Periostin directly and indirectly promotes tumor lymphangiogenesis of head and neck cancer

Background

Metastasis to regional lymph nodes via lymphatic vessels plays a key role in cancer progression. Tumor lymphangiogenesis is known to promote lymphatic metastasis, and vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis during the process of metastasis. We previously identified periostin as an invasion- and angiogenesis-promoting factor in head and neck squamous cell carcinoma (HNSCC). In this study, we discovered a novel role for periostin in tumor lymphangiogenesis.

Methods and Findings

Periostin overexpression upregulated VEGF-C mRNA expression in HNSCC cells. By using conditioned media from periostin-overexpressing HNSCC cells, we examined tube formation of lymphatic endothelial cells. Conditioned media from periostin-overexpressing cells promoted tube formation. To know the correlation between periostin and VEGF-C, we compared Periostin expression with VEGF-C expression in 54 HNSCC cases by immunohistochemistry. Periostin expression was correlated well with VEGF-C expression in HNSCC cases. Moreover, correlation between periostin and VEGF-C secretion was observed in serum from HNSCC patients. Interestingly, periostin itself promoted tube formation of lymphatic endothelial cells independently of VEGF-C. Periostin-promoted lymphangiogenesis was mediated by Src and Akt activity. Indeed possible correlation between periostin and lymphatic status in periostin-overexpressing xenograft tumors and HNSCC cases was observed.

Conclusions

Our findings suggest that periostin itself as well as periostin-induced upregulation of VEGF-C may promote lymphangiogenesis. We suggest that periostin may be a marker for prediction of malignant behaviors in HNSCC and a potential target for future therapeutic intervention to obstruct tumoral lymphatic invasion and lymphangiogenesis in HNSCC patients.     

Lysophosphatidic Acid Enhances Vascular Endothelial Growth Factor-C Expression in Human Prostate Cancer PC-3 Cells

Clinical evidence suggests that lymphangiogenesis and lymphatic metastasis are important processes during the progression of prostate cancer. Vascular endothelial growth factor (VEGF)-C was shown to be a key regulator in these processes. Our previous studies demonstrated that lysophosphatidic acid (LPA), a low-molecular-weight lipid growth factor, enhances VEGF-C expression in human endothelial cells. We previously demonstrated that the LPA receptor plays an important role in lymphatic development in zebrafish embryos. However, the effects of LPA on VEGF-C expression in prostate cancer are not known. Herein, we demonstrate that LPA up-regulated VEGF-C expression in three different human prostate cancer cell lines. In PC-3 human prostate cancer cells, the enhancing effects of LPA were mediated through both LPA1 and LPA3. In addition, reactive oxygen species (ROS) production and lens epithelium-derived growth factor (LEDGF) expression were involved in LPA(1/3)-dependent VEGF-C expression. Furthermore, autotaxin (ATX), an enzyme responsible for LPA synthesis, also participates in regulating VEGF-C expression. By interrupting LPA(1/3) of PC-3, conditioned medium (CM) -induced human umbilical vein endothelial cell (HUVEC) lymphatic markers expression was also blocked. In summary, we found that LPA enhances VEGF-C expression through activating LPA(1/3)-, ROS-, and LEDGF-dependent pathways. These novel findings could potentially shed light on developing new strategies for preventing lymphatic metastasis of prostate cancer.     

LPA1/3 signaling mediates tumor lymphangiogenesis through promoting CRT expression in prostate cancer

Lysophosphatidic acid (LPA) is a bioactive lipid growth factor which is present in high levels in serum and platelets. LPA binds to its specific G-protein-coupled receptors, including LPA₁ to LPA₆, thereby regulating various physiological functions, including cancer growth, angiogenesis, and lymphangiogenesis. Our previous study showed that LPA promotes the expression of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C in prostate cancer (PCa) cells. Interestingly, LPA has been shown to regulate the expression of calreticulin (CRT), a multifunctional chaperone protein, but the roles of CRT in PCa progression remain unclear. Here we investigated the involvement of CRT in LPA-mediated VEGF-C expression and lymphangiogenesis in PCa. Knockdown of CRT significantly reduced LPA-induced VEGF-C expression in PC-3 cells. Moreover, LPA promoted CRT expression through LPA receptors LPA₁ and LPA₃, reactive oxygen species (ROS) production, and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Tumor-xenografted mouse experiments further showed that CRT knockdown suppressed tumor growth and lymphangiogenesis. Notably, clinical evidence indicated that the LPA-producing enzyme autotaxin (ATX) is related to CRT and that CRT level is highly associated with lymphatic vessel density and VEGF-C expression. Interestingly, the pharmacological antagonist of LPA receptors significantly reduced the lymphatic vessel density in tumor and lymph node metastasis in tumor-bearing nude mice. Together, our results demonstrated that CRT is critical in PCa progression through the mediation of LPA-induced VEGF-C expression, implying that targeting the LPA signaling axis is a potential therapeutic strategy for PCa.     

In vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis

Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo.     

Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells

Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation.     

Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.     

T1alpha/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cells

The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1alpha/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.     

CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

Renal lymphangiogenesis is a new field of international nephrology in recent years and plays an important role in the progression of chronic renal disease. CD137 was originally described as a surface molecule present on activated T and NK cells and detected on hypoxic endothelial cells and inflamed blood vessels, but its function on lymphatic endothelial cells remains unclear. We investigated the relationships among CD137, lymphangiogenesis and macrophages, which are involved in interstitial fibrosis. Similar to other chronic inflammatory diseases, we found lymphangiogenesis and expression of CD137 in the renal tissue of patients with IgA nephropathy. CD137-positive lymphatic vessels were involved in the development process of IgA nephropathy and positively correlated with serum creatinine, serum urea nitrogen, serum uric acid, and urinary 24 h total protein. The expression of these indicators was negatively correlated with eGFR, plasma albumin, and HB. In mouse models of UUO, we verified that CD137 expression was significantly elevated during lymphangiogenesis and that its ligand CD137L was released by macrophages after VEGF-C stimulation in the kidney. In vitro, recombinant CD137L significantly enhanced LEC proliferation, migration and tube formation, and these effects were inhibited by CD137 siRNA. Mechanistically, the CD137L interaction with CD137 induced the transition from LC3-I to LC3-II and the expression of Atg5, Atg7, Atg12 and p62 proteins by activating the PI3K/AKT/mTOR pathway to promote autophagy. Knockdown of Atg5 and Atg7 blocked CD137L-induced autophagy. Thus, we propose that CD137L secretion by macrophages interacts with CD137 on lymphatic endothelial cells to prompt lymphangiogenesis in the kidney, which further drives fibrogenic responses. Our findings suggest that inhibition of the CD137-CD137L pathway is a novel therapeutic approach for obstructive nephropathy.     

Expression of vascular endothelial growth factor C in human pterygium

Vascular endothelial growth factor C (VEGF-C) and its receptor VEGFR-3 mediate lymphangiogenesis. In this study, we analyzed the expression of VEGF-C and VEGFR-3 as well as lymphatic vessels in the pterygium and normal conjunctiva of humans. Fifteen primary nasal pterygia and three normal bulbar conjunctivas, surgically removed, were examined in this study. The lymphatic vessel density (LVD) and blood vessel density were determined by the immunolabeling of D2-40 and CD31, markers for lymphatic and blood vessels, respectively. VEGF-C and VEGFR-3 expression in pterygial and conjunctival tissue proteins was detected by Western blotting and were evaluated using immunohistochemistry. The LVD was significantly higher in the pterygium than normal conjunctiva (p < 0.05). Western blot demonstrated high-level expression of VEGF-C and VEGFR-3 in the pterygium compared with normal conjunctiva. VEGF-C immunoreactivity was detected in the cytoplasm of pterygial and normal conjunctival epithelial cells. The number of VEGF-C-immunopositive cells in pterygial epithelial cells was significantly higher than in normal conjunctival cells (p < 0.05). VEGFR-3 immunoreactivity was localized in the D2-40-positive lymphatic endothelial cells. The present findings suggest the potential role of VEGF-C in the pathogenesis and development of a pterygium through lymphangiogenesis and the VEGF-C/VEGFR-3 pathway as a novel therapeutic target for the human pterygium.     

Co-expression of α9β1 integrin and VEGF-D confers lymphatic metastatic ability to a human breast cancer cell line MDA-MB-468LN

Introduction and Objectives

Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice.

Results

A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells.

