The Pivotal Role Of S1P1/3Mediated Regulation On Cardiac Microvessels In Diabetic Heart I/R Injury

【Background】By2010, the prevalence of diabetes in the adult populations of the UnitedKingdom, the United States, mainland China, and the United Arab Emirates hadexceeded7%,11%,15%, and17%respectively, which is associated with aseveral fold increase in the risk of developing cardiovascular disease. Althoughgreat efforts, including revascularization, have been made on coronary artery, therate of cardiovascular accident remains high.There appears considerable evidences provide further support for a key roleof cardiac microvascular dysfunction in diabetic pathogenesis. Furthermore, it isproved that VEGF family members could promote either vascular permeability orangiogenesis, which cannot explain all the phenomena of cardiac microvascularcomplications induced by diabetes. And our previous studies also suggests thatthere exist unknown factors to be elucidated in the physiopathologic process of diabetic cardiac microvascular complications.As one pleiotropic mediator in various pathological conditions, S1P and itsreceptors(S1P1-S1P5) are contained in body fluids and tissues at differentconcentrations. Especially, the S1P1and S1P3subtypes expressed on endothelialcells are proved to make excessive contribution to the vascular stabilizationbesides VEGF mediated regulation, suggesting their threshold role in theprogression of diabetic microvascular complications in heart. Meanwhile, diversesignaling related to the function of FTY720has been attributed, in part, to theinactivation of a family of protein kinase C (PKC). Our prior study hasestablished the key role of PKCβⅡon diabetic heart disease. The next logicalstep is to prove the effect of S1P1/3on diabetic microvascular regulation and theof PKCβⅡ in this process.【Objectives】The purpose of the present report is to provide a detailed study on:1. the role of S1P1/3and its possible mechanism in diabetic heart disease, andpotential protection of FTY720on cardiac microvessels;2. the role of S1P1/3and its possible mechanism in heart I/R injury in diabetes,and the potential protection of FTY720post-treatment on cardiacmicrovessels;【Methods】1. Part1in vivo (n=10):1) Diabetic rat was induced with a single intraperitoneal injection ofstreptozotocin (50mg/kg); FTY720(1mg/kg, i.p., q.d) was performed from the4thweek according to experimental designing. All rats were harvested atthe8thweek;2) All rats were divided into four groups: Con, DM, DM+VE, DM+FTY720;3) Cardiac vascular endothelial cell quantity and apoptosis analysis wereobtained by histopathologic analysis including Tunel, CD31and DAPI;4) After vascular cast mould, cardiac microvascular angiogenesis observationswere performed under scaning electron microscope;5) With lanthanum as tracer, cardiac microvascular permeability was observedunder electron microscope;6) Laser capture microdissection was utilized to obtain distinct populations ofendothelium cells for subsequent molecular analysis;7) The expression of S1P1and S1P3in heart were preliminarily tested byimmunofluorescence;8) RT-PCR was carried through for S1P1, S1P3and PKCβⅡ.in vitro (all experiments was repeated with3times):1) Cardiac microvascular endothelial cells(CMECs) were isolated and culturedin different groups; FTY720(10nmol/L) and PKCβⅡ over-expression wasperformed according to experimental designing;2) The group of in vitro experiment included control group(Con), high glucosemedium (25mmol/L)(HG), high glucose+vehicle medium(HG+Ve), highglucose+FTY720(HG+FTY720)medium and CMECs with PKCβⅡoverexpressed in high glucose+FTY720(HG+FTY720+PKCβⅡ);3) CMEC apoptosis assessment was performed by Tunel and DAPI;4) Migration of CMECs were tested by Transwell;5) Permeability of CMECs monolayer was tested by In vitro vascularpermeability assay kit; 6) Via Subcellular Protein Fractionation Kit for culltured cells, membrane andnuclear were separated;7) S1P1, S1P3, PKCβⅡ expressional level were analyzed by Western blot.2. Part2in vivo (n=10):1) Diabetic rat was induced with a single intraperitoneal injection ofstreptozotocin (50mg/kg); I/R operation was performed at the8thweek andFTY720(1mg/kg, i.p.) was performed10min before reperfusion accordingto experimental designing. All rats were harvested after3h reperfusion;2) All rats were divided into four groups: DM+Sham, DM+I/R,DM+I/R+Ve,DM+I/R+FTY720);3) Via retrograde cannulation from right carotid artery to left vetircle,hemodynamic properties of rats were recorded;4) Cardiac vascular endothelial cell quantity and apoptosis analysis wereobtained by histopathologic analysis including Tunel, CD31and DAPI;5) With lanthanum as tracer, cardiac microvascular permeability was observedunder electron microscope6) Double staining of Thioflavin S and Evans blue was used to assessedmicrovascular occlusive region7) Laser capture microdissection was utilized to obtain distinct populations ofendothelium cells for subsequent