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. 2013 Dec;3(4):936–951. doi: 10.1086/674754

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© 2014 by the Pulmonary Vascular Research Institute. All rights reserved.

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Sickle cell disease (SCD) lungs overexpress markers of pulmonary arterial hypertension (PAH). A, Western immunoblot of lysates from lung tissue were performed. Representative blots from 3 control (CTRL), 1 SCD-associated PAH, and 4 non-SCD PAH lungs are presented. Densitometry is presented as the mean ratio of total protein compared with β-actin ± standard deviation (SD). Single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL, and double asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL and SCD PAH. B, Pulmonary arterial smooth muscle cells from healthy CTRL and SCD pulmonary arteries were serum starved and treated in growth factor– and serum-free medium containing 0.1% bovine serum albumin with thrombospondin 1 (TSP1; 2.2 nmol/L) or hypoxia (1% O₂) with or without a CD47 blocking antibody (Ab; clone B6H12, 1 μg/mL). Cell lysates were prepared and Western immunoblots preformed. Representative data from 3 separate experiments are shown. Densitometry is presented as the mean ratio of total protein to β-actin ± SD. For TSP1, single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL, and the asterisk with a pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL and SCD-normoxia (Nx); for CD47, single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL; for endothelin receptor A (ETA), single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, the pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-hypoxia (Hx), and the pound sign with a percentage sign indicates a statistically significant difference (P < 0.05) compared with SCD-Nx, SCD-TSP1, and SCD-Hx; and for endothelin 1 (ET-1), single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, the pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-Hx, and double asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL. C, Total RNA was extracted and reverse transcribed to complementary DNA, and reverse-transcription polymerase chain reaction was performed. Results were normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase 1 and are presented as mean ± SD (from 3 experiments). For TSP1 messenger RNA (mRNA), single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, double asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL, the single pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-TSP1 and CTRL-Hx, and the double pound sign indicates a statistically significant difference (P < 0.05) compared with SCD-Nx, SCD-TSP1, and SCD-Hx; for CD47 mRNA, asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx; for ET-1 mRNA, single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, and double asterisks indicate a statistically significant difference (P < 0.05) compared with SCD-Nx; and for EDNRA mRNA, the single asterisk indicates a statistically significant difference (P < 0.05) compared with CTRL-Nx, the pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-TSP1, and double asterisks indicate a statistically significant difference (P < 0.05) compared with SCD-Nx and SCD-TSP1.

Inline graphic — Sickle cell disease (SCD) lungs overexpress markers of pulmonary arterial hypertension (PAH). A, Western immunoblot of lysates from lung tissue were performed. Representative blots from 3 control (CTRL), 1 SCD-associated PAH, and 4 non-SCD PAH lungs are presented. Densitometry is presented as the mean ratio of total protein compared with β-actin ± standard deviation (SD). Single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL, and double asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL and SCD PAH. B, Pulmonary arterial smooth muscle cells from healthy CTRL and SCD pulmonary arteries were serum starved and treated in growth factor– and serum-free medium containing 0.1% bovine serum albumin with thrombospondin 1 (TSP1; 2.2 nmol/L) or hypoxia (1% O₂) with or without a CD47 blocking antibody (Ab; clone B6H12, 1 μg/mL). Cell lysates were prepared and Western immunoblots preformed. Representative data from 3 separate experiments are shown. Densitometry is presented as the mean ratio of total protein to β-actin ± SD. For TSP1, single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL, and the asterisk with a pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL and SCD-normoxia (Nx); for CD47, single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL; for endothelin receptor A (ETA), single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, the pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-hypoxia (Hx), and the pound sign with a percentage sign indicates a statistically significant difference (P < 0.05) compared with SCD-Nx, SCD-TSP1, and SCD-Hx; and for endothelin 1 (ET-1), single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, the pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-Hx, and double asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL. C, Total RNA was extracted and reverse transcribed to complementary DNA, and reverse-transcription polymerase chain reaction was performed. Results were normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase 1 and are presented as mean ± SD (from 3 experiments). For TSP1 messenger RNA (mRNA), single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, double asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL, the single pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-TSP1 and CTRL-Hx, and the double pound sign indicates a statistically significant difference (P < 0.05) compared with SCD-Nx, SCD-TSP1, and SCD-Hx; for CD47 mRNA, asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx; for ET-1 mRNA, single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, and double asterisks indicate a statistically significant difference (P < 0.05) compared with SCD-Nx; and for EDNRA mRNA, the single asterisk indicates a statistically significant difference (P < 0.05) compared with CTRL-Nx, the pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-TSP1, and double asterisks indicate a statistically significant difference (P < 0.05) compared with SCD-Nx and SCD-TSP1.