Rieske Iron-Sulfur Protein Mediates Pulmonary Hypertension Following Nicotine/Hypoxia Coexposure

Abstract

The high mortality rate in patients with chronic obstructive pulmonary disease (COPD) may be due to pulmonary hypertension (PH). These diseases are highly associated with cigarette smoke and its key component nicotine. Here, we created a novel animal model of PH using coexposure to nicotine (or cigarette smoke) and hypoxia. This heretofore unreported model showed significant early-onset pulmonary vasoremodeling and PH. Using newly generated mice with complementary smooth muscle–specific Rieske iron-sulfur protein (RISP) gene knockout and overexpression, we demonstrate that RISP is critically involved in promoting pulmonary vasoremodeling and PH, which are implemented by oxidative ataxia telangiectasia-mutated–mediated DNA damage and NF-κB–dependent inflammation in a reciprocal positive mechanism. Together, our findings establish for the first time an animal model of hypoxia-induced early-onset PH in which mitochondrial RISP-dependent DNA damage and NF-κB inflammation play critical roles in vasoremodeling. Specific therapeutic targets for RISP and related oxidative stress–associated signaling pathways may create unique and effective treatments for PH, chronic obstructive pulmonary disease, and their complications.

Clinical Relevance

Given the dramatic increase in nicotine usage over the years and the severe role of cigarette smoking in chronic obstructive pulmonary disease (COPD), we believe our novel animal model of pulmonary hypertension (PH) using nicotine (or cigarette smoke) and hypoxia coexposure will be widely used in the near future. Our data further reveals that Rieske iron-sulfur protein, ataxia telangiectasia-mutated, NF-κB and their associated signaling molecules may become novel specific and effective targets for treating PH, COPD, and PH in COPD.

Chronic obstructive pulmonary disease (COPD) is the third-leading cause of death worldwide, and as many as 90% COPD cases can be attributed to cigarette or e-cigarette smoking (1, 2). Inhalation of nicotine, the major active component of cigarettes and e-cigarettes, replicates all major clinical features of COPD (3) and also causes pulmonary hypertension (PH) (4). The development of PH can eventually lead to heart failure and may account for the high mortality rate among patients with COPD (5). There is still no cure for these common, devastating diseases; moreover, the molecular mechanisms underlying the development and progression of COPD and consequent PH remain poorly understood.

Inhalation of nicotine or cigarette smoke (CS) is used to create a standard model of COPD (3, 6), and hypoxia is applied to create a standard model of PH (7, 8). In this study, we used nicotine/hypoxia (N/H) coexposure to develop and characterize a new animal model of PH. We hypothesized that this coexposure model would authentically illustrate the major features of PH and allow us to examine the synergistic effect of N/H on the pulmonary vasculature seen in patients with COPD and PH.

Recent studies by Schumacker and colleagues and our laboratory have established that Rieske iron-sulfur protein (RISP) of mitochondrial complex III is a main factor in reactive oxygen species (ROS) generation in pulmonary artery (PA) smooth muscle (SM) cells (PASMCs) (9–13). Additionally, these studies demonstrated that SM-specific RISP knockdown attenuated hypoxia-induced increases in right ventricular systolic pressure (RVSP), weight, and PH in vivo, whereas RISP overexpression produced the opposite effects. To reinforce the role of RISP, in the present study, we used TurboKnockout gene-targeting techniques to generate RISP-floxed mice and bred these floxed mice with SM myosin heavy chain 11 Cre recombinase mice to produce our SM-specific RISP-knockout (KO) mice. As a complementary approach, we further generated RISP gene overexpression (OE) mice using CRISPR-Cas9/Rosa26-sgRNA technology. Using these two complementary SM-specific RISP-KO and OE mouse strains, we determined a critical role of this molecule in PA vasoremodeling and PH in an N/H coexposure animal model.

Oxidative stress from the overproduction of ROS can lead to DNA damage; however, the role of ROS-induced DNA damage in PH, COPD, and COPD-associated PH remains poorly understood (14, 15). DNA damage response signaling is tightly orchestrated, with ataxia telangiectasia-mutated (ATM) kinase a central regulator (16, 17). Accordingly, we examined whether RISP-mediated mitochondrial ROS cause ATM-regulated DNA damage in PASMCs, leading to N/H-induced PA vasoremodeling and PH.

