bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation of the Statin Paradox in Different Populations of VICs
Authors: Monzack Elyssa L. , McCoy Chloe M. , Duxstad Kelsey A. , Masters Kristyn S. 
Published: July 2012

AbstractWhile numerous clinical studies have examined the effect of HMG-CoA reductase inhibitors (statin drugs) on calcific aortic valve disease (CAVD), their conflicting results have yielded many questions regarding the nature of statin-valve interactions. One step toward better understanding this relationship is to examine the effects of statin treatment on heart valves on a cellular level. Previous work found statin treatment to have a "paradoxical" effect in vitro, decreasing osteoblastic markers in valvular myofibroblasts, while increasing those same markers in osteoblast precursor cells. This finding that statins may be able to selectively induce bone formation only in a cell type that is already prone to mineralization leads to the question of how statin treatment will affect valvular interstitial cells (VICs), a heterogeneous cell population which is capable of differentiating into an osteoblast-like phenotype, termed obVICs. In this study, we set out to determine whether obVICs would respond to statin treatment in the same manner as myofibroblasts, or if obVICs would increase bone marker expression in a manner similar to a bone-derived cell type. This work was also complemented by a gene expression analysis of calcified human valves from individuals who were or were not taking a statin drug.
Porcine VICs were cultured in vitro, with or without 1 uM simvastatin, in either control or mineralization medium, where the control medium yields a heterogeneous population that is predominantly myofibroblasts, while the mineralization medium drives VICs toward an obVIC phenotype. Gene expression analysis included multiple myofibroblastic and osteoblastic markers and was conducted daily over an 8-day time course, yielding information about not only expression levels, but also their temporal dynamics. Gene expression profiles were compared between VICs and an osteoblastic cell line (MC3T3-E1) to assess similarities. Myofibroblastic and osteoblastic genes were also analyzed in aortic valves from human patients (+/- statin) undergoing aortic valve replacement surgery. Statin treatment increased osteoblastic gene expression in VICs cultured in mineralization medium (obVICs), but the same effect was not obtained in control medium. This finding suggests that VICs are capable of responding to statin treatment in a manner similar to bone cells, but only when VIC cultures are driven toward an osteoblastic phenotype. The MC3T3-E1 cells also increased osteoblastic gene expression upon statin treatment, although their basal level of osteogenic activity was substantially greater than that found in any of the obVIC cultures. Analysis of human valve data is ongoing. Overall, this study suggests that different subpopulations of VICs exhibit different and temporally dynamic responses to statin treatment, further complicating the ability to predict a clinical effect of statin drugs on CAVD.
Lysophosphatidylcholine Alters Valvular Interstitial Cell Mineralization
Authors: Wiltz Dena C. , Morrisett Joel D. , Grande-Allen K. Jane 
Published: July 2012

