Cell therapy has shown excellent promise as a treatment for BPD with benefits documented in pre-clinical studies using MSCs [ 34 ], hAECs [ 21 ] and endothelial progenitor cells [ 35 ]. hAECs have been shown previously to improve lung structures in hyperoxia-induced neonatal lung injury [ 22 ]. Could might suggest that hAECs could be useful for BPD, until now there is a lack of sufficient information necessary to inform clinical translation. Particularly, dose effects, timing of cell delivery and paths of administration have not been explored. The long-term associated with hAEC administration had also not been studied. In this particular study, we used a clinically relevant model in order to mimic BPD-like lung injury. We used a mixed approach for disease genesis that includes prenatal inflammation and sustained postnatal hyperoxia. Using this approach we were able to demonstrate a dose-dependent improvement in the lung tissue-to-airspace ratio in hAEC-treated animals. We showed that both intratracheal and intravenous administration of hAECs were equally efficacious when administered after lung inflammation was established. Moreover, we demonstrated that hAEC treatment improved lung architecture and reduced the initial spike in IL-1β and TNFα levels regardless of whether it was administered within 12 hours or 4 days of exposure to hyperoxia. These findings were also associated with a mitigation of pulmonary vessel loss and peripheral pulmonary arterial remodelling that is common associated with BPD. Additionally , early hAEC treatment appeared to be better than later administration.
A previous study reported that three repeated high doses of hAECs (1. 5 × 10 6 per dose) delivered intraperitoneally improved lung structure (tissue-to-airspace ratio and secondary septal crest) within hyperoxia-induced lung injury [ 22 ]. However , we were capable to normalise lung structure using a single dose of a hundred, 000 hAECs administered intratracheally or intravenously, suggesting these routes of administration may be more suitable in this context. This particular observation is important given that most extremely preterm infants showing with severe respiratory distress are likely to be intubated for respiratory system support [ 36 ], and certainly to be on 4 fluid support. Furthermore, this single hAEC dose can resolve lung injury regardless of whether cells were administered in the early stages of pulmonary inflammation or after pulmonary irritation is well underway. Promisingly, these beneficial effects had been sustained into adolescence and early adulthood. Given that the only real clinical trial assessing hAECs for experimental BPD utilizes a single dose of hAEC administered intravenously (1 × 10 6 cells/kg entire body weight), the dose effects seen in this study shows that dose escalation studies in future hAEC clinical tests are certainly warranted. Furthermore, the effect of early hAEC treatment preventing experimental BPD progression in this study shows that early hAEC delivery to infants at high risk associated with BPD should also be considered in future clinical trials.
The anti-inflammatory associated with hAECs in this setting of experimental BPD were furthermore assessed. Here we report that antenatal inflammation plus postnatal hyperoxia increased pulmonary infiltration of CD103 + CD11c + dendritic cells, NK cells and interstitial macrophages. This obtaining supports the observations by Nold et al. [ 37 ] where antenatal inflammation was induced simply by maternal systemic injection of LPS and combined with postnatal hyperoxia. In that study the authors observed increased macrophage and dendritic cell infiltration at PND28, suggesting that will temporal changes in immune cell recruitment occur all through postnatal exposure to hyperoxia. Unfortunately, we were unable to comment on modifications to the alveolar macrophages in our current study since the bronchioalveolar lavage fluid was collected prior to lung tissue selection for flow cytometric analysis. Nevertheless, the study described right here enabled us to chart the ontogeny of lung inflammation. Accordingly, we were able to assess the impact that various timings of administration have on lung injury plus repair, in relation to the ontogeny of inflammation. Levels of IL-1β and TNF-α were elevated in the injured group right after hyperoxia exposure as observed in other neonatal lung injuries studies [ 8 , 37 ]. This was diminished by 7 times in both early and late treatment groups. By 14 days, IL-1β levels were reduced dramatically but continued to be higher in the injured group. Similar to a previous getting by Nold et al. [ 37 ] i was unable to detect TNF-α by 14 days. Given that IL-1β and TNF-α are mainly secreted from activated macrophages and monocytes [ 38 ], and hAECs decreased the particular infiltration of interstitial macrophages, it was perhaps unsurprising that will early administration of hAECs mitigated this effect.
We further noticed that the levels of MCP-1 and MIP-2— the rodent homologue of MCP-1— were significantly increased after 14 times of postnatal hyperoxia exposure. This correlates with clinical research that reported elevated levels of MCP-1 and IL-8 within the lavage fluid of BPD infants [ 39 , 40 ]. MCP-1 is known to recruit monocytes to sites of inflammation [ 41 ], and activated macrophages and monocytes are proven to secrete MIP-2/IL-8, which is chemotactic for polymorphonuclear leukocytes [ 42 ]. Additionally , LIF which was also elevated by day time 14, is a pro-inflammatory cytokine known to potentiate macrophage aggregation and activation in vitro [ 43 ]. In this research, we observed that hAEC administration reduced levels of LIF, MCP-1 and MIP-2 regardless of the timing of hAEC management. Together, these findings indicate that macrophages are likely a vital population through which hAECs mediate neonatal lung repair. Curiously, RANTES, a cytokine released by T cells plus associated with reduced risk of clinical BPD [ 44 ], was significantly elevated in the late hAEC treatment team, suggesting that hAECs may be beneficial even after lung injury is certainly underway. It is unclear why RANTES was not elevated within the early hAEC treatment group. One possible explanation is the fact that we may have missed the critical window for calculating this elevation in RANTES levels, given a prior report that RANTES was significantly higher in preterm babies with reduced risk of BPD on PND7, but not PND14 [ 45 ]. Future studies using fresh models of BPD should include detailed studies into the relationship in between RANTES and severity of lung disease, and should evaluate its potential as a biomarker for disease severity.
While it was encouraging that inflammatory markers were reduced in both early plus late hAEC treatment groups, it is unlikely that decrease of inflammation alone could wholly account for the reparative or protective effects of hAECs. Thus, we sought to determine whether hAECs were able to augment repair by promoting endogenous reparative responses. Previously, both mesenchymal stromal cells (MSCs) and MSC-conditioned media were reported to increase the number of BASCs in the terminal bronchioles of neonatal mice subjected to persistent hyperoxic insult [ 17 ]. We observed a similar searching for in our current study, suggesting that hAECs may switch on the bronchioalveolar junction stem cell niche as part of the pro-reparative actions in a manner akin to that observed in MSCs. However , the activation of AT2 cells, another progenitor cell in the distal lungs, did not change across therapy groups. One possibility could be that the AT2 cells had been activated early on and this progenitor cell population had came back to a quiescent state by PND14. Another possibility which usually remains is that the mechanisms involved in AT2 cell and BASC proliferation and differentiation are different [ 16 , 46 – 48 ] so that while hAECs are able to augment the BASC response they may be unable to initiate the same cascade of events in AT2 cells.
Provided the important physical relationship between the endothelial cells and stem/progenitor cells in lung repair, it was pertinent to investigate the particular relative impact of hAEC treatment on each of these cellular types. When the BASC organoids were established stem/progenitor cellular material from each experimental group using healthy mouse lung endothelial cells as supporting stroma, we did not notice any differences in either the colony size or phenotype across all experimental groups. Our findings suggest that improved activation of the stem/progenitor cell compartment induced by hAEC treatment in vivo is likely due to an effect on the lung endothelial cells rather than the stem/progenitor cells themselves. As mentioned, endothelial cells have a significant influence on alveologenesis in vivo [ 23 ]. Likewise, stromal cells (mouse lung endothelial cells) are necessary to support BASC organoid cultures and can considerably influence BASC proliferation and differentiation in vitro [ 16 ]. By controlling for the contribution of the lung endothelial cells in our organoid cultures, we showed that the development characteristics of the stem/progenitor cell population remained unchanged simply by hAEC treatment, thereby suggesting that the endothelial cells had been the target cells of the beneficial effects of hAECs.
Vascular maldevelopment is another pathological hallmark of BPD where disrupted angiogenesis results in back simplification and vascular muscularisation and ultimately leads to supplementary pulmonary hypertension. Consistent with previous reports [ 49 ], we all observed a reduction in the numbers of small pulmonary vessels (diameter < 50 μ m) and associated back simplification following antenatal inflammation and postnatal hyperoxia. It was mitigated in both early and late hAEC treatment organizations. This is likely attributed to the secretion of pro-angiogenic aspects including epidermal growth factor (EGF), angiogenin, vascular endothelial growth factor (VEGF), platelet-derived growth factor B (PDGFB) and angiogenin as reported previously by us while others [ 9 , 50 ]. Vascular muscularisation begins in the peripheral pulmonary arteries, resulting in thicker and stiffer pulmonary artery wall space with increased pulmonary vascular resistance. Thickening and muscularisation associated with pulmonary vessels can be detected as early as PND14 in our fresh model of BPD. These effects were diminished in both earlier and late hAEC treatment groups. When pulmonary vascular muscularisation progresses, it can cause secondary pulmonary hypertension, which usually happens to more than half of babies with severe BPD in certain studies [ 32 ], thereby placing BPD survivors on continued cardiovascular risk [ 51 ].
