Community pharmacists' comfort levels with and barriers to application of an expanded scope of practice in Québec.

Background: In recent years, community pharmacists have seen their profession transition from a dispensing-focused role to a rapidly evolving clinically oriented practice. In Québec, Bill 31, adopted in 2020, increased the clinical opportunities for pharmacists with independent prescribing privileges in various defined clinical situations. As this expanded role can lead to different barriers, it is crucial to explore pharmacists' comfort levels with implementing such changes in their practice. Methods: A web-based survey was conducted from March 25 to May 28, 2021, among community pharmacists in Québec. We collected data with a questionnaire developed for this study. Questions were grouped into 4 domains: (1) characteristics of the respondents; (2) workload and work setting; (3) comfort level with, and barriers to, adjusting medications and following up pharmacologic treatments (86 clinical situations evaluated); and (4) general barriers and facilitators to implementation. Results: A total of 146 community pharmacists completed the questionnaire. Most were women (71.9%), younger than 50 years of age (86.2%), had a bachelor's degree (64.4%) as their highest academic level and had more than 10 years of experience as pharmacists (56.8%). Most of them worked exclusively in a community pharmacy (86.3%). Among the 86 clinical situations evaluated, there were 16 in which at least 80% of respondents felt comfortable. The main barriers identified were a lack of knowledge, experience and dedicated time and difficulties integrating these activities into the workflow; facilitators were having an adequate environment and resources. Conclusion: This study shows community pharmacists can confidently adjust pharmacotherapy for several conditions. However, they must have adequate time and resources. Also, the more complex the clinical situations were, the less comfortable community pharmacists felt adjusting pharmacotherapy. This study identified several areas where continuing education, training and mentoring could be offered and where the work environment and organization could be improved.

Pharmacists practising in family medicine groups: An evaluation 2 years after experiencing a virtual community of practice.

BACKGROUND: In 2018, a virtual community of practice (CoP) for pharmacists working in family medicine groups (FMGs) in Quebec province was developed. The aim of this CoP-called Réseau Québécois des Pharmaciens GMF (RQP GMF)-was to foster best practices by supporting FMG pharmacists. This study assesses the processes and outcomes of this CoP 2 years after its creation. METHODS: We performed a cross-sectional web-based study from March to May 2020. All FMG pharmacists who were registered as members of the RQP GMF (n = 326) were sent an invitation via a newsletter. The link to the questionnaire was also publicized in the CoP Facebook group. The questionnaire comprised a 38-item validated instrument assessing 8 dimensions of the CoP. A descriptive analysis was performed. RESULTS: A total of 112 FMG pharmacists (34.4%) completed the questionnaire. Respondents agreed that the RQP GMF was a joint enterprise (mean score, 4.18/5), that members shared their knowledge (mean score, 3.94/5) and engaged mutually (mean score, 3.50/5) and that the RQP GMF provided support (mean score, 3.92/5) and capacity building (mean score, 4.01/5). In general, they were satisfied with the implementation process (mean score, 3.68/5) and with activities proposed (mean score, 3.79/5). A lower proportion of respondents agreed that their participation in the RQP GMF generated external impacts, which led to a smaller mean score (3.37/5) for this dimension. CONCLUSION: The RQP GMF, one of the first communities of practice for pharmacists practising in family medicine groups, attained most of the objectives initially intended by the CoP. These results will facilitate the adaptation of processes and activities to better fulfil members' needs. Can Pharm J (Ott) 2021;154:xx-xx.

Pharmacists practising in family medicine groups: What are their activities and needs?

