Cell and Tissue Research

, Volume 328, Issue 2, pp 317–328

Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas

  • Noora Vainionpää
  • Ralf Bützow
  • Mika Hukkanen
  • David G. Jackson
  • Taina Pihlajaniemi
  • Lynn Y. Sakai
  • Ismo Virtanen
Regular Article

DOI: 10.1007/s00441-006-0366-2

Cite this article as:
Vainionpää, N., Bützow, R., Hukkanen, M. et al. Cell Tissue Res (2007) 328: 317. doi:10.1007/s00441-006-0366-2

Abstract

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin α5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin α5 chain and Lutheran glycoprotein, a receptor for the laminin α5 chain. In normal lymphatic capillaries, the presence of primarily α4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin α5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.

Keywords

Basement membrane Lymphatic vessel Endothelium Laminin Ovarian carcinoma Human 

Introduction

Lymphatic vessels transport excess fluid and macromolecules from the extracellular space of tissues and are passageways for the cells of the immune system. The impairment of these functions leads to lymphoedema and disturbances in immune responses (Oliver and Alitalo 2005; Randolph et al. 2005; Zawieja 2005). Lymphatic vessels are the main routes for the intravasation and dissemination of cancer cells, with lymph node metastasis being a prognostic factor for the clinical outcome in human carcinomas (Cao 2005; Oliver and Alitalo 2005; Wilting et al. 2005).

The formation of vessels and the function of endothelium are regulated by growth factors and interactions of endothelial cells with the surrounding extracellular matrix (ECM; Davis and Senger 2005; Ji 2006; Kalluri 2003). Basement membranes (BM), specialized sheets of ECM located adjacent to endothelia, epithelia and certain single cells support cells within tissues and maintain tissue architecture. The interaction of cells with BM proteins leads to the activation of intracellular signalling cascades and regulates cell behaviour. BMs consist of independent networks of laminins and type IV collagens bound together by nidogen. In addition, they comprise many proteoglycans, such as collagen type XVIII and perlecan (Davis and Senger 2005; Kalluri 2003; Yurchenco et al. 2004).

Among BM constituents, laminins are crucial for BM assembly and have a developmentally regulated, tissue-specific distribution and function (Yurchenco et al. 2004). Each laminin is a trimer of α, β and γ chains. Five α chains, three β chains and three γ chains have been characterized and form at least 15 different laminin isoforms (Aumailley et al. 2005; Miner and Yurchenco 2004). Blood vessel endothelial BMs are known to contain mainly laminin α4, α5, β1, β2 and γ1 chains, constituents of laminins (Lm)-411, Lm-421, Lm-511 and Lm-521 (Davis and Senger 2005; Hallmann et al. 2005; Patarroyo et al. 2002). Absence of these laminins in knock-out mice leads to certain defects in blood vessel formation (Miner et al. 1998; Miner and Li 2000; Thyboll et al. 2002).

Lymphatic vessels are fluid conduits lined by endothelial cells but differ from blood vessels in many structural aspects: initial lymphatic vessels are blind-ended structures, the lumina of the vessels are wider and more irregular, the endothelial cells are attenuated and the lymphatic capillaries are not enveloped by pericytes (Ji 2006; Leak and Burke 1966; Schmid-Schönbein 1990). Lymphatic endothelia are attached to fibrillin anchoring filaments, which have been suggested to function in resisting the collapse of the vessels and in the propulsion of the lymph within the vessels (Leak and Burke 1966; Solito et al. 1997). BMs of lymphatic endothelia appear discontinuous in electron microscopy (Leak and Burke 1966). Gene array analyses have shown that the expression of genes coding for BM proteins is lower in lymphatic endothelial cells than in endothelial cells of blood vessels (Hirakawa et al. 2003; Petrova et al. 2002; Podgrabinska et al. 2002). A general lack of immunoreactivity for laminin has been described in lymphatic vessels (Barsky et al. 1983; Wigle et al. 2002). Immunoreactivity for type IV collagens, however, has been found in the BM region of lymphatic endothelium in normal human skin (Sauter et al. 1998).

