We have carried out a comprehensive molecular mapping of PG-M/versican isoforms V0 -V3 in adult human tissues and have specifically investigated how the expression of these isoforms is regulated in endothelial cells in vitro. A survey of 21 representative tissues highlighted a prevalence of V1 mRNA; demonstrated that the relative frequency of expression was V1 > V2 > V3 > V2; and showed that <15% of the tissues transcribed significant levels of all four isoforms. By employing novel and previously described anti-versican antibodies we verified a ubiquitous versican deposition in normal and tumor-associated vascular structures and disclosed differences in the glycanation profiles of versicans produced in different vascular beds. Resting endothelial cells isolated from different tissue sources transcribed several of the versican isoforms but consistently failed to translate these mRNAs into detectable proteoglycans. However, if stimulated with tumor necrosis factor-␣ or vascular endothelial growth factor, they altered their versican expression by de novo transcribing the V3 isoform and by exhibiting a moderate V1/V2 production. Induced versican synthesis and de novo V3 expression was also observed in endothelial cells elicited to migrate in a wound-healing model in vitro and in angiogenic endothelial cells forming tubule-like structures in Matrigel or fibrin clots. The results suggest that, independent of the degree of vascularization, human adult tissues show a limited expression of versican isoforms V0, V2, and V3 and that endothelial cells may contribute to the deposition of versican in vascular structures, but only following proper stimulation.

EMILIN-1 (Elastin MicrofibrilInterface Located ProteIN), the prototype of the EMILIN family, consists of a cysteine-rich domain (EMI domain) at the N terminus, an extended region with a high potential coiled-coil structure, a short collagenous stalk, and a self-interacting globular gC1q-l domain. EMILIN-1 is an adhesive extracellular matrix constituent associated with elastic fibers, detected also in the proximity of cell surfaces. To localize the cell attachment site(s), monoclonal antibodies (mAbs) against EMILIN-1 or the gC1q-1 domain were used to inhibit cell attachment to EMILIN-1. Thus, one mAb mapping to the gC1q-1 domain caused complete inhibition of cell attachment. EMILIN-1 and gC1q-1 displayed a comparable dose-dependent ability to promote cell adhesion. Adhesion kinetics was similar to that of fibronectin (FN), reaching the maximum level of attachment at 20 min, but in the absence of cations adhesion was negligible. The relative adhesion strength to detach 50% of the cells was similar for EMILIN-1 and gC1q-1 (250 -270 ؋ g) but lower than that for FN (> >500). Cell adhesion to EMILIN-1 or gC1q-1 was completely blocked by a function-blocking ␤ 1 integrin subunit mAb. In contrast, adhesion to the complement C1q component was totally unaffected. Among the various function-blocking mAbs against the ␣ integrin subunits only the anti-␣ 4 fully abrogated cell adhesion to gC1q-1 and up to 70% to EMILIN-1. Furthermore, only K562 cells transfected with the ␣ 4 integrin chain, but not wild type K562, were able to adhere to EMILIN-1 and were specifically inhibited by anti-␣ 4 function-blocking mAb. Finally, cells attached to EMILIN-1 or gC1q-1, compared with cells plated on FN or vitronectin, which appeared well spread out on the substrate with prominent stress fibers and focal contacts, were much smaller with wide ruffles and a different organization status of the actin cytoskeleton along the cell periphery. This pattern was in accord with the ability of EMILIN-1 to promote cell movement.

The primary structure of human Elastin microfibril interface-located protein (EMILIN), an elastic fiber-associated glycoprotein, consists of a globular C1q domain (gC1q) at the C terminus, a short collagenous stalk, a long region with a high potential for forming coiled-coil ␣ helices, and a cysteine-rich N-terminal sequence. It is not known whether the EMILIN gC1q domain is involved in the assembly process and in the supramolecular organization as shown for the similar domain of collagen X. By employing the yeast two-hybrid system the EMILIN gC1q domains interacted with themselves, proving for the first time that this interaction occurs in vivo. The gC1q domain formed oligomers running as trimers in native gels that were less stable than the comparable trimers of the collagen X gC1q domain since they did not withstand heating. The collagenous domain was trypsin-resistant and migrated at a size corresponding to a triple helix under native conditions. In reducing agarose gels, EMILIN also migrated as a trimer, whereas under non-reducing conditions it formed polymers of many millions of daltons. A truncated fragment lacking gC1q and collagenous domains assembled to a much lesser extent, thus deducing that the C-terminal domain(s) are essential for the formation of trimers that finally assemble into large EMILIN multimers.Elastin microfibril interface-located protein (EMILIN) belongs to the C1q/TNF 1 superfamily of proteins (1) and is a considerable component of the elastic fiber system. Elastic fibers confer the properties of resiliency and elastic recoil to connective tissues and are secreted in the extracellular matrix (ECM) of many tissues in various forms. In the elastic ligaments they are deposited as solid branching and unbranching fine and thick rod-like fibers, in the blood vessels as concentric sheets of lamellae, in the elastic cartilages as a three-dimensional meshwork of fine fibrils, and in skin and lung as a combination of these (2). As previously studied in the chick model, the major characteristics of EMILIN, first extracted by means of denaturating and reducing agents, are as follows. It is preferentially extracted from tissues using buffers containing guanidine HCl and reducing agents; it forms a fibrillar network in the ECM of in vitro grown smooth muscle cells and fibroblasts and in the ECM of several tissues including blood vessels, skin, heart, lung, kidney, and cornea; it codistributes with elastin in most sites (3-7); it is a component of elastic fibers being mainly localized at the interface between amorphous elastin and the surrounding microfibrils (8); finally and more important for the functional significance of EMILIN, the process of in vitro elastin deposition was perturbed by the addition of anti-EMILIN antibodies to the culture medium suggesting that this protein plays a leading role during the elastic fiber formation process (8).Recently, the primary structure of the human EMILIN has been elucidated (1). The mature form of this multimodular protein consists of 996 amino acids and d...

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Objective The study aimed determining whether assessment of COMP degradation products could serve as a serological disease course and therapeutic response predictor in arthritis. Methods We generated a panel of monoclonal antibodies against COMP fragments and developed a novel capture ELISA for detecting COMP fragments in patients with osteoarthritis (OA) and rheumatoid arthritis (RA). This test was also used to monitor COMP fragments in surgically induced OA, collagen induced arthritis (CIA), and TNF transgenic animal models. Results Compared with a commercial COMP ELISA kit that detected no significant difference in COMP levels between OA and control groups, a significant increase of the COMP fragments were noted in the serum of OA patients assayed by this newly established ELISA. In addition, serum COMP fragment levels were well correlated with severity in OA patients and the progression of surgically induced OA in murine models. Furthermore, the serum levels of COMP fragments in RA patients, mice with CIA, and TNF transgenic mice were significantly higher when compared with their controls. Interestingly, treatment with TNFα inhibitors and methotrexate led to a significant decrease of serum COMP fragments in RA patients. Additionally, administration of Atsttrin (Tang, et al, Science, 2011 332(6028):478) also resulted in a significant reduction in COMP fragments in arthritis mice models. Conclusion A novel sandwich ELISA is capable of reproducibly measuring serum COMP fragments in both arthritic patients and rodent arthritis models. This test also provides a valuable means to utilize serum COMP fragments for monitoring the effects of interventions in arthritis.