Empagliflozin restores the integrity of the endothelial glycocalyx in vitro.

The antihyperglycemic agent empagliflozin not only improves glycemic control but has also been associated with clinically meaningful reductions in cardiovascular events. Studies have shown that empagliflozin significantly reduces cardiovascular death and heart failure-associated hospitalizations. Given that endothelial dysfunction is closely linked with the pathogenesis of atherosclerotic cardiovascular disease, we hypothesized that the cardiovascular benefits observed with empagliflozin may be a result of its positive impact on the health of the endothelial glycocalyx (GCX), a critical component for the endothelium homeostasis. Human abdominal aortic endothelial cells (HAAECs) were either statically cultured or subjected to a steady wall shear stress of 10 dyne/cm2. Empagliflozin (50 µM, 24 h) restored heparinase III-mediated GCX disruption and the normal mechanotransduction responses in GCX-compromised HAAECs while reducing the attachment of all-trans retinoic acid-transformed NB4 cells to HAAECs. The current body of work suggests that the cardioprotective properties previously reported for empagliflozin may in part be due to the ability of empagliflozin to preserve and restore the structural integrity of the GCX, which in turn helps to maintain vascular health by promoting an anti-inflammatory endothelium, in the presence of a pro-inflammatory environment. Further studies are needed to fully understand the mechanisms underlying the cardiovascular benefits of empagliflozin.

Simultaneous imaging of blood flow dynamics and vascular remodelling during development.

Normal vascular development requires blood flow. Time-lapse imaging techniques have revolutionised our understanding of developmental biology, but measuring changes in blood flow dynamics has met with limited success. Ultrasound biomicroscopy and optical coherence tomography can concurrently image vascular structure and blood flow velocity, but these techniques lack the resolution to accurately calculate fluid forces such as shear stress. This is important because hemodynamic forces are biologically active and induce changes in the expression of genes important for vascular development. Regional variations in shear stress, rather than the overall level, control processes such as vessel enlargement and regression during vascular remodelling. We present a technique to concurrently visualise vascular remodelling and blood flow dynamics. We use an avian embryonic model and inject an endothelial-specific dye and fluorescent microspheres. The motion of the microspheres is captured with a high-speed camera and the velocity of the blood flow in and out of the region of interest is quantified by micro-particle image velocitymetry (µPIV). The vessel geometry and flow are used to numerically solve the flow physics with computational fluid dynamics (CFD). Using this technique, we can analyse changes in shear stress, pressure drops and blood flow velocities over a period of 10 to 16 h. We apply this to study the relationship between shear stress and chronic changes in vessel diameter during embryonic development, both in normal development and after TGFß stimulation. This technique allows us to study the interaction of biomolecular and biomechanical signals during vascular remodelling using an in vivo developmental model.

Flow dynamics control the location of sprouting and direct elongation during developmental angiogenesis.

Angiogenesis is tightly controlled by a number of signalling pathways. Although our understanding of the molecular mechanisms involved in angiogenesis has rapidly increased, the role that biomechanical signals play in this process is understudied. We recently developed a technique to simultaneously analyse flow dynamics and vascular remodelling by time-lapse microscopy in the capillary plexus of avian embryos and used this to study the hemodynamic environment present during angiogenic sprouting. We found that sprouts always form from a vessel at lower pressure towards a vessel at higher pressure, and that sprouts form at the location of a shear stress minimum, but avoid locations where two blood streams merge even if this point is at a lower level of shear stress than the sprouting location. Using these parameters, we were able to successfully predict sprout location in quail embryos. We also found that the pressure difference between two vessels is permissive to elongation, and that sprouts will either change direction or regress if the pressure difference becomes negative. Furthermore, the sprout elongation rate is proportional to the pressure difference between the two vessels. Our results show that flow dynamics are predictive of the location of sprout formation in perfused vascular networks and that pressure differences across the interstitium can guide sprout elongation.

Blood flow can signal during angiogenesis not only through mechanotransduction, but also by affecting growth factor distribution.

Growth factors, such as VEGF, promote the sprouting of new blood vessels. Growth factors are generally produced far from the endothelium, and the transport of these proteins is often assumed to occur through diffusion. When sprouting occurs in a perfused vascular bed, however, interstitial flow is present that can modify protein transport. We recently developed a technique to analyze flow dynamics and vascular remodeling simultaneously in avian embryos. In this study, we extend our technique to model interstitial flow through the porous matrix of the mesenchymal tissue and use this to investigate how flow in the blood vessels affects the distribution of growth factors in the mesenchyme, using VEGF as a prototypical angiogenic molecule. We find that flow controls sprouting location and elongation, both through the direct action of mechanical force and through indirect effects on growth factor distribution. Most importantly, we find that the distribution of VEGF is regulated by interstitial flow, and the effect of diffusion of VEGF is negligible.

