Angiotensin receptor agonistic autoantibody-mediated tumor necrosis factor-alpha induction contributes to increased soluble endoglin production in preeclampsia

Abstract

Background: Preeclampsia is a prevalent life-threatening hypertensive disorder of pregnancy. The circulating antiangiogenic factor, soluble endoglin (sEng), is elevated in the blood circulation of women with preeclampsia and contributes to disease pathology; however, the underlying mechanisms responsible for its induction in preeclampsia are unknown.

Methods and results: Here, we discovered that a circulating autoantibody, the angiotensin receptor agonistic autoantibody (AT(1)-AA), stimulates sEng production via AT(1) angiotensin receptor activation in pregnant mice but not in nonpregnant mice. We subsequently demonstrated that the placenta is a major source contributing to sEng induction in vivo and that AT(1)-AA-injected pregnant mice display impaired placental angiogenesis. Using drug screening, we identified tumor necrosis factor-alpha as a circulating factor increased in the serum of autoantibody-injected pregnant mice contributing to AT(1)-AA-mediated sEng induction in human umbilical vascular endothelial cells. Subsequently, among all the drugs screened, we found that hemin, an inducer of heme oxygenase, functions as a break to control AT(1)-AA-mediated sEng induction by suppressing tumor necrosis factor-alpha signaling in human umbilical vascular endothelial cells. Finally, we demonstrated that the AT(1)-AA-mediated decreased angiogenesis seen in human placenta villous explants was attenuated by tumor necrosis factor-alpha-neutralizing antibodies, soluble tumor necrosis factor-alpha receptors, and hemin by abolishing both sEng and soluble fms-like tyrosine kinase-1 induction.

Conclusions: Our findings demonstrate that AT(1)-AA-mediated tumor necrosis factor-alpha induction, by overcoming its negative regulator, heme oxygenase-1, is a key underlying mechanism responsible for impaired placental angiogenesis by inducing both sEng and soluble fms-like tyrosine kinase-1 secretion from human villous explants. Our results provide important new targets for diagnosis and therapeutic intervention in the management of preeclampsia.

Figure 1. IgG from women with PE induces sEng secretion in pregnant but not non-pregnant mice via AT₁- receptor activation

IgG from women with PE or normotensive pregnant women was introduced by intra-orbital injection into pregnant mice or non-pregnant mice for five days with a single injection or double injection. (A) Plasma was collected at different time points as indicated and the concentration of sEng was determined by ELISA. Data are expressed as mean ± SEM. * P < 0.05 versus gestation day 18 pregnant mice injected with normotensive IgG. (B) Co-injection of losartan or the 7-aa epitope peptide inhibited the increase of sEng production by IgG from women with preeclampsia. * P < 0.01 versus gestation day 13 pregnant mice injected with IgG from women with preeclampsia. ** P < 0.05 versus gestation day 18 pregnant mice injected with preeclamptic IgG. (C) No effect on sEng production by IgG from women with preeclampsia in non-pregnant mice. Data are expressed as mean ± SEM. N=8 for each group.

Figure 2. Placenta contributes to sEng production in response to IgG from women with PE

(A) Semi-quantitative RT-PCR was used to quantify endoglin mRNA abundance from mouse placentas. L: losartan, 7-aa, seven amino acid epitope peptide. (B) The ratio of Eng mRNA/β-actin mRNA was obtained by performing densitometric analysis of multiple agarose gels (n=8 mice for each group). * P < 0.05 versus mice injected with IgG from normotensive pregnant women; ** P < 0.05 versus preeclamptic IgG injection. Data are expressed as mean ± SEM. (C) The expression levels of Eng and sEng protein were analyzed by western blot. The ratio of sEng (D) or Eng (E) protein to β-actin is used to represent the sEng and Eng expression levels. N=8 mice for each group. * P < 0.05 versus mice injected with IgG from normotensive pregnant women. Data are expressed as mean ± SEM.

Figure 3. CD-31 immunohistochemical staining of mouse placentas

(A) CD-31 (PECAM-1) staining of the placentas of mice injected with IgG from women with preeclampsia (PE) and normotensive pregnancy (NT) in absence or presence of Losartan or 7-aa epitope peptide. Inset: Junctional (J) and Labryinth (L) zone border. Four placenta were chosen from each category (n=8 mice). Scale bar: 50µm. (B) Quantification of CD-31 (PECAM-1) staining. Mean scores are represented ± SEM. N=8 mice for each category. * p<0.01versus normotensive IgG injection; **p<0.05 versus preeclamptic IgG injection.

