Kottamia Faint Imaging Spectro-Polarimeter (KFISP): opto-mechanical design, software control and performance analysis.

In this paper we describe the Kottamia Faint Imaging Spectro-Polarimeter (KFISP) that has been recently developed and designed to be mounted at the Cassegrain focus of the 1.88 m telescope at Kottamia Astronomical Observatory (KAO), Egypt. The optical design of KFISP is developed such that it can be used in various modes of operation. These are: direct imaging, spectroscopic, polarimetric imaging, and spectro-polarimetric. The KFISP is an all-refractive design to meet the polarimetric requirements and includes a focal reducer with a corrector section, collimator section, parallel beam section (containing various imaging components), and camera section. The corrector section gives an unvignetted Field-of-View of 8' × 8' and the collimator section has a focal length of 305 mm and matches the focal ratio of the input beam. The parallel beam section is 200 mm long and near the middle of it there is an image of the telescope pupil. The camera section includes 5 elements and has a focal length of 154.51 mm which gives an instrument effective final focal ratio of f/6.14 (acting as a telescope focal reducer of 1:2 ratio). The KFISP contains an internal calibration system which hosts the calibration light injection system, an integrating sphere equipped with the required calibration light sources. The opto-mechanical parts of KFISP contain a double-layered carbon fiber strut structure and comprises its subsystems of slit and guider assemblies, filter wheel drawer, grism wheel drawer, polarimetric components cubical box, and CCD camera which is integrated with camera optics. The CCD camera has 2048 × 2048 pixels with 13.5-micron square pixel size. The camera is cooled by liquid Nitrogen and is fixed to the KFISP through the integrated camera lens. The KFISP has been fully commissioned, mounted and is being tested in all modes of operation. In this paper we introduce the ambitious scientific goals, the optical setups of KFISP, its opto-mechanical implementation and the performance analysis of the instrument. In addition, we describe the camera system, its performance, and its software control. Finally, we present a sample of the first light observations obtained from the instrument.

Correction to: Drug Delivery from an Innovative LAMA/LABA Co-suspension Delivery Technology Fixed-Dose Combination MDI: Evidence of Consistency, Robustness, and Reliability.

This article was originally published Online First without open access. After publication it was discovered that the author had ordered open access during the production process. The incorrect license was assigned to this paper due to a technical error.

Drug Delivery from an Innovative LAMA/LABA Co-suspension Delivery Technology Fixed-Dose Combination MDI: Evidence of Consistency, Robustness, and Reliability.

To ensure consistency of clinical outcomes, orally inhaled therapies must exhibit consistent delivered dose and aerosol properties at the time of manufacturing, throughout storage, and during various patient-use conditions. Achieving consistency across these scenarios has presented a significant challenge, especially for combination products that contain more than one drug. This study characterized the delivered dose and aerosol properties of glycopyrrolate/formoterol fumarate metered dose inhaler (GFF MDI; Bevespi Aerosphere™). GFF MDI, a fixed-dose combination (FDC) of a long-acting muscarinic antagonist, glycopyrrolate (18 µg, equivalent to glycopyrronium 14.4 µg), and a long-acting ß2-agonist, formoterol fumarate (9.6 µg; equivalent to formoterol fumarate dihydrate 10 µg), is formulated using innovative co-suspension delivery technology, which suspends micronized drug crystals with spray-dried phospholipid porous particles in hydrofluoroalkane propellant. In this study, delivered dose uniformity was assessed through the labeled number of doses, and aerosol properties, such as percent fine particle fraction (FPF) and mass median aerodynamic diameter, were determined by cascade impaction. GFF MDI achieved reproducible dose delivery and an FPF greater than 55%, whether formulated and delivered as a monocomponent or dual FDC. The performance of GFF MDI was maintained across various manufacturing batches, under extended storage, and with variations in flow rate. Furthermore, unlike a GFF drug crystal-only suspension, drug delivery remained consistent for GFF MDI when simulated patient-handling errors were applied, such as reduced shake energy and delays between shaking and actuation. These results demonstrate that co-suspension delivery technology overcomes well-known sources of variability in MDI drug delivery.

