Targeted inhibition of complement activation prevents features of preeclampsia in mice

Preeclampsia is a major cause of maternal and neonatal morbidity and mortality. In mouse models, complement activation in the placenta is associated with abnormal placental development and miscarriage, and inhibiting complement prevents fetal injury. We mated two mouse strains, DBA/2 and CBA/J, expecting that the pregnancies might show features of preeclampsia and of immunologically mediated pregnancy loss. Along with placental dysfunction, these matings resulted in proteinuria, elevated BUN, fibrin deposition, and glomerular endotheliosis. We blocked placental complement activation throughout pregnancy by administering a single dose of the C3 inhibitor CR2-Crry given on day 5 of the pregnancy. This procedure specifically targets the sites of complement activation without inducing any systemic effects. Placental complement inhibition prevented oxidative stress and placental dysfunction, as well as proteinuria and renal pathologic features of preeclampsia. Thus, local blockade of complement activation at the maternal–fetal interface rescues preeclampsia in mice, and identifies new treatments. Hence, complement triggers a feed-forward cycle of placental damage, antiangiogenic factor production, and maternal vascular damage in patients. Preeclampsia is a major cause of maternal and neonatal morbidity and mortality. In mouse models, complement activation in the placenta is associated with abnormal placental development and miscarriage, and inhibiting complement prevents fetal injury. We mated two mouse strains, DBA/2 and CBA/J, expecting that the pregnancies might show features of preeclampsia and of immunologically mediated pregnancy loss. Along with placental dysfunction, these matings resulted in proteinuria, elevated BUN, fibrin deposition, and glomerular endotheliosis. We blocked placental complement activation throughout pregnancy by administering a single dose of the C3 inhibitor CR2-Crry given on day 5 of the pregnancy. This procedure specifically targets the sites of complement activation without inducing any systemic effects. Placental complement inhibition prevented oxidative stress and placental dysfunction, as well as proteinuria and renal pathologic features of preeclampsia. Thus, local blockade of complement activation at the maternal–fetal interface rescues preeclampsia in mice, and identifies new treatments. Hence, complement triggers a feed-forward cycle of placental damage, antiangiogenic factor production, and maternal vascular damage in patients. Preeclampsia is a major cause of maternal and neonatal morbidity and mortality.1.Ilekis J.V. Reddy U.M. Roberts J.M. Preeclampsia—a pressing problem: an executive summary of a National Institute of Child Health and Human Development workshop.Reprod Sci. 2007; 14: 508-523Crossref PubMed Scopus (101) Google Scholar Although typically diagnosed with the onset of clinical manifestations, proteinuria and hypertension after 20 weeks’ gestation, the syndrome begins much earlier in pregnancy with abnormal placental development. Uterine spiral arteries fail to remodel into dilated, flaccid vessels, which results in underperfusion of the intervillous space and placental hypoxia.2.Roberts J.M. Gammill H.S. Preeclampsia: recent insights.Hypertension. 2005; 46: 1243-1249Crossref PubMed Scopus (517) Google Scholar Placental oxidative stress and inflammation lead to the release of antiangiogenic factors into the maternal circulation.3.Gilbert J.S. Babcock S.A. Granger J.P. Hypertension produced by reduced uterine perfusion in pregnant rats is associated with increased soluble fms-like tyrosine kinase-1 expression.Hypertension. 2007; 50: 1142-1147Crossref PubMed Scopus (240) Google Scholar, 4.Makris A. Thornton C. Thompson J. et al.Uteroplacental ischemia results in proteinuric hypertension and elevated sFLT-1.Kidney Int. 2007; 71: 977-984Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 5.Nevo O. Soleymanlou N. Wu Y. et al.Increased expression of sFlt-1 in in vivo and in vitro models of human placental hypoxia is mediated by HIF-1.Am J Physiol Regul Integr Comp Physiol. 