Impaired activity of adherens junctions contributes to endothelial dilator dysfunction in ageing rat arteries

Abstract

Key points: Ageing-induced endothelial dysfunction contributes to organ dysfunction and progression of cardiovascular disease. VE-cadherin clustering at adherens junctions promotes protective endothelial functions, including endothelium-dependent dilatation. Ageing increased internalization and degradation of VE-cadherin, resulting in impaired activity of adherens junctions. Inhibition of VE-cadherin clustering at adherens junctions (function-blocking antibody; FBA) reduced endothelial dilatation in young arteries but did not affect the already impaired dilatation in old arteries. After junctional disruption with the FBA, dilatation was similar in young and old arteries. Src tyrosine kinase activity and tyrosine phosphorylation of VE-cadherin were increased in old arteries. Src inhibition increased VE-cadherin at adherens junctions and increased endothelial dilatation in old, but not young, arteries. Src inhibition did not increase dilatation in old arteries treated with the VE-cadherin FBA. Ageing impairs the activity of adherens junctions, which contributes to endothelial dilator dysfunction. Restoring the activity of adherens junctions could be of therapeutic benefit in vascular ageing.

Abstract: Endothelial dilator dysfunction contributes to pathological vascular ageing. Experiments assessed whether altered activity of endothelial adherens junctions (AJs) might contribute to this dysfunction. Aortas and tail arteries were isolated from young (3-4 months) and old (22-24 months) F344 rats. VE-cadherin immunofluorescent staining at endothelial AJs and AJ width were reduced in old compared to young arteries. A 140 kDa VE-cadherin species was present on the cell surface and in TTX-insoluble fractions, consistent with junctional localization. Levels of the 140 kDa VE-cadherin were decreased, whereas levels of a TTX-soluble 115 kDa VE-cadherin species were increased in old compared to young arteries. Acetylcholine caused endothelium-dependent dilatation that was decreased in old compared to young arteries. Disruption of VE-cadherin clustering at AJs (function-blocking antibody, FBA) inhibited dilatation to acetylcholine in young, but not old, arteries. After the FBA, there was no longer any difference in dilatation between old and young arteries. Src activity and tyrosine phosphorylation of VE-cadherin were increased in old compared to young arteries. In old arteries, Src inhibition (saracatinib) increased: (i) 140 kDa VE-cadherin in the TTX-insoluble fraction, (ii) VE-cadherin intensity at AJs, (iii) AJ width, and (iv) acetylcholine dilatation. In old arteries treated with the FBA, saracatinib no longer increased acetylcholine dilatation. Saracatinib did not affect dilatation in young arteries. Therefore, ageing impairs AJ activity, which appears to reflect Src-induced phosphorylation, internalization and degradation of VE-cadherin. Moreover, impaired AJ activity can account for the endothelial dilator dysfunction in old arteries. Restoring endothelial AJ activity may be a novel therapeutic approach to vascular ageing.

Keywords: NO; VE-cadherin; adherens junctions; aging; endothelium.

Figure 1. Immunoblot analysis of VE‐cadherin expression in young (yng) and old arteries

A, aortas and tail arteries were processed using the single lysis approach with SDS‐containing lysis buffer and representative blots are presented. Upper panel, two prominent bands for VE‐cadherin were observed at 140 and 115 kDa: levels of the 140 kDa species were highest in young compared to old aortas and tail arteries, whereas levels of the 115 kDa species were highest in old compared to young aortas. Lower panel, expression of claudin‐5, a transmembrane protein involved in endothelial tight junctions, was higher in old compared to young arteries. B, aortas and tail arteries were processed using sequential lysis in TTX‐containing buffer (TTX‐soluble fraction) followed by SDS‐containing buffer (TTX‐insoluble fraction). Representative blots are presented. The influence of ageing on levels of the 140 and 115 kDa VE‐cadherin species were the same as described in A. In young and old arteries, the 140 kDa species was captured in the TTX‐insoluble fraction (SDS), whereas the 115 kDa species was present in the TTX‐soluble pool (TTX). C, cell surface proteins in young aortas were labelled using Sulfo‐NHS‐SS‐Biotin and captured with immobilized NeutrAvidin gel. A representative blot from three experiments is presented. The 140 kDa but not the 115 kDa species was localized to the cell surface.

Figure 2. Endothelial adherens junctions in young (left) and old (right) tail arteries, assessed by immunofluorescence staining of VE‐cadherin (red; DRAQ5 nuclear staining: light blue)

Images are maximal projections of Z‐stacks comprising the entire endothelial layer. The intensity of VE‐cadherin staining at adherens junctions and the width of the junctions were decreased in old compared to young arteries. Indeed, there are numerous areas in old endothelium with severe thinning or disruption of the junctions (^*). Fluorescence images are presented in their original unprocessed state. White scale bar is 10 μm.

