| BackgroundIschemic stroke leads to high rates of disability and death, thereby imposing an enormous medical and social burden. Currently available therapies for injured cerebrovascular systems are insufficient. Meanwhile, stem cell transplantation can be used to repair ischemic vascular damage and thus offers new prospects for stroke therapy. As neither immune rejection, nor the ethical questions, the bone marrow mesenchymal stem cells (MSCs), which can be drawn from oneself, are the ideal seed cells.Cerebrovascular injury induced by ischemia-reperfusion has a key function in determining the survival of nerve cells after recanalization in focal ischemia by clogging microcirculation and disrupting the blood-brain barrier integrity. Recent reports have shown that MSCs participate in neovascularization and attenuate ischemic injury after focal cerebral ischemia. However, whether MSCs transdifferentiate into and replace lost cerebrovascular cells or perform paracrine function by secreting proangiogenic factors are unclear. Therefore, the mechanism of action of MSCs especially in the interaction between brain microvessel and grafted MSCs need to be further investigated. Among the many complex mechanisms underlying vascular endothelium ischemia-reperfusion injury, mitochondrial damage appears to contribute significantly to these pathological processes. Mitochondria are essential organelles that play prominent roles in biological processes such as aerobic metabolism, oxidative phosphorylation, and cell death pathways. Endothelial mitochondria have been recognized as playing critical roles in the signaling cellular responses to environmental cues, which may determine the endothelial function and fate, thereby influencing angiogenesis in ischemia-reperfusion injury. Limited experimental data are available on the effects of stem cells on injured mitochondria in endothelial cells.Recent studies have discovered highly sensitive nanotubular structures named tunneling nanotubes (TNTs) that bridge adjacent animal cells, enabling them to form complex networks. As a novel mechanism of cell-cell communication, TNTs facilitate the exchange of cellular components and signaling molecules between connected cells such as plasma membrane components, calcium ions, pathogens, and organelles, including mitochondria. We hypothesized that stem cells and post-ischemic endothelial cells interact with each other through the formation of TNTs and that this novel mechanism might be responsible for the beneficial effects exerted by engrafted stem cells.The discovery of TNT reveals a new style for intercellular communication, which has sparked the rethink the way of cell interaction. Until now the research on TNT is still in the preliminary stage, many problems has yet to be further investigated. Our findings challenge the classical view on stem cell transplantation for stroke therapy and may also have implications in the treatment of other ischemic diseases. Part I:Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube-mediated mitochondrial transferObjectives1. To observe the TNT connections between the HUVECs and the MSCs.2. To explore the formation mechanism of TNTs between the HUVECs and the MSCs.3. To investigate the transfer of the mitochondria between the HUVECs and the MSCs through the TNT.4. To explore the protective effect of the MSCs on the injured HUVECs via TNT-mediated mitochondrial transfer.Methods1. Cell culture, identification, label, and lentiviral transduction.The human MSCs were isolated from the bone marrow of the subjects using a density gradient. The HUVECs were obtained from the Key Laboratory of Cardiovascular Proteomics of Shandong Province. The MSCs were analyzed by fluorescence-activated cell sorting (FACS) to evaluate the cell surface markers. The MSCs expressed the antigens CD105, CD29, and CD44; however, they were negative for CD45and CD34. The HUVECs and MSCs were labeled for distinction before the co-cultivation. The MSCs were incubated with lentiviral vector pWPT-enhanced green fluorescence protein (EGFP). To investigate the mitochondrial transfer, the MSC nuclei were stained with Hoechst33342before co-cultivation. The mitochondria of the MSCs and the HUVECs were labeled. The pDsRed2-Mito vector and pAcGFPl-Mito vector was used to label the mitochondria of the HUVECs and MSCs, respectively.2. Functional Measurements of Mitochondrial Activity in the MSCs and the HUVECs. The measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are indicators