The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gröger, M.
Right arrow Articles by Petzelbauer, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gröger, M.
Right arrow Articles by Petzelbauer, P.
The Journal of Immunology, 2004, 173: 7161-7169.
Copyright © 2004 by The American Association of Immunologists

IL-3 Induces Expression of Lymphatic Markers Prox-1 and Podoplanin in Human Endothelial Cells1

Marion Gröger*,{dagger}, Robert Loewe*, Wolfgang Holnthoner*, Robert Embacher*,{dagger}, Manuela Pillinger*,{dagger}, G. Scott Herron{ddagger}, Klaus Wolff*,{dagger} and Peter Petzelbauer2,*,{dagger}

* Department of Dermatology, Division of General Dermatology, Medical University of Vienna, and {dagger} Ludwig Boltzmann Institute for Angiogenesis, Microcirculation and Inflammation, Vienna, Austria; and {ddagger} Department Dermatology, Palo Alto Medical Clinic, Palo Alto, CA 94301


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Factors determining lymphatic differentiation in the adult organism are not yet well characterized. We have made the observation that mixed primary cultures of dermal blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) grown under standard conditions change expression of markers during subculture: After passage 6, they uniformly express LEC-specific markers Prox-1 and podoplanin. Using sorted cells, we show that LEC but not BEC constitutively express IL-3, which regulates Prox-1 and podoplanin expression in LEC. The addition of IL-3 to the medium of BEC cultures induces Prox-1 and podoplanin. Blocking IL-3 activity in LEC cultures results in a loss of Prox-1 and podoplanin expression. In conclusion, endogenous IL-3 is required to maintain the LEC phenotype in culture, and the addition of IL-3 to BEC appears to induce transdifferentiation of BEC into LEC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-3 is a broadly acting regulatory protein of hemopoietic cell differentiation. It stimulates proliferation, survival, and differentiation of pluripotent hemopoietic stem cells, and promotes expansion of cells that differentiate into mature cell lineages. Interestingly, IL-3 also has the capacity to divert differentiation of already committed cells. In the presence of IL-3, cells of the osteoclast lineage differentiate into cells of the macrophage lineage (1). IL-3 signals to cells via the IL-3R, which is composed of an IL-3-specific {alpha}-chain and a common {beta}-chain shared with the GM-CSFR {alpha}-chain and the IL-5R {alpha}-chain (2, 3).

Endothelial cells (EC)3 express IL-3R {alpha}- and {beta}-chains, and expression can be increased by stimulation with TNF-{alpha} and/or IFN-{gamma} (4). EC respond to IL-3 by expressing IL-8 and E-selectin (5, 6). Moreover, IL-3 promotes endothelial tube formation and directional migration (7). Cultured EC have been identified as a source of IL-3 (8). Whether EC-derived IL-3 has autocrine functions is unknown, as is the question of a role for IL-3 in EC differentiation.

EC invest two distinct vascular trees, one building the transport system for blood and the other for lymph. They differ by morphology and phenotype as reviewed recently by several authors (9, 10, 11, 12, 13). Established markers for human lymphatic cells are, for example, Prox-1 (14), podoplanin (15, 16) (also called T1{alpha} (17) or gp36 (18)), vascular endothelial growth factor (VEGF)R-3 (19, 20), and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) (21). Also for blood EC (BEC), lineage-specific genes have been identified by transcriptional profiling (22, 23, 24), among constitutively expressed markers are N-cadherin (22, 23, 24), pathologische anatomie Leiden-endothelium Ag (PAL-E) (for microvessels) (16, 25), or CXCR4 (22, 23). During embryogenesis, factors governing lymphatic differentiation are well described. The transcription factor Prox-1 is the earliest detectable marker of lymphatic differentiation and is essential for differentiation of lymphatic EC (LEC) from the anterior cardinal vein endothelium (14, 26, 27). Lymphangiogenesis is regulated by growth factors VEGF-C and VEGF-D and their receptor VEGFR-3 (20, 28, 29, 30). The transmembrane mucoprotein podoplanin was shown to be important for the correct lymphatic network formation (26). In the adult organism, the situation is more complex and less defined. For example, in some tumors and in inflammation, the lymphatic growth factor receptor VEGFR-3 is expressed on proliferating lymphatic and blood vessels, resulting in VEGF-C-induced lymph and blood vessel formation (31, 32, 33, 34). This raises the question whether additional factors exist, which determine differentiation of LEC within the adult organism. In this study, we demonstrate that IL-3 regulates expression of LEC-specific markers Prox-1 and podoplanin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

HUVEC were isolated as described (35) and cultured in IMDM with 20% FCS, glutamine (2 mM; Invitrogen Life Technologies, Carlsbad, CA), penicillin (100 U/ml), streptomycin (100 µg/ml; both Invitrogen Life Technologies), and heparin/ECGS (PromoCell, Heidelberg, Germany).

Foreskin-derived microvascular EC were isolated and cultured as described (36). Briefly, foreskins were treated with dispase (Invitrogen Life Technologies) for 20 min at 37°C, followed by mechanically scraping EC with a cell scraper. Cells were then seeded into fibronectin-coated wells and cultured in EC growth medium (MV/ECGS; PromoCell). At passage 1, BEC and LEC were separated by magnetic sorting with an anti-podoplanin serum (15, 16).

