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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 6, 2000 as doi:10.1096/fj.00-0289fje. |
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Department of Ophthalmology, The Queen’s University of Belfast, Northern Ireland; and
* Department of Geriatrics, Mount Sinai Medical Center, New York, New York USA
2Correspondence: Department of Ophthalmology, The Queen’s University of Belfast, Royal Victoria Hospital, Belfast BT12 6BA, Northern Ireland, U.K. E-mail: a.stitt{at}qub.ac.uk
SPECIFIC AIMS
Advanced glycation end-products (AGEs) are important in the pathogenesis of diabetic retinopathy. The current study has investigated the binding and uptake of these adducts in the specialized retinal microvascular endothelium in an attempt to understand the nature of AGE interaction with the cell surface and how this may be related to vascular cell dysfunction. The interaction of AGEs with receptors localized to caveolin-rich membrane domains in these cells in vitro has also been examined.
PRINCIPAL FINDINGS
1. AGEs bind preferentially to receptors in caveolin-rich membrane
fractions and are internalized within caveolae organelles in retinal
microvascular endothelial cells
Through microsequence analysis of proteins from a wide range of
caveolin-rich membrane domains, one of the receptors for AGEs has been
previously localized to plasma membrane in macrovascular endothelial
caveolae . This implied that AGEs might interact with at least some of
their receptors through a caveolae-dependent mechanism, which could
have major implications for binding, uptake, and signal transduction
properties of ligand receptor interactions. Furthermore, caveolae have
particular significance for the highly specialized, continuous
endothelium of the retinal vasculature since these organelles are
thought to have a unique role in regulating uptake and transcytosis of
proteins across the blood neuronal barriers.
In this aspect of the study, bovine retinal microvascular endothelial
cells were exposed to gold conjugates of AGE-modified albumin (AGE-BSA)
and showed binding to caveolae on the apical plasma membrane as
recognized by their characteristic flask shape and size (
50–100 nm)
(Fig. 1A
). AGEs were also internalized within caveolae-sized
vesicles and sequentially appeared in endosomes, multivesicular
organelles, and lysosome-like structures (Fig. 1B
, C
).
Binding of AGEs could be effectively displaced by coincubation with a
x100 excess of unconjugated AGE-BSA but not native BSA. Incubation
with gold-conjugated native BSA showed binding and internalization
within clathrin-coated pits (Fig. 1D
) indicative of a
receptor-mediated endocytic uptake mechanism. Control RMECs exposed to
unconjugated gold colloid showed no discernible internalization of the
probe.
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Caveolin-rich membrane fractions were separated according to standard TritonX-100 (Tx-100) solubility procedures (caveolin-rich fractions are insoluble in this detergent due to there high cholesterol and glycosphingolipid content). Ligand dot blotting demonstrated significantly higher 125 I-AGE-BSA binding to Tx-100-insoluble (caveolin-enriched) fractions when compared to the Tx-100-soluble fraction (caveolae-depleted) (P < 0.02). A 100-fold excess of 125I-BSA failed to compete with this binding.
2. Components of the AGE receptor complex are enriched within
caveolae on the apical plasma membrane and colocalize with caveolin-1
Western analysis showed the presence of the AGE receptor
components AGE-R1 (50 kDa), AGE-R2 (80 kDa), and AGE-R3 (32
kDa) in whole plasma membrane extracts (Fig. 2A
). Further western analysis demonstrated the purity of the
TX-100-soluble and insoluble protein extracts by showing enrichment of
caveolin-1 and endothelial nitric oxide synthase (both markers for
caveolae) in the caveolin-rich fractions (Fig 2B
). When both
fractions were immunoblotted for AGE receptor complex components, it
was apparent that AGE-R2 and AGE-3 immunoreactivity was significantly
enriched in the caveolae fraction, with virtually none in the depleted
fraction (Fig. 2B
). AGE-R1 immunoreactivity was not
confirmed in either fraction, which may reflect damage to or masking of
the OST-48 epitope during the intensive extraction procedures.
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Ultrastructural immunogold labeling of intact cells also showed AGE receptor complex components within caveolae structures and related endosomal compartments. Controls using preimmune serum and primary antibody omission were negative.
