Mesenchymal Stem Cells Repair The Injured Cardiac Function Post Myocardial Infarction: An Experimental Study

Background and Aims:Ischemic heart disease?IHD?,also known as coronary atherosclerotic heart disease?CHD?,which can lead to myocardial infarction?MI?and heart failure?HF?,has become the leading cause of morbidity and mortality worldwide.During AMI,large numbers of cardiomyocytes are lost and replaced by non-contractile scar tissue because of the extremely limited regenerative ability of cardiomyocytes,thus leading to cardiac structural and functional remodeling,finally resulting in HF.Stem cells-based therapy provides a promising therapeutic strategy for promoting the recovery of impaired cardiac function post MI through paracrine,immunoregulation,proliferation and differentiation.In the past decades,multiple preclinical and clinical studies found that the regenerative stem cells or progenitor cells had showed good therapeutic effects in MI.However,the therapeutic effects remain inadequate for clinical practice.The poor long-term survival of the engrafted stem cells limits the therapeutic efficacy,which is not enough to replace the loss of heart function.A large number of previous studies have shown that more than 90%of the engrafted stem cells might not survive within the first 24 h.The main causes of the low survival outcome of transplanted stem cells include:the local microenvironment,such as necrotic tissue accumulation,acute inflammation,oxidative stress and ischemic anoxia post MI/reperfusion,is not conducive to the colonization and survival for transplanted cells;the scavenging effect of mononuclear macrophage system on the necrotic tissue post MI and the transplanted cells;mechanical extrusion of incessantly beating heart and flushing out through needle puncture hole lead to acute loss of the engrafted stem cells.Based on the above background,in order to improve the survival rate of the engrafted stem cells,the following two aspects were investigated in the present study.First,we used the ERS inhibitor PBA to inhibit the MI-activated ERS,which is the key mechanism to regulate the acute inflammation and oxidative stress,in order to improve myocardial AMI microenvironment and increase the survival of the transplanted stem cells.Second,we used a tissue engineering melatonin sustained-release cardiac patch as the carrier of stem cells through the epicardial pathway,in order to facilitate the transplanted stem cells to avoid direct contact with the harmful microenvironment and improve their survival rate.ERS is characterized by the accumulation of unfolded/misfolded proteins.Environmental insults or increased protein synthesis often lead to protein misfolding in the organelle.If the ER function is severely impaired because of excessive or prolonged exposure to stress,then the inflicted cells may undergo programmed cell death.Many studies have shown the association between ERS and inflammation and oxidative stress as well as apoptosis.Studies have confirmed the association between ERS and inflammation and ERS has also been linked to inflammatory basis of metabolic disease.In addition,PBA,an ERS inhibitor,was found to be beneficial to septic shock via inhibition of ERS-mediated oxidative stress,apoptosis,and cytokine release.Moreover,a study indicated that ERS was activated in MI and ERS signaling system had an important potential to act as regulator for myocardial survival during ischemia.In our previous study,we found that chitosan/silk fibroin modified nanofibrous cardiac patch,as the stem cell carrier,can improve the stem cell retention and survival rate post AMI through the simulation of the ECM,and the patch mechanical support can delay the progress of HF post AMI.Our previous research also confirmed melatonin can inhibit cell apoptosis and improve the survival rate of transplanted stem cells via inhibition of inflammation and oxidative stress through SIRT1 signal pathway.Because the infarcted and ischemic area post AMI is relatively isolated from the normal blood circulation and the epicardial cardiac patch in the early stage of transplantation is also in the isolated state with the body's blood circulation,systemic application of melatonin is difficult to achieve effective level in either myocardial injection or patch contained stem cells,even if the blood concentration is in the effective range.Hence,in the present study,a melatonin sustained release cardiac patch,as carrier for transplantated stem cells,was prepared to improve AMI myocardial microenvironment by local administration of melatonin and to promote the survival of the dying cardiomyocytes.On the other hand,the cardiac patch improved the survival rate of transplanted cells by the cyto-protective effect of stem cell under anoxic condition.The melatonin sustained-release patch was fabricated through the electrospinning method.PVP as the core,PCL as the outer layer,melatonin was loaded in the inside to prepare of a multilayer nanofiber to achieve melatonin sustained-release for continuous local administration to improve the survival rate of transplanted cells in order to facilitate the stem cells to play a more effective treatment effect.Methods:1.Fluc⁺–eGFP⁺transgenic mice were bred on a C57BL/6a background to stably express both firefly luciferase?Fluc?and enhanced green fluorescence protein?eGFP?in all tissues and organs and 8 week old male mice were used for BMSCs isolation.Fluc can be used for in vivo tracing of transplanted stem cells by BLI.GFP can be used as a histological marker for the outcome of transplanted stem cells.Because of GFP,the morphology and GFP activity of cells can be directly observed by fluorescence microscope.BLI can analyze the linear relationship between Fluc signal intensity and cell number,and cultured BMSCs biomarkers were identified by flow cytometry.2.Syngeneic male C57BL/6a mice with the same genetic background as the transgenic mice were randomized into five groups?wild type,8 week old,n=20 each?:Sham group,MI group,MI+BMSCs group,MI+PBA group and MI+BMSCs+PBA group ?PBA in the drinking water,500mg/kg/day,starting the day of MI model and continuing for 4 weeks?.Mouse MI model was established by ligation of LAD with 6-0 silk.Sham operated control group underwent the same surgical procedures except that the suture placed under the LAD was not tied.1×10⁶ BMSCs were intra-myocardially injected into the peri-infarct areas at three different sites with a total volume of 20ul in each mouse.Control animals received a same volume of PBS injection instead.3.For in vivo cell viability,engrafted BMSCs were detected on day 1,7,14,21and 28 days post operation by BLI.To evaluate the BMSCs viability in the heart tissue after BMSCs transplantation,immunofluorescence stain of cTn I/GFP?7 days post-operation?was performed.Transthoracic echocardiography?TTE?was recorded at 1,7,14,21,and 28 days post operation for cardiac function.ERS-related CHOP was assessed by nuclear CHOP level with immunofluorescence stain with primary antibody 3 days post-operation.ERS biomarkers,including ATF4,CHOP,GRP78 and GRP94,were examined to evaluate the ERS activation 3 days post-operation.TUNEL was performed to determine the apoptosis followed the manufacturer's instructions 3 days post-operation.The amount of IL-6,TNF-?and IL-10 in heart tissues was examined 3 days post-operation.Myocardial reactive oxygen species?ROS? generation in myocardium was examined 3 days post-operation using the ROS fluorescent probe-DHE by confocal microscope via in-situ DHE staining.Myocardial fibrosis was examined by Masson's trichrome stain to label collagen scar tissue and cardiac muscle 28 days post-operation.To evaluate the angiogenesis in the heart tissue after BMSCs transplantation,immunofluorescence stain of CD31?28 days post-operation?was performed.4.BMSCs were treated with hypoxia/serum deprivation?H/SD?.Briefly,BMSCs were exposed to hypoxia?94%N₂,5%CO₂,and 1%O₂?in an anaerobic system at 37? for 6 hours.In the control group,BMSCs were maintained at normoxia?95%air,5%CO₂? for equivalent periods.The application dose of PBA was 2mmol/L.Because CHOP is the key downstream factor of ERS,BMSCs were transfected by lentivirus to up-regulate the CHOP expression to study the impact on the effect of PBA.Immediately after H/SD,cell supernatant was collected to measure inflammatory factors and ROS related cytokine.In vitro assessment of BMSCs viability was detected by BLI.Immediately after H/SD,cells were collected to measure ROS generation.Production of intracellular ROS was assessed by measuring the sensitive ROS fluorescent probe-DCFHDA.WB of NF-?B-p65 and gp91phox levels was to assess inflammation and oxidative stress,respectively.5.The melatonin sustained-release patch was fabricated through the coaxial electrospinning method.PVP as the core,PCL as the outer layer,melatonin was loaded inside to prepare a multilayer nanofiber to achieve melatonin sustained-release.The patches,cut into 96-well plate size,were infiltrated in BMSCs medium.The supernatant was collected in 12,24,36,48,60,72,84,96 hours later,respectively.Then the concentration of melatonin was detected with ELISA kit,and then the melatonin release curve was drawn.In order to verify the cyto-protective effect of the patch,H/SD model is also