Elevated vasoinhibins may contribute to endothelial cell dysfunction and low birth weight in preeclampsia

Laboratory Investigation (2007) 87, 1009–1017 & 2007 USCAP, Inc All rights reserved 0023-6837/07 $30.00 Elevated vasoinhibins may contribute to endothelial cell dysfunction and low birth weight in preeclampsia Carmen González1,*, Adalberto Parra2,*, Jorge Ramı́rez-Peredo2, Celina Garcı́a1, José Carlos Rivera1, Yazmı́n Macotela1, Jorge Aranda1, Maria Lemini1, José Arias2, Francisco Ibargüengoitia2, Gonzalo Martı́nez de la Escalera1 and Carmen Clapp1 Vasoconstriction and defective placental angiogenesis are key factors in the etiology of preeclampsia. Prolactin levels are elevated in maternal blood throughout pregnancy and the human decidua produces prolactin that is transported to the amniotic fluid. Prolactin is cleaved to yield vasoinhibins, a family of peptides that inhibit angiogenesis and nitric oxidedependent vasodilation. Here, we conducted a case–control study to measure vasoinhibins in serum, urine, and amniotic fluid obtained from women with severe preeclampsia. We show that all three biological fluids contained significantly higher levels of vasoinhibins in preeclamptic women than in normal pregnant women. Amniotic fluid from preeclamptic women, but not from normal women, inhibited vascular endothelial growth factor-induced endothelial cell proliferation and nitric oxide synthase activity in cultured endothelial cells, and these actions were reversed by antibodies able to neutralize the effects of vasoinhibins. Furthermore, amniotic fluid does not appear to contain neutral prolactin-cleaving proteases, suggesting that vasoinhibins in amniotic fluid are derived from prolactin cleaved within the placenta. Also, cathepsin-D in placental trophoblasts cleaved prolactin to vasoinhibins, and its activity was higher in placental trophoblasts from preeclamptic women than from normal women. Importantly, birth weight of infants in preeclampsia inversely correlated with the extent to which the corresponding AF inhibited endothelial cell proliferation and with its concentration of prolactin þ vasoinhibins. These data demonstrate that vasoinhibins are increased in the circulation, urine, and amniotic fluid of preeclamptic women and suggest that these peptides contribute to the endothelial cell dysfunction and compromised birth weight that characterize this disease. Laboratory Investigation (2007) 87, 1009–1017; doi:10.1038/labinvest.3700662; published online 6 August 2007 KEYWORDS: angiogenesis; cathepsin-D; nitric oxide; preeclampsia; 16K prolactin; VEGF Preeclampsia affects about 5% of all pregnancies and results in substantial maternal and neonatal morbidity and mortality.1 Although the etiology of preeclampsia remains unclear, the syndrome may be initiated by placental factors causing endothelial cell dysfunction at the fetomaternal interface and in the systemic maternal circulation.1,2 Poor placental and decidual vascularization results in inadequate placental development and may restrict fetal growth, whereas dysregulation of the maternal vascular endothelium leads to hypertension and proteinuria—the clinical manifestations of preeclampsia.1,2 Vascular endothelial growth factor (VEGF) is a major promoter of angiogenesis and vasodilation in the placenta.2 The actions of VEGF are partially mediated by the production of endothelium-derived nitric oxide (NO),3 a potent vasorelaxant that regulates systemic blood pressure, vascular permeability, and angiogenesis.4 Decreased levels of VEGF and NO are seen not only during preeclampsia, but also before the onset of clinical symptoms.5–8 Moreover, interference with placental VEGF and NO compromises normal angiogenesis and leads to a poorly perfused fetoplacental unit, hypertension, proteinuria, and fetal growth restriction,6,9–11 suggesting that blockage of VEGF and NO has a causal role in preeclampsia. Prolactin (PRL), originally identified as a lactotrophic hormone secreted by the pituitary gland, is also synthesized 1 Departamento de Neurobiologı́a Celular y Molecular, Instituto de Neurobiologı́a, Universidad Nacional Autónoma de México (UNAM), Campus UNAM-Juriquilla, Querétaro, México and 2Instituto Nacional de Perinatologı́a Isidro Espinosa de los Reyes, Secretarı́a de Salud, México DF, México Correspondence: Dr C Clapp, PhD, Departamento de Neurobiologı́a Celular y Molecular, Instituto de Neurobiologı́a, Universidad Nacional Autónoma de México (UNAM), Campus UNAM-Juriquilla, Boulevard Juriquilla 3001, 76230 Querétaro, Qro, México. E-mail: *These two authors contributed equally to this work. Received 4 May 2007; revised 7 July 2007; accepted 9 July 2007 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 87 October 2007 1009 Elevated vasoinhibins in preeclampsia C González et al in numerous extrapituitary tissues, including the decidual cells in the uterus, from which it is transported to the amniotic fluid (AF) where it reaches high levels.12,13 Proteolysis of PRL by cathepsin-D or by matrix metalloproteases produces vasoinhibins, a family of peptides that act on endothelial cells to inhibit vasodilation and angiogenesis and to promote apoptosis-mediated vascular regression.14 Vasoinhibins suppress VEGF-induced NO synthase (NOS) activity in endothelial cells, and exogenous NO reverses inhibition by vasoinhibins of VEGF-induced endothelial cell proliferation and acetylcholine-induced vasodilation.15 The increased presence of PRL in maternal blood throughout pregnancy16 and its synthesis by decidual cells, coupled to the fact that vasoinhibins impair VEGF-dependent activation of endothelial NOS, angiogenesis, and vasodilation, suggest that vasoinhibins may play a role in preeclampsia. The purpose of this study was to determine whether vasoinhibins are present in the serum, urine, and AF from patients with severe preeclampsia, and if they could reduce the proangiogenic actions of VEGF, inhibit NOS activity, and contribute to reducedbirth weight. MATERIALS AND METHODS Study Population The study encompassed 21 pregnant women without history of diabetes mellitus, thyroid, liver, or chronic renal disease attending the Obstetrics Outpatient Department of the ‘Instituto Nacional de Perinatologı́a Isidro Espinosa de los Reyes’ in Mexico City. All women provided written, informed consent before collection of samples. The Institutional Review Board approved the collection and use of the samples, and the study was conducted according to the third edition of the Guidelines on the Practice of Ethical Committees in Medical Research issued by the Royal College of Physicians of London. The control group included eight clinically healthy, normotensive women between 18 and 38 years of age, with full-term (Z36 weeks of gestation) uneventful pregnancies (all singleton), who were undergoing cesarean section for obstetric reasons. The preeclamptic group included 13 previously normotensive women, 16–40 years old with severe preeclampsia diagnosed between 28 and 39 weeks of gestation (all singleton), (...truncated)