Introduction

Preeclampsia, estimated to affect 5–7% of all pregnancies worldwide, is normally characterized by hypertension, endothelial dysfunction, angiogenic imbalance and proteinuria. Though preeclampsia is a leading cause of maternal death and a major contributor to maternal and perinatal morbidity, the paucity of knowledge of its causal mechanisms remains a major obstacle to developing specific prevention and/or treatment modalities. Since the spontaneous development of preeclampsia is essentially limited to the human species, the study of preeclampsia in humans is of critical importance to identify biomarkers and potential pathogenic factors that correlate with the progression of the syndrome. However, experimental studies in pregnant women have obvious limitations that prevent complete investigation of many pathophysiological mechanisms involved in this syndrome. Moreover, studies in humans often limit the ability to establish cause and effect relationships. In contrast, experimental studies in animal models, despite their limitations, allow investigators to directly test whether certain factors found in preeclamptic women can indeed lead to hypertension and other manifestations of preeclampsia.

Here we review some of the more widely studied and/or recently developed animal models used to investigate mechanisms that link placental pathology with maternal endothelial activation/dysfunction and vascular abnormalities of preeclampsia. A wide variety of animal models have been used to investigate pathophysiological mechanisms of preeclampsia. These mechanisms and resulting models include hypoxia (reduced uterine perfusion pressure models), impaired angiogenesis (sFlt-1 and sEng infusion models), excessive maternal immune activation (TNF-α and AT1-AA infusion) and genetic mouse models that target specific pathogenic pathways.

Models Used to Investigate Links between Placental Ischemia and Endothelial and Cardiovascular Dysfunction

Preeclampsia develops during pregnancy and remits after delivery, implicating the placenta as a central culprit in the disease. An initiating event in preeclampsia is postulated to be reduced placental perfusion that leads to widespread dysfunction of the maternal vascular endothelium by mechanisms that remain to be fully elucidated. Experimental induction of chronic uteroplacental ischemia appears to be the most promising animal model to study potential mechanisms of preeclampsia since reductions in uteroplacental blood flow in a variety of animal models lead to a hypertensive state that closely resembles preeclampsia in women, including endothelial dysfunction, angiogenic imbalance, and proteinuria. In early studies of the relationship between placental blood flow and preeclampsia, Ogden et al. observed that partially occluding the infrarenal aorta of pregnant but not non-pregnant dogs resulted in hypertension. In subsequent studies, the bilateral ligation of the utero-ovarian arteries and placement of non-constrictive bands around the uterine arteries resulted in hypertension in dogs only after they achieved pregnancy. The animals uniformly developed hypertension and proterinuria. This hypertensive state persisted until the postpartum period, at which time pressures returned to normal. These findings were confirmed by studies where precise constriction of the aorta below the dog’s renal arteries resulted in hypertension and proteinuria. The investigators claimed that the animals also displayed glomerular endothelial lesions similar to those classically described in the kidneys of preeclamptic women. Losonczy et al. constricted the aortas of rabbits and demonstrated elevated blood pressure in chronically catheterized pregnant animals. These studies demonstrated elevated arterial pressures accompanied by increased peripheral resistance, a feature commonly observed in preeclamptic women.

The relationship between reduced uteroplacental perfusion and hypertension during pregnancy has also been demonstrated in sub-human primates. Cavanagh and co-workers studied baboons using interventions similar to those used by Hodari; ligation of the utero-ovarian arteries and placement of bands around the uterine arteries. Females bred after the surgery exhibited higher blood pressures than non-pregnant or sham-operated control pregnant animals, and were also reported to manifest glomerular endothelial swelling. An aortic constriction model was also reported in rhesus monkeys to study the effects of reduced uteroplacental perfusion from early pregnancy through delivery. In this latter model, the degree of aortic constriction was precisely controlled, and progressive hypertension, proteinuria, and glomerular endotheliosis developed. More recently, Makris et al. produced a uteroplacental ischemia model in radiotelemetered pregnant baboons by selective ligation of one uterine artery resulting in a 40% decrease in uteroplacental blood flow as determined by angiography. Hypertension, proteinuria, and increased production of antiangiogenic markers by the placenta and peripheral blood mononuclear cells were reported in the pregnant animals compared to control animas who underwent a sham procedure ( Fig. 10.1 ). Endothelial histological changes consistent with those seen in human preeclampsia were also reported.

