Primary spontaneous pneumothorax (PSP), characterized by the presence of air in the pleural space without apparent lung disease, affects adolescents and young adults with a significant recurrence rate (1, 2). PSP can be potentially life threatening, and the recurrence of PSP is a major long-term complication that warrants surgical intervention. Clinically, low body mass index (BMI), large-sized pneumothorax, and conservative or non-surgical treatment are risk factors for PSP recurrence (3, 4). However, a detailed understanding of the pathologic features associated with PSP and its recurrence remains lacking.

Pathological examination provides direct and powerful evidence of pathological changes and molecular insights into the pathophysiology of diseases. PSP occurs mainly from the pleural porosity of blebs or bullae. Several factors including distal airway anomaly or inflammation, apical ischemia, or abnormal connective tissue are related to the formation of blebs/bullae (5). A recent study has reported that matrix metalloproteinases (MMPs) particularly play a role in contributing to the susceptibility of the formation of bullae and areas of pleural porosity with spontaneous pneumothorax (6). However, the mechanism by which MMPs associate with the pathologic features of PSP and its recurrence remains unknown.

This study aimed to determine the histopathologic features involved in the pathogenesis of PSP and its recurrence through histological analysis of tissue slides obtained from surgery. It also aimed to determine the association of these pathologic findings with MMPs expression for PSP recurrence.

PSP recurrence is a serious complication and that needs to be addressed. Histopathological examination provides unique insights into the pathogenesis and recurrence of PSP. This study has demonstrated an association between the decreased macrophage accumulation and granulation tissue formation and PSP recurrence. Overexpression of MMP-2 and MMP-9 activities appear to be associated these pathologic findings contributing to healing impairment and recurrence of PSP.

The histopathologic findings corresponding to the most common cause of pneumothorax are blebs and bullae as in this study (5). Spontaneous pneumothorax is presumed to result from the pleural porosity and air leakage of the wall of the bullae. Pathologic changes induced by cigarette smoke may lead to localized emphysema with subsequent formation of blebs or bullae (9). However, the prevalence of smoking is relatively low in this study, supporting that the formation of bullae in PSP is smoke related but not crucial (4). By contrast, a pathway-based microarray analysis revealed a breakdown of cell-extracellular matrix (ECM) interactions in the lung contributing to the formation of bullae for PSP (6).

Fibrosis, a pathological process occurring after insult to tissue injury, is characterized by the accumulation of excess ECM components with scant capillaries (10). Repeated injuries, repair, and chronic inflammation are susceptible to fibrosis in which connective tissue replaces normal parenchymal tissue leading to the formation of permanent fibrotic scar (11), which particularly explain its strong correlation with repeated surgical intervention in this study. However, an occult, repetitive lung injury with repair could be hypothesized in patients with PSP because of the high prevalence of fibrosis in this study.

Some pathologic changes are reactive and reflect responses to the injury produced by the pneumothorax. An exuberant hyperplasia of mesothelial cells in serous membranes or pneumocytes lining pulmonary alveoli is frequently related to localized atelectasis or acute lung injury (12). Multinucleated giant cells are commonly associated with blebs and persistent air leak of pleural injury (13). Chronic inflammation associated with pleural porosity contributing to air leakage can be rich in eosinophil, lymphocyte, or plasma cell aggregation as in this study. However, these cell infiltrates are inflammatory characteristics but not specific to PSP.

PSP is associated with diffuse abnormalities within the pleura, a thin tissue covered by a layer of mesothelial cells surrounding the lungs (14). Mesothelial cells possessing multiple intercellular junctions that anchor these cells link the ECM via integrins (15). Biologically, MMPs are enzymes that degrade the ECM and remodel the connective tissue. In this study, MMPs, especially MMP-2 and MMP-9, were highly expressed and strongly correlated with mesothelial cell hyperplasia in patients with PSP. These findings support the idea that an imbalance of cell–ECM interactions and MMP overexpression contribute to pleural porosity, allowing air leakage into the pleural space in PSP (6).

Granulation tissue is characterized by fibrovascular proliferation, which participates in tissue repair mechanism through the production of connective tissue protein matrix and growth of microscopic blood vessels (16). Furthermore, macrophages are the main immune cells in the granulation tissue to phagocytize damaged tissue while healing the wound. MMPs also play an important role in the formation of granulation tissue by acting on the basement membranes of arterioles. In this study, the presence of macrophage accumulation and granulation tissue was strongly associated with MMPs in patients with PSP. Furthermore, the low frequency of these pathologic findings with high risk of recurrence in patients with PSP suggests that MMPs affecting healing capacity play a crucial role in wound healing and subsequent recurrence.

MMPs are proteolytic enzymes that participate in injury and repair mechanisms by regulating immune cell influx, facilitating the migration of fibroblasts and keratinocytes, and remodeling scar tissue (17). However, uncontrolled activity of these proteases can cause healing impairments. Non-healing wounds are characterized by elevated levels and activities of MMPs, especially collagenases and gelatinases, and concomitant abnormally low levels of TIMPs (18, 19). The present study found a significantly higher activity of gelatinases (MMP-2 and MMP-9) associated with impaired granulation tissue formation, which is related to PSP recurrence. This result indicates that a reduction in MMP-2 and MMP-9 activities could promote healing and prevent PSP recurrence.

A major limitation of our study is its retrospective review of tissue section slides, which may show associations among variables but can rarely establish causal relationships. The relatively small sample size for the correlations between MMPs and pathologic findings of PSP may also not be representative of the entire population. Clinically, it is hindered to obtain lung tissue from adolescent healthy volunteers as controls and serum levels of MMPs and TIMPs may not reflect lung tissue levels and/or action. However, a comprehensive histological analysis of lung tissue from PSP patients with a link to the biological role of serum MMPs provides novel insights into the pathogenesis of PSP and its recurrence.

A combination of histopathological examination of the lung tissues and biological activities of MMP analysis highlights the importance of an imbalance in cell-ECM interactions associated with PSP. A low prevalence of macrophage accumulation and granulation tissue is associated with increased risk of PSP recurrence. Overexpression of MMP-2 and MMP-9 activities may particularly play a role in healing impairment, leading to subsequent recurrence. However, further studies are warranted to address cause-effect relationships in studies of impact.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (No. 103-6472C). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

C-YC, T-PC, and T-YH designed and supervised the study. J-RC and S-YY provided samples and expert assistance in pathology analysis. C-JW performed experiments and analyzed the data. All authors contributed to the article and approved the submitted version.

This work was supported by CMRPG2D0331-2 and CMRPG2B0391-2 from the Chang Gung Medical Foundation, Chang Gung University, Taiwan.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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