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

Kriehuber E, Breiteneder-Geleff S, Groeger M, Soleiman A, Schoppmann SF, Stingl G, Kerjaschki D, Maurer D - J. Exp. Med. (2001)

Bottom Line: Recently, the 38-kD mucoprotein podoplanin was found to be expressed by microvascular LECs but not BECs in vivo.LECs as well as BECs formed junctional contacts with similar molecular composition and ultrastructural features.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, University of Vienna Medical School, Austria.

ABSTRACT
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.

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BECs and LECs form separate homotypic layers in mixed EC cultures. Mixed BECs and LECs were allowed to reach confluence and then were cultured for four additional days. (A–D) Cells were fixed and stained with anti-podoplanin Abs (FITC) and anti-CD31 (TRITC). In the double exposure shown in A, podoplanin+/CD31+ ECs (yellow) appear to cover the monolayer of podoplanin−/CD31+ ECs (red). In B–D, vertical optical sectioning by confocal microscopy directly shows that podoplanin+/CD31+/LECs (green; C and D) are positioned on top of podoplanin−CD31+ BECs (red; B and D). The blue line indicates the position of the culture dish. In E, EC bilayers were fixed and processed for anti-podoplanin immunogold labeling and vertical sections were analyzed by electron microscopy. LECs as identified by their intense labeling with 10-nm gold particles are found on top of nonlabeled BECs. Original magnification in A–D: ×1,000; E: ×40.000.
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Figure 6: BECs and LECs form separate homotypic layers in mixed EC cultures. Mixed BECs and LECs were allowed to reach confluence and then were cultured for four additional days. (A–D) Cells were fixed and stained with anti-podoplanin Abs (FITC) and anti-CD31 (TRITC). In the double exposure shown in A, podoplanin+/CD31+ ECs (yellow) appear to cover the monolayer of podoplanin−/CD31+ ECs (red). In B–D, vertical optical sectioning by confocal microscopy directly shows that podoplanin+/CD31+/LECs (green; C and D) are positioned on top of podoplanin−CD31+ BECs (red; B and D). The blue line indicates the position of the culture dish. In E, EC bilayers were fixed and processed for anti-podoplanin immunogold labeling and vertical sections were analyzed by electron microscopy. LECs as identified by their intense labeling with 10-nm gold particles are found on top of nonlabeled BECs. Original magnification in A–D: ×1,000; E: ×40.000.

Mentions: When cultures containing BECs and LECs were grown to confluence and replated for 12 h, islands of LECs formed that were surrounded by BECs (Fig. 2 C). EC bilayer formation occurred, when the culture was prolonged for 3 d after the cells had reached confluence. These consisted of plastic-adherent podoplanin−/CD31+ BECs and podoplanin+/CD31+ LECs on top (Fig. 6). In contrast, bilayer formation was never observed in monotypic cultures of FACS®-sorted LECs and BECs (Fig. 2A and Fig. B).


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

Kriehuber E, Breiteneder-Geleff S, Groeger M, Soleiman A, Schoppmann SF, Stingl G, Kerjaschki D, Maurer D - J. Exp. Med. (2001)

BECs and LECs form separate homotypic layers in mixed EC cultures. Mixed BECs and LECs were allowed to reach confluence and then were cultured for four additional days. (A–D) Cells were fixed and stained with anti-podoplanin Abs (FITC) and anti-CD31 (TRITC). In the double exposure shown in A, podoplanin+/CD31+ ECs (yellow) appear to cover the monolayer of podoplanin−/CD31+ ECs (red). In B–D, vertical optical sectioning by confocal microscopy directly shows that podoplanin+/CD31+/LECs (green; C and D) are positioned on top of podoplanin−CD31+ BECs (red; B and D). The blue line indicates the position of the culture dish. In E, EC bilayers were fixed and processed for anti-podoplanin immunogold labeling and vertical sections were analyzed by electron microscopy. LECs as identified by their intense labeling with 10-nm gold particles are found on top of nonlabeled BECs. Original magnification in A–D: ×1,000; E: ×40.000.
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Related In: Results  -  Collection

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Figure 6: BECs and LECs form separate homotypic layers in mixed EC cultures. Mixed BECs and LECs were allowed to reach confluence and then were cultured for four additional days. (A–D) Cells were fixed and stained with anti-podoplanin Abs (FITC) and anti-CD31 (TRITC). In the double exposure shown in A, podoplanin+/CD31+ ECs (yellow) appear to cover the monolayer of podoplanin−/CD31+ ECs (red). In B–D, vertical optical sectioning by confocal microscopy directly shows that podoplanin+/CD31+/LECs (green; C and D) are positioned on top of podoplanin−CD31+ BECs (red; B and D). The blue line indicates the position of the culture dish. In E, EC bilayers were fixed and processed for anti-podoplanin immunogold labeling and vertical sections were analyzed by electron microscopy. LECs as identified by their intense labeling with 10-nm gold particles are found on top of nonlabeled BECs. Original magnification in A–D: ×1,000; E: ×40.000.
Mentions: When cultures containing BECs and LECs were grown to confluence and replated for 12 h, islands of LECs formed that were surrounded by BECs (Fig. 2 C). EC bilayer formation occurred, when the culture was prolonged for 3 d after the cells had reached confluence. These consisted of plastic-adherent podoplanin−/CD31+ BECs and podoplanin+/CD31+ LECs on top (Fig. 6). In contrast, bilayer formation was never observed in monotypic cultures of FACS®-sorted LECs and BECs (Fig. 2A and Fig. B).

Bottom Line: Recently, the 38-kD mucoprotein podoplanin was found to be expressed by microvascular LECs but not BECs in vivo.LECs as well as BECs formed junctional contacts with similar molecular composition and ultrastructural features.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, University of Vienna Medical School, Austria.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus