key: cord-0322703-relr7nin
authors: Kamga Gninzeko, Franck J.; Tho, Cindy K.; Valentine, Michael S.; Wandling, Emily N.; Heise, Rebecca L.
title: Mechanical Ventilation induced DNA Damage and P21 in an Acute Aging Model of Lung Injury
date: 2022-03-08
journal: bioRxiv
DOI: 10.1101/2022.03.08.483505
sha: cbf0129a42d21d75c76b097184742a614be596a1
doc_id: 322703
cord_uid: relr7nin

Acute respiratory distress syndrome (ARDS) is a form of acute lung injury which leads to a paucity of oxygen. To remedy ARDS, patients are put on mechanical ventilators; however, the stretch resulting from the mechanical ventilator can lead to ventilator-induced lung injury or VILI. VILI is exacerbated by age. The combined effects of ARDS and mechanical ventilation can result in a hostile environment which may lead to senescence (stable cell cycle arrest). The role of senescence in VILI is poorly understood. Senescence is characterized by increased cyclin-dependent kinase inhibitors P16 and P21; however, P21 has been shown to occur in early senescence. We hypothesized that mechanical ventilation would lead to DNA damage and senescence-like phenotype. Both in vivo and in vitro models of VILI were used to investigate senescence and its mechanism in VILI. Mechanical ventilation increased senescence-associated markers such as DNA damage marker characterized by ɣH2AX, P21, senescence-associated secretory phenotype IL6, and decreased proliferation. Moreover, mechanical ventilation led to increased apoptosis. Lung sections were stained for KRT8 proteins, markers of transiently differentiated alveolar type 2 (AT2) cells which were reported to be more prone to DNA damage. Age and mechanical ventilation increased KRT8 positive cells. Finally, we probed a potential mediator of the stretch induced senescence in vitro by inhibiting P38-MAPK, which can be activated by DNA damage response, leading to increased P21. Inhibiting P38 decreased in P21 but did not decrease ɣH2AXThese findings suggest that mechanical ventilation may lead to a senescence-like phenotype involving the P38-MAPK pathway.

Acute respiratory distress syndrome (ARDS) is a form of acute lung injury which leads to a paucity of oxygen. To remedy ARDS, patients are put on mechanical ventilators; however, the stretch resulting from the mechanical ventilator can lead to ventilatorinduced lung injury or VILI. VILI is exacerbated by age. The combined effects of ARDS and mechanical ventilation can result in a hostile environment which may lead to senescence (stable cell cycle arrest). The role of senescence in VILI is poorly understood. Senescence is characterized by increased cyclin-dependent kinase inhibitors P16 and P21; however, P21 has been shown to occur in early senescence. We hypothesized that mechanical ventilation would lead to DNA damage and senescence-like phenotype. Both in vivo and in vitro models of VILI were used to investigate senescence and its mechanism in VILI. Mechanical ventilation increased senescence-associated markers such as DNA damage marker characterized by ɣH2AX, P21, senescence-associated secretory phenotype IL6, and decreased proliferation. Moreover, mechanical ventilation led to increased apoptosis. Lung sections were stained for KRT8 proteins, markers of transiently differentiated alveolar type 2 (AT2) cells which were reported to be more prone to DNA damage. Age and mechanical ventilation increased KRT8 positive cells. Finally, we probed a potential mediator of the stretch induced senescence in vitro by inhibiting P38-MAPK, which can be activated by DNA damage response, leading to increased P21. Inhibiting P38 decreased in P21 but did not decrease ɣH2AXThese findings suggest that mechanical ventilation may lead to a senescence-like phenotype involving the P38-MAPK pathway.

Acute respiratory distress syndrome (ARDS) is characterized by a leaky alveolar barrier and inflammation. ARDS has a high mortality rate and disproportionally affects the elderly population with a higher mortality and incidence rate [1] . There are signs of hindered gas exchange amongst ARDS patients [2] , leading to mechanical ventilation.

Despite the benefits of mechanical ventilation at mitigating the effects of ARDS and facilitating the gas exchange, the constant strain and stress created by the former due to the air moving in and out of the alveoli may worsen the injury and lead to ventilationinduced lung injury, VILI [3] . VILI is characterized by biotrauma due to the increased inflammation; volutrauma due to the overextension of the alveoli caused by air going in and out of the lungs during ventilation; and atelectrauma caused by the constant collapse and reopening of the alveoli [4] . These characteristics of VILI, especially biotrauma and volutrauma, can result in cascades of events that can cause the alveoli cells to become damaged and change phenotype, creating a hostile environment. The hostile environment created by VILI coupled with inflammation observed in ARDS may lead to senescence.