Inflammation, a process highly regulated by NF-κB signaling (18), was found to be upregulated in bronchial biopsies from cigarette-smoking patients with COPD (19, 20). However, the role of NF-κB–dependent inflammatory signaling in patients with COPD or PH is not well established. Thus, in this study, we intended to elucidate the interplaying roles of RISP, ATM-mediated DNA damage, and NF-κB–mediated inflammation in N/H-induced PA vasoremodeling and PH. The findings from this study demonstrate a novel murine model of PH. This heretofore unreported animal model exhibited significant early-onset pulmonary vasoremodeling and PH. A series of our further investigations thoroughly illustrate that mitochondrial RISP plays a role in the development of pulmonary vasoremodeling and PH. The role of RISP is implemented by oxidative ATM-dependent DNA damage and NF-κB–reliant inflammation. The implementation of these two signaling processes is exclusively accomplished in their reciprocal positive mechanism. Furthermore, RISP, DNA damage, NF-κB, and related inflammation molecules may become specific targets to treat COPD, PH, and COPD-associated PH.

Methods

A detailed description of study methods is provided in the data supplement.

Conditional Smooth Muscle RISP-KO and OE Mice

SM-specific RISP-KO (smMHC^Cre/+/RISP^+/−) mice were produced by breeding RISP-floxed (RISP^lox/lox) mice with myh11-cre mice. The production of RISP^lox/lox mice carrying a loxP-flanked exon 2 of mouse RISP gene is described in the data supplement. GFP-labeled myh11-cre mice were generated as previously reported (21). An SM-specific RISP OE mouse was generated by crossing an smMHC^Cre/+ mouse with a knock-in mouse with a CMV promoter, a loxP-stop-loxP element, and a RISP coding gene linked to the IRES-dTomato element in the Rosa26 locus. Detailed descriptions are provided in the data supplement.

Inducible SM RISP-KO Mouse

SM-specific RISP KO (SM-CreERT2^Cre/+/RISP^lox/lox) was generated by cross-breeding a tamoxifen-induced myh11 Cre mouse with a RISP^lox/lox mouse. Further details are provided in the data supplement.

Conditional SM ATM-KO Mouse

SM-specific ATM-KO (smMHC^Cre/+/ATM^lox/lox) mice were produced by cross-breeding ATM^lox/lox mice with myh11-cre mice. Further details are included in the data supplement.

Exposure Models

For nicotine exposure, mice were exposed to nicotine (18 mg/ml) for 1 h/d for 1 or 3 weeks in an unrestricted whole-body exposure system (Buxco Research Systems). For hypoxia, mice were placed in a normobaric chamber for 1 or 3 weeks. The oxygen concentration in the chamber was maintained at 10% (Biospherix Ltd.). For CS, mice were placed in a microprocessor-controlled CS machine (Teague Enterprises) that produced side-stream smoke from filtered 3R4F research cigarettes (University of Kentucky). Mice were exposed to CS for 5 h/d for 1 week or 2 months. For N/H and CS/hypoxia coexposures, mice were exposed to nicotine (1 h) or CS (5 h) and then hypoxia for 23 or 19 hours, respectively.

Ammonium Pyrrolidine Dithiocarbamate Treatment

Ammonium pyrrolidinedithiocarbamate (PDTC; P8765; Sigma Aldrich) was dissolved in 0.9% saline solution and administered at 50 mg/kg via a subcutaneous injection once per day for 1 week. Treatment with PDTC occurred daily before nicotine inhalation. Control mice were treated identically but injected with 0.9% saline solution instead of PDTC.

Statistics

A significance threshold of 0.05 was used for all tests. Power analyses were performed with the goal to detect a minimal 10% difference between groups for RVSP and Fulton index measurements. For multiple comparisons, a two-way ANOVA was performed to assess statistically significant differences. Post hoc analysis was performed using Tukey’s multiple comparisons correction unless otherwise stated. Exact P values are given for nonsignificant findings in the text and figure legends. Further details on statistics are provided in the data supplement.

