Increasing tPA activity in astrocytes induced by multipotent mesenchymal stromal cells facilitate neurite outgrowth after stroke in the mouse

Abstract

We demonstrate that tissue plasminogen activator (tPA) and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS) after treatment of stroke with multipotent mesenchymal stromal cells (MSCs). In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo) significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ) compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1) expression in astrocytes under normal and oxygen and glucose deprivation (OGD) conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke.

Figure 1. tPA and PAI-1 mRNA and protein levels in treated astrocytes.

qRT-PCR shows mRNA levels of tPA and PAI-1 in normal cultured astrocytes (A), astrocytes co-cultured with MSCs (A-M), OGD astrocytes (AO) and OGD astrocytes co-cultured with MSCs (AO-M) (a). OGD treatment significantly increased tPA and PAI mRNA levels in astrocytes. MSC co-culture significantly increased tPA mRNA level in both normal and OGD astrocytes whereas MSC co-culture significantly decreased PAI-1 mRNA level (b) in OGD astrocytes. Western blot shows protein levels of tPA and PAI-1 in normal cultured astrocytes (A), astrocytes co-cultured with MSCs (A-M), OGD astrocytes (AO) and OGD astrocytes co-cultured with MSCs (AO-M) (c). OGD treatment increased tPA and PAI protein level and co-culture MSCs increased tPA protein level whereas MSCs decreased PAI-1 protein level (d). *P<0.05, **P<0.01, compared with group A; ++P<0.01, compared with group AO.

Figure 2. tPA activity of astrocyte lysates and conditional media.

Zymography (a, b) shows that MSCs significantly increased tPA activity in normal cultured astrocytes and in OGD treated astrocytes (c), and similar results were obtained using the conditioned media harvested from the concomitant groups (d). Marker: prestained protein marker; A: normal cultured astrocytes; A-M: normal astrocytes co-cultured with MSCs; AO: OGD astrocytes; AO-M: OGD astrocytes co-cultured with MSCs. *P<0.05, **P<0.01, compared with group A; ##P<0.01, compared with group AO.

Figure 3. Neurite outgrowth in primary cultured cortical neuron after conditional media treatment.

Fluorescence microscopy (a) shows cortical neurite outgrowth. Control: primary cultured cortical neurons with neurobasal medium; medium from normal cultured astrocytes (A) increased neurite number and total length compared to those in control group; medium from OGD astrocytes (AO) significantly decreased neurite number and total length; media from normal astrocytes co-cultured with MSCs (A-M) and OGD astrocytes co-cultured with MSCs (AO-M) increased neurite total length compared to that in A and AO groups, respectively, and increased neurite number in AO-M group compared with AO group. tPA neutralizing antibody specifically antagonized tPA effects of AM and AO-M groups in neurite number and total length (b, c). Western blot shows that tPA expression in astrocytes was substantially down regulated by tPA siRNA (d). Medium from tPA knock-down astrocytes significantly reduced the neurite number and total length of cultured neurons compared with that from normal astrocytes or negative control siRNA transfected astrocytes (b, c). A-MT: medium from normal astrocytes co-cultured with MSCs, t-PA neutralizing antibody presented; AO-MT: medium from OGD astrocytes co-cultured with MSCs, t-PA neutralizing antibody presented; t-PA: 15nM rh-t-PA alone; AC: medium from astrocytes transfected with negative control siRNA; AS: medium from astrocytes transfected with tPA siRNA. Scale bars = 50 µm. *P<0.05, ** P<0.01, compared with control group; #P<0.05, ##P<0.01, compared with A group; ++P<0.01, compared with A-M group; ∧∧P<0.01, compared with AO group; ††P<0.01, compared with AO-M group; &&P<0.01, compared with AC group. Data are presented as Mean±SE, (neurons n = 50/group, Adjusted p-value = 0.0042).

Figure 4. tPA and PAI-1 levels and tPA activity in mice subjected to MCAo with or without MSC treatment.

Western blot shows tPA protein level (a) and zymography shows tPA activity (b) in MCAo mice with or without MSC treatment. tPA and PAI-1 expression was significantly increased in the IBZ of mice subjected to MCAo compared with normal mice. tPA expression was significantly increased and PAI-1 expression was significantly decreased in the IBZ of MCAo mice after MSC treatment compared with MCAo alone mice (c). MSC treatment significantly increased the activity of tPA in the IBZ compared with MCAo alone mice (d). N: normal mouse brain tissue; C: IBZ tissue from MCAo mice; M: IBZ tissue from MCAo mice after MSC treatment. **P<0.01, compared with normal mice; #P<0.05, ##P<0.01, compared with control MCAo mice.

Figure 5. tPA and PAI-1 level in MCAo mouse IBZ astrocytes respond to MSC.

(5a) presents the individual IBZ astrocytes dissected using LCM (before and after dissection), and tPA and PAI-1 mRNA level in these astrocytes with or without MSC treatment are shown in (b). MSC treatment significantly increased tPA mRNA level and concomitantly decreased PAI-1 mRNA level. Scale bars = 50 µm. *P<0.05 compared with MCAo mice.

Figure 6. MSCs promote neurite outgrowth, synaptic plasticity and cell survival.

Double staining (Bielshowsky, black for axons, indicated by green arrow; Luxol fast blue for myelin, indicated by red arrow.) shows axonal and myelin fibers in the striatum (b–c) along the IBZ after MCAo compared to normal brain (a). The integrated density (indicated the axonal and myelin fibers number) of white matter bundles in IBZ of striatum was decreased compared with that in normal brain, The axonal fibers and myelin of the striatum were enhanced by MSCs (c, d, Adjusted p-value = 0.0167). Immunofluorescent staining (e–g) shows synaptic regeneration (indicated by synaptophysin). Synaptophysin expression significantly increased in MSC treated rats (g, h, Adjusted p-value = 0.025). Apoptosis neuron (indicated by white arrow) is shown with double staining with TUNEL and MAP 2 (i–k), MSCs decreased the apoptosis neuron number (k, l, Adjusted p-value = 0.01667). Scale bars = 25 µm. **P<0.01, compared with normal mice; ##P<0.01, compared with control MCAo alone mice.