Chris Kiani

Structure and function of aggrecan

ABSTRACTAggrecan is the major proteoglycan in the articular cartilage. This molecule is important in the proper functioning of articular cartilage because it provides a hydrated gel structure (via its interaction with hyaluronan and link protein) that endows the cartilage with load-bearing properties. It is also crucial in chondroskeletal morphogenesis during development. Aggrecan is a multimodular molecule expressed by chondrocytes. Its core protein is composed of three globular domains (G1, G2, and G3) and a large extended region (CS) between G2 and G3 for glycosaminoglycan chain attachment. G1 comprises the amino terminus of the core protein. This domain has the same structural motif as link protein. Functionally, the G1 domain interacts with hyaluronan acid and link protein, forming stable ternary complexes in the extracellular matrix. G2 is homologous to the tandem repeats of G1 and of link protein and is involved in product processing. G3 makes up the carboxyl terminus of the core protein. It enhances glycosaminoglycan modification and product secretion. Aggrecan plays an important role in mediating chondrocyte-chondrocyte and chondrocyte-matrix interactions through its ability to bind hyaluronan.

β1-Integrin–collagen interaction reduces chondrocyte apoptosis

We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death (apoptosis). Moreover, we showed that extracellular matrix (ECM) molecules in the culture media provide vital chondrocyte–matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin β1 suggest that the collagen–chondrocyte interactions are mediated through integrin β1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte–matrix network.

Versican enhances locomotion of astrocytoma cells and reduces cell adhesion through its G1 domain

Versican is a large extracellular proteoglycan and is expressed in a variety of tissues including the central nervous system. A malignant astrocytoma cell line U87 with high motility expressed a higher level of versican than another malignant astrocytoma cell line U343 with lower motility. We observed that the U87 cells were less adherent to tissue culture plates than the U343 cells. To investigate the role of versican in astrocytoma cell migration, we generated recombinant products of a mini-versican construct expressed in COS-7 cells. We found that the mini-versican products enhanced astrocytoma cell migration. Furthermore, enhanced migration was promoted by the G1 domain but not the G3 domain of versican. We introduced culture medium containing products of the mini-versican, the G1, and the G3 constructs separately into the astrocytoma cell lines U87 and U343. The mini-versican and the G1 construct, but not the G3 construct, were shown to reduce astrocytoma cell adhesion. The present data suggest that versican exerts its effect on astrocytoma cell migration and adhesion through the G1 domain.

Promotion of chondrocyte proliferation by versican mediated by G1 domain and EGF-like motifs.

We have previously demonstrated that versican stimulated NIH3T3 fibroblast proliferation. Since versican is expressed in cartilage, we investigated whether versican plays a role in chondrocyte proliferation. We developed a technique to stably express a recombinant versican mini‐gene in chicken chondrocytes, and its effect on chondrocyte proliferation was analyzed by the increase in cell number. The effect of cell adhesion on cell proliferation was tested. Finally, the versican mini‐gene was truncated to assess the role of EGF‐like motifs in cell proliferation. Expression of the recombinant versican mini‐gene stimulated chondrocyte proliferation. Antisense oligonucleotides complementary to versican inhibited chondrocyte proliferation. The G1 domain of versican stimulated chondrocyte proliferation by destabilizing chondrocyte adhesion. Furthermore, deletion of the two EGF‐like motifs from the G3 domain also reduced the function of versican in stimulating cell proliferation. Versican enhances chondrocyte proliferation through a mechanism involving its G1 and G3 domains. This finding may have implications for our understanding of the pathogenesis of various joint diseases. J. Cell. Biochem. 73:445–457, 1999.

