Anti-oxidative and anti-inflammatory effects of Ginkgo biloba extract (EGb761) on hindlimb skeletal muscle ischemia-reperfusion injury in rats.

In posterior spine surgery, retractors exert pressure on paraspinal muscles, elevating intramuscular pressure and compromising blood flow, potentially causing muscle injury during ischemia-reperfusion. Ginkgo biloba extract (EGb 761), known for its antioxidant and free radical scavenging properties and its role in treating cerebrovascular diseases, is investigated for its protective effects against muscle ischemia-reperfusion injury in vitro and in vivo. Animals were randomly divided into the control group, receiving normal saline, and experimental groups, receiving varying doses of EGb761 (25/50/100/200 mg/kg). A 2-h hind limb tourniquet-induced ischemia was followed by reperfusion. Blood samples collected pre-ischemia and 24 h post-reperfusion, along with muscle tissue samples after 24 h, demonstrated that EGb761 at 1000 µg/mL effectively inhibited IL-6 and TNF-α secretion in RAW 264.7 cells without cytotoxicity. EGb761 significantly reduced nitric oxide (NO) and malondialdehyde (MDA) levels, myeloperoxidase (MPO) activity, and increased glutathione (GSH) levels compared to the control after 24 h. Muscle tissue sections revealed more severe damage in the control group, indicating EGb761's potential in mitigating inflammatory responses and oxidative stress during ischemia-reperfusion injury, effectively protecting against muscle damage.

A subpopulation of tissue remodeling monocytes stimulates revascularization of the ischemic limb.

Despite decades of effort aimed at developing clinically effective cell therapies, including mixed population mononuclear cells, to revascularize the ischemic limb, there remains a paucity of patient-based studies that inform the function and fate of candidate cell types. In this study, we showed that circulating proangiogenic/arteriogenic monocytes (PAMs) expressing the FcγIIIA receptor CD16 were elevated in patients with chronic limb-threatening ischemia (CLTI), and these amounts decreased after revascularization. Unlike CD16-negative monocytes, PAMs showed large vessel remodeling properties in vitro when cultured with endothelial cells and smooth muscle cells and promoted salvage of the ischemic limb in vivo in a mouse model of hindlimb ischemia. PAMs showed a propensity to migrate toward and bind to ischemic muscle and to secrete angiogenic/arteriogenic factors, vascular endothelial growth factor A (VEGF-A) and heparin-binding epidermal growth factor. We instigated a first-in-human single-arm cohort study in which autologous PAMs were injected into the ischemic limbs of five patients with CLTI. Greater than 25% of injected cells were retained in the leg for at least 72 hours, of which greater than 80% were viable, with evidence of enhanced large vessel remodeling in the injected muscle area. In summary, we identified up-regulation of a circulatory PAM subpopulation as an endogenous response to limb ischemia in CLTI and tested a potentially clinically relevant therapeutic strategy.

Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis.

BACKGROUND: Angiogenesis is critical for forming new blood vessels from antedating vascular vessels. The endothelium is essential for angiogenesis, vascular remodelling and minimisation of functional deficits following ischaemia. The insulin-like growth factor (IGF) family is crucial for angiogenesis. Insulin-like growth factor-binding protein 5 (IGFBP5), a binding protein of the IGF family, may have places in angiogenesis, but the mechanisms are not yet completely understood. We sought to probe whether IGFBP5 is involved in pathological angiogenesis and uncover the molecular mechanisms behind it. METHODS AND RESULTS: IGFBP5 expression was elevated in the vascular endothelium of gastrocnemius muscle from critical limb ischaemia patients and hindlimb ischaemic (HLI) mice and hypoxic human umbilical vein endothelial cells (HUVECs). In vivo, loss of endothelial IGFBP5 (IGFBP5EKO) facilitated the recovery of blood vessel function and limb necrosis in HLI mice. Moreover, skin damage healing and aortic ring sprouting were faster in IGFBP5EKO mice than in control mice. In vitro, the genetic inhibition of IGFBP5 in HUVECs significantly promoted tube formation, cell proliferation and migration by mediating the phosphorylation of IGF1R, Erk1/2 and Akt. Intriguingly, pharmacological treatment of HUVECs with recombinant human IGFBP5 ensued a contrasting effect on angiogenesis by inhibiting the IGF1 or IGF2 function. Genetic inhibition of IGFBP5 promoted cellular oxygen consumption and extracellular acidification rates via IGF1R-mediated glycolytic adenosine triphosphate (ATP) metabolism. Mechanistically, IGFBP5 exerted its role via E3 ubiquitin ligase Von Hippel-Lindau (VHL)-regulated HIF1α stability. Furthermore, the knockdown of the endothelial IGF1R partially abolished the reformative effect of IGFBP5EKO mice post-HLI. CONCLUSION: Our findings demonstrate that IGFBP5 ablation enhances angiogenesis by promoting ATP metabolism and stabilising HIF1α, implying IGFBP5 is a novel therapeutic target for treating abnormal angiogenesis-related conditions.

