Endothelial Transcytosis in Acute Lung Injury: Emerging Mechanisms and Therapeutic Approaches

Abstract

Acute Lung Injury (ALI) is characterized by widespread inflammation which in its severe form, Acute Respiratory Distress Syndrome (ARDS), leads to compromise in respiration causing hypoxemia and death in a substantial number of affected individuals. Loss of endothelial barrier integrity, pneumocyte necrosis, and circulating leukocyte recruitment into the injured lung are recognized mechanisms that contribute to the progression of ALI/ARDS. Additionally, damage to the pulmonary microvasculature by Gram-negative and positive bacteria or viruses (e.g., Escherichia coli, SARS-Cov-2) leads to increased protein and fluid permeability and interstitial edema, further impairing lung function. While most of the vascular leakage is attributed to loss of inter-endothelial junctional integrity, studies in animal models suggest that transendothelial transport of protein through caveolar vesicles, known as transcytosis, occurs in the early phase of ALI/ARDS. Here, we discuss the role of transcytosis in healthy and injured endothelium and highlight recent studies that have contributed to our understanding of the process during ALI/ARDS. We also cover potential approaches that utilize caveolar transport to deliver therapeutics to the lungs which may prevent further injury or improve recovery.

Keywords: PV-1; Src signaling; acute lung injury; caveolae (caveolin-1); endocytosis; endothelial permeability.

FIGURE 1

Caveolar neck protein PV1 regulates vesicle shape and internalization. Gold-labeled (Au) albumin was infused into control (PV1^fl/fl) and PV1 deficient (PV1^iΔEC) mice for 15 min. Lungs were subsequently harvested, minced, fixed, and further prepared for electron microscopy. (A) Endothelial cells contain an abundant number of caveolar vesicles (black arrowheads). Most vesicles are present on the luminal and abluminal membranes. Here, caveolae are observed filled with Au-Albumin present in the capillary lumen (Cap.). An internalized vesicle is noted (arrow) inside the cell and will ultimately migrate to the abluminal surface to deposit its content. (B) Loss of PV1 in endothelial cells increases vesicle internalization, noted by fewer caveolae at the cell membrane and increased number of vesicles (arrows). Loss of PV1 also increases caveolae clusters (yellow arrowheads). (C) A survey of lung endothelial cells reveals caveolae that feature diaphragms (blue arrowhead) comprised of PV1 oligomers and caveolae without diaphragms (black arrowhead). (D) Loss of PV1 results in loss of diaphragms and change in caveolar shape (red arrowheads). Observable changes include increased vesicle neck width, increased vesicle depth, and increased vesicle filling. Scale bar, 0.5 μm.

FIGURE 2

LPS-mediated inflammatory signaling increases transcytosis in endothelial cells. (1) LPS present in the lumen binds to TLR4 in caveolar microdomains. (2) TLR4 signaling activates MyD88 and Src kinase, resulting in caveolin-1 phosphorylation at tyrosine-14 and reduction in caveolin-1 oligomer stability. The vesicle is internalized via dynamin-mediated fission and TLR4 is subsequently degraded or recycled. (3) NF-κB is activated downstream of MyD88, resulting in translocation of NF-κB into the nucleus and p65/RelA-mediated upregulation of caveolin-1 expression. LPS also reduces PV1 expression, however, the mechanism through which this occurs is unclear. (4) Upregulation of caveolin-1 increases formation of vesicles, while less caveolar diaphragms are generated due to lack of PV1, which is essential for diaphragm formation. (5) Caveolae traffic to the cell membrane and invaginate, exhibiting wider neck and bulb diameter in the absence of diaphragms resulting in greater filling of the vesicle. Vesicles subsequently undergo fission, contributing to increased protein permeability of the endothelial barrier resulting in protein rich edema formation and ALI.