AbstractCalcific aortic valve disease (CAVD) is a condition of the heart characterized by thickening and calcification of the aortic valve and can lead to aortic stenosis, narrowing of the aortic valve that can obstruct left ventricular outflow. CAVD is thought to have similarities with atherosclerosis, in which the aortic wall demonstrates thickening due to plaque buildup. A notable similarity seen between CAVD and atherosclerosis is the accumulation of lipids in the tissues. One important chemical component involved in atherosclerosis is lysophosphatidylcholine (LPC), a phospholipid derived from phosphatidylcholine. LPC concentrations have been shown to increase in atherosclerotic conditions, and induce expression of osteogenic factors by vascular smooth muscle cells. The potential for LPC to affect valve cell calcification, however, has not been previously investigated. In addition, calcification of cells from different valves warrants investigation because the aortic valve becomes more bone-like and experiences onset of calcification sooner than the mitral valve during the calcification process.
This study investigated the effect of LPC on the propensity for calcification by porcine valve interstitial cells (VICs) from aortic and mitral valves. On day 0 VICs were seeded at a density of 50 000 cells/cm2 in low serum media. On day 1, the media is changed to media containing LPC in concentrations ranging from 0 to 100 µM. The cells are cultured for 8 days and then assessed for mineralization using histological stains (Alizarin Red S for calcium deposition and Von Kossa for phosphate deposition) and biochemical assays (Alkaline phosphatase activity). Significance (p <0.05) was determined using Analysis of Variance followed by Tukey post-hoc testing.
Interestingly, mineralization in the VIC cultures was decreased as LPC concentration increased from 0 to 1 µM. At 10 µM, however, an increase in mineralization was observed compared to the 1 µM cultures. VICs in 100 µM LPC media began to detach within 24 hours of LPC media application. Also, VICs from different valves displayed different levels of calcification at each condition. LPC alters mineralization in VIC cultures from both aortic and mitral valves, in a concentration dependent manner. Extremely high concentrations of LPC (at and above 100 µM) can be toxic to VICs. There is a unique behavior of VICs with addition of varying concentrations of LPC, most notably that low concentrations (below 10 µM) actually reduced mineralization. Other factors, such as effects of LPC on VIC proliferation and apoptosis, will be important to investigate in future work. This study demonstrates that LPC affects the mineralization potential of valvular cells in a way that is distinct from vascular cell types.
Decellularized Heart Valve Prostheses and the Anticalcific Potential of Simvastatin
Authors: Assmann Alexander , Munakata Hiroshi , Horstkötter Kim , Delfs Christofer , Poudel-Bochmann Bhawana , Boeken Udo , Akhyari Payam , Lichtenberg Artur 
Published: July 2012

AbstractDecellularization is a proven approach to decelerate the degenerative processes which lead to the failure of heart valve implants. In order to further delay the calcifying in vivo degeneration of tissue-engineered grafts, antiarteriosclerotic and antiinflammatory substances may be advantageous, and some in vitro reports on HMGCoA reductase inhibitors have shown encouraging results, whereas clinical trials have failed to prove a positive in vivo result.
Male Wistar rats (recipients) underwent an interventional generation of aortic insufficiency grade II – III (day -14) and were fed with a procalcific diet of high-dose vitamin D, cholesterol and calciumphosphate, additionally supplemented with simvastatin (group S; n = 6). Identically treated animals fed with the same diet, not supplemented with simvastatin, served as controls (group C; n = 6). Aortic conduits of Sprague-Dawley rats (donors) were decellularized according to a detergent-based protocol and infrarenally implanted (day 0) in an end-to-side manner in the recipients. Echocardiography, doppler sonography of the implant and blood serum analyses were conducted at days -14, 0 and 28. Graft explantation, histological and immunohistochemical analyses as well as quantitative real time PCR were performed after 4 weeks.
Acute AI grade II – III with echocardiographically confirmed reversed diastolic flow in the whole aorta caused significant left ventricular (LV) dilatation as well as decrease of LV ejection fraction (p <0.001 at day 28) and resulted in a mortality of 8%. Sonographic competence of the conduit perfusion and overall survival of the transplanted rats were 100%. After 4 weeks of simvastatin treatment, calcification of the implants was significantly lower in group C (p < 0.01), whereas especially the aortic valve and the ascending aorta were strained by a decreased calcium burden (vonKossa staining). Histological evaluation of the conduit implants revealed an intimal hyperplasia, involving α-smooth muscle actin expressing cells, with an increased intima-to-media ratio (p < 0.05) in the aortic walls of group S and in the aortic valves of group C. RNA analysis by quantitative PCR was performed to confirm these results. The present study in a standardized rat transplantation model failed to show an early benefit of the HMGCoA reductase inhibitor simvastatin to diminish the calcification of decellularized aortic conduit implants. Furthermore, existing literature in this field is contradictory, and therefore, further experiments with more detailed analyses and long-term observations are warranted.
Contribution of Specific Glycosaminoglycans to the Relaxation Properties of the Aortic Valve
Authors: Borghi Borghi , Ivan Carubelli , Chester Adrian H. , Yacoub Magdi H. 
Published: July 2012

