Mechanisms Of The Crosstalk Between Activated-Valve Interstitial Cells And M2 Macrophages In Promoting Aortic Valve Fibrosis And Calcification

BackgroundCalcific aortic valve disease(CAVD)is a relatively common valvular lesion.The onset of calcification of the valve leads to a restriction of leaflet motion and ultimately to aortic stenosis and hemodynamic changes.However,there are still no effective drugs or clinical markers for early diagnosis and treatment of CAVD,and research on CAVD is still a long way to go.The development of CAVD is regulated by the stimulation of injury factors,interactions between cells,extracellular matrix remodeling,and the complex immune network,The impact on disease development is multifaceted.Current research on CAVD is mainly focused on the osteogenic transformation of valve interstitial cells(VICs),which are activated from their original quiescent state and stimulated by injury factors and may further differentiate into osteoblasts on the basis of activated VICs.Activation of VICs is regulated by various factors,including cytokine release subsequent to endothelial injury,ECM remodeling,and the modulation of other cells within the valve leaflet.In response to the injury microenvironment,VICs are successfully activated into stretchier phenotype,which in turn secrete more cytokines that affect other cells surrounded and promote ECM remodeling.As a hallmark in the early course of valvular fibrosis,its regulation of CAVD is multifaceted,and its specific role in the early course of the disease deserves to be explored.The process of fibrosis and calcification progression has a close associations with the immune inflammatory response,and macrophages(Mφ),mast cells,and lymphocytes are all involved in the regulation of the immune network.As an important part of the immune response,macrophage chemotaxis,adhesion,and polarization are inextricably linked to CAVD progression.The role of M2 type Mφ,which are mainly involved in fibrosis progression and chronic inflammatory response,is less studied in CAVD,and their mutual regulatory relationship with mesenchymal cells in the early course of the disease may bring new directions for the study of CAVD.Objectives(A)To clarify the regulation of Mφ recruitment and polarization by activated VICs;(B)To clarify the mechanism of VICs activation and osteogenic differentiation by M2 type Mφ;(C)To clarify that activated VICs and Mφ act together to promote valve fibrosis and calcification in vivo CAVD model.Methods(A)Clinical data of CAVD and control patients and collection of clinical specimensBaseline data were collected from 15 patients who underwent aortic valve replacement and 15 patients who underwent heart transplantation between March 2021 and March 2022 in the Cardiac Surgery Department of Changhai Hospital.The patients’ aortic valve tissues were collected for histology staining and molecular test.Basic morphological changes of the valves were observed by H&E staining.Masson staining was used to detect valve fibrosis.Alizarin red staining and von Kossa staining were used to evaluate valve calcification.Immunohistochemical were used to detect activation of VICs and expression of Mφ in the valves,and Western-blot were used to observe the expression of osteoblast-associated proteins in the valves.The correlations between osteoblasts or activated VICs and Mφ were observed by q PCR.(B)Isolation,extraction and identification of porcine aortic valve interstitial cells and activation inductionExtraction and identification of VICs: A two-step method was used to isolate porcine aortic valve interstitial cells.Immunofluorescence(if)staining was used to label Vimentin,α-MSA and CD 31 to observe the purity of the extracted cells.Induction of fibrosis model of VICs: recombinant TGF-β1 was used to induce VICs with a concentration gradient for 3days,if staining was used to observe the α-SMA fluorescence signal intensity,and the appropriate gradient was determined by the results.Regulation of Smad2/3/ HA/CD44 on VICs activation: Western-blot was used to observe the changes in Smad2/3 phosphorylation levels after TGF-β1 induction.Bioinformatics analysis was used to detect differential gene expression in P-VICs under the same treatments in the GEO database.Western-blot was used to detect the changes in hyaluronan synthase 2(HAS2)expression.Finally,observe the activation of VICs after the inhibition of Smad2/3 pathway,HAS2,and interference with hyaluronan(HA)receptor CD 44 by Western-blot.(C)Regulation of macrophage recruitment and polarization by activated VICsMacrophage migration: q PCR and Western-blot were performed to observe the differences in macrophage chemokine expression in activated VICs and the differences in expression of related chemokines after inhibition of the AKT pathway.The changes in the migration ability of Mφ after activation of VICs and after inhibition of AKT pathway and related chemokine expression were observed by wound healing assay and Transwell assay.Changes in the proliferation ability of Mφ under related stimulation were observed by Ed U and CCK-8.Macrophage adhesion: Western-blot was used to observe the expression of related adhesion molecules in activated VICs.Cell adhesion assay was used to observe macrophage adhesion function after VICs activation and after inhibition of related pathways and adhesion molecules.Macrophage polarization: Mφ were treated with conditioned mediums of VICs and the polarization of Mφ was observed by flow immunophenotyping,Western-blot and real-time quantitative PCR.