CYCLIC STRETCH MEDIATES CHANGES IN AMYLOID PRECURSOR PROTEIN EXPRESSION AND NITRIC OXIDE SIGNALLING IN HUMAN CEREBRAL ENDOTHELIAL CELLS PRE-EXPOSED TO A GLYCOSPHINGOLIPID INHIBITOR

Abstract

Amyloid β (Aβ) deposits, a hallmark of Alzheimer's disease (AD), are aggregates of Aβ peptides that are cleaved of amyloid precursor protein (APP). Elevated pulsatility of arterial pressure and deregulation of ceramide and nitric oxide (NO) metabolism are independently associated with AD (Nation et al. 2013; Takasugi et al. 2015; Austin et al. 2013). Glycosphingolipid inhibitors that inhibit the enzyme glucosylceramide synthase by blocking ceramide glycosylation are known to increase gamma secretase-mediated-Aβ release (Takasugi et al. 2015). This study investigates whether pulsatile stretch of human cerebral microvascular endothelial cells (hCMEC) pre-treated with the glycosphingolipid inhibitor, D-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) alters expression and/or phosphorylation of APP and endothelial NO synthase (eNOS). hCMECs were pre-treated with 20 μM D-PDMP for 2 hours prior to 15% cyclic stretch for 18 hours at 1 Hz (n=7–8). Protein expression and phosphorylation (phospho-eNOS, Ser1177) were quantified using western blots. Results were analyzed using one way ANOVA (mean ± SEM, % control). APP expression significantly increased in response to stretch in ECs pre-treated with D-PDMP (170.2±27%, p<0.01) compared to those that have been stretched without pre-treatment (114.5±23%). eNOS phosphorylation was down-regulated in ECs subjected to stretch (9.9±4%) compared to vehicle control (100.0±0%, p<0.0001) and PDMP-treated-static control (107.0±17%, p<0.01). In ECs pre-treated with D-PDMP prior to stretch, phospho-eNOS levels significantly decreased (40.3±15%) compared to vehicle control (100.0±0%, p<0.05) and PDMP-treated-static control (107.0±17%, p<0.01). APP expression increased in cerebral ECs pre-exposed to D-PDMP after being exposed to cyclic stretch. eNOS phosphorylation was down-regulated by cyclic stretch. D-PDMP pre-treatment appeared to have a restoring effect on peNOS, although was not statistically significant. Glucosyl and/or lactosyl ceramide synthases may have regulatory roles in modulating APP expression and NO signalling in cerebral ECs and may have implications on Aβ load, NO production and related vascular pathologies in the context of AD.