The effect of simvastatin treatment on endothelial cell response to shear stress and tumor necrosis factor alpha stimulation.

BACKGROUND: Statin drugs are one of the most commonly prescribed pharmaceuticals by physicians. By blocking the rate-limiting step in the cholesterol biosynthesis pathway, statins inhibit cholesterol synthesis, which benefits patient health. However, since many other important cellular processes are regulated within this pathway, they may also be influenced by statin therapy. These pleiotropic effects of statins have not been fully investigated, but are believed to positively influence endothelial cells (ECs), which line the vasculature in a confluent monolayer. Few studies have considered the effect of blood flow on ECs and how this may augment EC response to statins. METHODS: In this study, the effect of statin treatment on ECs is investigated for cells stimulated with tumor necrosis factor alpha (TNF-α), an inflammatory cytokine that promotes an atheroprone endothelium. Additionally, ECs are exposed to a physiologically relevant wall shear stress (WSS) of 12.5 dynes/cm(2) using a three-dimensional tissue culture model to provide a realistic hemodynamic environment. ECs are analyzed for morphology using light microscopy as well as cytoskeletal structure and alignment using confocal microscopy. Statistical analysis is performed on the results using both the one-way analysis of variance with Bonferroni post-tests and the two-tailed t test. RESULTS: We have shown that statin treatment caused cells to adapt to a rounded, atheroprone morphology, with a significantly higher shape index. Oppositely, TNF-α stimulation caused cells to elongate to an atheroprotective morphology, with a significantly lower shape index. WSS and TNF-α were unable to reverse any statin-induced cell rounding or F-actin disruption. CONCLUSION: Further work is therefore needed to determine why statin drugs cause cells to have an atheroprone morphology, but an atheroprotective genotype, and why TNF-α stimulation causes an atheroprotective morphology, but an atheroprone genotype. Despite the morphological changes due to statins or stimulation, ECs still respond to WSS. Understanding how statins influence ECs will allow for more targeted treatments for hypercholestemia and potentially other diseases.

Leaching of the plasticizer di(2-ethylhexyl)phthalate (DEHP) from plastic containers and the question of human exposure.

Di(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizer to render poly(vinyl chloride) (PVC) soft and malleable. Plasticized PVC is used in hospital equipment, food wrapping, and numerous other commercial and industrial products. Unfortunately, plasticizers can migrate within the material and leach out of it over time, ending up in the environment and, frequently, the human body. DEHP has come under increased scrutiny as its breakdown products are believed to be endocrine disruptors and more toxic than DEHP itself. DEHP and its breakdown products have been identified as ubiquitous environmental contaminants, and daily human exposure is estimated to be in the microgram per kilogram level. The objective of this review is to summarize and comment on published sources of DEHP exposure and to give an overview of its environmental fate. Exposure through bottled water was examined specifically, as this concern is raised frequently, yet only little exposure to DEHP occurs through bottled water, and DEHP exposure is unlikely to stem from the packaging material itself. Packaged food was also examined and showed higher levels of DEHP contamination compared to bottled water. Exposure to DEHP also occurs in hospital environments, where DEHP leaches directly into liquids that passed through PVC/DEHP tubing and equipment. The latter exposure is at considerably higher levels compared to food and bottled water, specifically putting patients with chronic illnesses at risk. Overall, levels of DEHP in food and bottled water were below current tolerable daily intake (TDI) values. However, our understanding of the risks of DEHP exposure is still evolving. Given the prevalence of DEHP in our atmosphere and environment, and the uncertainty revolving around it, the precautionary principle would suggest its phaseout and replacement. Increased efforts to develop viable replacement compounds, which necessarily includes rigorous leaching, toxicity, and impact assessment studies, are needed before alternative plasticizers can be adopted as viable replacements.

