Combination of Pioglitazone, a PPARγ Agonist, and Synthetic Surfactant B-YL Prevents Hyperoxia-induced Lung Injury in Adult Mice

doi:10.21203/rs.3.rs-1762648/v1

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Combination of Pioglitazone, a PPARγ Agonist, and Synthetic Surfactant B-YL Prevents Hyperoxia-induced Lung Injury in Adult Mice

https://doi.org/10.21203/rs.3.rs-1762648/v1

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Introduction: Acute lung injury and acute respiratory distress syndrome are characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. We previously reported that a combination of aerosolized pioglitazone (PGZ) and synthetic lung surfactant B-YL peptide as surfactant protein B mimic prevents hyperoxia-induced neonatal rat lung injury, but this has not been tested in adult lung injury.

Method: We characterize effects of hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-β signaling pathways, which are critical mediators of hyperoxia-induced lung injury, 2) aberrations of lung homeostasis and injury repair following 24 or 72-hour exposure of adult mice lung explants to hyperoxia, and 3) whether these hyperoxia-induced aberrations can be blocked by concomitant treatment with PGZ and B-YL combination.

Results: Our study reveals that hyperoxia exposure of adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates β-catenin and LEF-1) and TGF-β (upregulation of key TGF-β signaling intermediates TGF-β type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, MCP-1, IL-1β, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1 and PECAM-1) markers. All of these changes were largely prevented by PGZ+B-YL combination.

Conclusions: The effectiveness of the PGZ+B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo sets the stage to test this promising therapeutic approach for adult lung injury in vivo.

Hyperoxia

Lung injury

Pioglitazone

Surfactant

Acute lung injury (ALI) and its severe form, i.e., acute respiratory distress syndrome (ARDS), are serious conditions and often life-threatening. However, currently, there is no effective treatment for both, which presently consists of providing only supportive respiratory care instead of treating the underlying cause [1]. Part of the reason for the failure in developing effective treatment for ALI/ARDS has been the failure to target fundamental molecular signaling pathways underlying ALI and ARDS pathogenesis. ALI/ARDS can result from direct lung injury, e.g., pneumonia, exposure to supra-physiologic oxygen concentrations (hyperoxia), inhalation of cigarette smoke and other toxic substances, and from indirect lung injury, e.g., sepsis and severe trauma with shock. Both ALI and ARDS are characterized by alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction [2–5]. Molecularly, activation of TGF-β and Wnt signaling, inhibition of peroxisome proliferator-activated receptor-gamma (PPARγ) signaling, and inactivation of surfactant activity by reactive oxygen species are critical components of ALI and ARDS pathogenesis that have not been previously targeted simultaneously.

Using a neonatal rat model, under both in-vitro and in-vivo conditions, we previously demonstrated that a combination of nebulized pioglitazone (PGZ), PPARγ agonist, and a novel synthetic lung surfactant with B-YL peptide as surfactant protein B (SP-B) mimic prevents hyperoxia-induced neonatal lung injury more than either modality alone [6]. Whether this strategy will also be effective for adult ALI is not known. To address this question, we hypothesized that by blocking Wnt and TGF-β signaling pathways’ activation, which are critical mediators of hyperoxia-induced lung injury, combined PGZ and B-YL treatment would block hyperoxia-induced adult lung injury.

Lung explant hyperoxia exposure model

Adult male (3 to 6-month-old) C57BL/6 mice were sacrificed using euthasol to harvest lungs and placed in 15% fetal bovine serum (FBS)/Waymouth medium on ice. Lungs were cut into ~ 1-millimeter cubes aseptically and cultured in six-well plates with 15% FBS/Waymouth medium while rocking on an oscillating platform (3 cycles/min). Lungs were incubated at 37°C under one of the following four conditions: 1) untreated control, 2) B-YL (100 mg/kg) only, 3) PGZ (1 mg/kg) only, and 4) B-YL (100 mg/kg) + PGZ (1 mg/kg). Lung explants were exposed to either 21%O2 (normoxia) or 95%O2 (hyperoxia) for 24 or 72h using sealed chambers (Billups-Rothenberg Inc., Del Mar, CA).

At 24 or 72h, lungs were harvested and snap-frozen in liquid nitrogen and processed for western blotting and q-RT-PCR for lung homeostasis, injury repair, and inflammatory markers.