2006; 291: R1085-R1093Crossref PubMed Scopus (239) Google Scholar Clinical manifestations, widespread endothelial cell dysfunction presenting as proteinuria, hypertension, hemolysis, elevated liver enzymes, low platelet counts (HELLP syndrome), and/or seizures, represent the maternal response to this excess of antiangiogenic factors.6.Young B.C. Levine R.J. Karumanchi S.A. Pathogenesis of preeclampsia.Annu Rev Pathol. 2010; 5: 173-192Crossref PubMed Scopus (469) Google Scholar One such antiangiogenic factor is soluble fms-like tyrosine kinase 1 (sFlt-1), a secreted splice variant of vascular endothelial growth factor (VEGF) receptor-1 that sequesters circulating VEGF and placental growth factor and prevents their interaction with endogengous receptors.7.Maynard S.E. Min J.Y. Merchan J. et al.Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Crossref PubMed Scopus (2875) Google Scholar Elevated levels of sFlt-1 are found in the circulation of pregnant women destined for preeclampsia.8.Levine R.J. Maynard S.E. Qian C. et al.Circulating angiogenic factors and the risk of preeclampsia.N Engl J Med. 2004; 350: 672-683Crossref PubMed Scopus (2563) Google ScholarPatients with cancer, who are treated with inhibitors of VEGF often develop proteinuria (21–64%) and may develop hypertension (3–36%)9.Zhu X. Wu S. Dahut W.L. et al.Risks of proteinuria and hypertension with bevacizumab, an antibody against vascular endothelial growth factor: systematic review and meta-analysis.Am J Kidney Dis. 2007; 49: 186-193Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar supports this pathogenic mechanism. We and others have suggested a relationship between the activation of the complement system and angiogenic factor imbalance linked to the placental dysfunction.10.Girardi G. Yarilin D. Thurman J.M. et al.Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction.J Exp Med. 2006; 203: 2165-2175Crossref PubMed Scopus (361) Google Scholar, 11.Lynch A.M. Gibbs R.S. Murphy J.R. et al.Complement activation fragment Bb in early pregnancy and spontaneous preterm birth.Am J Obstet Gynecol. 2008; 199 (e351-358): 354Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar In mouse models, complement deposition in the placenta is associated with the abnormal placental development and miscarriage, while inhibiting complement rescues pregnancies.12.Girardi G. Bulla R. Salmon J.E. et al.The complement system in the pathophysiology of pregnancy.Mol Immunol. 2006; 43: 68-77Crossref PubMed Scopus (137) Google Scholar, 13.Salmon J.E. Girardi G. Antiphospholipid antibodies and pregnancy loss: a disorder of inflammation.J Reprod Immunol. 2008; 77: 51-56Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar In patients, complement activation localizes to villous trophoblast injury in vivo and modulates trophoblast function in vitro.14.Rampersad R. Barton A. Sadovsky Y. et al.The C5b-9 membrane attack complex of complement activation localizes to villous trophoblast injury in vivo and modulates human trophoblast function in vitro.Placenta. 2008; 29: 855-861Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Preeclamptic placentas are characterized by the marked deposition of terminal complement complex (C5b-9).12.Girardi G. Bulla R. Salmon J.E. et al.The complement system in the pathophysiology of pregnancy.Mol Immunol. 2006; 43: 68-77Crossref PubMed Scopus (137) Google Scholar Our finding, that women with evidence of activation of the alternative pathway early in pregnancy are at increased risk for preeclampsia, suggests that complement contributes to the disease pathogenesis.11.Lynch A.M. Gibbs R.S. Murphy J.R. et al.Complement activation fragment Bb in early pregnancy and spontaneous preterm birth.Am J Obstet Gynecol. 2008; 199 (e351-358): 354Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Finally, recent reports of severe preeclampsia in patients with genetic defects in complement regulation, taken together with observation that complement regulatory proteins are highly expressed on trophoblasts, emphasize the importance of complement to this syndrome.15.Fang C.J. Richards A. Liszewski M.K. et al.Advances in understanding of pathogenesis of aHUS and HELLP.Br J Haematol. 