Angiotensin II type 1 receptor antibodies and increased angiotensin II sensitivity in pregnant rats

Abstract

Pregnant women who subsequently develop preeclampsia are highly sensitive to infused angiotensin (Ang) II; the sensitivity persists postpartum. Activating autoantibodies against the Ang II type 1 (AT(1)) receptor are present in preeclampsia. In vitro and in vivo data suggest that they could be involved in the disease process. We generated and purified activating antibodies against the AT(1) receptor (AT(1)-AB) by immunizing rabbits against the AFHYESQ epitope of the second extracellular loop, which is the binding epitope of endogenous activating autoantibodies against AT(1) from patients with preeclampsia. We then purified AT(1)-AB using affinity chromatography with the AFHYESQ peptide. We were able to detect AT(1)-AB both by ELISA and a functional bioassay. We then passively transferred AT(1)-AB into pregnant rats, alone or combined with Ang II. AT(1)-AB activated protein kinase C-α and extracellular-related kinase 1/2. Passive transfer of AT(1)-AB alone or Ang II (435 ng/kg per minute) infused alone did not induce a preeclampsia-like syndrome in pregnant rats. However, the combination (AT(1)-AB plus Ang II) induced hypertension, proteinuria, intrauterine growth retardation, and arteriolosclerosis in the uteroplacental unit. We next performed gene-array profiling of the uteroplacental unit and found that hypoxia-inducible factor 1α was upregulated by Ang II plus AT(1)-AB, which we then confirmed by Western blotting in villous explants. Furthermore, endothelin 1 was upregulated in endothelial cells by Ang II plus AT(1)-AB. We show that AT(1)-AB induces Ang II sensitivity. Our mechanistic study supports the existence of an "autoimmune-activating receptor" that could contribute to Ang II sensitivity and possible to preeclampsia.

Figure 1

A, Autoantibodies against angiotensin II receptor 1 (AT₁-AAs) from preeclamptic patients and antibodies against the AT₁ receptor (AT₁-AB) increased the cardiomyocyte spontaneous beating rate. Irbesartan (Irb; 1 µmol/L) and the AFHYESQ peptide inhibited. B, AT₁-ABs (1 µg) were used in different concentrations to detect AT₁, α-1, or β-1 receptor autoantibodies (α1-AB, β1-AB, and AT₁-AB). A strong dose-dependent signal for AT₁-AB was only present in the AT₁-receptor autoantibody ELISA; α- and β-adrenergic receptors were not recognized. C, AT₁-AB activated protein kinase C (PKC)-α in Chinese hamster ovary (CHO)/AT₁ receptor (AT₁R) cells; irbesartan (1 µmol/L) inhibited. Yellow shows PKC-α activation; red is more intense activation. D, Extracellular-regulated kinase (ERK) 1/2 was activated by AT₁-AB. CHO/AT₁R cells were treated for 15 minutes with 25 and 50 µg of AT₁-AB, respectively. Lanes 1 and 2 represent untreated cells. Eukaryotic initiation factor 4E (elF4E) and ERK1/2 antibody were loading controls.

Figure 2

A, Immunoblot for rabbit IgG. Rabbit IgG was detected in the serum of antibodies against the angiotensin II (Ang II) type 1 receptor (AT₁-ABs)–treated rats on day 11 and day 13 of pregnancy. B, ELISA for AT₁-AB shows that detection was possible on day 20 in the serum of AT₁-AB–treated rats. C, Telemetric blood pressures show that mean arterial blood pressure increased with Ang II infusion only when AT₁-ABs were present. D, Acute blood pressure response to Ang II (100 ng/kg per minute) infusion as bolus is shown. Ang II induced a higher blood pressure response in AT₁-AB pretreated rats vs control rats.

Figure 3

A, Fetal weights were significantly decreased in the angiotensin II (Ang II) plus antibodies against the Ang II type 1 receptor (AT₁-ABs) treated group. B, Embryo brain:liver ratio on day 20 showed that Ang II plus AT₁-ABs gave higher values than all of the other groups, indicating intrauterine growth restriction (IUGR) in this group. C, Trophoblast invasion into the spiral arteries in the Ang II plus AT₁-AB group was reduced compared with control. AT₁-AB alone also reduced the spiral artery trophoblast/vessel contour. D, Atherosis (arteriolosclerosis) lesions were increased in the Ang II plus AT₁-AB group. Left and middle show a cross-section of spiral arteries containing endovascular trophoblast. Between both lumens, atherotic lesions are seen with necrotic cells surrounded by interstitial trophoblasts. D, Atherosis was increased in the Ang II plus AT₁-AB group. Right is shown a different cross-section of a spiral artery with advanced lesions (*).

Figure 4

A, Endothelin 1 (ET-1) protein levels in the endothelial cell supernatant are shown. Angiotensin II (Ang II) and antibodies against the Ang II type 1 receptor (AT₁-AB) induced ET-1 production. Ang II plus AT₁-AB resulted in a strong ET-1 increase. B, Dot plots of log-2 signal values of rat hypoxia-inducible factor (HIF) 1α are shown. HIF-1α was differentially expressed at false discovery rate 3.02*10⁻⁴. HIF-1α was induced in the mesometrial triangle (maternal part of the rat placenta) from rats treated with Ang II plus AT₁-AB. C, HIF-1α was expressed in human placental villous explants. Ang II or AT₁-AB induced protein expression of HIF-1α. However, Ang II plus AT₁-AB increased HIF-1α further.