In this study, we have evaluated the distribution of BM proteins in lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-immunoreactive lymphatic vessels of normal human skin, digestive tract and ovary. Ovarian carcinomas have a high tendency to form lymphatic metastases (Ayhan et al. 2005) and possess intratumoural lymphatic vessels (Ueda et al. 2005; Yokoyama et al. 2003). To compare the distribution of BM constituents in lymphatic capillaries of normal and carcinoma tissues, we have also studied the distribution of BM proteins in LYVE-1-immunoreactive lymphatic capillaries of ovarian carcinomas.

Materials and methods

Tissues

Specimens of normal human adult skin (n = 8), oesophagus (n = 4), stomach (n = 4), small intestine (n = 4) and colon (n = 4) were obtained from surgical operations undertaken at Jorvi Hospital (Espoo, Finland) and were retrieved from the files of the Institute of Biomedicine/Anatomy with institutional permission. Specimens of normal human ovary (n = 5) and ovarian carcinomas (n = 15) were obtained from surgical operations at Helsinki University Central Hospital, Department of Obstetrics and Gynaecology (Helsinki, Finland) and retrieved from the files of the Department of Pathology with institutional permission. Histopathological evaluation of the specimens was performed by a pathologist who examined sections stained with haematoxylin-eosin. Normal tissue specimens were found free of any pathological processes. The carcinoma specimens were classified and graded according to the World Health Organization classification. They included 10 endometrioid carcinomas (six grade 1, two grade 2, two grade 3), three serous carcinomas (two grade 1, one grade 2) and two mucinous carcinomas (one grade 1, one grade 2). The tissues were frozen in liquid nitrogen and stored at −75°C until use.

Antibodies

The following mouse monoclonal antibodies (MAbs) were used: 161EB7 against the human Lm α1 chain (Virtanen et al. 2000), 5H2 against the Lm α2 chain (Leivo and Engvall 1988), BM-2 against the Lm α3 chain (Rousselle et al. 1991), 168FC10 against the human Lm α4 chain (Petäjäniemi et al. 2002), 4C7 against the human Lm α5 chain (Engvall et al. 1986; Tiger et al. 1997), 114DG10 against the human Lm β1 chain (Virtanen et al. 1997), S5F11 against the human Lm β2 chain (Wewer et al. 1997), 6F12 against the human Lm β3 chain (Marinkovich et al. 1992), 113BC7 against the human Lm γ1 chain (Määttä et al. 2001), D4B5 against the human γ2 chain (Mizushima et al. 1998), M3F7 against human collagen type IV (α1/2; Foellmer et al. 1983), NP32 against human collagen type VII (Sakai et al. 1986a), N2 against human collagen type XVIII (Valtola et al. 1999), A9 against human nidogen-1 (Katz et al. 1991) and 201 against fibrillin-1 (Sakai et al. 1986b). For the identification of lymphatic vessel endothelia, we used two different rabbit antisera against LYVE-1 (Banerji et al. 1999; Acris-antibodies, Hiddenhausen, Germany), rabbit antiserum to podoplanin (Breiteneder-Geleff et al. 1999) and MAb to vascular endothelial growth factor receptor-3 (VEGFR-3; Ludwid Institute for Cancer Research, and Licentia, Zürich, Switzerland). Blood vessel endothelia were identified with tetramethylrhodamine-isothiocyanate-coupled Ulex europaeus-I agglutinin (TRITC-UEA-I; Holthöfer et al. 1982; Vector Laboratories, Burlingame, Calif.), MAb EC-1 to platelet endothelial cell adhesion molecule -1 (PECAM-1/CD31; Tani et al. 1996) and MAb C8 to factor-VIII-related antigen (FVIIIR:Ag; Meyer et al. 1984). Lutheran glycoprotein was identified with MAb BRIC221 (Serotec, Oxford, UK). In addition, we used MAb hSM-V against smooth muscle myosin (Sigma, St. Louis, Mo.), MAb 10D6 against CD163 (Novocastra Laboratories, Newcastle upon Tyne, UK) and MAb KP1 against CD68 (NeoMarkers, Fremont, Calif.).

