Shira Natanson-Yaron

A Novel Human-Specific Soluble Vascular Endothelial Growth Factor Receptor 1: Cell Type-Specific Splicing and Implications to Vascular Endothelial Growth Factor Homeostasis and Preeclampsia

A human-specific splicing variant of vascular endothelial growth factor (VEGF) receptor 1 (Flt1) was discovered, producing a soluble receptor (designated sFlt1-14) that is qualitatively different from the previously described soluble receptor (sFlt1) and functioning as a potent VEGF inhibitor. sFlt1-14 is generated in a cell type-specific fashion, primarily in nonendothelial cells. Notably, in vascular smooth muscle cells, all Flt1 messenger RNA is converted to sFlt1-14, whereas endothelial cells of the same human vessel express sFlt1. sFlt1-14 expression by vascular smooth muscle cells is dynamically regulated as evidenced by its upregulation on coculture with endothelial cells or by direct exposure to VEGF. Increased production of soluble VEGF receptors during pregnancy is entirely attributable to induced expression of placental sFlt1-14 starting by the end of the first trimester. Expression is dramatically elevated in the placenta of women with preeclampsia, specifically induced in abnormal clusters of degenerative syncytiotrophoblasts known as syncytial knots, where it may undergo further messenger RNA editing. sFlt1-14 is the predominant VEGF-inhibiting protein produced by the preeclamptic placenta, accumulates in the circulation, and hence is capable of neutralizing VEGF in distant organs affected in preeclampsia. Together, these findings revealed a new natural VEGF inhibitor that has evolved in humans, possibly to protect nonendothelial cells from adverse VEGF signaling. Furthermore, the study uncovered the identity of a VEGF-blocking protein implicated in preeclampsia.

Protein F2, a novel fibronectin‐binding protein from Streptococcus pyogenes, possesses two binding domains

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)‐binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn‐binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene (prtF2) from GAS strain 100076 encoding a novel Fn‐binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high‐affinity Fn binding. prtF2‐related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn‐binding protein in GAS. Protein F2 is highly homologous to Fn‐binding proteins from Streptococcus dysgalactiae and Strep‐tococcus equisimilis, particularly in its carboxy‐terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non‐repeated stretch of approximately 100 amino acids and is located 100 amino acids amino‐terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.

Local Retention Versus Systemic Release of Soluble VEGF Receptor-1 Are Mediated by Heparin-Binding and Regulated by Heparanase

Rationale: The vascular endothelial growth factor (VEGF) decoy receptor soluble VEGF-R1 (sVEGF-R1) is thought to protect the cells that produce it from adverse VEGF signaling. To accomplish this role, a mechanism for pericellular retention of sVEGF-R1 is required. Local retention may also prevent the accumulation of high circulating levels of sVEGF-R1 and resulting interference with homeostatic VEGF functions in remote organs. Objective: To reveal natural storage depots of sVEGF-R1 and determine mechanisms underlying its pericellular retention. To uncover natural mechanisms regulating its systemic release. Methods and Results: We show that both the canonical and human-specific isoforms of sVEGF-R1 are strongly bound to heparin. sVEGF-R1 produced by vascular smooth muscle cells is stored in the vessel wall and can be displaced from isolated mouse aorta by heparin. Another major reservoir of sVEGF-R1 is the placenta. Heparin increases the level of sVEGF-R1 released by cultured human placental villi, and pregnant women treated with low molecular weight heparin showed markedly elevated levels of sVEGF-R1 in the circulation. Heparanase is expressed in human placenta at the same locales as sVEGF-R1, and its transgenic overexpression in mice resulted in a marked increase in the levels of circulating sVEGF-R1. Conversely, heparanase inhibition, by either a neutralizing antibody or by inhibition of its maturation, reduced the amounts of sVEGF-R1 released from human placental villi, indicating a natural role of heparanase in sVEGF-R1 release. Conclusions: Together, the findings uncover a new level of regulation governing sVEGF-R1 retention versus release and suggest that manipulations of the heparin/heparanase system could be harnessed for reducing unwarranted release of sVEGF-R1 in pathologies such as preeclampsia.

Inhibitory NK receptor recognition of HLA-G: regulation by contact residues and by cell specific expression at the fetal-maternal interface.

