Induction of fibroblast apoptosis by anti-CD44 antibody: implications for the treatment of fibroproliferative lung disease.

Fibroblast migration and proliferation within the alveolar wall and airspace after lung injury can lead to the development of fibrosis. Fibroblast cell surface CD44 is an adhesion receptor for provisional matrix proteins and mediates fibroblast invasion into fibrin matrices. Here we show that incubation of cultured fibroblasts with an anti-CD44 monoclonal antibody induces fibroblast detachment from the substratum and morphological changes compatible with apoptosis. In addition, we show that anti-CD44 monoclonal antibody rapidly induces fibroblast apoptosis within fibrin matrices. The effect of anti-CD44 antibody on induction of fibroblast apoptosis occurred within 8 hours and was dose dependent. Anti-CD44 antibody also induced fibroblast apoptosis in suspension. Furthermore, fibroblasts plated on anti-CD44-antibody-coated surfaces initially attached and spread on the antibody; however, after an 8-hour incubation time, many of the cells developed characteristic morphological features of apoptosis. Collectively, these data indicate that apoptosis did not result solely due to detachment from the substratum. Our results identify a new function for fibroblast cell surface CD44 related to the control of cell viability. We suggest this function may be important in fibroblast population control and could potentially be exploited in designing anti-fibrotic therapies.

Acute lung injury fibroblast migration and invasion of a fibrin matrix is mediated by CD44.

Fibrosis results when myofibroblasts invade the wound fibrin provisional matrix. Extracellular matrix receptors on the cell surface mediate cell adhesion, migration, and invasion. Recent work with transformed cells indicates that these cells use the cell surface matrix receptor CD44 for migration and invasion. In this study, we examine whether lung fibroblasts, isolated from patients dying with acute alveolar fibrosis, use CD44 to invade a fibrin matrix. Consistent with a role for CD44 in mediating fibroblast invasion and subsequent tissue fibrosis, immunohistochemical analysis of lung tissue from patients who died from acute alveolar fibrosis after lung injury reveals CD44-expressing mesenchymal cells throughout newly formed fibrotic tissue. PCR, Western, and immunoprecipitation analysis demonstrate that the 85-kD CD44 isoform is expressed by acute lung injury fibroblasts. Consistent with a role in mediating matrix adhesion and migration ultrastructurally, CD44 was found uniformly over the cell surface and was found densely labeling filopodia and lamellipodia, highly motile structures involved in cell migration. To determine if lung injury fibroblasts use CD44 to invade fibrin, a fibrin gel model of fibrosis was used. By blocking the function of CD44 with monoclonal antibodies, fibroblast invasion into a fibrin matrix was inhibited. To examine the mechanism by which CD44 mediates fibroblast invasion, the role of CD44 in fibroblast migration and adhesion was evaluated. Anti-CD44 antibody blocked fibroblast migration on the provisional matrix proteins fibronectin, fibrinogen, and hyaluronic acid. Additionally, fibroblast CD44 mediated adhesion to the provisional matrix proteins fibronectin, fibrin, and hyaluronic acid, but not to laminin, a component of the basement membrane. These findings support the hypothesis that fibroblast CD44 functions as an adhesion receptor for provisional matrix proteins and is capable of mediating fibroblast migration and invasion of the wound provisional matrix resulting in the formation of fibrotic tissue.

CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix.

Microvascular endothelial cell invasion into the fibrin provisional matrix is an integral component of angiogenesis during wound repair. Cell surface receptors which interact with extracellular matrix proteins participate in cell migration and invasion. Malignant cells use CD44-related chondroitin sulfate proteoglycan (CSPG) as a matrix receptor to mediate migration and invasion. In this study, we examine whether cell surface CSPG can mediate similar events in nonmalignant wound microvascular endothelial cells or whether use of CSPG for migration and invasion is a property largely restricted to malignant cells. After inhibiting CSPG synthesis with p-nitrophenyl beta-d xylopyranoside (beta-d xyloside), wound microvascular endothelial cells were capable of attaching and spreading on the surface of a fibrin gel; however, their ability to invade the fibrin matrix was virtually eliminated. To begin to examine the mechanism by which endothelial cells use CSPG to invade fibrin matrices, cell adhesion and migration on fibrinogen was examined. Endothelial cell adhesion and migration on fibrinogen were inhibited by both beta-d xyloside and after cleavage of chondroitin sulfate from the core protein by chondroitinase ABC. We have determined that wound microvascular endothelial cells express the majority of their proteoglycan as CSPG and that the CSPG core protein is immunologically related to CD44. PCR studies show that these cells express both the "standard" (CD44H) isoform and an isoform containing the variably spliced exon V3. In addition, anti-CD44 antibody blocks endothelial cell migration on fibrinogen. Affinity chromatography studies reveal that partially purified microvascular endothelial cell CSPG binds fibrinogen. These findings suggest that CD44-related CSPG, a molecule implicated in the invasive behavior of tumor cells, is capable of binding fibrinogen/fibrin, thereby mediating endothelial cell migration and invasion into the fibrin provisional matrix during wound repair.