Recombinant osteopontin in cerebral vasospasm after subarachnoid hemorrhage

Abstract

Objective: Osteopontin (OPN), a pleiotropic extracellular matrix glycoprotein, has been reported to be protective against ischemic lesions, but effects of OPN on vascular functions have not been investigated. The aim of this study was to assess whether recombinant OPN (r-OPN) could prevent cerebral vasospasm after subarachnoid hemorrhage (SAH) in rats.

Methods: r-OPN was administered intraventricularly to rats undergoing SAH by endovascular perforation, and its protective effects were evaluated by measuring the diameter of cerebral arteries and neurobehavioral testing. Western blotting and immunofluorescence were performed to explore the underlying mechanisms. An integrin receptor antagonist GRGDSP or mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1 small interfering RNA (siRNA) was also administered to r-OPN-treated SAH rats, and those effects were evaluated.

Results: Pre-SAH administration of r-OPN prevented vasospasm and neurological impairments at 24-72 hours post-SAH. r-OPN enhanced an endogenous MAPK inhibitor, MKP-1, and suppressed the phosphorylation of MAPKs, caldesmon, and heat shock protein 27 in the spastic cerebral arteries at 24 hours post-SAH. Immunofluorescence revealed that MKP-1 was induced in the arterial smooth muscle layer. GRGDSP prevented r-OPN-induced MKP-1 upregulation, and MKP-1 siRNA abolished both MAPK inactivation and anti-vasospastic effects by r-OPN. Post-SAH r-OPN treatment also prevented vasospasm.

Interpretation: r-OPN induced MKP-1 in the spastic cerebral arteries via binding to L-arginyl-glycyl-L-aspartate-dependent integrin receptors and prevented vasospasm after SAH. Therapeutic induction of MKP-1 may be a novel approach for the prevention and treatment of cerebral vasospasm.

Fig 1

Experimental designs. Experiment 1 (A) is designed to examine effects of pre-subarachnoid hemorrhage (SAH) treatment of 2 dosages of recombinant osteopontin (OPN) on vasospasm; experiments 2 (B) and 3 (C), to examine if an L-arginyl-glycyl-L-aspartate-dependent integrin receptor antagonist GRGDSP or MKP-1 small interfering RNA (siRNA) blocks the effects of OPN treatment, respectively; and experiment 4 (D), to examine effects of post-SAH OPN treatment on vasospasm. All treatments are performed by the intracerebroventricular infusion (ICV). Angio., angiography.

Fig 2

Effects of pre-subarachnoid hemorrhage (SAH) recombinant osteopontin (OPN) treatment on vasospasm. A, representative India ink angiograms; B–E, vessel diameter of each cerebral artery at 24 and 72 hours post-surgery. Pre-SAH OPN treatment significantly prevents vasospasm in the bilateral internal carotid arteries (B), left middle cerebral artery (C), left anterior cerebral artery (D) and basilar artery (E) associated with the improvement of neurobehavioral scores (ANOVA). Arrow, left middle cerebral artery; arrow head, left anterior cerebral artery; double arrow, left internal carotid artery; P, O 0.02, O 0.1, rats treated with phosphate-buffered saline (PBS), OPN (0.02μg) or OPN (0.1μg), respectively; n=6 per group except for n=7 in PBS- and 0.1μg of OPN-treated SAH rats at 72 hours post-SAH. Data are expressed as mean±standard deviation. ANOVA, *P<0.05, ^#P<0.05 vs. sham-operated rats treated with PBS or 0.1μg of OPN, respectively.

Fig 2

Effects of pre-subarachnoid hemorrhage (SAH) recombinant osteopontin (OPN) treatment on vasospasm. A, representative India ink angiograms; B–E, vessel diameter of each cerebral artery at 24 and 72 hours post-surgery. Pre-SAH OPN treatment significantly prevents vasospasm in the bilateral internal carotid arteries (B), left middle cerebral artery (C), left anterior cerebral artery (D) and basilar artery (E) associated with the improvement of neurobehavioral scores (ANOVA). Arrow, left middle cerebral artery; arrow head, left anterior cerebral artery; double arrow, left internal carotid artery; P, O 0.02, O 0.1, rats treated with phosphate-buffered saline (PBS), OPN (0.02μg) or OPN (0.1μg), respectively; n=6 per group except for n=7 in PBS- and 0.1μg of OPN-treated SAH rats at 72 hours post-SAH. Data are expressed as mean±standard deviation. ANOVA, *P<0.05, ^#P<0.05 vs. sham-operated rats treated with PBS or 0.1μg of OPN, respectively.

Fig 3

Representative Western blots (A) and effects of pre-subarachnoid hemorrhage (SAH) recombinant osteopontin (OPN) treatment on mitogen-activated protein kinase phosphatase-1 (MKP-1; B), phosphorylated c-Jun N-Terminal kinase (p-JNK; C), p38 (p-p38; D), extracellular signal-regulated kinase1/2 (p-ERK1/2; E), caldesmon (p-caldesmon; F) or heat shock protein 27 (p-HSP27; G) levels in cerebral arteries at 24 hours post-SAH. Pre-SAH 0.1μg of OPN significantly induces MKP-1 while reduces phosphorylated JNK, p38, ERK1/2, caldesmon and HSP27 levels in the spastic cerebral arteries associated with the improvement of vasospasm (ANOVA). Expression levels of each protein are expressed as a ratio of β-tubulin levels for normalization and as mean±standard deviation. N=4 in phosphate-buffered saline (PBS)- or OPN (0.1μg)-treated sham-operated rats; n=6 in PBS- or OPN (0.02 or 0.1μg)-treated SAH rats.

Fig 4

Immunofluorescence for smooth muscle cells (MYH11) and mitogen-activated protein kinase phosphatase-1 (MKP-1) in the intracranial internal carotid artery at 24 hours after subarachnoid hemorrhage (SAH). Pre-SAH 0.1μg of osteopontin (OPN) induces MKP-1 in the smooth muscle layer of the post-SAH arteries, in which vasospasm is improved. Sham-PBS, Sham-operated rats treated with phosphate-buffered saline (PBS); SAH-PBS or -OPN0.1, SAH rats treated with PBS or 0.1μg of OPN.

Fig 5

Representative Western blots (A) and effects of GRGDSP treatment on mitogen-activated protein kinase phosphatase-1 (MKP-1) levels (B) in cerebral arteries in subarachnoid hemorrhage (SAH) rats treated with 0.1μg of osteopontin (SAH-OPN0.1) at 24 hours post-SAH. An L-arginyl-glycyl-L-aspartate-dependent integrin receptor antagonist GRGDSP abolishes osteopontin-induced MKP-1 upregulation (unpaired t tests) irrespective of similar severity of SAH. MKP-1 levels are expressed as a ratio of β-tubulin levels for normalization and as mean±standard deviation. PBS, phosphate-buffered saline.

Fig 6

Effects of mitogen-activated protein kinase phosphatase-1 (M) small interfering RNA (siRNA) treatment on cerebral vasospasm in subarachnoid hemorrhage (SAH) rats treated with 0.1μg of osteopontin (SAH-OPN) at 24 hours post-SAH. MKP-1 siRNA abolishes osteopontin-induced improvement of vasospasm in all cerebral arteries (ANOVA; A, internal carotid artery; B, middle cerebral artery; C, anterior cerebral artery; D, basilar artery) irrespective of similar severity of SAH. Data are expressed as mean±standard deviation. C, control; sham ICV, sham intracerebroventricular infusion (burr hole only).