Abstract-Angiotensin II (Ang II), a circulating hormone that can be synthesized locally in the vasculature, has been implicated in diabetes-associated vascular complications. This study was conducted to determine whether high glucose (HG) (Ϸ23.1 mmol/L), a diabetic-like condition, stimulates Ang II generation and the underlying mechanism of its production in rat vascular smooth muscle cells. The contribution of various enzymes involved in Ang II generation was investigated by silencing their expression with small interfering RNA in cells exposed to normal glucose (Ϸ4.1 mmol/L) and HG. Angiotensin I (Ang I) was generated from angiotensinogen by cathepsin D in the presence of normal glucose or HG. Although HG did not affect the rate of angiotensinogen conversion, it decreased expression of angiotensinconverting enzyme (ACE), downregulated ACE-dependent Ang II generation, and upregulated rat vascular chymasedependent Ang II generation. The ACE inhibitor captopril reduced Ang II levels in the media by 90% in the presence of normal glucose and 19% in HG, whereas rat vascular chymase silencing reduced Ang II production in cells exposed to HG but not normal glucose. The glucose transporter inhibitor cytochalasin B, the aldose reductase inhibitor alrestatin, and the advanced glycation end product formation inhibitor aminoguanidine attenuated HG-induced Ang II generation. HG caused a transient increase in extracellular signal-regulated kinase (ERK)1/2 phosphorylation, and ERK1/2 inhibitors reduced Ang II accumulation by HG. These data suggest that polyol pathway metabolites and AGE can stimulate rat vascular chymase activity via ERK1/2 activation and increase Ang II production. In addition, decreased Ang II degradation, which, in part, could be attributable to a decrease in angiotensin-converting enzyme 2 expression observed in HG, contributes to increased accumulation of Ang II in vascular smooth muscle cells by HG. (Circ Res. 2007;101:455-464.)Key Words: ACE Ⅲ vascular chymase Ⅲ angiotensin Ⅲ VSMC Ⅲ high glucose A ngiotensin (Ang) II, a biologically active component of the renin-angiotensin system (RAS), plays an important role in regulating salt, water, and vascular homeostasis. 1 According to the classical view of RAS, Ang II is cleaved from angiotensin I (Ang I) by angiotensin-converting enzyme (ACE), 2 which is localized on the surface of endothelial cells but also has been shown to be present in the media of aorta and to a lesser degree in adventitia. 3 A soluble form of ACE can be found in plasma. 4 Ang I is formed from the circulating precursor angiotensinogen (AGT) secreted from the liver and is cleaved by renin secreted from the juxtaglomerular cells of the kidney. 5 The presence of local RAS has also been demonstrated in several tissues including vascular wall. 6 AGT is the only known precursor of Ang I and Ang II and is synthesized in vascular smooth muscle cells (VSMCs). 7 Evidence for the presence of renin in vascular tissue has been documented, 8 but renin-like activity in aortic tissue falls to very low ...

Abstract-Cytochrome P450 1B1, expressed in vascular smooth muscle cells, can metabolize arachidonic acid in vitro into several products including 12-and 20-hydroxyeicosatetraenoic acids that stimulate vascular smooth muscle cell growth. This study was conducted to determine whether cytochrome P450 1B1 contributes to angiotensin II-induced rat aortic smooth muscle cell migration, proliferation, and protein synthesis. Angiotensin II stimulated migration of these cells, measured by the wound healing approach, by 1. Metabolism of arachidonic acid to 5-, 12-, 15-, and 20-hydoxyeicosatetraenoic acids in these cells was not altered, but angiotensin II-and arachidonic acid-induced reactive oxygen species production and extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activity were inhibited by 2,4,3Ј,5Ј-tetramethoxystilbene and cytochrome P450 1B1 small hairpin RNA (shRNA) and by Tempol, which inactivates reactive oxygen species. Tempol did not alter cytochrome P450 1B1 activity. These data suggest that angiotensin II-induced vascular smooth muscle cell migration and growth are mediated by reactive oxygen species generated from arachidonic acid by cytochrome P450 1B1 and activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. (Hypertension. 2010;55:1461-1467.) Key Words: angiotensin II Ⅲ CYP1B1 Ⅲ vascular smooth muscle cell growth Ⅲ ROS T he renin-angiotensin system is one of the major components of the mechanisms that contribute to the regulation of blood volume and vascular resistance. 1 Angiotensin II (Ang II), the main biologically active agent of this system, also stimulates vascular smooth muscle cell (VSMC) hypertrophy and/or hyperplasia and inflammation and contributes to the development of hypertension, atherosclerosis, heart failure, and restenosis after vascular injury. [1][2][3][4][5][6] The pathophysiological actions of Ang II are mediated by activation of Ն1 serine-threonine and tyrosine kinase, generation of oxygen radicals, 7-9 and/or release of arachidonic acid (AA) by cytosolic phospholipase A 2 (cPLA 2 ) and production of its metabolites, 12-hydroxyeicosatetraenoic acid (12-HETE) and 20-HETE, generated via lipoxygenase and/or cytochrome P450 (CYP) 4A, respectively. 10 -18 Both 12-and 20-HETE promote VSMC migration, hyperplasia, and/or hypertrophy. 11,19 -22 CYP enzymes that metabolize xenobiotics, including polycyclic aromatic hydrocarbons and endobiotics, such as fatty acids and retinoids, are also expressed in extrahepatic tissues, including the cardiovascular system. 23-27 CYP1A1-encoded enzymes are expressed in vascular endothelium and smooth muscle cells, with much higher levels of activity in endothelial cells, whereas CYP1B1 is highly expressed in VSMCs and, to a lesser degree, in endothelial cells, 28,29 but shear stress upregulates mRNA and protein levels of CYP1A1 and CYP1B1 in endothelial cells. 30 Whether CYP1A1 and CYP1B1 contribute to the vascular function is not known. Recombinant CYP1B1 has been shown to me...

