Diabetic Lower Limb Ischemia Research Articles

Adipose-derived stem cell-secreted exosomes enhance angiogenesis by promoting macrophage M2 polarization in type 2 diabetic mice with limb ischemia via the JAK/STAT6 pathway

Diabetic lower limb ischemia is an intractable disease that leads to amputation and even death. Recently, adipose-derived stem cell-secreted exosomes (ADSC-Exo) have been reported as a potential therapeutic approach, but its specific mechanism of action is unknown. Studies have found that exosomes derived from stem cells can reduce inflammation and promote tissue repair. Macrophages play an important role in the development and repair of inflammation in lower limb ischemic tissue, but the specific regulation of ADSC-Exo in macrophages has rarely been reported. The present study aimed to verify whether ADSC-Exo could promote angiogenesis by regulating macrophages to reduce the level of inflammation in diabetic ischemic lower limbs. In this study, adipose-derived stem cells (ADSCs) were obtained and identified, and ADSC-Exos were isolated using ultracentrifugation and characterized using transmission electron microscopy, nanoparticle tracking analysis, and western blotting analysis. The uptake of ADSC-Exos by macrophages was observed using immunofluorescence, and macrophage polarization induced by ADSC-Exos was identified by flow cytometry, immunofluorescence and ELISA. The effects of ADSC-Exos on the proliferation, apoptosis, migration and adhesion of macrophages were evaluated using CCK-8 assay, flow cytometry, Transwell assay, scratch and adhesion experiments, and ELISA assay. The polarization-related JAK/STAT6 signaling pathway was explored by using western blotting. A lower limb ischemic model of type 2 diabetic mice was established and ADSC-Exos was intramuscularly injected into the mice. The blood flow in the lower limbs was assessed using a laser Doppler flowmeter, while the level of angiogenesis was determined using immunohistochemistry and immunofluorescence. The results of this study prove that ADSC-Exos induced M2-phenotype polarization of macrophages via the JAK/STAT6 signaling pathway can promote the proliferation, migration and adhesion of M2 macrophages, inhibit the apoptosis of macrophages, and promote the angiogenesis and revascularization of ischemic lower limbs in type 2 diabetic mice. Thus, this study provides a theoretical and experimental basis for the clinical treatment of diabetic lower limb ischemic disease.

Exosomes derived from adipose-derived stem cells overexpressing glyoxalase-1 protect endothelial cells and enhance angiogenesis in type 2 diabetic mice with limb ischemia

BackgroundDiabetic limb ischemia is a clinical syndrome and refractory to therapy. Our previous study demonstrated that adipose-derived stem cells (ADSCs) overexpressing glyoxalase-1 (GLO-1) promoted the regeneration of ischemic lower limbs in diabetic mice, but low survival rate, difficulty in differentiation, and tumorigenicity of the transplanted cells restricted its application. Recent studies have found that exosomes secreted by the ADSCs have the advantages of containing parental beneficial factors and exhibiting non-immunogenic, non-tumorigenic, and strong stable characteristics.MethodsADSCs overexpressing GLO-1 (G-ADSCs) were established using lentivirus transfection, and exosomes secreted from ADSCs (G-ADSC-Exos) were isolated and characterized to coculture with human umbilical vein endothelial cells (HUVECs). Proliferation, apoptosis, migration, and tube formation of the HUVECs were detected under high-glucose conditions. The G-ADSC-Exos were injected into ischemic hindlimb muscles of type 2 diabetes mellitus (T2DM) mice, and the laser Doppler perfusion index, Masson’s staining, immunofluorescence, and immunohistochemistry assays were adopted to assess the treatment efficiency. Moreover, the underlying regulatory mechanisms of the G-ADSC-Exos on the proliferation, migration, angiogenesis, and apoptosis of the HUVECs were explored.ResultsThe G-ADSC-Exos enhanced the proliferation, migration, tube formation, and anti-apoptosis of the HUVECs in vitro under high-glucose conditions. After in vivo transplantation, the G-ADSC-Exo group showed significantly higher laser Doppler perfusion index, better muscle structural integrity, and higher microvessel’s density than the ADSC-Exo and control groups by Masson’s staining and immunofluorescence assays. The underlying mechanisms by which the G-ADSC-Exos protected endothelial cells both in vitro and in vivo might be via the activation of eNOS/AKT/ERK/P-38 signaling pathways, inhibition of AP-1/ROS/NLRP3/ASC/Caspase-1/IL-1β, as well as the increased secretion of VEGF, IGF-1, and FGF.ConclusionExosomes derived from adipose-derived stem cells overexpressing GLO-1 protected the endothelial cells and promoted the angiogenesis in type 2 diabetic mice with limb ischemia, which will be a promising clinical treatment in diabetic lower limb ischemia.

