The Effect Of Substance P On Combined Transplantation Of Bone Marrow MSC And EPC After Ischemia Injury

Ischemic stroke is a complex entity with multiple etiologies and variable clinical manifestations.Middle cerebral artery(MCAO)is the most affected artery in cerebral vascular disease.Thrombosis can form in the extracranial and intracranial arteries when the intima is roughened and plaque forms along the injured vessel.Blood flow through the extracranial and intracranial systems decreases,and the collateral circulation maintains function.When the compensatory mechanism of collateral circulation fails,perfusion is compromised,leading to decreased perfusion and cell death.At the beginning of the disturbance of cerebral blood circulation,the cellular energy storage is depleted.Depleted energy deletes the ion gradient on both of extra-/intracellular environment.That disturbs the ion-homeostasis and cytotoxic neurotransmitter regulation as well.The sequential problems eventually lead to neuronal inflammation,which induces other destruction including blood-brain barrier(BBB)vasculature.BBB damage leads to increased vascular permeability,edema,and inflammatory cells infiltration,which cause a cascade of inflammatory secondary tissue damages.Early repair of BBB may protect brain tissue from further damage.BBB is composed with endothelial cells,pericytes and astrocytes.In the BBB of central nervous system(CNS),inner endothelial tube is tightly encircled by pericytes,which are sharing a basement membrane with endothelial cells and form direct synaptic-like peg-socket focal contacts between two cell types through N-cadherin and connexins.Under physiological conditions,selective permeability of BBB function strictly limit inflammatory the cells,inflammatory factors and neurotoxic substances enter into CNS;at the same time discharge metabolic substances into the pheripheral circulation,thereby maintain brain microenvironment homeostasis.After ischemic stroke,disruption of oxygen and glucose causes pericytes detachment from endothelial cells which leads increased permeability,brain edema,in turn,cause nerve inflammation.The only approved treatment for stroke is using the thrombolytic drug,recombinant tissue plasminogen activator(rt PA),to enhance the brain perfusion.The application rt PA is limited by the narrow time window which leads to only 5% of stroke patients received the treatment.Endogeneous cell replacement is not enough to repair adult CNS in patients with stroke because of the limited renewal ability and slow turnover of neural cells.Stem cell therapy has emerged as a novel and promising candidate approach for the treatment of stroke,probably by neuroprotection and neurorepairment via secreting various neural trophic factors and replacing damaged neurons.Isolated stem cells and progenitors from bone marrow,mainly refer to MSC,HSC,and EPC,have great advantages for therapeutic candidates on account of the easy collection and fast expansion in ex vivo culture.MSC transplanted into ischemic stroke indirectly promoted local blood flow through the secretion of cytokines;reduced the apoptosis of neurons,and promoted angiogenesis.At the same time,MSC was found to act as pericyte-like cell near the vascular structure,and very few transplanted MSC found expressing neuronal markers.EPC transplanted into ischemic stroke model enhanced endothelial cells repaire and angiogenesis.Many stem cell therapies using either MSC or EPC have been attempted in animal stroke model as well as in clinical trials to restore functional BBB and interrupt damage expansion at early stage of the stroke but no successful outcome has been reported yet.Considering that BBB is mainly composed of inner tubular endothelial cells,outward-encircling pericytes,and astrocyte end foot,more than one cell type may be required as reparative stem or precursor cells for the repair of BBB in the stroke.The high inflammatory environment of ischemic cerebral infarction has resulted in low grafting and survival of transplanted cells.To avoid the toxicity of the ischemic lesions and efficiently deliver stem cells into the brain,we selected the contralateral transplantation into the corpus callosum.To accelerate the migrating action,we added neuropeptide substance P(SP).SP was firstly know as main mediator of nociceptive signaling,however we firstly identified a new role of SP in tissue repair by mobilizing the bone marrow stem cells into the injury site as well as modulating the inflammatory immune responses.Importantly,SP enhanced immune suppressive actions of MSCs when ex vivo cultured.Therefore,we highly expect SP can facilitate stem cell homing to injury site and promote tissue repair with enhanced immune suppressive functions when co-injected with stem cells.However,dual cell therapy of both BM-EPC and BM-MSC anticipating tight blood vessel formation and their cooperated roles have been rarely studied.In this study,the effects of SP on co-cultured MSC and EPC were studied in vitro and in vivo after transplantation into rat model of ischemic stroke.Part I: The effect of SP on co-cultured MSC and EPC tube forming abilityMethod1.Isolation and characterization of bone marrow MSC and EPCBone marrow isolation of MSC and EPC.Characterization of MSC and EPC was detected using immunofluorescence staining and FACS of MSC and EPC marker expression.2.The effect of SP on MSC,EPC and co-cultured MSC and EPC tube forming abilityThe effects of SP on MSC,EPC and co-cultured MSC and EPC were detected using Matrigel tube foramtion at different time points.The localization of MSC and EPC in Matrigel tube formation was performed using fluorescence detection.3.The mechanism of SP promoted tube forming ability under co-culture of MSC and EPC in vitro(1)The effect of SP on tube forming related factor expression level: ELISA was applied to detect the secretion level of VEGF and PDGF-BB.Western blot was applied to detect the junction protein N-cadherin expression level.