Combination Of Danhong Injection And Adipose-derived Stem Cells Transplantation Promote Angiogenesis In Ischemic Hind Limb Of Diabetic Nude Mice

Backgroud: Adipose-derived stem cells (ADSCs) are widely used in basic researchand clinical intervention of patients with ischemic disease as seed cells for wide sourcesand weak immune rejection after transplantation. However common comorbidities ofischemic diseases include diabetes, coronary heart disease, hypertension, etc. High bloodpressure, glucose and lipids will lead to low homing rate and survival of transplanted cellsand not satisfactory clinical efficacy. In particular, long term high blood glucose in vivowould accumulate as advanced glycation end products (AGEs), which would causeoxidative stress and pro-inflammatory response. The above pathological responses wouldexert negative effects on the repair capability, homing and survival of stem cells. Theory ofgenerating vessels of Traditional Chinese Medicine (TCM) is considered closely relatedwith therapeutic angiogenesis. Results of many studies showed that injection of Danhong(DH) could significantly improve such patients’ chest pain and stroke.Objections: In this study, firstly ADSCs were challenged with AGEs, key pathogenicfactor for diabetes, to explore the protective effects of Danhong injection in maintainingstem cells’ function and promoting angiogenesis, and then such protective effects wereconfirmed and relative mechanisms were investigated in hindlimb ischemia modelgenerating by femoral artery excision in streptozotocin-treated nude mice by combingDanhong injection with ADSCs transplantation. Data from our study will not only enrichbasic theory system about treatment of TCM combination with stem cell transplantation forangiogenesis, but also provide new evidence for relative clinical application.Methods:1. Isolation, culture in vitro and identification of adipose tissue-derivedstem cells: The stromal cells were obtained by combination with enzymatically digestionand centrifugation, and then were suspended in DMEM/F12with10%FBS for culturing invitro. Changes of cultured cells’ morphology and emergence of cell colonies were observed.After80%confluence was reached, the cells were enzymatically digestion with0.1%trypsin and resuspended in new culture dishes for amplification. The cells were identifiedto induce differentiation towards adipocytes, osteocytes and chondrocytes.2. Negative effects of CML-BSA on function and angiogenesis of ADSCs andprotective effects of DH: After reaching80%confluence, ADSCs were synchronized for24hours with DMEM/F12without FBS, then exposed to5different interventions respectively for24hours, including PBS,60μg/ml BSA,60μg/ml CML-BSA,100μl/ml DH and60μg/ml CML-BSA+100μl/ml DH. The proliferation capability of such cells wereevaluated using WST-1assay, the apoptotic rates were investigated by Annexin V-FITCApoptosis Detection Kit and Caspase-Glo3/7Assay, migration ability were explored bytranswell assay, secreted VEGF in culture supernatant were measured by ELISA, andangiogenesis of such cells was observed in matrigel in vitro. Then, Hydrogen sulfide (H2S)was measured by reversed-phase high performance liquid chromatography (RP-HPLC)-fluorescence quantitation method. The expression of cystathionine gamma lyase (CSE),which is a major sources for endogenous enzymatic production of H2S.3. Establishment of hind limb ischemia model in diabetic nude mice: streptozontion(150mg/kg) was injected intraperitoneally in nude mice and blood glucose levels weremonitored after2weeks. Diabetes modeling success was identified by random bloodglucose levels higher than16mmol/L twice. After2weeks, hind limb ischemia model wasestablished by ligation and excision of femoral artery, arteria saphena, circumflex ofexternal iliac artery and muscular branched of femoral artery. Lower limb perfusions ofsuch mice were assessed using laser doppler perfusion imager system (LDPI).4. Efficacy of DH combined with ADSCs transplantation on angiogenesis in diabetichind limb ischemia model: Forty nude mice were randomly divided into four groups: groupI (n=10): diabetic hind limb ischemia model, group II (n=10): ADSCs transplantation,group III (n=10): DH (2μl/g×7days) ip, group IV (n=10): combination of DH (2μl/g×7days) ip and ADSCs transplantation. After2weeks, perfusions, vessel regeneration, andlocal VEGF expression of lower limbs among different groups were evaluated with LDPI,X-ray arterography, real-time PCR and ELISA respectively. And microvascular densities oflower limb muscle were investigated using HE and CD31staining. The expression of CSEwas measured by Real-Time PCR. H2S level of vein blood was measured byRP-HPLC-fluorescence quantitation method.Results：1. Identification of cultured adipose tissue-derived stem cells:1)Morphology:cultured cells began to attach to the dish bottom after6hours, and obvious cell coloniesformed after3-day culture and most of cells showed polarized growth and acquired afibroblastic-like morphology;2) in vitro differentiation: cells were induced to differentiatetowards adipocytes with IBMX, insulin and dexamethasone and quantified with oil-redstaining, osteocytes with dexamethasone, β-glycerophpsphate, and l-ascorbicacid-2-phosphate and quantified with alizarin red, chondrocytes with TGF-β, dexamethasone and insulin and quantified with alcian blue.2. Negative effects of CML-BSA on function and angiogenesis of ADSCs and theprotective effects of Danhong injection: Compared with the BSA control group,proliferation, migration and secretion capability of ADSCs were inhibited by stimuli withCML-BSA (P<0.05), but the apoptosis of such cells were promoted. Moreover, CML-BSAsignificantly reduced CSE and H2S expression (P<0.05). DH could promote proliferation,migration and secretion capability but inhibit apoptosis of ADSCs (P<0.05) vs PBS.Meanwhile, DH could increase CSE and H2S expression (P<0.05), and furthermorepartially reverse the negative effects of CML-BSA on ADSCs (P<0.05).3. Establishment of hind limb ischemia model in diabetic nude mice:2weeks afterSTZ injection, random blood glucose levels of nude mice significantly increased(20.33±1.93mmol/l), which implied successful diabetes modeling. And then, the perfusionsof operated left hind limbs in mice were significant lower than those of right ones withLDPI, which confirmed that the diabetic hind limb ischemia model was established.4. Efficacy of DH combined with ADSCs transplantation on angiogenesis in diabetichind limb ischemia model: Only ADSCs transplantation or DH ip could significantlyincrease perfusion, promote the formation of collateral circulation, enhance local VEGF,CSE, and H2S expression after2-week intervention compared with control group. Andmeanwhile capillary density of the ischemic hind limb showed similar trend. Whilecombination of DH and ADSCs transplantation could increase the protective effects furthervs DH ip or ADSCs transplantation alone.Conclusion:1. CML-BSA could significantly inhibit proliferation, migration, butpromote apoptosis, reduce VEGF expression and secretion of ADSCs, and impair ADSCsangiogenesis of ADSCs. DH injection would partially reverse the negative effects ofCML-BSA. Endogenous H2S pathway might play a key role in explaining the pathologicand physiologic processes.2. ADSCs transplantation or DH ip alone could improve ischemia perfusion andvascular tube-like generation in diabetic hind limb models. And, combination of DH ip andADSCs transplantation could exert more protective effects via promotion of releasingendogenous H2S.