A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1

Abstract

Antibody conjugates directed against intercellular adhesion molecule(ICAM-1) or platelet-endothelial cell adhesion molecule (PECAM-1) have formed the basis for drug delivery vehicles that are specifically recognized and internalized by endothelial cells. There is increasing evidence that ICAM-1 and PECAM-1 may also play a role in cell scavenger functions and pathogen entry. To define the mechanisms that regulate ICAM-1 and PECAM-1 internalization, we examined the uptake of anti-PECAM-1 and anti-ICAM-1 conjugates by endothelial cells. We found that the conjugates must be multimeric, because monomeric anti-ICAM-1 and anti-PECAM-1 are not internalized. Newly internalized anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with either clathrin or caveolin, and immunoconjugate internalization was not reduced by inhibitors of clathrin-mediated or caveolar endocytosis, suggesting that this is a novel endocytic pathway. Amiloride and protein kinase C (PKC) inhibitors, agents known to inhibit macropinocytosis,reduced the internalization of clustered ICAM-1 and PECAM-1. However,expression of dominant-negative dynamin-2 constructs inhibited uptake of clustered ICAM-1. Binding of anti-ICAM-1 conjugates stimulated the formation of actin stress fibers by human umbilical vein endothelial cells (HUVEC). Latrunculin, radicicol and Y27632 also inhibited internalization of clustered ICAM-1, suggesting that actin rearrangements requiring Src kinase and Rho kinase (ROCK) were required for internalization. Interestingly, these kinases are part of the signal transduction pathways that are activated when circulating leukocytes engage endothelial cell adhesion molecules, suggesting the possibility that CAM-mediated endocytosis is regulated using comparable signaling pathways.

Matrix metalloproteinase 12 silencing: A therapeutic approach to treat pathological lung tissue remodeling?

“…Nanoparticules coupled to antibodies against endothelial cell adhesion molecules ICAM-1 and PECAM-1 have been shown to be internalized [124,125]. This observation could be applied to antibodies or aptamers -siRNA conjugates.…”

Factors modulating the delivery and effect of enzymatic cargo conjugated with antibodies targeted to the pulmonary endothelium

“…These conjugates are indicated as anti-TM 34 /GOX, anti-TM 201 /GOX or IgG/GOX in the text. In an alternative method, GOX and targeting anti-TM were co-immobilized on the surface of 100 nm diameter polystyrene beads, to produce anti-TM/bead/GOX with final size ~200 nm in diameter, as described [26].…”

Expression of inappropriate cadherins by epithelial tumor cells promotes endocytosis and degradation of E-cadherin via competition for p120ctn

“…The pellets were resuspended in 2 Â Laemmli buffer and subjected to SDS-PAGE (Laemmli, 1970). Potassium depletion was carried out as described (Subtil et al, 1994) using a calcium containing buffer (Muro et al, 2003) to preserve cadherin cellcell contacts, for 1.5 h. Texas red-labeled transferrin (Molecular Probes, Eugene, OR, USA) was added 15 min before stopping the experiment.…”