Shin-ichiro Miura

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.

Ligand-independent signals from angiotensin II type 2 receptor induce apoptosis.

Conventional models of ligand–receptor regulation predict that agonists enhance the tone of signals generated by the receptor in the absence of ligand. Contrary to this paradigm, stimulation of the type 2 (AT2) receptor by angiotensin II (Ang II) is not required for induction of apoptosis but the level of receptor protein expression is critical. We compared Ang II‐dependent and ‐independent AT2 receptor signals involved in regulating apoptosis of cultured fibroblasts, epithelial cells and vascular smooth muscle cells. We found that induction of apoptosis—blocked by pharmacological inhibition of p38 mitogen‐activated protein kinase and caspase 3—is a constitutive function of the AT2 receptor. Biochemical and genetic studies suggest that the level of AT2 receptor expression is critical for physiological ontogenesis and its expression is restricted postnatally, coinciding with cessation of developmental apoptosis. Re‐expression of the AT2 receptor in remodeling tissues in the adult is linked to control of tissue growth and regeneration. Therefore, we propose that overexpression of the AT2 receptor itself is a signal for apoptosis that does not require the renin–angiotensin system hormone Ang II.

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.

Role of Aromaticity of Agonist Switches of Angiotensin II in the Activation of the AT1 Receptor

We have shown previously that the octapeptide angiotensin II (Ang II) activates the AT1 receptor through an induced-fit mechanism (Noda, K., Feng, Y. H., Liu, X. P., Saad, Y., Husain, A., and Karnik, S. S. (1996)Biochemistry 35, 16435–16442). In this activation process, interactions between Tyr4 and Phe8 of Ang II with Asn111 and His256 of the AT1receptor, respectively, are essential for agonism. Here we show that aromaticity, primarily, and size, secondarily, of the Tyr4side chain are important in activating the receptor. Activation analysis of AT1 receptor position 111 mutants by various Ang II position 4 analogues suggests that an amino-aromatic bonding interaction operates between the residue Asn111 of the AT1 receptor and Tyr4 of Ang II. Degree and potency of AT1 receptor activation by Ang II can be recreated by a reciprocal exchange of aromatic and amide groups between positions 4 and 111 of Ang II and the AT1 receptor, respectively. In several other bonding combinations, set up between Ang II position 4 analogues and receptor mutants, the gain of affinity is not accompanied by gain of function. Activation analysis of position 256 receptor mutants by Ang II position 8 analogues suggests that aromaticity of Phe8 and His256 side chains is crucial for receptor activation; however, a stacked rather than an amino-aromatic interaction appears to operate at this switch locus. Interaction between these residues, unlike the Tyr4:Asn111 interaction, plays an insignificant role in ligand docking.

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.

Transactivation of KDR/Flk-1 by the B2 Receptor Induces Tube Formation in Human Coronary Endothelial Cells

Abstract—Endothelial cells (ECs) are the critical cellular element responsible for postnatal angiogenesis. Vascular endothelial growth factor (VEGF) stimulates angiogenesis via the activation of kinase insert domain–containing receptor/fetal liver kinase-1 (KDR/Flk-1) in ECs. In addition, transactivation of KDR/Flk-1 by the bradykinin (BK) B2 receptor induces the activation of endothelial nitric oxide synthase (eNOS). These findings indicate that the precise role of BK in angiogenesis is likely to be more complex than initially thought, and it questions the importance of BK in angiogenic processes. Therefore, we examined whether transactivation by BK induced tube formation. We developed an in vitro model of human coronary artery EC (HCEC) tube formation on a matrix gel. We demonstrated that BK dose-dependently induced tube formation. Although a lower concentration of BK and VEGF did not separately induce tube formation, the formation was induced by a combination of lower concentrations of BK and VEGF, suggesting that VEGF and BK had a synergistic effect. The effect was blocked by a B2 receptor antagonist (HOE140) and specific inhibitors of VEGF receptor tyrosine kinases (Tki) and NOS. In addition, BK induced tyrosine phosphorylation of the KDR/Flk-1 receptor, as did VEGF itself. The transactivation was also blocked by HOE140 and Tki. Our results showed that, in HCECs, stimulation of the B2 receptor leads to the transactivation of KDR/Flk-1, as well as to eNOS activation, which induces tube formation. To our knowledge, this is a novel mechanism in which transactivation of KDR/Flk-1 by a G protein–coupled receptor, B2 receptor, may be a potent signal for tube formation.

Review: Angiotensin II type 1 receptor blockers: class effects versus molecular effects

Highly selective angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are now available. The AT1 receptor is a member of the G protein-coupled receptor (GPCR) superfamily and block the diverse effects of Ang II. Several ARBs are available for clinical use. Most ARBs have common molecular structures (biphenyl-tetrazol and imidazole groups) and it is clear that ARBs have ‘class effects’. On the other hand, recent clinical studies have demonstrated that not all ARBs have the same effects, and some benefits conferred by ARBs may not be class effects, and instead may be ‘molecular effects’. In addition, each ARB has been clearly shown to have specific molecular effects in basic experimental studies, and these effects may be due to small differences in the molecular structure of each ARB. However, it is controversial whether ARBs have molecular effects in a clinical setting. Although the presence of molecular effects for each ARB based on experimental studies may not directly influence the clinical outcome, this possibility has not been adequately evaluated. This review focuses on the class effects versus molecular effects of ARBs from bench to bedside.

INFLUENCE OF WORKLOAD ON THE ANTIHYPERTENSIVE EFFECT OF EXERCISE

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