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Comparison of Serotonin-Regulated Calcific Processes in Aortic and Mitral Valvular Interstitial Cells.

ABSTRACT: Calcification is an important pathological process and a common complication of degenerative valvular heart diseases, with higher incidence in aortic versus mitral valves. Two phenotypes of valvular interstitial cells (VICs), activated VICs and osteoblastic VICs (obVICs), synergistically orchestrate this pathology. It has been demonstrated that serotonin is involved in early stages of myxomatous mitral degeneration, whereas the role of serotonin in calcific aortic valve disease is still unknown. To uncover the link between serotonin and osteogenesis in heart valves, osteogenesis of aortic and mitral VICs was induced in vitro. Actin polymerization and serotonin signaling were inhibited using cytochalasin D and serotonin inhibitors, respectively, to investigate the role of cell activation and serotonin signals in valvular cell osteogenesis. To evaluate calcification progress, calcium and collagen deposits along with the expression of protein markers, including the rate-limiting enzyme of serotonin synthesis [tryptophan hydroxylase 1 (TPH1)], were assessed. When exposed to osteogenic culture conditions and grown on soft surfaces, passage zero aortic VICs increased extracellular collagen deposits and obVIC phenotype markers. A more intense osteogenic process was observed in aortic VICs of higher passages, where cells were activated prior to osteogenic induction. For both, TPH1 expression was upregulated as osteogenesis advanced. However, these osteogenic changes were reversed upon serotonin inhibition. This discovery provides a better understanding of signaling pathways regulating VIC phenotype transformation and explains different manifestations of degenerative pathologies. In addition, the discovery of serotonin-based inhibition of valvular calcification will contribute to the development of potential novel therapies for calcific valvular diseases.

SUBMITTER: Wang X

PROVIDER: S-EPMC8340088 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Mediastinal ionizing radiotherapy is associated with an increased risk of valvular disease, which demonstrates pathological hallmarks similar to calcific aortic valve disease (CAVD). Despite advances in radiotherapy techniques, the prevalence of comorbidities such as radiation-associated valvular disease is still increasing due to improved survival of patients receiving radiotherapy. However, the mechanisms of radiation-associated valvular disease are largely unknown. CAVD is considered to be an actively regulated disease process, mainly controlled by valvular interstitial cells (VICs). We hypothesize that radiation exposure catalyzes the calcific response of VICs and, therefore, contributes to the development of radiation-associated valvular disease. Methods and Results: To delineate the relationship between radiation and VIC behavior (morphology, calcification, and matrix turnover), two different in vitro models were established: (1) VICs were cultured two-dimensional (2D) on coverslips in control medium (CM) or osteogenic medium (OM) and irradiated with 0, 2, 4, 8, or 16 Gray (Gy); and (2) three-dimensional (3D) hydrogel system was designed, loaded with VICs and exposed to 0, 4, or 16 Gy of radiation. In both models, a dose-dependent decrease in cell viability and proliferation was observed in CM and OM. Radiation exposure caused myofibroblast-like morphological changes and differentiation of VICs, as characterized by decreased αSMA expression. Calcification, as defined by increased alkaline phosphatase activity, was mostly present in the 2D irradiated VICs exposed to 4 Gy, while after exposure to higher doses VICs acquired a unique giant fibroblast-like cell morphology. Finally, matrix turnover was significantly affected by radiation exposure in the 3D irradiated VICs, as shown by decreased collagen staining and increased MMP-2 and MMP-9 activity. Conclusions: The presented work demonstrates that radiation exposure enhances the calcific response in VICs, a hallmark of CAVD. In addition, high radiation exposure induces differentiation of VICs into a terminally differentiated giant-cell fibroblast. Further studies are essential to elucidate the underlying mechanisms of these radiation-induced valvular changes.

