Yoshinari Uehara

Testosterone up-regulates scavenger receptor BI and stimulates cholesterol efflux from macrophages

By lowering high density lipoprotein (HDL) cholesterol, testosterone contributes to the gender difference in HDL cholesterol and has been accused to be pro-atherogenic. The mechanism by which testosterone influences HDL cholesterol is little understood. We therefore investigated the effect of testosterone on the gene expression of apolipoprotein A-I (apoA-I), hepatic lipase (HL), scavenger receptor B1 (SR-BI), and the ATP binding cassette transporter A1 (ABCA1), all of which are important regulators of HDL metabolism. In both cultivated HepG2 hepatocytes and primary human monocyte-derived macrophages, testosterone led to a dose-dependent up-regulation of SR-BI, which was assessed on both the mRNA and the protein levels. As a functional consequence, we observed an increased HDL(3)-induced cholesterol efflux from macrophages. At supraphysiological dosages, testosterone also increased the expression of HL in HepG2 cells. Testosterone had no effect on the expression of apoA-I in HepG2 cells and ABCA1 in either HepG2 cells or macrophages. These data suggest that testosterone, despite lowering HDL cholesterol, intensifies reverse cholesterol transport and thereby exerts an anti-atherogenic rather than a pro-atherogenic effect.

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.

The Central Helices of ApoA-I Can Promote ATP-binding Cassette Transporter A1 (ABCA1)-mediated Lipid Efflux AMINO ACID RESIDUES 220–231 OF THE WILD-TYPE ApoA-I ARE REQUIRED FOR LIPID EFFLUX IN VITRO AND HIGH DENSITY LIPOPROTEIN FORMATION IN VIVO

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by ∼80–92% by apoA-I[Δ(185–243)], only by 15% by apoA-I[Δ(1–41)] or apoA-I[Δ(1–59)], and was restored to 75–80% of the wild-type apoA-I control value by double deletion mutants apoA-I[Δ(1–41)Δ(185–243)] and apoA-I[Δ(1–59)Δ(185–243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[Δ(232–243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30–40% and 50–65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[Δ(1–41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[Δ(1–41)Δ(185–243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220–231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.

Chymase inhibition suppresses high-cholesterol diet-induced lipid accumulation in the hamster aorta

OBJECTIVE The role of chymase (a mast cell-derived angiotensin II-forming serine proteinase) in aortic lipid deposition was investigated using an orally active, non-peptide chymase inhibitor, SUN-C8257. METHODS Male golden Syrian hamsters, 8 weeks old, were fed with a standard rodent meal supplemented with or without 0.5% cholesterol and 10% coconut oil for 12 weeks. The hamsters fed high cholesterol diet were further separated into two groups treated with or without SUN-C8257 for 12 weeks. The aortic lipid deposition was visualized by Oil red O staining and planimetrically measured. Immunohistochemical staining for angiotensin II (Ang II) of the aortic root region was performed. Aortic Ang II-forming activity was measured using Ang I as a substrate. Plasma total-, low-density lipoprotein (LDL)-, high-density lipoprotein (HDL)-cholesterol and triglyceride were quantified by enzymatic methods. Plasma Ang I and Ang II were measured by radioimmunoassay. RESULTS After 12 weeks of high cholesterol diet, aortic chymase activity in the untreated group increased significantly and showed a positive correlation with plasma total- and LDL-cholesterol. This group of hamsters developed marked lipid deposits in the aortic intima. However, treatment with SUN-C8257 significantly suppressed aortic lipid deposition without changing body weight, blood pressure, plasma LDL-cholesterol and Ang II levels. The level of the adventitial Ang II-immunoreactivity was markedly inhibited in the group treated with SUN-C8257. CONCLUSION Our results suggest that arterial chymase may participate in the acceleration of lipid deposition in arterial walls exposed to high plasma cholesterol and that inhibition of arterial chymase may retard the progression of atherosclerosis.

Pioglitazone enhances cholesterol efflux from macrophages by increasing ABCA1/ABCG1 expressions via PPARγ/LXRα pathway: Findings from in vitro and ex vivo studies

