Ginkgolide B caused the activation of the Akt/eNOS pathway through the antioxidant effect of SOD1 in the diabetic aorta.

Ginkgo biloba extract (GBE) helps lower cardiovascular disease risk. Diabetes mellitus (DM)-induced endothelial dysfunction is a critical and initiating factor in the beginning of diabetic vascular complications. It was reported that GBE causes an endothelial-dependent relaxation. This study was designed to figure out the molecular basis on which GBE protects from endothelial dysfunction in diabetes because the underlying mechanisms are unclear. Studies were performed in a normal control group and streptozotocin/nicotinamide-induced DM group. In aortas, notably diabetic aortas, GBE, and ginkgolide B (GB), a constituent of GBE, produced a dose-dependent relaxation. The relaxation by GB was abolished by prior incubation with L-NNA (an endothelial nitric oxide synthase (NOS) inhibitor), LY294002 (a phosphoinositide 3-kinase (PI3K) inhibitor), and Akt inhibitor, confirming the essential role of PI3K/Akt/eNOS signaling pathway. We also demonstrated that GB induced the phosphorylation of Akt and eNOS in aortas. The superoxide dismutase1 (SOD1) expression level decreased in DM aortas, but GB stimulation increased SOD activity and SOD1 expression in DM aortas. Our novel findings suggest that in DM aortas, endothelial-dependent relaxation induced by GB was mediated by activation of SOD1, resulting in activation of the Akt/eNOS signaling pathway.

Indoxyl sulfate decreases uridine adenosine tetraphosphate-induced contraction in rat renal artery.

The protein-bound uremic toxin indoxyl sulfate has negative effects on a variety of physiological activities including vascular function. Uridine adenosine tetraphosphate (Up4A), a new dinucleotide molecule affects vascular function including induction of vasocontraction, and aberrant responsiveness to Up4A is evident in arteries from disorders such as hypertension and diabetes. The link between indoxyl sulfate and the Up4A-mediated response is, however, unknown. We used Wistar rat's renal arteries to see if indoxyl sulfate will affect Up4A-mediated vascular contraction. In renal arteries of indoxyl sulfate, the contractile response generated by Up4A was dramatically reduced compared to the non-treated control group. Indoxyl sulfate increased endothelin-1-induced contraction but had no effect on phenylephrine, thromboxane analog, or isotonic K+-induced renal arterial contractions. UTP, ATP, UDP, and ADP-produced contractions were reduced by indoxyl sulfate. CH223191, an aryl hydrocarbon receptor (AhR) antagonist, did not reverse Up4A, and UTP contraction decreases caused by indoxyl sulfate. The ectonucleotidase inhibitor ARL67156 prevents indoxyl sulfate from reducing Up4A- and UTP-mediated contractions. In conclusion, we discovered for the first time that indoxyl sulfate inhibits Up4A-mediated contraction in the renal artery, possibly through activating ectonucleotidase but not AhR. Indoxyl sulfate is thought to play a function in the pathophysiology of purinergic signaling.

Methylglyoxal impairs ATP- and UTP-induced relaxation in the rat carotid arteries.

Although methylglyoxal (MGO), a highly reactive dicarbonyl compound, influences the functioning of the vasculature, modulating its effects on vascular reactivity to various substances remains unclear, especially purinoceptor ligands. Therefore, we sought to investigate the direct effects of MGO on relaxation induced by adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) in isolated rat carotid arteries. When carotid arteries were exposed to MGO (420 µM for 1 h), relaxation induced by acetylcholine or sodium nitroprusside was not affected by MGO. However, ATP- and UTP-induced relaxation was impaired by MGO compared with the control. In both ATP- and UTP-induced relaxation, endothelial denudation, incubation with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine or the selective P2Y purinoceptor 2 (P2Y2) receptor antagonist AR-C118925XX reduced relaxation in both the control and MGO groups, while the differences between the control and MGO groups were eliminated. The cyclooxygenase (COX) inhibitor indomethacin inhibited the differences in ATP/UTP-mediated relaxations between the control and MGO groups. Moreover, N-acetyl-L-cysteine (NAC), an antioxidant, could augment carotid arterial relaxation induced by ATP/UTP in the presence of MGO. MGO increased arachidonic acid-induced contraction, which was suppressed by NAC. Following both ATP/UTP stimulation, MGO increased the release of prostanoids. These results suggest that MGO impaired ATP- and UTP-induced relaxation in carotid arteries, which was caused by suppressed P2Y2 receptor-mediated signaling and reductions in endothelial NO. Moreover, MGO partially contributed to COX-derived vasoconstrictor prostanoids through increased oxidative stress.

