DOI QR코드

DOI QR Code

LymphanaxTM Enhances Lymphangiogenesis in an Artificial Human Skin Model, Skin-lymph-on-a-chip

스킨-림프-칩 상에서 LymphanaxTM 의 림프 형성 촉진능

  • Phil June Park (Basic Research & Innovation Division, AMOREPACIFIC R&I Center) ;
  • Minseop Kim (KU-KIST Graduate School of Converging Science and Technology, Korea University) ;
  • Sieun Choi (KU-KIST Graduate School of Converging Science and Technology, Korea University) ;
  • Hyun Soo Kim (Basic Research & Innovation Division, AMOREPACIFIC R&I Center) ;
  • Seok Chung (KU-KIST Graduate School of Converging Science and Technology, Korea University)
  • 박필준 (아모레퍼시픽 R&I 센터) ;
  • 김민섭 (고려대학교 KU-KIST 융합대학원) ;
  • 최시은 (고려대학교 KU-KIST 융합대학원) ;
  • 김현수 (아모레퍼시픽 R&I 센터) ;
  • 정석 (고려대학교 KU-KIST 융합대학원)
  • Received : 2024.05.28
  • Accepted : 2024.06.17
  • Published : 2024.06.30

Abstract

The cutaneous lymphatic system in humans plays a crucial role in draining interstitial fluid and activating the immune system. Environmental factors, such as ultraviolet light and natural aging, often affect structural changes of such lymphatic vessels, causing skin dysfunction. However, some limitations still exist because of no alternatives to animal testing. To better understand the skin lymphatic system, a biomimetic microfluidic platform, skin-lymph-on-a-chip, was fabricated to develop a novel in vitro skin lymphatic model of humans and to investigate the molecular and physiological changes involved in lymphangiogenesis, the formation of lymphatic vessels. Briefly, the platform involved co-culturing differentiated primary normal human epidermal keratinocytes (NHEKs) and dermal lymphatic endothelial cells (HDLECs) in vitro. Based on our system, LymphanaxTM, which is a condensed Panax ginseng root extract obtained through thermal conversion for 21 days, was applied to evaluate the lymphangiogenic effect, and the changes in molecular factors were analyzed using a deep-learning-based algorithm. LymphanaxTM promoted healthy lymphangiogenesis in skin-lymphon-a-chip and indirectly affected HDELCs as its components rarely penetrated differentiated NHEKs in the chip. Overall, this study provides a new perspective on LymphanaxTM and its effects using an innovative in vitro system.

인체 피부 림프계는 간질액을 배출하고 면역 시스템을 활성화하는 중요한 역할을 한다. 자외선과 자연적인 노화와 같은 환경 요인들은 종종 이러한 림프관의 구조적 변화를 일으키며, 이로 인해 피부 기능 장애를 발생시키기도 한다. 그러나 이러한 연구를 위한 동물 실험 대체 방안이 없기 때문에 여전히 연구를 진행하기엔 적합하지 않은 제한 사항들이 존재한다. 인체 피부 림프계를 더 잘 이해하고 림프관 형성에 관련된 분자 및 생리학적 변화를 조사하기 위해, 생체 모방 미세유체 플랫폼인 'skin-lymph-on-a-chip'을 제작하여, 새로운 체외 인체 피부 림프 모델을 개발하였다. 간단히 말해, 이 플랫폼은 체외에서 분화된 일차 정상 인간 표피 각질형성세포(NHEKs)와 피부 림프 내피세포(HDLECs)를 공동 배양하는 것을 의미한다. 약 500 시간 동안 자연 발효를 통해 확보하고 집약된 인삼 뿌리 추출물인 LymphanaxTM의 림프관 형성 효과를 평가하기 위해 해당 시스템에 적용하였고, 분자 수준 요인들의 변화는 딥러닝 기반 알고리즘을 사용하여 분석하였다. 결론적으로, LymphanaxTM는 skin-lymph-on-a-chip에서 건강한 림프관 형성을 촉진하였고, 그 성분들이 칩내에서 분화된 NHEKs를 거의 침투하지 않는 결과를 통해, HDELCs에 간접적으로 영향을 미쳤음을 확인하였다. 전반적으로, 이 연구는 기존과 차별화된 체외 인체 피부 림프모델 시스템의 확보와 더불어 이를 통한 LymphanaxTM의 림프 활성화 효과에 대한 새로운 관점을 제공한다.

Keywords

References

  1. 1 .K. N. Margaris and R. A . Black, Modelling the lymphatic system: Challenges and opportunities, J. R. Soc. Interface, 9(69), 601 (2012).
  2. M. Yousef, D. Silva, N. B. Chacra, N. Davies, and R. Lobenberg, The lymphatic system: A sometimes-forgotten compartment in pharmaceutical sciences, J. Pharm. Sci., 24, 533 (2021).
  3. A.W. Lund, T.R. Medler, S.A. Leachman, and L.M. Coussens, Lymphatic vessels, inflammation, and immunity in skin cancer, Cancer Discov., 6(1), 22 (2016).
  4. S. Eyerich, K. Eyerich, C. Traidl-Hoffmann, and T. Biedermann, Cutaneous barriers and skin immunity: Differentiating a connected network, Trends. Immunol., 39(4), 315 (2018).
  5. S. A. Stacker, S. P. Williams, T. Karnezis, R. Shayan, S. B. Fox, and M. G. Achen, Lymphangiogenesis and lymphatic vessel remodelling in cancer, Nat. Rev. Cancer, 14(3), 159 (2014).
  6. B. C. Y. Tse, A. L. Ferguson, Y. C. Koay, G. E. Grau, A. S. Don, and S. N. Byrne, Exposure to solar ultraviolet radiation establishes a novel immune suppressive lipidome in skin-draining lymph nodes, Front. Immunol., 13, 1045731 (2023).
  7. M. Sawane, and K. Kajiya, Ultraviolet light-induced changes of lymphatic and blood vasculature in skin and their molecular mechanisms, Exp. Dermatol., 1, 22 (2012).
  8. D. A. Glencross, T. R. Ho, N. Camina, C. M. Hawrylowicz, and P. E. Pfeffer, Air pollution and its effects on the immune system, Free Radic. Biol. Med., 151, 56 (2020).
  9. J. C. Fussell, and F. J. Kelly, Oxidative contribution of air pollution to extrinsic skin ageing, Free Radic. Biol. Med., 151, 111 (2020).
  10. P. Puri, S. K. Nandar, S. Kathuria, and V. Ramesh, Effects of air pollution on the skin: A review, Indian J. Dermatol. Venereol. Leprol., 83(4), 415 (2017).
  11. B. Thompson, K. Gaitatzis, X. J. de Jonge, R. Blackwell, and L. A. Koelmeyer, Manual lymphatic drainage treatment for lymphedema: a systematic review of the literature, J. Cancer Surviv., 15(2), 244 (2021).
  12. S. G. Rockson, Lymphedema, Am. J. Med., 110(4), 288 (2001).
  13. J. S. Drouin, L. Pfalzer, J. M. Shim, and S. J. Kim, Comparisons between manual lymph drainage, abdominal massage, and electrical stimulation on functional constipation outcomes: A randomized, controlled trial, Int. J. Environ. Res. Publ ic Heal th, 17(11), 3924 (2020).
  14. R. Su, F. Wang, and M. C. McAlpine, 3D printed microfluidics: advances in strategies, integration, and applications, Lab Chip, 23(5), 1279 (2023).
  15. M. Singh, Y. Tong, K. Webster, E. Cesewski, A. P. Haring, S. Laheri, B. Carswell, T. J. O'Brien, C. H. Aardema, R. S. Senger, J. L. Robertson, and B. N. Johnson, 3D printed conformal microfluidics for isolation and profiling of biomarkers from whole organs, Lab Chip, 17(15), 2561 (2017).
  16. P. Prabhakar, R. Sen, N. Dwivedi, R. Khan, P. Solanki, A. Srivastava, and C. Dhand, 3D-printed microfluidics and potential biomedical applications, Front. Nanotechnol., 3, 609355 (2021).
  17. S. Khunmanee and H. Park, Three-dimensional culture for in vitro folliculogenesis in the aspect of methods and materials, Tissue Eng. Part B Rev., 28, 1242 (2022).
  18. M. Ravi, V. Paramesh, S. R. Kaviya, E. Anuradha, F. D. P. Solomon, 3D cell culture systems: advantages and applications, J. Cell Physiol., 230(1), 16 (2015).
  19. J. Ahn, K. Ohk, J. Won, D. H. Choi, Y. H. Jung, J. H. Yang, Y. Jun, J. A. Kim, S. Chung, and S. H. Lee, Modeling of three-dimensional innervated epidermal like-layer in a microfluidic chip-based coculture system, Nat. Commun., 14(1), 1488 (2023).
  20. Y. Cho, K. Na, Y. Jun, J. Won, J. H. Yang, and S. Chung, Three-dimensional in vitro lymphangiogenesis model in tumor microenvironment, Front. Bioeng. Biotechnol., 9, 697657 (2021).
  21. J. Kim, S. Y. Cho, S. H. Kim, D. Cho, S. Kim, C. W. Park, T. Shimizu, J. Y. Cho, D. B. Seo, and S. S. Shin, Effects of Korean ginseng berry on skin antipigmentation and antiaging via FoxO3a activation, J. Ginseng Res., 41(3), 277 (2017).
  22. S. Y. Han, J. Kim, E. Kim, S. H. Kim, D. B. Seo, J. H. Kim, S. S. Shin, and J. Y. Cho, AKT-targeted antiinflammatory activity of Panax ginseng calyx ethanolic extract, J. Ginseng Res., 42(4), 496 (2018).
  23. W. Choi, H. S. Kim, S. H. Park, D. Kim, Y. D. Hong, J. H. Kim, and J. Y. Cho, Syringaresinol derived from Panax ginseng berry attenuates oxidative stress-induced skin aging via autophagy, J. Ginseng Res., 46(4), 536 (2022).
  24. B. K. Shin, S. W. Kwon, and J. H. Park, Chemical diversity of ginseng saponins from Panax ginseng, J. Ginseng Res., 39(4), 287 (2015).
  25. Y. H. Jung, K. Y. Park, J. H. Jeon, Y. S. Kwak, Y. B. Song, J. J. Wee, M. H. Rhee, and T. W. Kim, Red ginseng saponin fraction an isolated from Korean red ginseng by ultrafiltration on the porcine coronary artery, J. Ginseng Res., 35(3), 325 (2011).
  26. M. L. Xu, H. J. Kim, Y. R. Choi, and H. J. Kim, Intake of Korean red ginseng extract and saponin enhances the protection conferred by vaccination with inactivated influenza a virus, J. Ginseng Res., 36(4), 396 (2012).
  27. S. Duan S, J. R. Liu, X. Wang, X. M. Sun, H. S. Gong, C. W. Jin, and S. H. Eom, Thermal control using far-infrared irradiation for producing deglycosylated bioactive compounds from Korean ginseng leaves, Molecules, 27(15), 4782 (2022).
  28. I. H. Baik, K. H. Kim, and K. A. Lee, Antioxidant, anti-inflammatory and antithrombotic effects of ginsenoside compound K enriched extract derived from ginseng sprouts, Molecules, 26(13), 4102 (2021).
  29. H. J. Oh, H. Jin, and B. Y. Lee, The non-saponin fraction of Korean red ginseng ameliorates sarcopenia by regulating immune homeostasis in 22-26-month-old C57BL/6J mice, J. Ginseng Res., 46(6), 809 (2022).
