bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurements and modelling of ozone in the Mediterranean MBL: an investigation of the importance of ship emissions to local ozone production
Authors: Hedgecock I. M. , Gencarelli C. N. , Sch{ü}rmann G. J. , Sprovieri F. , Pirrone N.
Published: July 2012

Abstract. Elevated concentrations of ground level ozone are both hazardous to human health and detrimental to agricultural production. The Mediterranean Basin, due to its position under the descending branch of the Hadley Cell circulation during the summer months, enjoys periods of stable, sunny and warm weather which provide ideal conditions for the production of ozone. The presence of major population centres and numerous industrialised areas in the coastal zone result in both a continual supply of ozone precursor compounds and also a significant number of people to suffer the consequences of high ozone concentrations. Using the WRF/Chem model validated with data obtained from seven oceanographic measurement campaigns, performed between 2000 and 2010, aboard the Italian Research Council's R. V. Urania, and also from a number of EMEP monitoring stations located around the Mediterranean Basin, the importance of emissions from maritime traffic in the region has been investigated. The model results indicate that over large areas of the Mediterranean emissions from shipping contribute between 5 and 10 ppb to the ground level O3 daily average concentration during the summer. The contribution to the hourly average O3 is up to 40 ppb in some particularly busy shipping lanes. Importantly the results suggest that in a number of coastal areas the contribution from ship emissions to the local O3 concentration can make the difference between complying with the EU Air Quality standard of a maximum 8 h mean of 120 μg m−3 and exceeding it.
Organic nitrogen in precipitation across Europe
Authors: Cape J. N. , Tang Y. S. , González-Benítez J. , Mitošinková M. , Makkonen U. , Jocher M. , Stolk A.
Published: July 2012

Abstract. Measurements of total nitrogen and inorganic nitrogen in precipitation samples from NitroEurope sites across Europe permit the calculation of organic nitrogen concentrations and wet deposition, by difference. The contribution of organic N to total N in precipitation ranged from only a few % to around 40% across sites from Northern Finland to Italy, similar to results from previous individual studies. This paper presents the absolute and relative contributions of organic N to wet N deposition across Europe, and examines seasonal trends. There were only weak correlations with other solutes in precipitation. These simple statistics indicate that sources of organic N in precipitation vary across Europe, and that no single source is responsible. The organic N contributes to total N deposition, yet this input is rarely quantified in nitrogen budgets.
MAESPA: a model to study interactions between water limitation, environmental drivers and vegetation function at tree and stand levels, with an example application to [CO2] × drought interactions
Authors: Duursma R. A. , Medlyn B. E.
Published: July 2012

Abstract. Process-based models (PBMs) of vegetation function can be used to interpret and integrate experimental results. Water limitation to plant carbon uptake is a highly uncertain process in the context of environmental change, and many experiments have been carried out that study drought limitations to vegetation function at spatial scales from seedlings to entire canopies. What is lacking in the synthesis of these experiments is a quantitative tool incorporating a detailed mechanistic representation of the water balance that can be used to integrate and analyse experimental results at scales of both the whole-plant and the forest canopy. To fill this gap, we developed an individual tree-based model (MAESPA), largely based on combining the well-known MAESTRA and SPA ecosystem models. The model includes a hydraulically-based model of stomatal conductance, root water uptake routines, drainage, infiltration, runoff and canopy interception, as well as detailed radiation interception and leaf physiology routines from the MAESTRA model. The model can be applied both to single plants of arbitrary size and shape, as well as stands of trees. The utility of this model is demonstrated by studying the interaction between elevated [CO2] (eCa) and drought. Based on theory, this interaction is generally expected to be positive, so that plants growing in eCa should be less susceptible to drought. Experimental results, however, are varied. We apply the model to a previously published experiment on droughted cherry, and show that changes in plant parameters due to long-term growth at eCa (acclimation) may strongly affect the outcome of Ca × drought experiments. We discuss potential applications of MAESPA and some of the key uncertainties in process representation.
Synthesis, self-assembly and lipoplex formulation of two novel cyclic phosphonate lipids
Authors: Yeh Jennifer , Tamarkina Elena , Abdul Khalique Nada , Raju Liji , Pungente Michael D.
Published: July 2012

