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1.  Simulation of Early Calcific Aortic Valve Disease in a 3D platform: A Role for Myofibroblast Differentiation 
Calcific aortic valve disease (CAVD) is the most prevalent valve disease in the Western world. Recent difficulty in translating experimental results on statins to beneficial clinical effects warrants the need for understanding the role of valvular interstitial cells (VICs) in CAVD. In two-dimensional culture conditions, VICs undergo spontaneous activation similar to pathological differentiation, which intrinsically limits the use of in vitro models to study CAVD. Here, we hypothesized that a three-dimensional (3D) culture system based on naturally derived extracellular matrix polymers, mimicking the microenvironment of native valve tissue, could serve as a physiologically relevant platform to study the osteogenic differentiation of VICs.
Principal results
Aortic VICs loaded into 3D hydrogel constructs maintained a quiescent phenotype, similar to healthy human valves. In contrast, osteogenic environment induced an initial myofibroblast differentiation (hallmarked by increased alpha smooth muscle actin [α-SMA] expression), followed by an osteoblastic differentiation, characterized by elevated Runx2 expression, and subsequent calcific nodule formation recapitulating CAVD conditions. Silencing of α-SMA under osteogenic conditions diminished VIC osteoblast-like differentiation and calcification, indicating that a VIC myofibroblast-like phenotype may precede osteogenic differentiation in CAVD.
Major conclusions
Using a 3D hydrogel model, we simulated events that occur during early CAVD in vivo and provided a platform to investigate mechanisms of CAVD. This novel approach can provide important insight into valve pathobiology and serve as a promising tool for drug screening.
PMCID: PMC4906202  PMID: 26996755
Aortic valve stenosis; calcific aortic valve disease; tissue engineering; hydrogels; valvular interstitial cells
2.  Conceptual model for early health technology assessment of current and novel heart valve interventions 
Open Heart  2016;3(2):e000500.
The future promises many technological advances in the field of heart valve interventions, like tissue-engineered heart valves (TEHV). Prior to introduction in clinical practice, it is essential to perform early health technology assessment. We aim to develop a conceptual model (CM) that can be used to investigate the performance and costs requirements for TEHV to become cost-effective.
After scoping the decision problem, a workgroup developed the draft CM based on clinical guidelines. This model was compared with existing models for cost-effectiveness of heart valve interventions, identified by systematic literature search. Next, it was discussed with a Delphi panel of cardiothoracic surgeons, cardiologists and a biomedical scientist (n=10).
The CM starts with the valve implantation. If patients survive the intervention, they can remain alive without complications, die from non-valve-related causes or experience a valve-related event. The events are separated in early and late events. After surviving an event, patients can experience another event or die due to non-valve-related causes. Predictors will include age, gender, NYHA class, left ventricular function and diabetes. Costs and quality adjusted life years are to be attached to health conditions to estimate long-term costs and health outcomes.
We developed a CM that will serve as foundation of a decision-analytic model that can estimate the potential cost-effectiveness of TEHV in early development stages. This supports developers in deciding about further development of TEHV and identifies promising interventions that may result in faster take-up in clinical practice by clinicians and reimbursement by payers.
PMCID: PMC5073474  PMID: 27843569
Cost-Effectiveness; Conceptual Model; Tissue-Engineered Heart Valves; Early Health Technology Assessment
3.  Valvular Interstitial Cells Suppress Calcification of Valvular Endothelial Cells 
Atherosclerosis  2015;242(1):251-260.
Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. We previously proposed that valvular endothelial cells (VECs) replenish injured adult valve leaflets via endothelial-to-mesenchymal transformation (EndMT); however, whether EndMT contributes to valvular calcification is unknown. We hypothesized that aortic VECs undergo osteogenic differentiation via an EndMT process that can be inhibited by valvular interstitial cells (VICs).
Approach and Results
VEC clones underwent TGF-β1-mediated EndMT, shown by significantly increased mRNA expression of the EndMT markers α-SMA (5.3±1.2), MMP-2 (13.5±0.6) and Slug (12±2.1) (p<0.05), (compared to unstimulated controls). To study the effects of VIC on VEC EndMT, clonal populations of VICs were derived from the same valve leaflets, placed in co-culture with VECs, and grown in control/TGF-β1 supplemented media. In the presence of VICs, EndMT was inhibited, shown by decreased mRNA expression of α-SMA (0.1±0.5), MMP-2 (0.1±0.1), and Slug (0.2±0.2) (p<0.05). When cultured in osteogenic media, VECs demonstrated osteogenic changes confirmed by increase in mRNA expression of osteocalcin (8.6±1.3), osteopontin (3.7±0.3), and Run×2 (5.5±1.5). The VIC presence inhibited VEC osteogenesis, demonstrated by decreased expression of osteocalcin (0.4±0.1) and osteopontin (0.2±0.1) (p<0.05). Time course analysis suggested that EndMT precedes osteogenesis, shown by an initial increase of α-SMA and MMP-2 (day 7), followed by an increase of osteopontin and osteocalcin (day 14).