  30. S. H. Youn, S. M. Lee, C. K. Han, G. In, C. K. Park, and S. H. Hyun, Immune activity of polysaccharide fractions isolated from Korean red ginseng, Molecules, 25(16), 3569 (2020).
  31. J. Yu, L. Mao, L. Guan, Y. Zhang, and J. Zhao, Ginsenoside Rg1 enhances lymphatic transport of intrapulmonary silica via VEGF-C/VEGFR-3 signaling in silicotic rats, Biochem. Biophys. Res. Commun., 472(1), 182 (2016).
  32. L. H. Quan, J. Y. Piao, J. W. Min, H. B. Kim, S. R. Kim, D. U. Yang, and D. C. Yang, Biotransformation of ginsenoside Rb1 to prosapogenins, gypenoside XVII, ginsenoside Rd, ginsenoside F2, and compound K by leuconostoc mesenteroides DC102, J. Ginseng Res., 35(3), 344 (2011).
  33. M. M. Zhang, G. M. Huo, J. Cheng, Q. P. Zhang, N. Z. Li, M. X. Guo, Q. Liu, G. H. Xu, J. X. Zhu, C. F. Li, F. Zhou, and L. T. Yi, Gypenoside XVII, an active ingredient from Gynostemma pentaphyllum, inhibits C3aRassociated synaptic puning in stressed mice, Nutrients, 14(12), 2418 (2022).
  34. D. H. Choi, H. W. Liu, Y. H. Jung, J. Ahn, K. A. Kim, D. Oh, Y. Jeong, M. Kim, H. Yoon, B. Kang, E. Hong, E. Song, and S. Chung, Analyzing angiogenesis on a chip using deep learning-based image processing, Lab Chip, 23(3), 475 (2023).
  35. I. Wada, S. Nakao, M. Yamaguchi, Y. Kaizu, M. Arima, S. Sawa, and K. H. Sonoda, Retinal VEGF-A overexpression is not sufficient to induce lymphangiogenesis regardless of VEGF-C upregulation and Lyve1+ macrophage infiltration, Invest. Ophthalmol. Vis. Sci., 62(13), 17 (2021).
  36. O. Mece, D. Houbaert, M.L. Sassano, T. Durre, H. Maes, M. Schaaf, S. More, M. Ganne, M.G..Caballero, M. Borri, J. Verhoeven, M. Agrawal, K. Jacobs, G. Bergers, S. Blacher, B. Ghesquiere, M. Dewerchin, J. V. Swinnen, S. Vinckier, M. S. Soengas, P. Carmeliet, A. Noel, and P. Agostinis, Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis, Nat. Commun., 13(1), 2760 (2022).
  37. Y. Zhang, C. Zhang, L. Li, X. Liang, P. Cheng, Q. Li, X. Chang, K. Wang, S. Huang, Y. Li, Y. Liu, and G. Xu, Lymphangiogenesis in renal fibrosis arises from macrophages via VEGF-C/VEGFR3-dependent autophagy and polarization, Cell Death Dis., 12(1), 109 (2021).
  38. S. Lutter, S. Xie, F. Tatin, and T. Makinen, Smooth muscle-endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation, J. Cell Biol., 197(6), 837 (2012).
  39. J.D. Bos, and M. M. Meinardi, The 500 Dalton rule for the skin penetration of chemical compounds and drugs, Exp. Dermatol., 9(3), 165 (2000).
  40. M. Kim, S. Choi, D. H. Choi, J. Ahn, D. Lee, E. Song, H. S. Kim, M. Kim, S. Choi, S. Oh, M. Kim, S. Chung, and P. J. Park, An advanced 3D lymphatic system for assaying human cutaneous lymphangiogenesis in a microfluidic platform, NPG Asia Mater., 16, 7 (2024).