The non-classical HLA-G protein is distinguished from the classical MHC class I molecules by its expression pattern, low polymorphism and its ability to form complexes on the cell surface. The special role of HLA-G in the maternal-fetal interface has been attributed to its ability to interact with specific receptors found on maternal immune cells. However this interaction is restricted to a limited number of receptors. In this study we elucidate the reason for this phenomenon by comparing the specific contact residues responsible for MHC-KIR interactions. This alignment revealed a marked difference between the HLA-G molecule and other MHC class I molecules. By mutating these residues to the equivalent classical MHC residues, the HLA-G molecule regained an ability of interacting with KIR inhibitory receptors found on NK cells derived either from peripheral blood or from the decidua. Functional NK killing assays further substantiated the binding results. Furthermore, double immunofluorescent staining of placental sections revealed that while the conformed form of HLA-G was expressed in all extravillous trophoblasts, the free heavy chain form of HLA-G was expressed in more distal cells of the column, the invasion front. Overall we suggest that HLA-G protein evolved to interact with only some of the NK inhibitory receptors thus allowing a control of inhibition, while permitting appropriate NK cell cytokine and growth factor production necessary for a viable maternal fetal interface.

Characterization of a Mouse-Passaged, Highly Encapsulated Variant of Group A Streptococcus in In Vitro and In Vivo Studies

JRS4(HE), a highly encapsulated, mouse-passaged variant of group A streptococcal strain JRS4, was characterized. The mucoid phenotype of JRS4(HE) was preserved after extensive passage in vitro. The level and size of csrRS transcript in JRS4(HE) was similar to that of JRS4, yet JRS4(HE) expressed high levels of has and sagA and exhibited an increased activity of streptolysin S. These findings indicate that the CsrRS repressor system was inactive in JRS4(HE). JRS4(HE) adhered to HEp-2 cells at the stationary phase but did not internalize these cells. At midlogarithmic phase, JRS4(HE) neither adhered to nor internalized cells, because of an increased amount of hyaluronic acid. Mice injected subcutaneously with JRS4(HE) developed large, deep necrotic lesions. In contrast, mice challenged with JRS4 developed small, superficial lesions. Despite the use of a high inoculum, mice challenged with JRS4(HE) did not develop a lethal bacteremic infection. It is concluded that inactivation of CsrRS in vivo is insufficient to cause a spreading necrotic disease.

Modeling of Human Cytomegalovirus Maternal-Fetal Transmission in a Novel Decidual Organ Culture

ABSTRACT Human cytomegalovirus (HCMV) is the leading cause of congenital infection, associated with severe birth defects and intrauterine growth retardation. The mechanism of HCMV transmission via the maternal-fetal interface is largely unknown, and there are no animal models for HCMV. The initial stages of infection are believed to occur in the maternal decidua. Here we employed a novel decidual organ culture, using both clinically derived and laboratory-derived viral strains, for the ex vivo modeling of HCMV transmission in the maternal-fetal interface. Viral spread in the tissue was demonstrated by the progression of infected-cell foci, with a 1.3- to 2-log increase in HCMV DNA and RNA levels between days 2 and 9 postinfection, the expression of immediate-early and late proteins, the appearance of typical histopathological features of natural infection, and dose-dependent inhibition of infection by ganciclovir and acyclovir. HCMV infected a wide range of cells in the decidua, including invasive cytotrophoblasts, macrophages, and endothelial, decidual, and dendritic cells. Cell-to-cell viral spread was revealed by focal extension of infected-cell clusters, inability to recover infectious extracellular virus, and high relative proportions (88 to 93%) of cell-associated viral DNA. Intriguingly, neutralizing HCMV hyperimmune globulins exhibited inhibitory activity against viral spread in the decidua even when added at 24 h postinfection—providing a mechanistic basis for their clinical use in prenatal prevention. The ex vivo-infected decidual cultures offer unique insight into patterns of viral tropism and spread, defining initial stages of congenital HCMV transmission, and can facilitate evaluation of the effects of new antiviral interventions within the maternal-fetal interface milieu.