WHAT IS KNOWN AND OBJECTIVE: Pharmacists' responsibilities and practices have expanded over the years to be more clinical. Working in other settings and collaborating with other healthcare professionals can lead to new needs that are unmet by actual training. This study was performed to describe the characteristics and practices of pharmacists working in family medicine groups (FMGs) and to assess their needs to develop a practice-based network fostering best practices. METHODS: A Quebec province-wide cross-sectional study was performed from May to August 2018. Pharmacists practising in FMGs were identified through phone calls to all listed FMGs and via direct emails. All identified pharmacists were emailed an invitation to complete an online questionnaire comprising questions to assess their sociodemographic characteristics, to describe their FMG and to assess their needs to reach an optimal practice. The link to the questionnaire was also publicized in a Facebook group of FMG pharmacists and by several professional organizations. A descriptive analysis was performed and discussed with two committees: a working group of FMG pharmacists and an advisory committee comprising key stakeholders. RESULTS AND DISCUSSION: A total of 299 FMG pharmacists were identified, and 178 (59.5%) completed the online questionnaire. Most were women (71.9%), were less than 40 years old (71.9%) and also practised as community pharmacists (76.4%). Reviewing medication to optimize pharmacotherapy and answering questions related to specific issues were the most frequent activities, with 86.0% and 90.4% of pharmacists, respectively, reporting that they performed these often or very often. The most frequently mentioned needs were training and mentorship adapted to the FMG practice and improvement in the understanding that other healthcare professionals have about the role of the FMG pharmacist. Performing comprehensive medication assessments and developing thorough pharmaceutical care plans were among the clinical competencies that pharmacists wanted to develop. Scientific and interprofessional communication was also among the abilities they wished to optimize. WHAT IS NEW AND CONCLUSION: This study provided unique information about pharmacists practising in FMGs and elicited several needs. The results will inform the development of a practice-based network aimed at fulfilling these needs.

The Creation of a Practice-Based Network of Pharmacists Working in Family Medicine Groups (FMG).

A needs assessment study of pharmacists working in family medicine groups (FMG) demonstrated the necessity to build a practice-based network. This network would foster a faster integration into FMG and a more efficient collaborative practice. It would also take advantage of an existing practice-based research network (PBRN)-the STAT (Soutien Technologique pour l'Application et le Transfert des pratiques novatrices en pharmacie) network. A working group of nine FMG pharmacists from the different regions of the province of Quebec, Canada, and a committee of partners, including the key pharmacy organizations, were created. Between January 2018 and May 2019, nine meetings took place to discuss the needs assessment results and deploy an action plan. The practice-based network first year activities allowed identifying pharmacists working in FMGs across the province. A directory of these pharmacists was published on the STAT network. The vision, mission, mandate, name («Réseau Québécois des Pharmaciens GMF¼) and logo were developed. The first few activities include: Bi-monthly newsletters; a mentorship program; short evidence-based therapeutic letters (pharmacotherapeutic capsules) and a start-up kit to facilitate integration of these pharmacists. The Quebec FMG pharmacist practice-based network has been launched. It is planned to evaluate the members' satisfaction in late Spring 2020 with regards to activities and resources provided.

Effect of BMP-9 on endothelial cells and its role in atherosclerosis.

Atherosclerosis is an inflammatory disease involving dysfunction of endothelial cells (EC) and enhanced permeability of the endothelium to oxidized low-density lipoprotein and the transmigration of monocytes from the blood to the intima where they are transformed into foam cells after lipid engulfment. Changes in the composition of the basement membrane leading to increased fibronectin deposition also occur and modify EC-extracellular matrix (ECM) mechanotransduction. The release of lipids due to foam cell apoptosis, as well as the migration of vascular smooth muscle cells from the media to the intima and their proliferation, increase the stiffness of arteries at later stages of atherosclerosis. EC dysfunction also involves other factors, including soluble cytokines and growth factors (GF) such as bone morphogenetic proteins (BMP). BMP-9 is a potent circulatory GF which has been shown to affect EC behavior. However, to date, few studies have investigated its role in atherosclerosis. The present review describes the histology and homeostasis of arteries by explaining EC function/dysfunction and discusses BMP-9 effect on EC behavior, considering factors engaged in the development of atherosclerosis.

Effect of manufacturing and experimental conditions on the mechanical and surface properties of silicone elastomer scaffolds used in endothelial mechanobiological studies.

BACKGROUND: Mechanobiological studies allow the characterization of cell response to mechanical stresses. Cells need to be supported by a material with properties similar to the physiological environment. Silicone elastomers have been used to produce various in vitro scaffolds of different geometries for endothelial cell studies given its relevant mechanical, optical and surface properties. However, obtaining defined and repeatable properties is a challenge as depending on the different manufacturing and processing steps, mechanical and surface properties may vary significantly between research groups. METHODS: The impact of different manufacturing and processing methods on the mechanical and surface properties was assessed by measuring the Young's modulus and the contact angle. Silicone samples were produced using different curing temperatures and processed with different sterilization techniques and hydrophilization conditions. RESULTS: Different curing temperatures were used to obtain materials of different stiffness with a chosen silicone elastomer, i.e. Sylgard 184®. Sterilization by boiling had a tendency to stiffen samples cured at lower temperatures whereas UV and ethanol did not alter the material properties. Hydrophilization using sulphuric acid allowed to decrease surface hydrophobicity, however this effect was lost over time as hydrophobic recovery occurred. Extended contact with water maintained decreased hydrophobicity up to 7 days. Mechanobiological studies require complete cell coverage of the scaffolds used prior to mechanical stresses exposure. Different concentrations of fibronectin and collagen were used to coat the scaffolds and cell seeding density was varied to optimize cell coverage. CONCLUSION: This study highlights the potential bias introduced by manufacturing and processing conditions needed in the preparation of scaffolds used in mechanobiological studies involving endothelial cells. As manufacturing, processing and cell culture conditions are known to influence cell adhesion and function, they should be more thoroughly assessed by research groups that perform such mechanobiological studies using silicone.

Conjugation of multivalent ligands to gold nanoshells and designing a dual modality imaging probe.

Design and synthesis of branched tetraethylene glycol (TEG) based ligands for subsequent conjugation to gold nanoshells are reported. TEG enhances the aqueous solubility of hollow gold nanoshells (HAuNShs), and the branched architecture provides stability. An examination of the supernatant of the surface displacement reaction shows that the structure of the ligand plays an important role in the functionalization of HAuNShs. The binding of multivalent ligands leads to rupturing of the gold nanoshell architecture; most probably due to the large dendron not compensating the replacement of small citrate capping agents. The construction of a probe with dual imaging capabilities is demonstrated by covalent linking of a dendron containing Cy5.5A dye to gold nanoshells. It leads to fluorescence quenching of Cy5.5A by the gold nanoshells, as evidenced in solution and in cellular internalization studies with J774 and bEnd.3 cells.

Miktoarm star conjugated multifunctional gold nanoshells: synthesis and an evaluation of biocompatibility and cellular uptake.

A simple and highly versatile click chemistry based synthetic strategy to develop an ABC type miktoarm star ligand that is conjugated to gold nanoshells (GNS) is reported. The surface functionalized multifunctional GNS contain lipoic acid (LA) as a model therapeutic agent, poly(ethylene glycol) (PEG350) as a solubilizing and stealth agent, and tetraethylene glycol (TEG) with a terminally conjugated thiol moiety. These GNS have an average size of 40 nm, a shell thickness of 6 nm, a well-defined crystal structure lattice (111), and a surface absorption plasmon band in the near infrared (NIR) region. The miktoarm star and GNS functionalized with this ligand are non-cytotoxic for up to 5 µg mL-1 concentrations, and human umbilical vein endothelial cells internalize more than 85% of these GNS at 5 µg mL-1. Our results establish that the biocompatible miktoarm star ligand provides a useful platform to synthetically articulate the introduction of multiple functions onto GNS, and enhance their scope by combining their inherent imaging capabilities with efficient delivery and accumulation of active therapeutic agents.

VCAM-1-targeting gold nanoshell probe for photoacoustic imaging of atherosclerotic plaque in mice.

The development of molecular probes and novel imaging modalities, allowing better resolution and specificity, is associated with an increased potential for molecular imaging of atherosclerotic plaques especially in basic and pre-clinical research applications. In that context, a photoacoustic molecular probe based on gold nanoshells targeting VCAM-1 in mice (immunonanoshells) was designed. The molecular probe was validated in vitro and in vivo, showing no noticeable acute toxic effects. We performed the conjugation of gold nanoshells displaying near-infrared absorption properties with VCAM-1 antibody molecules and PEG to increase their biocompatibility. The resulting immunonanoshells obtained under different conditions of conjugation were then assessed for specificity and sensitivity. Photoacoustic tomography was performed to determine the ability to distinguish gold nanoshells from blood both in phantoms and in vivo. Ex vivo optical projection tomography of hearts and aortas from atherosclerotic and control mice confirmed the selective accumulation of the immunonanoshells in atherosclerotic-prone regions in mice, thus validating the utility of the probe in vivo in small animals for pre-clinical research. These immunonanoshells represent an adequate mean to target atherosclerotic plaques in small animals, leading to new tools to follow the effect of therapies on the progression or regression of the disease.

Regional variations in canine descending aortic tissue mechanical properties change with formalin fixation.

BACKGROUND/INTRODUCTION: Diseases of the aorta can alter the local mechanical properties of the tissue, leading to aneurysms and plaque instability. Local tissue properties are best evaluated from surgical samples or autopsy tissue using mechanical testing ex vivo. We examined whether formalin-fixed tissues preserve regional and local variations in tissue properties when compared to fresh tissues in order to determine if fixed tissue can be used to infer mechanical changes associated with tissue remodeling. METHODS: Equibiaxial mechanical tests were performed on canine descending thoracic aorta to quantify the regional and local tissue stiffness. Samples were taken from four locations along the aorta and from the lateral and medial side at each location. Half of the samples were randomly formalin fixed and used to measure the effect of fixation on local thickness, stiffness, and anisotropy. RESULTS: In fresh tissue, regional differences in tissue stiffness and thickness are present. Aortic tissue stiffens and thins along the aorta. Fixation did not alter thickness, significantly increased tissue stiffness, and altered the directional dependency of the mechanical properties (anisotropy) at low strain. Formalin fixation altered local stiffness of the aorta near the aortic arch. CONCLUSION: The changes in mechanical properties along the aorta were preserved in formalin-fixed samples. However, our results show that formalin fixation can change the variation in tissue stiffness and significantly affects the anisotropic properties of vascular tissues. Formalin fixation introduces spurious changes in mechanical properties, and we therefore strongly recommend the use of fresh aortic tissues for biomechanical analysis.

Low-cost three-dimensional imaging system combining fluorescence and ultrasound.

In this paper, we present a dual-modality imaging system combining three-dimensional (3D) continuous-wave transillumination fluorescence tomography with 3D ultrasound (US) imaging. We validated the system with two phantoms, one containing fluorescent inclusions (Cy5.5) at different depths, and another varying-thickness semicylindrical phantom. Using raster scanning, the combined fluorescence/US system was used to collect the boundary fluorescent emission in the X-Y plane, as well as recovered the 3D surface and position of the inclusions from US signals. US images were segmented to provide soft priors for the fluorescence image reconstruction. Phantom results demonstrated that with priors derived from the US images, the fluorescent reconstruction quality was significantly improved. As further evaluation, we show pilot in vivo results using an Apo-E mouse to assess the feasibility and performance of this system in animal studies. Limitations and potential to be used in artherosclerosis studies are then discussed.

Differential response of endothelial cells to simvastatin when conditioned with steady, non-reversing pulsatile or oscillating shear stress.

Few studies have investigated whether fluid mechanics can impair or enhance endothelial cell response to pharmacological agents such as statin drugs. We evaluated and compared Kruppel-like factor 2 (KLF2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) expression in human abdominal aortic endothelial cells (HAAEC) treated with increasing simvastatin concentrations (0.1, 1 or 10 µM) under static culture and shear stress (steady, non-reversing pulsatile, and oscillating). Simvastatin, steady flow, and non-reversing pulsatile flow each separately upregulated KLF2, eNOS, and TM mRNA. At lower simvastatin concentrations (0.1 and 1 µM), the combination of statin and unidirectional steady or pulsatile flow produced an overall additive increase in mRNA levels. At higher simvastatin concentration (10 µM), a synergistic increase in eNOS and TM mRNA expression was observed. In contrast, oscillating flow impaired KLF2 and TM, but not eNOS expression by simvastatin at 1 µM. A higher simvastatin concentration of 10 µM overcame the inhibitory effect of oscillating flow. Our findings suggest that oscillating shear stress renders the endothelial cells less responsive to simvastatin than cells exposed to unidirectional steady or pulsatile flow. Consequently, the pleiotropic effects of statins in vivo may be less effective in endothelial cells exposed to atheroprone hemodynamics.