Conclusion

Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.     

CEACAM1 resists hypoxia-induced inhibition of tube formation of human dermal lymphatic endothelial cells

Tube formation is one of the fundamental events required by angiogenesis and lymphangiogenesis. To date, there is little knowledge on the effects of hypoxia on tube formation of human dermal lymphatic endothelial cells (HDLECs). In this study, we found that tube formation of HDLECs was inhibited under hypoxic condition with decreased expressions of VEGF-D, CEACAM1 and Prox1 genes. However, hypoxia-induced inhibition of tube formation of HDLECs was reversed by conditional media from hypoxic tumor cells. After knockdown of CEACAM1 by siRNA transfection, tube formation of HDLECs was increased with elevated Prox1 expression, suggesting that CEACAM1 downregulates Prox1 and plays an inhibitory role in tube formation of HDLECs. Since the expressions of CEACAM1 and Prox1 were both decreased by hypoxia, there are additional mechanisms downregulating Prox1 expressions during hypoxia-inhibited tube formation of HDLECs.     

Lymphangiogenesis in vitro: Formation of lymphatic capillary-like channels from confluent monolayers of lymphatic endothelial cells

Summary Lymphatic endothelial cells grown long term in culture form lymphatic capillarylike tubes. Examination by light and transmission electron microscopy showed that these structures were closed loops composed of one to several cells connected by intercellular junction to form a luminal space. This first demonstration of lymphangiogenesis in confluent monolayer cultures of lymphatic endothelial cells (a) showed that collagen type I accelerated lymphatic capillary tube formation, whereas fibronectin and matrigel had no effect; b) provided a model to study lymphatic endothelial cell function and differentiation; and c) offered a possibility to distinguish differences between the process of lymphangiogenesis and angiogenesis by testing various factors and conditions that effect endothelial cell behavior.     

Multiple mechanisms are responsible for transactivation of the epidermal growth factor receptor in mammary epithelial cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.     

Hepatocyte growth factor promotes lymphatic vessel formation and function

The lymphatic vascular system plays a pivotal role in mediating tissue fluid homeostasis and cancer metastasis, but the molecular mechanisms that regulate its formation and function remain poorly characterized. A comparative analysis of the gene expression of purified lymphatic endothelial cells (LEC) versus blood vascular endothelial cells (BVEC) revealed that LEC express significantly higher levels of hepatocyte growth factor receptor (HGF-R). Whereas little or no HGF-R expression was detected by lymphatic vessels of normal tissues, HGF-R was strongly expressed by regenerating lymphatic endothelium during tissue repair and by activated lymphatic vessels in inflamed skin. Treatment of cultured LEC with HGF promoted LEC proliferation, migration and tube formation. HGF-induced proliferation of LEC did not require vascular endothelial growth factor receptor-3 activation, and HGF-induced cell migration was partially mediated via integrin alpha-9. Transgenic or subcutaneous delivery of HGF promoted lymphatic vessel formation in mice, whereas systemic blockade of HGF-R inhibited lymphatic function. These results identify HGF as a novel, potent lymphangiogenesis factor, and also indicate that HGF-R might serve as a new target for inhibiting pathological lymphangiogenesis.     

Vascular endothelial growth factor (VEGF) receptor-2 signaling mediates VEGF-C(deltaNdeltaC)- and VEGF-A-induced angiogenesis in vitro

Angiogenesis and lymphangiogenesis are regulated by members of the vascular endothelial growth factor (VEGF) family of cytokines, which mediate their effects via tyrosine kinase VEGF receptors -1, -2, and -3. We have used wild-type and mutant forms of VEGFs -A, -B, and -C, a pan-VEGFR tyrosine kinase inhibitor (SU5416) as well as neutralizing anti-VEGFR-2 antibodies, to determine which VEGF receptor(s) are required for bovine endothelial cell invasion and tube formation in vitro. This was compared to the ability of these cytokines to induce expression of members of the plasminogen activator (PA)-plasmin system. We found that cytokines which bind VEGFR-2 (human VEGF-A, human VFM23A, human VEGF-C(deltaNdeltaC), and rat VEGF-C(152)) induced invasion, tube formation, urokinase-type-PA, tissue-type-PA, and PA inhibitor-1, invasion and tube formation as well as signaling via the MAP kinase pathway were efficiently blocked by SU5416 and anti-VEGFR-2 antibodies. In contrast, cytokines and mutants which exclusively bind VEGFR-1 (human VFM17 and human VEGF-B) had no effect on invasion and tube formation or on the regulation of gene expression. We were unable to identify cytokines which selectively stimulate bovine VEGFR-3 in our system. Taken together, these findings point to the central role of VEGFR-2 in the angiogenic signaling pathways induced by VEGF-C(deltaNdeltaC) and VEGF-A.     