Pregnant baboons were subjected to unilateral uterine artery ligation (UPI) on gestational day 135 of a normal 182 day gestation, resulting in ~30–50% decrease in placental perfusion. Within 3 days, blood pressure in both sham and UPI animals showed an increase in both systolic and diastolic blood pressure both awake (A,B) and while asleep (C,D) as determined by implanted radiotelemetry. This effect was persistent through 2 weeks post-surgery.

Reprinted with permission from .

Eder and MacDonald reduced utero placental perfusion pressure in gravid rats, the intervention starting on the 14th day of a normally 21 day gestation. More recently, Granger and colleagues modified and further characterized this rat model in order to examine potential pathophysiological mechanisms that mediate the hypertension during chronic reductions in uteroplacental perfusion pressure (RUPP). They reduced uterine perfusion pressure in the gravid rat by approximately 40% by placing a silver clip around the aorta below the renal arteries. Because this procedure causes an adaptive increase in uterine blood flow via the ovarian artery, they also placed a silver clip on both the right and left uterine arcade at the ovarian end just before the first segmental artery. They reported that reducing uteroplacental perfusion pressure results in significant and consistent elevations in arterial pressure of 20–30 mm Hg as compared to control pregnant rats at day 19 of gestation. Reducing uteroplacental perfusion pressure in non-pregnant rats had no effect on blood pressure. RUPP-induced hypertension was also associated with proteinuria, reductions in renal plasma flow and glomerular filtration rate, and a hypertensive shift in the pressure–natriuresis relationship. Moreover, they found important evidence indicating that endothelial function is significantly impaired in the RUPP hypertensive rat. They compared the relaxation responses to acetylcholine between aortic vessel strips of pregnant and RUPP hypertensive rats, observing that endothelial-dependent vasodilatation was significantly attenuated in the RUPP hypertensive rats. In addition, the production of nitric oxide is reduced in vascular tissue while the syntheses of thromboxane, endothelin, and 8-isoprostane, a marker of oxidative stress, are elevated in the RUPP hypertensive rat as compared to normal pregnant rats at day 19 of gestation. Moreover, immune factors such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and angiotensin II type I receptor autoantibodies (ATI-AA) are significantly elevated in the plasma of the RUPP hypertensive rat. Intrauterine growth restriction is also present in the RUPP hypertensive rats since the average pup size in this group is smaller than in normal pregnant rats. Thus, RUPP-induced hypertension in the pregnant rat has many of the features of preeclampsia in women ( Table 10.1 ).

While uteroplacental ischemic models have many of the features of human preeclampsia they do have limitations. Although the rat RUPP model exhibits increases in protein excretion and consistent increases in blood pressure in response to reductions in uterine perfusion pressure, the proteinuric response is somewhat variable in the pregnant rat. The reason for the variability in the rat RUPP model is unknown but may be due to the short time frame (4–5 days) of exposure to placental ischemia. In contrast, proteinuria in the baboon uteroplacental ischemic model has a robust increase within the second and third week of uterine artery occlusion ( Fig. 10.1 ). However, the primate model has a long gestation, very costly and labor-intensive, and has been used by a limited number of laboratories.

While animal models of placental ischemia do have some limitations, studies in a variety of animal species suggest the reduced uteroplacental perfusion models have many features of preeclampsia in women. These models, along with appropriate pharmacological tools, provide an opportunity for investigators to quantify the relative importance of complex pathophysiological factors in mediating cardiovascular and renal dysfunction in response to placental ischemia during pregnancy. The models also provide an important tool to test the efficacy of novel therapeutic approaches for the prevention and treatment of preeclampsia.

Animal Models Used to Study Role of Angiogenic Factors (See also Chapter 6 )

One of the most promising systems to receive scrutiny in recent preeclampsia research is the vascular endothelial growth factor (VEGF) signaling pathway. VEGF, a powerful proangiogenic protein, is an essential factor in the maintenance of endothelial cell health. Additionally, VEGF is necessary for the maintenance of glomerular ultrastructure through the maintenance of its fenestrated endothelium. Transgenic VEGF knockout in glomerular podocytes resulted in proteinuria and glomerular endotheliosis, two common findings in preeclampsia. Similar findings are seen in cancer patients treated with VEGF monoclonal antibodies, as hypertension and proteinuria are common side effects. It is clear, then, that proper levels of VEGF are necessary for endothelial and vascular health.

One factor shown to interfere with VEGF signaling is the soluble form of the VEGF receptor, sFlt-1. sFlt-1 is an alternately spliced variant of the full-length receptor in which the transmembrane and cytosolic domains have been excised, leaving only the extracellular recognition domain. This recognition domain acts as a VEGF antagonist by binding free VEGF, and making it unavailable for proper signaling. Of particular interest for preeclampsia, sFlt-1 is positively regulated by hypoxia, specifically through the actions of HIF-1α, and has been shown to be produced by both placental trophoblasts and human placental villous explants in response to low oxygen tension. While sFlt-1 production is regulated by the hypoxia-inducible factor-1, studies using animal models (RUPP, TNF or AT1-AA infusion models) suggest that factors such as tumor necrosis factor and the agonistic autoantibody to the angiotensin II type I receptor also appear to be involved in the regulation of sFlt-1 production.

Several experimental models have demonstrated a causative role for sFlt-1 in the pathology of preeclampsia. Viral ectopic expression of sFlt-1 in pregnant rats led to a preeclampsia-like state, with hypertension, glomerular endotheliosis, and proteinuria ( Fig. 10.2 ). Models of reduced uterine perfusion pressure in both nonhuman primates and rats show significant increases in circulating sFlt-1, and concurrent decreases in bioavailable circulating VEGF. In agreement with the viral expression experiments, direct infusion of sFlt-1 into pregnant mice or rats induces preeclamptic-like symptoms, including hypertension and reduced fetal weight. As with several other experimental forms of hypertension, endothelin is one of the major effector molecules in sFlt-1-induced hypertension, as administration of an ET _A receptor antagonist completely normalizes blood pressure.

Adenoviral vectors encoding either Fc protein or sFlt-1 were injected into pregnant rats on gestation day 8 or 9 of a normal 21 day gestation. On gestational days 16 or 17, direct blood pressure measurements were performed on anesthetized animals. Animals receiving the Ad-sFlt-1 had significantly elevated blood pressure compared to the Ad-Fc controls (A). At the same time point, urine samples were obtained, and the urinary albumin/creatinine ratio was determined by ELISA. Dramatic increases in urinary albumin/creatinine were noted in the Ad-sFlt-1 group compared to the Ad-Fc controls (B), suggesting renal injury.

Adapted from .

It does not appear that the pathological manifestations seen in the sFlt-1-induced preeclampsia-like syndrome in animals are a direct result of circulating sFlt-1 but rather are due to the reduced availability of free circulating VEGF. When excess VEGF is administered simultaneously with the dose of sFlt-1 known to cause a preeclampsia-like status in pregnant rats the hypertensive response is muted, and there is less evidence of abnormal renal function, than when the antiangiogenic protein was administered alone. Additionally, VEGF administration in rats with placental ischemia restores normal blood pressure, renal function, and vascular activity. These and other data from animal models have led to the hypothesis that the ratio between sFlt-1 and VEGF is critical to maintain a healthy endothelium and normal vascular activity. The role of these proteins in the development of preeclampsia is a promising avenue in the search for new therapeutics.

Soluble endoglin (sEng), an antiangiogenic factor and soluble receptor for transforming growth factor-beta (TGF-β), is also implicated in the pathogenesis of PE. sEng is present during normal pregnancy, but is upregulated significantly in preeclampsia and correlates with disease severity. Utilizing adenoviral delivery of sEng in pregnant rats, Venkatesha et al. reported the development of hypertension, intrauterine growth restriction, and focal endotheliosis. These findings were more severe in rats which received the combination of both adenoviral sEng and sFlt-1 whereas animals also presented with hemolysis, elevated liver enzymes, and low platelets. The authors suggested that this model may reflect the syndrome of HELLP (hemolysis, elevated liver enzymes, and low platelets) seen in some PE women and could be utilized to investigate the pathophysiology of HELLP.