It was with these long-term risks in your mind that we assessed secondary pulmonary hypertension at 6 plus 10 weeks, which represents mouse adolescence and earlier adulthood. Here, a decreased PAT/PET ratio detected on echocardiography is indicative of pulmonary hypertension [ 52 ]. All of us report that the experimental model of BPD used in our present study resulted in pulmonary hypertension as evident in reduced PAT/PET ratios in both adolescent and adult mice. As the PAT/PET ratio was improved in both early and past due hAEC treatment groups this was only significant and totally reversed in the early treatment group. Further to this, we all observed evidence of right ventricular hypertrophy in both adolescence plus early adulthood which was ameliorated by both treatment groupings at week 6. However , this effect only persisted in the early hAEC treatment group. A similar study simply by Hansmann et al. [ 34 ] showed the fact that administration of MSC-conditioned media mitigated peripheral pulmonary artery muscularisation, pulmonary hypertension and right ventricular hypertrophy caused by postnatal hyperoxia (FiO 2 = 0. 75). However , it is important to note some essential differences between our current study and that conducted simply by Hansmann et al. In their study, the newborn rodents were exposed to postnatal hyperoxia, in the absence of an antenatal insult, for only 2 weeks before recovering within normoxia for 4 weeks prior to performing echocardiography. This therefore stands to reason that our current experimental model of BPD, which usually combines a longer period of postnatal hyperoxia with antenatal irritation, may more closely replicate the clinical disease.
Long-term decline within lung function has been well documented in adult plus adolescent survivors of BPD, including patients who just presented with mild respiratory distress [ 30 , 53 , 54 ]. We noticed evidence of persistent increased airway responsiveness in the adolescent as well as the adult. Outcomes from the lung function tests indicated that will intra-amniotic LPS and neonatal hyperoxia exposure led to improved lung resistance and decreased compliance. This pathology failed to self-resolve and in fact persisted following a 2-week recovery time period in normoxia. This is in keeping with a previous report simply by Regal et al. [ 55 ] which demonstrated that changes in lung resistance and compliance persisted in mice exposed to 70% hyperoxia for 7 times followed by 14 days of recovery in normoxia. Notably the particular beneficial effects of hAEC treatment in this study were furthermore persistent, particularly in the early hAEC treatment group, in spite of continued chronic hyperoxia exposure for 4 weeks following an intervention.
There are numerous of potential reasons for the apparent longevity of an one dose of hAECs. Firstly, it is possible that the cells had been administered within an ideal therapeutic window. Our study shows that the ideal therapeutic window of BPD is closer to the particular initiation of injury. Conversely, the longevity of helpful effects appears to be compromised when the hAECs are introduced right after injury is established. This is similar to the therapeutic window for corticosteroid therapy for clinical BPD. Systemic postnatal corticosteroids utilized during the early stages of clinical management (≤ 7 days) can prevent the development of BPD [ 56 ], whilst postnatal steroids used later (> 7 days) only provide symptomatic relief of chronic lung illnesses [ 57 ].
Another potential explanation is that hAEC administration may have epigenetic changes. Epigenetic modifications such as DNA methylation and histone modifications (e. g. acetylation, methylation, phosphorylation, ubiquitination) get a new chromatin conformation, resulting in activation or repression of gene expression [ 58 ]. hAEC treatment may result in epigenetic changes in targeted immune cells and/or local lung stem cells. For example , epigenetic changes associated with Wnt/β -catenin signalling may have influence inflammation or lung development. Wnt signalling is known to control leukocyte recruitment and macrophage service [ 59 ]; it is essential to endothelial cell movement plus proliferation in angiogenesis and vascular remodelling following damage [ 60 ]. Furthermore, key regulators of the Wnt whistling pathway are crucial to maintenance of epithelial stem cells [ 61 – 63 ]. These observations suggest that Wnt/β – catenin signalling is a point of intersection for inflammation, angiogenesis, alveologenesis and endogenous stem cellular maintenance, which are key mechanisms shown in hAEC save of BPD lung injury.
In general, we have demonstrated that BPD rodents had secondary pulmonary hypertension and decreased lung perform, which was consistent with the long-term adverse effects observed clinically. Coupled with our observations that neonatal hAEC treatment improved the particular long-term outcomes of experimental BPD in lung functionality and secondary cardiovascular changes, future clinical trials ought to include long-term follow-up.
There have been three clinical trials registered with ClinicalTrials. gov thus far, investigating the potential of MSCs (PNEUMOSTEM® ) as a therapy for BPD, with only one trial completed to date (NCT01297205). This phase 1b trial recruited nine preterm babies born between 23 and 29 weeks, with a delivery weight between 500 and 1250 g, and all babies required ventilation (> 12 breaths/min, > 25% oxygen) but were stable within 24 hrs of enrolment. The other two clinical trials (NCT01828957, NCT02381366) had similar inclusion criteria. Critically, these criteria never fulfil the NIH classifications for BPD, as not every babies requiring ventilation support will develop BPD. With this in mind, the particular phase 1b clinical trial we have commenced aims in order to assess the safety of hAEC administration to babies along with established BPD, with the supportive information that late management of hAECs improved functional outcomes after experimental BPD. However , given that our findings indicate greater benefits from previously interventions, we anticipate that future hAEC trials is going to be designed with this in mind.