Fibrinogen triggers astrocyte scar formation by promoting the availability of active TGF-beta after vascular damage

Abstract

Scar formation in the nervous system begins within hours after traumatic injury and is characterized primarily by reactive astrocytes depositing proteoglycans that inhibit regeneration. A fundamental question in CNS repair has been the identity of the initial molecular mediator that triggers glial scar formation. Here we show that the blood protein fibrinogen, which leaks into the CNS immediately after blood-brain barrier (BBB) disruption or vascular damage, serves as an early signal for the induction of glial scar formation via the TGF-beta/Smad signaling pathway. Our studies revealed that fibrinogen is a carrier of latent TGF-beta and induces phosphorylation of Smad2 in astrocytes that leads to inhibition of neurite outgrowth. Consistent with these findings, genetic or pharmacologic depletion of fibrinogen in mice reduces active TGF-beta, Smad2 phosphorylation, glial cell activation, and neurocan deposition after cortical injury. Furthermore, stereotactic injection of fibrinogen into the mouse cortex is sufficient to induce astrogliosis. Inhibition of the TGF-beta receptor pathway abolishes the fibrinogen-induced effects on glial scar formation in vivo and in vitro. These results identify fibrinogen as a primary astrocyte activation signal, provide evidence that deposition of inhibitory proteoglycans is induced by a blood protein that leaks in the CNS after vasculature rupture, and point to TGF-beta as a molecular link between vascular permeability and scar formation.

Figure 1.

Fibrinogen depletion decreases astrogliosis and neurocan expression. A, SWI, a model of cortical trauma. B, Three days after SWI, immunolabeling for fibrin (red) and GFAP (green) revealed perivascular fibrin colocalizing with reactive astrocytes in brain coronal sections of mice (top). Uninjured mice show no fibrinogen deposition in the brain (bottom). C, Low-magnification images of Fib ^−/− and ancrod-depleted WT mice show reduced astrogliosis demonstrated by immunolabeling of GFAP astrocytes (green) 3 d after SWI. Box indicates area of quantification. D, Fib ^−/− and ancrod-treated WT mice show reduced astrogliosis and neurocan expression 3 d after SWI, shown by immunolabeling of astrocytes for GFAP (green) and neurocan (red). Uninjured WT mice served as controls. E, Quantification revealed lower levels of GFAP and neurocan in Fib ^−/− and ancrod-treated mice than in WT mice (n = 5 per group) after SWI. Values are mean ± SEM. *p < 0.001. Scale bars: B, 75 μm; C, 500 μm; D, 60 μm.

Figure 2.

Fibrinogen regulates neurocan expression in primary astrocytes. A, Increased expression of neurocan mRNA in primary astrocytes after fibrinogen treatment as determined by quantitative PCR and normalized to GAPDH. Results are from three independent experiments performed in duplicate. B, C, Potent and rapid fibrinogen-induced neurocan protein secretion by primary astrocytes with TGF-β (B) or EGF (C) as positive controls. D, Fibrinogen-induced neurocan expression is concentration-dependent. Primary astrocytes were treated for 2 d with fibrinogen (0.1–2.5 mg/ml). TGF-β served as a positive control. Representative blots are shown from three independent experiments. Values are mean ± SEM of three independent experiments. *p < 0.01.

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.

Figure 4.

Fibrinogen-bound latent TGF-β is activated by astrocytes. A, Fibrinogen-induced Smad2 phosphorylation is blocked by anti-TGF-β neutralizing antibody. B, Plasma-isolated fibrinogen was tested for active and latent TGF-β with TGF-β isoform-specific ELISAs. C, Delayed fibrinogen-induced Smad2 phosphorylation in primary astrocytes. Astrocytes were treated with fibrinogen or TGF-β (positive control) for the indicated times or left untreated, and lysates were blotted for P-Smad2. TGF-β induces Smad2 phosphorylation (P-Smad2) as early as 10 min, whereas fibrinogen took 30 min. D, Western blotting of fibrinogen isolated from plasma reveals LAP1 and latent LTBP1. E, Plasma-isolated fibrinogen was immunoprecipitated with an antibody against human fibrinogen and immunoblotted with an antibody against LTBP1, revealing a complex of LTBP1 with fibrinogen. F, TGF-β ELISA revealed active TGF-β in supernatants of primary astrocytes treated with fibrinogen for 1 h. Results are from three independent experiments performed in duplicate. G, Immunolabeling for active TGF-β (green) and GFAP (red) revealed active TGF-β formation in astrocytes 1 h after fibrinogen treatment. H, Stereotactic injection of fibrinogen in mouse cortex results in active TGF-β formation, compared with control ACSF injection. Representative immunostaining is shown (n = 5 mice per group). Values are mean ± SEM. *p < 0.05. Scale bars: G, 13.5 μm; H, 90 μm.

