Combination of Linagliptin and Empagliflozin Preserves Cardiac Systolic Function in an Ischemia-Reperfusion Injury Mice With Diabetes Mellitus.

BACKGROUND: Sodium-glucose co-transporter 2 inhibitor (SGLT2i) and dipeptidyl peptidase 4 inhibitor (DPP4i) are oral hypoglycemic agents. Although SGLT2i has been shown having the beneficial effects on heart failure in basic and clinical studies, the combined effects of SGLT2i and DPP4i have not been established well. We investigated the effects of SGLT2i and DPP4i against diabetes mice model of myocardial ischemia-reperfusion injury. METHODS: Streptozotocin-induced diabetic C57BL/6J mice were divided into control (vehicle), empagliflozin (30 mg/kg/day), linagliptin (3 mg/kg/day) and combination (30 mg/kg/day and 3 mg/kg/day, respectively) groups. After 7 days of drug administration, 30 min of myocardial ischemia was performed. We investigated body weight, heart weight, blood glucose, and cardiac functions by pressure-volume Millar catheter followed by 28 days of additional drug administration. RESULTS: Blood glucose levels, body weight, and heart weight were not significantly different between the groups. In Millar catheter analysis, left ventricular volume at the peak left ventricular ejection rate which is one of the cardiac systolic parameters in combination group was significantly preserved than that in control (P = 0.036). The cardiac index in the combination group tended to be preserved compared to that in the control (P = 0.06). The pathological fibrotic area in the left ventricle in the combination group also tended to be smaller (P = 0.08). CONCLUSIONS: Combination therapy with linagliptin and empagliflozin preserved cardiac systolic function on the diabetes mice model of myocardial ischemia-reperfusion injury independent of blood glucose levels.

Unique mode of binding between angiotensin II type 1 receptor and its blockers.

BACKGROUND: We hypothesized that the 5-oxo-1,2,4-oxadiazole moiety of azilsartan (AZL), which represents a small difference in the molecular structures of AZL and candesartan (CAN) [angiotensin II type 1 receptor (AT1R) blockers], may be responsible for the molecular effects of AZL. METHODS: We examined the binding affinities of AZL and CAN to AT1R, along with their ability to block receptor activity. A competition binding study, inositol phosphate (IP) production assay and extracellular signal-regulated kinase (ERK) assay were performed using wild-type (WT) and mutants AT1R-transfected cells. RESULTS: The binding affinities of CAN and AZL were reduced by > 5-fold for Y35F, W84F, R167K, K199Q and I288A compared with WT. In addition, AZL showed a > 5-fold reduction in its binding affinity to V108A. CAN and AZL exhibited > 20-fold and > 100-fold reductions in binding affinity to R167K, respectively. The loss of binding affinity of AZL to R167K was greater than that of CAN. CAN-7H exhibited a > 10-fold reduction in binding affinity to R167K compared with CAN. On the other hand, the binding affinity of AZL-7H to R167K was comparable to that of AZL. While 10-6M CAN and CAN-7H partly blocked Ang II-induced IP production in R167K, 10-6M AZL and AZL-7H did not. In addition, 10-6M CAN, but not 10-6M AZL, partly blocked Ang II-induced ERK activation in R167K. CONCLUSIONS: The interaction between 5-oxo-1,2,4-oxadiazole in AZL and Arg167 in the AT1R appears to be more important than the interaction between the tetrazole ring in CAN and Arg167.

Advanced glycation of high-density lipoprotein and the functionality of aldosterone release in type 2 diabetes.

Patients with type 2 diabetes mellitus (DM) exhibit modification of high-density lipoprotein (HDL), which is likely to have an important role in the development of atherosclerotic cardiovascular disease (ASCVD). Excess production of aldosterone (Ald) results in hypertension as well as ASCVD. However, the biological activity of modified HDL in steroidogenesis is not clear. We measured the accumulation of thiobarbituric acid-reactive substances (TBARSs) and Nɛ-(carboxymethyl)lysine (CML) levels (markers of oxidation and glycoxidation, respectively) in isolated HDL from 41 patients with type 2 diabetes mellitus (DM group) and 41 age- and gender-matched patients in a non-DM group. We quantified angiotensin II-sensitized and -nonsensitized Ald release using a validated living adrenocortical cell assay. TBARS levels in isolated HDL were similar between patients in the DM and non-DM groups, whereas the CML content of HDL in the DM group was lower than that in the non-DM group, irrespective of higher blood glucose and hemoglobin A1c levels. There was no difference in the HDL-induced ex vivo Ald release between the groups. Although sustained hyperglycemia was not a determinant of HDL-induced Ald release, the degree of HDL glycoxidation was inversely associated with HDL-induced Ald release (r=-0.40, P<0.001). In conclusion, in vivo advanced glycoxidation of HDL had an inverse effect on HDL-induced Ald release, independent of the prevalence of type 2 DM.

The angiotensin II type 1 receptor-neprilysin inhibitor LCZ696 blocked aldosterone synthesis in a human adrenocortical cell line.