Empagliflozin reduces endoplasmic reticulum stress associated TXNIP/NLRP3 activation in tunicamycin-stimulated aortic endothelial cells.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have proven to be of therapeutic significance for cardiovascular diseases beyond the treatment of type 2 diabetes. Recent studies have demonstrated the beneficial effects of SGLT2i on endothelial cell (EC) dysfunction, but the underlying cellular mechanisms remain to be clarified. In this study, we sought to understand the effect of empagliflozin (EMPA; Jardiance®) on cell homeostasis and endoplasmic reticulum (ER) stress signaling. ER stress was induced by tunicamycin (Tm) in human abdominal aortic ECs treated with EMPA over 24 h. Tm-induced ER stress caused increases in the protein expression of thioredoxin interacting protein (TXNIP), NLR-family pyrin domain-containing protein 3 (NLRP3), C/EBP homologous protein (CHOP), and in the ratio of phospho-eIF2α/eIF2α. EMPA (50-100 µM) resulted in a dampened downstream activation of ER stress as seen by the reduced expression of CHOP and TXNIP/NLRP3 in a dose-dependent manner. Nuclear factor erythroid 2-related factor 2 (nrf2) translocation was also attenuated in EMPA-treated ECs. These results suggest that EMPA improves redox signaling under ER stress which in turn attenuates the activation of TXNIP/NLRP3.

Ultrasoft edge-labelled hydrogel sensors reveal internal tissue stress patterns in invasive engineered tumors.

Measuring internal mechanical stresses within 3D tissues can provide important insights into drivers of morphogenesis and disease progression. Cell-sized hydrogel microspheres have recently emerged as a powerful technique to probe tissue mechanobiology, as they can be sufficiently soft as to deform within remodelling tissues, and optically imaged to measure internal stresses. However, measuring stresses at resolutions of ∼10 Pa requires ultrasoft, low-polymer content hydrogel formulations that are challenging to label with sufficiently fluorescent materials to support repeated measurements, particularly in optically dense tissues over 100 µm thick, as required in cancer tumor models. Here, we leverage thermodynamic partitioning of hydrogel components to create "edge-labelled" ultrasoft hydrogel microdroplets, in a single polymerization step. Bright and stable fluorescent nanoparticles preferentially polymerize at the hydrogel droplet interface, and can be used to repeatedly track sensor surfaces over long-term experiments, even when embedded deep in light-scattering tissues. We utilize these edge-labelled microspherical stress gauges (eMSGs) in inducible breast cancer tumor models of invasion, and demonstrate distinctive internal stress patterns that arise from cell-matrix interactions at different stages of breast cancer progression. Our studies demonstrate a long-term macroscale compaction of the tumor during matrix encapsulation, but only a short-term increase in local stress as non-invasive tumors rapidly make small internal reorganizations that reduce the mechanical stress to baseline levels. In contrast, once invasion programs are initiated, internal stress throughout the tumor is negligible. These findings suggest that internal tumor stresses may initially prime the cells to invade, but are lost once invasion occurs. Together, this work demonstrates that mapping internal mechanical stress in tumors may have utility in advancing cancer prognostic strategies, and that eMSGs can have broad utility in understanding dynamic mechanical processes of disease and development.

Thoracic Aortic Aneurysm Risk Assessment: A Machine Learning Approach.

Background: Traditional methods of risk assessment for thoracic aortic aneurysm (TAA) based on aneurysm size alone have been called into question as being unreliable in predicting complications. Biomechanical function of aortic tissue may be a better predictor of risk, but it is difficult to determine in vivo. Objectives: This study investigates using a machine learning (ML) model as a correlative measure of energy loss, a measure of TAA biomechanical function. Methods: Biaxial tensile testing was performed on resected TAA tissue collected from patients undergoing surgery. The energy loss of the tissue was calculated and used as the representative output. Input parameters were collected from clinical assessments including observations from medical scans and genetic paneling. Four ML algorithms including Gaussian process regression were trained in Matlab. Results: A total of 158 patients were considered (mean age 62 years, range 22-89 years, 78% male), including 11 healthy controls. The mean ascending aortic diameter was 47 ± 10 mm, with 46% having a bicuspid aortic valve. The best-performing model was found to give a greater correlative measure to energy loss (R2 = 0.63) than the surprisingly poor performance of aortic diameter (R2 = 0.26) and indexed aortic size (R2 = 0.32). An echocardiogram-derived stiffness metric was investigated on a smaller subcohort of 67 patients as an additional input, improving the correlative performance from R2 = 0.46 to R2 = 0.62. Conclusions: A preliminary set of models demonstrated the ability of a ML algorithm to improve prediction of the mechanical function of TAA tissue. This model can use clinical data to provide additional information for risk stratification.

Improving the 3D Printability of Sugar Glass to Engineer Sacrificial Vascular Templates.

