Hiroki Manabe

Vitamin E protects against polymorphonuclear leukocyte-dependent adhesion to endothelial cells.

We evaluated the effects of α‐Toc on surface expression of CD11b/CD18 on polymorphonuclear leukocytes (PMN) stimulated with N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) and oxidized low‐density lipoprotein (oxLDL). Incubation of PMN with fMLP (1 μM) or oxLDL (100 μg/mL) increased CD11b/CD18 expression; pretreatment with α‐Toc reduced in a dose‐dependent manner. PMN obtained from healthy adults ingesting 600 mg α‐Toc per day for 10 days were similarly incubated with fMLP or oxLDL; the surface level of CD11b/CD18 was inversely correlated with serum α‐Toc concentrations. Adherence of PMN to human umbilical vein endothelial cells was increased by fMLP or oxLDL stimulation but reduced by α‐Toc pretreatment or anti‐CD18 monoclonal antibodies. cAMP‐dependent protein kinase (PKA) and protein kinase C (PKC) activity in PMN was also assayed. A PKC inhibitor, but not a PKA inhibitor, suppressed CD11b/CD18 up‐regulation, and α‐Toc slightly decreased fMLP‐ and oxLDL‐induced activation of PKC. These results suggest that α‐Toc may prevent inflammation by both reducing CD11b/CD18 up‐regulation and decreasing PMN‐dependent adherence to EC. J. Leukoc. Biol. 65: 757–763; 1999.

α-Tocopherol prevents apoptosis of vascular endothelial cells via a mechanism exceeding that of mere antioxidation

Alpha-tocopherol has been reported to exert an anti-atherogenesis effect. We attempted to clarify the effect of alpha-tocopherol-both as an antioxidant and as a nonantioxidant--on apoptosis induced by oxidized low-density lipoprotein (LDL) or oxysterols. Oxidized LDL and oxysterols induced necrosis and/or apoptosis of vascular endothelial cells. The induction of apoptosis was associated with increased caspase-3 activity and the generation of intracellular reactive oxygen species, both the effects of which were attenuated by alpha-tocopherol. Apoptosis was also decreased by beta-tocopherol or intracellular radical scavengers, but these suppressive effects were less than those of alpha-tocopherol. Neither beta-tocopherol nor the scavengers had pronounced effect on caspase-3 activity, but each of them decreased the generation of reactive oxygen species to the same extent as alpha-tocopherol. Our study suggests that alpha-Toc protects against apoptosis not only by scavenging reactive oxygen species, but also by inhibiting caspase activity, which means that its activity may exceed that of a mere antioxidant.

Methylprednisolone inhibits neutrophil-endothelial cell interactions induced by interleukin-1 β under flow conditions

The effects of methylprednisolone (m-PSL) on IL-1beta-induced neutrophil-endothelial cell interactions, which are normally mediated by increased expression of both intercellular adhesion molecule-1 (ICAM-1) and E-selectin on endothelial cells, were examined using an in vitro flow system. Human neutrophilic polymorphonuclear leukocytes (PMN) were perfused at a shear stress of 1 dyne/cm2 on human umbilical vein endothelial cells (HUVEC) pretreated with IL-1beta (20 U/mL) for 4 hours. Many PMN adhered to IL-1-stimulated HUVEC and then migrated beneath endothelial cell monolayers. Treatment of HUVEC with m-PSL inhibited adherence and migration of PMN in a dose dependent manner. M-PSL also inhibited IL-1beta-induced upregulation of E-selectin and ICAM-1 on HUVEC in a dose dependent manner. These results suggest that m-PSL works as an anti-inflammatory agent through inhibiting PMN-endothelial cell interactions.

7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species

Abstract The aim of the present study was to assess the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), monocytic adhesion of human aortic endothelial cells (HAECs), and the production of intracellular reactive oxygen species (ROS), when HAECs were stimulated by 7-ketocholesterol. 7-ketocholesterol enhances surface expression of ICAM-1 and VCAM-1 as determined by EIA, induces their mRNA expression by RT-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells. We confirmed up-regulation of ROS production of HAECs treated with 7-ketocholesterol. Although the surface expression of ICAM-1 and VCAM-1 on HAECs treated with 7-ketocholesterol increased in a time-dependent manner, α-tocopherol inhibited this increase of the surface expression of ICAM-1 and VCAM-1. In the monocytic adhesion assay, adhesion of U937 to HAECs treated with 7-ketocholesterol was enhanced, but monoclonal anti-ICAM-1 and VCAM-1 antibodies reduced the endothelial adhesiveness. In conclusion, this study suggests that the endothelial adhesiveness to monocytic cells that was increased by 7-ketocholesterol was associated with enhanced expression of ICAM-1 and VCAM-1 mediated by ROS production.

