Figure 5: ABCC4 Contributes to Endothelial and Monocyte Activation and Increased S1P Release From Activated Platelets in HIV

(A) Immunofluorescence microscopy of platelet adhesion to HUVECs in subjects with HIV. Stained platelets (green) were left untreated (Untreated) or treated with Ceefourin2 (Ceefourin, 10 μmol/l) for 30 min. Thrombin (0.05 U/ml, 5 min) was then added (Activated). HUVEC nuclei are stained with DAPI (blue). The images are representative of 3 subjects for each group. Values are represented as percentage of respective controls. **p < 0.01 and *p < 0.05. Magnification = 20×. (B and C) Gene expression analysis of HUVEC inflammatory genes IL-8, ICAM-1, and MCP1, and THP-1 markers IL-6 and MCP-1. HUVECs and THP-1 were cocultured with untreated or pre-treated (Ceefourin, 10 μmol/l) platelets from HIV-infected subjects at basal (upper graphs) and after stimulation with thrombin (0.05 U/ml, bottom graphs). Results were normalized on 18S5 RNA. Values represent fold change after normalization to basal or activated condition in 3 different subjects. **p < 0.01 versus Basal and *p < 0.05 versus Activated. (D) Sphingosine-1-phosphate (S1P) in platelet supernatants of HIV (n = 16) and controls (C) (n = 8) at the basal state was analyzed by high-performance liquid chromatography-mass spectrometry (HPLCMS/MS) and normalized for protein content. *p < 0.05. (E) S1P quantification in platelet supernatants of subjects with HIV (n = 17) and healthy controls (n = 7) before and after stimulation with thrombin (0.05 U/ml, 5 min). Levels are reported after normalization to respective basal levels. *p < 0.05. (F) Platelets from healthy subjects (n = 5) were left untreated or stimulated with thrombin in presence or absence of Ceefourin (10 μmol/l). S1P levels were quantified in platelet supernatants, and values expressed as percentage of resting platelets. *p < 0.05. (G) S1P quantified in plasma of subjects with HIV (n = 40) and controls (n = 16) by HPLCMS/MS. Abbreviations as in Figure 1.

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