Protein extraction and Western blotting

Liquid nitrogen flash-frozen lungs were homogenized with tissue homogenizer (Fisher Scientific, Pittsburg, PA) in RIPA (Radioimmunoprecipitation assay) lysis buffer containing 1mM EDTA and EGTA (Boston Bioproducts, Ashland, MA), 1mM PMSF, and complete proteinase inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Fifty micrograms of total protein for each sample were denatured using SDS-PAGE sample buffer and electrophoresed in 10% SDS polyacrylamide gel. Next, samples were transferred onto 0.45µm nitrocellulose membrane and blocked with 5% milk in TBS-Tween (TBST) and then probed with primary antibodies [surfactant protein-C (SP-C;1:200, cat#SC-13979), CCTα (1:200, cat#SC-376107), C/EBPα (1:150, cat#SC-365318), BCL-2 associated X protein (BAX;1:150, cat#SC-493), β-catenin (1:2500, cat#SC-7963) (all from Santa Cruz Biotechnology, Santa Cruz, CA), LEF-1 (1:800, cat#14972), BCL-2 (1:500, cat#26593), SMAD3 (1:800, cat#25494-1-AP), SMAD7 (1:800, cat#25840-1-AP), Fibronectin (1:3000, cat#15613), PPARγ (1:1000, cat#16643) (all from Proteintech, Rosemont, IL), calponin (1:2500, cat#2687, MilliporeSigma, Burlington, MA), ALK5 (1:500, cat#AB31013, Abcam, Cambridge, MA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH;1:3000, cat#MAB374, MilliporeSigma, Burlington, MA)] overnight at 4°C followed by appropriate secondary antibody and Pierce Super-Signal Chemiluminescent substrate (Thermo Fischer Scientific, Rancho Palos Verdes, CA). ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantitate protein bands. Bands were measured by densitometry and expressed relative to accompanying GAPDH bands as relative units.

RNA isolation and qRT-PCR

Tissue RNA extraction and qRT PCR were performed according to previously described method [7]. Primer sequences were: MCP-1 (sense5'-AGTAGGCTGGAGAGCTACAA-3'; anti-sense5'-GTATGTCTGGACCCATTCCTTC-3'), TGF- β1 (sense5'-CGAAGCGGACTACTATGCTAAA-3'; anti-sense5'-TCCCGAATGTCTGACGTATTG-3'), TGF- β3 (sense5'-CGCTACATAGGTGGCAAGAA-3'; anti-sense5'-CAAGTTGGACTCTCTCCTCAAC-3'), TNFα (sense5'-TTGTCTACTCCCAGGTTCTCT-3'; anti-sense5'-GAGGTTGACTTTCTCCTGGTATG-3'), IL-6 (sense5’-GAGTGGCTAAGGACCAAGACC-3’; anti-sense5’-AACGCACTAGGTTTGCCGA-3’), IL-1β (sense5’-AGCTTCAGGCAGGCAGTATC-3’; anti-sense5’-AAGGTCCACGGGAAAGACAC-3); PECAM-1 (sense5’-CACCCATCACTTACCACCTTATG-3’; anti-sense5’- TGTCTCTGGTGGGCTTATCT-3’), FLK-1 (sense5’-CGACATAGCCTCCACTGTTTAT-3’; anti-sense5’-TGTTCTTGTTCTCGGTGATGT-3’), FLT-1 (sense5’-CAACGTCACAGTCACCCTAAA-3’; anti-sense5’- CTCTCCTACTGTCCCATGTTATTC-3’), VEGF-A (sense5’-GCAGACCAAAGAAAGACAGAAC-3’; anti-sense5’-CAGTGAACGCTCCAGGATTTA-3’). Normalization control was Tuba1a: 5'-CTCTCTGTGGATTACGGAAAGAAG-3' (forward), 5'-GGTGGTGAGGATGGAATTGTAG-3' (reverse) [8]. All reactions were run in triplicate; relative expression was determined using comparative cycle threshold method (2 − ΔΔCT), recommended by supplier (Applied Biosystems). Abundance values were expressed as fold changes compared with corresponding control group.

Synthetic B-YL peptide surfactant

B-YL surfactant consists of 3% of synthetic SP-B peptide mimic B-YL formulated in mixture of synthetic surfactant lipids (DPPC:POPC:POPG 5:3:2 by weight%). B-YL is sulfur-free and oxidant resistant [6, 9]. Combining B-YL surfactant and PGZ does not alter the bioactivity of both components [6].

Statistical analysis

Student t-test and ANOVA were used to detect group differences, and p-value of < 0.05 was considered statistically significant (N = 3–4 from at least three separate experiments). The results are expressed as means +/-SEM; data are shown using GraphPad Prism software.

