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Biology of Reproduction 62, 691-697 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Human Endometrial Endothelial Cells: Isolation, Characterization, and Inflammatory-Mediated Expression of Tissue Factor and Type 1 Plasminogen Activator Inhibitor1

Frederick Schatza, Carl Soderlandc, Karen D. Hendricks-Muñozb, Rene P. Gerretsb, and Charles J. Lockwood2,a

a Department of Obstetrics and Gynecology and b Department of Pediatrics, New York University School of Medicine, New York, New York 10016 c Cell Systems, Kirkland, Washington 98034


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Binding of Ulex europaeus lectin to microvessels was used to isolate endothelial cells from cycling human endometrium. Cultured human endometrial endothelial cells (HEECs) exhibited endothelial cell-specific characteristics such as tube formation on a basement membrane matrix and sequestration of acetylated low-density lipoprotein. Markers for potentially contaminating epithelial, stromal, smooth muscle, and bone marrow-derived cells were not detected in the HEEC cultures. Basal and proinflammatory-stimulated immunostaining profiles for endothelial cell-specific adhesion markers, as exemplified by Von Willebrand's factor and E-selectin, were similar for cultured HEECs and human umbilical venous cord endothelial cells (HUVECs). However, HUVECs expressed several extracellular matrix proteins that were absent from cultured HEECs. In the latter, the protein kinase C agonist phorbol myristate acetate transiently enhanced tissue factor (TF) mRNA levels and elicited a more prolonged elevation in TF protein levels, but did not affect plasminogen activator inhibitor-1 (PAI-1) mRNA and protein levels. Inappropriate expression of TF, which initiates hemostasis by generating thrombin, and of PAI-1, which regulates hemostasis by acting as the primary inhibitor of fibrinolysis, can each lead to thrombosis. The differential regulation of TF and PAI-1 expression revealed in the current study emphasizes the importance of using HEECs to evaluate mechanisms regulating the hemostatic/thrombotic balance in human endometrium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During human implantation, blastocyst-derived trophoblasts breach endometrial blood vessels and establish the primordial uteroplacental circulation. Concomitantly, endovascular trophoblasts begin to replace the spiral arterial endothelial lining and remodel the intima and media of these vessels [1]. These processes provide the developing embryo with a requisite source of oxygen and nutrients, and are therefore key to reproductive success. However, they present contradictory hemostatic challenges. Thus, decidual hemorrhage can stem from inadequate hemostasis in these remodeled vessels. Alternatively, continued embryo survival requires maintenance of blood fluidity, thereby avoiding thrombosis and the sequelae of fetal growth retardation and stillbirth. A dearth of in vitro studies with human endometrial endothelial cells (HEECs) limits understanding of their physiological anticoagulant function as well as their pathological role in endometrial vascular thrombosis.

The current study sought to isolate HEECs from specimens of cycling tissue, with the goal of developing a relevant in vitro model for evaluating HEEC physiology and pathology. By examining morphological characteristics and antigen expression, it aimed to validate the endothelial nature of the cultured HEECs and to establish their homogeneity by ruling out the presence of extraneous cell types. An extensive number of concepts in vascular biology have relied on the in vitro behavior of human umbilical venous cord endothelial cells (HUVECs). However, HUVECs are derived from a type of vessel that is not affected by atherosclerosis, the most common of human vascular disorders [2]. The protein kinase C agonist phorbol myristate acetate (PMA) induces the expression of tissue factor (TF) and plasminogen activator (PA) inhibitor 1 (PAI-1) in cultured HUVECs and in endothelial cells derived from other large vessels such as saphenous veins and bovine aortas [3–7].

