Figure 5: ABCC4 Contributes to Endothelial and Monocyte Activation and Increased S1P Release From Activated Platelets in HIV
2019-06-11T07:07:04Z (GMT) by
(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.