Relationship between nulliparity and preeclampsia may be explained by altered circulating soluble fms-like tyrosine kinase 1

Objective: To test the hypothesis that the risk of preeclampsia in nulliparous women may be due to an anti-angiogenic state. Methods: Maternal serum samples obtained in the third trimester from nulliparous (n = 86) and multiparous (n = 165) singleton uncomplicated pregnancies were analyzed for levels of angiogenic factors – soluble fms like tyrosine kinase 1 (sFlt1) and placental growth factor (PlGF) by enzyme-linked immunosorbent assay (ELISA). Results: For nulliparous and multiparous pregnancies, serum sFlt1 levels were 12 732 ± 832 and 10 162 ± 666 (p = 0.020), serum PlGF levels were 215 ± 15 and 249 ± 14 (p = 0.093) (all reported as mean SD in pg/ml) and mean ratios of sFlt1/PlGF were 93 ± 12 and 62 ± 5 (p = 0.023), respectively. Adjustment for maternal age and fetal birth weight did not alter the results. Conclusions: Nulliparous pregnancies had higher circulating sFlt1 levels and sFlt1/PlGF ratios than multiparous pregnancies, suggesting an association with an angiogenic imbalance. Taken together with the pathogenic role of anti-angiogenic factors in preeclampsia, our data may be one explanation for the epidemiological observation that nulliparity is a risk factor for the development of preeclampsia.

Human trophectoderm apposition is regulated by interferon γ-induced protein 10 (IP-10) during early implantation.

INTRODUCTION The first step in human implantation is the attraction of the blastocyst to the endometrium. We aimed to study attraction of the human blastocyst to the endometrium, and how this process is accomplished by chemokines secreted by the endometrium. MATERIALS AND METHODS Blastocyst trophectoderm cells and other trophoblast lineage cells were subjected to attraction assays by IP-10 and other chemokines using transwell migration and chemotaxis assays. Chemokine expression and secretion were investigated using immunohistochemistry, ELISA, FACS analysis, and RT-PCR on material from flushing of the uterine cavity in endometrial biopsies. Chemokine receptor expression by blastocyst trophectoderm following PGD biopsy, trophectoderm derived from hES, placental villi, and other trophoblast lineage cells were characterized by the same methods. RESULTS IP-10 dramatically attracted trophectoderm derived from hES cells and other lineages by interaction with CXCR3 chemokine receptors, as shown by both chemotaxis and transwell migration. High levels of IP-10 were detected throughout the menstrual cycle at flushing of the uterine cavity. Immunohistochemistry, FACS analysis, and RT-PCR of endometrial biopsy detected IP-10 in glandular and stromal cells of the endometrium. High levels of IP-10 were detected in condition medium of the endometrial stromal and glandular cells. Of all of the chemokine/chemokine receptor combinations examined, the IP-10/CXCR3 interaction was the only cytokine that was significantly elevated. DISCUSSION While they await the wandering blastocyst, IP-10 is produced by many cells of the endometrium, but not by endometrial natural killer cells. CONCLUSION Endometrial IP-10 may specifically attract human blastocyst trophectoderm cells early in implantation.

Spatiotemporal Expression of Heparanase During Human and Rodent Ovarian Folliculogenesis

Abstract Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate proteoglycans (HSPGs), major components of the basement membrane (BM) and extracellular matrix (ECM). Heparanase activity results in release of HSPG-bound molecules, including basic fibroblast growth factor (FGF2). Structural and functional development of the corpus luteum (CL) involves tissue remodeling, active angiogenesis, and steroid production. Heparanase-induced ECM and BM breakdown as well as FGF2-stimulated endothelial proliferation may have an important role in the regulation of luteal function. Heparanase mRNA was detected by reverse-transcription-polymerase chain reaction in granulosa cells recovered from follicular fluid of in vitro fertilization patients. Using sulfate-labeled ECM, heparanase enzymatic activity was determined in human luteinized granulosa cells. Employing immunohistochemistry, heparanase protein was localized predominantly in the theca interna cell layer of the mature antral follicle, whereas in human corpora lutea, both luteinized granulosa and theca cells were immunostained for heparanase. During luteolysis, heparanase was identified in macrophages surrounding the forming corpus albicans. In serially sectioned ovaries from unstimulated rats as well as from eCG-treated rats, expression of heparanase was noted exclusively in the ovarian steroid-producing interstitial tissue. Following an ovulatory dose of hCG, heparanase was immunostained also in lutein cells of the forming corpora lutea. Temporal expression of heparanase in granulosa cells during the luteal phase and in macrophages during luteal regression supports the hypothesis that heparanase plays a role in human ovarian ECM remodeling and may potentiate cellular migration and growth factor bioavailability.