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

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Sickle cell disease (SCD)–associated pulmonary arterial hypertension (PAH) pulmonary artery smooth muscle cells (PASMCs) produce more superoxide (O₂^•−) and display enhanced proliferation and increased matrix protein gene expression. A, PASMCs isolated from healthy control (CTRL) and SCD proximal pulmonary arteries were serum starved and treated in growth factor– and serum-free medium containing 0.1% bovine serum albumin (BSA) incubated in normoxia (Nx) or hypoxia (1% O₂) for 3 hours. Superoxide production was measured in the 28,000 g membrane fraction and calculated from the initial linear rate of SOD-inhibitable cytochrome c reduction. Data represent the mean ratio of superoxide to total protein ± standard deviation (SD). Asterisks indicate a statistically significant difference (P < 0.05) compared with normoxic CTRL cells. B, PASMCs isolated from healthy CTRL and SCD proximal pulmonary arteries were serum starved and treated in growth factor– and serum-free medium containing 0.1% BSA with thrombospondin 1 (TSP1; 2.2 nmol/L) or hypoxia (1% O₂) with or without CD47 blocking antibody (clone B6H12, 1 μg/mL) for 24 hours. Cell lysates were prepared and Western immunoblots performed. Representative data from 3 different experiments are shown. Densitometry is presented as the mean ratio of total protein to β-actin ± SD. Asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL, and the pound sign indicates a statistically significant difference (P < 0.05) compared with SCD-normoxia (Nx), SCD-TSP1, and SCD-hypoxia (Hx). C, PASMCs isolated from CTRL and SCD pulmonary arteries were plated at subconfluent density and grown in standard smooth muscle cell medium (Lonza) or under serum-free conditions, and proliferation was measured at 48 hours. Data are presented as mean ± SD (from 3 experiments). For starved, the single asterisk indicates a statistically significant difference (P < 0.05) compared with control 1, and the double asterisk indicates a statistically significant difference (P < 0.05) compared with control 1 and control 2; for replete, the triple asterisk indicates a statistically significant difference (P < 0.05) compared with control 1 and control 2. D, Cells from SCD or CTRL pulmonary arterial segments were treated as in A and collected in lysis buffer, RNA was extracted and reverse transcribed to complementary DNA, and reverse-transcription polymerase chain reaction was performed for the indicated collagen genes. Results were normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase 1 and are presented as mean ± SD (from 3 experiments). For collagen Iα1 (Col Iα1) messenger RNA (mRNA), single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, and double asterisk indicates a statistically significant difference (P < 0.05) compared with CTRL-TSP1; for collagen IIIα1 (Col IIIα1) mRNA, the single asterisk indicates a statistically significant difference (P < 0.05) compared with CTRL-Nx and CTRL-TSP1, 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-Nx, CTRL-TSP1, and CTRL-Hx; and for collagen IVα1 (Col IVα1) mRNA, single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx and CTRL-TSP1, the pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-Hx, and the double pound sign indicates a statistically significant difference (P < 0.05) compared with SCD-Nx and SCD-TSP1.

Inline graphic — Sickle cell disease (SCD)–associated pulmonary arterial hypertension (PAH) pulmonary artery smooth muscle cells (PASMCs) produce more superoxide (O₂^•−) and display enhanced proliferation and increased matrix protein gene expression. A, PASMCs isolated from healthy control (CTRL) and SCD proximal pulmonary arteries were serum starved and treated in growth factor– and serum-free medium containing 0.1% bovine serum albumin (BSA) incubated in normoxia (Nx) or hypoxia (1% O₂) for 3 hours. Superoxide production was measured in the 28,000 g membrane fraction and calculated from the initial linear rate of SOD-inhibitable cytochrome c reduction. Data represent the mean ratio of superoxide to total protein ± standard deviation (SD). Asterisks indicate a statistically significant difference (P < 0.05) compared with normoxic CTRL cells. B, PASMCs isolated from healthy CTRL and SCD proximal pulmonary arteries were serum starved and treated in growth factor– and serum-free medium containing 0.1% BSA with thrombospondin 1 (TSP1; 2.2 nmol/L) or hypoxia (1% O₂) with or without CD47 blocking antibody (clone B6H12, 1 μg/mL) for 24 hours. Cell lysates were prepared and Western immunoblots performed. Representative data from 3 different experiments are shown. Densitometry is presented as the mean ratio of total protein to β-actin ± SD. Asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL, and the pound sign indicates a statistically significant difference (P < 0.05) compared with SCD-normoxia (Nx), SCD-TSP1, and SCD-hypoxia (Hx). C, PASMCs isolated from CTRL and SCD pulmonary arteries were plated at subconfluent density and grown in standard smooth muscle cell medium (Lonza) or under serum-free conditions, and proliferation was measured at 48 hours. Data are presented as mean ± SD (from 3 experiments). For starved, the single asterisk indicates a statistically significant difference (P < 0.05) compared with control 1, and the double asterisk indicates a statistically significant difference (P < 0.05) compared with control 1 and control 2; for replete, the triple asterisk indicates a statistically significant difference (P < 0.05) compared with control 1 and control 2. D, Cells from SCD or CTRL pulmonary arterial segments were treated as in A and collected in lysis buffer, RNA was extracted and reverse transcribed to complementary DNA, and reverse-transcription polymerase chain reaction was performed for the indicated collagen genes. Results were normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase 1 and are presented as mean ± SD (from 3 experiments). For collagen Iα1 (Col Iα1) messenger RNA (mRNA), single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx, and double asterisk indicates a statistically significant difference (P < 0.05) compared with CTRL-TSP1; for collagen IIIα1 (Col IIIα1) mRNA, the single asterisk indicates a statistically significant difference (P < 0.05) compared with CTRL-Nx and CTRL-TSP1, 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-Nx, CTRL-TSP1, and CTRL-Hx; and for collagen IVα1 (Col IVα1) mRNA, single asterisks indicate a statistically significant difference (P < 0.05) compared with CTRL-Nx and CTRL-TSP1, the pound sign indicates a statistically significant difference (P < 0.05) compared with CTRL-Hx, and the double pound sign indicates a statistically significant difference (P < 0.05) compared with SCD-Nx and SCD-TSP1.