Elsevier

Journal of Ethnopharmacology

Volume 196, 20 January 2017, Pages 213-224
Journal of Ethnopharmacology

Davallia bilabiata exhibits anti-angiogenic effect with modified MMP-2/TIMP-2 secretion and inhibited VEGF ligand/receptors expression in vascular endothelial cells

https://doi.org/10.1016/j.jep.2016.12.019Get rights and content

Highlights

  • D. bilabiata inhibited angiogenesis both in vitro and in vivo.

  • The extracellular MMP-2 activity was reduced by D.bilabiata both in vitro and in vivo dose-dependently.

  • D. bilabiata inhibited MMP-2, and MMP-14 and enhanced TIMP-2 and RECK in mRNA level.

  • D. bilabiata induced the dose-dependent decrease of MMP-2 and increase of TIMP-2 secretion.

  • The gene expressions of VEGF-A, -B, -C, -D and VEGFR-1, -2, -3 were all inhibited by D. bilabiata.

Abstract

Ethnopharmacological relevance

Davallia bilabiata Hosokawa (D. bilabiata), also called GuSuiBu, is popularly used as a substitute for Drynaria fortunei J. Sm for rheumatoid and degenerative arthritis in traditional Chinese medicine. Little is known about the underlying mechanisms of anti-angiogenesis responsible for arthritis in D. bilabiata which needs to be elucidated.

Aim of the study

The present study is intended to investigate the anti-angiogenic effect of D. bilabiata associated with the modulation of matrix metalloproteinases (MMPs) and down regulation of vascular endothelial growth factor (VEGF) ligand/receptors both in vivo and in vitro.

Materials and methods

We investigated the potential anti-angiogenic effect of D. bilabiata by the in vivo neovascularization of chick chorioallantoic membranes (CAM) assay, and the in vitro migration and matrix-induced tube formation assay using human umbilical vascular endothelial cells (HUVECs). The expressions of MMP-2, TIMP-2, RECK and VEGF/VEGFR were analyzed by real-time RT-PCR or Western blot method.

Results

One major compound from water extract of D. bilabiata was identified as Epicatechin 3-O-β-D-allopyranoside. D. bilabiata was confirmed to inhibit in vivo angiogenesis by CAM assay. D. bilabiata also exhibited in vitro anti-angiogenic and anti-regrowth effects as demonstrated by tube formation assay, transwell migration assay and wound healing assay. The mRNA expressions of MMP-2, and MMP-14 were decreased. On the contrary, tissue inhibitor of metalloproteinase-2 (TIMP-2), reversion-inducing cysteine-rich protein with kazal motifs (RECK) were increased by D. bilabiata. The extracellular MMP-2 activity was found to be reduced both in vitro and in vivo by D. bilabiata as determined by gelatin zymography. Results from western blot analysis and ELISA further demonstrated the decrease of MMP-2 and increase of TIMP-2 secretion after D. bilabiata treatment. The gene expressions of VEGF-A, -B, -C, -D and VEGFR-1, -2, -3 were all inhibited by D. bilabiata.

Conclusion

We concluded that the anti-angiogenic effect of D. bilabiata was associated with the decreased MMP-2 activity mediated by the upregulation of TIMP-2 and RECK, and the suppression of VEGF/VEGFRs expression.

Introduction

Trauma and bone diseases are both causes of joint inflammation to induce intra-articular effusion, synovial thickening, periarticular soft-tissue inflammation (bursitis or tendonitis), and eventually bony enlargement in most forms of arthritis. Arthritis is characterized by an inflammatory response associated with increased neovasculization to lead to reorganization of articular blood vessels. The synovium is inflamed and infiltrated by blood derived cells, including lymphocytes and macrophages (Szekanecz and Koch, 2009). The development of the pannus is characterized by an increased density of sub-lining blood vessels. The diseases in which synovial inflammation is common include rheumatoid arthritis (RA) and osteoarthritis, seronegative spondyloarthropathies, and septic arthritis (Walsh and Pearson, 2001).

