Altered vascular smooth muscle function in the ApoE knockout mouse during the progression of atherosclerosis

Abstract

Objectives: Relaxation of vascular smooth muscle (VSM) requires re-uptake of cytosolic Ca(2+) into the sarcoplasmic reticulum (SR) via the Sarco/Endoplasmic Reticulum Ca(2+) ATPase (SERCA), or extrusion via the Plasma Membrane Ca(2+) ATPase (PMCA) or sodium Ca(2+) exchanger (NCX). Peroxynitrite, a reactive species formed in vascular inflammatory diseases, upregulates SERCA activity to induce relaxation but, chronically, can contribute to atherogenesis and altered vascular function by escalating endoplasmic reticulum stress. Our objectives were to determine if peroxynitrite-induced relaxation and Ca(2+) handling processes within vascular smooth muscle cells were altered as atherosclerosis develops.

Methods: Aortae from control and ApoE(-/-) mice were studied histologically, functionally and for protein expression levels of SERCA and PMCA. Ca(2+) responses were assessed in dissociated aortic smooth muscle cells in the presence and absence of extracellular Ca(2+).

Results: Relaxation to peroxynitrite was concentration-dependent and endothelium-independent. The abilities of the SERCA blocker thapsigargin and the PMCA inhibitor carboxyeosin to block this relaxation were altered during fat feeding and plaque progression. SERCA levels were progressively reduced, while PMCA expression was upregulated. In ApoE(-/-) VSM cells, increases in cytosolic Ca(2+) [Ca(2+)]c in response to SERCA blockade were reduced, while SERCA-independent Ca(2+) clearance was faster compared to control.

Conclusion: As atherosclerosis develops in the ApoE(-/-) mouse, expression and function of Ca(2+) handling proteins are altered. Up-regulation of Ca(2+) removal via PMCA may offer a potential compensatory mechanism to help normalise the dysfunctional relaxation observed during disease progression.

Keywords: Atherosclerosis; Ca(2+); PMCA; Peroxynitrite; SERCA.

Fig. 1

Protein nitrosylation is increased in atherosclerotic ApoE^−/− mouse thoracic aortae. (a, b) Representative cross-sectional images of nitrotyrosine stained C57/BL-6 and 4 month hfd ApoE^−/− mouse thoracic aortae (200× magnification). (c, d) Smooth muscle actin staining. (e) Mean DAB staining/nuclear area (%) for nitrotyrosine and actin. (f) H&E stained aorta from 4 month hfd ApoE^−/−. L = lumen, SM = smooth muscle, P = plaque, FC = fibrous cap, +ve = areas of positive staining ***p < 0.001 vs. C57/BL-6, n = 5 mice per group.

Fig. 2

Mouse thoracic aorta relaxes to ONOO⁻ in a dose-dependent manner. (a) Representative trace of 3x10^-8 M U46619-induced contraction of mouse aorta and subsequent relaxation to increasing doses of ONOO⁻ (1 × 10⁻⁶ – 5x10⁻⁴ M). (b) ONOO⁻-induced relaxation in C57/BL-6, 2 and 4 month hfd ApoE^−/− aortae. All data presented are from endothelial denuded vessels. ***p < 0.001 for 2 month hfd ApoE^−/− vs. C57/BL-6, minimum of 7 mice per group.

Fig. 3

Expression levels of SERCA2b are progressively reduced while PMCA levels are increased in aortic smooth muscle from ApoE^−/− mice. (a, b) Representative immunoblots and averaged protein expression data for SERCA2b, pan-PMCA and GAPDH. Protein loads were 2.5, 5, 7.5 μg for SERCA and 10, 15, 20 μg for PMCA. Slopes of plotted band densities were standardised to GAPDH. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. C57/BL-6, minimum of 5 independent experiments.

Fig. 4

Efficacy of TG and CE is temporally altered as atherosclerosis progresses. (a–c) ONOO⁻-induced relaxation following 3 μM TG pre-incubation. ***p < 0.001 vs. control, †††p < 0.001 vs. TG treated C57/BL-6, ‡‡‡p < 0.001 vs. TG treated 2mo hfd ApoE^−/−. (d–f) ONOO⁻-relaxation following pre-incubation with 10 μM CE. ***p < 0.001 vs. control, ††p < 0.01 vs. CE treated C57/BL-6, ‡‡‡p < 0.001 vs. CE treated 2mo hfd ApoE^−/−. All experiments performed following endothelial denudation. Minimum of 5 mice per group.

Fig. 5

Ca²⁺ extrusion is upregulated in atherosclerotic mouse aortae. (a) Typical experimental protocol. (b–d) Representative responses to 10 mM caffeine and 1  μM TG (in the presence and absence extracellular Ca²⁺). (e) Mean peak Ca²⁺ responses (ΔF/F₀). (f) Decay times (s) of Ca²⁺ transients. *p < 0.05, **p < 0.01 and ***p < 0.001 vs C57/BL-6. The average of multiple cells from a single mouse was used as 1 data point, n = 5–6 mice per group.