VEGF deprivation affects endoglin expression in trophoblast cells and natural killers

Vascular Endothelial Growth Factors (VEGFs) are a group of proteins involved in differentiation of various cell types including endothelial cells, monocytes, macrophages, stem cells, tumor cells, vascular smooth muscle cells, trophoblast cells, and other cell populations that express VEGF receptors. Pathological conditions, such as abnormalities in placental development, can be caused by altered production and signaling of VEGFs. Trophoblast cells play a significant role in placental formation and are essential for angiogenesis due to their secretion and reception of VEGF. However, there is a lack of information in the literature regarding the influence of VEGF on functional characteristics of trophoblast cells. Maternal immune cells, particularly natural killer (NK) cells, have been shown to affect the activity of trophoblasts during pregnancy. Given the high abundance of NK cells in decidual tissue, it is important to evaluate their potential influence on phenotype of trophoblast cells. In this study, we investigated the expression of MICA, MICB, and CD105 proteins by NK cells and trophoblast cells. MICA and MICB are stress markers that allow us to assess cell viability. CD105 is a receptor expressed on the surface of various cell types. It plays a role in signal transmission from TGF-β family proteins. In particular, endoglin has been shown to regulate signaling from TGF-β by directing signals through the SMAD2/3 or SMAD1/5/8 pathways. According to the literature, endoglin inhibits the SMAD3- mediated signaling. However, similar effects of endoglin have not been confirmed for NK cells and trophoblasts. The studies of endoglin expression levels are of importance, since the signals from TGF-β are essential for differentiation of trophoblast cells. Disruption of TGF-β signaling can lead to pregnancy complications and miscarriage. We have demonstrated that VEGF plays a role in regulating the activity of trophoblasts and NK cells. In particular, treatment with neutralizing monoclonal antibodies to VEGF-A was associated with reduced expression of CD105, a VEGF coreceptor, on trophoblasts and NK cells under co-culture conditions. However, pretreatment of trophoblasts with anti-VEGF antibodies did not alter their resistance to the cytotoxic activity of NK cells. Taken together, these findings suggest that inhibition of VEGF signaling results in significant changes in reception of TGF-β family proteins by trophoblasts and natural killer cells.

1. Ander S.E., Diamond M.S., Coyne C.B. Immune responses at the maternal-fetal interface. Sci. Immunol., 2019, Vol. 4, no. 31, eaat6114. doi: 10.1126/sciimmunol.aat6114.

2. Apps R., Gardner L., Traherne J., Male V., Moffett A. Natural-killer cell ligands at the maternal-fetal interface: UL-16 binding proteins, MHC class-I chain related molecules, HLA-F and CD48. Hum. Reprod., 2008, Vol. 23, no. 11, pp. 2535-2548.

3. Apte R.S., Chen D.S., Ferrara N. VEGF in Signaling and Disease: Beyond Discovery and Development. Cell, 2019, Vol. 176, no. 6, pp. 1248-1264.

4. Cabrera-Sharp V., Read J.E., Richardson S., Kowalski A.A., Antczak D.F., Cartwright J.E., Mukherjee A., de Mestre A.M. SMAD1/5 signaling in the early equine placenta regulates trophoblast differentiation and chorionic gonadotropin secretion. Endocrinology, 2014, Vol. 155, no. 8, pp. 3054-3064.

5. Chen D.B., Zheng J. Regulation of placental angiogenesis. Microcirculation, 2014, Vol. 21, no. 1, pp. 15-25.

6. Chen W.S., Kitson R.P., Goldfarb R.H. Modulation of human NK cell lines by vascular endothelial growth factor and receptor VEGFR-1 (FLT-1). In Vivo, 2002, Vol. 16, no. 6, pp. 439-445.

7. Clark D.E., Smith S.K., He Y., Day K.A., Licence D.R., Corps A.N., Lammoglia R., Charnock-Jones D.S. A vascular endothelial growth factor antagonist is produced by the human placenta and released into the maternal circulation. Biol. Reprod., 1998, Vol.59, no. 6, pp. 1540-1548.