Effectiveness of distal arterial bypass with porcine decellularized vascular graft for treating diabetic lower limb ischemia.

Application of tissue engineered vascular grafts for small-diameter artery reconstruction has been a much anticipated advance in vascular surgery. The aim of this study is to assess the effectiveness of small-diameter decellularized vascular grafts in below-knee bypass surgery for diabetic lower extremity ischemia. Three patients with diabetic lower limb ischemia were admitted to the Department of Vascular Surgery, Xuanwu Hospital, Capital Medical University between May, 2010 and June, 2010. Decellularized porcine arteries with modified surface were implanted in the lower extremity for below-knee arterial revascularization. Imaging examination was performed for assessment of graft mechanical stability and patency at 1 month and 6 months after implantation. At 6 months after implantation, all three grafts were patent with no stenosis or aneurysm formation of the grafts were found on imaging assessment with primary patency rate of 100% (3/3) both at 1 month and 6 months after graft insertion. Decellularized vascular graft with surface modification for the small-diameter artery reconstruction had good clinical results after 6 months follow-up in three patients with diabetic lower limb ischemia.

Role of Cilostazol in Rats with Diabetic Lower Limb Ischemia and Its Influences on Changes in Matrix Metallopeptidase-9 and Matrix Metalloproteinase-8

Objective: To assess cilostazol’s effect on rats with diabetic lower limb ischemia. Methods: 36 Sprague-Dawley rats were randomly separated into normal group (n = 12), model group (n = 12) and cilostazol group (n = 12). The model of diabetic lower limb ischemia was prepared and normal saline was intraperitoneally injected postoperatively into model group. Besides, cilostazol was administrated through intraperitoneal injection after establishment of model in cilostazol group. After intervention for 6 weeks, doppler blood flow imager was employed to detect blood flow, and the expressions of MMP-8 and MMP-9 were assessed via immunohistochemistry, western blotting or quantitative polymerase chain reaction (qPCR). TUNEL assay was performed to evaluate cell apop-tosis. Results: Doppler blood flow imager showed that compared with normal group, perfusion unit (PU) index was significantly reduced in the other two groups (P < 0.05) with higher value in cilosta-zol group (P < 0.05). The immunohistochemistry results revealed that in comparison with those in normal group, MMP-8 and MMP-9 positive staining was significantly elevated in the other two groups (P < 0.05) with higher level in cilostazol group than model group. In addition, MMP-8 and MMP-9 expressions were significantly elevated in other two groups (P < 0.05), with higher level in cilostazol group (P < 0.05). Moreover, the vascular wall smooth muscle cells in the other two groups had a significantly elevated apoptosis rate (P < 0.05) with higher apoptosis in cilostazol group (P < 0.05). Conclusion: Cilostazol up-regulates MMP-8 and MMP-9 to increase the apoptosis of vascular wall smooth muscle cells, thereby improving the blood flow in rats with diabetic lower limb ischemia.

Hypoxia pretreatment of bone marrow mesenchymal stem cells facilitates angiogenesis by improving the function of endothelial cells in diabetic rats with lower ischemia.

