Endometrial Endothelial Cells ExpressEstrogen and Progesterone Receptors and

Exhibit a Tissue Specific Response toAngiogenic Growth Factors

M. LUISA IRUELA-ARISPE,JUAN CARLOS RODRIGUEZ-MANZANEQUE, AND

GRAZIELLA ABU-JAWDEHDepartment of Pathology, Harvard Medical School, and Beth Israel Deaconess

Medical Center, Boston, MA, USA

ABSTRACT

Objective: To develop a reliable method for the isolation and longterm cultureof microvessel endothelial cells from human endometrium and to evaluate theirresponse to angiogenic growth factors and steroid hormones in comparison toendothelial cells derived from other organs.Methods: Endometrial tissue from hysterectomy specimens were digested se-quentially with collagenase and trypsin, cultured for 24 h, then selected byadhesion to anti-CD-34 coated magnetic beads. Alternatively, anti-CD-34-coated beads could also be substituted by Ulex europaeus agglutinin-1, anti-PECAM, or anti-E-selectin-coated beads. Characterization of the isolated cul-tures included expression of endothelial cell markers, regulation of E-selectin inresponse to TNF-a, proliferative response to angiogenic growth factors, andexpression of progesterone and estrogen receptors. We also analyzed the relativebinding affinity of VEGF on endometrial endothelial cells in comparison to otherendothelial cell types.Results: Selection on anti-CD-34-coated beads eliminated contaminating cellsand resulted in a homogeneous population of human endometrial endothelialcells (HEEC), as assessed by expression of PECAM, von Willebrand’s factor, anduptake of acetylated-LDL. HEEC also upregulated E-selectin in response toTNF-a in a manner similar to that seen for other endothelial cell types. Expres-sion of progesterone and estrogen receptor was revealed by immunocytochem-istry and RT-PCR consistently until passage 5. Endometrial endothelial cellswere more responsive to growth stimulation by VEGF than were dermal endo-thelial cells isolated under similar conditions. Further characterization indicatedthat VEGF bound more avidly to HEEC than to other endothelial cell types.Conclusions: Human endometrial endothelial cells were isolated to homogene-ity by a two-part protocol and successfully passaged under culture conditionssimilar to those used for other endothelial cell types. The HEEC were veryresponsive to VEGF growth-stimulation likely due to elevated affinity, or in-creased levels of, KDR and FLT-1 on the cell surface. These results indicate thatHEEC are capable of maintaining a mature phenotype in culture and mightprovide a model for understanding the response of these cells to the recurrentcycles of proliferation imposed on the endometrium during menstruation.

KEY WORDS: angiogenesis, capillaries, microvessels, immunopurification, steroidhormones, VPF/VEGF

INTRODUCTION

Our understanding of endothelial cell physiologyand pathology depends, in large part, on studies per-formed in vitro with primary cultures of endothelialcells. Through studies of endothelial cells isolatedfrom various sources, it has become increasingly ap-parent that considerable differences exist between

Supported by the Pathology Foundation, Department of Pathol-ogy, Beth Israel Deaconess Medical Center, and from the NCIRO3#CA70559-01.For reprints of this article, contact Dr. Luisa Iruela-Arispe, De-partment of Molecular Cell and Developmental Biology, Molecu-lar Biology Institute, 611 Circle Drive East, Los Angeles, CA90095 USA.Received 19 June 1998; accepted 8 December 1998

Microcirculation (1999) 6, 127–140© 1999 Stockton Press All rights reserved 1073-9688/99 $12.00http://www.stockton-press.co.uk

macro- and microvascular endothelial cells. For in-stance, mediators that regulate a particular functionin umbilical-vein-derived endothelial cells are notfound to be equally effective on capillary endothe-lium (8,41). Others as well as we have shown thatcapillary endothelium express platelet-derivedgrowth factor BB (PDGF BB) receptors and respondto this growth factor, whereas large-vessel endothe-lium do not (4,5,6,58). Heterogeneity can also bemanifested by microenvironmental factors, becausethe response of endothelial cells is dependent on theextracellular context or stage of activation duringangiogenesis (23,30). For example, transforminggrowth factor-b1 inhibits proliferation of subconflu-ent endothelial cells, but stimulates mitosis when thesame cells have organized capillary-like structures invitro (36). In addition, a large number of studies hasrevealed organ-specific characteristics among micro-vascular endothelial cells. Clear differences existwith respect to cell-surface molecules (7,44), anti-genic determinants (3,42), permeability, and meta-bolic properties (15,19,60). These findings indicatesignificant heterogeneity among vascular beds, andsuggest that questions related to organ-specificphysiology might be better addressed by studies oforgan-specific endothelial cells.

In this article, we describe a procedure for the iso-lation of human endometrial endothelial cells. Thehuman endometrium is one of a limited number oforgans that undergo recurrent cycles of physiologicalvascular regeneration. Our purpose is to utilize en-dometrial endothelial cells in studies of angiogenicprogression and inhibition, as well as in the elucida-tion of other responses that are unique to endothelialcells from this tissue.

Endothelial microvascular cells have been isolatedfrom a variety of organs including skin (16,34,46),retina (10), synovium (37), brain (19), placenta(20,25), nasal mucosa (24), adrenal (22), mammarygland (32), heart (52), and lung (12,28,45). Theendothelium of human endometrium, however hasnot been studied in vitro. This tissue presents dy-namic physiological states on the vasculature, withrecurrent cycles of growth and growth-inhibitionthat provide an interesting model in which to askquestions related to the molecular regulation of an-giogenesis. We hypothesized that endometrial-derived endothelial cells are more responsive toregulation by angiogenic factors and inhibitors thanother endothelial cells. We also predict that thesecells express estrogen and progesterone receptorsand that its expression might provide modulation togrowth factor response. Here, we test this hypothesis

and evaluate the effect of several growth factors thatmight be active in regulating the growth of thesecells in vivo.

