Multipotent mesenchymal stromal cells increase tPA expression and concomitantly decrease PAI-1 expression in astrocytes through the sonic hedgehog signaling pathway after stroke (in vitro study)

Abstract

Multipotent mesenchymal stromal cells (MSCs) increase tissue plasminogen activator (tPA) activity in astrocytes of the ischemic boundary zone, leading to increased neurite outgrowth in the brain. To probe the mechanisms that underlie MSC-mediated activation of tPA, we investigated the morphogenetic gene, sonic hedgehog (Shh) pathway. In vitro oxygen and glucose deprivation and coculture of astrocytes and MSCs were used to mimic an in vivo ischemic condition. Both real-time-PCR and western blot showed that MSC coculture significantly increased the Shh level and concomitantly increased tPA and decreased plasminogen activator inhibitor 1 (PAI-1) levels in astrocytes. Inhibiting the Shh signaling pathway with cyclopamine blocked the increase of tPA and the decrease of PAI-1 expression in astrocytes subjected to MSC coculture or recombinant mouse Shh (rm-Shh) treatment. Both MSCs and rm-Shh decreased the transforming growth factor-β1 level in astrocytes, and the Shh pathway inhibitor cyclopamine reversed these decreases. Both Shh-small-interfering RNA (siRNA) and Glil-siRNA downregulated Shh and Gli1 (a key mediator of the Shh transduction pathway) expression in cultured astrocytes and concomitantly decreased tPA expression and increased PAI-1 expression in these astrocytes after MSC or rm-Shh treatment. Our data indicate that MSCs increase astrocytic Shh, which subsequently increases tPA expression and decreases PAI-1 expression after ischemia.

Figure 1

MSCs concomitantly increase Shh and tPA expressions in cultured astrocytes. qRT-PCR and western blot show the (A) mRNA and (B) protein levels of tPA and Shh in cultured astrocytes. OGD treatment increased the Shh and tPA levels compared with normal cultured astrocytes. MSC coculture increased the Shh and tPA levels in both normal cultured and OGD cultured astrocytes. (C) The Shh expression in cultured MSCs was obviously lower than in astrocytes under different culture conditions. ^*P<0.05, ^**P<0.01, compared with group A; ⁺P<0.05, ⁺⁺P<0.01, compared with group AO. A: normal astrocytes; AM: normal astrocytes cocultured with MSCs; AO: OGD astrocytes; AOM: OGD astrocytes cocultured with MSCs; M: MSCs; MO: OGD MSCs; MOA: OGD MSCs cocultured with astrocytes. MSC, mesenchymal stromal cell; OGD, oxygen and glucose deprivation; qRT-PCR, quantitative real-rime PCR; Shh, sonic hedgehog; tPA, tissue plasminogen activator.

Figure 2

MSCs stimulated the Shh signaling pathway and thereby regulates tPA and PAI-1 expressions in cultured astrocytes. qRT-PCR and western blot show the (A) mRNA and (B) protein levels of tPA in cultured astrocytes. MSC treatment and addition of exogenous rm-Shh increased the tPA level in cultured astrocytes. The Shh signaling pathway inhibitor cyclopamine blocked the tPA increase in astrocytes with MSC coculture and rm-Shh treatment. (C) Western blot shows PAI-1 protein level in cultured astrocytes. MSC treatment and addition of exogenous rm-Shh decreased PAI-1 expression in cultured astrocytes. The Shh signaling pathway inhibitor cyclopamine increased PAI-1 expression in astrocytes with MSC coculture and rm-Shh treatment. ^*P<0.05, ^**P<0.01, compared with normal astrocytes; ^P<0.05, ^^P<0.01 compared with OGD astrocytes; ^#P<0.05, ^##P<0.01, compared with MSC treatment; ⁺⁺P<0.01, compared with rm-Shh treatment. A: astrocytes; AM: astrocytes cocultured with MSCs; AMC: astrocytes cocultured with MSCs and cyclopamine added; AS: astrocytes treated with rm-Shh; ASC: rm-Shh treated astrocytes with cyclopamine added. MSC, mesenchymal stromal cell; PAI-1, plasminogen activator inhibitor 1; qRT-PCR, quantitative real-rime PCR; rm-Shh, recombinant mouse Shh; Shh, sonic hedgehog; tPA, tissue plasminogen activator.

Figure 3

Shh affected PAI-1 expression in astrocytes by regulating the TGF-β1 signaling pathway. ELISA detection shows that MSCs and rm-Shh significantly decreased TGF-β1 expression in cultured astrocytes. (A) The Shh signaling pathway inhibitor cyclopamine increased TGF-β1 level in astrocytes cocultured with MSCs. (B) Exogenous addition of rh-TGF-β1 significantly increased PAI-1 expression in astrocytes, and the TGF-β activity neutralizer TGF-β1 LAP specifically downregulated the PAI-1 expression. ^*P<0.05, ^**P<0.01, compared with normal cultured astrocytes; ^#P<0.05, compared with MSC treatment; ^P<0.05 compared with TGF-β1 treatment. C: control (normal cultured astrocytes); L: LAP treatment; T: TGF-β1 treatment; T+L: TGF-β1 and LAP treatment; Shh: rm-Shh treatment; MSCs: MSC treatment. MSC, mesenchymal stromal cell; PAI-1, plasminogen activator inhibitor 1; rm-Shh, recombinant mouse Shh; Shh, sonic hedgehog; TGF-β1, transforming growth factor-β1; ELISA, enzyme-linked immunosorbent assay.

Figure 4

Shh signaling pathway-mediated induction of tPA expression and inhibition of TGF-β/PAI-1 expression through Gli1. Transfected with (A) Shh-siRNA downregulated Shh expression in cultured astrocytes and concomitantly tPA expression was decreased and PAI-1 increased in these astrocytes. (B) Gli1 expression was downregulated after transfected Gli1-siRNA into astrocytes. (C) The downregulated expression of Gli1 inhibited tPA increase and inhibited TGF-β and PAI-1 decrease after MSCs or rm-Shh treatment. ^*P<0.05, ^**P<0.01, compared with vector-transfected astrocytes; ⁺P<0.05, compared with rm-Shh or MSCs treatment, respectively. MSC, mesenchymal stromal cell; PAI-1, plasminogen activator inhibitor 1; rm-Shh, recombinant mouse Shh; Shh, sonic hedgehog; TGF-β1, transforming growth factor-β1; tPA, tissue plasminogen activator.