Antibodies

First step Abs were as follows: anti-Prox-1 (RDI, Flanders, NJ), anti-macrophage mannose receptor (BD Pharmingen, San Jose, CA), PAL-E (RDI), anti-N-cadherin (BD Pharmingen), FITC-labeled CD31 (Immunotech, Marseille, France), anti-IL-3R {alpha}-chain Ab for FACS (clone 9F5), and blocking anti-IL-3R {alpha}-chain Ab (clone 7G3; both BD Pharmingen). As a specificity control for the anti-podoplanin serum, we raised a mouse monoclonal anti-human podoplanin Ab using peptide GASTGQPEDDTETTGLEGG (aa 22–40) as the Ag; a rabbit anti-human podoplanin serum was a gift from Dr. D. Kerjaschki (Department of Pathology, Medical University of Vienna). Isotype controls were purchased from Sigma-Aldrich (St. Louis, MO). Second step Abs were as follows: tetramethylrhodamine isothiocyanate-labeled goat anti-rabbit F(ab)2 (Jackson ImmunoResearch Laboratories, West Grove, PA). PE-labeled goat anti-rabbit F(ab)2 (BD Pharmingen), Alexa 488-labeled anti-rabbit IgG (Molecular Probes, Leiden, The Netherlands), FITC-labeled anti-mouse-IgG (Sigma-Aldrich), and HRP-labeled anti-mouse or anti-rabbit IgG (Bio-Rad, Hercules, CA).

FACS and laser scan analysis

For FACS, cells were detached from culture dishes using trypsin/EDTA. For laser scan imaging, cells were directly fixed on culture dishes with acetone/methanol (1:1) at –20°C for 10 min. Samples were then incubated with indicated first-step Abs followed by the appropriate fluorescence-labeled second-step Abs. Isotype-matched control Abs were used in parallel. Bound fluorescence was analyzed by FACScan (BD Biosciences, Mountain View, CA) or laser scan microscope (LSM 520; Zeiss, Oberkochen, Germany).

RT-PCR

Cells were lysed in lysis buffer (10 mM NaOH-HEPES (pH 7.8), 1.5 mM MgCl, 10 mM KCl, 1 mM DTT, 0.1% Nonidet P-40, and 1 U of RNase inhibitor (Roche, Basel, Switzerland)), and mRNA was isolated by using oligo(dT) beads (Dynal, Oslo, Norway). RNA was reverse transcribed with Superscript Reverse Transcriptase (Invitrogen Life Technologies) and hexamer primers (Roche) for 90 min at 42°C. One microliter of cDNA was then used for PCR with primers for podoplanin (forward, 5'-GAA GGT GTC AGC TCT GCT CT-3'; reverse, 5'-ACG TTG GCA GGG CGT AA-3'; 35 cycles), Prox-1 (forward, 5'-AAG ACA GAG CCT CTC CTG AAT C-3'; reverse, 5'-TTG CAC TTC CCG AAT AAG GTG AT-3'; 30 cycles), Lyve-1 (forward, 5'-GTG CTT CAG CCT GGT GTT G-3'; reverse, 5'-GCT TGG ACT CTT GGA CTC TTC-3'; 30 cycles), flt-4 (forward, 5'-AGC TCT CAG AGC TCA GAA GAG-3'; reverse, 5'-TTC TCT CTC TCT GCT TCA GCT-3'; 30 cycles). Primers for GAPDH were purchased from BD Clontech (Palo Alto, CA). As control for genomic DNA contamination, PCR was performed without reverse transcription. PCR conditions were 94°C for 30 s, 56°C for 40 s, and 72°C for 45 s. IL-3 mRNA was analyzed by real-time PCR using primers from assay 4327037T (Applied Biosystems, Foster City, CA) according to the instructions of the manufacturer.

ELISA

Cell culture supernatants were collected, protease inhibitors added (PMSF, leupeptin, and aprotinin), and analyzed using an IL-3 ELISA kit from R&D Systems (Minneapolis, MN) according to the instructions of the manufacturer. OD450 was measured.

Western blot

Western blotting was performed as described previously (37). Briefly, following cell lysis with Tris lysis buffer (containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM CaCl2, 1 mM PMSF, 10 µg/ml aprotinin, 15 µg/ml leupeptin, 1% Nonidet P-40, and 1% Triton X-100), proteins were separated on SDS polyacrylamide gels and blotted on polyvinylidene difluoride membranes. Blots were then incubated with indicated first-step Abs, followed by incubation with appropriate HRP-labeled second-step Abs. Signals were visualized by chemiluminescence (Super ECL System; Amersham Biosciences, Buckinghamshire, U.K.) and documented on Hyperfilm MP (Amersham Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Skin-derived EC acquire a lymphatic phenotype during subculture

Normal human skin contains approximately equal numbers of podoplanin-positive lymphatic vessels and podoplanin-negative blood vessels (an example of an immunofluorescence staining of human skin is shown in Fig. 1A). As a consequence, mechanically scraped EC consisted of LEC and BEC at a ratio of ~1:1 (ranging from 2:1 to 1:2). A FACS analysis using anti-podoplanin Abs of primary isolated cells (passage 0 cells) is shown in Fig. 1B. During subculture, the numbers of podoplanin-negative cells continuously decreased, and numbers of podoplanin-positive cells increased (Fig. 1B). Passage 6 cells consisted of 100% podoplanin-positive cells. As a positive control, we used CD31 Abs, which reacted equally with LEC and BEC (Fig. 1B).



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 1. A, Immunofluorescence double-staining of normal human skin samples with anti-podoplanin serum and CD31 Abs. Double-positive vessels represent LEC and are marked by arrowheads. The two right panels show staining with the corresponding negative controls. B, FACS analysis for podoplanin and CD31 expression of freshly isolated EC cultures (p0), passage 1 cells (p1), and passage 6 cells (p6) stained with anti-podoplanin serum (left panel), CD31 (middle panel), and controls (right panels). C, Marker profile of sorted BEC and LEC (passage 4 cells). FACS histograms are shown at the left side, Western blots at the right side, and RT-PCR at the bottom. Podoplanin was analyzed with two Abs, an anti-podoplanin serum or a mouse mAb (marked by asterisks). Note that immunoblots with anti-podoplanin serum reveal two bands of 38 and 28 kDa (18 ), whereas the mouse mAb detects a 38-kDa band only.

