Lymphatic microvessel density and lymphovascular invasion assessed by anti-podoplanin immunostaining in human breast cancer.

BACKGROUND: The development of an antibody against podoplanin has enabled us to selectively stain lymphatic vessels in breast cancer samples for the first time. MATERIALS AND METHODS: We investigated lymphatic vessels in 45 specimens of invasive breast cancer by immunostaining for podoplanin. Lymphatic microvessel density (LMVD) was correlated with various clinical and histopathological parameters. LMVD was also compared to blood microvessel density (BMVD), assessed by CD34 -immunostaining. RESULTS: LMVD as well as lymphovascular invasion (LVI) correlated significantly with the lymph node status (p=0.001/ p=0.035). Logistic regression revealed that LVI was the more important factor for development of lymph node metastasis (p=0.043). There was no significant association between various clinical and histopathological parameters and LMVD or LVI, nor was a correlation found between LMVD and BMVD (p=0.121). CONCLUSION: High LMVD and the presence of LVI are strongly associated with lymph node metastasis in breast cancer.

Orbital lymphangioma with positive immunohistochemistry of lymphatic endothelial markers (vascular endothelial growth factor receptor 3 and podoplanin).

BACKGROUND: Existence of true orbital lymphangiomas has been questioned in recent years. Therefore an orbital lymphangioma was analyzed with two new specific markers of lymphatic endothelium. METHODS: Case-report with clinicopathological, immunohistochemical, and ultrastructural findings. A 25-year-old man presented with recurrent lower lid "hematomas" and a pea-sized tumor palpable in the left lower lid. Magnetic resonance imaging showed an inferonasally located orbital tumor which extended to the posterior pole of the eye. The highly vascularized tumor was excised by medial orbitotomy. RESULTS: Histopathologically, the mass consisted of large, erythrocyte-filled cavernous vessels without evidence of smooth muscle cells or pericytes surrounding them. Numerous lymph follicles and small arterioles were scattered between them. Immunohistochemically, endothelial cells lining the lumina of the cavernous vessels were partly positive for podoplanin and vascular endothelial growth factor receptor 3 (flt-4), two markers of lymphatic endothelium. These markers did not react with endothelial cells lining the arterioles. Ultrastructurally, cavernous vessels displayed features characteristic of lymphatic vessels, and the smaller vessels demonstrated signs of arterioles. CONCLUSION: Ultrastructural analysis and immunohistochemistry using two new markers of lymphatic endothelium suggest a lymphatic nature of large vessels in an orbital lymphangioma. A greater series of vascular orbital tumors must be studied with these new lymph endothelial markers to confirm the existence of true orbital lymphangiomas and to analyze different profiles of lymph endothelial marker expression.

Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages.

A plexus of lymphatic vessels guides interstitial fluid, passenger leukocytes, and tumor cells toward regional lymph nodes. Microvascular endothelial cells (ECs) of lymph channels (LECs) are difficult to distinguish from those of blood vessels (BECs) because both express a similar set of markers, such as CD31, CD34, podocalyxin, von Willebrand factor (vWF), etc. Analysis of the specific properties of LECs was hampered so far by lack of tools to isolate LECs. Recently, the 38-kD mucoprotein podoplanin was found to be expressed by microvascular LECs but not BECs in vivo. Here we isolated for the first time podoplanin(+) LECs and podoplanin(-) BECs from dermal cell suspensions by multicolor flow cytometry. Both EC types were propagated and stably expressed VE-cadherin, CD31, and vWF. Molecules selectively displayed by LECs in vivo, i.e., podoplanin, the hyaluronate receptor LYVE-1, and the vascular endothelial cell growth factor (VEGF)-C receptor, fms-like tyrosine kinase 4 (Flt-4)/VEGFR-3, were strongly expressed by expanded LECs, but not BECs. Conversely, BECs but not LECs expressed VEGF-C. LECs as well as BECs formed junctional contacts with similar molecular composition and ultrastructural features. Nevertheless, the two EC types assembled in vitro in vascular tubes in a strictly homotypic fashion. This EC specialization extends to the secretion of biologically relevant chemotactic factors: LECs, but not BECs, constitutively secrete the CC chemokine receptor (CCR)7 ligand secondary lymphoid tissue chemokine (SLC)/CCL21 at their basal side, while both subsets, upon activation, release macrophage inflammatory protein (MIP)-3alpha/CCL20 apically. These results demonstrate that LECs and BECs constitute stable and specialized EC lineages equipped with the potential to navigate leukocytes and, perhaps also, tumor cells into and out of the tissues.

Inflammatory stromal reaction correlates with lymphatic microvessel density in early-stage cervival cancer.

