Altered Hypoxic-Adenosine Axis and Metabolism in Group III Pulmonary Hypertension

Abstract

Group III pulmonary hypertension (PH) is a highly prevalent and deadly lung disorder with limited treatment options other than transplantation. Group III PH affects patients with ongoing chronic lung injury, such as idiopathic pulmonary fibrosis (IPF). Between 30 and 40% of patients with IPF are diagnosed with PH. The diagnosis of PH has devastating consequences to these patients, leading to increased morbidity and mortality, yet the molecular mechanisms involved in the development of PH in patients with chronic lung disease remain elusive. Our hypothesis was that the hypoxic-adenosinergic system is enhanced in patients with group III PH compared with patients with IPF with no PH. Explanted lung tissue was analyzed for markers of the hypoxic-adenosine axis, including expression levels of hypoxia-inducible factor (HIF)-1A, adenosine A2B receptor, CD73, and equilibrative nucleotide transporter-1. In addition, we assessed whether altered mitochondrial metabolism was present in these samples. Increased expression of HIF-1A was observed in tissues from patients with group III PH. These changes were consistent with increased evidence of adenosine accumulation in group III PH. A novel observation of our study was of evidence suggesting altered mitochondrial metabolism in lung tissue from group III PH leading to increased succinate levels that are able to further stabilize HIF-1A. Our data demonstrate that the hypoxic-adenosine axis is up-regulated in group III PH and that subsequent succinate accumulation may play a part in the development of group III PH.

Keywords: adenosine A2B receptor; group III pulmonary hypertension; hypoxia-inducible factor-1A; idiopathic pulmonary fibrosis; succinate.

Figure 1.

Fibrosis and vascular remodeling in idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH). (A) Masson’s trichome–stained lung sections showing parenchymal areas from three distinct patients with IPF (top three panels) or IPF plus PH (bottom three panels). Scale bar represents 100 μm. (B) Ashcroft scores from upper and lower lobes from patients with IPF or IPF plus PH. (C) Collagen 1A1 (COL1A1) expression levels relative to 18s ribosomal RNA (18srRNA) expression levels from patients with IPF or IPF plus PH. (D) Masson’s trichome–stained lung sections showing representative vessels from three distinct patients with IPF (top three panels) or IPF plus PH (bottom three panels). Scale bar represents 100 μm. (E) Vessel remodeling assessed by measuring the distance between the adventitia and the lumen. (F) Mean pulmonary arterial pressure (mPAP) levels from patients with IPF or IPF plus PH. Results are presented as means ± SEM. Significance level: ***P < 0.001 refers to t-test comparisons between IPF and IPF plus PH groups.

Figure 2.

Representations of the molecular markers of hypoxia in PH secondary to IPF. (A) Diagram of the hypoxia pathway. (B) Immunoblot for hypoxia-inducible factor (HIF)-1A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from lung lysates from IPF and IPF plus PH lung tissue. (C) Immunofluorescence for α-smooth muscle actin (α-SMA; green signals), HIF-1A (red signals), and counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue signals). Scale bar represents 50 μm. Stabilization and nuclear localization of HIF-1A is shown in purple (white arrowheads). HIF-1A levels determined from ELISA from normal controls, IPF, and IPF plus PH lung tissue (D). Expression levels normalized to control lung of CD73 (E), GLUT1 (F), NOS3 (G), PAI1 (H), and PDK1 (I) from patients with IPF and patients with IPF plus PH. Results are presented as means ± SEM. (D) Significance levels: ***P < 0.001 and *P < 0.05 refers to ANOVA comparisons between control and IPF or IPF plus PH groups. ^#P< 0.05 refers to ANOVA comparisons between IPF and IPF plus PH groups. (E–I) Significance level: *P < 0.05 refers to t-test comparisons between IPF and IPF plus PH groups.

Figure 3.

Succinate metabolism is altered in IPF plus PH. (A) Immunoblot from lung lysates comparing control against IPF and patients with IPF plus PH for succinate dehydrogenase (SDHA; succinate dehydrogenase complex, subunit A) and GAPDH as a control. (B) SDH activity in isolated mitochondrial extracts and (C) succinate levels determined from lung lysates from control subjects, patients with IPF, and patients with IPF plus PH. (D) Simple linear regression with 95% confidence interval (CI) showing the relationship between HIF-1A and succinate levels (r² = 0.1524 and P = 0.0329). (E) Immunoblot from lung lysates comparing control subjects against patients with IPF and patients with IPF plus PH for prolyl-hydroxylase-1 (PHD1) and GAPDH as a control. Results are presented as means ± SEM. Significance levels: ***P < 0.001 and *P < 0.05 refers to ANOVA comparisons between control and IPF or IPF plus PH groups. ^##0.001 < P <0.01 and ^#P < 0.05 refers to ANOVA comparisons between IPF and IPF plus PH groups.

Figure 4.

Markers of mitochondrial stability. Transcript expression levels of peroxisome proliferator-activated receptor γ, coactivator 1 α (PPARGC1A; A) and mitofusin 2 (MFN2; B) from normal, IPF, and IPF plus PH lung samples. Results are presented as means ± SEM. Significance levels: ***P < 0.001 refers to ANOVA comparisons between control and IPF or IPF plus PH groups.

Figure 5.

Adenosine metabolism markers and their correlation to mPAP. (A) Schematic of extracellular adenosine metabolism and the sequential activity of CD39 and CD73, along with adenosine deaminase (ADA), to degrade ATP to the final product of inosine. (B) Simple linear regression with 95% CI showing relationship between mPAP and cellular expression of CD39 (r² = 0.2208 and P = 0.057). (C) Simple linear regression with 95% CI showing relationship between mPAP and CD73 (r² = 0.2593 and P = 0.044). (D) Simple linear regression with 95% CI showing relationship between mPAP and ADA (r² = 0.2503 and P = 0.041). (E) Immunohistochemistry for CD73 (blue signals) and αSMA; red signals from representative lung sections of a control subject (left), a patient with IPF (middle), or a patient with IPF plus PH (right). The scale bar represents 75 μm. The solid arrowheads point to areas rich in CD73 signals. Simple linear regression with 95% CI showing the relationship between HIF-1A levels and CD73 activity (r² = 0.4031 and P = 0.0082) (F). Ado, adenosine; Ino, inosine.

Figure 6.

Equilibrative nucleotide transporter (ENT) 1 involvement in patients with IPF and patients with IPF plus PH. (A) Immunoblot for ENT1 and GAPDH from normal (control), IPF, and IPF plus PH lung lysates. (B) Immunohistochemistry for ENT1 staining (brown signals) showing its presence around vessels in control, IPF, and IPF plus PH lung sections. The scale bar represents 100 μm. The arrows point to the localization of ENT1 signals; “V” denotes a vessel.

Figure 7.

Simple linear regression with 95% CI to evaluate relationship between different adenosine receptors and mPAP. (A) Relationship between adenosine receptor (ADORA) 1 and mPAP (r² = 0.0485 and P = 0.38). (B) Association between ADORA2A and mPAP (r² = 0.1220 and P = 0.155). (C) Correlation between ADORA3 and mPAP (r² = 0.0554 and P = 0.38). (D) Correlation between ADORA2B and mPAP (r² = 0.1685 and P = 0.037). (E) Correlation between ADORA2B and 6-minute walk distance (6MWD) (r² = 0.2422 and P = 0.023). (F) Immunoblot of patients with IPF and patients with IPF plus PH evaluating ADORA2B, with β-actin as a control.