While compelling data derived from animal and human studies suggest an important role for angiogenic imbalance in the pathophysiology of preeclampsia, there are many unanswered questions and many opportunities for future research in animal models. For example, the molecular mechanisms involved in the regulation of sFlt-1 production have yet to be fully elucidated. Moreover, while sFlt-1 appears to play an important role in the pathogenesis of preeclampsia, research delineating the roles of specific inhibitors of sFlt-1 production had just started as of 2014. No doubt the finding of ways to inhibit sFlt-1 or stimulate greater production of VEGF and PlGF is of critical importance, with animal models needed to advance such research.

Models Used to Investigate the Role of Immune Mechanisms in Preeclampsia

There is a growing interest in maternal immunity and inflammatory processes as mediators of the clinical manifestations of preeclampsia. Preeclampsia is associated with a dysregulation of natural killer cells, activation of CD4 T lymphocytes, the release of proinflammatory factors such as TNF-α and IL-6 and IL-17, and the production of angiotensin II type-1 receptor autoantibody (AT1-AA). However, the importance of these immune factors in the pathogenesis of preeclampsia in humans is unknown. In recent years, a number of animal models were developed in an attempt to dissect the relative importance of each of these immune factors.

Of interest is work suggesting that the inflammatory response is triggered by particles, ranging from large deported multinuclear fragments to sub-cellular components, shed from the syncytial surface of the human placenta. Using an in vitro model (see cover photomicrograph and Chapter 6 ), Chamley and co-workers observed that when syncytial knots are shed by an apoptosis-like programmed cell death process, then phagocytosed by macrophages, the macrophages produce a tolerogenic response. However, necrotic syncytial knots, when phagocytosed, appear to be immunostimulatory. While there is a strong correlation between trophoblast debris and immune activation in preeclampsia, it is unclear whether these particles could indeed elicit a pathogenic response in vivo . To address this issue, Chamley et al. recently reported chronically administered necrotic trophoblast debris to pregnant rats and observed increased blood pressure relative to pregnant controls in late gestation. This report appears to be the first demonstration that necrotic trophoblast debris can alter blood pressure in vivo and, while the mechanism for this response remains to be determined, this observation is consistent with the growing body of in vitro evidence suggesting that necrotic trophoblast debris can contribute to the hypertension of preeclampsia.

In normal pregnancies, there is a heightened inflammatory state when compared to non-pregnant women, but in preeclampsia there is further elevation of inflammation markers, e.g., IL-6 and TNF-α, when compared to healthy pregnancies. While inflammatory cytokines such as IL-6 and TNF-α are elevated in preeclamptic women, the importance of these cytokines in mediating the cardiovascular and renal dysfunction has yet to be fully elucidated. Serum levels of TNF-α and IL-6 are elevated in RUPP rats and chronic infusion of TNF-α or IL-6 into pregnant rats increases arterial pressure and decreases renal plasma flow and glomerular filtration rate. Blockade of TNF-α with the soluble receptor Etanercept attenuated the associated hypertension and decreased tissue ET-1 expression in RUPP rats. These findings indicate that TNF-α and IL-6 may play a role in mediating the hypertension and reduction in renal hemodynamics observed during RUPP in pregnant rats.

Similar findings were recently demonstrated in a primate model of cytokine-induced preeclampsia. Hennessey and co-workers reported that continuous administration of TNF-α to pregnant baboons for 2 weeks at mid-gestation caused increased blood pressure, proteinuria, and plasma sFlt-1. The source of circulating sFlt-1 was determined to be at least partially placental, confirming its role as a link between placental dysfunction and the maternal syndrome. The placenta accrete seen in one baboon in the treatment group potentially suggests a fundamental disruption of placental structure and function as a result of TNF-α infusion.

Numerous antiinflammatory cytokines are decreased in women with preeclampsia but the role of these cytokines in blood pressure regulation during pregnancy is unknown. Mitchell and co-workers recently addressed this issue by examining whether the lack of the potent antiinflammatory cytokine interleukin-4 (IL-4) would be sufficient to elicit a preeclampsia-like syndrome in mice, and when coupled with immune system activation would further augment these symptoms. They reported that pregnant IL-4-deficient mice exhibited altered splenic immune cell subsets, increased levels of proinflammatory cytokines, placental inflammation, mild hypertension, endothelial dysfunction, and proteinuria compared to pregnant control mice. Compared to pregnant control mice treated with with the Toll-like receptor 3 agonist polyinosinic:polycytidylic (poly I:C).which exhibit preeclampsia-like symptoms, poly I:C-treated pregnant IL-4-deficient mice exhibited a further increase in proinflammatory cytokine levels, which was associated with augmented SBP and endothelial dysfunction. Collectively, these data show that the absence of IL-4 is sufficient to induce mild preeclampsia-like symptoms in mice due to excessive inflammation. Thus, the antiinflammatory effects of IL-4 may be important in preventing hypertension during pregnancy.

