Cellular, pharmacological, and biophysical evaluation of explanted lungs from a patient with sickle cell disease and severe pulmonary arterial hypertension

Abstract

Pulmonary hypertension is recognized as a leading cause of morbidity and mortality in patients with sickle cell disease (SCD). We now report benchtop phenotyping from the explanted lungs of the first successful lung transplant in SCD. Pulmonary artery smooth muscle cells (PASMCs) cultured from the explanted lungs were analyzed for proliferate capacity, superoxide (O2 (•-)) production, and changes in key pulmonary arterial hypertension (PAH)-associated molecules and compared with non-PAH PASMCs. Upregulation of several pathologic processes persisted in culture in SCD lung PASMCs in spite of cell passage. SCD lung PASMCs showed growth factor- and serum-independent proliferation, upregulation of matrix genes, and increased O2 (•-) production compared with control cells. Histologic analysis of SCD-associated PAH arteries demonstrated increased and ectopically located extracellular matrix deposition and degradation of elastin fibers. Biomechanical analysis of these vessels confirmed increased arterial stiffening and loss of elasticity. Functional analysis of distal fifth-order pulmonary arteries from these lungs demonstrated increased vasoconstriction to an α1-adrenergic receptor agonist and concurrent loss of both endothelial-dependent and endothelial-independent vasodilation compared with normal pulmonary arteries. This is the first study to evaluate the molecular, cellular, functional, and mechanical changes in end-stage SCD-associated PAH.

Keywords: CD47; endothelin 1; lung transplant; matrix; pulmonary arterial hypertension; sickle cell disease; superoxide; thrombospondin 1.

Figure 1

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.

Figure 2

Sickle cell disease (SCD)–associated pulmonary arterial hypertension (PAH) lungs display general vasculopathy and upregulation of markers of PAH. A, Light microscopic examination of hematoxylin-eosin (right) and Verhoeff’s (left) stain of tissue sections of SCD-associated PAH whole lung demonstrated marked arterial thickening, inflammation, and hypertrophic collagen. In sections stained with Verhoeff’s, elastic fibers are black, collagen fibers are pink/red, and cell cytoplasm is yellow. Magnification, ×10; scale bars = 200 μM. B, Representative immunofluorescent cross sections of proximal pulmonary arteries, fifth-order pulmonary arteries, and peripheral parenchyma from control non-PAH and SCD-associated PAH lungs. The top two rows display thrombospondin 1 (TSP1; red) and endothelin 1 (ET-1; green), and the bottom two rows display CD47 (green) and endothelin receptor A (ETA; red). Magnification, ×20. PA: pulmonary arteries.

Figure 3

Vascular remodeling in sickle cell disease (SCD)–associated pulmonary arterial hypertension (PAH) contributes to abnormal biomechanics. A, Upper panels of control and SCD-associated pulmonary arterial tissue samples after Movat’s pentachrome staining demonstrating increased neointimal formation. Representative sections are presented. M: vascular media; Adv: vascular adventitia; NI: neointima. Lower panels show control and SCD-associated pulmonary arterial samples after 2-photon second-harmonic-generation microscopy, wherein elastin is shown in green and collagen is shown in red. The white circle depicts clipped medial elastin; the single arrow points to inner media collagen deposition, and the double-arrow points to outer media collagen deposition and highlights clipping and fragmentation of media elastin and accumulation of collagen in the inner and outer regions of the vascular media of the SCD sample. B, Stress-versus-strain behavior of fresh SCD-associated pulmonary arterial compared with non-PAH control (CTRL) pulmonary arterial segment. ε_trans is the strain of transition from the elastin-dominant region (A) to the collagen-dominant region (B), associated with increased collagen engagement. C, Tissue-stored energy of fresh SCD-associated PAH pulmonary arterial segments compared with non-PAH control pulmonary arterial segments. PA: pulmonary arteries.

Figure 4

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.

Figure 5

Distal fifth-order pulmonary arteries from end-stage sickle cell disease (SCD)–associated pulmonary arterial hypertension (PAH) lungs display increased vasoconstriction and loss of endothelial-dependent and endothelial-independent vasodilation. A, Vasoconstriction of control and SCD lung distal fifth-order pulmonary arteries was determined to a log dose of phenylephrine (PE) with results normalized to KCl response. Data are presented as mean ± standard deviation (SD; from 4 rings per treatment point). Asterisks indicate a statistically significant difference (P < 0.05) compared with control vessel. Control and SCD lung fifth-order pulmonary arteries were treated with a PE dose producing 80% maximum contraction (EC₈₀) and then challenged with log dose concentrations of acetylcholine (Ach; B; 10⁻⁸ to 10⁻⁵ M; Sigma-Aldrich) or sodium nitroprusside (SNP; C; 10⁻⁹ to 10⁻⁶ M; Sigma-Aldrich), and vasodilation was determined. Data are presented as mean ± SD (from 4 rings at each treatment point). Asterisks indicate a statistically significant difference (P < 0.05) compared with control vessel.

Figure 6

Reverse-transcription polymerase chain reaction validation of microarray data of changes in RNA expression in sickle cell disease (SCD) lung tissue of selected genes. Data represent the mean ± standard deviation of 5 technical replicates of the SCD lung tissue and 5 technical replicates each of 4 normal control lung tissue specimens. Asterisks indicate a statistically significant difference (P < 0.01) compared with control (CTRL).