Since TF is the primary mediator of hemostasis [8] and PAI-1 affects hemostasis by acting as the fast inactivator of fibrinolysis [9], we examined PMA effects on TF and PAI-1 expression in cultured HEECs in order to initiate direct studies of the hemostatic-thrombotic balance in human endometrium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues

Specimens of cycling endometrium were obtained from consenting patients undergoing hysterectomy for myomas and were transported to a sterile laminar flow hood. A small portion of each specimen was formalin-fixed and histologically dated by the criteria of Noyes et al. [10], and endothelial cells were isolated from the remainder. A portion of some specimens were frozen in OCT (Miles Inc., Elkhart, IN) in cold 2-methyl butane and were sectioned for fluorescent lectin staining.

Lectin Staining of Endometrium

Frozen sections of human endometrium were incubated for 30 min at 4°C with fluorescein-labeled lectins (Sigma, St. Louis, MO) diluted 1:50 in PBS and then washed with buffer (2 mM Tris-HEPES, 140 mM NaCl at pH 7.4) to remove free lectin. The slides were fixed in absolute ethanol for 40 min, air-dried, examined by fluorescence microscopy, and photographed.

Cell Isolation and Culture

HEECs Endometrial specimens were dissected free of visible vascular tissue, minced in Ca2+/Mg2+-free PBS (Cell Systems Corp., Kirkland, WA) + 1% dialyzed fetal bovine serum (FBS) (HyClone, Logan, UT), and digested with type VII high-purity collagenase (Sigma) and dispase (Boehringer Mannheim, Indianapolis, IN) in the presence of deoxyribonuclease (DNase) I (Sigma) and 1% dialyzed fetal bovine serum (FBS). The digest was filtered through a 70-µm cell strainer (Falcon; Becton-Dickinson, Franklin Lakes, NJ) to remove undigested tissue fragments. The filtrate was treated with 1 mg/ml of biotinylated UEA-1 (Ulex europaeus) lectin (Sigma), which binds selectively to microvascular endothelial cells in human endometrial sections (see Fig. 1). Biotinylated UEA-1 labeled cells were separated from nonlabeled cells by panning on activated surface/AIS MicroCELLector flasks (Applied Immune Sciences, Menlo Park, CA) coated with streptavidin (Sigma). The reaction mixture contained 107 cells incubated at 4°C with 0.1 ml UEA-1.



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  		FIG. 1. Fluorescence microscopy of a luteal-phase (Day 24) endometrial specimen after incubation with fluorescein-labeled lectins. A) Dolichos bifoloris agglutinin. x320. B) UEA-1. x320. (Published at 87%.)

Cells retained on the MicroCELLector flasks were released and cultured in CS-C Complete Medium (phenol red-free DMEM:Ham's F-12 [1:1] with 15 mM HEPES, containing endothelial cell growth factor) supplemented with 15% stripped fetal calf serum in flasks coated with attachment factor (Cell Systems). A typical yield from 1.5 g of tissue exceeded 5 x 105 UEA-1-positive cells in a primary culture. The HEECs were grown to confluence in a 37°C, 95% air:5% CO2 incubator, with the medium changed every two days, and they were harvested with the CS-C Passage Reagent Group, which involves the sequential use of EDTA, trypsin-EDTA, and trypsin inhibitor solutions (Cell Systems). Cells were split 1: 6 for passaging.

Human endometrial stromal cells Endometrial stromal cells were isolated to homogeneity from fragments of cycling endometrium and cultured as previously described [11, 12]. Briefly, the fragments were digested with type 1 collagenase (Worthington Biochemical Corp., Freehold, NJ); then the digestate was filtered through a 38-µm stainless steel sieve to remove the glands. The filtrate was centrifuged on a 30–60% Percoll gradient to produce a stromal cell-enriched layer at the interface. The stromal cells were purified to virtual homogeneity by exploiting their more rapid adherence to the surface of polystyrene tissue culture dishes (30 min in a 37°C, 95% air:5% CO2 incubator) than that of contaminating cell types.

HUVECs and human vascular smooth muscle cells HUVECs and vascular smooth muscle cells, which were derived from umbilical arteries, were obtained from Cell Systems and cultured as described for HEECs.