 
IL-3 is constitutively expressed in LEC

The molecular characterization of sorted LEC and BEC is shown in Fig. 1C. LEC are characterized by the expression of podoplanin, Flt-4, LYVE-1, Prox-1, and mannose receptor, and BEC by PAL-E and N-cadherin expression. To determine factors inducing podoplanin expression of mixed (nonsorted) LEC/BEC in culture, we screened cell culture supernatants of sorted LEC and BEC for cytokines. We found IL-3 consistently present in LEC and absent in BEC supernatants (Fig. 2A). HUVEC were analyzed for comparison and also found negative for IL-3 (see also Nilsen et al. (8)). Primary cultures, which, due to the isolation procedure, consisted of a mixture of LEC and BEC (mixed LEC/BEC), had slightly higher IL-3 release than in sorted LEC (by Student’s t test, the difference compared with LEC was NS). IL-3 expression could be induced following stimulation with TNF-{alpha}/IFN-{gamma} in LEC and in BEC, but IL-3 levels were ~10 times higher in stimulated LEC than in stimulated BEC (Fig. 2A). To confirm results, IL-3 mRNA was analyzed by real-time PCR: IL-3 mRNA was detectable in unstimulated LEC after a mean of 37 cycles, and was absent in unstimulated BEC even after 45 cycles (GAPDH was detectable after 21 cycles). Fig. 2B gives an example of a representative semiquantitative RT-PCR for IL-3.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 2. A, IL-3 secretion into the culture medium of HUVEC, sorted BEC and LEC (passage 4 cells), and primary cultures of freshly isolated skin-derived EC consisting of a mixture of BEC and LEC (mixed BEC and LEC) as determined by ELISA. Basal IL-3 release under standard conditions is shown in the left panel. The right panels show IL-3 release following stimulation with TNF-{alpha}/IFN-{gamma} (10 ng/ml each) for 24 h (mean ± SEM of three independent experiments performed each in triplicate; the difference between BEC and LEC is significant, p < 0.01; the difference between mixed cultures and LEC is NS). B, Semiquantitative RT-PCR for IL-3 mRNA expression in BEC and LEC is shown (35 cycles). As a control, GAPDH mRNA expression is shown (20 cycles). C, IL-3R {alpha}-chain expression as determined by FACS is shown. The left panels show IL-3R {alpha}-chain expression of indicated cells grown under standard conditions. The right panels show IL-3R {alpha}-chain expression following stimulation with TNF-{alpha}/IFN-{gamma} (10 ng/ml each) for 12 h. Thin lines are isotype controls; bold lines are staining with the anti-IL-3R{alpha} Ab.

 
It was previously shown that HUVEC express IL-3R {alpha}- and {beta}-chain, and that expression is induced by TNF-{alpha}/IFN-{gamma} (4). In this study, we show that also LEC and BEC express IL-3R (slightly higher in LEC than in BEC), and that expression was enhanced by TNF-{alpha}/IFN-{gamma}. Fig. 2C shows results for the {alpha}-chain; expression of the {beta}-chain followed the pattern of the {alpha}-chain (data not shown) (6, 38, 39). As a negative control, dermal fibroblasts were analyzed in parallel (Fig. 2C).

IL-3 up-regulates podoplanin and Prox-1 expression

Cell surface expression of podoplanin was analyzed by FACS. Following IL-3 stimulation, podoplanin expression was enhanced in LEC (3-fold). In BEC and HUVEC, IL-3 induced de novo expression of podoplanin, albeit to a weaker extent (2-fold above baseline; Fig. 3). As a negative control, IL-3 was heat inactivated, which did not up-regulate podoplanin expression (Fig. 3). Also TNF-{alpha}/IFN-{gamma} induced podoplanin expression in LEC, BEC, and HUVEC. The addition of 1 µg/ml anti-IL-3R {alpha}-chain Ab (clone 7G3, blocks signal transduction of the IL-3R (40)) inhibits TNF-{alpha}/IFN-{gamma}-induced podoplanin expression (Fig. 3), which indicates that a TNF-{alpha}/IFN-{gamma}-induced IL-3 release is responsible for the observed podoplanin up-regulation. Following prestimulation with TNF-{alpha}/IFN-{gamma}, subsequent IL-3 stimulation further enhanced podoplanin expression in LEC (4-fold). Also in BEC and HUVEC, TNF-{alpha}/IFN-{gamma} prestimulation followed by IL-3 was additive; podoplanin expression was further induced (Fig. 3). Dermal fibroblasts analyzed as a negative control neither expressed podoplanin at baseline nor following TNF-{alpha}/IFN-{gamma} and IL-3 stimulation (Fig. 3).



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 3. IL-3 induces podoplanin as determined by FACS analysis. Sorted LEC, sorted BEC, HUVEC (passage 4 cells), or dermal fibroblasts were cultured for 24 h in medium alone or in medium supplemented with IL-3 (25 ng/ml; 24 h), heat-inactivated IL-3, TNF-{alpha}/IFN-{gamma} (10 ng/ml each; 24 h), TNF-{alpha}/IFN-{gamma} plus anti-IL-3R{alpha} Ab 7G3 (1 µg/ml), or TNF-{alpha}/IFN-{gamma} (12 h) followed by IL-3 (24 h). Staining was performed with the control serum (row at the top) or with anti-podoplanin serum (all others). Geometric mean fluorescence values are inserted into each panel and represent the mean of three independent experiments.

