Data_Sheet_1_t-PA Suppresses the Immune Response and Aggravates Neurological Deficit in a Murine Model of Ischemic Stroke.docx

Introduction: Acute ischemic stroke (AIS) is a potent trigger of immunosuppression, resulting in increased infection risk. While thrombolytic therapy with tissue-type plasminogen activator (t-PA) is still the only pharmacological treatment for AIS, plasmin, the effector protease, has been reported to suppress dendritic cells (DCs), known for their potent antigen-presenting capacity. Accordingly, in the major group of thrombolyzed AIS patients who fail to reanalyze (>60%), t-PA might trigger unintended and potentially harmful immunosuppressive consequences instead of beneficial reperfusion. To test this hypothesis, we performed an exploratory study to investigate the immunomodulatory properties of t-PA treatment in a mouse model of ischemic stroke.Methods: C57Bl/6J wild-type mice and plasminogen-deficient (plg−/−) mice were subjected to middle cerebral artery occlusion (MCAo) for 60 min followed by mouse t-PA treatment (0.9 mg/kg) at reperfusion. Behavioral testing was performed 23 h after occlusion, pursued by determination of blood counts and plasma cytokines at 24 h. Spleens and cervical lymph nodes (cLN) were also harvested and characterized by flow cytometry.Results: MCAo resulted in profound attenuation of immune activation, as anticipated. t-PA treatment not only worsened neurological deficit, but further reduced lymphocyte and monocyte counts in blood, enhanced plasma levels of both IL-10 and TNFα and decreased various conventional DC subsets in the spleen and cLN, consistent with enhanced immunosuppression and systemic inflammation after stroke. Many of these effects were abolished in plg−/− mice, suggesting plasmin as a key mediator of t-PA-induced immunosuppression.Conclusion: t-PA, via plasmin generation, may weaken the immune response post-stroke, potentially enhancing infection risk and impairing neurological recovery. Due to the large number of comparisons performed in this study, additional pre-clinical work is required to confirm these significant possibilities. Future studies will also need to ascertain the functional implications of t-PA-mediated immunosuppression for thrombolyzed AIS patients, particularly for those with failed recanalization.</p

Bone turnover markers in rats: Percentage change from baseline.

Bone turnover markers in rats: Percentage change from baseline.</p

Trabecular bone variables: Ultra-distal femurs of stroke and control rats.

<p>Trabecular bone variables: Ultra-distal femurs of stroke and control rats.</p

MMP-9 levels are increased in spinal cord extracts from wild-type and t-PA^-/- mice following induction of EAE.

Spinal cord extracts (20 μg per lane) from MOG35-55 immunized t-PA-/- and wild-type mice at NSS stage 3 of EAE were subjected to SDS-PAGE electrophoresis and gelatin zymography. As shown, both wild-type and t-PA-/- mice demonstrated an increase in proMMP-9 but there was no significant change in levels of MMP-2. The increase in proMMP-9 was not seen in t-PA-/- mice at an early stage of EAE (pre-onset, NSS stage 1.5). As a control for active MMP-9 activity, conditioned media from HT1080 fibrosarcoma cells were included that constitutively express pro-MMP-9 (left lane of B). pro-MMP-9 in the HT1080 samples was activated using amino-phenyl mercuric acetate (APMA) to convert pro-MMP-9 into its active form (shown on the gel). The signal intensities (in arbitrary units; “au”) for MMP-9 in wild-type (A) and t-PA-/- mice (B) were quantitated by densitometry and presented in 4C and 4D, respectively.</p

Retention of animals and bones through study.

<p>Bones from the animal that died during surgery were not collected. Bones from the other two animals that died prematurely are included in the primary analysis.</p

Fibrinogen levels are increased in spinal cord extracts of wild-type, t-PA^-/- and T4+ mice following induction of EAE.