Another factor produced by the maternal immune system that has received a great deal of attention in recent years is the angiotensin-1 receptor autoantibody (AT1-AA), which has been identified in the circulation of preeclamptic women. The AT1-AA has been purified and investigators have shown that AT1-AA signaling, via the AT1 receptor, results in a variety of physiological effects. AT1-AA induces signaling in vascular cells and trophoblasts including transcription factor activation. The signaling increases TNF-α and reactive oxygen species generation, both of which have been implicated in preeclampsia. Although these novel findings implicate AT1-AA in the pathogenesis of hypertension during preeclampsia, the specific mechanisms that lead to excess production of these autoantibodies and the mechanisms whereby AT1-AA increases blood pressure during pregnancy remain unclear.

Utilizing a bioassay for AT1-AA, LaMarca and colleagues determined that reductions in placental perfusion in pregnant rats had a profound effect to stimulate AT1-AA production. In contrast, the AT1-AA was not detected in normal pregnant rats. In addition, they were able to suppress the production of AT1-AA in the RUPP model of placental ischemia by administration of CD20 blockade. CD20 blockade depletes B lymphocytes and suppresses secretion of antibody. AT1-AA suppression via B cell depletion results in a blunted blood pressure response to placental ischemia. Collectively, these novel findings suggest that a reduction in placental perfusion may be an important stimulus for AT1-AA production. In addition to placental ischemia as a stimulus for the AT1-AA, chronic infusion of TNF-α in normal pregnant rats results in AT1-AA production. The findings suggest the immune mechanisms stimulated by placental ischemia may play an important role in AT1-AA production.

In support of a role for AT1-AA in causing hypertension, the LaMarca lab reported that infusion of purified rat AT1-AA, isolated from serum collected from a pregnant transgenic rat overproducing components of the renin-angiotensin system, into pregnant rats from day 12 to day 19 of gestation, increased serum AT1-AA and blood pressure. Zhou et al. also demonstrated that immunoglobulins isolated from preeclamptic women increase systolic pressure in pregnant mice. This phenotype was ameliorated with co-injection of an AT1 receptor antagonist or the seven-amino-acid peptide that selectively blocks the actions of the AT1-AA. While these collective findings suggest that AT1-AA has important hypertensive actions, the contribution of AT1-AA to the pathophysiology of hypertension in response to placental ischemia or preeclampsia remains an important area of investigation. Although animal studies suggest that increasing plasma AT1-AA concentrations in pregnant rats to levels observed in preeclamptic women or placental ischemic rats result in increased arterial pressure, the quantitative importance of AT1-AA in the pathophysiology of preeclampsia in humans has yet to be fully elucidated. Clinical studies utilizing specific antagonists of the AT1-AA or studies that block the formation of AT1-AA in preeclamptic women are the only approaches to truly determine the role of AT1-AA in human preeclampsia.

Utilizing adoptive transfer methods and pharmacological tools in the RUPP model, a role for CD4+T cells in the hypertension in response to placental ischemia has been identified. Adoptively transferred CD4+T cells from RUPP rats increased blood pressure in normal pregnant rats. Moreover, the hypertension that developed in response to adoptive transfer of RUPP CD4+T cells was associated with elevated TNF, sFlt-1, AT1-AA, and endothelin in normal pregnant recipient rats, none of which were elevated in normal pregnant control rats. To further establish a potential role of CD4+T cells in the pathophysiology of preeclampsia, the LaMarca lab suppressed T cell activity by administration of abatacept (Orencia), which is a fusion molecule of CTLA-4. CTLA-4 is a marker on T cells used to stimulate an immune response to antigens. Administration of Orencia on gestational day 13 decreased T cells and the blood pressure response to RUPP in pregnant rats, supporting the hypothesis that T cells are important in causing hypertension in response to placental ischemia. (See also Chapter 8 , Chapter 15 regarding discussion of immunology and volume regulation in normal pregnancy and preeclampsia.)