Bronchopulmonary dysplasia is the clinical condition with the highest mortality and morbidity among the long-term complications of prematurity. It is important because it causes frequent hospitalizations, growth retardation, and neuropsychomotor developmental delays
1,2. In vitro studies have shown that CC16, a natural, potent immunosuppressive and anti-inflammatory agent, inhibits monocyte and polymorphic leukocyte chemotaxis and phagocytosis. It has many anti-inflammatory properties, such as inhibiting the release of substances that initiate inflammation and inhibit neutrophil activation
4.
As gestational age decreases, CC16 levels in serum and bronchoalveolar lavage fluid decrease, and there are ongoing studies on whether this predisposes individuals to the development of BPD12.
Rallis et al.4, examined the effect of perinatal factors on cord blood CC16 levels in a retrospective cohort study of 60 newborns born in 2023 with a gestational age (GA) <34 weeks. In neonates with a GA<32 weeks, cord blood CC16 concentrations were significantly lower in neonates with a GA between 320/7 and 33/6/7 weeks than in neonates with a GA between 32 and 33 weeks (7.6±2.9 ng/mL compared with 5.4±2.5, p=0.039). In this study, there was no significant difference between the groups in CC16 levels measured on day 1. On day 3, CC16 levels were increased but not significantly so in the BPD group, and CC16 levels were generally lower in the control group. Although this difference was not statistically significant, it may be explained by the fact that all of the patients had RDS and received mechanical ventilation support. In the literature, there are studies in which the CC16 level on the 1st postnatal day was found to be low, and there are also studies in which it was found to be high13.
Sarafidis et al.13, studied CC16 levels by taking serum samples in the first 2 hours after birth, at 72 hours, 14th day, and 36 weeks of gestation or at discharge in a study conducted with 35 neonates born at less than 31 weeks of gestation with or without ventilation support and found that CC16 levels were very low in the group without ventilation support compared to the group with ventilation support. Similarly, in the present study, the development of BPD and CC16 levels were significantly greater in the ventilated group with severe RDS.
In this study, the decrease observed in the patient group and the control group on day 14 may be explained by the improvement in RDS findings and the alleviation of inflammation. In addition, the increase in apoptosis induced by caspase-3 on day 14 and the decrease in the number of damaged cells may be the reasons for the low CC16 level. In patients with bronchopulmonary dysplasia, the increase in day 28 values may be explained by the chronic process of damage, alveolar damage caused by mechanical ventilation, and increased permeability of the bronchoalveolar-blood barrier. This hypothesis may explain the relationship with the development of bronchopulmonary dysplasia.
In 2017, Guzmán-Bárcenas et al.14 examined CC16 levels in bronchoalveolar lavage fluid on days 1 and 7 in term and preterm infants intubated for respiratory distress and reported that CC16 levels were lower in the group that developed BPD than in the control group, and the CC16 levels measured on day 7 were lower than those measured on day 1. This result may lead to the hypothesis that 'The lack of a pulmonary protective effect due to low CC16 levels leads to the development of BPD’.
A study published by Jinle Lin et al.15 in 2020 demonstrated the protective effect of rCC16 on lipopolysaccharide-induced pneumocyte apoptosis and the prevention of cellular damage. Rallis et al.4 examined CC16 levels in cord blood and reported that CC16 levels were significantly lower in newborns with BPD than in those without BPD.
CC16 may be an important marker of pulmonary damage and the development of BPD.
TGF-β1, which is involved in cellular proliferation and maturation, is a multifunctional protein7. It regulates cell proliferation, differentiation and matrix production; embryonic development; wound healing; and pulmonary vascularization16. Premature exposure to septation by disrupting normal molecular pathways. Importantly, TGF-β signaling is a key growth factor known to affect injury, mesenchymal homeostasis, and lung development17-20. Although there are high levels of TGF-β in aspirates, the findings of animal studies linking altered TGF-β increases to alveolar development are contradictory. Chronic hyperoxia impairs septation and increased TGF-β results in alveolar disruption21-25.
Kotecha et al.26 measured activated and total TGF-β1 levels in BALF in a study involving 15 patients diagnosed with RDS, 18 patients who developed BPD, and 7 patients who did not develop BDP and who were born at less than the 36th gestational week. TGF-β1 levels were 30±21.1 ng/ml in infants with BPD, 6.5±2.4 ng/ml in patients with RDS, and 2.8±2.3 ng/ml in the control group. In the group with BPD, the increase was greatest on the 4th day. According to the results of Kotecha's study (26), TGF-β1 can be used as an early marker in the development of BPD. In this study, TGF-β1 levels on day 1 were significantly greater in the BPD group than in the control group. This result was consistent with the literature22,27.