8. Clark D.E., Smith S.K., Licence D., Evans A.L., Charnock-Jones D.S. Comparison of expression patterns for placenta growth factor, vascular endothelial growth factor (VEGF), VEGF-B and VEGF-C in the human placenta throughout gestation. J. Endocrinol., 1998, Vol.159, no. 3, pp. 459-467.

9. Eidukaite A., Siaurys A., Tamosiunas V. Differential expression of KIR/NKAT2 and CD94 molecules on decidual and peripheral blood CD56bright and CD56dim natural killer cell subsets. Fertil. Steril., 2004, Vol. 81 Suppl. 1, pp. 863-868.

10. Fitzpatrick T.E., Lash G.E., Yanaihara A., Charnock-Jones D.S., Macdonald-Goodfellow S.K.,Graham C.H. Inhibition of breast carcinoma and trophoblast cell invasiveness by vascular endothelial growth factor. Exp. Cell Res., 2003, Vol. 283, no. 2, pp. 247-255.

11. Fong G.H., Rossant J., Gertsenstein M., Breitman M.L. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature, 1995, Vol. 376, no. 6535, pp. 66-70.

12. Garcia J., Hurwitz H.I., Sandler A.B., Miles D., Coleman R.L., Deurloo R., Chinot O.L. Bevacizumab (Avastin®) in cancer treatment: A review of 15 years of clinical experience and future outlook. Cancer Treat. Rev., 2020, Vol. 86, 102017. doi: 10.1016/j.ctrv.2020.102017.

13. Gong J.H., Maki G., Klingemann H.G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia, 1994, Vol. 8, no. 4, pp. 652-658.

14. Guo B., Slevin M., Li C., Parameshwar S., Liu D., Kumar P., Bernabeu C., Kumar S. CD105 inhibits transforming growth factor-beta-Smad3 signalling. Anticancer Res., 2004, Vol.24, no. 3a, pp. 1337-1345.

15. Haider S., Lackner A.I., Dietrich B., Kunihs V., Haslinger P., Meinhardt G., Maxian T., Saleh L., Fiala C., Pollheimer J., Latos P.A., Knofler M. Transforming growth factor-beta signaling governs the differentiation program of extravillous trophoblasts in the developing human placenta. Proc. Natl. Acad. Sci. U S A, 2022, Vol. 119, no. 28, e2120667119. doi: 10.1073/pnas.2120667119.

16. Hurwitz H.I., Fehrenbacher L., Hainsworth J.D., Heim W., Berlin J., Holmgren E., Hambleton J., Novotny W.F., Kabbinavar F. Bevacizumab in combination with fluorouracil and leucovorin: an active regimen for first-line metastatic colorectal cancer. J. Clin. Oncol., 2005, Vol. 23, no. 15, pp. 3502-3508.

17. Ito N., Wernstedt C., Engstrom U., Claesson-Welsh L. Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules. J. Biol. Chem., 1998, Vol. 273, no. 36, pp. 23410-23418.

18. Jackson M.R., Carney E.W., Lye S.J., Ritchie J.W. Localization of two angiogenic growth factors (PDECGF and VEGF) in human placentae throughout gestation. Placenta, 1994, Vol.15, no. 4, pp. 341-353.

19. Jin X., Mao L., Zhao W., Liu L., Li Y., Li D., Zhang Y., Du M. Decidualization-derived cAMP promotes decidual NK cells to be angiogenic phenotype. Am. J. Reprod. Immunol., 2022, Vol.88, no. 3, e13540. doi: 10.1111/aji.13540.

20. Kendall R.L., Wang G., Thomas K.A. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem. Biophys. Res. Commun., 1996, Vol. 226, no. 2, pp. 324-328.

21. Kohler P.O., Bridson W.E. Isolation of hormone-producing clonal lines of human choriocarcinoma. J. Clin. Endocrinol. Metab., 1971, Vol.32, no. 5, pp. 683-687.

22. Laakkonen J.P., Lahteenvuo J., Jauhiainen S., Heikura T., Yla-Herttuala S. Beyond endothelial cells: Vascular endothelial growth factors in heart, vascular anomalies and placenta. Vascul. Pharmacol., 2019, Vol. 112, pp. 91-101.

23. Lebrin F., Goumans M.J., Jonker L., Carvalho R.L., Valdimarsdottir G., Thorikay M., Mummery C., Arthur H.M., ten Dijke P. Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction. EMBO J., 2004, Vol. 23, no. 20, pp. 4018-4028.

24. Li D.Y., Sorensen L.K., Brooke B.S., Urness L.D., Davis E.C., Taylor D.G., Boak B.B., Wendel D.P. Defective angiogenesis in mice lacking endoglin. Science, 1999, Vol. 284, no. 5419, pp. 1534-1537.

25. Li Y., Zhu H., Klausen C., Peng B., Leung P.C. Vascular Endothelial Growth Factor-A (VEGF-A) Mediates Activin A-Induced Human Trophoblast Endothelial-Like Tube Formation. Endocrinology, 2015, Vol. 156, no. 11, pp. 4257-4268.

26. Maynard S.E., Min J.Y., Merchan J., Lim K.H., Li J., Mondal S., Libermann T.A., Morgan J.P., Sellke F.W., Stillman I.E., Epstein F.H., Sukhatme V.P., Karumanchi S.A. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J. Clin. Invest., 2003, Vol.111, no. 5, pp. 649-658.

27. Melder R.J., Koenig G.C., Witwer B.P., Safabakhsh N., Munn L.L., Jain R.K. During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium. Nat. Med., 1996, Vol. 2, no. 9, pp. 992-997.

28. Melincovici C.S., Bosca A.B., Susman S., Marginean M., Mihu C., Istrate M., Moldovan I.M., Roman A.L., Mihu C.M. Vascular endothelial growth factor (VEGF) – key factor in normal and pathological angiogenesis. Rom. J. Morphol. Embryol., 2018, Vol. 59, no. 2, pp. 455-467.

29. Mikhailova V., Khokhlova E., Grebenkina P., Salloum Z., Nikolaenkov I., Markova K., Davidova A., Selkov S., Sokolov D. NK-92 cells change their phenotype and function when cocultured with IL-15, IL-18 and trophoblast cells. Immunobiology, 2021, Vol. 226, no. 5, 152125. doi: 10.1016/j.imbio.2021.152125.

30. Naderan M., Sabzevary M., Rezaii K., Banafshehafshan A., Hantoushzadeh S. Intravitreal anti-vascular endothelial growth factor medications during pregnancy: current perspective. Int. Ophthalmol., 2021, Vol. 41, no. 2, pp. 743-751.

31. Nickel J., Ten Dijke P.,Mueller T.D. TGF-beta family co-receptor function and signaling. Acta Biochim. Biophys. Sin., 2018, Vol. 50, no. 1, pp. 12-36.

32. Papadopoulos N., Martin J., Ruan Q., Rafique A., Rosconi M.P., Shi E., Pyles E.A., Yancopoulos G.D., Stahl N., Wiegand S.J. Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis, 2012, Vol. 15, no. 2, pp. 171-185.

33. Rajagopalan S., Long E.O. KIR2DL4 (CD158d): An activation receptor for HLA-G. Front. Immunol., 2012, Vol. 3, 258. doi: 10.3389/fimmu.2012.00258.

34. Sandler A., Gray R., Perry M.C., Brahmer J., Schiller J.H., Dowlati A., Lilenbaum R., Johnson D.H. Paclitaxelcarboplatin alone or with bevacizumab for non-small-cell lung cancer. N. Engl. J. Med., 2006, Vol. 355, no. 24, pp. 2542-2550.

35. Scherner O., Meurer S.K., Tihaa L., Gressner A.M., Weiskirchen R. Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling. J. Biol. Chem., 2007, Vol.282, no. 19, pp. 13934-13943.

36. Schiessl B., Innes B.A., Bulmer J.N., Otun H.A., Chadwick T.J., Robson S.C., Lash G.E. Localization of angiogenic growth factors and their receptors in the human placental bed throughout normal human pregnancy. Placenta, 2009, Vol. 30, no. 1, pp. 79-87.

37. Sharkey A.M., Charnock-Jones D.S., Boocock C.A., Brown K.D.,Smith S.K. Expression of mRNA for vascular endothelial growth factor in human placenta. J. Reprod. Fertil., 1993, Vol. 99, no. 2, pp. 609-615.

38. Sharma S., Godbole G.,Modi D. Decidual Control of Trophoblast Invasion. Am. J. Reprod. Immunol., 2016, Vol. 75, no. 3, pp. 341-350.

39. Simons M., Gordon E., Claesson-Welsh L. Mechanisms and regulation of endothelial VEGF receptor signalling. Nat. Rev. Mol. Cell Biol., 2016, Vol. 17, no. 10, pp. 611-625.

40. Tan H.X., Yang S.L., Li M.Q., Wang H.Y. Autophagy suppression of trophoblast cells induces pregnancy loss by activating decidual NK cytotoxicity and inhibiting trophoblast invasion. Cell Commun. Signal., 2020, Vol. 18, no. 1, 73. doi: 10.1186/s12964-020-00579-w.

41. Trembath A.P., Markiewicz M.A. More than Decoration: roles for natural killer group 2 member d ligand expression by immune cells. Front. Immunol., 2018, Vol. 9, 231. doi: 10.3389/fimmu.2018.00231.

42. Vinnars M.T., Bjork E., Nagaev I., Ottander U., Bremme K., Holmlund U., Sverremark-Ekstrom E., MinchevaNilsson L. Enhanced Th1 and inflammatory mRNA responses upregulate NK cell cytotoxicity and NKG2D ligand expression in human pre-eclamptic placenta and target it for NK cell attack. Am. J. Reprod. Immunol., 2018, Vol. 80, no. 1, e12969. doi: 10.1111/aji.12969.

43. Wallace A.E., Fraser R., Cartwright J.E. Extravillous trophoblast and decidual natural killer cells: a remodelling partnership. Hum. Reprod. Update, 2012, Vol. 18, no. 4, pp. 458-471.

44. Waltenberger J., Claesson-Welsh L., Siegbahn A., Shibuya M., Heldin C.H. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J. Biol. Chem., 1994, Vol. 269, no. 43, pp. 26988-26995.

45. Wang J., Ding J., Zhang S., Chen X., Yan S., Zhang Y., Yin T. Decreased USP2a Expression Inhibits Trophoblast Invasion and Associates With Recurrent Miscarriage. Front. Immunol., 2021, Vol. 12, 17370. doi: 10.3389/fimmu.2021.717370.

46. Wang X.Q., Zhou W.J., Hou X.X., Fu Q., Li D.J. Trophoblast-derived CXCL16 induces M2 macrophage polarization that in turn inactivates NK cells at the maternal-fetal interface. Cell. Mol. Immunol., 2018, Vol. 15, no. 12, pp. 1038-1046.

47. Wu D., Luo S., Wang Y., Zhuang L., Chen Y., Peng C. Smads in human trophoblast cells: expression, regulation and role in TGF-beta-induced transcriptional activity. Mol. Cell. Endocrinol., 2001, Vol. 175, no. 1-2, pp. 111-121.

48. Yang F., Zheng Q., Jin L. Dynamic Function and Composition Changes of Immune Cells During Normal and Pathological Pregnancy at the Maternal-Fetal Interface. Front. Immunol., 2019, Vol. 10, 2317. doi: 10.3389/fimmu.2019.02317.

49. Yi Y., Cheng J.C., Klausen C., Leung P.C.K. TGF-beta1 inhibits human trophoblast cell invasion by upregulating cyclooxygenase-2. Placenta, 2018, Vol. 68, pp. 44-51.

50. Zhao J., Schlosser H.A., Wang Z., Qin J., Li J., Popp F., Popp M.C., Alakus H., Chon S.H., Hansen H.P., Neiss W.F., Jauch K.W., Bruns C.J., Zhao Y. Tumor-derived extracellular vesicles inhibit natural killer cell function in pancreatic cancer. Cancers, 2019, Vol. 11, no. 6, 874. doi: 10.3390/cancers11060874.

51. Zhou Y., McMaster M., Woo K., Janatpour M., Perry J., Karpanen T., Alitalo K., Damsky C., Fisher S.J. Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome. Am. J. Pathol., 2002, Vol. 160, no. 4, pp. 1405-1423.