of aerobic respiration and glycolysis, were conducted with an XF24Extracellular Flux Analyzer. The mitochondrial function of the MSCs and the HUVECs were analyzed, and the results were normalized to the cell number.3.In Vitro Ischemia-Reperfusion Model and Co-culture ModelIn vitro ischemia-reperfusion was simulated by conducting oxygen glucose deprivation (OGD) and reoxygenation (RO) on the HUVECs in an anoxia chamber. After150min of the OGD, the RO was performed by reinstating the cells under the normoxic conditions and the pre-OGD medium. At4h of RO, an equal number of MSCs were directly added to the damaged HUVECs.4. The laser scanning confocal microscopy was conducted to investigate the TNT-like structures connections and the transport of mitochondria.The laser scanning confocal microscopy was conducted during and after the in vitro ischemia to investigate the morphological changes and possible interactions among the co-cultured HUVECs and MSCs. The TNT-like structures connections between the HUVECs and the MSCs and the membrane protrusions (MPs) were counted after24h of co-culture.5. The exchange rate of mitochondria between the HUVECs and the MSCs was analyzed using FACS analysis.To investigate the mitochondrial transfer further, the exchange rate of mitochondria between the HUVECs and the MSCs was analyzed after co-culture for48h using FACS analysis. We could also distinguish the two different cells by their Hoechst33342label and different morphologies using FACS.6. To investigate the protective effects of MSCs, the cell viability, apoptosis, and mitochondrial activity the of the HUVECs was analyzed respectively.To provide direct evidence for the functional role of mitochondria transfer via TNT-like structures, we generated MSCs with mitochondrial dysfunction using mtDNA depletion by cell treatment with ethidium bromide, which do not affect the formation of TNTs and the transfer of other molecules and organelles. After co-culture for48h, the cell apoptosis of the HUVECs we analyzed using the FITC Annexin V Apoptosis Detection Kit I (BD PharmingenTM, San Diego, CA, USA). The cell viability of the HUVECs was estimated using the Cell Counting Kit-8(CCK-8; Dojindo, Japan). The mitochondrial function of the HUVECs was analyzed using the XF24Extracellular Flux Analyzer.7. The possiblity of cell fusion, the paracrine function and the passive phagocytosis of mitochondria was respectively analyzed.The MSC conditioned media were collected after48h of culture, and the concentration of the MSC conditioned media cytokines was measured using ELISA kits [vascular endothelial growth factor,(VEGF); platelet-derived growth factor BB,(PDGF-BB); fibroblast growth factor2,(FGF-2)].To investigate the possiblity of cell fusion, the nuclear DNA and mitochondrial DNA (mtDNA) extracted from the HUVECs, the MSCs, and rescued HUVECs were assayed by direct sequencing of the PCR products and the GeneScan analysis based on the short tandem repeat sequences.To investigate the possibility that the HUVECs phagocytose MSC debris containing mitochondria and thus acquire double-labeled fluorescent dyes, the mitochondria isolated from MSCs and human platelets were used as sources of membrane-bound functional mitochondria. These were then co-cultured with the injured HUVECs for48h.Results1. MSCs have superior mitochondrial function compared to HUVECs.2. MSCs and endothelial cells can exchange mitochondria via the TNT-like structures (the basal level of bi-directional exchange of mitochondria occurs with equal frequencies).3. OGD/RO stress-induced mitochondrial transfer through the TNT-like structures becomes frequent and almost unidirectional from the MSCs to the injured endothelial cells.4. The TNT formations between the MSCs and HUVECs are substantially reduced after the LatA or Annexin V treatment. The formation of the TNT-like structures connecting the endothelial cells to the MSCs is dependent on the F-actin polymerization of and the exofacial PS domains.5. Mesenchymal stem cells can rescue aerobic respiration and protect the endothelial cells from apoptosis via tunneling nanotube like structure-mediated mitochondrial transfer. The addition of LatA or Annexin V partially suppresses this effect. The TNT-mediated protection on aerobic respiration is completely abrogated by co-cultures with the MSCs having mitochondrial dysfunction,(no statistical differences comparing with the OGD/RO group), while that the protective effect on HUVECs viability is partially suppressed (significant differences comparing with the OGD/RO group, p<0.05), suggesting that the transfer of functional mitochondria from MSCs to HUVECs was required to rescue the endothelial cells from damage, but not unique.6. The cell fusion, paracrine function, and passive phagocytosis of mitochondria can be excluded as the key mechanism for protective effects of MSCs. The potential side effects of LatA or Annexin V on the cultures of the MSCs or HUVECs were investigated. The addition of LatA or Annexin V caused negligible changes in the cell viability of MSCs or HUVECs based on the CCK-8assay and also affected the MSCs paracrine function of several primary proangiogenic factors (VEGF, PDGF-BB, FGF-2) insignificantly by ELISA analysis, suggesting that the TNT-like structures might be essential, whereas the soluble cytokines were less likely to act as the principal rescue factors.The nuclear DNA of the rescued HUVECs from five independent samples was conformably from the HUVECs and did not contain the nuclear genome from the MSCs. Cell fusion can be excluded as the mechanism for the mitochondrial transfer.The HUVECs became senescent, and double-labeled cellswere not observed. No significant changes in cell viabilityand mitochondrial activity were observed in the co-cultures compared with the injured HUVEC culture alone, which may exclude the possibility that the HUVECs recovered from injury through passive phagocytosis of the mitochondria. Part Ⅱ:Mesenchymal stem cells protect mitochondrial function of cerebral microvasculature and promote recovery from ischemic strokeObjectives1. To investigate the effect of MSCs on the infarct cortex.2. To explore the effect of MSCs on the neurobehavioral functions after stroke.3. To explore the therapeutic effect of MSCs on the angiogenesis in ischemia-reperfusion mjury.4. To investigate the effect of MSCs on mitochondrial activity of micro vascular fragments in ischemic cerebral hemisphere.5. To explore the role of TNT-mediated mitochondrial transfer in MSC grafting for the treatment of ischemic stroke in vivo.Methods1. The middle cerebral artery occlusion (MCAO) and reperfusion model.MCAO and reperfusion surgery were carried out as previously described. A nylon filament with a rounded tip was inserted into the right internal carotid artery at approximately18.5mm to20.0mm to occlude the origin of the right middle cerebral artery. After120min of MCAO, the filament was withdrawn to restore the blood flow (both occlusion and reperfusion were confirmed by laser Doppler). After the surgery, the rats were tested for neurological deficits according to Longa and Bederson’s five score regulation. The rats with scores of2or3were kept for the subsequent experiments.2. The culture, identification, label and transplantation of MSCs.The rat MSCs were isolated and cultured as previously described. To investigate the mitochondria transfer, MSC nuclei were stained with Hoechst33342, and the mitochondria of the MSCs were also labeled using pDsRed2-Mito vector. The transduced cells were purified by FACS, and then expanded. At24h after brain ischemia and reperfusion, randomly assigned animals received IA injection of MSCs, as previously described. The right carotid artery was again exposed, whereas the external carotid artery and the pterygopalatine and superior thyroid arteries were ligated or coagulated. Up to5X105MSCs in10μL of PBS were injected into the common carotid artery using a10u L syringe with a33G microneedle. No difference in mortality and morbidity was found in the different experimental groups.3. Analysis of Infarct Volume.2,3,5-triphenyltetrazolium chloride (TTC) staining for determining the therapeutic effect of MSCs on the infarct cortex. Rats (n=5per group) were anesthetized with chloral hydrate (0.4g/kg body weight, intraperitoneally) and decapitated at7days after MSC transplantation. The brains were removed and sliced into2mm-thick coronal sections, and then immersed in2%2,3,5-triphenyltetrazolium chloride (TTC; Sigma, USA) in PBS for20min. The size of the infarct area was analyzed through the digital images of the brain slices using ImageJ software. The infarct volume percentage was calculated as a percentage of the contralateral hemisphere.4. Behavioral tests.Two behavioral tests were performed to evaluate the motor function before MCAO and at1,7,14, and28days after MSC transplantation (n=20per group). A rotarod test evaluated the coordinated movements of the limbs and the body through balance on a rotarod, which was slowly accelerated from4rpm to40rpm within4min. The length of time that the animals stayed on the rod was recorded. Data were calculated as the percentage of mean duration (10trials) on the rotor-rod compared with the baseline values. Another behavioral test was performed using the running wheel systems. Rats were transferred to individual cages containing an exercise wheel coupled to a bicycle computer. Total running distance, average speed, and maximum speed were recorded at different time points. 