(D)Effect of M2-type Mφ on the activation of VICs and osteogenic transformationInduction of macrophage polarization: Mφ were induced to M2 type by IL-4,and the induction was observed by flow immunophenotyping and Western-blot.Regulation of activation and osteogenic transformation of VICs: The effect of M2 Mφ on activation and osteogenic transformation of VICs was observed by conditioned culture and co-culture.Calcification of VICs after conditioned-calcification induction medium treatment was observed by Alizarin red staining at different culture times and the expression of relevant osteogenic markers was observed by Western-blot.(E)Observe calcification progression in CAVD miceIn vivo calcification model establishment: a model of aortic valve calcification was formed by feeding Apo E-/-mice with a high-fat diet for 2 months.Model establishment of CAVD was observed by ultrasound.Histological staining evaluation and if staining: Masson staining was used to observe the fibrosis of the valve and Von Kossa was used to detect the calcification.Changes in extracellular matrix components were observed by HA and CD44 if staining.Calcification was observed by co-staining with osteogenic-related proteins and macrophage markers.Results(A)Fibrogenesis and calcification in clinical specimens and the relationship between fibrosis or calcification with MφImmunohistochemical staining revealed that macrophage expression and VICs activation were upregulated in CAVD.Western-blot revealed that expression of osteoblast marker and activated VICs marker elevated in CAVD.HA content in the extracellular matrix was also increased.Real-time quantitative PCR revealed that osteogenic marker has a correlation with both types of macrophage expression,and only the M2 type is correlated to the activation of VICs and fibrogenesis.(B)Identification of P-VICs and fibrosis induction of themIf staining showed that most of the isolated cells were Vimentin-positive cells,and only a few α-SMA fluorescence signals were detected.Western-blot showed an increase in AKT pathway and HAS2 expression,and differential expression analysis of similarly treated VICs in the GEO database revealed that HAS2 expression was significantly upregulated after TGF-β1 induction,and Elisa also suggested an increase in expression of HA in the supernatant of activated VICs.Activation of VICs was inhibited after inhibition of Smad2/3,HAS2,and interference with CD44.(C)Effects of activated VICs on macrophage recruitment and polarizationReal-time quantitative PCR showed that the expression of macrophage chemokines CCL2 and CCL5 were increased after the activation of VICs,with CCL5 being more significantly increased.The migration assay revealed that macrophage migration activity was enhanced after activation of VICs which can be weakened by the inhibition of AKT pathway and interference with CCL5.Ed U staining revealed an increase in macrophage proliferation after a short period of conditioned culture and CCK8 showed significant macrophage proliferation after a long period of conditioned culture.CD44 was observed to elevate after VICs activation and so as the adhesion function of Mφ.Mφ polarized toward the M2 type after culture in conditioned medium of activated VICs.(D)M2-type Mφ promote the activation and accelerate the osteogenic differentiation of VICsAfter culturing VICs with M2-type conditioned medium,it was found that the activation of VICs was elevated.Co-culture system also suggested that M2-type Mφ could promote the activation of VICs,while neither conditioned cultivation nor co-culture for could directly lead to the osteogenic transformation of VICs.Followed by the induction of M2 macrophage-conditioned calcification induction medium of VICs,both alizarin red staining and Western-blot suggested that it could promote osteogenic transformation of VICs.(E)Inhibition of VICs activation and clearance of Mφ in high-fat diet fed Apo E-/-miceUltrasound results showed that high-fat diet fed Apo E-/-mice showed an increase in aortic peak flow velocity.After TGF-β1 inhibition and macrophage depletion,the aortic valve peak flow velocity increased compared with the CAVD group.Von Kossa staining showed significant calcified nodules in the aortic valve of CAVD model mice,while after TGF-β1 inhibition and macrophage depletion,calcified nodules were significantly reduced.HA-CD 44 expression was reduced compared with the CAVD group,and so as the osteogenic protein expression and M2 macrophage markers.ConclusionFibrogenesis can be clearly observed in the course of CAVD,and HA content in the extracellular matrix also increases.VICs activation during fibrogenesis can promote the recruitment of Mφ and polarization to M2 type,and conversely,M2 type macrophages can promote activation of VICs.Excessively activated VICs can accelerate the development of valve calcification.