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5th Biennial Conference on Heart Valve Biology and Tissue Engineering
- Conference date: 18-20 May 2012
- Location: Mykonos Island, Greece
- Volume number: 2012
- Published: 01 May 2012
1 - 20 of 86 results
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Regulation of Abnormal Atrioventricular Valve Development
Authors: Huseyin C. Yalcin, Sarah Ajaeb and Jonathan T. ButcherAbstractHemodynamic forces play an essential role in driving the morphogenesis of the embryonic heart and its valves. We previously found that genes that regulate heart morphogenesis like BMP2 and VEGFA, have hemodynamically specific patternings. However, how mechanical signals like wall shear stress (WSS) affect these expression levels to regulate heart morphogenesis is not fully understood. A detailed understanding of the hemodynamic environment and gene expression patterns inside normally and abnormally growing embryonic hearts would help inform the clinical progression of congenital heart defects and develop treatment strategies to restore these defects. We previously developed a system to quantify local fluid forces within growing embryonic heart where we combined imaging modalities with computational fluid dynamics (CFD), and quantified the hemodynamics within the AV canal and OFT in chicks. In the current study, we analyzed how altered hemodynamics drives changes in local gene expression and downstream morphogenesis of AV valves. Perturbed blood flow was created via either left atrial ligation (LAL) at HH24 or right atrial ligation (RAL) at HH25, to constrict blood flow on respective side of the heart. Hemodynamic environment in the AV canal for operated embryos were compared to control groups via doppler ultrasound at HH31. Gene expression levels were analyzed using RT-PCR. Heart morphology was analyzed at HH31 via micro-CT and histology. RT-PCR results at HH25 showed that LAL caused a drastic decrease in gene expression levels in LV myocardium (VEGFA expression 18.2±5.6%, BMP2 expression 21.7±1.4% compared to controls) and in AV cushions (VEGFA expression 9.8±8.1%, BMP2 expression 3.5±1.9 %), whereas in RV myocardium, expression levels were not affected significantly (VEGFA expression 77.5±5.8% , BMP2 expression 66.0±10.0%). Doppler ultrasound showed that neither peak blood velocities nor time averaged velocities in right and left AV canal are different for both LAL and RAL at HH31, suggesting WSS levels are not different between experimental groups (max shear stress 287dynes/cm2) at that stage. For LAL embryos, left AV valve area was smaller (48±4% vs. 67±6%, p<0.05, valve areas normalized to total left and right AV valve areas) and right valve area was bigger (52±4% vs. 33±6%, p<0.05) compared to controls, whereas in RAL embryos, opposite was true at HH31, right AV valve are was smaller (12±5 % vs. 33±6%, p<0.05) and left AV valve area was larger (88±5% vs %67±6, p<0.05). The ratio of right ventricle to left ventricle was significantly larger in LAL embryos compared to RAL embryos (1.1 vs 0.55). Our results show that morphological abnormalities follow the alterations in gene expression levels which are caused by changes in mechanical signals due to altered hemodynamics. We found that at later stages hemodynamics is restored to keep the blood circulation normal, with an altered morphology.
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Contribution of epicardially derived cells to the leaflets of the murine AV valves
Authors: John Burch, Maurice Van den Hoff, Marie Lockhart, Laura Briggs and Andy WesselsAbstractTo determine the spatiotemporal contribution of epicardially derived cells (EPDCs) to the leaflets of the developing atrioventricular (AV) valves in the murine heart we have used a mWt1/IRES/GFP-Cre mouse and traced the fate of EPDCs from embryonic day (ED)10 until birth. Migration of EPDCs into the mesenchyme of the AV cushions starts around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets that derive from the lateral atrioventricular cushions, i.e. the mural leaflet of the left AV valve and the lateral leaflet of the right AV valve. In these developing leaflets the EPDCs eventually largely replace the endocardially-derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the respective leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in heart development.
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Matrix-controlled In-growth Selectivity as a Principle of Heart Valve Regeneration
Authors: Neil Davies, Mona Bracher, Michelle Sun, Deon Bezuidenhout and Peter ZillaAbstractSpontaneous scaffold based tissue regeneration may be a promising alternative to in vitro tissue engineered heart valve prostheses. Selective promotion of ingrowth of desired cells versus suppression of potentially harmful cell invasion would be a key principle of such an approach. We have begun to demonstrate the utility of hydrogels engineered with specific adhesive and degradation peptide sites towards achieving this long-term goal. Polyethylene glycol hydrogels that through Michaels addition chemistry allow for both polymerization via enzymatically (matrix metalloproteinase (MMP)) degradable peptides and appendage of adhesive peptides were developed. Hydrogels were crosslinked with either a relatively enzymatically promiscuous peptide (MMP-pep) or sequences specifically degradable by either MMP-14 or MMP-9 (MMP-14pep, MMP-9pep). MMPs are of interest as there is evidence of cell specific expression. For example in vascular cells under physiological conditions MMP-9 is expressed at low levels except in macrophages. Adhesive peptides utilised were RGD, YIGSR and/or PHRSN. A range of primary vascular cells (smooth muscle cells (SMC), endothelial cells (EC), valvular intestitial cells (VIC) and fibroblasts (FB)) were cultured individually or as spheroids in 2 or 3-D. Migration and invasion were then assessed by time-lapse phase and confocal microscopy. ECs were shown to migrate significantly more rapidly on a surface containing YIGSR/RGD relative to other peptide combinations whilst the migration of SMC was unaffected. Hydrogels polymerized with MMP-14pep or MMP-9pep were shown to be highly preferentially cleaved by their relative enzymes. VICs, Fb and SMC primarily utilised MMPs to invade the hydrogels as demonstrated by inhibition of sprout formation by the MMP inhibitor, GM6001. VICs and FB invaded the gels in the following order, MMP-pep>>MMP-14>MMP-9. However, SMC showed a marked preference for invasion into MMP-14pep crosslinked hydrogels above that observed in their MMP-pep and MMP-9pep counterparts. Confocal microscopy analysis also showed a significantly more branched and interconnected pattern of invasion for SMC in MMP-14pep hydrogels. Thus we have begun to show that through engineering of extracellular matrix mimics with precise enzymatic and adhesive recognition sites it is possible to selectively influence specific cell types invasive behaviour suggesting that cell-specific ingrowth scaffolds may be achievable.
