Valve endothelial-interstitial interactions drive emergent complex calcific lesion formation in vitro.

OBJECTIVE: Calcific aortic valve disease (CAVD) is an actively regulated degenerative disease process. Clinical lesions exhibit marked 3D complexity not represented in current in vitro systems. We here present a unique mechanically stressed 3D culture system that recapitulates valve interstitial cell (VIC) induced matrix calcification through myofibroblastic activation and osteoblastic differentiation. We test the hypothesis that valve endothelial (VEC) - interstitial collaborative interactions modulate the risk and complexity of calcific pathogenesis within mechanically stressed and pro-inflammatory environments. APPROACH AND RESULTS: Porcine aortic valve endothelial and interstitial cells (VEC and VIC) were seeded in a mechanically constrained collagen hydrogels alone or in co-culture configurations. Raised 3D VIC-filled lesions formed within 7 days when cultured in osteogenic media (OGM), and surprisingly exacerbated by endothelial coculture. We identified a spatially coordinated pro-endochondral vs. pro-osteogenic signaling program within the lesion. VEC underwent Endothelial-to-Mesenchymal Transformation (EndMT) and populated the lesion center. The spatial complexity of molecular and cellular signatures of this 3D in vitro CAVD system were consistent with human diseased aortic valve histology. SNAI1 was highly expressed in the VEC and subendothelial direct VIC corroborates with human CAVD lesions. Spatial distribution of Sox9 vs. Runx2 expression within the developed lesions (Sox9 peri-lesion vs. Runx2 predominantly within lesions) mirrored their expression in heavily calcified human aortic valves. Finally, we demonstrate the applicability of this platform for screening potential pharmacologic therapies through blocking the canonical NFκB pathway via BAY 11-7082. CONCLUSIONS: Our results establish that VEC actively induce VIC pathological remodeling and calcification via EndMT and paracrine signaling. This mechanically constrained culture platform enables the interrogation of accelerated cell-mediated matrix remodeling behavior underpinned by this cellular feedback circuit. The high fidelity of this complex 3D model system to human CAVD mechanisms supports its use to test mechanisms of intercellular communication in valves and their pharmacological control.

Crystallinity of hydroxyapatite drives myofibroblastic activation and calcification in aortic valves.

Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease. STATEMENT OF SIGNIFICANCE: We implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease.

Valve interstitial cell tensional homeostasis directs calcification and extracellular matrix remodeling processes via RhoA signaling.

AIMS: Valve interstitial cells are active and aggressive players in aortic valve calcification, but their dynamic mediation of mechanically-induced calcific remodeling is not well understood. The goal of this study was to elucidate the feedback loop between valve interstitial cell and calcification mechanics using a novel three-dimensional culture system that allows investigation of the active interplay between cells, disease, and the mechanical valve environment. METHODS & RESULTS: We designed and characterized a novel bioreactor system for quantifying aortic valve interstitial cell contractility in 3-D hydrogels in control and osteogenic conditions over 14 days. Interstitial cells demonstrated a marked ability to exert contractile force on their environment and to align collagen fibers with the direction of tension. Osteogenic environment disrupted interstitial cell contractility and led to disorganization of the collagen matrix, concurrent with increased αSMA, TGF-ß, Runx2 and calcific nodule formation. Interestingly, RhoA was also increased in osteogenic condition, pointing to an aberrant hyperactivation of valve interstitial cells mechanical activity in disease. This was confirmed by inhibition of RhoA experiments. Inhibition of RhoA concurrent with osteogenic treatment reduced pro-osteogenic signaling and calcific nodule formation. Time-course correlation analysis indicated a significant correlation between interstitial cell remodeling of collagen fibers and calcification events. CONCLUSIONS: Interstitial cell contractility mediates internal stress state and organization of the aortic valve extracellular matrix. Osteogenesis disrupts interstitial cell mechanical phenotype and drives disorganization, nodule formation, and pro-calcific signaling via a RhoA-dependent mechanism.

Distance learning improves attainment of professional milestones in the early years of surgical training.

OBJECTIVES: To assess the impact of a surgical sciences e-learning programme in supporting the academic development of surgical trainees during their preparation for professional examination. BACKGROUND: In 2007, a 3-year online part-time Master of Surgical Sciences (MSc) degree programme was launched, utilizing an innovative platform with virtual case scenarios based on common surgical conditions addressed by the curriculum relating to the Membership Examination of the Royal Colleges of Surgeons (MRCS). Multiple-choice questions with feedback and discussion boards facilitated by expert clinical tutors provided formative assessment. Summative assessment comprised written examination at the end of each of the first 2 years (equivalent to MRCS level), culminating in submission of a research dissertation in year 3 toward an MSc. METHODS: Students' age, gender, and level at entry to the programme were documented. Anonymized student feedback from 2008 to 2012 was examined using online questionnaires, and performance in the MSc programme was compared to MRCS examination outcomes for students who had consented to release of their results. RESULTS: A total of 517 surgical trainees from 40 countries were recruited over the 6-year period, and 116 MSc students have graduated to date. Of 368 students, 279 (76%) were foundation doctors (interns) and had not commenced formal surgical training on enrolling in the MSc programme. However, level at entry did not influence performance (P > 0.05 across all 3 years). Average pass rates since the programme launched, for those students completing all of the required assessments, were 84% ± 11% in year 1, 85% ± 10% in year 2, and 88% ± 7% in year 3 of the MSc programme. MSc students had significantly higher MRCS pass rates than nonenrolled trainees (67% vs 51%, P < 0.01, n = 352). There was a significant correlation between MRCS examination performance and overall performance in the MSc (R = 58%; P < 0.01, n = 37). Of 248 respondents, 202 (81%) considered that the MSc would improve their chances of gaining a surgical training post, and 224 (90%) would recommend the programme to their peers. CONCLUSIONS: The online MSc programme supports academic development of trainees in the early years of surgical training, is well received by students, and is associated with improved success in their professional examination.

On the prediction of monocyte deposition in abdominal aortic aneurysms using computational fluid dynamics.

In abdominal aortic aneurysm disease, the aortic wall is exposed to intense biological activity involving inflammation and matrix metalloproteinase-mediated degradation of the extracellular matrix. These processes are orchestrated by monocytes and rather than affecting the aorta uniformly, damage and weaken focal areas of the wall leaving it vulnerable to rupture. This study attempts to model numerically the deposition of monocytes using large eddy simulation, discrete phase modelling and near-wall particle residence time. The model was first applied to idealised aneurysms and then to three patient-specific lumen geometries using three-component inlet velocities derived from phase-contrast magnetic resonance imaging. The use of a novel, variable wall shear stress-limiter based on previous experimental data significantly improved the results. Simulations identified a critical diameter (1.8 times the inlet diameter) beyond which significant monocyte deposition is expected to occur. Monocyte adhesion occurred proximally in smaller abdominal aortic aneurysms and distally as the sac expands. The near-wall particle residence time observed in each of the patient-specific models was markedly different. Discrete hotspots of monocyte residence time were detected, suggesting that the monocyte infiltration responsible for the breakdown of the abdominal aortic aneurysm wall occurs heterogeneously. Peak monocyte residence time was found to increase with aneurysm sac size. Further work addressing certain limitations is needed in a larger cohort to determine clinical significance.

Comparison of patient-specific inlet boundary conditions in the numerical modelling of blood flow in abdominal aortic aneurysm disease.

Three inlet boundary condition datasets were derived from phase-contrast MRI: (i) centre line velocity data converted to two-dimensional (2D) velocity profile using Womersley equations (Womersley), (ii) 2D velocity profile with one axial component of velocity (1CV), (iii) 2D velocity profile with three components of velocity (3CV). Computational fluid dynamics was performed using a rigid wall approach with geometry data extracted from the computed tomography dataset. Helical flow was present in the 1CV and 3CV simulations, with more complex patterns for the 3CV case. The Womersley method produced simplified flow patterns with an absence of helical flow. Mean values of quantitative indices (helical flow index, mean wall shear stress, oscillatory index) were compared with the 3CV inlet data. These were lower for both the Womersley inlet data (28%, 71%, 56%) and the 1CV inlet data (9%, 24%, 69%). It was concluded that inlet methods based on centre line velocity, such as might be obtained from Doppler ultrasound, lead to significantly simplified abdominal aortic aneurysm haemodynamics and thus are not recommended. Single velocity component (axial) data from MRI might suffice when general flow characteristics and spatial wall shear stress are required. Ideally 2D MRI velocity profiles with 3-velocity component data are preferred to fully account for helical flow.

In vivo mononuclear cell tracking using superparamagnetic particles of iron oxide: feasibility and safety in humans.

BACKGROUND: Cell therapy is an emerging and exciting novel treatment option for cardiovascular disease that relies on the delivery of functional cells to their target site. Monitoring and tracking cells to ensure tissue delivery and engraftment is a critical step in establishing clinical and therapeutic efficacy. The study aims were (1) to develop a Good Manufacturing Practice-compliant method of labeling competent peripheral blood mononuclear cells with superparamagnetic particles of iron oxide (SPIO), and (2) to evaluate its potential for magnetic resonance cell tracking in humans. METHODS AND RESULTS: Peripheral blood mononuclear cells 1-5 × 10(9) were labeled with SPIO. SPIO-labeled cells had similar in vitro viability, migratory capacity, and pattern of cytokine release to unlabeled cells. After intramuscular administration, up to 10(8) SPIO-labeled cells were readily identifiable in vivo for at least 7 days using magnetic resonance imaging scanning. Using a phased-dosing study, we demonstrated that systemic delivery of up to 10(9) SPIO-labeled cells in humans is safe, and cells accumulating in the reticuloendothelial system were detectable on clinical magnetic resonance imaging. In a healthy volunteer model, a focus of cutaneous inflammation was induced in the thigh by intradermal injection of tuberculin. Intravenously delivered SPIO-labeled cells tracked to the inflamed skin and were detectable on magnetic resonance imaging. Prussian blue staining of skin biopsies confirmed iron-laden cells in the inflamed skin. CONCLUSIONS: Human peripheral blood mononuclear cells can be labeled with SPIO without affecting their viability or function. SPIO labeling for magnetic resonance cell tracking is a safe and feasible technique that has major potential for a range of cardiovascular applications including monitoring of cell therapies and tracking of inflammatory cells. Clinical Trial Registration- URL: http://www.clinicaltrials.gov; Unique identifier: NCT00972946, NCT01169935.

The mechanobiology of mitral valve function, degeneration, and repair.

In degenerative valve disease, the highly organized mitral valve leaflet matrix stratification is progressively destroyed and replaced with proteoglycan rich, mechanically inadequate tissue. This is driven by the actions of originally quiescent valve interstitial cells that become active contractile and migratory myofibroblasts. While treatment for myxomatous mitral valve disease in humans ranges from repair to total replacement, therapies in dogs focus on treating the consequences of the resulting mitral regurgitation. The fundamental gap in our understanding is how the resident valve cells respond to altered mechanical signals to drive tissue remodeling. Despite the pathological similarities and high clinical occurrence, surprisingly little mechanistic insight has been gleaned from the dog. This review presents what is known about mitral valve mechanobiology from clinical, in vivo, and in vitro data. There are a number of experimental strategies already available to pursue this significant opportunity, but success requires the collaboration between veterinary clinicians, scientists, and engineers.

Cyclic strain anisotropy regulates valvular interstitial cell phenotype and tissue remodeling in three-dimensional culture.

Many planar connective tissues exhibit complex anisotropic matrix fiber arrangements that are critical to their biomechanical function. This organized structure is created and modified by resident fibroblasts in response to mechanical forces in their environment. The directionality of applied strain fields changes dramatically during development, aging, and disease, but the specific effect of strain direction on matrix remodeling is less clear. Current mechanobiological inquiry of planar tissues is limited to equibiaxial or uniaxial stretch, which inadequately simulates many in vivo environments. In this study, we implement a novel bioreactor system to demonstrate the unique effect of controlled anisotropic strain on fibroblast behavior in three-dimensional (3-D) engineered tissue environments, using aortic valve interstitial fibroblast cells as a model system. Cell seeded 3-D collagen hydrogels were subjected to cyclic anisotropic strain profiles maintained at constant areal strain magnitude for up to 96 h at 1 Hz. Increasing anisotropy of biaxial strain resulted in increased cellular orientation and collagen fiber alignment along the principal directions of strain and cell orientation was found to precede fiber reorganization. Cellular proliferation and apoptosis were both significantly enhanced under increasing biaxial strain anisotropy (P<0.05). While cyclic strain reduced both vimentin and alpha-smooth muscle actin compared to unstrained controls, vimentin and alpha-smooth muscle actin expression increased with strain anisotropy and correlated with direction (P<0.05). Collectively, these results suggest that strain field anisotropy is an independent regulator of fibroblast cell phenotype, turnover, and matrix reorganization, which may inform normal and pathological remodeling in soft tissues.

Abdominal aortic aneurysm growth predicted by uptake of ultrasmall superparamagnetic particles of iron oxide: a pilot study.

BACKGROUND: Abdominal aortic aneurysms are a major cause of death. Prediction of aneurysm expansion and rupture is challenging and currently relies on the simple measure of aneurysm diameter. Using MRI, we aimed to assess whether areas of cellular inflammation correlated with the rate of abdominal aortic aneurysm expansion. METHODS AND RESULTS: Stable patients (n=29; 27 male; age, 70±5 years) with asymptomatic abdominal aortic aneurysms (4.0 to 6.6 cm) were recruited from a surveillance program and imaged using a 3-T MRI scanner before and 24 to 36 hours after administration of ultrasmall superparamagnetic particles of iron oxide (USPIO). The change in T2* value on T2*-weighted imaging was used to detect accumulation of USPIO within the abdominal aortic aneurysm. Histological examination of aneurysm tissue confirmed colocalization and uptake of USPIO in areas with macrophage infiltration. Patients with distinct mural uptake of USPIO had a 3-fold higher growth rate (n=11, 0.66 cm/y; P=0.020) than those with no (n=6, 0.22 cm/y) or nonspecific USPIO uptake (n=8, 0.24 cm/y) despite having similar aneurysm diameters (5.4±0.6, 5.1±0.5, and 5.0±0.5 cm, respectively; P>0.05). In 1 patient with an inflammatory aneurysm, there was a strong and widespread uptake of USPIO extending beyond the aortic wall. CONCLUSIONS: Uptake of USPIO in abdominal aortic aneurysms identifies cellular inflammation and appears to distinguish those patients with more rapidly progressive abdominal aortic aneurysm expansion. This technique holds major promise as a new method of risk-stratifying patients with abdominal aortic aneurysms that extends beyond the simple anatomic measure of aneurysm diameter. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00794092.

Early career choices and successful career progression in surgery in the UK: prospective cohort studies.

BACKGROUND: Changes to the structure of medical training worldwide require doctors to decide on their career specialty at an increasingly early stage after graduation. We studied trends in career choices for surgery, and the eventual career destinations, of UK graduates who declared an early preference for surgery. METHODS: Postal questionnaires were sent, at regular time intervals after qualification, to all medical qualifiers from all UK medical schools in selected qualification years between 1974 and 2005. They were sent in the first year after qualification, at year three and five years after qualification, and at longer time intervals thereafter. RESULTS: Responses were received from 27,749 of 38,280 doctors (73%) at year one, 23,468 of 33,151 (71%) at year three, and 17,689 of 24,870 (71%) at year five. Early career preferences showed that surgery has become more popular over the past two decades. Looking forward from early career choice, 60% of respondents (64% of men, 48% of women) with a first preference for a surgical specialty at year one eventually worked in surgery (p < 0.001 for the male-female comparison). Looking backward from eventual career destinations, 90% of responders working in surgery had originally specified a first choice for a surgical specialty at year one. 'Match' rates between eventual destinations and early choices were much higher for surgery than for other specialties. Considering factors that influenced early specialty choice 'a great deal', comparing aspiring surgeons and aspiring general practitioners (GPs), a significantly higher percentage who chose surgery than general practice specified enthusiasm for the specialty (73% vs. 53%), a particular teacher or department (34% vs. 12%), inclinations before medical school (20% vs. 11%), and future financial prospects (24% vs. 13%); and a lower percentage specified that hours and working conditions had influenced their choice (21% vs. 71%). Women choosing surgery were influenced less than men by their inclinations before medical school or by their future financial prospects. CONCLUSIONS: Surgery is a popular specialty choice in the UK. The great majority of doctors who progressed in a surgical career made an early and definitive decision to do so.

The effect of surgical subspecialization on outcomes in peptic ulcer disease complicated by perforation and bleeding.

BACKGROUND: Emergency surgical services in Edinburgh were restructured in July 2002 to deliver subspecialist management of colorectal and upper-gastrointestinal emergencies on separate sites. The effect of emergency subspecialization on outcome from perforated and bleeding peptic ulceration was assessed. METHODS: All patients admitted with complicated peptic ulceration (January 2000-February 2005) were identified from a prospectively compiled database. RESULTS: Perforation: 148 patients were admitted with perforation before the service reorganization (period A - 31 months) of whom 126 (85.1%) underwent surgery; 135 patients were admitted in period B (31 months) of whom 114 (84.4%) were managed operatively. The in-hospital mortality was lower in period B (14/135, 10.4%) than period A (30/148, 20.3%; P = 0.023; relative risk (RR), 0.51; 95% confidence interval (CI), 0.28-0.91). There was a significantly higher rate of gastric resection in the second half of the study (period A 1/126 vs. period B 8/114; P = 0.015; RR, 8.84; 95% CI, 1.48-54.34). Length of hospital stay was similar for both groups. Bleeding: 51 patients underwent operative management of bleeding peptic ulceration in period A and 51 in period B. There were no differences in length of stay or mortality between these two groups. CONCLUSION: Restructuring of surgical services with emergency subspecialization was associated with lower mortality for perforated peptic ulceration. Subspecialist experience, intraoperative decision-making, and improved postoperative care have all contributed to this improvement.