Research Article
Lysophosphatidic acid up-regulates vascular endothelial growth
factor-C and lymphatic marker expressions in human endothelial
cells
C.-I. Lina,†, C.-N. Chenc,d,g,†, M.-T. Huange, S.-J. Leea,b, C.-H. Lina, C.-C. Changfand H. Leea,b,c,d,*
a
Institute of Zoology, National Taiwan University, 1 Roosevelt Rd., Sec. 4, Taipei 106 (Taiwan), Fax: +8862-2363-6837, e-mail: [email protected]
b
Department of Life Science, National Taiwan University, Taipei (Taiwan)
c
Angiogenesis Research Center, National Taiwan University, Taipei (Taiwan)
d
Department of Surgery, National Taiwan University Hospital, Taipei (Taiwan)
e
Department of Pediatrics, National Taiwan University Hospital, Taipei (Taiwan)
f
Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, National Taiwan University, Taipei (Taiwan)
g
Division of Mechanics, Research Center for Applied Sciences, Academia Sinica, Taipei (Taiwan) Received 4 June 2008; received after revision 21 June 2008; accepted 1 July 2008
Online First 21 July 2008
Abstract. Lysophosphatidic acid (LPA) is a low-molecular-weight lipid growth factor, which binds to G-protein-coupled receptors. Previous studies have shown that LPA enhances vascular endothelial growth factor-A (VEGF-A) expression in cancer cells and promotes angiogenesis process. However, the roles of LPA in lymphatic vessel formation and lymphangio-genesis have not been investigated. Here, we demon-strated that LPA up-regulated VEGF-C mRNA and protein expressions in human umbilical vein endothe-lial cells (HUVECs). Furthermore, the expression levels of lymphatic markers, including Prox-1,
LYVE-1 and podoplanin, were enhanced in LPA-stimulated tube forming endothelial cells in vitro and in vivo. Moreover, we showed that pretreatment with MAZ51, a VEGFR-3 kinase inhibitor, and introduc-tion of VEGFR-3 siRNA suppressed LPA-induced HUVEC tube formation and lymphatic marker ex-pressions. These results demonstrated that LPA enhances expression of lymphatic markers through activating VEGF-C receptors in endothelial cells. This study provides basic information that LPA might be a target for therapeutics against lymphangiogenesis and tumor metastasis.
Keywords. LPA, VEGF-C, endothelial cells, lymphatic markers, lymphangiogenesis.
Introduction
Lysophosphatidic acid (LPA) is a low-molecular-weight phospholipid present in plasma and tissues at
the micromolar level [1]. By interacting with cell surface G-protein-coupled receptors (GPCRs) of the endothelial differentiation gene (Edg) subfamily [2], LPA modulates various physiological functions such as cell proliferation, differentiation, and stimulation of tumor metastasis [3, 4]. Until now, five LPA receptors, LPA1–5, have been identified. In previous
† These authors contribute equally to this work.
* Corresponding author. 1420-682X/08/172740-12 DOI 10.1007/s00018-008-8314-9 Birkhuser Verlag, Basel, 2008
studies, LPA was shown to regulate human endothelial cell proliferation [3, 5], migration [3, 6], capillary-like tube formation in vitro [7, 8], activation of proteases [9], and expression of inflammation-related genes [10]. These results suggested that LPA may play an important role in regulating vessel formation in endothelial cells.
Both angiogenesis and lymphangiogenesis are events dependent on vessel formation [11, 12]. Accumulating evidence indicates that LPA plays an important role in the angiogenesis process [13, 14]. However, the importance of LPA in lymphangiogenesis has not been studied. The formation of new lymphatic vessels depends on the effects of various lymphangiogenic factors [15, 16]. Vascular endothelial growth factor (VEGF)-C has been implicated as being a potent stimulator of both angiogenesis and lymphangiogen-esis [17]. By binding with VEGFR-2 and VEGFR-3, VEGF-C exerts its biological functions in various cell types [18]. Many reports indicated that VEGF-C is a ligand for VEGFR-3, while proteolytically processed VEGF-C binds to both VEGFR-2 and VEGFR-3 [19]. Since VEGFR-2 is thought to be the main mediator of angiogenesis and VEGFR-3 is crucial for lymphangiogenesis [20, 21], the promotion by VEGF-C of endothelial cell participation in the lymphangio-genesis process is predominantly mediated through binding with VEGFR-3 [22]. Previous studies indi-cated that VEGF-C largely acts via VEGFR-2 in skin [23] and is predominantly mediated through VEGFR-3 in cancer cells [24], implying that VEGF-C might regulate cellular functions through different VEGF receptors in different cell types. VEGF-C has been reported to be expressed by several cancer cells such as ovarian carcinoma cells [25], lung adenocarcinoma cells [26], breast cancer cells [27], and head-and-neck squamous carcinoma cells [28], and to promote lymphatic metastasis progression, thus promoting tumor malignancy [29].
The lack of specific lymphatic markers has in the past hampered progress with lymphangiogenesis studies. However, the recent identification of specific lym-phatic markers has greatly contributed to diagnoses of lymphatic disorders and to lymphangiogenesis re-search [30]. Among these markers, Prox-1, LYVE-1, and podoplanin are commonly used to determine the effects of VEGF-C on lymphangiogenesis [31, 32]. These three lymphatic markers have also been used to promote lymphatic metastasis of tumors in clinical research [33].
