Role of Myeloperoxidase, Oxidative Stress, and Inflammation in Bronchopulmonary Dysplasia

Abstract

Bronchopulmonary dysplasia (BPD) is a lung complication of premature births. The leading causes of BPD are oxidative stress (OS) from oxygen treatment, infection or inflammation, and mechanical ventilation. OS activates alveolar myeloid cells with subsequent myeloperoxidase (MPO)-mediated OS. Premature human neonates lack sufficient antioxidative capacity and are susceptible to OS. Unopposed OS elicits inflammation, endoplasmic reticulum (ER) stress, and cellular senescence, culminating in a BPD phenotype. Poor nutrition, patent ductus arteriosus, and infection further aggravate OS. BPD survivors frequently suffer from reactive airway disease, neurodevelopmental deficits, and inadequate exercise performance and are prone to developing early-onset chronic obstructive pulmonary disease. Rats and mice are commonly used to study BPD, as they are born at the saccular stage, comparable to human neonates at 22-36 weeks of gestation. The alveolar stage in rats and mice starts at the postnatal age of 5 days. Because of their well-established antioxidative capacities, a higher oxygen concentration (hyperoxia, HOX) is required to elicit OS lung damage in rats and mice. Neutrophil infiltration and ER stress occur shortly after HOX, while cellular senescence is seen later. Studies have shown that MPO plays a critical role in the process. A novel tripeptide, N-acetyl-lysyltyrosylcysteine amide (KYC), a reversible MPO inhibitor, attenuates BPD effectively. In contrast, the irreversible MPO inhibitor-AZD4831-failed to provide similar efficacy. Interestingly, KYC cannot offer its effectiveness without the existence of MPO. We review the mechanisms by which this anti-MPO agent attenuates BPD.

Keywords: N-acetyl-lysyltyrosylcysteine amide; bronchopulmonary dysplasia; cellular senescence; endoplasmic reticulum stress; myeloperoxidase.

Figure 1

Developmental stages of the lungs. Lung development can be divided into five stages, including embryonic (0–8 weeks), pseudoglandular (8–16 weeks of gestation), canalicular (16–24 weeks of gestation), saccular (24–36 weeks of gestation), and alveolar (36 weeks of gestational to 2–8 years of age). Biologically, neonates born after 36 weeks of gestation should be able to breathe without treatment or support. The oxygen requirement after 36 weeks post-conceptionally is thus chosen as the definition for BPD for extremely premature infants born before 32 weeks.

Figure 2

Pathologic findings of human BPD. As compared to the thin-wall and well-septated alveoli in normal lungs (A), BPD lungs are characterized by alveolar simplification, decreased septation, thickened alveolar walls, decreased capillary counts, alveolar hyperinflation mixed with collapse, and superimposed inflammatory cell infiltration (B). The pathologic findings reasonably explain the lung stiffness, poor oxygenation, restricted airway, and thickened secretion clinicians encountered during the care of BPD infants.

Figure 3

Rat pups upregulate lung antioxidative proteins after exposure to HOX. Rat pups can upregulate the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidative enzyme expression. Rat pups were exposed to >90% O₂ (HOX) from postnatal day 1 (P1) to P10 and then recovered to room air afterward. Three representative Nrf2-mediated proteins—thioredoxin-1 (TRX), glutathione-S-transferase (GST), and heme-oxygenase-1 (HO1)—in the lungs were quantified by immunoblots. All three proteins are upregulated as early as P4. The increased GST and HO1 expressions persisted throughout HOX exposure. The upregulated HO1 persisted even after the pups recovered to room air for 11 days (P21). (Reprinted from reference [94] with permission).

Figure 4

MPO contributes to BPD onset. HOX activates alveolar macrophages to recruit the circulating neutrophils, as evidenced by increased lung MPO expression at P4 (A). Neutrophil infiltration might contribute to BPD onset, as morphometric BPD changes are not seen at this point (B). **: p < 0.01 (n = 4).

Figure 5

HOX exposure causes the BPD phenotype in rat pups. Alveolar simplification, reductions in radial alveolar counts and secondary septations, and infiltration of inflammatory cells are seen in the lungs under H&E stains (A). The decreased blood vessel counts in the HOX-exposed lungs indicate impaired angiogenesis under the immunohistochemistry stains using an antibody against the rat endothelial cell antigen (B). N: NOX, H: HOX. Red arrow: endothelial cells in blood vessels. Scale bar = 100 μm.

Figure 6

OS is increased in BPD rat lungs. (A) The levels of Cl-Tyr are 2.6-fold higher in BPD lung lysates, indicating an HOCl-mediated OS. (B) The levels of 3-NT are 1.9-fold higher in BPD lung lysates, indicating a peroxynitrite- or reactive nitrogen species-mediated OS. (C) The integrated signals of 8-OH-dG are 1.8-fold higher in BPD lung, indicating OS-mediated DNA damage. (Reprinted from reference [95]. Used with permission). Scale bar = 200 μm. *: p < 0.05 (n = 12).

Figure 7

Summary of the MPO activities. MPO comprises two heavy chains and two light chains with a heme core. Iron can have multiple redox states as a transition metal; hence, there are three compounds for MPO. Through the halogenation and peroxidase cycles, compound I converts the chloride anion into hypochlorous acid, where hydrogen peroxide is available (reaction 2). There are two conditions in which MPO functions as catalase (reactions 1 and 5; green ovals with dotted outlines).

