Valvular Interstitial Cells Research Articles

Biofabrication of poly(l-lactide-co-ε-caprolactone)/silk fibroin scaffold for the application as superb anti-calcification tissue engineered prosthetic valve

In this study, electrospun scaffolds were fabricated by blending poly(l-lactide-co-ε-caprolactone) (PLCL) and silk fibroin (SF) with different ratios, and further the feasibility of electrospun PLCL/SF scaffolds were evaluated for application of tissue engineered heart valve (TEHV). Scanning electron microscopy (SEM) results showed that the surface of PLCL/SF electrospun scaffolds was smooth and uniform while the mechanical properties were appropriate as valve prosthesis. In vitro cytocompatibility evaluation results demonstrated that all of the PLCL/SF electrospun scaffolds were cytocompatible and valvular interstitial cells (VICs) cultured on PLCL/SF scaffolds of 80/20 & 70/30 ratios exhibited the best cytocompatibility. The in vitro osteogenic differentiation of VICs including alkaline phosphatase (ALP) activity and quantitative polymerase chain reaction (qPCR) assays indicated that PLCL/SF scaffolds of 80/20 & 90/10 ratios behaved better anti-calcification ability. In the in vivo calcification evaluation model of rat subdermal implantation, PLCL/SF scaffolds of 80/20 & 90/10 ratios presented better anti-calcification ability, which was consistent with the in vitro results. Moreover, PLCL/SF scaffolds of 80/20 & 70/30 ratios showed significantly enhanced cell infiltration and M2 macrophage with higher CD206+/CD68+ ratio. Collectively, our data demonstrated that electrospun scaffolds with the PLCL/SF ratio of 80/20 hold great potential as TEHV materials.

LncRNA RMST Promotes Progression of Calcification of Aortic Valve Via the miR-195-5p/Smad7 Axis

Calcified aortic valve disease (CAVD) is a cardiovascular disease closely related to cardiovascular morbidity and mortality. Osteogenesis differentiation of valvular interstitial cells (VICs) is the key process of CAVD. In this study, we investigated whether the circulating exosomes participated in the pathogenesis and progression of CAVD and explored the underlying mechanism. We constructed the CAVD model in rats to perform in-vivo experiments, and isolated the VICs to perfume in-vitro studies. A series of methods were used including exosomes extraction and identification, RNA sequencing, ALP activity assay, alizarin red staining, qRT-PCR, western blot, and luciferase reporter assay. RNA sequencing showed that the circulating exosomes from CAVD patients and the normal people exhibited aberrant long non-coding RNA level, among which RMST was significantly upregulated in the CAVD-derived circulating exosomes and in the calcified aortic valve tissues. ALP activity assay and alizarin red staining showed that RMST promoted the VICs calcification. Luciferase reporter assay indicated that RMST acted as the miR-195-5p sponge, and miR-195-5p could target the 3’UTR of Smad7. Further functional experiments revealed that RMST was enriched in the circulating exosomes, leading to the upregulation of Smad7 in through sponging miR-195-5p to promote osteogenic differentiation in VICs. Moreover, adipose-derived exosomes showed the same effect as the CAVD-derived circulating exosomes. In conclusion, we found that circulating exosomes from CAVD patients promoted the VICs calcification through delivering the lncRNA RMST which acted as the miR-195-5p sponge to regulate the Smad7 expression. These results provided a new understanding of the molecular mechanism of the progression of CAVD. Funding: This work was supported by National Natural Science Foundation of China (grant no. 81974033). Declaration of Interest: None to declare. Ethical Approval: The present research program had been reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Nanjing Medical University.