Background: Synthetic cationic lipids hold much potential as gene packaging and delivery agents for the treatment of inherited and acquired life threatening diseases, such as cancer, AIDS, cardiovascular diseases, and certain autoimmune disorders. Methods: We report the synthesis, self-assembly as characterized by critical micelle concentrations and plasmid DNA gel retardation using two novel cyclic, phosphonate cationic lipids 2a and 2b, which were synthesized by derivatizing two diastereomeric macrocyclic phosphonates 1a and 1b with a 2-carbon hydroxylamine linker, N, N-dimethylethanolamine (3). Results: The production of cyclic phosphonate lipids 2a and 2b in 73% and 60% yields, respectively, was achieved using classical synthetic methods involving nucleophilic substitution at the phosphorus centre. The characterization of these lipids with Mass Spectrometry, 1H, 13C and 31P Nuclear Magnetic Resonance supported the proposed stereochemistry and molecular structure of C17H36NO3P for these lipids. Critical micelle concentrations (CMC) for 2a and 2b were found to be 1.2 mM and 1.4 mM, respectively, at 25°C, providing evidence for the self-assembling ability of these novel cyclic lipids. Finally, lipid-DNA complexes (lipoplexes) formulated at various N/P (+/ − ) molar charge ratios and containing the co-lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were shown to retard DNA in an agarose gel retardation assay. Conclusions: The synthesis, aggregation and DNA binding properties of these novel cyclic phosphonate lipids suggest that they may have utility serving as gene packaging and delivery agents.
The role of Insulin-like Growth Factor-1 in Calcification of Human Aortic Valves
Authors: Poudel-Bochmann Bhawana , Barth Mareike , Boeken Udo , Albert Alexander , Lichtenberg Artur , Akhyari Payam
Published: July 2012

Insulin-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.
Reversible Effects of Polyunsaturated Fatty Acids on Activation of Interstitial Cells From Porcine Aortic Valves
Authors: Witt Wolfgang , Jannasch Anett , Matschke Klaus , Waldow Thomas
Published: July 2012

Valvular 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.
Pentraxin 3 is a Potential Diagnostic Marker for Aortic Stenosis
Authors: Davison Steven , Brinkman-Ferguson Erin , Claude Andrew , Dycus David , Lodato Dena , Cooper Robert , Warnock James N.
Published: July 2012

Pentraxin 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.
C-type Natriuretic Peptide Modulates TGF-β1-induced Synthesis of Proteoglycan by VICs
Authors: Blaser Mark , Kwon Jung Woo , Sider Krista L. , Kwong Andrea V. , Wei Kuiru , Simmons Craig A.
Published: July 2012

Aortic 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.
Contribution of epicardially derived cells to the leaflets of the murine AV valves
Authors: Burch John , Van den Hoff Maurice , Lockhart Marie , Briggs Laura , Wessels Andy
Published: July 2012

To 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.
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

While 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

Calcific 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

Decellularization 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

The 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

Although 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

The 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

Incubating 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

When 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

Aortic 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.
Pressure and Angiotensin II Influence the Mechanical Properties of Aortic Valves
Authors: Myles Valtresa , Liao Jun , Warnock James N.
Published: July 2012

Elevated cyclic pressure and angiotensin II (Ang II) both promote aortic valve collagen synthesis, an early hallmark of aortic sclerosis. In the current study, it was hypothesized that the increased collagen production induced by either elevated pressure or Ang II would increase tissue stiffness.
Porcine aortic valve leaflets were randomly assigned to four groups; leaflets in group 1 were treated with 10-6M Ang II, leaflets in group 2 were exposed to 80 or 120 mmHg cyclic pressure, leaflets in group 3 were treated with Ang II and exposed to cyclic pressure and leaflets in group 4 were the control. Biaxial testing was performed after 24 and 48 hours on 10mm x 10mm samples of tissue dissected from the central region of the leaflets. Four fiducial markers arranged in an approximately 4 mm x 4 mm square were placed in the center of the extracted portion of the leaflets to track tissue strain. A membrane tension (force/unit length) was applied along each axis and increased slowly from a pre-stress tension of ~0.5 N/m to a peak tension of 60 N/m. The samples were preconditioned for ten contiguous cycles, following an equibiaxial protocol of TCC:TRR = 60:60 N/m, where TCC and TRR are the tensions applied in the circumferential and radial directions, respectively. Tissue extensibility was characterized by maximum stretch along the circumferential direction (λcc) and maximum stretch along the radial direction (λrr), at an equibiaxial tension of 60 N/m. Leaflet stiffness was greater in the circumferential direction than in the radial direction, which is consistent with previous studies. The peak stretches of native valves were calculated as 1.05±0.02 and 1.40±0.02 in the circumferential and radial directions, respectively. There was no significant difference in the stiffness of native valves compared to those exposed to 0mmHg(-Ang II) or 80mmHg(-Ang II) for 24 or 48 hours. Elevated pressure increased stiffness in both directions after 24 and 48 hours. After 24 and 48 hours Ang II significantly increased stiffness in the radial direction. The combination of Ang II and elevated pressure increased stiffness in radial direction after 24 and 48 hours. Increased stiffness may be due to remodeling of the ECM. Excessive collagen production is known to hinder valve function, eventually resulting in aortic stenosis. In conclusion, the results of the present study demonstrated that both elevated pressure and Ang II play a role in the increased stiffness of aortic valve leaflets.