ICAM-1 recycling in endothelial cells: a novel pathway for sustained intracellular delivery and prolonged effects of drugs

Abstract

IntroductionIntercellular adhesion molecule-1 (ICAM-1) is an Ig family transmembrane glycoprotein constitutively exposed on the luminal surface of endothelial cells (ECs). 1-3 ICAM-1 represents an attractive target for drug delivery to ECs, since it is up-regulated and functionally involved in vascular inflammation, oxidant stress, and thrombosis. [4][5][6][7] Antibodies to ICAM-1 are being explored as therapeutics and affinity carriers in cell cultures, animal models, and early clinical studies. [8][9][10][11][12][13] In addition to acting as delivery vehicles, antibody blocking of ICAM-1 suppresses leukocyte adhesion to ECs, providing an anti-inflammatory benefit to the effects of drugs. 14,15 Targeting nanocarriers (NCs) to EC determinants decreases the clearance of drugs from the bloodstream and permits site-specific delivery, increasing therapeutic capacity and reducing side effects (Muzykantov 16 ). Internalization and proper subcellular processing of drugs also are critical in the rational design of drug delivery systems (Muro et al 17 ). For instance, intracellular targeting of antioxidants in ECs may help to detoxify oxidants produced within the cell body and decrease elimination of drugs that otherwise shed from the EC surface. [18][19][20][21][22] ICAM-1 targeting offers the possibility of intracellular drug delivery, given that ECs internalize multimeric anti-ICAM conjugates and anti-ICAM/NCs via a unique, newly defined pathway, cell adhesion molecule (CAM)-mediated endocytosis. 23 ICAM-1 engagement by multimeric ligands triggers signaling via protein kinase C, Src family kinases, and Rho-dependent kinase, also involves dynamin and amiloride-sensitive Na ϩ /H ϩ exchangers, leading to rapid reorganization of the actin cytoskeleton and formation of endocytic compartments. 23 Intracellular delivery of an antioxidant enzyme, catalase, to ECs via CAM-mediated endocytosis may help contain vascular oxidant stress by minimizing catalase shedding from the cell surface. Endocytosed catalase does not escape endosomes but retains enzymatic activity within these organelles. Due to the high diffusion rate of H 2 O 2 across cellular membranes, catalase within endocytic vesicles intercepts intracellular oxidants and provides antioxidant protection. Decay of this antioxidant effect occurs approximately 2-3 hours after internalization, due to pH-dependent proteolytic degradation following delivery to lysosomes. 13 This time frame is sufficient to protect lung vasculature from acute oxidant stress in animal models. 11,24,25 By analogy with classical endocytic receptors, internalized ICAM-1 could follow nanoparticle trafficking to lysosomes or dissociate from anti-ICAM in a sorting prelysosomal compartment. The latter scenario, including recycling of internalized ICAM-1 molecules to the cell surface, could provide a pathway for recurrent drug delivery permitting sustained effects. However, the fate of ICAM-1 molecules involved in endocytosis is not known. The only pathway for endothelial ICAM-1 turnover identified to da...

PLGA Nanoparticle−Peptide Conjugate Effectively Targets Intercellular Cell-Adhesion Molecule-1

“…Then, the co-localization of nanoparticles within Lysotracker-labeled cellular compartments was determined. The results were confirmed by labeling lysosomes with Texas Red dextran (10,000 kDa) following a similar protocol (7,29).…”

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

“…We tested distribution of the radiolabeled TM antibodies and GOX conjugates one hour after IV injection. Previous studies of immunotargeting of radiolabeled conjugates directed to highly accessible endothelial determinants such as TM, angiotensin-converting enzyme (ACE), PECAM-1, intercellular adhesion molecule 1 (ICAM-1) and GP90 showed that their maximum uptake in the lungs occurs within 15-30 min, followed by a short 15-30 min stable period and subsequent reduction of the pulmonary level observed at times more than one hour after IV injection [22,25,27,[46][47][48][49][50][51][52]. Therefore, the kinetics of lung edema development showing a maximum 4 hour after injection of a lower dose of the conjugate selected to avoid overt early lethality (Fig.4C) is not due to delayed accumulation of GOX in the lungs.…”

Control of intracellular trafficking of ICAM-1-targeted nanocarriers by endothelial Na+/H+exchanger proteins

“…Cells were seeded onto 12-mm 2 gelatin-coated coverslips in 24-well plates and were treated overnight with TNF-␣ before experiments. TNF-␣ treatment upregulates ICAM-1 expression; therefore, it enhances anti-ICAM/NC binding to HUVEC, yet it does not affect their internalization kinetics or trafficking (37,41).…”

“…Internalization of anti-CAM/NC occurs via a common pathway, CAM endocytosis, in a variety of CAM-positive cell types (22,36,37,40,41). Human umbilical vein EC (HUVEC) and an endothelial-like cell line, EAhy926 (13), were selected to study the uptake and trafficking of anti-ICAM/NC since they provided insights that have subsequently correlated with in vivo models for endothelial targeting of anti-ICAM/NC (37). HUVEC (Clonetics, San Diego, CA) and EAhy926 were cultured at 37°C, 5% CO 2, and 95% relative humidity in supplemented medium 199 or DMEM (GIBCO-BRL, Grand Island, NY), respectively (36).…”

“…Reporter cargoes and experimental therapeutics, such as catalase, conjugated with anti-ICAM or loaded onto ICAM-targeted nanocarriers (NC), 1) bind to and protect EC against oxidants; 2) accumulate in the pulmonary endothelium after systemic injection; and 3) undergo enhanced targeting to inflamed endothelium (2,(35)(36)(37).…”