 
FACS data were confirmed by podoplanin Western blots using whole-cell lysates of LEC and BEC (Fig. 4A). Prox-1 expression was analyzed by immunofluorescence. In unstimulated LEC, Prox-1 was found in the nucleus in 100% of cells. In contrast, in unstimulated BEC, Prox-1 was undetectable, but was inducible upon stimulation with IL-3 (Fig. 4B). Podoplanin and Prox-1 mRNA expression was analyzed by semiquantitative RT-PCR and were readily detectable in LEC at baseline and induced following IL-3 stimulation (Fig. 4C). In contrast, unstimulated BEC and HUVEC were negative for podoplanin and Prox-1 mRNA, but was inducible following IL-3 stimulation (Fig. 4C). As a negative control, dermal fibroblasts were analyzed and found negative for podoplanin and Prox-1 mRNA (Fig. 4C).



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 4. A, Western blotting for IL-3-induced podoplanin expression. IL-3 (25 ng/ml) was added to the medium for 24 h. Cells were then lysed, and immunoblots were performed with the polyclonal rabbit anti-podoplanin serum, which detects two different gylcosylation forms, 28 and 38 kDa, respectively. As a loading control, immunoblots for CD31 expression are shown. B, Immunofluorescence laser scan images for Prox-1 expression. Prox-1 is seen in a nuclear localization (red) in untreated LEC, and immunofluorescence is enhanced following IL-3 stimulation. In contrast, Prox-1 is absent in unstimulated BEC and induced following IL-3 stimulation. To visualize cell margins, cells were double-stained with CD31 Abs in green. C, Semiquantitative RT-PCR for podoplanin and Prox-1 expression in unstimulated cells or following IL-3-stimulation for indicated times is shown. Fibroblasts were used as a negative control.

 
Anti-IL-3R {alpha}-chain Abs inhibit Prox-1 and podoplanin expression

To analyze the role of endogenous IL-3, sorted LEC were cultured in the presence of an anti-IL-3R {alpha}-chain Ab (1 µg/ml) (40). In the presence of this blocking Ab, podoplanin expression in LEC decreased (as analyzed by immunofluorescence LSM imaging; Fig. 5A). After 24 h of treatment of LEC, islands of cells emerged, which had lost podoplanin expression, whereas other islands showed persisting podoplanin expression. Islands of podoplanin-positive cells became continuously smaller, and after 168 h, podoplanin expression was almost completely lost in all LEC (Fig. 5A). In the presence of a control Ab, cells remained positive for podoplanin (Fig. 5A, first panel). Results were confirmed by FACS analysis. Fig. 5B gives the cumulative result of three independent experiments. After 168 h of anti-IL-3R{alpha} Ab treatment, ~90% of cells were negative for podoplanin expression, whereas an isotype-matched control Ab had no effect (Fig. 5B). After 168 h, the anti-IL-3R{alpha} Ab was removed by repeated washing and replaced by fresh medium. As shown in Fig. 5C, cells remained negative for podoplanin even after withdrawal of the Ab. Results from immunofluorescence and FACS were confirmed by RT-PCR; podoplanin and Prox-1 mRNA were undetectable following 72-h treatment with anti-IL-3R{alpha} Ab (Fig. 5B, inset).



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 5. Anti-IL-3R{alpha} Abs inhibit podoplanin and Prox-1 expression. A, LEC were cultured in the presence of anti-IL-3R{alpha} Ab 7G3 (1 µg/ml) for indicated times, and then double-stained with anti-podoplanin (red) and CD31 (green) Abs and analyzed by laser scan microscopy. The left image shows persisting podoplanin expression in the presence of a control Ab. After 24-h treatment with anti-IL-3R{alpha} Abs, islands of cells emerged, which had lost podoplanin expression, whereas surrounding cells showed persisting podoplanin expression. After 168 h, podoplanin expression was almost completely lost in all LEC (right panel). B, FACS analysis for podoplanin expression in LEC treated with anti-IL-3R{alpha} or isotype control Abs. Geometric mean fluorescence levels from three independent experiments are shown and expressed as the percentage of inhibition of podoplanin expression compared with untreated cells (mean ± SD). The corresponding RT-PCR for podoplanin and Prox-1 mRNA expression is shown as an inset in B. C, Representative FACS histogram for podoplanin expression in untreated LEC (first panel) and anti-IL-3R{alpha} Ab-treated LEC (168 h; second panel). Thereafter, the Ab was washed out, and cells were cultured in medium alone for another 48 and 168 h, respectively. Podoplanin expression remained negative even after withdrawal of the Ab (third and fourth panels). D, Anti-IL-3R{alpha} Ab treatment changes cell morphology. Transmission microscopic images of LEC (first image) and BEC (third image) treated with a control Ab. The image in the middle shows LEC treated with 1 µg/ml anti-IL-3R{alpha} Ab for 48 h. The asterisk denotes flattened cells resembling LEC, and the circles denote more spindled cells resembling BEC.

 
Anti-IL-3R{alpha} Ab treatment also altered cell morphology of cultured LEC. A representative example of transmission light microscopy images of LEC (cultured with isotype Ab), BEC (cultured with isotype Ab), and LEC cultured with anti-IL-3R{alpha} Ab is shown in Fig. 5D. LEC appeared as flattened cells, whereas BEC had a more spindled morphology. In the presence of anti-IL-3R{alpha}, a population of spindled cells emerged comparable to those seen in pure BEC cultures. These islands correspond to the podoplanin-negative cells shown in Fig. 5A.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Prox-1 is the earliest lymphatic marker during embryonic development (14, 26) and is thought to be the critical gene programming the lymphatic phenotype (14, 23, 27, 41). Podoplanin expression occurs later and appears to be important for the lymphatic network formation (23, 26, 41). Both molecules are constitutively expressed on lymphatic endothelium in the adult organism, but factors that induce or maintain their expression are currently unknown. In this study, we identified for the first time a soluble factor, IL-3, which is expressed in cultured LEC but not in BEC, and which is required to maintain expression of LEC-specific markers Prox-1 and podoplanin. Blocking IL-3 effects leads to a loss of expression of these molecules in cultured LEC.

