Abstract
The molecular and cellular mechanisms by which hypertension enhances atherosclerosis are poorly understood. Angiotensin II (Ang II) has been implicated in the regulation of cellular lipoxygenases (LO), which are thought to play a role in atherogenesis by inducing oxidative modification of low density lipoprotein (LDL). We sought to test the hypothesis that Ang II would stimulate murine macrophage LO activity (which has both 12- and 15-LO activity). Competitive binding studies revealed the presence of Ang II AT1 receptors on mouse peritoneal macrophages (MPM) and J-774 cells, but not on the RAW cell line. Valsartan, a specific AT1 receptor antagonist inhibited Ang II binding, whereas PD 123319, an AT2 receptor antagonist did not. Incubation of MPM or J-774 cells with Ang II (10 pM to 1 microM) for 24 h led to a 2.5-3.5-fold increase in LO activity, measured as generated 13-HODE or 12(S)-HETE. This stimulation was inhibited by valsartan, but not by PD 123319. In contrast, Ang II did not stimulate LO activity in RAW macrophages. Semiquantitative reverse transcriptase-polymerase chain reaction showed a 2-3-fold increase in LO mRNA in MPM, but not in RAW cells after treatment with Ang II. Ang II also induced an increase in 12-LO protein. In addition, pretreatment of J-774 cells with Ang II increased in a dose-dependent manner the ability of the cells to modify LDL, resulting in greater chemotactic activity for monocytes, typical of minimally modified LDL. This stimulation was inhibited by AT1 receptor blockade. In summary, these data suggest that Ang II increases macrophage LO activity via AT1 receptor-mediated mechanisms and this further increases the ability of the cells to generate minimally oxidized LDL. These studies provide a link between hypertension and the associated increased atherosclerosis observed in hypertensive patients.
Highlights
These data suggest that Angiotensin II (Ang II) increases macrophage LO activity via AT1 receptor-mediated mechanisms and this further increases the ability of the cells to generate minimally oxidized low density lipoprotein (LDL)
We determined the parameters of Ang II binding to mouse peritoneal macrophages (MPM), J-774, and RAW cell lines
Ang II-specific binding on J-774 and on MPM could be dose-dependently competed by valsartan, an AT1 receptor antagonist with an IC50 of 1 nM, whereas PD 123319, an AT2 receptor blocker was not able to inhibit [125I-Sar1,Ile8]Ang II binding even at millimolar concentrations (Fig. 1B)
Summary
Materials—Ang II (human), FMLP, indomethacin, Tyrode’s salts, and glutaraldehyde were purchased from Sigma. [Sar1,Ile8]Ang II was from Bachem Fine Chemicals (Torrance, CA). Cells were treated with Ang II for 24 h prior to incubation with radiolabeled linoleic acid. 125I-Angiotensin II Binding—[125I-Sar1,Ile8]Ang II binding and competition assays were performed on adherent cells as described previously with minor modifications (28). The cells were incubated with increasing concentrations of [125I-Sar1,Ile8]Ang II for 60 min at room temperature. The supernatants of the treated cells, in the absence or presence of LDL, with and without pretreatment with Ang II, as well as native LDL alone, were placed in the lower compartment of a chemotactic chamber which was separated from the upper compartment by a 5-m pore-sized polycarbonate membrane (Poretics, Livermore, CA). Purified reticulocyte 15-LO was included as positive control; non-immune sheep IgG was used as negative control This antibody has previously been shown to cross-react with both human and mouse 12/ 15-LO (38). Data are represented as mean Ϯ S.E., and p Ͻ 0.05 was considered statistically significant
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