Many reports have shown that LPA up-regulates VEGF-A, another VEGF family member recognized as a potent angiogenic factor expressed by ovarian cancer cells, and which stimulates the angiogenesis process [34]. However, the role of LPA in regulating
VEGF-C and the subsequent lymphangiogenesis process has not been studied. In the present study, we first observed that LPA up-regulated VEGF-C mRNA and protein expressions in human umbilical vein endothelial cells (HUVECs) in a dose- and time-dependent fashion. Moreover, LPA also enhanced the mRNA and protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HU-VECs. In addition, LPA induced endothelial cell tube formation and elevations of Prox-1, LYVE-1, and podoplanin expressions in these LPA-stimulated en-dothelial cells in which tubes formed in vitro and in vivo. LPA also stimulated endothelial cell prolifer-ation during the lymphangiogenesis process. More-over, LPA-induced lymphatic marker expressions could be blocked by pretreatment with the VEGFR-3 kinase inhibitor and introduction of VEGFR-VEGFR-3 siRNA. Our findings first demonstrate that LPA might be a potent lymphangiogenic factor, which promotes VEGF-C expression in endothelial cells, thereby stimulating lymphatic markers expression and facilitating the lymphangiogenesis process.
Materials and methods
Reagents and antibodies. LPA and MAZ51 were purchased from Sigma (St. Louis, MO). Normal mouse and goat immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Human recombinant VEGF-A and VEGF-C were purchased from R&D systems (Min-neapolis, MN). Human recombinant EGF was pur-chased from PeproTech (Rocky Hill, NJ). Fetal bovine serum (FBS) and M199 were purchased from Hyclone (Logan, UT). Trypsin-EDTA was purchased from Gibco BRL (Grand Island, NY). Endothelial cell growth medium (EGM) was purchased from Cell Applications (San Diego, CA). Penicillin, streptomy-cin, and L-glutamine were purchased from Invitrogen (Carlsbad, CA).
Cell culture. HUVECs were isolated from fresh umbilical cords by treatment with 1 % collagenase (Sigma) in phosphate-buffered saline (PBS) at 37 8C for 10 min. After elution with M199 containing 20 % FBS, HUVECs were cultured on 0.04 % gelatin-coated (Sigma) 10-cm plates (Greiner Bio-One, Kremsmuen-ster, Austria) in M199 medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine (Invitrogen), 10 % (v/v) FBS, and 25 % (v/v) EGM. Cells underwent one passage weekly. Cells were subcultured after trypsinization [in a 0.5 % (v/v) trypsin solution, supplemented with 0.2 % (v/v) EDTA] and used throughout passages 2–4.
Reverse-transcription polymerase chain reaction (RT-PCR). Total cellular RNA was extracted from HUVECs using the TRIzol reagent (Gibco), and a Superscript kit (Gibco) was used for the RT synthesis of cDNA. PCR amplification was performed using the oligonucleotide primers of human VEGF-C (5’-CTCACTTCCTGCCGATGC-3’ and 5’-GTTCGCACHTUNGTRENNUNGT-ACHTUNGTRENNUNGGACHTUNGTRENNUNGCCTGACACTG-3’), LPA1
(5’-CACHTUNGTRENNUNGGACHTUNGTRENNUNGGACHTUNGTRENNUNGAACHTUNGTRENNUNGGACHTUNGTRENNUNGACTG-ACHTUNGTRENNUNGACACHTUNGTRENNUNGTACHTUNGTRENNUNGGACHTUNGTRENNUNGTACHTUNGTRENNUNGCACHTUNGTRENNUNGAACHTUNGTRENNUNGGACHTUNGTRENNUNGCA-3’ and 5’-GACHTUNGTRENNUNGGACHTUNGTRENNUNGTACHTUNGTRENNUNGCACHTUNGTRENNUNGCACHTUNGTRENNUNGAGAACTAT-ACHTUNGTRENNUNGGACHTUNGTRENNUNGCACHTUNGTRENNUNGCACHTUNGTRENNUNGGACHTUNGTRENNUNGAACHTUNGTRENNUNGGACHTUNGTRENNUNGA-3’), LPA3 (5’-T
ACHTUNGTRENNUNGTACHTUNGTRENNUNGAACHTUNGTRENNUNGGACHTUNGTRENNUNGCTGCTGCCACHTUNGTRENNUNGG-ACHTUNGTRENNUNGAACHTUNGTRENNUNGTACHTUNGTRENNUNGTACHTUNGTRENNUNGTACHTUNGTRENNUNGCACHTUNGTRENNUNGTACHTUNGTRENNUNGT-3’ and 5’-AACHTUNGTRENNUNGTACHTUNGTRENNUNGGACHTUNGTRENNUNGAACHTUNGTRENNUNGTACHTUNGTRENNUNGGAGGAAGGCCA-ACHTUNGTRENNUNGTACHTUNGTRENNUNGGACHTUNGTRENNUNGAACHTUNGTRENNUNGG-3’), Prox-1 (5’-AACHTUNGTRENNUNGAACHTUNGTRENNUNGGACHTUNGTRENNUNGAACHTUNGTRENNUNGCAGAGCCTCTCC-ACHTUNGTRENNUNGTGAAACHTUNGTRENNUNGTACHTUNGTRENNUNGC-3’ and 5’-TACHTUNGTRENNUNGTACHTUNGTRENNUNGGACHTUNGTRENNUNGCACHTUNGTRENNUNGACTTCCCGAATAAACHTUNGTRENNUNGG-ACHTUNGTRENNUNGGACHTUNGTRENNUNGTACHTUNGTRENNUNGGACHTUNGTRENNUNGAACHTUNGTRENNUNGT-3’), LYVE-1 (5’-GACHTUNGTRENNUNGTACHTUNGTRENNUNGGACHTUNGTRENNUNGCACHTUNGTRENNUNGTACHTUNGTRENNUNGTCAGCCTGG-ACHTUNGTRENNUNGTACHTUNGTRENNUNGGACHTUNGTRENNUNGTACHTUNGTRENNUNGTACHTUNGTRENNUNGG-3’ and 5’-GACHTUNGTRENNUNGCTTGGACTCTTGGACTCT-ACHTUNGTRENNUNGTC-3’), and podoplanin (5’-CCACHTUNGTRENNUNGAGGAGAGCA-ACAACTCAA-3’ and 5’-GATGCGAATGCACHTUNGTRENNUNGCACHTUNGTRENNUNGTACHTUNGTRENNUNGGACHTUNGTRENNUNGT-ACHTUNGTRENNUNGTACHTUNGTRENNUNGACACHTUNGTRENNUNGAACHTUNGTRENNUNGC-3’) with 35 cycles of 30 s at 94 8C, 30 s at 60 8C, and 2 min at 72 8C. The primers used to amplify GAPDH were 5’-dAACHTUNGTRENNUNGCACHTUNGTRENNUNGCACHTUNGTRENNUNGAACHTUNGTRENNUNGCACHTUNGTRENNUNGAGTTCATGCCATCAC and 5’-dTCCACCACCCTGTTGCTGACHTUNGTRENNUNGTA with 35 cy-cles of 30 s at 94 8C, 30 s at 55 8C, and 2 min at 72 8C. PCR products were resolved on 2 % agarose gels, stained with ethidium bromide, and photographed. Enzyme-linked immunosorbent assay (ELISA). The VEGF-C concentrations in the culture supernatant were measured by ELISA kits purchased from R&D Systems. An antibody specific for the cytokine to be studied was coated onto the wells of a 96-well ELISA plate. Samples, including standards with known cyto-kine contents, control specimens, and unknowns, were pipetted into these wells, followed by the addition of a biotinylated second antibody. During the first incuba-tion, the cytokine antigen simultaneously bound to the captured antibody on one site and to the solution phase biotinylated antibody on a second site. After removal of any excess of the secondary antibody, conjugated streptavidin peroxidase (Chemicon International, Te-mecula, CA) was added. This enzyme bound to the biotinylated antibody to complete the four-member sandwich. After a second incubation and washing to remove all unbound enzyme, a substrate solute was added, which was acted upon by the bound enzyme to produce color. The intensity of this colored product was directly proportional to the concentration of cytokines present in the original specimen.
Cyflow analysis. Sub-confluent HUVECs were starved for 16 h and treated as indicated. Suspensions of 106
cells were permeabilized with methanol (–20 8C). Permeabilized cells in 200 ml PBS with 0.1 % fatty acid-free bovine serum albumin (BSA) were mixed with 2 ml of the antibodies against human Prox-1, LYVE-1, or podoplanin and were incubated for 1 h at
4 8C. Antibody-conjugated cells were washed with PBS three times and incubated with FITC-conjugated secondary antibodies (Pierce Chemical, Rockford, IL) for 2 h at 4 8C. Fluorescence signals were determined by CyFlow
SL (Partec, Mnster, Ger-many) and analyzed by WinMDI version 2.8 software. In vitro Matrigel tube formation assay. Matrigel (BD Pharmingen, San Diego, CA) at 0.4 ml/well was plated evenly in a 24-well plate, and incubated at 37 8C for 30 min before seeding the HUVECs (0.5105
cells/ well). Tube formation was studied over 6 h and photo-graphed by phase-contrast microscopy. The original magnification used was 100. The Matrigel was fixed with methanol (–20 8C), blocked, permeabilized, and stained with a mouse anti-human antibody (clone: WM59) against PECAM-1 (BD Pharmingen) and a goat anti-human Prox-1 antibody (clone: AF2727, R&D systems) followed by incubation with an FITC-conjugated goat anti-mouse secondary antibody (DAKO, Carpinteria, CA) or an AlexaFluor-555-con-jugated donkey anti-goat secondary antibody (Molec-ular Probes, Eugene, OR). The Matrigel was also stained with goat anti-human LYVE-1 (clone: AF2089, R&D systems) or mouse anti-human podoplanin (clone: 4D5a55E6, Santa Cruz Biotechnology) fol-lowed by incubation with an AlexaFluor-555-conjugat-ed donkey anti-goat secondary antibody (Molecular Probes) or Cy5-conjugated goat anti-mouse secondary antibody (DAKO). After a series of further washes with PBS, samples were mounted on glass slides and viewed using a Zeiss fluorescence microscope (Ober-kochen, Germany). The original magnification used was 100.