Immunohistochemisty

For immunohistochemistry, cryosections (6–7 μm thick) were fixed in acetone at −20°C for 10 min. The specimens were first exposed to MAbs, followed by Alexa Fluor 568 goat anti-mouse IgG (Molecular Probes, Eugene, Ore.) or Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes). For double-labelling, the specimens were then exposed either to rabbit antisera and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes) or to TRITC-UEA-I for 30 min. The specimens were embedded in sodium veronal-glycerol buffer (pH 8.4) or polyvinylalcohol mounting medium and examined either with a Leica Aristoplan microscope (Leica Microsystems, Wetzlar, Germany) equipped with appropriate filters or by laser scanning confocal microscopy with a Leica TCS SP2 system (Leica Microsystems) and argon and krypton laser excitation lines at 488 nm and 568 nm. Image stacks were collected by using sequential scanning through the sections at a standardized 120-nm z-sampling density. The results are shown as maximum intensity projections of four section stacks. Negative controls involved the omission of the primary antibodies. In positive controls, all antibodies showed immunoreactivity in the specific locations of tissues at which immunoreactivity for the corresponding proteins had previously been described.

Results

Identification of lymphatic vessels

First, we evaluated methods for identifying lymphatic capillaries and for distinguishing them from blood capillaries. Previous studies have shown that lymphatic capillaries differ from blood capillaries with respect to their morphological characteristics including a wider lumen, irregular endothelial morphology and the lack of smooth-muscle-like cells in their intima (Ji 2006; Leak and Burke 1966; Schmid-Schönbein 1990). Lymphatic capillaries also differ from larger lymphatic vessels, precollectors and collecting vessels, with regard to their lack of smooth-muscle-like cells (Schmid-Schönbein 1990). Although lymphatic endothelial cells are known to express blood vessel endothelial markers PECAM-1/CD31 and FVIIIR:Ag and UEA-I-binding sites to some extent, previous studies have suggested that the distribution of PECAM-1 is different (Sauter et al. 1998; Sleeman et al. 2001) and that the reactivity for FVIIIR:Ag and UEA-I is weaker in lymphatic vessels than in blood vessels (Banerji et al. 1999; Gnepp 1987; Sleeman et al. 2001). Lymphatic vessels can be identified on the basis of their immunoreactivity for lymphatic endothelial markers, such as LYVE-1, podoplanin and VEGFR-3 (Banerji et al. 1999; Jackson 2003; Sleeman et al. 2001). In addition to lymphatic vessels, LYVE-1 immunoreactivity has been detected in liver and spleen sinusoids and in tissue macrophages (Banerji et al. 1999; Jackson 2003), podoplanin immunoreactivity in kidney podocytes, osteoblastic cells, type I pneumocytes and cells of the choroid plexus (Sleeman et al. 2001) and VEGFR-3 immunoreactivity in cells of the haematopoietic system, developing blood vessels, fenestrated capillaries and blood capillaries of tumours and chronic wounds (Sleeman et al. 2001), amongst others.

In the present study, LYVE-1-immunoreactive vessels were found in all specimens. All vessels presenting LYVE-1 immunoreactivity (Fig. 1a) showed thin irregular vessel walls, weak reactivity for FVIIIR:Ag (Fig. 1b) and TRITC-UEA-1 (Fig. 1c) and net-like immunoreactivity for PECAM-1 (Fig. 1d). Blood vessels possessed thicker vessel walls, stronger reactivity for FVIIIR:Ag (not shown) and TRITC-UEA-I (Fig. 1c), more uniform reactivity for PECAM-1 (Fig. 1d) and a lack of immunoreactivity for LYVE-1 (not shown). Some LYVE-1 immunoreactivity was found in single cells that were distinguishable from lymphatic capillaries by their morphology and immunoreactivity for macrophage marker CD68 and occasionally also for CD163 (not shown). Fibrillin fibres, found as an abundant network in ECM, ran parallel with LYVE-1-immunoreactive lymphatic endothelium (Fig. 1e,f). All LYVE-1-immunoreactive vessels lacked immunoreactivity for smooth muscle myosin (Fig. 1g,h), which was used to identify smooth-muscle-like cells (Diaz-Flores et al. 1991), whereas immunoreactivity for smooth muscle myosin was found in all blood vessels (not shown). Podoplanin-immunoreactive vessels of the skin showed similar characteristics to those of the LYVE-1-immunoreactive vessels, whereas the podoplanin antiserum did not detect lymphatic vessels in the gastrointestinal tract, ovary or ovarian carcinoma specimens (not shown). MAb against VEGFR-3 did not give immunoreactivity with lymphatic vessels in our specimens but, in double-labelling with LYVE-1 antiserum, it showed immunoreactivity with a few cell-like structures near LYVE-1-immunoreactive lymphatic vessels, such as the lacteals of intestinal villi (not shown). Therefore, we used double-labelling with LYVE-1 antiserum to show the distribution of BM proteins in lymphatic vessels.
Fig. 1

Identification of the lymphatic vessels. Lymphatic capillaries of human skin (arrows) showed thin irregular vessel walls, immunoreactivity for lymphatic endothelial marker LYVE-1 (a) and, in double-labelling experiments, weak immunoreactivity for FVIIIR:Ag (b). In double-labelling with UEA-I (c) and PECAM-1 (d), reactivity for UEA-I was clearly weaker in lymphatic capillaries (long arrows) than in blood capillaries (short arrows), whereas immunoreactivity for PECAM-1 showed a scattered net-like distribution in lymphatic capillaries (long arrows) and a more uniform pattern in blood capillaries (short arrows). Immunoreactivity for fibrillin-1 (e) was found in ECM as an abundant network, which was found in contact with the epithelium (arrowheads). As shown by double-labelling with LYVE-1 (f), fibrillin-1 fibres were also found in colocalization with lymphatic endothelium (long arrows). In further double-labelling experiments, LYVE-1-immunoreactive lymphatic vessels (g) lacked immunoreactivity for smooth muscle (SM) myosin (h). Bar 25 μm

BM proteins in lymphatic vessels of skin, ovary and digestive tract

The results of the distribution of BM proteins in lymphatic vessels are summarized in Table 1. In skin (Fig. 2), fibrillar immunoreactivity for the Lm α4, β1, β2 and γ1 chains was colocalized with LYVE-1 immunoreactivity. The immunoreactivity for the Lm β1 chain was weaker than that for the Lm β2 chain. All skin lymphatic capillaries lacked immunoreactivity for the Lm α1, α2, α3, α5, β3 and γ2 chains. Among other BM proteins (Fig. 3), immunoreactivity for nidogen-1, collagen type IV (α1/2) and collagen type XVIII was found in lymphatic endothelium, whereas the lymphatic capillaries lacked immunoreactivity for collagen type VII. The immunoreactivity for BM constituents in lymph vessels was much weaker than that found in blood vessels (Fig. 2, Lm α4 chain; others not shown). In normal ovary, immunoreactivity for BM proteins in lymphatic vessels was comparable with that found in the skin (Fig. 3, Lm α5 chain; others not shown). In most specimens of the digestive tract, lymphatic vessels showed either a lack of immunoreactivity (three oesophagus, one stomach and two small intestine specimens) or traces of immunoreactivity (one oesophagus, three stomach, one small intestine and three colon specimens) for the Lm α5 chain (Fig. 3), although immunoreactivity for the Lm α5 chain was seen in one small intestine and one colon specimen. In all specimens of the digestive tract, immunoreactivity for other BM constituents in lymphatic vessels was comparable with that found in the skin (not shown).
Table 1

