Abstract
A hallmark characteristic of both Alzheimer’s Disease (AD) and Type II Diabetes Mellitus (T2DM) is injury and dysfunction of the vascular endothelium. In the brain microvasculature, vascular endothelial cells work in concert with neurons and astrocytes to form a specialized structure called the blood-brain barrier (BBB), which regulates the movement of substances between the circulation and neural environment. Injury to the BBB can result in dysfunction and the weakened barrier allows improper passage of substances, ultimately resulting in a damaged neural environment that may contribute to the development of AD. The exact mechanisms by which this dysfunction of the endothelium occurs remain elusive, but emerging research has suggested that high circulating insulin levels, such as often occur during T2DM, may play a role. Moreover, the Notch signaling pathway is involved in endothelial function of the systemic vasculature under normal physiological and pathological states, and may also be involved in endothelial dysfunction in the brain. This research work investigated how supraphysiological insulin levels and Notch signaling affect the structural integrity and permeability of the brain endothelium. To carry out this work, the well-characterized human cerebral microvascular endothelial cell line (hCMEC/d3) was used to assess alterations in Notch signaling, expression of the tight junctional protein Zonula Occludens 1 (ZO-1), and endothelial barrier permeability during hyperinsulinemic conditions, as well as the potential involvement of Notch signaling in insulin-induced changes to the brain endothelium. More specifically, qRT-PCR and western blotting methods were used to assess gene and protein expression, respectively; and the Transwell permeable support system was employed to analyze endothelial barrier permeability using a fluorescently-conjugated dextran tracer molecule. The results from this work indicated that hyperinsulinemia increases the gene and protein expression of Notch1, while decreasing the protein expression of ZO-1 in human brain endothelial cells. In addition to this, our results also showed that inhibition of the Notch1 receptor, particularly during hyperinsulinemic conditions, increases the expression of ZO-1 protein in human brain endothelial cells. Finally, our study revealed that hyperinsulinemic conditions do not alter permeability at the brain endothelium, but permeability decreases following Notch1 inhibition during both normal physiological and hyperinsulinemic states. Overall, our research study provides insight about the impact of both hyperinsulinemia and Notch signaling on brain endothelial structure and function that may have relevance for understanding the pathophysiology of T2DM and AD.