OBJECTIVE Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, reportedly reduces cardiovascular events in diabetic patients. ATP cassette binding transporters (ABC) A1 and G1 are pivotal molecules for cholesterol efflux (ChE) from macrophages and high density-lipoprotein biogenesis, and the A1 transporter is regulated by a PPARγ-liver receptor X (LXR) pathway. Also, pioglitazone induces ABCG1 expression, though the exact mechanism remains unclear. We therefore investigated the effects of pioglitazone on ABCA1/G1 expression in vitro and ex vivo. METHODS The effects of pioglitazone on ChE and ABCA1/G1 expressions in macrophages were assessed. Then, mRNA was quantified in macrophages when PPARγ/LXR inhibition by siRNA or overexpression of oxysterol sulfotransferase was performed. ABCA1/G1 promoter activity with mutated LXR-responsive elements was also measured. As an ex vivo study, 15 type 2 diabetic patients were administered pioglitazone or placebo, and ChE assays and protein expressions were determined using macrophages cultured with the corresponding sera. RESULTS Pioglitazone increased LXRα/ABCA1/G1 expressions, which enhanced ChE from macrophages. Inhibition of PPARγ/LXR pathways revealed that LXR was primarily involved in pioglitazones transactivation of ABCA1 but only partially involved for ABCG1. Promoter assays showed that ABCG1 was regulated more by the promoter in intron 4 than that upstream of exon 1 but both promoters were responsive to LXR activation. Sera obtained after pioglitazone treatment promoted ChE and ABCA1/G1 expressions in macrophages. CONCLUSION Pioglitazone enhanced ChE from macrophages by increasing ABCA1/G1 in LXR-dependent and -independent manners. Our comparable in vitro and ex vivo results shed new light on pioglitazones novel anti-atherogenic property.

Genetic analysis of the epithelial sodium channel in Liddle's syndrome.

Background Liddles syndrome is an autosomal inheritable disorder that causes hypertension due to excess function of sodium channel. Objective To analyze the DNA sequence of the amiloride-sensitive epithelial sodium channel (ENaC) in three patients who had low-renin hypertension with hypokalemia. The patients included a 24-year-old woman and her 20-year-old brother whose mother was hypertensive. The third patient was a 15-year-old girl with no family history of hypertension. Methods The DNA sequence of the ENaC was analyzed as follows. Venous blood samples were collected from the patients and total genomic DNA was prepared by standard methods. Specific primers were used for direct polymerase chain reaction; one set of primers for amplifying the C terminus (codon 523–638) of the b subunit of ENaC, and two sets of primers for amplifying the C terminus (codons 525–587 and 568–650) of the γ subunit of ENaC. Polymerase chain reaction products were purified and subjected to direct DNA sequence analysis. Results Direct sequence analysis demonstrated the presence of a single-base substitution in one segment of the b subunit of ENaC, a C→T transition that changed the encoded Pro (CCC) at codon 616 to Ser (TCC) in the siblings (cases 1 and 2). In case 3, we found a missense mutation of Pro (CCC) to Leu (CTC) at codon 616. Case 3 is considered to be sporadic, since DNA sequencing of the PY motif of her parents gave normal results. Conclusions The DNA sequences of the ENaC in three patients with Liddles syndrome were analyzed. In one family case, we found a new missense mutation of Pro (CCC) to Ser (TCC) at codon 616 in the β subunit of ENaC. A genetic analysis of the amiloride-sensitive epithelial sodium channel is recommended in assessing patients with low-renin, salt-sensitive hypertension whose blood pressure is not responsive to spironolactone treatment.

Free Fatty Acid Causes Leukocyte Activation and Resultant Endothelial Dysfunction Through Enhanced Angiotensin II Production in Mononuclear and Polymorphonuclear Cells

Release of free fatty acid (FFA) from adipose tissue is implicated in insulin resistance and endothelial dysfunction in patients with visceral fat obesity. We demonstrated previously that increased FFA levels cause endothelial dysfunction that is prevented by inhibition of the renin-angiotensin system (RAS) in humans. However, the mechanisms for FFA-mediated activation of RAS and the resultant endothelial dysfunction were not elucidated. We investigated effects of elevated FFA on activity of circulating and vascular RAS, angiotensin II–forming activity of leukocytes, and leukocyte activation of normotensive subjects. We showed that increased FFA levels significantly enhanced angiotensin II–forming activity in human mononuclear (mean fold increase: 3.5 at 180 minutes; P=0.0016) and polymorphonuclear (2.0; P=0.0012) cells, whereas parameters of the circulating and vascular RAS were not affected. We also showed that FFA caused angiotensin II– dependent leukocyte activation, which impaired endothelial function partly via increased myeloperoxidase release and presumably enhanced adhesion of leukocytes. We propose that the enhanced production of angiotensin II by FFA in mononuclear and polymorphonuclear cells causes activation of leukocytes that consequently impairs endothelial function. RAS in leukocytes may regulate the leukocyte-vasculature interaction as the mobile RAS in humans.

