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Ann Anat 190 (2008) 238—245

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RESEARCH ARTICLE

Immunohistochemical expression of vascularendothelial growth factor A (VEGF), and itsreceptors (VEGFR1, 2) in normal and pathologicconditions of the human thymus

Anca Maria Cimpeana, Marius Raicaa,�, Svetlana Encicab,Remus Corneac, Viorica Bocana

aDepartment of Histology, ‘‘Victor Babes-’’ University of Medicine and Pharmacy, Timis-oara,Eftimie Murgu Square nr. 2, Timis-oara 300041, RomaniabDepartment of Pathology, ‘‘Niculae Stancioiu’’ Heart Institute, Cluj Napoca, RomaniacDepartment of Pathology, Clinic Hospital Nr. 1, Timisoara, Romania

Received 5 February 2007; accepted 28 May 2007

KEYWORDSHuman thymus;VEGF;VEGFR 1 and 2;Epithelial cells;Thymoma

ee front matter & 2008aanat.2007.05.003

ing author. Tel.: +40256ess: raica@umft.ro (M.

SummaryVascular endothelial growth factor A (VEGF-A) is an angiogenic growth factor that isa primary stimulant of the vascularization of solid tumors. In the tumormicroenvironment, an upregulation of both VEGF and its receptors occurs, leadingto a high concentration of occupied receptors on tumor vascular endothelium.Also, VEGF is involved in the development of the normal vascular network of thethymus. Little is known about VEGF expression in normal and malignant thymictissue. Our purpose was to study the pattern and localization of VEGF expression inbenign conditions of the thymus and thymoma to determine a possible correlationwith VEGF receptors VEGFR1, VEGFR2 and microvascular density. All caseswere positive for VEGF and VEGFR1, 2 in the epithelial cells, in a cytoplasmic,granular pattern. In the normal thymus, there were positive epithelial cells withsubcapsular distribution and Hassall’s corpuscle epithelial cells. In acute thymicinvolution, the positive fields were correlated with dilation and stasis of bloodvessels and lymphocyte depletion. Rare positive cells were found in other types ofinvolution; the myasthenic thymus showed an intense and diffuse reaction inlymphoid follicles of the medulla. A strong reaction for VEGF was observed in type B3thymomas in neoplastic epithelial cells, normal endothelial cells, plasma within theblood vessels and focally in the stroma adjacent to the tumor. Receptors for VEGF

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204476.Raica).

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Expression of VEGF-A and VEGFR1, 2 in human thymus 239

were positive in neoplastic epithelial cells and endothelium. We hypothesized thatVEGF acts as an immunoregulatory factor in the normal thymus and as proangiogenicand autocrine factor in thymomas.& 2008 Elsevier GmbH. All rights reserved.

Introduction

It has been demonstrated that the thymus takesits origin from the endoderm of the third brachialpouch (Blackburn and Manley, 2004). The interac-tions between lymphoid cells and thymic epithelialcells of the stroma provide the morphological andfunctional basis for the development of this organ(Hale, 2004). Vascular endothelial growth factor(VEGF) plays an important role in angiogenesis. It isinvolved in wound healing and the reproduc-tive cycle, mechanically induced changes of thecartilage in osteoarthitris (Pufe et al., 2005) andtumor angiogenesis and progression. VEGF is in-volved in the organogenesis of the first threepharyngeal pouches and in the pathogenesis of diGeorge’s syndrome in mice but failed humanembryo (Stalmans et al., 2003). The vascularnetwork of the normal thymus consists of branchingand anastomosing blood vessels but the molecularbasis involved in typical vasculature formation isunknown. Muller et al. (2005) reported VEGFA geneexpression in fetal and adult thymic epithelial cellsin mice. In humans, the expression of VEGF A innormal and malignant thymic tissues is less studied.In human fetal thymuses aged between 16 and 22weeks of gestation, messenger RNA encoding VEGFA was minimally detected. The immunohistochem-ical expression of VEGF peptide has a cytoplasmicpattern and is localized mainly in the epithelialcells and myocytes, including smooth muscle cellslining blood vessels but not in the endothelial cells(Shifren et al., 1994). There are no published dataabout expression of VEGF A in the normal humanthymus in postnatal life and in benign lesions of thethymus, e.g. acute thymic involution, myastheniagravis or age-related thymic involution. The corre-lation between VEGF A expression as a potentangiogenic factor and development of tumor bloodvessels in human thymomas and thymic carcinomawas partially described by Tomita et al. (2002).Because of the limitations in the number of cases,the expression of VEGF in thymomas and thymiccarcinoma has not been studied as a target forantibodies-based therapy. All the reasons men-tioned above lead us to describe the immunohisto-chemical expression of VEGF in normal humanthymus, related benign lesions and thymomas.