It should be noted that, during embryogenesis, IL-3 does not appear to be the critical factor for the formation of the lymphatic tree, because IL-3-deficient animals have no severe lymphatic dysfunction. However, current reports have not specifically searched for subtle changes in lymphatic function (42, 43); thus, the role of IL-3 during development has to remain open.

Another issue concerns the question of why the expression of IL-3 has not yet been identified as a LEC-specific gene in previous gene expression arrays (22, 23, 24). On the one hand, this might be due to the reported instability of IL-3 mRNA, which complicates detection of IL-3 mRNA (44, 45). In contrast, this seeming discrepancy might be due to the very low IL-3 mRNA levels in unstimulated LEC cultures, which may have escaped detection in arrays. However, constitutive IL-3 expression levels are generally low in most cell types previously analyzed (46), and even such low concentrations are biologically active in settings of an autocrine IL-3 release. IL-3 supplemented to the medium requires much higher concentrations to achieve biological activity (46).

In our assays, committed cells were used, which were isolated and sorted from human foreskins and thus already differentiated into either a BEC or a LEC phenotype. Even in such differentiated cells, IL-3 was able to induce Prox-1 and podoplanin expression. Although IL-3-induced expression levels of Prox-1 and podoplanin were much lower in BEC than in LEC, it involved the whole cell population, excluding growth selection as the basis of this phenomenon. Moreover, phenotypic changes appeared within 12–24 h, and population doubling times in EC are 36–48 h, which also argues against a growth selection phenomenon. We also show that first-line inflammatory cytokines TNF-{alpha}/IFN-{gamma} induce podoplanin expression, and this also depended on IL-3 (because it is inhibited by anti-IL-3R{alpha} Abs). Moreover, TNF-{alpha}/IFN-{gamma} prestimulation was additive to subsequent IL-3-induced Prox-1 and podoplanin expression. This additive effect is most likely based on TNF-{alpha}/IFN-{gamma}-induced IL-3R expression. Such a mechanism has been previously described for IL-3-induced MHC class II expression in HUVEC, where TNF-{alpha}/IFN-{gamma} prestimulation increased IL-3R expression and subsequently MHC class II expression induced by IL-3 (4).

To date, IL-3 is the first soluble factor identified that is able to induce Prox-1 and podoplanin expression in differentiated EC. This IL-3 effect was seen in LEC and in BEC, which raises the intriguing question whether IL-3-treated BEC transdifferentiate into LEC. Based on current knowledge, the answer is complex, because (first) cultured LEC have a certain heterogeneous expression of lymphatic markers (e.g., LYVE-1 or VEGFR-3 may be absent). This phenomenon was reviewed by Saharinen et al. (47) and interpreted as the appearance of different LEC subpopulations. Second, the situation is more complicated in experiments where a phenotypic switch from BEC into LEC was induced by, for example, overexpressing Prox-1 (23, 41) or by infection with Karposi sarcoma-associated herpes virus (HHV8) (48, 49). Prox-1 overexpression in BEC induced only ~20% of lymphatic genes and repressed only ~40% of blood vessel genes. This is also true for HHV8 infection of blood vessel endothelium, where some, but not all, lymphatic markers were up-regulated, and some blood-specific markers disappeared. Surprisingly, even Prox-1 was not induced in all experiments (48, 49). Thus the definition of a true LEC or BEC phenotype still floats with accumulating data on these cells (reviewed by Saharinen et al. (47)). In other words, we think that the question whether IL-3 induces a true BEC-to-LEC conversion or a hybrid form cannot be answered at present.

With regard to in vivo effects of IL-3, others have shown new vessel formation in mice injected with IL-3-containing Matrigel plugs, but at the time of this report, phenotypic markers for LEC were yet not well characterized, and thus distinctions between LEC and BEC were not made (7, 50). Moreover, this type of experiment would not differentiate between lymphangiogenesis from pre-existing lymph vessels and lymph vessel formation through transdifferentiation or a combination thereof. Anyway, the possibility for IL-3 to induce lymphangiogenesis raises the question whether increased IL-3 activity in tumors is associated with a high metastatic potential. This was shown for rat mammary adenocarcinoma cell clones, which were highly metastatic when they produced high levels of IL-3, but were poorly metastatic when they had no IL-3 activity (51). As a natural source of IL-3, tumor-infiltrating lymphocytes have to be considered (50, 52). Finally, the effects of the proinflammatory cytokines TNF-{alpha} and IFN-{gamma} on Prox-1 and podoplanin expression raises the possibility that lymphangiogenesis observed in situations of chronic inflammation (53, 54) is in part mediated through transdifferentiation of BEC into LEC.

In conclusion, we show that endogenous IL-3 is required to maintain the LEC phenotype in culture, and the addition of IL-3 to BEC induces expression of LEC-specific markers. Our data support the possibility that BECs can reprogram their phenotype into LECs, and IL-3 may contribute to lymphangiogenesis in tumors or in settings of chronic inflammation.


