By integrating subtractive phage display with MS we identified JUP and its smaller isoforms as potential biomarkers of atherosclerosis. In fact, we provide three lines of evidence for this conclusion. First, by using a hypothesis-free proteomics approach we identified JUP-81 and JUP-63, a protein encoded by the related cDNA FLJ60424, in secretomes of atherosclerotic plaques. The fact that these proteins are released from the endarterectomised tissue into the culture medium indicates that they might well be released in vivo into the blood stream as well, which is a prerequisite for any potential blood biomarker of atherosclerotic processes. In line with this initial discovery, atherosclerotic but not control secretomes contain proteins with apparent molecular weights of 30, 55, 63 and 81 kD that immunoreact with our discovering phage display antibody scFv 25G5, as well as independent commercial antibodies against JUP. Immunoreactivity of several antibodies including the scFv screening antibody with the JUP isoforms could be reduced by pre-incubation of the antibodies with JUP, indicating that these bands do represent JUP isoforms. Second, compared to healthy controls, median concentrations of JUP-81 were increased by factors higher than 2 and 14 in plasma of patients with stable CAD and ACS, respectively. Third, macrophages of endarterectomized plaques as well as monocytes, macrophages and fibrin platelet clots of coronary thrombi of ACS patients show a strong anti-JUP-immunoreactivity. Lysates of thrombi contained the same 55 kD JUP-antigen which was detected in atherosclerotic secretomes as well as cultivated monocyte-derived macrophages.
JUP is a protein component of desmosomes, which are junction complexes with essential structural functions in tissues that experience mechanical stress 
. Desmosomes connect neighbouring cells through their transmembrane cadherins (desmocollin and desmoglein). The cytoplasmic tails of cadherins are connected through JUP, plakophilin and desmoplakin to the intermediate filaments. The essential physiological role of JUP for regular function of desmosomes in the myocardium is indicated by the findings of premature cardiac death of JUP knockout mice 
and arrhytmogenic right ventricular cardiomyopathy in patients carrying mutations in the JUP gene 
. Furthermore, the importance of JUP for endothelial integrity is indicated by the results of several in vitro
. Recent data by Sun et al.
demonstrated that overexpression of the disintegrin and metalloproteinase 15 (ADAM15), a metalloprotease that was recently identified as a regulator of endothelial permeability, led to dissociation of gamma-catenins (JUP) from VE-cadherin 
. At least in theory, the increased JUP plasma levels in ACS and CAD patients may hence be explained by either myocardial or endothelial damage or both. In agreement herewith, concentrations of JUP-81 in human plasma correlated with other biomarkers of myocardial damage (troponin T, r
0.44) and dysfunction (NT-proBNP, r
0.41) as well as endothelial dysfunction (sVCAM1, r
0.31). In addition, we found that macrophages of endarterectomized plaques and coronary thrombi, as well as macrophages differentiated either from peripheral blood monocytes or THP1 monocytes in vitro
, express JUP isoforms. This finding was unexpected, since macrophages are single cells that do not form continuous and adherent stretches of cells, which are interconnected by desmosomes or junctions. In addition, JUP isoforms could be detected in plasma from patients with PAOD (having peripheral atherosclerotic plaques without suffering a myocardial infarction), but not in a swine model in which non-atherosclerotic myocardial infarction was induced by ligation. Taken together, these findings indicate that JUP isoforms are produced by macrophages in the atherosclerotic plaques, thrombi and differentiated from monocytes in vitro
, rather than being released from the myocardium.
In addition to JUP-81, a 63 kD JUP-homologue encoded by cDNA FLJ60424, was identified by both MS and Western blotting of atherosclerotic secretomes. Both the 81 kD- and 63 kD-proteins were immunoprecipitated from secretomes with scFv 25G5 and were recognized by several commercial antibodies with epitopes in the N-terminus of JUP-81. Furthermore, reactivity of scFv 25G5 with JUP-63 could be competed by pre-incubation of the antibody with recombinant JUP-81. This suggests that scFv 25G5 recognizes an epitope that is shared by JUP-81 and JUP-63 and is thus likely to be located in the common N-terminal part of both proteins. Currently, nothing is known about the 63 kD JUP isoform, since it has only been described as a coding DNA submitted into the databases of EMBL 
, GenBank 
and DDBJ 
. Cloning and expression of JUP-81 and JUP-63 in bacterial or mammalian cells are needed to gain more insight into the location of the epitope that is recognized by 25G5 and to generate antibodies and standards for sandwich ELISAs to quantify these proteins in patient samples. In addition, high-affinity antibodies that specifically recognize JUP-63 will be essential to characterize its tissue expression pattern and function.
It is not clear whether JUP-55 and JUP-30 are degradation products of JUP-81 and/or JUP-63, or if these two proteins represent additional, as yet unknown (alternative splicing) variants of JUP. Blast database searches with the JUP sequence identified highly identical, smaller sequences that could represent the 30 kD band, but no sequence was found that could correspond to a protein band of 55 kD. Mapping with commercial antibodies revealed that JUP-55 is located in the N-terminal part of JUP and that JUP-30 may be located somewhere between amino acid residues 50 and 545 (). In further studies, JUP isoforms (fragments) will be identified by mass spectrometry to gain information about their sequences and origin.
In conclusion, this is the first report of JUP and its isoforms in the context of atherosclerosis and cardiovascular disease. The increased JUP concentrations in plasma of patients with stable CAD and ACS suggest JUP and its isoforms as potential biomarkers for atherosclerosis. The development of quantitative, high-throughput immunoassays for JUP and for the JUP isoforms, as well as their subsequent application in different clinical studies are needed to validate JUP as a biomarker for diagnosis, prognosis or monitoring of atherosclerosis.