The isolation of capillary-derived endothelial cells isa technically challenging proposition. This is due tothe frequent contamination with neighboring celltypes in the tissue, primarily pericytes, fibroblasts,and smooth muscle cells. A number of techniqueshave been used to increase the ratio of endothelialcells to other contaminating cell types in primarycultures. These include differential plating (10,13),mechanical weeding (46), media rich in D-valine(16), and filtration of microvessels through nylonmembranes (10). More recently, flow cytometry (28)and binding to paramagnetic spheres (Dynabeads)coated with endothelial cell surface markers(31,32,37,55) have been used with great success.

We have studied the feasibility of using paramag-netic spheres coated with anti-CD-34, and other en-dothelial-specific cell surface markers, to isolate ho-mogeneous endothelial cultures from the human en-dometrium. Here, we describe this method andpresent data regarding the expression of endothelialcell markers in the purified cell populations. In ad-dition, we demonstrate that human endometrial en-dothelial cells (HEEC) are more responsive to VEGFthan are other endothelial cells from various tissues,and further demonstrate that the effect is likely dueto an increased affinity, or elevated number ofVEGF receptors, on the cell surface. These data pro-vide evidence that cultured endometrial endothelialcells constitute a practical model system in which toexplore questions related to the recurrent nature ofthe angiogenic response typical of the human endo-metrium and establish clear functional differencesbetween HEEC and endothelial cells from other or-gans.

MATERIALS AND METHODS

Isolation of Endothelial Cells

Endometria from pre-menopausal hysterectomyspecimens, removed for reasons other than endome-trial abnormalities, were used for the isolation ofendothelial cells. The uteri were from patients 30 to50 y old and were processed under sterile conditions30 min to 2 h after surgery. The use of human speci-mens for these experiments was reviewed and ap-proved by the Committee on Clinical Investigations,Beth Israel Deaconess Medical Center, Boston, Mas-sachusetts.

Briefly, incisions were made along the lateral aspects

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of the uterus and strips of endometrium were re-moved from the upper portion of the posterior wall.The strips of endometrium were divided into cubesof 0.5–0.7 cm in diameter. Tissue cubes were theneither processed for RNA extraction, fixed for histol-ogy/immunocytochemistry, or immersed in Hankssolution for isolation of cells.

For cell isolation, tissue cubes were rinsed in phenolred-free DMEM containing antibiotics (penicillinand streptomycin at 100 mg/mL) and amphoteri-cin-B (Gibco/BRL, Grand Island, NY). Collagenase(Sigma, St. Louis, MO) at a final concentration of0.1 mg/mL was added to the suspension and keptunder constant agitation at room temperature for1–2 h. Released cells were spun and pellets wererinsed 3 times by resuspension and centrifugation infresh DMEM. A second mild digestion with trypsin-EDTA was frequently necessary to further break upthe microvessels. Filtration through nylon mesh (70mM) (Fisher, Santa Clara, CA) increased the efficacyof attachment of endothelial cells to the coated fi-bronectin dishes, by removal of partially digestedmatrix fragments and epithelial components. Afterdigestion, cells were washed in EBM-2 (Clonetics,Walkersville, MI) containing 1% FCS, by three se-quential resuspension and centrifugation cycles. Thefinal pellet was reconstituted in 3 mL of EBM-2 andplated on petri dishes previously coated with fibro-nectin (Sigma) (50 mg/mL) on the same mediasupplemented with 10% FCS, 100 U/mL penicillin,and 100 mg/mL streptomycin. After 2 h, nonadher-ent cells were removed. Adherent cells were washedand grown until confluency. These cultures were se-rially subcultured, and the purity of the culturedcells was examined by acetylated-LDL endocytosis(BTI, Stoughton, MA).

Further purification was accomplished by an affinitymethod using antibodies directed against the cell-surface protein CD-34 (Affinity Bioreagents, Nes-hanic Station, NJ). Cultured cells were incubatedwith anti-CD-34-coated magnetic beads for 10–15min (for preparation of the beads, see below). Afterseveral thorough washes with serum-free EBM, cul-tures were released from the substrate by brief treat-ment with trypsin, and cells with attached beadswere isolated using a magnetic particle concentrator.

Isolated cells were then plated and cultured inEBM-2 supplemented with 15% FCS, 1 mg/mL hy-drocortisone acetate, 5 × 10−5 M N6, 28-0-dibu-tyrul-adenosine 38:58-cyclic monophosphate(Sigma), 1000 U/mL penicillin, and 100 mg/mLstreptomycin (Gibco/BRL). After 1–2 days or when

cultures reached 80% confluency, a second magneticpurification was performed. After two immune-magnetic purifications, we consistently found that100% of the cells were able to endocytose Ac-LDL.For subcultures, confluent HEEC were split at a 1:4ratio and maintained for four additional passages(5–6 total).

Human dermal endothelial cells (HDEC) were a giftfrom Michael Detmar (Department of Pathology,Beth Israel Deaconess Medical Center, Boston,MA) (55). Human umbilical vein endothelial cells(HUVEC) were a gift from Don Senger (Departmentof Pathology, Beth Israel Deaconess Medical Center,Boston, MA). Human coronary endothelial cells(HCEC) were obtained from Clonetics. Experimentswere performed with cultures between passages 3and 5. Subculturing ratio was always 1:4. Severalindependent isolates were used for each experimentto ensure consistency in the results.