   Acknowledgments
 
We thank Dr. D. Kerjaschki (Department of Pathology, Medical University of Vienna) for critical discussions and for the anti-podoplanin serum.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a research grant from the Stavros Niarchos Foundation. Back

2 Address correspondence and reprint requests to Dr. Peter Petzelbauer, Department of Dermatology, Division of General Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail address: Peter.Petzelbauer{at}meduniwien.ac.at Back

3 Abbreviations used in this paper: EC, endothelial cell; BEC, blood EC; LEC, lymphatic EC; VEGF, vascular endothelial growth factor; LYVE, lymphatic vessel endothelial hyaluronan receptor; PAL-E, pathologische anatomie Leiden-endothelium Ag. Back

Received for publication May 26, 2004. Accepted for publication October 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Khapli, S. M., L. S. Mangashetti, S. D. Yogesha, M. R. Wani. 2003. IL-3 acts directly on osteoclast precursors and irreversibly inhibits receptor activator of NF-{kappa}B ligand-induced osteoclast differentiation by diverting the cells to macrophage lineage. J. Immunol. 171:142.[Abstract/Free Full Text]
  2. Geijsen, N., L. Koenderman, P. J. Coffer. 2001. Specificity in cytokine signal transduction: lessons learned from the IL-3/IL-5/GM-CSF receptor family. Cytokine Growth Factor Rev. 12:19.[Medline]
  3. Martinez-Moczygemba, M., D. P. Huston. 2003. Biology of common {beta} receptor-signaling cytokines: IL-3, IL-5, and GM-CSF. J. Allergy Clin. Immunol. 112:653.[Medline]
  4. Korpelainen, E. I., J. R. Gamble, W. B. Smith, M. Dottore, M. A. Vadas, A. F. Lopez. 1995. Interferon-{gamma} upregulates interleukin-3 (IL-3) receptor expression in human endothelial cells and synergizes with IL-3 in stimulating major histocompatibility complex class II expression and cytokine production. Blood 86:176.[Abstract/Free Full Text]
  5. Brizzi, M. F., G. Garbarino, P. R. Rossi, G. L. Pagliardi, C. Arduino, G. C. Avanzi, L. Pegoraro. 1993. Interleukin 3 stimulates proliferation and triggers endothelial-leukocyte adhesion molecule 1 gene activation of human endothelial cells. J. Clin. Invest. 91:2887.
  6. Korpelainen, E. I., J. R. Gamble, W. B. Smith, G. J. Goodall, S. Qiyu, J. M. Woodcock, M. Dottore, M. A. Vadas, A. F. Lopez. 1993. The receptor for interleukin 3 is selectively induced in human endothelial cells by tumor necrosis factor {alpha} and potentiates interleukin 8 secretion and neutrophil transmigration. Proc. Natl. Acad. Sci. USA 90:11137.[Abstract/Free Full Text]
  7. Dentelli, P., L. Del Sorbo, A. Rosso, A. Molinar, G. Garbarino, G. Camussi, L. Pegoraro, M. F. Brizzi. 1999. Human IL-3 stimulates endothelial cell motility and promotes in vivo new vessel formation. J. Immunol. 163:2151.[Abstract/Free Full Text]
  8. Nilsen, E. M., F. E. Johansen, F. L. Jahnsen, K. E. Lundin, T. Scholz, P. Brandtzaeg, G. Haraldsen. 1998. Cytokine profiles of cultured microvascular endothelial cells from the human intestine. Gut 42:635.[Abstract/Free Full Text]
  9. Alitalo, K., P. Carmeliet. 2002. Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell 1:219.[Medline]
  10. Baldwin, M. E., S. A. Stacker, M. G. Achen. 2002. Molecular control of lymphangiogenesis. BioEssays 24:1030.[Medline]
  11. Jackson, D. G.. 2003. The lymphatics revisited: new perspectives from the hyaluronan receptor LYVE-1. Trends Cardiovasc. Med. 13:1.[Medline]
  12. Oliver, G., M. Detmar. 2002. The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature. Genes Dev. 16:773.[Free Full Text]
  13. Pepper, M. S., M. Skobe. 2003. Lymphatic endothelium: morphological, molecular and functional properties. J. Cell Biol. 163:209.[Abstract/Free Full Text]
  14. Wigle, J. T., N. Harvey, M. Detmar, I. Lagutina, G. Grosveld, M. D. Gunn, D. G. Jackson, G. Oliver. 2002. An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J. 21:1505.[Medline]
  15. Breiteneder-Geleff, S., A. Soleiman, H. Kowalski, R. Horvat, G. Amann, E. Kriehuber, K. Diem, W. Weninger, E. Tschachler, K. Alitalo, D. Kerjaschki. 1999. Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am. J. Pathol. 154:385.[Abstract/Free Full Text]
  16. Kriehuber, E., S. Breiteneder-Geleff, M. Groeger, A. Soleiman, S. F. Schoppmann, G. Stingl, D. Kerjaschki, D. Maurer. 2001. Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J. Exp. Med. 194:797.[Abstract/Free Full Text]
  17. Ma, T., B. Yang, M. A. Matthay, A. S. Verkman. 1998. Evidence against a role of mouse, rat, and two cloned human T1{alpha} isoforms as a water channel or a regulator of aquaporin-type water channels. Am. J. Respir. Cell Mol. Biol. 19:143.[Abstract/Free Full Text]
  18. Zimmer, G., F. Oeffner, V. Von Messling, T. Tschernig, H. J. Groness, H. D. Klenk, G. Herrler. 1999. Cloning and characterization of gp36, a human mucin-type glycoprotein preferentially expressed in vascular endothelium. Biochem. J. 341:277.