Results

Creation of a Novel Murine Model of PH Using N/H Coexposure

Male and female C57BL/6 mice (12–16 wk old) were exposed to nicotine (18 mg/ml) inhalation, hypoxia exposure, or N/H coexposure for 3 weeks as diagrammed in Figure 1A. The results revealed that, following nicotine inhalation, hypoxia, and N/H coexposure, male mice displayed increased RVSP, Fulton index, and PA medial wall thickness (MWT), indicating the development of PH. In contrast, female mice showed a response to only hypoxia exposure (Figures 1B–1E). Accordingly, male animals were exclusively used in all the remaining experiments. In male mice, we observed that there were no significant differences in RVSP, Fulton index, and PA MWT between nicotine, hypoxia, or N/H coexposure (Figures 1F–1H). Validation of nicotine inhalation and ventricular hypertrophy were, respectively, assessed by increases in serum cotinine levels (Figure E1A in the data supplement) and RV B-type natriuretic peptide concentrations (see Figure E1B) in male and/or female control and nicotine-exposed mice. These findings are consistent with a previous report (4), providing additional evidence that nicotine indeed enhances hypoxic cellular responses.

Figure 1.

Development and characterization of a novel animal model of early-stage pulmonary hypertension (PH) following nicotine and hypoxia (N/H) coexposure. Mice were exposed to 3 weeks of nicotine (18 mg/ml) for 1 h/d, 5 d/wk; hypoxia (10% O₂, 24 h/d); or a coexposure of 1 h/d nicotine and 23 h/d hypoxia. (A) Schematic diagrams illustrating exposure models. Male and female mice were used to determine (B) right ventricular (RV) systolic pressure (RVSP) measurements using right heart catheterization, (C) Fulton index (weight ratio of RV to left ventricle and septum) as an indicator of RV hypertrophy, and (D and E) quantified relative medial wall thickness (MWT) of pulmonary arteries (PAs) at various diameters using Weigert’s Resorcin-Fuchsin (WRF) staining in (D) male mice and (E) female mice. Male mice were used to assess (F) RVSP, (G) Fulton index, and (H) PA relative MWT quantification. Representative images with WRF staining are shown. (I) RVSP, (J) Fulton index, and (K) relative MWT of PAs and representative images of WRF staining of untreated (“U”), nicotine inhalation (“N”), hypoxia exposure (“H”), and N/H coexposure (CO) for 1 week. Data are presented as mean ± SEM. Numbers in parentheses indicate the number of animals used per group. *P < 0.05 versus the untreated control group using one- or two-way ANOVA test followed by a post hoc t test with Tukey’s multiple comparisons correction. COPD = chronic obstructive pulmonary disease; RV/LV+S = weight ratio of right ventricle to left ventricle + septum. Scale bars, 50 μm.

Next, we asked whether the synergistic effect of N/H coexposure occurs in the early stages of PH development. In this set of experiments, male mice were exposed to nicotine, hypoxia, or N/H coexposure for only 1 week. Interestingly, we found that the coexposure significantly increased all markers of PH (RVSP, Fulton index, and PA MWT) compared with nicotine or hypoxia exposure alone (Figures 1I–1K).

We further tested whether nicotine at a lower dose (9 mg/ml) would also synergistically cause PH with hypoxia. The results indicated that nicotine at 9 mg/ml was not sufficient to augment RVSP or Fulton index with hypoxia (see Figures E1C and E1D). Thus, we continued to use nicotine at 18 mg/ml, a clinically relevant concentration (22), throughout all the relevant experiments. Moreover, the shorter coexposure (1 wk) would allow us to investigate the synergistic effect of nicotine and hypoxia in early-onset PH compared with the individual effect seen following the longer exposure (i.e., 3 wk).

RISP of Mitochondrial Complex III Is a Major Factor in N/H Coexposure–induced PH

Our previous study identified that RISP gene knockdown blocked chronic hypoxia-induced PH (9); similarly, Waypa and colleagues (the same group as Schumacker and colleagues) reported that RISP KO inhibited acute hypoxia-evoked PH (10). To address the role of RISP in PH and COPD, we generated SM-specific RISP-KO and OE mice (Figure E2). Nicotine-induced ROS generation was blocked in pulmonary arterial mitochondria isolated from SM-specific heterozygous KO (hetKO) for RISP in mice. Increases in RVSP following N/H coexposure for 1 week were partially blocked in SM-specific RISP hetKO mice compared with control (wild-type [WT]) mice (Figures 2A–2C). Moreover, the increase in RVSP was completely abrogated in tamoxifen-inducible SM-specific RISP-KO mice (Figures 2D and 2E). In contrast, nicotine-induced ROS generation was significantly increased in SM-specific RISP OE PA mitochondria. Moreover, SM-specific RISP OE exacerbated the increase in RVSP (Figures 2G–2I), but only in male mice, with no effect in female mice. We further revealed that female mice had higher RISP protein expression (see Figure E2A). Additionally, we found that PA remodeling mirrored the RVSP measurements in genetically altered RISP mice following N/H coexposure (Figures 2F and 2J). We validated that RISP KO did not affect protein expression of mitochondrial protein COX1 (cytochrome c oxidase 1), an important subunit of complex IV involved in ATP synthesis (see Figure E2D). These findings indicate that RISP plays an important role in PA remodeling and PH.

