Adiponectin-Induced Dilation of Isolated Porcine Retinal Arterioles via Production of Nitric Oxide From Endothelial Cells

Abstract

Purpose.: Adiponectin, an important adipocytokine secreted by adipocytes, has anti-inflammatory and atheroprotective effects on vascular tissue via the adiponectin receptor (adipoR). However, the action of adiponectin in the retinal microcirculation is unknown. We examined the direct effect and underlying mechanism of the vasomotor action of adiponectin in porcine retinal arterioles.

Methods.: Porcine retinal arterioles (internal diameter, 60–90 μm) were isolated, cannulated, and pressurized (55 cmH₂O) without flow in this in vitro study. Videomicroscopic techniques were used to record changes in diameter in response to adiponectin.

Results.: The retinal arterioles dilated in a dose-dependent (0.125–7.5 μg/mL) manner in response to adiponectin. The vasodilation decreased significantly after removal of the endothelium. N^G-nitro-L-arginine methyl ester (a nitric oxide [NO] synthase inhibitor), 1H-1,2,4-oxadia-zolo[4,3-a]quinoxalin-1-one (a soluble guanylyl cyclase inhibitor), but not wortmannin (a phosphatidylinositol 3-kinase inhibitor) inhibited the effect of adiponectin-induced vasodilation comparable with that of denudation. Pretreatment with compound C, an activated protein kinase (AMPK) inhibitor, partially but significantly reduced vasodilation. Incubation with GW6471, a peroxisome proliferator-activated receptor blocker, did not significantly inhibit vasodilation by adiponectin. AdipoR1 and adipoR2 immunoreactions were observed in the endothelium of retinal arterioles.

Conclusions.: Adiponectin elicits mainly endothelium-dependent dilation of the retinal arterioles. Endothelium-dependent vasodilation likely induced by adiponectin results from NO via activation of guanylyl cyclase that is partially dependent on AMPK activity. Understanding the effect of adiponectin on the retinal vasculature may help improve potential therapies for retinal vascular disorders, especially diabetic retinopathy in patients with type 2 diabetes mellitus.

Introduction

Many clinical reports have shown that obesity, which is the accumulated state of excessive adipose tissue, is a major risk factor for cardiovascular diseases. ¹ However, adipocytes not only are storage reservoirs for fat, but also are active endocrine organs that play multiple metabolic roles in regulating systemic physiology. ² Adiponectin is an important adipocytokine secreted by adipocytes and abundant plasma protein in the circulation. ^3,4 Since low plasma adiponectin concentrations (hypoadiponectinemia) are associated with endothelial dysfunction, ⁵ which is involved in macrovascular and microvascular diseases, ^6,7 adiponectin may play a protective role in the pathogenesis of vascular diseases. Indeed experimental studies have shown that adiponectin inhibits inflammation, which occurs during the early stage of atherosclerosis ⁸ in vitro ^9,10 and in vivo, ¹¹ via activation of adiponectin receptors (adipoR) expressed in multiple tissues including liver, ¹² skeletal muscle, ¹³ macrophages, ¹⁴ and vascular endothelial cells. ¹⁵  

Although the results of previous clinical studies that examined the relationship between plasma adiponectin concentrations and severity of diabetic retinopathy (DR) remain inconsistent, ^16–18 some experimental studies have reported that adiponectin generated nitric oxide (NO), ^19,20 a powerful vasodilator of retinal arterioles, ²¹ in endothelial cells. These experimental observations suggested that additional investigation is needed to assess the relation of adiponectin with DR. Furthermore, numerous studies have shown that retinal blood flow (RBF) is reduced at an early stage in patients with type 1 diabetes ^22,23 and in streptozotocin-induced diabetic rat models. ²⁴ Recently, our previous clinical study found that RBF decreased before the development and progression of DR in patients with type 2 diabetes. ²⁵ Therefore, if adiponectin has a potential to cause vasodilation of retinal vessels, it is likely that a decreased serum adiponectin level may impair the RBF, resulting in development of retinopathy, in patients with type 2 diabetes mellitus. However, it has not been fully clarified whether adiponectin per se can influence the retinal microcirculation. In the current study, we examined the direct effect of adiponectin on the retinal arterioles and investigated the underlying signaling mechanisms involved in this vasomotor activity using isolated vessels. 