Methylglyoxal augments uridine diphosphate-induced contraction via activation of p38 mitogen-activated protein kinase in rat carotid artery.

The methylglyoxal elicits diverse adverse effects on the body. Uridine diphosphate, an extracellular nucleotide, plays an important role as a signaling molecule controlling vascular tone. This study aimed to evaluate the relationship between methylglyoxal and uridine diphosphate-induced carotid arterial contraction in rats. Additionally, we examined whether p38 mitogen-activated protein kinase (MAPK) would involve such responses. Organ baths were conducted to determine vascular reactivity in isolated carotid arterial rings, and western blotting was used for protein analysis. Treatment with methylglyoxal to carotid arterial rings showed concentration-dependent augmentation to uridine diphosphate-induced contraction in the absence and presence of NG-nitro-L-arginine, which is a nitric oxide synthase inhibitor, whereas, methylglyoxal did not affect serotonin- or isotonic high K+-induced contraction in the presence of a nitric oxide synthase inhibitor. Under nitric oxide synthase inhibition, SB203580, which is a selective p38 MAPK inhibitor, suppressed uridine diphosphate-induced contraction in both the control and methylglyoxal-treated groups, and the difference in uridine diphosphate-induced contraction was abolished by SB203580 treatment. The levels of phosphorylated p38 MAPK were increased by methylglyoxal in carotid arteries, not only under the basal condition but also under uridine diphosphate stimulation. The suppression of uridine diphosphate-induced contraction by a highly selective cell-permeable protein kinase C inhibitor bisindolylmaleimide I was observed in the methylglyoxal-treated group but not in the controls. Moreover, methylglyoxal-induced augmentation of uridine diphosphate-induced contraction was prevented by N-acetyl-L-cysteine. These results suggest that methylglyoxal could enhance uridine diphosphate-induced contraction in rat carotid arteries and may be caused by activation of p38 MAPK and protein kinase C and increased oxidative stress.

Indoxyl sulfate enhances endothelin-1-induced contraction via impairment of NO/cGMP signaling in rat aorta.

The microbiome-derived tryptophan metabolite, indoxyl sulfate, is considered a harmful vascular toxin. Here, we examined the effects of indoxyl sulfate on endothelin-1 (ET-1)-induced contraction in rat thoracic aortas. Indoxyl sulfate (10-3 M, 60 min) increased ET-1-induced contraction but did not affect isotonic high-K+-induced contraction. The ET-1-induced contraction was enhanced by endothelial denudation in both control and indoxyl sulfate-treated groups. BQ123 (10-6 M), an ETA receptor antagonist, reduced the ET-1-induced contraction in both control and indoxyl sulfate groups. BQ788 (10-6 M), an ETB receptor antagonist, increased the contraction in the control group but had no effect on the indoxyl sulfate group. Conversely, indoxyl sulfate inhibited relaxation induced by IRL1620, an ETB receptor agonist. L-NNA, an NO synthase (NOS) inhibitor, increased the ET-1-induced contractions in both the control and indoxyl sulfate groups, whereas L-NPA (10-6 M), a specific neuronal NOS inhibitor, did not affect the ET-1-induced contraction in both groups. However, ODQ, an inhibitor of soluble guanylyl cyclase, increased the ET-1-induced contraction in both groups. Organic anion transporter (OAT) inhibitor probenecid (10-3 M) and antioxidant N-acetyl-L-cysteine (NAC; 5 × 10-3 M) inhibited the effects of indoxyl sulfate. A cell-permeant superoxide scavenger reduced the ET-1-induced contraction in the indoxyl sulfate group. The aortic activity of SOD was reduced by indoxyl sulfate. The present study revealed that indoxyl sulfate augments ET-1-induced contraction in rat aortae. This enhancement may be due to the impairment of NO/cGMP signaling and may be attributed to impairment of the antioxidant systems via cellular uptake through OATs.

