Ginsenoside Rb1 promotes angiogenesis potentially by activating the JAK-STAT3 signalling pathway

Conventional revascularization strategies for ischemic heart disease (IHD) are designed to prompt reperfusion of the coronary artery to the salvaged cardiomyocytes. However, these strategies may cause myocardial reperfusion injuries. Therefore, a safe and effective strategy needs to be developed to improve the conventional strategies. Here, we investigated the pro-angiogenic effect of Ginsenoside Rb1 (Rb1) to provide the experimental basis for angiogenesis-mediated drug therapy of IHD. Thus, Human umbilical vein endothelial cells (HUVECs) were treated with either a vehicle or Rb1 at 4, 8, 12 or 16 ?M for 24 h. A model of hindlimb ischemia was established using C57BL/6J mice. In sham-operated mice, only the femoral artery was isolated without ligation whereas the other operations and supplementation control group were consistent. The mice in the supplementation group were injected with Rb1 (50 mg/kg body wt./day) for 7 days. The results indicated that Rb1 promotes cell proliferation, adhesion, migration and tube formation in the HUVECs in a dose-dependent manner. The ED50 of Rb1 to improve cell adhesion is 8 ?M. In mice, Rb1 promoted angiogenesis after the ligation of the femoral artery and ameliorated the ischemic conditions. Intriguingly, more blood flow recovery was observed in the Rb1 supplemented mice than in the vehicle-treated mice (0.85 ± 0.05 vs. 0.71±0.10 on day 3; 0.94±0.10 vs. 0.75±0.08 on day 7). In HUVECs, Rb1 increased the phosphorylation of STAT3 and JAK, which may be the mechanism through which Rb1 mitigates IHD. Moreover, our results confirmed that Rb1mitigates IHD potentially by activating the JAK-STAT3 pathway. Further clinical trials are warranted to verify the clinical implications of Rb1.

Application of temperature-sensitive chitosan hydrogel encapsulated exosomes in ischemic diseases / 中国组织工程研究

BACKGROUND: Exosomes derived from mesenchymal stem cells can reduce myocardial ischemia and reperfusion injury, but there are some problems such as short half-life, fast clearance and low targeting. OBJECTIVE: To modify and encapsulate exosomes with temperature-sensitive chitosan hydrogel to increase the retention rate of exosomes in the body, and to achieve better therapeutic effect. METHODS: Cell transfection method was used to knock down piR823 in exosomes derived from umbilical cord mesenchymal stem cells, and the effect of knockdown of piR823 exosomes on the proliferation and apoptosis of C2C12 cells was detected. Chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel was prepared and mixed directly with exosomes to prepare chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel encapsulated with exosomes. The gel-forming properties, rheology and in vitro sustained release properties of the hydrogel after encapsulation of exosomes were tested. Thirty C57BL/6J mice were taken to establish hind limb ischemia models, and randomly divided into five groups. The gastrocnemius of group A was injected with PBS; group B was injected with chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel; and group C was injected with exosomes-encapsulated chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel; group D was injected with exosomes; group E was injected with exosomes knocking down piR823. Limb function and recovery, blood flow, grip strength, exercise endurance and muscle regeneration were detected in each group. RESULTS AND CONCLUSION: (1) Exosomes knocking down piR823 inhibited the proliferation of C2C12 cells; normal exosomes inhibited hydrogen peroxide-induced apoptosis of C2C12 cells. The inhibitory effect of exosomes on apoptosis was weakened after piR823 was knocked down. (2) The hydrogel encapsulating exosomes had gel-forming properties, but the gel-forming time was shortened, and it could slowly and continuously release exosomes for more than 30 days. (3) After 28 days of hindlimb ischemia, the blood flow recovery of the left limb in group C was better than that in groups B and D (P < 0.05); it in group D was better than group E (P < 0.05); the grip strength, endurance, running time and distance of group C were better than those of groups D and B (P < 0.05), and above indexes in group D were better than in group E (P < 0.05). Muscle regeneration was better in group C than in groups B and D (P < 0.05), and it in group D was better than group E (P < 0.05). (4) The results showed that encapsulation of exosomes by chitosan/β-sodium glycerophosphate temperature-sensitive hydrogel prolonged the residence time of exosomes in vivo, significantly enhanced blood perfusion and recovery of tissue function after ischemia, and the treatment effect was more significant.