Senescence is an irreversible terminal cell cycle arrest process in which proliferating cells stop responding to replication-promoting stimuli. However, evidence suggests that senescence can occur prematurely in response to injury [5] . Because of its heterogenicity across different tissues and conditions, senescence is a complex phenomenon that is poorly understood. Furthermore, more is known about senescence in chronic lung diseases than acute. In senescence, cyclin-dependent kinase inhibitors such as P21 and P16 are upregulated. While P21 plays an essential role in early senescence, P16 is accumulated at a later stage of senescence [6] . There is also evidence that P21, not P16, is upregulated with VILI [7] . Senescent cells also produce autocrine and paracrine signaling collectively known as senescence-associated secretory phenotype, SASP. SASP mediators include pro-inflammatory cytokines such as IL-6, which can exacerbate inflammation [8] .

Though some parts of the senescence mechanism have been investigated, more still needs to be done to fully understand the mechanism of senescence in VILI and other diseases. One of the mechanisms of senescence is the DNA damage response pathway which could be activated via mechanotransduction. Cyclic stretch like the one generated by mechanical ventilation can break DNA strands down via MAPK activation [9] , leading to upregulation of DNA damage proteins such as ɣH2AX [10] . Activation of ɣH2AX, in turn, will lead to the activation of P21 [11] . P38-MAPK is also known to activate the P53-P21 pathway resulting in cellular senescence [11] . However, this pathway has not been investigated in the context of senescence in acute diseases or VILI.

In this research, we are investigating whether mechanical ventilation promotes senescence-like phenotypes. We also examine whether stretch induces DNA damage response, leading to senescence. Finally, we are using a P38-MAPK inhibitor to understand the mechanism of senescence in VILI. We hypothesized that mechanical ventilation would lead to DNA damage and senescence-like phenotype via P38-MAPK activation, and blocking it will reduce this phenotype.

Animal: Male young (8-10 weeks) and old (20-22 months) C57BL/6 mice were acquired from the National Institute on aging and housed at the VCU vivarium. All procedures performed on the mice were approved by VCU Institutional Animal Care and Use Committee (IACUC). Mice were mechanically ventilated at 0 PEEP and 35 and 45 cmH2O for young and old mice, respectively, for 2 hours. Lung mechanics were measured at 30-minute increments for the duration of ventilation.

Sample Collection: At the end of the 2-hour mechanical ventilation, blood was collected from the vena cava and centrifuged to obtain plasma. As previously described, gravityassisted bronchoalveolar lavage (BAL) was performed [12] . Briefly, PBS was instilled into the mice lungs and collected and repeated twice. The BAL fluid (BALF) was then centrifuged, the supernatant was transferred onto a new tube for later processing. The cells were resuspended then cytospun onto a microscope slide. Western Blot: Western blot was performed according to the manufacturer's protocol.

Briefly, BCA was performed on the freshly isolated proteins using a Pierce BCA protein assay kit (Sigma, 23227) to determine the protein concentration. Electrophoresis was performed using 30 ug of proteins loaded into each 10-well gel; proteins were then transferred from the gel onto a membrane. The membrane was blocked for 1 hour, followed by primary antibody incubation overnight at 4 C. The next day, the membrane was rinsed and incubated in a solution of HRP bound secondary antibody for 1 hour, followed by rinsing and incubation in Pierce ECL western blotting substrate TUNEL staining: Click-iT TM Plus TUNEL assay for in situ apoptosis detection with Alexa Fuor TM 647 was performed according to the manufacturer protocol (ThermoFisher Scientific, C10619). Briefly, slides were deparaffinized using xylenes, decreasing percentages of ethanol, saline solution, and PBS. The tissue slides were fixed and permeabilized using 4% paraformaldehyde and proteinase K. Then, TdT reaction was performed by incorporating EdUTP into dsDNA strand breaks. Finally, fluorescence Click-iT TM plus reaction was performed to detect EdUTP; then the slides were mounted using ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher Scientific, P36931).

Statistical analysis: Subjects were randomly assigned to different groups. Power analysis was estimated based on previous work. A minimum of 3 mice per group was used. One-way and two-way ANOVA followed by posthoc Tukey's multiple comparison test were performed when appropriate to compare multiple groups. A T-test was also performed to compare two groups. GraphPad Prism 6 was used for statistical analysis.

