Keijiro Saku

Effects of angiotensin II type 1 receptor antagonist on electrical and structural remodeling in atrial fibrillation

UNLABELLED The purpose of the present study was to evaluate the effect of angiotensin II type 1 receptor (AT1R) antagonist on chronic structural remodeling in atrial fibrillation (AF). BACKGROUND We previously reported that an AT1R antagonist, candesartan, prevents acute electrical remodeling in a rapid pacing model. However, the effect of candesartan on chronic structural remodeling in AF is unclear. METHODS Sustained AF was induced in 20 dogs (10 in a control group and 10 in a candesartan group) by rapid pacing of the right atrium (RA) at 400 beats/min for five weeks. Candesartan was administered orally (10 mg/kg/day) for one week before rapid pacing and was continued for five weeks. The AF duration, atrial effective refractory period (AERP) at four sites in the RA, and intra-atrial conduction time (CT) from the RA appendage to the other three sites were measured every week. RESULTS The mean AF duration in the control group after five weeks was significantly longer than that with candesartan (1,333 +/- 725 vs. 411 +/- 301 s, p < 0.01). The degree of AERP shortening after five weeks was not significantly different between the two groups. The CT from the RA appendage to the low RA after five weeks with candesartan was significantly shorter than that in the control (43 +/- 14 vs. 68 +/- 10 ms, p < 0.05). The candesartan group had a significantly lower percentage of interstitial fibrosis than the control group (7 +/- 2% vs. 16 +/- 1% at the RA appendage, p < 0.001). CONCLUSIONS Candesartan can prevent the promotion of AF by suppressing the development of structural remodeling.

Serum Lysophosphatidic Acid Is Produced through Diverse Phospholipase Pathways

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities that accounts for many biological properties of serum. LPA is thought to be produced during serum formation based on the fact that the LPA level is much higher in serum than in plasma. In this study, to better understand the pathways of LPA synthesis in serum, we evaluated the roles of platelets, plasma, and phospholipases by measuring LPA using a novel enzyme-linked fluorometric assay. First, examination of platelet-depleted rats showed that half of the LPA in serum is produced via a platelet-dependent pathway. However, the amount of LPA released from isolated platelets after they are activated by thrombin or calcium ionophore accounted for only a small part of serum LPA. Most of the platelet-derived LPA was produced in a two-step process: lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine, and lysophosphatidylserine, were released from activated rat platelets by the actions of two phospholipases, group IIA secretory phospholipase A2(sPLA2-IIA) and phosphatidylserine-specific phospholipase A1 (PS-PLA1), which were abundantly expressed in the cells. Then these lysophospholipids were converted to LPA by the action of plasma lysophospholipase D (lysoPLD). Second, accumulation of LPA in incubated plasma was strongly accelerated by the addition of recombinant lysoPLD with a concomitant decrease in LPC accumulation, indicating that the enzyme produces LPA by hydrolyzing LPC produced during the incubation. In addition, incubation of plasma isolated from human subjects who were deficient in lecithin-cholesterol acyltransferase (LCAT) did not result in increases of either LPC or LPA. The present study demonstrates multiple pathways for LPA production in serum and the involvement of several phospholipases, including PS-PLA1, sPLA2-IIA, LCAT, and lysoPLD.

Mechanism of action of gemfibrozil on lipoprotein metabolism.

Gemfibrozil is a potent lipid regulating drug whose major effects are to increase plasma high density lipoproteins (HDL) and to decrease plasma triglycerides (TG) in a wide variety of primary and secondary dyslipoproteinemias. Its mechanism of action is not clear. Six patients with primary familial endogenous hypertriglyceridemia with fasting chylomicronemia (type V lipoprotein phenotype) with concurrent subnormal HDL cholesterol levels (HDL deficiency) were treated initially by diet and once stabilized, were given gemfibrozil (1,200 mg/d). Each patient was admitted to the Clinical Research Center with metabolic kitchen facilities, for investigation of HDL and TG metabolism immediately before and after 8 wk of gemfibrozil treatment. Gemfibrozil significantly increased plasma HDL cholesterol, apolipoprotein (apo) AI, and apo AII by 36%, 29%, and 38% from base line, respectively. Plasma TG decreased by 54%. Kinetics of apo AI and apo AII metabolism were assessed by analysis of the specific radioactivity decay curves after injection of autologous HDL labeled with 125I. Gemfibrozil increased synthetic rates of apo AI and apo AII by 27% and 34%, respectively, without changing the fractional catabolic rates. Stimulation of apo AI and apo AII synthesis by gemfibrozil was associated with the appearance in plasma of smaller (and heavier) HDL particles as assessed by gradient gel electrophoresis and HDL composition. Postheparin extra-hepatic lipoprotein lipase activity increased significantly by 25% after gemfibrozil, and was associated with the appearance in plasma of smaller very low density lipoprotein particles whose apo CIII:CII ratio was decreased. These data suggest that gemfibrozil increases plasma HDL levels by stimulating their synthesis. Increased transport (turnover) of HDL induced by gemfibrozil may be significant in increasing tissue cholesterol removal in these patients.