Figure 2.

Rieske iron-sulfur protein (RISP)–mediated reactive oxygen species (ROS) regulates PH development following CO. (A) Representative Western blot images and quantification of RISP expression in isolated PAs from wild-type (WT, control) and smooth muscle (SM)–specific conditional RISP heterozygous knockout (hetKO) mice (yellow), (D) SM-specific tamoxifen-inducible RISP-KO (indKO) mice SM (red), or (G) SM-specific RISP overexpression (OE) mice (gray). ROS measurements using dichlorofluorscein (DCF) fluorescence with or without nicotine stimulation in PA mitochondria isolated from WT, (B) RISP hetKO, or (H) RISP OE mice. RVSP of “U” mice or mice exposed to 1 week CO using WT, (C) RISP hetKO, (E) RISP indKO, or (I) RISP OE mice. (F and J) Representative WRF staining images and quantification of PA relative MWT. Data are presented as mean ± SEM. Numbers in parentheses indicate the number of animals used per group. *P < 0.05 versus the untreated control group; ^†P < 0.05 versus the untreated RISP hetKO, indKO group, or OE group; and ^#P < 0.05 versus 1-week CO control group using a two-tailed t test (A, D, and G) or one- or two-way ANOVA followed by post hoc test using Tukey’s multiple comparisons correction (B, C, E, F, and H–J). Scale bars, 50 μm.

SM-Specific RISP Deletion Attenuates Nicotine/Hypoxia-induced ATM-mediated DNA Damage

It is well established that overproduced ROS can lead to oxidative stress and cause DNA damage (23, 24). Thus, we examined the role of nicotine and hypoxia on DNA damage by assessing protein expression of γ-histone 2AX (γ-H2AX), a commonly used marker for double-strand DNA breaks (25), in PAs isolated from N/H coexposure mice. The data revealed significantly increased γ-H2AX protein expression following coexposure (Figure 3). We then investigated whether ATM was a central molecule to involve the DNA damage in response to N/H coexposure. Our data unveiled that ATM-mediated signaling was activated, as shown by increased expression of p-Chk2 (phosphorylated checkpoint kinase-2), a well-known effector protein of ATM signaling after double-strand DNA breaks (16). We also found that p-Chk2 expression was similar between male and female mice.

Figure 3.

RISP-generated ROS mediates PH by oxidative stress–induced DNA damage–associated ATM (ataxia telangiectasia-mutated) signaling. (A) Representative Western blot images and quantification of RISP, γ-H2AX (a biomarker for double-strand DNA damage) protein expression, and p-Chk2 (a downstream molecule of ATM DNA damage signaling) protein expression in PA tissue isolated from control or SM-specific RISP hetKO mice after no treatment (“U”) or 1 week CO. (B) Expression of 8-OHdG in serum from control or SM-specific RISP hetKO mice following CO as measured by ELISA. Data are presented as mean ± SE. Numbers in parentheses represent the number of samples tested per group. *P < 0.05 versus the untreated control group and ^#P < 0.05 versus the control group after 1 week CO, two-tailed t test (B) or two-way ANOVA (A) followed by post hoc test using Tukey’s multiple comparisons correction. γ-H2AX = phosphorylated H2AX; p-Chk2 = phosphorylated Chk2.

To test the involvement of RISP signaling in ATM-mediated DNA damage, we examined the expression of DNA damage markers in PA tissues isolated from SM-specific RISP hetKO and WT mice following N/H coexposure. The reduction in RISP expression largely inhibited the markers of DNA damage. Additionally, RISP hetKO significantly reduced circulating 8-OHdG, a biomarker for oxidative DNA damage (26), following N/H coexposure compared with WT mice.

