Although replete with cytotoxic machinery, uterine NK (uNK) cells remain tolerant at the maternal-fetal interface. The mechanisms that facilitate the uNK cell tolerance are largely unknown. Here we demonstrate that VEGF C, a pro-angiogenic factor produced by uNK cells, is responsible for their non-cytotoxic activity. VEGF C-producing uNK cells support endovascular processes as demonstrated in a three dimensional co-culture model of capillary tube formation on matrigel. Peripheral blood NK cells fail to produce VEGF C and remain cytotoxic. This response can be reversed by exogenous VEGF C. We show that cytoprotection by VEGF C can be related to induction of the “transporter associated with antigen processing (TAP)-1” expression and MHC class I assembly in target cells. siRNA-mediated silencing of TAP-1 expression abolished the VEGF C-imparted protection. Overall, these results demonstrate that empowerment of uNK cells with angiogenic factors keeps them non-cytotoxic. This phenotype is critical to their pregnancy compatible immuno-vascular role during placentation and fetal development.

Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.

Hypoxia has been implicated in the pathogenesis of preeclampsia, a hypertensive disorder of pregnancy. However, in vivo evidence and mechanistic understanding remain elusive. Preeclampsia is associated with impaired placental angiogenesis. We have recently shown that interleukin (IL)-10 can support trophoblast-driven endovascular cross-talk. Accordingly, we hypothesize that pathologic levels of oxygen coupled with IL-10 deficiency induce severe preeclampsia-like features coupled with elevated production of anti-angiogenic factors, apoptotic pathways, and placental injury. Exposure of pregnant wild type and IL-10−/− mice to 9.5% oxygen resulted in graded placental injury and systemic symptoms of renal pathology, proteinurea (wild type 645.15±115.73 versus 198.09±93.45; IL-10−/− 819.31±127.85 versus 221.45±82.73 µg/mg/24 hours) and hypertension (wild type 118.37±14.45 versus 78.67±14.07; IL-10−/− 136.03±22.59 versus 83.97±18.25 mmHg). Recombinant IL-10 reversed hypoxia-induced features in pregnant IL-10−/− mice confirming the protective role of IL-10 in preeclampsia. Hypoxic exposure caused marked elevation of soluble fms-like tyrosine kinase 1 (110.8±20.1 versus 44.7±11.9 ng/ml) in IL-10−/− mice compared to their wild type counterparts (81.6±13.1 versus 41.2±8.9 ng/ml), whereas soluble endoglin was induced to the similar levels in both strains (~380±50 versus 180±31 ng/ml). Hypoxia-induced elevation of p53 was associated with marked induction of pro-apoptotic protein Bax, down-regulation of Bcl-2, and trophoblast-specific apoptosis in utero-placental tissue. Collectively, we conclude that severe preeclampsia pathology could be triggered under certain threshold oxygen levels coupled with intrinsic IL-10 deficiency which lead to excessive activation of anti-angiogenic and apoptotic pathways.

Preeclampsia is a major pregnancy complication with potential short- and long-term consequences for both mother and fetus. Understanding its pathogenesis and causative biomarkers is likely to yield insights for prediction and treatment. Herein, we provide evidence that transthyretin, a transporter of thyroxine and retinol, is aggregated in preeclampsia and is present at reduced levels in sera of preeclamptic women, as detected by proteomic screen. We demonstrate that transthyretin aggregates form deposits in preeclampsia placental tissue and cause apoptosis. By using in vitro approaches and a humanized mouse model, we provide evidence for a causal link between dysregulated transthyretin and preeclampsia. Native transthyretin inhibits all preeclampsia-like features in the humanized mouse model, including new-onset proteinuria, increased blood pressure, glomerular endotheliosis, and production of anti-angiogenic factors. Our findings suggest that a focus on transthyretin structure and function is a novel strategy to understand and combat preeclampsia.