Effects of rosuvastatin on electronegative LDL as characterized by capillary isotachophoresis: the ROSARY Study

Electronegative LDL, a charge-modified LDL (cm-LDL) subfraction that is more negatively charged than normal LDL, has been shown to be inflammatory. We previously showed that pravastatin and simvastatin reduced the electronegative LDL subfraction, fast-migrating LDL (fLDL), as analyzed by capillary isotachophoresis (cITP). The present study examined the effects of rosuvastatin on the more electronegative LDL subfraction, very-fast-migrating LDL (vfLDL), and small, dense charge-modified LDL (sd-cm-LDL) subfractions. Patients with hypercholesterolemia or those who were being treated with statins (n = 81) were treated with or switched to 2.5 mg/d rosuvastatin for 3 months. Rosuvastatin treatment effectively reduced cITP cm-LDL subfractions of LDL (vfLDL and fLDL) or sdLDL (sd-vfLDL and sd-fLDL), which were closely related to each other but were different from the normal subfraction of LDL [slow-migrating LDL (sLDL)] or sdLDL (sd-sLDL) in their relation to the levels of remnant-like particle cholesterol (RLP-C), apolipoprotein (apo) C-II, and apoE. The percent changes in cm-LDL or sd-cm-LDL caused by rosuvastatin were correlated with those in the particle concentrations of LDL or sdLDL measured as LDL-apoB or sdLDL-apoB and the levels of HDL-C, RLP-C, apoC-II, and apoE. In conclusion, rosuvastatin effectively reduced both the vfLDL subfraction and sd-cm-LDL subfractions as analyzed by cITP.

Increased Chymase Activity in Internal Thoracic Artery of Patients With Hypercholesterolemia

Apart from ACE, various angiotensin II (Ang II)-forming serine proteinases (eg, chymase, kallikrein, and cathepsin G) are known to exist in human tissues, but their clinical significance or the regulatory mechanisms that control their activities are not well established. A recent clinical study has shown that chymase activity was significantly increased in human atherosclerotic or aneurysmal aorta. The association between vascular Ang II-forming activities (AIIFAs) in the human internal thoracic artery (ITA) and various clinical parameters was studied with the use of ITAs obtained from 32 patients who underwent coronary artery bypass graft surgery. Total and ACE- and chymase-dependent AIIFAs in homogenates of ITAs were determined. Total AIIFA was 8.67+/-0.86 (nmol Ang II formed. min(-1). mg protein(-1) [U]), and approximately 95% of the activities were due to chymase. Serum total cholesterol level, but no other risk factors, significantly correlated with chymase- (r=0. 60, P<0.001) and ACE- (r=0.35, P<0.05) dependent AIIFAs, respectively. LDL cholesterol level was also correlated with chymase-dependent AIIFAs (r=0.47, P<0.05). Mast cells identified through the use of toluidine blue or immunohistochemical staining appeared in the adventitia but not in the intima or media of ITAs. Our results suggest that an increased plasma LDL cholesterol level may induce increased arterial chymase and ACE activity.

Antiarrhythmogenic Effect of Reconstituted High-Density Lipoprotein Against Ischemia/Reperfusion in Rats

OBJECTIVES This study analyzed the antiarrhythmogenic effect of reconstituted high-density lipoprotein (rHDL) against ischemia/reperfusion in vivo. BACKGROUND Recent studies have suggested that a reduction in the plasma HDL level may contribute to cardiac sudden death. Although there are currently only a few therapeutic strategies for increasing HDL, an exciting new therapeutic option, rHDL, has recently been developed to prevent coronary artery disease. METHODS To analyze the suppression of reperfusion arrhythmia by rHDL (apolipoprotein A-I with 1-palmitoyl-2-oleoyl-phosphatidyl-choline), 92 male Wistar rats were divided into 10 groups: rats that had been pre-treated with or without rHDL, apolipoprotein A-I, or 1-palmitoyl-2-oleoyl-phosphatidyl-choline in the presence or absence of inhibitors of Akt protein kinase, nitric oxide (NO), or extracellular-signal-regulated kinase (ERK) administered intravenously before left coronary artery occlusion. We also used human coronary artery endothelial cells and adenosine triphosphate-binding cassette transporter (ABC) A1-, ABCG1-, or scavenger receptor class B, type I-transfected ldlA7 cells systems. RESULTS The duration of ventricular tachycardia or ventricular fibrillation after reperfusion in rHDL-pre-treated rats was much shorter than that in untreated rats. Apolipoprotein A-I or 1-palmitoyl-2-oleoyl-phosphatidyl-choline alone had no effect. The effect of rHDL was blocked by inhibitors of Akt, NO, and ERK. Plasma NO concentration in the rHDL group was significantly higher. In addition, rHDL activated phospho(p)-Akt, p-ERK, and p-endothelial NO synthesis in endothelial cells. The rHDL activated p-ERK in ABCA1- or ABCG1-transfected but not scavenger receptor class B, type I-transfected ldlA7 cells. CONCLUSIONS The rHDL-induced NO production, probably mediated by ABCA1 or ABCG1 through an Akt/ERK/NO pathway in endothelial cells, may suppress reperfusion-induced arrhythmias. The HDL-based therapy may hold the promise of reducing the incidence of such arrhythmias after ischemia/reperfusion.