Immunoreactivity for basement membrane (BM) constituents in lymphatic vessels of various tissues (+ immunoreactivity in all specimens, (+) heterogeneous immunoreactivity in some of the specimens, lack of immunoreactivity in all specimens)

 BM constituent

Skin

Digestive tract

Ovary

Ovarian carcinoma

Lm α1

(+)

Lm α2

(+)

Lm α3

Lm α4

+

+

+

+

Lm α5

(+)

+

Lm β1

+

+

+

+

Lm β2

+

+

+

+

Lm β3

Lm γ1

+

+

+

+

Lm γ2

Collagen (α1/2) IV

+

+

+

+

Collagen VII

Collagen XVIII

+

+

+

+

Nidogen-1

+

+

+

+

Fig. 2

Distribution of laminins in lymphatic capillaries of skin. Lymphatic endothelium was visualized by double-labelling with LYVE-1 antiserum. Lymphatic capillaries lacked immunoreactivity for the Lm α1 (a, b), α2 (c, d) and α3 chains (e, f) but showed immunoreactivity for the Lm α4 chain (g, h, long arrows), which was weaker in the lymphatic capillaries than in blood capillaries (short arrows). Immunoreactivity for the Lm α5 chain (i, j) was found in blood endothelium (short arrows) but not in lymphatic endothelium (long arrows). Lymphatic endothelium showed weaker immunoreactivity for the Lm β1 chain (k, l) than for the β2 chain (m, n). Lymphatics lacked immunoreactivity for the Lm β3 chain (o, p). Lymphatic endothelium showed immunoreactivity for the Lm γ1 chain (q, r) but lacked immunoreactivity for the Lm γ2 chain (s, t). Bars 25 μm

Fig. 3

Distribution of BM constituents in lymphatic capillaries. Lymphatic endothelium was visualized by double-labelling with LYVE-1. Lymphatic capillaries of skin showed immunoreactivity for nidogen (a, b) and α1/2 chains of collagen (Coll) type IV (c, d). The skin lymphatics lacked immunoreactivity for collagen type VII (e, f) but immunoreactivity for collagen type XVIII was found in co-alignment with lymphatic endothelium (g, h). In the ovary, lymphatic capillaries lacked immunoreactivity for the Lm α5 chain (i, j). In the stomach (k, l), traces of immunoreactivity for the Lm α5 chain were found in lymphatic capillaries (long arrows), whereas strong uniform immunoreactivity was found in blood capillaries (short arrows). Bars 25 μm

In order to obtain a better spatial conception of the relationship between lymphatic endothelial cells and the BM proteins, we also studied BM protein distribution in lymphatic capillaries by laser scanning confocal microscopy. The results revealed (Fig. 4) that fragmentary immunoreactivity for the Lm α4, β1, β2 and γ1 chains, nidogen-1, collagen type IV (α1/2) and collagen type XVIII occurred in contact with the lymphatic endothelium. No immunoreactivity for the Lm α5 chain was found in the lymphatics of skin and ovary but patches of immunoreactivity were found in some lymphatics of the digestive tract.
Fig. 4

BM protein distribution in lymphatic vessels as shown by double-labelling with LYVE-1 and revealed by laser scanning confocal microscopy. In skin, immunoreactivity for the Lm α4 (a), β1 (b), β2 (c) and γ1 (d) chains, nidogen (e), collagen type IV (α1/2) (f) and collagen type XVIII (g) was found as a discontinuous layer in contact with LYVE-1-immunoreactive lymphatic endothelium. Lymphatic capillaries of skin (h) and ovary (i) lacked immunoreactivity for the Lm α5 chain, whereas patches of immunoreactivity for the Lm α5 chain were found (j, arrows) in lymphatic capillaries of the gastrointestinal tract. In ovarian carcinomas (ca), immunoreactivity for the Lm α5 chain was found adjacent to the lymphatic endothelium (k, arrows). Bar 20 μm