The immune tolerance and de novo vascularization are two highly intriguing processes at the maternal-fetal interface that appear to be central for normal pregnancy outcome. Immune tolerance occurs despite the local presence of an active maternal immune system including macrophages, dendritic cells and specialized CD56 bright CD16 − uterine NK cells (65-70%). Recent observations indicate that the phenotypic and functional repertoire of uNK cells is distinct from peripheral blood NK (pNK) and endometrial NK cells (eNK) cells, challenging the understanding of their temporal occurrence and function. Origin and specialized programming of uNK cells continue to be debated. uNK cells, replete with an armamentarium to kill the foreign, tolerate the conceptus and facilitate pregnancy. Why do these uNK cells remain non-cytotoxic? Are these NK cells "multitasking" in nature harboring beneficial and detrimental roles in pregnancy? Are there distinct subpopulations of NK cells that may populate the decidua? We propose that the endometrium/decidua functions as an "inducible tertiary lymphoid tissue" that supports the recruitment and expansion of CD56 bright CD16 − NK cells, and induces transcriptional upregulation of angiogenic machinery in response to exposure to local hormonal factors, cytokine milieu and perhaps hypoxia. The angiogenic features of uNK cells could further result in a "multitasking" phenotype that still remains to be characterized. This paper discusses the factors and pathways that bridge the angiogenic and non-cytotoxic response machineries at the maternalfetal interface.

Early diagnosis and treatment of preeclampsia would significantly reduce maternal and fetal morbidity and mortality. However, its etiology and prediction have remained elusive. Based on the hypothesis that sera from patients with preeclampsia could function as a "blueprint" of causative factors, we describe a serum-based pregnancy-specific mouse model that closely mirrors the human condition as well as an in vitro predictive assay. We show that a single administration of human preeclampsia serum in pregnant IL-10-/- mice induced the full spectrum of preeclampsia-like symptoms, caused hypoxic injury in uteroplacental tissues, and elevated soluble fms-like tyrosine kinase 1 and soluble endoglin, markers thought to be related to the disease. The same serum sample(s) induced a partial preeclampsia phenotype in wild-type mice. Importantly, preeclampsia serum disrupted cross talk between trophoblasts and endothelial cells in an in vitro model of endovascular activity. Disruption of endovascular activity could be documented in serum samples as early as 12 to 14 weeks of gestation from patients who subsequently developed preeclampsia. These results indicate that preeclampsia patient sera can be used to understand the pregnancy-specific disease pathology in mice and can predict the disorder.

The placental-decidual interaction through invading trophoblasts determines whether a physiological transformation of the uterine spiral arteries is established or not. Trophoblast-orchestrated artery remodeling is central to normal placentation. Dysregulated uteroplacental interaction and vascular remodeling are thought to be associated with the molecular events underlying the pathology of late pregnancy anomalies including preeclampsia. Although the exact gestational age at which trophoblast invasion ceases is not known, it remains unclear whether late pregnancy trophoblasts retain the ability to transform the uterine arteries. Here, we have developed a dual cell, in vitro culture system that mimics the vascular remodeling events during normal pregnancy. We demonstrate that first and third trimester trophoblasts respond differentially to interactive signals from endothelial cells when cultured on matrigel. Term primary trophoblasts or immortalized third trimester extravillous TCL1 trophoblasts not only fail to respond to signals from endothelial cells but also inhibit endothelial cell tube formation. In contrast, HTR8 cells, representing a first trimester trophoblast cell line with invasive properties, undergo spontaneous migration and synchronize with the endothelial cells in a capillary network. This disparity in behavior was confirmed in vivo using a matrigel plug assay. Poor expression of VEGF C and VEGF receptors coupled with high E-cadherin expression by term primary trophoblasts and TCL1 cells contributed to their restricted interactive and migratory properties. We further show that the kinase activity of VEGF R2 is essential for proactive cross-talk by HTR8 cells. This unique behavior of first trimester trophoblasts in the presence of endothelial cells offers a potential approach to study cell-cell interactions and to decipher modulatory components in the serum samples from adverse pregnancy outcomes.

Human chorionic gonadotropin (hCG) is crucial for successful pregnancy. Its many functions include angiogenesis and immune regulation. Despite years of research, the etiology of preeclampsia remains unknown. Marked by insufficient trophoblast invasion and poor spiral artery remodeling, preeclampsia has also been linked to immune dysregulation. Here we discuss the roles of hCG in the context of endovascular cross-talk between trophoblasts and endothelial cells and immune tolerance. We propose that functional and glycosylation modifications of hCG may contribute to the pathogenesis of preeclampsia.