This work reports on an effort to decipher the alignment of brain microvasculature endothelial cells to physical constrains generated via adhesion control on hydrogel surfaces and explore the corresponding responses upon glucose level variations emulating the hypo- and hyperglycaemic effects in diabetes. We prepared hydrogels of hyaluronic acid a natural biomaterial that does not naturally support endothelial cell adhesion, and specifically functionalised RGD peptides into lines using UV-mediated linkage. The width of the lines was varied from 10 to 100 µm. 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 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.