Ageing‐induced internalization and degradation of VE‐cadherin leads to vascular dysfunction

With increasing age, the arterial system undergoes deterioration which contributes to cardiovascular disease, a leading cause of morbidity and mortality worldwide. Agedependent deterioration of the vascular network is partly mediated by endothelial cell dysfunction, which is characterized by reduced nitric oxide (NO) levels, impaired endothelial cell barrier, increased susceptibility to apoptosis, and an overall pro-inflammatory phenotype. The protective role of endothelial cells in the vasculature is thought to be mediated by increased clustering of VE-cadherins at the adherens junctions, which promotes protective signalling by amplifying endothelial NO-mediated dilatation and increasing vascular stability. VE-cadherins are calcium-dependent glycoproteins that play a vital role in proper endothelial cell-to-cell contact and allow transfer of intracellular signals from one cell to another. It has been suggested that they may play a protective role under pathological conditions, but their role in ageing-induced vascular disease has not been thoroughly investigated. In a recent issue of The Journal of Physiology, Chang et al. (2017) investigated the association between ageing and vascular dysfunction of the arterial system. The authors aimed to determine the contribution of altered endothelial adherens junctions in vascular disease. They provided direct evidence of ageing-induced molecular changes that promote endothelial dysfunction through VE-cadherin internalization and degradation. Specifically, weaker endothelial cell-to-cell contacts were associated with impaired vessel response to acetylcholine, resulting in an overall impairment of the vascular system. The study provides novel insight into the mechanisms by which ageing promotes vascular disease and highlights potential therapeutic targets for future investigations. In the study, Chang et al. (2017) successfully showed that two VE-cadherin species (115 and 140 kDa), had significantly different expression profiles in young (3–4 months of age) and old (22–24 months of age) rat arteries. They used a novel single lysis approach by utilizing a sodium dodecyl sulfate (SDS) lysis buffer to obtain total artery lysates. They found that the 140 kDa VE-cadherin expression was higher in aortas and tail arteries of young rats, whereas the expression of the 115 kDa VE-cadherin was higher in older rat aortas and was completely absent in the tail arteries. To further study the two VE-cadherin species, the authors used a sequential lysis approach, wherein arteries were lysed in a Tris–HCl and Triton X-100 (TTX) buffer to obtain a TTX-soluble fraction, followed by the addition of SDS to obtain a TTX-insoluble SDS-soluble fraction. Immunoblots after sequential lysis revealed that the 140 kDa VE-cadherin species was predominantly found in the TTX-insoluble fraction, whereas the 115 kDa species was in the TTX-soluble fraction, in both young and old rats. Moreover, cell surface labelling analysis via biotinylation of surface proteins and subsequent elution purification with a dithiothreitol-containing buffer revealed that the 140 kDa species was predominantly located on the endothelial cell surface. The authors hypothesized that the 140 kDa VE-cadherin species, localized at the endothelial cell surface, may be necessary for establishing proper contact between endothelial cells at adherens junctions. They further reasoned that with increasing age, the 140 kDa protein is likely internalized and degraded into the 115 kDa species, thus contributing to weaker adherens junctions. In fact, these findings are in line with a previous study by Lee et al. (1999) showing that mature adherens junctions are associated with the movement of VE-cadherin from the TTX-soluble to the TTX-insoluble fraction. Furthermore, studies in cultured endothelial cells have shown that internal degradation of VE-cadherin generates a fragment that is approximately 25 kDa smaller than the native protein, thereby further reinforcing the notion that the 140 kDaVE-cadherin species is degraded into the non-functional 115 kDa species with increasing age. Immunofluorescence analysis of VEcadherins in tail arteries demonstrated that the width and peak intensities of adherens junctions were significantly lower in old rats, compared to younger ones. In addition, the arteries of old rats showed areas of severe endothelial thinning and disrupted adherens junctions, further implicating the role of VE-cadherins in ageing-induced vascular disease. These findings are in direct contrast to a previous study by Huynh et al. (2011) which showed that ageing mouse aortas had a larger width of endothelial adherens junctions. The authors reason that the likely contradiction is a result of differences in data analysis methodology. Huynh and colleagues measured the widest part of the endothelial junctions, while Chang et al. (2017) utilized a non-selective approach for determining junctional width. Therefore, the findings reported by Chang et al. could have been underestimated. Additionally, Chang and colleagues evaluated vessel function by measuring dilatation to acetylcholine in both old and young tail arteries. Their results revealed that arteries isolated from old rats exhibited a decrease in maximum dilatation and a reduced sensitivity in response to acetylcholine. Furthermore, the inhibition of NO synthase resulted in reduced dilatation response and impaired sensitivity in both old and young arteries. However, the residual dilatation, after NO synthase inhibition, was greater in old rats compared to younger ones. Collectively, the data demonstrated that old arteries exhibited impaired endothelial adherens junctions and more vascular dysfunction. Due to differences in anatomy, structure and VE-cadherin expression between aortas and tail arteries, it would be of interest to see whether the same effects are observed on aortas. Moreover, rescue experiments could be performed to further show the definite role of VE-cadherin clustering at adherens junctions in proper artery function. To directly implicate VE-cadherins in vascular dysfunction, Chang et al. used a function-blocking antibody, specific to