Figure 8

Early expression of inflammatory proteins in the BPD rat lungs. The HMGB1 levels significantly increase at P4, when MPO levels also increase. The TLR4, RAGE, and GSNOR levels do not show a significant change. This finding suggests both HMGB1 and MPO are involved in BPD onset. **: p < 0.01 (n = 4).

Figure 9

Increased expression of inflammatory proteins during BPD progression. Under HOX exposure, the expressions of HMGB1 (A), TLR4 (B), and RAGE (C) are all increased at P10. (Reprinted from reference [95] with permission). *: p < 0.05 (n = 12).

Figure 10

GSNOR contributes to the BPD progression. (A) GSNOR expression increased by 30% in HOX rat BPD lungs. (B) Daily treatment with N6022, a GSNOR inhibitor, effectively attenuates the BPD changes in HOX-exposed rat lungs, suggesting a contributory role of GSNOR in BPD progression. *: p < 0.05 (n = 12).

Figure 11

HMGB1 in vitro treatment upregulates GSNOR expression, ER stress markers, and cellular senescence markers. (A) Two concentrations (100 ng/mL and 200 ng/mL) are used to treat RALC, RLMVEC, and PASMC for 48 h. Immunoblots quantify the expression of GSNOR. GSNOR expression increases in all three lung cell types, with RLMVEC having the highest response. (B) Changes in ER stress are studied in RLMVEC. All ER stress markers increase after 200 ng/mL HMGB1 treatment for 48 h. (C) Change in cellular senescence is also studied in RLMVEC with 200 ng/mL HMGB1 treatment for 48 h. The commonly used markers for cellular senescence increase after HMGB1 treatment. RLAC: rat lung alveolar cells; RLMVEC: rat lung microvascular endothelial cells; PASMC: pulmonary artery smooth muscle cells from rat pups. * p < 0.05 (n = 3~4).

Figure 12

The BPD-sterile inflammatory pathway. Exposing rat pups to hyperoxia elicits oxidative stress-mediated lung responses that culminate in the BPD phenotype. The figure summarizes our recent findings. These responses interact with each other to form a complex relationship. There are at least four sources of OS, including hyperoxia (a), myeloperoxidase (b), endoplasmic reticulum stress (c), and cellular senescence (d). The OS coming from HOX can be reduced clinically, to a limited extent, through the judicious use of oxygen. Specific inhibitors can target the other three OSs. However, MPO-mediated OS seems upstream of the other two and could be the best therapeutic target. This is a newly edited figure with cellular senescence included. (Reprinted from reference [94] with permission). ↑: increase; ↓: decrease.

Figure 13

KYC protection of HOX-exposed lungs. KYC daily intraperitoneal injection of 10 mg/kg improves the alveolar formation (A), decreases MPO(+) inflammatory cell infiltration (red arrows) (B), and immunofluorescence stain of MPO (red) distribution (C), apoptosis (D), ER stress (E), and cellular senescence (F). The MPO immunofluorescence (red) stain shows reduced MPO release into the tissue. The nuclei are stained with DAPI (blue). The in situ TUNEL stain represents DNA damage and is used to estimate apoptosis. The dilated ER structure under the electron microscope indicates that KYC attenuates ER stress. GL-13 stain, a modified lipofuscin stain equivalent to the acidic β-galactosidase activity stain, and commonly used markers (p16, p21, p53, γH2AX or H2AFX, SerpinE1, and SPP1) are used to quantify cellular senescence. LB: lamella body; M: mitochondria (Reprinted from references [94,112] with permission). (A,C,D) Scale bar = 100 μm; (B) Scale bar = 200 μm; (E) Scale bar = 500 μm; (F) Scale bar = 50 μm. *: p < 0.05 (n = 6).

Figure 14

KYC attenuates inflammatory signaling and augments antioxidative capacity in HOX-exposed lungs. (A) The major DAMP protein HMGB1 and its two main receptors, TLR4 and RAGE, decrease in BPD lungs after KYC treatment. (B) Immunoprecipitation of the BPD lung lysates by HMGB1 antibody shows decreased bindings of TLR4 and RAGE with HMGB1 on immunoblots by KYC. (C) Using the same immunoprecipitation strategy, we see oxidized (ox-) and acetylated (Ac-) HMGB1 increases in BPD lungs by KYC. (D) Although the increased Nrf2 expression in room air-exposed (NOX, left panel) lungs is not as dramatic in HOX-exposed (right panel) lungs, KYC increases the antioxidative capacity of neonatal lungs by upregulating Nrf2 expression in both oxygen environments. (Reprinted from reference [95] with permission). *: p < 0.05, (n = 12 and 4 for (A) and (B), respectively).

Figure 15

Irreversible MPO inhibition does not attenuate BPD. Rat pups received 15 mg/kg of AZD4831 at P2, P4, P6, P8, and P10. The irreversible MPO inhibitor does not affect the morphology of rat lungs raised in room air or the BPD lungs. Scale bar = 100 μm. *: p < 0.054 (n = 9).