Microangiopathy with impaired angiogenesis and excessive accumulation of extracellular matrix may cause severe hypoxia in SSc [53
]. However, what is the exact role played by hypoxia in the pathogenesis of SSc? Is it just the consequence of microangiopathy and fibrosis or does it contribute to the progression of SSc?
DNA microarray studies revealed the first causal links between hypoxia and fibrosis [50
]. Manalo and coworkers [50
] detected a striking number of genes encoding collagens or collagen-modifying enzymes that were induced in pulmonary endothelial cells after 24 hours at 1% oxygen. These genes included collagen (COL)1A2, COL4A1, COL4A2, COL5A1, COL9A1 and COL18A1, as well as procollagen prolyl hydroxylases (P4HA1 and P4HA2), lysyl oxidase (LOX) and lysyl hydroxylases (procollagen lysyl hydroxylase and procollagen lysyl hydroxylase 2). Similar links between hypoxia and fibrosis have also been found in other models and organs, for example kidney [70
], liver [72
] and lung [73
]. Together, these findings indicate that hypoxia could promote extracellular matrix production and that it may actively be involved in the pathogenesis of profibrotic disorders such as SSc.
We could demonstrate that hypoxia induced several extra-cellular matrix proteins, including fibronectin-1, thrombo-spondin-1, proα 2(I) collagen (COL1A2), IGF-binding protein 3 (IGFBP-3) and TGF-β-induced protein (TGF-βi) in cultured dermal fibroblasts [74
]. Type 1 collagens and fibronectins are the major matrix proteins within fibrotic lesions [52
]. Thrombospondin-1 also accumulates in SSc and modulates angiogenesis. TGF-βi is an extracellular matrix protein that is known to be highly expressed in arteriosclerotic plaques [75
] and in zones of thickened extracellular matrix in the bladder [76
]. IGFBP-3 directly induces the synthesis of fibronectin in lung fibroblasts [77
] and protects IGF-1 from degradation. IGF-1 itself stimulates collagen synthesis and downregulates the production of collagenases in fibroblasts [77
Induction and production of these extracellular matrix proteins in response to hypoxia was time dependent and inversely correlated with oxygen levels [74
]. Most of these proteins were significantly upregulated after 24 hours of oxygen deprivation, with a further significant increase after 48 hours. The expression of fibronectin-1, thrombospondin-1, COL1A2 and IGFBP-3 was significantly enhanced at 8% oxygen concentration and increased further with lower oxygen levels, reaching a maximum at 1% oxygen. Of note, severe and chronic hypoxia, as may be found in the skin of SSc patients [54
], was associated with the most marked effects on the induction of extracellular matrix proteins.
These results were confirmed in a mouse model of systemic normobaric hypoxia [74
]. Consistent with the results obtained in vitro
, extracellular matrix proteins were upregulated in mice exposed to hypoxia after 24 hours compared with control mice breathing air with 21% oxygen. Prolonged exposure for 48 hours resulted in further upregulation of fibronectin 1, thrombospondin 1 and COL1A2, whereas TGF-βi and IGFBP3 mRNA levels decreased slightly. Because TGF-β is a major stimulus for the induction of extracellular matrix proteins in SSc [52
], its role for hypoxia-dependent fibrogenesis was also studied in dermal SSc fibroblasts. Neutralizing antibodies against TGF-β completely abrogated the induction of COL1A2, fibronectin 1, thrombospondin 1 and TGF-βi in SSc fibroblasts that were cultured under hypoxic conditions for 48 hours [74
]. These findings suggest that inhibition of TGF-β-dependent pathways may prevent the profibrotic effects of hypoxia.
Consistent with the results on TGF-β signalling, the expression of the fibrogenic cytokine connective tissue growth factor (CTGF) was also shown to be upregulated in SSc in response to hypoxia [79
]. CTGF is a critical mediator of TGF-β-induced skin fibrosis in SSc [80
]. Its serum levels are elevated in SSc patients and have been suggested to correlate with skin fibrosis [81
]. Hong and coworkers [79
] found increased levels of CTGF mRNA and protein in fibroblasts exposed to 1% of oxygen or treated with cobalt chloride, a chemical stabilizer of HIF-1α. The induction of CTGF in response to hypoxia depended on HIF-1α [79
]. Because the authors concentrated on short-term hypoxia of up to 4 hours, it remains unclear whether CTGF is also induced by chronic hypoxia and by HIF-1α-independent mechanisms in SSc.
Thus, accumulating evidence suggests that hypoxia might be actively involved in pathogenesis of SSc by stimulating the release of extracellular matrix protein. This could result in a vicious circle of hypoxia and fibrosis. Hypoxia stimulates the production and accumulation of extracellular matrix. The resulting tissue fibrosis inhibits diffusion of oxygen, causing further tissue hypoxia, which stimulates further the production of extracellular matrix (Figure ). Activation of TGF-β-dependent pathways appears to play a central role in the induction of extracellular matrix proteins by hypoxia, and inhibition of TGF-β signalling might prevent hypoxia-induced tissue fibrosis. However, further studies are needed to characterize further the role played by hypoxia in SSc and to identify the molecular mechanisms activated by hypoxia in SSc.