In vivo Matrigel plug assay. Eight-week-old BALB/c mice were given a subcutaneous injection at the abdominal midline with 0.4 ml Matrigel supplemented with media, and 5 mM LPA. After 7 days, the mice were killed, and the plugs were removed and then processed for hematoxylin and eosin (H&E) staining. Images were photographed by phase-contrast microscopy. The vessels penetrating the Matrigel were subjected to an immuno-histochemical assay and visualized by Zeiss fluorescence microscopy. The original magnification used was 40. BALB/c mice were obtained from laboratory animal center of National Taiwan University medical school (Institutional Animal Care and Use Committee appro-val no: 096046).
Immunohistochemical assay. Subcutaneously injected Matrigel plugs were dissected away from BALB/c mice and fixed in 4 % paraformaldehyde before being immunostained for Prox-1 and PECAM-1. Briefly, Matrigel plug sections were blocked and permeabilized
in PBS containing 20 % fetal calf serum and 0.5 % Triton X-100. After incubation, the mouse anti-human antibody (clone: Mec13.3) against PECAM-1 (BD Pharmingen) and rabbit anti-mouse Prox-1 antibody (clone: AB5475, Chemicon International) were fol-lowed by incubation with the FITC-conjugated goat anti-rat secondary antibody (DAKO) and AlexaFluor-555-conjugated goat anti-rabbit secondary antibody (Molecular Probes). Sections were also stained with goat anti-mouse LYVE-1 (Clone: AF2125, R&D Systems) or goat anti-mouse podoplanin (Clone: AF3244, R&D Systems) followed by incubation with an AlexaFluor-555-conjugated donkey anti-goat sec-ondary antibody (Molecular Probes). After three washes with PBS, samples were mounted on glass slides and viewed using a Zeiss fluorescence micro-scope.
Cell proliferation detection by 5-bromo-2’-deoxyur-idine (BrdU). To detect proliferating cells during the course of tube formation, media or 5 mM of LPA-treated HUVECs seeded on Matrigel-coated plates were incubated with 10 mM of the thymidine analog BrdU (Sigma), which is selectively incorporated into cellular DNA during the S-phase. After 24 h of incubation, samples were stained with the FITC-conjugated anti-BrdU antibody (BD Pharmingen) and then visualized by Zeiss fluorescence microscope. siRNA transfections. siRNAs targeting VEGFR-3 were obtained from Santa Cruz Biotechnology. Se-quences of 21-nucleotide scrambled siRNA were UUCUCCGAACGUGUUCACGUdTdT, and AC-GUGACACGUUCGGAGAAdTdT. HUVECs were transfected using an optimized protocol for electro-poration of HUVECs with the Nucleofector appara-tus (Amaxa Biosystems, Kçln, Germany). Cells at 80 % confluence were trypsinized and centrifuged. Cells (1106
) were resuspended in 100 ml of supple-mented HUVECs NucleofectorTM solution (Amaxa Biosystems) and electroporated in the presence of 2 mg of various siRNA oligonucleotides or constructs. Transfected cells were seeded onto gelatin-coated plastic dishes and used after 24 h.
Statistical analysis. Significant differences between treatment groups were tested using analysis of var-iance (ANOVA) followed by Duncans new multiple-range tests (StatView; Abacus Concept, Berkeley, CA). Each experiment was repeated at least three times. A value of p<0.05 was considered statistically significant.
Results
LPA enhances VEGF-C mRNA expression in HU-VECs. LPA promotes angiogenesis through enhanc-ing the expressions of various genes, includenhanc-ing VEGF-A [13, 34]. We further investigated if LPVEGF-A up-regulates mRNA expression of the lymphangiogenic factor VEGF-C. LPA up-regulated VEGF-C mRNA expression in HUVECs in a concentration-dependent manner (Fig. 1A). The expression of VEGF-C mRNA appeared at 1 mM and peaked at 5 mM LPA treatment, and the effect was sustained at 10 mM of LPA treat-ment. Thus, we used 5 mM LPA for the rest of the experiments. Because the enhancement of VEGF-C mRNA expressions by LPA treatment in HUVECs was concentration dependent, we next investigated whether the effects of LPA on VEGF-C mRNA levels in HUVECs were time dependent (Fig. 1C). HU-VECs were incubated with 5 mM LPA for different time intervals as indicated. The enhanced VEGF-C mRNA expressions in HUVECs were first observed as early as 1 h after treatment was initiated, peaked at 2 h, were sustained at a high level until 4 h, and then declined thereafter. These results indicated that LPA enhances VEGF-C mRNA expressions in HUVECs. LPA enhances VEGF-C protein expression in HU-VECs. Since mRNA levels were increased by LPA, we further investigated if the elevated mRNA level of VEGF-C was also correlated with protein expression levels. The total VEGF-C protein levels in LPA-treated cells were detected using ELISA. Our results showed that LPA enhanced VEGF-C protein expres-sions in concentration- (Fig. 1B) and time-dependent (Fig. 1D) manners. Consistent with the RT-PCR results, the enhancement effects of LPA on VEGF-C protein expression in HUVECs peaked at a concen-tration of 5 mM, and the effect was sustained at 10 mM of LPA treatment. In the time-course experiments, the enhancement effects of LPA on VEGF-C protein expressions peaked at 8 h, and the effect was sustained at 24 h after treatment (Fig. 1D). These results indicated that LPA enhances mRNA and protein expressions of VEGF-C in HUVECs.
