Combination treatment with N-acetyl-seryl-aspartyl-lysyl-proline and tissue plasminogen activator provides potent neuroprotection in rats after stroke

Abstract

Background and purpose: N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), an endogenously produced circulating peptide in humans and rodents, exerts anti-inflammatory and cardioprotective activities in various cardiovascular diseases.

Methods: The present study evaluated the neuroprotective effect of AcSDKP alone and in combination with thrombolytic therapy in a rat model of embolic focal cerebral ischemia.

Results: We found that treatment with AcSDKP alone at 1 hour or the combination treatment with AcSDKP and tissue plasminogen activator (tPA) at 4 hours after stroke onset substantially increased AcSDKP levels in plasma and cerebrospinal fluid and robustly reduced infarct volume and neurological deficits, without increasing the incidence of brain hemorrhage compared with ischemic rats treated with saline, AcSDKP alone at 4 hours, and tPA alone at 4 hours. Moreover, the combination treatment considerably reduced the density of nuclear transcription factor-κB (NF-κB), transforming growth factor β (TGF-β), and plasminogen activator inhibitor-1 (PAI-1) positive cerebral blood vessels in the ischemic brain, all of which were associated with reduced microvascular fibrin extravasation and platelet accumulation compared with tPA monotherapy. In vitro, AcSDKP blocked fibrin-elevated TGF-β1, PAI-1, and NF-κB proteins in primary human brain microvascular endothelial cells.

Conclusions: Our data indicate that AcSDKP passes the blood-brain barrier, and that treatment of acute stroke with AcSDKP either alone at 1 hour or in combination with tPA at 4 hours of the onset of stroke is effective to reduce ischemic cell damage in a rat model of embolic stroke. Inactivation of TGF-β and NF-κB signaling by AcSDKP in the neurovascular unit may underlie the neuroprotective effect of AcSDKP.

Keywords: capillary permeability; ischemia; stroke.

Figure 1. Infarct volume and neurological functional outcome

Panel A shows the effects of AcSDKP alone and in combination with tPA on infarct volume assessed 7 days after MCAO. Panel B, C, and D show the neurological functional outcome measured by neurological severity score, foot-fault test, and adhesive removal test at 1 (black bar) and 7 (open bar) days after stroke onset, respectively.

Figure 2. MRI measurement

Panels A and B show T2 weighted MRI at 1, 72and 144 h after MCAO of a representative rat treated with tPA (A) and the combination of AcSDKP and tPA (B) 4 h after stroke. Panels C and D are H&E stained sections obtained from the same representative rats 7 days after MCAO, respectively. Bar graph shows quantitative data of T2 values.

Figure 3. Plasma and CSF AcSDKP level

Bar graph shows plasma and CSF AcSDKP level measured by EIA in normal and ischemic rats 24h after initiation of treatment.

Figure 4. Fibrin and platelet accumulation

Panel A shows immunoreactivity of fibrin (red) and EBA (green) in representative rats treated with tPA monotherapy and the combination of AcSDKP and tPA. Bar graph B shows the quantitative data of extravascular fibrin leakage in the ischemic brain. Panel C show thrombocyte immunoreactivity (green) in EBA immunoreactive vessels (red) in the representative rats treated with tPA monotherapy and the combination of AcSDKP and tPA. Panel D shows the quantitative data of thrombocyte immunoreactive vessels in the ischemic brain. Bars=100 μm.

Figure 5. Cerebrovascular TGFβ1, p65, and PAI-1 expression

Panel A shows immunoreactivity of TGFβ1 in a representative normal, ischemic rats treated with tPA monotherapy, and ischemic rats treated with the combination of AcSDKP and tPA. Panel B shows colocalization of pSmad2/3 (red) with TGFβ1 immunoreactive vessels (green) in a representative ischemic rat treated with saline. Panel C shows double immunofluorescent staining of PAI-1 (green) with EBA (red) from a representative normal rat, and in ischemic rats treated with saline, tPA, or AcSDKP+tPA. Panel D shows p65 immunoreactivities from a representative normal rat, and in ischemic rats treated with saline, tPA, or AcSDKP+tPA. Bar graphs show the quantitative data of TGFβ1, pSmad2/3, PAI-1, and p65 immunoreactive vessels in the ischemic brain, respectively. Bars=50 μm.

Figure 6. Effects of AcSDKP on cerebral endothelial cells

Panel A shows the representative Western blots of TGFβ1, PAI-1, and p65 in HBECs cultured with fibrin in the presence or absence of AcSDKP. Bar graphs show the quantitative data of TGFβ1(B), PAI-1(C), and p65(E) protein levels on HBECs, respectively. *p<0.05 vs Control. ⁺p<0.05 vs Fibrin.