Results:

To assess the effects of mechanical ventilation on the lung structure and physiology, lung histology, lung mechanics perturbation maneuvers, and PMN (Polymorphonuclear leukocytes) count were performed. H&E-stained lung sections showed that highpressure MV caused increased structural damage on the lungs of both young and old mice. Furthermore, when considering age alone, old mice inherently showed more significant structural damage signs than young mice without mechanical ventilation ( figure 1A) . BCA results showed increased protein in the BALF with age and ventilation.

These results suggest that mechanical ventilation and age are cofactors for lung injury ( figure 1B) .

Similarly, the count of PMN on BALF showed a significant increase in PMN intrusion in the alveoli space with mechanical ventilation in both age groups. At baseline, without mechanical ventilation, old mice showed significantly higher number of PMN in the BALF compared to the young non-ventilated. However, in the old group who did not receive mechanical ventilation, we did not see much increase in PMN compared to the young non mechanically ventilated (figure 1C). 

P21, a protein involved in the cell cycle, was analyzed. There was an increase in P21 gene expression with mechanical ventilation. Young and old mice that were mechanically ventilated had a significant increase in P21 compared to the young nonventilated. Mechanically ventilated old mice also showed a significant increase in P21 compared to the old non-ventilated. These results suggest that mechanical ventilation plays a role in P21 regulation. However, we did not observe a significant increase in P21 gene expression with age alone; this could be because P21 is an early marker of senescence ( figure 4B ).

Ki67, a proliferation marker, did not increase with age or mechanical ventilation. Ki67 level was significantly lower in the young mice when mechanically ventilated compared to the young non-ventilated. This is consistent with the P21 marker increase suggesting that mechanical ventilation and age initially lead to increased P21 gene expression and the lack of proliferation ( figure 4C ).

IL6, a pro-inflammatory marker part of the SASP, was also probed in the BALF. It has been previously shown that when cells become senescent, they produce cytokines which include IL6 [8] . Mechanically ventilated mice showed an increase in IL6 level (figure 4D); this is on par with P21 gene expression groups. In all, these results suggest that increased DNA damage, high P21 gene expression levels, and reduced Ki67

(proliferation), all hallmarks of cellular senescence, also lead to increase SASP. There are mainly two cell types in the alveolar epithelium, alveolar type 1 and type 2 (AT1 and AT2). It has been shown that due to mechanical cues and in certain diseases, AT2 differentiate into AT1 [13] . It has also been shown that before differentiating to AT1 due to various stimuli, AT2 cells go through a transient state which is positive for Krt8 [14] . These transient KRT8+ cells are also more prone to DNA damage [15] . high pressure.

We first wanted to know if human SAEC undergoes DNA damage in response to cyclic stretch, potentially leading to cellular senescence. Compared to the static group, there was more DNA damage manifested by an increase ɣH2AX in the stretch SAEC group (figure 6 A). Similarly, there was an increase in P21 in stretch SAEC compared to the static group ( figure 6B ). This is consistent with the results obtained in the mechanically ventilated old mice; The SAEC were from patients aged 51-66 years. As in old mice, there was an increase in ɣH2AX with mechanical stretch compared to the static group.

In addition, it was only when mechanically ventilated that the old mice showed an increase in P21.

Since it had been reported that activation of P38-MAPK leads to an increase in P21, we inhibited P38-MAPK using a P38 inhibitor. However, when given the P38 inhibitor, there 

With the advent of novel respiratory diseases, including the current pandemic caused by COVID-19, more and more patients are ending up on mechanical ventilators, which lead to further injuries such as VILI. Despite being a lifeline for some patients, mechanical ventilators can leave lifelong damages [16] that are still not fully understood. In this study, we have developed both in vitro and in vivo models to simulate different damages caused by mechanical ventilation. We used these models to study the different mechanisms potentially involved in VILI, which can better understand the disease for future therapies.

It has been extensively documented that the shear forces generated by mechanical ventilation exacerbate lung injury [17] . The characteristics of these lung injuries include the changes in lung structure. For instance, other studies, including ours, have shown that after mechanical ventilation, the airspace of mice becomes enlarged [18] - [20] . This is in line with what we observed in the lung histology of the mechanically ventilated mice. This airspace enlargement is also more pronounced in old mice generally, which is then exacerbated with mechanical ventilation (figure 1 A) due to the change in the lung's parenchyma and alveolarization with age [21] . These structural changes in the lung could also lead to a change in the lung's mechanics.