A prominent obstacle in scaling up tissue engineering technologies for human applications is engineering an adequate supply of oxygen and nutrients throughout artificial tissues. Sugar glass has emerged as a promising 3D-printable, sacrificial material that can be used to embed perfusable networks within cell-laden matrices to improve mass transfer. To characterize and optimize a previously published sugar ink, we investigated the effects of sucrose, glucose, and dextran concentration on the glass transition temperature (Tg), printability, and stability of 3D-printed sugar glass constructs. We identified a sucrose ink formulation with a significantly higher Tg (40.0 ± 0.9°C) than the original formulation (sucrose-glucose blend, Tg = 26.2 ± 0.4°C), which demonstrated a pronounced improvement in printability, resistance to bending, and final print stability, all without changing dissolution kinetics and decomposition temperature. This formulation allowed printing of 10-cm-long horizontal cantilever filaments, which can enable the printing of complex vascular segments along the x-, y-, and z-axes without the need for supporting structures. Vascular templates with a single inlet and outlet branching into nine channels were 3D printed using the improved formulation and subsequently used to generate perfusable alginate constructs. The printed lattice showed high fidelity with respect to the input geometry, although with some channel deformation after alginate casting and gelation-likely due to alginate swelling. Compared with avascular controls, no significant acute cytotoxicity was noted when casting pancreatic beta cell-laden alginate constructs around improved ink filaments, whereas a significant decrease in cell viability was observed with the original ink. The improved formulation lends more flexibility to sugar glass 3D printing by facilitating the fabrication of larger, more complex, and more stable sacrificial networks. Rigorous characterization and optimization methods for improving sacrificial inks may facilitate the fabrication of functional cellular constructs for tissue engineering, cellular biology, and other biomedical applications.

Use of inert gas jets to measure the forces required for mechanical gene transfection.

BACKGROUND: Transferring genes and drugs into cells is central to how we now study, identify and treat diseases. Several non-viral gene therapy methods that rely on the mechanical disruption of the plasma membrane have been proposed, but the success of these methods has been limited due to a lack of understanding of the mechanical parameters that lead to cell membrane permeability. METHODS: We use a simple jet of inert gas to induce local transfection of plasmid DNA both in vitro (HeLa cells) and in vivo (chicken chorioallantoic membrane). Five different capillary tube inner diameters and three different gases were used to treat the cells to understand the dependency of transfection efficiency on the dynamic parameters. RESULTS: The simple setup has the advantage of allowing us to calculate the forces acting on cells during transfection. We found permeabilization efficiency was related to the dynamic pressure of the jet. The range of dynamic pressures that led to transfection in HeLa cells was small (200 ± 20 Pa) above which cell stripping occurred. We determined that the temporary pores allow the passage of dextran up to 40 kDa and reclose in less than 5 seconds after treatment. The optimized parameters were also successfully tested in vivo using the chorioallantoic membrane of the chick embryo. CONCLUSIONS: The results show that the number of cells transfected with the plasmid scales with the dynamic pressure of the jet. Our results show that mechanical methods have a very small window in which cells are permeabilized without injury (200 to 290 Pa). This simple apparatus helps define the forces needed for physical cell transfection methods.

Effects of di-(2-ethylhexyl) phthalate and four of its metabolites on steroidogenesis in MA-10 cells.

Phthalate plasticizers are used in the plastics industry to aid in processing and impart flexibility to plastics. Due to the broad use of plastics, and the tendency of plasticizers to leach out of polymers, plasticizers have become ubiquitous in the environment. Concerns about the testicular toxicity of phthalate plasticizers, in particular di-(2-ethylhexyl) phthalate (DEHP), have arisen due to their ability to cause male reproductive tract abnormalities in animal models. It has been assumed that the DEHP metabolite, mono-(2-ethylhexyl) phthalate (MEHP), is the active compound, however, metabolites such as 2-ethylhexanol, 2-ethylhexanal and 2-ethylhexanoic acid, have not been thoroughly investigated. The aim of this study was to evaluate the anti-androgenic potential of these metabolites in vitro with a mouse Leydig tumor cell line, MA-10 cells. DEHP, MEHP and 2-ethylhexanal were found to decrease cell viability, as well as steroidogenic potential. The latter was assessed using an enzyme-linked immunosorbent assay (ELISA) to quantify steroid production and quantitative real-time polymerase chain reaction (qRT-PCR) to assess gene expression analysis of key steroidogenic enzymes. 2-Ethylhexanal proved to be the most potent steroidogenic disruptor, offering intriguing implications in the search for the mechanism of phthalate testicular toxicity. Overall, the study suggests the involvement of multiple active metabolites in the testicular toxicity of DEHP.