Development of a Bi-Layered Cryogenic Electrospun Polylactic Acid Scaffold to Study Calcific Aortic Valve Disease in a 3D Co-Culture Model

Calcified aortic valve disease (CAVD) is the most prevalent valve disease in the elderly in the western world. Targeted pharmacological therapies are limited since the underlying mechanisms of CAVD are not well understood. Appropriate 3D in vitro models could potentially improve our knowledge of the disease. Here, we developed a 3D in vitro heart valve model that resembles the morphology of the valvular extracellular matrix and mimics the mechanical and physiological behavior of the aortic valve fibrosa and spongiosa. We employed cryogenic electrospinning to engineer a bi-layered cryogenic electrospun scaffold (BCES) with defined morphologies that allowed valvular endothelial cell (VEC) adherence and valvular interstitial cell (VIC) ingrowth into the scaffold. Using a self-designed cell culture insert allowed us to establish the valvular co-culture simultaneously by seeding VICs on one side and VECs on the other side of the electrospun scaffold. Proof-of-principle calcification studies were successfully performed using an established osteogenic culture protocol and the here designed 3D in vitro heart valve model.

The pathomechanism of human myxomatous valvular degeneration at the mechanical and cellular level.

The purpose of this study was to explore the pathomechanism of human myxomatous valve degeneration by investigating changes in the phenotype of valvular cells, the metabolism of the extracellular matrix and their mechanical properties. Mitral valve specimens were harvested from patients who had undergone valve replacement, and divided into two groups: patients with a myxomatous mitral valve and a control group. Histological investigation showed that the morphology of the extracellular matrix was looser and less coordinated in myxomatous valves than in controls. α-SMA (α-smooth muscle actin) and Vimentin were positive and DNA (deoxyribonucleic acid) assay of leaflets and expression of SMemb (embryonic smooth muscle myosin heavy chain), MMP-13 (matrix Metalloproteinases-13), MMP-1 mRNA (messenger Ribonucleic Acid) of the myxomatous valves were increased while the hydroxyproline content, expression of TIMP-1 (tissue inhibitor of metalloproteinase-1) mRNA and mechanical properties were decreased compared with controls. Compared to the quiescent interstitial cells in non-myxomatous valves, interstitial cells in myxomatous valves exhibit myofibroblast activation and express excessive levels of matrix metalloproteinases. The balance between MMP/TIMP was disrupted. We conclude that overactivation of VICs (Valvular interstitial cells) and the imbalance of MMP/TIMP could be important features of the pathomechanism of myxomatous mitral valve degeneration.

Evogliptin Suppresses Calcific Aortic Valve Disease by Attenuating Inflammation, Fibrosis, and Calcification.

Calcific aortic valve disease (CAVD) accompanies inflammatory cell infiltration, fibrosis, and ultimately calcification of the valve leaflets. We previously demonstrated that dipeptidyl peptidase-4 (DPP-4) is responsible for the progression of aortic valvular calcification in CAVD animal models. As evogliptin, one of the DPP-4 inhibitors displays high specific accumulation in cardiac tissue, we here evaluated its therapeutic potency for attenuating valvular calcification in CAVD animal models. Evogliptin administration markedly reduced calcific deposition accompanied by a reduction in proinflammatory cytokine expression in endothelial nitric oxide synthase-deficient mice in vivo, and significantly ameliorated the mineralization of the primary human valvular interstitial cells (VICs), with a reduction in the mRNA expression of bone-associated and fibrosis-related genes in vitro. In addition, evogliptin ameliorated the rate of change in the transaortic peak velocity and mean pressure gradients in our rabbit model as assessed by echocardiography. Importantly, evogliptin administration in a rabbit model was found to suppress the effects of a high-cholesterol diet and of vitamin D2-driven fibrosis in association with a reduction in macrophage infiltration and calcific deposition in aortic valves. These results have indicated that evogliptin prohibits inflammatory cytokine expression, fibrosis, and calcification in a CAVD animal model, suggesting its potential as a selective therapeutic agent for the inhibition of valvular calcification during CAVD progression.

Vascular endothelial growth factor on Runt-related transcript factor-2 in aortic valve cells.