The structural damage and increased alveolar size, factors related to airspace enlargement, increase lung volume and compliance [22] . The increased alveolar space and the structural damage observed in their histology (figure 2) could explain why the old mice have inherently a higher lung single compartment static compliance compared to the young mice. However, other studies have shown that mechanical ventilation leads to a decrease in compliance [23] , as observed in the young group in this study.

Multiple studies have shown that structural lung damage leads to fluid exudation in the alveolar space [24] - [27] . This exudative phase is the cause of acute inflammation and leads to the release of the pro-inflammatory cytokine, which will then recruit immune cells to the site of the injury [28] . Amongst the recruited immune cells, PMN appears always to be recruited at a higher amount with ventilation. Bobba et al. have shown that

PMNs are mechanosensitive, and they release cytokines in response to barotrauma, increased pressure usually associated with mechanical ventilation that causes alveolar damage [29] . The presence of PMN with mechanical ventilation correlate to our data where there was a high rise of PMN with ventilation in both young and old group.

In addition to causing structural lung damage and recruitment of PMN, Blazquez-Prieto et al. have shown that when mechanically ventilated, mice exhibited a change in the nuclear envelope which coexisted with an increase ɣH2AX, a marker of DNA damage [7] . Moreover, increased accumulation of ɣH2AX has been associated with apoptosis [30] and cellular senescence [31] . Indeed, when cells sense DNA damage, they try to repair it by activating DNA damage response and increasing ɣH2AX, which could lead cells to cell cycle arrest and adapting senescence and a pro-inflammatory phenotype (SASP) [32] as a protective mechanism acutely to prevent further injury. However, activation of DNA damage response can also lead to apoptosis to clear out damaged cells [33] . [38] . Another characteristic of senescent cells is a lack of proliferation due to cell cycle arrest. With increased senescence markers, there is a decrease in Ki67 [39] . This is in line with our findings where mechanical ventilation combined with age and age alone; there was a decrease in proliferation marked by a decrease in Ki67 ( figure 4C ); further signs that mechanical ventilation and age lead to senescence-like phenotype.

The potential role for P38 TGF-β, which is involved in multiple physiological functions and diseases [40] - [43] , has been shown to play an important role in pulmonary senescence [44] , [45] . The sequestered TGF-β on the extracellular matrix is released during a stretch. Due to that release compounded with the injurious state of the environment that already exists, TGF-β becomes activated [46] . The activated TGF-β will cause a cascade of events, including activating the P38-MAPK pathway [47] . Activation of the P38-MAPK pathway plays an essential role in regulating the cell cycle by increasing P53, increasing P21 [48] . Though some parts of this mechanism have been extensible studied, the role of p38 inhibition has not been examined in an aging model of VILI. In the quest to investigate the mechanism of these senescence-like phenotypes in VILI, we used a P38

inhibitor to control the level of P21 observed with mechanical stretch ventilation.

Inhibiting P38 in this study led to a decrease in P21 but not ɣH2AX because cyclic stretch leads to DNA damage that activates the P38-MAPK pathway.

Limitations:

The markers of senescence are very heterogeneous dynamic. Though markers traditionally associated with senescence were upregulated, we cannot be certain this is a chronic or irreversible phenotype as these markers can be found at other cellular states and conditions [49] . The hallmarks of senescence observed in this study could be a protective mechanism started by the cells to prevent further injuries [7] . They may revert when the acute injury is under control if the immune system has not already cleared out those cells. Further studies need to be done to confirm the state of senescence of the cells when stretched, such as survival study with long-term recovery.

Though the study of the P38 mechanism performed on primary human lung cells isolated from the distal part of the alveoli provided some valuable information on how P21 is upregulated, more in vivo studies need to be performed t. We could not obtain cells isolated from young patients; this mechanism needs to be verified using cells from young patients to confirm if the mechanism holds with age.

Conclusion:

In conclusion, we have shown that high-pressure mechanical ventilation and age lead to structural damage at tissue, cellular, and proteins levels. These damages were correlated with the increase of KRT8 positive cells, which are susceptible to DNA damage and could play a vital role in senescence. We have also shown signs of senescence-like phenotypes with mechanical ventilation and with age. Finally, we have provided evidence that p38 may be a therapeutic target in stretch-induced senescence.

These findings provide a better understanding of injuries resulting during mechanical ventilation and VILI and could be used for better-targeted therapies from injuries created by ventilation.

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