Moreover, receptors for VEGF (VEGFR1 and VEGFR2)have been characterized and described in varioustypes of tumors, e.g. brain tumors (Lungu et al.,2007) in both endothelial and tumor cells andin ovarian carcinoma and melanoma A375 celllines (Masood et al., 2001). These receptorsregulate physiological and pathological angiogen-esis as well. VEGFR2 is a direct signal transducerfor pathological angiogenesis including cancer anddiabetic retinopathy; thus, VEGFR2 itself and itssignaling appear to be critical targets for suppres-sion of these diseases. VEGFR1 plays a dual role, anegative role in angiogenesis in the embryo mostlikely by trapping VEGF-A and a positive role inadulthood in a tyrosine kinase-dependent manner(Shibuya, 2006). Receptors for VEGF have not beencharacterized in the human thymus and its roles inpathologic conditions are not known.

Material and methods

Our study included 17 patients between 1 monthand 50 years of age. The thymuses were removedduring surgery performed for cardiovascular mal-formations (n ¼ 7), myasthenia gravis symptoms(n ¼ 3) or abnormal mediastinal masses (n ¼ 7)detected by radiographic or CT scan exam. Biopsieswere fixed in buffered formalin for 24 h and paraffinembedded according to standard techniques. Themicroscopic diagnosis was established on 5-mm-thick sections, stained with hematoxylin and eosin.The pathologic diagnosis of thymomas was per-formed according to WHO classification (Rosai,1999). Additional sections from each case wereused to perform immunohistochemistry for thedetection of VEGF and VEGFR1, 2 expression.Antibody types, clones and detection systems aresummarized in Table 1. Assessment of results wascarried out with the Nikon Eclipse 600 microscope.We evaluated the presence of positive epithelialcells, their distribution and correlation betweenhistopathology and expression of VEGF. We alsoanalyzed the VEGFR1 and VEGFR2 expression innormal and pathological conditions of the thymus.We scored the intensity of reaction as 0 (noreaction found), +1 (weak reaction), +2 (moderate)and +3 (strong staining). Kidney tissue represented

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Table 1. Antibody types and technical characteristics of immunohistochemical methods

Ab. Clone Dilution Antigen retrieval Peroxidase block Incubationtime

Detectionsystem

Chromogennuclear stain

MoaHu VEGF (Dako) VG1 1:30 150, HIER, high pHretrieval solution(Dako)

3% hydrogenperoxide indistilled water

1 h, RT LSAB+ DAB modifiedlillehematoxylin

VEGFR1(N-term)rabbit monoclonal(epitomics)

1:250 100, HIER, citratepH 6 (Dako)

3% hydrogenperoxide indistilled water

300, RT Catalyzedsignalamplification

DAB modifiedlillehematoxylin

VEGFR2 rabbitpolyclonal (novusbiologicals)

1:75 100, HIER, citratepH 6 (Dako)

3% hydrogenperoxide indistilled water

300, RT Catalyzedsignalamplification

DAB modifiedlillehematoxylin

CD 105 (endoglin),mouse monoclonalDako

SN6h 1:50 Proteinase K, 100 RT(Dako)

3% hydrogenperoxide indistilled water

300, RT LSAB+ DAB modifiedlillehematoxylin

CD34 mousemonoclonal (Dako)

QBEnd10

Ready-to-use

200, HIER, citratepH 6 (Dako)

3% hydrogenperoxide indistilled water

300, RT ENVISION DAB modifiedlillehematoxylin

Figure 1. Positive vessel for VEGFR 2 at corticomedullaryjunction from human thymus. (IHC, anti-VEGFR2, DAB,400� ).

A.M. Cimpean et al.240

a positive control for VEGF and staining of thymussmall blood vessels endothelium for VEGFR1 and 2(Figure 1). We performed vessel counting at 400�magnification, using five consecutive fields withhigh microvascular density.

Results

On routine hematoxylin and eosin stain, threenormal thymuses were diagnosed with well-knownhistology of cortex and medulla and presence ofHassall corpuscles inside the latter. We also foundone case of acute thymic involution, one thymoli-

poma, three age-related involutions, three myas-thenic thymuses and six thymomas (2 type A, 1 typeAB, 1 type B2 and 2 type B3).