Calcific aortic valve disease is associated with ageing and high mortality. However, no effective pharmacological treatment has been developed. Vascular endothelial growth factor (VEGF) and its receptor are overexpressed in the calcified aortic valve tissue. However, the role of VEGF in calcific aortic valve disease pathogenesis and its underlying mechanisms remain unclear. Runt-related transcription factor 2 expression and calcium-related signalling were investigated in porcine valvular interstitial cells with or without human VEGF-A recombinant protein (VEGF165 , 1-100ng/mL) treatment and/or calmodulin-dependent kinase II (CaMKII) inhibitor (KN93, 10µmol/L) and inositol triphosphate receptor inhibitor (2-aminoethyldiphenyl borate, 30µmol/L) for 5days. VEGF165 -treated cells had higher Runt-related transcription factor 2 expression and CaMKII/ adenosine 3',5'-monophosphate response element-binding protein (CREB) signalling activation than did control cells. KN93 reduced Runt-related transcription factor 2 expression and CREB phosphorylation in VEGF165 -treated cells. The 2-aminoethyldiphenyl borate also reduced Runt-related transcription factor 2 expression in VICs treated with VEGF165 . VEGF upregulated Runt-related transcription factor 2 expression in VICs by activating the IP3R/CaMKII/CREB signalling pathway.

Calcium Signaling Regulates Valvular Interstitial Cell Alignment and Myofibroblast Activation in Fast‐Relaxing Boronate Hydrogels

Front Cover: Valvular interstitial cell (VIC) alignment and myofibroblast activation are investigated using a Poly(ethylene glycol) (PEG) based fastrelaxing boronate hydrogel with tunable viscoelasticity. VIC spreading and alignment correlates with higher extents of stress relaxation, signaling through transient receptor potential vanilloid 4 (TRPV4) calcium channel. A potential crosstalk between viscoelasticity, calcium signaling, and mechanotransduction is proposed, which may provide insights for treatment of valvular tissue fibrosis. This cover art depicts the different cellular behaviors when VICs are encapsulated in 3D hydrogel systems with different viscoelasticity. This is reported by Hao Ma, Laura J. Macdougall, Andrea GonzalezRodriguez, Megan E. Schroeder, Dilara Batan, Robert M. Weiss, and Kristi S. Anseth in article 2000268.

Acetylsalicylic acid prevents the pro-calcific differentiation of valvular interstitial cells through the production of anti-inflammatory lipoxins

Background and Aims: To date, no pharmacological therapy has proven to slow down the progression of calcific aortic valve disease (CAVD). Our specific aim is to study the effects of acetylsalicylic acid (ASA) and ASA-triggered lipoxins (ATL) on valvular interstitial cells (VIC) calcification.

Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve.

Calcific aortic valve disease (CAVD), a degenerative disease characterized by inflammation, fibrosis and calcification, is accelerated in diabetes. Hyperglycemia contributes to this process by mechanisms that still need to be uncovered. We have recently developed a 3D model of the human aortic valve based on gelatin methacrylate and revealed that high glucose (HG) induced osteogenic molecules and increased calcium deposits in a pro-osteogenic environment. To further understand the events leading to calcification in diabetic conditions in CAVD, we analyzed here the inflammatory and remodeling mechanisms induced by HG in our 3D model. We exposed valvular endothelial cells (VEC) and interstitial cells (VIC) to normal glucose (NG) or HG for 7 and 14 days, then we isolated and separated the cells by anti-CD31 immunomagnetic beads. The changes induced by HG in the 3D model were investigated by real-time polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. Our results showed that HG induced expression of different cytokines, cell adhesion molecules and matrix metalloproteinases in VEC and VIC. In addition, protein kinase C was increased in VEC and VIC, indicating molecular mechanisms associated with HG induced inflammation and remodeling in both valvular cells. These findings may indicate new biomarkers and targets for therapy in diabetes associated with CAVD.

