Pleiotropic Effects of the Protease-Activated Receptor 1 (PAR1) Inhibitor, Vorapaxar, on Atherosclerosis and Vascular Inflammation

Figure 1. PAR1 activation corresponds to endothelial activation and vascular inflammation in patients with atherosclerotic disease. Active (thrombin-cleaved) PAR1 was detected via flow cytometry on circulating PBMCs. Biomarkers indicative of (A) endothelial activation (VCAM-1, ICAM-1, E-selectin) and (B) vascular inflammation (TNF-α, IL-6, CRP) were measured with ELISA. Results are expressed as single values (n = 190), Pearson correlation coefficients, and linear regression lines with 95% CI.

Figure 2. Myocardial PAR1 expression correlates with marker of thrombo-inflammation in patients with coronary atherosclerosis. Relative PAR1 gene expression in EMBs: (A) relative to inflammatory marker VCAM-1, TF (both relative gene expression), and distribution of CD11b+/Mac-1+ macrophages (IHC); and (B) relative to TLR2 and TLR4 gene expression. Results are expressed as single values (n = 12), Pearson correlation coefficients, and linear regression lines with 95% CI.

Figure 3. PAR1 is present in human carotid atherosclerotic lesions and colocalizes with TLR2 and TLR4. Representative HE-staining images of human atherosclerotic plaque obtained by CEA (A,B, left panels). Displayed are the specimen from two patients. Corresponding immunofluorescence staining (A,B, right panels) reveals (A) intraplaque expression of PAR1 (green) within the endothelial layer (arrowheads) and the immune cell-infiltrating area (asterisk). (B) Colocalization of PAR1 (green) with TLR2 (magenta) and TLR4 (red). Scale bars (A) 160 µm/250 µm and (B) 400 µm/100 µm.

Figure 4. Vorapaxar reduces de novo atherosclerosis in ApoEko mice fed an atherogenic diet. ApoEko mice received a WD (0.21% cholesterol) (control) ± vorapaxar (10 mg/kg WD) for 16 weeks. (A) Quantification of plaque areas in aortic arch (arrows), expressed as % Oil red O-positive area. (B) Plaque area (%) (asterisks) within aortic sinus (HE-staining images). Representative images, scale bars (A) 500 µm and (B) 400 µm. Results are expressed as single values (n = 6), mean with SD.

Figure 5. PAR1 inhibition with vorapaxar attenuates vascular inflammation. (A) Reduced aortic transcription of VCAM-1, E-selectin, and TF. (B) Corresponding immunofluorescence staining of VCAM-1 (red). (C,D) Endothelial cells were isolated from ApoEko mice on a WD (control) ± vorapaxar (10 mg/kg WD) after 16 weeks. (C) Diminished relative gene expression of VCAM-1, E-selectin, and TF. (D) Vorapaxar treatment reduced expression of TLR2 and TLR4 in isolated endothelial cells and (E) within the atherosclerotic plaque covering endothelial layer (arrows) and subepithelial layer (reduced red IHC signal, right panel). (F) Quantification of relative mean TLR2 and TLR4 intensity within the atherosclerotic plaque covering the endothelial layer. Representative images, scale bars (B) 130 µm and (E) 40 µm. Asterisks indicate luminal side. Results are expressed as single values (n = 6), mean with SD.

Figure 6. Vorapaxar treatment is associated with an anti-inflammatory cell and cytokine profile within atherosclerotic plaques. (A,B) Intraplaque distribution of pro-inflammatory CD68-positive cells (% in relation to total plaque area) and the ratio of CD80+/CD206+ cells (M1/M2 macrophages) was reduced under vorapaxar treatment. (C) Aortic transcription of chemotactic CCL2, proinflammatory cytokines TNF-α and IL-1β, and innate immune receptors TLR2 and TLR4 decreased in the vorapaxar group. (D) Presence of CD3+ T cells and aortic gene expression of inflammatory T-cell cytokines (IL-2, IL-12B, IFN-γ) were found to be decreased under the treatment with the PAR1 inhibitor. (E,F) Reduction of the necrotic core size (% in relation to total plaque area) (asterisk in the corresponding image) and apoptotic plaque area (active caspase-3-positive plaque area) in vorapaxar-treated mice. Representative images, scale bars 400 µm. Results are expressed as single values (n = 6), mean with SD.

Figure 7. PAR1 inhibition with vorapaxar reduced thrombogenicity in mice. (A) In ApoEko mice, addition of vorapaxar reduced circulating markers of platelet activation (P-selectin, PF4/CXCL4) and thrombin activity (TAT). (B) Net thrombogenic potential of plaques from either the control or the vorapaxar-treated group. PRP or PPP from B6 wt mice was stimulated with homogenized aortic plaque material. Platelet activation marker (P-selectin, PF4/CXCL4) and thrombin activity (TAT) were measured in supernatant. Results are expressed as single values (n = 6), mean with SD.