The I allele of the angiotensin-converting enzyme gene is associated with an increased percentage of slow-twitch type I fibers in human skeletal muscle

The insertion (I) allele of the human angiotensin‐converting enzyme (ACE) gene is associated with lower serum and tissue ACE activity, and with greater endurance performance and enhanced mechanical efficiency of trained muscle. We tested the hypothesis that the ACE‐I allele may be associated with increased slow‐twitch fiber, which is more efficient than fast‐twitch fiber in low‐velocity contraction, by examining the association between the ACE genotype and skeletal muscle fiber (SMF) types in 41 untrained healthy young volunteer subjects (31 males, 10 females, age 24 ± 3 years). Skeletal muscle samples were taken from the left vastus lateralis using the needle‐biopsy method. Slow‐twitch type I fibers and fast‐twitch type IIa and IIb fibers were classified histochemically based on staining for myosin adenosine triphosphatase (ATPase) activity at different pH values. Amylase‐periodic acid‐Schiff staining was used to visualize capillaries around fibers. ACE‐II subjects had significantly (p < 0.01) higher percentages of type I fibers (50.1 ± 13.9%vs 30.5 ± 13.3%) and lower percentages of type IIb fibers (16.2 ± 6.6%vs 32.9 ± 7.4%) than ACE‐DD subjects. The linear trends for decreases in type I fibers and increases in type IIb fibers from ACE‐II → ID → DD genotypes were significant as assessed by an analysis of variance. The ratio of type I:II fibers also differed according to the ACE genotype. A multivariate logistic regression analysis showed that the ACE‐I allele had significant additive and recessive (codominant) effects on the increased type I fibers and the ratio of type I:II fibers. No specific pattern of capillarization was observed among the three ACE genotypes. In conclusion, the ACE‐I allele was associated with increased type I SMF, which may be a mechanism for the association between the ACE genotype and endurance performance.

Conformational switch of angiotensin II type 1 receptor underlying mechanical stress-induced activation

The angiotensin II type 1 (AT1) receptor is a G protein‐coupled receptor that has a crucial role in the development of load‐induced cardiac hypertrophy. Here, we show that cell stretch leads to activation of the AT1 receptor, which undergoes an anticlockwise rotation and a shift of transmembrane (TM) 7 into the ligand‐binding pocket. As an inverse agonist, candesartan suppressed the stretch‐induced helical movement of TM7 through the bindings of the carboxyl group of candesartan to the specific residues of the receptor. A molecular model proposes that the tight binding of candesartan to the AT1 receptor stabilizes the receptor in the inactive conformation, preventing its shift to the active conformation. Our results show that the AT1 receptor undergoes a conformational switch that couples mechanical stress‐induced activation and inverse agonist‐induced inactivation.

Plasma fibrinogen levels as an independent indicator of severity of coronary atherosclerosis.

The relationship between plasma fibrinogen levels and the severity of coronary atherosclerosis was examined in 229 patients, aged 25-82 years (162 men and 67 women), undergoing coronary angiography. Severity of coronary atherosclerosis was assessed in terms of the number of vessels with a 75% or greater stenosis and Gensinis severity score. Fibrinogen levels increased progressively with the severity of coronary atherosclerosis, determined by both the number of involved vessels and Gensinis severity score in men, and the relationships were statistically significant. Similar patterns were noted among women, but the trends were not statistically significant. The association was evident even after adjustment for age, hypertension, total cholesterol, cigarette smoking, alcohol intake, high density lipoprotein cholesterol and body mass index. These results provide evidence that in the Japanese also plasma fibrinogen levels can serve as an independent indicator of the progression of coronary atherosclerosis.

