ABSTRACT
Circulating endothelial progenitor cells (EPCs) play a key role in the maintenance of endothelial homoeostasis and promote vascular repair. They may also be of predictive value for cardiovascular events. Reduced EPC number and functional activity have been associated with several cardiovascular risk factors, but their relationship with hypertension remains unclear. The objective of this study was to investigate if number and function of circulating EPCs are reduced in patients with refractory hypertension (RHT). Circulating EPCs (CD34+ CD133+/CD45+) were isolated from peripheral blood by flow cytometry in 39 RHT and 30 normotensive controls. EPC number was also determined in vitro after 7 days in culture. After age adjustment, EPC concentration was significantly reduced in RHT as compared with controls (mean (95% CI), 33.8 (18.1-49.6) vs 69.1 (50.7-87.5) EPCs per 10(5) peripheral mononuclear cells (MNCs), respectively; P=0.014). After in vitro culture, EPCs were also reduced in patients as compared with controls (mean (95% CI), 142.3 (49.5-235.0) vs 611.0 (480.2-741.8) EPCs per field, respectively, P<0.001). In multiple linear regression analysis, circulating EPCs were significantly reduced by 56.3% in RHT as compared with control (P=0.006), independently of all other known risk factors. Moreover, RHT had a high independent predictive value for lower EPC proliferation. The number of EPCs per field was reduced by 76.7% in RHT with respect to controls (P<0.001). In summary, the number of circulating EPCs after culture is reduced in patients with RHT, which may be related to the increased rate of endothelial dysfunction, atherosclerotic disease and cardiovascular events observed in this population.
Subject(s)
Endothelial Cells/cytology , Hypertension/blood , Stem Cells/cytology , Adult , Cardiovascular Diseases/blood , Case-Control Studies , Cells, Cultured , Chi-Square Distribution , Down-Regulation , Endothelium, Vascular/cytology , Female , Flow Cytometry , Humans , Male , Middle Aged , Regression Analysis , Risk FactorsABSTRACT
We report here that junctional adhesion molecule (JAM) interacts with calcium/calmodulin-dependent serine protein kinase (CASK), a protein related to membrane-associated guanylate kinases. In Caco-2 cells, JAM and CASK were coprecipitated and found to colocalize at intercellular contacts along the lateral surface of the plasma membrane. Association of JAM with CASK requires the PSD95/dlg/ZO-1 (PDZ) domain of CASK and the putative PDZ-binding motif Phe-Leu-Val(COOH) in the cytoplasmic tail of JAM. Temporal dissociation in the junctional localization of the two proteins suggests that the association with CASK is not required for recruiting JAM to intercellular junctions. Compared with mature intercellular contacts, junction assembly was characterized by both enhanced solubility of CASK in Triton X-100 and reduced amounts of Triton-insoluble JAM-CASK complexes. We propose that JAM association with CASK is modulated during junction assembly, when CASK is partially released from its cytoskeletal associations.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases , Cell Adhesion Molecules/metabolism , Nucleoside-Phosphate Kinase/metabolism , Base Sequence , Caco-2 Cells , Cytoplasm/enzymology , DNA Primers , Guanylate Kinases , Humans , Junctional Adhesion Molecules , Protein Binding , Subcellular Fractions/enzymologyABSTRACT
We review here our work on the molecular and functional organization of endothelial cell-to-cell junctions. The first part of the review is dedicated to VE-cadherin, characterized by our group few years ago. This protein is a member of the large family of transmembrane adhesion proteins called cadherins. It is endothelial cell specific and plays a major role in the organization of adherens junctions. Inactivation of VE-cadherin gene or in vivo truncation of its cytoplasmic tail leads to a lethal phenotype due to the lack of correct organization of the vasculature in the embryo. We found that the defect was due to apoptosis of endothelial cells, which became unresponsive to the survival signal induced by vascular endothelial cell growth factor. Our data indicate that VE-cadherin may act as a scaffolding protein able to associate vascular endothelial cell growth factor receptor and to promote its signaling. In the second part of the review we consider another protein more recently discovered by us and called junctional adhesion molecule (JAM). This protein is a small immunoglobulin which is located at tight junctions in the endothelium and in epithelial cells. Evidence is discussed indicating that JAM takes part in the organization of tight junctions and modulates leukocyte extravasation through endothelial intercellular junctions in vitro and in vivo. The general role of tight junctions in endothelial cells is also discussed.