Effect of PGZ and B-YL on lung homeostasis markers

We determined the effect of PGZ and B-YL under normoxic conditions on key epithelial (SP-C and CCTα) and mesenchymal (PPARγ) markers of lung homeostasis. With PGZ + B-YL treatment, SP-C and CCTα protein levels increased at both 24 and 72h (Fig. 1a and 1b). PGZ alone treatment also increased CCTα at both time points, while SP-C protein levels were increased only at 72h. Notably, the increases were more pronounced with PGZ + B-YL versus PGZ alone. In contrast, although PPARγ protein levels were not affected by any treatment at 24h, it increased equally with PGZ alone and PGZ + B-YL treatments at 72h. Taken together, effect of PGZ and B-YL treatment on lung homeostasis markers in adult lungs are similar to those in neonatal lungs [6].

Effect of PGZ and B-YL surfactant on hyperoxia-induced activation of TGF-β and Wnt signaling pathways

Since TGF-β and Wnt signaling activations are critical mediators of injury repair response in almost all organs, including the lung, we next evaluated activation of these pathways in hyperoxia-induced adult lung injury and how these are affected by various treatments. We determined that levels of key canonical Wnt pathway proteins β-catenin and LEF-1 upregulated with hyperoxia and this increase was attenuated with PGZ alone and PGZ + B-YL combined treatments (Fig. 2a). Similarly, hyperoxia exposure activated TGF-β signaling as evidenced by increased protein levels of ALK5 and SMAD 3 (Fig. 2b). Concomitant PGZ + B-YL treatment blocked increases in both proteins, while PGZ alone blocked increase in ALK5 only. Although there was a trend towards increase in SMAD 7 protein levels with hyperoxia exposure with or without any treatment, it did not reach statistical significance in any experimental group. TGF-β activation on exposure to hyperoxia and its blockage by PGZ + B-YL was also supported by mRNA levels of TGF-β1 at 24 and 72h and TGF-β3 at 24h (Fig. 2c).

We next examined protein levels of lung myogenic proteins calponin and fibronectin, both of which are targets of TGF-β and Wnt signaling. Hyperoxia-induced increase in protein levels of both calponin and fibronectin was blocked by PGZ alone and PGZ + B-YL treatments, albeit with a more prominent effect in the PGZ + B-YL treatment group (Fig. 3).

Since PPARγ signaling, the main intervention strategy used in this study by using a PPARγ agonist, antagonizes Wnt and TGF-β activations under both homeostasis and injury repair conditions [10], we determined protein levels of key PPARγ signaling intermediates PPARγ and C/EBPα (Fig. 4). On exposure of adult mouse lung explants to hyperoxia, C/EBPα protein level decreased significantly; however, in contrast to findings in several other lung injury models, PPARγ protein levels did not decrease. Importantly, however, compared to controls, with PGZ + B-YL treatment, PPARγ protein levels increased. Notably, both PGZ and PGZ + B-YL treatments blocked hyperoxia-induced decreases in C/EBPα levels.

Effect of PGZ and B-YL on hyperoxia-induced alterations of inflammatory cytokines and cellular apoptosis

Hyperoxia exposure resulted in significant alterations in inflammatory cytokines such as IL-6, MCP-1, IL-1β, and TNFα, which are known mediators of lung injury (Fig. 5a). At 24h, compared to controls, although there was no increase in IL-6 expression in the hyperoxia exposed group, treatment with B-YL alone, PGZ alone, and with PGZ + B-YL decreased it. At 72h, hyperoxia increased IL-6 expression, which was blunted with B-YL and PGZ treatments singly as well as in combination. MCP-1 expression increased with hyperoxia exposure at both 24 and 72h, but this increase was not blocked with any treatment. With hyperoxia, IL-1β expression decreased, but with PGZ + B-YL treatment, IL-1β decreased further at 24h; however, following 72h hyperoxia exposure, its level increased, an effect, blocked in the PGZ only and PGZ + B-YL treatment groups. TNFα expression decreased at 24h, but increased at 72h in the hyperoxia exposure group, effects that were not affected with any treatment. Additionally, hyperoxia-induced alterations in mRNA levels of inflammatory cytokines were also accompanied by increased apoptosis, as determined by protein levels of BCL-2 and BAX (Fig. 5b). Per other data on protective effects of PGZ and PGZ + B-YL, hyperoxia-induced increase in apoptosis was also blocked by PGZ alone and PGZ + B-YL treatments.

Lastly, as shown in Fig. 5c, determination of endothelial markers indicated increased mRNA expression of VEGF-A at both 24h and 72h, with blockage of this increase with all interventions at 24h, but with no effect at 72h. VEGF-receptor (FLT-1) mRNA expression increased at 24h but decreased at 72h, with blockage of hyperoxia-induced increase at 24h in all treatment groups without any effect on 72h mRNA levels in the PGZ only and PGZ + B-YL treatment groups. There was no effect of hyperoxia on FLK-1, another VEGF receptor, mRNA levels with or without any treatment at either the 24h or 72h timepoint. Lastly, PECAM-1, a key endothelial marker increased at 24h, an effect that was blocked in all treatment groups; however, there was no effect of hyperoxia on PECAM-1 mRNA expression at 72h with or without any intervention.

Acute lung injury and ARDS are serious conditions; both can be life-threatening, yet there is no effective treatment. This ex-vivo study confirmed previously known activations in TGF-β and Wnt signaling pathways in adult lung injury. It also showed that hyperoxia-induced activation in these pathways in adult lung injury was effectively blocked by combined treatment with PGZ and B-YL. The PGZ + B-YL combination also attenuated the hyperoxia-induced increase in inflammatory cytokines, cellular apoptosis, myogenic protein levels, and alterations in endothelial injury markers. Additionally, PGZ + B-YL treatment increased protein levels of PPARγ, SP-C, and CCTα in normoxia, indicating its pro-homeostatic effect. Although PGZ alone also had significant lung protectant and pro-homeostatic effects, overall, these effects were more pronounced with the PGZ + B-YL combination versus PGZ alone. These data suggest possible effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult lung injury, likely mediated via blockage of TGF-β and Wnt signaling activations, both of which are critical mediators of adult lung injury.

As supported by multiple human and animal studies, TGF-β activation plays a central role in injury repair in almost all organs, including in the lung [11–13]. In addition, activation of Wnt signaling in adult lung injury is supported by numerous experimental models of ALI and adult patients with ARDS [14–16]. Importantly, TGF-β activation and its downstream interaction with canonical Wnt pathway intermediates result in disrupted homeostatic epithelial-mesenchymal interactions [6, 17–21]. Although a complex network of signaling molecules is involved in these interactions, the primary TGF-β signaling intermediates include ALK5 and SMAD 3 and SMAD 7. The major Wnt signaling pathway intermediates include Wnt receptors, Frizzled and LRP 5/6, and intracellular mediators such as Disheveled, Axin, APC, and GSK-3β. Interactions of these intermediates lead to β-catenin stabilization, resulting in its nuclear translocation where it binds to LEF-1 to induce target gene transcription. Therefore, in this study, we examined key signaling molecules of TGF-β (ALK 5, SMAD 3 and SMAD 7) and Wnt (β-catenin and LEF-1) pathways and their downstream targets, including cellular apoptosis (BCL-2 and BAX), inflammatory markers (IL-6, IL-1β, MCP, TNFα), myogenic markers (fibronectin and calponin) and endothelial (VEGF-A, PECAM-1, FLT, FLK) markers. In addition, expression of key Wnt pathway antagonists PPARγ and C/EBPα was determined.

As in our prior study that examined neonatal rat lung injury [6], in this study, using adult mouse lung explants, we observed that hyperoxia-induced TGF-β activation (upregulation of ALK5 and SMAD 3 protein levels) was effectively blocked by PGZ + B-YL combination. In contrast to our previous study, an increase in SMAD 7 protein level, which is an autoinhibitory response to TGF-β activation, was not observed; this is likely due to a relatively shorter duration of hyperoxia exposure (72-hours) in this study versus the 7-day exposure in the previous study.