5. Analysis of Microvessel Density.Microvessel density was measured as previously described. The rats were anesthetized and received IA injections fluorescein isothiocyanate-dextran amine (FD-2000S) to label the blood vessels at15days after MSC transplantation. The rat brains were rapidly fixed in4%paraformaldehyde and cryo-cut at30μm. Under a confocal microscope, we observed the microvessel densities in both the ischemic core and the peri-ischemic region. Microvessel density, the proportion of the area occupied by the labeled microvessels to the whole picture in pixels, was measured using ImageJ. The location of the DsRed2+/Hoechst33342+cells and DsRed2+/Hoechst33342-cells was also observed under the confocal microscope.6. Brain Microvascular Fragment Isolation and the mitochondrial activity measurements.The brain microvascular fragments of the right ischemic cerebral hemisphere of the rats were isolated using the methods described in our previous study. Fresh rat right cerebral cortices were cut into uniform1mm3sections. The sections were subsequently digested in a type II collagenase solution and collagenase/dispas solution. The microvascular fragments were purified twice in20%bovine serum albumin and33%continuous Percoll density gradient centrifugation. Purified microvascular fragments were plated on collagen type IV and fibronectin-coated XF24tissue culture plate. The mitochondrial function of the HUVECs was analyzed using the XF24Extracellular Flux Analyzer. OCR and ECAR were normalized to protein concentration for microvascular fragments. Protein measurements of the microvascular fragments were performed using a BCA Protein Assay Kit.Results1. MSCs can decrease the infarct area of stroke rats significantly (p<0.05).2. MSCs can improve the motor function of stroke rats significantly (p<0.05).3. MSCs can promote angiogenesis in ischemic penumbra of stroke rats significantly (p<0.05). 4. MSCs can protect the mitochondrial function of damaged cerebral microvasculature significantly (p<0.05).5. The the addition of LatA or Annexin V does not affect the number of DsRed2+/Hoechst33342+cells (all of the transplanted MSCs, including the transdifferentiated MSCs and the MSCs occurring cell fusion with the host cells) in the peri-infarct area, suggesting that LatA or Annexin V had little impact on the homing, retention, or migration of MSCs to the peri-infarct area. However, The addition of LatA or Annexin V can significantly decrease the number of DsRed2+/Hoechst33342-cells, and the beneficial effect of MSC grafting for the treatment of ischemic stroke is also significantly suppressed by LatA or Annexin V (p<0.05), suggesting that the beneficial effect may be principally based on TNT-mediated mitochondrial transfer, not on the paracrine, cell fusion or transdifferentiation mechanism.Conclusions1. MSCs have superior mitochondrial function compared to HUVECs. MSCs and endothelial cells can exchange mitochondria via the TNT-like structures (the basal level of bi-directional exchange of mitochondria occurs with equal frequencies).2. OGD/RO stress-induced mitochondrial transfer by TNT formation becomes frequent and almost unidirectional from MSCs to injured endothelial cells, thereby resulting in rescue of aerobic respiration and protection of endothelial cells from apoptosis.3. TNT formation might represent a defense and rescue mechanism. The formation of TNTs connecting endothelial cells to MSCs is dependent on F-actin polymerization of and the exofacial PS domains.4. MSC transplantation in vivo indirectly supports the function of TNT-mediated mitochondrial transfer in protecting the brain microvascular system from ischemic-reperfusion injury, enhancing angiogenesis, reducing infarct volume, and improving functional recovery. While the cell fusion, paracrine function, passive phagocytosis of mitochondria, and the transdifferentiation can be excluded as the key mechanism for protective effects of MSCs. |
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