Figure 4. TNF-α is the serum factor in autoantibody-injected pregnant mice that is responsible for induction of sEng production by endothelial cells

(A) HUVEC cells were treated with Ang II, IgG from preeclamptic or normotensive women and the concentration of sEng in cell culture supernatant was determined. Data are expressed as mean (± SEM) of ≥3 experiments performed in duplicate (n=12–14 patient’s IgG for each group). (B) HUVEC cells treated with serum from control pregnant mice, pregnant mice injected with IgG from normotensive (NT) or preeclamptic women (PE) or coinjected with preeclamptic IgG and losartan (PE+Losartan), 7-aa epitope peptide (PE+7-aa) at day 5 following the initial injections. HUEVC cells also treated with anti-TNF-α, Enbrel or hemin for 30 minitues before adding serum from pregnant mouse injected with IgG from women with preeclampsia (mouse serum (PE)). After 24 hour the sEng concentration in cell culture supernatants were measured. * P < 0.05 versus cells treated with serum from mouse injected with normotensive IgG. ** P < 0.05 versus cells treated with serum from mouse injected with preeclamptic IgG. Data are expressed as mean ± SEM. N =8 mice’s serum for each group. (C) HUVEC cells were treated with TNF-α in the presence of TNF-α neutralizing antibody, Enbrel or hemin for 24 hours. Concentration of sEng in cell culture media was determined by ELISA. * P < 0.01 versus control untreated cells. ** P < 0.05 versus cells treated TNF-α alone. Data are expressed as mean (± SEM) of ≥3 experiments performed in duplicate (n=9 for each group). (D) IgG from normotensive or preeclamptic pregnant women were introduced into pregnant mice at gestation days 13 and 14. Mouse serum was collected at gestation days 18 and levels of TNF-α were measured by ELISA. Data are expressed as mean ± SEM, n=5–8 for each group. * P < 0.05 versus mice treated with normotensive IgG. ** P < 0.05 versus mice treated with preeclamptic IgG alone. (E) Placentas were collected (n=8–10 for each group) at gestation day 18 (5 days following the initial IgG injection). Real-time PCR was performed to quantify the TNF-α mRNA abundance. Data are expressed as mean ± SEM. * P < 0.001 versus normotensive IgG injection. ** P < 0.01 versus preeclamptic IgG injection..

Figure 5. AT₁-AA-mediated TNF-α induction contributes to impaired placenta angiogenesis by inducing both sEng and sFlt-1 secretion from human villous placental explants

Normal human placental villous explants were collected and treated with IgG from women with preeclampsia or normotensive pregnant individuals in the presence or absence of various reagents for 72 hours. (A) At the end of treatment, human placental villous explants were collected, fixed and stained with (H&E), anti-human Eng and anti-human CD31 antibody. Scale bar, 100 µm (H&E and CD31) or 200 µm (Eng). Syncytiotrophoblast (syn) is indicated by arrow head. (B–C) Expression of endoglin (B) and CD31 (D) were quantified using Image-Pro Plus image analysis sosftware. (E–F) Cell culture supernatants were collected for sEng (E) and sFlt-1 (F) measurements by ELISA. Data are expressed as mean ± SEM of ≥4 experiments performed in duplicate (n=12–14 patient’s IgG for each category). * P < 0.001 versus villous explants treated with normotensive IgG. ** P < 0.05 versus villi treated with preeclamptic IgG.

Figure 5. AT₁-AA-mediated TNF-α induction contributes to impaired placenta angiogenesis by inducing both sEng and sFlt-1 secretion from human villous placental explants

Normal human placental villous explants were collected and treated with IgG from women with preeclampsia or normotensive pregnant individuals in the presence or absence of various reagents for 72 hours. (A) At the end of treatment, human placental villous explants were collected, fixed and stained with (H&E), anti-human Eng and anti-human CD31 antibody. Scale bar, 100 µm (H&E and CD31) or 200 µm (Eng). Syncytiotrophoblast (syn) is indicated by arrow head. (B–C) Expression of endoglin (B) and CD31 (D) were quantified using Image-Pro Plus image analysis sosftware. (E–F) Cell culture supernatants were collected for sEng (E) and sFlt-1 (F) measurements by ELISA. Data are expressed as mean ± SEM of ≥4 experiments performed in duplicate (n=12–14 patient’s IgG for each category). * P < 0.001 versus villous explants treated with normotensive IgG. ** P < 0.05 versus villi treated with preeclamptic IgG.

Figure 5. AT₁-AA-mediated TNF-α induction contributes to impaired placenta angiogenesis by inducing both sEng and sFlt-1 secretion from human villous placental explants

Normal human placental villous explants were collected and treated with IgG from women with preeclampsia or normotensive pregnant individuals in the presence or absence of various reagents for 72 hours. (A) At the end of treatment, human placental villous explants were collected, fixed and stained with (H&E), anti-human Eng and anti-human CD31 antibody. Scale bar, 100 µm (H&E and CD31) or 200 µm (Eng). Syncytiotrophoblast (syn) is indicated by arrow head. (B–C) Expression of endoglin (B) and CD31 (D) were quantified using Image-Pro Plus image analysis sosftware. (E–F) Cell culture supernatants were collected for sEng (E) and sFlt-1 (F) measurements by ELISA. Data are expressed as mean ± SEM of ≥4 experiments performed in duplicate (n=12–14 patient’s IgG for each category). * P < 0.001 versus villous explants treated with normotensive IgG. ** P < 0.05 versus villi treated with preeclamptic IgG.

Figure 6. Working model of AT₁-AA-mediated TNF-α induction in impaired placental angiogenesis in preeclampsia

This diagram represents a possible signaling cascade by which the autoantibody-mediated TNF-α induction overcomes HO-1 (heme oxygenase 1), its negative regulator, and contributes to increased sEng and sFlt-1 secretion and subsequent impaired placental angiogenesis, a major feature in preeclampsia. This implies blockade of TNF-α and AT₁ receptor or increasing HO-1 signaling may be potential therapeutic strategies in the management of this serious disorder of pregnancy for both mom and babies.