Laminar shear stress prevents simvastatin-induced adhesion molecule expression in cytokine activated endothelial cells.

In addition to lowering cholesterol, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, have been shown to modulate gene expression in endothelial cells. The effect of statins on cell adhesion molecule expression is unclear and largely unexplored in endothelial cells exposed to shear stress, an important regulator of endothelial cell function. In this study, the effect of simvastatin on vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression was evaluated in human abdominal aortic endothelial cells (HAAEC) conditioned with various levels of laminar wall shear stress with or without tumor necrosis factor alpha (TNFα). As expected, TNFα alone greatly enhanced both VCAM-1 and ICAM-1 mRNA and protein. In static culture, simvastatin potentiated the TNFα-induced increase in VCAM-1 and ICAM-1 mRNA but not total protein at 24 h. Mevalonate, a precursor to cholesterol biosynthesis, eliminated the effect of simvastatin. Exposure of endothelial cells to elevated levels of laminar shear stress during simvastatin treatment prevented the potentiating effect of simvastatin on cell adhesion molecule mRNA. A shear stress of 12.5 dyn/cm² eliminated the increase in VCAM-1 by simvastatin, while 25 dyn/cm² was needed for ICAM-1. We conclude that simvastatin enhances VCAM-1 and ICAM-1 gene expression in TNFα-activated endothelial cells through inhibition of HMG-CoA reductase. High levels of laminar shear stress prevented the upregulation of VCAM-1 and ICAM-1 by simvastatin suggesting that an induction of cell adhesion molecules by statins may not occur in endothelial cells exposed to shear stress from blood flow.

Endothelial cell morphologic response to asymmetric stenosis hemodynamics: effects of spatial wall shear stress gradients.

Endothelial cells are known to respond to hemodynamic forces. Their phenotype has been suggested to differ between atheroprone and atheroprotective regions of the vasculature, which are characterized by the local hemodynamic environment. Once an atherosclerotic plaque has formed in a vessel, the obstruction creates complex spatial gradients in wall shear stress. Endothelial cell response to wall shear stress may be linked to the stability of coronary plaques. Unfortunately, in vitro studies of the endothelial cell involvement in plaque stability have been limited by unrealistic and simplified geometries, which cannot reproduce accurately the hemodynamics created by a coronary stenosis. Hence, in an attempt to better replicate the spatial wall shear stress gradient patterns in an atherosclerotic region, a three dimensional asymmetric stenosis model was created. Human abdominal aortic endothelial cells were exposed to steady flow (Re=50, 100, and 200 and tau=4.5 dyn/cm(2), 9 dyn/cm(2), and 18 dyn/cm(2)) in idealized 50% asymmetric stenosis and straight/tubular in vitro models. Local morphological changes that occur due to magnitude, duration, and spatial gradients were quantified to identify differences in cell response. In the one dimensional flow regions, where flow is fully developed and uniform wall shear stress is observed, cells aligned in flow direction and had a spindlelike shape when compared with static controls. Morphological changes were progressive and a function of time and magnitude in these regions. Cells were more randomly oriented and had a more cobblestone shape in regions of spatial wall shear stress gradients. These regions were present, both proximal and distal, at the stenosis and on the wall opposite to the stenosis. The response of endothelial cells to spatial wall shear stress gradients both in regions of acceleration and deceleration and without flow recirculation has not been previously reported. This study shows the dependence of endothelial cell morphology on spatial wall shear stress gradients and demonstrates that care must be taken to account for altered phenotype due to geometric features. These results may help explain plaque stability, as cells in shoulder regions near an atherosclerotic plaque had a cobblestone morphology indicating that they may be more permeable to subendothelial transport and express prothrombotic factors, which would increase the risk of atherothrombosis.

The response of human aortic endothelial cells in a stenotic hemodynamic environment: effect of duration, magnitude, and spatial gradients in wall shear stress.

Inflammation plays a key role in the development and stability of coronary plaques. Endothelial cells alter their expression in response to wall shear stress (WSS). Straight/tubular and asymmetric stenosis models were designed to study the localized expression of atheroprone molecules and inflammatory markers due to the presence of the spatial wall shear stress gradients created by an eccentric plaque. The effects of steady wall shear stress duration (0-24 h) and magnitude (4.5-18 dynes/cm(2)) were analyzed in human abdominal aortic endothelial cells through quantitative real-time polymerase chain reaction (PCR) and immunofluorescence analysis in straight/tubular models. Regional expression was assessed by immunofluorescence and confocal microscopy in stenosis models. Under steady fully developed flow, endothelial cells exhibited a sustained increase in levels of atheroprotective genes with WSS duration and magnitude. The local response in the stenosis model showed that expression of endothelial nitric oxide synthase and Kruppel-like factor 2 is magnitude rather than gradient dependent. A WSS magnitude dependent transient increase in translocation of transcription factor nuclear factor kappaB was observed. Intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin exhibited a sustained increase in protein expression with time. The mRNA levels of these molecules were transiently upregulated and this was followed by a decrease in expression to levels lower than static controls. Regionally, increased inflammatory marker expression was observed in regions of WSS gradients both proximal and distal to the stenosis when compared with the uniform flow regions, whereas the atheroprotective markers were expressed to a greater extent in regions of elevated WSS magnitudes. The results from the straight/tubular model cannot explain the regional variation seen in the stenosis models. This may help explain the localization of inflammatory cells at the shoulders of plaques in vivo.

Effect of simvastatin on Kruppel-like factor2, endothelial nitric oxide synthase and thrombomodulin expression in endothelial cells under shear stress.

AIMS: Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) and fluid wall shear stress have been reported to modulate the expression of genes related to inflammation, blood coagulation, thrombosis, and vascular constriction in cultured endothelial cells. In this study, we investigated the combined effect of laminar shear stress (LSS) and statins on endothelial cell gene expression. MAIN METHODS: Kruppel-like factor 2 (KLF2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) mRNA and protein expression were evaluated in human abdominal aortic endothelial cells (HAAEC) treated with simvastatin (0.1, 1 or 10 microM) at various levels of LSS (0, 1.25, 12.5 or 25 dynes/cm(2)). KEY FINDINGS: As expected, simvastatin and LSS separately enhanced KLF2, eNOS, and TM mRNA expressions. The combination of simvastatin and LSS resulted in significantly higher mRNA levels of all three genes compared to cells treated with LSS only. The highest KLF2, eNOS, and TM mRNA levels were detected at 10 microM simvastatin and 25 dynes/cm(2). Under these conditions, eNOS and TM protein levels were also elevated. Combining LSS and simvastatin produced an overall additive increase in KLF2, eNOS, and TM mRNA. Treatment of the endothelial cells with 10 microM simvastatin and 200 microM mevalonate completely eliminated the effect of simvastatin. SIGNIFICANCE: Our results suggest an additive increase in KLF2, eNOS, and TM expressions when simvastatin and LSS are combined. These results may help to explain the proposed non-lipid lowering benefits of statins observed in the clinic.

Neutrophil adhesion on endothelial cells in a novel asymmetric stenosis model: effect of wall shear stress gradients.

Leukocytes play a pivotal role in the progression of atherosclerosis. A novel three-dimensional in vitro asymmetric stenosis model was used to better investigate the role of local hemodynamics in the adhesion of leukocytes to an established plaque. The adhesion of a human promyelocytic cell line (NB4) on a human abdominal aortic endothelial cell (EC) monolayer was quantified. NB4 cells were circulated over TNF-alpha stimulated and nonstimulated ECs for 1 or 6 h at 1.25 or 6.25 dynes/cm(2) and compared to static conditions. Cytokine stimulation increased significantly EC expression of intercellular adhesion molecule and vascular cell adhesion molecule. Under static conditions, neutrophils adhered overall more than under flow, with decreased adhesion with increasing shear. Adhesion was significantly higher in the recirculation region distal to the stenosis than in the inlet. Preshearing the ECs decreased the expression of cell adhesion molecules in inflamed endothelium and significantly decreased adhesion. However, the ratio of adhesion between the recirculation zone and the inlet increased, hence exhibiting an increased regional difference. This work suggests an important role for neutrophil-EC interactions in the atherosclerotic process, especially in wall shear stress gradient regions. This is important clinically, potentially helping to explain plaque stability.

