Schematic diagram of the hierarchy and interplay between ET-1, TGF-β and CTGF. CTGF, connective tissue growth factor; ET, endothelin; NF-κB, nuclear factor-κB; TGF, transforming growth factor. Reproduced from [6] by permission of the publisher (Taylor & Francis Ltd, ).

Synergistic effects of TGF-β and ET-1 in fibroblast activation. Signal intensities from Western blots of fibroblasts isolated from co-cultures of HaCaT keratinocytes and fibroblasts after 7 days in Dulbecco's modified Eagle medium/0.5% foetal calf serum. α-Smooth muscle actin (α-SMA) expression increased in co-cultures (see lane 1) as compared with fibroblasts alone (compare dotted line). Addition of an anti-transforming growth factor (TGF)-β antibody or the endothelin (ET)-1 inhibitor PD 156252 resulted in reduced α-SMA expression. Addition of anti-TGF-β and PD 156252 blocked α-SMA expression to almost basal levels [33].

Effects of endothelin include stimulating myofibroblast formation, leading to a concomitant increase in collagen production and fibrosis. Binding of endothelin (ET)-1 to ET-1 receptor subtype A (ET_A) and ET_B has different effects in different cell types. The binding of ET-1 to smooth muscle cell ET_A and ET_B receptors leads to vasoconstriction and mitogenesis, and activation of ET_B receptors on endothelial cells promotes the release of nitric oxide and prostacyclin, and plays a minor role in endothelial dependent vasodilatation. In fibroblasts ET-1 results in the increased production of collagen and leads to fibrosis. Reproduced with permission from Galiè et al. Cardiovascular Research ^© Elsevier 2004 [4].

TGF-β plays a pivotal role in the induction of ET-1. (a) Stellate (top panel) and sinusoidal endothelial (bottom panel) cells were isolated from normal rat livers or those injured by bile duct ligation (BDL). Transforming growth factor (TGF)-β₁ was applied at the indicated concentration in serum-free medium for 24 hours. Endothelin (ET)-1 was measured by radioimmunoassay. *P < 0.05 versus no TGF-β₁ (n = 5). (b) Stellate and endothelial cells were isolated from normal rat livers, and those after BDL and those after soluble TGF-β₁ receptor (STR) antagonist during BDL (BDL + STR). *P < 0.05 versus normal and *P < 0.05 versus BDL (n = 8). Reproduced with permission from Shao et al. Mol Biol Cell 2003 ^© The American Society for Cell Biology [9].

The Tsk-1 model of cutaneous fibrosis exhibits increased MAGP-2, loss of fibulin-5 and increased fibulin-2 matrix. (a) Tissue sections from tight skin (Tsk) mice (panels 1 and 3) and control (panels 2 and 4) mice were immunostained for microfibril-associated glycoprotein (MAGP)-2. MAGP-2 is indicated by arrowheads. MAGP-2 was detected at higher levels in all dermal layers (panel 1 versus panel 2). Hypodermis (H) is shown (panel 3 versus panel 4). PC, panniculus carnosus; D, dermis. (b) Control (left panel) and Tsk (right panel) mice skin sections from 6-week-old mice were stained in parallel for fibulin-5 by immunohistochemistry. Positive staining is indicated by arrows at the muscle-connective tissue (M-CT) interface. Hypodermal connective tissue (HD) and hypodermal muscle (M) are indicated. Original magnification: 400×. (c) Control (panels 1 and 3) and Tsk (panels 2 and 4) mouse skin sections from 6-week-old mice were stained in parallel for fibulin-2 by immunohistochemistry. Positive staining is indicated by arrows (M-CT interface) and in panel 4 around hair follicles (F). (a) Reproduced with permission from Lemaire et al. Arthritis Rheum 2004 ^© John Wiley & Sons/American College of Rheumatology [18]. (b) and (c) Reproduced with permission from [19].