LPA enhances Prox-1, LYVE-1, and podoplanin mRNA and protein expressions in HUVECs. We next determined whether LPA-induced VEGF-C mRNA expression contributes to lymphangiogenesis. The mRNA expression level of the three lymphatic markers, Prox-1, LYVE-1, and podoplanin, were up-regulated by 5-mM LPA treatment at 8 and 12 h (Fig. 1C). By Cyflow analysis, we observed that 5 mM LPA, 10 ng/ml EGF, and 100 ng/ml VEGF-C en-hanced Prox-1, LYVE-1, and podoplanin protein
expressions in HUVECs. However, treatment with VEGF-A (100 ng/ml) showed no enhancement effects on these lymphatic marker expressions in HUVECs (Fig. 2). In the presence of control antibodies, media-and LPA-treated cells were negative for these lym-phatic marker expressions (Fig. 2). These results confirmed the specificity of antibodies against human Prox-1, LYVE-1, and podoplanin. Consistent with the RT-PCR findings (Fig. 1C), these results further confirmed that LPA might be a specific lymphangiogenic factor, which stimulates lymphatic marker expression in human endothelial cells.
LPA induces HUVEC tube formation and specific lymphatic marker expressions in vitro. To determine whether LPA-induced VEGF-C mRNA expression has any physiological significance, we next investigated LPA-induced HUVEC tube formation in vitro. HU-VECs were treated with media or LPA (5 mM) and then seeded onto Matrigel-coated plates. We observed that LPA profoundly enhanced HUVEC tube formation in vitro (Fig. 3A). Using an in vitro Matrigel tube formation assay followed by immunostaining with a specific lymphatic marker, Prox-1, we found a signifi-cant increase in Prox-1 staining in tube-forming
Figure 1. Lysophosphatidic acid (LPA) up-regulates vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) in a dose- and time-dependent manner. (A) HUVECs were incubated with LPA for 4 h at various concentrations as indicated. RNAs from treated cells were harvested and subjected to RT-PCRs using specific primer sets for human VEGF-C or GAPDH. The reaction products were separated on 2 % agarose gels and photographed. (B) Levels of VEGF-C in supernatant of cultured HUVECs were measured by ELISA analysis after incubation with LPA for 24 h at various concentrations as indicated. (C) HUVECs were also incubated with LPA (5 mM) for various times as indicated and then subjected to RT-PCRs using specific primer sets for human VEGF-C, Prox-1, LYVE-1, Podoplanin or GAPDH as described above. Histograms represent quantification of RT-PCR corrected with GAPDH and analyzed by PhosphorImagerusing ImageQuaNTsoftware. All data are relative multiples of
expression compared to untreated cells. (D) Patterns of VEGF-C protein expressions were determined after HUVECs were treated with 5 mM LPA for various times as indicated and were measured by ELISA analysis as described above. All ELISA analysis data are expressed as the mean SE. * Statistically different as compared to the control (p<0.05). Similar experiments were repeated three times, and a representative result is shown.
HUVECs in response to LPA (Fig. 3B). Moreover, we also observed profound increases in LYVE-1 and podoplanin staining in LPA-induced tube-forming HUVECs (Fig. 3C). In addition, we found that Prox-1 signals were detected in cell nucleus, whereas LYVE-1 and podoplanin were expressed in cell membrane as well as PECAM-1 (Fig. 3B, C), which is consist with previous findings [35]. By staining with PECAM-1, a well-known endothelial cell marker, we observed that only LPA-treated HUVECs expressed PECAM-1 (Fig. 3B). In contrast, media-treated samples displayed less green signals due to treatment of media stimulating no HUVEC branch formation. These results demon-strated that LPA might be a potent regulator, which enhances human endothelial cells tube formation and specific lymphatic marker expressions in vitro.
LPA induces HUVEC tube formation and specific lymphatic marker expressions in vivo. Since LPA enhances human endothelial cell tube formation and specific lymphatic marker expressions in vitro (Fig. 3), we further investigated the effects of LPA on endo-thelial cell tube formation and specific lymphatic marker expressions in vivo. Matrigel-containing media or LPA (5 mM) was injected subcutaneously into the abdominal area of BALB/c mice. The vascularized plugs were removed and processed by H&E staining to identify if the area was covered by vessels. As shown in Figure 4A, new vessel formation was observed in LPA-containing Matrigel plugs. These results confirmed that LPA can induce endo-thelial cell tube formation in vivo. Moreover, LPA also enhanced Prox-1, LYVE-1, and podoplanin expres-sions by endothelial cells in Matrigel plugs removed from the mouse abdominal subcutaneous midline
Figure 2. LPA up-regulates Prox-1, LYVE-1, and podoplanin protein expressions in HUVECs. (A) HUVECs were treated with media, LPA (5 mM), endothelial growth factor (EGF; 10 ng/ml), VEGF-C (100 ng/ml), or VEGF-A (100 ng/ml) for 8 h. Cells were dissociated by trypsinization and fixed with a 4 % paraformaldehyde solution. Fixed cells were incubated with a goat anti-human Prox-1 antibody for 1 h at 4 8C, then treated with an FITC-conjugated anti-goat secondary antibody for 30 min at 4 8C and analyzed by Cyflow. Media- and LPA-treated cells were also stained with a control antibody, normal goat IgG followed by an FITC-conjugated anti-goat secondary antibody, which was used for a negative control. (B) Trypsinized cells were incubated with the goat anti-human LYVE-1 or mouse anti-human podoplanin antibody or control IgG for 1 h at 4 8C, then treated with an FITC-conjugated secondary antibody for 30 min at 4 8C and analyzed by Cyflow. Media- and LPA-treated cells were also stained with control antibodies, normal goat IgG, or mouse goat IgG followed by FITC-conjugated anti-goat or anti-mouse secondary antibody, which was used as a negative control.
