No abstract

Cardiovascular tissue injury in ischemia/reperfusion has been shown to be prevented by angiotensin-converting enzyme (ACE) inhibitors. However, the mechanism on endothelial cells has not been assessed in detail. Cultured human aortic endothelial cells (HAEC) were exposed to hypoxia with or without reoxygenation. Hypoxia enhanced apoptosis along with the activation of caspase-3. Reoxygenation increased lactate dehydrogenase release time-dependently, along with an increase of intracellular oxygen radicals. ACE inhibitor quinaprilat and bradykinin significantly lessened apoptosis and lactate dehydrogenase release with these effects being diminished by a kinin B2 receptor antagonist and a nitric oxide synthase inhibitor. In conclusion, hypoxia activated the suicide pathway leading to apoptosis of HAEC by enhancing caspase-3 activity, while subsequent reoxygenation induced necrosis by enhancing oxygen radical production. Quinaprilat could ameliorate both apoptosis and necrosis through the upregulation of constitutive endothelial nitric oxide synthase via an increase of bradykinin, with the resulting increase of nitric oxide.

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.

Alpha-tocopherol supplementation is reported to protect against cardiovascular disease and to influence cells involved in atherogenesis, such as monocytes. Interactions between monocytes and vascular endothelial cells occur early in atherogenesis, and adhesion is mediated by integrins. We evaluated the effects of alpha-tocopherol on expression of Mac-1 (CD11b/CD18) by monocytes after stimulation with oxidized low-density lipoprotein (LDL), which is implicated as a potent chemotactic agent in atherogenesis. Incubation of whole blood with oxidized LDL (100 microg/ml) increased Mac-1 expression on monocytes, and preincubation with alpha-tocopherol reduced this upregulation in a concentration dependent manner. In another experiment, whole blood was obtained from healthy adult volunteers after 10 days of alpha-tocopherol administration (600 mg/day) and was incubated with oxidized LDL (100 microg/ml). There was a decrease in the upregulation of Mac-1 compared with that measured before administration. Adherence of oxidized LDL-stimulated monocytes to human umbilical vein endothelial cells was reduced by pretreatment with alpha-tocopherol, and was also inhibited by an anti-CD18 monoclonal antibody. Experiments with protein kinase C inhibitors suggested that reduction of Mac-1 upregulation by alpha-tocopherol was secondary to a decrease of protein kinase C activity. In conclusion, alpha-tocopherol suppressed the upregulation of Mac-1 expression on monocytes by oxidized LDL.

Summary Quinaprilat, an angiotensin-converting enzyme (ACE) inhibitor, is commonly used for the therapy of hypertension, but the influence of ACE inhibitor for the atherogenesis has not been clear. Then we investigated the suppressive effect of ACE inhibitor on the expression of adhesion molecules and the production of cytokines which play an important role in the early development of atherosclerosis in endothelial cells (ECs). Quinaprilat treatment significantly reduced the activation of nuclear factor-KB (NF-KB), the surface expression of vascular cell adhesion molecule-1 (VCAM-1), the production of cytokines such as monocyte chemoattractant protein-1 and interleukin-6, and the number of mononuclear leukocytes adhering to ECs induced by tumor necrosis factor-a (TNF-a) and oxidized low-density lipoprotein (oxLDL). This suppressive effect was diminished by both a kinin B2 receptor antagonist and a nitric oxide synthase (NOS) inhibitor. These results indicated that quinaprilat reduced the activation of NF-KB under the stress of chemoattractants such as TNF-a and oxLDL through the increase of endothelial nitric oxide (NO) produced by activated endothelial nitric oxide synthase (eNOS) via a secondary accumulation of bradykinin in ECs. These results suggest an anti-atherogenic effect of ACE inhibitor.