Heart Valve Interstitial Cells Research Articles

Directed Differentiation of Human Induced Pluripotent Stem Cells to Heart Valve Cells.

A main obstacle in current valvular heart disease research is the lack of high-quality homogeneous functional heart valve cells. Human induced pluripotent stem cells (hiPSCs)-derived heart valve cells may help with this dilemma. However, there are no well-established protocols to induce hiPSCs to differentiate into functional heart valve cells, and the networks that mediate the differentiation have not been fully elucidated. To generate heart valve cells from hiPSCs, we sequentially activated the Wnt, BMP4, VEGF (vascular endothelial growth factor), and NFATc1 signaling pathways using CHIR-99021, BMP4, VEGF-165, and forskolin, respectively. The transcriptional and functional similarity of hiPSC-derived heart valve cells compared with primary heart valve cells were characterized. Longitudinal single-cell RNA sequencing was used to uncover the trajectory, switch genes, pathways, and transcription factors of the differentiation. An efficient protocol was developed to induce hiPSCs to differentiate into functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells. After 6-day differentiation and CD144 magnetic bead sorting, ≈70% CD144+ cells and 30% CD144- cells were obtained. On the basis of single-cell RNA sequencing data, the CD144+ cells and CD144- cells were found to be highly similar to primary heart valve endothelial cells and primary heart valve interstitial cells in gene expression profile. Furthermore, CD144+ cells had the typical function of primary heart valve endothelial cells, including tube formation, uptake of low-density lipoprotein, generation of endothelial nitric oxide synthase, and response to shear stress. Meanwhile, CD144- cells could secret collagen and matrix metalloproteinases, and differentiate into osteogenic or adipogenic lineages like primary heart valve interstitial cells. Therefore, we identified CD144+ cells and CD144- cells as hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells, respectively. Using single-cell RNA sequencing analysis, we demonstrated that the trajectory of heart valve cell differentiation was consistent with embryonic valve development. We identified the main switch genes (NOTCH1, HEY1, and MEF2C), signaling pathways (TGF-β, Wnt, and NOTCH), and transcription factors (MSX1, SP5, and MECOM) that mediated the differentiation. Finally, we found that hiPSC-derived valve interstitial-like cells might derive from hiPSC-derived valve endothelial-like cells undergoing endocardial-mesenchymal transition. In summary, this is the first study to report an efficient strategy to generate functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells from hiPSCs, as well as to elucidate the differentiation trajectory and transcriptional dynamics of hiPSCs differentiated into heart valve cells.

Clinical and Experimental Study of High Mobility Group Box-2 and Valvular Calcification in Elderly Patients with Degenerative Heart Valve Disease

Introduction: The aim of this study was to investigate the relationship between the high mobility group box-2 (HMGB2) and valve calcification in senile degenerative heart valve disease (SDHVD). Methods: According to the echocardiographic results, patients with calcified heart valves were used as the experimental group and patients without calcified heart valves were used as the control group; blood was drawn for testing, and serum levels of HMGB2 were measured by an enzyme-linked immunosorbent assay. Human heart valve interstitial cells (hVICs) cultured in vitro were randomly divided into two groups. The calcification group was cultured with a medium containing calcification induction solution and cells were induced on days 1, 3, and 5, and the control group was cultured with a standard medium. Expression of bone morphogenetic protein 4 (BMP-4) and HMGB2 in both groups was detected by Western blot. RT-PCR was performed to detect the expression of the HMGB2 gene during calcification. The hVICs were cultured in vitro for 4 days with different concentrations of exogenous HMGB2 (0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 2 μg/mL), while the control group was cultured with a standard medium and the expression of BMP-4 and NF-κB P65 was detected by Western blot. Results: The serum level of HMGB2 was 7.90 (5.92, 12.39) μg/L, higher than that of 7.06 (5.06, 9.73) μg/L in the valve calcification group in elderly patients with degenerative valve disease (p = 0.005); the differences were statistically significant. In in vitro experiments, the cellular calcification protein BMP-4 and the HMGB2 protein were higher in the calcification group compared to the control group (p < 0.05). Exogenous stimulation of hVICs with HMGB2 was able to upregulate the expression of BMP-4 and NF-κB P65 (p < 0.05). Conclusions: HMGB2 is correlated with valvular calcification in senile degenerative heart valve disease. The HMGB2 protein may promote the process of SDHVD valve calcification by activating the NF-κB pathway and upregulating the expression of BMP-4.