Endothelial dysfunction induced by unordered metabolism results in vascular reconstruction challenges in diabetic lower limb ischemia (DLLI). Mesenchymal stem cells (MSCs) are multipotent secretory cells that are suitable for clinical DLLI treatment, but their use has been hampered by poor survival after injection. Hypoxia can significantly enhance the capacity of MSCs to secrete angiogenic factors. We investigated transient hypoxia pretreatment of MSCs to facilitate revascularization in DLLI. Rat bone marrow MSCs (BM-MSCs) were cultured at different oxygen concentrations for varying time periods. The results indicated that transient pretreatment (5% O2, 48 h) not only increased the expression of VEGF-1α, ANG, HIF-1α and MMP-9 in BM-MSCs as assessed by real-time RT-PCR, but also increased the expression of Bcl-2 as determined by western blotting. The transplantation of pretreated BM-MSCs into rats with DLLI demonstrated accelerated vascular reconstruction when assayed by angiography and immunohistochemistry. CM-Dil-labeled tracer experiments indicated that the survival of BM-MSCs was significantly improved, with approximately 5% of the injected cells remaining alive at 14 days. The expression levels of VEGF-1α, MMP-9 and VEGF-R were significantly increased, and the expression of pAKT was up-regulated in ischemic muscle. Double immunofluorescence studies confirmed that the pretreated BM-MSCs promoted the proliferation and inhibited the apoptosis of endothelial cells. In vitro, pretreated BM-MSCs increased the migratory and tube forming capacity of endothelial cells (ECs). Hypoxia pretreatment of BM-MSCs significantly improved angiogenesis in response to tissue ischemia by ameliorating endothelial cell dysfunction and is a promising therapeutic treatment for DLLI.

In vitro differentiation into endothelial-like cells from bone marrow mesenchymal stem calls in diabetic rats

Objective To investigate the methods for separation and culture of bone marrow mesenchymal stem cells (BMSCs) in vitro in diabetic rats and the capability of differentiation into endotheliallike cells.Methods Rat diabetic model (DM) was induced by injection of stretopzin (STZ) in SD rats.After 12 weeks of DM duration,BMSCs were separated from bone marrow with density gradient centrifugation,wall sticking screening and amplified in vitro.The surface markers of BMSCs were evaluated with fluorescence-activated cell sorter (FACS).BMSCs of the third passage were obtained and divided into two groups.In the induction group,the cells were induced to differentiate in M199 medium containing 20％ fatal bovine serun ( FBS),vascular endothelial growth factor ( VEGF,10 μg/L) and basic fibroblast growth factor (bFGF,2 μg/L).No cytokine was added in the blank control group.Two weeks later,the cell morphology was observed under the phase contrast microscopy.The expression levels of VEGFR-2 were detected by using immunohistochemistry.EPC-specific marker CD34 was evaluated with FACS,and release of nitrous oxide (NO) from culture cells was also detected by NO detection reagent.Weibel-Palade (W-P) bodies in the cytoplasm were observed under the transmission electron microscopy.Results Rat diabetic model in SD rats that were injected with STZ intraperitoneally was established.Cultured BMSCs at the third passage were positive for CD44 and CD90 [ (97.8 ± 0.9 ) ％ and (96.8 ± 1.4) ％,respectively ],but negative for CD11 b/c and CD34 [ ( 13.2 ± 0.6 ) ％ and ( 1.2 ± 0.5 ) ％,respectively ].On the 14 day in the induction group,the cell morphology observed under the phase contrast microscopy was not distinctly different.Differentiation antigens VEGFR2 and CD34 in the induction group were higher [ (97.1 ± 1.0) ％,(65.0 ± 3.9) ％ ] than in the blank control group [ (7.0 ± 1.0 ) ％,( 0.9 ± 0.3 ) ％ ] ( P ＜ 0.05 ).The content of nitrous oxide in the induction group was also obviously increased as compared with the blank control group [ (94.14 ± 3.25 ) vs.( 70.37 ± 2.1 0) μmol/L,P ＜ 0.05 ).W-P bodies in the cytoplasm of the cultured cells were not found in the two groups.Conclusion BMSCs in diabetic rats can be isolated by density gradient centrifugation and adherent culture.VEGF and bFGF can induce differentiation of BMSCs in diabetic rats into endothelial-like cells in vitro.It is suggested that BMSCs may be used as a new type of seed cells for the diabetic lower limb ischemia disease. Key words: Diabetic; Bone marrow mesenchymal cell; Induction differentiation; Endothelial-like cell

Preliminary evaluation of clinical effects of below-knee arterial bypass on diabetic lower limb ischemia

