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From the Department of Clinical Pathology,*
the
Department of Dermatology
Division of
Immunology, Allergy and Infectious Diseases, and the Department of
Internal Medicine III,
Division of
Nephrology and Dialysis, University of Vienna, Vienna, Austria, and the
Department of Pathology,§
Haartman Institute,
Molecular/Cancer Biology Laboratory, University of Helsinki,
Helsinki, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Recently, we have observed in rat and human kidneys5 that podoplanin, a ~38-kd glomerular podocyte membrane mucoprotein, was localized in endothelia of small vessels with topographic and morphological features of lymphatic capillaries. Furthermore, a podoplanin-related rat glycoprotein was found in the endothelium of presumptive lymphatic vessels.6 Another protein shown to be expressed uniquely on lymphatic endothelia was the receptor for the vascular endothelial growth factor C, designated vascular endothelial growth factor receptor-3 (VEGFR-3) or flt-4.7-9 Thus we have reasoned that, if antibodies specific for human podoplanin and flt-4 immunolabel the same type of vessel, they could serve as selective immunohistochemical markers in pathologically altered tissues. Here we have confirmed and extended this hypothesis in an immunohistochemical pilot study of normal endothelium, benign vascular tumors, and a small number of angiosarcomas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Paraffin blocks of 40 cases of vascular tumors and five cases of
gastrointestinal Kaposi's sarcomas from HIV-infected patients (Tables 1 and 2)
were obtained from the archive of the Institute of Clinical Pathology,
University of Vienna. Native renal cortex was dissected from kidneys
removed because of hypernephroid carcinoma (n =
10); native lung tissue was dissected from lobectomy specimens removed
because of non-small cell carcinoma (n = 5).
Cutaneous lymphangiomas (n = 3), normal lymph
nodes (n = 3), and normal skin
(n = 5) were obtained from the Department of
Surgery, University of Vienna. Two fetal hygroma colli were from
spontaneous abortions in the 22d and 23rd gestational week.
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Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG F(ab')2, FITC- and tetramethylrhodamine-conjugated goat anti-rabbit IgG F(ab')2 were obtained from Jaxell-Accurate (Westbury, NY). Texas red-conjugated goat anti-mouse IgG, biotinylated horse anti-mouse IgG, goat anti-rabbit IgG, and streptavidin-biotin complex peroxidase system were purchased from Vector Laboratories (Burlingame, CA). Goat anti-rabbit and goat anti-mouse IgG conjugated to 5- or 10-nm gold particles were from Amersham (Auroprobe, Buckinghamshire, UK). Alkaline phosphatase-conjugated rabbit anti-mouse IgG and alkaline phosphatase-conjugated goat anti-rabbit Fc fragment specific IgG were from Promega (Madison, WI). Ulex europaeus I lectin (UEA I), rabbit polyclonal antibodies directed to human factor VIII-related antigen and to UEA I lectin and mouse monoclonal antibody directed to CD31 (clone JC 70A) were from Dako (Glostrup, Denmark). Mouse monoclonal antibody to CD34 (clone QBEnd/10) was from Novocastra Laboratories (Newcastle on Tyne, UK). Mouse monoclonal anti-human endothelial antibody (clone PAL-E) was obtained from Harlan Sera Lab (Crawley Down, UK). Mouse monoclonal antibodies to human VEGFR-3 were prepared as described.9 WGA-sepharose 4B was purchased from Pharmacia LKB (Uppsala, Sweden). Sodium dodecyl sulfate (SDS), acrylamide, TEMED, ammonium persulfate, high- and low molecular weight standards, Tween 20 and PDG-6 columns were from BioRad Laboratories (Richmond, CA). Lowicryl K4M was obtained from Chemische Werke Lowi (Waldkraiburg, Germany). Diaminobenzidine (DAB) was from Serva (Heidelberg, Germany). Leupeptin, antipain, pepstatin, Triton X-100, dithiothreitol (DTT), ammonium sulfate, ethylendiamine tetraacetic acid (EDTA), heparin, protease type XIV from Streptomyces griseus, phosphotungstic acid, and 4',6-diamidino-2-phenylindole were from Sigma (Deisenhofen, Germany). Pefablock SCR was from Pentapharm (Basel, Switzerland). Nitro blue tetrazolium and 5'-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) was obtained from Kirkegaard and Perry (Gaithersburg, MD). Nitrocellulose (0.45 µm pore size) was from Schleicher and Schuell (Dassel, Germany). Human umbilical vein endothelial cells (HUVEC) and endothelial cell growth supplement were purchased from Technoclone, Vienna. PenStrep, M199 medium and fetal calf serum were obtained from GIBCO BRL (Gaithersburg, MD).
Antibodies Specific for Human Podoplanin and Podocalyxin
Rabbit anti-human podoplanin IgG was raised against the recombinant human homologue of the rat 43-kd glycoprotein podoplanin (Kowalski H, Soleiman A, Endres D, Breiteneder-Geleff S, Kerjashki D, manuscript in preparation). Rabbit sera were affinity-purified using nitrocellulose strips containing recombinant protein, as described.10 Mouse monoclonal anti-podocalyxin antibody (clone GB6) was produced using as antigen a wheat germ agglutinin-binding fraction of a Triton X-100 lysate of isolated human glomeruli, as described for rat podocalyxin.11
Immunofluorescence
Unfixed 4-µm-thick cryostat sections of normal human kidney, placenta, and skin were incubated with rabbit anti-human podoplanin IgG (0.5 µg/ml), monoclonal mouse anti-podocalyxin IgG (10 µg/ml), or monoclonal mouse anti-VEGFR-3 IgG (1µg/ml) followed by FITC-conjugated goat anti-rabbit or rabbit anti-mouse F(ab')2 fragments. For double immunofluorescence, cryostat or deparaffinized, rehydrated paraffin sections were incubated with 10% egg albumin for 15 minutes, followed by a mix of antibodies specific for human podoplanin, human VEGFR-3, and individual endothelial markers (CD31, podocalyxin (clone GB6), and PAL-E) for 14 hours at 4°C. Sections were then incubated with FITC-conjugated goat anti-rabbit IgG and Texas red-conjugated goat anti-mouse IgG, and examined in an Axiophot microscope equipped for epifluorescence (Zeiss, Oberkochen, Germany). Negative controls included omission of first antibodies or substitution of primary antibodies by irrelevant rabbit or mouse IgG.
Immunoperoxidase on Paraffin Sections
Three-µm-thick deparaffinized serial sections of skin, lymph node, and vascular tumors were directly incubated with anti-CD34 IgG. For immunostaining with other endothelial probes pretreatment of sections was required; protease digestion (Sigma protease XIV, 0.1%) was used for CD31, podocalyxin, factor VIII-related antigen, and UEA I. Microwave pretreatment (600 W for 10 minutes) was performed for anti-human podoplanin IgG. Sections were incubated with primary antibodies for 1 hour at 20°C, followed by biotinylated horse anti-mouse IgG or goat anti-rabbit IgG for 30 minutes and detection by a streptavidin-peroxidase complex, according to the manufacturer's instructions. Peroxidase reaction product was visualized by DAB and H2O2 .
The approximate relative number of podoplanin-positive tumor cells was estimated in paraffin sections of immunoperoxidase-labeled sections. The number of reactive cells was counted in 10 medium-power fields per tumor (magnification, x40) of nonnecrotic areas, and the identity of cells was subsequently confirmed by high magnification (x460). The number of tumor cells expressing CD31 and podocalyxin were similarly determined. Relative numbers of immunoreactive cells were expressed as percentages of the total number of tumor cells.
Immunoelectron Microscopy
Normal skin was fixed in 4% paraformaldehyde in 20 mmol/L phosphate-buffered saline (PBS) for 6 hours at 20°C and embedded in Lowicryl K4M.12 Ultrathin sections were incubated in PBS/1% egg albumin, followed by rabbit anti-podoplanin IgG (5 µg/ml) and goat anti-rabbit IgG conjugated to 10-nm gold particles. Other sections were double-labeled with rabbit anti-podoplanin IgG and monoclonal mouse anti-podocalyxin IgG, followed by goat anti-rabbit IgG conjugated to 5-nm gold particles and goat anti-mouse IgG conjugated to 10-nm gold particles.
Cell Culture
HUVECs were cultured in M199 medium, supplemented with 20% fetal calf serum, 1% PenStrep, 0.1% heparin, and 50 µg/ml endothelial cell growth supplement, and used up to passage 4.
Immunoblotting
Membrane protein fractions were prepared from tissue samples of hygroma colli (n = 2), lymphangioma (n = 3), human placenta (n = 5), human lung (n = 5), and isolated human glomeruli, and subconfluent HUVECs as described.13 Briefly, tissues were frozen in liquid N2, powdered, and suspended in PBS containing protease inhibitors (2 mg/L pepstatin, antipain, leupeptin, and 10 mg/L Pefablock). Cultured cells were rinsed in PBS and scraped from the dishes. Tissues and cells were incubated in Na2CO3, pH 11.5, and the resulting membrane vesicle fraction was processed for immunoblotting as described,5 using polyclonal rabbit anti-human podoplanin antibody (0.5µg/ml), or monoclonal anti-podocalyxin antibody GB6 (1µg/ml) as primary antibodies.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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The specificity of affinity purified rabbit anti-human podoplanin
IgG used for immunohistochemistry was confirmed by immunoblotting of
membrane protein fractions of human renal glomeruli, lung and
placentary membrane protein fractions. In these samples, a 38-kd
protein characteristic for human podoplanin was specifically labeled
(Figure 1
, lanes D-F).
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. Double labeling with
anti-podoplanin IgG and antibodies to VEGFR-3, at present the only
established marker for lymphatic endothelium, resulted in perfect
overlap of the fluorescence signals (Figure 2, C
-E).
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. By
contrast, anti-podocalyxin IgG and CD31 immunolabeled HEVs (Figure 3B)
,
while sinus endothelial cells were only weakly stained.
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. Endothelia of blood
vessels were consistently devoid of gold particles (Figure 4B)
.
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Podoplanin Is Expressed in Endothelial Cells of Benign Lymphatic Tumors
Podoplanin was localized in endothelia of 11 lymphangiomas of
skin, oral mucosa, and mesenterium, whereas adjacent blood vessels were
not labeled (Figure 5D)
. Anti-podoplanin
IgG specifically labeled a ~38-kd protein by immunoblotting on
membrane protein fractions of two lymphangiomas, indicating the
presence of authentic podoplanin (Figure 1G)
. Controls included
omission of the primary antibody, or replacement by other
endothelium-specific proteins such as podocalyxin. Anti-podocalyxin IgG
was calibrated by immunoblotting of human glomerular lysates, a major
source of this protein, and in HUVECs (Figure 6
, lane D), and specifically labeled a
protein doublet with an apparent molecular mass of 170 kd (Figure 6
,
lane B).
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. Immunostaining of consecutive sections with vascular endothelial
markers (CD31, CD34, factor VIII-related antigen, UEA I, podocalyxin)
resulted not only in complementary labeling of tumor blood vessels, but
also in variable labeling of lymphatic endothelium (Figure 5, B and C)
. Collectively, these results indicated that podoplanin was specifically expressed by endothelia of benign lymphatic tumorous lesions, similar to nonneoplastic lymphatic endothelium.
Angiosarcomas Coexpress Podoplanin and Markers of Blood Vessel Endothelium
Eight common angiosarcomas (cutaneous, soft tissue, and visceral
type) and three epitheloid cutaneous angiosarcomas (Table 2)
were
immunostained with anti-podoplanin IgG. All were high-grade tumors (G3)
and contained nonvasoformative or poorly vasoformative areas (Figure 7A)
. Podoplanin was variably expressed in
10 of 11 tumors in 5–70% of tumor cells (Table 2)
.
Podoplanin-expressing tumor cells were arranged in clusters of 5 to 30
cells that were confluent in tumors with high levels of expression, and
frequently formed abortive lumina with occasional erythrocytes.
Isolated, single podoplanin-containing tumor cells were also
occasionally encountered, some of which showed atypical mitotic figures
(Figure 7B
, inset). Invariably, podoplanin was detected in highest
concentrations at tumor cells' membranes (Figure 7, B and C)
.
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. There was no correlation of podoplanin expression
and histological type of angiosarcoma. Localization of vascular
endothelial markers in consecutive paraffin sections showed some
topographic, but no quantitative, association with podoplanin (Figure 7, D
-F).
To determine precisely whether podoplanin-producing tumor cells
expressed endothelial antigens of blood vessels simultaneously, double
immunofluorescence with IgGs specific for podoplanin and podocalyxin
was performed. Both markers labeled at least 30% of tumor cells and
were concentrated in clusters (Figure 8)
,
in agreement with the results obtained by immunoperoxidase labeling.
Most tumor cells (>70%) coexpressed podoplanin and podocalyxin
(Figure 8C)
, whereas only a small fraction expressed podoplanin only,
and none podocalyxin alone. Podoplanin was localized also with this
technique in highest concentration on cell surfaces, while podocalyxin
and CD31 were predominately found in the cytoplasm of tumor cells
(Figure 8, A and B)
. Tumor cells located in the black background in
these preparations failed to stain with either marker.
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or the oral mucosa. In all cases the atypical endothelial cells lining
these spaces expressed podoplanin on their cell surface (100% of
podoplanin-positive tumor cells, Figure 9B
). In consecutive sections
the Kaposi's sarcoma spindle cells revealed moderate to strong
reactivity for the endothelial markers CD31 and CD34 (Figure 9, C and D)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Podoplanin was originally found on the surface of rat glomerular epithelial cells (podocytes) and linked to flattening of foot processes (hence the name "podoplanin") that occurs in glomerular diseases and is related to proteinuria. Database comparison of the DNA-deduced amino acid sequence of rat podoplanin has revealed its identity with a glycoprotein in rat bone (designated E11) that was also observed in vessels of the connective tissue that showed features of lymphatic capillaries.6 Additional related proteins were found in rat type I alveolar cells, in mouse osteoblasts, osteocytes, and type I epithelial cells of thymus, and in canine MDCK cells, where they serve as receptors for influenza C viruses.15-18 Podoplanin and its relatives share a high level of identity in their transmembrane domains, but little conserved sequence in their ectodomains apart from several conserved O-glycosylation sites. Thus, podoplanin shows features of a membrane mucoprotein of currently unknown biological function. As heavily O-glycosylated mucoproteins were recently identified as counterreceptors for selectins that mediate adhesion of inflammatory cells,19 it is possible that podoplanin plays a similar role in lymphatic endothelia.
Evidence that podoplanin is specifically expressed by lymphatic endothelia was based on the following findings: (1) Affinity-purified rabbit anti-podoplanin IgG specifically and exclusively immunolabeled endothelial cells of vessels that were clearly distinct from PAL-E-labeled blood vessels. (2) Podoplanin-positive vessels were also selectively stained with anti-VEGFR-3 IgG in double-labeling experiments. (3) Dermal lymphatic capillaries were identified by ultrastructural morphological criteria and found by immunoelectron microscopy to be selectively labeled by anti-podoplanin antibodies. (4) In benign lymphatic tumorous lesions (lymphangioma, hygroma), podoplanin was localized in endothelial cells. (5) Immunoblotting of lymphangiomas with anti-podoplanin IgG revealed a ~38-kd protein that was identical with podoplanin in human lung and isolated glomeruli. Collectively these data indicate that podoplanin is expressed in lymphatic endothelia and thus provides a valid marker for the study of the phenotypic characteristics of neoplastic endothelial cells.
Immunocytochemical localization of podoplanin in benign lymphatic tumorous lesions (lymphangiomas, hygromas) resulted in intense continuous labeling of endothelial cells lining the ectatic vascular spaces. Podoplanin was also detected by immunoblotting as a 38-kd protein in membrane protein fractions of lymphangiomas. Conversely, endothelial cells of adjacent nonneoplastic blood vessels were strongly labeled with established endothelial markers, such as CD31, and also with anti-podocalyxin antibodies, but not with anti-podoplanin IgG. Thus, it appears that immunostaining of lymphatic and blood endothelial markers were, in essence, mutually exclusive on lymphangioma cells. This contrasts with Kaposi's sarcoma, in which cells lining the proliferative vascular channels reacted with both lineage markers. The observation that in the early stages of tumor growth virtually all of the malignant endothelial cells expressed podoplanin provides further evidence that Kaposi's sarcoma spindle cells are related to lymphatic endothelium.9,20
Malignant tumors derived from endothelial cells are known to express endothelial antigens in varying quantities. This appears to be related to the ratio of vasoformative to poorly vasoformative to solid areas within a given tumor and also to the sites of tumor origin.2,3,21-25 Generally, in vascular tumors the degree of differentiation is inversely related to the lesion's expression of markers specific for the assumed lineage. As a diagnostic aid to distinguish angiosarcoma from other types of sarcomas, a large panel of antibodies specific for vascular endothelia such as factor VIII-related antigen, CD31, and CD34 were used.26,27 However, this approach fails to disclose mixed phenotypic lineages of hemangiosarcoma or lymphangiosarcoma components in individual high-grade sarcomas,1-3 and in particular in the rare tumors arising from areas rich in lymphatic vessels and/or persistent chronic lymphedema (lymphedema-associated angiosarcoma).14,28,29
Here we have used podoplanin as a specific marker of lymphatic endothelium to unravel the relative fractions of lymphatic and vascular differentiated tumor cells in common and epitheloid G3 angiosarcomas and in early Kaposi's sarcomas. Angiosarcoma cells were identified by cellular atypia, atypical mitotic figures and loss of cellular cohesion, and shedding of the tumor cells into the vascular clefts. These criteria clearly excluded the possibility that reactive nontumorous lymphatic vessels were mistaken for tumor cells. Because the sample of cases is very small (n = 16) in this preliminary trial, it is premature to speculate about the expression of podoplanin (ie, contribution of the lymphatic phenotype) as a criterion for classifying angiosarcomas. Intriguingly, the large majority of podoplanin-expressing tumor cells also coexpressed vascular endothelial markers podocalyxin and CD31. These findings indicate that a variable fraction of angiosarcoma cells within any given tumor displays the characteristics of both endothelial phenotypes. It is not clear whether this indicates different sites of origin or dysregulated phenotypic expression due to malignant transformation. An alternative explanation could be that the tumor cells derive from immature endothelial precursor cells.
Collectively, the results of this study establish that podoplanin is a valid marker for lymphatic endothelia. Using specific antibodies as immunohistochemical tools, we have found that podoplanin expression is preserved in endothelia of all benign lymphatic tumorous lesions and all Kaposi's sarcomas examined. By contrast, 10 of 11 G3 angiosarcomas contained only variable fractions of podoplanin-expressing tumor cells that also coexpressed markers of blood vessel phenotype. It remains to be determined in a larger number of cases whether lymphatic differentiation and podoplanin expression could serve as reliable criteria for recognizing subgroups of G3 angiosarcomas of clinical and biological relevance. The expression of podoplanin in a subset of angiosarcoma tumor cells could suggest their derivation from a common precursor of lymphatic and blood vascular endothelial cells.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgements |
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Footnotes |
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Supported by SFB 005, Projects 7 and 11, from the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung (to D. K. and E. T.). H. K. was supported by a fellowship from the Leukemia Society of America (Grant # 5015–94).
Accepted for publication November 5, 1998.
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