Reconstituted High-Density Lipoprotein Stimulates Differentiation of Endothelial Progenitor Cells and Enhances Ischemia-Induced Angiogenesis

Background—Plasma high-density lipoprotein (HDL) levels have an inverse correlation with incidence of ischemic heart disease as well as other atherosclerosis-related ischemic conditions. However, the molecular mechanism by which HDL prevents ischemic disease is not fully understood. Here, we investigated the effect of HDL on differentiation of endothelial progenitor cells and angiogenesis in murine ischemic hindlimb model. Methods and Results—Intravenous injection of reconstituted HDL (rHDL) significantly augmented blood flow recovery and increased capillary density in the ischemic leg. rHDL increased the number of bone marrow–derived cells incorporated into the newly formed capillaries in ischemic muscle. rHDL induced phosphorylation of Akt in human peripheral mononuclear cells. rHDL (50 to 100 &mgr;g apolipoprotein A-I/mL) promoted differentiation of peripheral mononuclear cells to endothelial progenitor cells in a dose-dependent manner. The effect of rHDL on endothelial progenitor cells differentiation was abrogated by coadministration of LY294002, an inhibitor of phosphatidylinositol 3-kinase. rHDL failed to promote angiogenesis in endothelial NO–deficient mice. Conclusions—rHDL directly stimulates endothelial progenitor cell differentiation via phosphatidylinositol 3-kinase/Akt pathway and enhances ischemia-induced angiogenesis. rHDL may be useful in the treatment of patients with ischemic cardiovascular diseases.

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.

Mast cell chymase limits the cardiac efficacy of Ang I–converting enzyme inhibitor therapy in rodents

Ang I-converting enzyme (ACE) inhibitors are widely believed to suppress the deleterious cardiac effects of Ang II by inhibiting locally generated Ang II. However, the recent demonstration that chymase, an Ang II-forming enzyme stored in mast cell granules, is present in the heart has added uncertainty to this view. As discussed here, using microdialysis probes tethered to the heart of conscious mice, we have shown that chronic ACE inhibitor treatment did not suppress Ang II levels in the LV interstitial fluid (ISF) despite marked inhibition of ACE. However, chronic ACE inhibition caused a marked bradykinin/B2 receptor-mediated increase in LV ISF chymase activity that was not observed in mast cell-deficient KitW/KitW-v mice. In chronic ACE inhibitor-treated mast cell-sufficient littermates, chymase inhibition decreased LV ISF Ang II levels substantially, indicating the importance of mast cell chymase in regulating cardiac Ang II levels. Chymase-dependent processing of other regulatory peptides also promotes inflammation and tissue remodeling. We found that combined chymase and ACE inhibition, relative to ACE inhibition alone, improved LV function, decreased adverse cardiac remodeling, and improved survival after myocardial infarction in hamsters. These results suggest that chymase inhibitors could be a useful addition to ACE inhibitor therapy in the treatment of heart failure.

High Density Lipoprotein–Induced Angiogenesis Requires the Activation of Ras/MAP Kinase in Human Coronary Artery Endothelial Cells

Objective—Plasma high density lipoprotein (HDL) levels have been shown to be inversely correlated with coronary artery disease, but the mechanisms of the direct protective effect of HDL on endothelial cells (ECs) are not fully understood. In this study, we investigated the role of the HDL-mediated promotion of angiogenesis in human coronary artery ECs (HCECs). Methods and Results—We developed an in vitro model of HCEC tube formation on a matrix gel. We optimized the maximum dose of HDL required to induce tube formation in initial experiments, in which the dose response showed that the maximum effective dose of HDL was 100 &mgr;g/mL. PD98059, an inhibitor of p42/44 mitogen-activated protein kinase (MAPK) activity, but not SB203580, an inhibitor of p38 MAPK activity, suppressed HDL-induced tube formation. Dominant-negative Ras N17 inhibited HDL-induced tube formation. HDL activated Ras according to a ras pull-down assay, and this effect was inhibited by pertussis toxin. Moreover, HDL activated phospho(p)-p42/44 MAPK, whereas Ras N17 blocked HDL-induced pp42/44 MAPK. Conclusions—These results indicate that HDL induced a potent signal through a Ras/MAPK pathway mediated by a pertussis toxin–sensitive G-protein coupled receptor to the angiogenic phenotype in HCECs.