Figure 5.

Fibrinogen is necessary for active TGF-β formation and signaling in the CNS. A, Immunolabeling for GFAP (green) and active TGF-β (red) revealed astrocyte-specific expression of active TGF-β after SWI (yellow). B, Decreased levels of active TGF-β immunolabeling (red) in Fib ^−/− and ancrod-treated mice 3 d after SWI. Uninjured WT brain served as a negative control. C, Lower levels of active TGF-β after SWI in Fib ^−/− and ancrod-treated mice than in WT controls (n = 5 per group). D, Brain lysates of Fib ^−/− and ancrod-treated WT mice show reduced Smad2 phosphorylation 3 d after SWI. Brain lysates of uninjured WT mice do not show P-Smad2 activation. Lysates from two mice per experimental treatment are shown. Values are mean ± SEM. *p < 0.001. Scale bar, 60 μm.

Figure 6.

Synergism between fibrinogen and active TGF-β in the induction of astrogliosis and neurocan deposition. A, Fib ^−/− mice show reduced astrogliosis and neurocan expression demonstrated by immunostaining for GFAP and neurocan, 3 d after TGF-β injection followed by SWI. Uninjured WT mice served as controls. Stereotactic injection of TGF-β induced a similar increase in astrogliosis and neurocan expression in Fib ^−/− and WT controls injected with ACSF. B, Quantification revealed significant lower levels of GFAP and neurocan in Fib ^−/− mice than in WT mice (n = 4 per group) after TGF-β injection and SWI. Values are mean ± SEM. *p < 0.001. Scale bar, 90 μm.

Figure 7.

Fibrinogen regulates astrocyte-induced scar formation through the TGF-β receptor pathway in vivo. A, Stereotactic injection of fibrinogen into the cortex. B, Astrocyte immunolabeling (green) revealed reactive astrocytosis in fibrinogen-injected mice; injection of albumin, laminin, and fibronectin had no effect. C, Quantification revealed a 3.4-fold increase in astrogliosis after fibrinogen injection. n = 5 mice per condition. D, TGF-β receptor inhibitor injection in mice abolished fibrinogen-induced astrogliosis and neurocan deposition demonstrated by immunostaining for GFAP (green, top row) and neurocan (red, bottom row). E, Quantification revealed a sevenfold increase in GFAP and a tenfold increase in neurocan immunoreactivity in fibrinogen-injected mice compared with controls (n = 5 per group). TGF-β receptor inhibitor in fibrinogen-injected mice (n = 5) revealed a 2.5-fold decrease in GFAP immunoreactivity and a threefold decrease in neurocan immunoreactivity compared with fibrinogen-injected mice (n = 5). Values are mean ± SEM. *p < 0.001. Scale bars: B, 90 μm; D, 60 μm.

Figure 8.

Fibrinogen induces scar formation as a carrier of latent TGF-β. A, Fibrinogen fractions isolated from plasma. Fraction I-2 is intact 340 kDa fibrinogen composed of three pairs of nonidentical polypeptide chains, designated Aα (63.5 kDa), Bβ (56 kDa), and γ (47 kDa). Fraction I-9 lacks residues of the C terminus of the Aα chain. B, Western blotting of plasma fractions I-2 and I-9 shows no LTBP1 in fraction I-9. Coomassie staining revealed the absence of full-length fibrinogen Aα chain in fraction I-9. C, Immunolabeling of astrocytes (GFAP, green) and neurocan (red) shows less astrogliosis and lower neurocan expression after stereotactic injection of fraction I-9 than after injection of fraction I-2. ACSF-injected mice served as controls. D, GFAP and neurocan levels are lower in mice injected with fraction I-9 than in those injected with fraction I-2 (n = 6 per group). E, TGF-β ELISA revealed reduced latent TGF-β1 in plasma-isolated fibrinogen fraction I-9. Values are mean ± SEM of three independent experiments performed in duplicate. *p < 0.001. Scale bar, 90 μm.

Figure 9.

Proposed model for the role of fibrinogen-bound latent TGF-β activated by reactive astrocytes after injury. In the uninjured CNS, fibrinogen-bound latent TGF-β circulates within the blood stream. BBB disruption or vascular rupture after trauma leads to leakage of fibrinogen-bound latent TGF-β into the CNS. Fibrinogen-bound latent TGF-β interacts with local perivascular astrocytes, leading to active TGF-β formation, which induces reactive astrocytosis by regulating the TGF-β/Smad signaling pathway, resulting in scar formation. Local provisional fibrin matrix thus functions as a primary astrocyte activation signal initiating scar formation in the CNS by regulating the bioavailability of active TGF-β at sites of vascular damage.