Caveolae-deficient Endothelial Cells Show Defects in the Uptake and Transport of Albumin in Vivo

Abstract

The role of endothelial cell caveolae in the uptake and transport of macromolecules from the blood-space to the tissue-space remains controversial. To address this issue directly, we employed caveolin-1 gene knock-out mice that lack caveolin-1 protein expression and caveolae organelles. Here, we show that endothelial cell caveolae are required for the efficient uptake and transport of a known caveolar ligand, i.e. albumin, in vivo. Caveolin-1-null mice were perfused with 5-nm gold-conjugated albumin, and its uptake was followed by transmission electron microscopy. Our results indicate that gold-conjugated albumin is not endocytosed by Cav-1-deficient lung endothelial cells and remains in the blood vessel lumen; in contrast, gold-conjugated albumin was concentrated and internalized by lung endothelial cell caveolae in wild-type mice, as expected. To quantitate this defect in uptake, we next studied the endocytosis of radioiodinated albumin using aortic ring segments from wild-type and Cav-1-null mice. Interestingly, little or no uptake of radioiodinated albumin was observed in the aortic segments from Cav-1-deficient mice, whereas aortic segments from wild-type mice showed robust uptake that was time-and temperature-dependent and competed by unlabeled albumin. We conclude that endothelial cell caveolae are required for the efficient uptake and transport of albumin from the blood to the interstitium.

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

“…First, we demonstrated that LacCer internalization occurred by a clathrin-independent mechanism using multiple pathway-specific inhibitors as well as a DN construct of Eps15 that blocks clathrin-dependent endocytosis (Figure 2), consistent with our previous results in HSFs (Puri et al, 2001). Second, we showed that the fluorescent LacCer analog colocalized with fluorescently labeled albumin (Figure 3), which is reported to be internalized via caveolae in other cell types (Shubert et al, 2001). Furthermore, both the LacCer analog and fluorescent albumin extensively colocalized with DsRed-cav-1 in RFs at an early stage (2 min) of internalization (Figure 4).…”

“…To further characterize the endocytosis of LacCer, we carried out colocalization experiments using labeled albumin, which internalizes via caveolae in some cell types (Shubert et al, 2001). In preliminary studies, we found that internalization (5 min at 37°C) of fluorescent albumin was inhibited by 88.9 Ϯ 6.8% in cells pretreated with nystatin, similar to that seen using LacCer and CtxB (Figure 2A), but was inhibited by only 7.2 Ϯ 1.7% following pretreatment with CPZ.…”

“…However, more recent studies have shown that CtxB can be significantly internalized by clathrin-dependent endocytosis in some cell types (Shogomori and Futerman, 2001;Torgersen et al, 2001). Another potential marker for caveolar uptake is labeled albumin, which is reported to be internalized primarily by caveolae (Schnitzer et al, 1994;Shubert et al, 2001).…”

“…There is currently a great deal of interest in the study of caveolar-mediated endocytosis because this pathway appears to be important for cell entry and intracellular delivery of some bacterial toxins, viruses, and bacteria as well as some growth factors and other circulating proteins (Schnitzer et al, 1994;Gleizes et al, 1996;Anderson, 1998;Lobie et al, 1999;Okamoto et al, 2000;Shin et al, 2000;Pelkmans et al, 2001;Shubert et al, 2001;Marjomaki et al, 2002). Caveolar endocytosis is a clathrin-independent, dynamin-dependent process (Henley et al, 1998;Oh et al, 1998); however, the molecular machinery that differentiates caveolar endocytosis from other forms of clathrin-independent endocytosis is not yet fully defined.…”

Clustering Induces a Lateral Redistribution of α2β1 Integrin from Membrane Rafts to Caveolae and Subsequent Protein Kinase C-dependent Internalization

“…Caveolin-deficient mice have given novel insight into the function of caveolae. Caveolin-1 gene knockout leads to pulmonary defects, vasoconstriction, or dilatation abnormalities and resistance to diet-induced obesity (Drab et al, 2001;Razani et al, 2001;Schubert et al, 2001;Sotgia et al, 2002;Razani et al, 2002). Simian virus 40 (SV40) has been shown to be internalized through caveolae and detailed studies focused on the virus entry mechanism have produced a lot of new information about the biology of caveolae (Pelkmans et al, 2001.…”