Biomechanical forces promote embryonic haematopoiesis.

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41(+)c-Kit(+) haematopoietic progenitor cells, concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the para-aortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.

Metroticket approach in a retrospective cohort study to predict overall survival after surgical resection for intermediate stage hepatocellular carcinoma.

BACKGROUND: Surgical resection is a curative therapy for early-stage hepatocellular carcinoma (HCC) patients meeting the Milan criteria as well as a widely used therapy in intermediate-stage HCC. However, intermediate-stage HCC encompasses a wide spectrum of disease and there is a lack of good predictive models for the long-term clinical outcome of HCC patients currently. Here, we adopt Mazzaferro's Metroticket 2.0 to create a robust survival prediction model for intermediate-stage HCC patients undergoing surgical resection. Our algorithm considers age, AFP levels, ALBI score, and nodule size/number to generate survival estimates in an accessible graph format. Importantly, our model surpasses the American Joint Committee on Cancer staging model and was validated with independent US patient data. METHODS: We conducted a retrospective analysis of OS and RFS in early- and intermediate-stage HCC patients treated with liver resection, including a training cohort in Singapore and a validation cohort in North Carolina, USA. RESULTS: We recorded 278 deaths (35.0%) and 428 patients (53.9%) in the first 5-years after surgical resection; higher ALBI score, higher lnAFP, more advanced age and higher tumour burden index were identified as significant parameters. The overall predictive capability of our model, with the inclusion of AFP, is reflected with a UNO's C-statistic of 0.655, which is 1.11 times better than the 0.5895 C-statistic of the 8th AJCC TNM Staging model. CONCLUSIONS: Our modified Metroticket model allows for more granular and better-informed prognostication. This will help surgeons and patients make accurate comparisons between the clinical outcomes of surgical resection and other non-surgical treatments.

Extending Surgical Resection for Hepatocellular Carcinoma Beyond Barcelona Clinic for Liver Cancer (BCLC) Stage A: A Novel Application of the Modified BCLC Staging System.

Objective: We aimed to prognosticate survival after surgical resection of HCC stratified by stage with amalgamation of the modified Barcelona Clinic Liver Cancer (BCLC) staging system and location of tumour. Methods: This single-institutional retrospective cohort study included patients with HCC who underwent surgical resection between 1st January 2000 to 30th June 2016. Participants were divided into 6 different subgroups: A-u) Within MC with Unilobar lesions; A-b) Within MC + Bilobar lesions; B1-u) Out of MC + within Up-To-7 + Unilobar lesions; B1-b) Out of MC + within Up-to-7 + Bilobar lesions; B2-u) Out of MC + Out of Up-To-7 + Unilobar lesions; B2-b) Out of MC + Out of Up-To-7 + Bilobar lesions. A separate survival analysis was conducted for solitary HCC lesions according to three subgroups: A-S (Within MC); B1-S (Out of MC + within Up-To-7); B2-S (Out of MC + out of Up-To-7). Results: A total of 794 of 1043 patients with surgical resection for HCC were analysed. Groups A-u (64.6%), A-b (58.4%) and B1-u (56.2%) had 5-year cumulative overall survival (OS) rates above 50% after surgical resection and median OS exceeding 60 months (P = 0.0001). The 5-year cumulative recurrence-free survival rates (RFS) were 40.4% (group A-u), 38.2% (group A-b), 36.3% (group B1-u), 24.6% (group B2-u), and 7.3% (group B2-b)(P=0.0001). For solitary lesions, the 5-year OS for the subgroups were A-S (65.1%), B1-S (56.0%) and B2-S (47.1%) (P = 0.0003). Compared to A-S, there was also a significant trend towards relatively poorer OS as the lesion sizes increased in B1-S (HR 1.46, 95% CI 1.03-2.08) and B2-S (HR 1.65, 95% CI 1.25-2.18). Conclusion: We adopted a novel approach combining the modified BCLC B sub-classification and dispersion of tumour to show that surgical resection in intermediate stage HCC can be robustly prognosticated. We found that size prognosticates resection outcomes in solitary tumours.

Consistent Pulmonary Drug Delivery with Whole Lung Deposition Using the Aerosphere Inhaler: A Review of the Evidence.

Metered dose inhalers (MDIs) are one of the most common device types for delivering inhaled therapies. However, there are several technical challenges in development and drug delivery of these medications. In particular, suspension-based MDIs are susceptible to suspension heterogeneity, in vitro drug-drug interactions, and patient handling errors, which may all affect drug delivery. To overcome these challenges, new formulation approaches are required. The AerosphereTM inhaler, formulated using co-suspension delivery technology, combines drug crystals with porous phospholipid particles to create stable, homogenous suspensions that dissolve once they reach the airways. Two combination therapies using this technology have been developed for the treatment of COPD: glycopyrrolate/formoterol fumarate (GFF MDI; dual combination) and budesonide/glycopyrrolate/formoterol fumarate (BGF MDI; triple combination). Here, we review the evidence with a focus on studies assessing dose delivery, lung deposition, and effects on airway geometry. In vitro assessments have demonstrated that the Aerosphere inhaler provides consistent dose delivery, even in the presence of simulated patient handling errors. Combination therapies delivered with this technology also show a consistent fine particle fraction (FPF) and an optimal particle size distribution for delivery to the central and peripheral airways even when multiple drugs are delivered via the same inhaler. Studies using gamma scintigraphy and functional respiratory imaging have demonstrated that GFF MDI is effectively deposited in the central and peripheral airways, and provides clinically meaningful benefits on airway volume and resistance throughout the lung. Overall, studies suggest that the Aerosphere inhaler, formulated using co-suspension delivery technology, may offer advantages over traditional formulations, including consistent delivery of multiple components across patient handling conditions, optimal particle size and FPF, and effective delivery to the central and peripheral airways. Future studies may provide additional evidence to further characterize the clinical benefits of these technical improvements in MDI drug delivery.

Functional endothelialized microvascular networks with circular cross-sections in a tissue culture substrate.

Functional endothelialized networks constitute a critical building block for vascularized replacement tissues, organ assist devices, and laboratory tools for in vitro discovery and evaluation of new therapeutic compounds. Progress towards realization of these functional artificial vasculatures has been gated by limitations associated with the mechanical and surface chemical properties of commonly used microfluidic substrate materials and by the geometry of the microchannels produced using conventional fabrication techniques. Here we report on a method for constructing microvascular networks from polystyrene substrates commonly used for tissue culture, built with circular cross-sections and smooth transitions at bifurcations. Silicon master molds are constructed using an electroplating process that results in semi-circular channel cross-sections with smoothly varying radii. These master molds are used to emboss polystyrene sheets which are then joined to form closed bifurcated channel networks with circular cross-sections. The mechanical and surface chemical properties of these polystyrene microvascular network structures enable culture of endothelial cells along the inner lumen. Endothelial cell viability was assessed, documenting nearly confluent monolayers within 3D microfabricated channel networks with rounded cross-sections.

Cell migration into scaffolds under co-culture conditions in a microfluidic platform.