AbstractThe aortic valve (AV) is characterized by a complex mechanical behavior which is closely linked to its structural components. The central layer of the AV is rich in glycosaminoglycans (GAGs) which play an important role in the biomechanics of the AV. In this study the effect of selective GAGs depletion on time dependent mechanical behavior of porcine AV was analyzed.
Fresh strips of porcine AV cusps were cut in either in the radial or in the circumferential direction and mounted on a tensile testing system (Bose Electroforce) for mechanical testing. Three groups of valves were treated enzymatically, in order to remover either all the GAGs (group 1), the sulphated GAGs only (group 2) or the non-sulphated GAGs only (group 3). Each group had a control group. Mechanical tests were performed on each strip and stress relaxation kinematics as well as relaxation percentage were compared between treated and untreated specimens. Tensile and stress relaxation tests were performed on the strips under physiological load levels. The reduced stress relaxation function was fitted to the experimental data using a two phase (τ1 and τ2) exponential decay model. Relaxation percentage was significantly lower in group 1 for both circumferential (group 1: 20.58% vs control 28.34%, p = 0.004) and radial strips (group 1: 18.89% vs control 28.85%, p = 0.006). In this group, the early relaxation value (τ1) markedly decreased in the radial direction (group 1: 7.86s vs control: 10.35s, p = 0.0041) while no statistical difference was achieved in the circumferential direction. When looking at selective GAGs depletion, an effect of the hyaluronic acid depletion (group 3) was seen on the excursion of circumferentially oriented strips (group 2: 23.15% vs control: 27.76%, p = 0.049). No major effect was seen comparing the results of group 2 with its control. No effect on t2 was found. Histology confirmed the successful GAGs depletion. The presence of GAGs influences the biomechanics of the AV in terms of time dependent mechanical properties. The presence of hyaluronic acid has a distinctive effect on the relaxation excursion of the cusps while no effect was apparent for sulphated GAGs. These results provide further insight into the relationship between structure and fuction in the AV.
Characterization of Sex-Related Differences in Valvular Interstitial Cells
Authors: McCoy Chloe , Nicholas Dylan Q. , Masters Kristyn S. 
Published: July 2012

AbstractAlthough the pathogenesis of calcific aortic valve disease (CAVD) is not well understood, males have been identified as having a two-fold increased risk for the disease when compared to females. In this study, we examined gene expression profiles in healthy pigs and measured markers of disease in vitro to determine whether the differences in clinical risk between males and females translate into measurable intrinsic differences on the cellular scale. In addition, we also investigated potential sex-related differences in cellular response to TGF-β1, an inflammatory stimulus known to be elevated in calcified human aortic valve explants.
mRNA was isolated from three male and three female porcine aortic valves (denuded of endothelial cells) and hybridized to Affymetrix® GeneChip Porcine Genome microarrays. Mean expression values of each probe set in the male samples were compared with those in the female samples and biological processes were analyzed from the dataset for overrepresentation using Gene Ontology term enrichment analysis. From the microarrays there were 183 genes identified as being significantly (fold change>2; P<0.05) different in healthy male versus female aortic valve leaflets. Within this significant gene list there were 298 overrepresented biological processes, several of which are relevant to pathways identified in CAVD pathogenesis. In particular, pathway analysis indicated that cellular proliferation, apoptosis, cell migration, ossification, and extracellular matrix reorganization were all significantly represented in the data set. In vitro culture of male and female porcine valve cells also revealed intrinsic differences between sexes, with male cells exhibiting higher proliferation, apoptosis, and expression of αSMA after five days of culture. When exposed to TGF-β1, male cells grown in serum-free culture were found to be more sensitive to the inflammatory cytokine, with dramatically decreased apoptosis and proliferation compared to a marginal decrease in apoptosis and no change in proliferation in female cells. These data suggest that some sex-related propensity for CAVD may be present on the cellular level in healthy subjects, possibly resulting in a differential response to systemic factors that promote disease onset and progression. These results also offer motivation for further sex-related studies with valve cells to better determine possible genetic contributors and to explore sex-related susceptibilities for valve calcification.
Influence of Scaffold Structure on Cell Behaviour for Valve Tissue Engineering
Authors: Carubelli Ivan , Sohier Jerome , Ashwell Alice , Sarathchandra Padmini , Chester Adrian H, , Yacoub Magdi H. 
Published: July 2012

AbstractThe success of a tissue engineered heart valve is dependent on developing the right structure, the right interactions between the cells and the right matrix and mechanical force. Different materials have been used as scaffold, however the best processing method have not been established. We have used 2 different type of scaffold: collagen based scaffold and a nanofibrillar scaffold made from poly(ε-caprolactone) (PCL). Different seeding methods have been tested for cell compatibility of these scaffolds with human adipose derived mesenchymal stem cells (hADSCs) and with human telomerase immortalized bone marrow derived stem cells (hTERT).
Nanofibrillar scaffolds have been produced by jet-spraying the polymer on a metal grid. Collagen scaffolds were made by freeze drying a 1% bovine collagen solution chemically crosslinked with EDC-NHS. Morphological evaluations of the structures were performed using scanning electron microscopy. 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. Two different type of cells (600000/scaffolds) were seeded using different seeding methods to find the best condition: top seeding for 2 hours and then dynamically cultured using a rotary mixer, directly dynamically seeded for different time period and with different volume (5, 10 and 25 ml). DNA quantification using Hoescht 3258 and DAPI staining were used to evaluate proliferation and penetration inside the scaffold. Immunohistochemistry was used to check collagen production. PCL scaffolds were composed of non woven nanofibers (600 nm average diameter) assembled in a highly open structure. Collagen scaffold showed an interconnected porous structure with average pore size of 100 um. Nanofibrillar scaffolds showed higher elastic modulus compared to collagen (200 KPa compared to 150 KPa). The different cell seeding approaches had an effect on cellular distribution and cell number. With 10 ml of volume cells attached more after 24 hours compared to 5 ml with no further difference compared to 25 ml. Top-seeded matrices resulted in a high cell concentration on the seeded surface while rotary seeding allowed cells to attach on both scaffold sides but in fewer numbers. Regardless of seeding method, cells proliferated extensively (up to 10 and 2-fold DNA increase for hTERT and hADSC respectively ) on both scaffold, but proliferation was up to twice higher within nanofibrillar structures compared to collagen scaffolds. Both cell types were able to populate the entire area of both scaffolds over 10 and 14 culture days for hADSC and hTERT respectively. Both cell type produced their own ECM within the scaffolds as indicated by collagen I positive staining. Jet-sprayed PCL nanofibrillar scaffolds are a promising alternative to collagen scaffolds for cellular infiltration and proliferation.
Non-Cytotoxic Cross-Linking of Bioactive Porcine Matrices
Authors: Somers Pamela , De Somer Filip , Cornelissen Ria , Thierens Hubert , Van Nooten Guido 
Published: July 2012