A recent clinical study indicated that an angiotensin II (Ang II) type 1 (AT1) receptor-neprilysin inhibitor (ARNi) designated LCZ696 (sacubitril/valsartan, as combined sodium complex) was superior to enalapril at reducing the risks of death and hospitalization due to heart failure. Therefore, we investigated the possible mechanisms of the beneficial effect of LCZ696, in which the inhibition of neprilysin enhances atrial natriuretic peptide (NP) or brain NP (ANP or BNP)-evoked signals that can block Ang II/AT1 receptor-induced aldosterone (Ald) synthesis in human adrenocortical cells. The binding affinity of valsartan+LBQ657 (active moiety of sacubitril) to the AT1 receptor was greater than that of valsartan alone in an AT1 receptor-expressing human embryonic kidney cell-based assay. There was no difference in the dissociation from the AT1 receptor between valsartan+LBQ657 and valsartan alone. In Ang II-sensitized human adrenocortical cells, ANP or BNP alone, but not LBQ657 or valsartan alone, significantly decreased Ald synthesis. The level of suppression of Ald synthesis by ANP or BNP with LBQ657 was greater than that by ANP or BNP without LBQ657. The suppression of ANP was blocked by inhibitors of regulator of G-protein signaling proteins and cyclic GMP-dependent protein kinase. The inhibition of neprilysin did not change the mRNA levels of the AT1 receptor, ANP receptor A, regulator of G-protein signaling protein, renin or 3ß-hydroxysteroid dehydrogenases. In conclusion, the inhibition of neprilysin by LBQ657 enhances the NP-evoked signals that can block Ang II/AT1 receptor-induced Ald synthesis in human adrenocortical cells.

Comparison of aldosterone synthesis in adrenal cells, effect of various AT1 receptor blockers with or without atrial natriuretic peptide.

Bifunctional angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) that can block the activation of not only AT1 receptor, but also neprilysin, which metabolizes vasoactive peptides including atrial natriuretic peptide (ANP), are currently being developed. However, the usefulness of the inactivation of ANP in addition to the AT1 receptor with regard to aldosterone (Ald) synthesis is not yet clear. We evaluated the inhibitory effects of various ARBs combined with or without ANP on Ang II-induced adrenal Ald synthesis using a human adrenocortical cell line (NCI-H295R). Ang II increased Ald synthesis in a dose- and time-dependent manner. Ald synthesis induced by Ang II was completely blocked by azilsartan, but not PD123319 (AT2 receptor antagonist). CGP42112 AT2 receptor agonist did not affect Ald synthesis. While most ARBs block Ang II-induced Ald synthesis to different extents, azilsartan and olmesartan have similar blocking effects on Ald synthesis. The different effects of ARBs were particularly observed at 10(-7) and 10(-8 )M. ANP attenuated Ang II-induced Ald synthesis, and ANP-mediated attenuation of Ang II-induced Ald synthesis were blocked by inhibitors of G-protein signaling subtype 4 and protein kinase G. ANP (10(-8) and 10(-7 )M) without ARBs inhibited Ald synthesis, and the combination of ANP (10(-7 )M) and ARB (10(-8 )M) had an additive effect with respect to the inhibition of Ald synthesis. In conclusions, ARBs had differential effects on Ang II-induced Ald synthesis, and ANP may help to block Ald synthesis when the dose of ARB is not sufficient to block its secretion.

Ability of the new AT1 receptor blocker azilsartan to block angiotensin II-induced AT1 receptor activation after wash-out.

INTRODUCTION: The recently approved angiotensin II (Ang II) type 1 (AT1) receptor blocker (ARB) azilsartan strongly reduces blood pressure (BP) in patients with hypertension. We previously reported that azilsartan showed unique binding behavior to the AT1 receptor because of its 5-oxo-1,2,4-oxadiazole moiety. However, the ability of azilsartan to block Ang II-dependent AT1 receptor activation is not yet clear. MATERIALS AND METHODS: Azilsartan and a derivative of azilsartan (azilsartan-7H) that lacks a carboxyl group at the benzimidazole ring were used. Ang II-induced inositol phosphate (IP) production and extracellular signal-regulated kinase (ERK) activation were analyzed in a cell-based wash-out assay. RESULTS: Azilsartan, but not azilsartan-7H, completely blocked Ang II-induced IP production and ERK activation. Our previous report demonstrated that azilsartan mainly interacts with Tyr(113), Lys(199), and Gln(257) in the AT1 receptor. The interactions between azilsartan and Tyr(113) and Gln(257), but not Lys(199), were critical for blocking Ang II-induced IP production and ERK activation after wash-out. CONCLUSIONS: Although our findings regarding the molecule-specific effects of azilsartan are based on basic research, they may lead to an exciting insight into the mechanism of azilsartan.

Lower level of low-density lipoprotein cholesterol by statin prevents progression of coronary restenosis after successful stenting in acute myocardial infarction.

