Activated Factor X Induces Endothelial Cell Senescence Through IGFBP-5

Uncontrolled coagulation contributes to the pathophysiology of several chronic inflammatory diseases. In these conditions, senescent cells are often observed and is involved in the generation of inflammation. The coincidence of hyper-coagulation, cell senescence, and inflammation suggests the existence of a common underlying mechanism. Recent evidence indicates that activated coagulation factor X (FXa) plays a role in the processes beyond blood coagulation. This non-hematologic function entails the mediation of inflammation and tissue remodeling. We therefore tested the hypothesis that FXa induces cell senescence resulting in tissue inflammation and impaired tissue regeneration. Human umbilical vein endothelial cells were stimulated with FXa for 14 days. The proliferation of cells treated with FXa was significantly smaller, and the fraction of senescence-associated β-galactosidase-positive cells was increased as compared to the control group. RT-qPCR array revealed that FXa increased the expression of IGFBP-5, EGR-1, p53, and p16INK4a. Inhibition of FXa by a direct FXa inhibitor, rivaroxaban, or IGFBP-5 by siRNA decreased FXa-induced cell senescence, restoring cell proliferation. Moreover, in an ischemic hind limb mouse model, FXa inhibited neovascularization by endothelial progenitor cell. However, rivaroxaban significantly restored FXa-induced impaired angiogenesis. In summary, FXa induced endothelial cell senescence through IGFBP-5, resulting in impaired angiogenesis.


Animal Preparation
All experimental procedures were reviewed and approved by the Institutional Animal Committee at the Department of Veterinary Science of Osaka University School of Medicine (approved number; 25-043-06) and follow the recommendations of the guidelines for animal experimentation at research institutes (Ministry of Education, Culture, Sports, Science and Technology, Japan), guidelines for animal experimentation at institutes (Ministry of Health, Labor and Welfare, Japan), and guidelines for proper conduct for animal experimentation (Science Council of Japan). C57BL6 mice aged 6-10 weeks were anesthetized with isoflurane for operative resection of one femoral artery, as described previously 1 . The ischemic/nonischemic limb blood flow ratio was measured using laser Doppler imaging (LDI, Moor LDI-Mark 2, Moor Instruments, UK). Tissue sections from the adductor brevis muscles of ischemic limbs were harvested on day 14. The sections were stained with specific antibodies. A total of 8 different fields (in 3 cross sections from 4-5 animals) were randomly selected, and CD31-positive capillary density was determined by confocal microscopy.

Reagents and antibodies
Human FXa was purchased from BioVision Inc, California, USA. Mouse FXa was purchased from Abcam plc, Cambridge, UK. Rivaroxaban was kindly donated from Bayer pharma AG, Leverkusen, Germany. IGFBP-5 antibody was from R&D Systems, Minnesota, USA. EGR-1 antibodies were from Santa Cruz Biotechnology, Inc. Texas, USA. p53 and p16 INK4a antibody was obtained from Cell Signaling, technology, Massachusetts, USA. Anti-mouse CD31 antibody was from BD Bioscience, California, USA. Anti-GFP antibody was from Abcam plc, Cambridge, UK. IGFBP-5 plasmid (pcDNA3-IGFBP5-V5) was purchased from addgene, Cambridge, MA, USA, and siRNA for PAR1/2 and IGFBP-5 were from Santa Cruz Biotechnology, Inc. Texas, USA. Human recombinant IGFBP-5 was from R&D Systems.

Cell culture
Human umbilical vein endothelial cells (HUVEC, passage 5 to 9) purchased from Lonza were cultured in endothelial basal medium-2 (EBM-2) (Clonetics, Walkersville, Maryland, USA) supplemented with EGM and 5 % fetal bovine serum (FBS). HUVECs were stimulated by FXa (1 or 10 nM) with or without rivaroxaban (10 µM) every other days for 14 days. Overexpression of IGFBP-5 or knockdown experiments by siRNA was performed for 10 days in HUVEC. For endothelial progenitor cells (EPC) culture, bone marrow mononucleotide cells were isolated from C57BL/6-Tg (CAG-EGFP) mouse as described previously 2 and cultured in EBM-2 supplemented with EGM and 10% FBS. After 7 days in culture, EPCs were stimulated by FXa (1 or 10 nM) with or without rivaroxaban (10 µM) every other days for 14 days. One day after femoral artery resection, mice were received 2×10 5 culture-expanded EPC was injected intravenously.

Proliferation assay and Matrigel tubular assay,
Mitogenic activity was measured with MTS assay kit (Promega, Madison, Wisconsin, USA). We conducted Matrigel tube formation assay as described previously. 2 Briefly, 9×10 4 HUVEC were plated and incubated at 37 °C for the 24 hours on a growth factor-reduced Matrigel-coated 48 well dish. Tubular length was measured by using image J soft wear.

Analysis of senescence marker of HUVEC
Several human senescence markers of HUVEC was measured by RT² Profiler™ PCR Array Human Aging, QIAGEN (California, USA) as manufactures instruction. Each category consist of mixed three samples. Therefore, we validated it by RT-PCR or western botting later.

Isolation of total RNA and RT-PCR
Total RNA was isolated using RNeasy Mini Kit (QIAGEN, Hilden, Germany). DNase treated total RNA was reverse-transcribed with the High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA) to produce complementary DNA (cDNA). Reverse transcription-generated cDNA encoding the target genes was amplified and quantified by the ViiA-7™ real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the primer set shown below.