Intro: Degenerative joint disease inflicts over 60% of the aging population. Lack of innate healing by articular cartilage has been attributed to a decreased proliferation of mature chondrocytes, the avascular state of the tissue and a dedifferentiation into a fibroblastic-like cell. This change in phenotype leads to a cell with decreased responsiveness to anabolic cytokines and increased responsiveness to catabolic cytokines corresponding to a shift in collagen production from type II to type I and III. This altered phenotype is a well established characteristic of OA chondrocytes. Despite the conflicting reports on the action of TGF-beta on ECM in vitro, in vivo animal studies suggested that it promotes healing of full and partial thickness articular defects. In addition, heterozygote Smad 3 knockout mice and transgenic mice expressing a dominant negative form of RII develop degenerative joint disease. In mice with a papain induced OA, an inhibition of endogenous TGF-beta by addition of a scavenger soluble form of the type II signaling receptor results in an enhanced proteoglycan loss and impaired cartilage repair. These models provide a convincing link between dysregulation of the TGF-beta signaling cascade and articular cartilage injury. Although clearly implicated in the pathophysiology of cartilage injury, little is known about TGF-beta action in human chondrocytes. Even less has been elucidated regarding TGF-beta receptors on these cells. We have extensively characterized the TGF-beta receptor profile on human chondrocytes and identified several novel receptors and heteromeric receptor complexes. Among them, endoglin was found to be of particular interest as human heterozygotes develop HHT I with abnormal angiogenesis and homozygote lethal mutants demonstrate severely disturbed vasculogenesis. The role of endoglin on cartilage an avascular tissue is intriguing. Thus our objectives were to delineate the role of endoglin in human chondrocytes and to determine if endoglin expression is related to the state of cell differentiation and ECM production.
Methods: Human chondrocytes and human articular cartilage obtained intraoperatively were cultured as serial passage monolayers, in 3-D alginate beads, or as explants and morphology followed and documented with photomicroscopy. Endoglin expression was determined using Western blot and affinity labeling techniques. The effect of endoglin on TGF-beta signaling and type II collagen production was determined using overexpression and morpholinos antisense technology. TGF-beta responsiveness was determined by PAI-1 (Plasminogen Activator Inhibitor-1)-promoter-luciferase reporter activity in OA and normal cells and correlated to endoglin expression and cell phenotype.
Results: We demonstrate the progressive dedifferentiation of immortalized and primary human chondrocytes in monolayer culture and their redifferentiation potential in 3-D alginate bead culture system. Using this system we show that endoglin expression increases as cells dedifferentiate from early and recovered to late passages. In addition we show that the morphology of OA cells is consistent with a dedifferentiated phenotype and that these cells are less responsive to TGF-beta than normal primary cells. Moreover we show that endoglin expression on dedifferentiated chondrocytes is markedly enhanced. We provide evidence that endoglin inhibits TGF-beta signaling in human chondrocytes and more importantly inhibits type II collagen synthesis.
Conclusions: Understanding the factors regulating chondrocyte phenotype is the initial step towards cartilage regeneration. We have demonstrated that endoglin expression correlates with the state of differentiation of the chondrocyte and for the first time illustrated the inhibitory function of endoglin in these cells using morpholinos antisense technology. We have established a critical link between endoglin expression, phenotype, and their responsiveness to TGF-beta and ultimately ECM regulation. Local modulation of endoglin and thus TGF-beta action in chondrocytes may provide a novel mechanism to enhance cartilage repair.