Capillary morphogenesis is a complex cellular process that occurs in response to external stimuli. A number of assays have been used to study critical regulators of the process, but those assays are typically limited by the inability to control biochemical gradients and to obtain images on the single cell level. We have recently developed a new microfluidic platform that has the capability to control the biochemical and biomechanical forces within a three dimensional scaffold coupled with accessible image acquisition. Here, the developed platform is used to evaluate and quantify capillary growth and endothelial cell migration from an intact cell monolayer. We also evaluate the endothelial cell response when placed in co-culture with physiologically relevant cell types, including cancer cells and smooth muscle cells. This resulted in the following observations: cancer cells can either attract (MTLn3 cancer cell line) endothelial cells and induce capillary formation or have minimal effect (U87MG cancer cell line) while smooth muscle cells (10T 1/2) suppress endothelial activity. Results presented demonstrate the capabilities of this platform to study cellular morphogenesis both qualitatively and quantitatively while having the advantage of enhanced imaging and internal biological controls. Finally, the platform has numerous applications in the study of angiogenesis, or migration of other cell types including tumor cells, into a three-dimensional scaffold or across an endothelial layer under precisely controlled conditions of mechanical, biochemical and co-culture environments.

An Open-Label Study Evaluating the Performance of the Dose Indicator in a Metered Dose Inhaler Delivering Glycopyrrolate and Formoterol Fumarate in Patients with Moderate-to-Very Severe Chronic Obstructive Pulmonary Disease.

BACKGROUND: GFF MDI is a glycopyrrolate/formoterol fumarate fixed-dose combination metered dose inhaler formulated using co-suspension delivery technology. This open-label, single-arm multicenter study (NCT02268396) evaluated the accuracy, reliability, and functionality of the GFF MDI AeroCount® dose indicator when used by patients with chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: The study enrolled subjects (40-80 years of age) with an established clinical history (≥6 months) of COPD, who completed an electronic diary twice daily to record study-drug administration time, the number of actuations used, and pre- and post-dose dose indicator readings. The primary endpoint was the percentage of devices for which the number of subject-reported actuations was consistent (±20 actuations) with the dose indicator-based actuation count (equal to 130 minus the dose indicator reading) at the end of the treatment period (4 weeks). Safety was monitored throughout the study. RESULTS: A total of 138 subjects with moderate-to-very severe COPD (50.7% male; mean [standard deviation (SD)] age 62.1 [8.3] years) were enrolled and treated. Subject-reported actuation count and dose indicator-based actuation counts were consistent for 96.4% (132/137) of devices at the end of the treatment period (4 weeks) in the intent-to-treat (ITT) population and for all devices in the per-protocol (PP) population. The mean (SD) dose indicator-based actuation and subject-reported actuation counts in the ITT population (n = 137) were 113.4 (18.9) and 117.0 (19.0), respectively, with a mean (SD) difference of 3.6 (7.9). The mean (SD) dose indicator-based actuation and subject-reported actuation counts in the PP population (n = 112) were 116.8 (8.7) and 119.7 (8.1), respectively. There were no unexpected safety findings. CONCLUSIONS: This study supported the accuracy, reliability, and utility of the dose indicator integrated into the GFF MDI device when used by patients with COPD.

Perceiving patterns in random series: dynamic processing of sequence in prefrontal cortex.

We demonstrate that regions within human prefrontal cortex develop moment-to-moment models for patterns of events occurring in the sensory environment. Subjects viewed a random binary sequence of images, each presented singly and each requiring a different button press response. Patterns occurred by chance within the presented series of images. Using functional magnetic resonance imaging (fMRI), we identified activity evoked by viewing a stimulus that interrupted a pattern. Prefrontal activation was evoked by violations of both repeating and alternating patterns, and the amplitude of this activation increased with increasing pattern length. Violations of repeating patterns, but not of alternating patterns, activated the basal ganglia.

Infraclavicular and supraclavicular approaches to brachial plexus for ambulatory elbow surgery: A randomized controlled observer-blinded trial.

STUDY OBJECTIVE: To compare the effectiveness of supraclavicular and infraclavicular approaches to brachial plexus block for elbow surgery. DESIGN: Prospective, parallel arm, observer-blinded, randomized controlled trial. SETTING: This study occurred in a designated block room at St. Joseph's hospital, a large academic tertiary hospital in London, Canada. PATIENTS: 150 adult ASA class I-III patients undergoing elective ambulatory elbow surgery. INTERVENTIONS: Patients were randomized to receive either an ultrasound-guided infraclavicular or a supraclavicular block with ropivacaine. MEASUREMENTS: Both groups were assessed for performance and sensory block onset times. Motor block, effective surgical anesthesia, procedure-related pain, axillary nerve block and ulnar nerve sparing were additional outcomes. We analyzed continuous and non-continuous variables with the independent t-test and chi-square test respectively and considered statistical significance when type 1 error was under 0.05. MAIN RESULTS: We observed similar mean block procedure times at 285 (±128) seconds in infra and 307 (±138) seconds in supra group (p = 0.3). The mean time of sensory block onset in both groups was similar: Infra 20.4 (±7.9) and supra 18.9 (±7.1) min (p = 0.4). Conversion to general anesthesia (4.2 vs 5.5%; p = 0.73) and the need for local anesthetic supplement (4.2 vs 4.1%; p = 0.98) was similar in both groups. We observed an increased incidence of paresthesia in the supra group (8.3 vs 23.2%; p = 0.014). CONCLUSION: We found that both blocks were equally effective for elbow surgery with similar procedure and block onset times and failure rates. Lower incidence of paresthesia was associated with the infraclavicular block with no change in other complications compared to the supraclavicular technique.

Role of fenestration and resection for symptomatic solitary liver cysts.

BACKGROUND: Symptomatic liver cysts can be managed surgically by fenestration or by hepatic resection. The present study was designed to investigate the current role of each surgical technique in the management of this benign condition. METHODS: Forty consecutive patients with symptomatic liver cyst (non-parasitic and non-malignant) surgically treated by fenestration or resection were identified from a prospectively collected database. An analysis of primary outcome measures including operative parameters, morbidity and mortality rates, length of postoperative stay and recurrence rates in months was carried out. RESULTS: The laparoscopic fenestration group had the best perioperative outcome. At median follow up of 20 months, there were no recurrences in the resection group but recurrence occurred in 6/27 (22%) in the fenestration group. Four of these recurrences were asymptomatic and were managed conservatively while two symptomatic recurrences required a resection. CONCLUSION: Laparoscopic fenestration is the best treatment for symptomatic liver cysts as the primary operation. It is associated with the lowest blood loss, lowest morbidity and shortest hospital stay. Liver resection is best reserved for recurrent symptomatic cysts and cystic lesions suspicious of tumours where it can be safely performed and associated with a zero recurrence rate.

Dental age estimation standards for a Western Australian population.

Age estimation in the juvenile skeleton primarily relies on the assessment of the degree of dental and skeletal development relative to full maturity. The timing of the mineralization and eruption of the teeth is a sequential process that, compared to skeletal growth and development, is less affected by extrinsic influences such as nutrition and/or chronic illness. Accordingly, radiographic visualization and analysis of different tooth formation stages are the foundation for a number of widely applied age estimation standards. Presently, however, there is a relative paucity of contemporary dental age estimation standards for a Western Australian population. To that end, the aim of the present study is to develop statistically quantified radiographic age estimation standards for a Western Australian juvenile population. A total of 392 digital orthopantomograms (202 male and 190 female) of Western Australian individuals are analyzed. Following, Moorrees et al. (J. Dent. Res. 42 (1963a) 490-502; Am. J. Phys. Anthropol. 21 (1963) 205-213), dental development and root resorption was assessed. Alveolar eruption was analyzed following Bengston (Northwest Univ. Bull. 35 (1935) 3-9). Stages of dental development were used to formulate a series of age estimation polynomial regression models; prediction accuracy (±0.998 to 2.183 years) is further validated using a cross-validation (holdout) sample of 30 film orthopantomograms. A visual atlas of dental development and eruption was subsequently designed for the pooled sex sample. The standards presented here represent a non-invasive and statistically quantified approach for accurate dental age estimation in Western Australian juvenile individuals.

Faces evoke spatially differentiated patterns of BOLD activation and deactivation.

Using fMRI techniques sensitive to blood oxygen-level dependent (BOLD) contrast, we measured brain activity in participants (n=8) as they viewed images of faces presented periodically within a continuously changing montage of common objects. Consistent with prior studies, we identified regions of ventral extrastriate cortex, primarily in the fusiform and inferior temporal gyri and nearby cortex, that were activated by faces as measured by an increase in BOLD signal. In addition, we made the novel observation that faces deactivated other areas of ventral extrastriate cortex, primarily in the lingual and parahippocampal gyri and medial to activations. These deactivated regions, identified by a decrease in BOLD signal, may reflect populations of neurons that decrease their activity when faces appear, possibly as a consequence of category-specific inhibition.

Age estimation standards for a Western Australian population using the dental age estimation technique developed by Kvaal et al.

In the present global socio-political scenario, an increasing demand exists for age estimation in living persons, such as refugees and asylum seekers, who seldom have any documentation for proof of identity. Age estimation in the living poses significant challenges because the methods need to be non-invasive, accurate and ethically viable. Methods based on the analysis of the pulp chamber are recommended for age estimation in living adults. There is, however, a paucity of studies of this nature and population specific standards in Western Australia. The aim of the present study is therefore, to test the reliability and applicability of the method developed by Kvaal et al. (1995) for the purpose of developing age estimation standards for an adult Western Australian population. A total of 279 digital orthopantomograms (143 female; and 136 male) of Australian individuals were analysed. A subset of the total sample (50) was removed as a cross-validation (holdout) sample. Following the method described in Kvaal et al. (1995), length and width measurements of the tooth and pulp chamber were acquired in maxillary central and lateral incisors; second premolars, mandibular lateral incisors; canines and first premolars. Those measurements were then used to calculate a series of ratios (length and width), which were subsequently used to formulate age estimation regression models. The most accurate model based on a single tooth was for the maxillary central incisor (SEE ±9.367 years), followed by the maxillary second premolar (SEE ±9.525 years). Regression models based on the measurement of multiple teeth improved age prediction accuracy (SEE ±7.963 years). The regression models presented here have expected accuracy rates comparable (if not higher than) to established skeletal morphoscopic methods. This method, therefore, offers a statistically quantified methodological approach for forensic age estimation in Western Australian adults.

Age estimation standards for a Western Australian population using the coronal pulp cavity index.

Age estimation is a vital aspect in creating a biological profile and aids investigators by narrowing down potentially matching identities from the available pool. In addition to routine casework, in the present global political scenario, age estimation in living individuals is required in cases of refugees, asylum seekers, human trafficking and to ascertain age of criminal responsibility. Thus robust methods that are simple, non-invasive and ethically viable are required. The aim of the present study is, therefore, to test the reliability and applicability of the coronal pulp cavity index method, for the purpose of developing age estimation standards for an adult Western Australian population. A total of 450 orthopantomograms (220 females and 230 males) of Australian individuals were analyzed. Crown and coronal pulp chamber heights were measured in the mandibular left and right premolars, and the first and second molars. These measurements were then used to calculate the tooth coronal index. Data was analyzed using paired sample t-tests to assess bilateral asymmetry followed by simple linear and multiple regressions to develop age estimation models. The most accurate age estimation based on simple linear regression model was with mandibular right first molar (SEE ±8.271 years). Multiple regression models improved age prediction accuracy considerably and the most accurate model was with bilateral first and second molars (SEE ±6.692 years). This study represents the first investigation of this method in a Western Australian population and our results indicate that the method is suitable for forensic application.

Microfabricated engineered particle systems for respiratory drug delivery and other pharmaceutical applications.

Particle Replication in Non-Wetting Templates (PRINT(®)) is a platform particle drug delivery technology that coopts the precision and nanoscale spatial resolution inherently afforded by lithographic techniques derived from the microelectronics industry to produce precisely engineered particles. We describe the utility of PRINT technology as a strategy for formulation and delivery of small molecule and biologic therapeutics, highlighting previous studies where particle size, shape, and chemistry have been used to enhance systemic particle distribution properties. In addition, we introduce the application of PRINT technology towards respiratory drug delivery, a particular interest due to the pharmaceutical need for increased control over dry powder characteristics to improve drug delivery and therapeutic indices. To this end, we have produced dry powder particles with micro- and nanoscale geometric features and composed of small molecule and protein therapeutics. Aerosols generated from these particles show attractive properties for efficient pulmonary delivery and differential respiratory deposition characteristics based on particle geometry. This work highlights the advantages of adopting proven microfabrication techniques in achieving unprecedented control over particle geometric design for drug delivery.

Hemodynamic environments from opposing sides of human aortic valve leaflets evoke distinct endothelial phenotypes in vitro.

The regulation of valvular endothelial phenotypes by the hemodynamic environments of the human aortic valve is poorly understood. The nodular lesions of calcific aortic stenosis (CAS) develop predominantly beneath the aortic surface of the valve leaflets in the valvular fibrosa layer. However, the mechanisms of this regional localization remain poorly characterized. In this study, we combine numerical simulation with in vitro experimentation to investigate the hypothesis that the previously documented differences between valve endothelial phenotypes are linked to distinct hemodynamic environments characteristic of these individual anatomical locations. A finite-element model of the aortic valve was created, describing the dynamic motion of the valve cusps and blood in the valve throughout the cardiac cycle. A fluid mesh with high resolution on the fluid boundary was used to allow accurate computation of the wall shear stresses. This model was used to compute two distinct shear stress waveforms, one for the ventricular surface and one for the aortic surface. These waveforms were then applied experimentally to cultured human endothelial cells and the expression of several pathophysiological relevant genes was assessed. Compared to endothelial cells subjected to shear stress waveforms representative of the aortic face, the endothelial cells subjected to the ventricular waveform showed significantly increased expression of the "atheroprotective" transcription factor Kruppel-like factor 2 (KLF2) and the matricellular protein Nephroblastoma overexpressed (NOV), and suppressed expression of chemokine Monocyte-chemotactic protein-1 (MCP-1). Our observations suggest that the difference in shear stress waveforms between the two sides of the aortic valve leaflet may contribute to the documented differential side-specific gene expression, and may be relevant for the development and progression of CAS and the potential role of endothelial mechanotransduction in this disease.

Biomechanical Regulation of Endothelium-dependent Events Critical for Adaptive Remodeling.

Alterations in hemodynamic shear stress acting on the vascular endothelium are critical for adaptive arterial remodeling. The molecular mechanisms regulating this process, however, remain largely uncharacterized. Here, we sought to define the responses evoked in endothelial cells exposed to shear stress waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells. A lumped parameter model of the human coronary collateral circulation was used to simulate normal and adaptive remodeling coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC), and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling to identify genes distinctly regulated by collateral flow. Analysis of these transcriptional programs identified several genes to be differentially regulated by collateral flow, including genes important for endothelium-smooth muscle interactions. In particular, the transcription factor KLF2 was up-regulated by the adaptive remodeling coronary collateral waveform, and several of its downstream targets displayed the expected modulation, including the down-regulation of connective tissue growth factor. To assess the effect of endothelial KLF2 expression on smooth muscle cell migration, a three-dimensional microfluidic assay was developed. Using this three-dimensional system, we showed that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared with control EC and that this reduction can be rescued by the addition of exogenous connective tissue growth factor. Collectively, these results demonstrate that collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling.