Uncontrolled expression of vascular endothelial growth factor and its receptors leads to insufficient skin angiogenesis in patients with systemic sclerosis.

Systemic sclerosis (SSc) skin lesions are characterized by disturbed vessel morphology with enlarged capillaries and an overall reduction in capillary density, suggesting a deregulated, insufficient angiogenic response. It has been postulated that this phenomenon is due to reduced expression of the potent angiogenic factor vascular endothelial growth factor (VEGF). In contrast to this hypothesis, we demonstrate that the expression of both VEGF and its receptors VEGFR-1 and VEGFR-2 is dramatically upregulated in skin specimens of SSc patients throughout different disease stages. Interestingly, upregulation of VEGF was not mediated by hypoxia-inducible transcription factor-1 (HIF-1) as indicated by only a weak expression of the oxygen-sensitive alpha-subunit of HIF-1 in the skin of SSc patients. This was unexpected on measuring low Po2 values in the SSc skin by using a polarographic oxygen microelectrode system. Considering our observation that PDGF and IL-1beta costimulated VEGF expression, we propose that chronic and uncontrolled VEGF upregulation that is mediated by an orchestrated expression of cytokines rather than VEGF downregulation is the cause of the disturbed vessel morphology in the skin of SSc patients. Consequently, for therapeutic approaches aiming to improve tissue perfusion in these patients, a controlled expression and timely termination of VEGF signaling appears to be crucial for success of proangiogenic therapies.

Bucillamine induces the synthesis of vascular endothelial growth factor dose-dependently in systemic sclerosis fibroblasts via nuclear factor-kappaB and simian virus 40 promoter factor 1 pathways.

The pathogenesis of systemic sclerosis (SSc) is characterized by activation of the immune system, impaired angiogenesis, and activated dermal fibroblasts. The effects of the immunosuppressive agent bucillamine (SA 96) on fibroblasts and angiogenic factors have not been examined. SA 96, and particularly its metabolite SA 981, increased the levels of vascular endothelial growth factor (VEGF) mRNA and protein dose-dependently in dermal fibroblasts from patients with SSc and healthy control subjects without influencing cell viability. SSc fibroblast cultures showed consistently a higher inducibility of VEGF than cultures from healthy control subjects. Preincubation with the SP-1 inhibitor mithramycin as well as blockade of nuclear factor (NF)-kappaB signaling with pyrrolidine dithiocarbamate treatment and IkappaB transfection reduced significantly the transcription of VEGF, indicating that both transcription factors contribute to the activation of VEGF by SA 981. Specific binding of NF-kappaB protein to its binding site after treatment with SA 981 was confirmed by electrophoretic mobility shift assay. In contrast, SA 981 did not influence the stability of VEGF mRNA as analyzed with actinomycin D assays. The study provides evidence for a role of NF-kappaB in the transcriptional regulation of the VEGF gene. SA 96 might have positive aspects on the impaired angiogenesis in patients with SSc.

Cytokine mRNA and protein expression in primary-culture and repeated-passage synovial fibroblasts from patients with rheumatoid arthritis.

Constitutive mRNA expression and secretion of proinflammatory and anti-inflammatory cytokines was comparatively analyzed in rheumatoid arthritis (RA) synovial fibroblasts (SFB), isolated from primary culture or derived by repeated passage; normal-skin fibroblasts were used as controls. First-passage RA-SFB (n = 3) secreted large amounts of IL-6 (15,800 +/- 2,110 pg/ml; mean +/- SEM), but only limited amounts of tumor necrosis factor (TNF)-alpha (22.1 +/- 1.1 pg/ml) or IL-10 (35.7 +/- 34.2 pg/ml; only one of three samples was positive). IL-1beta, IL-15, and IL-18 were not detectable at the protein level and showed very low mRNA levels by semiquantitative RT-PCR. In repeated-passage RA-SFB (tenth passage), protein secretion was significantly lower for IL-6 (one-twentieth of the initial level) and TNF-alpha (two-thirds), and markedly reduced for IL-10 (one-quarter, with only one of three samples positive). While the decrease of IL-10 protein from first to tenth passage was paralleled by a corresponding decrease of mRNA, the relative mRNA levels for IL-6 and TNF-alpha were actually increased (20-fold and 300-fold, respectively), indicating post-transcriptional and/or post-translational regulation of these cytokines. Due to highly variable levels among individual patients, however, no significant differences were observed for any cytokine mRNA between primary-culture and repeated-passage RA-SFB (ninth passage). Likewise, no significant differences were detectable between RA-SFB and normal-skin fibroblasts (primary-culture and repeated-passage). By producing high amounts of IL-6 and limited amounts of TNF-alpha, RA-SFB may contribute to the (im)balance of proinflammatory and anti-inflammatory cytokines in the inflamed joint.