2008; 143: 336-348Crossref PubMed Scopus (82) Google Scholar, 16Fakhouri F. Roumenina L. Provot F. et al.Pregnancy-associated hemolytic uremic syndrome revisited in the era of complement gene mutations.J Am Soc Nephrol. 2010; 21: 859-867Crossref PubMed Scopus (275) Google Scholar We considered the potential of complement inhibitor therapy to prevent preeclampsia in those at high risk, but were concerned that the prolonged systemic inhibition of complement interferes with host defense. Novel complement therapeutics that are targeted to the sites of complement activation provide an alternative approach17.Song H. He C. Knaak C. et al.Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.J Clin Invest. 2003; 111: 1875-1885Crossref PubMed Scopus (86) Google Scholar with limited immunosuppression, improved bioavailability, and enhanced efficacy in the experimental models of ischemia-reperfusion injury, arthritis, and lupus.18.Atkinson C. Song H. Lu B. et al.Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.J Clin Invest. 2005; 115: 2444-2453Crossref PubMed Scopus (141) Google Scholar, 19.Atkinson C. Qiao F. Song H. et al.Low-dose targeted complement inhibition protects against renal disease and other manifestations of autoimmune disease in MRL/lpr mice.J Immunol. 2008; 180: 1231-1238Crossref PubMed Scopus (51) Google Scholar, 20.Banda N.K. Levitt B. Glogowska M.J. et al.Targeted inhibition of the complement alternative pathway with complement receptor 2 and factor H attenuates collagen antibody-induced arthritis in mice.J Immunol. 2009; 183: 5928-5937Crossref PubMed Scopus (53) Google Scholar Whether this strategy could be successful in preeclampsia depends on how important complement activation at the maternal–fetal interface early in pregnancy is to the placental dysfunction, angiogenic dysregulation, and consequent maternal syndrome later in pregnancy. We address this question in an experimental model of preeclampsia triggered by immune and inflammatory mediators. DBA/2-mated CBA/J mice are well studied as a model of immunologically mediated pregnancy loss that shares features with human recurrent miscarriage.10.Girardi G. Yarilin D. Thurman J.M. et al.Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction.J Exp Med. 2006; 203: 2165-2175Crossref PubMed Scopus (361) Google Scholar, 21.Clark D.A. Chaouat G. Arck P.C. et al.Cytokine-dependent abortion in CBA × DBA/2 mice is mediated by the procoagulant fgl2 prothrombinase [correction of prothombinase].J Immunol. 1998; 160: 545-549PubMed Google Scholar We have shown that these matings are characterized by angiogenic dysregulation manifested as elevated levels of circulating sFlt-1, abnormal placental development, and fetal growth restriction, and that systemic complement inhibition reverses angiogenic imbalance, histological changes in placenta, and prevents fetal injury.10.Girardi G. Yarilin D. Thurman J.M. et al.Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction.J Exp Med. 2006; 203: 2165-2175Crossref PubMed Scopus (361) Google Scholar Because the maternal manifestations of preeclampsia, most notably proteinuria and hypertension, are mediated in part by antagonism of VEGF signaling by sFlt-1 produced by the placenta as a consequence of ischemia and inflammation,4.Makris A. Thornton C. Thompson J. et al.Uteroplacental ischemia results in proteinuric hypertension and elevated sFLT-1.Kidney Int. 2007; 71: 977-984Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 7.Maynard S.E. Min J.Y. Merchan J. et al.Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Crossref PubMed Scopus (2875) Google Scholar we hypothesized that CBA/J × DBA/2 matings would demonstrate features of preeclampsia and reveal mediators and mechanisms that activate the vicious cycle of placental damage, antiangiogenic factor production, and maternal vascular damage in patients. In this report, we provide the first evidence that DBA/2-mated CBA/J pregnancies have characteristics of human preeclampsia, including placental and renal manifestations. We show that targeted complement inhibition early in pregnancy in this mouse model prevents the placental and later maternal syndrome, and that such a therapy is as effective as administering VEGF to restore angiogenic balance. In initial studies, we compared placental and maternal phenotypes of DBA/2-mated CBA/J mice with control matings, BALB/c-mated CBA/J (Table 1). In CBA/J × DBA/2 mice, weight of surviving fetuses was decreased, fetal/placental weight ratios were reduced, and fetal resorptions were increased, consistent with the alterations in placental development we previously described.10.Girardi G. Yarilin D. Thurman J.M. et al.Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction.J Exp Med. 2006; 203: 2165-2175Crossref PubMed Scopus (361) Google Scholar These abortion-prone matings had elevated levels of circulating sFlt-1 as early as day 7 of pregnancy (Table 1), which continued to increase throughout the pregnancy and remained higher than CBA/J × BALB/c (P<0.05) until day 15 when mice were killed. CBA/J × DBA/2 mice had increased levels of placental isoprostane 8-iso-prostaglandin F2a (STAT-8, a marker of oxidative stress associated with impaired trophoblast invasion) and elevated circulating thrombin–antithrombin III complexes (TAT, a marker of activation of coagulation cascade increased in microvascular injury) confirming our previous studies (Table 1). Findings similar to those detailed in Table 1 have been described in patients with preeclampsia.22.Fareed J. Hoppensteadt D.A. Leya F. et al.Useful laboratory tests for studying thrombogenesis in acute cardiac syndromes.Clin Chem. 1998; 44: 1845-1853PubMed Google Scholar, 23.Redecha P. van Rooijen N. Torry D. et al.Pravastatin prevents miscarriages in mice: role of tissue factor in placental and fetal injury.Blood. 2009; 113: 4101-4109Crossref PubMed Scopus (96) Google ScholarTable 1Features of preeclampsia in CBA/J × DBA/2 matingsaAll assessments were performed on samples obtained on day 15 of pregnancy, except sFlt-1, which was measured on day 7 of pregnancy.Mice/groupCBA/J × DBA/2CBA/J × BALB/cP-valuePlacental function Fetal weight (mg)20–26288±3314±5<0.0001 Fetal/placenta ratio (mg/mg)20–262.4±0.052.7±0.05<0.0001 Fetal resorption frequency (%)20–2623±47±4<0.0025 STAT-8 (pg/ml)21–22171±24121±14<0.04Maternal syndrome sFlt-1 (pg/ml)41454±137425±56<0.0004 TAT (μg/l)593±2732±14<0.05 Urine albumin/creatinine (μg/mg)10359±102166±24<0.05 Blood BUN (mg/dl)14–1673.2±6.957.1±5.3<0.04Abbreviations: BUN, blood urea nitrogen; sFlt-1, fms-like tyrosine kinase 1; TAT, thrombin–anti-thrombin III.a All assessments were performed on samples obtained on day 15 of pregnancy, except sFlt-1, which was measured on day 7 of pregnancy. Open table in a new tab Abbreviations: BUN, blood urea nitrogen; sFlt-1, fms-like tyrosine kinase 1; TAT, thrombin–anti-thrombin III. To determine whether DBA/2-mated CBA/J mice develop the maternal features of preeclampia, we assessed renal function and kidney histopathology. Urinary protein and blood urea nitrogen (BUN) on day 15 of pregnancy (mid trimester) were higher in CBA/J × DBA/2 mice compared with CBA/J × BALB/c mice (urine albumin/creatinine ratio: 359±102 versus 166±24 μg/mg, n=10/group, P<0.05; BUN: 73±7 versus 57±5 mg/dl, n=14–16, P<0.05; Table 1 and Figure 1a and b). In grouped studies, 50% of CBA/J × DBA/2 mice had albumin/creatinine ratios greater than the mean level of CBA/J × BALB/c matings. Proteinuria tended to be related to the placental function, as defined by mean fetal weight at day 15 (Pearson r=-0.6, n=10 mice; P=0.068). Immunofluorescence studies of kidneys showed diffuse fibrin deposition within glomerular capillary walls and lumina in CBA/J × DBA/2 mice (mean fibrin score 1.5+ range 1–2+) compared with CBA/J × BALB/c (mean fibrin score 0.25+; range 0–0.5+), a finding described in patients with preeclampsia (Figure 1c). No glomerular intracapillary fibrin thrombi were detected by light microscopy. Electron microscopy revealed focal glomerular endothelial cell body swelling consistent with ‘endotheliosis’ associated with segmental foot process effacement (Figure 1d). No glomerular intracapillary fibrin tactoids were identified. This constellation of renal pathological findings was not present in CBA/2 × BALB/c matings and represents the classic findings in human preeclampsia.24.Sheehan H.L. Renal morphology in preeclampsia.Kidney Int. 1980; 18: 241-252Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 25.Gaber L.W. Spargo B.H. Lindheimer M.D. Renal pathology in pre-eclampsia.Baillieres Clin Obstet Gynaecol. 1994; 8: 443-468Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 26.Kincaid-Smith P. The renal lesion of preeclampsia revisited.Am J Kidney Dis. 1991; 17: 144-148Abstract Full Text PDF PubMed Scopus (64) Google Scholar To determine whether pregnancy causes hypertension in the CBA/J × DBA/2 crosses, arterial pressures of CBA/J mice were recorded with surgically implanted radiotelemeters 24 h/day beginning 3 days before mating and continuing through delivery. Mean arterial blood pressure calculated for 19 CBA/J × DBA/2 matings and 9 CBA/J × BALB/c matings were not different throughout pregnancy (Figure 2a). We considered the possibility that mice with severe placental dysfunction would become hypertensive and performed a subset analysis comparing blood pressure in the most divergent groups of mice defined by litter size, a proxy for placental function, because blood pressure was monitored. We compared mean arterial blood pressure in DBA/2 mated-CBA/J mice with fewer than five offspring to that of BALB/c-mated CBA/J mice with greater than seven offspring and again found no differences between the groups (Figure 2b). Although a hallmark of human preeclampsia, other informative genetic mouse models with reduced spiral artery modification and placental dysfunction also fail to develop hypertension27.Croy B.A. He H. Esadeg S. et al.Uterine natural killer cells: insights into their cellular and molecular biology from mouse modelling.Reproduction. 2003; 126: 149-160Crossref PubMed Scopus (173) Google Scholar, 28.Caron K. Hagaman J. Nishikimi T. et al.Adrenomedullin gene expression differences in mice do not affect blood pressure but modulate hypertension-induced pathology in males.Proc Natl Acad Sci USA. 2007; 104: 3420-3425Crossref PubMed Scopus (28) Google Scholar and 10–15% of patients with the most severe forms of preeclampsia are not hypertensive.29.Douglas K.A. Redman C.W. Eclampsia in the United Kingdom.Bmj. 1994; 309: 1395-1400Crossref PubMed Scopus (596) Google Scholar The strong evidence for placental dysfunction, taken together with our new findings of renal manifestations characteristic of preeclampsia, identify CBA/J × DBA/2 mice as a new model to explore underlying events that trigger some, but not all, cardinal features of preeclampsia. To determine whether there was systemic evidence for complement activation early in pregnancy, we measured blood levels of C3a, a marker of classical or alternative pathway activation, between days 3 and 7. Circulating C3a was significantly higher in CBA/2 × DBA/J compared with CBA/2 × BALB/c matings (422±43 versus 315±34, n=9–10 mice/group, P<0.05), at a time when there was no evidence of complement activation in the kidney (Figure 3, right panels). We have previously shown that excessive complement activation occurs at the maternal–fetal interface of the developing placenta by day 8 of pregnancy10.Girardi G. Yarilin D. Thurman J.M. et al.Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction.J Exp Med. 2006; 203: 2165-2175Crossref PubMed Scopus (361) Google Scholar and confirmed these findings in the current study (Figure 3, left panel). To determine whether blocking complement activation in the developing placenta early in pregnancy would prevent features of preeclampsia in DBA/2-mated CBA/J mice, we treated mice with CR2-Crry, a complement inhibitor specifically targeted to sites of complement activation that provides highly effective local protection without significant systemic inhibition.17.Song H. He C. Knaak C. et al.Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.J Clin Invest. 2003; 111: 1875-1885Crossref PubMed Scopus (86) Google Scholar, 18.Atkinson C. Song H. Lu B. et al.Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.J Clin Invest. 2005; 115: 2444-2453Crossref PubMed Scopus (141) Google Scholar, 30.Atkinson C. Zhu H. Qiao F. et al.Complement-dependent P-selectin expression and injury following ischemic stroke.J Immunol. 2006; 177: 7266-7274Crossref PubMed Scopus (68) Google Scholar CR2-Crry is composed of the complement receptor type 2 (CR2)-binding site for cell-bound C3 degradation products (iC3b/C3d) linked to the complement regulatory protein domain of the murine protein CR2-related gene/protein y (Crry), a murine C3 convertase inhibitor that blocks all complement activation pathways.31.Molina H. Wong W. Kinoshita T. et al.Distinct receptor and regulatory properties of recombinant mouse complement receptor 1 (CR1) and Crry, the two genetic homologues of human CR1.J Exp Med. 1992; 175: 121-129Crossref PubMed Scopus (118) Google Scholar A single dose of CR2-Crry (0.2 mg) administered i.v. on day 5 of pregnancy prevented complement deposition (Figure 3, left panels) and placental dysfunction characteristic of CBA/2 × DBA/J matings (Figure 4). In mice treated with CR2-Crry, there was no increase in placental STAT-8, a measure of oxidative stress associated with pregnancy complications23.Redecha P. van Rooijen N. Torry D. et al.Pravastatin prevents miscarriages in mice: role of tissue factor in placental and fetal injury.Blood. 2009; 113: 4101-4109Crossref PubMed Scopus (96) Google Scholar, 32.Walsh S.W. Vaughan J.E. Wang Y. et al.Placental isoprostane is significantly increased in preeclampsia.Faseb J. 2000; 14: 1289-1296Crossref PubMed Google Scholar, 33.Roberts J.M. Lain K.Y. Recent Insights into the pathogenesis of pre-eclampsia.Placenta. 2002; 23: 359-372Abstract Full Text PDF PubMed Scopus (485) Google Scholar (Figure 4a), and no increase in the circulating levels of the antiangiogenic factor sFlt-1 (Figure 4b). Levels of sFlt-1 in CBA/2 × DBA/J mice treated with CR2-Crry on day 5 were comparable with CBA/2 × BALB/c matings and remained lower than untreated CBA/2 × DBA/J matings through day 15, when the mice were killed (P<0.03). Similarly, in CR2-Crry-treated mice there was no elevation in plasma TAT levels on day 15 of pregnancy (Figure 4c). As predicted by the measures of improved placental function in CBA/2 × DBA/J mice, CR2-Crry prevented increased fetal resorption characteristic of these matings (Figure 4d). Fetal weights in CR2-Crry-treated pregnancies increased slightly (control versus CR2-Crry: 288±3 versus 294±4 mg, n=14–28, P=not significant). Studies of CR2-Crry in other experimental models show accumulation of the inhibitor at sites of tissue iC3b/C3d deposition and prolonged tissue half-life consistent with CR2 domain-mediated binding to tissue ligands.17.Song H. He C. Knaak C. et al.Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.J Clin Invest. 2003; 111: 1875-1885Crossref PubMed Scopus (86) Google Scholar, 18.Atkinson C. Song H. Lu B. et al.Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.J Clin Invest. 2005; 115: 2444-2453Crossref PubMed Scopus (141) Google Scholar, 19.Atkinson C. Qiao F. Song H. et al.Low-dose targeted complement inhibition protects against renal disease and other manifestations of autoimmune disease in MRL/lpr mice.J Immunol. 2008; 180: 1231-1238Crossref PubMed Scopus (51) Google Scholar Taken together with our previous report of C3 degradation fragments in the decidua and ectoplacental cone of DBA/J-mated CBA/2 mice,10.Girardi G. Yarilin D. Thurman J.M. et al.Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction.J Exp Med. 2006; 203: 2165-2175Crossref PubMed Scopus (361) Google Scholar the current findings indicate that early in pregnancy complement inhibition localized exclusively to the maternal–fetal interface is sufficient to prevent placental dysfunction and poor pregnancy outcomes associated with preeclampsia. Given that maternal manifestations of preeclampsia are considered to result from placental oxidative stress and vascular damage because of the dysregulation of angiogenic factors,33.Roberts J.M. Lain K.Y. Recent Insights into the pathogenesis of pre-eclampsia.Placenta. 