Tissue-Type Plasminogen Activator-A ^296–299 Prevents Impairment of Cerebral Autoregulation After Stroke Through Lipoprotein-Related Receptor–Dependent Increase in cAMP and p38

Abstract

Background and Purpose The sole FDA approved treatment for stroke is tPA, but its brief therapeutic window and complications of treatment constrain its use. One limitation may be its potential to exacerbate impairment of cerebral autoregulation after stroke. Vasodilation is maintained by elevations in cAMP. However, cAMP levels fall after stroke due to over-activation of NMDA receptors (NMDA-Rs) by toxic levels of glutamate, an effect that is exacerbated by tPA. Binding of wild type (wt-) tPA to the low-density lipoprotein-related receptor (LRP) mediates dilation. We propose that binding of wt-tPA to NMDA-Rs reduces cAMP and impairs vasodilation. We hypothesize that tPA-A296-299, a variant that is fibrinolytic but cannot bind to NMDA-Rs, preferentially binds to LRP and increases cAMP and p38, limiting autoregulation impairment after stroke. Methods Stroke was induced by photothrombosis in pigs equipped with a closed cranial window, CBF determined by microspheres, and CSF cAMP and p38 determined by ELISA. Results Stroke decreased CBF. CBF was reduced further during hypotension, indicating impairment of autoregulation. Autoregulation was further impaired by wt-tPA, which was prevented by MK801 and tPA-A296-299. Protection by tPA-A296-299 was blocked by anti-LRP Ab, the LRP antagonist RAP, and the p38 inhibitor SB 203580, but not by control IgG. Stroke reduced CSF cAMP, which was reduced further by wt-tPA, but augmented by tPA-A296-299. CSF p38 was unchanged by wt-tPA, increased by tPA-A296-299, and decreased by anti-LRP Ab and RAP. Conclusions tPA-A296-299 prevents impairment of cerebral autoregulation after stroke through a LRP-dependent increase in cAMP and p38.

tPA variant tPA‐A296–299 Prevents impairment of cerebral autoregulation and necrosis of hippocampal neurons after stroke by inhibiting upregulation of ET‐1

“…To follow up the implications of these findings, we next investigated the effects of impaired cerebral autoregulation on neuronal cell integrity in the hippocampus. Infarct volume as a percentage of hemispheric volume was 1 ± 1, 45 ± 5, 64 ± 5, and 4 ± 2% for sham control, stroke, stroke + wt‐tPA, and stroke + tPA‐A 296–299 animals, n = 5 (similar to previously published results; Armstead et al, ). Stroke increased the number of necrotic neurons in the CA1 and CA3 regions of the hippocampus, in the ipsilateral, but not in the contralateral hemisphere, further demonstrating the focal effects of stroke (Figure ).…”

“…An important observation in the present study is that stroke‐induced impairment of cerebral autoregulation is associated with neuronal cell necrosis in the CA1 and CA3 hippocampus. Recent studies have shown that focal photothrombotic stroke causes a local area of infarct in the piglet brain parenchyma (Armstead et al, ), as expected. However, the results of the present study surprisingly showed that such a local insult could have much more wide‐ranging injurious effects on tissue.…”

“…An important observation in the present study is that stroke-induced impairment of cerebral autoregulation is associated with neuronal cell necrosis in the CA1 and CA3 hippocampus. Recent studies have shown that focal photothrombotic stroke causes a local area of infarct in the piglet brain parenchyma(Armstead et al, 2016), as expected. However,FIG URE 1 Effect of photothrombosis (PTI) on hippocampal blood flow (ml/min.100g) during normotension and hypotension (moderate, severe) at 5h post insult in the ipsilateral vs contralateral hemisphere in the absence and presence of wt-tPA and tPA-A 296-299 (1 mg/kg iv), administered at 4h post insult, n 5 5.…”

“…This study also provides insight into the intravascular signaling pathway that impairs cerebrovascular autoregulation post stroke and administration of tPA. wt‐tPA binds to the lipoprotein‐related receptor (LRP), which mediates vasodilation and permeability of the NVU (Armstead et al, ), as well as to NMDA‐Rs, which inhibits (Figure ; Armstead et al, ). We propose that after stroke, the NMDA‐R dependent pathway shown on the right side of Figure predominates due to release of high local concentrations of endogenous (i.e., wt) tPA and endogenous glutamate and that this pathway is markedly enhanced when wt‐tPA is administered as therapy.…”

“…Based on our prior finding that the “docking site” in wt‐tPA is required to bind and activate NMDA‐Rs (Nassar et al, ), we constructed a variant (tPA‐K 296 A/H 297 A/R 298 A/R 299 A [tPA‐A 296–299 ]) that prevents the enzyme from binding to NMDA‐Rs, but retains fibrinolytic activity. Recent data show that, when given in a therapeutically relevant timeframe after stroke in pigs, tPA‐A 296–299 prevents acute impairment of cerebral autoregulation by increasing intracellular cAMP and blocking upregulation of phosphorylated JNK (Armstead, Riley, Yarovoi, Higazi, & Cines, ). We hypothesize that activation of NMDA‐Rs by wt‐tPA after stroke leads to upregulation of JNK followed by an increase in ET‐1, imparing cerebral autoregulation and leading to tissue injury.…”