Grain-Based Dietary Background Impairs Restoration of Blood Flow and Skeletal Muscle During Hindlimb Ischemia in Comparison With Low-Fat and High-Fat Diets

Background: Among vascular pathologies associated with obesity, peripheral artery disease (PAD) occupies the important position. In clinical practice, nutritional interventions are recommended for patients with PAD. In this work, we investigated how the different dietary backgrounds affect the regeneration rate of ischemic hindlimb in mice.Methods: Male C57BL/6J mice were housed on three types of diet: low-fat (LFD), high-fat (HFD), and grain-based diet (GBD) for 13 weeks. Metabolic parameters including FBG level, ITT, and GTT were evaluated. The blood flow was assessed by laser Doppler scanning on 7, 14, and 21 days after hindlimb ischemia. Necrotic area of m.tibialis, macrophage infiltration, and angiogenesis/arteriogenesis were evaluated by histology. Glucose uptake in recovered skeletal muscle was analyzed using [3H]-2-deoxyglucose, and GLUT1 and GLUT4 expression were assessed by Western blotting.Results: In our work, we developed three experimental groups with different metabolic parameters: LFD with normal glucose metabolism, GBD with mild hyperglycemia, and HFD with impaired glucose tolerance. GBD-fed mice had a tendency to increase necrosis of m. tibialis and significantly higher macrophage infiltration than LFD and HFD groups. Moreover, GBD-fed mice had a trend to decreased blood flow recovery and significantly impaired arteriogenesis. Recovered skeletal muscle of GBD-fed mice had lower glucose uptake and decreased level of GLUT4 expression.Conclusion: Thus, we conclude that dietary background and metabolic status determine the rate of post-ischemic regeneration including angiogenesis, skeletal muscle recovery and metabolic activity. The most effective regeneration is supported by LFD, while the lowest rate of regeneration occurs on GBD.

Nano-Sized Extracellular Matrix Particles Lead to Therapeutic Improvement for Cutaneous Wound and Hindlimb Ischemia

Cell-derived matrix (CDM) has proven its therapeutic potential and been utilized as a promising resource in tissue regeneration. In this study, we prepared a human fibroblast-derived matrix (FDM) by decellularization of in vitro cultured cells and transformed the FDM into a nano-sized suspended formulation (sFDM) using ultrasonication. The sFDM was then homogeneously mixed with Pluronic F127 and hyaluronic acid (HA), to effectively administer sFDM into target sites. Both sFDM and sFDM containing hydrogel (PH/sFDM) were characterized via immunofluorescence, sol–gel transition, rheological analysis, and biochemical factors array. We found that PH/sFDM hydrogel has biocompatible, mechanically stable, injectable properties and can be easily administered into the external and internal target regions. sFDM itself holds diverse bioactive molecules. Interestingly, sFDM-containing serum-free media helped maintain the metabolic activity of endothelial cells significantly better than those in serum-free condition. PH/sFDM also promoted vascular endothelial growth factor (VEGF) secretion from monocytes in vitro. Moreover, when we evaluated therapeutic effects of PH/sFDM via the murine full-thickness skin wound model, regenerative potential of PH/sFDM was supported by epidermal thickness, significantly more neovessel formation, and enhanced mature collagen deposition. The hindlimb ischemia model also found some therapeutic improvements, as assessed by accelerated blood reperfusion and substantially diminished necrosis and fibrosis in the gastrocnemius and tibialis muscles. Together, based on sFDM holding a strong therapeutic potential, our engineered hydrogel (PH/sFDM) should be a promising candidate in tissue engineering and regenerative medicine.

Ischemic-Trained Monocytes Improve Arteriogenesis in a Mouse Model of Hindlimb Ischemia