Identification of Gene Expression Differences between Lymphangiogenic and Non-Lymphangiogenic Non-Small Cell Lung Cancer Cell Lines

It is well established that lung tumors induce the formation of lymphatic vessels. However, the molecular mechanisms controlling tumor lymphangiogenesis in lung cancer have not been fully delineated. In the present study, we identify a panel of non-small cell lung cancer (NSCLC) cell lines that induce lymphangiogenesis and use genome-wide mRNA expression to characterize the molecular mechanisms regulating tumor lymphangiogenesis. We show that Calu-1, H1993, HCC461, HCC827, and H2122 NSCLC cell lines form tumors that induce lymphangiogenesis whereas Calu-3, H1155, H1975, and H2073 NSCLC cell lines form tumors that do not induce lymphangiogenesis. By analyzing genome-wide mRNA expression data, we identify a 17-gene expression signature that distinguishes lymphangiogenic from non-lymphangiogenic NSCLC cell lines. Importantly, VEGF-C is the only lymphatic growth factor in this expression signature and is approximately 50-fold higher in the lymphangiogenic group than in the non-lymphangiogenic group. We show that forced expression of VEGF-C by H1975 cells induces lymphangiogenesis and that knockdown of VEGF-C in H1993 cells inhibits lymphangiogenesis. Additionally, we demonstrate that the triple angiokinase inhibitor, nintedanib (small molecule that blocks all FGFRs, PDGFRs, and VEGFRs), suppresses tumor lymphangiogenesis in H1993 tumors. Together, these data suggest that VEGF-C is the dominant driver of tumor lymphangiogenesis in NSCLC and reveal a specific therapy that could potentially block tumor lymphangiogenesis in NSCLC patients.     

Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression

Lysophosphatidic acid (LPA) has been found to mediate myeloid differentiation, stimulate osteogenesis, alter cell proliferation and migration, and inhibit apoptosis in chondrocytes. The effect of LPA on the angiogenic capability of chondrocytes is not clear. This study aimed to investigate its effect on the angiogenic capability of human chondrocytes and the underlying mechanism of these effects. Human chondrocyte cell line, CHON-001, commercialized human chondrocytes (HC) derived from normal human articular cartilage, and human vascular endothelial cells (HUVECs) were used as cell models in this study. The angiogenic capability of chondrocytes was determined by capillary tube formation, monolayer permeability, cell migration, and cell proliferation. An angiogenesis protein array kit was used to evaluate the secretion of angiogenic factors in conditioned medium. Angiogenin, insulin-like growth factor-binding protein 1 (IGFBP-1), interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase (MMP)-9, and vascular endothelial growth factor (VEGF) mRNA and protein expressions were evaluated by Q-RT-PCR and EIA, respectively. LPA receptor (LPAR) expression was determined by RT-PCR. Signaling pathways were clarified using inhibitors, Western blot analysis, and reporter assays. The LPA treatment promoted the angiogenic capability of CHON-001 cells and HC, resulting in enhanced HUVEC capillary tube formation, monolayer permeability, migration, and cell growth. Angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF mRNA and protein expressions were significantly enhanced in LPA-treated chondrocytes. LPA2, 3, 4 and 6 were expressed in CHON-001 and HC cells. Pretreatment with the Gi/o type G protein inhibitor, pertussis toxin (PTX), and the NF-kB inhibitor, PDTC, significantly inhibited LPA-induced angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF expressions in chondrocytes. The PTX pretreatment also inhibited LPA-mediated NF-kB activation, suggesting the presence of active Gi/NF-kB signaling in CHON-001 and HC cells. The effect of LPA on the angiogenesis-inducing capacity of chondrocytes may be due to the increased angiogenesis factor expression via the Gi/NF-kB signaling pathway.