Collagen networks within 3D PEG hydrogels support valvular interstitial cell matrix mineralization

Enzymatically degradable hydrogels were designed for the 3D culture of valvular interstitial cells (VICs), and through the incorporation of various functionalities, we aimed to investigate the role of the tissue microenvironment in promoting the osteogenic properties of VICs and matrix mineralization. Specifically, porcine VICs were encapsulated in a poly(ethylene glycol) hydrogel crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinker (KCGPQG↓IWGQCK) and formed via a thiol-ene photoclick reaction in the presence or absence of collagen type I to promote matrix mineralization. VIC-laden hydrogels were treated with osteogenic medium for up to 15 days, and the osteogenic response was characterized by the expression of RUNX2 as an early marker of an osteoblast-like phenotype, osteocalcin (OCN) as a marker of a mature osteoblast-like phenotype, and vimentin (VIM) as a marker of the fibroblast phenotype. In addition, matrix mineralization was characterized histologically with Von Kossa stain for calcium phosphate. Osteogenic response was further characterized biochemically with calcium assays, and physically via optical density measurements. When the osteogenic medium was supplemented with calcium chloride, OCN expression was upregulated and mineralization was discernable at 12 days of culture. Finally, this platform was used to screen various drug therapeutics that were assessed for their efficacy in preventing mineralization using optical density as a higher throughput readout. Collectively, these results suggest that matrix composition has a key role in supporting mineralization deposition within diseased valve tissue.

The role of noncoding RNAs during aortic valve stenosis

Abstract Background Aortic valve stenosis (AVS) is the most common valve disease worldwide. Thought to be a purely degenerative disease, it is now clear that shear stress/endothelial dysfunction, lipid deposition and inflammation lead to calcification and stenosis of the valve. There is evidence, that extracellular vesicles (EVs) are actively involved in calcification processes. Practically all cells, including endothelial cells, can generate EVs, which can be shed into the blood stream and into the interstitial space. EVs contain lipids, proteins and nucleic acids, including noncoding RNAs (ncRNAs). EVs can be taken up by acceptor cells and their cargo, especially the ncRNA content, can change the phenotype of these cells. NcRNAs have been shown to have protective and damaging properties in AVS, which can lead to disease progression. EVs are actively involved in atherosclerosis and vascular calcification, but their role during AVS formation remains largely unknown. Purpose We hypothesize, that EV-derived ncRNAs play a crucial role during calcification of the aortic valve through regulation of endothelial to mesenchymal transition (EndMT) and calcification of valvular interstitial cells. Methods and results In initial screening experiments, we investigated ncRNA (micro RNA, miRNA and long noncoding RNA, lncRNA) content in aortic valve tissue from explanted human aortic valves from patients undergoing surgical aortic valve replacement. There is a differential expression of miRNAs and lncRNAs in aortic valve tissue from patients with AVS and patients without AVS. We could also show a differential packaging of ncRNAs into EVs generated from patient aortic valve tissues. Furthermore, ncRNA expression in aortic valve tissue is altered in a “wire-injury” mouse model of AVS. We can demonstrate in vitro that EVs and their content can be transferred from valvular endothelial cells (VECs) to valvular interstitial cells (VICs) and vice versa. Additionally, we have established an isolation method of VECs and VICs from human samples. To identify ncRNAs involved in EndMT, we are investigating the effect of laminar and pulsatile flow on the expression of ncRNAs in vitro. We can demonstrate that different flow patterns lead to a vast change in ncRNA expression in primary VECs. Downstream effects of identified ncRNAs are currently under investigation in our in vitro calcification and EndMT models. Transfection of VICs with miRNA mimics and inhibitors and lentiviral transduction of our identified targets lead to an altered calcification potential of valve cells. Conclusion The analysis of the cell type specific expression of ncRNAs and the intercellular communication via EVs will greatly help our understanding of the pathomechanisms leading to valvular calcification. Pathway analysis will generate new targets that could be used to develop therapeutics to ameliorate disease progression. EV-based miRNA mimics and inhibitors could be used to treat valvular calcification. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft, TRR259; Else-Kröner-Fresenius