Effects of growth differentiation factor 11 on angiogenesis in diabetic hindlimb ischemia / 中华内分泌代谢杂志

Objective To investigate the effects and possible mechanism of growth differentiation factor 11 (GDF11)on angiogenesis in diabetic hindlimb ischemia. Methods Sixty SD rats were used in this study. Diabetes was induced by intraperitoneal injection of streptozotocin. 3 days after streptozotocin administration, 40 rats with plasma glucose concentration≥16.7 mmol/L were selected in the subsequent experiments. 12 weeks after diabetes was induced,the left femoral artery and all the sides branches were dissected free and excised. After resection of the left femoral artery,rats were randomized to four groups:PBS group(n=10),GDF11 group(n=10),IgG Ab group (n=10),or GDF11 Ab group(n=10). After 0,7,and 14 days,the serial blood flows were measured by a Laser Doppler perfusion image(LDPI)analyzer. To detect capillary endothelial cell,the sections of muscles were reacted with anti-CD31 monoclonal antibodies,and subsequently reacted with Cy3-conjugated anti-rabbit IgG antibody. The expression levels of HIF1α and VEGF were detected by western blotting. Results In GDF11 group significantly increased the blood perfusion and capillary density of ischemia hindlimb of the diabetic rats were found,which was correlated to an increased level of HIF1α and VEGF. In contrast, GDF11 Ab could lead to the opposite effects. Conclusion GDF11 treatment promotes the recovery of diabetic hindlimb ischemia, which may be related to the improvement of expression of HIF1 alpha and VEGF.

Rat Model of Hindlimb Ischemia Induced via Embolization with Polyvinyl Alcohol and N-Butyl Cyanoacrylate

OBJECTIVE: To investigate the feasibility of a rat model on hindlimb ischemia induced by embolization from the administration of polyvinyl alcohol (PVA) particles or N-butyl cyanoacrylate (NBCA). MATERIALS AND METHODS: Unilateral hindlimb ischemia was induced by embolization with NBCA (n = 4), PVA (n = 4) or surgical excision (n = 4) in a total of 12 Sprague-Dawley rats. On days 0, 7 and 14, the time-of-flight magnetic resonance angiography (TOF-MRA) and enhanced MRI were obtained as scheduled by using a 3T-MR scanner. The clinical ischemic index, volume change and degree of muscle necrosis observed on the enhanced MRI in the ischemic hindlimb were being compared among three groups using the analysis of variance. Vascular patency on TOF-MRA was evaluated and correlated with angiographic findings when using an inter-rater agreement test. RESULTS: There was a technical success rate of 100% for both the embolization and surgery groups. The clinical ischemic index did not significantly differ. On day 7, the ratios of the muscular infarctions were 0.436, 0.173 and 0 at thigh levels and 0.503, 0.337 and 0 at calf levels for the NBCA, PVA and surgery groups, respectively. In addition, the embolization group presented increased volume and then decreased volume on days 7 and 14, respectively. The surgery group presented a gradual volume decrease. Good correlation was shown between the TOF-MRA and angiographic findings (kappa value of 0.795). CONCLUSION: The examined hindlimb ischemia model using embolization with NBCA and PVA particles in rats is a feasible model for further research, and muscle necrosis was evident as compared with the surgical model.

Effect of Nanoparticle with VEGF in Mouse Ischemic Hindlimb Model

PURPOSE: Vascular endothelial growth factor (VEGF) is one of the factors regulating angiogenesis. For angiogenesis, the local concentration of VEGF has to be maintained. Because of its short half-life, VEGF has been conjugated with nanoparticles. Some nanoparticles, such as poly (lactic-co-glycolic acid (PLGA)) or polyethylenimine (PEI) are commonly used in this field, but have weak points such as faster release than expected and cell toxicity. We investigated the effect of core/shell nanoparticles including lecithin lipid cores in the ischemic hindlimb model. METHODS: Mice were anesthetized and a region of the common femoral artery and vein was ligated and excised. Hindlimb ischemic mice (n=28) were divided randomly into four groups: Control group (normal saline, n=7), mouse VEGF group (mVEGF, n=7), nanoparticle including mVEGF group (N-mVEGF, n=7), and nanoparticle/hydrogel mouse VEGF group (NH-mVEGF, n=7). The drug was injected postoperatively into the thigh muscle of the ischemic limb. Perfusion, capillary number and H&E stain were assessed 28 d after treatment. RESULTS: The capillary number increased in N-mVEGF and mVEGF group (P=0.026). Improvements of ischemic limb perfusion were inferior in N-mVEGF, NH-mVEGF groups (P=0.006) compared to other groups. Mice received N-mVEGF, NH-mVEGF treatment showed significant inflammation in the H&E staining. CONCLUSION: Sustained VEGF delivery via core/shell nanoparticle with lecithin core did not show improved perfusion rate despite an increase in capillary number. Furthermore, vacuolization and induction of inflammation requiring a different composition of nanoparticle should be tested.

Genetic modification of bone marrow stem cells in treatment of hind limb ischemia / 国际外科学杂志

With the development of gene technology,it is gradually possible and much better for genetic modification of bone marrow stem cells in treatment of hindlimb ischemia. Gene modification of cells prior to their transplantation, especially stem cells, enhances their survival and increases their function in cell therapy. But this treatment is only used for the experimental stage. Here, we review the current uses of gene-modified bone marrow stem cells in vascular disease.

The protective effects of tumor necrosis factor receptor 1 deletion on hindlimb ischemia / 中华急诊医学杂志

Objective Signals in the tumor necrosis factor α TNFα pathway, including TNFα and tumor necrosis factor α receptor 1TNFR1, are upregulated after acute hindlimh ischemia. It is unclear, however, whether this upregulation is apathophysiological consequence or a compensatory response. In the present study, the effect of TNFR1 deletion in a mouse model of hindlimb ischemia was investigated.Methods Hindlimb ischemia was produced through ligation of the proximal and distal left femoral artery and its branches in TNFR1 knockout TNFR1-/- and wild type WT mice. Laser Doppler blood flow measurement showed that limb perfusion was significantly higher in TNFR1-/- mice than that in the WT mice at 1 day after operation.Results The calculated ischemic scores were 3.67 ± 0.52 in WT group and 1.83 ± 0.41 in TNFR1-/- group P ＜0.05. The rate of amputation was 50% in WT mice versus 0% in TNFR1-/- mice. There were less TUNEL positive cells in the calf muscle of TNFR1-/- mice than that of WT mice. Furthermore, DNA fragmentation was more obvious in WT mice.Western blot showed less expression of Bax and cleaved caspase 3 in TNFR1-/- mice 1 day after ischemia, suggesting a reduced apoptotic process in the absence of TNFR1.Conclusion In mice with hindlimb ischemia, knockout of TNFR1 prevents the activation of death-related proteins down streaming to TNF α and attenuated cell death including apoptosis. Systemic block of the TNFR1 might be a new interventional methods-to preserve the limbs from acute ischemic attack in patients with peripheral arterial obstructive disease.

Pathologic and VEGF changes of transplantation of endothelial progenitor cells for hindlimb ischemia in diabetic rabbit model / 中国实用内科杂志

Objective To explore the effects of transplantation of endothelial progenitor cells(EPC)from autologous bone marrow for hindlimb ischemia in diabetic rabbit model,and the difference between diabetes and nondiabetes.Methods Rabbits were randomly allocated into three groups for transplantation therapy,PBS control group(n=8,A group),EPC transplantation with diabetes group(n=14,B group),EPC transplantation without diabetes group(n=8,C group).Diabetic rabbit model was established by injecting alloxan,and hindlimb ischemia was induced by complete excision of femoral artery.Diabetic rabbits with hindlimb ischemia were therapied by transplanted ex vivo expanded EPC,and then effects were assessed by capillary density and capillary to muscle fiber ratio,and VEGF in muscle was also determined.Results Capillary density,capillary to muscle fiber ratio,and VEGF in muscle increased significantly in EPC transplantation group with or without diabetes after transplanting 14 days(P