GLP-1 modulates insulin-induced relaxation response through ß-arrestin2 regulation in diabetic mice aortas.

AIMS: Diabetes impairs insulin-induced endothelium-dependent relaxation by reducing nitric oxide (NO) production. GLP-1, an incretin hormone, has been shown to prevent the development of endothelial dysfunction. In this study, we hypothesized that GLP-1 would improve the impaired insulin-induced relaxation response in diabetic mice. We also examined the underlying mechanisms. METHODS: Using aortic rings from ob/ob mice, an animal model of obesity and type 2 diabetes, and from lean mice, vascular relaxation responses and protein expressions were evaluated using insulin, GLP-1, and pathway-specific inhibitors to elucidate the mechanisms of response. In parallel experiments, ß-arrestin2 siRNA-transfected aortas were treated with GLP-1 to evaluate its effects on aortic response pathways. RESULTS: When compared to that of untreated ob/ob aortas, GLP-1 increased insulin-induced vasorelaxation and NO production. AMPK inhibition did not alter this vasorelaxation in both GLP-1-treated lean and ob/ob aortas, while Akt inhibition reduced vasorelaxation in both groups, and co-treatment with GLP-1 and insulin caused Akt/eNOS activation. Additionally, GLP-1 decreased GRK2 activity and enhanced ß-arrestin2 translocation from the cytosol to membrane in ob/ob aortas. ß-Arrestin2 siRNA decreased insulin-induced relaxation both in lean aortas and GLP-1-treated ob/ob aortas. CONCLUSIONS: We demonstrated that insulin-induced relaxation is dependent on ß-arrestin2 translocation and Akt activation via GLP-1-stimulated GRK2 inactivation in ob/ob aortas. We showed a novel cross-talk between GLP-1-responsive ß-arrestin2 and insulin signalling in diabetic aortas.

Differential Contractile Reactivity to Nucleotides in Femoral Arteries of OLETF and LETO Rats.

Extracellular nucleotides play an important role in the regulation of vascular function, and an abnormal vascular function is an important participant in the development and progression of diabetic vascular complications. The purpose of this study was to determine whether contractile responses induced by extracellular nucleotides and a dinucleotide, uridine adenosine tetraphosphate (Up4A), in femoral arteries would be altered at the chronic stage of type 2 diabetes. We determined the changes in contractile reactivity induced by ATP, uridine triphosphate (UTP), uridine diphosphate (UDP), and Up4A in the femoral arteries of Otsuka Long-Evans Tokushima Fatty (OLETF) rats (aged male type 2 diabetic rats) and, Long-Evans Tokushima Otsuka (LETO) rats (controls for OLETF rats). ATP-induced contractions were greater in OLETF rats than in LETO rats. UTP-induced contractions were lower in OLETF rats than in LETO rats. UDP- and Up4A-induced contractions were similar between OLETF and LETO rats. The femoral artery contractile changes induced by the extracellular nucleotides and dinucleotide were similar when nitric oxide synthase was inhibited. These results suggest that the extent of femoral artery contractile reactivity to nucleotides/dinucleotides differs during long-term duration of type 2 diabetes.

Plant polyphenols Morin and Quercetin rescue nitric oxide production in diabetic mouse aorta through distinct pathways.