(2)The effect of NK-1R antagonist on PDGF-BB expression level: ELISA was applied after NK-1R antagonis to detect the secretion level of PDGF-BB.(3)The effect of PDGF-BB on SP enhanced tube forming ability: Matrigel tube forming assay was applied after using PDGF-BB neutralize antibody.The location of MSC and EPC was detected using fluorescence detection.Result1.Isolation and characterization of bone marrow MSC and EPCWe successfully isolated bone marrow MSC and EPC.Isolated MSC highly expressed MSC markers α-SMA、CD29、 NK-1R and CD106;low expression of CD4 and CD34.EPC highly expressed EPC markers UEA-1,CD34,and CD31.2.The effect of SP on MSC,EPC and co-cultured MSC and EPC tube forming abilityMSC was not able to form tubular structure with and without SP.SP treatment on EPC increased tube length started from 1.5h.Under co-culture condition,MSC incorporated into EPC tubular structure and with the presence of SP increased pericyte-like number of MSC.3.The mechanism of SP promoted tube forming ability under co-culture of MSC and EPC in vitro(1)The effect of SP on tube forming related factor expression level: SP treated MSC and EPC significantly increased VEGF,PDGF-BB,and junction protein N-cadherin expression level.(2)The effect of NK-1R antagonist on PDGF-BB secretion level: Secretion level of PDGF-BB was significantly decreased after applying of NK-1R antagonis.(3)The effect of PDGF-BB on SP enhanced tube forming ability: PDGF-BB neutralize antibody significantly reduced incorporated MSC into tubular structure.ConclusionSP enhance co-cultured MSC and EPC tube forming ability by increased production of PDGF-BB,incorporated MSC and further enhance junction protein N-cadherin expression.Part II: The effect of SP on combinatory transplantation of MSC and EPC after rat ischemia injuryMethod1.Induction of rat ischemic stroke and sample selectionUsing middle cerebral artery occlusion for inducing rat ischemic stroke.Sample selection based on cerebral blood flow and m NSS.2.The effect of SP combined with MSC and EPC on ischemic injury function recoveryFunction recovery was evaluated using m NSS test.Infarction area was measured using immunohistochemistry staining of MAP2.3.SP effect on MSC and EPC migration(1)SP effect on MSC and EPC migration: Using in vivo tracking and PKH labeling tracking to confirm MSC and EPC migration rount and efficiency.(2)Incorporated MSC and BBB forming astrocyteas: Immunofluorescence staining of astrocyte marker GFAP and detected location between incorporated MSC and GFAP positive astrocyte.4.The effect of SP combined with MSC and EPC on BBB restorationeEvaluation of BBB restoration using immunohistochemistry staining of BBB marker SMI71.The effect of SP was detected by quantification of SMI71 positive BBB desity,lenth and diameter.5.The effect of SP combined with MSC and EPC on inflammatory modulation and neurogenesis after ischemic injuryInflammatory regulation was detected using immunohistochemistry staining of reactive astrocyte marker GFAP and actived macrophage marker CD68;Neurogenesis was detected using immunohistochemistry staining of neuronal marker Neu N and proliferation cell marker PCNA.Result1.Induction of rat ischemic stroke and sample selectionRat ischemic stroke was induced using middle cerebral artery occlusion.Sample was selected using two standards: cerebral blood flow reduction during occlusion more than 40%,and m NSS between 5 to 10.2.The effect of SP combined with MSC and EPC on ischemic injury function recoveryMSC and EPC transplantation improved the function recovery 28 d post injury and this effect was futher enhanced by SP.MAP2 immunohistochemistry staining showed that with the presence of SP significantly protected tissue integrity,and reduced the infarction area.3.SP effect on MSC and EPC migration(1)SP effect on MSC and EPC migration:tracking of PKH labeling showed migration route of transplanted MSC and EPC was throught corpus callosum.As early as 3d post transplantation,MSC and EPC were able to be observed in ipsilateral area with MSC pericyte like coverage near vascular structure.With the presence of SP,early phase post trasnplantaion enhanced the migration on MSC into ipsilateral side and late phase increased the MSC incorporation into vascular structure.(2)Incorporated MSC and BBB forming astrocyteas: Incorporated MSC and GFAP positive astrocyte showed near vascular co-localiztion which indicate similarity of incorporated MSC with BBB structure.4.The effect of SP combined with MSC and EPC on BBB restorationeImmunohistochemistry staining of SMI71 showed most abundant in number,longest length BBB in penumbra when SP transplanted with MSC and EPC.5.The effect of SP combined with MSC and EPC on inflammatory modulation and neurogenesis after ischemic injurySP transplanted with MSC and EPC reduced GFAP positive glia scar formation and reduced actived macrophage number in penumbra area.At the same time increased the number of neurons in penumbra and the number of proliferating cells near subventricular zone.ConclusionSP combined with bone marrow derived MSC and EPC enhanced the ischemic recovery by promoting restoration of BBB,suppress the inflammatory cell activity and enhance the neurogenesis.