Lymphaticovenous anastomosis in rabbits: A novel live experimental animal model for supermicrosurgical training.

BACKGROUND: Lymphaticovenous anastomosis is widely used in lymphedema management. Although its effectiveness in reducing edema in patients can be clinically observed, evaluating the long-term outcomes of this technique can be complex. This study established an animal model to assess the outcomes of lymphaticovenous anastomosis technique at 15 and 30-days post-surgery using indocyanine green lymphography, Patent Blue V dye injection, and histopathological examination. METHODS: An experimental model was established in the hindlimbs of 10 rabbits using the popliteal vein and afferent lymphatic vessels in the popliteal area. The subjects were divided into two groups: the first group (n = 5) underwent patency assessment at 0 and 15 days, and the second group (n = 5) at 0 and 30-days, resulting in 20 anastomoses. Patency was verified at 0, 15, and 30-days using indocyanine green lymphography and Patent Blue V injection. Histopathological examinations were performed on the collected anastomosis samples. RESULTS: The patency rate was 90% (19/20) initially, 60% (6/10) at 15 days post-surgery, and 80% (8/10) at 30-days. The average diameter of lymphatic vessels and veins was 1.0 mm and 0.8 mm, respectively. The median number of collateral veins was 3; the median surgical time was 65.8 min. Histopathology revealed minimal endothelial damage and inflammatory responses due to the surgical sutures, with vascular inflammation and thrombosis in a single case. Local vascular neoformations were observed. CONCLUSION: This study highlights the reliability and reproducibility of using rabbits as experimental models for training in lymphaticovenous anastomosis technique owing to the accessibility of the surgical site and dimensions of their popliteal vasculature.

Gold Nanorod-Loaded Nano-Contrast Agent with Composite Shell-Core Structure for Ultrasonic/Photothermal Imaging-Guided Therapy in Ischemic Muscle Disorders.

Purpose: This study aims to broaden the application of nano-contrast agents (NCAs) within the realm of the musculoskeletal system. It aims to introduce novel methods, strategies, and insights for the clinical management of ischemic muscle disorders, encompassing diagnosis, monitoring, evaluation, and therapeutic intervention. Methods: We developed a composite encapsulation technique employing O-carboxymethyl chitosan (OCMC) and liposome to encapsulate NCA-containing gold nanorods (GNRs) and perfluoropentane (PFP). This nanoscale contrast agent was thoroughly characterized for its basic physicochemical properties and performance. Its capabilities for in vivo and in vitro ultrasound imaging and photothermal imaging were authenticated, alongside a comprehensive biocompatibility assessment to ascertain its effects on microcirculatory perfusion in skeletal muscle using a murine model of hindlimb ischemia, and its potential to augment blood flow and facilitate recovery. Results: The engineered GNR@OCMC-liposome/PFP nanostructure exhibited an average size of 203.18±1.49 nm, characterized by size uniformity, regular morphology, and a good biocompatibility profile. In vitro assessments revealed NCA's potent photothermal response and its transformation into microbubbles (MBs) under near-infrared (NIR) irradiation, thereby enhancing ultrasonographic visibility. Animal studies demonstrated the nanostructure's efficacy in photothermal imaging at ischemic loci in mouse hindlimbs, where NIR irradiation induced rapid temperature increases and significantly increased blood circulation. Conclusion: The dual-modal ultrasound/photothermal NCA, encapsulating GNR and PFP within a composite shell-core architecture, was synthesized successfully. It demonstrated exceptional stability, biocompatibility, and phase transition efficiency. Importantly, it facilitates the encapsulation of PFP, enabling both enhanced ultrasound imaging and photothermal imaging following NIR light exposure. This advancement provides a critical step towards the integrated diagnosis and treatment of ischemic muscle diseases, signifying a pivotal development in nanomedicine for musculoskeletal therapeutics.