Elastin Stabilizes an Infarct and Preserves Ventricular Function

Background—After a myocardial infarction, the injured region becomes fibrotic and the myocardial scar may expand if the ventricular wall lacks elasticity. Cardiac dilatation may precipitate the vicious cycle of progressive heart failure. The present study evaluated the functional benefits of increasing elastin within a myocardial scar using cell based gene therapy. Methods and Results—A myocardial infarction was generated by ligation of the left anterior descending artery in rats. Six days later, 2×106 syngeneic rat endothelial cells transfected with the rat elastin gene (elastin group, n=14) or an empty plasmid (control group, n=14) were transplanted into the infarct scar. Cardiac function, left ventricular (LV) volume, and infarct size were monitored over 3 months by echocardiography, Langendorff measurements, and planimetry. Elastin deposition was evaluated in the cells and in the infarct region by Western blot assay and by histological examination. Recombinant elastin was found in the scar in the elastin group but not the control group during the 3 months after cell transplantation. Histological assessment demonstrated organized elastic fibers within the infarct region. LV volume and infarct size were significantly smaller (P<0.05) in the elastin group than in the control group. Cardiac function evaluated by echocardiography and during Langendorff perfusion was significantly better (P<0.05) in the elastin group than in the control group. Conclusions—Expressing recombinant elastin within the myocardial scar reduced scar expansion and prevented LV enlargement after a myocardial infarction. Altering matrix remodeling after an infarct preserved the LV function for at least 3 months.

Identification of the Motif in Versican G3 Domain That Plays a Dominant-negative Effect on Astrocytoma Cell Proliferation through Inhibiting Versican Secretion and Binding

This study was designed to investigate the mechanisms by which mutant versican constructs play a dominant-negative effect on astrocytoma cell proliferation. Although a mini-versican or a versican G3 construct promoted growth of U87 astrocytoma cells, a mini-versican lacking epidermal growth factor (EGF) motifs (versicanΔEGF) and a G3 mutant (G3ΔEGF) exerted a dominant-negative effect on cell proliferation. G3ΔEGF-transfected cells formed smaller colonies, arrested cell cycle at G1 phase, inhibited expression of cell cycle proteins cdk4 and cyclin D1, and contained multiple nucleoli. In cell surface binding assays, G3 products expressed in COS-7 cells and bacteria bound to U87 cell surface. G3ΔEGF products exhibited decreased binding activity, but higher levels of G3ΔEGF products were able to inhibit the binding of G3 to the cell surface. G3ΔEGF expression inhibited secretion of endogenous versican in astrocytoma cells and also inhibited the secretion of mini-versican in COS-7 cells co-transfected with the mini-versican and G3ΔEGF constructs. The effect seems to depend on the expression efficiency of G3ΔEGF, and it occurred via the carbohydrate recognition domain.

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage‐dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell‐cell interaction, and cell spreading. Treatment with EGF increased cell adhesion‐regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion‐regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle‐associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF‐induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion. J. Cell. Biochem. 77:569–583, 2000.

The roles of matrix molecules in mediating chondrocyte aggregation, attachment, and spreading

The most abundant macromolecules in cartilage are hyaluronan, collagen, aggrecan, and link protein, which are believed to play roles in maintaining a unique three‐dimensional network for a functional joint. This study was designed to investigate the roles of the major extracellular molecules in mediating chondrocyte‐matrix interactions. We employed specific approaches to remove components individually or in combination: hyaluronan was digested with hyaluronidase; type II collagen was digested with collagenase; aggrecan expression was inhibited with antisense and β‐xyloside approaches; and link protein expression was inhibited with antisense oligonucleotides. Digestion of hyaluronan induced chondrocyte attachment to tissue culture plates, collagen‐coated plates, and fibroblast‐like chondrocyte cultures, and induced chondrocyte aggregation. Treated chondrocytes exhibited a fibroblast‐like morphology, and the effects of hyaluronidase were dose‐dependent. Conversely, the effect of collagenase on chondrocyte adhesion and aggregation was far less pronounced. Treatment with Arg‐Gly‐Asp peptide inhibited chondrocyte‐collagen interaction. Chondrocyte attachment was enhanced by antisense oligonucleotides complementary to aggrecan and link protein and by β‐xyloside treatment. Nevertheless, hyaluronan seems to predominate over the other molecules in mediating chondrocyte‐matrix interactions. J. Cell. Biochem. 79:322–333, 2000.