Keiko Ishida

Metformin normalizes endothelial function by suppressing vasoconstrictor prostanoids in mesenteric arteries from OLETF rats, a model of type 2 diabetes

We previously reported that in mesenteric arteries from aged Otsuka Long-Evans Tokushima fatty (OLETF) rats (a type 2 diabetes model) endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired while endothelium-derived contracting factor (EDCF)-mediated contraction is enhanced (Matsumoto T, Kakami M, Noguchi E, Kobayashi T, Kamata K. Am J Physiol Heart Circ Physiol 293: H1480-H1490, 2007). Here we investigated whether acute and/or chronic treatment with metformin might improve this imbalance between the effects of the above endothelium-derived factors in mesenteric arteries isolated from OLETF rats. In acute studies on OLETF mesenteric arteries, ACh-induced relaxation was impaired and the relaxation became weaker at high ACh concentrations. Both metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside [AICAR, an AMP-activated protein kinase (AMPK) activator that is also activated by metformin] 1) diminished the tendency for the relaxation to reverse at high ACh concentrations and 2) suppressed both ACh-induced EDCF-mediated contraction and ACh-stimulated production of prostanoids (thromboxane A2 and PGE2). In studies on OLETF arteries from chronically treated animals, metformin treatment (300 mg.kg(-1).day(-1) for 4 wk) 1) improved ACh-induced nitric oxide- or EDHF-mediated relaxation and cyclooxygenase (COX)-mediated contraction, 2) reduced EDCF-mediated contraction, 3) suppressed production of prostanoids, and 4) reduced superoxide generation. Metformin did not alter the protein expressions of endothelial nitric oxide synthase (eNOS), phospho-eNOS (Ser1177), or COX-1, but it increased COX-2 protein. These results suggest that metformin improves endothelial functions in OLETF mesenteric arteries by suppressing vasoconstrictor prostanoids and by reducing oxidative stress. Our data suggest that within the timescale studied here, metformin improves endothelial function through this direct mechanism, rather than by improving metabolic abnormalities.

Eicosapentaenoic Acid Improves Imbalance between Vasodilator and Vasoconstrictor Actions of Endothelium-Derived Factors in Mesenteric Arteries from Rats at Chronic Stage of Type 2 Diabetes

Accumulating evidence demonstrates that dietary intake of n-3 polyunsaturated fatty acids (PUFAs) is associated with a reduced incidence of several cardiovascular diseases that involve endothelial dysfunction. However, the molecular mechanism remains unclear. We previously reported that mesenteric arteries from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats exhibit endothelial dysfunction, leading to an imbalance between endothelium-derived vasodilators [namely, nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)] and vasoconstrictors [endothelium-derived contracting factors (EDCFs)] [namely cyclooxygenase (COX)-derived prostanoids] (Am J Physiol Heart Circ Physiol 293:H1480–H1490, 2007). We hypothesized that treating OLETF rats with eicosapentaenoic acid (EPA), a major n-3 PUFA, may improve endothelial dysfunction by correcting this imbalance. In OLETF rats [compared with age-matched control Long-Evans Tokushima Otsuka (LETO) rats]: 1) acetylcholine (ACh)-induced (endothelium-dependent) relaxation was impaired, 2) NO- and EDHF-mediated relaxations and nitrite production were reduced, and 3) ACh-induced EDCF-mediated contraction, production of prostanoids, and the protein expressions of COX-1 and COX-2 were all increased. When OLETF rats received chronic EPA treatment long-term (300 mg/kg/day p.o. for 4 weeks), their isolated mesenteric arteries exhibited: 1) improvements in ACh-induced NO- and EDHF-mediated relaxations and COX-mediated contraction, 2) reduced EDCF- and arachidonic acid-induced contractions, 3) normalized NO metabolism, 4) suppressed production of prostanoids, 5) reduced COX-2 expression, and 6) reduced phosphoextracellular signal-regulated kinase (ERK) expression. Moreover, EPA treatment reduced both ERK2 and nuclear factor (NF)-κB activities in isolated OLETF aortas. We propose that EPA ameliorates endothelial dysfunction in OLETF rats by correcting the imbalance between endothelium-derived factors, at least partly, by inhibiting ERK, decreasing NF-κB activation, and reducing COX-2 expression.

Involvement of NO and MEK/ERK pathway in enhancement of endothelin-1-induced mesenteric artery contraction in later-stage type 2 diabetic Goto-Kakizaki rat

Endothelin (ET)-1 is a likely candidate for a key role in diabetic vascular complications. However, no abnormalities in the vascular responsiveness to ET-1 have been identified in the chronic stage of type 2 diabetes. Our goal was to look for abnormalities in the roles played by ET receptors (ET(A) and ET(B)) in the mesenteric artery of the type 2 diabetic Goto-Kakizaki (GK) rat and to identify the molecular mechanisms involved. Using mesenteric arteries from later-stage (32-38 wk old) individuals, we compared the ET-1-induced contraction and the relaxation induced by the selective ET(B) receptor agonist IRL1620 between GK rats and control Wistar rats. Mesenteric artery ERK activity and the protein expressions for ET receptors and MEK were also measured. In GK rats (vs. age-matched Wistar rats), we found as follows. 1) The ET-1-induced contraction was greater and was attenuated by BQ-123 (ET(A) antagonist) but not by BQ-788 (ET(B) antagonist). In the controls, BQ-788 augmented this contraction. 2) Both the relaxation and nitric oxide (NO) production induced by IRL1620 were reduced. 3) ET-1-induced contraction was enhanced by N(G)-nitro-l-arginine (l-NNA; NO synthase inhibitor) but suppressed by sodium nitroprusside (NO donor). 4) The enhanced ET-1-induced contraction was reduced by MEK/ERK pathway inhibitors (PD-98059 or U0126). 5) ET-1-stimulated ERK activation was increased, as were the ET(A) and MEK1/2 protein expressions. 6) Mesenteric ET-1 content was increased. These results suggest that upregulation of ET(A), a defect in ET(B)-mediated NO signaling, and activation of the MEK/ERK pathway together represent a likely mechanism mediating the hyperreactivity to ET-1 examined in this study.

Enhancement of mesenteric artery contraction to 5-HT depends on Rho kinase and Src kinase pathways in the ob/ob mouse model of type 2 diabetes.

Background and purpose:  Arteries from hypertensive subjects are reportedly hyperresponsive to 5‐hydroxytryptamine (5‐HT), but it remains unclear whether this is true in chronic type 2 diabetes. We have assessed responses to 5‐HT shown by mesenteric arteries from type 2 diabetic ob/ob mice (27–32 weeks old) and have identified the molecular mechanisms involved.

Mechanisms underlying the losartan treatment-induced improvement in the endothelial dysfunction seen in mesenteric arteries from type 2 diabetic rats

It is well known that type 2 diabetes mellitus is frequently associated with vascular dysfunction and an elevated systemic blood pressure, yet the underlying mechanisms are not completely understood. We previously reported that in mesenteric arteries from established type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats, which exhibit endothelial dysfunction, there is an imbalance between endothelium-derived vasodilators [namely, nitric oxide (NO) and hyperpolarizing factor (EDHF)] and vasoconstrictors [contracting factors (EDCFs) such as cyclooxygenase (COX)-derived prostanoids]. Here, we investigated whether the angiotensin II receptor antagonist losartan might improve endothelial dysfunction in OLETF rats at the established stage of diabetes. In mesenteric arteries isolated from OLETF rats [vs. those from age-matched control Long-Evans Tokushima Otsuka (LETO) rats]: (1) the acetylcholine (ACh)-induced relaxation was impaired, (2) the NO- and EDHF-mediated relaxations were reduced, (3) the ACh-induced EDCF-mediated contraction and the production of prostanoids were increased, and (4) superoxide generation was increased. After such OLETF rats had received losartan (25 mg/kg/day p.o. for 4 weeks), their isolated mesenteric arteries exhibited: (1) improvements in ACh-induced NO- and EDHF-mediated relaxations, (2) reduced EDCF- and arachidonic acid-induced contractions, (3) suppressed production of prostanoids, (4) reduced PGE(2)-mediated contraction, and (5) reduced superoxide generation. Within the timescale studied here, losartan did not change the protein expressions of endothelial NO synthase, COX1, or COX2 in mesenteric arteries from either OLETF or LETO rats. Losartan thus normalizes vascular dysfunction in this type 2 diabetic model, and the above effects may contribute to the reduction of adverse cardiovascular events seen in diabetic patients treated with angiotensin II receptor blockers.

Involvement of CaM kinase II in the impairment of endothelial function and eNOS activity in aortas of Type 2 diabetic rats.

In the present sutdy, we have examined the relationship between the CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) pathway and endothelial dysfunction in aortas from GK (Goto-Kakizaki) Type 2 diabetic rats. The ACh (acetylcholine)-induced relaxation and NO production were each attenuated in diabetic aortas (compared with those from age-matched control rats). ACh-stimulated Ser(1177)-eNOS (endothelial NO synthase) phosphorylation was significantly decreased in diabetic aortas (compared with their controls). ACh markedly increased the CaMKII phosphorylation level within endothelial cells only in control aortas (as assessed by immunohistochemistry and Western blotting). ACh-stimulated Thr(286)-CaMKII phosphorylation within endothelial cells was significantly decreased in diabetic aortas (compared with their controls). The ACh-induced relaxations, NO production, eNOS phosphorylation, and CaMKII phosphorylation were inhibited by KN93 and/or by lavendustin C (inhibitors of CaMKII) in control aortas, but not in diabetic ones. Pre-incubation of aortic strips with a PP (protein phosphatase)-1 inhibitor, PPI2 (protein phosphatase inhibitor 2), or with a PP2A inhibitor, CA (cantharidic acid), corrected the above abnormalities in diabetic aortas. The expression of PP2A type A subunit was increased in diabetic aortas. The ACh-stimulated Thr(320)-phosphorylation level of PP1α was lower in diabetic aortas than in their controls, but the total PP1α protein level was not different. These results suggest that the aortic relaxation responses, NO production, and eNOS activity mediated by CaMKII phosphorylation are decreased in this Type 2 diabetic model, and that these impairments of CaMKII signalling may be, at least in part, due to enhancements of PP1α activity and PP2A expression.

Mechanisms underlying altered extracellular nucleotide-induced contractions in mesenteric arteries from rats in later-stage type 2 diabetes: effect of ANG II type 1 receptor antagonism

Little is known about the vascular contractile responsiveness to, and signaling pathways for, extracellular nucleotides in the chronic stage of type 2 diabetes or whether the ANG II type 1 receptor blocker losartan might alter such responses. We hypothesized that nucleotide-induced arterial contractions are augmented in diabetic Goto-Kakizaki (GK) rats and that treatment with losartan would normalize the contractions. Here, we investigated the vasoconstrictor effects of ATP/UTP in superior mesenteric arteries isolated from GK rats (37-42 wk old) that had or had not received 2 wk of losartan (25 mg·kg(-1)·day(-1)). In arteries from GK rats (vs. those from Wistar rats), 1) ATP- and UTP-induced contractions, which were blocked by the nonselective P2 antagonist suramin, were enhanced, and these enhancements were suppressed by endothelial denudation, by cyclooxygenase (COX) inhibitors, or by a cytosolic phospholipase A(2) (cPLA(2)) inhibitor; 2) both nucleotides induced increased release of PGE(2) and PGF(2α); 3) nucleotide-stimulated cPLA(2) phosphorylations were increased; 4) COX-1 and COX-2 expressions were increased; and 5) neither P2Y2 nor P2Y6 receptor expression differed, but P2Y4 receptor expression was decreased. Mesenteric arteries from GK rats treated with losartan exhibited (vs. untreated GK) 1) reduced nucleotide-induced contractions, 2) suppressed UTP-induced release of PGE(2) and PGF(2α), 3) suppressed UTP-stimulated cPLA(2) phosphorylation, 4) normalized expressions of COX-2 and P2Y4 receptors, and 5) reduced superoxide generation. Our data suggest that the diabetes-related enhancement of ATP-mediated vasoconstriction was due to P2Y receptor-mediated activation of the cPLA(2)/COX pathway and, moreover, that losartan normalizes such contractions by a suppressing action within this pathway.

Abnormalities of endothelium-dependent responses in mesenteric arteries from Otsuka Long-Evans Tokushima Fatty (OLETF) rats are improved by chronic treatment with thromboxane A2 synthase inhibitor.

Thromboxane A(2) (TXA(2)) is thought to contribute to the development of diabetic complications. We tested the hypothesis that the impaired endothelial function seen in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (a type 2 diabetic model) might be improved by chronic treatment with ozagrel, a TXA(2) synthase inhibitor. In mesenteric arteries from OLETF rats (40-46 weeks old) [vs. those from age-matched Long-Evans Tokushima Otsuka (LETO) rats]: (1) ACh-induced endothelium-dependent relaxation, NO-mediated relaxation, and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation were all reduced; (2) ACh-induced cyclooxygenase-dependent contraction was enhanced; (3) endothelium-derived contracting factor (EDCF)-mediated contraction was enhanced; (4) ACh-stimulated nitrite production was reduced but the nitrate/nitrite ratio was increased; and (5) ACh-stimulated production of TXA(2) was increased. Chronic treatment with ozagrel (100mg/kg/day for 4 weeks, starting when they were 36-42 weeks of age) partly corrected the above abnormalities. These results suggest that ozagrel has normalizing effects on endothelial functions in OLETF mesenteric arteries, at least partly by increasing endothelium-derived relaxing factors (i.e., NO and EDHF) signaling and reducing EDCF signaling.

Cilostazol improves endothelial dysfunction by increasing endothelium-derived hyperpolarizing factor response in mesenteric arteries from Type 2 diabetic rats.

Diabetes mellitus impairs endothelial function, an effect that can be considered a hallmark of the development of cardiovascular diseases in diabetics. Cilostazol, a selective phosphodiesterase 3 inhibitor, is currently used to treat patients with diabetic vascular complications. However, the effects of cilostazol on responses mediated by endothelium-derived relaxing [in particular, nitric oxide (NO) and hyperpolarizing factors (EDHF)] and contracting factors remain unclear. Here, we hypothesized that cilostazol could improve endothelial dysfunctions in mesenteric arteries isolated from type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Using cilostazol-treated (100 mg/kg/day for 4 weeks) or -untreated OLETF and control (Long Evans Tokushima Otsuka) rats, we examined the acetylcholine-induced endothelium-dependent responses and the cell-permeant cyclic adenosine monophosphate (cAMP) analog-induced relaxations in the superior mesenteric artery. We also determined blood parameters in these animals. In OLETF rats, chronic treatment with cilostazol reduced the blood levels of triglyceride, non-esterified fatty acids, and leptin, and increased antioxidant capacity, but did not alter the blood glucose or insulin levels. In studies on mesenteric arteries from cilostazol-treated OLETF animals, the cilostazol treatment improved: (a) the acetylcholine-induced EDHF-mediated relaxation and (b) the cAMP-mediated relaxation. However, cilostazol did not alter the NO-mediated relaxation or the endothelium-derived contracting factor-mediated contraction. These results suggest that cilostazol improves endothelial functions in OLETF mesenteric arteries by increasing EDHF signaling, and that it normalizes some metabolic abnormalities in OLETF rats. On that basis, cilostazol may prove to be a potent drug for the clinical treatment of diabetic vasculopathy.

Mechanisms underlying reduced P2Y1‐receptor‐mediated relaxation in superior mesenteric arteries from long‐term streptozotocin‐induced diabetic rats

Extracellular nucleotides activate cell‐surface purinergic (P2) receptors, contribute to the local regulation of vascular tone and play important roles in pathophysiological states. However, little is known about the vasodilator effects of P2Y1‐receptor activation in diabetic states. We hypothesized that in a model of established type 1 diabetes, long‐term streptozotocin (STZ)‐induced diabetic rats, the arterial relaxation elicited by a P2Y1‐receptor agonist would be impaired.