Carnitine palmitoyltransferase-I (CPT-I) catalyzes the rate-controlling step of fatty acid oxidation. CPT-I converts long-chain fatty acyl-CoAs to acylcarnitines for translocation across the mitochondrial membrane. The mRNA levels and enzyme activity of the liver isoform, CPT-I␣, are greatly increased in the liver of hyperthyroid animals. Thyroid hormone (T3) stimulates CPT-I␣ transcription far more robustly in the liver than in nonhepatic tissues. We have shown that the thyroid hormone receptor (TR) binds to a thyroid hormone response element (TRE) located in the CPT-I␣ promoter. In addition, elements in the first intron participate in the T3 induction of CPT-I␣ gene expression, but the CPT-I␣ intron alone cannot confer a T3 response. We found that deletion of sequences in the first intron between ؉653 and ؉744 decreased the T3 induction of CPT-I␣. Upstream stimulatory factor (USF) and CCAAT enhancer binding proteins (C/EBPs) bind to elements within this region, and these factors are required for the T3 response. The binding of TR and C/EBP to the CPT-I␣ gene in vivo was shown by the chromatin immunoprecipitation assay. We determined that TR can physically interact with USF-1, USF-2, and C/EBP␣. Transgenic mice were created that carry CPT-I␣-luciferase transgenes with or without the first intron of the CPT-I␣ gene. In these mouse lines, the first intron is required for T3 induction as well as high levels of hepatic expression. Our data indicate that the T3 stimulates CPT-I␣ gene expression in the liver through a T3 response unit consisting of the TRE in the promoter and additional factors, C/EBP and USF, bound in the first intron.

In rat diabetic animal models, ANG(1-7) treatment prevents the development of cardiovascular complications. Angiotensin-converting enzyme (ACE)2 is a major ANG(1-7)-generating enzyme in vascular smooth muscle cells (VSMCs), and its expression is decreased by a prolonged exposure to high glucose (HG), which is reflected by lower ANG(1-7) levels. However, the underlying mechanism of its downregulation is unknown and was the subject of this study. Rat aortic VSMCs were maintained in normal glucose (NG) or HG ( approximately 4.1 and approximately 23.1 mmol/l, respectively) for up to 72 h. Several PKC and NADPH oxidase inhibitors and short interfering (si)RNAs were used to determine the mechanism of HG-induced ACE2 downregulation. Cell lysates were subjected to Western blot analysis, real-time quantitative PCR, and ANG(1-7) radioimmunodetection. At 72 h of HG exposure, ACE2 mRNA, protein, and ANG(1-7) levels were decreased (0.17 +/- 0.01-, 0.47 +/- 0.03-, and 0.16 +/- 0.01-fold, respectively), and the expression of NADPH oxidase subunit Nox1 was increased (1.70 +/- 0.2-fold). The HG-induced ACE2 decrease was reversed by antioxidants and Nox1 siRNA as well as by inhibitors of glycotoxin formation. ACE2 expression was PKC-betaII dependent, and PKC-betaII protein levels were reduced in the presence of HG (0.32 +/- 0.03-fold); however, the PKC-betaII inhibitor CG-53353 prevented the HG-induced ACE2 loss and Nox1 induction, suggesting a nonspecific effect of the inhibitor. Our data suggest that glycotoxin-induced Nox1 expression is regulated by conventional PKCs. ACE2 expression is PKC-betaII dependent. Nox1-derived superoxides reduce PKC-betaII expression, which lowers ACE2 mRNA and protein levels and consequently decreases ANG(1-7) formation.