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Investigating the Contribution of Pathological Levels of Cyclic Strain on Vascular and Valvular Calcification
Authors: Zannatul Ferdous, Hanjoong Jo and Robert NeremAbstractAortic stenosis and atherosclerosis tend to coexist in most patients with cardiovascular disease; however, the causes and mechanisms of calcification are still not clearly understood. To understand the contributions of physiological (10%) and pathological (5%, 15%) levels of cyclic strain in calcification, we used a model system of tissue-engineered collagen gels containing human aortic smooth muscle cells (HASMC) and human aortic valvular interstitial cells (HAVIC), both isolated from non-calcific heart transplant tissues. The tissue engineered collagen gels were cultured in standard osteogenic media for three weeks in a custom designed bioreactor and all assessments were performed at the end of the culture period. The major finding of this study was that bone morphogenic protein (BMP) -2, -4 and transforming growth factor (TGF)-β1 mRNA expression significantly changed in response to the magnitude of strain in valvular cells, while the least expression was observed for the representative 10% physiological strain. On the other hand, these mRNA expressions in vascular cells responded to strain, but did not vary due to the magnitude (5% versus 10% versus 15%) of strain. When the BMP-2 and BMP-4 protein expression was detected using immunostaining, we observed that only valvular cells showed greater BMP-2 expression for 5% and 15% strain when compared to 10% strain within the same cell type. Our results suggest that cell mediated differences exist between vascular and valvular cells in their response to different levels of cyclic strain.
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The Role of Integrins on the Regulation of Contractility in Mitral Valve
Authors: Marzieh Zamani, Padmini Sarathchandra, Najma Latif, Magdi H. Yacoub and Adrian H. ChesterAbstractThe mechanical properties of heart valves are modulated by the contractile response of valve interstitial cells (VICs). It is unknown how the contractile responses of VICs are translated to the extra cellular matrix to alter the stiffness of the valve tissue. We have studied the signalling mechanisms of integrin subunits and specifically α2β1 integrin, a predominant collagen binding molecule, in mediating the contractility of porcine mitral valve tissue and cells. The expression of integrins was analysed by immunostaining of cells. The contractility of porcine mitral VICs in the presence and absence of a α2β1 integrin blocking antibody and the effect of different signalling inhibitors was analysed in a collagen gel. The effect of modulators of contractility was determined on mitral valve tissue incubated in organ baths which challenged with endothelin-1 (10-10-10-7M) following 24 hours incubation with α2β1 integrin blocking antibody. Immunohistochemistry confirmed positive staining for α2β1 integrin and the expression of α1, α2, α3 and β1 in the VICs. The α2β1 blocking antibody was able to significantly reduce the contraction of collagen gels from 63.6±7.8 to 23.4±4.3 % (p<0.001) of control (no cells in gel). PF573228, the inhibitor of focal adhesion kinase, caused a reduction of gel contraction by 30% of the control. The maximum contractile response of cusp tissue to ET-1 maintained under isometric conditions in tissue baths was significantly reduced from 9.5±5.0 to 3.0±2.5mN/g (p<0.001) wet weight by the presence of the α2β1 integrin blocking antibody. These results demonstrate a role for α2β1 integrin in linking the contractile response of valve cells to the extra cellular matrix and highlight further the complex interactions between the cells and the extra cellular matrix of the valve.
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Modeling Flow-Responsive Gene Regulation In Native Valve Tissue
Authors: Arpi Siyahian, Fariba Chalajour, Xiaoyuan Ma, Frank L. Hanley and R. Kirk RiemerAbstractA variety of approaches to heart valve tissue engineering are underway by many groups of investigators. We believe that a thorough understanding of the biophysical and biomolecular interactions that mediate normal leaflet homeostasis will inform improved approaches to the engineering of truly regenerative heart valve grafts. However, our understanding of the fundamental biology of normal valve tissue is still quite limited. This informational gap has led us to develop an ex vivo system for long-term valve culture in order to study the mechanobiology of native tissue. Using this system, we have cultured trileaflet rat heart valves with and without flow-induced valve cycling. We report here the preliminary findings about the effects of both ex vivo culture and of valve cycling on pulmonary leaflet cell gene expression following 7 days of flow culture. Expression of leaflet mRNA was queried using Affymetrix whole genome microarrays and the results analyzed using GeneSpring, EXPLAIN and MetaCore (GeneGO) software. Our analysis revealed 2147 genes whose expression was altered under flow conditions, and 1918 genes under static conditions. 1501 genes were common to both conditions. The effects of culture on gene expression affected multiple pathways, but the most active gene groups involved pathways for cytoskeletal remodeling, development, and cell adhesion. The genes involved in these pathways were altered in both culture conditions. Changes in cytoskeletal and ECM remodeling genes were more prominent in the static condition, while those involved in VEGF signaling were seen under flow condition. Our previously reported histological analyses of cultured rat pulmonary valve leaflet tissue revealed that flow promotes the maintenance of tissue integrity while the lack of flow leads to extracellular matrix remodeling and fibrinoid tissue formation. These gene expression analyses confirm the expected role of flow in maintaining leaflet tissue integrity as evidenced by its induction of VEGF signaling genes, while the lack of flow induces changes in genes involved in extracellular matrix and cytoskeletal remodeling.