An AT1-receptor antagonist and an angiotensin-converting enzyme inhibitor protect against hypoxia-induced apoptosis in human aortic endothelial cells through upregulation of endothelial cell nitric oxide synthase activity.

The protective effects and roles of AT1-receptor antagonists (AT1-RA) or angiotensin-converting enzyme inhibitors (ACEI) on vascular endothelial cell (EC) injury during hypoxia are not entirely known. Therefore, we investigated these effects and mechanisms in human aortic (HA) EC. DNA fragmentation, Lactate dehydrogenase (LDH) release, and caspase-3 activity were measured in cultured HAEC after exposure to hypoxia in the presence or absence of an AT1-RA (candesartan, CS) and/or an ACEI (temocaprilat, TC). Next, we investigated endothelial cell nitric oxide synthase (ecNOS) and inducible (i) NOS to determine the role of the bradykinin(BK)-NO pathway in the protective effect on ACEI and AT1-RA in the setting of hypoxia-induced apoptosis. Exposure to hypoxia increased DNA fragmentation in HAEC associated with the activation of caspase-3, but did not affect LDH release. In addition, hypoxia induced ecNOS mRNA but not mRNA iNOS. CS and/or TC reduced apoptosis induced by hypoxia in a dose-dependent manner, and significantly increased BK and ecNOS expression. This effect was attenuated by the kinin B2 receptor antagonist, HOE 140, and the NOS inhibitor, N-nitro-l-arginine methylester (L-NMMA). Hypoxia activates the pathway leading to apoptosis by enhancing caspase-3 activity. Both CS and TC can ameliorate hypoxia-induced apoptosis in HAEC through inhibiting caspase-3 activation by enhancing ecNOS activity, via the accumulation of BK.

Inhibitory Effects of Red Wine Extracts on Endothelial-Dependent Adhesive Interactions with Monocytes Induced by Oxysterols

Red wine polyphenolic compounds have been demonstrated to possess antioxidant properties, and several studies have suggested that they might constitute a relevant dietary factor in the protection from coronary heart disease. The aim of the present study is to examine whether red wine extracts (RWE) can ameliorate oxysterol-induced endothelial response, and whether inhibition of adhesion molecule expression is involved in monocyte adhesion to endothelial cells. Surface expression and mRNA levels of adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1) were determined by ELISA and RT-PCR performed on human aortic endothelial cells (HAEC) monolayers stimulated with 7beta-hydroxycholesterol or 25-hydroxycholesterol. Incubation of HAEC with oxysterols (10 microM) increased expression of adhesion molecules in a time-dependent manner. Pretreatment of HAEC with RWE at final concentrations of 1, 10, and 100 ng/ml significantly inhibited the increase of surface protein expression and mRNA levels. Adherence of monocytes to oxysterol-stimulated HAEC was increased compared to that of unstimulated cells. Treatment of HAEC with RWE significantly inhibited adherence of monocytes. These results suggest that RWE works as an anti-atherogenic agent through the inhibition of endothelial-dependent adhesive interactions with monocytes induced by oxysterols.

The inhibitory effect of alacepril, an angiotensin-converting enzyme inhibitor, on endothelial inflammatory response induced by oxysterol and TNF-α

Abstract The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-α. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-α-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-α. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-α.

Effect of α-Tocopherol on Expression of Intercellular Adhesion Molecule-1 and Vascular Adhesion Molecule-1 on Human Vascular Endothelial Cells

α-Tocopherol (α-Toc) has been reported to be an antioxidative agent, but the effect of α-Toc on inflammatory reactions has not been well investigated. Endothelial-dependent interactions with leukocytes are crucial events in an inflammatory pathway. We examined the effect of α-Toc on the expression of adhesion molecules, which are indispensable to endothelial-dependent interactions with leukocytes, on human umbilical vein endothelial cells (HUVECs) stimulated by interleukin-1β (IL-1β). HUVECs were pretreated with α-Toc for 24h before stimulation with IL-1β (20U/ml). Surface expression of intercellular (ICAM-1) and vascular (VCAM-1) adhesion molecules on HUVECs was assessed by enzyme-linked immunosorbent assay 8h after incubation with IL-1β. Although IL-1β significantly increased the expression of ICAM-1 and VCAM-1 on HUVECs, pretreatment with α-Toc reduced the expression of ICAM-1 and VCAM-1 on HUVECs in a dose-dependent manner. These findings indicate that a-Toc may work as an antiinflammatory agent through inhibiting the up-regulation of adhesion molecules on endothelial cells.