Sirolimus-FKBP12.6 impairs endothelial barrier function through protein kinase C-α activation and disruption of the p120-vascular endothelial cadherin interaction

Abstract

Objective: Sirolimus (SRL) is an immunosuppressant drug used to prevent rejection in organ transplantation and neointimal hyperplasia when delivered from drug-eluting stents. Major side effects of SRL include edema and local collection of intimal lipid deposits at drug-eluting stent sites, suggesting that SRL impairs endothelial barrier function (EBF). The aim of this study was to address the role of SRL on impaired EBF and the potential mechanisms involved.

Approach and results: Cultured human aortic endothelial cells (HAECs) and intact human and mouse endothelium was examined to determine the effect of SRL, which binds FKBP12.6 to inhibit the mammalian target of rapamycin, on EBF. EBF, measured by transendothelial electrical resistance, was impaired in HAECs when treated with SRL or small interfering RNA for FKBP12.6 and reversed when pretreated with ryanodine, a stabilizer of ryanodine receptor 2 intracellular calcium release channels. Intracellular calcium increased in HAECs treated with SRL and normalized with ryanodine pretreatment. SRL-treated HAECs demonstrated increases in protein kinase C-α phosphorylation, a calcium sensitive serine/threonine kinase important in vascular endothelial (VE) cadherin barrier function through its interaction with p120-catenin (p120). Immunostaining of HAECs, human coronary and mouse aortic endothelium treated with SRL showed disruption of p120-VE cadherin interaction treated with SRL. SRL impairment of HAEC EBF was reduced with protein kinase C-α small interfering RNA. Mice treated with SRL demonstrated increased vascular permeability by Evans blue albumin extravasation in the lungs, heart, and aorta.

Conclusions: SRL-FKBP12.6 impairs EBF by activation of protein kinase C-α and downstream disruption of the p120-VE cadherin interaction in vascular endothelium. These data suggest this mechanism may be an important contributor of SRL side effects related to impaired EBF.

Keywords: PKC; endothelium; sirolimus.

Figure 1

SRL Activates PKCα, Disrupts the Interaction of p120 with VE Cadherin and the Endothelial Barrier. (A) Human aortic endothelial cells (HAECs) were immunoblotted for phosphorylated PKCα (Ser 657) after treatment with SRL at the indicated range of doses for 24 hours (1 - 500 nmol/L). Densitometry was performed (mean ± SD, n = 3, * p < 0.05). (B) HAECs were treated with SRL (500 nmol/L) from 30 minutes to 24 hours and immunoblotted for pPKCα at the times shown. Densitometry was performed (mean ± SD, n = 3, * p < 0.05). (C) p120 was immunoprecipitated from HAEC lysates at the indicated times after SRL treatment (500 nmol/L) and precipitates were immunoblotted for pPKCα and VE-cadherin. Total cell lysates were also immunoblotted (IB) for the respective antibodies and representative examples shown. (D) Densitometry was performed for the association of p120 with pPKCα and VE-cadherin (mean ± SD, n = 3, * and # p < 0.05 compared to 0 hrs). (E) Immunofluorescent imaging of HAECs with VE Cadherin (green) and p120 (red) was performed after no SRL treatment (cont) and 24 hours treatment (SRL) shown in 20x. White arrows denoted intraendothelial deposits with increased p120 and VE cadherin content. White bar indicates 20 mm. (F) Representative 2-D florescent intensity plots for immunoflourescent images each treatment group (shown in E) with pearson correlation coefficients (r) for the co-localization of p120 and VE cadherin pixels shown in inset (mean ± SD, p < 0.01 for cont v. SRL, n > 4 fields). (G) VE cadherin protein expression (relative to endothelial beta actin expression)levels were decreased atafter 24 hours of SRL treatment as measured by immunoblotting. Densitometry was performed for the relative expression of VE cadherin to beta actin (mean ± SD, n = (experiments repeated 4, * p < 0.05). times) (H) Interendothelial (IE) gap areas were assessed after SRL treatment at the indicated times by immunoflorescent imaging of membrane VE cadherin (mean ± SD, n > 4 fields, * p < 0.05 compared to t = 0).

Figure 2

SRL-FKBP12.6 Impairs Endothelial Barrier Function (EBF) by Modulating Intracellular Calcium Concentration via RyR2 Channels. (A) Treatment with SRL impairs EBF as measured by reduced normalized transendothelial electrical resistance (TEER) while PKCα siRNA reduces SRL-induced decrease in TEER (mean ± SEM, n = 3 wells, normalization was to each individual baseline value, t = 0). (B) Treatment with FKBP12.6 siRNA decreases TEER which is reversed by a RyR2 stabilization with ryanodine (50 μmol/L for 1 hour) pre-treatment (mean ± SEM, n = 3 wells). (C) Pre-treatment with ryanodine reduces the SRL-induced decrease in TEER (mean ± SEM, n = 3 wells). (D) Ryanodine pre-treatment prevents SRL-induced increase in HAEC transwell permeability (mean ± SD, n = 4 wells, * p < 0.05 compared to change in FITC-Dextran in control wells). (E-F) SRL induces an intracellular calcium leak which is ameliorated by ryanodine pre-treatment in HAECs (mean emission ratio [F/Fo = Fluo3/Fura Red] ± SD, n > 10 cells, * < 0.05 v. SRL + Ryan). (G) Treatment of HAEC with Torin2, a selective ATP-competitive inhibitor of mTOR, does not impair EBF compared with control (mean ± SEM, n = 3 wells). (H) Representative western blots of activated S6K (pS6K) and 4E-BP (p4E-BP) representing major downstream signaling products of the mTOR pathway after Torin2 treatment for 24 hours (n = 4).

Figure 3

SRL Induces Increased Vascular Permeability and Disrupts the Interaction of VE-cadherin and p120 in vivo and ex vivo. (A) Intraperitoneal injection of SRL (1 mg/kg/day for 3 days) and vehicle-treated mouse myocardium showing increased Evans blue albumin (EBA) extravasation both in the micro- and microvasculature stained with CD31 (green) at 4x magnification. Gross example of the heart and lungs in each group are also shown. White bar indicates 100 μm. (B) Vascular permeability of different endothelial beds (aortic, heart, lungs) was increased in SRL-treated mice when compared to vehicle as measured by EBA content in the respective tissue homogenate (mean ± SD, n = 6 mice,* p < 0.05 compared to vehicle (cont)). (C) SRL treatment disrupts the interaction of p120 (red) and VE cadherin (green) in C57BL/6 mice aortic endothelium (MAE) when compared with vehicle treated mice (top) shown in 20x. White bar indicates 10 μm. (D) Human coronary endothelium treated ex vivo with SRL (500 nmol/L) for 24 hours or vehicle showing increased p120 (red) decreased co-localization with VE cadherin (green) in SRL-treated arteries compared with vehicle (cont). Magnification at 40x with white bar indicating 10 μm.

Figure 4

Proposed Mechanism of Sirolimus to Impair Endothelial Barrier Function. (A) Sirolimus (SRL) displaces FKBP12.6 from RyR2 calcium release channel (blue oval) in vascular endothelial cells results in increased intracellular release of free Ca²⁺ from the endoplasmic reticulum. (B) PKCα is activated and destabilized the p120-VE cadherin interaction. (C) p120 and eventually VE cadherin move from the membrane to the intracellular space leading to impaired endothelial barrier function.