Materials and Methods

Animal Preparation

All animal procedures were approved by the Animal Care Committee of Asahikawa Medical University and 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, 25–35 kg) after they were killed in a local abattoir and transported to the laboratory in a moist chamber on ice. 

Isolation and Cannulation of Microvessels

The techniques used to identify, isolate, cannulate, pressurize, and visualize the retinal vasculature have been described previously. ^26–29 Briefly, the anterior segment and vitreous body were removed. The posterior segment was placed in a cooled dissection chamber (∼8°C) in physiologic salt solution (PSS). Single second-order retinal arterioles (90–130 μm in situ) were dissected with microdissection forceps and the isolated retinal arterioles 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, two independent pressure reservoirs were used to pressurize the vessel to 55 cmH₂O intraluminal pressure without flow. ³⁰ The internal diameter of the isolated vessels was recorded continuously using videomicroscopic techniques during the experiment. ²⁶  

Experimental Protocols

Cannulated arterioles were bathed in PSS at 36°C to 37°C to allow development of basal tone. After a stable basal tone developed (∼30–40 minutes), the dose-dependent vasodilation to various concentrations of adiponectin (dose range, 0.125–7.5 μg/mL) was constructed based on evidence that the plasma levels of adiponectin exceeded 3 μg/mL in clinically normal subjects. ⁴ After the control responses were completed, the vessels were washed with PSS to allow redevelopment of basal tone. The vasodilation elicited by adiponectin was reexamined after 30 minutes to confirm the reproducibility of the response. The vessels were exposed to each concentration of agonists for 10 to 15 minutes until a stable diameter was established. 

To elucidate the signaling mechanisms involved in the retinal arteriolar dilation induced by adiponectin, the following series of experiments was performed. The role of the endothelium in the adiponectin-induced dilation was evaluated by comparing the responses before and after removal of the endothelium by luminal perfusion of nonionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (0.4%) as described previously. ^28,29,31 To ensure that the vascular smooth muscle function was not compromised by CHAPS, the dose-dependent vessel dilation in response to the endothelium-independent vasodilator sodium nitroprusside (SNP; 0.1–100 μM) was examined before and after denudation. Only vessels that exhibited normal basal tone showed no vasodilation in response to the endothelium-dependent vasodilator bradykinin (10 nM), ³² and the vessels with unaltered vasodilation in response to SNP after removal of the endothelium were accepted for further study with adiponectin. 

The involvement of endothelium-derived vasodilators (i.e., prostaglandins, NO, and cytochrome P450 [CYP] metabolites) in mediating the vascular response was assessed in the presence of known effective concentrations of the specific enzyme inhibitors indomethacin (10 μM), ^27,33 N^G-nitro-L-arginine methyl ester (L-NAME; 10 μM) ^26,27 ; sulfaphenazole, the CYP 2C9 inhibitor (10 μM) ³⁴ ; and miconazole, the nonselective CYP inhibitor (10 μM), ³⁵ respectively. The role of guanylyl cyclase/cyclic guanosine monophosphate (cGMP) signaling was assessed by treating the vessels with the soluble guanylyl cyclase inhibitor 1H-1,2,4-oxadia- zolo[4,3-a]quinoxalin-1-one (ODQ; 0.1 μM). ^27,28 To probe the involvement of phosphatidyl inositol (PI) 3-kinase and AMP activated-protein kinase (AMPK), which is expressed in endothelial and smooth muscle cells, ^36,37 we studied the adiponectin-induced response after incubation of the vessels with the PI3-kinase inhibitor wortmannin (0.1 μM) ³⁸ and the AMPK inhibitor compound C (10 μM). ³⁹ Moreover, because peroxisome proliferator-activated receptor (PPAR)-α also is expressed in endothelial ⁴⁰ and smooth muscle cells, ⁴¹ we examined the role of PPAR-α on the effect of adiponectin using the isolated vessels pretreated with the PPAR-α antagonist GW6471 (3 μM) ⁴² before we examined the adiponectin-induced response. SNP (0.1–10 μM) was used to probe endothelium-independent NO-mediated vasodilation. 