The data indicate that EndMT may precede VEC osteogenesis. This study shows that VICs inhibit VEC EndMT and osteogenesis, indicating the importance of VEC–VIC interactions in valve homeostasis.
PMCID: PMC4546848  PMID: 26232165
Calcific aortic valve disease; Valvular endothelial cells; Valvular interstitial cells; Calcification; Endothelial-to-mesenchymal transformation
4.  Directing valvular interstitial cell myofibroblast-like differentiation in a hybrid hydrogel platform 
Advanced healthcare materials  2014;4(1):121-130.
Three dimensional (3D) hydrogel platforms are powerful tools, providing controllable, physiologically relevant microenvironments that could aid in understanding the role of various environmental factors in directing valvular interstitial cell (VIC) phenotype. Continuous activation of VICs and their transformation from quiescent fibroblast to activated myofibroblast phenotype is considered to be an initiating event in the onset of valve disease. However, relative contribution of changes in VIC phenotype are poorly understood since most 2-dimensional (2D) culture systems lead to spontaneous VIC myofibroblastic activation. Here, a hydrogel platform composed of photocrosslinkable versions of native valvular extracellular matrix components –methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) – is proposed as a 3D culture system to study VIC phenotypic changes. Our results showed that VIC myofibroblast-like differentiation, determined by α-SMA, MMP-9, and Collagen type I expression, occurs spontaneously in mechanically soft GelMA hydrogels. In contrast, VICs encapsulated in HAMA-GelMA hybrid hydrogels, does not occur spontaneously and require exogenous delivery of TGFβ1, indicating that hybrid hydrogels can be used to study cytokine-dependent transition of encapsulated VICs. This study demonstrated that a hybrid hydrogel platform can be used to maintain a quiescent VIC phenotype and study the effect of pathological environmental cues on VIC activation, which will aid in understanding pathobiology of valvular disease.
PMCID: PMC4276556  PMID: 24958085
5.  Prosthetic aortic valve selection: current patient experience, preferences and knowledge 
Open Heart  2015;2(1):e000237.
Current clinical practice guidelines advocate shared decision-making (SDM) in prosthetic valve selection. This study assesses among adult patients accepted for aortic valve replacement (AVR): (1) experience with current clinical decision-making regarding prosthetic valve selection, (2) preferences for SDM and risk presentation and (3) prosthetic valve knowledge and numeracy.
In a prospective multicentre cohort study, AVR patients were surveyed preoperatively and 3 months postoperatively.
132 patients (89 males/43 females; mean age 67 years (range 23–86)) responded preoperatively. Decisional conflict was observed in 56% of patients, and in 25% to such an extent that it made them feel unsure about the decision. 68% wanted to be involved in decision-making, whereas 53% agreed that they actually were. 69% were able to answer three basic knowledge questions concerning prosthetic valves correctly. 56% were able to answer three basic numeracy questions correctly. Three months postsurgery, 90% (n=110) were satisfied with their aortic valve prosthesis, with no difference between mechanical and bioprosthetic valve recipients.
In current clinical practice, many AVR patients experience decisional conflict and suboptimal involvement in prosthetic valve selection, and exhibit impaired knowledge concerning prosthetic valves and numeracy. Given the broad support for SDM among AVR patients and the obvious need for understandable information, to-be-developed tools to support SDM in the setting of prosthetic valve selection will help to improve quality of decision-making, better inform and actively involve patients, and reduce decisional conflict.
Trial registration number
PMCID: PMC4395830  PMID: 25893105
6.  Sequential use of human-derived medium supplements favours cardiovascular tissue engineering 
For clinical application of tissue engineering strategies, the use of animal-derived serum in culture medium is not recommended, because it can evoke immune responses in patients. We previously observed that human platelet-lysate (PL) is favourable for cell expansion, but generates weaker tissue as compared to culture in foetal bovine serum (FBS). We investigated if human serum (HS) is a better human supplement to increase tissue strength. Cells were isolated from venous grafts of 10 patients and expanded in media supplemented with PL or HS, to determine proliferation rates and expression of genes related to collagen production and maturation. Zymography was used to assess protease expression. Collagen contraction assays were used as a two-dimensional (2D) model for matrix contraction. As a prove of principle, 3D tissue culture and tensile testing was performed for two patients, to determine tissue strength. Cell proliferation was lower in HS-supplemented medium than in PL medium. The HS cells produced less active matrix metallo-proteinase 2 (MMP2) and showed increased matrix contraction as indicated by gel contraction assays and 3D-tissue culture. Tensile testing showed increased strength for tissues cultured in HS when compared to PL. This effect was more pronounced if cells were sequentially cultured in PL, followed by tissue culture in HS. These data suggest that sequential use of PL and HS as substitutes for FBS in culture medium for cardiovascular tissue engineering results in improved cell proliferation and tissue mechanical properties, as compared to use of PL or HS apart.
