Studies in mice and humans show fibroblasts that overexpress HAS2 develop an aggressive phenotype.
Scientists have implicated an interplay between overexpression of hyaluronan synthase 2 (HAS2) and CD44—the major cell surface receptor for hyaluronan (HA)—in the mechanism that triggers myofibroblasts to develop an aggressive phenotype that ultimately leads to severe lung fibrosis. Studies by a Duke University School of Medicine-led team showed that fibroblasts isolated from engineered mice overexpressing HAS2 exhibited a greater invasive capacity, whereas deletion of HAS2 in mesenchymal cells stopped the invasive phenotype, prevented myofibroblast accumulation, and inhibited the development of lung fibrosis. In addition, blocking CD44 prevented both the invasive phenotype of myofibroblasts and progressive fibrosis in vivo.
Reporting in the Journal of Experimental Medicine, Paul Noble, M.D., and colleagues say their findings were supported by subsequent studies in human patients with idiopathic pulmonary fibrosis (IPF). Fibroblasts isolated from these individuals also exhibited an invasive phenotype that was dependent on HAS2 and CD44, and could be blocked by anti-CD44 antibodies.
The Duke team suggests that understanding the mechanisms leading to an invasive phenotype could lead to new approaches to treating disorders such as IPF that are characterized by severe tissue fibrosis. The work is described in a paper titled “Severe lung fibrosis requires an invasive fibroblast phenotype regulated by hyaluronan and CD44.”
The mechanisms underlying progressive tissue fibrosis have to date remained incompletely understood. What is known, the Duke researchers note, is that human patients with IPF demonstrate an accumulation of extracellular matrix in the gas-exchange regions of the lung, and the disease is defined by the accumulation of myofibroblasts in fibroblastic foci, accompanied by destruction of the alveolar basement membrane. Separate lines of research have also shown that fibroblasts and myofibroblasts from IPF patients demonstrate distinct properties similar to those of metastatic cancer cells, including the ability to invade extracellular matrix.
Accumulation of HA produced by mesenchymal cells is also a characteristic of disorders associated with progressive tissue fibrosis, and the glycosaminoglycan is, coincidentally, produced by a variety of tumor cells. Its production in tumor cells has led to the proposition that HA contributes to tumor metastasis through interactions with its cell surface receptor CD44, which has been shown to play a role in inflammatory cell recruitment, activation, and tumor growth and metastasis.
This collection of findings led the Duke team to hypothesize that in IPF, interactions between HA and CD44 may similarly drive the process by which fibroblasts develop an aggressive phenotype—including the ability to invade basement membrane—similar to that of metastatic cancer cells that overexpress HA.
The researchers first characterized the role of HAS2 expression by myofibroblasts in the pathogenesis of pulmonary fibrosis, using a mouse model that overexpresses human HAS2 in α-smooth muscle actin (ASMA)-expressing myofibroblasts. These transgenic ASMA-HAS2 animals were found to demonstrate increased HA deposition around large airways and blood vessels at baseline, and accumulated increased concentrations of HA in the lung interstitium and alveolar space after challenge with the lung-damaging drug bleomycin. The mortality rate of ASMA-HAS2 transgenic mice was also higher after lung injury. Interestingly, the fibrotic response to bleomycin in the ASAM-HAS2 animals showed evidence of progressive fibrosis and accumulation of myofibroblasts in lung tissue, at the time points at which the fibrotic response in control mice was starting to abate. A fibrodestructive response in the periphery of the lung was observed in the ASMA-HAS2 transgenic mice similar to that observed in lung tissue from patients with IPF.
Conversely, HAS2-knockout mice developed significantly less fibrosis after lung injury, and at sites of lung remodelling there was significantly less accumulation of myofibroblasts, HA, and collagen, compared with both wild-type or ASMA-HAS2 transgenic mice.
The team then moved on to evaluate the ability of fibroblasts to invade Matrigel (a composite matrix with basement membrane constituents). Fibrotic fibroblasts from ASMA-HAS2 transgenic mice spontaneously invaded Matrigel and demonstrated increased levels of HAS2 mRNA in comparison with noninvading fibroblasts from the same animals. Fibroblasts from bleomycin-treated ASMA-HAS2 mice demonstrated an even greater invasive capacity. In contrast, fibroblasts isolated from the HAS2 knockout animals were far less invasive. These cells were also markedly deficient in HA production and demonstrated reduced ability to form characteristic HA surface coats and extrude HA. These findings support a role for HAS2 in the development of an invasive myofibroblast phenotype, the researchers note.
Bringing a potential role for CD44 into the mix, the Duke team found that administering anti-CD44 antibodies to ASMA-HAS2 transgenic mice at the time of lung injury significantly blocked lung collagen accumulation, while fibroblasts isolated from CD44-null mice after bleomycin treatment demonstrated significantly impaired invasive capacity. Similar results were obtained with fibroblasts isolated from ASMA-HAS2 mice that lacked the gene for CD44. Even treating bleomycin-challenged wild-type mice with the CD44 antibodies blunted fibroblast invasion.
Overall, the data strongly suggest that the invasive phenotype requires both HAS2 and CD44, because targeting either by different approaches inhibited tissue invasion, the team remarks. In order to extrapolate their findings in mice to humans, the researchers isolated primary lung fibroblasts from patients with IPF and analyzed their invasive capacity. They found the cells demonstrated a striking increase in invasive capacity compared with fibroblasts from normal lung tissue, and the invasive IPF fibroblasts also demonstrated increased expression of HAS2 mRNA.
Significantly, when HSA2 gene expression was knocked down in primary human cells using an siRNA, the capacity to invade matrix was markedly inhibited. The same effect was observed when IPF fibroblasts were treated with an antibody against human CD44.
The next stage in the research was to investigate the interplay between HAS2 and CD44 by examining gene expression. Invasive fibroblasts from ASMA-HAS2 transgenic mice demonstrated upregulation of HAS2 and CD44, but also significant upregulated expression of matrix metalloproteinases (MMPs) that promote fibroblast migration and invasion, and downregulation of tissue inhibitor metalloproteinase (TIMP) that inhibit inhibit cell invasion of matrix. Similar patterns of gene expression were observed in invasive human IPF fibroblasts.
“These data suggest that HAS2 plays a key role in promoting fibrogenesis in several ways,” the researchers conclude. “First, HAS2 overexpression increased CD44 expression and HA production. Second, HAS2 overexpression promoted activation of a gene program that facilitates emergence of an invasive phenotype. The resultant imbalance in the ratio of MMP/TIMP expression in fibrotic fibroblasts provides a mechanism to promote tissue invasion.”
Further studies will be required to determine whether the development of an invasive fibrotic phenotype requires growth factors such as TGF-β or PDGF, Dr. Noble and colleagues admit. Nevertheless, they add, “The finding that coordinated gene expression with upregulation of matrix-degrading enzymes and downregulation of inhibitors of these enzymes occurs in both mouse and man suggests that this approach could be used to identify therapeutic targets.”