Diabetic vascular complications are associated with endothelial dysfunction. Various plant-derived polyphenols benefit cardiovascular function by protecting endothelial nitric oxide (NO) production through as yet unclear mechanisms. This study compared the effects of two structurally similar polyphenols, Morin (MO) and Quercetin (QU), on endothelial function in isolated aorta from control and streptozotocin (STZ)-induced diabetic mice. Vascular function under treatment with MO, QU, and various signaling pathway modulators was measured by isometric tension in an organ bath system, NO production by chemical assay and HPLC, and changes in protein signaling factor expression or activity by western blotting (WB). Both polyphenols acted as potent vasodilators and this effect was associated with increased phosphorylation of Akt and endothelial NO synthase (eNOS). An Akt inhibitor blocked MO- and QU-induced vasorelaxation as well as Akt phosphorylation. However, inhibitors of phosphoinositide 3-kinase (PI3K) and AMP-activated protein kinase (AMPK) suppressed only QU-induced vasorelaxation, NO production, and AMPK phosphorylation. These results suggested that plant polyphenols MO and QU both promote eNOS-mediated NO production and vasodilation in diabetic aorta, MO via Akt pathway activation and QU via PI3K/Akt and AMPK pathway activation. Elucidation of these pathways may define effective therapeutic targets for diabetic vascular dysfunction.

Impaired UTP-induced relaxation in the carotid arteries of spontaneously hypertensive rats.

Uridine 5'-triphosphate (UTP) has an important role as an extracellular signaling molecule that regulates inflammation, angiogenesis, and vascular tone. While chronic hypertension has been shown to promote alterations in arterial vascular tone regulation, carotid artery responses to UTP under hypertensive conditions have remained unclear. The present study investigated carotid artery responses to UTP in spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY). Accordingly, our results found that although UTP promotes concentration-dependent relaxation in isolated carotid artery segments from both SHR and WKY after pretreatment with phenylephrine, SHR exhibited significantly lower arterial relaxation responses compared with WKY. Moreover, UTP-induced relaxation was substantially reduced by endothelial denudation and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine in both SHR and WKY. The difference in UTP-induced relaxation between both groups was abolished by the selective P2Y2 receptor antagonist AR-C118925XX and the cyclooxygenase (COX) inhibitor indomethacin but not by the thromboxane-prostanoid receptor antagonist SQ29548. Furthermore, we detected the release of PGE2, PGF2α, and PGI2 in the carotid arteries of SHR and WKY, both at baseline and in response to UTP. UTP administration also increased TXA2 levels in WKY but not SHR. Overall, our results suggest that UTP-induced relaxation in carotid arteries is impaired in SHR perhaps due to impaired P2Y2 receptor signaling, reductions in endothelial NO, and increases in the levels of COX-derived vasoconstrictor prostanoids.

Mechanisms underlying suppression of noradrenaline-induced contraction by prolonged treatment with advanced glycation end-products in organ-cultured rat carotid artery.

We investigated the direct effects of prolonged exposure to advanced glycation end-products (AGEs) on noradrenaline-induced contraction of rat carotid artery smooth muscle. Noradrenaline-induced contraction of endothelium-denuded carotid artery rings was suppressed by AGE-bovine serum albumin (AGE-BSA) pretreatment (0.01 and 0.1 mg/mL for 23 ± 1 h) compared with vehicle pretreatment (control), whereas isotonic-K+-induced contraction was not significantly altered by AGE-BSA pretreatment. This reduction in noradrenaline-induced contraction by AGE-BSA (0.1 mg/mL) was reversed by iberiotoxin, an inhibitor of large-conductance calcium-activated potassium (BKCa) channels, but not by inhibitors of other K channels [4-AP (Kv inhibitor), TRAM-34 (IKCa inhibitor), or glibenclamide (KATP inhibitor)]. Acute incubation of carotid arterial rings with H2O2 had also reduced noradrenaline-induced contraction in control arteries, but it had no effect on noradrenaline-induced contraction in AGE-BSA-pretreated arteries. Alternatively, acute incubation with the H2O2 scavenger catalase increased noradrenaline-induced contraction of AGE-BSA-pretreated arteries but had no effect on noradrenaline-induced contraction of control arteries. Noradrenaline-induced contraction in the presence of H2O2 was increased by co-treatment with iberiotoxin. The AGE-BSA-mediated suppression of noradrenaline-induced contraction was prevented by the organic cation transporter 3 (OCT3) inhibitor corticosterone, whereas the expression of OCT3 protein was similar between control and AGE-BSA-treated endothelium-denuded carotid arteries. These findings suggest that noradrenaline-induced arterial contraction is reduced by prolonged AGE-BSA exposure due to activation of BKCa channels via H2O2 generation and increased OCT3-mediated noradrenaline transport activity.