All drugs were administered extraluminally unless otherwise stated. Each pharmacologic inhibitor was incubated with the vessels for at least 30 minutes. 

Measurement of Nitrite/Nitrate

The stable NO end products nitrite and nitrate, collectively NOx, were measured by high-performance liquid chromatography (ENO-20; Eicom, Kyoto, Japan). We collected samples from the chamber 15 minutes after adiponectin 5 μg/mL administration. After injection of 10 μL of the pretreated sample into the system, NOx production was measured using the Griess method. ⁴³  

Immunohistochemistry

To identify and localize the vascular adipoR, retinal arterioles were embedded and frozen in OCT compound (Tissue-Tek; Sakura Finetek, Torrance, CA). Frozen sections (10-μm thick) were fixed in 4% paraformaldehyde and immunolabeled with specific primary antibodies (adipoR1 and adipoR2; Phoenix Pharmaceuticals, Belmont, CA) and either an anti-α-smooth muscle actin (SMA) antibody (Sigma-Aldrich, St. Louis, MO) or an anti-endothelial NO synthase (eNOS; Santa Cruz Biotechnology, Santa Cruz, CA) antibody. The slides then were incubated with Cy3-labeled or fluorescein-labeled (GE Healthcare Life Sciences, Piscataway, NJ) secondary antibodies and observed for red (Cy3) and green (fluorescein) images and analyzed using a confocal microscope (Fluoview FV 1000; Olympus, Tokyo, Japan). To exclude nonspecific staining, negative controls were performed by omitting the primary antibody and included in every staining series. Merged images were created with Java-based imaging software (ImageJ; National Institutes of Health, Bethesda, MD). 

Chemicals

Adiponectin was obtained from R&D Systems (Minneapolis, MN). Compound C was obtained from Calbiochem (San Diego, CA). Other drugs were obtained from Sigma-Aldrich. L-NAME and SNP were dissolved in PSS. Indomethacin, sulfaphenazole, miconazole, and ODQ were dissolved in ethanol. Wortmannin, GW6471, and compound C 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%. ²⁷ Vehicle control studies indicated that this final solvent concentration did not affect the arteriolar function. 

Data Analysis

At the end of each experiment, the vessel was relaxed in ethylenediaminetetraacetic acid (1 mM) calcium-free PSS, to obtain its maximal diameter at 55 cmH₂O intraluminal pressure. ^26,28 All diametric changes in response to agonists were normalized to this maximal vasodilation and expressed as a percentage of the maximal dilation. ^26,28 Data are reported as the mean ± standard error mean (SEM); n represents the number of vessels studied. Statistical comparisons of the changes in resting tone by antagonists were performed using the Student's t-test. Statistical differences in NOx production between adiponectin and vehicle treatment were examined using the Mann-Whitney U test. Two-way analysis of variance (ANOVA), followed by the Bonferroni multiple-range test, was used to determine the significance of the difference between control and experimental interventions. One-way AVOVA followed by Dunnett's 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 Adiponectin

The basal tone in all vessels (n = 70) ranged from 55% to 78% (average, ∼68% ± 1%) of their maximal diameters in a 36°C to 37°C bath temperature with 55 cmH₂O intraluminal pressure. The average resting and maximal diameters of the vessels were 77 ± 1 μm and 113 ± 2 μm, respectively. Adiponectin produced consistent dose-dependent dilation of the retinal arterioles, and the vasodilatory response reached its maximal level within 10 to 15 minutes. The threshold concentration for vasodilation was 0.5 μg/mL and the highest concentration (7.5 μg/mL) elicited approximately 40% of the maximal dilation (Fig. 1). A higher concentration (10 μg/mL) of adiponectin did not elicit further dilation of the retinal arterioles (data not shown). Further study showed that adiponectin-induced dilation was reproducible and did not deteriorate after repeated applications (Fig. 1). 

Figure 1

 

Response of isolated retinal arterioles to adiponectin. The dose-dependent vasodilatory effect of adiponectin is examined (first trial, resting diameter, 77 ± 4 μm; maximal diameter, 109 ± 4 μm; n = 8) and repeated after a 30-minute washout period (second trial, resting diameter, 77 ± 4 μm; maximal diameter, 109 ± 4 μm; n = 8). *P < 0.05 versus baseline.

Role of Endothelium

Eight vessels were subjected to the denudation protocol. After perfusion with CHAPS, two of the eight vessels lost basal tone and two showed partial inhibition by the endothelium-dependent vasodilator bradykinin. These apparently damaged or partially denuded vessels were excluded from further study. The remaining four vessels maintained basal tone (control, 67% ± 1% versus denudation, 65% ± 2%; P = 0.21) and the vasodilation induced by bradykinin (10 nM) was abolished (control, 87% ± 2% versus denudation, 2% ± 2%). These vessels also exhibited normal vasodilation in response to SNP (Table). In these accepted denuded vessels, the dilation in response to the lower concentrations of adiponectin (<5 μg/mL) was almost abolished, and the response to the highest adiponectin concentration decreased from 50% to 15% (P = 0.001, Fig. 2). 

Figure 2

 

Effect of the removal of the endothelium by perfusion with 0.4% CHAPS. *P < 0.05 versus control.

Table

 

Diametric Responses of the Retinal Arterioles to SNP

	Vessels, n	SNP, μM
		0.1	1	10	100
Control	10	5.1 ± 0.7	23.4 ± 3.5	49.6 ± 3.1	85.4 ± 3.2
Denudation	4	7.4 ± 0.8	32.2 ± 3.1	53.8 ± 3.0	86.9 ± 3.7
L-NAME	4	6.2 ± 1.7	22.8 ± 4.9	47.4 ± 6.7	81.6 ± 10.9
Compound C	5	3.6 ± 0.5	23.0 ± 6.9	48.0 ± 4.4	82.4 ± 4.5
Compound C + L-NAME	4	4.2 ± 1.8	21.9 ± 2.1	46.5 ± 2.8	86.7 ± 3.6

 Data are expressed as the mean ± SEM. Based on two-way ANOVA, compared with controls, the responses to SNP are unaffected by any perturbations.

Role of Endothelium-Derived Factors

Inhibition of cytochrome P450 epoxygenase and prostaglandins by sulfaphenazole, miconazole, and indomethacin, respectively, did not affect the vasodilatory response to adiponectin (Fig. 3A). The NOS inhibitor L-NAME inhibited adiponectin-induced vasodilation (P < 0.001, Fig. 3A) comparable with that produced by denudation (L-NAME versus denudation; P > 0.05; Figs. 2, 3A). These agents did not significantly alter the basal tone. 

Figure 3

 

(A) Effect of incubation with the NOS inhibitor L-NAME (10 μM), the cyclooxygenase inhibitor indomethacin (10 μM), the CYP2C9 inhibitor sulfaphenazole (10 μM), and the nonselective CYP inhibitor miconazole (10 μM). *P < 0.05 versus control. (B) The NOx production response to adiponectin (5 μg/mL) or vehicle was examined 15 minutes after injection of adiponectin or vehicle into the chamber. *P < 0.05 versus vehicle.

NO Production

To assess whether NO increased in the isolated retinal arteriole as a result of adiponectin, we measured NOx in the chamber after adiponectin administration. The NOx levels in the chamber significantly increased after adiponectin compared with the vehicle (Fig. 3B). 

Role of Guanylyl Cyclase

ODQ significantly reduced the vasodilatory response to adiponectin in a manner similar to L-NAME (Fig. 4). The basal tone was not significantly altered by ODQ. 

Figure 4

 

Effect of incubation with the soluble guanylyl cyclase inhibitor ODQ (0.1 μM). *P < 0.05 versus control.