used in this part,and the specific steps are described as described.In vitro experiments were divided into six groups:normal control group ?Con?;BMSCs were cultured in normal conditions with non-melatonin patch?non-mel patch?and melatonin patch?Mel patch?,hypoxia BMSCs?H/SD?;BMSCs were cultured under hypoxic condition with non-melatonin patch?H/SD+non-mel patch? and melatonin patch?H/SD+mel patch?.Immediately after H/SD,cell supernatant was collected to measure inflammatory factors and ROS related cytokine,as well as paracrine factors bFGF,VEGF and SDF-1.In vitro assessment of BMSCs viability was detected by BLI.6.Wild type SD rats?male,140-160g?were randomized into six groups?each n=20?: Sham group,MI group?MI model was established by ligation of LAD?,non-mel patch group,mel patch group,BMSCs+non-mel patch and BMSCs+mel patch group ?Patches with or without BMSCs were adhered onto the epicardium over the infarcted region respectively,patch size:9×9 mm²,BMSCs count:2×10⁵,co-cultured for 24 h?.Myocardial apoptosis in the peri-infarct area was determined by TUNEL staining 24 hours post-operation.Viability of engrafted BMSCs was tracked using BLI via built-in Fluc in vivo 1,7,14,21 and 28 days post-operation.The amount of IL-6,TNF-?and IL-10 in heart tissues was examined 3 days post-operation.Myocardial reactive oxygen species?ROS?generation in myocardium was examined 3 days post-operation using the ROS fluorescent probe-DHE by confocal microscope via in-situ DHE staining.Cardiac function was monitored by transthoracic echocardiography?TTE?.Histopathological stainings?Masson's Trichrome,cTn I/GFP and CD31 immunofluorescence?were performed to evaluate myocardium fibrosis,BMSCs viability and neovascularization.Results1.BMSCs isolated from Fluc⁺–eGFP⁺transgenic mice displayed a fibroblast-like morphology and showed green fluorescence under a microscope.Fluc-signal intensity,as measured by BLI,exhibited a linear correlation with the number of BMSCs.Flow cytometry confirmed that the BMSCs show the distinct MSC surface markers CD44?CD29 and CD90 and negative for CD31?CD34 and CD45.2.BLI signal intensity in the BMSCs+PBA group experienced a progressive decline during the following 4 weeks and was still detectable till 4 weeks after BMSCs engraftment.However,Fluc signal of BMSCs group were undetectable 4 weeks post-operation.In keeping with the BLI,immunofluorescence displayed more GFP-positive BMSCs within the ischemic heart in PBA-treated group on POD7 compared with BMSCs group.The combined therapy of BMSCs and PBA significantly lowered AI compared with BMSCs group and PBA group.4 weeks later,combined therapy of BMSCs and PBA resulted in further significant improvement of EF and FS compared with BMSCs group and PBA group.Masson's trichrome stain showed that myocardial fibrosis was reduced in BMSCs group and PBA group and combined therapy of BMSCs and PBA more significantly decreased myocardial fibrosis post MI.CD31-positive tubular structures showed that the density of neovascularization in the peri-infarcted area markedly increased in BMSCs and PBA groups than that in the MI group and combined therapy of BMSCs and PBA showed a much more pronounced improvement.Either BMSCs or PBA was able to reverse the increase of pro-inflammatory cytokines TNF-?and IL-6 and further enhance the increase of anti-inflammatory cytokine IL-10 in infarcted myocardium,while combined administration of BMSCs and PBA further strengthened this trend.Immunofluorescence staining of CHOP and WB of ERS biomarkers indicated that PBA decreased the MI-activated ERS.DHE staining showed that either BMSCs or PBA significantly lowered MI-induced increase of DHE fluorescence intensity,while combined therapy of BMSCs and PBA further decreased DHE fluorescence intensity.3.TUNEL assay showed that the apoptotic index?AI?was promoted in BMSCs subjected to H/SD.PBA can attenuate H/SD-induced apoptosis and this effect was partly counteracted by CHOP up-regulation by lentivirus.BLI depicted that the suppressed BMSC viability under H/SD injury can be reversed by PBA,while the CHOP up-regulation counteracted the effect?versus the H/SD group,P<0.05?.Similar to the results of animal experiments,PBA can relieve the release of pro-inflammatory cytokines?IL-6 and TNF-??induced by H/SD and further increased the release of anti-inflammatory cytokines?IL-10?.These effects were counteracted by CHOP up-regulation by lentivirus.Flow cytometry showed that H/SD injury greatly increased the ROS generation and PBA can reduce the H/SD-induced ROS generation.However,CHOP up-regulation by lentivirus counteracted the effect.WB showed that PBA can reduce