Increased Mutagen Sensitivity and DNA Damage in Pulmonary Arterial Hypertension

Abstract

Rationale: Pulmonary arterial hypertension (PAH) is a serious lung condition characterized by vascular remodeling in the precapillary pulmonary arterioles. We and others have demonstrated chromosomal abnormalities and increased DNA damage in PAH lung vascular cells, but their timing and role in disease pathogenesis is unknown.

Objectives: We hypothesized that if DNA damage predates PAH, it might be an intrinsic cell property that is present outside the diseased lung.

Methods: We measured DNA damage, mutagen sensitivity, and reactive oxygen species (ROS) in lung and blood cells from patients with Group 1 PAH, their relatives, and unrelated control subjects.

Measurements and main results: Baseline DNA damage was significantly elevated in PAH, both in pulmonary artery endothelial cells (P < 0.05) and peripheral blood mononuclear cells (PBMC) (P < 0.001). Remarkably, PBMC from unaffected relatives showed similar increases, indicating this is not related to PAH treatments. ROS levels were also higher (P < 0.01). DNA damage correlated with ROS production and was suppressed by antioxidants (P < 0.001). PBMC from patients and relatives also showed markedly increased sensitivity to two chemotherapeutic drugs, bleomycin and etoposide (P < 0.001). Results were consistent across idiopathic, heritable, and associated PAH groups.

Conclusions: Levels of baseline and mutagen-induced DNA damage are intrinsically higher in PAH cells. Similar results in PBMC from unaffected relatives suggest this may be a genetically determined trait that predates disease onset and may act as a risk factor contributing to lung vascular remodeling following endothelial cell injury. Further studies are required to fully characterize mutagen sensitivity, which could have important implications for clinical management.

Keywords: BMPR2; DNA damage; genetic susceptibility; reactive oxygen species.

Figures

Figure 1.

DNA damage assays in pulmonary artery endothelial cells (PAEC). (A) Micronucleus (MN) is visualized as additional discrete nuclear material (arrow) in a binucleate (BN) cell in which cytokinesis has been blocked with cytochalasin B. (B) The number of MN per 1,000 BN cells was scored in control PAEC (n = 4), pulmonary arterial hypertension (PAH) PAEC with a chromosomal abnormality (n = 4), and PAH PAEC that were cytogenetically normal on single-nucleotide polymorphism microarray analysis (n = 4). One-way analysis of variance was statistically significant (P = 0.003), and chromosomally abnormal PAEC were significantly different than control cells in post hoc pairwise testing. **P < 0.01. (C and D) Immunofluorescent analysis of histone H2AX phosphorylation (γH2AX) was performed by confocal microscopy. Results are expressed as the ratio of the area of γH2AX staining to total nuclear area for at least 70 cells per case. PAH PAEC (n = 5) showed significantly more γH2AX staining than control cells (n = 3); t test, *P < 0.05. Mean ± SD. Representative images are shown for control and patient cells. APAH = associated PAH; HPAH = heritable PAH; IPAH = idiopathic PAH.

Figure 2.

DNA damage assays in peripheral blood cells. (A) The number of micronuclei (MN) per 1,000 binucleate (BN) cells was scored by light microscopy in lymphoblastoid cell lines from control subjects (n = 7), patients with pulmonary arterial hypertension (n = 21), and unaffected relatives of patients (n = 8), including two relatives carrying hereditary pulmonary arterial hypertension mutations. One-way analysis of variance was statistically significant (P < 0.001). Patients and relatives had a significantly higher number of MN than control subjects in post hoc testing. **P < 0.01; ***P < 0.001. (B) Results were validated by flow cytometry on fresh peripheral blood mononuclear cells from an independent group of control subjects (n = 7), relatives (n = 9), and patients (n = 23). Number of MN was counted per 50,000 intact nuclei. Kruskal-Wallis, P < 0.001; post hoc pairwise tests, *P < 0.05; ***P < 0.001. Mean ± SD.

Figure 3.

Increased reactive oxygen species (ROS) in pulmonary arterial hypertension pulmonary artery endothelial cells and lymphoblastoid cell lines. (A) Pulmonary artery endothelial cells from control subjects (n = 4) and patients (n = 8) were incubated with CellROX Green reagent and analyzed by flow cytometry for 20,000 events. The percentage of ROS-positive cells was significantly higher in pulmonary arterial hypertension cases than control subjects. Mean ± SD; ***P < 0.001. (B) Rate of ROS production in lymphoblastoid cell lines from patients (n = 10), relatives (n = 6), and control subjects (n = 7) was analyzed by flow cytometry. Results are expressed as the change in the percentage of ROS-positive cells among 20,000 counts after 1 hour in antioxidant-free medium compared with baseline, average of at least two independent experiments. Mean ± SD; one-way analysis of variance, P = 0.001; **P < 0.01 (post hoc pairwise tests).

Figure 4.

Effect of antioxidant on DNA damage in peripheral blood cells. Fresh peripheral blood mononuclear cells from control subjects (n = 9), patients (n = 21), and first-degree relatives (n = 10) were incubated with 200 μM N-acetylcysteine (NAC) during the 72-hour micronucleus (MN) protocol. The number of MN per 1,000 binucleate (BN) cells was scored by light microscopy and analyzed by two-way analysis of variance with repeated measures. There was a statistically significant interaction between treatment and patient groups (P < 0.001). In post hoc analyses, NAC significantly reduced the number of MN in cells from patients and relatives but not control subjects; ns = not significant; ***P < 0.001.

Figure 5.

DNA damage induced by genotoxic drugs in peripheral blood mononuclear cells. Induction of micronuclei (MN) by the genotoxic drugs etoposide and bleomycin was measured in peripheral blood mononuclear cells from control subjects (n = 7), relatives (n = 9), and patients (n = 23) by flow cytometry for 50,000 intact nuclei. Untreated cells are the same results as shown in Figure 2B. Mean ± SD; two-way analysis of variance interaction, P < 0.001. Post hoc tests were performed for drug versus untreated cells in each group; ns = not significant; **P < 0.01, ***P < 0.001.

Figure 6.

Model illustrating the potential role of DNA damage in pulmonary arterial hypertension (PAH). Individuals with normal genotype have low levels of reactive oxygen species (ROS) and DNA damage and normal restoration of the pulmonary vasculature after endothelial injury. Individuals with the PAH-susceptible genotype produce higher levels of ROS (red triangles) and have higher baseline levels of DNA damage, both in the lung and in white blood cells (blue circles). This may lead to an increase in the number of pulmonary artery endothelial cells with unrepaired genomic damage (highlighted in gray). On endothelial cell injury and death, cells with preexisting genomic damage may be resistant to apoptosis and primed for hyperproliferation. If the injury itself includes ROS generation or other genotoxic damage, cells with the PAH-susceptible genotype are also more sensitive to the DNA-damaging effects, thus amplifying the extent of the injury compared with normal cells. As vascular remodeling and pulmonary hypertension progresses, hypoxia, abnormalities of mitochondrial metabolism, inflammation, and oxidative stress may lead to additional DNA damage and create a vicious cycle of ongoing endothelial and smooth muscle cell dysfunction.