Novel plasminogen activator inhibitor-1-derived peptide protects against impairment of cerebrovasodilation after photothrombosis through inhibition of JNK MAPK

Abstract

The sole FDA-approved treatment for acute stroke is recombinant tissue-type plasminogen activator (rtPA). However, rtPA aggravates the impairment of cerebrovasodilation induced by global hypoxia/ischemia; this impairment is attenuated by the preinjury treatment with the plasminogen activator inhibitor derivative EEIIMD. MAPK (a family of kinases, p38, and JNK) is upregulated after cerebral ischemia. In this study, we determined whether the novel plasminogen activator inhibitor-derived peptide, Ac-RMAPEEIIMDRPFLYVVR-amide, (PAI-1-DP) given 30 min before or 2 h after, focal central nervous system injury induced by photothrombosis would preserve responses to cerebrovasodilators and the role of p38 and JNK MAPK in such effects. Cerebrospinal fluid JNK and p38 levels were elevated by photothrombotic injury, an effect potentiated by rtPA. Cerebrovasodilation was blunted by photothrombosis and reversed to vasoconstriction by rtPA but restored to dilation by PAI-1-DP pre- and posttreatment. PAI-1-DP blocked JNK, but preserved p38 MAPK upregulation after photothrombosis. The JNK MAPK antagonist SP600125 prevented, and the p38 antagonist SB203580 potentiated, impaired cerebrovasodilation after photothrombosis. These data indicate that rtPA impairs cerebrovasodilation after injury by activating JNK, while p38 MAPK is protective, and that the novel peptide PAI-1-DP protects by inhibiting activation of JNK by rtPA. JNK MAPK inhibitors, including PAI-1-DP, may offer a novel approach to increase the benefit-to-risk ratio of thrombolytic therapy and enable its use in central nervous system ischemic disorders.

Fig. 1.

Pretreatment phosphorylation of JNK MAPK in cerebrospinal fluid (CSF) prior to photothrombotic injury (PTI) (0 min) and as a function of time (hour) after PTI in vehicle or pretreated with recombinant tissue-type plasminogen activator (rtPA; 2 mg/kg iv), Ac-RMAPEEIIMDRPFLYVVR-amide [PAI-1-derived peptide (PAI-1-DP)], U0126, SB203580, SP600125, or D-JNKI1 (ERK, p38, and JNK MAPK inhibitors, all 1 mg/kg iv), n = 5 per group. Data are expressed as %control by ELISA determination of phospho-MAPK and total MAPK isoforms and subsequent normalization to total form. Pretreatment was 30 min before PTI. *P < 0.05 vs. corresponding time 0; +P < 0.05 vs. corresponding PTI-nontreated value.

Fig. 2.

Posttreatment phosphorylation of JNK MAPK in CSF prior to PTI (0 min) and as a function of time (hour) after PTI in vehicle, or posttreated with rtPA (2 mg/kg iv), PAI-1-DP, U0126, SB203580, SP600125, or D-JNKI1 (ERK, p38, and JNK MAPK inhibitors, all 1 mg/kg iv), n = 5 per group. Data expressed as %control by ELISA determination of phospho-MAPK and total MAPK isoforms and subsequent normalization to total form. Posttreatment time was 2 h after PTI. *P < 0.05 vs. corresponding time 0 value; +P < 0.05 vs. corresponding PTI nontreated value.

Fig. 3.

Pretreatment phosphorylation of p38 MAPK in CSF prior to PTI (0 min) and as a function of time (hour) after PTI in vehicle, or pretreated with rtPA (2 mg/kg iv), PAI-1-DP, U0126, SB203580, SP600125, or D-JNKI1 (ERK, p38, and JNK MAPK inhibitors, all 1 mg/kg iv), n = 5 per group. Data expressed as %control by ELISA determination of phospho-MAPK and total MAPK isoforms and subsequent normalization to total form. Pretreatment was 30 min before PTI. *P < 0.05 vs. corresponding time 0 value; +P < 0.05 vs. corresponding PTI nontreated value.

Fig. 4.

Posttreatment phosphorylation of p38 MAPK in CSF prior to PTI (0 min) and as a function of time (hour) after PTI in vehicle, or posttreated with rtPA (2 mg/kg iv), PAI-1-DP, U0126, SB203580, SP600125, or D-JNKI1 (ERK, p38, and JNK MAPK inhibitors, all 1 mg/kg iv), n = 5 per group. Data expressed as %control by ELISA determination of phospho-MAPK and total MAPK isoforms and subsequent normalization to total form. Posttreatment time was 2 h after PTI. *P < 0.05 vs. corresponding time 0 value; +P < 0.05 vs. corresponding PTI nontreated value.

Fig. 5.

Pretreatment influence of hypotension (moderate, severe) on pial artery diameter in newborn pigs before (control), after PTI, or pretreated with rtPA (2 mg/kg iv), PAI-1-DP, U0126, SB203580, SP600125, or D-JNKI1 (ERK, p38, and JNK MAPK inhibitors, all 1 mg/kg iv), n = 5 per group. Pretreatment time was 30 before PTI. *P < 0.05 vs. corresponding control value; +P < 0.05 vs. corresponding nontreated PTI value; #P < 0.05 vs. corresponding PTI+rtPA value.

Fig. 6.

Posttreatment influence of hypotension (moderate, severe) on pial artery diameter in newborn pigs before (control), after PTI, or posttreated with rtPA (2 mg/kg iv), PAI-1-DP, U0126, SB203580, SP600125, or D-JNKI1 (ERK, p38, and JNK MAPK inhibitors, all 1 mg/kg iv), n = 5 per group. Posttreatment time was 2 h after PTI. *P < 0.05 vs. corresponding control value; +P < 0.05 vs. corresponding nontreated PTI value; #P < 0.05 vs. corresponding PTI+rtPA value.

Fig. 7.

Pretrement influence of hypercapnia (Low, High) on pial artery diameter in newborn pigs before (control), after PTI, or pretreated with rtPA (2 mg/kg iv), PAI-1-DP (PAI-1-DP), U0126, SB203580, SP600125 or D-JNKI1 (ERK, p38, and JNK MAPK inhibitors, all 1 mg/kg iv), n = 5 per group. Pretreatment time was 30 before PTI. *P < 0.05 vs. corresponding control value; +P < 0.05 vs. corresponding nontreated PTI value; #P < 0.05 vs. corresponding PTI +rtPA value.

Fig. 8.

Posttreatment influence of hypercapnia (Low, High) on pial artery diameter in newborn pigs before (control), after PTI, or posttreated with rtPA (2 mg/kg iv), PAI-1-DP, U0126, SB203580, SP600125, or D-JNKI1 (ERK, p38, and JNK MAPK inhibitors, all 1 mg/kg iv), n = 5 per group. Posttreatment time was 2 h after PTI. *P < 0.05 vs. corresponding control value; +P < 0.05 vs. corresponding nontreated PTI value; #P < 0.05 vs. corresponding PTI +rtPA value.