Glucagon-like peptide-1 increased the vascular relaxation response via AMPK/Akt signaling in diabetic mice aortas.

The incretin glucagon-like peptide-1 (GLP-1) elicits direct favorable effects on the cardiovascular system. This study aimed to evaluate the acute effects of GLP-1 on improving aortic endothelial dysfunction in diabetic mice. Additionally, we examined whether GLP-1 elucidated the underlying mechanisms. Using the diabetic mouse models induced by nicotinamide and streptozotocin, we investigated the functional changes in the aorta caused by GLP-1. Organ baths were performed for vascular reactivity in isolated aortic rings, and western blotting was used for protein analysis. The diabetic aortas showed enhanced GLP-1-induced relaxation response and nitric oxide (NO) production. However, the pretreatment of GLP-1 did not significantly change the endothelial-dependent relaxation response to acetylcholine and -independent relaxation response to sodium nitroprusside. On the other hand, the GLP-1-induced relaxation response and NO production were abolished by the endothelial NO synthase inhibitor, GLP-1 receptor antagonist, Akt inhibitor, and AMP-activated protein kinase (AMPK) inhibitor. Finally, in diabetic mice, considerable increases in phosphorylation of Akt and AMPK were found in aortas stimulated with GLP-1, both of which were decreased by pretreatment with the AMPK inhibitor. GLP-1 significantly enhanced endothelial-dependent relaxation in diabetic aortas. The effect may be mediated through activation of the AMPK/Akt pathway via a GLP-1 receptor-dependent mechanism.

Effect of Equol on Vasocontractions in Rat Carotid Arteries Treated with High Insulin.

Previous research has indicated that high insulin affects vascular function. Equol is an active metabolite of daidzein, an isoflavone produced from soy by intestinal microbial flora, with beneficial effects on the vascular system. This study investigated whether equol was beneficial for vascular function under high insulin conditions. Using organ culture techniques, rat carotid arteries were treated for 23 ± 1 h with a vehicle, high insulin (100 nM), or equol (100 µM) plus high insulin (100 nM). Vascular isometric forces were measured by the organ bath technique. In each endothelium-intact ring, the contractions induced by high-K+, noradrenaline, or by serotonin (5-HT) were similar for the vehicle, insulin, and equol + insulin treatments. Contractions induced by a selective 5-HT2A receptor agonist (TCB2) increased with insulin treatment (vs. vehicle), but less so with equol + insulin. Under basal conditions, a selective 5-HT2B receptor agonist (BW723C86) did not induce contraction; following precontraction by a thromboxane analog, it induced contraction but not relaxation. These responses were similar across the three treatments. Acetylcholine-induced relaxations were also similar for the three treatments. In the endothelium-denuded preparations, 5-HT-induced contraction was augmented with insulin treatment (vs. vehicle) but less so by equol + insulin treatment. These differences in 5-HT-induced contractions were eliminated by iberiotoxin, a large-conductance calcium-activated K+ channel (BKCa) inhibitor. These results suggest that equol exerts a preventive effect on the enhancement of 5-HT-induced contraction by high insulin (possibly mediated by the 5-HT2A receptor), and that these effects may be attributed to the activation of BKCa channels in vascular smooth muscle.

ERK-containing microparticles from a diabetic mouse induce endothelial dysfunction.

