The Role Of Keratin1 In Hypoxic Pulmonary Hypertension

Pulmonary hypertension(PH)is a disease that seriously endangers human health due to the progressive increasing of pulmonary vascular resistance and increasing pulmonary circulation pressure.The pathological features show that the thickened blood vessels are surrounded by a large number of inflammatory cells,neutral,Granulocytes,macrophages,etc.which participate in the process of vessel remodeling.Keratin 1(Krt1)is a cytoskeletal fiber-forming protein found in skin and other epithelial tissues that can form a network of filaments that are not easily destroyed in cells and was considered to be very inert.More and more studies have shown that keratin is not only involved in the formation of cytoskeleton,but may play an important role in signal transduction and information integration in cells,but the role of Krt1 in PH is unclear.In this study,we constructed rat PH models by continuous induction of hypoxia to explore the expression of Krt1 and its possible role in PH.At the same time,the effect of Krt1 on PH was studied by tracheal injection of adeno-associated virus carried with Krt1 gene to rat lung.The possible effects of Krt1 on PH were elucidated from animal level,cell level and molecular level,which provided a new theoretical basis and therapeutic target for PH pathogenesis.OBJECTIVE: To investigate whether Krt1 can activate Toll-like receptor 7(TLR7)in PASMCs,and activing NF-κB that mediates the expression of inflammatory factors S100a8 and S100a9 in the process of vascular remodeling.METHODS: Rats were induced to PH by exposuring to hypoxia three weeks.(1)Screening of Krt1 gene by RNA-seq,TMT and RRBS multidimensional sequencing analysis;(2)Rats injected with adeno-associated virus carried with Krt1 gene was detected to explore Krt1 protective effect on PH;(3)Hemodynamic detection method was used to measure right ventricular systolic pressure(RVSP)and right ventricular mass index(RVMI);(4)HE staining and Masson staining were used to detect vascular remodeling;(5)RT-PCR,Western blot,immunofluorescence and ISH were used to determine the Krt1 expression and localization in rat pulmonary artery specimens;(6)Wound healing were used to determine the proliferation and migration levels,NF-κB protein expression of PASMCs after interference and overexpression of Krt1 was analyzed by western blot.The expression of TLR7,S100a8 and S100a9 m RNA was detected by RT-PCR.RESULTS:(1)Compared with the Control,the RVSP and RVMI of the hypoxic model rats were significantly increased and the blood vessels were thickened,which suggesting the model was successfully prepared;(2)Sequencing analysis and verification results showed that Krt1 expression was significantly decreased in hypoxic PH.Krt1 m RNA expression was negatively correlated with RVSP and RVMI in hypoxic 0,1,2,and 3 weeks rat models(p< 0.05);(3)After the tracheal injection of adeno-associated virus carried with Krt1 gene into rats lungs,RVSP and RVMI decreased,and the degree of vascular remodeling was relieved;(4)Interference with Krt1 can promote the migration and phenotypic transformation of PASMCs;(5)Interfering with Krt1 expression,NF-κB,TLR7,S100a8,S100a9 expression were enhanced,TLR7 inhibitor IRS can inhibit the expression of related immune inflammatory factors;overexpressing Krt1,the expression level of immune inflammatory factors is lower compared with PASMCs in hypoxic condition,suggesting that Krt1 activates TLR7 and mediates the expression of inflammatory factors S100a8 and S100a9 in PASMCs via NF-κB.Conclusion:Down-regulation of Krt1 in hypoxic PH promote pulmonary vascular remodeling by activating immune inflammatory factors TLR7,S100a8,S100a9,which providing a new theoretical basis and therapeutic target for the PH pathogenesis.