A new mouse model to explore therapies for preeclampsia

Abstract

Background: Pre-eclampsia, a pregnancy-specific multisystemic disorder is a leading cause of maternal and perinatal mortality and morbidity. This syndrome has been known to medical science since ancient times. However, despite considerable research, the cause/s of preeclampsia remain unclear, and there is no effective treatment. Development of an animal model that recapitulates this complex pregnancy-related disorder may help to expand our understanding and may hold great potential for the design and implementation of effective treatment.

Methodology/principal findings: Here we show that the CBA/J x DBA/2 mouse model of recurrent miscarriage is also a model of immunologically-mediated preeclampsia (PE). DBA/J mated CBA/J females spontaneously develop many features of human PE (primigravidity, albuminuria, endotheliosis, increased sensitivity to angiotensin II and increased plasma leptin levels) that correlates with bad pregnancy outcomes. We previously reported that antagonism of vascular endothelial growth factor (VEGF) signaling by soluble VEGF receptor 1 (sFlt-1) is involved in placental and fetal injury in CBA/J x DBA/2 mice. Using this animal model that recapitulates many of the features of preeclampsia in women, we found that pravastatin restores angiogenic balance, ameliorates glomerular injury, diminishes hypersensitivity to angiotensin II and protects pregnancies.

Conclusions/significance: We described a new mouse model of PE, were the relevant key features of human preeclampsia develop spontaneously. The CBA/J x DBA/2 model, that recapitulates this complex disorder, helped us identify pravastatin as a candidate therapy to prevent preeclampsia and its related complications. We recognize that these studies were conducted in mice and that clinical trials are needed to confirm its application to humans.

Figure 1. Preeclamptic features in CBA/J x DBA/2 matings.

Increased fetal resorption frequency (A) and decreased litter size (B)) were observed only in first pregnancies in DBA/2 mated CBA/J mice. Normal pregnancies (not different from control matings CBA/J x BALB/c) were observed in second (2^nd P) and third (3^rd P) pregnancies with the same male. Data are mean values plus or minus SD. (C) In CBA/J x DBA/2 mice, a time-course increase in urine albumin to creatinine ratio (ACR), that reaches statistic significance at day 10 of pregnancy, was observed (*p<0.05 vs CBA/J x DBA/2). Albuminuria was not observed in control matings CBA/J x BALB/c mice. N = 6-8 mice/experimental group (Di) Transmission electron micrographs of glomeruli from DBA/J mated CBA/J mice (original magnification 10,000x) show swelling of glomerular endothelial cells and loss of fenestrations. Numerous red blood cells trapped between swollen endothelial cells were observed occluding the glomerular capillaries. No signs of endothelial injury, well preserved fenestrations and widely patent glomerular capillary lumina are observed in kidneys from CBA/J x BALB/c mice (control group) (Ei). (Dii) Kidney perfusion studies reveal diminished blood perfusion (the fluorescent tracer did not accumulate in the glomerular capillaries) in glomeruli from CBA/J x DBA/2 mice with abnormal pregnancies compared to control CBA/J x BALB/c matings (Eii). (Diii) Increased fibrin staining in glomeruli from CBA/J x DBA/2 mice when compared to CBA/J x BALB/c mice (Eiii). (Div and Eiv) Jones methenamine silver staining. Diffusely and irregularly thickened glomerular basement membranes were observed in some glomeruli in CBA/J x DBA/2 mice (20%) (Fig 1Div) compared to BALB/c mated CBA/2 females (Fig 1Eiv). Kidneys from 5-6 mice were studies in each experimental group.

Figure 2. MAP and contractile response to angiotensin II in CBA/J x DBA/2 mice.

(A) No significant increase in MAP was observed in CBA/J x DBA/2 compared to CBA/J x BALB/c control matings. Data were expressed as change in MAP from mating day. CBA/J x DBA/2 mice showed an increase response to an acute a bolus injection of AngII compared to CBA/J x BALB/c mice (*p<0.01). The response to Ang II was expressed as the mean change in MAP from basal values before Ang II injection. (B) Aortic rings from CBA/J x DBA/2 and CBA/J x BALB/c contracted in response to Ang II (* p<0.05). Aortic rings from CBA/J x DBA/2 mice showed increased reduction of the inner diameter in response to Ang II (# different from CBA/J x BALB/c. P<0.05). N = 8–10 mice/experimental group. (C) Staring at day 11 of pregnancy, plasma leptin levels were increased in CBA/J x DBA/2 mice when compared to control CBA/J x BALB/c matings (* p<0.01). This increase was maintained until delivery. Leptin levels in CBA/J x DBA/2 and control matings drop to pre-pregnancy values after parturition. N = 6–8 mice/experimental group.

Figure 3. Pravastatin prevents glomerular injury and hypersensitivity to angiotensin II in CBA/J x DBA/2 mice.

(A) Increased ACR levels were observed in CBA/J x DBA/2 mice along pregnancy (* p<0.01). Treatment with pravastatin abrogated albuminuria in CBA/J x DBA/2 mice (B) CBA/J x DBA/2 mice treated with pravastatin showed no signs of renal endothelial damage. (Bi) EM studies (original magnification 10,000 x) showed well preserved endothelial cells and open capillaries lumen in CBA/J x DBA/2 mice treated with pravastatin. (Bii) Increased fibrin deposition was not observed in glomerular capillaries from DBA/2 mated CBA/J mice that received pravastatin. (Biii) Pravastatin restored renal blood flow in CBA/J x DBA/2 mice. (Biv) Jones methenamine silver staining shows no signs of endotheliosis in CBA/J x DBA/2 mice treated with pravastatin. (C) Aortic rings from CBA/J x DBA/2 mice treated with pravastatin did not show increased contractile response in response to Ang II when compared to untreated CBA/J x DBA/2 mice (*p<0.05). N = 5–8 mice/experimental group.

Figure 4. Pravastatin restores angiogenic balance in CBA/J x DBA/2 mice.

(A) Pravastatin restored free plasma VEGF levels in CBA/J x DBA/2 mice (*p<0.05). (B) SM9-1 trophoblast cells incubated with pravastatin showed increase VEGF release in a dose dependent manner (*p<0.05). Values are expressed as percentage of control values.(C) Pravastatin (5- 10-20 µg/ml) increased cell proliferation in SM9-1 trophoblasts in a dose-dependent manner. (D) CBA/J x DBA/2 mice treated with VEGF 121 showed no signs of endotheliosis. (Di) electron microscopy studies show well preserved glomerular endothelial cells in CBA/J x DBA/2 mice treated with VEGF121.original magnification 10,000 x (Dii) Adequate blood perfusion comparable to CBA/J x BALB/c control matings was observed in CBA/J x DBA/2 mice treated with VEGF121. (Diii) Increased fibrin deposition was not observed in glomerular capillaries from DBA/2 mated CBA/J mice that received VEGF121. (E) Pravastatin ameliorated ACR in CBA/J x DBA/2 mice at day 15 of pregnancy (* p<0.01). N = 6–8 mice/experimental group.