Photodynamic Regulation Of Adipose-derived Mesenchymal Stem Cells In The Treatment Of Lower Limb Ischemia In Mice On High Fat Diet

Objective: In recent years,the secondary complications of diabetes have become a global public health problem,with peripheral arterial disease(PAD)being a common and serious complication of this metabolic disorder.Currently,therapies of PAD rely on targeted medical,genetic therapies,mechanical revascularization through either percutaneous or surgical approaches.However,the application of these therapies is limited,so it is urgent to seek more effective therapies.There is increasing evidence that the injection of Adipose-derived mesenchymal stem cells(ADSCs)enables repair and regeneration of neovasculature.Therapeutic persistence and reduced primitive stem cell function following cell delivery remains a critical hurdle for the clinical translation of stem cells in current approaches.Therefore,We specially developed device with greater refined wavelengths of light to improve the therapeutic effect of ADSCs.In summary,we proposed a scientific hypothesis: Photodynamics may promote the paracrine and endothelialization of ADSCs,mediate angiogenesis and glucose metabolism,and improve the ischemic disease of diabetic lower limbs.This study aimed to explore whether photodynamics could enhance the treatment of lower limb ischemia by ADSCs in mice on high-fat diet,and whether it could promote the formation of lower limb ischemic angiogenesis and glucose uptake in mice.Methods: 1.High-fat fed mice model : C57BL/6J mice were fed a high-fatand high-sugar diet for 14 weeks,with adequate water and feed.2.Extraction of ADSCs: Subcutaneous adipose tissue were digested with type 1 collagenase by incubating in a shaker at 37°C for 30 min,then centrifuged the mixture at370 g for 10 min.Cells were suspended in ADSCs growth medium.3.Photoactivation of ADSCs: Cultured ADSCs were transferred into a sterile syringe and subjected to light treatment for 30 min using a cell therapy transformation apparatus-HarmoneyRegena device,which integrates monochromatic lights of three different wavelengths,including 575-595 nm(5-20 mW),630-635 nm or 660-670 nm(10-100 mW),and 510-540 nm(10-60mW).4.Characterization of ADSCs: ADSCs at passage 3 were collected and the expressions of CD29,CD90 and CD105 were detected by flow cytomlantationetry.5.Cell proliferation: The photoactivated ADSCs were further cultured to observe the proliferation of cells on days 3,5 and 7 after activation.6.Establishment of hindlimb ischemia model: The mice were intraperitoneally injected with 1% sodium pentobarbital.The Femoral Artery Ligation(FAL)was performed on the left leg of every mouse.7.Transplantation of ADSCs: Almost 500,000 ADSCs were infused intravenously via the tail vein into the high-fat fed mice.8.Laser doppler imaging: Perfusion of the lower extremities of the mice was measured immediately before and after surgery and then at specific time points(3,7,14,21,28 and 38 days post surgery)using a scanning moorLDI2-HIR high-resolution laser Doppler imager.The blood flow perfusion ratio of the ischemic/non-ischemic hindlimb was used as an index for post-ischemia recovery of perfusion.9.Immunofluorescenceassay: To determine the ischemia-induced angiogenesis,the ischemic muscles were prepared into pathological paraffin sections,and the expression of the smooth muscle actin(α-actin)and platelet endothelial cell adhesion molecule-1(PECAM-1,also known as CD31)was detected by immunofluorescence staining.10.Real-time fluorescence quantitative PCR detection: Hindlimb muscles were collected 38 days post-ischemia and RNA was extracted using Trizol reagent.Detect vascular growth factors and inflammatory factors mRNA expression levels in hindlimb muscles.11.Glucose tolerance test(GTT)and Insulin tolerance test(ITT):Mice were fasted for 12 hours and 6 hours on the 22 nd and 25 th day after the operation,respectively.Glucose solution(2g/kg)and insulin solution(0.75IU/Kg)were intraperitoneal injected into the mice.Blood samples were then obtained from the caudal vein,and the blood glucose levels were measured at 0,30,60,and120 min after injection using an Accu-Check glucometer(Roche Diagnostics).12.ADSCs endothelialization detection: Expression of CD31 and CD34 in ADSCs at 3,7 and 14 days after photoactivation was detected by flow cytometry.13.Proteomics analysis: The proteome of hADSCs CM was analyzed based on the protein intensities measured by LC/MS-MS.Results: 1.ADSCs were positive for mesenchymal stem cell markers CD29,CD90,CD105,which were consistent with mesenchymal stem cell characteristics.The phenotype of the Lighted-ADSCs remained largely unchanged,similar to Normal-ADSCs.2.Cell numbers measured for Lighted-ADSCs increased significantly in comparison to values obtained forNormal-ADSCs.This suggests that photodynamics can promote ADSCs proliferation.3.Transplantation of ADSCs did not affect body fat and lipid levels in mice on a high-fat diet.4.Doppler flow scanning and immunofluorescence staining indicated that Lighted-ADSCs promoted the recovery of ischemic lower limb perfusion in mice on high-fat diet,and increased the angiogenesis of ischemic lower limb muscles.5.Real-time quantitative PCR showed that photodynamic increased the expression of VEGF-A,VEGF-C,PDGF-B and ANGPTL4.6.Real-time quantitative PCR showed that photodynamic decreased the expression of M1 inflammatory factors and increased the expression of M2 inflammatory factors,which promoting the polarity transformation of macrophages in ischemic muscle.7.Transplantation of ADSCs can improve the abnormal glucose and insulin metabolism induced by high-fat diet,improve glucose tolerance and enhance insulin sensitivity.8.Photodynamics increased the expression of CD31 and CD34 in ADSCs,leading to the proliferation of ADSCs-derived endothelial progenitor cells(EPCs).9.Photoactived hADSCs CM retained a more complete pro-angiogenic activity with significant upregulation of angiogenesis related proteins.Conclusion:1.Photodynamics promoted the proliferation of ADSCs and can promote the differentiation of ADSCs into endothelial progenitor cells.2.Photodynamics can enhance the therapeutic effect of ADSCs on lower limb ischemia,improve blood perfusion and promote angiogenesis.3.ADSCs transplantation improved the metabolic microenvironment,glucose toleranceand insulin sensitivity of mice on high-fat diet.4.Photodynamics significantly upregulation of angiogenic proteins secreted by hADSCs.5.Photodynamics can effectively improve the role of ADSCs in the treatment of lower limb ischemia,which is conducive to the clinical application of ADSCs...