(Fig. 4B, C). Similar to in vitro Matrigel tube forma-tion results, we observed that less PECAM-1 signals could be detected in media-treated samples. More-over, Prox-1 was expressed in cell nucleus, whereas LYVE-1 and podoplanin were expressed on cell membrane (Fig. 4B, C). These results suggest that LPA might regulate the formation of lymphatic vessel endothelial cells in vivo.
LPA stimulates endothelial cell lymphatic marker expression and cell proliferation in vitro. Here we further verify whether LPA stimulates tube-formed endothelial cell proliferation. As shown in Figure 5, significant BrdU incorporation was observed in Prox-1-positive cells in response to 5 mM LPA treatment. These results indicated that LPA might modulate specific lymphatic marker expressions and prolifera-tion in human endothelial cells.
Enhancement by LPA of Prox-1, LYVE-1, and podoplanin expressions in HUVECs is mediated through a VEGF-C-dependent mechanism. Since
LPA induces VEGF-C mRNA and protein expression in HUVECs (Fig. 1A, B) and LPA also enhances Prox-1, LYVE-1, and podoplanin mRNA and protein expressions in HUVECs (Figs. 1, 2), we further investigated if the effect of LPA on these lymphatic markers expression is mediated through the induction of VEGF-C expression in endothelial cells. We addressed this question using MAZ51, a VEGFR-3 kinase inhibitor [36]. By Cyflow analysis, pretreat-ment with 10 mM MAZ51 for 12 h significantly sup-pressed the enhancement effects of LPA on Prox-1, LYVE-1, and podoplanin protein expressions in HUVECs (Fig. 6). These results indicated that en-hancement of Prox-1, LYVE-1, and podoplanin ex-pressions in endothelial cells by LPA is VEGF-C dependent. To further clarify the role of VEGF-C/ VEGFR-3 axis on LPA-induced HUVEC tube for-mation and lymphatic marker expression, HUVECs were transfected with scrambled or VEGFR-3 siRNA followed by media or 5-mM LPA treatment, and then subjected to in vitro Matrigel tube formation assay. Our results showed that introduction of VEGFR-3
Figure 3. LPA stimulates endothelial cell tube formation and specific lymphatic markers expression in vitro. (A) HUVECs were starved and treated with media or 5 mM LPA for 8 h and then seeded onto Matrigel-coated plates. Images were taken at 6 h after plating and visualized by phase-contrast microscopy. The histogram represents branches from each cell, which were counted from three representative 100 fields/well. * Statistically different as compared to the control (p<0.05). (B) Serum-starved HUVECs were treated with media or LPA (5 mM) for 8 h and then seeded on Matrigel-coated plates. At 6 h after plating, the Matrigel of each experiment was fixed and subjected to an immunocytochemical assay. Staining of a mouse anti-human PECAM-1 or a goat anti-human Prox-1 primary antibody followed by an FITC-conjugated anti-mouse or an Alexa-555-conjugated donkey anti-goat secondary antibody is shown. (C) Media or LPA-treated cells seeded on Matrigel-coated plates were also stained with a goat anti-human LYVE-1 or mouse anti-human podoplanin antibody followed by an AlexaFluor-555-conjugated donkey anti-goat or Cy5-conjugated goat anti-mouse secondary antibody. All images were visualized by fluorescence microscopy (original magnification, 100).
siRNA profoundly suppressed PECAM-1 signals in both LPA- and media-treated samples. Moreover, we found that Prox-1 signals detected in LPA-treated samples were significantly inhibited by VEGFR-3 siRNA (Fig. 6B). These results further confirmed that
LPA-stimulated endothelial tube formation and lym-phatic marker expression are mediated through a VEGF-C/VEGFR-3-dependent mechanism.
Pretreatment of MAZ51 suppressed LPA-induced HUVEC tube formation and Prox-1 expression in vitro. To further investigate if VEGF-C/VEGFR-3 axis mediates LPA-enhanced endothelial tube for-mation, HUVECs were pretreated with vehicle or MAZ51 (10 mM) for 12 h followed by treatments with media, LPA (5 mM), or VEGF-A (100 ng/ml) for 8 h. Treated cells were subjected to in vitro Matrigel tube formation assay. Our data showed that pretreatment of MAZ51 significantly
sup-pressed LPA- but not VEGF-A-enhanced
HUVEC tube formation in vitro (Fig. 7A). Further-more, our results demonstrated that MAZ51 pro-foundly suppressed PECAM-1 signals in LPA- but not VEGF-A-treated samples. In addition, we found that Prox-1 signals were significantly en-hanced in LPA- but not VEGF-A-treated samples and these enhancement effects were remarkably abrogated by MAZ-51 (Fig. 7B). These findings suggested that VEGR-3 is required for LPA but not
Figure 4. LPA stimulates endothelial cell tube formation and specific lymphatic markers expression in vivo. (A) Matrigel plugs were generated by a subcutaneous injection of Matrigel containing media or LPA (5 mM) into the abdominal region of BALB/c mice. The plugs were removed 7 days later and processed for hematoxylin and eosin (H&E) staining. The histogram represents the number of vessels penetrating the Matrigel, which were counted from three representative fields. * Statistically different as compared to the control (p<0.05). (B) Sections of the Matrigel plugs were incubated with a rat anti-mouse PECAM-1 or a rabbit anti-mouse Prox-1 primary antibody followed by an FITC-conjugated goat anti-rat or an Alexa-555-conjugated goat anti-rabbit secondary antibody (C) Sections were also stained with a goat anti-mouse LYVE-1 or podoplanin antibody followed by AlexaFluor-555-conjugated donkey anti-goat secondary antibodies. All images were visualized by fluorescence microscopy (original magnification, 100).