Dual-laser confocal microscopy showed a marked degree of colocalization between caveolin-1 and the components of the AGE-RC from an intracellular and plasma membrane perspective. Upon superimposition of the different colored labeling patterns, there were significant areas of colocalization between caveolin-1 and AGE-R1, R2, and R3.
CONCLUSION AND SIGNIFICANCE
The location of receptors on the plasma membrane of vascular endothelium has an important bearing on the intracellular fate of ligand within the cell. Furthermore, the downstream signaling and subsequent cell responses after receptor–ligand interaction is dependent on the spatial proximity of signal transducing molecules. In the current study it has been shown that AGEs interact with their receptors within caveolae on the RMEC apical plasma membrane, a phenomenon that has implications for the binding, internalization, trafficking, and signal transduction of AGE ligands in the retinal vasculature.
Many gold conjugates, such as insulin and low density lipoproteins are internalized by a receptor-mediated endocytosis mechanism through clathrin-coated pits, processed in the endosomal system with a proportion transcytosed, and released at the basal PM. Physiological/aphysiological modification of albumin changes its receptor recognition and renders it susceptible to scavenger receptor interaction, which may account for the change in binding/internalization characteristics of the molecule. Indeed, a caveolae-mediated internalization and enhanced uptake of glycated albumin by capillary and aortic endothelium of kidney vasculature in diabetic mice has been reported. In the current study, where the albumin was advanced glycated, there was no apparent uptake of the ligand through clathrin-coated pits, suggesting that a very large proportion of AGE molecules enter the retinal microvascular endothelium through caveolae. The failure of BSA to compete with AGE binding to caveolae suggests albumin receptors do not recognize AGE-modified forms of the protein.
The receptor-mediated removal of senescent, highly modified molecules from the circulation is an important function of many cells and tissues, including the vascular endothelium. It is thought that AGE receptor systems have evolved to provide specific removal pathways for these molecules, which are subsequently destroyed by lysosomal degradation. In diabetes, where AGEs occur at markedly elevated levels, the enhanced receptor-mediated removal systems may result in sequestration of high levels of AGEs within intracellular compartments. In the retina, AGEs are known to accumulate in retinal vascular cells during diabetes and in normoglycemic rats infused with preformed AGEs. The receptor-mediated sequestration of highly reactive species within intracellular compartments is likely to have a serious effect on biochemistry and physiology.
Caveolae are composed primarily of glycosphingolipids, cholesterol, and
the integral membrane protein caveolin-1 while they also serve to
compartmentalize many intermediates involved in signal transduction.
The localization of the AGE receptor complex to caveolae, as reported
in the current study, attests to the complexity of the potential
signaling cascades, which could control AGE-mediated cell growth
patterns, the abnormal expression of important genes, and the
initiation of cell death pathways. The protein kinase C substrate
nature of the AGE-R2 component of the AGE receptor complex and its
ability to be phosphorylated after AGE exposure suggest that it may be
intimately linked with receptor-mediated signal transduction. The
spatial organization and molecular interaction of the three-component
AGE receptor complex is likely to occur within the caveolae compartment
(Fig. 3
). AGE-R3 (also known as Mac-2 or galectin-3) does not have a
membrane-spanning domain, and is thought to associate with the other
receptor components on the plasma membrane and play a vital role in
initiating coherence of the complex. It seems reasonable to assume that
caveolar localization of these individual components would appreciably
enhance the formation of the complex, as is the case for several other
caveolae-localized components (Fig. 3)
.
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The rapidly intensifying research into caveolae has brought about a changing view of this membrane domain. The role of these organelles in transcytosis, potocytosis, and vascular leakage along with their enrichment for receptor proteins and signaling molecules makes them important for the study of human disease. With regard to diabetic retinopathy and its pathogenesis, caveolae may play a previously unrecognized role, not the least in excessive vasopermeability culminating in overt breakdown of the blood retinal barrier. The knowledge that AGE receptors are localized in specialized membrane domains and that AGEs are sequestered into cells within caveolae warrants further investigation of these important organelles within the context of diabetic vascular complications.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0289fje To cite this article, use (October 6, 2000) FASEB J. 10.1096/fj.00-0289fje 
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