In wound healing epidermal-dermal interactions are known to regulate keratinocyte proliferation and differentiation. To find out how fibroblasts respond to epithelial stimuli, we characterized fibroblasts in monolayer co-culture with keratinocytes. On co-culture numerous extracellular matrix- and smooth muscle cell-associated gene transcripts were up-regulated in fibroblasts, suggesting a differentiation into myofibroblasts. Increased alpha-smooth muscle actin (alpha-SMA) protein expression in co-cultured fibroblasts started at approximately day 4, was serum-independent, but required endogenous transforming growth factor (TGF)-beta. In co-cultures, TGF-beta neutralizing monoclonal antibody strongly reduced alpha-SMA induction. Endogenous TGF-beta production and activation were increased at 24 and 48 hours, requiring, like alpha-SMA induction, close keratinocyte-fibroblast proximity. As myofibroblast differentiation only started after 4 days, we analyzed the presence of endogenous inhibitors at early time points. Blocking keratinocyte-derived interleukin (IL)-1 using IL-1 receptor antagonist, alpha-SMA expression in co-cultures was potentiated. Conversely, adding exogenous IL-1alpha completely suppressed endogenous alpha-SMA induction. In co-cultured fibroblasts strong nuclear factor-kappaB binding activity was observed from 2 hours, decreasing at 2 and 4 days, suggesting an early, IL-1-mediated inhibition of TGF-beta signaling in co-cultured fibroblasts. This biphasic differentiation event is regulated by the balance of endogenous TGF-beta and IL-1 activity and is reminiscent of myofibroblast differentiation at early and later stages of wound healing.

The individual susceptibility to pulmonary fibrosis (PF) remains a mystery, suggesting a role for genetic predisposition. The pathogenesis of PF involves a multitude of factors mediating crosstalk between various tissue components. Some factors, such as transforming growth factor beta, are recognized as key elements in the process, whereas the role of others, such as connective tissue growth factor (CTGF), is unclear. We investigated if Balb/c mice, known to be fibrosis resistant partly due to lack of CTGF induction upon stimulation with bleomycin, can be transformed into fibrosis-sensitive individuals by generation of a CTGF-rich environment using transient overexpression of CTGF by adenoviral gene transfer (AdCTGF). We show that AdCTGF is not sufficient to cause fibrosis, and that bleomycin challenge results in inflammation, but not fibrosis, in Balb/c mouse lungs. This inflammation is accompanied by lower levels of CTGF and tissue inhibitor of metalloproteinase-1 gene expression compared with fibrosis-prone C57BL/6 mice. However, concomitant administration of AdCTGF and bleomycin leads to a persistent upregulation of tissue inhibitor of metalloproteinase-1 gene and a significant fibrotic response in Balb/c similar to that in C57BL/6 mice. We propose that CTGF is an important mediator in the pathogenesis of PF in that it provides a local microenvironment in the lung that causes individual susceptibility. CTGF should be considered as a novel drug target and as a potential marker for identifying individuals at risk.

The incidence of finding evidence of both emphysema and pulmonary fibrosis in the same patient has received increased attention. Several investigators have found on biopsy the presence of emphysema of the upper zones and diffuse parenchymal disease with fibrosis of the lower zones of the lung, especially associated with current or previous heavy smokers. Believed previously to be two different disease mechanisms, there are now data to implicate some common pathways of cell and molecular activation leading to the different morphologic and physiologic outcomes. According to a current view, emphysema may originate from a protease/antiprotease imbalance, whereas a role for antiproteases has been proposed in the modulation of fibrosis. Overexpression of transforming growth factor ␤ (TGF-␤) in experimental rodent models leads to progressive pulmonary fibrosis, accompanied with marked upregulation of protease inhibitors, such as tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator inhibitor-1 (PAI-1) genes, along with excessive matrix accumulation. It may be that a "matrix degrading" pulmonary microenvironment, one in which metalloproteinase activities prevail, favors the development of emphysema, whereas a "matrix nondegrading" microenvironment, with enhanced presence of TIMPs, would lead to matrix accumulation and fibrosis. Surprisingly, although Smad3 null mice, deficient in TGF-␤ signal transmission, are resistant to bleomycin-and TGF-␤-mediated fibrosis, they develop spontaneous age-related airspace enlargement, consistent with emphysema, with a lack of ability to repair tissue damage appropriately. A common element is tissue damage and repair, with TGF-␤ and the Smad signaling pathway playing prominent molecular roles. Both changes can be followed in experimental models with noninvasive imaging and physiologic measurements.

Platelets are a major source of transforming growth factor-beta1 (TGF-beta1) in the circulation as they release latent growth factor in response to activation. We report here that human platelets, when stimulated with thrombin, activated a significant proportion of the latent TGF-beta released. Latent TGF-beta activation was independent of cytokine release, since activation was delayed compared to platelet degranulation. Activation occured in releasates and did not require the continuous presence of platelets. Classical mechanisms of latent TGF-beta activation were not involved, since activation was not affected by gene deletion and/or inhibitors of the known TGF-beta activators/co-factors, thrombospondin-1 (TSP-1), mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR), plasminogen/plasmin, or several other candidate proteases. In contrast, latent TGF-beta activation was significantly inhibited by the furin inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone and L-hexaarginine. We show that platelets contain a furin-like enzyme which is released upon platelet activation. We conclude that, following activation, platelets release and activate latent TGF-beta1 via mechanisms involving the release and activity of a furin-like proprotein convertase. This novel mechanism of latent TGF-beta activation might represent an important mediator and therapeutic target of platelet TGF-beta1 functions, for example, in early wound repair, fibrosis, or arteriosclerosis.

Airway remodeling, including subepithelial fibrosis, is a characteristic feature of asthma and likely contributes to the pathogenesis of airway hyperresponsiveness. We examined expression of genes related to airway wall fibrosis in a model of chronic allergen-induced airway dysfunction using laser capture microdissection and quantitative real-time PCR. BALB/c mice were sensitized and subjected to chronic ovalbumin exposure over a 12-wk period, after which they were rested and then harvested 2 and 8 wk after the last exposure. Chronic allergen-exposed mice had significantly increased indices of airway remodeling and airway hyperreactivity at all time points, although no difference in expression of fibrosis-related genes was found when mRNA extracted from whole lung was examined. In contrast, fibrosis-related gene expression was significantly upregulated in mRNA obtained from microdissected bronchial wall at 2 wk after chronic allergen exposure. In addition, when bronchial wall epithelium and smooth muscle were separately microdissected, gene expression of transforming growth factor-beta1 and plasminogen activating inhibitor-1 were significantly upregulated only in the airway epithelium. These data suggest that transforming growth factor-beta1 and other profibrotic mediators produced by airway wall, and specifically, airway epithelium, play an important role in the pathophysiology of airway remodeling.

The currently accepted approach to treatment of idiopathic pulmonary fibrosis (IPF) is based on the assumption that it is a chronic inflammatory disease, and most available antiinflammatory drugs target numerous biological processes involving multiple genes, but are not often beneficial. More novel therapeutic strategies take recent findings about the underlying molecular mechanisms of fibrogenesis into account, and ongoing and as yet unpublished clinical trials in IPF aim to block single gene targets believed to play major roles in disease progression. Characterization of the mechanisms involved in the pathogenesis of IPF has largely come from the use of animal disease models in rodents. Most data suggest, from among the different factors, a prominent role for the transforming growth factor (TGF)-beta1 and platelet-derived growth factor pathways. Inflammation is a critical element of the initiation of fibrosis and data indicate that the Smad pathway is a necessary link to fibrosis through TGF-beta and Smad3 signaling, which introduces matrix regulation as a new target for therapeutic intervention. Regardless, gene targeted therapy has numerous pitfalls that have to be addressed before we see a real therapeutic advance.

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Altered gene expression of important profibrotic mediators in the different cellular lung compartments in patients with UIP likely plays an important role in pathogenesis of the deranged extracellular matrix deposition and subsequent fibrosis in this condition.