Empagliflozin mitigates endothelial inflammation and attenuates endoplasmic reticulum stress signaling caused by sustained glycocalyx disruption.

The disruption of the endothelial cell (EC) glycocalyx (GCX) leads to cellular dysfunction promoting inflammation and cardiovascular disease progression. Recent studies have shown that empagliflozin (EMPA; Jardiance), a sodium-glucose cotransporter 2 inhibitor used in the treatment of type 2 diabetes, can improve EC functions impacted by GCX disruption although the exact cellular mechanisms remain to be elucidated. In this study, the effect of EMPA on EC inflammatory response induced by sustained GCX disruption was investigated. Human aortic ECs were cultured under shear (10 dyne/cm2) for 24 h with or without sustained degradation of heparan sulfate (HS). HS degradation increased inflammatory cell adhesion to ECs. EMPA (50 µM) normalized adhesion levels under sustained HS degradation. Protein expressions of eNOS, phospho-eNOS Ser1177 and ICAM-1 remained unchanged between conditions. Transcriptome analysis revealed the induction of the unfolded protein response (UPR) through the increased expression of ATF3, ATF4, DDIT3 (CHOP), EIF2AK3 (PERK), HSPA5 (Grp78), PPP1R15A (GADD34) and TRIB3 which was in part downregulated by EMPA. mRNA and protein expression of thioredoxin interacting protein (TXNIP) was also downregulated by EMPA. Mitigation of oxidative stress with N-Acetyl-L-cysteine resulted in similar reduction in inflammatory cell adhesion compared to EMPA which could indicate a potential mechanism by which EMPA normalized the inflammatory response. In conclusion, this study demonstrated the potential of EMPA to resolve the inflammatory response of ECs caused by sustained GCX disruption while altering UPR signaling under endoplasmic reticulum stress.

Wall stresses of early remodeled pulmonary autografts.

OBJECTIVE: The Ross procedure is an excellent option for children or young adults who need aortic valve replacement because it can restore survival to that of the normal aged-matched population. However, autograft remodeling can lead to aneurysmal formation and reoperation, and the biomechanics of this process is unknown. This study investigated postoperative autograft remodeling after the Ross procedure by examining patient-specific autograft wall stresses. METHODS: Patients who have undergone the Ross procedure who had intraoperative pulmonary root and aortic specimens collected were recruited. Patient-specific models (n = 16) were developed using patient-specific material property and their corresponding geometry from cine magnetic resonance imaging at 1-year follow-up. Autograft ± Dacron for aneurysm repair and ascending aortic geometries were reconstructed to develop patient-specific finite element models, which incorporated material properties and wall thickness experimentally measured from biaxial stretching. A multiplicative approach was used to account for prestress geometry from in vivo magnetic resonance imaging. Pressure loading to systemic pressure (120/80) was performed using LS-DYNA software (LSTC Inc, Livermore, Calif). RESULTS: At systole, first principal stresses were 809 kPa (25%-75% interquartile range, 691-1219 kPa), 567 kPa (485-675 kPa), 637 kPa (555-755 kPa), and 382 kPa (334-413 kPa) at the autograft sinotubular junction, sinuses, annulus, and ascending aorta, respectively. Second principal stresses were 360 kPa (310-426 kPa), 355 kPa (320-394 kPa), 272 kPa (252-319 kPa), and 184 kPa (147-222 kPa) at the autograft sinotubular junction, sinuses, annulus, and ascending aorta, respectively. Mean autograft diameters were 29.9 ± 2.7 mm, 38.3 ± 5.3 mm, and 26.6 ± 4.0 mm at the sinotubular junction, sinuses, and annulus, respectively. CONCLUSIONS: Peak first principal stresses were mainly located at the sinotubular junction, particularly when Dacron reinforcement was used. Patient-specific simulations lay the foundation for predicting autograft dilatation in the future after understanding biomechanical behavior during long-term follow-up.

3D printed ascending aortic simulators with physiological fidelity for surgical simulation.