3D-Printed proangiogenic patches of photo-crosslinked gelatin and polyurethane hydrogels laden with vascular cells for treating vascular ischemic diseases.

Engineering vascularized tissues remains a promising approach for treating ischemic cardiovascular diseases. The availability of 3D-bioprinted vascular grafts that induce therapeutic angiogenesis can help avoid necrosis and excision of ischemic tissues. Here, using a combination of living cells and biodegradable hydrogels, we fabricated 3D-printed biocompatible proangiogenic patches from endothelial cell-laden photo-crosslinked gelatin (EC-PCG) bioink and smooth muscle cell-encapsulated polyurethane (SMC-PU) bioink. Implantation of 3D-bioprinted proangiogenic patches in a mouse model showed that EC-PCG served as an angiogenic capillary bed, whereas patterned SMC-PU increased the density of microvessels. Moreover, the assembled patterns between EC-PCG and SMC-PU induced the geometrically guided generation of microvessels with blood perfusion. In a rodent model of hindlimb ischemia, the vascular patches rescued blood flow to distal tissues, prevented toe/foot necrosis, promoted muscle remodeling, and increased the capillary density, thereby improving the heat-escape behavior of ischemic animals. Thus, our 3D-printed vascular cell-laden bioinks constitute efficient and scalable biomaterials that facilitate the engineering of vascular patches capable of directing therapeutic angiogenesis for treating ischemic vascular diseases.

Nitric oxide releasing nanofiber stimulates revascularization in response to ischemia via cGMP-dependent protein kinase.

Nitric oxide (NO) promotes angiogenesis via various mechanisms; however, the effective transmission of NO in ischemic diseases is unclear. Herein, we tested whether NO-releasing nanofibers modulate therapeutic angiogenesis in an animal hindlimb ischemia model. Male wild-type C57BL/6 mice with surgically-induced hindlimb ischemia were treated with NO-releasing 3-methylaminopropyltrimethoxysilane (MAP3)-derived or control (i.e., non-NO-releasing) nanofibers, by applying them to the wound for 20 min, three times every two days. The amount of NO from the nanofiber into tissues was assessed by NO fluorometric assay. The activity of cGMP-dependent protein kinase (PKG) was determined by western blot analysis. Perfusion ratios were measured 2, 4, and 14 days after inducing ischemia using laser doppler imaging. On day 4, Immunohistochemistry (IHC) with F4/80 and gelatin zymography were performed. IHC with CD31 was performed on day 14. To determine the angiogenic potential of NO-releasing nanofibers, aorta-ring explants were treated with MAP3 or control fiber for 20 min, and the sprout lengths were examined after 6 days. As per either LDPI (Laser doppler perfusion image) ratio or CD31 capillary density measurement, angiogenesis in the ischemic hindlimb was improved in the MAP3 nanofiber group; further, the total nitrate/nitrite concentration in the adduct muscle increased. The number of macrophage infiltrations and matrix metalloproteinase-9 (MMP-9) activity decreased. Vasodilator-stimulated phosphoprotein (VASP), one of the major substrates for PKG, increased phosphorylation in the MAP3 group. MAP3 nanofiber or NO donor SNAP (s-nitroso-n-acetyl penicillamine)-treated aortic explants showed enhanced sprouting in an ex vivo aortic ring assay, which was partially abrogated by KT5823, a potent inhibitor of PKG. These findings suggest that the novel NO-releasing nanofiber, MAP3 activates PKG and promotes therapeutic angiogenesis in response to hindlimb ischemia.

Dapagliflozin Improves Angiogenesis after Hindlimb Ischemia through the PI3K-Akt-eNOS Pathway.

Recently, the vascular protective effect of anti-diabetic agents has been receiving much attention. Sodium glucose cotransporter 2 (SGLT2) inhibitors had demonstrated reductions in cardiovascular (CV) events. However, the therapeutic effect of dapagliflozin on angiogenesis in peripheral arterial disease was unclear. This study aimed to explore the effect and mechanism of dapagliflozin on angiogenesis after hindlimb ischemia. We first evaluated the effect of dapagliflozin on post-ischemic angiogenesis in the hindlimbs of rats. Laser doppler imaging was used to detect the hindlimb blood perfusion. In addition, we used immunohistochemistry to detect the density of new capillaries after ischemia. The relevant signaling pathways of dapagliflozin affecting post-ischemic angiogenesis were screened through phosphoproteomic detection, and then the mechanism of dapagliflozin affecting post-ischemic angiogenesis was verified at the level of human umbilical vein endothelial cells (HUVECs). After subjection to excision of the left femoral artery, all rats were randomly distributed into two groups: the dapagliflozin group (left femoral artery resection, receiving intragastric feeding with dapagliflozin (1 mg/kg/d), for 21 consecutive days) and the model group, that is, the positive control group (left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days). In addition, the control group, that is the negative control group (without left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days) was added. At day 21 post-surgery, the dapagliflozin-treatment group had the greatest blood perfusion, accompanied by elevated capillary density. The results showed that dapagliflozin could promote angiogenesis after hindlimb ischemia. Then, the ischemic hindlimb adductor-muscle tissue samples from three rats of model group and dapagliflozin group were taken for phosphoproteomic testing. The results showed that the PI3K-Akt-eNOS signaling pathway was closely related to the effect of dapagliflozin on post-ischemic angiogenesis. Our study intended to verify this mechanism from the perspective of endothelial cells. In vitro, dapagliflozin enhanced the tube formation, migration, and proliferation of HUVECs under ischemic and hypoxic conditions. Additionally, the dapagliflozin administration upregulated the expression of angiogenic factors phosphorylated Akt (p-Akt) and phosphorylated endothelial nitric oxide synthase (p-eNOS), as well as vascular endothelial growth factor A (VEGFA), both in vivo and in vitro. These benefits could be blocked by either phosphoinositide 3-kinase (PI3K) or eNOS inhibitor. dapagliflozin could promote angiogenesis after ischemia. This effect might be achieved by promoting the activation of the PI3K-Akt-eNOS signaling pathway. This study provided a new perspective, new ideas, and a theoretical basis for the treatment of peripheral arterial disease.

miR-709 exerts an angiogenic effect through a FGF2 upregulation induced by a GSK3B downregulation.

The aim of this study was to identify angiogenic microRNAs (miRNAs) that could be used in the treatment of hindlimb ischemic tissues. miRNAs contained in extracellular vesicles (EVs) deriving from the plasma were analyzed in C57BL/6 mice, which have ischemia tolerance, and in BALB/c mice without ischemia tolerance as part of a hindlimb ischemia model; as a result 43 angiogenic miRNA candidates were identified. An aortic ring assay was employed by using femoral arteries isolated from BALC/c mice and EVs containing miRNA; as a result, the angiogenic miRNA candidates were limited to 14. The blood flow recovery was assessed after injecting EVs containing miRNA into BALB/c mice with hindlimb ischemia, and miR-709 was identified as a promising angiogenic miRNA. miR-709-encapsulating EVs were found to increase the expression levels of the fibroblast growth factor 2 (FGF2) mRNA in the thigh tissues of hindlimb ischemia model BALB/c mice. miR-709 was also found to bind to the 3'UTR of glycogen synthase kinase 3 beta (GSK3B) in three places. GSK3B-knockdown human artery-derived endothelial cells were found to express high levels of FGF2, and were characterized by increased cell proliferation. These findings indicate that miR-709 induces an upregulation of FGF2 through the downregulation of GSK3B.

Systematic review and meta-analysis of the effect of bone marrow-derived cell therapies on hind limb perfusion.

Preclinical and clinical studies on the administration of bone marrow-derived cells to restore perfusion show conflicting results. We conducted a systematic review and meta-analysis on preclinical studies to assess the efficacy of bone marrow-derived cells in the hind limb ischemia model and identify possible determinants of therapeutic efficacy. In vivo animal studies were identified using a systematic search in PubMed and EMBASE on 10 January 2022. 85 studies were included for systematic review and meta-analysis. Study characteristics and outcome data on relative perfusion were extracted. The pooled mean difference was estimated using a random effects model. Risk of bias was assessed for all included studies. We found a significant increase in perfusion in the affected limb after administration of bone marrow-derived cells compared to that in the control groups. However, there was a high heterogeneity between studies, which could not be explained. There was a high degree of incomplete reporting across studies. We therefore conclude that the current quality of preclinical research is insufficient (low certainty level as per GRADE assessment) to identify specific factors that might improve human clinical trials.

Carbon Monoxide Alleviates Post-ischemia-reperfusion Skeletal Muscle Injury and Systemic Inflammation.

Restoration of blood flow in skeletal muscle after a prolonged period of ischemia induces muscular ischemia-reperfusion injury, leading to local injury/dysfunction in muscles followed by systemic inflammatory responses. However, preventive/curative agents for skeletal muscle ischemia injury are unavailable in clinics to date. Increasing evidence has validated that carbon monoxide (CO) prevents the progression of ischemia-reperfusion injury in various organs owing to its versatile bioactivity. Previously, we developed a bioinspired CO donor, CO-bound red blood cells (CO-RBC), which mimics the dynamics of RBC-associated CO in the body. In the present study, we have tested the therapeutic potential of CO-RBC in muscular injury/dysfunction and secondary systemic inflammation induced by skeletal muscle ischemia-reperfusion. The results indicate that CO-RBC rather than RBC alone suppressed elevation of plasma creatine phosphokinase, a marker of muscular injury, in rats subjected to both hind limbs ischemia-reperfusion. In addition, the results of the treadmill walking test revealed a significantly decreased muscular motor function in RBC-treated rats subjected to both hind limbs ischemia-reperfusion than that in healthy rats, however, CO-RBC treatment facilitated sustained muscular motor functions after hind limbs ischemia-reperfusion. Furthermore, CO-RBC rather than RBC suppressed the production of tumour necrosis factor (TNF)-α and interleukin (IL)-6, which were upregulated by muscular ischemia-reperfusion. Interestingly, CO-RBC treatment induced higher levels of IL-10 compared to saline or RBC treatments. Based on these findings, we suggest that CO-RBC exhibits a suppressive effect against skeletal muscle injury/dysfunction and systemic inflammatory responses after skeletal muscle ischemia-reperfusion.

Alginate-Encapsulated Mesenchymal Stromal Cells Improve Hind Limb Ischemia in a Translational Swine Model.

BACKGROUND: Cellular therapies have been investigated to improve blood flow and prevent amputation in peripheral artery disease with limited efficacy in clinical trials. Alginate-encapsulated mesenchymal stromal cells (eMSCs) demonstrated improved retention and survival and promoted vascular generation in murine hind limb ischemia through their secretome, but large animal evaluation is necessary for human applicability. We sought to determine the efficacy of eMSCs for peripheral artery disease-induced limb ischemia through assessment in our durable swine hind limb ischemia model. METHODS AND RESULTS: Autologous bone marrow eMSCs or empty alginate capsules were intramuscularly injected 2 weeks post-hind limb ischemia establishment (N=4/group). Improvements were quantified for 4 weeks through walkway gait analysis, contrast angiography, blood pressures, fluorescent microsphere perfusion, and muscle morphology and histology. Capsules remained intact with mesenchymal stromal cells retained for 4 weeks. Adenosine-induced perfusion deficits and muscle atrophy in ischemic limbs were significantly improved by eMSCs versus empty capsules (mean±SD, 1.07±0.19 versus 0.41±0.16, P=0.002 for perfusion ratios and 2.79±0.12 versus 1.90±0.62 g/kg, P=0.029 for ischemic muscle mass). Force- and temporal-associated walkway parameters normalized (ratio, 0.63±0.35 at week 3 versus 1.02±0.19 preligation; P=0.17), and compensatory footfall patterning was diminished in eMSC-administered swine (12.58±8.46% versus 34.85±15.26%; P=0.043). Delivery of eMSCs was associated with trending benefits in collateralization, local neovascularization, and muscle fibrosis. Hypoxia-cultured porcine mesenchymal stromal cells secreted vascular endothelial growth factor and tissue inhibitor of metalloproteinase 2. CONCLUSIONS: This study demonstrates the promise of the mesenchymal stromal cell secretome at improving peripheral artery disease outcomes and the potential for this novel swine model to serve as a component of the preclinical pipeline for advanced therapies.

Endothelial ELABELA improves post-ischemic angiogenesis by upregulating VEGFR2 expression.