Uncontrolled TGF release may predispose patients to lung tissue damage, the suppression of alveolarization, and the development of BPD. Gauldie et al.22 first intranasally transferred the activated TGF-β1 gene into adult rats via adenovirus, and it was histologically determined that progressive interstitial fibrosis developed in the adult rat lung within a few weeks after transfer. Then, the activated TGF-β1 gene was transferred into one-day-old rats treated with the same vector via the same route, and BALF and lung tissue samples were collected for histological examination of 46 samples on days 3, 7, and 28. In the gene transfer group, an increase in TGF-β1 levels was observed in the BALF on all days, starting from day 3. Deterioration of the alveolar sac in the lung tissue on the 3rd day and alveolar septation, thick and hypercellular septae, and abnormal capillary development were observed on the 28th day. Considering the results of the study, it has been shown that uncontrolled release of TGF-β1 causes severe pulmonary damage and leads to the progression of fibrosis. Similar to the results of Gauldie et al.22, the TGF-β1 levels in the patient group were significantly greater in the BD group than in the control group on day 3.
Jonsson et al.25 measured TGF-β1 levels in BALF in a study conducted in premature infants. In serial samples, TGF-β1 levels were increased in patients without BPD. Similarly, in the present study, TGF-β1 levels were increased on the 14th and 28th days in the BPD group. Similar to previous studies, it was thought that high levels of TGF-β1, compared to those in the control group, may predispose individuals to the development of BPD by causing tissue damage.
In a study published in 2019 titled ‘Mesenchymal stromal cells and TGF-β1 in tracheal aspirate of premature infants: Early predictors for bronchopulmonary dysplasia?8 TGF-β1 levels in tracheal aspirate samples obtained in the first week of life were high in patients who developed BPD, and it was concluded that TGF-β1 could be used as an early marker. The relationship between the development of BPD and TGF-β has been established in studies conducted for many years3.
All cells in the human body are in a constant state of construction and destruction. Apoptosis plays an important role in maintaining the natural balance and the continuation of cellular development and differentiation25,27. Caspase molecules are essential proteins that play a key role in the apoptotic process25,28-30.
Mokres et al.31 evaluated angiogenesis, apoptosis via activated caspase-3, VEGF receptor-1 and receptor-2 levels, TGF-β activation, and elastin levels in the lung tissue of neonatal rats receiving oxygen and ventilation support. On the sixth day, the lung tissue was examined. Compared to those in the control group, in the lungs of rats receiving ventilation and oxygen support, a 3-fold increase in alveolar area, a 50% decrease in endothelial surface area and alveolar number, a 5-fold increase in apoptosis, a greater than 50% increase in lung elastin tissue, and a thickening of the alveolar wall structure were observed. In this study, there was no difference in caspase-3, 8, or 9 levels on day 1 in the BPD group. No comparison could be made in the control group since day 1 values were not analyzed. The reason for the lack of difference in the day 1 results may be that the immature lung is exposed to barotrauma and oxytrauma for a shorter period, and therefore, the cellular response is delayed.
The suppression of long noncoding RNA was experimentally shown to be protective against BPD in a study conducted by Tao et al. 32 using caspase 3 inhibition and miRNA 421. In this study, they prevented the development of BPD by inhibiting caspase-3.
Guthmann et al.33 studied TNF-α, caspase-3, caspase-8, and caspase-9 in alveolar type II cells after 24 and 48 hours of O2 exposure in rats. Immediately after birth, the groups given 100% O2 for 24 and 48 hours were compared with the group breathing room air. Type II cells and alveolar macrophages were obtained from all 3 groups by bronchoalveolar lavage and grown in cell culture. TNF-α, caspase-3, and caspase-8 levels were increased in both groups exposed to hyperoxia compared to those in the normoxic group. No difference was observed in caspase-9 activity. After 24 and 48 hours of hyperoxia, caspase-3 and caspase-8 activities decreased in normoxic rats. There was no significant difference in caspase-9 levels. In this study, when the changes in caspase levels in the BPD group over time were analyzed, caspase-3, caspase-8, and caspase-9 levels were close to each other on the first day, whereas increases in the levels of all three caspases were observed on the 3rd day. On the 14th day, a decrease in the expression of all three caspases was observed. On the 28th day, caspase-3 levels continued to decrease, while caspase-8 and caspase-9 levels increased. Collectively, the caspase results revealed that apoptosis increased in the patient group, apoptosis was triggered on the 14th day, and the extrinsic pathway was more effective.
The limitation of this study may be the fewer patients and the fact that newborns born in a single province were not included in the study. Multicenter studies with a higher number of cases will be more decisive on the subject.
In this study, the relationships between bronchopulmonary dysplasia and CC16, TGF-β1, caspase-3, caspase-8, caspase-9, and CC16 levels were found to be elevated at all stages of life, especially from the third day of life, in infants with BPD. Similarly, TGF-β1 levels are elevated at all times in patients with BPD. Considering the results and the available literature, these two parameters may play important roles in the progression to BPD and may be important indicators for early diagnosis of the disease and determination of patient prognosis.
Caspase activity increased on the third day of life in patients with BPD and remained high in the following days, and the extrinsic pathway was more active, especially with caspase-8 activation. Caspase inhibitors, which are among the treatment examples developed by interfering with apoptosis, may be an alternative to medical treatment methods for BPD.
Many studies have been conducted to protect preterm infants from BPD, which is an important cause of mortality and morbidity. With the evaluation of the results obtained and the development of effective treatment methods, it will be possible to predict BPD, which is an important problem in premature infants, in the early period and to take important steps in the treatment and prevention of its complications.