BM proteins in lymphatic vessels of ovarian carcinomas

In agreement with previous studies (Ueda et al. 2005; Yokoyama et al. 2003), LYVE-1-immunoreactive lymphatic vessels were found in the large peripheral stromal areas in all ovarian carcinomas. In endometrioid ovarian carcinomas, many lymphatic vessels were also found, within the tumours, in thin cords of stroma between the islets of carcinoma cells (Fig. 5).
Fig. 5

BM protein distribution in lymphatic capillaries of ovarian endometrioid carcinoma as shown by double-labelling with LYVE-1 antiserum (arrows position of lymphatic vessels). In the tumours in which immunoreactivity for the Lm α1 (a, b) or α2 (c, d) chain was found in the stroma (st), patches of immunoreactivity for these Lm chains were also detected in some lymphatic vessels. Immunoreactivity for the Lm α4 (e, f) and α5 (g, h) chains was found in the lymphatic endothelium. In the lymphatic endothelium, immunoreactivity for the Lm β1 chain (i, j) was pronounced when compared with that for the Lm β2 chain (k, l). Immunoreactivity for the Lm γ1 chain (m, n), nidogen (o, p), collagen type IV (α1/2) (q, r) and collagen type XVIII (s, t) was also found in the lymphatic endothelium. Bar 25 μm

In endometrioid ovarian carcinomas (Fig. 5), most lymphatic vessels lacked immunoreactivity for the Lm α1 and α2 chains. In some specimens in which the stroma showed immunoreactivity for the Lm α1 chain (5/10 of specimens) or the Lm α2 chain (3/10 of specimens), patchy immunoreactivity for these laminin chains was also detected in the BM region of some lymphatic capillaries. The BM region of carcinoma lymphatic vessels showed immunoreactivity for Lm α4, α5, β1, β2 and γ1 chains, nidogen-1, collagen type IV (α1/2) and collagen type XVIII. Immunoreactivity for the Lm β1 chain was prominent when compared with that for the Lm β2 chain. By laser scanning confocal microscopy, the immunoreactivity for BM proteins appeared as a layer in contact with the lymphatic endothelium (Fig. 4k, Lm α5 chain; others not shown). All lymphatic capillaries in carcinomas lacked immunoreactivity for the Lm α3, β3 and γ2 chains and for collagen type VII (not shown). The BM protein distribution in lymphatic vessels of serous and mucinous carcinomas was comparable (not shown).

Lutheran glycoprotein in lymphatic vessels

Because immunoreactivity for the Lm α5 chain suggested variability in the laminin content in the lymphatic vessels of various tissues, we wanted to analyse this aspect in more detail. Therefore, we studied the distribution of Lutheran glycoprotein, the only adhesion receptor thought to bind specifically to the Lm α5 chain (Kikkawa and Miner 2005), in lymphatic vessels. Lymphatic capillaries of skin, ovary and gastrointestinal tract lacked immunoreactivity for Lutheran glycoprotein, whereas immunoreactivity for this glycoprotein was detected in lymphatic capillaries of ovarian carcinomas (Fig. 6).
Fig. 6

Distribution of Lutheran glycoprotein (Lu) in lymphatic vessels. Lymphatic endothelium was visualized by double-labelling with LYVE-1. In human skin (a, b), ovary (c, d) and gastrointestinal tract (e, f), the lymphatic capillaries lacked immunoreactivity for Lutheran glycoprotein (arrows). In ovarian carcinoma (g, h), immunoreactivity for Lutheran glycoprotein was colocalized with the lymphatic endothelium (arrows). Bars 25 μm

Discussion

Interactions of cells with BM proteins are central for blood vessel formation, maintenance and function, and for the transmigration of inflammatory cells through blood vessel endothelium (Davis and Senger 2005; Hallmann et al. 2005; Patarroyo et al. 2002; Sixt et al. 2001; Wang et al. 2006). A critical role for BM proteins has also been suggested with regard to the formation and function of the lymphatic capillaries (Ji 2006; Pepper and Skobe 2003), although the distribution of BM proteins, particularly various laminin isoforms, in lymphatic vessels has remained elusive.