PMCID: PMC3822844  PMID: 21645237
tissue engineering; heart valve; human; serum; platelet-lysate; cardiovascular
7.  Aortic root dimension changes during systole and diastole: evaluation with ECG-gated multidetector row computed tomography 
Cardiac pulsatility and aortic compliance may result in aortic area and diameter changes throughout the cardiac cycle in the entire aorta. Until this moment these dynamic changes could never be established in the aortic root (aortic annulus, sinuses of Valsalva and sinotubular junction). The aim of this study was to visualize and characterize the changes in aortic root dimensions during systole and diastole with ECG-gated multidetector row computed tomography (MDCT). MDCT scans of subjects without aortic root disease were analyzed. Retrospectively, ECG-gated reconstructions at each 10% of the cardiac cycle were made and analyzed during systole (30–40%) and diastole (70–75%). Axial planes were reconstructed at three different levels of the aortic root. At each level the maximal and its perpendicular luminal dimension were measured. The mean dimensions of the total study group (n = 108, mean age 56 ± 13 years) do not show any significant difference between systole and diastole. The individual dimensions vary up to 5 mm. However, the differences range between minus 5 mm (diastolic dimension is greater than systolic dimensions) and 5 mm (vice versa). This variability is independent of gender, age, height and weight. This study demonstrated a significant individual dynamic change in the dimensions of the aortic root. These results are highly unpredictable. Most of the healthy subjects have larger systolic dimensions, however, some do have larger diastolic dimensions.
PMCID: PMC3230759  PMID: 21359833
Aortic valve; Electrocardiogram (ECG)-gated imaging techniques; Multidetector row computed tomography
8.  Ischemia of the lung causes extensive long-term pulmonary injury: an experimental study 
Respiratory Research  2008;9(1):28.
Lung ischemia-reperfusion injury (LIRI) is suggested to be a major risk factor for development of primary acute graft failure (PAGF) following lung transplantation, although other factors have been found to interplay with LIRI. The question whether LIRI exclusively results in PAGF seems difficult to answer, which is partly due to the lack of a long-term experimental LIRI model, in which PAGF changes can be studied. In addition, the long-term effects of LIRI are unclear and a detailed description of the immunological changes over time after LIRI is missing. Therefore our purpose was to establish a long-term experimental model of LIRI, and to study the impact of LIRI on the development of PAGF, using a broad spectrum of LIRI parameters including leukocyte kinetics.
Male Sprague-Dawley rats (n = 135) were subjected to 120 minutes of left lung warm ischemia or were sham-operated. A third group served as healthy controls. Animals were sacrificed 1, 3, 7, 30 or 90 days after surgery. Blood gas values, lung compliance, surfactant conversion, capillary permeability, and the presence of MMP-2 and MMP-9 in broncho-alveolar-lavage fluid (BALf) were determined. Infiltration of granulocytes, macrophages and lymphocyte subsets (CD45RA+, CD5+CD4+, CD5+CD8+) was measured by flowcytometry in BALf, lung parenchyma, thoracic lymph nodes and spleen. Histological analysis was performed on HE sections.
LIRI resulted in hypoxemia, impaired left lung compliance, increased capillary permeability, surfactant conversion, and an increase in MMP-2 and MMP-9. In the BALf, most granulocytes were found on day 1 and CD5+CD4+ and CD5+CD8+-cells were elevated on day 3. Increased numbers of macrophages were found on days 1, 3, 7 and 90. Histology on day 1 showed diffuse alveolar damage, resulting in fibroproliferative changes up to 90 days after LIRI.
The short-, and long-term changes after LIRI in this model are similar to the changes found in both PAGF and ARDS after clinical lung transplantation. LIRI seems an independent risk factor for the development of PAGF and resulted in progressive deterioration of lung function and architecture, leading to extensive immunopathological and functional abnormalities up to 3 months after reperfusion.
PMCID: PMC2335107  PMID: 18366783

Results 1-8 (8)