Mechanisms Of BFGF Regulating Aortic Valve Interstitial Cells Calcification By Inhibiting Endoplasmic Reticulum Stress

Background:The incidence rate of calcific aortic valve disease(CAVD)is higher in the population,although only 0.2% in adults.This proportion increased to 9.8% for middle-aged and elderly people over 50 years old.Aortic valve fibrosis and calcification can lead to aortic stenosis and increase blood flow velocity,with increased left ventricular afterload leading to heart failure.A growing number of studies have shown that aortic Valve calcification is not only a passive calcification process that occurs with aging,but also involves pathological changes similar to active atherosclerosis,such as chronic inflammation,lipoprotein deposition and valve interstitial cells(VICs)osteogenic transdifferentiation.Among them,the phenotypic transdifferentiation of VICs under the stimulation of various pathological factors,such as myofibroblasts and osteoblasts,is an important internal mechanism of the occurrence and development of CAVD.Currently,valve replacement is recognized as the most effective treatment for CAVD.However,replacement valves have certain disadvantages.Mechanical valves require long-term clotting monitoring and lifelong oral anticoagulants,with the attendant side effects of a higher risk of bleeding or embolism.Biological valve is easy to decay in the long term,and its service life is limited.However,CAVD patients tend to be older and often suffer from underlying diseases such as diabetes and hypertension,and the high surgical risk makes some patients lose the opportunity for surgical intervention.Therefore,exploring the potential pathogenesis of CAVD and developing new effective preventive or therapeutic drugs have significant social significance and economic value.Basic fibroblast growth factor(bFGF),also known as fibroblast growth factor 2(FGF-2),is a member of the FGF polypeptide growth factor family.It has a wide range of biological regulatory effects,regulating cell proliferation,adhesion,differentiation and apoptosis.Currently,bFGF related drugs have been partially applied in clinical practice,mainly used to promote wound healing,vascular remodeling,reduce scar formation and other surgical diseases.BFGF has been shown to regulate osteogenic responses by regulating a variety of cell proliferation and transdifferentiation processes.It is interesting that bFGF can significantly reduce the activity of Alkaline phosphatase(ALP)and calcification process of dental pulp stem cells in osteogenic medium.In the process of inducing bone marrow stromal cell calcification,bFGF intervention can significantly improve the osteogenic calcification ability of bone marrow stromal cells.It is noteworthy that bFGF regulation of osteogenic response is often diverse.Studies have shown that bFGF can significantly increase collagen fiber synthesis in local tissues,accelerate microcapillary remodeling,and ultimately promote calcification reaction.Currently,there are few studies on the regulatory role of bFGF in the pathogenesis of cardiovascular diseases,and there are few reports on its effect on VICs calcification in CAVD.Therefore,this study focused on the effect of bFGF on VICs osteogenic calcification in CAVD and its potential mechanism,in order to provide new ideas for the study of potential drug therapeutic targets for CAVD.Objectives:1.To investigate the expression of bFGF in the aortic valve and serum of CAVD patients;2.To elucidate the effects of bFGF on VICs morphology,calcification,proliferation and apoptosis at the cellular level;3.The effect of bFGF on osteogenic calcification of VICs in CAVD rats was investigated from animal level;4.RNA-seq technique was used to explore the potential mechanisms of bFGF inhibiting osteogenic differentiation of VICs.Methods:1.Collection and treatment of clinical valve and serum specimens: Aortic valve specimens and clinical non-calcified valve specimens were collected from Patients with CAVD,and valvular calcification caused by congenital,rheumatic and bacterial valvular disease were excluded.Valves in the control group were mainly from patients with normal valves undergoing heart transplantation in our hospital.Valve specimens obtained clinically were preserved and treated according to the operation requirements.The valve prepared for pathological section was placed in formalin solution for fixation for later immunohistochemical staining and other experiments.Valves that require tissue protein extraction are immediately placed in liquid nitrogen.In order to detect the bFGF level in serum of patients with CAVD,venous blood samples of patients with CAVD before surgery were also collected in this study.Patients with coronary atherosclerosis were excluded from the control group in order to exclude other calcification disorders.The venous blood of the enrolled patients was collected before surgery and placed at room temperature for 15 minutes for natural coagulation,followed by centrifugation for20 min at 2000-3000r/min.The supernatant was collected and stored at-80℃ for examination.The serum bFGF content was detected by ELISA.Through the above experiments,the expression level of bFGF in the aortic valve and serum of CAVD patients was clarified.2.Isolation and culture of p-VICs and establishment of cell calcification model: The primary porcine aortic valve used in this experiment was obtained from adult commercial pigs slaughtered by Shanghai Songlin meat food company.Aortic VICs were obtained by type II collagenase secondary digestion method.This separation method has been used in Changhai Cardiothoracic Surgery Laboratory for a long time.It has been proved that this method can obtain VICs with stable and ideal purity,so there is no need to obtain cell phenotype identification step again.The isolated cells avoided contamination and were passed to the 2nd to 6th generations for subsequent cytological experiments.Cell calcification model construction: P-VICs obtained by the above methods were cultured in osteogenic induction medium(50 μg/m L ascorbic acid,10-7 mol/L insulin,2 mmol/L sodium dihydrogen phosphate and 5% fetal bovine serum DMEM complete medium).Our laboratory has confirmed that p-VICs cultured in this medium for 7 days can form stable calcified nodules.Meanwhile,bFGF at different concentration gradients(0,10,50,100 ng/ m L)was administered to intervene the process of VICs