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Lung Endothelial Transcytosis

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Transcytosis of macromolecules through lung endothelial cells is the primary route of transport from the vascular compartment into the interstitial space. Endothelial transcytosis is mostly a caveolae‐dependent process that combines receptor‐mediated endocytosis, vesicle trafficking via actin‐cytoskeletal remodeling, and SNARE protein directed vesicle fusion and exocytosis. Herein, we review the current literature on caveolae‐mediated endocytosis, the role of actin cytoskeleton in caveolae stabilization at the plasma membrane, actin remodeling during vesicle trafficking, and exocytosis of caveolar vesicles. Next, we provide a concise summary of experimental methods employed to assess transcytosis. Finally, we review evidence that transcytosis contributes to the pathogenesis of acute lung injury. © 2020 American Physiological Society. Compr Physiol 10:491‐508, 2020.

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Figure 1. Figure 1. Transcytosis of Au‐albumin in mouse lung. (A) Au‐albumin uptake from the capillary lumen is transported via caveolae toward the subendothelial space. Caveolae reside attached to the plasma membrane and as free intracellular vesicles within lung endothelial cells. Vesicular invaginations of the plasma membrane mediate tracer albumin uptake (black arrows) leading to internalization and exocytosis (arrowhead). The majority of albumin in healthy endothelial cells is transported through caveolae, while paracellular transport is restricted by junctional proteins (green arrow). (B) Absence of Au‐albumin transport in caveolin‐1 knockout (Cav‐1−/−) mouse lung endothelium. Genetic deletion of caveolin‐1 resulted in the elimination of caveolae and thereby vesicular uptake and transport of Au‐albumin tracer. Caveolin‐1 deficiency is associated with increased eNOS activity and loss of junctional integrity (green arrow) resulting in paracellular transport of Au‐albumin in Cav‐1−/− lung endothelium but not healthy controls.
Figure 2. Figure 2. Mechanism of receptor‐mediated endocytosis. (1) Serum albumin binds and activates albumin‐binding protein gp60 in caveolae and on the plasma membrane proper. (2) Ligation of gp60 results in its clustering and Gβγ and nitric oxide‐mediated activation of Src kinase. (3) Src subsequently phosphorylates caveolin‐1 (Y14), destabilizing caveolin‐1 oligomers and causing vesicle swelling. Caveolin‐1 Y14 phosphorylation subsequently inhibits further nitric oxide production, thereby limiting local Src activation in caveolae. Finally, phosphorylated dynamin‐2 is recruited from the cytosol to the caveolar neck via the SH3 domain of intersectin‐1. (4) Dynamin‐2 subsequently oligomerizes and initiates GTP‐dependent fission of caveolae from the plasma membrane. These events result in internalization of caveolae.
Figure 3. Figure 3. Actin dynamics during caveolae internalization. Rat lung microvascular endothelial cells demonstrating actin organization at baseline (A) and following exposure to albumin (B). Actin patches (arrowheads) visible following albumin exposure. (C) Caveolae neck proteins, filamin A, and Rho GTPases work in concert to promote actin polymerization and actin filament stability, thus maintaining a linear array of caveolae at the plasma membrane. (D) Receptor activation by macromolecules results in caveolin‐1 phosphorylation and PKC‐mediated phosphorylation of pacsin2 and filamin A, promoting vesicle internalization. Phospho‐caveolin interacts with several effectors of caveolae internalization, including Cdc42, filamin A, and RalA. Cdc42‐GDP binding to phospho‐caveolin prevents its conversion to the GTP bound state, reducing actin polymerization. RalA is recruited to caveolae along with filamin A, resulting in downstream activation of PLD2, production of phosphatidic acid, and subsequent endocytosis.

Figure 1. Transcytosis of Au‐albumin in mouse lung. (A) Au‐albumin uptake from the capillary lumen is transported via caveolae toward the subendothelial space. Caveolae reside attached to the plasma membrane and as free intracellular vesicles within lung endothelial cells. Vesicular invaginations of the plasma membrane mediate tracer albumin uptake (black arrows) leading to internalization and exocytosis (arrowhead). The majority of albumin in healthy endothelial cells is transported through caveolae, while paracellular transport is restricted by junctional proteins (green arrow). (B) Absence of Au‐albumin transport in caveolin‐1 knockout (Cav‐1−/−) mouse lung endothelium. Genetic deletion of caveolin‐1 resulted in the elimination of caveolae and thereby vesicular uptake and transport of Au‐albumin tracer. Caveolin‐1 deficiency is associated with increased eNOS activity and loss of junctional integrity (green arrow) resulting in paracellular transport of Au‐albumin in Cav‐1−/− lung endothelium but not healthy controls.

Figure 2. Mechanism of receptor‐mediated endocytosis. (1) Serum albumin binds and activates albumin‐binding protein gp60 in caveolae and on the plasma membrane proper. (2) Ligation of gp60 results in its clustering and Gβγ and nitric oxide‐mediated activation of Src kinase. (3) Src subsequently phosphorylates caveolin‐1 (Y14), destabilizing caveolin‐1 oligomers and causing vesicle swelling. Caveolin‐1 Y14 phosphorylation subsequently inhibits further nitric oxide production, thereby limiting local Src activation in caveolae. Finally, phosphorylated dynamin‐2 is recruited from the cytosol to the caveolar neck via the SH3 domain of intersectin‐1. (4) Dynamin‐2 subsequently oligomerizes and initiates GTP‐dependent fission of caveolae from the plasma membrane. These events result in internalization of caveolae.

