Figure 3.

Fibrinogen regulates astrocyte-induced formation of inhibitory ECM through the TGF-β receptor pathway in vitro. A, Fibrinogen-induced inhibitory CSPG expression by primary astrocytes is not regulated through the EGFR pathway. Conditioned medium from fibrinogen-treated and untreated astrocytes was blotted with anti-CSPG antibody. Astrocytes were incubated with EGFR inhibitor 1 h before stimulation with fibrinogen. B, Fibrinogen does not induce phosphorylation of the EGFR in astrocytes. Serum-starved astrocytes were treated with fibrinogen for the indicated times or left untreated, and lysates were blotted for P-EGFR. EGF served as a positive control. C, Treatment of primary astrocytes with a TGF-β receptor type I inhibitor blocked fibrinogen-induced neurocan expression. TGF-β served as a positive control. D, TGF-β receptor type I inhibitor blocks fibrinogen-induced Smad2 phosphorylation. E, Neurite outgrowth assay with astrocyte-conditioned medium. Conditioned medium (CM) from fibrinogen-treated primary astrocytes aged for 30 d inhibited neurite outgrowth of cortical neurons. This inhibitory effect was abolished by pretreatment with TGF-β receptor type I inhibitor. F, ChABC digestion of conditioned medium from fibrinogen-treated primary astrocytes aged for 40 d in culture reduced the inhibition of neurite outgrowth of cortical neurons. G, Quantification of neurite outgrowth and neurite length revealed an inhibitory effect of fibrinogen-treated astrocyte-CM in astrocytes aged for 30 d. The TGF-β receptor inhibitor abolished this effect. Values are mean ± SEM of at least three different experiments. A minimum of 900–1000 neurons per condition were analyzed. *p < 0.001. Representative blots are shown from three independent experiments. Scale bar, 40 μm.