BACKGROUND: Post-ischemic angiogenesis is critical for perfusion recovery and tissue repair. ELABELA (ELA) plays an essential role in embryonic heart development and vasculogenesis. However, the mechanism of ELA on post-ischemic angiogenesis is poorly characterized. METHODS: We first assessed ELA expression after hind limb ischemia (HLI) in mice. We then established a HLI model in tamoxifen-inducible endothelial-ELA-specific knockout mice (ELAECKO) and assessed the rate of perfusion recovery, capillary density, and VEGFR2 pathway. Knockdown of ELA with lentivirus or siRNA and exogenous addition of ELA peptides were employed to analyze the effects of ELA on angiogenic capacity and VEGFR2 pathway in endothelial cells in vitro. The serum levels of ELA in healthy people and patients with type 2 diabetes mellitus (T2DM) and diabetic foot ulcer (DFU) were detected by a commercial ELISA kit. RESULTS: In murine HLI models, ELA was significantly up-regulated in the ischemic hindlimb. Endothelial-specific deletion of ELA impaired perfusion recovery and angiogenesis. In physiologic conditions, no significant difference in VEGFR2 expression was found between ELAECKO mice and ELAWT mice. After ischemia, the expression of VEGFR2, p-VEGFR2, and p-AKT was significantly lower in ELAECKO mice than in ELAWT mice. In cellular experiments, the knockdown of ELA inhibited endothelial cell proliferation and tube formation, and the addition of ELA peptides promoted proliferation and tube formation. Mechanistically, ELA upregulated the expression of VEGFR2, p-VEGFR2, and p-AKT in endothelial cells under hypoxic conditions. In clinical investigations, DFU patients had significantly lower serum levels of ELA compared to T2DM patients. CONCLUSION: Our results indicated that endothelial ELA is a positive regulator of post-ischemic angiogenesis via upregulating VEGFR2 expression. Targeting ELA may be a potential therapeutic option for peripheral arterial diseases.

Novel Angiogenesis Role of GLP-1(32-36) to Rescue Diabetic Ischemic Lower Limbs via GLP-1R-Dependent Glycolysis in Mice.

BACKGROUND: Restoring the capacity of endothelial progenitor cells (EPCs) to promote angiogenesis is the major therapeutic strategy of diabetic peripheral artery disease. The aim of this study was to investigate the effects of GLP-1 (glucagon-like peptide 1; 32-36)-an end product of GLP-1-on angiogenesis of EPCs and T1DM (type 1 diabetes) mice, as well as its interaction with the classical GLP-1R (GLP-1 receptor) pathway and its effect on mitochondrial metabolism. METHODS: In in vivo experiments, we conducted streptozocin-induced type 1 diabetic mice as a murine model of unilateral hind limb ischemia to examine the therapeutic potential of GLP-1(32-36) on angiogenesis. We also generated Glp1r-/- mice to detect whether GLP-1R is required for angiogenic function of GLP-1(32-36). In in vitro experiments, EPCs isolated from the mouse bone marrow and human umbilical cord blood samples were used to detect GLP-1(32-36)-mediated angiogenic capability under high glucose treatment. RESULTS: We demonstrated that GLP-1(32-36) did not affect insulin secretion but could significantly rescue angiogenic function and blood perfusion in ischemic limb of streptozocin-induced T1DM mice, a function similar to its parental GLP-1. We also found that GLP-1(32-36) promotes angiogenesis in EPCs exposed to high glucose. Specifically, GLP-1(32-36) has a causal role in improving fragile mitochondrial function and metabolism via the GLP-1R-mediated pathway. We further demonstrated that GLP-1(32-36) rescued diabetic ischemic lower limbs by activating the GLP-1R-dependent eNOS (endothelial NO synthase)/cGMP/PKG (protein kinase G) pathway. CONCLUSIONS: Our study provides a novel mechanism with which GLP-1(32-36) acts in modulating metabolic reprogramming toward glycolytic flux in partnership with GLP-1R for improved angiogenesis in high glucose-exposed EPCs and T1DM murine models. We propose that GLP-1(32-36) could be used as a monotherapy or add-on therapy with existing treatments for peripheral artery disease. REGISTRATION: URL: www.ebi.ac.uk/metabolights/; Unique identifier: MTBLS9543.

TRIM2 Selectively Regulates Inflammation-Driven Pathological Angiogenesis without Affecting Physiological Hypoxia-Mediated Angiogenesis.