BACKGROUND: In early-stage cervical cancer, high lymphatic microvessel density (LMVD) indicates favorable prognosis. This unexpected finding was thought to be an effect of local immunological response, although no data supported this thesis. MATERIALS AND METHODS: LMVD and lymphovascular invasion (LVI) were assessed in 85 specimens of cervical cancer stage pT1b by immunostaining for podoplanin, a marker for lymphatic endothelia. Local immunological response, evident by inflammatory stromal reaction (ISR), was determined in H&E-stained slides and rated from grade 1 (absent or weak) to 3 (strong) RESULTS: A good correlation of LMVD and ISR was found (p=0.002). While a strong correlation between LMVD and the presence of LVI was found (p<0.001), no association between LMVD and pelvic lymph node involvement (p=0.732) was observed. ISR indicated favourable prognosis of patients (p=0.0247, log-rank test). CONCLUSION: Our findings suggest that ISR might play a role in the induction of lymphangiogenesis in early stage cervical cancer.

Vascular endothelial growth factor accelerates renal recovery in experimental thrombotic microangiopathy.

BACKGROUND: Renal microvascular injury characterizes thrombotic microangiopathy (TMA). The possibility that angiogenic growth factors may accelerate recovery in TMA has not been studied. METHODS: TMA was induced in rats by the selective right renal artery perfusion of antiglomerular endothelial cell IgG (30 mg/kg). Twenty-four hours later, rats received vascular endothelial growth factor (VEGF121, 100 microg/kg/day) or vehicle (control) daily until day 14. To evaluate renal function, the unperfused left kidney was removed at day 14, and rats were sacrificed at day 17. RESULTS: The induction of TMA was associated with loss of glomerular and peritubular capillary endothelial cells and decreased arteriolar density at day 1. Some spontaneous capillary recovery was present by day 17; however, repair was incomplete, and severe tubulointerstitial damage occurred. The lack of complete microvascular recovery was associated with reduced VEGF immunostaining in the outer medulla. VEGF-treated rats had more glomeruli with intact endothelium, less glomerular ischemia (collapsed glomeruli), and greater peritubular capillary density with less peritubular capillary loss. This was associated with less tubulointerstitial fibrosis, less cortical atrophy, and improved renal function. CONCLUSIONS: VEGF accelerates renal recovery in this experimental model of TMA. These studies suggest that angiogenic growth factors may provide a new therapeutic strategy for diseases associated with endothelial cell injury.

[Focal proliferation in reactive hyperplastic parathyroid tissue in end-stage renal failure--implication for autotransplantation after total parathyroidectomy]. / Fokale Proliferationen in reaktiv hyperplastischen Nebenschilddrüsen bei terminaler Niereninsuffizienz-Implikation für die Autotransplantation nach totaler Parathyreoidektomie.

Total parathyroidectomy with simultaneous autotransplantation may be associated with recurrence of graft-dependent hyperfunction due to excessive proliferation. We performed macroscopic tissue selection with a stereomicroscope prior to autotransplantation, which resulted in very low recurrence rates. As this technique greatly depends on experience, we investigated the possibility of additionally using proliferation staining (PCNA, MIB-1) for the detection of dysfunctional tissue. Selected tissue from 26 patients was investigated. Serial sections of freshly removed parathyroid tissue were correlated with their macroscopic appearance, HE and immunohistochemically stained paraffin sections, and with semithin Epon sections. The asymptotic growth mode of clonal proliferating regions was reflected by highest staining intensity (1-5%) in small to medium sized foci (diffuse, up to 3 mm in diameter) and very low staining in large areas (diffuse or nodular, 5-15 mm in diameter, from 0.03 to 0.003% positive cells). Thus, very large dysfunctional regions with (almost) no proliferation could not be detected by this method. However, they were very evident on macroscopic investigation. In conclusion, multiple fulminant recurrence after parathyroidectomy can be prevented by selecting tissue after proliferation staining. This may allow a delayed autotransplantation after total parathyoidectomy for those surgeons lacking experience in macroscopic tissue classification.

Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi's sarcoma tumor cells.

