Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2

<div><p>Apolipoprotein E3 (apoE3) is thought to protect against atherosclerosis by enhancing reverse cholesterol transport. However, apoE3 also has cholesterol-independent effects that contribute to its anti-atherogenic properties. These include altering extracellular matrix protein synthesis and inhibiting vascular smooth muscle cell proliferation. Both of these cholesterol-independent effects result from an apoE3-mediated induction of cyclooxygenase-2 (Cox2). Nevertheless, how apoE3 regulates Cox2 remains unknown. Here, we show that apoE3 inhibits the activation of Rho, which reduces the formation of actin stress fibers and focal adhesions and results in cellular softening. Inhibition of Rho-Rho kinase signaling or direct cellular softening recapitulates the effect of apoE3 on Cox2 expression while a constitutively active Rho mutant overrides the apoE3 effect on both intracellular stiffness and Cox2. Thus, our results describe a previously unidentified mechanism by which an atheroprotective apolipoprotein uses Rho to control cellular mechanics and Cox2.</p></div

ApoE3 reduces intracellular stiffness through the Rho signaling pathway.

<p>(A) VSMCs in 10% FBS were treated with apoE3 or Y27632, or their respective vehicle controls (Ctrl), for 24 hr. Intracellular stiffness was determined by AFM. <i>n = 4</i>. (B) VSMCs were plated on fibronectin-coated low or high stiffness hydrogels in 10% FBS with or without apoE3 for 24 hr, and intracellular stiffness was determined by AFM. <i>n = 4</i>. (C) VSMCs infected with adenoviruses encoding LacZ or Rho^V14 were incubated with 10% FBS in the absence or presence of apoE3 for 24 hr, and intracellular stiffness was measured by AFM. <i>n = 4</i>. Data information: Graphs show mean + SEM. *<i>p</i><0.05.</p

ApoE3 decreases Rho activity and F-actin stress fibers in human VSMCs.

<p>(A-B) Starved VSMCs were incubated with 10% FBS in a culture dish (A) or on fibronectin-coated low or high stiffness hydrogels (B) with or without apoE3 treatment for 9 hr. Rho activity was measured. <i>n = 4</i>. (C) VSMCs were incubated with 10% FBS in the absence or presence of apoE3 for 24 hr. Scale bar = 50 μm. Fixed cells were incubated with FITC-conjugated phalloidin to stain f-actin. (D) Average phalloidin intensity of single cells was analyzed using ImageJ and normalized to control cells. <i>n = 4</i> independent experiments with 5–10 cells analyzed per experiment. Data information: Graphs show mean + SEM. *<i>p</i><0.05 or **<i>p</i><0.01.</p

ApoE3 inhibits Rho-GTP to regulate Cox2.

<p>A working model depicting how apoE3 regulates Rho and intracellular stiffness and how these effects regulate mechanosensitive Cox2 expression.</p