Goat anti-rabbit Alexa Fluor 568 (Invitrogen) was dissolved in 50% blocking buffer in PBS and incubated for 2?h at room temperature (RT)
Goat anti-rabbit Alexa Fluor 568 (Invitrogen) was dissolved in 50% blocking buffer in PBS and incubated for 2?h at room temperature (RT). 100?m. We evaluated cell alignment by measuring the nuclei, cell, and F-actin orientations, and the nuclei and cell eccentricity via immunofluorescent staining and image analysis. We Picroside II found that the brain microvascular endothelial cells aligned and elongated to these physical constraints for all line widths. In addition, we also observed that varying the cell medium glucose levels affected the cell alignment along the patterns. We believe our results may provide a platform for further studies on the impact of altered glucose levels in cardiovascular disease. strong class=”kwd-title” Subject terms: Biomaterials – cells, Cell biology Introduction Endothelial cells line blood vessels throughout the body, in different microenvironments ranging from large arteries to microvascular veins, and substantial differences have been observed comparing endothelial cells from different sources1. Brain microvascular endothelial cells constitute a vital part of the blood brain barrier with the role to provide a protective environment for the brain. These endothelial cells are part of the brain microvasculature that constitute a unique subset of non-fenestrated vessels, Picroside II that allow them to control the transport of molecules between the brain and the rest of the body2. This control is achieved by various means, where the tight junction proteins binding the brain microvascular endothelial cells tightly together play a specifically important role as they hinder paracellular flux of harmful substrates to reach the brain during normal homeostasis3. Different disease states, among these diabetes, have been shown to affect the endothelial cell functions and thus might ultimately alter the barrier properties4. Microfabrication techniques such as micropatterning of surfaces or microfluidics have been extensively utilized to study endothelial cells from various different regions of the body1,5C7. It is Picroside II well-studied and demonstrated that cell adhesion can Picroside II be spatially controlled by micropatterning cell adhesion peptides or proteins on otherwise inert surfaces8. Similarly, endothelial cells have been seen to respond to micropatterned lines by elongating and aligning with the line direction, organizing their nuclei and actin fibres in parallel with the micropatterns and adopting an atheroprotective phenotype5C7,9C12. Furthermore, it has been shown that alignment on micropatterned surfaces alone is enough to influence gene expression of e.g. inflammatory genes and that cell shape determines cellular function13,14. These studies have however mostly included endothelial cells from larger vessels e.g. aortic cells or umbilical vein endothelial cells whereas the corresponding effects on brain microvasculature endothelial cells have not been studied in such detail. Some important differences between large vein endothelial cells and microvasculature endothelial cells have been reported, where e.g. human umbilical cord endothelial vein cells (HUVECs) have been observed to align with flow-induced shear stresses15, whereas brain microvascular endothelial cells neither align nor elongate, or transitions from cobblestone to spindle-like morphology under shear stress or in response to curvature16C18. These different responses occurred despite HUVECs and the brain microvascular cell line b.End3 display similar responses to shear stress in CASP8 terms of connexin37 expression, highly expressed in the healthy atheroprotective phenotype19. In light of these differences in response to shear stress and curvature, we wanted to investigate the response of brain microvascular endothelial cells on micropatterned lines. Here, we patterned arginyl-glycyl-aspartic acid (RGD) peptide lines on hyaluronic acid hydrogels. We show the response of b.End3?cells, a mouse brain microvascular endothelial cell line, on different line widths (10C100?m). Furthermore, we study the alignment in response to altered glucose levels to simulate hypo- and hyperglycaemia or altered blood glucose levels associated with diabetes. As several studies report that altered glucose levels have an effect on endothelial cell alignment in response to fluid-induced shear stress20C22 we wanted to understand if this would also affect the alignment of the brain microvascular endothelial cells when cultured on micropatterned lines. Results and discussion In this paper we have investigated how brain microvascular endothelial cells respond to micropatterns of various widths. In addition, we report the behaviour of these cells under various glucose concentrations, emulating different blood sugar levels associated with diabetes. Brain microvascular endothelial cells adhere to micropatterned lines on hyaluronic acid acrylamide hydrogels First, we wanted to study the behaviour of brain Picroside II microvascular endothelial cells when.