Recombinant Osteopontin Stabilizes Vascular Smooth Muscle Cell Phenotype Via Integrin Receptor/Integrin-linked Kinase/Rac-1 Pathway After Subarachnoid Hemorrhage In Rats

Part ?: The time course of the vascular smooth muscle phenotypic transformation after experimental subarachnoid hemorrhageObjective: This study aimed to establish subarachnoid hemorrhage(SAH)model and determine the time course of the vascular smooth muscle phenotypic transformation after experimental SAH.Methods: 36 rats were randomly assigned into 5 groups: sham(n=8);6 hours after SAH(n=6);12 hours after SAH(n=6);24 hours after SAH(n=8)and 72 hours after SAH(n=8).Western blots were used to detect the protein expression of ?-SMA and SMemb in the cerebral vessels of each group.Double immunohistochemistry staining of SMemb and von Willebrand factor were also performed in sham(n=2)and 24 hours(n=2)and 72 hours(n=2)after SAH.Results: We investigated whether the marker proteins of vascular smooth muscle phenotype would respond to early brain injury after SAH.Western blot analysis was performed to determine the protein expression of ?-SMA and SMemb at 6 hours,12 hours,24 hours,and 72 hours after SAH.Results showed that the ?-SMA level decreased as early as 6 hours after SAH and had its significant depression at 24 hours.Its level continued to decrease at 72 hours,whereas it had no significant difference at 24 hours.In addition,the expression of SMemb was significantly increased in the cerebral vessels at 24 hours compared with sham animals.To further identify the result,immunofluorescence analysis was performed for SMemb expression of the middle cerebral artery and the basilar artery.The double immunofluorescence staining revealed that the SMemb immunoreactivity increased on the wall of cerebral vessels at 24 and 72 hours after SAH.Conclusion: The phenotypic transformation of vascular smooth muscle may be involved in the pathophysiology of early brain injury in experimental SAH.Part II: Research on the effects of recombinant osteopontin on vascular smooth muscle phenotypic transformation after experimental subarachnoid hemorrhageObjective: This part was designed to investigate the effects of recombinant osteopontin(rOPN)on vascular smooth muscle phenotypic transformation after experimental subarachnoid hemorrhage(SAH).Methods: In the outcome evaluation,30 rats were randomly divided into 4 groups: sham(n=8),SAH(n=8),SAH+vehicle(5 ?L of sterile phosphate buffered saline(PBS))(n=6),and SAH+0.02 ng/?L rOPN(0.1 ng in 5 ?L of sterile PBS;n=8).Vehicle or rOPN was injected intracerebroventricularly 3 hours after SAH onset.Neurological scores,hematoxylin and eosin staining were assessed at 24 hours after SAH.Double immunohistochemistry staining of SMemb and von Willebrand factor were also performed in sham(n=2),SAH(n=2),and SAH+0.02 ng/?L rOPN(n=2).Results: We test whether rOPN attenuates the vascular smooth muscle phenotypic transformation after SAH.SAH animals demonstrated severe behavior deficits compared with sham animals.The rOPN group had a significant improvement in neurological functions compared with the SAH group.Consistent with neurological improvement,we performed immunofluorescence analysis and hematoxylin and eosin staining for the BA,the SMemb immunoreactivity increased at 24 hours after SAH,and the rOPN can reduce the immunoreactivity of the SMemb.In addition,the cross-sectional area of the basilar artery in the rOPN group was also significantly increased,and the wall thickness was significantly reduced compared with SAH.Conclusion: rOPN prevented the vascular smooth muscle phenotypic transformation and improved the neurological outcome after SAH in rats.Part ?: Research on the mechanisms of recombinant osteopontin on vascular smooth muscle phenotypic transformation after experimental subarachnoid hemorrhageObjective: This part aimed to investigate the mechanisms of recombinant osteopontin on vascular smooth muscle phenotypic transformation after experimental subarachnoid hemorrhage(SAH).Methods: Forty-eight rats were randomly assigned into 8 groups for the mechanism study: sham(n=6),SAH+vehicle(5 ?L of sterile PBS;n=6),SAH+0.02 ng/?L rOPN(0.1 ng in 5 ?L of sterile PBS;n=6),SAH+0.02 ng/?L rOPN(0.1 ng in 5 ?L sterile PBS)+ GRGDSP(100 pmol/1?L;n=6),SAH+0.02 ng/?L rOPN(0.1 ng in 5 ?L of sterile PBS)+Fib-14(0.2 mg/5 ?L;n=6),SAH+0.02 ng/?L rOPN(0.1 ng in 5 ?L of sterile PBS)+500 pmol scrambled small interfering RNA(si RNA;in 5 ?L of sterile PBS;n=6),SAH+0.02 ng/?L rOPN(0.1 ng in 5 ?L of ster-ile PBS)+500 pmol ILK si RNA(in 5 ?L of sterile PBS;n=6),and SAH+0.02 ng/?L rOPN(0.1 ng in 5 ?L of sterile PBS)+NSC2376619(30 ?g/5 ?L;n=6).Scrambled si RNA or ILK si RNA was intracerebroventricularly injected 48 hours before SAH.GRGDSP is an arginylglycylaspartic acid(RGD)-dependent integrin receptor antagonist.Fib-14 is the focal adhesion kinase(FAK)inhibitor,and NSC23766 is the specific Rac-1 inhibitor.All 3 were administered 1 hour before SAH induction.Neurological scores were performed 24 hours after SAH.Western blots and Rac-1-GTP-binding assay of cerebral vessels were conducted 24 hours after SAH in all groups(n=6).Results: First,we determined which receptor was involved in the beneficial effects observed by rOPN.The integrin receptor antagonist GRGDSP(100 pmol in 1 ?L)was administered.GRGDSP abolished the rOPN-induced improvement of neurological scores.To further evaluate the vascular smooth muscle phenotypic transformation,Western blot of ILK and p-FAK was conducted.The results revealed that rOPN treatment stabilized vascular smooth muscle phenotype by significantly improving the expression of ILK and p-FAK and GRGDSP reversed the expression of rOPN on ILK and p-FAK.Then,we determined whether the integrin signaling mediators,FAK and ILK,were able to transduce the integrin-dependent regulation mechanisms.The inhibition of FAK(Fib-14;0.2 mg dissolved in 5 ?L of PBS)administered 1 hour before SAH induction neither significantly improved the neurological defect nor modified the marker proteins of phenotype.Finally,a set of si RNAs directed against ILK were mixed and administered,and the knockdown efficiency was validated by western blot analysis.ILK levels were significantly reduced compared with the negative control si RNA groups.ILK in vivo knockdown sufficiently abolished the protective effect of rOPN treatment as shown in modified Garcia test.To further identify downstream pathways of ILK,the levels of active Rac-1 were determined.rOPN protects the vascular smooth muscle phenotype by upregulating the levels of active Rac-1,and ILK si RNA pretreatment reserved the upregulated level of active Rac-1 compared with the rOPN group,whereas scrambled si RNA did not show those effects.To strengthen the role of Rac-1 in the pathway,the inhibitor of Rac-1(NSC23766;30 ?g dissolved in 5?L of PBS)was administered.NSC23766 and ILK in vivo si RNA administrtion both decreased the levels of ?-SMA and increased SMemb compared with the rOPN treatment group,whereas scrambled si RNA did not show those effects.Conclusion: rOPN prevented the phenotypic transformation of vascular smooth muscle after SAH in rats via the integrin receptor/ILK/Rac-1 pathway.Part ?: Research on the therapeutic effect of intranasal delivery of rOPN on brain injury after experimental subarachnoid hemorrhage in ratsObjective: This part aimed to investigate the therapeutic effect of intranasal delivery of rOPN on brain injury after experimental subarachnoid hemorrhage(SAH)in rats.Methods: To propose a clinically translational route for rOPN administration as a therapy against early brain injury after SAH,intranasal administration was performed at different time points(1 hour,3 hours,and 6 hours)after SAH onset.Seventy-eight rats were randomly assigned into 5 groups,such as sham(n=18),SAH + Vehicle(50?l PBS;n=18),SAH + 0.1?g/?l rOPN(5 ?g in 50 ?l PBS,1h;n=12),SAH + 0.1?g/?l rOPN(5 ?g in 50 ?l PBS,3h;n=18),SAH + 0.1?g/?l rOPN(5 ?g in 50 ?l PBS,6h;n=12).Neurological Scores,including modified Garcia test and beam balance test,SAH grading scores,brain swelling,brain water content and India ink angiography were assessed at 24 hours after SAH in all groups.Results: To investigate the clinical translational treatment with rOPN,the nasal administration of rOPN(rOPN dissolved in PBS was administered alternately into the left and right nares.rOPN(5 ?g)in a total volume of 50 ?L was administered intranasally to each animal.)was performed at 3 different time points(1 hour,3 hours,and 6 hours)after SAH induction.There was a significant improvement in the neurological score at 1 hour and 3 hours in the treatment group.Brain swelling is represented by wet brain weight,brain volume(size),and cerebral blood volume(hemoglobin content).Both brain wet weight and brain volume were significantly increased 24 hours after SAH.The SAH group showed increased brain water content 24 hours after SAH in both hemispheres.rOPN treatment at 1 hour and 3 hours significantly reduced the brain water content in bilateral cerebral hemispheres,whereas treatment at 6 hour did not have the effect.rOPN treatment at 1 hour and 3 hours both attenuated increases in wet brain weight and brain volume compared with the SAH group.A marked increase in cerebral blood volume was noted after SAH,and cerebral blood volume in rOPN treatment at 1 hour and 3 hours were both significantly lower.India ink angiography was performed to measure the lumen diameter within each vascular segment of intracranial cerebral arteries.The 3-hour treatment group of rOPN significantly increased the lumen diameter in the left internal carotid artery,left middle cerebral artery,left anterior cerebral artery,and basilar artery 24 hours post SAH compared with the SAH group.Conclusion: Intranasal delivery of rOPN ameliorated neurological deficits and improved the brain swelling,brain edema,and lumen diameter 24 hours after SAH.