Immunomagnetic isolation of HEEC was also per-formed, with equal success, using anti-CD-31 orPECAM antibodies, anti-E-selectin-coated beads, orUlex europaeus agglutinin-1, which binds specifi-cally to terminal L-fucosyl residues on the surface ofhuman endothelial cells (37). For E-selectin, pre-treatment of cultures with TNF-a (50 ng/ml)(TNF-a was a kind gift from Dr. Michael Detmar,Beth Israel Deaconess Medical Center, Boston, MA)for 6 h was required for upregulation of this mol-ecule at the cell surface.

Preparation of Coated Magnetic Beads

Magnetic beads (15 mg), coated with sheep anti-mouse IgG or tosylactivated (for conjugation withlectin) (Dynal, Lake Success, NY), were incubatedend-over-end with one of the following antibodies toendothelial cell surface markers: (a) mouse anti-human E-selectin monoclonal antibody (Genzyme,Cambridge, MA); (b) mouse anti-human CD34monoclonal antibody (Affinity Bioreagents); (c)mouse anti-human PECAM monoclonal antibody; or(d) Ulex europaeus lectin (Vector Labs, Burlingame,CA), and were stored at 4 °C for up to 1 mo. Incu-bation took place overnight at 4 °C, after whichmagnetic beads were washed 4 times with serum-free media containing 0.2 mg/mL BSA under sterileconditions using a magnetic particle concentrator.

Characterization of Endothelial Cells

Cells were plated on fibronectin-coated coverslips,washed with Hanks solution, and fixed in cold 4%paraformaldehyde for 30 min at 4 °C. Fixed cells

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were rinsed with PBS, blocked with 10% normalgoat serum (Sigma) for 2 h, and incubated for 1 hwith primary antibodies. Control coverslips were in-cubated with buffer alone.

The preparations were then washed 5 times withPBS and incubated for 1 h with the appropriate bio-tinylated secondary antibody (Vector) at 1:200 di-lution. Cells were again washed 5 times with PBSand incubated with avidin-fluorescein isothiocya-nate (Vector) at 1:200 dilution. Cells were washed asabove, mounted in 50% glycerol in PBS, and pho-tographed with a Zeiss photomicroscope with anEcktakrome film 1600 ASA.

Purified cultures were also examined for the pres-ence of scavenger receptors for acetylated low den-sity lipoprotein using 1,18dioctadecyl-3,3,38,38-tetramethyl-indocarbocyanine perchlorate acety-lated low density lipoprotein (Dil-Ac-LDL)(Biochemical Technologies, Inc., Stoughton, MA).Cells were incubated in DMEM containing Dil-Ac-LDL (10 mg/mL) at 37 °C for 4 h. Thereafter, cellswere fixed for 30 min in 3% paraformaldehyde so-lution, mounted, and observed by fluorescent mi-croscopy.

For immunocytochemical studies, human dermalendothelial cells and stromal endometrial cells wereused as positive and negative controls, respectively.

Isolation of Total RNA and Northern Blot Analysis

Total RNA was isolated by the single-step procedureof Chomcznski and Sacchi (14) and the integrity ofthe samples was assessed on agarose minigels. TotalRNA was transferred to nylon membranes (Nytran)(Amersham, Arlington Heights, IL), linked to themembranes by ultraviolet light (Stratalinker), andprehybridized at 42 °C for 2–5 h in a solution con-taining 50% formamide, 6× SSPE, 1× Denhart’s so-lution, 0.1% SDS, and 100 mg/mL of heat-denatured salmon sperm DNA. Hybridization with[32P]-labeled cDNA proceeded in the same solutionat 42 °C for 12–18 h (32). Stringency of washesvaried according to the probe. Membranes were thenexposed to Kodak X-Omat AR film for 1–3 days.Probes were prepared from cDNA restriction frag-ments labeled with [32P]-dCTP by random primingusing a Multiprime kit (Amersham) and purified onSephadex G-50 (Promega, Madison, WI). Loadingand transfer efficiency were evaluated with a probeto 28S rRNA. Densitometry or Phosphor imagerscans of these signals were used to normalize thevalues obtained from other probes.

The expression of E-selectin mRNA was investigated

on cultures treated with TNF-a (50 ng/mL) for 30min, 2 h, 4 h, and 6 h.

Cell Proliferation Assays

Confluent cultures were incubated for 48 h in theabsence of serum or growth factors. Under theseconditions, we have found that endothelial cells en-ter a state of mitotic quiescence. Quiescent cells werethen reseeded on plates previously coated with Vit-rogen (50 mg/mL) and treated with 1% FCS alone orin the presence of VEGF (Peprotech, Rocky Hill,NJ), FGF-2 (a kind gift from Dr. Gera Neufelt, Is-rael), or PDGF-BB (Peprotech). Incubation timesvaried as indicated in the figure legends. For assayslonger than 48 h, media was replaced every 2 days.At the end of treatment, cells were either countedwith hemocytometer or coulter counter, or pulsedwith 1 mCi/mL of [3H]-thymidine (NEN, Boston,MA). Thereafter, cultures were washed twice withserum-free DMEM, fixed with cold 10% trichloro-acetic acid for 10 min, washed with cold ethanol,and air dried. Incorporation of [3H]-thymidine intoacid-insoluble material was determined by scintilla-tion counting.