Role of PI3kinase and AMPK

Compound C, but not wortmannin, partly but significantly reduced the adiponectin-induced vasodilation (Fig. 5), suggesting that adiponectin elicited vasodilation of the retinal arterioles by AMPK. These agents did not significantly affect the basal tone. 

Figure 5

 

Effect of incubation with the PI3-kinase inhibitor wortmannin and the AMPK inhibitor compound C. *P < 0.05 versus control.

Role of PPARα Pathway

Administration of the PPARα inhibitor did not affect the basal tone of the retinal arterioles (data not shown). In addition, GW6471 did not significantly affect vasodilation in response to adiponectin (Fig. 6), indicating that adiponectin-induced dilation of the retinal arterioles was not involved with PPARα in the vessel wall. 

Figure 6

 

Effect of incubation with the PPARα inhibitor GW6471 (5 μM).

Response to SNP

SNP (0.1–100 μM) was used to probe endothelium-independent NO-mediated vasodilation. The vascular response to SNP was examined in the presence of various interventions as mentioned previously (Table). 

Localization of AdipoR1 and AdipoR2

In the retinal arterioles, adipoR1 and adipoR2 immunoreactions occurred in the vascular endothelium but not the vascular smooth muscle layer (Fig. 7). No staining signals were detected in the negative control experiments in which the specific antibody was omitted (data not shown). 

Figure 7

 

Immunohistochemical detection of adipoRs in the isolated retinal arterioles. (A) In the presence of anti-adipoR1 receptor (green) or eNOS (red) antibodies, immunostaining is seen in the endothelium for adipoR1 and anti-eNOS. The merged image shows overlapping staining (yellow) in the endothelial layer. (B) In the presence of anti-adipoR2 (green) and anti-eNOS (red) antibodies, high levels of immunostaining are seen in the endothelium for both proteins, which is confirmed by overlapping staining (yellow). (C) In the presence of anti-adipoR1 (green) and anti-α-SMA (red) antibodies, selective immunostaining is seen in the endothelium for adipoR1 and in smooth muscle for eNOS, respectively. The merged image shows no overlapping staining. (D) In the presence of anti-adipoR2 (green) and anti-SMA (red) antibodies, high levels of immunostaining are seen in the endothelium for adipoR2 and in smooth muscle for eNOS, respectively. The merged image shows no overlapping staining. Arrowheads indicate the endothelial cells. Arrows indicate the vascular smooth muscle cells. Data represent three separate experiments. Scale bars: 50 μm.

Discussion

It is unclear if the serum adiponectin level is associated with the pathogenesis of DR. ^16–18 Because adiponectin induced production of NO in the vascular endothelial cells ^19,20 and elicited approximately 35% to 40% vasodilation in murine mesenteric arteries at the same concentration, ²⁰ it is possible that adiponectin may be involved in RBF regulation. The current study found that adiponectin elicited dose-dependent vasodilation of the retinal arterioles, with approximately 40% of the dilation at the high concentration (7.5 μg/mL; Fig. 1). This was the first study to report the vasodilatory action of adiponectin in the retinal arterioles. Since plasma adiponectin levels generally range from 2 to 17 μg/mL in normal subjects, ⁴ the 7.5 μg/mL serum adiponectin level among the normal concentrations might be sufficient to cause vasodilation of the retinal arterioles (Fig. 1). 

In isolated rat aorta, ⁴⁴ endothelial ablation significantly abolished vasodilation in response to globular adiponectin, indicative of the C-terminal globular region of adiponectin. ⁴⁵ We found that adiponectin-induced dilation, with the exception of the high concentration (Fig. 2), was greatly inhibited in the retinal arterioles after the endothelium was removed, suggesting that adiponectin mainly elicits endothelial-dependent vasodilation of isolated retinal vessels. 

