Inhibition of Platelets by Clopidogrel Suppressed Ang II‐Induced Vascular Inflammation, Oxidative Stress, and Remodeling

Figures

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  Figure 1. Ang II infusion induced platelet activation and accumulation within the vessel. A, Representative images of flow cytometry displaying activated platelets, determined by the population of platelets expressing P‐selectin relative to the whole population of platelets in the fresh blood from saline‐infused or Ang II‐infused vessels at days 1, 3, or 7, respectively. B, Quantitation of P‐selectin‐positive platelets. C, Quantitation of mean fluorescent intensity of CD41. D, Representative images of IHC staining showing platelet accumulation (CD41) within the vessels with saline infusion or Ang II infusion at days 1, 3, and 7. Black arrow indicates aggregates of platelets, while red arrow indicates colocalization of platelets and nuclei. E and F, Western blot analysis for the abundance of platelets (CD41) in purified platelets (first lane, positive control) or vessels from mice subjected to Ang II or saline. Ang II indicates angiotensin II; IHC, immunohistochemistry. *P<0.05, **P<0.01, ***P<0.001 vs saline; #P < 0.05; n=4 to 6 per group.

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  Figure 2. Clopidogrel abolished Ang II‐induced platelet activation, and vascular inflammatory responses and remodeling. A through C, systolic blood pressure, bleeding time, and P‐selectin of groups with saline or Ang II infusion, with or without clopidogrel treatment. D, Representative images of IHC staining of CD41 in the vessel sections. E, Quantitation of CD41. F, Representative images of H&E staining of the vessels. G, Wall thickness of each group was analyzed. H, Representative images of Masson staining of the vessels. I, The percentage of fibrotic area was analyzed. J, Representative images of IHC staining of Mac‐3 on vessel sections. K, Mac‐3 positive cells were analyzed. L, mRNA levels of IL‐1β, IL‐6, TNF‐α, and VCAM‐1 in vessels. M, mRNA levels of collagen I and III in vessels. N, Plasma levels of IL‐1β, IL‐6, TNF‐α, and MCP‐1. A indicates adventitia; Ang II, angiotensin II; E, endangium; IHC, immunohistochemistry; IL‐1β, interleukin‐1β; IL‐6, interleukin‐6; M, media; MCP‐1, monocyte chemotactic protein 1; TNF‐α, tumor necrosis factor‐α; VCAM‐1, vascular cell adhesion molecule 1. *P<0.05, **P<0.01 vs saline+vehicle; # P<0.05, ## P<0.01 vs Ang II+vehicle; n=4 to 6 per group.

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  Figure 3. Clopidogrel reduced vascular oxidative stress in response to Ang II. A, Dihydroethidine staining of vessels with or without clopidogrel treatment following saline or Ang II infusion. Oxidative stress was stained by dihydroethidine, which was indicated by red fluorescence. The elastic lamina was indicated by green fluorescence. B, The fluorescent intensity of dihydroethidine was analyzed. C, mRNA levels of NOX‐1, NOX‐2, and NOX‐4 in vessels. D, The protein levels of NOX‐1, 2, and 4 in vessels were examined by using Western blot. E through G, Quantitation of NOX‐1, 2, and 4. A indicates adventitia; Ang II, angiotensin II; DHE, dihydroethidine; E, endangium; M, media; NOX‐1, NADPH oxidase 1; NOX‐2, NADPH oxidase 2; NOX‐4, NADPH oxidase 4. E: endangium; M: media; A: adventitia. *P<0.05, **P<0.01 vs saline+vehicle; # P<0.05, ## P<0.01 vs Ang II+vehicle; n=4 to 6 per group.

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  Figure 4. Clopidogrel suppressed Ang II‐evoked P‐M binding both in circulation and vessel. A, Time course of platelet‐monocyte, platelet‐neutrophil, and platelet‐lymphocyte binding in the blood of mice with saline or Ang II infusion determined by flow cytometry. B, Time course of platelet‐macrophage binding in vessels of mice with saline or Ang II infusion determined by flow cytometry. C, Representative images of flow cytometry displaying P‐M binding in blood, determined by the population of monocytes expressing P‐selectin relative to the whole population of monocytes. D, Quantitation of circulating P‐M binding. E, Representative images of flow cytometry displaying infiltrated leukocytes, determined by CD45‐positive cells in the vessels. F, Quantitation of infiltrated leukocytes in vessels. G, Representative images of flow cytometry displaying infiltrated P‐M binding in vessels, determined by the population of cells expressing P‐selectin and F4/80 relative to the whole population of F4/80 macrophages. H, Quantitation of infiltrated P‐M binding in the vessel samples. Ang II indicates angiotensin II; P‐M, platelet‐monocyte. *P<0.05, **P<0.01 vs saline+vehicle; ***P<0.001; # P<0.05 vs Ang…

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  Figure 5. Platelets promoted monocyte activation in response to Ang II stimulation. A, Representative images showed adhesive monocytes to HUVECs under the different conditions. HUVECs were stained by MitoGreen, determined by green fluorescence. THP‐1 cells were stained by Dil, determined by red fluorescence. B, Quantitation of adhesive monocytes. C and D, mRNA levels of IL‐1β and IL‐6 in vessels. Ang II indicates angiotensin II; IL‐1β, interleukin‐1β; IL‐6, interleukin‐6; HUVECS, human umbilical vein endothelial cells. *P<0.05 vs vehicle+monocytes alone; # P<0.05 vs Ang II+monocytes alone; n=5 to 6 per group.

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  Figure 6. Clopidogrel treatment prevented vascular dysfunction in response to Ang II. A, Concentration‐response curves of endothelium‐dependent (Ach) in mice with saline or Ang II infusion, with or without clopidogrel treatment. B, Concentration‐response curves of endothelium‐independent (SNP) relaxation in mice with saline or Ang II infusion, with or without clopidogrel treatment. Ang II indicates angiotensin II; Ach, acetylcholine; SNP, sodium nitroprusside. *P<0.05 vs saline+vehicle; # P<0.05 vs Ang II+clopidogrel; n=5 to 6 per group.

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  Figure 7. Effects of ACEI and ARB on platelet activation and vascular accumulation in mice with Ang II infusion. A, Quantitation of P‐selectin‐positive platelets in the blood from mice with saline or Ang II infusion, with or without ACEI treatment. B, Quantitation of P‐selectin‐positive platelets in the blood from mice with saline or Ang II infusion, with or without ARB treatment. C, Quantitation of circulating P‐M binding in the blood from mice with saline or Ang II infusion, with or without ACEI treatment. D, Quantitation of circulating P‐M binding in the blood from mice with saline or Ang II infusion, with or without ARB treatment. E, IHC staining of platelets in vessel sections of mice with or without ARB/ACEI treatment in response to saline or Ang II infusion. F, Quantitation of platelet accumulation in vessel samples. Ang II indicates angiotensin II; ACEI, angiotensin‐converting enzyme inhibitor; ARB, angiotensin receptor blocker; IHC, immunohistochemistry; P‐M, platelet‐monocyte. **P<0.01 vs saline+vehicle; # P<0.05 vs Ang II+vehicle; n=4 to 6 per group.

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  Figure 8. The potential role of platelets in Ang II‐infused vessel. High levels of Ang II induced platelet activation. Activated platelets subsequently bind to monocyte in the circulation, promoting monocyte activation. P‐M binding infiltrates into the vessel and then enhances vascular inflammatory responses. Ang II indicates angiotensin II; P‐M, platelet‐monocyte.