To evaluate medium-long term patency of below-knee bypass on patients who suffered from diabetic lower limb ischemia. Clinical and follow-up data of 51 patients was retrospectively analyzed who underwent 56 below-knee bypass because of diabetes from November 2001 to December 2006. There were 35 male and 16 female with an average age of 68 years. They endured 26 months ischemic time lag in average, and had suffered from diabetes for 11 years. All of the patients were performed bypass to below-knee (posterior tibial, anterior tibial or peroneal) arteries. Kaplan-meier method was applied. The subgroups of different operative methods and different out-flow vessels were compared by Log-rank tests. An average follow-up time of 23 months was achieved, and lost-follow-up rate was 15%. The total 1- and 5-year primary patent rates were 68% and 54% respectively, secondary patent rate were 70% and 60% respectively, limb salvage rates were 69% and 65%, survival rates were 82% and 60%. One year (3 years) of patent rate(s) of operative method subgroups of femoral-popliteal-infrageniculate bypass with composite grafts, femoral/popliteal-infrageniculate bypass with artificial grafts and femoral/popliteal-infrageniculate bypass with autologous veins were 70% (50%), 33% (33%) and 70% (70%) respectively. One year (4 years) of patent rate(s) of out-flow vessel subgroups of posterior tibial artery, anterior tibial artery and peroneal artery were 65% (60%), 80% (53%) and 77% (66%) respectively. However, both subgroups did not show any statistic differences by log-rank tests. Partial or whole autologous veins as bypass grafts should be chosen when infrageniculate bypass is considered in diabetic patients. Considerable patent rates are acceptable no matter what kinds of out-flow vessels are chosen.

Effect of Lower Extremity Bypass Surgery on Inflammatory Reaction and Endothelial Dysfunction in Type 2 Diabetic Patients

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia and dyslipidemia. The abnormalities in nutrient metabolism and elevated inflammatory mediators resulting from DM lead to impairment of wound healing and vulnerability to infection and foot ulcers. Diabetic lower limb ischemia often leads to limb necrosis. Lower extremity bypass surgery (LEBS) is indicated to prevent limb loss in patients with critical leg ischemia. This study investigated the alteration of inflammatory and endothelium dysfunction markers before and after LEBS in DM patients. Twenty one type 2 DM patients with LEBS were included. Blood was drawn before and at 1 day and 7 days after surgery in the patients. Plasma soluble cellular adhesion molecule levels and blood leukocyte integrin expressions were measured. Also, plasma concentrations of endothelin-1 and nitric oxide were analyzed to evaluate the vascular endothelial function. The results showed that there were no significant differences in plasma cellular adhesion molecules, endothelin-1 and nitric oxide levels, nor did any differences in leukocyte integrin expressions before and after the operation. These results suggest that the efficacy of LEBS on alleviating inflammatory reaction and improving endothelial function in DM patients was not obvious.

Diabetic Critical Ischemia of Lower Limbs: Distal Arterial Revascularisation

Objectives: To evaluate the outcomes of ischemic diabetic foot lesions for which distal arterial bypass grafting was considered as the first-line vascular procedure.Patients and Methods: Between November 2004 and November 2006, 19 lower limbs of 17 diabetic patients with lower limb critical ischemia were operated in our department. The bypass grafts included five femoro-popliteal bypass grafts below knee and 14 distal bypass grafts. The 14 distal bypass grafts included; seven venous grafts on the dorsalis pedis artery, four femorotibial pTfE grafts and three femoroperoneal grafts. Arterio-venous fistula was applied to the distal anastomosis site in three limbs.Results: At the time of discharge, the graft patency rate was 93.75%. The mean follow up period was 24 months. The primary cumulative patency rate was 63% at 2 year. The corresponding secondary patency rate was 87%. Among six ischemic ulcers, four ulcers healed within 2 to12 months (66.6%). At 24 months, the cumulative rate of limb salvage was 76% and that of survival was 74%.Conclusion: Distal arterial bypass in diabetic lower limb ischemia improves blood circulation that accelerates foot ulcer healing. It can also avoid amputation or lower its level, and thus improving the patient’s quality of life.