The major findings of the present study are as follows: (1) vascular endothelium–dependent relaxation in response to acetylcholine in KKAy mice was improved strongly by azilsartan compared to candesartan cilexetil; (2) the ratio of Ser1177/Thr495 phosphorylation of eNOS, a putative marker for eNOS activation was impaired in KKAy mice, and the healthy ratio was effectively restored by azilsartan compared to candesartan cilexetil; (3) the differences in the expression levels of MCP1, F4/80, Nox2, and Nox4 of the aortic wall and in the expression of TNFα in the perivascular fat were attenuated by azilsartan compared to candesartan cilexetil. These data suggest that azilsartan restores endothelial function more effectively than does candesartan cilexetil, by normalizing eNOS function and by reducing inflammation and oxidative stress in diabetic mice.
Vascular endothelial dysfunction is implicated in the pathogenesis of cardiovascular diseases  and is well known to occur in obesity  and type 2 diabetes . The present study compared the efficacy of azilsartan and candesartan cilexetil on vascular reactivity, which reflects eNOS inactivation [24–26], and we measured phosphorylation of vascular eNOS as an indicator of eNOS activity. Enzymatic activity of eNOS is regulated by multiple phosphorylation of specific sites on the eNOS protein . When Ser1177 is phosphorylated, nitric oxide production is increased 2- to 3-fold. In contrast, Thr495 is a negative regulatory site of eNOS in that phosphorylation leads to decreased enzymatic activity . It has been reported that phosphorylation of Ser1177-eNOS is decreased in diabetic rats  and diabetic patients . Inversely, eNOS phosphorylation at Ser495 was reported to be increased in db/db diabetic mice . These data are consistent with our results showing that diabetic KKAy mice have a tendency for lower phosphorylation at Ser1177 and stronger phosphorylation at Thr495; these mice show a significantly decreased phosphorylation ratio of Ser1177 to Thr495 on eNOS compared to control C57BL6J mice. The Ser1177/Thr495 ratio was effectively normalized (increased) by azilsartan treatment compared to candesartan cilexetil. The kinases AMPK, Akt, protein kinase A (PKA), calmodulin/Ca2+ dependent protein kinase (CaMKII), protein kinase G (PKG) and the phosphatase protein phosphatase 2A (PP2A) have all been implicated in the regulation of eNOS-Ser1177 phosphorylation , while protein kinase C (PKC) has been shown to phosphorylate eNOS-Thr495. In addition, there is evidence for co-ordination between dephosphorylation of eNOS-Thr495 and activating phosphorylation of eNOS-Ser1177 . The mechanism(s) for the alteration of phosphorylation at Ser1177 and Thr495 in the diabetic state are largely unknown, however, altered signaling in AMPK, Akt, PKA, CaMKII, PKG, PP2A, and PKC in the diabetic state could be involved in the deregulated eNOS phosphorylation.
In the aortic wall of KKAy mice, mRNA expression levels of MCP1 and F4/80 tended to be increased, suggestive of overproduction of proinflammatory cytokines by activated macrophages/monocytes. Such proinflammatory cytokines frequently cause overproduction of ROS via excessive stimulation of reduced nicotinamide adenine dinucleotide phosphate . Cellular sources of ROS include NADPH-dependent oxidases, xanthine oxidase, lipoxygenases, mitochondrial oxidases, and NO synthases . NADPH oxidase (Nox) is a major source of ROS in diabetic humans  and diabetic animals . Seven isoforms of Nox have been described in mammals . Each isoform contains a core catalytic subunit, i.e., Nox1–Nox5 and dual oxidase (DUOX) 1 and DUOX 2 . Each Nox catalytic isoform contains up to 5 regulatory subunits that determine 1) maturation and expression of Nox and DUOX subunits in biological membranes, 2) enzyme activation, and 3) spatial organization. In our study, mRNA expression levels of Nox2 (gp91phox), the major catalytic component of endothelial NADPH oxidase, and Nox4, a component of endothelial and smooth muscle NADPH oxidase , were increased, suggesting that ROS production is increased via overexpression of Nox2 and Nox4 in KKAy mice. Reportedly, a siRNA-mediated knockdown of Nox2 (which is upregulated in diabetic endothelial cells) reduces ROS production and improves vascular function . It has also been reported that Nox4 is upregulated in an animal diabetic model  or as a result of hyperglycemia , with a concomitant increase in ROS. Taken together, upregulation of Nox2 and/or Nox4 in the aorta may be linked to ROS overproduction and vascular dysfunction in our murine model of diabetes.
