Expression And Function Of IL-33/ST2 In Mouse Vascular Endothelial Cells

Background: Interleukin33,a cytokine in the IL-1 family,was first identified in human endothelial cells as a "high endothelial vein nuclear factor"(NF-HEV)and is expressed mainly in epithelial and endothelial cells.ST2,also known as IL1RL1,is also a member of the IL-1 superfamily.It is the only known receptor for IL-33 and is mainly expressed on immune cells.Endothelial cells,also known as vascular endothelial cells,have many important functions such as forming the inner wall of blood vessels.There is a special kind of vascular endothelial cells in endothelial cells,cerebrovascular endothelial cells,which are one of the cells of blood-brain barrier and have special filtration function.IL-33 is constitutively expressed at high levels in central nervous system tissues,especially in CNS resident cells such as astrocytes,which release cytokines that act on brain microvascular endothelial cells.Brain microvascular endothelial cells are not only a member of endothelial cells but also an important component of CNS,and their IL-33 expression remains unclear.At the same time,the effect of brain microvascular endothelial cells on the release of IL-33 by CNS resident cells also needs to be further explored.In addition,the heart is also an organ with high constitutive expression of IL-33.As an important link between the cardiovascular system and the immune system,it is not clear whether IL-33 is expressed or regulated by the cardiovascular endothelial cells.Objective: To explore the expression of IL-33 and its receptor ST2 in mouse brain and cardiovascular endothelial cells,and the effect of IL-33 on microvascular endothelial cells in mouse brain.Methods: 1.The expression of IL-33 and ST2 in endothelial cells and the expression of IL-33/ST2 in brain microvascular endothelial cells were retrieved by biochemistry analysis: The human protein atlas,Single cell expression atlas and Vascular Single cells databases were used for information retrieval.2.Isolation and culture of mouse primary cerebral microvascular endothelial cells: The cerebral cortex of 8-week-old C57BL/6 and BALB/c mice was isolated and cultured in vitro after two-step digestion.The primary cerebral microvascular endothelial cells of mice were obtained and their purity was detected by immunofluorescence method and flow cytometry.3.Expression of IL-33/ST2 in mouse vascular endothelial cells:(1)The expression of IL-33 in primary cerebral microvascular endothelial cells of C57BL/6 and BALB/c mice was detected by Western blot and immunofluorescence,the expression of ST2 was detected by Western blot and RT-PCR,and the expression of IL-33 in endothelial cells was detected by RT-PCR under the stimulation of inflammation-related factors.(2)Expression of IL-33 and ST2 in mouse cardiovascular endothelial cells was detected by immunohistochemistry.(3)The expression of IL-33 in b End.3 cell lines was detected by Western blot,immunofluorescence and RT-PCR,the expression of ST2 in b End.3 cell lines was detected by Western blot,and the expression of IL-33 in b End.3 cell lines.4.The role of IL-33/ST2 in mouse brain microvascular endothelial cells: The expressions of VCAM-1,Claudin-5,IL-33 and ST2 in C57BL/6 mouse primary brain microvascular endothelial cells and b End.3 cell lines were detected by Western blot.The expression of VCAM-1 and Claudin-5 in primary brain microvascular endothelial cells and b End.3 cell line of C57BL/6 mouse was detected by Q-PCR,and the expression of CD31 in b End.3 cell line was detected by immunofluorescence assay.Results: 1.Expression of IL-33 and ST2 in endothelial cells: Biocredit analysis showed that many human endothelial cells expressed IL-33 and ST2 simultaneously,while many mouse endothelial cells did not express IL-33/ST2 or had low expression of IL-33/ST2.C57BL/6 mouse brain endothelial cells did not express IL-33 and had low expression of ST2.2.High purity mouse primary brain microvascular endothelial cells were successfully cultured.3.Expression of IL-33/ST2 in mouse vascular endothelial cells: IL-33 was not constitutively expressed in primary cultured brain microvascular endothelial cells of C57BL/6 mice,nor could it be induced by TNF-α and HMGB1,but constitutively expressed ST2.The primary cultured brain microvascular endothelial cells of BALB/c mice also showed no constitutive expression of IL-33 but constitutive expression of ST2.Cardiovascular endothelial cells of C57BL/6 mice expressed ST2 but not IL-33.b End.3 cell lines expressed IL-33 and ST2 constitutively,and IL-33 expression did not change significantly under TNF-α and HMGB1 stimulation.4.Effect of IL-33/ST2 on microvascular endothelial cells in mouse brain: The low concentration of IL-33 had no significant effect on the expression of VCAM-1,Claudin-5,IL-33 and ST2 in primary brain microvascular endothelial cells and Bend.3 cell lines of C57BL/6 mice.High concentration of IL-33 increased the expression of tight junction proteins Claudin-5 and CD31 in primary endothelial cells and b End.3 cells,but had no significant effect on the expression of VCAM-1,IL-33 and ST2.Conclusion: 1.Adult mouse cerebral microvascular endothelial cells and cardiovascular endothelial cells did not express IL-33 but express ST2.2.Immortalized mouse brain microvascular endothelial cell lines expressed IL-33 and ST2.3.IL-33 increased the expression of tight junction in mouse cerebral microvascular endothelial cells.