Design and Analysis of a Biodegradable Polycaprolactone Flow Diverting Stent for Brain Aneurysms

The flow diverting stent (FDS) has become a promising endovascular device for the treatment of aneurysms. This research presents a novel biodegradable and non-braided Polycaprolactone (PCL) FDS. The PCL FDS was designed and developed using an in-house fabrication unit and coated on two ends with BaSO4 for angiographic visibility. The mechanical flexibility and quality of FDS surfaces were examined with the UniVert testing machine, scanning electron microscope (SEM), and 3D profilometer. Human umbilical vein endothelial cell (HUVEC) adhesion, proliferation, and cell morphology studies on PCL FDS were performed. The cytotoxicity and NO production by HUVECs with PCL FDS were also conducted. The longitudinal tensile, radial, and bending flexibility were found to be 1.20 ± 0.19 N/mm, 0.56 ± 0.11 N/mm, and 0.34 ± 0.03 N/mm, respectively. The FDS was returned to the original shape and diameter after repeated compression and bending without compromising mechanical integrity. Results also showed that the proliferation and adhesion of HUVECs on the FDS surface increased over time compared to control without FDS. Lactate dehydrogenase (LDH) release and NO production showed that PCL FDS were non-toxic and satisfactory. Cell morphology studies showed that HUVECs were elongated to cover the FD surface and developed an endothelial monolayer. This study is a step forward toward the development and clinical use of biodegradable flow diverting stents for endovascular treatment of the aneurysm.

Fluid flow exposure promotes epithelial-to-mesenchymal transition and adhesion of breast cancer cells to endothelial cells

Background Mechanical interactions between tumor cells and microenvironments are frequent phenomena during breast cancer progression, however, it is not well understood how these interactions affect Epithelial-to-Mesenchymal Transition (EMT). EMT is associated with the progression of most carcinomas through induction of new transcriptional programs within affected epithelial cells, resulting in cells becoming more motile and adhesive to endothelial cells. Methods MDA-MB-231, SK-BR-3, BT-474, and MCF-7 cells and normal Human Mammary Epithelial Cells (HMECs) were exposed to fluid flow in a parallel-plate bioreactor system. Changes in expression were quantified using microarrays, qPCR, immunocytochemistry, and western blots. Gene–gene interactions were elucidated using network analysis, and key modified genes were examined in clinical datasets. Potential involvement of Smads was investigated using siRNA knockdown studies. Finally, the ability of flow-stimulated and unstimulated cancer cells to adhere to an endothelial monolayer, migrate and invade membrane pores was evaluated in flow and static adhesion experiments. Results Fluid flow stimulation resulted in upregulation of EMT inducers and downregulation of repressors. Specifically, Vimentin and Snail were upregulated both at the gene and protein expression levels in flow stimulated HMECs and MDA-MB-231 cells, suggesting progression towards an EMT phenotype. Flow-stimulated SNAI2 was abrogated with Smad3 siRNA. Flow-induced overexpression of a panel of cell adhesion genes was also observed. Network analysis revealed genes involved in cell flow responses including FN1, PLAU, and ALCAM. When evaluated in clinical datasets, overexpression of FN1, PLAU, and ALCAM was observed in patients with different subtypes of breast cancer. We also observed increased adhesion, migration and invasion of flow-stimulated breast cancer cells compared to unstimulated controls. Conclusions This study shows that fluid forces on the order of 1 Pa promote EMT and adhesion of breast cancer cells to an endothelial monolayer and identified biomarkers were distinctly expressed in patient populations. A better understanding of how biophysical forces such as shear stress affect cellular processes involved in metastatic progression of breast cancer is important for identifying new molecular markers for disease progression, and for predicting metastatic risk.

Development of personalized cell-populated vascular graft in vitro

Aim. To create a personalized cell-populated small-diameter vascular prosthesis in a pulsating bioreactor.Methods. Tubular grafts were made by electrospinning from mixtures of biodegradable polymers, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(εcaprolactone) (PCL). The inner surface is modified with fibrin. Tubular scaffolds were colonized with cultured colony-forming endothelial cells and grown under static conditions for 2 days. Then, the cell-populated prostheses continued to be cultivated for 5 days in a pulsating bioreactor system with a final shear stress of 2.85 dynes/cm².Results. The advantages of the cultivation of cell-populated vascular prostheses in a pulsating bioreactor have been revealed. The selected mode of cultivation of cellpopulated vascular prostheses under conditions of a pulsating flow with a shear stress of 2.85 dynes/cm² did not have a damaging effect on the integrity of the endothelial monolayer. Moving unidirectional mechanical stimuli of chaotic orientation fibers of F-actin changed to a predominant orientation in the direction of flow, and also increased the expression of F-actin, Talin focal adhesion protein, and specific endothelial markers CD309, CD31, vWF.Conclusion. The creation of a personalized cell-populated small-diameter vascular prosthesis with a functional endothelial monolayer is possible due to the use of autologous endothelial cells, autologous fibrin, and cultivation under conditions of a pulsating flow.

Comparison of Leading Biosensor Technologies to Detect Changes in Human Endothelial Barrier Properties in Response to Pro-Inflammatory TNFα and IL1β in Real-Time

Electric Cell-Substrate Impedance Sensing (ECIS), xCELLigence and cellZscope are commercially available instruments that measure the impedance of cellular monolayers. Despite widespread use of these systems individually, direct comparisons between these platforms have not been published. To compare these instruments, the responses of human brain endothelial monolayers to TNFα and IL1β were measured on all three platforms simultaneously. All instruments detected transient changes in impedance in response to the cytokines, although the response magnitude varied, with ECIS being the most sensitive. ECIS and cellZscope were also able to attribute responses to particular endothelial barrier components by modelling the multifrequency impedance data acquired by these instruments; in contrast the limited frequency xCELLigence data cannot be modelled. Consistent with its superior impedance sensing, ECIS exhibited a greater capacity than cellZscope to distinguish between subtle changes in modelled endothelial monolayer properties. The reduced resolving ability of the cellZscope platform may be due to its electrode configuration, which is necessary to allow access to the basolateral compartment, an important advantage of this instrument. Collectively, this work demonstrates that instruments must be carefully selected to ensure they are appropriate for the experimental questions being asked when assessing endothelial barrier properties.

Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.

Enhanced endothelial barrier function by monoclonal antibody activation of vascular endothelial cadherin

Excessive vascular permeability is a serious complication of many inflammatory disease conditions. We have developed monoclonal antibodies that inhibit increases in endothelial monolayer permeability induced by several signaling factors by activating VE-cadherin mediated adhesion and stabilizing cell junctions. These antibodies and/or the mechanisms they reveal may lead to important therapeutics to treat vascular leakiness and inflammation.

ADAM10-Mediated Cleavage of ICAM-1 Is Involved in Neutrophil Transendothelial Migration

To efficiently cross the endothelial barrier during inflammation, neutrophils first firmly adhere to the endothelial surface using the endothelial adhesion molecule ICAM-1. Upon actual transmigration, the release from ICAM-1 is required. While Integrin LFA1/Mac1 de-activation is one described mechanism that leads to this, direct cleavage of ICAM-1 from the endothelium represents a second option. We found that a disintegrin and metalloprotease 10 (ADAM10) cleaves the extracellular domain of ICAM-1 from the endothelial surface. Silencing or inhibiting endothelial ADAM10 impaired the efficiency of neutrophils to cross the endothelium, suggesting that neutrophils use endothelial ADAM10 to dissociate from ICAM-1. Indeed, when measuring transmigration kinetics, neutrophils took almost twice as much time to finish the diapedesis step when ADAM10 was silenced. Importantly, we found increased levels of ICAM-1 on the transmigrating neutrophils when crossing an endothelial monolayer where such increased levels were not detected when neutrophils crossed bare filters. Using ICAM-1-GFP-expressing endothelial cells, we show that ICAM-1 presence on the neutrophils can also occur by membrane transfer from the endothelium to the neutrophil. Based on these findings, we conclude that endothelial ADAM10 contributes in part to neutrophil transendothelial migration by cleaving ICAM-1, thereby supporting the release of neutrophils from the endothelium during the final diapedesis step.