Myostatin mediates abdominal aortic atherosclerosis progression by inducing vascular smooth muscle cell dysfunction and monocyte recruitment

Figure 1. Expression of Myostatin (Mstn) in human peri-renal aortic specimens evaluated by immunohistochemistry. Representative pictures of normal aortas (A), non-progressive intimal lesions (B) and progressive atherosclerotic lesions (C). (Magnification: x100; Bars = 50 μM). The arrows indicate positive areas; (D) Image analysis of Mstn immunopositivity plotted in relation to the type of atherosclerosis (Norm: Normal aorta; AIT: adaptive intimal thickening; IX: Intimal xanthoma; PIT: pathological intimal thickening; EFA: Early fibroatheroma; LFA: late fibroatheroma; TCFA: thin cap fibroatheroma; PR: plaque rupture; HR: healed plaque rupture; FCP: fibrotic calcified plaque). Mstn expression significantly correlates with lesion type progression (R = 0.627, p = 0.0001).

Figure 2. Myostatin (Mstn) immunostaining in progressive atherosclerotic lesions. Panel A: late fibroatheroma, LFA; panel B: thin cap fibroatheroma, TCFA; panel C: plaque rupture, PR; panel D: healed plaque rupture, HR. Boxes indicate segments with Mstn positivity in correspondence of neovasa (NV), macrophages (Φ) and infiltrating cells (IC); magnification: 4-100x. Panel A and D: the first left image is stained with Movat pentachrome.

Figure 3. Immunohistochemical double staining of aortic cellular components associated with Myostatin (Mstn). Panel A: Mstn (brown) and α smooth muscle actin (α-SMA) (red); merged image shows that Mstn colocalizes with α-SMA. Panel B: staining with Mstn (brown), CD45 (red) and α-SMA (brown). Panel C: merged images of Mstn and CD45, Mstn and α-SMA (Mstn brown; CD45 and α-SMA red) and CD45 and α-SMA (brown and red respectively), Mstn colocalizes with CD45 and α-SMA and the latter with CD45; fields correspond to boxes in panel B (Magnification: 100x panel A,C; 20x panel B).

Figure 4. Effects of Myostatin (Mstn) on A7r5 cell cycle (A), proliferation index (B) and cell size (C). A 24 hour Mstn treatment caused an increase of G0/G1 phase as revealed by propidium iodide staining and flow-cytometry reading. At 48 hours theproliferation rate as revealed by CFSE-DA assay and the cell size were reduced. Data shown as mean ± SEM (A,B) and median ± IQR (C). *p < 0.05, **p < 0.01 vs. untreated cells (n = 3).

Figure 5. Effects of Myostatin (Mstn) on VSMC phenotype. (A) Smoothelin mRNA expression analyzed by real-time RT-PCR. All data were first normalized to GAPDH mRNA and values were expressed as fold increase ± SEM versus untreated cells. **p = 0.0054. (B) Evaluation of cytoskeleton organization by immunostaining: α smooth muscle actin (α-SMA) network in Mstn treated cells is deranged respect to control cells (magnification 100x). (C) F-actin stress fiber organization visualized with phalloidin-FITC in the absence or presence of Mstn. A 24 hour Mstn treatment affected F-actin organization. Arrows indicate cortical F-actin distribution (magnification 40x). (D) Mstn induces VSMC migration. **p < 0.01 vs. untreated cells. (E) Mstn acts as a chemoattractant stimulus on VSMCs. *p < 0.05 vs. untreated cells. (F) Effects of Mstn on MCP-1 and CCR2 mRNA levels determined by real time PCR after 4 and 24 hour exposition. Values are expressed as fold increase ± SEM versus untreated cells. *p < 0.05, **p < 0.01. (G) MCP-1 and CCR2 protein expression as evaluated by western blot and immunocytochemistry respectively, after a 48 hour treatment.

Figure 6. Effects of Myostatin (Mstn) on monocytes. (A) In THP-1 cell line, Mstn induces MCP-1 and α-smooth muscle actin (α-SMA) mRNAs. (B) In human freshly isolated monocytes, Mstn overexpressed MCP-1 and α-SMA mRNAs; (C) MCP-1 increased Mstn mRNA expression; mRNA levels were determined by real time PCR. All data were first normalized to β-actin mRNA and expressed as fold increase ± SEM versus untreated cells (D) MCP-1 and CCR2 protein expression; MCP-1 was evaluated in THP-1 cells by immunocytochemistry and in freshly isolated human monocytes by western blot after a 16 hour and one hour of Mstn treatment respectively. CCR2 membrane expression was evaluated on THP-1 cells after 8 hours of Mstn exposure. (E) Migration assay of THP-1 cells toward control medium, Mstn enriched medium (500 ng/ml), MCP-1 (as positive control). (F) Chemotaxis of freshly isolated monocytes, toward a Mstn-enriched medium at different concentrations. MCP-1 was used as positive control. (G) Mstn exposition caused F-actin polymerization distributed mainly in the cortical region, as demonstrated by AlexaFluor 488-Phalloidin staining (magnification 40x). *p < 0.05, **p < 0.01, ***p < 0.001 vs. untreated cells.

Figure 7. Effect of Myostatin (Mstn) on MAPKinase phosphorylation in A7r5 VSMCs and human freshly isolated monocytes. Cells were exposed to Mstn for 15, 45, 60 minutes and cell lysates were analyzed by western blot. Membranes were stripped and reprobed for non-phosphorylated MAPKs. Mstn induces phosphorylation of JNK and p38 in A7r5 cells (A), and of JNK in monocytes (B). All results represent mean ± SEM obtained from three independent experiments. *p < 0.05, **p < 0.01 vs T0. p-JNK = Phosphorylated JNK, p-ERK = Phosphorylated ERK, p-p38 = Phosphorylated p38.