the H/SD-induced overexpression of NF-?B-p65 and gp91phox,while CHOP up-regulation by lentivirus counteracted the effect.4.Melatonin release curve showed that the patch was still sustained-release melatonin until 108 hours.Similar to the results of animal experiments,PBA can relieve the release of pro-inflammatory cytokines?IL-6 and TNF-??induced by H/SD and further increased the release of anti-inflammatory cytokines?IL-10?.These effects were counteracted by CHOP up-regulation by lentivirus.TUNEL staining showed reduced apoptotic index in MI+patch/melatonin/BMSCs group.ELISA showed that after H/SD treatment,BMSCs?H/SD and H/SD+non-mel group patch?supernatant of inflammatory factors IL-6,TNF-a and IL-10 levels were elevated,and BMSCs co-cultured with melatonin sustained-release patch?H/SD+mel patch?,compared to H/SD+non-mel patch and and H/SD,the proinflammatory cytokines IL-6 and TNF-? decreased,while anti-inflammatory factor IL-10 increased.Compared with Con group,the levels of SOD,GSH and MDA in supernatant decreased in H/SD treated BMSCs ?H/SD and H/SD+non-mel patch group?,and more significantly than that in BMSCs co-cultured with melatonin sustained release patch?H/SD+mel patch?.The levels of paracrine factors bFGF,VEGF and SDF-1 under hypoxia stimulation?H/SD and H/SD+non-mel patch group?were slightly higher than those in normal control group,while BMSCs co-cultured with melatonin sustained release patch?H/SD+mel patch? increased significantly.BLI showed that the Fluc signal of BMSCs in H/SD+mel patch group was stronger than that of H/SD and H/SD+non-mel patch groups.5.BLI showed Fluc signal of BMSCs+mel-patch group decreased significantly weaker than BMSCs+non-mel patch group over the 4 weeks post-operation.The immunofluorescence staining of tissue GFP was consistent with BLI:GFP positive cells of BMSCs+mel-patch rats were more than that in BMSCs+non-mel patch rats.Echocardiography showed that there was no significant difference in left ventricular ejection fraction?EF?and fraction shortening?FS?between all groups on baseline and POD 1.However,4 weeks later,EF and FS were improved in both BMSCs+mel-patch group and BMSCs+non-mel patch BMSCs+non-mel patch BMSCs+non-mel patch group in comparison to that of MI group.However,4 weeks later,EF and FS were improved in BMSCs+mel-patch group more significantly.3 days post the operation,the apoptosis index and DHE fluorescence intensity were higher in the BMSCs+non-mel patch group than in the BMSCs+mel patch group.ELISA results showed that the levels of IL-6 and TNF-?in BMSCs+mel-patch were lower than those of BMSCs+non-mel patch group,while the levels of anti-inflammatory factor IL-10 were higher than those of BMSCs+non-mel patch group.28 days after operation,the degree of cardiac fibrosis in group BMSCs+mel patch was significantly lower than that in group BMSCs+non-mel patch,and the neovascularization of the tissue was higher than that of the non-mel patch group.Conclusions1.In this study,we verified the activation of ERS in MI by histology and protein experiments in a mouse model of MI.We found that anti-ERS by the chemical chaperones PBA can alleviate inflammation,oxidative stress and apoptosis post MI via inhibiting PERK–eIF2?–ATF4–CHOP signaling pathway.This is crucial for the survival of the engrafted BMSCs,which is also verified by the BMSCs tracing in vivo. In addition,we found that PBA could improve the survival of BMSCs treated with hypoxia/serum deprivation?H/SD?via inhibition of the activated ERS and up-regulation of CHOP by lentivirus can counteract this benefit,which further confirmed that the protective effect of PBA is achieved by inhibiting ERS via PERK signaling pathway.Our data support anti-ERS treatment as a promising strategy and a novel therapeutic target for MSC-based MI therapy.2.In this study,we demonstrate that the melatonin sustained-release cardiac patch improved retention and viability of the engrafted BMSCs.In vitro experiments showed that the patch had cytoprotective effect for BMSCs on anoxic condition and increased BMSCs's anoxia tolerance.Animal experiments confirmed that melatonin sustained-release patch as a BMSC carrier could increase the survival rate and promote the recovery of the injured cardiac function.The patches with BMSCs have the therapeutic effect on improving cardiac function and slowing the progression of HF.The mechanism of the therapeutic effect may be associated with alleviating cardiac fibrosis,inhibiting apoptosis and promoting angiogenesis.Our findings suggest the melatonin sustained-release cardiac patch as an effective and recommended strategy for stem cell-based therapy.