AbstractIncubating a porcine aortic valve matrix with a platelet gel (PG) concentrate creates a bioactive matrix which is loaded with growth factors. These matrices can be repopulated with mesenchymal stem cells. However, these recellularized matrices still elicit a host immune response. Therefore, the aim of this study was to evaluate the cytotoxicity and cross-linking effect of naturally organic compounds such as quercetin, tannic acid, caffeic acid and catechin on these matrices and to investigate the effect of these cross-linkers on the in vitro growth factor release rate. Porcine aortic heart valves were decellularized using a detergent/enzymatic treatment. Cytotoxicty of the cross-linkers was evaluated by cell culture media supplementation of 10, 100, 1000, 5000, 10000 and 20000µg/mL. These concentrations were also used to cross-link the acellular matrices. Mechanical strength of the leaflets was investigated. Also the effect of these cross-linkers on the growth factor release from the PG loaded scaffolds was evaluated by ELISA assays. Results showed that proliferation of porcine mesenchymal stem cells increased significantly with increasing concentrations of quercetin, tannic acid, caffeic acid and catechin. All compounds, except tannic acid, significantly increased mechanical strength of the matrices. Moreover, tensile strength of quercetin cross-linked matrices was comparable to the commercially available 0.625% glutaradehyde fixed valves. Furthermore, cross-linking of the matrices resulted in a decreased burst release of growth factors during the first 4 hours but prolonged the release after 24 hours when compared to non-cross-linked matrices.
Natural compounds such as quercetin, caffeic acid and catechin can be used to cross-link porcine aortic valve matrices. Moreover, the in vitro release of growth factors can be prolonged which can be very advantageous in the recellularization of these scaffolds.
Taurodeoxycholate Overcomes Limitations of Deoxycholate for Xenogenic Cell Removal
Authors: Buratto Edward J. , Gastaldello Alessandra , Naso Filippo , Bottio Tomaso , Tarzia Vincenzo , Dal Lin Carlo , Ortolani Fulvia , Bonetti Antonella , Marchini Maurizio , Spina Michel , Gandaglia Alessandro , Gerosa Gino 
Published: July 2012

AbstractWhen replacement of heart valves is required there is almost no alternative to overcome the shortcomings of the conventional substitutes and the clinical outcomes of recently devised cell-depleted tissue engineered xenogeneic constructs are still controversial. Particularly, osmotic shock- and deoxycholate (DOC)-based acellular preparations that gained approval for use in surgical practice, are reported to have been fully or partly unsuccessful. The formers leading to patient deaths and the others resulting in either a high number of explantations or in successful outcomes at midterm follow-up according to different reports. Experimental evidence obtained in the present investigation indicated that inconsistent clinical outcomes of deoxycholate (DOC)-based heart valve preparations might have been related at least in part to incomplete or variable removal of xenogenic cell material following DOC solubility limitations. Therefore we explored alternatively the efficiency of taurodeoxycholate (TDOC), the highly soluble conjugated form of DOC, associated with Triton X 100 (TRI). Characterization of the resulting acellular scaffold, included shape, volume and mass analysis, quantification of residual xenoantigen alpha-Gal, histology, immunofluorescence, scanning and transmission electron microscopy as well as pulse duplicator testing at systemic pressures. In contrast to previous DOC and combined SDS (sodium dodecyl sulfate)-DOC procedures, adoption of TDOC resulted in complete removal of alpha-Gal xenoantigen, with apparent reduction of laminin and enhanced fibronectin detection by immunofluorescence. Besides cell removal from leaflet, sinus and aortic wall, detailed morphological investigation revealed unconventional aspects of the stromal matrix distribution in native and treated samples. In native samples GAG concentration in spongiosa resulted apparently comparable to that in fibrosa layer while collagen and elastic fibres, respectively, exhibited a peculiar interconnected distribution throughout the valve layers. After TRI-TDOC treatment total leaflet hydration was unchanged while mass, area and thickness decreased. The general hydrodynamic performance of the TRI-TDOC-scaffold well accorded with substantial maintenance of matrix architecture while increased post-treatment gradients and regurgitant volumes correlated with loss of ECM components and partial leaflet retraction. Considering the remarkable cell-removal efficiency and the solubility properties, TDOC is worth of further investigation in the perspective to replace DOC for obtaining xenogenic valve scaffolds free of cell remnants and detergent residues with the aim to restore valvular function in vivo or after dynamic cell culture in vitro.
Investigating the Contribution of Pathological Levels of Cyclic Strain on Vascular and Valvular Calcification
Authors: Ferdous Zannatul , Jo Hanjoong , Nerem Robert 
Published: July 2012

AbstractAortic 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.