Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis

Nuria Beneit, José Luis Martín-Ventura, Carlota Rubio-Longás, Óscar Escribano, Gema García-Gómez, Silvia Fernández, Giorgio Sesti, Marta Letizia Hribal, Jesús Egido, Almudena Gómez-Hernández, Manuel Benito

Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. We previously demonstrated that overexpression of insulin receptor isoform A (IRA) and insulin-like growth factor-I receptor (IGF-IR) confers a proliferative and migratory advantage to vascular smooth muscle cells (VSMCs) promoting plaque growth in early stages of atherosclerosis. However, the role of insulin receptor (IR) isoforms, IGF-IR or insulin-like growth factor-II receptor (IGF-IIR) in VSMCs apoptosis during advanced atherosclerosis remains unclear. We evaluated IR isoforms expression in human carotid atherosclerotic plaques by consecutive immunoprecipitations of insulin receptor isoform B (IRB) and IRA. Western blot analysis was performed to measure IGF-IR, IGF-IIR, and α-smooth muscle actin (α-SMA) expression in human plaques. The expression of those proteins, as well as the presence of apoptotic cells, was analyzed by immunohistochemistry in experimental atherosclerosis using BATIRKO; ApoE-/-mice, a model showing more aggravated vascular damage than ApoE-/-mice. Finally, apoptosis of VSMCs bearing IR (IRLoxP+/+VSMCs), or not (IR-/-VSMCs), expressing IRA (IRA VSMCs) or expressing IRB (IRB VSMCs), was assessed by Western blot against cleaved caspase 3. We observed a significant decrease of IRA/IRB ratio in human complicated plaques as compared to non-complicated regions. Moreover, complicated plaques showed a reduced IGF-IR expression, an increased IGF-IIR expression, and lower levels of α-SMA indicating a loss of VSMCs. In experimental atherosclerosis, we found a significant decrease of IRA with an increased IRB expression in aorta from 24-week-old BATIRKO; ApoE-/-mice. Furthermore, atherosclerotic plaques from BATIRKO; ApoE-/-mice had less VSMCs content and higher number of apoptotic cells. In vitro experiments showed that IGF-IR inhibition by picropodophyllin induced apoptosis in VSMCs. Apoptosis induced by thapsigargin was lower in IR-/-VSMCs expressing higher IGF-IR levels as compared to IRLoxP+/+VSMCs. Finally, IRB VSMCs are more prone to thapsigargin-induced apoptosis than IRA or IRLoxP+/+VSMCs. In advanced human atherosclerosis, a reduction of IRA/IRB ratio, decreased IGF-IR expression, or increased IGF-IIR may contribute to VSMCs apoptosis, promoting plaque instability and increasing the risk of plaque rupture and its clinical consequences.