Table Serum lipid Profiles of the rabbits in the four groups at week 16.

<p>Group A2 is Akt inhibitor triciribine treatment group, Group B2 is mTOR inhibitor rapamycin treatment group, Group C2 is mTOR-siRNA group and Group D2 is the control group.</p><p>TC, total cholesterol; TG, triglyceride; HDL, high density lipoprotein; LDL, low density lipoprotein.</p

Cytokines secreted by macrophages and effect of different inhibitors on Akt and mTOR molecules.

<p>(A, B) ELISA assay of cytokines in rabbit primary macrophage supernate showed an increased level of IL-10 and decreased level of IFN-γ in group A1 than E1. On the contrary, level of IL-10 was lower in group B1, C1 and D1 compared to group E1 whereas IFN-γ was higher. (C) QRT PCR showed that mRNA level declined in each group compared to group E1. (D, E) Western blot revealed that total Akt and total mTOR did not change among the five groups while phosphorylation of the Akt and mTOR reduced significantly in the experimental groups compared to the control. However, Akt in group C1 and D1 were hyperphosphorylated. A1: LY294002; B1: triciribine; C1: rapamycin; D1: mTOR-siRNA; E1: control. ^*<i>P</i><0.01 vs control, ^#<i>P</i><0.05 vs control.</p

Autophagy was induced in the presence of API-2, rapamycin and mTOR-siRNA respectively and inhibited in response to LY294002.

<p>Rabbit peritoneal macrophages were obtained to verify if blockage of PI3K, Akt, mTOR molecules respectively could promote autophagy. (A, B) Cell immunofluorescence staining of LC3-II among different groups (×400) showed that increased LC3-II punctate dots appeared in B1, C1 and D1 groups while decreased punctate dots in group A1 compared to group E1. (C) Expression of autophagy related gene Beclin 1 detected by QRT PCR showed remarkable higher Beclin 1 mRNA in group B1, C1 and D1 and lower in group A1 than group E1. (D) Western blot showed that expression of autophagy protein Beclin 1 and Atg5-Atg12 conjugation increased in group B1, C1 and D1 and decreased in group A1 than group E1. (E) Transmission electron microscope showed an intact nonpyknotic nucleus and numerous vacuoles, large cytoplasmic inclusions and myeline figure in the cytoplasm in group B1, C1 and D1, whereas group A1 and E1 did not display vacuolization but a little was observed (base level autophagy). Endoplasmic reticulum and mitochondria displayed varying degrees of swelling in group B1, C1 and D1. The “N” represents the nuclear. A1: LY294002; B1: triciribine; C1: rapamycin; D1: mTOR-siRNA; E1: control. ^*<i>P</i><0.01 vs control, ^#<i>P</i><0.05 vs control.</p

Macrophage autophagy induced by drugs and mTOR-siRNA alleviated vulnerability index.

<p>The vulnerability index can be calculated as: (macrophage staining %+ lipid staining %)/(smooth muscle cell %+ collagen fiber %). So we conducted Sirius red (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090563#pone-0090563-g005" target="_blank">Fig. 5</a>) and oil red O staining together with IH for macrophages (RAM-11) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090563#pone-0090563-g005" target="_blank">Fig. 5</a>) and smooth muscle cells (α-SMA). (A, B) Oil Red O Staining was taken for lipid content evaluation and was expressed as a percentage verse the whole vessel lumen. Our result revealed much smaller plaque in aorta abdominals in group A2, B2 and C2 compared to group D2. (C) IH analysis of α-SMA amount did not detect difference in plaque area among the four groups. L represents for vascular lumen, P represents for plaque. (D) Vulnerability was calculated by macrophage, lipid, α-SMA and collagen. It was significantly lower in the experimental group than the control group. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control.</p

Macrophage autophagy promotes vulnerable plaque stability via decreased plaque burden and macrophages.

<p>IVUS measurement was taken to measure lumen area (LA), external elastic membrane area (EEMA), plaque area (PA) and plaque burden (PB) (PA =  EEMA – LA, PB% =  PA/EEMA ×100%). (A) IVUS image showed that the plaque area was huge in group D2 than that of A2, B2 and C2. (B) PA and PB calculated by LA and EEMA. We can see PA in the abdominal aorta in group A2, B2 and C2 was significantly lower than that in group D2, and PB% was remarkably reduced in group A2, B2 and C2 compared to group D2. However, the three groups did not differ in PB%. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control.</p

Effect of Pharmacological triggers in the experiment showed the rupture of plaque incidence higher in control group.

<p>IH staining and transmission electron microscope showed mTOR decreased and autophagy increased in experimental group. Pharmacological triggers were done by 0.15·kg⁻¹ of Chinese Russell's viper venom injecting intraperitoneally, 30 min later, 0.02 mg·kg⁻¹ histamine was injected intravenously. (A) Haematoxylin and eosin staining of the cross section of the abdominal aorta in a rabbit of group D2 showing plaque rupture and thrombosis. (B, C) IH staining showed mTOR decreased obviously in group A2, B2 and C2 than D2. (D) Transmission electron microscope analysis showed macrophage autophagy was enhanced in group A2, B2 and C2 compared to group D2. Arrows in picture B2, C2 and d2 represent vacuoles with cytoplasmic inclusions and myeline figure. The “N” represents the nuclear. (E) IH analysis of Atg5-Atg12 conjugation found percentage of positive-stain cells was significantly increased in group A2, B2 and C2 in comparison to group D2. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control, ^#<i>P</i><0.05 vs control.</p

Sirius red staining for collagen and IH for macrophages in plaque.

<p>(A) The interstitial collagen content of aorta abdominals plaques was evaluated by Sirius red staining. Collagens are identified by birefringence under polarized light illumination following standard sirius red procedure. (B) IH for macrophage in plaque detected increased macrophages expressed in control group and less in group A2, B2 and C2. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control.</p