Figure 5. Effects of LPA on HUVEC lymphatic marker expression and cell proliferation in vitro. BrdU were added to starved HUVECs for 16 h, and cultures were treated with media or LPA (5 mM) for 8 h. Treated cells were seeded on Matrigel-coated plates and stained with a goat anti-human Prox-1 primary antibody followed by an Alexa-555-conjugated donkey anti-goat secondary antibody or FITC-conjugated BrdU antibody. All images were visualized by fluorescence microscopy (original magnification, 100).
Figure 6. LPA-induced Prox-1, LYVE-1, and podoplanin protein expressions in HUVECs were inhibited by VEGFR-3 kinase inhibitor and VEGFR-3 siRNA. (A) HUVECs were pretreated with vehicle control or 10 mM of MAZ51 for 12 h. Treated cells were then treated with control media or 5 mM LPA for 8 h. Cells were dissociated by trypsinization and fixed by a 4 % paraformaldehyde solution. Fixed cells were incubated with a goat anti-human Prox-1 antibody for 1 h at 4 8C, then treated with an FITC-conjugated anti-goat secondary antibody for 30 min at 4 8C and analyzed by Cyflow. Trypsinized cells were also incubated with the goat anti-human LYVE-1 or mouse anti-human podoplanin antibody at 4 8C, then treated with an FITC-conjugated secondary antibody for 30 min at 4 8C and analyzed by Cyflow. (B) HUVECs were transfected with scrambled or VEGFR-3 siRNA. At 24 h after transfection, HUVECs were treated with media or LPA (5 mM) for 8 h and then seeded on Matrigel-coated plates. At 6 h after plating, Matrigel of each experiment was permeabilized with methanol (–20 8C) and subjected to an immunocytochemical assay. Staining of a mouse anti-human PECAM-1 or a goat anti-human Prox-1 primary antibody followed by an FITC-conjugated anti-mouse or an Alexa-555-conjugated donkey anti-goat secondary antibody is shown.
Figure 7. Pretreatment with the VEGFR-3 kinase inhibitor sig-nificantly suppressed LPA-in-duced HUVEC tube formation and Prox-1 expression in vitro. (A) HUVECs were pretreated with vehicle control or 10 mM of MAZ51 for 12 h. Treated cells were treated with media, LPA (5 mM), or VEGF-A (100 ng/ml) for 8 h and then seeded on Ma-trigel-coated plates. At 6 h after plating, images were taken and visualized by phase-contrast mi-croscopy. (B) Matrigel of each experiment was permeabilized with methanol (–20 8C) and sub-jected to immunocytochemical assay. Staining of a mouse human PECAM-1 or a goat anti-human Prox-1 primary antibody followed by an FITC-conjugated anti-mouse or an Alexa-555-con-jugated donkey anti-goat secon-dary antibody is shown.
VEGF-A to stimulate endothelial cell tube forma-tion and subsequent lymphatic marker expressions.
Discussion
Many growth factors have been reported to stimulate the differentiation of various endothelial cell types into lymphatic endothelial cells. Interleukin-3 (IL-3) is a potential stimulator for promoting non-lymphatic en-dothelial cell differentiation into lymphatic enen-dothelial cells that positively express Prox-1. IL-3 therefore regulates non-lymphatic endothelial cell differentiation into lymphatic endothelial cells, thus promoting pro-gression of the lymphangiogenesis process [35]. These studies demonstrated that not only lymphatic endothe-lial cells but also other types of endotheendothe-lial cells can be used to study lymphangiogenesis. Groger et al. [35] also reported that the basal mRNA expression levels of podoplanin and LYVE-1 are relative high in lymphatic endothelial cells, while mRNA expression levels of these two lymphatic markers are low in HUVECs. Our results also confirmed that basal mRNA expression levels of Prox-1, LYVE-1 and podoplanin are low in HUVECs (Fig. 1). These findings suggested that HU-VECs, an easily obtained endothelial cell type, is suitable for studying lymphatic marker expressions in response to agonists. In the present study, we found that LPA induced VEGF-C mRNA expression in endothe-lial cells, and subsequently Prox-1, LYVE-1, and podoplanin expressions in endothelial cells in vitro and in vivo. Our previous study showed that LPA modulates endothelial cell proliferation [18]. In this study, we further demonstrated that LPA stimulates lymphatic marker expressions in these proliferating endothelial cells (Fig. 5).