Objective: Monocytes, which play an important role in arteriogenesis, can build immunologic memory by a functional reprogramming that modifies their response to a second challenge. This process, called trained immunity, is evoked by insults that shift monocyte metabolism, increasing HIF (hypoxia-inducible factor)-1α levels. Since ischemia enhances HIF-1α, we evaluate whether ischemia can lead to a functional reprogramming of monocytes, which would contribute to arteriogenesis after hindlimb ischemia. Methods and Results: Mice exposed to ischemia by 24 hours of femoral artery occlusion (24 hours trained) or sham were subjected to hindlimb ischemia one week later; the 24-hour trained mice showed significant improvement in blood flow recovery and arteriogenesis after hindlimb ischemia. Adoptive transfer using bone marrow-derived monocytes (BM-Mono) from 24-hour trained or sham donor mice, demonstrated that recipients subjected to hindlimb ischemia who received 24 hours ischemic-trained monocytes had remarkable blood flow recovery and arteriogenesis. Further, ischemic-trained BM-Mono had increased HIF-1α and GLUT-1 gene expression during femoral artery occlusion. Circulating cytokines and GLUT-1 were also upregulated during femoral artery occlusion.Transcriptomic analysis and confirmatory qPCR performed in 24 hours trained and sham BM-Mono revealed that among the 15 top differentially expressed genes, 4 were involved in lipid metabolism in the ischemic-trained monocytes. Lipidomic analysis confirmed that ischemia training altered the cholesterol metabolism of these monocytes. Further, several histone-modifying epigenetic enzymes measured by qPCR were altered in mouse BM-Mono exposed to 24 hours hypoxia. Conclusions: Ischemia training in BM-Mono leads to a unique gene profile and improves blood flow and arteriogenesis after hindlimb ischemia.

Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3β/NRF2 Pathway in Diabetic Hindlimb Ischemia

Background. Peripheral arterial disease (PAD) is a typical disease of atherosclerosis, most commonly influencing the lower extremities. In patients with PAD, revascularization remains a preferred treatment strategy. Buyang Huanwu decoction (BHD) is a popular Chinese herbal prescription which has showed effects of cardiovascular protection through conducting antioxidant, antiapoptotic, and anti-inflammatory effects. Here, we intend to study the effect of BHD on promoting revascularization via the Akt/GSK3β/NRF2 pathway in diabetic hindlimb ischemia (HLI) model of mice. Materials and Methods. All db/db mice ( n = 60 ) were randomly divided into 6 groups by table of random number. (1) Sham group ( N = 10 ): 7-0 suture thread passed through the underneath of the femoral artery and vein without occlusion. The remaining 5 groups were treated differently on the basis of the HLI (the femoral artery and vein from the inguinal ligament to the knee joint were transected and the vascular stump was ligated with 7-0 silk sutures) model: (2) HLI+NS group ( N = 15 ): 0.2 ml NS was gavaged daily for 3 days before modeling and 14 days after occlusion; (3) HLI+BHD group ( N = 15 ): 0.2 ml BHD (20 g/kg/day) was gavaged daily for 3 days before modeling and 14 days after occlusion; (4) HLI+BHD+sh-NC group ( N = 8 ): local injection of adenovirus vector carrying the nonsense shRNA (Ad-GFP) in the hindlimbs of mice before treatment; (5) HLI+BHD+sh-NRF2 group ( N = 8 ): knockdown of NRF2 in the hindlimbs of mice by local intramuscular injection of adenovirus vector carrying NRF2 shRNA (Ad-NRF2-shRNA) before treatment; and (6) HLI+BHD+LY294002 group ( N = 4 ): intravenous injection of LY294002 (1.5 mg/kg) once a day for 14 days on the basis of the HLI+BHD group. Laser Doppler examination, vascular cast, and immunofluorescence staining were applied to detect the revascularization of lower limbs in mice. Western blot analysis was used to detect the expression of vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor- (TNF-) α, heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone-1 (NQO-1), catalase (CAT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphorylated protein kinase B (p-AKT), and phosphorylated glycogen synthase kinase-3 beta (p-GSK3β). HE staining was used to assess the level of muscle tissue damage and inflammation in the lower extremities. Local multipoint injection of Ad-NRF2-shRNA was used to knock down NRF2, and qPCR was applied to detect the mRNA level of NRF2. The blood glucose, triglyceride, cholesterol, MDA, and SOD levels of mice were tested using corresponding kits. The SPSS 20.0 software and GraphPad Prism 6.05 were used to do all statistics. Values of P < 0.05 were considered as statistically significant. Results and Conclusions. BHD could enhance the revascularization of lower limbs in HLI mice, while BHD has no effect on blood glucose and lipid level in db/db mice ( P > 0.05 ). BHD could elevate the protein expression of VEGF, HO-1, NQO-1, and CAT ( P < 0.05 ) and decrease the expression of IL-1β, IL-6, and TNF-α ( P < 0.05 ) in HLI mice. Meanwhile, BHD could activate NRF2 and promote the phosphorylation of AKT/GSK3β during revascularization ( P < 0.05 ). In contrast, knockdown of NRF2 impaired the protective effects of BHD on HLI ( P < 0.05 ). LY294002 inhibited the upregulation of NRF2 activated by BHD through inhibiting the phosphorylation of the AKT/GSK3β pathway ( P < 0.05 ). The present study demonstrated that BHD could promote revascularization on db/db mice with HLI through targeting antioxidation, anti-inflammation, and angiogenesis via the AKT/GSK3β/NRF2 pathway.

