Focus on Early Events: Pathogenesis of Pulmonary Arterial Hypertension Development

Abstract

Significance: Pulmonary arterial hypertension (PAH) is a progressive disease of the lung vasculature characterized by the proliferation of all vascular wall cell types, including endothelial, smooth muscle, and fibroblasts. The disease rapidly advances into a form with extensive pulmonary vascular remodeling, leading to a rapid increase in pulmonary vascular resistance, which results in right heart failure. Recent Advances: Most current research in the PAH field has been focused on the late stage of the disease, largely due to an urgent need for patient treatment options in clinics. Further, the pathobiology of PAH is multifaceted in the advanced disease, and there has been promising recent progress in identifying various pathological pathways related to the late clinical picture. Critical Issues: Early stage PAH still requires additional attention from the scientific community, and although the survival of patients with early diagnosis is comparatively higher, the disease develops in patients asymptomatically, making it difficult to identify and treat early. Future Directions: There are several reasons to focus on the early stage of PAH. First, the complexity of late stage disease, owing to multiple pathways being activated in a complex system with intra- and intercellular signaling, leads to an unclear picture of the key contributors to the pathobiology. Second, an understanding of early pathophysiological events can increase the ability to identify PAH patients earlier than what is currently possible. Third, the prompt diagnosis of PAH would allow for the therapy to start earlier, which has proved to be a more successful strategy, and it ensures better survival in PAH patients.

Keywords: hemolysis; inflammation; metabolism; oxidative stress; pulmonary hypertension; vascular damage.

FIG. 1.

Illustration based on longitudinal MCT model that explains the selection of early time-points for the study. Early time-points for this model are indicated in green, where RVSP is slightly raised but without compensatory effect from the heart. Late pulmonary hypertension (red) is showed as a pronounced increase in both RVSP and Fulton index. MCT, monocrotaline; PH, pulmonary hypertension; RVSP, right ventricular systolic pressure. Color images are available online.

FIG. 2.

The generalized metabolic disturbances in PAH lungs indicated by the untargeted metabolic profiling. A central role of mitochondrial metabolism in reprogramming is attributed to a decrease in oxidative phosphorylation due to respiratory chain deficiency and reduced glucose oxidation due to pyruvate dehydrogenase deficiency. Reduction in fatty acid transport leads to a decrease in fatty acid oxidation. TCA cycle metabolites flux is upregulated by anaplerotic reactions that are required for the building blocks production for cellular proliferation instead of energy metabolism. This mitochondrial metabolic shift is accompanied with an increase in glycolysis for the energy demand and downstream of glycolysis metabolic pathways such as pentose phosphate pathway, nucleotide biosynthesis, and increased glycosylation, leading to proliferation and ECM remodeling. Another point of dysregulation is the nitric oxide and urea cycles, leading to vasoconstriction, increased reactive oxygen species/reactive nitrogen species, and polyamines production. Overall metabolic shift in lungs favors proliferation and dysfunction of cellular roles in all varieties of vascular cells. ECM, extracellular matrix; NO, nitric oxide; PAH, pulmonary arterial hypertension; TCA, tricarboxylic acid. Color images are available online.

FIG. 3.

Correlation between RVPSP and lung weight as a measure of vascular leakage. Both parameters were measured at the early stages (0–14 days) of MCT (A) and SU/hypoxia models (B). In SU/hypoxia rats, PAH was induced by a single injection of Sugen 5416 (50 mg/kg, subcutaneous) followed by exposure to hypoxia (10% O₂), as previously published (145). In the MCT group, the disease was initiated by MCT injection (60 mg/kg intraperitoneal.). Lungs from the MCT model were stained with carbonic anhydrase IX, the marker of hypoxia, that clearly indicates the presence of hypoxia in vessels surrounded by perivascular edema (arrows indicate vessel) (C). RVPSP, right ventricle peak systolic pressure; SU, Sugen SU5614. Color images are available online.

FIG. 4.

Apoptosis is the primary fate of endothelial cell death resulting in oxidized HMGB1 release and increases oxidative potential in the media. (A) HPAECs (ScienCell, Carlsbad, CA) were cultured in ECM growth media supplemented with 5% FBS and penicillin–streptomycin in a humidified incubator (21% O₂, 5% CO₂) at 37°C. To induce cell death, cells were treated with Sugen 5416 (20 μM) in 0.5% FBS for 24 h, as published (101). Apoptosis and necrosis were quantified by using Apoptosis and Necrosis Quantification Kit (Biotum, Fremont, CA) according to the manufacturer's protocol, as published (136). Sugen treatment stimulated apoptosis (Early and Late) but not necrosis in HPAEC. (B) Media from untreated HPAEC, HPAEC with apoptosis induced as described in (A) and necrosis induced by three to four cycles of cell freezing and thawing, as published (137) were used for Western blot analysis as previously described (137) to measure reduced and oxidized forms of HMGB1. Necrotic but not apoptotic cells produced a marked extracellular HMGB1 signal and showed an accumulation of the reduced form of HMGB1 in cell culture media. The difference in the total protein in the media related to the difference in FBS amount used in the experiment—negative control (media that were not used for cell culturing) and apoptotic media contain 0.5% FBS, untreated and necrotic media—5% FBS. The difference in total media proteins does not correlate with the HMGB1 signal that is absent in the negative control, and the media collected from untreated cells show a light signal from oxidized HMGB1 in the apoptotic media and a strong but mostly reduced signal in the necrotic media. (C) Same media samples collected for (B) were used to measure plasma redox homeostasis by analyzing ORP and total antioxidant capacity (Cap) electrochemically by using RedoxSys^® Diagnostic System, as reported (137). Apoptotic media showed a significant increase in oxidative potential and a decrease in antioxidant capacity, indicating the development of oxidative stress in apoptotic cells and the release of oxidized cellular content. Necrotic media show a significant reduction in ORP signal, suggesting that necrosis produces release cellular components in a reduced state. N = 4; *p < 0.05 versus untreated; ^#p < 0.05 versus apoptosis. FBS, fetal bovine serum; HMGB1, high mobility group box 1; HPAEC, human pulmonary artery endothelial cell; ORP, oxidation–reduction potential. Color images are available online.

FIG. 5.

Apoptosis of vascular cells stimulates anti-inflammatory M2 macrophages, which release proliferative factors for damage resolution and activate vascular remodeling. In contrast, necrotic damage of vascular cells stimulates M1 macrophages with proinflammatory properties, increasing perivascular inflammation, vascular permeability, and fibrosis. Color images are available online.

FIG. 6.

Plasma creatinine levels in PAH patients (N = 30) correlate significantly (p < 0.0001) with the level of hemolysis. Free hemoglobin levels were measured as recently published (144).

FIG. 7.

The markers of early pathological events related to oxidative stress, vascular damage, inflammation, hemolysis, and metabolic reprogramming could lead to the earlier detection of PAH. The particular sequence of the events shown is approximate, as they could vary in different patients and simultaneously co-occur, thus, cross-amplifying each other. However, multiple reports suggested preceding the symptom onset. Therefore, identification of specific biomarkers or their combination could not only decrease the time between symptom onset and diagnosis based on echocardiography (Echo), RHC, and circulating pro-BNP but also identify patients at risk even during the asymptomatic phase of PAH. DAMPs, damage-associated molecular patterns; pro-BNP, probrain natriuretic peptide; RHC, right heart catheterization. Color images are available online.