Characterization of Cultured Cells

Morphology Second-passage HEECs were suspended in CS-C Complete Medium and seeded (3000 cells/mm2) on either 25-mm cell culture inserts coated with Matrigel (Collaborative Biomedical Products Inc., New Bedford, MA), or on attachment factor-coated plastic (Cell Systems), and allowed to grow to confluence. Scanning electron microscopy (SEM) was carried out with a Hitachi (Tokyo, Japan) S530 Scanning Electron Microscope, after the cultures were postfixed in 3% gluteraldehyde:1% OsO4.

Antigen expression Cultured HEECs, HUVECs, human vascular smooth muscle cells, and human endometrial stromal cells were each fixed for 5 min in ice-cold absolute methanol and then incubated with primary monoclonal antibodies that were obtained from either Becton-Dickinson or Sigma, and used at a dilution of 1:250 in PBS. Indirect fluorescence was carried out after incubation with fluorescein-conjugated second antibody (Dako, Copenhagen, DK) used at a dilution of 1:50. A semiquantitative scoring system with a range of staining intensity—none (-), weak (+), moderate (++), strong (+++), and intense (++++)—was used to assess relative immunofluorescence.

Regulation of TF and PAI-1 expression Confluent HEECs were starved for 3 h in M 199 (Gibco, Grand Island, NY) in a 37°C, 95% air:5% CO2 incubator. The medium was exchanged for a serum-free defined medium lacking growth factor (SF-4Z0-500-S; Cell Systems) containing PMA (Sigma) or 0.1% ethanol, the vehicle used to dissolve PMA. When the incubations were terminated, the collected media were centrifuged, and the supernatants and harvested cell pellets were stored at -70°C. Parallel cultures were frozen on dry ice and stored at -70°C for RNA extraction.

Northern blot analysis Total RNA was extracted from cultured HEECs by guanidinium thiocyanate-chloroform. Approximately 15 µg total RNA from each of the experimental cultures, and molecular weight RNA standards (Boehringer-Mannheim) were separated on a 1% agarose gel containing 2.2 M formaldehyde, then transferred to a Zeta-Probe nylon membrane (Bio-Rad Labs., Hercules, CA). Levels of TF and PAI-1 mRNA were detected using previously described probes [11, 13], which were labeled with [32P]deoxy-CTP to high specific activity by random priming with a Boehringer-Mannheim kit. Hybridization was performed by standard methods, and the washed filters were exposed to Kodak XAR film (Eastman Kodak, Rochester, NY). Total RNA loads were standardized by reprobing the stripped membranes with a 32P-labeled probe for actin.

Biochemical assays Sensitive ELISAs (American Diagnostica, Greenwich, CT) were used to measure immunoreactive TF levels in the cell pellets and PAI-1 levels in the cell-conditioned medium as described [11, 13]. Protein and DNA content were measured in aliquots of the cell pellets by the Bio-Rad assay (Bio-Rad Labs.) and the method of Hinegardner [14], respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
UEA-1 Expression in Human Endometrial Vessels and in Cultured HEECs

Lectin binding to specific surface determinants has proven useful in cell isolation. Figure 1A indicates that Dolichos biflorus agglutinin, which binds to endothelial cells of developing brain vasculature [15], stained the apical surface of human endometrial glands but did not significantly stain either the stromal cells or vessels. By contrast, Figure 1B reveals that the vasculature displayed prominent staining for UEA-1, which binds to blood vessels in bone marrow [16]. Only minimal UEA-1 staining was evident in either glands or stroma.

As described in Materials and Methods, binding of UEA-1 to endometrial microvasculature was used to isolate HEECs. Figure 2A indicates that fluorescein-conjugated UEA-1 decorated the surface of endothelial cell-enriched endometrial fragments obtained after panning. Moreover, the uniform fluorescence shown in Figure 2B indicates that recognition of UEA-1 was preserved in second-passage HEECs grown on attachment factor-coated tissue culture plastic.