The BM region of all lymphatic vessels showed immunoreactivity for the Lm α4, β1, β2 and γ1 chains, suggesting the presence of laminin isoforms Lm-411 and Lm-421 (Aumailley et al. 2005; Miner and Yurchenco 2004). Because the α4 chain laminins are truncated lacking the entire N-terminal short arm of the α chain, which has been shown to be involved in laminin network self-assembly (Yurchenco et al. 2004), the presence of only α4 chain laminins in BM region of most lymphatic vessels may compromise the formation of endothelial BMs and may therefore in part explain the incomplete formation of a classical electron-dense BM, as previously defined by electron microscopy (Leak and Burke 1966), at this site. However, this requires confirmation by immuno-electron microscopy, once appropriate antibodies become available for such investigations.

The absence of the N-terminal short arm does not, however, prevent the interaction of α4 chain laminins with cell surface receptors, which predominantly interact with the C-terminal LG-domains of laminin α chains (Aumailley et al. 2005; Miner and Yurchenco 2004; Yurchenco et al. 2004). Although Lm-411 is a poor adhesion substratum for blood vessel endothelial cells (Doi et al. 2002), it seems to support endothelial cell survival and migration, as well as leukocyte migration and proliferation (DeHahn et al. 2004; Doi et al. 2002; Geberhiwot et al. 2001). The α4 chain laminins are key promoters of leukocyte extravasation in a mouse model for autoimmune encephalomyelitis (Sixt et al. 2001) and for tumour invasion in human gliomas (Khazenzon et al. 2003). The presence of α4 chain laminins in the BM region of lymphatic capillaries suggests that they could influence the function of lymphatic endothelial cells and facilitate cell migration through lymphatic endothelium.

The lymphatic capillaries of the studied ovarian carcinomas showed consistent immunoreactivity also for the laminin α5 chain, a constituent of laminin isoforms Lm-511 and Lm-521. The α5 chain laminins, which comprise the N-terminal short arm, might facilitate laminin polymerization and BM formation in these vessels (Yurchenco et al. 2004). Lm-511 is a better adhesion substratum for blood vessel endothelial cells than Lm-411 (Doi et al. 2002). In contrast to Lm-411, Lm-511 seems to prevent the transmigration of lymphocytes through endothelial BMs in a mouse model for autoimmune encephalomyelitis (Sixt et al. 2001). Leukocyte migration through venular walls seems to occur only at sites of low Lm-511 expression (Wang et al. 2006). The presence of α5 chain laminins in the BM region of tumour lymphatic vessels suggests that these laminins could inhibit cell migration through the endothelium in these vessels.

Among receptors for the Lm α5 chain, Lutheran glycoprotein is the only receptor that, according to current knowledge, has only one ligand, the Lm α5 chain (Kikkawa and Miner 2005; Miner and Yurchenco 2004). In tissues, Lutheran glycoprotein is only found at sites at which the Lm α5 chain is found in adjacent BMs; the distribution of Lutheran glycoprotein may thus reflect specific functions of Lm α5 chain (Kikkawa and Miner 2005; Moulson et al. 2001). Lutheran glycoprotein seems to function, for example, in the adhesion of erythrocytes and blood vessel endothelial cells (El Nemer et al. 1998; Vainionpää et al. 2006). Our results show that Lutheran glycoprotein is present in lymphatic vessels of ovarian carcinomas but not in those of normal tissues, emphasizing the difference between these vessels suggested by the distribution of the Lm α5 chain.