Angiogenesis is a critical physiological response to ischemia but becomes pathological when dysregulated and driven excessively by inflammation. We recently identified a novel angiogenic role for tripartite-motif-containing protein 2 (TRIM2) whereby lentiviral shRNA-mediated TRIM2 knockdown impaired endothelial angiogenic functions in vitro. This study sought to determine whether these effects could be translated in vivo and to determine the molecular mechanisms involved. CRISPR/Cas9-generated Trim2-/- mice that underwent a periarterial collar model of inflammation-induced angiogenesis exhibited significantly less adventitial macrophage infiltration relative to wildtype (WT) littermates, concomitant with decreased mRNA expression of macrophage marker Cd68 and reduced adventitial proliferating neovessels. Mechanistically, TRIM2 knockdown in endothelial cells in vitro attenuated inflammation-driven induction of critical angiogenic mediators, including nuclear HIF-1α, and curbed the phosphorylation of downstream effector eNOS. Conversely, in a hindlimb ischemia model of hypoxia-mediated angiogenesis, there were no differences in blood flow reperfusion to the ischemic hindlimbs of Trim2-/- and WT mice despite a decrease in proliferating neovessels and arterioles. TRIM2 knockdown in vitro attenuated hypoxia-driven induction of nuclear HIF-1α but had no further downstream effects on other angiogenic proteins. Our study has implications for understanding the role of TRIM2 in the regulation of angiogenesis in both pathophysiological contexts.

The comparison of adipose-derived stromal cells (ADSCs) delivery method in a murine model of hindlimb ischemia.

BACKGROUND: Adipose-derived stromal cells (ADSCs) demonstrate ability to promote tissue healing and down-regulate excessive inflammation. ADSCs have been used to treat critical limb ischemia in preclinical and clinical trials, but still, there is little known about their optimal delivery strategy. To date, no direct analysis of different methods of ADSCs delivery has been performed in the hindlimb ischemia model. Therefore, in this study we focused on the therapeutic efficacy of different ADSCs delivery methods in a murine model of hindlimb ischemia. METHODS: For the hADSCs isolation, we used the subcutaneous adipose tissue collected during the surgery. The murine hindlimb ischemia was used as a model. The unilateral femoral artery ligation was performed on 10-12-week-old male C57BL/6. ADSCs were delivered directly into ischemic muscle, into the contralateral muscle or intravenously. 7 and 14 days after the surgery, the gastrocnemius and quadriceps muscles were collected for the immunohistochemical analysis. The results were analyzed with relevant tests using the Statistica software. RESULTS: Our research revealed that muscle regeneration, angiogenesis, arteriogenesis and macrophage infiltration in murine model of hindlimb ischemia differ depending on ADSCs delivery method. We have demonstrated that intramuscular method (directly into ischemic limb) of ADSCs delivery is more efficient in functional recovery after critical limb ischemia than intravenous or contralateral route. CONCLUSIONS: We have noticed that injection of ADSCs directly into ischemic limb is the optimal delivery strategy because it increases: (1) muscle fiber regeneration, (2) the number of capillaries and (3) the influx of macrophages F4/80+/CD206+.

Poly(ethylenimine)-Cyclodextrin-Based Cationic Polymer Mediated HIF-1α Gene Delivery for Hindlimb Ischemia Treatment.

Hindlimb ischemia is a common disease worldwide featured by the sudden decrease in limb perfusion, which usually causes a potential threat to limb viability and even amputation or death. Revascularization has been defined as the gold-standard therapy for hindlimb ischemia. Considering that vascular injury recovery requires cellular adaptation to the hypoxia, hypoxia-inducible factor 1 α (HIF-1α) is a potential gene for tissue restoration and angiogenesis. In this manuscript, effective gene delivery vector PEI-ß-CD (PC) was reported for the first application in the hindlimb ischemia treatment to deliver HIF-1α plasmid in vitro and in vivo. Our in vitro finding demonstrated that PC/HIF-1α-pDNA could be successfully entered into the cells and mediated efficient gene transfection with good biocompatibility. More importantly, under hypoxic conditions, PC/HIF-1α-pDNA could up-regulate the HUEVC cell viability. In addition, the mRNA levels of VEGF, Ang-1, and PDGF were upregulated, and transcriptome results also demonstrated that the cell-related function of response to hypoxia was enhanced. The therapeutic effect of PC/HIF-1α-pDNA was further estimated in a murine acute hindlimb ischemia model, which demonstrated that intramuscular injection of PC/HIF-1α-pDNA resulted in significantly increased blood perfusion and alleviation in tissue damage, such as tissue fibrosis and inflammation. The results provide a rationale that HIF-1α-mediated gene therapy might be a practical strategy for the treatment of limb ischemia.

Effects of Pemafibrate on Reducing Oxidative Stress and Augmenting Angiogenesis in Ischemic Limb Tissue.

