Fig 1.
tPA promotes C3 cleavage through a plasmin-mediated extrinsic pathway.
(A) C3 protein was incubated with plasmin. Plasmin-mediated C3a production increased in a dose-dependent manner. ***P<0.001 vs. control (n = 3). (B) tPA promotes C3 cleavage in human plasma, and this process is suppressed by α2-antiplasmin but not by CD35. *P<0.05, **P<0.01, ***P<0.001 vs. controls (n = 3). (C) Following intravenous tPA infusion in C57BL/6 mice in the absence of ischemia, a significant increase in plasma C3a levels was observed. *P<0.05 (n = 3).
Fig 2.
C3aR is strongly expressed in ischemic brain tissue and endothelial cells subjected to OGD.
(A) WB for C3aR on brain homogenates obtained from mice subjected to MCAO. C3aR protein is strongly upregulated in the ischemic hemisphere. **P<0.01 (n = 5). (B) Western blot for C3aR performed on lysates of bEnd.3 cells subjected to OGD demonstrates significantly increased C3aR protein expression relative to non-ischemic controls. *P<0.05 (n = 3). (C) Immunocytochemical staining (40X) demonstrates baseline C3aR expression on non-ischemic bEnd.3 cells. (D) Following OGD, enhanced C3aR expression (green) is noted. Scale bars = 20 μm.
Fig 3.
C3a does not directly cause neuronal or endothelial cell death but modulates endothelial cell permeability in vitro.
Recombinant mouse C3a was incubated with PCN and bEnd.3 cells ± OGD. OGD increases LDH release by PCN (A) and bEnd.3 cells (B). However, C3a did not affect LDH production by either cell type. **P< 0.01, ***P< 0.001 vs. controls (n = 4–6). We next assessed the effect of C3a on permeability of a monolayer of bEnd.3 cells subjected to OGD. (C) In this model, C3a alone as well as OGD increased permeability compared to control non-ischemic cells. (D) C3a also increased monolayer permeability relative to OGD alone. *P< 0.05, **P< 0.01 vs. controls (n = 4).
Fig 4.
tPA promotes C3 cleavage in ischemic mouse brain through an MBL-independent pathway.
Brain homogenates were obtained from mice subjected to MCAO who received i.v. tPA at reperfusion. (A) In wild-type mice, a significant increase in C3a concentration was observed in the ischemic hemisphere, with further increases in C3a after tPA administration. (B) MBL-null mice did not demonstrate significant C3 cleavage in the ischemic hemisphere; however, tPA does promote a dramatic increase in brain C3a in these mice. *P<0.05, **P<0.01, ***P<0.001 vs. controls (n = 7).
Fig 5.
Both tPA and C3aRA confer robust neuroprotection following transient MCAO.
(A) Following transient MCAO, C3aRA, tPA and the combination of C3aRA/tPA significantly reduced infarct volume relative to vehicle-treated controls. (B) Representative TTC-stained coronal sections of brains from mice in each cohort. (C) tPA alone or tPA/C3aRA improves neurological function relative to vehicle-treated controls. (D) No significant differences in cerebral blood flow were noted among treatment groups. (E) No significant differences in mortality were noted among treatment groups. **P< 0.01, ***P<0.001 vs. controls (n = 10).
Fig 6.
C3aRA ameliorates tPA-mediated brain edema and hemorrhage following MCAO.
(A) tPA administration significantly increases relative cerebral edema, and this increase was ameliorated by C3aRA. (B) tPA significantly increased cerebral hemorrhage following MCAO, an effect that was suppressed by antagonism of the C3a receptor. *P<0.001, **P<0.01, ***P<0.001 vs. controls (n = 10).
Fig 7.
tPA-mediated complement cascade activation in stroke.
Schematic depicting the interplay between cerebral ischemia and tPA, resulting in complement C3a generation, and subsequent brain injury.