Pro-inflammatory factors such as IL-1b and tumor necrosis factor-a (TNF-a) have been shown to en-hance VEGF-C expression in HUVECs [37]. In this study, we also demonstrated that LPA stimulated VEGF-C mRNA and protein expressions in HU-VECs (Fig. 1). Since LPA has been reported to be a proinflammatory factor [38], our data suggest that these proinflammatory factors might be potent regu-lators of VEGF-C expression in human endothelial cells. In our previous study, we demonstrated that LPA stimulates IL-1b expression in HUVECs [39], imply-ing that LPA might also regulate VEGF-C expression through up-regulating IL-1b expression in human endothelial cells. In addition, proinflammatory factors including TNF-a and interferon-g (IFN-g) enhance Prox-1 and podoplanin expressions in endothelial cells, and these enhancement effects are dependent on IL-3 [40, 41]. These findings further illustrate that proinflammatory factors may play critical roles in
normal endothelial cells differentiating into lymphatic endothelial cells, which is consistent with our current results. One recent study reported that elevation of circulating interleukin-8 (IL-8) is highly correlated to VEGF-C but not VEGF-A level elevation in meta-static esophageal squamous carcinoma patient [42]. Since our previous study found that LPA enhances IL-8 expression in HUVECs in an IL-1-dependent manner [39], we suggested that LPA induces IL-1-mediated IL-8 expression in human endothelial cells might contribute to VEGF-C rather than VEGF-A production and subsequent lymphatic markers ex-pressions in human endothelial cells.
The role of VEGF-C in association with VEGFR-3 on promoting lymphangiogenesis process has been well established [19, 22]. Our results showed that LPA-induced Prox-1, LYVE-1, and podoplanin protein expressions in HUVECs were significantly suppressed by pretreatment with MAZ51, a VEGFR-3 kinase inhibitor (Fig. 6). These results suggested that LPA might up-regulate these lymphatic marker expres-sions through up-regulating VEGF-C expression in endothelial cells.
VEGF-A is a well-known key regulator of angiogenesis process [43, 44]. Using an in vitro Matrigel tube formation assay and in vivo Matrigel plug assay, VEGF-Awas shown to significantly enhance HUVECs tube formation in vitro [45, 46] and vessels penetrating the Matrigel in vivo [47, 48]. Moreover, pre-incubation of VEGFR-2 kinase inhibitor, ZD6474 largely sup-pressed VEGF-A-induced endothelial cell tube for-mation in vitro and in vivo [48], implying that VEGF-A/VEGFR-2 axis might play critical role on modulat-ing endothelial cell tube formation and subsequent angiogenesis process. Our results demonstrated that VEGF-C but not VEGF-A showed enhancement effects on protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin in HUVECs (Fig. 2). Consistent with previous study [41], our observation suggest that involvement of VEGF-C via VEGFR-3 might be the major signaling pathway mediating LPA-stimulated endothelial cell tube formation and the subsequent lymphangiogenesis process, while the VEGF-A/VEGFR-2 axis might not regulate lymphatic marker expressions in endothelial cells.
As shown in Figure 7A, we demonstrated that mor-phology of LPA- and VEGF-A-enhanced endothelial cell tubes are different, which is consistent with the results in previous study that morphology of VEGF-C- and VEGF-A-enhanced endothelial cell tubes differed [49]. Moreover, our data revealed that MAZ51 significantly suppressed LPA-enhanced Prox-1 expression in endothelial cells (Fig. 7B), fur-ther suggesting that LPA might be a lymphangiogenic factor.
Many agonists have been reported to up-regulate both VEGF-A and VEGF-C. Mancino et al. [50] reported that estrogen can stimulate both A and VEGF-C in the human cholangiocarcinoma cell line, HuH-28 cells. In addition, IL-1a and IL-6 have been reported as agonists for both VEGF-A and VEGF-C expression in CAPAN-1 cells, a pancreatic cancer cell line [51]. Ristimaki et al. [37] further indicated that IL-1b up-regulates both VEGF-A and VEGF-C expression in HUVECs. These studies suggested that various pro-inflammatory cytokines stimulate VEGF-A and VEGF-C expression in various types of cells. Previous studies reported that LPA could stimulate VEGF-A expression in cancer cells [13, 34]. In the present study, we further observed that LPA stimulated VEGF-C expression in HUVECs, suggesting that LPA might affect angiogenesis and lymphangiogenesis process. In summary, this study first demonstrates that LPA might enhance VEGF-C mRNA and protein expres-sions and subsequent mRNA and protein expresexpres-sions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in human endothelial cells. In addition, LPA also enhanced endothelial cell tube formation and lymphatic marker expressions in vitro and in vivo, implying that LPA might up-regulate VEGF-C ex-pression, which contributes to lymphatic marker expression in endothelial cells, thus promoting pro-gression of the lymphangiogenesis process. LPA also stimulated endothelial cell proliferation as the lym-phangiogenesis process proceeds. Furthermore, LPA might enhance lymphatic marker expressions through up-regulating VEGF-C expression in endothelial cells, therefore facilitating progression of the lym-phangiogenesis process. Our study clarifies the role of LPA in lymphangiogenesis, and these findings also suggest some potential targets for anti-lymphangio-genesis therapies.
Acknowledgements. We thank Dr. F-J. Hsieh at National Taiwan University Hospital who provided umbilical cords for the endo-thelial cell preparation (Institutional Review Board approval no: 9561709146). This work was supported by grants (NSC96-2311-B-002-018-MY2) (to H. Lee) from the National Science Council, Taiwan, and (96-EC-17-A-19-S1 – 016) from the Department of Industrial Technology, Ministry of Economic Affairs, Taiwan (to H. Lee and C-N. Chen).
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