OBJECTIVE: It is unclear whether the reduction of coronary restenosis by statins is due to a decrease in low-density lipoprotein (LDL) cholesterol and/or pleiotropic effects. Therefore, we performed quantitative coronary angiography (QCA) and analyzed the lipid profile and changes in adhesion molecules and chemokines caused by statin in patients with acute myocardial infarction (AMI). METHODS: The subjects included AMI patients who had initial coronary angiograms and significant coronary stenosis and were implanted with a stent. After stent implantation, patients were treated either with (n = 36) or without (n = 14) statin. The primary end-point for this study was the absolute changes in the lipid profile, C-reactive protein (CRP), adhesion molecules, chemokines and stenosis measured by QCA between the post-stent and follow-up angiogram at 6 months after stenting. RESULTS: Treatment with statin reduced % coronary diameter stenosis (DS) and was associated with a greater reduction in LDL cholesterol at 6 months after stenting in patients with acute myocardial infarction (AMI), while there were no differences in adhesion molecules, chemokines, CC chemokine receptor or CXC chemokine receptor. Interestingly, changes in % DS between before and after statin treatment at 6 months (Delta%DS) were positively correlated with DeltaLDL cholesterol, and patients who had an LDL cholesterol level of less than 80 mg/dl had a significantly lower Delta%DS. In addition, Delta%DS was significantly related only to the reduction in LDL cholesterol as assessed by a stepwise multivariable regression analysis. CONCLUSION: These results suggest that the lower level of LDL cholesterol is the most critical factor in preventing coronary restenosis.

Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor.

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity.

Efficacy of a multicomponent program (patient-centered assessment and counseling for exercise plus nutrition [PACE+ Japan]) for lifestyle modification in patients with essential hypertension.

With conventional lifestyle modification programs, it can be difficult for hypertensive individuals to modify their lifestyles and maintain the changes. We assessed whether a multicomponent program (patient-centered assessment and counseling for exercise plus nutrition [PACE+ Japan]) based on behavior theory and social cognitive theory would be effective for treating patients with essential hypertension. We examined 57 outpatients aged 62+/-10 years with essential hypertension irrespective of antihypertensive drug treatment. Participants were randomly divided into two groups: 1) a PACE+ Japan follow-up group (n =18), who were given an action-plan sheet and systemic health counseling by a physician and counselor every 4 weeks for 24 weeks, and 2) a PACE+ Japan-only group (n =20), who were given an action-plan sheet but did not receive counseling. An age- and sex-matched control group (n =19) was also selected. The decrease in systolic blood pressure (SBP) (Delta SBP=SBP at 24 weeks minus that at 0 weeks) in the PACE+ Japan follow-up group was significantly greater than that in the control group. In addition, the Delta percentage of Fat (%Fat) and Delta urinary sodium extraction (U-Na) in the PACE+ Japan follow-up group were significantly greater than those in the control group. With regard to changes in total energy expenditure, exercise energy expenditure and total energy intake between 0 weeks and 24 weeks, significant improvements were observed for the PACE+ Japan follow-up group. Delta U-Na was determined to significantly predict Delta SBP as assessed by stepwise selection. In addition, the partial correlation coefficient of Delta SBP with Delta U-Na was 0.361 (p =0.011) as assessed by a multiple regression analysis. Therefore, PACE+ Japan follow-up counseling was associated with a reduction in SBP, which in turn was associated with reduction in U-Na. This new program may be effective for reducing blood pressure in hypertensives.

CXCR3 chemokine receptor-plasma IP10 interaction in patients with coronary artery disease.

In rat models of transplant vasculopathy, the strongest staining of CXCR3 is observed in the innermost layer of the neointima and because neointimal hyperplasia is seen after coronary angioplasty, the CXC chemokines may be targets for preventing stenosis. The expression of leukocyte surface chemokine receptors (CCR2/CCR5/CXCR2/CXCR3), as determined by flow cytometry, and plasma concentrations of monocyte chemoattractant protein (MCP)-1 and interferon-inducible protein (IP)10, as determined by enzyme immunoassays, were investigated in 55 patients with coronary artery disease (CAD) who underwent percutaneous transluminal coronary angioplasty (PTCA) and 20 patients without significant coronary stenosis based on the results of coronary catheterization during the same period (C group). The patients with CAD were divided into 3 groups: 20 with de novo stenosis (D group), 15 with restenosis (R group) and 20 without restenosis (N group) after PTCA. CXCR3 expression on lymphocytes, but not monocytes, in the R group was significantly lower than that in the C group. Although the plasma concentrations of IP10 in the D and N groups did not differ from that in the C group, the concentration in the R group was significantly higher. Increased plasma concentrations of IP10 were accompanied by a compensatory decrease in the CXCR3 expression on lymphocytes, but not monocytes, suggesting that a high plasma concentration of IP10 strongly induces monocytes signaling. The CXCR3 - plasma IP10 chemokine receptor - chemokine interaction on monocytes may affect the development of coronary restenosis, but not de novo stenosis, in patients with CAD.