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  		FIG. 2. Direct fluorescence showing stepwise purification of HEECs from tissue. A) The surfaces of endometrial fragments obtained after panning are decorated with fluorescein-conjugated UEA-1 with the nuclei excluded. x100. B) Monolayers of second-passage HEECs labeled with rhodamine-conjugated UEA-1 lectin. x400. (Published at 87%.)

Characterization of Cultured HEECs

Phase-contrast microscopy (Fig. 3A) and SEM (Fig. 3C) indicated that the HEECs formed flattened monolayers on attachment factor-coated tissue culture plastic. By contrast, parallel cultures grown on Matrigel, a basement membrane extract, organized into networks of tube-like structures as evidenced by both phase-contrast microscopy (Fig. 3B) and SEM (Fig. 3D). Figure 4 reveals intense localization of the endothelial cell-specific marker, Di-I-Ac-LDL in both the monolayers (Fig. 4A) and the capillary-like tubes (Fig. 4B).



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  		FIG. 3. Effects of cell culture substrate on organization of HEECs. Passage 2 HEECs were grown in parallel for 5 days on Cell Systems attachment factor-coated plastic or on Matrigel (see Materials and Methods) and examined by either phase contrast microscopy or SEM. A) Phase contrast microscopy of HEECs cultured on attachment factor-coated culture plastic. x100. B) Phase contrast microscopy of HEECs cultured on Matrigel. x400. C) SEM of HEECs cultured on attachment factor-coated culture plastic. x2000. D) SEM of HEECs cultured on Matrigel. x8000. (Published at 87%.)



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  		FIG. 4. Fluorescence microscopy of HEECs labeled with DI-I-Ac-LDL. The cultures described in Figure 3 were incubated with a 1:50 dilution of the rhodamine-labeled adduct in CS-C complete medium for 4 h, coverslipped, and visualized while the cells were viable. A) Second-passage HEECs cultured on attachment factor-coated tissue culture plastic. x200. B) Second-passage HEECs cultured on Matrigel. x800. (Published at 68%.)

Table 1 shows the results of subjecting monolayer cultures of HEECs, HUVECs, human endometrial stromal cells, and vascular smooth muscle cells to immunostaining with panels of fluorescein-labeled monoclonal antibodies. Both HEECs and HUVECs displayed similar staining patterns for an array of endothelial cell specific surface markers. These included Von Willebrand's factor (VWF), as well as adhesion molecules involved in leukocyte trafficking. Of the latter group, the expression of E-selectin, intercellular adhesion molecule 1 (ICAM-1), very late activation antigen 4 (VLA-4), lymphocyte function-associated antigen 1ß (LFA-1ß), and vascular cell adhesion molecule 1 (VCAM-1) was either enhanced or induced by a pro-inflammatory cytokine in both HEECs and HUVECs. By contrast, HUVECs strongly expressed several extracellular matrix (ECM) proteins (i.e, collagen IV, chondroitin sulfate, heparan sulfate, and fibronectin) that were absent from, or barely detected in, the cultured HEECs. Table 1 also indicates that human endometrial stromal cells stained for fibronectin, and vascular smooth muscle cells stained for vinculin and fibronectin, whereas the HEECs failed to immunostain for either marker. Moreover, neither the epithelial cell marker cytokeratin nor markers of bone marrow-derived cells were detected in the cultured HEECs.


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  		TABLE 1. Characterization of antigen expression in cultured HEECs by immunofluorescence.a