calcification.Western Blot,PCR,flow cytometry,alizarine red staining and other experimental methods were used to explore the effects of bFGF on VICs morphology,calcification,proliferation,apoptosis and other biological functions and related mechanisms.3.Establishment of aortic valve calcification rat model: Male SD rats aged 3-4weeks were divided into control group,calcification model group and calcification+bFGF intervention group,with 5 rats in each group.Valve calcification induced diet rich in high fat and cholesterol components(common mixed feed: lard: cholesterol: Pig bile salt=88.5:10:1:0.5),the control group was fed with ordinary mixed feed for 3 months.Meanwhile,in order to ensure the modeling effect,25000IU/kg vitamin D solution was intraperitoneously injected one day after one month of high-fat diet,and the dosage of vitamin D was carefully controlled to avoid poisoning to death of rats.Cardiac ultrasound results of rats were collected after 3 months of continuous feeding,and then cardiac samples were obtained to prepare valve tissue sections for subsequent immunohistochemistry,Tunel,immunofluorescence,Alizarin red,HE staining and other experiments to explore the effect of bFGF on valve calcification in vivo.4.Explore the potential mechanism of bFGF inhibiting VICs calcification by bioinformatics methods : In vitro pig valvular interstitial cells,calcification and calcification + bFGF group were set up,the extraction of total RNA and sent to the easy line biotechnology company transcriptome sequencing,screening of differentially expressed m RNA and bioinformatics analysis,to select the core molecule or signaling pathways for validation,to further explore the potential mechanism of bFGF regulating valve calcification.5.Statistical analysis methods: IBM SPSS software(Version 21)was used for statistical analysis.All data were statistically analyzed using one-way ANOVA,Bonferroni correction for multiple groups or Graph Pad Prism 8.0(Graph Pad Software,CA,USA)T test for two groups.The mean value of continuous variables is expressed as mean ± standard error.All in vitro experiments were performed with 3 replicates per group.The difference was statistically significant(P < 0.05).Results:1.The expression of bFGF increased in the valve of CAVD patients,accompanied by the increase of serum contentWestern Blot and immunohistochemical results showed that bFGF was widely expressed in human aortic valve,and the expression of bFGF was increased in calcified valve compared with normal valve.Alizarin red staining revealed calcified valves with calcium nodules,thickening and structural disorder.Serum bFGF content in CAVD patients was also slightly increased by ELISA.2.bFGF can improve the proliferation of VICs and inhibit the occurrence of VICs calcification,apoptosis and senescencePorcine VICs were isolated by collagenase digestion method and CAVD cell model was constructed.The effects of bFGF on the morphology,calcification,proliferation and apoptosis of VICs after osteogenic induction were detected.The results showed that the calcification induction degree of VICs was significantly reduced under the action of bFGF.Western Blot and immunofluorescence experiments showed that the expressions of osteogenic markers such as Runx2,OPN and SP7 decreased.The phenotypic markerα-SMA was significantly decreased in myofibroblasts.The results indicated that the typical fusiform changes of VICs were observed after adding bFGF.CCK-8,Western Blot,flow cytometry and senescence staining showed that bFGF could significantly increase the proliferation activity of VICs,accelerate cell cycle,delay cell senescence,and inhibit apoptosis.3.bFGF intervention in CAVD rats reduced the degree of valve calcification and the proportion of apoptotic cellsThe rat model of CAVD was established by high-fat and high-cholesterol diet +Vit D intraperitoneal injection to further verify the role of bFGF in the occurrence and development of aortic valve calcification.Cardiac ultrasound showed that the aortic valve differential pressure and peak flow rate decreased after bFGF intervention,and the differences were statistically significant.Alizarin red staining and immunohistochemical staining showed that bFGF could significantly reduce the formation of calcified aortic valve nodules and inhibit the expression of osteogenic markers like Runx2 and OPN.Masson staining suggested that collagen fibers were involved in the normal structure of the aortic valve,but there was no significant difference between the groups,indicating that bFGF was not involved in the generation of collagen fibers in the valve tissue.Tunel staining and method analysis showed that aortic valve calcification was also accompanied by apoptosis,and this pathological change could also be inhibited by bFGF,which was consistent with cytological results.4.bFGF may inhibit endoplasmic reticulum stress-induced apoptosis by activating AKT and ERK pathways to inhibit VICs calcificationmRNA sequencing and bioinformatics analysis showed that there were many differentially expressed genes in VICs under the action of bFGF,indicating that bFGF had a wide influence on the biological functions of VICs.KEGG pathway analysis suggested that bFGF could activate ERK and AKT pathways with decreased expression of ERS-related proteins,suggesting that ERS,AKT and ERK pathways may play an inportant role as the potential mechanism of bFGF inhibiting VICs osteogenesis and transdifferentiation.Subsequently,two classical signal inhibitors were added:LY294002(PI3K/ AKT pathway inhibitor)and PD98059(ERK1/2 pathway inhibitor).Western Blot showed that when LY294002 and PD98059 were added,Western blot showed that when LY294002 and PD98059 were added,the expression of ATF6,CHOP,Casease-3increased,the expression of anti apoptotic protein Bcl2 decreased,and the expression of osteogenic transdifferentiation protein Runx2 and OPN increased,suggesting that the blocking of AKT and ERK pathways can partially eliminate the calcification inhibition of bFGF on VICs,accompanied by the enhanced ERS.Conclusions:The expression of bFGF was increased in calcified aortic valve tissues,and exogenous bFGF had extensive effects on VICs morphology,proliferation,apoptosis,calcification and other biological functions.bFGF can inhibit the apoptosis induced by endoplasmic reticulum stress by activating AKT and ERK pathways,and then inhibit the calcification of VICs.