Endothelial dysfunction is a hallmark of diabetic vascular complications. Microparticles (MPs) are small vesicles shed from the surface of blood and vascular cells that act as stimuli and during apoptosis. Circulating MPs of diabetic rats have been shown to induce endothelial dysfunction. However, the underlying mechanisms require further study. In this study, we investigated how MPs from diabetic mice affect endothelial function. MPs were collected from streptozotocin-induced diabetic mice and Institute of Cancer Research (ICR) mice as controls. The levels of MPs were assessed and characterized by flow cytometry, enzyme-linked immunosorbent assay and dot blotting. Normal mice aortas were incubated with MPs and expressions of enzymes and vascular relaxation were analyzed. We found that (1) circulating MPs level increased in diabetic mice; (2) MPs impaired endothelial-dependent relaxation in mice aorta, but diabetic mice-derived MPs (diabetes mellitus (DM) MPs) were easier to attach to the endothelial cells than were control MPs; (3) DM MPs had more extracellular signal-regulated kinase (ERK)1/2 than did control mice-derived MPs, and they induced ERK1/2 activation in mice aortas; (4) DM MPs decreased endothelial nitric oxide synthase (eNOS) in mice aortas, and eNOS was emitted from endothelial cells to blood in the shape of endothelial MPs. DM MPs significantly altered endothelial function by activation of ERK1/2, which might provide a therapeutic target for diabetic vascular complications.

Inactivation of MAPK in epididymal fat and amelioration of triglyceride secretion by injection of GRK2 siRNA in ob/ob mice.

Abnormal G protein-coupled receptor kinase 2 (GRK2) accumulation has a crucial role in the development of insulin resistance and diabetes. Although GRK2 siRNA transfection in the liver improves insulin resistance-related vascular complications, the effects of GRK2 siRNA in lipid metabolism and obesity remain unknown. To investigate how GRK2 siRNA affects obesity, ob/ob mice were transfected with GRK2 siRNA, mainly in the liver, by using a hydrodynamic-based procedure. Epididymal fat, glucose, triglyceride, non-esterified fatty acid (NEFA), and alanine transaminase activity were higher in the control siRNA-transfected ob/ob mice than in the control siRNA-transfected Lean mice, but these parameters were reduced by GRK2 siRNA transfection into the ob/ob mice. GRK2 expression in epididymal fat was not altered among the 3 groups, although hepatic GRK2 expression was higher in the control siRNA-transfected ob/ob mice than in the control siRNA-transfected Lean mice. Additionally, we found that Akt interacted with GRK2 in the liver. Furthermore, phosphorylation levels of ERK1/2 and JNK were higher in the epididymal fats from the control siRNA-transfected ob/ob mice than in those from the control siRNA-transfected Lean mice, but they were lowered by transfection with GRK2 siRNA. The study results showed that GRK2 siRNA improved blood triglyceride levels and abnormal or excessive activity of mitogen-activated protein kinases in epididymal fat. This effect may be promoted by inhibition of the NEFA production pathway in the liver. Therefore, the interaction of organs (hepatic GRK2-epididymal fat) may help improve insulin resistance and diabetes-associated pathophysiology.

Co-treatment with clonidine and a GRK2 inhibitor prevented rebound hypertension and endothelial dysfunction after withdrawal in diabetes.

Hypertension and diabetes are associated with a risk of cardiovascular disease. Clonidine is currently used as a fourth-line drug therapy for hypertension because of its rebound hypertensive effect and short half-life. The purpose of this study was to investigate the combined effect of an antihypertensive drug (clonidine) and a G-protein-coupled receptor kinase 2 (GRK2) inhibitor on rebound hypertension and endothelial dysfunction. The clonidine and/or GRK2 inhibitor were administered by continuous infusion for 14 days by using an osmotic pump that was implanted subcutaneously. To test the effects of GRK2 inhibitotr, we measured blood pressure by using a tail-cuff system in diabetic mice in which rebound hypertension was induced by withdrawal after clonidine treatment and measured vascular responses in isolated aortas from these mice. The mice were then euthanized 7 days later. We observed that, in diabetes mellitus (DM) mice, blood pressure began to decline after 3 days of clonidine or clonidine + GRK2-inhibitor infusion. However, 15 days after initiation of treatment, the blood pressure of the clonidine only-treated DM mice began to increase and resulted in a high final blood pressure. At 21 days, clonidine withdrawal triggered rebound hypertension together with impaired endothelium-dependent relaxation, increased GRK2 activity, and reduced Akt/endothelial NO synthase (eNOS)/NO production in aortas. Conversely, withdrawal of the combination clonidine/GRK2-inhibitor treatment did not cause rebound hypertension, and normal induction of endothelium-dependent relaxation, decreased GRK2 activity, and increased Akt/eNOS were observed in aortas from DM mice. These results suggest that suppression of GRK2 activity affects rebound hypertension-associated vascular endothelial dysfunction by targeting the Akt/eNOS signaling pathway.

