Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines.

OBJECTIVES: Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS: Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS: Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION: Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.

Activation of the Thromboxane A2 Receptor by 8-Isoprostane Inhibits the Pro-Angiogenic Effect of Vascular Endothelial Growth Factor in Scleroderma.

The pathogenesis of scleroderma (SSc) includes components of autoimmunity, vascular dysfunction, and accumulation of extracellular matrix. 8-isoprostane, an oxidized lipid created by oxidative stress, activates the thromboxane A2 receptor (TXAR) and the Rho-associated kinase (ROCK) pathway. In this study, we determined whether the TXAR was activated by 8-isoprostane in SSc endothelial cells (ECs) and whether this pathway inhibited VEGF-induced angiogenesis. Elevated 8-isoprostane was observed in plasma and conditioned media from SSc patients. SSc-conditioned media inhibited EC tube formation, whereas addition of vitamin E, by reducing 8-isoprostane, increased tube formation. VEGF did not induce angiogenesis in SSc ECs, but vitamin E or TXAR inhibition restored its effect. The expression of TXAR, RhoA, and ROCK1/2 was elevated in SSc ECs. ROCK activity and 8-isoprostane-induced ROCK activation were significantly higher in SSc ECs, whereas VEGF had no effect. The hyper-activation of the TXAR leads to inhibition of VEGF-induced angiogenesis, as inhibition of the TXAR pathway results in a blockade of 8-isoprostane-induced ROCK activation and restoration of VEGF activity. These results suggest that the TXAR pathway has a crucial role in angiogenesis and that 8-isoprostane is not just a by-product of oxidative stress but also has a significant role in the impaired angiogenesis that characterizes SSc.

Lipoic acid plays a role in scleroderma: insights obtained from scleroderma dermal fibroblasts.

INTRODUCTION: Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and organs. Increase in oxidative stress and platelet-derived growth factor receptor (PDGFR) activation promote collagen I (Col I) production, leading to fibrosis in SSc. Lipoic acid (LA) and its active metabolite dihydrolipoic acid (DHLA) are naturally occurring thiols that act as cofactors and antioxidants, and are produced by lipoic acid synthetase (LIAS). The goal of this study was to examine whether LA and LIAS was deficient in SSc patients and determine the effect of DHLA on the phenotype of SSc dermal fibroblasts. N-acetylcysteine (NAC), a commonly used thiol antioxidant, was included as a comparison. METHODS: Dermal fibroblasts were isolated from healthy subjects and patients with diffuse cutaneous SSc. Matrix metalloproteinase (MMPs), tissue inhibitors of MMPs (TIMP), plasminogen activator inhibitor-1 (PAI-1) and LIAS were measured by ELISA. The expression of Col I was measured by immunofluorescence, hydroxyproline assay, and quantitative PCR. PDGFR phosphorylation and α-smooth muscle actin (α-SMA) was measured by Western blotting. Student's t-tests were performed for statistical analysis and p-values of less than 0.05 with two-tailed analysis were considered statistically significant. RESULTS: The expression of LA and LIAS in SSc dermal fibroblasts was lower than normal fibroblasts, however LIAS was significantly higher in SSc plasma and appeared to be released from monocytes. DHLA lowered cellular oxidative stress, and decreased PDGFR phosphorylation, Col I, PAI-1, and α-SMA expression in SSc dermal fibroblasts. It also restored the activities of phosphatases that inactivated the PDGFR. SSc fibroblasts produced lower levels of MMP-1 and 3, and DHLA increased them. In contrast, TIMP-1 levels were higher in SSc but DHLA had minimal effect. Both DHLA and NAC increased MMP-1 activity when SSc cells were stimulated with PDGF. In general, DHLA showed better efficacy than NAC in most cases. CONCLUSIONS: DHLA not only acts as an antioxidant but also an antifibrotic since it has the ability to reverse the profibrotic phenotype of SSc dermal fibroblasts. Our study suggests that thiol antioxidants, including NAC and LA/DHLA, could be beneficial for patients with SSc.