Figure 3. Dilatation of young and old rat isolated tail arteries to the endothelial agonist acetylcholine (A) or the NO donor DEA NONOate (B)

Responses to acetylcholine (A) were assessed under control conditions (upper) and after inhibition of NO synthase with l‐NAME (lower). Dilator responses were analysed in pressurized arteries (P _TM of 60 mmHg) during stable constriction to phenylephrine (C). Data are expressed relative to baseline diameter (B) and presented as means ± SEM for A, n = 15 (control old), n = 15 (control young), or n = 8–9 (l‐NAME‐treated arteries); B, n = 6. Ageing decreased dilatation to acetylcholine under control conditions. After l‐NAME, the residual dilatation to acetylcholine (1 μm) was greater in old compared to young arteries. Ageing did not significantly affect dilatation to NONOate. See Table 1 for analyses.

Figure 4. Effects of a function blocking antibody to VE‐cadherin (VEC FBA) compared to a control antibody (Control) on dilatation to acetylcholine in young and old rat isolated tail arteries

Dilatation was analysed in paired pressurized arteries (P _TM of 60 mmHg) during stable constriction to phenylephrine (C). One artery of each pair was treated with a VE‐cadherin function blocking antibody (BV13, 50 μg ml⁻¹) and the other with a control antibody (50 μg ml⁻¹). Data are expressed relative to baseline diameter (B) and presented as means ± SEM for n = 6. The VE‐cadherin FBA inhibited dilatation to acetylcholine in young but not old arteries. Indeed, after the FBA, there was no longer any significant difference in dilatation to acetylcholine between young and old arteries. See Table 1 for analyses.

Figure 5. Tyrosine phosphorylation and ageing arteries

A, Src kinase activity, assessed by immunoblot analysis of total compared to Tyr⁴¹⁶ phosphorylation, was increased in old compared to young (yng) tail arteries. Arteries were processed using the single lysis approach and a representative blot is presented. B, tyrosine phosphorylation of VE‐cadherin in old and young aortas was assessed by immunoprecipitation of tyrosine‐phosphorylated proteins followed by immunoblots for VE‐cadherin. This approach was performed on TTX‐soluble (TTX) and TTX‐insoluble (SDS) fractions, obtained using the sequential lysis approach. Representative blots for old and young arteries are presented. Tyrosine phosphorylation was detected in the 115 kDa but not 140 kDa VE‐cadherin species, and levels of tyrosine‐phosphorylated 115 kDa protein were increased in old compared to young aortas.

Figure 6. Effect of the Src inhibitor saracatinib on levels of VE‐cadherin (upper panel) and on Src activity (lower panels) in old tail arteries

Paired old tail arteries were incubated under control conditions (C) or in the presence of saracatinib (Sar, 5 μm, 90 min, at a P _TM of 60 mmHg), and then processed using the sequential lysis approach to obtain TTX‐soluble (TTX) and TTX‐insoluble (SDS) fractions. Representative blots are presented. Saracatinib reduced Src activity, as assessed by immunoblot analysis of Tyr⁴¹⁶‐phosphorylated compared to total Src. The Src inhibitor also increased levels of the 140 kDa VE‐cadherin species, which was associated with the TTX‐insoluble fraction.

Figure 7. Effect of the Src inhibitor saractinib on endothelial adherens junctions in old tail arteries, assessed by immunofluorescence staining of VE‐cadherin (green; DRAQ5 nuclear staining: light blue)

Paired old tail arteries were incubated under control conditions or in the presence of saracatinib (5 μm, 90 min, at a P _TM of 60 mmHg) before being processed for staining. Images are maximal projections of Z‐stacks comprising the entire endothelial layer. Saracatinib increased the intensity of VE‐cadherin staining at adherens junctions and the width of the junctions. Fluorescent images are presented in their original unprocessed state. White scale bar is 10 μm.

Figure 8. Effects of the Src inhibitor saracatinib on dilatation to acetylcholine in young (bottom) and old (top, middle) rat isolated tail arteries

Dilatation was analysed in pressurized arteries (P _TM of 60 mmHg) during stable constriction to phenylephrine (C). Data are expressed relative to baseline diameter (B) and presented as means ± SEM for n = 6. Saracatinib (5 μm) increased dilatation to acetylcholine in old (top) but not young (bottom) arteries. After saracatinib, there was no longer any significant difference in dilatation to acetylcholine between young and old arteries. When old arteries were treated with VE‐cadherin FBA (BV13, 50 μg ml⁻¹), which did not affect dilatation in old arteries (see Fig. 4), the Src inhibitor no longer had any significant effect on dilatation to acetylcholine in old arteries (middle). See Table 1 for analyses.