RISP Regulates NF-κB Inflammation in PH and COPD

COPD is significantly exacerbated by inflammation due to cigarette smoking (27). The role of inflammation and its underlying mechanisms in PH are not well understood. Here, we examined the involvement of NF-κB, a major inflammatory signaling pathway in PH, after N/H coexposure. To first elucidate whether NF-κB has a functional role in PH, we treated mice with PDTC (50 mg/kg), a thiol compound known to inhibit NF-κB activation (28, 29), before each nicotine inhalation session (Figure 4A). We validated NF-κB inhibition by assessing protein expression of the NF-κB inhibitory protein IκBα (Figure 4B). We found that inhibition of NF-κB blocked increases in RVSP, Fulton index, and PA remodeling following N/H coexposure (Figures 4C–4E). These results indicate that NF-κB inflammatory signaling contributes to the development of PH and its associated vasoremodeling.

Figure 4.

NF-κB inflammation contributes to PH via RISP signaling. (A) Schematic diagram illustrating 1-week CO model with NF-κB pharmacological inhibition using ammonium pyrrolidinedithiocarbamate (PDTC; 50 mg/kg). (B) Representative Western blot images and quantification of IκBα (P = 0.2808) protein expression in PA tissue isolated from “U” control mice or PDTC-treated mice exposed to control (“C”) or 1 week of CO. (C) RVSP, (D) Fulton index, and (E) representative images of WRF staining and quantification of PA relative MWT of untreated mice or mice exposed to 1 week of CO in the absence or presence of PDTC. (F) Representative Western blot images and quantification of NF-κB inhibitory protein IκBα expression in PA tissue isolated from WT or SM-specific RISP hetKO mice following no treatment (“U”) or 1 week of CO. Data are presented as mean ± SEM. Numbers in parentheses indicate the number of animals or samples used per group. *P < 0.05 versus the untreated control group and ^#P < 0.05 versus 1-week CO control group using one-way ANOVA followed by post hoc t test with Tukey’s multiple comparisons correction (B–D) or two-way ANOVA followed by post hoc test using Tukey’s multiple comparisons correction (E and F). Scale bars, 50 μm.

To determine the contribution of RISP-mediated ROS signaling on NF-κB inflammation, we compared expression of NF-κB inhibitory protein IκBα in PAs from SM-specific RISP hetKO and WT mice following N/H coexposure. The data displayed significantly higher IκBα protein expression in PAs from hetKO mice compared with WT mice (Figure 4F), suggesting that RISP signaling may regulate the NF-κB inflammation that contributes to N/H-induced PH.

NF-κB Inflammation and ATM-mediated DNA Damage Create a Positive Feedback Mechanism in Promoting PH

NF-κB–dependent inflammatory signaling causes the recruitment of inflammatory cells like macrophages and neutrophils (30); these cells can cause oxidative stress and DNA damage (31). Thus, we asked whether the observed DNA damage resulted from increased inflammation. Using SM-specific ATM-KO mice, we found that RVSP and Fulton index did not increase following N/H coexposure (Figures 5A–5C). Similarly, the ATM-KO mice did not display the increased PA remodeling that was seen in the control mice following coexposure (Figure 5D). IκBα protein expression was unchanged in ATM-KO mice following N/H coexposure (Figure 5E). In addition, PDTC also blocked N/H coexposure–induced increases in inflammation and DNA damage markers (Figure 5F). Collectively, these data suggest that ATM-mediated DNA damage and NF-κB–dependent inflammation form a positive feedback mechanism in promoting the development of N/H-induced PA vasoremodeling and PH.

Figure 5.

NF-κB–mediated inflammation and ATM-regulated DNA damage create a positive feedback mechanism to mediate N/H-evoked PH. (A) Representative Western blot images and quantification of ATM protein expression in PA tissues isolated from WT or SM-specific ATM-KO mice. (B) RVSP, (C) Fulton index, and (D) representative images using WRF staining and quantification of PA relative MWT of WT or SM-specific ATM KO after 1 week of CO or no treatment (“U”). (E) Representative Western blot images and quantification of NF-κB inhibitory protein IκBα in PA tissue isolated from ATM-KO mice following no treatment or 1 week of CO. (F) Representative Western blot images and quantification of γ-H2AX protein expression in PA tissues isolated from untreated control mice or PDTC-treated mice without exposure (control, “C”) or following 1 week of CO. Data are presented as mean ± SEM. Numbers in parentheses indicate the number of animals or samples used per group. *P < 0.05 using two-tailed t test (A and E), one-way ANOVA (F), or two-way ANOVA (B–D) followed by post hoc test with Tukey’s multiple comparisons correction. Scale bars, 50 μm.

