Angiotensin II regulation of ovine fetoplacental artery endothelial functions: interactions with nitric oxide

Abstract

During normal pregnancy, elevated angiotensin II (Ang II) concentrations in the maternal and fetal circulations are associated with dramatic increases in placental angiogenesis and blood flow. Much is known about a local renin-angiotensin system within the uteroplacental vasculature. However, the roles of Ang II in regulating fetoplacental vascular functions are less well defined. In the fetal placenta, the overall in vivo vasoconstrictor responses of the blood vessels to Ang II infusion is thought to be less than that in its maternal counterpart, even though infused Ang II induces vasoconstriction. Recent data from our laboratories suggest that Ang II stimulates cell proliferation and increases endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) in ovine fetoplacental artery endothelial cells. These data imply that elevations of the known vasoconstrictor Ang II in the fetal circulation may indeed play a role in the marked increases in fetoplacental angiogenesis and that Ang II-elevated endothelial NO production may partly attenuate Ang II-induced vasoconstriction on vascular smooth muscle. Together with both of these processes, the high levels of Ang II in the fetal circulation may serve to modulate overall fetoplacental vascular resistance. In this article, we review currently available data on the expression of Ang II receptors in the ovine fetal placenta with particular emphasis on the effects of Ang II on ovine fetoplacental endothelium. The potential cellular mechanisms underlying the regulation of Ang II on endothelial growth and vasodilator production are discussed.

Figure 1. Immunohistochemical localization of AT₁-R and eNOS in cotyledons (COT), fetoplacental arteries (FPA), and isolated fetoplacental artery endothelial (OFPAE) cells from late pregnant ewes

The tissue sections and OFPAE cells were stained with an antibody against AT₁-R or eNOS (from Zheng et al. 1999b, 2000). Note that positive AT₁-R and eNOS brownish staining in COT and FPA was primarily present in endothelial cells. *, microvessels; VC, villous core.

Figure 2. Expression of eNOS and iNOS proteins as well as NO production in ovine COT from day 110–142 pregnant ewes

Proteins (50 μg lane⁻¹) from COT were separated and eNOS and iNOS were detected using specific antibodies (from Zheng et al. 2000). Quantitative data normalized to α-actin are from three blots of three experiments and are expressed as means ± s.e.m. Controls: proteins (5 μg) from ovine fetoplacental artery endothelial cells and mouse macrophages, respectively, for eNOS and iNOS. Total NO (nitrate and nitrite) levels in COT conditioned media were determined using a NO analyser (NOA 280, Silvers Instruments) based on a reaction of conversion of nitrite and nitrate to NO. Total NO production was calculated by a standard curve generated with sodium nitrate as the standard and normalized by the protein content of corresponding samples. Data have been subtracted from those in unconditioned media controls and are expressed as means ± s.e.m. Means with different letters differ significantly (P < 0.05).

Figure 3. Expression of AT₁-R and AT₂-R on OFPAE cells and their effects on Ang II-stimulated OFPAE cell proliferation

Upper panel: binding of ¹²⁵I-labelled Ang II to OFPAE cells in the absence (total binding) or presence of unlabelled DuP 753 or PD 123319 (from Zheng et al. 1999b). Data are expressed as mean (±s.e.m.) binding of ¹²⁵I-Ang II alone and are representative of data obtained in three experiments. Lower panel: effects of Ang II in the absence or presence of unlabelled DuP 753 or PD 123319 on proliferation of OFPAE cells (from Zheng et al. 1999b). Data are expressed as mean (±s.e.m.) percentage of control. *Significant difference from control (P < 0.05).

Figure 4. Effect of Ang II on eNOS protein expression and NO production in OFPAE cells

Cells were treated with Ang II (10 nm) for 24 h. Media were collected for detecting total NO (nitrate and nitrite) levels. Proteins separated were used for measuring changes in eNOS protein. For eNOS protein, data are expressed as mean (±s.e.m.) percentage of control value (in absence of Ang II). For NO levels, data have been subtracted from those in unconditioned media controls and are expressed as mean ± s.e.m.*Significant difference from control (P < 0.05). (from Zheng et al. 2005.)

Figure 5. Ang II activation of ERK_1/2 in OFPAE cells

A, translocalization of phosphorylated ERK_1/2 induced by Ang II in OFPAE cells. After 16 h of serum starvation, cells were treated with Ang II (10 nm) for 0, 1, 10 or 15 min or 10 min after 1 h of pretreatment with PD 98059 (50 μm). Cells were fixed and stained with a phospho-specific ERK_1/2 antibody. Note: brown staining indicates positive phosphorylated ERK_1/2; blue nuclear staining is haematoxylin counterstaining.B, Western blot analysis for Ang II-induced phosphorylation of ERK_1/2. Cells were treated with Ang II (10 nm) for 10 min alone or with PD 98059 (50 μm, 1 h pretreatment). For Western blot analysis, proteins were separated, and phosphorylated ERK_1/2 was detected using a phospho-specific ERK or total ERK antibody. pERK_1/2, phosphorylated ERK_1/2; tERK_1/2, total ERK_1/2; Ctrl, control; PD, PD 98059; Std, standard phosphorylated (2 ng protein) or total (10 ng protein) ERK_1/2. (Zheng et al. 2005.)

Figure 6. Effects of PD 98059 on Ang II-increased eNOS protein expression and NO production in OFPAE cells

Cells were treated with Ang II (10 nm) for 24 h in the absence or presence of PD 98059 (50 μm; 1 h of pretreatment). Media were collected for total NO detection, and proteins were used for detecting eNOS protein. For eNOS protein, data are expressed as mean (±s.e.m.) percentage of control (in absence of Ang II). For NO levels, data have been subtracted from those in unconditioned media controls, normalized by the protein content of corresponding wells. Means with different letters differ significantly (P < 0.05). (from Zheng et al. 2005.)