  19. Kaipainen, A., J. Korhonen, T. Mustonen, V. W. van Hinsbergh, G. H. Fang, D. Dumont, M. Breitman, K. Alitalo. 1995. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc. Natl. Acad. Sci. USA 92:3566.[Abstract/Free Full Text]
  20. Karkkainen, M. J., T. Makinen, K. Alitalo. 2002. Lymphatic endothelium: a new frontier of metastasis research. Nat. Cell Biol. 4:E2.[Medline]
  21. Banerji, S., J. Ni, S. X. Wang, S. Clasper, J. Su, R. Tammi, M. Jones, D. G. Jackson. 1999. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J. Cell Biol. 144:789.[Abstract/Free Full Text]
  22. Hirakawa, S., Y. K. Hong, N. Harvey, V. Schacht, K. Matsuda, T. Libermann, M. Detmar. 2003. Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells. Am. J. Pathol. 162:575.[Abstract/Free Full Text]
  23. Petrova, T. V., T. Makinen, T. P. Makela, J. Saarela, I. Virtanen, R. E. Ferrell, D. N. Finegold, D. Kerjaschki, S. Yla-Herttuala, K. Alitalo. 2002. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J. 21:4593.[Medline]
  24. Podgrabinska, S., P. Braun, P. Velasco, B. Kloos, M. S. Pepper, D. G. Jackson, M. Skobe. 2002. Molecular characterization of lymphatic endothelial cells. Proc. Natl. Acad. Sci. USA 99:16069.[Abstract/Free Full Text]
  25. Schlingemann, R. O., G. M. Dingjan, J. J. Emeis, J. Blok, S. O. Warnaar, D. J. Ruiter. 1985. Monoclonal antibody PAL-E specific for endothelium. Lab. Invest. 52:71.[Medline]
  26. Schacht, V., M. I. Ramirez, Y. K. Hong, S. Hirakawa, D. Feng, N. Harvey, M. Williams, A. M. Dvorak, H. F. Dvorak, G. Oliver, M. Detmar. 2003. T1{alpha}/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J. 22:3546.[Medline]
  27. Wilting, J., M. Papoutsi, B. Christ, K. H. Nicolaides, C. S. von Kaisenberg, J. Borges, G. B. Stark, K. Alitalo, S. I. Tomarev, C. Niemeyer, J. Rossler. 2002. The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues. FASEB J. 16:1271.[Abstract/Free Full Text]
  28. Karkkainen, M. J., P. Haiko, K. Sainio, J. Partanen, J. Taipale, T. V. Petrova, M. Jeltsch, D. G. Jackson, M. Talikka, H. Rauvala, et al 2004. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat. Immunol. 5:74.[Medline]
  29. Makinen, T., L. Jussila, T. Veikkola, T. Karpanen, M. I. Kettunen, K. J. Pulkkanen, R. Kauppinen, D. G. Jackson, H. Kubo, S. Nishikawa, et al 2001. Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat. Med. 7:199.[Medline]
  30. Veikkola, T., L. Jussila, T. Makinen, T. Karpanen, M. Jeltsch, T. V. Petrova, H. Kubo, G. Thurston, D. M. McDonald, M. G. Achen, et al 2001. Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J. 20:1223.[Medline]
  31. Kubo, H., T. Fujiwara, L. Jussila, H. Hashi, M. Ogawa, K. Shimizu, M. Awane, Y. Sakai, A. Takabayashi, K. Alitalo, et al 2000. Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis. Blood 96:546.[Abstract/Free Full Text]
  32. Paavonen, K., P. Puolakkainen, L. Jussila, T. Jahkola, K. Alitalo. 2000. Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing. Am. J. Pathol. 156:1499.[Abstract/Free Full Text]
  33. Partanen, T. A., K. Alitalo, M. Miettinen. 1999. Lack of lymphatic vascular specificity of vascular endothelial growth factor receptor 3 in 185 vascular tumors. Cancer 86:2406.[Medline]
  34. Partanen, T. A., J. Arola, A. Saaristo, L. Jussila, A. Ora, M. Miettinen, S. A. Stacker, M. G. Achen, K. Alitalo. 2000. VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues. FASEB J. 14:2087.[Abstract/Free Full Text]
  35. Petzelbauer, P., C. A. Watson, S. E. Pfau, J. S. Pober. 1995. IL-8 and angiogenesis: evidence that human endothelial cells lack receptors and do not respond to IL-8 in vitro. Cytokine 7:267.[Medline]
  36. Petzelbauer, P., J. R. Bender, J. Wilson, J. S. Pober. 1993. Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture. J. Immunol. 151:5062.[Abstract]
  37. Matsumura, T., K. Wolff, P. Petzelbauer. 1997. Endothelial cell tube formation depends on cadherin 5 and CD31 interactions with filamentous actin. J. Immunol. 158:3408.[Abstract]
  38. Colotta, F., F. Bussolino, N. Polentarutti, A. Guglielmetti, M. Sironi, E. Bocchietto, M. De Rossi, A. Mantovani. 1993. Differential expression of the common {beta} and specific {alpha} chains of the receptors for GM-CSF, IL-3, and IL-5 in endothelial cells. Exp. Cell Res. 206:311.[Medline]
  39. Korpelainen, E. I., J. R. Gamble, M. A. Vadas, A. F. Lopez. 1996. IL-3 receptor expression, regulation and function in cells of the vasculature. Immunol. Cell Biol. 74:1.[Medline]
  40. Sun, Q., J. M. Woodcock, A. Rapoport, F. C. Stomski, E. I. Korpelainen, C. J. Bagley, G. J. Goodall, W. B. Smith, J. R. Gamble, M. A. Vadas, A. F. Lopez. 1996. Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor {alpha}-chain and functions as a specific IL-3 receptor antagonist. Blood 87:83.[Abstract/Free Full Text]