Despite intensive research over the past decade, the exact lineage relationship of Kaposi's sarcoma (KS) tumor cells has not yet been settled. In the present study, we investigated the expression of two markers for lymphatic endothelial cells (EC), ie, vascular endothelial growth factor receptor-3 (VEGFR-3) and podoplanin, in AIDS and classic KS. Both markers were strongly expressed by cells lining irregular vascular spaces in early KS lesions and by tumor cells in advanced KS. Double-staining experiments by confocal laser microscopy established that VEGFR-3-positive and podoplanin-positive cell populations were identical and uniformly expressed CD31. By contrast, these cells were negative for CD45, CD68, and PAL-E, excluding their hemopoietic and blood vessel endothelial cell nature. Podoplanin expression in primary KS tumor lysates was confirmed by Western blot analysis. Both splice variants of VEGFR-3 were found in KS-tumor-derived RNA by RT-PCR. In contrast to KS tumor cells in situ, no expression of VEGFR-3 and podoplanin was detected in any of four KS-derived spindle cell cultures and in one KS-derived autonomously growing cell line (KS Y-1). Our findings that KS tumor cells express two lymphatic EC markers in situ strongly suggest that they are related to or even derived from the lymphatic EC lineage. Lack of these antigens on cultured cells derived from KS lesions indicates that they might not represent tumor cells that grow in tissue culture, but rather other cell types present in KS lesions.

Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.

Angiosarcomas apparently derive from blood vessel endothelial cells; however, occasionally their histological features suggest mixed origin from blood and lymphatic endothelia. In the absence of specific positive markers for lymphatic endothelia the precise distinction between these components has not been possible. Here we provide evidence by light and electron microscopic immunohistochemistry that podoplanin, a approximately 38-kd membrane glycoprotein of podocytes, is specifically expressed in the endothelium of lymphatic capillaries, but not in the blood vasculature. In normal skin and kidney, podoplanin colocalized with vascular endothelial growth factor receptor-3, the only other lymphatic marker presently available. Complementary immunostaining of blood vessels was obtained with established endothelial markers (CD31, CD34, factor VIII-related antigen, and Ulex europaeus I lectin) as well as podocalyxin, another podocytic protein that is also localized in endothelia of blood vessels. Podoplanin specifically immunolabeled endothelia of benign tumorous lesions of undisputed lymphatic origin (lymphangiomas, hygromas) and was detected there as a 38-kd protein by immunoblotting. As paradigms of malignant vascular tumors, poorly differentiated (G3) common angiosarcomas (n = 8), epitheloid angiosarcomas (n = 3), and intestinal Kaposi's sarcomas (n = 5) were examined for their podoplanin content in relation to conventional endothelial markers. The relative number of tumor cells expressing podoplanin was estimated and, although the number of cases in this preliminary study was limited to 16, an apparent spectrum of podoplanin expression emerged that can be divided into a low-expression group in which 0-10% of tumor cells contained podoplanin, a moderate-expression group with 30-60% and a high-expression group with 70-100%. Ten of eleven angiosarcomas and all Kaposi's sarcomas showed mixed expression of both lymphatic and blood vascular endothelial phenotypes. By double labeling, most podoplanin-positive tumor cells coexpressed endothelial markers of blood vessels, whereas few tumor cells were positive for individual markers only. From these results we conclude that (1) podoplanin is a selective marker of lymphatic endothelium; (2) G3 angiosarcomas display a quantitative spectrum of podoplanin-expressing tumor cells; (3) in most angiosarcomas, a varying subset of tumor cells coexpresses podoplanin and endothelial markers of blood vessels; and (4) all endothelial cells of Kaposi's sarcomas expressed the lymphatic marker podoplanin.

[Podoplanin--a specific marker for lymphatic endothelium expressed in angiosarcoma]. / Podoplanin--ein spezifischer Marker für Lymphgefässendothelien wird in Angiosarkomen exprimiert.

AIMS: Angiosarcomas apparently derive from endothelia of the blood vasculature, however occasionally their histologic features suggest mixed origin from blood and lymphatic endothelia. In the absence of specific positive markers for lymphatic endothelia the precise distinction between these components was not possible so far. Here we provide evidence that podoplanin, a approximately 38 kD membrane glycoprotein of podocytes is a specific marker of lymphatic endothelium that was used to identify the relative fraction of tumor cells with lymphatic or blood vascular endothelial phenotype in vascular tumors. METHODS: Podoplanin was localized in normal human skin and kidney cortex by immunohistochemistry on paraffin sections, double immunofluorescence on frozen sections with PAL-E, immunoelectron microscopy and by immunoblotting. 45 vascular tumors (29 benign lesions, 11 angiosarcomas and 5 gastrointestinal Kaposi's sarcomas) were evaluated for podoplanin expression. Complementary staining was obtained with established endothelial markers (CD 31, CD 34, Factor VIII related antigen, UEA I) and with podocalyxin, another podocytic protein mainly present in endothelia of blood vessels. RESULTS: In human tissues podoplanin is specifically expressed in the endothelium of lymphatics, but not in blood vasculature or in hemangiomas. This expression is preserved in endothelia of all benign lymphatic tumorous lesions and all Kaposi's sarcomas examined. By contrast 10 out of 11 G3 angiosarcomas contained only variable fractions of podoplanin-expressing tumor cells. Most tumor cells coexpressed podoplanin and markers of blood vessel phenotype. CONCLUSIONS: (1) Podoplanin is a selective marker of lymphatic endothelium; (2) G3 angiosarcomas display a quantitative spectrum of podoplanin-expressing tumor cells; (3) In the majority of angiosarcomas tumor cells coexpress podoplanin and endothelial markers of blood vessels; (4) All endothelial cells of Kaposi's sarcomas expressed the lymphatic marker podoplanin.