Concentration and time effects of dextran exposure on endothelial cell viability, attachment, and inflammatory marker expression in vitro.

Dextran is commonly used to alter growth medium rheological properties for in vitro flow experiments in order to match physiological parameters. Despite its acceptance in literature, few studies have examined dextran effects on cells. In this study, we investigated changes in endothelial cell function due to dextran, under static and flow conditions, in a concentration and time-dependent manner. Dextran increased endothelial cell viability, decreased their ability to attach to culture plates and decreased leukocyte adhesion to endothelial cells. Under static conditions, dextran increased protein and mRNA expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in a concentration and time-dependent manner and caused the nuclear translocation of NF-kappaB. Steady laminar wall shear stress modulated the effects of dextran on ICAM-1, VCAM-1, and NF-kappaB expression in straight/tubular in vitro models. When the expression was normalized to their respective time matched static dextran control, it did not affect the ability to detect changes caused by shear on the mRNA expression of ICAM-1 and VCAM-1. This study demonstrates that dextran can alter endothelial cell function and therefore, caution is advised and time matched dextran controls are necessary when using dextran for dynamic cell studies.

The development of 3-D, in vitro, endothelial culture models for the study of coronary artery disease.

The response of the vascular endothelium to wall shear stress plays a central role in the development and progression of atherosclerosis. Current studies have investigated endothelial response using idealized in vitro flow chambers. Such cell culture models are unable to accurately replicate the complex in vivo wall shear stress patterns arising from anatomical geometries. To better understand this implication, we have created both simplified/tubular and anatomically realistic in vitro endothelial flow models of the human right coronary artery. A post-mortem vascular cast of the human left ventricular outflow tract was used to create geometrically accurate silicone elastomer models. Straight, tubular models were created using a custom made mold. Following the culture of human abdominal aortic endothelial cells within the inner lumen, cells were exposed to steady flow (Re = 233) for varying time periods. The resulting cell morphology was analyzed in terms of shape index and angle of orientation relative to the flow direction. In both models a progressive elongation and alignment of the endothelium in the flow direction was observed following 8, 12, and 24 hours. This change, however, was significantly less pronounced in the anatomical model (as observed from morphological variations indicative of localized flow features). Differences were also observed between the inner and outer walls at the disease-prone proximal region. Since morphological adaptation is a visual indication of endothelial shear stress activation, the use of anatomical models in endothelial genetic and biochemical studies may offer better insight into the disease process.

Local mechanical and structural properties of healthy and diseased human ascending aorta tissue.

OBJECTIVE: This study investigates the mechanics and histology of healthy and dilated human ascending aortas (AA). The regional variation in mechanical response and tissue structure were compared. METHODS: Rings of human AA from healthy (n=5), dilated tricuspid aortic valve (TAV, n=5), and dilated bicuspid aortic valve (BAV, n=6) patients were mechanically tested. Each aortic ring was sectioned into quadrants-anterior, posterior, medial (inner curvature) and lateral (outer curvature). Low- and high-stress elastic moduli were calculated from the equibiaxial stress strain curve to determine the local mechanical properties. Histological analysis was used to quantify the percent composition of elastin, collagen, and smooth muscle cells. RESULTS: BAV tissue was thinnest and contained the largest percent composition of collagen. Both TAV and BAV tissue had significantly less elastin than healthy tissue. At low strain in the circumferential direction, TAV tissue was on average the least stiff. The elastic modulus was dependent on quadrant and tissue type but not direction (isotropic). Generally, the lateral quadrant tissue was the stiffest and the medial quadrant the least stiff. There were no apparent local variations in the tissue histology. CONCLUSIONS: Local variations in tissue thickness and mechanical properties were evident in all samples analyzed and may be linked to the type of aortic valve present.