Hyaluronic acid regulates heart valve interstitial cell contraction in fibrin-based scaffolds

Heart valve disease is associated with high morbidity and mortality worldwide resulting in hundreds of thousands of heart valve replacements each year. Tissue engineered heart valves (TEHVs) have the potential to overcome the major limitations of traditional replacement valves; however, leaflet retraction has led to the failure of TEHVs in preclinical studies. As native unmodified hyaluronic acid (HA) is known to promote healthy tissue development in native heart valves, we hypothesize that adding unmodified HA to fibrin-based scaffolds common to tissue engineering will reduce retraction by increasing cell-scaffold interactions and density of the scaffolds. Using a custom high-throughput culture system, we found that incorporating HA into millimeter-scale fibrin-based cell-populated scaffolds increases initial fiber diameter and cell-scaffold interactions, causing a cascade of mechanical, morphological, and cellular responses. These changes lead to higher levels of scaffold compaction and stiffness, increased cell alignment, and less bundling of fibrin fibers by the cells during culture. These effects significantly reduce scaffold retraction and total contractile force each by around 25%. These findings increase our understanding of how HA alters tissue remodeling and could inform the design of the next generation of tissue engineered heart valves to help reduce retraction. Statement of significanceTissue engineered heart valves (TEHVs) have the potential to overcome the major limitations of traditional replacement valves; however, leaflet retraction induced by excessive myofibroblast activation has led to failure in preclinical studies. Developing valves are rich in hyaluronic acid (HA), which helps maintain a physiological environment for tissue remodeling without retraction. We hypothesized that adding unmodified HA to TEHVs would reduce retraction by increasing cell-scaffold interactions and density of the scaffolds. Using a high-throughput tissue culture platform, we demonstrate that HA incorporation into a fibrin-based scaffold can significantly reduce tissue retraction and total contractile force by increasing fiber bundling and altering cell–mediated matrix remodeling, therefore increasing gel density and stiffness. These finding increase our knowledge of native HA's effects within the extracellular matrix, and provide a new tool for TEHV design.

Protective Effects of Fucoxanthin on Hydrogen Peroxide-Induced Calcification of Heart Valve Interstitial Cells.

Cardiovascular diseases such as atherosclerosis and aortic valve sclerosis involve inflammatory reactions triggered by various stimuli, causing increased oxidative stress. This increased oxidative stress causes damage to the heart cells, with subsequent cell apoptosis or calcification. Currently, heart valve damage or heart valve diseases are treated by drugs or surgery. Natural antioxidant products are being investigated in related research, such as fucoxanthin (Fx), which is a marine carotenoid extracted from seaweed, with strong antioxidant, anti-inflammatory, and anti-tumor properties. This study aimed to explore the protective effect of Fx on heart valves under high oxidative stress, as well as the underlying mechanism of action. Rat heart valve interstitial cells under H2O2-induced oxidative stress were treated with Fx. Fx improved cell survival and reduced oxidative stress-induced DNA damage, which was assessed by cell viability analysis and staining with propidium iodide. Alizarin Red-S analysis indicated that Fx has a protective effect against calcification. Furthermore, Western blotting revealed that Fx abrogates oxidative stress-induced apoptosis via reducing the expression of apoptosis-related proteins as well as modulate Akt/ERK-related protein expression. Notably, in vivo experiments using 26 dogs treated with 60 mg/kg of Fx in combination with medical treatment for 0.5 to 2 years showed significant recovery in their echocardiographic parameters. Collectively, these in vitro and in vivo results highlight the potential of Fx to protect heart valve cells from high oxidative stress-induced damage.

KPT-330 Prevents Aortic Valve Calcification via a Novel C/EBPβ Signaling Pathway.

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Smooth Muscle α-Actin Expression in Mitral Valve Interstitial Cells is Important for Mediating Extracellular Matrix Remodeling.

Background: Mitral valve prolapse (MVP) affects 3–6% of the total population including those with connective tissue disorders. Treatment is limited, and patients commonly require surgery which can be impermanent and insuperable. Abnormal prolapse of mitral valve leaflets into the left atria is caused by disturbances to the composition and organization of the extracellular matrix (ECM), that weaken biomechanics. This process, known as myxomatous degeneration is characterized by an abnormal accumulation of proteoglycans, in addition to collagen fiber disruption and elastic fiber fragmentation. The underlying mechanisms that promote myxomatous degeneration to the point of biomechanical failure are unknown, but previous histological studies of end-stage diseased tissue have reported abnormal α-smooth muscle actin (SMA) in a subset of heart valve interstitial cells (VICs); however, the contribution of these abnormal cells to MVP pathogenesis has not been extensively examined. Methods: In vivo and in vitro approaches were used. Mice harboring a Fbn1C1039G mutation mimic human Marfan Syndrome and develop MVP. Using these mice, temporal and spatial changes in SMA expression relative to myxomatous degeneration were examined using histological techniques. In parallel in vitro experiments, SMA expression was downregulated in primary porcine mitral VICs directly using siRNA, and indirectly using the actin depolymerizing agent Latrunculin A. In addition, the regulation of SMA in VICs by mechanical stiffness was explored relative to ECM remodeling. Results: We show, in mitral valves from Fbn1C1039G/+ mice, that abnormal increases in SMA expression in VICs are evident during early postnatal stages of disease, prior to significant myxomatous degeneration as indicated at later stages by increased proteoglycans and collagen type I (Col1a1). Furthermore, abnormal SMA expression continues to increase during the course of pathogenesis and is localized to the mid belly region of the mitral valve leaflets from 10 weeks. Using an in vitro approach, we demonstrate that reduced SMA function by direct siRNA or indirect Latrunculin A treatment attenuates proteoglycan and Col1a1 expression in porcine mitral VICs. While upstream, we provide insights to show that SMA is regulated by mechanical tension in VICs to promote changes in ECM homeostasis. Conclusions: Together, our data show that in VICs, SMA, an actin binding protein, is important for mediating ECM remodeling associated with phenotypes observed in myxomatous degeneration, and its expression is regulated by mechanical tension. These novel insights could inform the development of future non-surgical therapeutics to halt the progression of mitral valve degeneration thereby avoiding end-stage prolapse.

A Multi-Scale Modeling Approach to Determine 3D Heart Valve Interstitial Cell Biophysical Behavior in a Hydrogel Environment

A Multi-Scale Modeling Approach to Determine 3D Heart Valve Interstitial Cell Biophysical Behavior in a Hydrogel Environment

Human interstitial cellular model in therapeutics of heart valve calcification.

Calcific aortic valve disease is a common, severe heart condition that is currentlywith no proven, effective drug treatment and requires a surgical valve replacement or an entire heart explanation. Thus, developing novel, targeted therapeutic approaches becomes a major goal for cardiovascular disease research. To achieve this goal, isolated heart valve interstitial cells could be an advanced model to explore molecular mechanisms and measure drug efficacy. Based on this progress, molecular mechanisms that harbor components of inflammation and fibrosis coupled with proteins, for example, BMP-2, TLRs, RANKL, Osteoprotegerin, have been proposed. Small molecules or antibodies targeting these proteins have shown promising efficacy for either reversing or slowing down calcification development in vitro. In this review, we summarize these potential therapeutics with some highlights of interstitial cellular models.

Hypoxia-mediated regulation of the secretory properties of mitral valve interstitial cells.

The sophisticated function of the mitral valve depends to a large extent on its extracellular matrix (ECM) and specific cellular components. These are tightly regulated by a repertoire of mechanical stimuli and biological pathways. One potentially important stimulus is hypoxia. The purpose of this investigation is to determine the effect of hypoxia on the regulation of mitral valve interstitial cells (MVICs) with respect to the synthesis and secretion of extracellular matrix proteins. Hypoxia resulted in reduced production of total collagen and sulfated glycosaminoglycans (sGAG) in cultured porcine MVICs. Increased gene expression of matrix metalloproteinases-1 and -9 and their tissue inhibitors 1 and 2 was also observed after incubation under hypoxic conditions for up to 24 h. Hypoxia had no effect on MVIC viability, morphology, or phenotype. MVICs expressed hypoxia-inducible factor (HIF)-1α under hypoxia. Stimulating HIF-1α chemically caused a reduction in the amount of sGAG produced, similar to the effect observed under hypoxia. Human rheumatic valves had greater expression of HIF-1α compared with normal or myxomatous degenerated valves. In conclusion, hypoxia affects the production of certain ECM proteins and expression of matrix remodeling enzymes by MVICs. The effects of hypoxia appear to correlate with the induction of HIF-1α. This study highlights a potential role of hypoxia and HIF-1α in regulating the mitral valve, which could be important in health and disease.NEW & NOTEWORTHY This study demonstrates that hypoxia regulates extracellular matrix secretion and the remodeling potential of heart valve interstitial cells. Expression of hypoxia-induced factor-1α plays a role in these effects. These data highlight the potential role of hypoxia as a physiological mediator of the complex function of heart valve cells.

On the Functional Role of Valve Interstitial Cell Stress Fibers: A Continuum Modeling Approach.

The function of the heart valve interstitial cells (VICs) is intimately connected to heart valve tissue remodeling and repair, as well as the onset and progression of valvular pathological processes. There is yet only very limited knowledge and extant models for the complex three-dimensional VIC internal stress-bearing structures, the associated cell-level biomechanical behaviors, and how they change under varying activation levels. Importantly, VICs are known to exist and function within the highly dynamic valve tissue environment, including very high physiological loading rates. Yet we have no knowledge on how these factors affect VIC function. To this end, we extended our previous VIC computational continuum mechanics model (Sakamoto, et al., 2016, "On Intrinsic Stress Fiber Contractile Forces in Semilunar Heart Valve Interstitial Cells Using a Continuum Mixture Model," J. Mech. Behav. Biomed. Mater., 54(244-258)). to incorporate realistic stress-fiber geometries, force-length relations (Hill model for active contraction), explicit α-smooth muscle actin (α-SMA) and F-actin expression levels, and strain rate. Novel micro-indentation measurements were then performed using cytochalasin D (CytoD), variable KCl molar concentrations, both alone and with transforming growth factor β1 (TGF-β1) (which emulates certain valvular pathological processes) to explore how α-SMA and F-actin expression levels influenced stress fiber responses under quasi-static and physiological loading rates. Simulation results indicated that both F-actin and α-SMA contributed substantially to stress fiber force generation, with the highest activation state (90 mM KCL + TGF-β1) inducing the largest α-SMA levels and associated force generation. Validation was performed by comparisons to traction force microscopy studies, which showed very good agreement. Interestingly, only in the highest activation state was strain rate sensitivity observed, which was captured successfully in the simulations. These unique findings demonstrated that only VICs with high levels of αSMA expression exhibited significant viscoelastic effects. Implications of this study include greater insight into the functional role of α-SMA and F-actin in VIC stress fiber function, and the potential for strain rate-dependent effects in pathological states where high levels of α-SMA occur, which appear to be unique to the valvular cellular in vivo microenvironment.

Upregulated Notch1 expression promotes bone morphogenetic protein-2/4 expression of calcified human heart valve interstitial cells

To observe the protein expression of Notch1 in the cultured calcified human heart valve interstitial cells (hVICs) in vitro and related mechanisms. hVICs were divided into two groups: control hVICs were cultured in conventional media for 14 days and calcified hVICs were cultured with calcification inducers: β-glycerophosphate (500 μl), ascorbic acid (200 μl), dexamethasone(100 μl) for 7 days. The calcified hVICs were further divided into calcified hVICs group and inhibited calcified hVICs by adding specific Notch1 inhibitor DAPT (50 μmol/L(4 μl/hole))groups and cultured for another 7 days. Inflammatory response of all groups were induced by lipopolysaccharide (LPS) for 8 to 12 hours. Western blot was used to detect the protein expression of Notch1, phosphorylation nuclear transcription factor κB (p-NF-κB), bone morphogenetic protein-2/4(BMP-2/4). ELISA was applied to detect the content of BMP-2 secretion of the groups. Von Kossa staining was used to observe of cellular calcification. (1)Von Kossa staining is positive in the induced calcification group, the expression of Notch1, p-NF-κB, BMP-2 and BMP-4 is significantly higher in the induced calcification group than in the control group (all P<0.05). The expression of BMP-2 is significantly higher in the induced calcification group than in control group ((88.23±3.28) pg/ml vs. (25.41±3.68) pg/ml, P=0.02). (2) After treatment with DAPT, the calcification and the expression of Notch1, p-NF-κB, BMP-2 and BMP-4 were significantly decreased compared to calcification group (all P<0.05). The expression of BMP-2 is (26.74±4.62) pg/ml in the calcification inhibition group and (80.41±2.96) pg/ml in calcified control group (P=0.02). Upregulated Notch 1 expression promotes BMP-2/4 secretion in LPS stimulated hVICs, and contributes to osteogenic changes in hVICs. Inhibiting Notch1 can decrease the BMP-2/4 secretion and calcification in hVICs, which may serve as a novel therapeutic option for treating calcific valve disease.

On intrinsic stress fiber contractile forces in semilunar heart valve interstitial cells using a continuum mixture model

Heart valve interstitial cells (VICs) play a critical role in the maintenance and pathophysiology of heart valve tissues. Normally quiescent in the adult, VICs can become activated in periods of growth and disease. When activated, VICs exhibit increased levels of cytokines and extracellular matrix (ECM) synthesis, and upregulated expression and strong contraction of α-smooth muscle actin (α-SMA) fibers. However, it remains unknown how expression and contraction of the α-SMA fibers, which vary among different VIC types, contribute to the overall VIC mechanical responses, including the nucleus and cytoskeleton contributions. In the present study, we developed a novel solid-mixture model for VIC biomechanical behavior that incorporated 1) the underlying cytoskeletal network, 2) the oriented α-SMA stress fibers with passive elastic and active contractile responses, 3) a finite deformable elastic nucleus. We implemented the model in a full 3D finite element simulation of a VIC based on known geometry. Moreover, we examined the respective mechanical responses of aortic and pulmonary VICs (AVICs and PVICs, respectively), which are known to have different levels of α-SMA expression levels and contractile behaviors. To calibrate the model, we simulated the combined mechanical responses of VICs in both micropipette aspiration (MA) and atomic force microscopy (AFM) experiments. These two states were chosen as the VICs were under significantly different mechanical loading conditions and activation states, with the α-SMA fibers inactivated in the MA studies while fully activated in the AFM studies. We also used the AFM to study the mechanical property of the nucleus. Our model predicted that the substantial differences found in stiffening of the AVIC compared to the PVICs was due to a 9 to 16 times stronger intrinsic AVIC α-SMA stress fiber contractile force. Model validation was done by simulating a traction force microscopy experiment to estimate the forces the VICs exert on the underlying substrate, and found good agreement with reported traction force microscopy results. Further, estimated nuclear stiffness for both AVICs and PVICs were similar and comparable to the literature, and were both unaffected by VIC activation level. These results suggest substantial functional differences between AVICs and PVICs at the subcellular level. Moreover, this first VIC computational biomechanical model is but a first step in developing a comprehensive, integrated view of the VIC pathophysiology and interactions with the valve ECM micro-environment based on simulation technologies.

Dynamic Heterogeneity of the Heart Valve Interstitial Cell Population in Mitral Valve Health and Disease.

The heart valve interstitial cell (VIC) population is dynamic and thought to mediate lay down and maintenance of the tri-laminar extracellular matrix (ECM) structure within the developing and mature valve throughout life. Disturbances in the contribution and distribution of valve ECM components are detrimental to biomechanical function and associated with disease. This pathological process is associated with activation of resident VICs that in the absence of disease reside as quiescent cells. While these paradigms have been long standing, characterization of this abundant and ever-changing valve cell population is incomplete. Here we examine the expression pattern of Smooth muscle α-actin, Periostin, Twist1 and Vimentin in cultured VICs, heart valves from healthy embryonic, postnatal and adult mice, as well as mature valves from human patients and established mouse models of disease. We show that the VIC population is highly heterogeneous and phenotypes are dependent on age, species, location, and disease state. Furthermore, we identify phenotypic diversity across common models of mitral valve disease. These studies significantly contribute to characterizing the VIC population in health and disease and provide insights into the cellular dynamics that maintain valve structure in healthy adults and mediate pathologic remodeling in disease states.

Application and improvement of calcific heart valve interstitial cell culture method

Objective To compare the quality of cells obtained by two different methods:the modified two-stage collagenase digestion method versus the traditional enzyme digestion method.Methods Cells obtained by the modified two-stage collagenase digestion method were studied as the experiment group,and those obtained bv the traditional enzyme digestion method served as the control group.The time when the cells began to adhere and the quantity of adherent cells after 24 h were observed.Immunocytochemical staining and flow cytometry were performed to analyze the phenotype of calcific valvular interstitial cells.Results The quantity of adherent cells after 24 h in the experiment group was four times of that in the control group.The difference was statistically significant (P ＜ 0.05).Flow cytometry confirmed alive primary valve interstitial cells accounted for 94.39％ in the experimental group.Cell phenotype detection revealed 99.2％ were positive for vimentin,and 53.3％ were positive for α-smooth muscle actin (α-SMA).Conclusion In terms of the quantity of cells,cell viability and cell phenotype,the cells obtained by the modified two-stage collagenase digestion method are superior to those obtained by traditional enzyme digestion and can be used in vitro experiments. Key words: Calcified; Valve interstitial cells; Cell culture

Dilated Cardiomyopathy in a Patient with a Primary Hepatic Neuroendocrine Carcinoma: A Case Report and Literature Review

Introduction: Primary Hepatic Neuroendocrine Carcinoma (PHNEC) has an indolent natural history resulting in devastating clinical syndromes. PHNEC (CHD) has been reported in up to 70% of cases of carcinoid syndrome. Most common clinical presentations of CHD include right-sided valve lesions. We present a case of PHNEC with dilated cardiomyopathy and severe left ventricular dysfunction. To our knowledge, this association has never been reported. Case Report: A 56-year-old male presented for 2-3 weeks of dyspnea and abdominal discomfort. 2-D echocardiogram revealed that hypokinesis of the left ventricular with four chambers enlargement, mitral regurgitation, pulmonary hypertension, and tricuspid regurgitation. Catheterizarion revealed three-vessel coronary artery disease, aortic insufficiency, and left ventricular dysfunction with EF of 15-20%. CT abdomen showed a mass in the liver. Biopsy reported a neuroendocrine carcinoma. Urine 5-hydoxyubdikeacetic acid (HIAA) was 23 mg/day (normal: 0-15 mg/day). An octreotide scan revealed that demonstrated uptake only in the liver. The tumor was diagnosed as a PHNEC. The patient was treated with octreotide acetate. His medications also included aspirin, lisinopril, lasix, spironolactone, and carvedilol. Subsequent 2-D echocardiogram after treatment for 6 months revealed LV function to EF of 40%. Discussion: CHD is associated with deposition of fibrous tissue on the endocardical surface. Development is thought to relate to the vasoactive substance secreted, such as serotonin (5-HT), by the tumor cell in the liver reaching the right heart. Study shows that serotonin (5-HT) may increase synthesis and upregulate tissue growth factor β, as well as stimulating collagen synthesis by heart valve interstitial cells. Most common deposition of carcinoid plaque has been found in tricuspid valve. Left-sided lesions of CHD only occur in up to 15% of all cases since serotonin is inactivated as it passes through lung parenchyma. Involvement of left side may be due to the presence of right-to-left shunt, bronchial carcinoid, or high level of circulating vasoactive substances. PHNEC has a unique specificity during its occurrence; the pathogenesis of CHD with PHNEC remains incompletely understood. Conclusion: Dilated cardiomyopathy with severe left ventricular dysfunction in a patient with a primary hepatic neuroendocrine carcinoma has been rarely reported. Further need of reporting similar cases is required, which may provide us a better understanding of the etiology and the best treatment/prevention options for PHNEC and CHD.

Calcific nodule morphogenesis by heart valve interstitial cells is strain dependent

Calcific aortic valve disease (CAVD) results in impaired function through the inability of valves to fully open and close, but the causes of this pathology are unknown. Stiffening of the aorta is associated with CAVD and results in exposing the aortic valves to greater mechanical strain. Transforming growth factor β1 (TGF-β1) is enriched in diseased valves and has been shown to combine with strain to synergistically alter aortic valve interstitial cell (AVIC) phenotypes. Therefore, we investigated the role of strain and TGF-β1 on the calcification of AVICs. Following TGF-β1 pretreatment, strain induced intact monolayers to aggregate and calcify. Using a wound assay, we confirmed that TGF-β1 increases tension in the monolayer in parallel with α-smooth muscle actin (αSMA) expression. Continual exposure to strain accelerates aggregates to calcify into mature nodules that contain a necrotic core surrounded by an apoptotic ring. This phenotype appears to be mediated by strain inhibition of AVIC migration after the initial formation of aggregates. To better interpret the extent to which externally applied strain physically impacts this process, we modified the classical Lamé solution, derived using principles from linear elasticity, to reveal strain magnification as a novel feature occurring in a mechanical environment that supports nodule formation. These results indicate that strain can impact multiple points of nodule formation: by modifying tension in the monolayer, remodeling cell contacts, migration, apoptosis, and mineralization. Therefore, strain-induced nodule formation provides new directions for developing strategies to address CAVD.

Fibroblast growth factor-2 promotes in vitro heart valve interstitial cell repair through the Akt1 pathway

Fibroblast growth factor-2 promotes in vitro heart valve interstitial cell repair. Fibroblast growth factor-2 acts through betaglycan which is known to bind both transforming growth factor-β and fibroblast growth factor-2 at different locations on the molecule. When fibroblast growth factor-2 binds to betaglycan, transforming growth factor-β binding to betaglycan is reduced, allowing for more transforming growth factor-β to be available to activate pSmad2/3 which then promotes repair. This study investigates another pathway through which fibroblast growth factor-2 regulates valve interstitial cell repair. We used an in vitro model of cell culture disruption. Confluent valve interstitial cell monolayers were disrupted, creating an experimental wound in the confluent monolayer, and incubated in treatments of exogenous fibroblast growth factor-2, anti-fibroblast growth factor receptor antibody, active Akt1, and Akt inhibitor. Valve interstitial cell monolayers were immunohistochemically stained and quantified for nuclear pSmad2/3 at the wound edge. The extent of closure was measured up to 96 h after disruption. Anti-fibroblast growth factor receptor antibody significantly increased both nuclear pSmad2/3 staining at the wound edge and wound closure compared to nontreated control. This increase was less than that seen when valve interstitial cells were treated with fibroblast growth factor-2 and combined treatments of fibroblast growth factor-2 and anti-fibroblast growth factor receptor antibody did not further increase nuclear pSmad2/3 staining compared to fibroblast growth factor-2 alone. This suggested that the regulation of wound closure by fibroblast growth factor-2 also involved pathways other than transforming growth factor-β/Smad signaling. Treatment with Akt1 significantly increased wound closure, while Akt inhibitor reduced closure as compared to nontreated valve interstitial cells. Fibroblast growth factor-2 and fibroblast growth factor-2 neutralizing antibody up-regulated and down-regulated phosphorylated Akt1 expression in valve interstitial cells, respectively. Fibroblast growth factor-2 promotes valve interstitial cell repair in two ways: the fibroblast growth factor-2/fibroblast growth factor-2 receptor interaction through the activation of Akt1 independent of the transforming growth factor-β/Smad2/3 signaling pathway and the previously described transforming growth factor-β/Smad signaling.

Aortic Valve Cyclic Stretch Causes Increased Remodeling Activity and Enhanced Serotonin Receptor Responsiveness

Increased serotonin (5-hydroxytryptamine [5HT]) receptor (5HTR) signaling has been associated with cardiac valvulopathy. Prior cell culture studies of 5HTR signaling in heart valve interstitial cells have provided mechanistic insights concerning only static conditions. We investigated the hypothesis that aortic valve biomechanics participate in the regulation of both 5HTR expression and interrelated extracellular matrix remodeling events. The effects of cyclic stretch on aortic valve 5HTR, expression, signaling, and extracellular matrix remodeling were investigated using a tensile stretch bioreactor in studies which also compared the effects of adding 5HT and (or) the 5HT-transporter inhibitor, fluoxetine. Cyclic stretch alone increased both proliferation and collagen in porcine aortic valve cusp samples. However, with cyclic stretch, unlike static conditions, 5HT plus fluoxetine caused the greatest increase in proliferation (p<0.0001), and also caused significant increases in collagen (p<0.0001) and glycosaminoglycans (p<0.0001). The DNA microarray data demonstrated upregulation of 5HTR2A and 5HTR2B (>4.5-fold) for cyclic stretch versus static (p<0.001), while expression of the 5HT transporter was not changed significantly. Extracellular matrix genes (eg, collagen types I, II, III, and proteoglycans) were also upregulated by cyclic stretch. Porcine aortic valve cusp samples subjected to cyclic stretch upregulate 5HTR2A and 2B, and also initiate remodeling activity characterized by increased proliferation and collagen production. Importantly, enhanced 5HTR responsiveness due to increased 5HTR2A and 2B expression results in a significantly greater response in remodeling endpoints (proliferation, collagen, and GAG production) to 5HT in the presence of 5HT transporter blockade.

Cell density regulates in vitro activation of heart valve interstitial cells

Valve interstitial cells, the most prominent cell type in the heart valve, are activated and express α-smooth muscle actin in valve repair and in diseased valves. We hypothesize that cell density, time in culture, and the establishment of cell-cell contacts may be involved in regulating valve interstitial cell activation in vitro. To study cell density, valve interstitial cells were plated at passages 3 to 5, at a density of 17,000 cells/22 × 22 mm(2) coverslip, and grown for 1, 2, 4, 7, and 10 days. Valve interstitial cells were stained for α-smooth muscle actin and viewed under confocal microscopy to characterize the intensity of staining. To study time in culture, valve interstitial cells were plated at a 10-fold higher density to achieve similar growth densities over a shorter time period compared with valve interstitial cells plated at low density. α-Smooth muscle actin staining was compared at the same time points between those plated at high and low densities. To confirm valve interstitial cell activation as indicated by α-smooth muscle actin staining, valve interstitial cells were stained for cofilin at days 2, 5, 8, and 14 days postplating. To study the association of transforming growth factor β with valve interstitial cell activation with respect to cell density, valve interstitial cells were stained for α-smooth muscle actin and transforming growth factor β at 2, 4, 6, and 8 days postplating. To study the activation of the transforming growth factor β signaling pathway, valve interstitial cells were stained for pSmad2/3 at days 2, 4, 6, 8, 10, and 12 days postplating. To study cell contacts and activation, subconfluent and confluent cultures of valve interstitial cells were stained for β-catenin, N-cadherin, and α-smooth muscle actin. Also, whole-cell lysates of subconfluent and confluent valve interstitial cell cultures were probed by Western blot analysis for phospho-β-catenin at Ser33/37/Thr41, which is the form of β-catenin targeted for proteosomal degradation. The percentage valve interstitial cells with high-intensity α-smooth muscle actin staining decreases significantly between days 1 and 4, and at confluency, most cells show absent or low-intensity staining, regardless of time in culture. Similar results are obtained with cofilin staining. Transforming growth factor β and nuclear pSmad2/3 staining in valve interstitial cells decreases concurrently with valve interstitial cell activation as cell density increases. Examining β-catenin and N-cadherin staining, single valve interstitial cells show no cell-cell contact with strong cytoplasmic staining, with some showing nuclear staining of β-catenin, while confluent monolayers show strong staining of fully established cell-cell contacts, weak cytoplasmic staining, and absent nuclear staining. The presence of cell-cell contacts is associated with a decreased α-smooth muscle actin. The level of phospho-β-catenin at Ser33/37/Thr41 is lower in confluent cultures compared with low-density subconfluent valve interstitial cell cultures. Cell-cell contacts may inhibit valve interstitial cell activation, while absence of cell-cell contacts may contribute to activation.

Parallel Functional Activity Profiling Reveals Valvulopathogens Are Potent 5-Hydroxytryptamine2BReceptor Agonists: Implications for Drug Safety Assessment

Drug-induced valvular heart disease (VHD) is a serious side effect of a few medications, including some that are on the market. Pharmacological studies of VHD-associated medications (e.g., fenfluramine, pergolide, methysergide, and cabergoline) have revealed that they and/or their metabolites are potent 5-hydroxytryptamine(2B) (5-HT(2B)) receptor agonists. We have shown that activation of 5-HT(2B) receptors on human heart valve interstitial cells in vitro induces a proliferative response reminiscent of the fibrosis that typifies VHD. To identify current or future drugs that might induce VHD, we screened approximately 2200 U.S. Food and Drug Administration (FDA)-approved or investigational medications to identify 5-HT(2B) receptor agonists, using calcium-based high-throughput screening. Of these 2200 compounds, 27 were 5-HT(2B) receptor agonists (hits); 14 of these had previously been identified as 5-HT(2B) receptor agonists, including seven bona fide valvulopathogens. Six of the hits (guanfacine, quinidine, xylometazoline, oxymetazoline, fenoldopam, and ropinirole) are approved medications. Twenty-three of the hits were then "functionally profiled" (i.e., assayed in parallel for 5-HT(2B) receptor agonism using multiple readouts to test for functional selectivity). In these assays, the known valvulopathogens were efficacious at concentrations as low as 30 nM, whereas the other compounds were less so. Hierarchical clustering analysis of the pEC(50) data revealed that ropinirole (which is not associated with valvulopathy) was clearly segregated from known valvulopathogens. Taken together, our data demonstrate that patterns of 5-HT(2B) receptor functional selectivity might be useful for identifying compounds likely to induce valvular heart disease.