The upregulation of ROS-producing Nox2 and Nox4 was decreased strongly by azilsartan as compared to candesartan cilexetil, according to the present results. We could not determine whether the greater inhibition of aortic Nox2 and Nox4 expression by azilsartan significantly sensitizes endothelial vasodilator response to acetylcholine. Angiotensin II levels are increased in patients with diabetes  and hyperglycemia potently upregulated expression of the angiotensin II type 1 receptor (AT1) ; thus, both could sensitize vascular cells to angiotensin II. Oak and Cai reported that streptozotocin-induced diabetes in mice is characterized by a marked increase in aortic ROS production, which is inhibited by NG-nitro-L-arginine methyl ester hydrochloride (L-NAME, inhibitor of nitric oxide synthase) in contrast to nondiabetic controls, indicating uncoupling of eNOS in the diabetic state . According to their data, angiotensin II receptor type 1 blocker candesartan decreased eNOS-derived ROS while augmenting nitric oxide bioavailability in diabetic aortas, which is suggestive of recoupling of eNOS. Nox activity was more than doubled in the endothelium-denuded diabetic aortas but this effect was attenuated by candesartan, indicating that Nox remains active in nonendothelial vascular tissues, although uncoupled eNOS is responsible for endothelial production of O2. They concluded that the dual effect on uncoupled eNOS and Nox might explain the high efficacy of angiotensin II antagonists in restoring endothelial function .
Going back to our results, because azilsartan has higher affinity for and slower dissociation from AT1 receptors  and shows stronger inverse agonism , these effects of azilsartan on AT1 receptor, as compared with candesartan cilexetil, may underlie the superior efficacy in diabetic vascular dysfunction via the dual effect on uncoupled eNOS and Nox. There is a report showing that angiotensin II-induced contraction was augmented in aorta rings isolated from diabetic rats and suggesting that the enhanced functional coupling of AT1 receptors results in supersensitivity to Ang II . The higher affinity of azilsartan for AT1 receptors may be beneficial for protecting angiotensin II-induced vascular remodeling in the diabetic condition . Clinical studies have exhibited that some benefits conferred by ARBs may not be class effects, but rather molecular effects . It was shown in a clinical study that in losartan users uric acid levels decrease from baseline, while they increase in users of other ARBs like valsartan, telmisartan, candesartan, and olmesartan . Among these ARBs, losartan uniquely exhibits a cis-inhibitory effect on the uptake of uric acid by the renal uric acid transporter (URAT1) . Partial chemical structures for the URAT1 competitive binding may involve an AT1 receptor-independent mechanism of action . In our study, plasma levels of uric acid increased comparably in KKAy mice treated with vehicle, candesartan cilexetil, or azilsartan as compared to C57BL/6 J, indicating no difference in uric acid metabolism between two ARBs. It has been reported that genetic disruption or pharmacological inhibition by telmisartan of the AT1R attenuates atherosclerosis and improves endothelial function in diabetic ApoE-/- mice via the PPARγ pathway . In 3 T3-L1 preadipocytes, azilsartan enhanced adipogenesis as well as effects on expression of PPARα, PPARδ, leptin, adipsin, and adiponectin . Azilsartan also potently inhibited vascular cell proliferation in the absence of exogenously supplemented angiotensin II or in cells lacking AT1 receptors . These findings suggest that azilsartan can function as a pleiotropic ARB with beneficial effects on actions that could involve more than just blockade of AT1 receptors and/or beyond their antihypertensive effects.
In the perivascular fat, mRNA expression levels of TNFα, MCP1, and Nox2 were increased in KKAy, and the overexpression of TNFα and Nox2 was attenuated only by azilsartan. Aortic expression of PPARγ2 was decreased in KKAy, but was not altered by candesartan cilexetil and azilsartan. We previously demonstrated that adiponectin secreted from perivascular adipose tissue has a protective role in neointimal formation after endovascular injury thanks to its anti-inflammatory properties, whereas perivascular adipose tissue–secreted TNFα plays an adverse atherogenic role in neointimal formation because of its proinflammatory effects [51, 52]. Kurata et al. reported that blockade of angiotensin II receptor ameliorates adipocytokine dysregulation and that such action is mediated, at least in part, by a reduction of oxidative stress in accumulated adipose tissue . In agreement with the previous report, mRNA expression of TNFα, MCP1, and Nox2 was increased in the perivascular fat from KKAy mice, and these anomalies in the expression of TNFα and Nox2 were attenuated only by azilsartan. Although the current study could not verify the direct link between vascular dysfunction and attenuation of adipocytokine dysregulation in perivascular fat, the possible role of perivascular fat in azilsartan-induced vascular remodeling should be assessed in future studies.
Although weight in the liver was increased, weight in the soleus and quadriceps muscle was decreased in the KKAy mice. A relative decrease of muscle mass as compared to body weight fails to metabolize abundant fat and worsens fat accumulation in the liver. Abundant fat with concomitant metabolic derangement underlies vascular dysfunction in the KKAy diabetic mice [53, 54]. In the soleus muscle of KKAy, mRNA expression levels of TNFα, MCP1, and Nox2 were increased and IRS-1 was decreased, but this overexpression was not attenuated by either candesartan cilexetil or azilsartan. It has been shown that azilsartan reduced left ventricular hypertrophy, cardiac fibrosis, plasminogen activator inhibitor-1 (PAI-1; a marker of profibrosis) in aortic banding mice fed high-fat diet , indicating that azilsartan may exert favorable biological effects in non-diabetic obese insulin-resistant condition, which shares a common mechanism such as enhanced ROS/inflammation signals with the current model.