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Inflammation Drives Endothelial-to-Mesenchymal Transition and Interstitial Calcification in 3D in Vitro Culture
Authors: Jennifer Richards, Emily Farrar, Gretchen Mahler and Jonathan ButcherAbstractCalcific aortic stenosis is a serious pathology that accounts for 43% of those suffering from heart valve disease. Valve disease initially presents with inflammation and endothelial dysfunction, which can lead to an endothelial-to-mesenchymal (EndMT) shift in the endothelial layer of the valve. It is not yet well understood how valve endothelial cells (VEC) regulate valve interstitial cells (VIC) in inflammatory or osteogenic environments. We here present a 3D culture system that models cellular responses and interactions when exposed to different environmental conditions. Porcine aortic VEC and/or VIC were cultured in mechanically constrained 3D type I collagen hydrogels for up to 14 days. Early disease studies introduced different dosages of TNFα to VEC on gel surfaces for up to 48 hours. EndMT was assessed via real time PCR and cell invasion within the matrix. Apoptosis and proliferation in VEC via TUNEL and anti-BrdU IHC staining were also evaluated in early inflammatory conditions. Later early-stage disease studies introduced osteogenic and inflammatory environments to 3D VIC/VEC gels through the addition of osteogenic differentiation factors into the culture media (OGM), or 30 ng/ml TNFα. Calcium deposition within the matrix was measured via Alizarin Red staining. Real time PCR was used to evaluate the expression of calcification-related genes such as osteocalcin and runx-2. Early inflammatory conditions as induced by addition of TNFα for 48 hours stimulated EndMT-like VEC activation, including matrix invasion and upregulation of αSMA and snail. Addition of TNFα increases VEC proliferation in a dose dependent manner. TNFα does not significantly increase apoptosis, even at the highest dosage levels. VIC gels cultured in OGM for 14 days significantly calcified and expressed osteocalcin and runx-2. Co-culture with VEC inhibited both matrix calcium deposition and osteoblastic differentiation. When 30 ng/ml TNFα was added to VIC 3D cultures, TNFa induced increased matrix calcium deposition in 14 days, which was then mitigated by co-culture with VEC. The inhibitory effect of VEC on VIC calcification in OGM was blocked when TNFα was introduced to the culture. These results demonstrate that inflammatory microenvironments induce calcification in valve interstitial cells. There is a protective role for the valve endothelium in VIC calcification, which could be inhibited by EndMT induced by the inflammatory conditions found in diseased valves. We found that apoptosis is not a driving factor in inflammatory activation of VEC or osteogenic differentiation of VIC. Targeting TNFα and EndMT signaling could be important therapeutic strategies. More generally, targeting VEC is a promising approach for early treatment of valve disease. This co-culture 3D system is a powerful tool to elucidate mechanisms of valve disease.
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Atypical expression and regulation of smooth muscle markers in human calcified valves
Authors: Najma Latif, Padmini Sarathchandra, Magdi H. Yacoub and Adrian H. ChesterAbstractCalcific aortic stenosis and atherosclerosis share many similar characteristics suggesting that similar mediators and pathogenic pathways are involved. In both conditions, the native cells are quiescent. However upon damage, both valve interstitial cells (VICs) and smooth muscle cells (SMCs) in the valve and the vascular wall respectively undergo remodelling and transformation. We hypothesise that the smooth muscle cell phenotype plays a role during the calcification process. 12 normal and 16 calcified valves were analysed for smooth muscle markers including smooth muscle α-actin, smooth muscle myosin, calponin, caldesmon, desmin, SM1 and SM2 by immunocytochemistry. The expression of myocardin-related transcription factors, (MRTFA/B) as key regulators of smooth muscle gene expression, was also evaluated. Normal human valves demonstrated the expression of smooth muscle markers localised to the base of the valve. Occasionally smooth muscle markers were seen in small groups of cells in the region from the base to the central region of the valve but never in the region from the central to the co-apting edge. Smooth muscle markers in all the calcified valves demonstrated an increased and aberrant expression. The expression of smooth muscle myosin, SM1, SM2, calponin and smooth muscle α-actin was found to be abundantly expressed around calcified nodules and distal to the nodules. The pattern of staining was frequently localised along the majority of the valve tissue as well as in isolated cells. Additionally, regions of the endothelium and endothelial cells lining the vessels were found to be positive for smooth muscle markers in 9/16 calcified valves. Normal valves did not demonstrate any expression of MRTFs. However, MRTF-A and MRTF-B were induced in calcified valves in a similar pattern to the expression of smooth muscle markers. They were also induced in the endothelium and the endothelial cells lining some vessels. Importantly, the expression of MRTFs was nuclear in both VICs and endothelial cells indicative of activation. TGFβ1 (10ng/ml) was able to induce and translocate the expression of MRTF-A to the nucleus in VICs within 4 hours in some isolates. Calcified valves harbour a greatly increased number of smooth muscle marker-positive VICs and endothelial cells. This aberrant expression in endothelial cells may emulate endothelial to mesenchymal transformation (EMT) during embyonic valvulogenesis. Concomittantly, calcified valves expressed MRTFs, key regulators of smooth muscle gene expression. The presence of smooth muscle cell markers and MRTFs in calcified valves suggest a role for this cell phenotype in the development of the calcification process.
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Characterization of Early Porcine Aortic Valve Disease
Authors: Krista L. Sider, Andrea V. Kwong, Bowen Wei and Craig A. SimmonsAbstractIn calcific aortic valve disease (CAVD), pathological differentiation of valve interstitial cells (VICs) and lesion formation occur focally in the fibrosa layer. We have shown that VIC pathological differentiation in vitro is sensitive to matrix stiffness. We therefore hypothesized that focal changes in the stiffness of the fibrosa with CAVD progression correlate with alterations in the extracellular matrix (ECM) and pathological cell phenotypes in vivo. Twenty-four male Yorkshire pigs were fed either a control or experimental diet supplemented with 12% lard and 1.5% cholesterol for 2 or 5 months (n = 6 per group). Effective moduli were measured in focal regions of the fibrosa (n = 295) and ventricularis (n = 142) layers of intact right coronary aortic valve leaflets by micropipette aspiration. Histology was conducted within radial center sections and selected focal test locations within the fibrosa from each leaflet to characterize local ECM and cell phenotypes. Early stage disease was induced by the experimental diet, as evidenced by more significant proteoglycan-rich lesions (~10 - 200 µm thick) onlayed on the fibrosa (p < 0.05, controlling for genetic variability). Interestingly, lipid deposition was seen in only 28% of regions with early lesions, and 15% of regions without early lesions. No osteoblastic or macrophage cells were present in control or experimental valves, with only slight myofibroblast presence at the base of some 5 month lesions. Chondrogenic Sox9-positive cells were observed extensively and were positively correlated with proteoglycan content (p < 0.0001). Regions within the fibrosa were significantly stiffer than those in the ventricularis in both control and experimental valves (p <0.001). Although there were no significant differences between the control and experimental overall tissue moduli at these early time points, modulus heterogeneity did increase with disease. Furthermore, lesions in the fibrosa had lower moduli than non-lesion fibrosa locations (p < 0.06). These soft lesions contained significantly more proteoglycan (p <0.001) and Sox9 expression (p = 0.014) than normal fibrosa tissue. In conclusion, ECM remodelling can occur in a porcine model of early CAVD in the absence of lipid deposition, inflammatory cells, osteoblasts, or myofibroblasts, but with significant proteoglycan-rich lesion and chondrogenic cell presence. Mechanically soft proteoglycan-rich lesions may support chondrogenic VIC differentiation, providing new insights into early CAVD pathogenesis.
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The role of Insulin-like Growth Factor-1 in Calcification of Human Aortic Valves
AbstractInsulin-like Growth Factor-1 (IGF-1) is a multi-functional protein that plays a role in survival, growth, proliferation and differentiation of various cell types. Particularly in the bone, IGF-1 is involved in tissue formation and growth, and the lack of expression of the gene leads to striking features such as short bone phenotype and low bone mineral density. In atherosclerosis, IGF system has been shown to stimulate vascular smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) synthesis contributing to maintenance of plaque stability. Consequently, a reduction of IGF-1 in atherosclerotic plaques has been suggested to increase SMC apoptosis and reduce ECM synthesis leading to weakening of the plaque. Despite extensive studies on its role in various organs and disease models, there is very little known about the effects of IGF-1 in human aortic valve (hAV) tissue that can suffer damaging effects such as cell death, differentiation and tissue mineralization, resulting into Aortic Valve Stenosis (AVS). The objective of the present study is therefore to investigate potential function of IGF-1 in this disease model. Human aortic valve samples were collected from patients undergoing aortic valve replacement or heart transplantation. The tissue expression of IGF-1 was analysed at mRNA level by quantitative Realtime PCR in calcified hAV (n=10-14) and non-calcified hAV (n=3-6), using the comparative Ct method. In vitro, the expression of endogenous IGF-1 was investigated on human valve interstitial cells (hVICs) upon administration of calcifying medium with or without transforming growth factor-beta1 (TGF-b1), a known pro-mineralization molecule (n=3). In addition to the expression of IGF-1, alkaline phosphatase (ALPL), TGF-b1 and osteopontin (OPN) were also examined at RNA level. IGF-1 expression was significantly up-regulated in the diseased valves compared to the controls (4.10 +/- 1.20 calcified vs. 1.10 +/- 0.76 control, p=0.0127). In vitro, there was an up-regulation of IGF-1 (p=0.06) and TGF-b1 expression (p=0.02) upon TGF-b1 stimulation. OPN expression was also highly up-regulated in calcifying media treated cultures, independent of TGF-b1 treatment. ALPL expression in contrast, was higher in normally growing cells compared to TGF-b1 and/or calcifying media treated cells (p=0.05). The significant elevation of IGF-1 in calcified valves in this small patient cohort strongly suggests that IGF-1 might be a key player in AVS disease. Whether the elevated IGF-1 expression is an effect or the cause of calcification is currently under investigation. Our preliminary data on TGF-b1 treatment of hVICs suggests that IGF-1 expression might be an effect of cells to counter apoptotic death conferred by TGF-b1 treatment. Further work on the detailed molecular mechanisms involved in IGF-1 action and its downstream signalling pathways is needed.
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Reversible Effects of Polyunsaturated Fatty Acids on Activation of Interstitial Cells From Porcine Aortic Valves
Authors: Wolfgang Witt, Anett Jannasch, Klaus Matschke and Thomas WaldowAbstractValvular interstitial cells (VICs) constitute the most prominent cell type in aortic valve cusps. VICs are a heterogeneous group of cells of multiple origins with mostly fibroblast properties. Like fibroblasts in general, VICs can acquire an activated state (myofibroblasts) upon exposure to several stimuli including mediators of inflammation, growth factors, mechanical stress or changes of ECM composition. An increase of activated VICs in adult valves is considered as indicator of pathological developments. Cultivation of VICs using standard 2D cell culture procedures also induces VIC activation resulting in contraction and spontaneous nodule formation. Our previous work has shown that this phenotype switch can be reversed by exposure to polyunsaturated fatty acids (PUFAs). In order to investigate underlying cellular mechanisms of the PUFA effects, VICs from porcine aortic valves were isolated and subcultured on collagen-coated surfaces. Spontaneous nodule formation after transfer to uncoated polystyrene was completely blocked by docosahexaenoic acid (DHA) and arachidoic acid (ARA) whereas eicosapentaenoic acid and a commercial extract from fish oil (Omacor) were less active. Oleic acid and palmitic acid were without effect. Treatment with ARA or DHA reduced the expression of myofibroblast marker proteins, α-smooth muscle actin (SMA) and myosin II, of a key enzyme of collagen synthesis (prolyl 4-hydroxylase), and of the phosphorylated (inactive) form of the F-actin severing protein, cofilin. In contrast, the abundance of fibroblast marker S100A4 was increased after treatment with PUFAs. The steady state level of active RhoA was reduced in the presence of DHA and ARA, and inhibition of RhoA or ROCK elicited the same effects as PUFAs. Finally, exposure to ARA and DHA reduced the G/F-actin ratio, and stabilization of F-actin with jasplakinolide blocked the effect of PUFAs on the expression of myofibroblast markers and on nodule formation. After culturing VICs with PUFAs for 14 days and subsequently in the absence of PUFAs for 4 days, cells regained the myofibroblast phenotype, showing the PUFA-induced phenotype switch was fully reversible. In conclusion, the results suggest that the differentiation of VICs to myofibroblasts can be truly reversed by certain PUFAs via the RhoA – ROCK – G-actin pathway, whereas an alternative mechanism, the preferential outgrowth of a undifferentiated subpopulation of VICs, seems to be unlikely since the PUFA effect was fully reversible.
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Pentraxin 3 is a Potential Diagnostic Marker for Aortic Stenosis
AbstractPentraxin 3 (ptx3) is a member of the long pentraxin family and is rapidly produced and released by many cell types, including endothelial cells, in response to primary inflammatory signals. In our preliminary work, PTX3 gene expression was significantly increased in aortic valve tissue following exposure to elevated cyclic pressure. Consequently, we hypothesized that ptx3 would be a useful biomarker for the early diagnosis of aortic valve sclerosis. Isolated aortic VICs were treated with Angiotensin II (300nM), TNF-alpha (10 ng/ml) or elevated cyclic pressure for six hours. The cell culture supernatant was collected and used to determine ptx3 protein expression using ELISA. Total RNA was isolated from the cells and PTX3 gene expression was determined using semi-quantitative RT-PCR. In addition to cell culture studies, ptx3 protein expression was determined in hypertensive New Zealand White rabbits. Rabbits underwent Goldblatt one-clip/one-kidney surgery to induce hypertension (n=5). Four sham models served as a control. Blood pressure, echocardiography data and serum samples were collected at 0, 2 and 4 months. After 4 months, rabbits were euthanized and aortic valve tissue was collected for histological and gene expression analysis. Data from the cell culture studies showed that elevated cyclic pressure caused an increase in PTX3 gene expression and ptx3 protein expression. However, no significant changes were observed in gene or protein expression from cells treated with Ang II or TNF-alpha. Data collected from the animal model showed that blood pressure increased significantly for the experimental group but not the control group. Levels of ptx3 protein were measured from the serum and showed a slight increase over the four-month course of the experiments. The data suggest that ptx3 expression is mechanosensitive in the aortic valve and is not stimulated by biochemical factors such as TNF-alpha or Ang II. The increase in ptx3 expression in hypertensive rabbits demonstrates that this could be a potential diagnostic marker for aortic stenosis.
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Effects of Finite Deformation on Extracellular Matrix Production and Mechanical Properties
AbstractWith our ability to incorporate viable cells distributed throughout the scaffold, we are provided a unique, controllable platform to develop a generalized finite deformation framework than can be used to gain an understanding of how the evolving extracellular matrix phase contributes to the construct gross mechanical behavior. The biosynthetic response of microintegrated VSMC’s was investigated low (15%), intermediate (30%), and high (50%) strain groups. These magnitudes were chosen as they correspond to a wide range of NAR deformations and physiologically relevant. A constant, quasi-static strain rate as applied sufficient to obtain a 1 Hz cycle duration. Culture durations of 7, 14, and 21 day time points were used for each strain level to quantify the ECM synthesis capacity of VSMC microintegrated in electrospun PEUU. A static group was also preformed at each time period. Results indicate that VSMC biosynthetic behavior is function of global strain with peaked soluble collagen synthesis was observed in specimens exposed to 30% strain. Our primary goal was to elucidate the mechanical behavior characteristics of the de-novo formed ECM. We determined the matrix mechanical contribution by assuming that the total mechanical response is simply the summation of the individual phases and any potential interactions that might arise between them. We thus quantified the collagen mass fraction and utilized an enzymatic technique to remove the ECM from the constructs, then retested them to obtain the degraded scaffold only responses. Results indicated that the newly formed matrix phase exhibit a highly anisotropic biaxial response, and was over 100 fold stiffer than similar ECM formed using stiff scaffolds previously studied in our lab. Moreover, the formed ECM had predicted mechanical properties similar to glutaraldehyde treated pericardium, a common heart valve biomaterial. Interestingly, peak biosynthetic activity correlates well with in vitro principle strain levels observed in PEUU tri-leaflet valves exposed to native ovine right side pressure. This suggests that physiologic hemodynamic conditions are optimal for the development of robust ECM accretion. To our knowledge, this is the first reported study to consider the effects of large deformation and the corresponding outcomes in terms of ECM mechanical integrity. Furthermore, the results reveal interesting insights into the functional role of the matrix accretion process in engineered tissues.
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Bio-inspired Anisotropic Nanofibrillar Matrices for Heart Valve Engineering
Authors: Jerome Sohier, Ivan Carubelli, Najma Latif, Padmini Sarathchandra, Adrian H. Chester and Magdi YacoubAbstractOur strategy for heart valve tissue engineering is the use of autologous cells to populate appropriate template matrices. In this context, an important goal is to devise a suitable biomimetic scaffold that supports proper cell growth and cell-matrix interactions by reproducing the specific anisotropic fibrillar structure of valves extracellular matrix (ECM). A novel type of highly porous anisotropic nanobrillar matrices was developed and evaluated with regards to structure, mechanical properties and ability to support human adipose derived stem cell (hADSC) colonization, growth and ECM production in vitro. Nanofibrillar structures were obtained by jet-spraying poly (ε-caprolactone) dissolved in chloroform on a variably rotating drum. Morphological evaluations of the structures were performed using scanning electron microscopy while porosity was calculated from polymer density, weight and volume. Elastic modulus of dry scaffolds (10x6x1 mm, n=5) was measured with a planar biaxial test bench with displacement rate of 0.05 mm/s. Human adipose derived stem cells (500,000) were top and rotary seeded on nanofibrillar discs (diameter 1 cm and thickness 0.8 mm) and cultured in 10 ml of complete medium under rotation (10 rpm) for 18 days. Histology (DAPI staining), DNA quantification and immunohistochemistry were used to characterize the resulting cellularized structures. The speed of drum rotation was adjusted up to 3000 rpm to produce highly aligned fibres (600 nm of average diameter) from the sprayed polymer. In conjunction with fibres anisotropy, the scaffolds Young's modulus was simultaneously increased (from 0.3 to 0.7 Mpa) and decreased (from 0.3 to 0.01 Mpa) longitudinally and orthogonally to fibre alignment, respectively. In addition, fibre alignment further increased scaffolds porosity from 97 % (isotropic) to 99%. Anisotropic matrices allowed a more extensive cellular invasion than isotropic scaffolds, possibly linked to their higher porosity and therefore open structure. hADSC proliferated significantly (up to 6-fold DNA increase), bridged the entire scaffolds thicknesses after 10 days and produced their own ECM as evidenced by collagen I production. This study highlights the potential of a newly developed highly porous anisotropic nanofibrillar matrices as substrate for tissue engineering, and in particular for heart valve engineering.
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PCL Scaffolds and Reduced In-Vitro Cell Expansion to Improve Engineered Valvular Tissue Formation
AbstractDifferent types of synthetic scaffolds are used for tissue engineering of heart valves. Tissue-engineered heart valves (TEHV), based on a rapid degrading polyglycolic acid (PGA) scaffold coated with poly-4-hydroxybutyrate (P4HB) and seeded with vascular derived cells, have shown promising in-vivo results. However, a major drawback of these TEHV is compaction and retraction of the leaflets causing regurgitation. It is hypothesized that this is a result of traction forces exerted by the cells, combined with an imbalance of the formed tissue and loss of mechanical integrity of the scaffold due to degradation. The aim of this study is to evaluate alternative approaches to overcome the compaction and retraction of TEHV without compromising on tissue composition and properties. The alternative approaches that are studied here are 1) the use of the slow degrading poly-ε-caprolactone (PCL) scaffold for prolonged mechanical integrity and 2) the use of lower passage vascular cells for enhanced tissue formation. Compaction, tissue formation, cell phenotype and mechanical properties of tissues based on passage 3, 5 and 7 vascular cells in PCL and PGA-P4HB scaffolds are compared. TEHV aim to be designed for humans, but since the ovine model is used to show proof of principle both human and ovine cells were used. Passage 3, 5 and 7 (p3, p5 and p7) human and ovine vascular-derived cells were seeded onto both PGA-P4HB and PCL scaffold strips (n=6 per passage and scaffold material), using fibrin as a cell carrier. After 4 weeks of culture under constrained static conditions, one strip was used for histology. The remaining strips were used for mechanical testing and biochemical assays as indicators for tissue strength and tissue formation, respectively. After 4 weeks, the tissues based on PGA-P4HB showed 50-60% compaction, while PCL-based tissues showed compaction of 0-10%. Cell passage number and species did not influence compaction. Tissue formation was comparable between passage numbers and scaffold materials in ovine while human p5 showed decreased tissue formation in both scaffold materials. Collagen content was increased with decreasing passage numbers in both species and scaffold materials. No differences in cell phenotype between the scaffold materials or cell passage numbers were observed. The Young’s modulus and the ultimate tensile strength of tissues of both species were higher in the lower passage groups of both scaffold groups. This study shows that PCL scaffolds may serve as alternative scaffold material for tissue engineering heart valves with minimal compaction and without compromising on tissue composition and properties. Cells from lower passages showed to improve tissue formation. Reducing cell expansion time will result in faster generation of TEHV, providing a more rapid treatment to patients.
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Fibrin Based Tissue Engineered Heart Valves Featuring the Sinuses of Valsalva
Authors: Petra Mela, Thomas Schermer, Julia Frese, Thomas Schmitz-Rode and Stefan JockenhoevelAbstractThe implementation of the sinuses of Valsalva in both the aortic and the pulmonary position is a crucial step towards the development of functional tissue engineered heart valves with optimal hemodynamic performance and reduced risk of thrombi formation. However, the implementation of these features is not standard in tissue engineered heart valves. In our laboratory we aim at the realization of autologous heart valves starting from materials isolated from the patient (fibrinogen and cells) and shaped into 3D geometries by moulding techniques. We present two new fabrication methods that result in the realization of heart valve scaffolds reproducing the complex geometry of semilunar valves. The first concept consists of a mould in two parts: a ventricular part and a vascular part which contains three removable bulbs representing the sinuses of Valsalva; in the second concept the vascular part features three flexible protrusions shaped as the sinuses of Valsalva which can be collapsed when a vacuum is applied. The moulds were designed with the 3D CAD software Pro/Engineer (PTC, Needham, MA, USA) and manufactured by rapid prototyping. The cell embedded fibrin gel valves were produced by polymerizing a fibrinogen solution in TBS (10 mg/ml) with CaCl2, thrombin and ovine umbilical cord derived fibroblasts (10x106 /ml) suspended in TBS. Afterwards, the obtained construct was placed in a static bioreactor to be cultured on the mould for 14 days in order to avoid cell-mediated tissue contraction before transferring it to a bioreactor for dynamic cultivation of a duration of 2 weeks. To release the fibrin scaffold from the mould after static cultivation the vascular part and subsequently the three removable sinuses of Valsalva were removed (first approach), or the vascular part was collapsed and taken out as one part (second approach). The constructs were successfully released without any tearing with both approaches despite the poor mechanical properties of the fibrin gel. Valved conduits including the sinuses of Valsalva were obtained without the need for suturing any of the parts together. After static and dynamic cultivation the conduits demonstrated good compliance. The tissue development was evaluated by histology (hematoxylin, eosin and immunohistochemistry) hydroxyproline assay and DNA assay. The presence of the sinuses of Valsalva in the aortic and the pulmonary root is fundamental for the correct functioning of semilunar heart valves. The implementation of the sinuses in tissue engineered valves will lead to an improvement of the valve function and an increased durability. Ongoing research focuses on the optimization of the cell source and of the conditioning protocol as to achieve optimal extracellular matrix synthesis and mechanical properties and minimize cell mediated tissue contraction.
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New Biomaterials in Heart Valve Tissue Engineering
AbstractIn situ tissue engineering may lead to a clinically and economically attractive new generation of heart valve replacement therapies, overcoming the limitations of currently used prostheses. Development of novel scaffolds is required for the proposed in situ tissue engineering. These scaffolds must be able to carry a full mechanical load immediately after implantation, and should subsequently provide all biological cues for the recruitment and attachment of specific circulating cells, and should also induce the growth of new tissue. The properties of the scaffold materials should be optimized with respect to mechanical properties and biodegradability characteristics. Synthetic biomaterials are tunable, which enables the creation of tailor made scaffolds suitable for in situ tissue engineering of heart valves. Two different approaches towards scaffold materials have been investigated, based on a biodegradable polyester (PCL or PLLCL), and either a quadruple hydrogen-bonding ureidopyrimidinone (UPy) unit or a bis-urea (BU) moiety. Both were investigated in vivo regarding biocompatibility and degradation kinetics. Thirty rats received the UPy and BU polyester based, disk-shaped implants subcutaneously. To investigate the effects of the implants over time, samples were explanted on day 2, 5, 10, 21 and 84. Disks with surrounding tissue were fixed in 10% neutral buffered formalin. After fixation, explants were dehydrated in graded alcohols and longitudinal sections were embedded in paraffin and stained. From each explant the foreign body response was quantified per high power field. Both the UPy and the BU-based materials proved biocompatible. In the acute phase all investigated biomaterials showed an infiltration of neutrophil granulocytes and mononuclear cells. In the chronic phase encapsulation by fibroblasts took place in all cases. Degradation rates were investigated by gel permeation chromatography (GPC) after 21 and 84 days. Little degradation was observed for the PCL-based polymers over the course of the experiment. The PLLCL-based polymers with a BU moiety, however, showed little at 21 days, but marked degradation at 84 days. Which of the explored materials is best suitable for in situ tissue engineering of heart valves will depend on further experiments, investigating how much time is needed for cell recruitment, adhesion, differentiation and tissue growth.
This research forms part of the Project P1.01 iValve of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation. The financial contribution of the Nederlandse Hartstichting is gratefully acknowledged.
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Human Valve Interstitial Cells Demonstrate Transdifferentiation Potential
Authors: Najma Latif, Padmini Sarathchandra, Adrian H Chester and Magdi H, YacoubAbstractAortic stenosis and valvular degeneration is characterized by lipid accumulation, presence of cartilage and calcification. The identity and cellular origins of the cells mediating these effects are unknown. We have investigated the potential of human valve interstitial cells (VICs) to transdifferentiate into osteogenic, adipogenic and chondrogenic-like lineages and examined the presence of resident stem cells. Transdifferentiation potential of VICs was assessed after removal of stem cells. Human aortic heart valves (n=8; mean age 64.7 ± 7.5 years) from patients undergoing transplantation, free from calcification and disease, and were used to isolate VICs. Differentiation was carried out by incubating VICs for 21 days with media containing ascorbate (50µg/ml), dexamethasone (10-8M) and β-glycerophosphate (10mM) for osteogenic differentiation, with ascorbate (50µg/ml), dexamethasone (10-7M) and indomethacin (50µg/ml) for adipogenic differentiation and with insulin transferrin selenium and TGFβ1 (10ng/ml) for chondrogenic differentiation. Immunocytochemistry and fluorescence-activated cell sorting (FACS) were used to assess stem cell populations and removal of sub-populations of VICs. Mesenchymal stem cells were isolated from bone marrow samples obtained from healthy human donors (n=6) and used as positive controls. We analysed the gene expression of some of the Wnt family as potential mediators of transdifferentiation. Incubation of VICs with osteogenic media induced alkaline phosphatase and osteocalcin expression in 34.2 ± 4.6% of VICs, with adipogenic media induced oil red O, SREBP and PPARγ expression in 13.8 ± 5.1% of VICs and with chondrogenic media changed the morphology in 41.7 ± 4.8% of VICs but did not induce collagen type II, type X or aggrecan expression. Cultured VICs expressed CD44 (94.0± 4.4%), CD73 (79.4 ± 15.4%) and CD105 (6.9 ± 2.6%) in common with mesenchymal stem cells. However very low percentages of stem cells were identified in cultured VICs, CD34 (2.8% ± 0.50), CD133 (1.84 ± 0.77%), c-kit (0.72% ± 0.21) and stro-1 (1.55 ± 0.93%). Valve leaflets demonstrated only occasional positive markers for stem cells. Cultured VICs were negative for CD31, CD14, CD45, Tie-2 and flk-1. Removal of these stem cells by FACS demonstrated that purified VICs retained their ability to transdifferentiate into osteogenic and adipogenic lineages to the same degree. Gene expression of the Wnt family showed the expression of Wnt2, Wnt2B, Wnt5B, Wnt 10B. A number of frizzled receptors were detected, FZD2-5 and FZD 6-10 as well as inhibitors DKK 1-3. The population of purified human VICs have the capacity to transdifferentiate into osteogenic, adipogenic and chondrogenic-like lineages. Human valve leaflets and cultures contain a resident stem cell population which can differentiate and thus contribute to valve IC population as well as to pathological phenotypes.
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C-type Natriuretic Peptide Modulates TGF-β1-induced Synthesis of Proteoglycan by VICs
Authors: Mark Blaser, Jung Woo Kwon, Krista L. Sider, Andrea V. Kwong, Kuiru Wei and Craig A. SimmonsAbstractAortic valve disease (AVD) is a cell-mediated pathology marked by unchecked remodeling of the extracellular matrix (ECM). This process is thought to be promoted by increased levels of TGF-β1 in the valve leaflets and the concomitant loss of protective factors such as C-type natriuretic peptide (CNP). Among its many functions, TGF-β1 stimulates the expression of proteoglycan (PG), in part via mitogen activated protein kinase (MAPK)-mediated Smad signalling. We and others have observed the formation of PG-rich lesions in early diseased aortic valves. Here we investigated the relationship between TGF-β1, CNP, and PG in a mouse model of early AVD and in vitro. Male wild-type (WT) C57Bl/6J mice were fed a control diet or BioServ F3282, a high-fat, high-carbohydrate diet (HF/HC) with 58.7% kcal from fat (cholesterol < w/w) for four months (n = 4-6 per group). Longitudinal aortic valve sections from formalin-fixed and paraffin-embedded hearts were stained by Movat’s pentachrome and immunostained for CNP and TGF-β1. Valve interstitial cells (VICs) were isolated from healthy porcine aortic valves and treated in vitro with 5 ng/ml TGF-β1 and/or 1 µM CNP or 10 µM U0126 (inhibitor of MEK, upstream of the MAPK Erk1/2) for up to 6 days. Erk1/2 phosphorylation was measured by Western blot, while PG expression in media collected from these cultures was assayed by Alcian Blue guanidine-HCL solubilisation. Mice on the HF/HC diet for four months became obese, developed mild hypercholesterolemia, and had early AVD, as demonstrated by hemodynamic dysfunction and significant thickening of the leaflets (data shown in another abstract). Thickening was due to PG deposition (11435 ± 7681 vs. 5448 ± 2948 µm2, p < 0.01), not increased collagen content (1729 ± 815 vs. 1771 ± 663 µm2, p = 0.87). TGF-β1 levels were elevated in leaflets of mice on the HF/HC diet, particularly in the region of PG lesions. The PG lesions in HF/HC mice also had lower CNP expression than non-lesion regions. In VICs, TGF-β1 induced a 29% increase of PG expression (p < 0.05), which was significantly reduced by the addition of CNP (p < 0.05). Interactions between TGF-β1 and CNP in PG synthesis were mediated in part via Erk1/2 signaling, as TGF-β1 increased Erk1/2 phosphorylation, CNP abrogated TGF-β1-induced Erk1/2 activation, and blocking Erk1/2 signaling with U0126 significantly reduced PG expression (p = 0.06). These studies demonstrate that in early AVD, thickened leaflets are PG-rich with elevated expression of TGF-β1 and down-regulated CNP. Furthermore, TGF-β1 induces PG expression in VICs, a process which is inhibited by CNP – possibly through Erk1/2. Insight into the mechanisms by which TGF-β1 and CNP regulate the formation of PG lesions helps further our understanding of early disease progression and may aid in identifying novel medical strategies to arrest this disease before a substantive and possibly untreatable burden of calcification occurs.
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The Role Of BMP/Wnt Signalling In Human Heart Valve Calcification
Authors: Paul Riem Vis, Adrian Chester, Najma Latif, Padmini Sarathchandra, Jolanda Kluin and Magdi YacoubAbstractAortic valve calcification is a complex process that is characterised by the expression of bone markers in the valve leaflet. The Wnt pathway and bone morphogenetic proteins (BMPs) have been implicated to play a role in vascular and heart valve calcification. This study examines the regulation of Wnt3a by BMPs and the functional effects mediated by Wnt3a in human valve interstitial cells (VICs). Treatment of VICs by BMPs up-regulated the expression of Wnt3a, ALP, RUNX2, and β-catenin, as was shown by Western Blot analysis. Analysis of human pathological specimens by immunohistochemistry showed an increased level of expression of Wnt3a, Msx2 and β-catenin was localised to peri-calcific regions of the tissue. The concentration-dependent proliferation or differentiation of VICs in response to Wnt3a was measured by the incorporation of [3H]-thymidine and activity of alkaline phosphatase (ALP) respectively. Wnt3a-treated VICs proliferated in a concentration-dependent manner. At higher concentrations, Wnt3a caused a significant increase in ALP activity and expression of RUNX2. Lastly, Western blot experiments showed that BMP2 induced RUNX2 up-regulation was mediated through β-catenin/Wnt-signalling and not Smad-signalling. This study demonstrates the important role of Wnt-signalling in valve calcification in response to BMPs. Wnt3a may induce valve calcification by regulating the proliferation and osteogenic differentiation of cells within the valve. These findings may help to identify new therapeutic.
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