PH-sensing G Protein-coupled Receptor GPR65 Promotes The Effect Of Adipose-Derived Stem Cells In Critical Ischemic Limbs

Backgrounds and Objectives:In peripheral arterial disease(PAD),the narrowed or occluded arteries leads to the insufficient blood supply to lower limbs.Atherosclerotic plaque in lower limbs or thrombosis in hypercoagulable state are the most common etiologies for it and severe limb ischemia(Critical limb ischemia,CLI)is the most advanced stage of it.The prognosis of CLI patients is such poor following with the amazing mortality and amputation rate.There are 20%-50% of no-option CLI patients for revascularization,which have to follow the therapy of amputation.Thus,there is an urgent need to develop a novel method of angiogenesis to improve the blood flow for these patients,reducing the bad mortality and amputation rate.And stem cell-based therapy with the high expectation from researchers and patients has been explored for two decades.Although a growing number of experimental data about stem cell-based therapy is always positive,the results from clinical trials failed the expectations.In most cases,the pain scores,walking distance without pain and percutaneous oxygen partial pressure in patients undergoing treatment have been improved,However,there was no benefits from the mortality and amputation rate for now.Which caused the poor result of clinical trials? A key point is the complicated microenvironment in vivo of CLI patients.Especially,for these CLI patients with long-term metabolic imbalances,such as atherosclerosis and diabetes,their environments in vivo have a such high possibility to trigger damages for transplanted stem cells.Acidosis,a typical pathological feature in hypoxia and inflammation,is the result of modification of metabolic pattern following with a lower pH in extracellular environments.Previous reports pointed out that,compared with healthy people,the pH in gastrocnemius surface of PAD patients was generally decreased in a severity-dependent manner.Besides,the pH in gastrocnemius muscle of CLI patients with gangrene was reduced to 6.85.Therefore,the level of pH could reflect the severity of lower limb ischemia partly,which means the pH in ischemic limbs from different CLI patients is unlike and the individual pH will cause different impairments to transplanted stem cells.In this way,we have to realize the all possible damages caused by extracellular acidosis for finding way to avoid ruins.For this,we focused on a member of proton-sensing G protein-coupled receptors family,GPR65.GPR65 is activated by acidic conditions of pH<7.4 and achieved the maximum activity between pH=6.2-6.8,which is consistent with the pathological microenvironment in CLI.There were some reports pointed out that GPR65 could avoid the acidosis-induced apoptosis through the Bcl-2 pathway effectively,and inhibit the immune and inflammatory responses significantly.Therefore,the purpose of this study was to explore the effect of GPR65 in acidosis-induced damages on adipose-derived stem cells(ADSCs)and check its function of improving the survival and biological effects under acidic microenvironments.Also,we would like to suggest a potential target gene to improve the efficacy of stem cell-based therapy in clinical applications via experiments in vitro and vivo.Methods:Firstly,we established the model of severe lower limb ischemia in nude mice and used microdialysis technology to detect the metabolic level in ischemic lower limb muscles 0.5-72 hours after the ischemic event,which determined the exist of acidosis ischemic limbs.Then,the primary adipose-derived stem cells(ADSCs)were successfully isolated from human adipose tissues with the nice potential of differentiation and stable passages.Next,in vitro experiment,we treated ADSCs with acidic conditions(pH=7.4/7.2/7.0/6.5)for 24/48 hours and explores the impairments of extracellular acidosis on ADSC via cell functions,cell subcellular morphology and transcriptional analysis.For the study of GPR65,we constructed the GPR65 overexpression(GPR65-OE)plasmid,and established GPR65-OE-ADSC successfully after virus packaging and infection.Subsequently,ADSCs were treated with above acidic conditions for 24/48 hours.We tried to observed the differences of cell function and cell subcellular morphology between GPR65-OE and Vector group,then we also performed RNA-Seq analysis to probe the potential mechanism of GPR65 effects.At last,we transplanted GPR65-modified ADSC into the lower limbs of severely ischemic nude mice to detect the blood perfusion,collateral circulation,and angiogenesis in ischemic lower limbs.We compared the results from PBS,ADSC,Vector and OE-GPR65 group to determine the function of GPR65 in improving the therapeutic efficacy of ADSC for critical limb ischemia.Results:The results from microdialysis analysis in critical ischemic lower limbs showed that the content of lactate in ischemic limb muscles was increased sharply 0.5-72 hours after the ischemic event,which reflected the acidic microenvironment in ischemic area.In addition,from 0.5-72 h after operation,the glucose level in ischemic limb muscles was keeping down,suggesting the energy metabolic imbalance in ischemic limbs.The data from vitro experiments showed that,extracellular acidosis inhibited the proliferation and viability of ADSCs,induced cell apoptosis,arrested cell cycle,promoted cell senescence progression and induced mitochondrial damage,while GPR65 gene modification promoted the proliferation and viability of ADSC in acidic environment,avoided the acid-induced apoptosis,boosted cell cycle,and developed mitochondrial function.Besides,the results of RNA-Seq analysis were consistent with these observed phenotypes,reminding that GPR65 overexpression could benefit cell cycle,stabilize chromatin and promote cell division,moreover,inhibit immune and inflammatory responses.At last,experiments in vivo confirmed that,compared with the PBS group,the ADSC group and Vector group,ADSC with GPR65-OE improved the blood perfusion,collateral circulation,and angiogenesis dramatically in ischemic limbs.Conclusion:The extracellular acidosis in microenvironment could induce the apoptosis of transplanted ADSC and inhibit its various biological effects significantly,while GPR65 could effectively avoided the acidosis-induced damages and improve the bioavailability of ADSC under acidic environments in vitro.Besides,in the critical ischemic limbs of nude mice,GPR65 modification improved the efficacy of ADSC-bases therapy powerfully.To sum up,GPR65 is a candidate target for improving the efficiency of adipose-derived stem cells therapy in CLI.