Lamellar granules are sphingolipid-enriched organelles, probably intimately related to the tubulo-vesicular elements of the trans-Golgi network, that deliver the precursors of stratum corneum barrier lipids to the extracellular compartment. Caveolins are cholesterol-binding scaffolding proteins that facilitate the assembly of cholesterol- and sphingolipid-enriched membrane domains known as caveolae. Similarities in the composition of lamellar granules and caveolae suggest that caveolins could be involved in lamellar granule assembly, trafficking, and/or function. In order to explore this relationship, we have examined the expression of caveolins in epidermis, keratinocyte cultures, and an isolated lamellar granule fraction using immunolabeling, immunoblotting, and northern blotting. Several antibodies show immunolocalization of caveolin-1 in the basal layer of human epidermis, with a decline in the suprabasal layers and a reemergence of expression at the stratum granulosum/stratum corneum junction. Two of three caveolin-2 antibodies show little basal staining, but strong signal throughout the rest of the epidermis, whereas a third shows a pattern like caveolin-1. An antibody against caveolin-3 shows a strong signal at the stratum granulosum/stratum corneum interface. Caveolins partially colocalize with glucocerebrosidase, an enzyme known to be critical for remodeling of extruded lamellar granule contents, with AE17, a previously described lamellar-granule-associated antibody, and with glucosylceramides, a major lipid component of lamellar granules. Caveolin-1 protein is present in undifferentiated low-calcium-grown keratinocyte cultures, decreases upon induction of differentiation, and then rises to levels above those seen in undifferentiated cultures, consistent with the immunofluorescence findings. Caveolin-1 mRNA expression parallels that of the protein. Caveolin-2 mRNA and protein expression were unchanged over the course of culture differentiation. Keratinocyte caveolin-1 mRNA expression is not induced by an increase in medium calcium level and is markedly reduced by phorbol-ester-mediated protein kinase C induction. Caveolin-1 is enriched in an isolated lamellar granule fraction that is also enriched, as we have previously described, in lysosomal acid lipase and glucocerebrosidase, and localizes to structures consistent with lamellar granules on immunoelectron microscopy. The differentiation-dependent expression of caveolin-1, the colocalization of caveolins with putative lamellar-granule-associated antigens, their enrichment in isolated lamellar granules, and their presence in lamellar-granule-like structures on immunoelectron microscopy, along with their known structural role in the assembly of glycosphingolipid- and cholesterol-enriched domains in other cell types, suggest that caveolins may play a role in lamellar granule assembly, trafficking, and/or function.

The large capillary mass of the newborn lung demands the presence of endothelial cell precursors in lung tissue before development of the pulmonary capillary bed. The objective of this investigation was to isolate and characterize putative endothelial cell precursors from developing human lung. CD34, a cell surface marker for hematopoietic progenitor cells, endothelial precursor cells, and small vessel endothelial cells, was employed as an immunological "handle" for the selection of the desired cells. When CD34+ cells were isolated from midtrimester human fetal lung tissue, then maintained in culture, the isolated cells expressed immunoreactivity for the endothelial cell marker von Willebrand factor and the vascular endothelial growth factor receptors KDR and Flt-1. However, only 5% or fewer of the cells expressed PECAM, an important factor in cell-cell interactions and a marker for endothelial cells associated with vessels. The CD34+ cells endocytosed acetylated low-density lipoprotein and formed capillary-like structures when incubated in a cushion of Matrigel. RT-PCR analysis of mRNA for endothelial cell-related proteins Flt-1, Tie-2, and endothelial nitric oxide synthase demonstrated expression of these mRNAs by the isolated cells for at least 16 cell passages. These observations demonstrate that capillary endothelial cell precursors can be isolated from developing human lung and maintained in cell culture. These cells represent a potentially important tool for investigating the regulation of mechanisms governing development of the air-blood barrier in the human lung.