Differential Expression of TF and PAI-1 by Cultured HEECs in Response to PMA

Figure 5A indicates that after a 2-h incubation with 25 ng/ml PMA, levels of immunoreactive (ir) TF were elevated in the HEEC cultures by 15-fold. After 19 h, basal TF levels were essentially unchanged, whereas PMA-enhanced levels of ir TF had decreased to about 40% of the levels attained at 2 h. By contrast, Figure 5B shows that HEEC-secreted levels of ir PAI-1 were unaffected by PMA at both time points. However, basal levels of PAI-1 had increased by 20-fold at 19 h compared with 2 h, thereby attesting to the viability of the HEECs during incubation in the serum-free defined medium. While the effects of PMA shown in Figure 5 were obtained with fifth-passage HEECs, enhanced TF levels and unaltered PAI-1 levels were also seen in confluent third- and sixth-passage HEECs. These results suggest that the HEEC phenotype is stable in vitro. Figure 6 indicates that PMA elicited a dose-dependent increase in TF levels in the HEEC monolayers and confirms that PMA did not alter levels of immunoreactive PAI-1. Western blotting of cell extracts and conditioned medium, respectively, confirmed the ELISA results for TF and PAI-1 (data not shown).



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  		FIG. 5. Effects of PMA on TF and PAI-1 protein expression in cultured HEECs. Confluent fifth-passage HEECs from 5 preparations were starved for 3 h and then incubated in parallel (see Materials and Methods) in a defined medium containing 0.1% ethanol (vehicle control) or 25 ng/ml PMA for 2 h and 19 h. A) Immunoreactive TF analyzed by ELISA in the cell pellet. B) ir PAI-1 analyzed by ELISA in the medium. *Control versus PMA, P < 0.01, n = 5 by Mann Whitney test. **Control versus PMA, P < 0.05, n = 5 by Mann Whitney test



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  		FIG. 6. Dose-response effects of PMA on TF and PAI-1 protein expression in cultured HEECs. Confluent fifth-passage HEECs were starved for 3 h in M199 (see Materials and Methods) and then incubated in a defined medium with either 0.1% ethanol (vehicle control) or PMA at the concentrations indicated by the abscissa. Ordinate: immunoreactive levels by ELISAs of TF in the cell pellet, and PAI-1 in conditioned medium supernatant (% of control)

Consistent with the PMA enhancement of TF protein expression, the Northern blot shown in Figure 7 indicates that PMA elicited a transient time-dependent elevation in steady-state levels of TF mRNA. A several-fold increase in TF mRNA levels was evident at 2 h of incubation. As expected, PMA did not affect levels of either the 2.2- or 3.2-kilobase species of PAI-1 mRNA.



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  		FIG. 7. Effects of PMA on TF and PAI-1 mRNA expression in cultured HEECs. Confluent fifth-passage HEECs were starved for 3 h, then incubated in parallel in a defined medium containing 0.1% ethanol (vehicle control) or 25 ng/ml PMA for up to 6 h. At each time point, total cell RNA was extracted and subjected to Northern blot analysis. Steady-state levels of actin mRNA were used to normalize differences in RNA loading


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study sought to characterize and culture endothelial cells isolated by binding of UEA-1 lectin to vessels in the endometrial layer with the goal of developing an in vitro model for evaluating the regulation of human endometrial hemostasis. Several criteria validated the endothelial nature of the HEECs. Unlike the flattened monolayers that formed on attachment factor-coated tissue culture plastic, HEECs organized into networks of tube-like structures on the basement membrane component substrate Matrigel. Tube formation by endothelial cells grown in contact with ECM components is thought to reflect angiogenesis [17–19]. By contrast, our previous studies showed that when grown in contact with Matrigel and/or collagen I gels, human endometrial glandular epithelial cells form polarized monolayers on the gel surface [20], whereas human endometrial stromal cells and decidualized stromal cells penetrated and formed clusters within the gel [21, 22]. Consistent with uptake and sequestration of acetylated low-density lipoprotein (LDL) by a well-described endothelial cell/megakaryocyte-specific mechanism [23], cultured HEECs stained intensely for the presence of the fluorescent adduct Di-I-Ac-LDL. Fluorescent microscopy of cultured HEECs performed after incubation with panels of monoclonal antibodies rigorously established the homogeneity of the cultured HEECs. Thus, prominent immunostaining was seen for such endothelial cell-specific surface markers as VWF, which regulates hemostasis by binding to platelet receptors [24], and mediators of leukocyte trafficking, such as E-selectin, ICAM-1, VLA-4, LFA-1ß, and VCAM-1 [25]. In contrast, cultured HEECs did not display markers of likely epithelial, stromal, smooth muscle, and bone marrow-derived cell contaminants. Moreover, consistent with their microvascular origin, HEECs did not express several ECM proteins that were readily detected in cultures of the large vessel-derived HUVECs. These observations underscore the importance of employing HEECs for studies of HEEC biology.