  41. Hong, Y. K., N. Harvey, Y. H. Noh, V. Schacht, S. Hirakawa, M. Detmar, G. Oliver. 2002. Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate. Dev. Dyn. 225:351.[Medline]
  42. Lantz, C. S., J. Boesiger, C. H. Song, N. Mach, T. Kobayashi, R. C. Mulligan, Y. Nawa, G. Dranoff, S. J. Galli. 1998. Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature 392:90.[Medline]
  43. Guthridge, M. A., F. C. Stomski, D. Thomas, J. M. Woodcock, C. J. Bagley, M. C. Berndt, A. F. Lopez. 1998. Mechanism of activation of the GM-CSF, IL-3, and IL-5 family of receptors. Stem Cells 16:301.[Abstract/Free Full Text]
  44. Hirsch, H. H., A. P. Nair, V. Backenstoss, C. Moroni. 1995. Interleukin-3 mRNA stabilization by a trans-acting mechanism in autocrine tumors lacking interleukin-3 gene rearrangements. J. Biol. Chem. 270:20629.[Abstract/Free Full Text]
  45. Stoecklin, G., X. F. Ming, R. Looser, C. Moroni. 2000. Somatic mRNA turnover mutants implicate tristetraprolin in the interleukin-3 mRNA degradation pathway. Mol. Cell. Biol. 20:3753.[Abstract/Free Full Text]
  46. Jiang, X., A. Lopez, T. Holyoake, A. Eaves, C. Eaves. 1999. Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia. Proc. Natl. Acad. Sci. USA 96:12804.[Abstract/Free Full Text]
  47. Saharinen, P., T. Tammela, M. Karkkainen, K. Alitalo. 2004. Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation. Trends Immunol. 25:387.[Medline]
  48. Hong, Y., K. Foreman, J. W. Shin, S. Hirakawa, C. L. Curry, D. R. Sage, T. Libermann, B. J. Dezube, J. D. Fingeroth, M. Detmar. 2004. Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus. Nat. Genet. 36:683.[Medline]
  49. Wang, H., M. W. B. Trotter, D. Lagos, D. Bourboulia, S. Henderson, T. Mäkinen, S. Elliman, A. M. Flanagan, K. Alitalo, C. Boshoff. 2004. Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma. Nat. Genet. 36:687.[Medline]
  50. Dentelli, P., A. Rosso, C. Calvi, B. Ghiringhello, G. Garbarino, G. Camussi, L. Pegoraro, M. F. Brizzi. 2004. IL-3 affects endothelial cell-mediated smooth muscle cell recruitment by increasing TGF{beta} activity: potential role in tumor vessel stabilization. Oncogene 23:1681.[Medline]
  51. McGary, C. T., M. E. Miele, D. R. Welch. 1995. Highly metastatic 13762NF rat mammary adenocarcinoma cell clones stimulate bone marrow by secretion of granulocyte-macrophage colony-stimulating factor/interleukin-3 activity. Am. J. Pathol. 147:1668.[Abstract]
  52. Kharkevitch, D. D., D. Seito, G. C. Balch, T. Maeda, C. M. Balch, K. Itoh. 1994. Characterization of autologous tumor-specific T-helper 2 cells in tumor-infiltrating lymphocytes from a patient with metastatic melanoma. Int. J. Cancer 58:317.[Medline]
  53. Geleff, S., S. F. Schoppmann, G. Oberhuber. 2003. Increase in podoplanin-expressing intestinal lymphatic vessels in inflammatory bowel disease. Virchows Arch. 442:231.[Medline]
  54. Kerjaschki, D., H. M. Regele, I. Moosberger, K. Nagy-Bojarski, B. Watschinger, A. Soleiman, P. Birner, S. Krieger, A. Hovorka, G. Silberhumer, et al 2004. Lymphatic neoangiogenesis in human kidney transplants is associated with immunologically active lymphocytic infiltrates. J. Am. Soc. Nephrol. 15:603.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
M. Oka, C. Iwata, H. I. Suzuki, K. Kiyono, Y. Morishita, T. Watabe, A. Komuro, M. R. Kano, and K. Miyazono
Inhibition of endogenous TGF-{beta} signaling enhances lymphangiogenesis
Blood, May 1, 2008; 111(9): 4571 - 4579.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
R. Sivakumar, N. Sharma-Walia, H. Raghu, M. V. Veettil, S. Sadagopan, V. Bottero, L. Varga, R. Levine, and B. Chandran
Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained Levels of Vascular Endothelial Growth Factors A and C Early during In Vitro Infection of Human Microvascular Dermal Endothelial Cells: Biological Implications
J. Virol., February 15, 2008; 82(4): 1759 - 1776.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
H J Van Kruiningen and J.-F. Colombel
The forgotten role of lymphangitis in Crohn's disease
Gut, January 1, 2008; 57(1): 1 - 4.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Zeng, K. Opeskin, J. Goad, and E. D. Williams
Tumor-Induced Activation of Lymphatic Endothelial Cells via Vascular Endothelial Growth Factor Receptor-2 Is Critical for Prostate Cancer Lymphatic Metastasis
Cancer Res., October 1, 2006; 66(19): 9566 - 9575.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
T. Kono, H. Kubo, C. Shimazu, Y. Ueda, M. Takahashi, K. Yanagi, N. Fujita, T. Tsuruo, H. Wada, and J. K. Yamashita
Differentiation of Lymphatic Endothelial Cells From Embryonic Stem Cells on OP9 Stromal Cells
Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2070 - 2076.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gröger, M.
Right arrow Articles by Petzelbauer, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gröger, M.
Right arrow Articles by Petzelbauer, P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS