Supplementary material
Supplementary material
Files in this Data Supplement:
- Online methods -
Immunohistochemical Analysis in Human Tissues
ELISA Analysis of CXCL12 Levels in Human Pulmonary Hypertensive Disease
Immunohistochemical Analysis in Murine Lungs
Immunohistochemical Analysis in Rat Lungs
Quantification of CXCR7 and CXCR4 staining
CXCL12 ELISA Analysis in Murine Samples
Western Blotting in Human Microvascular Endothelial Cells
Scratch Assay
Proliferation Assay
Migration Assay - Figure S1. Immunostaining in the vasculature of patients with usual interstitial pneumonia with pulmonary hypertension (UIP-PH) - CXCR7 (panels a & b), CXCR4 (panels d & e) and CXCL12 (panels g & h) protein is expressed (brown colour) in the endothelium of the pulmonary vasculature in UIP-PH. Positive protein staining in small ( <_10061549m diameter="diameter" _="_" column="column" _1="_1" and="and" large="large">100m diameter � column 2) remodeled vessels are shown. Positive staining was absent when isotype matched antibodies were tested on the tissue sections (IgG2A for CXCR7 (panel c), IgG2B for CXCR4 (panel f), IgG1 for CXCL12 (panel i). Arrows indicate endothelium of vessels, scale bar = 50m, x 40 objective.
- Figure S2. Immunostaining of infiltrating cells in patients with usual interstitial pneumonia with pulmonary hypertension (UIP-PH) - Representative images of infiltrating cells in the lung in IPAH showed positive staining with CXCR7 (panels a and b), CXCR4 (panels d and e) and CXCL12 (panels g & h) at low (x10 objective � column 1) or high (x40 objective � column 2) magnification. No staining was observed in these cells with appropriate isotype controls (IgG2A for CXCR7 (panel c), IgG2B for CXCR4 (panel f), IgG1 for CXCL12 (panel i). Scale bars are as indicated.
- Figure S3. Immunohistochemical localization of CXCR7 and CXCR4 in a rat model of hypoxic pulmonary hypertension - Basal expression of CXCR7 in normoxic lung tissue (panel a) increased with exposure to hypoxia (panel b). Stereological analysis indicated that the volume fraction of CXCR7-positive cells was significantly higher in hypoxic lungs (panel d). Similarly, the volume fraction of CXCR4-stained tissue in normoxic (panel e) and hypoxic (panel f) lungs showed that CXCR4 protein was significantly higher in the hypoxic lungs (panels h). No staining was observed with appropriate IgG controls (panel c, g and k). Representative images from n=6-8 lungs/group are shown; arrows indicate vessels; x 40 objective, scale bar = 50m. Data are presented as means � SEM (* = p<0.05).
- Figure S4. In vitro experiments with human pulmonary microvascular endothelial cells - (A) CXCL12 induced scratch wound healing in human pulmonary microvascular endothelial cells was inhibited by both CCX771 and CCX773. (B) CXCL12 (10nM) induced significant human pulmonary microvascular endothelial cell proliferation and blocking CXCR7 with either CCX771 or CCX773 inhibited this proliferative effect. (C) CXCL12 significantly induced pulmonary microvascular endothelial cell migration, an effect that was unaltered by either CXCR7 inhibitor, CCX771 and CCX773. All experiments were carried out n=6 independent times; * indicates significant difference between control and CXCL12; # indicates significant difference from all other groups (p<0.05).