Spinal cord extracts (50 μg) from wild-type mice (A), t-PA-/- mice (B), T4control mice (C) and T4+ mice (D) were subjected to SDS-PAGE and evaluated for fibrinogen accumulation by western blot analysis under both control (naïve) conditions (CON) and following EAE (NSS 2 or 3 as shown). As shown, all three chains of fibrinogen (α, β, γ) were increased in spinal cords after EAE and this occurred regardless of the absence or neuronal over-expression of t-PA. The lower panel of D is a western blot for fibrinogen in plasma samples (1 μl) from the same T4+ mice as evaluated in the upper panel. As shown, plasma levels of fibrinogen remained unchanged in mice subjected to EAE.</p

Fibrin zymographic analysis of t-PA activity in spinal cord extracts of mice subjected to EAE.

(A): Spinal cord extracts (50 μg protein loaded per lane) from MOG35-55 immunized t-PA-/- and wild-type mice at NSS stage 3 of EAE were subjected to SDS-PAGE electrophoresis and fibrin zymography. As shown, wild-type mice demonstrated an increase in t-PA activity following EAE. Also evident is the presence of a slower migrating fibrinolytic moiety present following EAE most likely representing t-PA:PAI-1 complex formation. (B): Fibrin zymogram performed using t-PA and u-PA standards showing that the plasminogen activator activity seen in spinal cord extracts from wild-type (WT) control mice co-migrates with t-PA (note that the samples run in lanes 5 and 6 in (B) were the same samples loaded to lanes 4 and 5 in (A). Extracts from t-PA-/- mice were also used as a control to indicate the absence of both t-PA and u-PA.</p

The Influence of Differentially Expressed Tissue-Type Plasminogen Activator in Experimental Autoimmune Encephalomyelitis: Implications for Multiple Sclerosis

<div><p>Tissue type plasminogen activator (t-PA) has been implicated in the development of multiple sclerosis (MS) and in rodent models of experimental autoimmune encephalomyelitis (EAE). We show that levels of t-PA mRNA and activity are increased ~4 fold in the spinal cords of wild-type mice that are mice subjected to EAE. This was also accompanied with a significant increase in the levels of pro-matrix metalloproteinase 9 (pro-MMP-9) and an influx of fibrinogen. We next compared EAE severity in wild-type mice, t-PA^-/- mice and T4+ transgenic mice that selectively over-express (~14-fold) mouse t-PA in neurons of the central nervous system. Our results confirm that t-PA deficient mice have an earlier onset and more severe form of EAE. T4+ mice, despite expressing higher levels of endogenous t-PA, manifested a similar rate of onset and neurological severity of EAE. Levels of proMMP-9, and extravasated fibrinogen in spinal cord extracts were increased in mice following EAE onset regardless of the absence or over-expression of t-PA wild-type. Interestingly, MMP-2 levels also increased in spinal cord extracts of T4+ mice following EAE, but not in the other genotypes. Hence, while the absence of t-PA confers a more deleterious form of EAE, neuronal over-expression of t-PA does not overtly protect against this condition with regards to symptom onset or severity of EAE.</p></div

t-PA activity and mRNA levels are increased in spinal cord extracts of T4+ mice.

A: Spinal cord extracts prepared from naïve T4 control and T4+ mice were evaluated for t-PA dependent plasminogen activation using an amidolytic assay (see methods). T4+ mice (n = 4) displayed a 14.58 ± 1.78 fold increase in plasminogen activation rates relative to their littermate T4control mice (n = 3). Data presented as Mean±SEM. *p(B): A ~4-fold increase in t-PA mRNA is also observed in spinal cord extracts from naïve T4control and T4+ mice (p(C): T4control mice immunized with the MOG35-55 peptide also displayed a ~4-fold increase in t-PA mRNA levels (assessed at clinical severity score of 3); p<0.01. Data presented as Mean±SEM.</p

Coronal section micro-CT images of ultra-distal femurs of left (non-paretic) legs of animals with sham surgery control and middle cerebral occlusion stroke (case).

<p>White boxes indicate the regions of analysis for bone density and bone micro-structural parameters. Image on left = Control (5035#682–1049). Image on right = Case (5041#689–1065).</p