CS, Similar to Nicotine, Produces Synergistic Effects on Hypoxia-elicited PH

We modified our coexposure animal model by using CS in combination with hypoxia to determine whether CS produced similar detrimental effects as nicotine inhalation. As shown in Figure 6, the altered markers of PH in mice following CS/hypoxia coexposure for 1 week were similar to those in mice after N/H coexposure (Figures 6A–6D). These data strongly indicate that nicotine, the major component of CS, is the important driving factor in PH development.

Figure 6.

Cigarette smoking (CS), similar to nicotine inhalation, causes early-onset PH. (A) Schematic diagram illustrating the exposure model using CS (5 h/d, 5 d/wk) for 1 week or 2 months. (B) RVSP, (C) Fulton index, and (D) representative images of WRF staining and quantification of PA relative MWT in mice following no treatment (“U”), 1 week CS alone, or 1 week CS/hypoxia coexposure (CO). (E) RVSP, (F) Fulton index, and (G) representative images of WRF staining and quantification of PA relative MWT of mice following 2-month exposure to CS alone, 2-month CS/hypoxia coexposure, or no treatment (“U”). Data are presented as mean ± SEM. Numbers in parentheses indicate the number of animals or samples used per group. *P < 0.05, one-way or two-way ANOVA followed by post hoc test with Tukey’s multiple comparisons correction. Scale bars, 50 μm.

It has been shown that mice develop emphysematous lungs in 2-month CS exposure models (32). Using this information, we examined the effect of the CS/hypoxia coexposure model by including CS exposure alone for 7 weeks followed by 1 week of CS/hypoxia coexposure. The results revealed that RVSP and Fulton index were significantly augmented compared with 1 week of N/H coexposure (Figures 6E–6G). However, compared with 2 months of CS exposure alone, there was no significant difference after the addition of hypoxia exposure. These data further suggest that the synergistic effect occurs in the early stages of PH.

Discussion

The popularity of nicotine consumption through e-cigarettes and vaping has skyrocketed (33). CS and nicotine use account for as many as 90% of COPD cases, and the high mortality rate of COPD patients is a result of the development of PH (34). The direct effects of nicotine, the major active and highly addictive component of CS, on the pulmonary vasculature are still largely unknown. Studies over the years have documented a number of animal models for PH or COPD. Hypoxia exposure in the absence or presence of SU5416 is widely used to create a model of PH, in addition to pharmacological approaches including monocrotaline. A number of COPD animal models of chronic exposure to CS and nicotine have been shown to produce COPD-like symptoms and features (3, 27, 34–36).

Despite the existence of many animal models in which to study COPD or PH, there are no models of PH in COPD, also known as COPD-associated PH or PH secondary to COPD (6, 8). In this study, for the first time, we report a novel murine model of PH following nicotine inhalation or combined CS/hypoxia exposure. This heretofore unreported animal model may advance research in the field by further elucidating the molecular pathogenesis underlying the disease development and progression and may also help to identify specific and effective therapeutic options for PH, COPD, and PH in COPD.

To consider sex as an important biological variable, we first examined the effect of nicotine inhalation and hypoxic exposure on PH in male and female animals. Our findings indicate that male mice, but not female mice, responded well to 3 weeks of nicotine exposure, as shown in Figure 1. These data are consistent with previous reports that have highlighted the protective role of estrogen in animal models of PH (37). Clinically, it has also been shown that the prevalence of PH is higher in women; however, the severity of PH is lower because female patients tend to exhibit better responses to treatments and present with better prognoses than male patients (38). Given this fact, we used male mice to test our proposed N/H coexposure model because they showed a response to nicotine and hypoxia. We noticed a distinct difference in responses between 1- and 3-week exposures. Most significantly, a 1-week single exposure to nicotine or hypoxia did not alter RVSP, Fulton index, or PA remodeling compared with 3-week single exposures (Figures 1I–1J). However, 3-week N/H coexposure did not further increase PH compared with the 1-week exposure. This suggests that the synergistic effect of nicotine and hypoxia on the PA vasculature occurs in the early stages of PH in COPD. In this never previously reported animal model, pulmonary vasoremodeling and PH have a significant early onset in 1 week. In contrast, nicotine inhalation or CS inhalation and hypoxic exposure need several weeks to show effects. This unique early-onset feature may lead to widespread use of this animal model in the near future.