Alteration of Vascular Responsiveness to Uridine Adenosine Tetraphosphate in Aortas Isolated from Male Diabetic Otsuka Long-Evans Tokushima Fatty Rats: The Involvement of Prostanoids.

We investigated whether responsiveness to dinucleotide uridine adenosine tetraphosphate (Up4A) was altered in aortas from type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats compared with those from age-matched control Long-Evans Tokushima Otsuka (LETO) rats at the chronic stage of disease. In OLETF aortas, we observed the following: (1) Up4A-induced contractions were lower than those in the LETO aortas under basal conditions, (2) slight relaxation occurred due to Up4A, but this was not observed in phenylephrine-precontracted LETO aortas, (3) acetylcholine-induced relaxation was reduced (vs. LETO), and (4) prostanoid release (prostaglandin (PG)F2α, thromboxane (Tx)A2 metabolite, and PGE2) due to Up4A was decreased (vs. LETO). Endothelial denudation suppressed Up4A-induced contractions in the LETO group, but increased the contractions in the OLETF group. Under nitric oxide synthase (NOS) inhibition, Up4A induced contractions in phenylephrine-precontracted aortas; this effect was greater in the LETO group (vs. the OLETF group). The relaxation response induced by Up4A was unmasked by cyclooxygenase inhibitors, especially in the LETO group, but this effect was abolished by NOS inhibition. These results suggest that the relaxant component of the Up4A-mediated response was masked by prostanoids in the LETO aortas and that the LETO and OLETF rats presented different contributions of the endothelium to the response.

Poly (I:C) impairs NO donor-induced relaxation by overexposure to NO via the NF-kappa B/iNOS pathway in rat superior mesenteric arteries.

Recent studies have suggested a link between vascular dysfunction and innate immune activation including toll-like receptors (TLRs), but the detailed mechanism remains unclear. Here we investigated whether poly (I:C) [a synthetic double-strand RNA recognized by TLR3, melanoma differentiation-associated gene 5 (MDA5), and retinoic acid-inducible gene I (RIG-I)] affected nitric oxide (NO)/cGMP-related vascular relaxation, one of the major cascades of relaxation, in rat superior mesenteric arteries. Using organ-cultured arteries, we found that poly (I:C) (30µg/mL for approximately 1 day) markedly reduced sodium nitroprusside (SNP)-induced relaxation (vs. vehicle); this was prevented by co-treatment with a TLR3 inhibitor. Relaxation induced by 8-Br cGMP (a phosphodiesterase (PDE)-resistant cGMP analogue) and the expression of proteins related to NO/cGMP signaling did not differ between vehicle- and poly (I:C)-treated groups. When PDEs were inhibited by IBMX (a nonselective PDE inhibitor), the SNP-induced relaxation was still greatly reduced in poly (I:C)-treated arteries (vs. vehicle). Poly (I:C) reduced SNP-stimulated cGMP production, but increased NO production and iNOS expression (vs. vehicle). The impairment of SNP-induced relaxation by poly (I:C) was prevented by co-treatment with either iNOS or a nuclear factor-kappa B (NF-κB) inhibitor. This effect induced by poly (I:C) appeared to be independent of oxidative stress. The SNP-induced relaxation was reduced in freshly isolated arteries by pre-incubation with SNP in a concentration-dependent manner. Poly (I:C) did not alter protein levels of TLR3, TRIF/TICAM-1, or phospho-IRF3/IRF3, whereas RIG-I and MDA5 were significantly upregulated (vs. vehicle). These results suggest that poly (I:C) impairs NO donor-induced relaxation in rat superior mesenteric arteries via overexposure to NO produced by the NF-κB/iNOS pathway.

Anti-Inflammatory Role of Langerhans Cells and Apoptotic Keratinocytes in Ultraviolet-B-Induced Cutaneous Inflammation.