New ex vivo calcification model for intact murine aortic valves

Abstract Introduction Calcific aortic valve disease (CAVD) is a common, progressive disease of the aortic valves for which no medical treatment exists and surgery is the only therapeutic solution. The discovery of novel pharmacological treatment for CAVD has been hampered due to the lack of suitable test-systems. Purpose We aimed to establish an ex vivo calcification model for intact mouse aortic valves. Methods We used the ex vivo flow model for mouse hearts (Miniature Tissue Culture System) to induce calcification with culture media supplemented with 1) β-glycerophosphate, ascorbic acid, and dexamethasone (OSM) or 2) inorganic phosphates (PI) and compared this with in vitro calcification of mouse valvular interstitial cells (mVICs). Results Ex vivo cultured aortic valve leaflets calcified in the presence of PI, but not OSM. RUNX was upregulated in both OSM and PI conditions, whereas alkaline phosphatase (ALP) and cyclooxygenase-2 (COX2) were differentially expressed. Apoptosis was not observed, together indicating that osteogenic calcification took place. Interestingly, both OSM and PI were able to induce calcification in vitro, revealing in vitro-ex vivo differences. In addition to the calcification in the aortic valves, endochondral calcification was present in the sinus near the hinge of the aortic valve in both PI and OSM conditions as evidenced by the expression of collagen II, aggrecan and ALP. Conclusions Together these data show that we have established an ex vivo calcification model for mouse aortic valves. Osteogenic calcification is induced in the aortic leaflets, whereas endochondral calcification is induced in the aortic sinus, reflecting the calcification found in human CAVD. Using this model, we can now study the initiation and progression of aortic valve calcification. Funding Acknowledgement Type of funding source: Other. Main funding source(s): Dutch Heart Foundation, grant number 2013T093. Grants received from GE Healthcare, Lantheus medical imaging, St Jude Medical, Medtronic, Boston Scientific, Biotronik, and Edwards Lifesciences

Tumor Necrosis Factor Alpha and Interleukin 1 Beta Suppress Myofibroblast Activation Via Nuclear Factor Kappa B Signaling in 3D-Cultured Mitral Valve Interstitial Cells

Mitral valve disease is a major cause of cardiovascular morbidity throughout the world. Many different mitral valve pathologies demonstrate a pronounced degree of fibrotic remodeling, often accompanied by an inflammatory state. Mitral valve fibrosis is mediated by valvular interstitial cells (VICs), which reside in the valve leaflets and show a tendency to differentiate into myofibroblast-like cells during disease conditions. In this study, we investigated the effects of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) on mitral VICs, since these pro-inflammatory cytokines have been shown to exert pleiotropic effects on various cell types in other fibrotic disorders. Using biomimetic three-dimensional culture systems, we demonstrated that TNF-α and IL-1β suppress myofibroblast differentiation in mitral VICs, as evidenced by gene and protein expression of alpha smooth muscle actin and smooth muscle 22 alpha. Addition of TNF-α and IL-1β also inhibited mitral VIC-mediated contraction of collagen gels. Furthermore, inhibition of NF-κB, which is downstream of TNF-α and IL-1β, reversed these effects. These results reveal targetable pathways that could enable the development of pharmaceutical treatments for alleviating fibrosis during mitral valve disease.

MiR-214 Attenuates Aortic Valve Calcification by Regulating Osteogenic Differentiation of Valvular Interstitial Cells

Calcific aortic valve disease (CAVD) is a common heart valve disease in aging populations, and aberrant osteogenic differentiation of valvular interstitial cells (VICs) plays a critical role in the pathogenesis of ectopic ossification of the aortic valve. miR-214 has been validated to be involved in the osteogenesis process. Here, we aim to investigate the role and mechanism of miR-214 in CAVD progression. miR-214 expression was significantly downregulated in CAVD aortic valve leaflets, accompanied by upregulation of osteogenic markers. Overexpression of miR-214 suppressed osteogenic differentiation of VICs, while silencing the expression of miR-214 promoted this function. miR-214 directly targeted ATF4 and Sp7 to modulate osteoblastic differentiation of VICs, which was proved by dual luciferase reporter assay and rescue experiment. miR-214 knockout rats exhibited higher mean transvalvular velocity and gradient. The expression of osteogenic markers in aortic valve leaflets of miR-214 knockout rats was upregulated compared to that of the wild-type group. Taken together, our study showed that miR-214 inhibited aortic valve calcification via regulating osteogenic differentiation of VICs by directly targeting ATF4 and Sp7, indicating that miR-214 may act as a profound candidate of targeting therapy for CAVD.

Aldo-keto reductase family 1 member B induces aortic valve calcification by activating hippo signaling in valvular interstitial cells

AimsCalcific aortic valve disease (CAVD) is a primary cause of cardiovascular mortality; however, its mechanisms are unknown. Currently, no effective pharmacotherapy is available for CAVD. Aldo-keto reductase family 1 member B (Akr1B1) has been identified as a potential therapeutic target for valve interstitial cell calcification. Herein, we hypothesized that inhibition of Akr1B1 can attenuate aortic valve calcification. Methods and resultsNormal and degenerative tricuspid calcific valves from human samples were analyzed by immunoblotting and immunohistochemistry. The results showed significant upregulation of Akr1B1 in CAVD leaflets. Akr1B1 inhibition attenuated calcification of aortic valve interstitial cells in osteogenic medium. In contrast, overexpression of Akr1B1 aggravated calcification in osteogenic medium. Mechanistically, using RNA sequencing (RNAseq), we revealed that Hippo-YAP signaling functions downstream of Akr1B1. Furthermore, we established that the protein level of the Hippo-YAP signaling effector active-YAP had a positive correlation with Akr1B1. Suppression of YAP reversed Akr1B1 overexpression-induced Runx2 upregulation. Moreover, YAP activated the Runx2 promoter through TEAD1 in a manner mediated by ChIP and luciferase reporter systems. Animal experiments showed that the Akr1B1 inhibitor epalrestat attenuated aortic valve calcification induced by a Western diet in LDLR−/− mice. ConclusionThis study demonstrates that inhibition of Akr1B1 can attenuate the degree of calcification both in vitro and in vivo. The Akr1B1 inhibitor epalrestat may be a potential treatment option for CAVD.

Aβ40 Promotes the Osteoblastic Differentiation of Aortic Valve Interstitial Cells through the RAGE Pathway.

Amyloid beta (Aβ) peptide 40 enhances the activation of receptor for advanced glycation end products (RAGE) in immune-inflammatory diseases. RAGE exhibits several effects in the setting of numerous cardiovascular events. We hypothesized that the Aβ40/RAGE pathway is involved in the osteoblastic differentiation of the valvular interstitial cell (VIC) phenotype, and RAGE knockout intervention could reduce the calcification of aortic valve interstitial cells (AVICs) by inhibiting the extracellular-regulated kinase1/2 (ERK1/2)/nuclear factor kappa-B (NF-κB) signaling pathway. To test this hypothesis, the activation of Aβ40/RAGE pathway in human calcific AVs was evaluated with immunohistochemical staining. Cultured calcific VIC models were used in vitro. The VICs were stimulated using Aβ40, with or without RAGE small interfering ribonucleic acid (siRNA), and ERK1/2 and NF-κB inhibitors for analysis. Our data revealed that Aβ40 induced the ERK1/2/NF-κB signaling pathway and osteoblastic differentiation of AVICs via the RAGE pathway in vitro.

Transforming growth factor-β1 promotes fibrosis but attenuates calcification of valvular tissue applied as a three-dimensional calcific aortic valve disease model

Calcific aortic valve disease (CAVD) is characterized by valvular fibrosis and calcification and driven by differentiating valvular interstitial cells (VICs). Expression data from patient biopsies suggest that transforming growth factor (TGF)-β1 is implicated in CAVD pathogenesis. However, CAVD models using isolated VICs failed to deliver clear evidence on the role of TGF-β1. Thus, employing cultures of aortic valve leaflets, we investigated effects of TGF-β1 in a tissue-based three-dimensional (3-D) CAVD model. We found that TGF-β1 induced phosphorylation of Mothers against decapentaplegic homolog (SMAD) 3 and expression of SMAD7, indicating effective downstream signal transduction in valvular tissue. Thus, TGF-β1 increased VIC contents of rough endoplasmic reticulum, Golgi, and secretory vesicles as well as tissue levels of RNA and protein. In addition, TGF-β1 raised expression of proliferation marker cyclin D1, attenuated VIC apoptosis, and upregulated VIC density. Moreover, TGF-β1 intensified myofibroblastic VIC differentiation as evidenced by increased α-smooth muscle actin and collagen type I along with diminished vimentin expression. In contrast, TGF-β1 attenuated phosphorylation of SMAD1/5/8 and upregulation of β-catenin while inhibiting osteoblastic VIC differentiation as revealed by downregulation of osteocalcin expression, alkaline phosphatase activity, and extracellular matrix incorporation of hydroxyapatite. Collectively, these effects resulted in blocking of valvular tissue calcification and associated disintegration of collagen fibers. Instead, TGF-β1 induced development of fibrosis. Overall, in a tissue-based 3-D CAVD model, TGF-β1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of VICs, thus triggering dominant fibrosis. Simultaneously, by inhibiting SMAD1/5/8 activation and canonical Wnt/β-catenin signaling, TGF-β1 attenuates osteoblastic VIC differentiation, thus blocking valvular tissue calcification. These findings question a general phase-independent CAVD-promoting role of TGF-β1.NEW & NOTEWORTHY Employing aortic valve leaflets as a tissue-based three-dimensional disease model, our study investigates the role of transforming growth factor (TGF)-β1 in calcific aortic valve disease pathogenesis. We find that, by activating Mothers against decapentaplegic homolog 3, TGF-β1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of valvular interstitial cells, thus triggering dominant fibrosis. Simultaneously, by inhibiting activation of Mothers against decapentaplegic homolog 1/5/8 and canonical Wnt/β-catenin signaling, TGF-β1 attenuates apoptosis and osteoblastic differentiation of valvular interstitial cells, thus blocking valvular tissue calcification. These findings question a general phase-independent calcific aortic valve disease-promoting role of TGF-β1.

Collagen Networks within 3D PEG Hydrogels Support Valvular Interstitial Cell Matrix Mineralization

Enzymatically degradable hydrogels were designed for the 3D culture of valvular interstitial cells (VICs), and through the incorporation of various functionalities, we aimed to investigate the role of the tissue microenvironment in promoting the osteogenic properties of VICs and matrix mineralization. Specifically, VICs were encapsulated in a poly(ethylene glycol) hydrogel crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinker (KCGPQG↓IWGQCK) and formed via a thiol-ene photoclick reaction in the presence or absence of collagen type I to promote matrix mineralization. VIC-laden hydrogels were treated with osteogenic medium for up to 15 days, and the osteogenic response was characterized by the expression of genetic markers, RUNX2 as an early marker of an osteoblast-like phenotype, and OCN, as a marker of a mature osteoblast-like phenotype. In addition, matrix mineralization was characterized histologically with a Von Kossa stain for calcium phosphate. Osteogenic response was further characterized biochemically with calcium assays, and physically via optical density measurements. When the osteogenic medium was supplemented with calcium chloride, OCN gene expression and mineralization were discernable within 12 days of culture. Finally, this platform was used to screen various drug therapeutics that were assessed for their efficacy in preventing mineralization using optical density as a higher throughput read out. Collectively, these results suggest that matrix composition has a key role in supporting mineralization deposition within diseased valve tissue.

Myofibroblastic differentiation of rat valvular interstitial cells in culture

Familial genetic and genome wide studies associated FLNA, DCHS1, DZIP1 and TNS1 genes with mitral valve prolapse (MVP). These genes point to the cellular mechanical stress response as a unifying common pathway in MVP. The myofibroblastic differentiation of the valve interstitial cells (VICs) has been described as one of the main mechanisms associated with MVP. We recently developed a unique knock-in (KI) rat model expressing the Filamin A FlnA-P637Q mutation identified MVP patients. To determine the primary cell culture conditions of the mitral VICs to limit myofibroblastic differentiation with the objective to analyze in vitro the impact of the FlnA-P637Q mutation on VICs responses to mechanical stress. Mitral valves were dissected from 3 week-old WT and KI rats, then grown on collagen coated dishes in either 4.5 g/l or 1 g/l glucose DMEM supplemented with 10 or 2% Fetal Calf Serum (FCS). Growth rate and myofibroblastic differentiation of the cells were analyzed by immunofluorescence, western blot and qPCR experiments. Our preliminary results show that WT and KI cells grow with similar growth rates (t1/2= 24 h) in 4.5 g/l glucose/10% FCS DMEM. In addition, immunofluorescence and qPCR experiments show that both cell types exhibit significant smooth muscle actin (SMA-ACTA2) expression as soon as they are grown in culture (“Passage” P0). SMA expression further increases (× 1.5) when cells are trypsinized and amplified once (P1) or twice (P2). On the other hand, growing the cells in 1 g/l glucose/2% FCS resulted in a 45% decrease of SMA expression at P0 and P1. SMA expression finally reaches, at P2, the level of high glucose culture condition. Our preliminary studies enabled us to establish culture conditions that limit myofibroblastic differentiation of WT and FlnA-P637Q mitral VICs at early “passages”. The response of these “quiescent” cells to mechanical stress and the impact of FlnA-P637Q mutation are now under study.

Secreted Factors From Proinflammatory Macrophages Promote an Osteoblast-Like Phenotype in Valvular Interstitial Cells.

Resident valvular interstitial cells (VICs) activate to myofibroblasts during aortic valve stenosis progression, which further promotes fibrosis or even differentiate into osteoblast-like cells that can lead to calcification of valve tissue. Inflammation is a hallmark of aortic valve stenosis, so we aimed to determine proinflammatory cytokines secreted from M1 macrophages that give rise to a transient VIC phenotype that leads to calcification of valve tissue. Approach and Results: We designed hydrogel biomaterials as valve extracellular matrix mimics enabling the culture of VICs in either their quiescent fibroblast or activated myofibroblast phenotype in response to the local matrix stiffness. When VIC fibroblasts and myofibroblasts were treated with conditioned media from THP-1-derived M1 macrophages, we observed robust reduction of αSMA (alpha smooth muscle actin) expression, reduced stress fiber formation, and increased proliferation, suggesting a potent antifibrotic effect. We further identified TNF (tumor necrosis factor)-α and IL (interleukin)-1β as 2 cytokines in M1 media that cause the observed antifibrotic effect. After 7 days of culture in M1 conditioned media, VICs began differentiating into osteoblast-like cells, as measured by increased expression of RUNX2 (runt-related transcription factor 2) and osteopontin. We also identified and validated IL-6 as a critical mediator of the observed pro-osteogenic effect. Proinflammatory cytokines in M1 conditioned media inhibit myofibroblast activation in VICs (eg, TNF-α and IL-1β) and promote their osteogenic differentiation (eg, IL-6). Together, our work suggests inflammatory M1 macrophages may drive a myofibroblast-to-osteogenic intermediate VIC phenotype, which may mediate the switch from fibrosis to calcification during aortic valve stenosis progression.