The current study also found that inhibition of NOS in adiponectin-induced vasodilation of the retinal arterioles was comparable with that resulting from endothelial disruption (Figs. 2, 3A). It has reported that adiponectin induced vasodilation by NO production in murine mesenteric arteries ²⁰ and phosphorylated eNOS in human umbilical vein endothelium cells (HUVECs) ^20,46 and bovine aortic endothelium cells (BAECs). ^19,47 We found that adiponectin increased the NOx level in the chamber with isolated retinal arterioles (Fig. 3B), suggesting that adiponectin can produce NO in the retinal arterioles. Taken together, these results supported our finding that endothelial-derived NO contributed to the adiponectin-induced vasodilation of the retinal arterioles. However, blockade of cyclooxygenase and cytochrome P450 epoxygenase did not alter retinal arteriolar dilation in response to adiponectin, indicating that adiponectin can elicit dilation of the retinal arterioles via activation of NO from the endothelium independent of prostaglandins and cytochrome P450 metabolites, which are the other two vasodilators secreted from the endothelium. 

It is widely accepted that cGMP elevation in vessels occurs in response to NO-mediated activation of soluble guanylyl cyclase and subsequently vasodilation, ⁴⁸ while it has reported that cGMP-independent mechanisms for activation of NO may also exist in rabbit aorta ⁴⁹ and rat middle cerebral arteries. ⁵⁰ Therefore, the signaling molecules responsible for the adiponectin-induced NO-mediated dilation in the retinal arterioles remain to be determined. In the current study, inhibition of soluble guanylyl cyclase by ODQ was associated with a significant decrease in the dilation of the retinal arterioles in response to adiponectin (Fig. 4), in the same manner as that of denudation (Fig. 2) and L-NAME (Fig. 3A). Moreover, because subsequent administration of ODQ to L-NAME-treated vessels did not further reduce the vasodilation (n = 3, data not shown), it seems that adiponectin mediates dilation of the retinal arterioles through activation of NO/cGMP signaling. 

AMPK, which plays a key role in modulating vascular reactivity in the endothelium ³⁶ and smooth muscle cells, ³⁷ is involved in NO production and subsequent dilation of the retinal arterioles. ²⁹ The current study showed that blockade of AMPK with compound C partially but significantly suppressed adiponectin-induced dilation of the retinal arterioles (Fig. 5), suggesting that AMPK might be involved in this dilation. Previous reports have described that transient transfection with a dominant-inhibitory mutant of AMPK partially inhibited NO production in response to adiponectin in BAECs, ¹⁹ whereas adenovirus-mediated overexpression of a dominant-negative version of AMPK completely blocked adiponectin-mediated NO release and phosphorylation of eNOS in HUVECs. ²⁰ Thus, although it remains unclear to what extent AMPK affects NO production in response to adiponectin, coadministration of L-NAME and compound C inhibited vasodilation of the retinal arterioles in response to adiponectin in the same manner as L-NAME (Fig. 5), indicating that dilation of the retinal arterioles by adiponectin may occur via activation of eNOS partially in an AMPK-dependent manner. 

We found that adiponectin rapidly elicited vasodilation of the retinal arterioles for 10 to 15 minutes (data not shown). Most previous reports have shown that adiponectin phosphorylated eNOS for 5 to 30 minutes in HUVECs ²⁰ and BAECs. ^19,47 Moreover, AMPK, which is involved in NO generation in response to adiponectin as mentioned previously, is phosphorylated by adiponectin within 15 to 30 minutes ²⁰ and 15 to 120 minutes in HUVECs. ⁴⁷ Since we showed that NO production increased in the chamber 15 minutes after adiponectin treatment (Fig. 3B), adiponectin can generate NO from the retinal endothelial cells for 10 to 15 minutes and subsequently dilate the isolated retinal arterioles. 

Despite the fact that inhibition of PI3-kinase-Akt with wortmannin significantly suppressed adiponectin-evoked phosphorylation of eNOS in BAECs, ¹⁹ we found that vasodilation of the retinal arterioles in response to adiponectin was not inhibited by wortmannin pretreatment (Fig. 5). Consistent with our findings, expressing dominant-negative Akt did not affect NO production in response to adiponectin in BAECs. ¹⁹ Collectively, although further investigations are needed to confirm the upstream kinases responsible for eNOS phosphorylation by adiponectin, PI3-kinase/Akt activity does not seem necessary for dilation of the retinal arterioles in response to adiponectin. 

