Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-21
2002-04-23
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S007100, C424S143100, C424S145100, C424S139100
Reexamination Certificate
active
06376199
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods and compositions for diagnosing and treating conditions requiring regulation of trophoblast invasion.
BACKGROUND OF THE INVENTION
During placental development the establishment of fetal-maternal interactions is critical for a successful human pregnancy (1). Abnormalities of placenta formation due to shallow trophoblast invasion have been linked to preeclampsia and fetal growth restriction (2). In contrast, uncontrolled trophoblast invasion and abnormal trophoblast growth are associated with hydatiform mole and choriocarcinoma. In the course of placenta formation, chorionic villous cytotrophoblasts undergo two morphologically distinct pathways of differentiation. The vast majority of cytotrophoblasts in both floating and anchoring villi fuse to form the syncytiotrophoblast layer, which permits gas and nutrient exchange for the developing embryo. A small percentage of cytotrophoblasts in anchoring villi break through the syncytium, at selected sites, and generate columns of non-polarized cells which migrate into the endometrium. These extravillous trophoblasts (EVT) invade deeply into the uterus reaching the first third of the myometrium at which point they invade the spiral arteries, replacing their endothelium and vascular wall. Invasion peaks at 12 weeks of gestation and rapidly declines thereafter. indicating that, unlike tumour invasion, it is spatially and temporally regulated (3). Trophoblast invasion in the decidua is accompanied by a complex modulation of the synthesis and degradation of extracellular matrix (ECM) proteins and in the expression of adhesion molecules (4-6). Along the invasive pathway, ECM proteins undergo changes in their spatial distribution with loss of laminin and appearance of fibronectin (3,4). EVT loose the expression of E-cadherins, responsible for cell-cell adhesion between polarized stem cytotrophoblasts, down-regulate &agr;
6
&bgr;
4
integrin, a laminin receptor, and acquire &agr;
5
&bgr;
1
integrin, a fibronectin recepor (7). Once the EVT invade the endometrium they express the &agr;
1
&bgr;
1
integrin, a collagen/laminin receptor. Thus, specific changes in ECM proteins and their receptors are associated with the acquisition of an invasive phenotype by the extravillous trophoblasts (4).
Preeclampsia occurs in 5-10% of pregnancies and is the leading cause of death and illness in women during pregnancy. Preeclamnpsia is also associated with considerable fetal
eonatal complications because of adverse intrauterine conditions and preterm delivery. There is currently no effective pharmacologic treatment for preeclampsia and the only remedy is to remove the placenta (and hence deliver the fetus preterm). Current protocols, including bedrest and antihypertensive drugs, seek to stabilize maternal/fetal condition until delivery is necessitated. It is estimated that around 200,000 children are born preterm in North America due to preeclampsia. Many of these babies will require costly intensive care at birth and if they survive may face a lifetime of chronic illness (e.g. lung disease) or disability (e.g. cerebral palsy, mental handicaps, blindness). These conditions represent a significant impact on subsequent requirements for community health care resources. Therefore, reducing the incidence of preeclampsia and preterm birth would have a tremendous positive impact on health care delivery.
SUMMARY OF THE INVENTION
The present inventors have studied the mechanisms that regulate trophoblast invasion. The inventors have found that antisense disruption of the expression of the TGF&bgr; receptor, endoglin, triggers invasion of cytotrophoblast from first trimester villous explants in vitro indicating that the TGF&bgr; receptor system, and in particular endoglin, plays a critical role in regulating this process. Significantly, the present inventors defined components that endogenously regulate trophoblast invasion. TGF-&bgr;
3
was found to be a major regulator of trophoblast invasion in vitro. In particular, the presence of TGF-&bgr;
3
and its receptors at 5-8 weeks at a time when there is no spontaneous trophoblast invasion and the absence of these molecules at 12-13 weeks when spontaneous invasion occurs, establishes a major role for TGF-&bgr;
3
as an endogenous inhibitor of trophoblast invasion. Down-regulation of TGF-&bgr;
3
(but not &bgr;
1
or &bgr;
2
) expression using antisense oligonucleotides, stimulated extravillous trophoblast cell (EVT) outgrowth/migration and fibronectin production in 5-8 villous explants indicating that TGF-&bgr;
3
acts to suppress in vivo trophoblast invasion. The effects of antisense treatment to TGF-&bgr;
3
are specific as they are prevented by addition of exogenous TGF-&bgr;
3
but not TGF-&bgr;
1
or TGF-&bgr;
2
. The stimulatory effects of TGF-&bgr;
3
are lost after 9 weeks of gestation which is compatible with TGF-&bgr;
3
being produced by the villi during a specific window of gestation within the first trimester (5-8 weeks) and that inhibition of its synthesis stimulates trophoblast differentiation. Addition of exogenous TGF-&bgr;
3
to the villous explants inhibits fibronectin synthesis.
The clinical importance of TGF-&bgr;
3
in regulating trophoblast invasion has been highlighted by the finding that TGF-&bgr;
3
is highly expressed in trophoblast tissue of preeclamptic patients when compared to that in age-matched control placenta while there was no change in the expression of either the &bgr;
1
or &bgr;
2
isoform. Fibronectin and &agr;
5
integrin expression were also greater in preeclamptic placenta, indicating that in preeclampsia, where there is shallow trophoblast invasion, trophoblast cells are arrested as an &agr;
5
integrin phenotype producing TGF-&bgr;
3
. These data are supported by the finding that villous explants from a control (non-preeclamptic placenta, 32 weeks of gestation) spontaneously formed columns of trophoblasts that invaded the surrounding Matrigel, while explants from a preeclamptic placenta did not.
In contrast to TGF-&bgr;
3
, activin, a TGF-&bgr; receptor, has been found to trigger trophoblast invasion. Follistatin an activin binding protein, inhibited the stimulatory effect of activin, and antibodies and antisense to endoglin.
Oxygen tension was also found to play a role in regulating trophoblast invasion. The expression of the hypoxia inducible factor, HIF-1&agr;, parallels that of TGF-&bgr;
3
in first trimester trophoblast (i.e. peaks at 6-8 weeks but decreases after 9-10 weeks when oxygen tension increases). Expression of HIF-1&agr; was dramatically increased in placentas of preeclamptic patients when compared to age-matched control tissue. Induction of HIF-1&agr; by low PO
2
(around 6-8 weeks) up regulates TGF-&bgr;
3
transcription and blocks trophoblast invasion. A failure of the system to down-regulate at 9-11 weeks (either due to a block in response to normoxia or the absence of an increase in oxygen tension) leads to shallow invasion and predisposes to preeclampsia.
In addition to endoglin, the present inventors have found that TGF-&bgr;
3
signals through a receptor complex which includes RI (ALK1), RII and endoglin. While TGF-&bgr; RI (ALK-5) and TGF-&bgr; R-II are expressed throughout the villi and decidua at 9-10 weeks gestation, they were found to be absent from the base of the proximal columns of the anchoring villi at the transition zone between the villous and the invading EVT exactly at the site where endoglin is up-regulated. This dramatic change in TGF-&bgr; receptor expression indicates that EVT within the columns in situ are not subject to the inhibitory actions of TGF&bgr;, but via R-I and R-II they come under the control of this ligand upon entering the decidua. In addition, antisense induced disruption of RI (ALK-1) and RII expression stimulated trophoblast outgrowth/migration and fibronectin synthesis. In contrast, antisense to RI (ALK-5) inhibited fibronectin synthesis.
Broadly stated the present invention relates to a method for detecting, preventing, and/or treating a condition requiring
Caniggia Isabella
Lye Stephen
Post Martin
Andres Janet L.
Eyler Yvonne
Merchant & Gould P.C.
The Hospital for Sick Children (HSC)
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