Anatomy and function of the lymphatic vessels in the parietal pleura and their plasticity under inflammation in mice.

Inflammatory pleuritis often causes pleural effusions, which are drained through lymphatic vessels (lymphatics) in the parietal pleura. The distribution of button- and zipper-like endothelial junctions can identify the subtypes of lymphatics, the initial, pre-collecting, and collecting lymphatics. Vascular endothelial growth factor receptor (VEGFR)-3 and its ligands VEGF-C/D are crucial lymphangiogenic factors. Currently, in the pleura covering the chest walls, the anatomy of the lymphatics and connecting networks of blood vessels are incompletely understood. Moreover, their pathological and functional plasticity under inflammation and the effects of VEGFR inhibition are unclear. This study aimed to learn the above-unanswered questions and immunostained mouse chest walls as whole-mount specimens. Confocal microscopic images and their 3-dimensional reconstruction analyzed the vasculatures. Repeated intra-pleural cavity lipopolysaccharide challenge induced pleuritis, which was also treated with VEGFR inhibition. Levels of vascular-related factors were evaluated by quantitative real-time polymerase chain reaction. We observed the initial lymphatics in the intercostals, collecting lymphatics under the ribs, and pre-collecting lymphatics connecting both. Arteries branched into capillaries and gathered into veins from the cranial to the caudal side. Lymphatics and blood vessels were in different layers with an adjacent distribution of the lymphatic layer to the pleural cavity. Inflammatory pleuritis elevated expression levels of VEGF-C/D and angiopoietin-2, induced lymphangiogenesis and blood vessel remodeling, and disorganized the lymphatic structures and subtypes. The disorganized lymphatics showed large sheet-like structures with many branches and holes inside. Such lymphatics were abundant in zipper-like endothelial junctions with some button-like junctions. The blood vessels were tortuous and had various diameters and complex networks. Stratified layers of lymphatics and blood vessels were disorganized, with impaired drainage function. VEGFR inhibition partially maintained their structures and drainage function. These findings demonstrate anatomy and pathological changes of the vasculatures in the parietal pleura and their potential as a novel therapeutic target.

Anatomy and pathology of lymphatic vessels under physiological and inflammatory conditions in the mouse diaphragm.

The lymphatic vessels in the parietal pleura drain fluids. Impaired drainage function and excessive fluid entry in the pleural cavity accumulate effusion. The rat diaphragmatic lymphatics drain fluids from the pleura to the muscle layer. Lymphatic subtypes are characterized by the major distribution of discontinuous button-like endothelial junctions (buttons) in initial lymphatics and continuous zipper-like junctions (zippers) in the collecting lymphatics. Inflammation replaced buttons with zippers in tracheal lymphatics. In the mouse diaphragm, the structural relationship between the lymphatics and blood vessels, the presence of lymphatics in the muscle layer, and the distributions of initial and collecting lymphatics are unclear. Moreover, the endothelial junctional alterations and effects of vascular endothelial growth factor receptor (VEGFR) inhibition under pleural inflammation are unclear. We subjected the whole-mount mouse diaphragms to immunohistochemistry. The lymphatics and blood vessels were distributed in different layers of the pleural membrane. Major lymphatic subtypes were initial lymphatics in the pleura and collecting lymphatics in the muscle layer. Chronic pleural inflammation disorganized the stratified layers of the lymphatics and blood vessels and replaced buttons with zippers in the pleural lymphatics, which impaired drainage function. VEGFR inhibition under inflammation maintained the vascular structures and drainage function. In addition, VEGFR inhibition maintained the lymphatic endothelial junctions and reduced the blood vessel permeability under inflammation. These findings may provide new targets for managing pleural effusions caused by inflammation, such as pleuritis and empyema, which are common pneumonia comorbidities.