Subject(s)
Endothelium, Vascular/metabolism , Endothelium, Vascular/physiology , Adherens Junctions , Animals , Antigens, CD , Apoptosis , Cadherins/genetics , Cadherins/metabolism , Cadherins/physiology , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Survival , Cytoplasm/metabolism , Embryo, Mammalian/metabolism , Endothelial Growth Factors/metabolism , Epithelial Cells/metabolism , Junctional Adhesion Molecules , Lymphokines/metabolism , Mice , Models, Biological , Phenotype , Tight Junctions/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Junctional adhesion molecule (JAM) is an integral membrane protein that belongs to the immunoglobulin superfamily, localizes at tight junctions, and regulates both paracellular permeability and leukocyte transmigration. To investigate molecular determinants of JAM function, the extracellular domain of murine JAM was produced as a recombinant soluble protein (rsJAM) in insect cells. rsJAM consisted in large part of noncovalent homodimers, as assessed by analytical ultracentrifugation. JAM dimers were also detected at the surface of Chinese hamster ovary cells transfected with murine JAM, as evaluated by cross-linking and immunoprecipitation. Furthermore, fluid-phase rsJAM bound dose-dependently solid-phase rsJAM, and such homophilic binding was inhibited by anti-JAM Fab BV11, but not by Fab BV12. Interestingly, Fab BV11 exclusively bound rsJAM dimers (but not monomers) in solution, whereas Fab BV12 bound both dimers and monomers. Finally, we mapped the BV11 and BV12 epitopes to a largely overlapping sequence in proximity of the extracellular amino terminus of JAM. We hypothesize that rsJAM dimerization induces a BV11-positive conformation which in turn is critical for rsJAM homophilic interactions. Dimerization and homophilic binding may contribute to both adhesive function and junctional organization of JAM.
Subject(s)
Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/metabolism , Animals , Blotting, Western , CHO Cells , Cell Membrane/metabolism , Cricetinae , Cross-Linking Reagents/pharmacology , DNA, Complementary/metabolism , Dimerization , Dose-Response Relationship, Drug , Endothelium/chemistry , Epitope Mapping , Epitopes , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Junctional Adhesion Molecules , Kinetics , Leukocytes/chemistry , Mice , Precipitin Tests , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sodium Chloride/pharmacology , Time Factors , Transfection , UltracentrifugationABSTRACT
Junctional adhesion molecule (JAM) is an integral membrane protein that has been reported to colocalize with the tight junction molecules occludin, ZO-1, and cingulin. However, evidence for the association of JAM with these molecules is missing. Transfection of Chinese hamster ovary cells with JAM (either alone or in combination with occludin) resulted in enhanced junctional localization of both endogenous ZO-1 and cotransfected occludin. Additionally, JAM was coprecipitated with ZO-1 in the detergent-insoluble fraction of Caco-2 epithelial cells. A putative PDZ-binding motif at the cytoplasmic carboxyl terminus of JAM was required for mediating the interaction of JAM with ZO-1, as assessed by in vitro binding and coprecipitation experiments. JAM was also coprecipitated with cingulin, another cytoplasmic component of tight junctions, and this association required the amino-terminal globular head of cingulin. Taken together, these data indicate that JAM is a component of the multiprotein complex of tight junctions, which may facilitate junction assembly.
Subject(s)
Cell Adhesion Molecules/metabolism , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Tight Junctions/metabolism , Animals , Cell Adhesion Molecules/genetics , Cell Line , Humans , Junctional Adhesion Molecules , Macromolecular Substances , Membrane Proteins/genetics , Microfilament Proteins , Microscopy, Fluorescence , Multiprotein Complexes , Occludin , Phosphoproteins/genetics , Precipitin Tests , Protein Binding , Transfection , Zonula Occludens-1 ProteinABSTRACT
Interendothelial tight junctions regulate paracellular permeability and maintain cell polarity. The assembly and remodeling of tight junctions are examined, focusing on the molecular interactions between tight junction components and their functional role in endothelial biology. The molecular structures of two subcellular organelles related to tight junctions, the intercalated disks in cardiomyocytes and the slit diaphragms in glomerular podocytes, are discussed.