ROS play an important role in the pathophysiology of many diseases. Studies have depicted increased ROS signaling in lung tissues from cigarette-smoking patients with COPD (39). In this study, we investigated the role of N/H coexposure on mitochondrial ROS signaling. The previous studies by Schumacker and colleagues and our team have characterized the role of RISP of mitochondrial complex III as a major factor in cellular ROS generation in PASMCs (9–11, 13). Here, we found that nicotine-induced ROS generation in isolated PA mitochondria and N/H-evoked increases in RVSP were largely inhibited in SM-specific RISP hetKO mice compared with control mice (Figures 2A–2C); moreover, we further demonstrate that pulmonary vasoremodeling and PH were completely blocked in SM-specific tamoxifen-inducible RISP-KO mice (Figures 2D–2F). Using SM-specific RISP deletion mice, Waypa and colleagues also found that these animals had significantly attenuated PH following acute hypoxia alone (10). In contrast, RISP OE resulted in an exacerbated effect, but only in male mice (Figures 2G–2J). We also found that female mice had higher RISP protein expression in PAs compared with male mice (see Figure E2A). Even with RISP OE, female mice did not respond to N/H coexposure (see Figures E2H and E2I). These data further support the idea that estrogen may play a protective role in female mice and suggest that N/H-induced effects work independently of RISP in female mice. The circulating oxidative DNA damage biomarker 8-OHdG was also markedly decreased in serum isolated from RISP hetKO mice following N/H coexposure compared with control mice (Figure 3B). Furthermore, other mitochondrial proteins (e.g., COX1) were not affected, supporting that RISP is specifically involved in PH development following N/H coexposure.

Nicotine inhalation and CS may lead to oxidative stress–induced DNA damage, which is often associated with cancers (40, 41). In view of the similar pathophysiologic responses between some cancers and PH (e.g., cell hyperproliferation) (42), we hypothesized that DNA damage could play an important role in N/H-induced PA vasoremodeling and PH. Our data indicate increased expression of ATM-mediated DNA damage markers (γ-H2AX and p-Chk2) in our model of PH in COPD (Figure 3). The expression of these DNA damage markers was significantly reduced in RISP hetKO mice. We further found that baseline p-Chk2 expression was similar in male and female mice (see Figure E2A). These data suggest that N/H-induced effects are independent of RISP-mediated ATM signaling in female subjects. Additionally, SM-specific ATM KO blocked the increases in PA remodeling, RVSP, and Fulton index following N/H coexposure (Figures 5B–5D). These data suggest that RISP-mediated mitochondrial ROS may cause ATM-regulated DNA damage that leads to the development of PH in COPD.

It is generally accepted that DNA damage–induced ATM activation leads to p53 activation. In turn, this activation would trigger cell cycle arrest to prevent the replication of damaged DNA. A study by Wakasugi and colleagues (43) demonstrated that, in hypoxia-induced PH, there was a significant reduction in p53 expression in the lungs; moreover, using SM-specific p53 loss or gain of function in mice, they observed that p53 has little role in PH pathogenesis. Our laboratory has previously demonstrated that hypoxia causes an increase in cyclin D1 (through NF-κB) and subsequent hyperproliferation of PASMCs (9). This previous report and the present study support the paradoxical role of DNA damage in the development of PH, again highlighting the hyperproliferative and antiapoptotic phenotype of PASMCs.

Nicotine inhalation and CS in COPD and PH can lead to chronic inflammation, which plays a significant role in COPD and PH (2, 27, 44). Increased inflammatory cells such as macrophages and neutrophils have been documented in sputum, BAL fluid, and bronchial biopsies of patients with COPD (45). Recruitment of inflammatory cells can increase ROS production and inflammation (46, 47). Here, we identified ROS-induced inflammation that contributes to N/H-induced PA vasoremodeling via NF-κB inflammatory signaling in mice following N/H coexposure (Figure 4).