Binding Assays

VEGF (Peprotech) was iodinated according to theprocedure of Hunter and Greenwood (33). Briefly,1mg of VEGF was mixed with 1mCi of Na[125I] and10mL of chloramine T (Sigma) (0.2 mg/mL) up to afinal volume of 150mL in 0.5M phosphate buffer pH7.6. Approximately 1 min later, the reaction wasstopped by the addition of 10mL of saturated acetyl-tyrosine and 10 mL of 10 mMKI. Free 125I was re-moved on a desalting PD10 column (Biorad) pre-equilibrated with PBS containing 1 mg/mL of gela-tin. Fractions were counted before and afterprecipitation with 10% trichloroacetic acid.

Endothelial cells (HEEC and HDEC) were seeded ondishes and allowed to grow to confluency (7 × 104

cells). Cells were then transferred to 4 °C andwashed with cold binding buffer (EBM-2 containing1 mg/mL gelatin). Binding was performed at 4 °Cfor 1 h. Experiments were done in triplicate. Com-petition was performed with increased levels ofunlabeled VEGF. Non-specific binding was deter-mined in the presence of an excess of unlabeledVEGF (500 ng/mL). At the end of the incubationperiod, cultures were washed 4 times with cold PBSand one last time with cold PBS containing 2M NaCl.At this point, cells were lysed with 500mL of 1%sodium dodecyl sulfate and 1mM EDTA. At saturat-ing concentrations, the nonspecific binding was less

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than 20%. Binding was analyzed according toScatchard’s procedure with the use of the ligand-fitting program.

RESULTS

We have developed a two-step procedure for the iso-lation of HEEC, which consists of differential platingand magnetic isolation with endothelial-specific cell-surface markers. The use of coated Dynabeads toenrich endothelial cell preparations dramatically im-proved the purity of the cultures. Rarely, a secondmagnetic isolation was necessary. After selection

with Dynabeads, cells coated with numerous mag-netic beads could be seen under contrast microscopy.Cells spread to the fibronectin-coated dishes within2 h. Presence of the beads did not inhibit adhesion orspreading, and the beads were rapidly diluted outand lost during the first two passages.

Using this technique, HEEC grew to confluency,showed contact inhibition, and presented a typicalcobblestone morphology. The use of EBM-2 mediasupplemented with 15% fetal calf serum, hydrocor-tisone, and cAMP was essential to prevent senes-cence and keep a doubling time of 30 h to 42 h forfour-to-five passages.

Figure 1A shows a phase-contrast micrograph of the

Figure 1. Isolation of endothelial cells from human en-dometrial specimens. (A) Phase- contrast micrograph ofcells isolated from the endometrium 24 h after initial plat-ing. Two morphologies are apparent: cobblestone (closedarrows), likely to represent endothelial cells and elongated(open arrows), likely from stromal cells. (B) Expression ofCD34 in human endometrium. Endothelial cells from cap-illaries are positive as indicated by the brown peroxidasereaction (arrows). (C) Confluent culture of HEEC afterselection with anti-CD-34-coupled magnetic beads. Notethe cobblestone appearance of the monolayer that is freeof beads after two passages. (D) Acetylated-LDL bindingof endothelial cells after immune-selection with CD34. Allcells in the culture are positive.

Figure 2. Expression and distribution of endothelial cellmarkers on endometrial tissue. Endometrial tissue wasfixed in paraformaldehyde and embedded in paraffin.Sections (5mm) were subjected to immunocytochemicalanalysis using the following primary antibodies: (A) anti-von Willebrand factor (vWF); (B) Biotinylated Ulex eu-ropeus lectin; (C) anti-CD-31; and (D) anti-CD-34. De-tection was accomplished by use of a biotinylated second-ary antibody followed by avidin-FITC. Staining withHoechst revealed nuclei in blue. Arrows indicate positivevessels.

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cultured cells after differential plating, but prior tomagnetic bead selection. Note two clearly distinctmorphologies: (a) cobblestone (closed arrow), pre-sumably endothelial cells; and (b) elongated (openarrow), presumably fibroblasts. By endocytosis ofAc-LDL, we verified that approximately 70% of thecells in most preparations were of endothelial origin.Further purification was accomplished by an affinitymethod using antibodies directed against the cell-surface protein CD-34, CD-31, or Ulex lectin. Asdepicted by Fig. 1B, CD-34 is specific to the endo-thelium. CD-34 is a cell-surface protein present inboth endothelial and hematopoietic cells, the latter-cell type was not present in our original cultures.Cultured cells were incubated with anti-CD-34 mag-netic beads and released from the substrate by brieftreatment with trypsin. Cells bound to beads weresubsequently plated and subcultured. Microscopicexamination post-confluency showed a clearly dis-tinct cobblestone monolayer (Fig. 1C). Cultureswere also incubated with acetylated-LDL. After im-mune-magnetic purification, we consistently foundthat 100% of the cells were able to uptake Ac-LDL,as shown by the punctuate fluorescence indicatingthe endocytosis of Ac-LDL into secondary lysosomes(Fig. 1D).

To confirm the purity of the endothelial isolationsand to characterize the cultures further, we per-formed a series of immunocytochemical analysis andfunctional assays. For the immunocytochemistry, weinitially tested the expression of the endothelialmarkers in the endometrial tissue. Figure 2 showsimmunolocalization of von Willebrand factor(vWF) (Fig. 2A), Ulex europeus lectin (Fig. 2B),CD-31 (Fig. 2C), and CD-34 (Fig. 2D). All antibod-ies provided vascular patterns although interestingdifferences were observed.