Angiogenesis is found to be a critical factor in the pathogenesis of inflammatory diseases such as arthritis and tumor progression (Szekanecz and Koch, 2009). Angiogenic factors include growth factors, pro-inflammatory cytokines, chemokines, extracellular matrix molecules, matrix-degrading proteolytic enzymes, cellular adhesion molecules (CAMs) and so on (Szekanecz et al., 2010). Vascular endothelial growth factor (VEGF), the main proangiogenic factor, is produced within synovia in response to pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) (Szekanecz et al., 2010). VEGF could induce neovascularization, stimulate endothelial cell proliferation, increase vascular permeability and stimulate leukocyte extravasation into inflammatory processes (Szekanecz et al., 2010, Szekanecz and Koch, 2009). Synovial inflammation is associated with chemokine-driven recruitment of inflammatory leukocytes into the synovia. This process involves CAMs, synovial matrix macromolecules, as well as matrix-degrading proteolytic enzymes in the synovial connective tissue during the perpetuation of angiogenesis (Fanjul-Fernandez et al., 2010, Szekanecz and Koch, 2009). Conversely, tissue inhibitors of metalloproteinases (TIMP) antagonize the effects of angiogenic proteases during angiogenesis. The RECK protein is a membrane-anchored glycoprotein normally expressed in all cells of the body (Alexius-Lindgren et al., 2014). The RECK protein has an important role in tissue remodeling and inhibits the activity of proteolytic enzymes including MMP-2, MMP-9 and MMP-14 (MT1-MMP) (Alexius-Lindgren et al., 2014). Currently, many slow-acting anti-rheumatoid agents including gold, penicillamine, sulfasalazine, methotrexate and antimalarials not only reduce endothelial cell proliferation in vitro but also inhibit angiogenesis in vivo (Hirata et al., 1989, Madhok et al., 1991, Matsubara et al., 1989, Matsubara and Ziff, 1987, Potvin et al., 1997).

Both of Davallia bilabiata Hosokawa and Davallia formosana Hayata (also called GuSuiBu) are mainly distributed in Taiwan and used for treating traumatism. There is a variety of GuSuiBu with the same name that have been documented and used in Taiwan as homonym. The rhizomes of D. formosana have been popularly and historically used to treat bone diseases including bone fracture, arthritis, and osteoporosis (Lin et al., 2013). D. formosana is used to improve the symptoms of arthritis and can inhibit osteoclasts via the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and effectively ameliorate ovariectomy-induced osteoporosis (Lin et al., 2013). So far, little is known about the mechanism of anti-angiogenesis responsible for arthritis in D. bilabiata. In the present study, we aimed to investigate the potential anti-angiogenic effect of D. bilabiata by the in vivo neovascularization of chick chorioallantoic membranes (CAM) assay, and the in vitro migration and matrix-induced tube formation assay using human umbilical vascular endothelial cells (HUVECs). The expression of MMP-2, TIMP-2, RECK and VEGF/VEGFR were also studied.

Section snippets

Preparation of water extract of Davallia bilabiata (D. bilabiata)

D. bilabiata used in this study was identified based on the definition described in Flora of Taiwan and authenticated by Dr. Rong-Chi Yang, the chief of the Chinese Herbal Pharmacy in Chang Gung Memorial Hospital. A sample of D. bilabiata is deposited in Set D, TN: No118607 in the herbarium of National Taiwan University. The extraction was prepared according to Taiwanese good manufacturing practice (GMP) methodologies and guidelines. Briefly, the whole plant was minced and extracted with

D. bilabiata inhibited in vivo angiogenesis on CAMs of chicken embryos

We used the CAM assay to study the in vivo effect of D. bilabiata on PMA-induced angiogenesis. As shown in Fig. 2A, the newly spouting vessels in the PMA-induced group were large and much more branches were observed compared to the control CAM treated with 1×PBS only under the stereomicroscope. The PMA-induced angiogenic response was effectively inhibited by D. bilabiata. Addition of 100 μl 0.1 and 0.5 mg/ml of D. bilabiata led to a decrease of approximate 31% and 76% in the angiogenic index

Discussion

D. bilabiata was widely used in traditional Chinese medicine as a substitute for D. fortunei to treat arthritis and bone injury. Previous study showed that D. fortunei has anti-angiogenic activity as the inhibitory activity on tube formation induced by HUVECs (Nam et al., 2003). However, the anti-angiogenic effect of D. bilabiata associated with arthritis is still not clear. In this study, we attempted to further clarify potential mechanisms underlying the anti-angiogenic effect of D. bilabiata

Conclusions

Collectively, this is the first time to demonstrate that D. bilabiata exerted anti-angiogenic effect both in vivo and in vitro. The inhibition of cell migration and angiogenesis by D. bilabiata was associated with the suppression of MMP-2 activity in vascular endothelial cells. The dual inhibitory mechanisms of D. bilabiata on the modulation of MMP-2/TIMP-2 balances and the suppression of VEGF ligand/receptors were thought to contribute to its anti-angiogenic effect more efficiently. Together

Authors’ contributions and conflicts

Chun-Ting Liu and Kuo-Wei Bi wrote the paper. Chao-Chun Huang reviewed and amended the text. Hsiao-Ting Wu and Hui-Ya Ho performed the experiments and analyzed the data. Jong-Hwei S. Pang and Sheng-Teng Huang designed and conducted the study as well as edited the whole article. All authors have read and approved the manuscript. The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the Ministry of Science and Technology of Taiwan (MOST 105-2320-B-039-035) and Chang Gung Memorial Hospital with CMRPG891121, 891122, 891123, 8E0641 and CMRPD1D0261. The authors would like to thank MEd. Jill Mc Callum and James Waddell for the critical reading and correction of our manuscript.

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