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.

Arterial stiffness forms the basis of cardiovascular diseases (CVD) and is also an independent predictor of CVD risk. Early detection and intervention of arterial stiffness are important for improving the global burden of CVD. Pulse wave velocity (PWV) is the gold standard for assessing arterial stiffness and the molecular mechanism of arterial stiffness remains to be studied. Extracellular matrix (ECM) remodeling is one of the major mechanisms of arterial stiffness. Partial quantitative changes of ECM proteins can be detected in plasma. Therefore, we examined the hypothesis that a discovery proteomic comparison of plasma proteins between high arterial stiffness (baPWV ≥ 1,400 cm/s) and normal arterial stiffness (baPWV < 1,400 cm/s) populations might identify relevant changed ECM proteins for arterial stiffness. Plasma samples were randomly selected from normal arterial stiffness (n = 6) and high arterial stiffness (n = 6) people. Isobaric tags for relative and absolute quantitation (iTRAQ) based quantitative proteomics technique was performed to find a total of 169 differentially expressed proteins (DEPs). Nine ECM proteins were included in all DEPs and were all up-regulated proteins. Fibulin-1 had the highest statistically fold-change (FC = 3.7, p < 0.0001) in the high arterial stiffness population compared with the control group during the nine ECM proteins. The expression of plasma fibulin-1 in normal arterial stiffness (n = 112) and high arterial stiffness (n = 72) populations was confirmed through enzyme-linked immunosorbent assay (ELISA). Similarly, ELISA results showed that plasma concentrations of fibulin-1 in the high arterial stiffness group were higher than those in the normal arterial stiffness group (12.69 ± 0.89 vs. 9.84 ± 0.71 μg/ml, p < 0.05). Univariate analysis of fibulin-1 with brachial-ankle pulse wave velocity (baPWV) indicated that fibulin-1 was positively correlated with baPWV in all participants (r = 0.32, p < 0.01) and a stronger positive correlation between baPWV and fibulin-1 in high arterial stiffness group (r = 0.64, p < 0.0001) was found. Multiple regression analysis of factors affecting baPWV showed that fibulin-1 was also a significant determinant of the increased ba-PWV (R 2 = 0.635, p = 0.001). Partial correlation analysis showed that baPWV increased with the growth of plasma fibulin-1(r = 0.267, p < 0.001). In conclusion, our results demonstrated that fibulin-1 is positively correlated with ba-PWV and an independent risk factor for arterial stiffness.

Objective:To analyze the composition and functional characteristics of the intestinal microflora in novel coronavirus pneumonia patients with arterial stiffness, in order to provide empirical evidence for rational use of intestinal microecological modulators.Methods:Patients with novel coronavirus pneumonia admitted to our hospital between April 5 and April 19, 2020 were enrolled as research subjects.They were divided into the stiffness group and the control group according to the results of arterial stiffness.Stool samples were collected within 7 days of admission.Intestinal flora DNA was analyzed and entered into a database, shotgun metagenomic sequencing was performed, and bioinformatics analysis was conducted based on sequencing results.Results:A total of 16 patients with novel coronavirus pneumonia were included in this study, including 7 in the stiffness group and 9 in the control group.Brachial-ankle pulse wave velocity and blood pressure were higher in the stiffness group than in the control group( P<0.05). Beta diversity analysis at the phylum level showed that there were significant differences in the composition of the intestinal flora between the two groups( P<0.05). Patients with arterial stiffness had a lower relative abundance than the controls in fecal Holdemanella, Mitsuokella, Deinococcus, Lachnospira, Turicibacter, Butyrivibrio, Sporomusa, and Halanaerobium, species associated with the production of short-chain fatty acids(SCFAs), regulation of energy metabolism, anti-radiation, anti-oxidative stress and anti-inflammatory effects.The Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis showed that the intestinal flora in the control group was mainly involved in the pathways of lipopolysaccharide biosynthesis, metabolism of SCFA and other amino acids, and membrane transport, while the intestinal flora in the stiffness group was mostly concerned with the pathways of amino acid metabolism and DNA damage repair. Conclusions:The use of microbial agents capable of increasing short-chain fatty acids in patients with novel caronavirus infection may contribute to the restoration of intestinal flora homeostasis.