Project description:Calcific aortic valve disease (CAVD) is characterized by progressive stiffening of aortic valve (AV) tissues, inducing stenosis and insufficiency. Bicuspid aortic valve (BAV) is a common congenital defect in which the AV has two leaflets rather than three, with BAV patients developing CAVD decades years earlier than in the general population. Current treatment for CAVD remains surgical replacement with its continued durability problems, as there are no pharmaceutical therapies or other alternative treatments available. Before such therapeutic approaches can be developed, a deeper understanding of CAVD disease mechanisms is clearly required. It is known that AV interstitial cells (AVICs) maintain the AV extracellular matrix and are typically quiescent in the normal state, transitioning into an activated, myofibroblast-like state during periods of growth or disease. One proposed mechanism of CAVD is the subsequent transition of AVICs into an osteoblast-like phenotype. A sensitive indicator of AVIC phenotypic state is enhanced basal contractility (tonus), so that AVICs from diseased AV will exhibit a higher basal tonus level. The goals of the present study were thus to assess the hypothesis that different human CAVD states lead to different biophysical AVIC states. To accomplish this, we characterized AVIC basal tonus behaviors from diseased human AV tissues embedded in 3D hydrogels. Established methods were utilized to track AVIC-induced gel displacements and shape changes after the application of Cytochalasin D (an actin polymerization inhibitor) to depolymerize the AVIC stress fibers. Results indicated that human diseased AVICs from the non-calcified region of TAVs were significantly more activated than AVICs from the corresponding calcified region. In addition, AVICs from the raphe region of BAVs were more activated than from the non-raphe region. Interestingly, we observed significantly greater basal tonus levels in females compared to males. Furthermore, the overall AVIC shape changes after Cytochalasin suggested that AVICs from TAVs and BAVs develop different stress fiber architectures. These findings are the first evidence of sex-specific differences in basal tonus state in human AVICs in varying disease states. Future studies are underway to quantify stress fiber mechanical behaviors to further elucidate CAVD disease mechanisms.

Project description:Background and aim of the studyAortic valve calcification is believed to involve the differentiation of valvular interstitial cells (VICs) into either a myofibroblastic or an osteoblast-like phenotype. Despite purported similarities between diseased VICs and osteoblasts, few studies have directly compared VICs and osteoblasts in side-by-side experiments. In the present study, VICs were compared against multiple osteoblastic cell types at different stages of differentiation. These findings may help to resolve whether VICs progress through a myofibroblastic phenotype before reaching an osteoblast-like stage.MethodsThree cell types representing a range of osteoblastic lineage commitment and differentiation were used in the phenotypic comparison against VICs. Specifically, VICs, embryonic fibroblasts (C3H10T1/2), pre-osteoblasts (MC3T3-E1), and mature primary osteoblasts were cultured on tissue-culture polystyrene in control or mineralization medium, and harvested for qPCR, DNA, and protein analysis at time points ranging from one to eight days.ResultsCulture of VICs in mineralization medium decreased the expression of alpha-smooth muscle actin (alpha-SMA; a myofibroblast marker), with no peak in alpha-SMA gene or protein expression in mineralization medium at any time point. The application of a mineralization medium led to increased expression levels of alkaline phosphatase (ALP; an early mineralization marker) for all cell types, although the magnitude of the increase in ALP was drastically smaller for VICs than for the osteogenic cell types. Only the osteogenic cell types demonstrated an appreciable increase in osteocalcin (an indicator of later-stage mineralization).ConclusionWhile the addition of mineralization medium generally increased the expression of osteogenic markers and decreased the expression of myofibroblastic markers, VICs displayed different levels and patterns of expression than the osteoblastic cell types used for comparison. Additionally, the lack of an alpha-SMA increase at any point after the addition of mineralization medium to VICs indicated that these cells may not need to progress through a myofibroblastic stage before reaching an osteoblast-like gene expression profile.

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Comparison of Serotonin-Regulated Calcific Processes in Aortic and Mitral Valvular Interstitial Cells.

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