OBJECTIVE: Oxidative damage is observed in the ischemic limbs of patients with arteriosclerosis obliterans. We investigated whether pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, reduced oxidative stress in ischemic limbs and consequently rescued limb damage in model mice. MATERIALS AND METHODS: We surgically induced hind-limb ischemia in mice and orally administered pemafibrate solution (P-05 group, 0.5 mg/kg/day; P-10 group, 1.0 mg/kg/day) or control solution (control group). Seven days after the surgery, differences in reactive oxygen species (ROS) contents, antioxidative enzyme and transcription factor expression, blood flow, and capillary density in ischemic limbs were assessed. RESULTS: Tissue ROS levels were lower in the P-05 and P-10 groups compared with those in the control group. Although the tissue expression levels of nuclear factor-erythroid 2-related factor 2 increased in the P-10 group compared with that in the control group, no corresponding changes were observed in the tissue expression of four antioxidative enzymes. The limb salvage rates and capillary densities in ischemic limbs were higher in the P-05 and P-10 groups than that in the control group. CONCLUSION: Pemafibrate treatment reduced oxidative stress and augmented angiogenesis in ischemic limbs, contributing to prevention of limb damage in mice.

Transmembrane stem factor nanodiscs enhanced revascularization in a hind limb ischemia model in diabetic, hyperlipidemic rabbits.

Therapies to revascularize ischemic tissue have long been a goal for the treatment of vascular disease and other disorders. Therapies using stem cell factor (SCF), also known as a c-Kit ligand, had great promise for treating ischemia for myocardial infarct and stroke, however clinical development for SCF was stopped due to toxic side effects including mast cell activation in patients. We recently developed a novel therapy using a transmembrane form of SCF (tmSCF) delivered in lipid nanodiscs. In previous studies, we demonstrated tmSCF nanodiscs were able to induce revascularization of ischemia limbs in mice and did not activate mast cells. To advance this therapeutic towards clinical application, we tested this therapy in an advanced model of hindlimb ischemia in rabbits with hyperlipidemia and diabetes. This model has therapeutic resistance to angiogenic therapies and maintains long term deficits in recovery from ischemic injury. We treated rabbits with local treatment with tmSCF nanodiscs or control solution delivered locally from an alginate gel delivered into the ischemic limb of the rabbits. After eight weeks, we found significantly higher vascularity in the tmSCF nanodisc-treated group in comparison to alginate treated control as quantified through angiography. Histological analysis also showed a significantly higher number of small and large blood vessels in the ischemic muscles of the tmSCF nanodisc treated group. Importantly, we did not observe inflammation or mast cell activation in the rabbits. Overall, this study supports the therapeutic potential of tmSCF nanodiscs for treating peripheral ischemia.

Microvascular permeability and texture analysis of bone marrow in diabetic rabbits with critical limb ischemia based on dynamic contrast-enhanced magnetic resonance imaging.

BACKGROUND: Early on in the development of diabetes, skeletal muscles can exhibit microarchitectural changes that can be detected using texture analysis (TA) based on volume transfer constant (Ktrans) maps. Nevertheless, there have been few studies and thus we evaluated microvascular permeability and the TA of the bone marrow in diabetics with critical limb ischemia (CLI). METHODS: Eighteen male rabbits were randomly assigned equally into an operation group with hindlimb ischemia and diabetes, a sham-operated group with diabetes only, and a control group. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) was performed on all rabbits at predetermined intervals (1, 5, 10, 15, 20, and 25 days post-surgery). The pharmacokinetic model was used to generate the permeability parameters, while the textural parameters were derived from the Ktrans map. Data analysis methods included the independent sample t-test, Mann-Whitney U test, repeated-measures analysis of variance, and Pearson correlation tests. RESULTS: The Ktrans values reached a minimum on day 1 after ischemia induction, then gradually recovered, but remained lower than those of the sham-operated group. The volume fraction only showed a significant difference between the operation group and the sham-operated group on day 5 post-surgery, but not in the extravascular extracellular space volume fraction at all time points. A significantly reduced Ktrans on day 1, a decreased number of bone trabeculae (Tb.N), and the area of bone trabeculae (Tb.Ar), and an increased microvessel density on day 25 in the operation group compared with the sham-operated group were observed. At each time point, there was a discernible difference between the two groups in the mean value, mean of positive pixels, and sumAverage. CONCLUSIONS: The early stages of diabetic bone marrow with CLI can be evaluated by DCE-MRI for microvascular permeability. Texture analysis based on DCE-MRI could act as an imaging discriminator and new radiological analysis tool for critical limb ischemia in diabetes mellitus.