Lack of detection of agonist activity by antibodies to platelet-derived growth factor receptor alpha in a subset of normal and systemic sclerosis patient sera.

OBJECTIVE: To investigate whether agonist anti-platelet-derived growth factor receptor alpha (anti-PDGFRalpha) antibodies are present in the serum of patients with systemic sclerosis (SSc; scleroderma). METHODS: Sera were obtained from healthy subjects and scleroderma patients. An electrochemiluminescence binding assay was performed for detection of serum autoantibodies to PDGFRalpha, PDGFRbeta, epidermal growth factor receptor (EGFR), and colony-stimulating factor receptor 1 (CSFR1). Serum immunoglobulin was purified by protein A/G chromatography. To assess Ig agonist activity, PDGFRalpha-expressing cells were incubated with pure Ig and the level of receptor phosphorylation determined in an enzyme-linked immunoassay, as well as by Western blotting. Ig agonist activity was also assessed in a mitogenic assay and by MAP kinase activation in a PDGFRalpha-expressing cell line. RESULTS: Sera from 34.3% of the healthy subjects and 32.7% of the SSc patients contained detectable autoantibodies to PDGFRalpha and PDGFRbeta, but not EGFR or CSFR1. Purified Ig from these sera was shown to retain PDGFR binding activity and, at 200-1,000 microg/ml, exhibited no agonist activity in a cell-based PDGFRalpha phosphorylation assay and did not stimulate a mitogenic response or MAP kinase activation in a PDGFRalpha-expressing cell line. Two purified Ig samples that were unable to bind PDGFRalpha did exhibit binding activity to a nonglycosylated form of PDGFRalpha. CONCLUSION: Although approximately one-third of sera from scleroderma patients contained detectable autoantibodies to PDGFR, these antibodies were not specific to scleroderma, since they were also detected in a similar percentage of samples from normal subjects. PDGFRalpha agonist activity was not demonstrated when purified Ig from these sera was tested in cell-based assays.

Recombinant semaphorin 6A-1 ectodomain inhibits in vivo growth factor and tumor cell line-induced angiogenesis.

The Semaphorins are a large family of transmembrane, GPI-anchored and secreted proteins that play an important role in neuronal and endothelial cell guidance. A human gene related to the class 6 Semaphorin family, Semaphorin 6A-1 (Sema 6A-1) was identified by homology-based genomic mining. Recent implication of Sema 3 family members in tumor angiogenesis and our expression analysis of Sema 6A-1 suggested that class 6 Semaphorin might effect tumor neovascularization. The mRNA expression of Sema 6A-1 was elevated in several renal tumor tissue samples relative to adjacent nontumor tissue samples from the same patient. Sema 6A-1 transcript was also expressed in the majority of renal clear cell carcinoma (RCC) cell lines and to a lesser extent in endothelial cells. To test the role of Sema 6A-1 in tumor angiogenesis, we engineered, expressed and purified the Sema 6A-1 soluble extracellular domain (Sema-ECD). The purified Sema-ECD was screened in a variety of endothelial cell-based assays both in vitro and in vivo. In vitro, Sema-ECD blocked VEGF-mediated endothelial cell migration. These effects were explained in part by our observation in endothelial cells that Sema-ECD inhibited VEGF-mediated Src, FAK and ERK phosphorylation. In vivo, mouse Matrigel assays demonstrated that the intraperitoneal administration of recombinant Sema-ECD inhibited both bFGF/VEGF and tumor cell line-induced neovascularization. These findings reveal a novel therapeutic utility for Sema 6A-1 (Sema-ECD) as an inhibitor of growth factor as well as tumor-induced angiogenesis.