1.The role and mechanisms of fibulin-1 in senescence-related calcification of vascular smooth muscle cells
Xiaolu LIANG ; Dan YAN ; Mandi LUO ; Lei RUAN ; Cuntai ZHANG
Chinese Journal of Geriatrics 2022;41(5):580-585
Objective:To investigate the role and mechanisms of fibulin-1 in senescence-related calcification of rat vascular smooth muscle cells induced by high-concentrationphosphate treatment.Methods:From September 2020 to September 2021, rat primary vascular smooth muscle cells were extracted from the thoracic aorta and abdominal aorta of 10 male SD rats aged 6 to 8 weeks.Phosphate(2.5 mmol/L Pi)was used to stimulate the calcification of vascular smooth muscle cells(VSMCs)in a model of stress-induced senescence-related calcification.Cellular senescence was assessed by SA-β-gal staining.Cellular calcification was determined by alizarin red staining and quantification of calcium deposition.Phenotypic transformation indexes and the expression of fibulin-1 during the process of calcification were detected by Western blot.The expression of fibulin-1 in primary rat vascular smooth muscle cells was knocked down by siRNA, the expression of pSmad3 was detected by immunofluorescence, and the effects of fibulin-1 on phenotypic transformation indexes of smooth muscle cells were detected by Western blot.The cells were cultured with recombinant fibulin-1 while transforming growth factor beta(TGF-β)inhibitor A83-01 and pSmad3 inhibitor SIS3 were also added.The senescence and calcification indexes of smooth muscle cells were detected by Western blot.Results:In the stress-induced aging model with phosphate stimulation of calcification in rat VSMCs, the expression of fibulin-1 was up-regulated( t=11.20, P<0.01), the expressions of MHC and SM22α was down-regulated( t=7.97, P<0.01; t=10.27, P<0.01), and the expression of osteoblastic phenotype markers OPN and Bmp2 and senescence marker P53 was up-regulated( t=4.79, P<0.01; t=9.56, P<0.01; t=14.07, P<0.01). Knockdown of fibulin-1 attenuated the degree of senescence and calcium deposition in VSMCs( t=12.90, P<0.05)and decreased the expression of OPN, Bmp2 and P53( t=5.92, P<0.05; t=10.15, P<0.01; t=8.28, P<0.01), at the same time, and TGF-β and pSmad3 expression was inhibited( t=12.90, P<0.01; t=7.46, P<0.01). After the addition of TGF-β/ smad3 pathway inhibitors, the stimulatory effect of recombinant fibulin-1 on phenotypic transformation and senescence protein expression inVSMCs was significantly reduced( t=4.52, P<0.01; t=9.82, P<0.01; t=3.85, P<0.05). Conclusions:Fibulin-1 can promote aging-related calcification of vascular smooth muscle cells through the TGF-β/smad3 signaling pathway.