Volume 68, Issue 1, Pages (July 2005)

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Volume 68, Issue 1, Pages 110-120 (July 2005) Shedding of endothelial protein C receptor contributes to vasculopathy and renal injury in lupus: In vivo and in vitro evidence1 Carlos A. Sesin, Xiaoming Yin, Charles T. Esmon, Jill P. Buyon, Robert M. Clancy Kidney International Volume 68, Issue 1, Pages 110-120 (July 2005) DOI: 10.1111/j.1523-1755.2005.00385.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Plasma levels of soluble endothelial protein C receptor (sEPCR). Distribution of soluble EPCR in 59 healthy controls and 81 systemic lupus erythematosus (SLE) patients. Individuals with soluble EPCR greater than mean control + 2 SD are indicated by bold symbols. Symbols above line indicate soluble EPCR levels greater than those of 75% of the control subjects (i.e., >200ng/mL). Kidney International 2005 68, 110-120DOI: (10.1111/j.1523-1755.2005.00385.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Serial studies of soluble endothelial protein C receptor (sEPCR) levels in plasma. Plasma levels of soluble EPCR were assessed in systemic lupus erythematosus (SLE) patients (A to C) and controls (D) at separate visits during an interval of 27 months. (A) Subset of SLE patients with soluble EPCR levels > control mean + 2 SD (>339ng/mL) (group A); data are longitudinal measurements (where available). (B) SLE patients with soluble EPCR >75% of controls but <mean control + 2 SD (group B). (C) SLE patients with soluble EPCR ≤75% of controls (≤200ng/mL) (group C). (D) Controls. Note: When soluble EPCR was plotted at each clinic visit for patients in group A, values were highly variable. Kidney International 2005 68, 110-120DOI: (10.1111/j.1523-1755.2005.00385.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Plasma levels of soluble E-selectin (sE-selectin). (A) Distribution of soluble E-selectin in 57 healthy controls and 63 systemic lupus erythematosus (SLE) patients. Individuals with soluble E-selectin levels greater than mean control + 2 SD are indicated by bold symbols. (B) Correlation of soluble E-selectin levels with levels of soluble endothelial protein C receptor (sEPCR). Kidney International 2005 68, 110-120DOI: (10.1111/j.1523-1755.2005.00385.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Membrane-bound endothelial protein C receptor (mEPCR) in human aortic endothelial cells (HAEC). HAEC were incubated with or without interferon-γ (IFN-γ) (100ng/mL, 24 hours), interleukin (IL)-1 (5ng/mL) in the absence and presence of metalloprotease inhibitors (CC1000, 10 nM). Resting and IFN-γ-treated HAEC were stained with mouse anti-EPCR or isotype and fluorescein isothiocyanate (FITC) antimouse IgG. (A) Cells were analyzed by FACS. (B) Cell fluids, from the HAEC experiment in (A), were analyzed for soluble EPCR (sEPCR) by a sandwich enzyme-linked immunosorbent assay (ELISA) method. Kidney International 2005 68, 110-120DOI: (10.1111/j.1523-1755.2005.00385.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Membrane-bound endothelial protein C receptor (mEPCR) in circulating endothelial cells from systemic lupus erythematosus (SLE) patients and controls. Circulating endothelial cells were isolated from peripheral blood as described. Representative stains of a patient with SLE (B to E) and controls (A and F). Venous blood was drawn into tubes with ethylenediaminetetraacetic acid (EDTA) and studied immediately. Circulating endothelial cells were isolated by immunomagnetic separation using P1H12-coated iron beads. The cell isolate was applied to silica and analyzed by scanning electron microscopy (A and B) or applied as a cytospin to a slide, fixed, stained with isotype control (C) or with anti-EPCR (D to F) and alkaline phosphatase-conjugated antimouse IgG, counterstained with hematoxylin. (D) A representative EPCR-negative circulating endothelial cell from an SLE patient. (E and F) Cells that stain positive for EPCR from SLE patient (E) and control (F). Kidney International 2005 68, 110-120DOI: (10.1111/j.1523-1755.2005.00385.x) Copyright © 2005 International Society of Nephrology Terms and Conditions