Fibrosis is one of the largest groups of diseases for which there is no therapy but is believed to occur because of a persistent tissue repair program. During connective tissue repair, “activated” fibroblasts migrate into the wound area, where they synthesize and remodel newly created extracellular matrix. The specialized type of fibroblast responsible for this action is the α-smooth muscle actin (α-SMA)–expressing myofibroblast. Abnormal persistence of the myofibroblast is a hallmark of fibrotic diseases. Proteins such as transforming growth factor (TGF)β, endothelin-1, angiotensin II (Ang II), connective tissue growth factor (CCN2/CTGF), and platelet-derived growth factor (PDGF) appear to act in a network that contributes to myofibroblast differentiation and persistence. Drugs targeting these proteins are currently under consideration as antifibrotic treatments. This review summarizes recent observations concerning the contribution of TGFβ, endothelin-1, Ang II, CCN2, and PDGF and to fibroblast activation in tissue repair and fibrosis and the potential utility of agents blocking these proteins in affecting the outcome of cardiac fibrosis.

The CCN family is a group of six secreted proteins that specifically associate with the extracellular matrix. Structurally, CCN proteins are modular, containing up to four distinct functional domains. CCN family members are induced by growth factors and cytokines such as TGFβ and endothelin 1 and cellular stress such as hypoxia, and are overexpressed in pathological conditions that affect connective tissues, including scarring, fibrosis and cancer. Although CCN family members were discovered over a decade ago, the precise biological role, mechanism of action and physiological function of these proteins has remained elusive until recently, when several key mechanistic insights into the CCN family emerged. The CCNs have been shown to have key roles as matricellular proteins, serving as adaptor molecules connecting the cell surface and extracellular matrix (ECM). Although they appear not to have specific high-affinity receptors, they signal through integrins and proteoglycans. Furthermore, in addition to having inherent adhesive abilities that modulate focal adhesions and control cell attachment and migration, they execute their functions by modulating the activity of a variety of different growth factors, such as TGFβ. CCN proteins not only regulate crucial biological processes including cell differentiation, proliferation, adhesion, migration, apoptosis, ECM production, chondrogenesis and angiogenesis, but also have more sinister roles promoting conditions such as fibrogenesis.

Fibrotic diseases are a significant global burden for which there are limited treatment options. The effector cells of fibrosis are activated fibroblasts called myofibroblasts, a highly contractile cell type characterized by the appearance of α-smooth muscle actin stress fibers. The underlying mechanism behind myofibroblast differentiation and persistence has been under much investigation and is known to involve a complex signaling network involving transforming growth factor-β, endothelin-1, angiotensin II, CCN2 (connective tissue growth factor), and platelet-derived growth factor. This review addresses the contribution of these signaling molecules to cardiac fibrosis.

The cytokine transforming growth factor beta (TGFbeta) is a major contributor to fibrogenic responses both in vitro and in vivo. TGFbeta possesses many functions; thus, broadly targeting TGFbeta signaling as an anti-fibrotic approach is anticipated to be problematic. Recent experiments, however, have begun to elucidate the signaling pathways through which TGFbeta activates a fibrotic program. This review critically evaluates the evidence supporting TGFbeta as a pro-fibrotic cytokine, with special attention to cardiac fibrosis, and suggests several possible points for selective drug intervention to combat chronic fibrotic disease.

Objective: Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor β (TGFβ) and is a mediator of some profibrotic effects of TGFβ in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin‐induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I α2) in this mouse model and in human pulmonary fibroblasts.Methods: Transgenic mice that were carrying luciferase and β‐galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts.Results: In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti‐CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by ∼25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycin‐treated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK‐1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c‐Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK‐1/2 and JNK signaling.Conclusion: Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation of Col1a2. Blocking strategies revealed the signaling mechanisms involved. These findings show CTGF to be a rational target for therapy in fibrotic diseases such as SSc.

The CCN (cyr61, ctgf, nov) family of modular proteins regulate diverse biological affects including cell adhesion, matrix production, tissue remodelling, proliferation and differentiation. Recent targeted gene disruption studies have demonstrated the CCN family to be developmentally essential for chondrogenesis, osteogenesis and angiogenesis. CCN2 is induced by agents such as angiotensin II, endothelin‐1, glucocorticoids, HGF, TGFβ, and VEGF, and by hypoxia and biomechanical and shear stress. Dysregulated expression of CCN2 has also been widely documented in many fibroproliferative diseases. This mini‐review will focus on CCN2, and the recent progress in understanding CCN2 gene regulation in health and disease. That CCN2 should be considered a novel and informative surrogate clinical bio‐marker for fibroproliferative disease is discussed.