Adiponectin can increase fatty acid combustion via PPARα activation, thereby decreasing triglyceride content in the liver and skeletal muscle. ^51,52 In the presence of GW6471, a PPARα antagonist, vasodilation of the retinal arterioles in response to adiponectin was unaffected (Fig. 6), suggesting that adiponectin may elicit dilation of the retinal arterioles independent of PPARα. 

Although many studies have indicated that adipoR is expressed in multiple tissues, ^12–15 no study has reported adipoR expression in the retinal vessels. We first confirmed that adipoR1 and adipoR2 are expressed in the vascular endothelium but not in the smooth muscle layer of the retinal arterioles (Fig. 7), which agrees with previous reports that adipoR1 and adipoR2 are expressed in HUVECs ^15,53 and BAECs. ^15,54 Because simultaneous downregulation of adipoR1 and adipoR2 but not each receptor alone significantly suppressed adiponectin-induced NO production in HUVECs, ²⁰ we speculate that both receptors may play an important role in the reaction of NO production to adiponectin in the retinal arterioles. 

The current study had two limitations. The first limitation was the difficulty in accurately determining the involvement of the adipoR in adiponectin-induced dilation of the retinal arterioles. A recent study reported that both adipoR1 and adipoR2 are linked to involvement of adiponectin-induced NO production in endothelial cells. ²⁰ However, because there is no commercially available blocker of adipoR1 and/or adipoR2, we could not clarify whether either receptor or both are associated with adiponectin-induced vasodilation of the retinal arterioles. Further studies are required to examine the implication of these receptors in this dilation. The second limitation was the difficulty in determining if adiponectin can exert slight but significant vasodilation of the retinal arterioles at less than normal plasma levels (0.5–2.5 μg/mL). This vasoreactivity of the retinal arterioles at a pathophysiologic concentration in vitro indicated that any substances that impaired the dilatory reaction to adiponectin may exist in vivo. The ratio of leptin to adiponectin is correlated with the carotid intima-media thickness, ⁵⁵ an independent predictor of cardiovascular diseases. In addition, leptin per se stimulated superoxide production in BAECs. ⁵⁶ Although these observations suggested that leptin affects vasodilation of the retinal arterioles in response to adiponectin, our preliminary experiment showed that adiponectin-induced dilation was unaltered by leptin (5 ng/mL) ⁵⁷ (data not shown), indicating that leptin likely does not have any effect on the adiponectin-induced dilation of the retinal arterioles. Thus, we did not identify the reason for vasodilation elicited by adiponectin at pathophysiologic concentrations (0.5–2.5 μg/mL) in the current study. Further elucidation of factors that affect the retinal vasodilatory response to adiponectin is needed. 

In summary, we showed that adiponectin, an adipocytokine, elicits potent dilation of the retinal arterioles with the endothelium-dependent pathway, which is mediated via activation of the eNOS signal pathway partly by AMPK for NO release and consequent activation of the soluble guanylyl cyclase/cGMP pathway (Fig. 8). In addition, adipoR1 and adipoR2 are expressed in the vascular endothelium but not in the smooth muscle layer of the retinal arterioles. Taken together, adiponectin induces vasodilation of the retinal arterioles through activation of adipoR1 and/or adipoR2 in the retinal endothelial cells. Because the RBF is impaired in early-stage DR in patients with type 2 diabetes mellitus, ²⁵ therapeutic interventions that enhance the actions of adiponectin may lead to a novel potential treatment. Further clinical study is needed to determine if elevation of the serum adiponectin level by pharmacologic interventions can improve the impaired RBF in patients with type 2 diabetes and retinal vascular disorders. 

Figure 8

 

Schematic illustration of proposed signaling mechanisms involved in retinal arteriolar dilation in response to adiponectin. Inhibition of these signaling pathways by their respective inhibitors is indicated by the vertical lines in reference to the direction of the straight line.

Acknowledgments

Supported by a Grant-in-Aid for Scientific Research (C) 18591904 (TN) and Young Scientists (B) 24791828 (TO) from the Ministry of Education, Science, and Culture, Tokyo, Japan. 

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