2002; 23: 359-372Abstract Full Text PDF PubMed Scopus (485) Google Scholar, 34.Fisher S.J. The placental problem: linking abnormal cytotrophoblast differentiation to the maternal symptoms of preeclampsia.Reprod Biol Endocrinol. 2004; 2: 53Crossref PubMed Scopus (169) Google Scholar, 35.Redman C.W. Sargent I.L. Latest advances in understanding preeclampsia.Science. 2005; 308: 1592-1594Crossref PubMed Scopus (1900) Google Scholar, 36.Maynard S. Epstein F.H. Karumanchi S.A. Preeclampsia and angiogenic imbalance.Annu Rev Med. 2008; 59: 61-78Crossref PubMed Scopus (237) Google Scholar we hypothesized that targeting inhibitors of complement to the placenta would not only prevent placental dysfunction but would also attenuate other features of preeclampsia in CBA/J × DBA/2 pregnancies. To investigate this possibility, we administered CR2-Crry on day 5 of pregnancy and measured urinary protein, BUN, and examined renal tissue on day 15. Targeted complement inhibition prevented renal lesions in CBA/J × DBA/2 mice; albumin/creatinine ratios, and BUN at day 15 were comparable with CBA/J × BALB/c mice (Figure 1a and b). Immunofluorescence staining of kidneys showed that the increase in glomerular capillary wall and luminal deposits of fibrin in CBA/J × DBA/2 mice (mean fibrin score 1.5+; range 1–2+) was markedly attenuated in mice treated with CR2-Crry (fibrin score 0.75+; range 0.5–1+; Figure 1c). Similarly, the endothelial cell swelling and areas of segmental foot process evident by electron microscopy in kidneys of CBA/J × DBA/2 mice, were averted in pregnancies treated with CR2-Crry (Figure 1d). The histologic changes in glomeruli are not related to local activation of complement. Immunofluorescence studies demonstrated minimal C3 (mainly in the interstitium) in kidneys of CBA/J × DBA/2, which did not differ from that in CBA/J × BALB/c mice (Figure 3, right panels). Taken together, our findings demonstrate that blockade of complement activation targeted exclusively to areas of complement activation is sufficient to prevent maternal features of preeclampsia. To confirm that complement inhibition prevents preeclamptic renal injury and proteinuria, we used a second strategy. We treated CBA/2 × DBA/J mice with a non-targeted systemic complement inhibitor, anti-C5 monoclonal antibody (mAb), that blocks cleavage of C5, a pivotal complement component that generates two effectors, C5a, a potent anaphylatoxin, and C5b, which initiates formation of the C5b-9 membrane attack complex. We have previously shown that C5a–C5a receptor interactions initiate angiogenic dysregulation and placental dysfunction in abortion-prone mice.10.Girardi G. Yarilin D. Thurman J.M. et al.Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction.J Exp Med. 2006; 203: 2165-2175Crossref PubMed Scopus (361) Google Scholar, 37.Girardi G. Berman J. Redecha P. et al.Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome.J Clin Invest. 2003; 112: 1644-1654Crossref PubMed Scopus (535) Google Scholar Consistent with our findings using CR2-Crry, anti-C5 mAb prevented proteinuria and decreased BUN in DBA/J-mated CBA/2 mice (Figure 1a and b). Placental oxidative stress was also reduced in anti-C5 mAb-treated mice (placental STAT-8: control versus anti-C5 mAb 121±14 versus 77±43 pg/ml, n=21 and 4 mice/group, respectively, P=0.057) supporting the concept that complement activation contributes to the placental injury that drives angiogenic dysregulation and promotes a feed-forward loop that culminates in fetal growth restriction or death and maternal vascular damage. Dysregulation of angiogenic factors presages preeclampsia in humans, but interventions to reverse the imbalance are not yet available.36.Maynard S. Epstein F.H. Karumanchi S.A. 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