Traditionally, endothelial cells have been character-ized immunocytochemically with antibodies againstvWF. Although antibodies against this protein gen-erally provide an excellent label of endothelial cellsin large blood vessels, binding to capillary endothe-lium has been inconsistent. This phenomenon wasclearly evident in the endometrium. Larger and me-dium-size vessels were well-stained, however thenumber of positive vessels was clearly lower than inadjacent sections stained with Ulex, CD-34, or CD-31. One can associate the inconsistency in staining topossible regulation of vWF during the endometrialcycle, as has been previously suggested (2,38), or tothe fact that vWF is differentially regulated in vas-cular beds of different size and organs, as indicatedby recent data on promoter expression (1). We tendto favor the second hypothesis, because analysis of

12 independent endometrial specimens from differ-ent stages of the endometrial cycle showed the samepattern (data not shown). In addition, we found thatanti-CD-31 did not stain subepithelial capillaries,while both Ulex and anti-CD-34 stained these ves-sels. PECAM-1/CD-31 is a member of the cell-adhesion molecule subfamily of Ig-like proteins andis constitutively expressed at the cell surface of en-dothelial cells and platelets (51). Nevertheless, thesubendothelial capillaries are considered to be sinu-soidal in nature, therefore, it is conceivable that thenature of their cell-juctional interactions is differentfrom that of continuous endometrial capillaries,which showed expression of PECAM-1. Both Ulexand CD-34 showed ubiquitous expression amongdifferent capillaries in the endometrium. In addition,dermal endothelial cells and endometrial stromalcells were used as positive and negative controls, re-spectively. Immunolocalization of vWF showed apunctuate intracellular pattern in most, but not all,endometrial and dermal-derived endothelial cells(Figs. 3A and B). This pattern was consistent withthe spotty distribution observed in the endometrialtissue sections (Fig. 2). PECAM staining was de-tected at the cell membrane and more intensely atthe cell–cell junctions (Figs. 3C and D). Expressionof CD-34 was consistent in all the cultured cells (Fig.3E). We also stained stromal fibroblasts, as controls;no signal was detected with antibodies toward CD-34, PECAM, or Ulex in these cells (Fig. 3F). Humanendometrial endothelial cells were also stained withdesmin and smooth muscle a-actin to preclude con-tamination by pericytes or smooth muscle cells (datanot shown).

Expression of E-selectin can provide further supportto the endothelial nature of our isolates. E-selectin isexpressed on endothelial cells and in platelets. Inmost endothelial cultures, E-selectin levels are low orundetectable (9), nevertheless, transcripts are sig-nificantly increased upon treatment with TNF-a(55). Human endometrial endothelial cell culturesshowed low-basal level of E-selectin mRNA, whichshowed a significant increase as early as 30 min aftertreatment with TNF-a (Fig. 4). Upregulationpeaked at 2 h and was still detected by 6 h. Cultureswere refractive to a second TNF-a treatment admin-istered within 24 h (data not shown). E-selectin pro-tein was also increased by TNF-a, but with slightlydelayed kinetics. Upregulation of E-selectin byTNF-a was also used successfully to purify HEECusing magnetic beads coated with E-selectin anti-bodies. The advantage of this procedure is that thebeads are quickly lost due to shedding of E-selectinfrom the endothelial surface. The potential criticism

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of this technique is that it would preferentially selectonly responsive endothelial populations.

Endometrial Endothelial Cells Express BothProgesterone and Estrogen Receptors

Investigation of steroid receptors on endometrial-derived endothelial cells is of particular interest inthis tissue. The cyclic nature of endometrial repairand descamation is temporarily related to the serumlevels of 17-b-estradiol and progesterone. It hasbeen repeatedly postulated that the angiogenic re-sponse of the endometrium is likely under hormonalcontrol, nevertheless, there are no studies to supportthis claim directly. The presence of estrogen andprogesterone receptors has been clearly and repeat-edly demonstrated on smooth muscle cells (43), infact, menstruation is believed to result from the con-striction of the smooth muscle cells from the spiralarteries in response to low levels of progesterone andestradiol. Identification of steroid receptors on endo-thelial cells, however, has followed a more contro-versial path, with studies supporting and contradict-ing their expression on the endothelium. To someextent, these controversies could be founded on in-trinsic organ-specific differences among vascular beds.

We performed immunohistochemical analysis ofprogesterone and estrogen receptor expression insections of endometrium and in isolated endothelialcells. Figure 5 shows immunolocalization of bothprogesterone (Fig. 5A) and estrogen (Fig. 5B) recep-tor on endothelial endometrial tissue sections. Theexpression is retained on the purified isolated cul-tures (Figs. 5C and D). Presence of these receptorswas also confirmed at the transcriptional level. Weconducted reverse transcriptase followed by PCRanalysis on a series of total RNA samples fromHEEC at different passage numbers. We were ableto detect a positive band for both estrogen and pro-gesterone receptors on HEEC cells (Fig. 5) consis-tently up to passage 5. The data confirms findingsfrom the immunocytochemical analysis, and alsodemonstrates that expression of these receptors islost upon serial passage in culture.

Human Endometrial Endothelial Cells ExpressReceptors for VEGF and Respond Rapidly toVEGF Stimulation

Expression of VEGF receptors KDR and FLT-1 islargely confined to the endothelium, although a fewexceptions to this rule have been noted in the litera-ture (11). To further support the endothelial natureof purified HEEC cultures, we tested for VEGF re-ceptor on endothelial and stromal cells isolated fromendometrium. Figure 6 shows Northern blot analysis

for VEGF and for its receptors, KDR and FLT-1.Surprisingly, levels of both KDR and FLT-1 mRNAwere elevated on HEEC when compared to dermalendothelial cells. In contrast, VEGF mRNA was de-tected at equivalent levels in both endometrial anddermal-derived fibroblasts.

We next evaluated the proliferative response ofHEEC to several angiogenic growth factors, VEGF,FGF-2, and PDGF-BB, and compared this responseto that of endothelial cells derived from other organs(HDEC, HCEC, and HUVEC). All cells were used atthe same passage number. Interesting differenceswere noted among the cell types (Fig. 7A and Table1). Clearly, HEEC were the most responsive toVEGF, followed by HDEC. The response of endo-metrial-derived cells to FGF-2 was not as impressiveas that of HDEC. The data suggest that clear differ-ences exist in the response of organ-specific endo-thelial cells to particular growth factors. Interest-ingly, HEEC did not respond to PDGF-BB. PDGF-breceptors have been identified in endothelial cellsfrom microvessels, being excluded from large vessels(4,5,6,58); therefore, it is not surprising that bothHUVEC and HCEC respond poorly to this growthfactor. Nevertheless, cells isolated from dermis andendometrium both contain a large component of mi-crovascular-derived endothelium. We examined theexpression of PDGF-b receptors in endometrial sec-tions by immunocytochemistry and found that themicrovasculature was devoid of signal (data notshown) providing an explanation to our findings.Therefore, the response of microvessel endothelialcells is potentially heterogeneous to this ligand, or atleast excluded from endometrial tissue.

Response of HEEC to VEGF was further evaluatedby cell-cycle analysis and binding assays. Humanendometrial endothelial cells and HDEC were madequiescent, and response to VEGF was evaluated bythymidine incorporation for 80 h (Fig. 7B). Humanendometrial endothelial cells entered S phase by 20h after treatment, in contrast to 30–35 h for HDEC.By 80 h, HEEC had completed two cell cycles, whileHDEC had completed only one.

Binding assays to VEGF were also performed onHEEC, HCEC, HDEC, and HUVEC (Fig. 8). Animpressive difference was detected between HEECand the other cell types. While complete competitionwas seen at 100 ng/mL of cold VEGF for HCEC,HUVEC, and HDEC; twice the amount of unlabeledcompetitor was required to eliminate binding of la-beled VEGF to HEEC. The data suggest an in-creased number of receptors for VEGF on HEEC (asalso indicated by Fig. 6) or a significantly higher

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affinity for this growth factor by a yet unknownmechanism.

Estrogen and Progesterone Modulate the Responseof HEEC to Growth Factors

To evaluate the functional significance of estrogenand progesterone receptors on HEEC, we treatedcultures with increasing concentrations of 17-b-estradiol and progesterone in the presence and ab-sence of VEGF and FGF-2. Figure 9 indicates theresults of six independent proliferation experimentsperformed on HEEC cultures from passages 3 to 5.Also, 17-b-estradiol potentiated, while progesteroneinhibited the proliferative response mediated byboth angiogenic growth factors. The effect mediatedby estradiol was significant at physiological levels(0.1 to 1 nM) and was as high as 75% over baseline.These results provide functional relevance to estra-diol on the vascular repair of the endometrium dur-ing the proliferative phase of the menstrual cycle. In

contrast, progesterone was inhibitory on endothelialcell proliferation. This finding is consistent with thesuppressive signals predominant during the secre-tory phase, when circulating levels of this hormoneare highest and when the vascular repair has ceased.

The positive effect of estradiol on endothelial cellproliferation has been reported for HUVEC andcoronary endothelial cells (40,50), however, the ef-fects were more modest than those observed onHEEC. We have also performed similar experimentson HDEC, HLEC, and HUVECs. In our hands, thestimulatory effect mediated by estradiol in those cul-tures was not always consistent, and when seen wasmodest (12–15%) (data not shown). The effect ofprogesterone, has not been previously reported onthe endothelium.DISCUSSION

Given the growing evidence that endothelial cellsisolated from different organs express unique prop-

Figure 3. Expression of endothelial cell markers by cultured endometrial endothelial cells. Human endometrial-derived (A, C, and E), dermal-derived (B, and D) endothelial cells or endometrial fibroblasts (F) were cultured on glasscover-slips, fixed in paraformaldehyde, and treated briefly with triton X-100. Cultures were then incubated with thefollowing antibodies: (A and B) vWF; (C and D) PECAM-1; and (E and F) CD-34. Immunocomplexes were detectedwith biotinylated secondary antibodies followed by avidin-FITC. Nuclei were stained with Hoechst. Arrows indicatepositive reaction.

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erties, it is reasonable to predict that different cap-illary beds might respond to tissue-specific cues thatregulate local patterns of growth and inhibition.Therefore, an in-depth study of organ-specific vas-cular beds can be expected to provide informationabout particular pathways that might have broadapplicability to the pharmacological regulation ofangiogenesis in that organ, without necessarily af-fecting microvessels in other tissues. In the presentstudy, we derived a simple protocol for the isolationof endothelial cells from the endometrium, demon-strated that these cells exhibit a significantly higherbinding for VEGF, and provide evidence for a func-tional modulation of steroid hormones on growthfactors’ function. The endothelial nature of the iso-lated cells has been characterized by several conven-tional markers and by functional assays. In general,HEEC, display features similar to endothelial cellisolated from other organs. However, they appear tohave an enhanced responsiveness to the angiogenicgrowth factor, VEGF, and do not respond to PDGF-BB. As other reports have indicated (2,38), we foundthat expression of von Willebrand factor is heterog-enous and identifies endothelial cells from large- andmedium-size vessels in the endometrium preferen-

Figure 4. Endometrial endothelial cells upregulate E-Selectin mRNA upon treatment with TNF-a. Human en-dometrial endothelial cells were cultured in the absence ofserum for 16 h. TNF-a at 50 ng/mL (+) or vehicle (−)were added to the cultures. Cells were harvested at 30min, 2 h, and 6 h. Total RNA was isolated, subjected toelectrophoretic separation, and transferred to Nytranmembranes. Hybridization with an E-selectin cDNA frag-ments revealed marked increase in expression upon treat-ment with TNFa. A cDNA fragment from 36B4 ribosomalprotein was used as control for loading and transfer effi-ciency.

Figure 5. Identification of progesterone and estrogen receptors on human endometrial endothelial cells. Endometrialtissue sections were incubated with an anti-progesterone receptor (A) or anti-estrogen receptor (B) antibody followedby biotinylated secondary antibodies. Immune complexes were revealed after treatment with diaminobenzidine as abrown color. Counterstaining was performed with a nuclear fast-red dye. Arrows in both panels indicate positiveendothelial cells. Isolated HEEC cultures were incubated with anti-progesterone receptor (C) or estrogen receptor (D)antibodies, followed by secondary conjugated to FITC. To validate the immunocytochemical results, total RNA isolatedfrom HEEC cultures was also subjected to reverse transcriptase for the generation of first-strand cDNAs followed byPCR with progesterone receptor (C, lanes 1–4) or estrogen receptor (D, lanes 5–8) specific primers. Lanes are PCRreactions from HEEC cultures at progressive passage number: 1 and 5: passage 2; 2 and 6: passage 5; 3 and 7: passage7; 4 and 8: passage 10.

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tially. In contrast, Ulex lectin and CD-34 showed amore homogeneous and consistent distribution inendometrial capillaries.

The endometrium is a unique organ that undergoesphysiologic cyclic remodeling. During its growth andmaturation, intense vascular growth takes place,supporting stromal and glandular expansion. Theendometrial vascular bed, and endometrial endothe-lial cells in particular, have not been well-characterized. Because these cells present uniquefeatures of recurrent growth and inhibition, we ra-tionalized that inherent differences must exist be-tween endometrial endothelial cells and endotheliumfrom other vascular beds.

The nature of the angiogenic stimulus in the endo-metrium is also poorly defined. Although a numberof growth factors known to be angiogenic in otherorgans have been identified in the human endome-trium, the specific effect of these cytokines on endo-metrial endothelial cells has never been investigated.Among the factors identified are FGF-2, VEGF,insulin-like growth factor, epidermal growth fac-tor, and transforming growth factor-b (29). Inter-estingly, the levels of such growth factors is notincreased, as would be expected, early in the prolif-

erative phase when vascular repair and angiogenesisfollow menstruation. Heparin-like activity has alsobeen detected in endometrial fluids with increasingconcentrations toward the end of the cycle in wom-en, which could enhance the action of specific hep-arin-dependent angiogenic agents (29). Most ofthese cytokines also influence fibroblast prolifera-tion, and in some cases regulate epithelial growthand differentiation (as transforming growth factor-b). Therefore, it is possible that the presence of somegrowth-stimulating factors might not be directly re-lated to the endometrial angiogenic wave. The iso-

Figure 6. Endometrial-derived endothelial cells expressKDR and FLT-1. Total RNA was isolated from endome-trial endothelial cells (HEEC), dermal endothelial cells(HDEC), dermal fibroblasts (HDF), and endometrial stro-mal (HES). All strains were used between passages 3 and5. Resulting Northern blots were hybridized with cDNAprobes to KDR; FLT-1; VPF/VEGF; and 28S ribosomalsubunit for evaluation of loading and transfer efficiency.Lanes: 1 4 HEEC; 2 4 HDEC; 3 4 HDF; 4 4 HES. Figure 7. Endometrial endothelial cells proliferate in re-

sponse to VEGF and FGF-2. (A) Endothelial cell cultures(HEEC, HCEC, HUVEC, and HDEC) at identical passagenumber were made quiescent by serum deprivation for 48h. Thereafter, 2 × 105 cells from each type were platedonto new dishes and treated with 1) 1% fetal calf serum(FCS); 2) 1% FCS + 20 ng/mL VPF/VEGF; 3) 1% FCS+ 20 ng/mL FGF-2; 4) 1% FCS + PDGF-BB (20 ng/mL).After 4 days of incubation, cells were counted by coultercounter. Values were normalized to control (DMEM) ±SEM. n 4 4 for all conditions. (B) Cell-cycle progressionanalysis of HEEC and HDEC. Cultures were made quies-cent by 48 h, serum deprivation after confluency. Cellswere passed into 12-well plates and harvested every 8 hfor a total of 120 h. A pulse of [3H]-thymidine was 4 hprior to harvest time. Values indicate incorporation ofTCA-precipitable counts ± SEM. n 4 5 for all conditions.

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lation of endometrial endothelial cells was a neces-sary step to evaluate the relative contribution ofthese growth factors to the cyclic vascular repair inthis tissue. In addition, the endometrium is, unlikeother organs, under the regulatory control of steroidhormones. The relative contribution of these hor-mones to the angiogenic response has been hypoth-esized, yet not investigated. Our results provide evi-dence that steroid hormones can modulate the actionof growth factors on HEEC. Therefore, although lev-els of angiogenic growth factors are not drasticallychanged through the proliferative and secretoryphases of the menstrual cycle, the contribution ofestrogen and progesterone helps to undestand how,in combination, hormones and growth factors regu-late vascular repair in the endometrium.

Our study also showed that HEEC respond to bothFGF-2 and VEGF, but not to PDGF-BB. A signifi-cantly higher growth response was seen in HEEC inresponse to VEGF. The finding led us to compare

affinity binding of this growth factor for HEEC andother endothelial cell types. As reported, impressivedifferences were found. Clearly, HEEC had a higherbinding capacity for VEGF than any of the otherendothelial cell types examined, providing an expla-nation for the marked proliferative response to thisgrowth factor.

VEGF binds to two receptor tyrosine kinases, FLT-1and KDR (17,49,56). Upon binding and formationof receptor dimers, activation is characterized by re-ceptor phosphorylation followed by initiation of thecell-cycle cascade. Our observation of higher bindinglevels for VEGF in HEEC suggests that these cells

Table 1. Relative proliferative increases of severalendothelial cell types by angiogenic growth factors

HEEC HCEC HUVEC HDEC

VEGF 72%* 28.6% 21.4% 51.2%FGF-2 30.2% 31.4% 39.3% 63.4%PDGF BB 4% 5.7% 3.6% 44%

*Percentages were calculated from data presented in Fig. 7A.

Figure 8. VEGF-binding profile to several endothelialcell types. Endothelial cell (HEEC, HCEC, HUVEC, andHDEC) and stromal fibroblasts (HSF) were plated on 48-well plates at equal number. Highest binding of 1 ng/mL[125I]-VEGF 165 to endothelial cultures is referred to as100%. VEGF binding was competed by increasingamounts of unlabeled VEGF.

Figure 9. Estrogen and progesterone modulate the pro-liferative responses of VEGF and FGF-2 in HEEC. Qui-escent HEEC cells from passage 3 to 5 were seeded on48-well plates and treated with 2 ng/mL FGF-2 and 25ng/mL VEGF in the presence of steroids (A: estradiol andB: progesterone) or vehicle, as indicated. Incubation withgrowth factors and hormones was performed for 48 h andlabeling with [3H]-thymidine (1mCi/mL) was performedduring the last 12 h of incubation. Values indicate incor-poration of TCA-precipitable counts ± SEM. n 4 6 for allconditions.

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have higher levels of receptors and are more respon-sive to growth stimulation than other cell types. Ourdata showed that in response to VEGF, HEEC enterS phase more quickly than HDEC, HUVEC, andHCEC suggesting a dramatically enhanced responseto this growth factor.

Most of our current knowledge of the endometrialvasculature is based on morphological studies(21,29,54). The structure of the primate endometri-al vasculature is unique when compared to othermammals. At the myoendometrial junction, radialarteries branch into 1) basal arteries, which supplythe basal endometrium, and 2) spiral arteries, whichsupply the functional endometrium (39). The basalarteries maintain the integrity of the basal layerthrough all phases of the menstrual cycle and appearto be unaffected by hormonal stimuli. In contrast,the spiral arteries appear responsive to steroids andundergo alterations in morphology and length thatare well-correlated with fluctuations in circulatinghormones (39). Falling levels of progesterone andestrogen promote contraction of the spiral arteriesand result in episodes of intermittent hypoxia in thefunctional endometrium. The end result is necrosisand exfoliation of the functional endometrium withloss of blood. Following the menstrual phase, thebasal microvessels give rise to a new capillary plexus.The cyclic nature of endothelial growth follows en-dometrial physiology and is thought to be regulated,at least indirectly by steroid hormones (21,54,57).Here, we demonstrate the presence of both estrogenand progesterone receptors in HEEC, indicating thatthese hormones play a direct role in the regulation ofendothelial cell function in the endometrium. Sev-eral reports in the literature have indicated that 17-b-estradiol stimulates both migration and prolifera-tion of human umbilical vein and human coronaryendothelial cells (40,59). A supporting role for es-tradiol in angiogenesis was also demonstrated usingin vitro assays (50). It is likely that this hormonedisplays similar functions on HEEC.

The prompt vascular-growth inhibition associatedwith the secretory phase of the endometrial cycle isalso unique to this organ. During the vasculargrowth, the rate of proliferation of endothelial cellshas been compared to that of tumor cells (54); how-ever, in contrast to tumor cells, vessel growth ceasesafter the reconstitution of the functional layer. Un-like any other organ examined, we found that ex-pression of thrombospondin-1 (TSP-1) is temporallyregulated in the endometrium, predominantly ex-pressed during the secretory phase, a time when cap-illary growth is suppressed (35). Equally, a novel

gene named METH-2, with significant homology toTSP-1 and angio-inhibitory properties (Vazquezand Iruela-Arispe, submitted), is also expressed in anarrow window during the early secretory phase(days 17–21) (Iruela-Arispe, unpublished observa-tions). Because of the potential importance of vas-cular regulation to control of several endometrialdisorders, the identification of specific regulators ofvascular growth and inhibition is of particular inter-est in this tissue.

An increasing body of literature provides examplesof biochemical and functional heterogeneity in en-dothelial surfaces. Data are now available that char-acterize differentiated microdomains in tissue vascu-lature (18,26,27,47,48,53,61). Given the propertiesof human endometrial endothelial cells, the expres-sion of unique genes in this microvasculature mightseem intrinsically obvious, and indicates a highprobability that a search for such genes will providesignificant insights into the function of this tissue. Atpresent, the literature offers no data on this issue.The identification of endometrial-specific genes,such as METH-2, that differentiate endometrial en-dothelial cells from endothelial cells of other vascu-lar beds can provide important basic information ofthe understanding of the pathologies uniquely ob-served in endometrial capillaries. In addition, re-search in this area can provide important tools forpharmacological targeting and for potential treat-ment of endometrial pathologies. The availability ofhuman endometrial endothelial cells will make studyof these, and other issues, more experimentally ac-cessible.

ACKNOWLEDGMENTS

The authors thank Dr. Michael Detmar, for humandermal endothelial cells; and Dr. Don Senger, for thehuman umbilical vein endothelial cells; Ms. SarahOikemus, for her excellent technical assistance; andMs. Carol Foss, for her secretarial assistance.

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