VEGF was positive in both normal and neoplastichuman thymus. The pattern of the immunostainingfor VEGF was granular cytoplasmic. Epithelial cellsof the stroma in the normal thymus expressedVEGF. Positive reaction was found as a subcapsularnetwork of epithelial cells (Figure 2a), isolatedepithelial cells in the cortex and weak to moderatein almost all epithelial cells of the thymus medulla.All types of Hassall corpuscles expressed VEGF. Forthe juvenile type, the reaction was present in theentire corpuscle (Figure 2b). The outer cells of theHassall corpuscles were positive for VEGF with thesame cytoplasmic, granular pattern in the periph-ery of mature and senescent types. In acute thymicinvolution, the positive islands of epithelial cellswere grouped around dilated blood vessels withsevere stasis. The positivity of epithelial cells toVEGF in this type of involution was correlated withlymphocyte-depleted areas (Figure 2c). The thymictissue from the cords of age-related thymic involu-tion contained few positive cells with weakintensity in the periphery, near the adipose tissuebetween these cords. The intensity of reaction washigher in the thymic tissue from myasthenia gravis,with diffuse distribution of positive epithelial cells.This is correlated with a high density of bloodvessels. There were no differences of expressionbetween thymoma-associated myasthenia gravisand myasthenia gravis without this association.Lymphoid follicles from the medulla also display

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Figure 2. Normal human thymus. Subcapsular (a, 400� ) positive epithelial cells for VEGF. Hassall’s corpuscles positivefor VEGF, juvenile type (b, 400� ). Thymic involution with positive epithelial cells for VEGF in lymphocyte-depletedarea (c, 400� ).

Figure 3. VEGF expression in thymomas. Spindle positive cells of type A thymoma (+1), (a, 200� ). Intenseimmunoreaction (+3) in almost all neoplastic cells from type B3 thymoma (b, 400� ). CD105 immunostaining for type Athymoma (c, 400� ) and B3 thymoma (d, 400� ).

Expression of VEGF-A and VEGFR1, 2 in human thymus 241

positive reaction for VEGF. All types of thymomasincluded in the study expressed VEGF. In type Athymoma the spindle cells were weak to moderatepositive for VEGF with diffuse distribution; the

same pattern was found in spindle areas from ABthymoma (Figure 3a). The majority of neoplasticepithelial cells from B2 thymoma showed moderateexpression of VEGF that was more intense around

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A.M. Cimpean et al.242

perivascular spaces. Thymoma type B3 was char-acterized by positive reaction in all neoplasticcells with strong, granular cytoplasmic pattern(Figure 3b). Focally, the stroma adjacent to thetumor was positive for VEGF, a feature alsodetected in the endothelium of large vesselsaround the tumor. In these zones we noticed thehighest vessel density for CD105 compared withother thymomas (Figure 3c and d). The remnantthymus near the type B3 thymoma expressed VEGFwith weak intensity, compared with the neoplastictissue and had the same distribution pattern like inthe normal thymus.

Normal thymus also expressed VEGFR1 andVEGFR2. All epithelial cells of the medulla werepositive for both receptors with +3 staining forVEGFR 1 and +1 for VEGFR 2. All types of Hassallcorpuscles were composed of positive epithelialcells. In senescent corpuscles, the peripheralepithelial cells were positive (+2) for VEGFR 1 andfor VEGFR 2 (+1). Thymic nurse cells from thecortex expressed VEGFR 1 with moderate intensityand also VEGFR 2, but weak and not constant.Subcapsular epithelial cells were negative for bothreceptors. Acute thymic involution shared intensepositive staining (+3) of VEGFR 2 on the endothe-lium of dilated vessels. We also detected thepresence of immunostaining in the stromal epithe-lial cells network of involuted thymus with moder-

Figure 4. VEGFR 1 and 2 in thymomas. Positive areas (+3) fortype AB thymomas (b, 400� ). Intense staining for VEGFR 1 (cthymoma.

ate intensity (+2). VEGFR 1 was weak positive withthe same distribution.

VEGFR 1 and VEGFR 2 were positive in neoplasticepithelial cells and vessels of endothelium of allthymomas. There were differences in expressionof receptors closely linked to the type of thymoma.In type AB thymoma we found an intense (+3)staining for VEGFR 1 in spindle neoplastic cellsfrom spindle areas, whereas lymphocytes werenegative (Figure 4a). For VEGFR 2 we observedfoci with +2 positive staining (Figure 4b), whichalternate with +1 positive zones. Scattered positivecells with macrophage-like morphology wereobserved in connective tissue septae betweenneoplastic areas. Type B3 thymomas had a diver-gent expression of VEGFR 1 and 2. All thymicepithelial cells were positive (+3) for VEGFR 1(Figure 4c) with the same intensity like VEGFexpression. Type 2 VEGF receptor was positive intype B3 thymoma scattered epithelial cells withweak (+1) intensity. Isolated groups of neoplasticcells showed moderate (+2) staining for VEGFR 2(Figure 4d).

Inside the capsule there were numerous invasivesites of thymoma, which shared positivity for bothreceptors, especially for VEGFR 1.

Differences between thymoma type, VEGF,VEGFR 1 and 2 expression and vessels count areshown in Table 2.

VEGFR1 (a, 400� ) and +2 immunostaining for VEGFR 2 in, 400� ) and moderate (d, 400� ) for VEGFR 2 in type B3

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Table 2. Comparative expression of VEGF and VEGFR1and 2 in thymomas and median range of microvasculardensity for CD34 and endoglin

VEGF VEGFR1 VEGFR2 CD34 CD105

Type A thymoma +/++ +++ + 48 20Type AB thymoma +/++ +++ + 53.6 20.8Type B2 thymoma ++ +++ ++ 44.2 24.2Type B3 thymoma +++ +++ +/++ 53 44.8

+, weakly positive; ++, moderately positive; +++, intenselypositive.

Expression of VEGF-A and VEGFR1, 2 in human thymus 243

Discussion

Discovered for the first time by two teams(Senger et al., 1983; Dvorak et al., 1995) as avascular permeability factor and as a mitogenendothelial factor by Napoleone Ferrara (Ferraraand Henzel, 1989), VEGF plays an important role inthe early development of the cardiovascular systemat the embryo and postnatal in normal andpathologic conditions. This growth factor is in-volved in the development and progression ofmalignant tumors and represents the only acceptedtarget for the antiangiogenic therapy using huma-nized monoclonal antibody treatment, like Bevaci-zumab in metastatic colon carcinoma (De Gramontet al., 2006). There are numerous studies about thispotent angiogenic factor in malignancies (Takahashiet al., 2007; Miyata et al., 2006; Ljungberg et al.,2006) but few in normal tissues. In some cancers(e.g. prostate), the expression of VEGF in locallyinvasive tumors has prognostic significance for theradiotherapy outcome (Green et al., 2007). Thereare few data about the expression of VEGF-C andVEGF-A in normal thymus after birth (Joory et al.,2006). The development of vascular network of thethymus depends on the high concentration of VEGFin epithelial cells of the thymus; after birth, thelevels of VEGF rapidly decrease in the normalhuman thymus. This is in accord with our findings inthe normal thymic tissue, in which we foundpositive reaction for VEGF, restricted to mostepithelial cells beneath the capsule and disposedas a network, few cell in the deep cortex and theepithelial cells of all Hassall corpuscles types,previously described (Raica et al., 2006). Juveniletype of Hassall corpuscles expressed VEGF in allepithelial cells from the center and the periphery,with the same moderate intensity. For other threetypes of Hassall corpuscles (immature, mature andsenescent), VEGF was positive only in the periph-eral cells. The maintenance of VEGF expression inthe Hassall corpuscles after birth correlates with its