Interestingly, in contrast to hyperoxia-induced PPARγ downregulation in the developing lung rat model, in adult mouse lung explants, PPARγ protein levels were unaffected at both timepoints examined, suggesting that alterations in TGF-β and Wnt signaling pathways likely play a more dominant role in the adult lung injury than in the neonatal lung injury. Nevertheless, the evidence from numerous adult organ injury models including the adult lung clearly reflects antagonistic effects of PPARγ and Wnt signaling pathways [10, 22, 23]. Therefore, it is not surprising that upregulation of PPARγ signaling (increase in PPARγ and C/EBPα protein levels) with both PGZ alone and PGZ + B-YL combined treatments was associated with blockage of hyperoxia-induced activation in Wnt signaling (increased β-catenin and LEF-1 protein levels) and increase in its downstream myogenic targets (fibronectin and calponin). Lastly, although molecular mechanisms involved remain incompletely understood and were not the focus of this study, hyperoxia-induced increases in inflammatory cytokines and apoptosis were effectively blocked by the PGZ + B-YL combination consistently at 72h but not at 24h, which may be a function of the shorter duration of treatment. Regarding the effects of hyperoxia on pulmonary endothelial markers, unlike our findings, hyperoxia has been shown to decrease VEGF and VEGF receptor (FLT-1) levels in other models. This difference also might be due to a relatively shorter duration of hyperoxia exposure and the use of lung explants in this study versus other in-vivo studies [24, 25]. However, similar to our observation, upregulation of PECAM-1 has been reported with hyperoxia-induced lung injury in an adult mouse model [26].

Although both surfactant and PPARγ agonists treatment have been tried individually in various ARDS models, benefits of either drug alone are limited. For example, exogenous surfactant treatment in patients with ALI/ARDS significantly lowered inspired oxygen need; however, there was no significant decrease in mortality [27]. The lack of mortality benefit may be explained at least partially due to only limited surfactant reaching the distal lung in these studies [28]. In addition, although PPARγ agonist administration has shown benefits in ameliorating pulmonary inflammation, e.g., a decrease in neutrophil influx and tissue injury in a LPS-induced lung injury model [29], sole treatment with PGZ did not have any benefit on endotoxin induced lung inflammation in humans [29, 30]. To promote injury repair and block lung surfactant dysfunction and oxidant damage simultaneously, we combined PGZ with a novel synthetic surfactant B-YL. This combined PGZ + B-YL approach targets key signaling pathways disrupted in ALI/ARDS and provides an oxidant-resistant surfactant B-YL. B-YL is an innovative surfactant developed to resist hyperoxia-induced inactivation; it is relatively stable and highly active in its native form. We previously demonstrated that combining B-YL with PGZ does not affect B-YL’s surfactant bioactivity, and B-YL does not affect PGZ’s PPARγ agonist activity. Moreover, when delivered via nebulization, combining B-YL with PGZ improved PGZ’s delivery by about 30% versus PGZ delivered alone [6].

In summary, this study, using the adult mouse lung explant model, demonstrates that combined PGZ and B-YL treatment mitigates hyperoxia-induced activation of TGF-β and Wnt signaling and the accompanying increase in inflammatory cytokines, myogenic proteins, and cellular apoptosis. Although PGZ alone also exhibited significant protective effects, overall, the effects were more pronounced with PGZ + B-YL combination versus PGZ alone. The effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult lung injury ex-vivo encourages testing this promising therapeutic approach in-vivo.

Funding:

This study was supported by grants from NIH (HD071731, HD127237, HL151769, and HL152915) and the Tobacco-Related Disease Research Program (23RT-0018, 27IP-0050, and T29IR0737).

Disclosures:

The authors have no relevant financial or non-financial interest to disclose.

Author Contributions:

Conceptualization & Methodology: Virender K. Rehan; Investigation/Data Analysis/Visualization: Chie Kurihara, Reiko Sakurai, Tsai-Der Chuang; Interpretation of results: Chie Kurihara, Reiko Sakurai, Tsai-Der Chuang, Frans J. Walther, Virender K. Rehan; Writing-original draft preparation: Chie Kurihara, Virender K. Rehan; Writing-review and editing: Chie Kurihara, Alan J. Waring, Frans J. Walther, Virender K. Rehan. All Authors read and approved the final manuscript.

Ethics Approval:

All animal procedures were performed following National Institutes of Health guidelines for the care and use of laboratory animals and approved by The Lundquist Institute Animal Care and Use Committee.

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Combination of Pioglitazone, a PPARγ Agonist, and Synthetic Surfactant B-YL Prevents Hyperoxia-induced Lung Injury in Adult Mice

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Abstract

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Introduction

Materials And Methods

Lung explant hyperoxia exposure model

Protein extraction and Western blotting

RNA isolation and qRT-PCR

Synthetic B-YL peptide surfactant

Statistical analysis

Results

Effect of PGZ and B-YL on lung homeostasis markers

Effect of PGZ and B-YL surfactant on hyperoxia-induced activation of TGF-β and Wnt signaling pathways

Effect of PGZ and B-YL on hyperoxia-induced alterations of inflammatory cytokines and cellular apoptosis

Discussion

Declarations

References

Additional Declarations

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