molecular analysis8) RT-PCR was used to detect the expressional level of S1P1, S1P3andPKCβⅡ. in vitro(all experiments was repeated with3times):1) Cardiac microvascular endothelial cells(CMECs) were isolated and culturedin different groups; PKCβⅡ over-expression, SI/R (30min/3h) and FTY720treatment (10nmol/L) before reperfusion was performed according toexperimental designing;2) The group of in vitro experiment included HG, HG+Sham, HG+SI/R,HG+SI/R+Ve), HG+SI/R+FTY720, HG+SI/R+FTY720+PKCβⅡ;3) CMEC apoptosis assessment was performed by Tunel and DAPI;4) Permeability of CMECs monolayer was tested by In vitro vascularpermeability assay kit;5) Via Subcellular Protein Fractionation Kit for culltured cells, membrane andnuclear were separated;6) S1P1, S1P3, PKCβⅡ expressional conditions were analyzed by Westernblot.【Results】1. Part1in vivo:1) The achievement ratio of diabetes was more than90%(random bloodglucose≥16.7mmol/L);2) Compared with Con group, decreased CMECs quantity and increasedapoptosis index were obtained in DM group. And FTY720significantlydecreased the apoptosis induced by diabetes;3) Mess pathological angiogenesis was clearly showed in DM group andsignificantly ameliorated by FTYT20;4) Cardiac microvascular permeability was increased obviously in DM groupand FTY720could improve the barrier function. 5) The immunofluorescence results showed upregulated S1P3anddownregulated S1P1in diabetic heart. And FTY720could decreased thesechanges.6) Upregulated S1P1and PKCβⅡ expression, and downregulated S1P3expression were obtained in DM group. And normalized expression wasdemonstrated in the group receiving FTY720.in vitro:1) CMECs were successfully cultured and identified by AcLDL staining.overexpression of PKCβII based on Lentivirus was assessed as successfulaccording to the result of western blot;2) Double staining revealed a higher apotosis index in the HG and HG+Vegroups. FTY720normalized the index. Overexpression of PKCβⅡsignificantly decreased the anti-apoptosis effect of FTY720;3) The data showed that administration of FTY720in medium reversedmigration induced by high glucose to almost a normal state. However,up-regulation of PKCβII suppressed the effect of FTY720;4) FTY720attenuated the permeability change induced by high glucose.However, the effect of FTY720was significantly decreased in the PKCβⅡoverexpression group5) In HG group, a significant decrease of S1P1, and S1P3translocation fromnuclear to membrane, and increase of PKCβII were demonstrated. FTY720attenuated the changes in S1P1, S1P3, and PKCβII induced by high glucose.However, PKCβⅡ overexpression weakened the effects of FTY720withoutany influence on S1P1and S1P3. 2. Part2in vivo:1) Compared with DM, diastolic and systolic dysfunction were weakened bycardiac I/R injury. FTY720could alleviate this injury;2) Compared with DM group, sharp decreased CMECs quantity and increasedapoptosis index were obtained in DM+I/R group. And FTY720significantlydecreased the apoptosis induced by I/R;3) Cardiac microvascular barrier function was weakened obviously by I/Rinjury and FTY720was helpful to recover the permeability;4) Microvascular occlusive region of DM+I/R group is larger than DM andDM+Sham group, which could be lessened by FTY720;5) Upregulated S1P1and PKCβⅡ expression, and downregulated S1P3expression were obtained in DM+I/R group. And these changes tended to benormalized in the group receiving FTY720.in vitro:1) Overexpression of PKCβII based on Lentivirus was assessed as successfulaccording to the result of western blot;2) Compared with HG group, a higher apoptosis index was obtained inHG+SI/R group. FTY720normalized this index. Overexpression ofPKCβⅡ significantly decreased the anti-apoptosis effect of FTY720;3) Compared with HG group, FTY720attenuated the permeability changeinduced by SI/R. However, the effect of FTY720was significantly decreasedby PKCβⅡ overexpression;4) Compared with HG group, these changes in HG+SI/R group weredemonstrated including: significantly decreased S1P1expression; increase ofS1P3on membrane and decrease of S1P3on nuclear; and upregulated PKCβII level. FTY720could attenuate these changes, however, which wasundermined by PKCβⅡ overexpression.【Conclusions】1. Our study represents that the deregulation of S1P1and S1P3is an importantsignal responsible for cardiac microvascular complications in diabetes.FTY720might be competent to serve as a potential therapeutic approach fordiabetic heart disease through regulating S1P1expression and S1P3translocation, then ameliorating cardiac microvascular barrier impairment andpathologic angiogenesis, which might be partly dependant onPKCβⅡ-mediated signaling pathway.2. Our findings also indicates an important role of S1P1and S1P3in diabeticheart I/R injury. Via S1P1/3-PKCβⅡ-dependent pathway, FTY720is helpfulto decrease vascular endothelial cell apoptosis and occlusive region, andimprove cardiac microvascular barrier function, furthermore, improve cardiacfunction.