Introduction: Three-dimensional (3D) printed multimaterial ascending aortic simulators were created to evaluate the ability of polyjet technology to replicate the distensibility of human aortic tissue when perfused at physiological pressures. Methods: Simulators were developed by computer-aided design and 3D printed with a Connex3 Objet500 printer. Two geometries were compared (straight tube and idealised aortic aneurysm) with two different material variants (TangoPlus pure elastic and TangoPlus with VeroWhite embedded fibres). Under physiological pressure, ß Stiffness Index was calculated comparing stiffness between our simulators and human ascending aortas. The simulators' material properties were verified by tensile testing to measure the stiffness and energy loss of the printed geometries and composition. Results: The simulators' geometry had no effect on measured ß Stiffness Index (p>0.05); however, ß Stiffness Index increased significantly in both geometries with the addition of embedded fibres (p<0.001). The simulators with rigid embedded fibres were significantly stiffer than average patient values (41.8±17.0, p<0.001); however, exhibited values that overlapped with the top quartile range of human tissue data suggesting embedding fibres can help replicate pathological human aortic tissue. Biaxial tensile testing showed that fiber-embedded models had significantly higher stiffness and energy loss as compared with models with only elastic material for both tubular and aneurysmal geometries (stiffness: p<0.001; energy loss: p<0.001). The geometry of the aortic simulator did not statistically affect the tensile tested stiffness or energy loss (stiffness: p=0.221; energy loss: p=0.713). Conclusion: We developed dynamic ultrasound-compatible aortic simulators capable of reproducing distensibility of real aortas under physiological pressures. Using 3D printed composites, we are able to tune the stiffness of our simulators which allows us to better represent the stiffness variation seen in human tissue. These models are a step towards achieving better simulator fidelity and have the potential to be effective tools for surgical training.

Distinct Expression of Nonmuscle Myosin IIB in Pulmonary Arteries of Patients With Aortic Stenosis vs Insufficiency Undergoing a Ross Procedure.

BACKGROUND: Clinical studies have revealed a greater risk of pulmonary autograft dilation after the Ross procedure in patients with preoperative aortic insufficiency (AI). The present study examined whether the morphologic, biomechanical, and cellular properties of the pulmonary artery (PA) from patients with AI were phenotypically different compared with patients diagnosed with aortic stenosis (AS). METHODS: PA segments were harvested from patients undergoing the Ross procedure for AS (n = 16) and AI (n = 6). Preoperative aortic annulus was significantly larger (P < 0.05) in patients with AI (28.5 ± 1.8 mm) vs AS (22.8 ± 1.2 mm). Morphologic, biomechanical, and cellular phenotypes of the PA were analyzed. RESULTS: Collagen and elastin content in the media of the PA wall were similar in patients with AS and AI. Elastic modulus and energy loss of the PA were not significantly different between the groups. In the media of the PA, expression of a panel of vascular smooth muscle cell-specific proteins were similar in patients with AS and AI. In contrast, nonmuscle myosin IIB protein levels in the PA of AS patients were significantly higher compared with AI patients, and immunofluorescence identified staining in α-smooth muscle actin-positive vascular smooth muscle cells. CONCLUSIONS: Despite similar morphological and biomechanical properties, the disparate expression of nonmuscle myosin IIB protein distinguishes the PA of patients with AI from patients with AS. The biological role in vascular smooth muscle cells and the potential contribution of nonmuscle myosin IIB to pulmonary autograft dilation in a subset of AI patients after the Ross procedure remain to be determined.

An in vitro Perfused Macroencapsulation Device to Study Hemocompatibility and Survival of Islet-Like Cell Clusters.

Transplantation of hydrogel-encapsulated pancreatic islets is a promising long-term treatment for type 1 diabetes that restores blood glucose regulation while providing graft immunoprotection. Most human-scale islet encapsulation devices that rely solely on diffusion fail to provide sufficient surface area to meet islet oxygen demands. Perfused macroencapsulation devices use blood flow to mitigate oxygen limitations but increase the complexity of blood-device interactions. Here we describe a human-scale in vitro perfusion system to study hemocompatibility and performance of islet-like cell clusters (ILCs) in alginate hydrogel. A cylindrical perfusion device was designed for multi-day culture without leakage, contamination, or flow occlusion. Rat blood perfusion was assessed for prothrombin time and international normalized ratio and demonstrated no significant change in clotting time. Ex vivo perfusion performed with rats showed patency of the device for over 100 min using Doppler ultrasound imaging. PET-CT imaging of the device successfully visualized metabolically active mouse insulinoma 6 ILCs. ILCs cultured for 7 days under static conditions exhibited abnormal morphology and increased activated caspase-3 staining when compared with the perfused device. These findings reinforce the need for convective transport in macroencapsulation strategies and offer a robust and versatile in vitro system to better inform preclinical design.

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.

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 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.