Epitope-specific antibodies to the 43-kD glomerular membrane protein podoplanin cause proteinuria and rapid flattening of podocytes.

The 43-kD integral membrane protein podoplanin is localized on the surface of rat podocytes, and transcriptionally downregulated in rat puromycin nephrosis. In this study, a single intravenous injection of polyclonal rabbit anti-podoplanin IgG resulted in selective binding of IgG to the entire podocyte's surface. Some IgG produced by different rabbits rapidly induced transient proteinuria (approximately 350 mg/24 h at day 1, normal levels around day 5), whereas other IgG were ineffective. All anti-podoplanin IgG shared a common binding site at amino acids 39 to 47 (DDMVNPGLE), whereas IgG inducing glomerular damage specifically bound to an additional epitope at amino acids 74 to 79 (PIEELP), as observed by a SPOTs analysis on overlapping synthetic peptides. Proteinuria was not prevented by complement depletion or by treatment with the oxygen radical scavenger dimethylthiourea. Injection of Fab fragments failed to induce glomerular pathology, indicating that dimerization of podoplanin by divalent IgG was required. Proteinuria was paralleled by extensive flattening of foot processes that was also induced by blood-free perfusion of isolated rat kidneys with anti-podoplanin IgG. Thus, glomerular changes were due to direct interaction of distinct epitope(s) of podoplanin and divalent IgG. These results provide evidence that podoplanin plays a role in maintaining the unique shape of podocyte foot processes and glomerular permeability.

Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis.

Puromycin aminonucleoside nephrosis (PAN), a rat model of human minimal change nephropathy, is characterized by extensive flattening of glomerular epithelial cell (podocyte) foot processes and by severe proteinuria. For comparison of expression of glomerular membrane proteins of normal and PAN rats, a membrane protein fraction of isolated rat glomeruli was prepared and monoclonal antibodies were raised against it. An IgG-secreting clone designated LF3 was selected that specifically immunolabeled podocytes of normal but not of PAN rats. The antigen of LF3 IgG was identified as a 43-kd glycoprotein. Molecular cloning of its cDNA was performed in a delta gt11 expression library prepared from mRNA of isolated rat glomeruli. The predicted amino acid sequence indicated a 166-amino-acid integral membrane protein with a single membrane-spanning domain, two potential phosphorylation sites in its short cytoplasmic tail, and six potential O-glycosylation sites in the large ectodomain. High amino acid sequence identities were found to membrane glycoproteins of rat lung and bone and mouse thymus epithelial cells as well as to a phorbol-ester-induced protein in a mouse osteoblast cell line and to a canine influenza C virus receptor. In PAN, expression of this 43-kd protein was selectively reduced to < 30%, as determined by quantitative immunogold electron microscopy, immunoblotting, and Northern blotting. These data provide evidence that transcription of the 43-kd transmembrane podocyte glycoprotein is specifically down-regulated in PAN. To indicate that this protein could be associated with transformation of arborized foot processes to flat feet (Latin, pes planus) we have called it podoplanin.

Quantitation of collagen fibril cross-section profiles in aging human veins.

The cross-section profiles and the diameter distribution of collagen fibrils were examined quantitatively in normal human internal jugular veins at different ages (first, fifth, and eighth decades). All fibrils showed a regular cross-striation pattern of native-type collagen fibrils irrespective of their cross-section profiles. Irregularly outlined ("dysplastic") fibrillar profiles were observed among the normally occurring circular cross-section profiles. The frequency of such unusual fibrils significantly increased with age. This increase was more pronounced in the tunica media as compared with the tunica adventitia. In the tunica media diameters of collagen fibrils also generally increased with age. In the tunica adventitia, on the other hand, fibrillar diameters were not significantly altered at different ages. The results of this study indicate that the frequency of both the irregularly outlined fibrillar cross-section profiles and increased fibrillar diameters depends on age in normal vascular walls. Therefore, it is concluded that the occurrence of "dysplastic" fibrils is a physiologic age-related phenomenon rather than a morphologic sign of pathologic alteration of collagen. The higher frequency of irregularly outlined collagen fibrils in the tunica media may indicate a higher and/or altered synthetic behavior of smooth muscle cells when compared with fibroblasts of the tunica adventitia.