Type IV collagens enable BMs to withstand mechanical stress (Yurchenco et al. 2004). Cleavage fragments of type IV collagen α chains, including arresten, canstatin and tumstatin, inhibit not only the proliferation and migration of blood vessel endothelial cells, but also angiogenesis (Ortega and Werb 2002). In addition to type IV collagens, collagen type XVIII is ubiquitously found in blood vessel endothelial BMs (Saarela et al. 1998). Its cleavage fragment, endostatin, inhibits the proliferation of blood vessel endothelial cells, angiogenesis and tumour growth (O’Reilly et al. 1997). Moreover nidogen, the linker between laminin and collagen IV networks in BMs (Kalluri 2003; Yurchenco et al. 2004), has been associated with capillary-like structure formation (Titz et al. 2004). Our data shows the consistent presence of collagen type IV (α1/2), collagen type XVIII and nidogen-1 in the BM region of normal lymphatic capillaries and lymphatic capillaries of ovarian carcinoma. This suggests that these proteins may have similar effects on lymphatic endothelia as those described for blood vessel endothelia.

In many previous studies, lymphatic vessels have been distinguished from blood vessels on the basis of their weak immunoreactivity for BM proteins (Barsky et al. 1983; Wigle et al. 2002). In most studies describing weak or lack of immunoreactivity for laminins in lymphatic vessels, antibodies against mouse laminin from Engelbreth-Holm-Swarm tumour have been used. The specificity of these antibodies in human tissues has been poorly characterized but they have been suggested to detect laminin γ1 chain (Erickson and Couchman 2000). Our results show that immunoreactivity for certain laminin chains is present in BM region of lymphatic endothelia but that the immunoreactivity is generally much weaker in lymphatic vessels than in blood vessels. These results thus agree with those of Sauter et al. (1998) who have found collagen type IV in the BM region of lymphatic vessels. On the other hand, the gene expression of collagen type IV α chains, collagen type XVIII, the Lm α3, α5, β1, β2 and γ2 chains and nidogen has been shown to be lower in lymphatic endothelial cells than in the endothelial cells of blood vessels (Hirakawa et al. 2003; Petrova et al. 2002; Podgrabinska et al. 2002). This is also in agreement with our results demonstrating clearly weaker immunoreactivity for these proteins in lymphatic vessels than in blood vessels.

In conclusion, α4 chain laminins, type IV and XVIII collagens and nidogen-1 were found in the BM region of normal and ovarian carcinoma lymphatic capillaries. The distribution of BM proteins may be the reason for the incomplete formation of a classical electron-dense BM, as previously defined by electron microscopy, around lymphatic capillaries. The differential distribution of α5 chain laminins and of their receptor, Lutheran glycoprotein, proposes a difference between lymphatic capillaries of normal tissues and carcinomas.

Acknowledgements

MAb M3F7 raised by Foellmer et al. (1983) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. We thank Profs. K. Alitalo, E. Engvall, D. Kerjaschki, J.H. Miner, K. Miyazaki, P. Rousselle and U. Wewer for antibodies. For technical assistance, we acknowledge Ms. Pipsa Kaipainen, Mr. Hannu Kamppinen, Mr. Reijo Karppinen, Ms. Marja-Leena Piironen, Ms. Outi Rauanheimo, Ms. Anne Reijula and Ms. Hanna Wennäkoski.

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Noora Vainionpää
    • 1
  • Ralf Bützow
    • 2
    • 6
  • Mika Hukkanen
    • 1
  • David G. Jackson
    • 3
  • Taina Pihlajaniemi
    • 4
  • Lynn Y. Sakai
    • 5
  • Ismo Virtanen
    • 1
  1. 1.Institute of Biomedicine/AnatomyUniversity of HelsinkiHelsinkiFinland
  2. 2.Department of PathologyHaartman Institute University of HelsinkiHelsinkiFinland
  3. 3.MRC Human Immunology UnitInstitute of Molecular Medicine, John Radcliffe HospitalOxfordUK
  4. 4.Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, Collagen Research UnitUniversity of OuluOuluFinland
  5. 5.Department of Biochemistry and Molecular Biology, Shriners Hospital for ChildrenOregon Health and Science UniversityPortlandUSA
  6. 6.Department of Obstetrics and Gynecology, Research Laboratory, BiomedicumHelsinki University Central HospitalHelsinkiFinland

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