Local Production of Activated Factor X in Atherosclerotic Plaque Induced Vascular Smooth Muscle Cell Senescence

Figure 1. In situ hybridization of coagulation FX in human atherosclerotic plaques. (A) In situ hybridization of human atherosclerotic plaques obtained from the patients subjected to carotid endarterectomy with an antisense probe to human FX. (B) This panel shows αSMA and DAPI staining. (C) The sense probe strand shows no specific hybridization. (D) Control carotid artery samples with antisense probe demonstrated much weaker signals compared to the samples from patients. (E,F) A higher-magnification image shows strong positive staining in VSMCs (E) and inflammatory cells (F).

Figure 2. Induction of SMC senescence by FXa through IGFBP-5. (A–C) Human aortic VSMCs were treated every other day for 14 days with FXa (50 nM), FXa (50 nM) with rivaroxaban (10 μM), or recombinant IGFBP-5 (rIGFBP-5, 100 ng/mL). Additionally, IGFBP-5 was overexpressed in human aortic VSMCs using pIGFBP5. (A) Senescent cells were detected by senescence associated β-galactosidase staining (SA-β-gal staining). (B) The fractions of SA-β gal-positive VSMCs. *, **P < 0.05 vs. control (CTRL) and FXa, respectively. n = 10. (C) Over-expression of IGFBP-5 increased the fractions of SA-β Gal-positive VSMCs. *P < 0.05 vs. control (CTRL). n = 10. (D–F) FXa significantly increased the expression of IGFBP-5 and p53, and similarly, rIGFBP-5 up-regulated p53 expression in human aortic VSMCs. However, rivaroxaban or IGFBP-5 siRNA remarkably reduced FXa-induced IGFBP-5 and p53 up-regulation. *, **P < 0.05 vs. control and FXa, respectively. n = 4.

Figure 3. Senescence-associated inflammatory responses and impaired the proliferation. (A) Relative mRNA expression of IL-1β/GAPDH, IL-6/GAPDH, MCP-1/GAPDH, and IGFBP-5/GAPDH in FXa treated human aortic VSMCs. *, **P < 0.05 vs. control siRNA and control siRNA + FXa 50 nM, respectively. n = 3. (B) FXa-induced inflammatory cytokine expression was reduced by the direct FXa inhibitor, ribaroxban. *, **P < 0.05 vs. control and FXa 50 nM, respectively. n = 5. (C) The ability of FXa-treated human aortic VSMCs to proliferate was measured by MTS assay. *, **P < 0.05 vs. control (CTRL) and FXa (50 nM), respectively. n = 6.

Figure 4. Distribution of FXa and IGFBP-5 staining area in human atherosclerotic plaques. (A) Human atherosclerotic plaques were stained with FXa (left), IGFBP-5 (middle), and Hematoxylin-Eosin staining (right). (B,C) Higher magnification of the FXa and IGFBP-5 positive area in VSMCs (B, black arrow) and inflammatory cells (C, white arrow).