involvement in the maturation of lymphocytesbefore birth in human thymus. These data supportthe hypothesis that Hassall corpuscles are activestructures that participate in the lymphocyteselection and maturation in the postnatal life(Watanabe et al., 2005). Some studies describedthe inhibitory effect of high levels of VEGF thatinterferes with the development of T cells fromearly hematopoietic progenitor cells of lympho-cytes into the thymus and the link between thymicatrophy and VEGF (Ohm et al., 2003). These datasupport our findings from acute thymic involution,where areas with intense positive staining for VEGFwere associated with lymphocyte depletion. Highnumber of blood vessels and their particularmorphology, with dilation and stasis in acute thymicinvolution, was linked to the vascular permeabilityproperties of VEGF. The expression pattern ofVEGF in the thymus from myasthenia gravis couldexplain the high microvascular density noticed inthe thymic tissue. It appears to be a correlationbetween tumor angiogenesis and the invasivenessin thymomas with a correlation between micro-vascular density, VEGF expression and Masaokaclinical stage (Papadopoulos and Thomas, 2005).There is a suggestion of the presence of parench-yma microvessels in invasive thymomas but not fornoninvasive thymomas. Our data suggest that thereis a strong correlation between expression of VEGFin thymomas and the microvascular density calcu-lated on slides stained for CD105. We found a highermicrovascular density for CD105 in type B3 thymo-mas, which had a strong immunoreactivity forVEGF. Expression of such a marker in the thymomacould be a target for antiangiogenic therapyfocusing on both the tumor blood vessels and cellsthat produce high levels of VEGF. Increasedcirculating levels of VEGF are found in patientswith thymic carcinoma but not thymomas (Sasakiet al., 2001). A phase II Study of Erlotinib plusBevacizumab given to patients with advancedthymomas and thymic carcinoma is going on inorder to determine the objective response rate tothis combination. One of the outcomes of this studyis to correlate the expression of VEGF in tumorsamples, circulating VCAM1 and urine VEGF levelspre-therapy with response to therapy. Most ofthe studies concerning VEGF expression in solidtumors used immunohistochemistry first to detectthe presence of this marker in the tumorsamples. These studies included colorectal cancer(Galizia et al., 2004; Ochs et al., 2004), breastcancer (Kostopoulos et al., 2006), prostate cancer(Strohmeyer et al., 2004) and renal cell carcinoma(Fukata et al., 2005). However, no morphologic orfunctional evidence concerning VEGF expression in

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normal thymus and thymomas has yet beenpublished. In our study, type B3 thymomas showedintense staining for VEGF A in all tumor epithelialcells. Our results suggest that VEGF could be apositive marker for the use of bevacizumab therapyin this type of thymoma.

Some human breast cancer epithelial cells havebeen shown to express VEGF receptors suggesting apotential autocrine-mediated growth stimulationof cancer by VEGF (Huh et al., 2005). In our study,all cases of thymoma expressed VEGF receptors inendothelial and tumor epithelial cells. The patternof expression pattern differed depending on thetype of thymoma. We found a correlation betweenthe strong expression of VEGF and VEGFR 1 in typeB3 thymomas. The pathways through which VEGF/VEGFR1 exert autocrine activity in tumor cells is atopic of research. In vitro, VEGFR 1 activation byVEGF leads to the activation of the mitogen-activated protein kinase (MAP kinase) signal trans-duction pathway in tumor cells (Hicklin and Ellis,2005; Fan et al., 2005). Activation of VEGFreceptors in tumor cells leads to phenotypicchanges including an increase in cell invasion andmigration. All thymomas included in our study,which were positive for VEGFR 1, had capsularinvasion. Thus, our results suggested that type 1receptor for VEGF might be involved in this processby enhancement of migration and invasion poten-tial of neoplastic thymic epithelial cells. Thispresumed mechanism of action of the VEGF/VEGFR1 complex could explain the paradoxicalbehavior of some thymomas which are of low-grademalignancy – type A and AB thymomas – but share acharacteristic extensive capsular invasion at thetime of diagnosis or recurrent events after surgicaltreatment and chemotherapy.

Kiseleva et al. (2005) reported that VEGF notonly acts as angiogenic factor, but also as animmunoregulatory factor. VEGF modulates prolif-eration of thymocytes and expression of mRNA type2 VEGF receptor. Reinders et al. (2003) describedthe involvement of VEGF in acute rejection ofallografts. These data, taken together with thereport of Watanabe et al. (2005), support ourfindings concerning the positive reaction of Has-sall’s corpuscles cells for VEGF and its receptors.

The positive Hassall’s corpuscles for VEGF re-ceptors found on normal thymus together with astrong reaction of the medulla thymic epithelialcells could be linked to the lymphocytic maturationand migration from the cortex.

VEGF may support the growth of some thymomasnot only by inducing angiogenesis but also by actingdirectly through VEGF receptor expressed on tumorcells. VEGF receptor expression detected on var-

ious tumor cells has been associated with tumorgrowth and invasiveness. However, the progno-stic significance of VEGF receptors expression ontumor cells remains unclear. Our results suggesta possible correlation between invasiveness andVEGF/VEGFR1 expression in thymomas.

In conclusion, we hypothesized that VEGF andVEGFR 1and 2 play an important role in normalthymus through immunomodulatory properties.Also, in the tumors of the thymus, VEGF acts as aproangiogenic factor through VEGFR2 and promotesmigration and invasion through VEGFR 1 expressedby tumor cells.

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