Characterization of Novel P-Selectin Targeted Complement Inhibitors in Murine Models of Hindlimb Injury and Transplantation

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterized in vitro and in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thrombotic vs. hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation.

Loss of Id3 (Inhibitor of Differentiation 3) Increases the Number of IgM-Producing B-1b Cells in Ischemic Skeletal Muscle Impairing Blood Flow Recovery During Hindlimb Ischemia

Objective: Neovascularization can maintain and even improve tissue perfusion in the setting of limb ischemia during peripheral artery disease. The molecular and cellular mechanisms mediating this process are incompletely understood. We investigate the potential role(s) for Id3 (inhibitor of differentiation 3) in regulating blood flow in a murine model of hindlimb ischemia (HLI). Approach and Results: HLI was modeled through femoral artery ligation and resection and blood flow recovery was quantified by laser Doppler perfusion imaging. Mice with global Id3 deletion had significantly impaired perfusion recovery at 14 and 21 days of HLI. Endothelial- or myeloid cell-specific deletion of Id3 revealed no effect on perfusion recovery while B-cell–specific knockout of Id3 (Id3 BKO ) revealed a significant attenuation of perfusion recovery. Flow cytometry revealed no differences in ischemia-induced T cells or myeloid cell numbers at 7 days of HLI, yet there was a significant increase in B-1b cells in Id3 BKO . Consistent with these findings, ELISA demonstrated increases in skeletal muscle and plasma IgM. In vitro experiments demonstrated reduced proliferation and increased cell death when endothelial cells were treated with conditioned media from IgM-producing B-1b cells and tibialis anterior muscles in Id3 BKO mice showed reduced density of total CD31 + and αSMA + CD31 + vessels. Conclusions: This study is the first to demonstrate a role for B-cell–specific Id3 in maintaining blood flow recovery during HLI. Results suggest a role for Id3 in promoting blood flow during HLI and limiting IgM-expressing B-1b cell expansion. These findings present new mechanisms to investigate in peripheral artery disease pathogenesis.

Astragaloside IV Promotes the Angiogenic Capacity of Adipose-derived Mesenchymal Stem Cells in a Hindlimb Ischemia Model by FAK Phosphorylation via CXCR2

BackgroundTherapeutic a­ngiogenesis by transplantation of autologous/allogeneic adipose stem cells (ADSCs) is a potential method for the treatment of severe limb ischemia (CLI). However, the therapeutic efficiency is limited by poor viability, adhesion, migration and differentiation after cell transplantation into the target area. Astragaloside IV (AS-IV), one of the main active components of Astragalus, has been widely used in the treatment of ischemic diseases and can promote cell proliferation and angiogenesis.MethodsADSCs were obtained and pretreated with the different concentration of AS-IV. In vitro, we analyzed the influence of AS-IV on ADSC proliferation, migration, angiogenesis and recruitment of human umbilical vein endothelial cells (HUVECs) and analyzed the relevant molecular mechanism. In vivo, we injected drug-pretreated ADSCs into a Matrigel or hindlimb ischemia model and evaluated the therapeutic effect by the laser Doppler perfusion index, immunofluorescence and histopathology. ResultsIn vitro experiments showed that AS-IV improved ADSC migration, angiogenesis and endothelial recruitment. The molecular mechanism may be related to the upregulation of CXCR2 to promote the phosphorylation of focal adhesion kinase (FAK). In vivo, Matrigel plug assay showed that ADSCs pretreated with AS-IV have stronger angiogenic potential. The laser Doppler perfusion index of the hindlimbs of mice in the ADSC/AS-IV group was significantly higher than the laser Doppler perfusion index of the hindlimbs of mice of the ADSC group and the control group, and the microvessel density was significantly increased.ConclusionAS-IV pretreatment can improve the migration, angiogenesis and endothelial cell recruitment of ADSCs by FAK phosphorylation via CXCR2, as well as the therapeutic effect on ischemic hindlimb model, which will bring new insights into the treatment of severe limb ischemia.