The mechanism by which injected HA exerts CD44-dependent anti-fibrotic effects in murine OA appears related to the finding [33
] that the fibroblast to myofibroblast transition in progressive murine lung fibrosis is also modulated by HA in a CD44-dependent fashion. In a similar way, it has been shown [34
] that HA exhibits a CD44-dependent protection against LPS-induced murine sepsis by binding to TLR4 and blocking excessive inflammatory cytokine production. In this context, we set out to determine whether the cartilage-protective effects of intra-articular HA operate thru a CD44-dependent modulation of the chondrogenic/fibrogenic gene response pathways and/or thru changes in the expression of the critical metalloproteinases, ADAMTS5 and MMP13. Based on the finding that cartilage degradation in the TTR model follows the formation of fibrotic tissue deposits, we hypothesized that it would be associated with the high expression of fibrogenic genes, relative to chondrogenic, and that HA-mediated protection would operate through a reversal to high chondrogenic expression.
To summarize the results (Table ), we found that in the acute phase of the model (Days 0 to 5), and before evidence of any cartilage lesions, there was a generalized increase in expression of both chondrogenic and fibrogenic genes in both tissue compartments (presumably a direct anabolic response to TGFbeta1 injection). After TTR, and in the presence of tissue fibrosis and cartilage erosion, the chondrogenic genes in both tissue compartments had essentially normalized to naïve levels, except for Col2a1 and Col10a1, which remained elevated in the meniscus/synovium. At the same time, the fibrogenic genes in both tissue compartments remained elevated or even increased further, particularly in the cases of Col3a1 and Col5a1. In addition, the expression levels of both Adamts5 and Mmp13 were markedly increased in both tissue compartments in the TTR model. These results are consistent with the idea that cartilage degradation is due to a high expression of fibrogenic genes relative to chondrogenic genes, and also due to a high expression of the metalloproteinases known to be involved in the degradative cascade.
Summary of tissue changes in the TTR model illustratesthe therapeutic effects of HA injection.
Most importantly, in terms of understanding the mechanism of HA-mediated protection, it was found that HA injection resulted in activation (relative to saline injection) of chondrogenic genes in the cartilage/subchondral bone and a diminution of fibrogenic genes in both tissue compartments. Further, HA injection resulted in a normalization of expression of Adamts5 and Mmp13 in both compartments. These results indicate that HA-mediated protection is due to a repression of fibrogenesis and an enhancement of chondrogenesis in the cartilage/subchondral bone along with a lowering of the expression of the relevant metalloproteinases in both compartments.
At the histological level, (Figure ), when TTR samples are examined together with the naïve and acute sections, it is apparent that TGFbeta1 treatment alone results in the appearance of cells with a fibrogenic pericellular/cell-associated matrix, and that during the biomechanical challenge of treadmill running, this cell population is markedly diminished as the surface layer is eroded. However, when HA is injected before treadmill usage (TTR+HA), the fibrogenic pool of cells and associated matrix remains intact. Taken together, the data suggest that HA prevents the catabolic response of chondrocytes which are surrounded by a fibrogenic ECM. In this context, treadmill running alone for 14 days results in only minimal cartilage thinning on the femoral condyles [26
] and, although treadmill alone stimulates Col1a1
(approximately four-fold) and Col2a1
(approximately seven-fold) expression (but not other collagens), this did not cause a detectable accumulation of fibrogenic collagens in the pericellular/cell-associated space (data not shown). It is likely that cartilage erosion in this model is ultimately driven by "catabolic" soluble mediators and one possible source is the mixture of cell types of the remodeled synovial lining. In this context Hematoxylin/Eosin histology of the perimeniscal synovium (Figure ) showed that HA prevented both fibrotic remodeling and neovascularization, in keeping with the observed inhibition of profibrotic gene expression (Table ). An associated effect of HA during treadmill running is inhibition of TGFbeta1-induced chondrogenic gene induction in the synovium/meniscus (Table ), which suggests that the elimination of both fibrotic and chondrogenic cells might promote restoration of the homeostatic adipose-rich stroma of the synovium.
It should be noted that with respect to changes in gene expression, the results were obtained with samples containing more than one type of tissue. In one case it was tibial and femoral cartilage with attached subchondral bone, and in the other it was the lateral and medial menisci with attached synovium. It is, therefore, not possible to discern which cell-types were most affected at the gene expression level by the injection of hyaluronan. However, the immunohistochemical studies did illustrate that in most cases all tissues exhibited protein changes consistent with the alterations in gene expression observed.
A more mechanistic insight into the effects of HA injection seen in this study is suggested by our studies with Cd44
-/- mice (Figure ). We have previously reported that CD44, together with pericellular aggrecan and HA can act as a potent inhibitor of profibrogenic TGFbeta1 signaling in dermal wound healing [17
]. This could at least in part be explained by a shift from an ALK5/SMAD2,3 (fibrogenic) to an ALK1/SMAD1,5,8 (chondrogenic) signaling response. The requirement for CD44 in HA-mediated protection from joint degradation in this OA model indicates that the injected HA interacts with cell surface CD44 and, thereby, mediates a switch from fibrogenesis to chondrogenesis in the cartilage and adipogenesis in the activated stromal cell population (Figure ). This hypothesis is strengthened by the knowledge that chondrogenesis and adipogenesis are known to be promoted by strong BMP/ALK1/Smad1/5/8 signaling [35
A proposed molecular mechanism for joint protection by HA
The current study shows that HA injection, when used soon after a joint insult, can inhibit the cascade of OA-like changes which occur in the cartilage/subchondral bone and meniscus/synovium (the data are summarized in Table ). This protective effect of HA has also been demonstrated convincingly in a range of other animal models of OA [3
]. However, uncovering the central process by which HA operates will clearly require further work to delineate its effects on the fate of proliferated synovial stromal cells and also on soluble mediator production during biomechanical stimulation. For example, HA may prevent the CD44-dependent transition of TGFbeta1-activated stromal cells to a stable myofibroblastic phenotype, much as demonstrated in human fibroblast differentiation [37
]. Indeed, such a process might prevent the appearance of contractile myofibroblasts in human OA cartilage [39
]. In addition, HA may reduce apoptosis [40
], which in turn could reduce fibrotic remodeling by modulating innate immune responses, as shown for alveolar epithelial cells in lung fibrosis [41