Induction of TF expression transforms the endothelial cell membrane from an anticoagulant to a pro-coagulant surface. TF is a glycoprotein comprising a hydrophilic extracellular domain, a membrane-spanning hydrophobic domain, and a cytoplasmic tail [8, 26]. It serves as the transmembrane cell receptor for factor VII and its activated form (VIIa). Binding to TF markedly accelerates factor VIIa-dependent activation of factors X and IX, which promotes conversion of prothrombin to thrombin [27]. TF is localized below, between, and on the surface [28, 29] of cultured endothelial cells. Upon exposure to plasma, endothelial cell-expressed TF generates thrombin, which forms fibrin on the endothelial cell surface [30]. The in vitro expression of TF in endothelial cells such as HUVECs is enhanced by diverse stimuli. Continuous exposure to various agonists initially results in up-regulation of TF mRNA and protein levels, followed by a decline in TF expression. This characteristic biphasic response is thought to constitute a mechanism that limits fibrin formation on the endothelial cell surface [2]. Thus, the observations in the current study that TF mRNA and protein levels were up-regulated in cultured HEECs after exposure to PMA, and that this up-regulation was followed by a rapid decline in TF mRNA levels and a slower decline in TF protein levels, provide an expected and important measure of endothelial cell function.

In several endothelial cell types, the expression of PAI-1, a member of the SERPIN family of antiproteases, is enhanced by growth factors and cytokines (reviewed in [2]). Endothelial cell-derived PAI-1 is deposited in the underlying ECM, where it is bound to plasma-derived vitronectin [31], which stabilizes the biological activity of PAI-1. Active PAI-1 promotes hemostasis by serving as the "fast inhibitor" of tissue-type PA, the primary fibrinolytic agent [9]. However, over-expression of PAI-1, especially in conjunction with over-expressed TF, is associated with thrombosis [32].

The current study indicates that PMA-enhanced TF expression in cultured HEECs was not accompanied by alterations in PAI-1 mRNA or protein expression. Thus, unlike TF, PAI-1 synthesis may not be mediated by protein kinase C in HEECs. As TF and PAI-1 affect the hemostatic/thrombotic balance by different mechanisms, the differential regulation of TF and PAI-1 in cultured HEECs emphasizes the relevance of these cells as a model with which to investigate the physiological regulation of, and pathological changes in, endometrial hemostasis. The latter includes nonpregnant conditions such as abnormal uterine bleeding due to fibroids, as well as uterine bleeding secondary to contraception or hormone replacement therapy. Moreover, abnormal hemostasis in HEECs is probably involved in perinatal and maternal morbidity and mortality associated with decidual hemorrhage as occurs in abruptions, and with various uteroplacental thrombotic conditions such as intrauterine fetal growth retardation, preeclampsia, and stillbirth.

This initial study stressed the use of cultured HEECs to investigate the regulation of hemostasis/thrombosis in human endometrium. However, the microvasculature is clearly involved in virtually every aspect of endometrial biology, with corresponding reproductive implications. Thus, by regulating uptake of oxygen, nutrients, and ovarian steroids, these vessels perforce control growth and differentiation of the functional endometrial layer. Moreover, elaboration of adhesion molecules on the endothelial cell surface is expected to control leukocyte trafficking into the endometrium [33]. Predictably, the resident endometrial leukocyte population will determine the composition of the cytokine milieu and thus affect such widespread processes as endovascular trophoblast invasion as well as the expression of proteases involved in degradation of the endometrial ECM leading to menstruation. The novel in vitro model described in the current study should permit direct investigations of these phenomena.