UV radiation, particularly UVB, is the major risk factor for the induction of skin cancer, and it induces skin inflammation and immunosuppression. Although reports documented that Langerhans cells (LCs) play various roles in photobiology, little is known about whether they contribute to UVB-induced cutaneous inflammation. Recently, the anti-inflammatory effect of apoptotic cells was noted. This study focuses on the roles of LCs and apoptotic cells in UVB-induced cutaneous inflammation. We show that LCs are essential for resolution of UVB-induced cutaneous inflammation. Administration of quinolyl-valyl-O-methylaspartyl-[2,6-difluophenoxy]-methyl ketone, a broad-spectrum caspase inhibitor with potent antiapoptotic properties, inhibited the formation of UVB-induced apoptotic cells and aggravated UVB-induced cutaneous inflammation in wild-type mice. In contrast, exacerbation of UVB-induced cutaneous inflammation following quinolyl-valyl-O-methylaspartyl-[2,6-difluophenoxy]-methyl ketone administration was not observed in LC-depleted mice. These results suggest that the interaction between LCs and apoptotic cells is critical for resolution of UVB-induced cutaneous inflammation. Interestingly, UVB-induced apoptotic keratinocytes were increased in LC-depleted mice. In addition, we revealed that UVB-induced apoptotic keratinocytes were phagocytosed by LCs ex vivo and that prolongation of UVB-induced cutaneous inflammation following treatment with Cytochalasin D, an inhibitor of phagocytosis, was partially attenuated in LC-depleted mice. Collectively, our findings demonstrate that the interaction between LCs and apoptotic cells, possibly via LC-mediated phagocytosis of apoptotic keratinocytes, has an essential anti-inflammatory role in the resolution of UVB-induced cutaneous inflammation.

UVB Exposure Prevents Atherosclerosis by Regulating Immunoinflammatory Responses.

OBJECTIVE: UVB irradiation is an established treatment for immunoinflammatory cutaneous disorders and has been shown to suppress cutaneous and systemic inflammatory diseases through modulation of the adaptive immune response. However, it remains unknown whether UVB irradiation prevents an immunoinflammatory disease of arteries such as atherosclerosis. APPROACH AND RESULTS: Here, we show that UVB exposure inhibits the development and progression of atherosclerosis in atherosclerosis-prone mice by expanding and enhancing the functional capacity of CD4+ forkhead box P3+ regulatory T cells and regulating proatherogenic T-cell responses. Experimental studies in Langerhans cell-depleted mice revealed that epidermal Langerhans cells play a critical role in UVB-dependent induction of CD4+ forkhead box P3+ regulatory T cells, suppression of proatherogenic T-cell responses, and prevention of atherosclerotic plaque development. CONCLUSIONS: Our findings suggest the skin immune system as a novel therapeutic target for atherosclerosis and provide a novel strategy for the treatment and prevention of atherosclerosis.

A Comparative Study of Vasorelaxant Effects of ATP, ADP, and Adenosine on the Superior Mesenteric Artery of SHR.

We investigated superior mesenteric arteries from spontaneously hypertensive rats (SHR) to determine the relaxation responses induced by ATP, ADP, and adenosine and the relationship between the relaxant effects of these compounds and nitric oxide (NO) or cyclooxygenase (COX)-derived prostanoids. In rat superior mesenteric artery, relaxation induced by ATP and ADP but not by adenosine was completely eliminated by endothelial denudation. In the superior mesenteric arteries isolated from SHR [vs. age-matched control Wistar Kyoto rats (WKY)], a) ATP- and ADP-induced relaxations were weaker, whereas adenosine-induced relaxation was similar in both groups, b) ATP- and ADP-induced relaxations were substantially and partly reduced by N(G)-nitro-L-arginine [a NO synthase (NOS) inhibitor], respectively, c) indomethacin, an inhibitor of COX, increased ATP- and ADP-induced relaxations, d) ADP-induced relaxation was weaker under combined inhibition by NOS and COX, and e) adenosine-induced relaxation was not altered by treatment with these inhibitors. These data indicate that levels of responsiveness to these nucleotides/adenosine vary in the superior mesenteric arteries from SHR and WKY and are differentially modulated by NO and COX-derived prostanoids.