubMed]
Mizutani M Kern TS Lorenzi M . Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy. J Clin Invest . 1996;97:2883–2890. [CrossRef] [PubMed]
Blanco-Rivero J Marquez-Rodas I Xavier FE Long-term fenofibrate treatment impairs endothelium-dependent dilation to acetylcholine by altering the cyclooxygenase pathway. Cardiovasc Res . 2007;75:398–407. [CrossRef] [PubMed]
Footnotes
 Supported by a Grant-in-Aid for Scientific Research (C) (18591904) from the Ministry of Education, Science, and Culture, Tokyo, Japan, Uehara Memorial Foundation, Takeda Foundation, and Akiyama Life Science Foundation (TN).
Footnotes
 Disclosure: T. Omae, None; T. Nagaoka, None; I. Tanano, None; T. Kamiya, None; A. Yoshida, None
Figure 1.
 
Response of isolated retinal arterioles to fenofibrate. The dose-dependent vasodilatory effect of fenofibrate was examined (first trial, resting diameter: 81 ± 4 μm; maximum diameter: 113 ± 5 μm; n = 7) and then repeated after a 30-minute washout period (second trial, resting diameter: 81 ± 4 μm; maximum diameter 113 ± 5 μm; n = 7).
Figure 1.
 
Response of isolated retinal arterioles to fenofibrate. The dose-dependent vasodilatory effect of fenofibrate was examined (first trial, resting diameter: 81 ± 4 μm; maximum diameter: 113 ± 5 μm; n = 7) and then repeated after a 30-minute washout period (second trial, resting diameter: 81 ± 4 μm; maximum diameter 113 ± 5 μm; n = 7).
Figure 2.
 
Role of endothelium in retinal arteriolar dilation in response to fenofibrate. The dose-dependent vasodilation in response to fenofibrate was examined before (control) and after the removal of the endothelium by perfusion with 0.4% CHAPS. *P < 0.05 versus control.
Figure 2.
 
Role of endothelium in retinal arteriolar dilation in response to fenofibrate. The dose-dependent vasodilation in response to fenofibrate was examined before (control) and after the removal of the endothelium by perfusion with 0.4% CHAPS. *P < 0.05 versus control.
Figure 3.
 
Role of endothelium-derived factor in the dilation of retinal arterioles in response to fenofibrate. The dose-dependent vasodilation induced by fenofibrate was examined before (control) and after incubation with the NOS inhibitor L-NAME (10 μM), the cyclooxygenase inhibitor indomethacin (10 μM), or the cytochrome-P450 epoxygenase inhibitor sulfaphenazole (10 μM). *P < 0.05 versus control.
Figure 3.
 
Role of endothelium-derived factor in the dilation of retinal arterioles in response to fenofibrate. The dose-dependent vasodilation induced by fenofibrate was examined before (control) and after incubation with the NOS inhibitor L-NAME (10 μM), the cyclooxygenase inhibitor indomethacin (10 μM), or the cytochrome-P450 epoxygenase inhibitor sulfaphenazole (10 μM). *P < 0.05 versus control.
Figure 4.
 
Role of guanylyl cyclase in the vasodilatory response to fenofibrate in isolated retinal arterioles. The dose-dependent vasodilatory response to fenofibrate was examined before (control) and after incubation with the soluble guanylyl cyclase inhibitor ODQ (0.1 μM). *P < 0.05 versus control.
Figure 4.
 
Role of guanylyl cyclase in the vasodilatory response to fenofibrate in isolated retinal arterioles. The dose-dependent vasodilatory response to fenofibrate was examined before (control) and after incubation with the soluble guanylyl cyclase inhibitor ODQ (0.1 μM). *P < 0.05 versus control.
Figure 5.
 
Role of the PI3-kinase and AMPK pathways in the vasodilatory response to fenofibrate in the endothelium of isolated retinal arterioles. The dose-dependent vasodilatory response was examined before (control) and after intraluminal incubation with the PI3-kinase inhibitor wortmannin and the AMPK inhibitor compound C. In addition, to investigate the involvement of AMPK in retinal smooth muscle arterioles, the dose-dependent vasodilatory response was examined before and after extraluminal pretreatment with compound C. *P < 0.05 versus control.
Figure 5.
 
Role of the PI3-kinase and AMPK pathways in the vasodilatory response to fenofibrate in the endothelium of isolated retinal arterioles. The dose-dependent vasodilatory response was examined before (control) and after intraluminal incubation with the PI3-kinase inhibitor wortmannin and the AMPK inhibitor compound C. In addition, to investigate the involvement of AMPK in retinal smooth muscle arterioles, the dose-dependent vasodilatory response was examined before and after extraluminal pretreatment with compound C. *P < 0.05 versus control.
Figure 6.
 
Role of PPARα pathway in the vasodilatory response to fenofibrate in isolated retinal arterioles. (A) The dose-dependent vasodilatory response to fenofibrate was examined before (control) and after extraluminal incubation with the PPARα inhibitor GW6471 (5 μM) and intraluminal incubation with GW6471 (5 μM). (B) To examine the dose-response effect of fenofibrate and WY14643, the retinal arterioles were treated with fenofibrate and WY14643, respectively (10 nM to 30 μM). *P < 0.05 versus control. # P < 0.05 versus baseline.
Figure 6.
 
Role of PPARα pathway in the vasodilatory response to fenofibrate in isolated retinal arterioles. (A) The dose-dependent vasodilatory response to fenofibrate was examined before (control) and after extraluminal incubation with the PPARα inhibitor GW6471 (5 μM) and intraluminal incubation with GW6471 (5 μM). (B) To examine the dose-response effect of fenofibrate and WY14643, the retinal arterioles were treated with fenofibrate and WY14643, respectively (10 nM to 30 μM). *P < 0.05 versus control. # P < 0.05 versus baseline.
Figure 7.
 
Schematic illustration of proposed signaling mechanisms involved in retinal arteriolar dilation in response to fenofibrate. Inhibition of these signaling pathways by their respective inhibitors is indicated by the vertical lines in reference to the direction of the straight line.
Figure 7.
 
Schematic illustration of proposed signaling mechanisms involved in retinal arteriolar dilation in response to fenofibrate. Inhibition of these signaling pathways by their respective inhibitors is indicated by the vertical lines in reference to the direction of the straight line.
Table.
 
Diameter Responses of Retinal Arterioles to SNP
Table.
 
Diameter Responses of Retinal Arterioles to SNP
n SNP (μM)
0.1 1 10 100
Control 19 5.9 ± 0.9 21.6 ± 2.1 50.5 ± 0.7 83.4 ± 1.7
Denudation 5 9.4 ± 3.1 31.5 ± 5.4 52.7 ± 5.0 82.0 ± 1.2
L-NAME 4 7.2 ± 2.7 30.3 ± 6.7 52.2 ± 6.4 82.8 ± 1.1
Wortmannin 4 4.5 ± 1.1 18.1 ± 3.1 50.6 ± 5.2 83.6 ± 3.7
Compound C
Intraluminal 5 6.3 ± 1.7 17.4 ± 3.4 42.1 ± 6.2 80.9 ± 5.1
Extraluminal 4 8.6 ± 2.5 30.0 ± 10.2 55.2 ± 10.5 87.3 ± 5.4
WY-14643 6 5.3 ± 1.2 25.1 ± 7.0 62.6 ± 3.8 89.3 ± 0.6
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×