   FOOTNOTES
 
First decision: 27 July 1999.

1 This work was supported in part by a grant from the National Institutes of Health RO1 HD33937-05 (C.J.L.) and M01 RR 00096 (GCRC). Back

2 Correspondence: Charles J. Lockwood, Department of Obstetrics and Gynecology, 550 First Avenue, New York University School of Medicine, New York, NY 10016. FAX: 212 263 8251; lockwc01{at}mcrcr.med.nyu.edu Back

Accepted: October 18, 1999.

Received: June 16, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pijnenborg R, Bland JM, Robertson WB, Brosens I. Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy. Placenta 1983; 4:397–414.[Medline]
  2. Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, Pober JS, Wick TM, Konkle BA, Schwartz BS, Barnathan ES, McCrae KR, Hug BA, Schmidt AM, Stern DM. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 1998; 91:3527–3561.[Free Full Text]
  3. Parry GC, Mackman N. Transcriptional regulation of tissue factor expression in human endothelial cells. Arterioscler Thromb Vasc Biol 1995; 15:612–621.[Abstract/Free Full Text]
  4. Grulich-Henn J, Muller-Berghaus G. Regulation of endothelial tissue plasminogen activator and plasminogen activator inhibitor type I synthesis by diacylglycerol, phorbol ester, and thrombin. Blut 1990; 61:38–44.[CrossRef][Medline]
  5. Santell L, Levin EG. Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits secretion of plasminogen activator inhibitor-1 from human endothelial cells. J Biol Chem 1988; 263:16802–16808.[Abstract/Free Full Text]
  6. Archipoff G, Beretz A, Freyssinet J-M, Klein-Soyer C, Brisson C, Cazenave J-P. Heterogenous regulation of constitutive thrombomodulin or inducible tissue-factor activities on the surface of human saphenous-vein endothelial cells in culture following stimulation by interleukin-1, tumour necrosis factor, thrombin or phorbol ester. Biochem J 1991; 273:679–684.
  7. Slivka SR, Loskutoff DJ. Regulation of PAI-1 synthesis by protein kinase c and cAMP on bovine aortic endothelial cells. Biochim Biophys Acta 1991; 1094:317–322.[Medline]
  8. Nemerson Y. Tissue factor and hemostasis. Blood 1988; 71:1–8.[Free Full Text]
  9. Mignatti P, Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 1993; 73:161–195.[Free Full Text]
  10. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1:3–25.
  11. Lockwood CJ, Nemerson Y, Guller S, Krikun G, Alvarez M, Hausknecht V, Gurpide E, Schatz F. Progestational regulation of human endometrial stromal cell tissue factor expression during decidualization. J Clin Endocrinol Metab 1993; 76:231–236.[Abstract]
  12. Arcuri F, Monder C, Lockwood CJ, Schatz F. Expression of 11ß-hydroxysteroid dehydrogenase during decidualization of human endometrial stromal cells. Endocrinology 1996; 137:595–600.[Abstract]
  13. Schatz F, Lockwood CJ. Progestin regulation of plasminogen activator inhibitor type 1 in primary cultures of endometrial stromal and decidual cells. J Clin Endocrinol Metab 1993; 77:621–625.[Abstract]
  14. Hinegardner RT. An improved fluorometric assay for DNA. Anal Biochem 1971; 39:197–201.[CrossRef][Medline]
  15. Auerbach R. Endothelial cell heterogeneity: its possible role in endometrial pathology. In: Alexander NJ, d'Arcangues C (eds.), Steroid Hormones and Uterine Bleeding. Washington, DC: AAAS Press; 1992; 171–177.
  16. Raffi S, Shapiro F, Rimarchin J, Nachman RL, Ferris B, Weksler B, Moore MAS, Asch AS. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 1994; 84:10–19.[Abstract/Free Full Text]
  17. Sankar S, Mahooti-Brooks N, Bensen L, McCarthy TL, Centrella M, Madri JA. Modulation of transforming growth factor ß receptor levels on microvascular endothelial cells during in vitro angiogenesis. J Clin Invest 1996; 97:1436–1446.[Medline]
  18. Schwartz MA, Schaller MD, Ginsburg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol 1995; 11:549–599.[CrossRef][Medline]
  19. Montesano R, Orci L, Vassalli P. In vitro organization of endothelial cells into capillary-like networks is promoted by collagen matrices. J Cell Biol 1983; 97:1648–1652.[Abstract/Free Full Text]
  20. Schatz F, Gordon RE, Laufer N, Gurpide E. Culture of human endometrial cells under polarizing conditions. Differentiation 1990; 42:184–190.[CrossRef][Medline]
  21. Schatz F, Gurpide E, Hausknecht V, Markiewicz L, Toth-Pal E, Cudemo V, Lockwood CJ. Ovarian steroid regulated expression of tissue factor and prostaglandin PGF2{alpha} in cultured human endometrial cells. In: Alexander NJ, d'Arqangues CD (eds.), Steroid Hormones and Uterine Bleeding. Washington, DC: AAAS Publications; 1992: 307–315.
  22. Schatz F, Papp C, Toth-Pal E, Lockwood CJ. Ovarian steroid modulated stromelysin-1 expression in human endometrial stromal and decidual cells. J Clin Endocrinol Metab 1994; 78:1467–1472.[Abstract]
  23. Gumkowski F, Kaminska G, Kaminski M, Morrissey LW, Auerbach R. Heterogeneity of mouse vascular endothelium. In vitro studies of lymphatic, large blood vessel and microvascular endothelial cells. Blood Vessels 1987; 24:11–23.[Medline]
  24. Dong JF, Hyun W, Lopez JA. Aggregation of mammalian cells expressing the platelet glycoprotein (GP) Ib-IX complex and the requirement for tyrosine sulfation of GPIba. Blood 1995; 86:4175–4183.[Abstract/Free Full Text]
  25. Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Am Rev Physiol 1995; 57:827–872.[CrossRef][Medline]
  26. Bach RR. Initiation of coagulation by tissue factor. CRC Crit Rev Biochem 1988; 23:339–368.[Medline]
  27. Carmeliet P, Collen D. Tissue factor. Int J Biochem Cell Biol 1998; 30:661–667.[CrossRef][Medline]
  28. Camera M, Giesen PLA, Fallon BM, Aufiero BM, Taubman M, Tremoli E, Nemerson Y. Cooperation between VEGF and TNF-{alpha} is necessary for exposure of active tissue factor on the surface of human endothelial cells. Arterioscler Thromb Vasc Biol 1999; 531–537.
  29. Narahara N, Enden T, Wiiger M, Prydz H. Polar expression of tissue factor in human umbilical vein endothelial cells. Arterioscler Thromb 1994; 14:1815–1820.[Abstract/Free Full Text]
  30. Stern D, Nawroth P, Handley D, Kisiel W. An endothelial cell-dependent pathway of coagulation. Proc Natl Acad Sci USA 1995; 82:2523–2527.
  31. Seiffert D, Wagner NN, Loskutoff DJ. Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor. J Cell Biol 1990; 111:1283–1291.[Abstract/Free Full Text]
  32. Estelles A, Gilabert J, Grancha S, Yammamoto K, Thinnes T, Espana F, Azner J, Loskutoff DJ. Abnormal expression of type I plasminogen activator inhibitor and tissue factor in severe preeclampsia. Thromb Haemostasis 1998; 79:500–508.[Medline]
  33. Tabibzadeh S, Kong QF, Babaknia A. Expression of adhesion molecules in human endometrial vasculature through out the menstrual cycle. J Clin Endocrinol Metab 1994; 79:1024–1032.[Abstract]



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