NF-κB, a family of transcription factors, plays a critical role in inflammation as a result of 1) increased production of inflammatory molecules like cytokines, chemokines, and adhesion molecules and 2) altered cell proliferation and apoptosis (9, 18, 30). Dysregulated inflammatory signaling can lead to inflammatory COPD and associated PH. In this study, we employed a potent NF-κB inhibitor, PDTC, to assess the functional role of NF-κB inflammatory signaling in PH. Our findings revealed that PDTC blocked increases in RVSP, Fulton index, and PA remodeling following N/H coexposure (Figures 4A–4E). It should be noted that, in addition to the inhibitory effect of NF-κB (29), PDTC may also act as an antioxidant to scavenge ROS (48, 49). Regardless, PDTC substantially dominates its inhibitory effect on NF-κB; thus, our results suggest that NF-κB–mediated inflammatory signaling is involved in N/H-induced PH. In SM-specific RISP hetKO mice, we observed the blockade of increased NF-κB activity, as determined by higher protein expression of IκBα following N/H coexposure (Figure 4F). These findings indicate the role of RISP in regulating NF-κB inflammatory signaling, contributing to PH in COPD.

Increased inflammatory signaling can cause ROS production and oxidative stress. DNA damage may also lead to an inflammatory response. The interplay between inflammation and DNA damage has not been thoroughly investigated in PH or COPD. In this study, we examined the interactive effect of NF-κB–dependent inflammation and ATM-mediated DNA damage using PDTC and SM-specific ATM-KO mice. As hypothesized, if DNA damage resulted from inflammation, we would expect inflammatory markers to be present even in the absence of DNA damage signaling. However, following N/H coexposure, NF-κB activity was unchanged in our ATM-KO mice (Figure 5E) but significantly increased in normal mice (Figure 6A). Additionally, the NF-κB inhibitor PDTC produced a similar effect (Figure 5F). PDTC blocks the accumulation of proinflammatory cytokines and subsequent inflammatory responses (28, 29). Collectively, our findings suggest that NF-κB–dependent inflammation and ATM-mediated DNA damage may positively interact with each other to mediate the development of N/H-induced PA vasoremodeling and PH and may be of interest for further investigation.

CS is one of the most widely recognized risk factors for COPD and other diseases. Although nicotine is the major component of cigarettes, cigarettes are composed of a toxic mix of more 7,000 chemicals, 70 of which are known carcinogens (50). Thus, we examined whether CS would further exacerbate PH compared with nicotine. Interestingly, the increased RVSP, Fulton index, and PA remodeling were not significantly different in mice between 1 week of CS/hypoxia exposure and N/H coexposure (Figure 6). These data suggest that nicotine is the important driving factor in the early stages of PH.

As summarized in Figure 7, our study establishes the first animal model of N/H-induced PH by demonstrating the synergistic effect of nicotine and hypoxia. This animal model may illustrate the early onset of PH in COPD, recapitulating the severity of continued nicotine or CS use for patients with COPD. Further investigations using our newly created complementary SM-specific RISP-KO and OE mice uncover the role of RISP-dependent mitochondrial ROS in N/H-induced PA vasoremodeling and PH. We provide evidence that RISP mediates ATM-associated DNA damage and NF-κB–regulated inflammation promoting N/H-induced PA vasoremodeling and PH in COPD, whereby the roles of ATM and NF-κB are implemented by their reciprocal positive feedback mechanism. Moreover, RISP, ATM, NF-κB, and their associated signaling molecules may become novel specific and effective targets in the treatment of PH, COPD, and PH in COPD.

Figure 7.

Schematic illustration of CS (or nicotine)/hypoxia coexposure–induced PH and the underlying signaling mechanisms. (A) Our findings provide the first evidence to demonstrate CS or nicotine plus hypoxia coexposure causes mitochondrial RISP-mediated ROS generation, oxidative stress–dependent DNA damage, ATM activation, NF-κB–regulated inflammation, and N/H-induced PA vasoremodeling and PH. (B) As proposed, SM-specific genetic and pharmacological inhibition (e.g., shRNA knockdown) of RISP, ATM, and NF-κB may become potent and specific therapeutic targets for treating PH and COPD. The image was created using BioRender.