Figure 3. Actin dynamics during caveolae internalization. Rat lung microvascular endothelial cells demonstrating actin organization at baseline (A) and following exposure to albumin (B). Actin patches (arrowheads) visible following albumin exposure. (C) Caveolae neck proteins, filamin A, and Rho GTPases work in concert to promote actin polymerization and actin filament stability, thus maintaining a linear array of caveolae at the plasma membrane. (D) Receptor activation by macromolecules results in caveolin‐1 phosphorylation and PKC‐mediated phosphorylation of pacsin2 and filamin A, promoting vesicle internalization. Phospho‐caveolin interacts with several effectors of caveolae internalization, including Cdc42, filamin A, and RalA. Cdc42‐GDP binding to phospho‐caveolin prevents its conversion to the GTP bound state, reducing actin polymerization. RalA is recruited to caveolae along with filamin A, resulting in downstream activation of PLD2, production of phosphatidic acid, and subsequent endocytosis.
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Teaching Material

Joshua H. Jones and Richard D. Minshall. Lung Endothelial Transcytosis. Compr Physiol 10 : 2020, 491-508.

Didactic Synopsis

Major teaching points

    1. Endothelial transcytosis is an energy-dependent process in which macromolecules in the blood are internalized via vesicles, trafficked across the cell, and ultimately released into the sub-endothelial space.
    2. Caveolae are 40-80 nm omega shaped vesicles that act as transcytotic carriers for macromolecular cargo.
    3. Cargo receptors that localize to caveolae enable specific uptake into endothelial cells.
    4. Receptor-mediated endocytosis entails ligand-receptor interactions that result in Src activation, caveolin-1 phosphorylation, caveolar vesicle swelling and ultimately vesicle release from the plasma membrane.
    5. Caveolae directly associate with the cellular cytoskeleton via actin binding proteins. Association with actin filaments is required for caveolae invagination from the plasma membrane.
    6. Caveolae internalization and trafficking require post-translational modification of caveolin-1 and actin binding proteins.
    7. Internalized caveolar vesicles eventually tether to the abluminal membrane, followed by vesicle fusion via SNARE proteins and exocytosis of cargo into the sub-endothelial space.
    8. Experimental evaluation of caveolar transcytosis often requires the use of multiple techniques, including transmission electron microscopy and transwell studies.
    9. Lipopolysaccharide, hydrogen peroxide, and activated neutrophils increase transcytotic events in lung endothelial cells, and thus transcytosis contributes to the pathogenesis of Acute Lung Injury.

Didactic Legends

The following legends to the figures that appear throughout the article are written to be useful for teaching.

Figure 1. Teaching Points: Caveolae (Figure 1A, black arrows) comprise the overwhelming majority of endocytic vesicles (vesicles that detach and internalize from the plasma membrane). These vesicles often transport macromolecules from the capillary lumen, into and through the endothelial cell, and finally into the sub-endothelial space. Healthy lung capillary endothelial cells typically restrict passage of large molecules between cells (Figure 1A, green arrow); hence the bulk of macromolecule transport occurs via transcellular transport (transcytosis). Caveolae are responsible for the majority of transcytosis in endothelial cells. Caveolae-associated proteins (e.g. caveolins, cavins, etc) are important for caveolae formation, shape, and function. Consequently, loss of these proteins can result in abnormal caveolae membrane tubulation or absence of caveolae altogether (e.g. caveolin-1 deletion). Loss of caveolae and/or their constituent proteins cause endothelial nitric synthase (eNOS) to become dysregulated, producing reactive oxide species that modify junctional proteins. Compromised junctional integrity results in paracellular transport (Figure 1B, green arrow)

Figure 2. Teaching Points: Caveolae-mediated transcytosis facilitates most of the macromolecule transport through endothelial cells. Transcytosis can be divided into three events: endocytosis, intracellular vesicular trafficking, and exocytosis. The first event, endocytosis, is critical for the initiation of transcytosis. Receptors and signaling effectors are concentrated in caveolae, facilitating efficient signal transduction. Binding of macromolecules stimulates NO production and Src activation, which facilitates caveolin-1 phosphorylation and vesicle swelling. Dynamin-2 behaves as "molecular scissors", facilitating budding of caveolae from the plasma membrane. Dynamin-2 is recruited from the cytosol to the caveolar neck region via Intersectin-1, forming oligomers and ultimately cleaving caveolae from the membrane surface into the cytosol of the cell.


Figure 3. Teaching Points: Actin filaments serve as "anchors" to caveolae, providing structural support for these vesicles while they are localized to the plasma membrane. In turn, caveolae-associated proteins contribute to actin assembly and stability, including pacsin2 and filamin A. Pacsin2 binds both EHD2 and F-actin and reportedly increases the stability of F-actin. Filamin A crosslinks actin, binds membrane receptors, and interacts directly with caveolin-1. PKC-mediated phosphorylation of pacsin2 induces its removal from caveolae, thereby weakening the interaction between caveolae and the cytoskeleton. The interaction between Filamin A and caveolin-1 is greater following receptor activation and filamin A phosphorylation promotes trafficking of vesicles from the membrane. Phospho-caveolin interacts with GDP bound Cdc42, restricting GTP loading and actin assembly. Caveolin-1 and filamin A recruit RalA from the cytosol, resulting in downstream phospholipase D2 (PLD2) activation, phosphatidic acid (PA) synthesis, and PA-mediated internalization of caveolae. PA has an important role in endocytosis, as inhibition of PLD2 (and thus PA production) diminishes vesicle internalization.

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How to Cite

Joshua H. Jones, Richard D. Minshall. Lung Endothelial Transcytosis. Compr Physiol 2020, 10: 491-508. doi: 10.1002/cphy.c190012