Model Of Critical Limb Ischemia Research Articles

Growth factor free, peptide-functionalized gelatin hydrogel promotes arteriogenesis and attenuates tissue damage in a murine model of critical limb ischemia

Critical limb ischemia (CLI) occurs when blood flow is restricted through the arteries, resulting in ulcers, necrosis, and chronic wounds in the downstream extremities. The development of collateral arterioles (i.e. arteriogenesis), either by remodeling of pre-existing vascular networks or de novo growth of new vessels, can prevent or reverse ischemic damage, but it remains challenging to stimulate collateral arteriole development in a therapeutic context. Here, we show that a gelatin-based hydrogel, devoid of growth factors or encapsulated cells, promotes arteriogenesis and attenuates tissue damage in a murine CLI model. The gelatin hydrogel is functionalized with a peptide derived from the extracellular epitope of Type 1 cadherins. Mechanistically, these “GelCad” hydrogels promote arteriogenesis by recruiting smooth muscle cells to vessel structures in both ex vivo and in vivo assays. In a murine femoral artery ligation model of CLI, delivery of in situ crosslinking GelCad hydrogels was sufficient to restore limb perfusion and maintain tissue health for 14 days, whereas mice treated with gelatin hydrogels had extensive necrosis and autoamputated within 7 days. A small cohort of mice receiving the GelCad hydrogels were aged out to 5 months and exhibited no decline in tissue quality, indicating durability of the collateral arteriole networks. Overall, given the simplicity and off-the-shelf format of the GelCad hydrogel platform, we suggest it could have utility for CLI treatment and potentially other indications that would benefit from arteriole development.

Functionally enhanced cell spheroids for stem cell therapy: Role of TIMP1 in the survival and therapeutic effectiveness of stem cell spheroids.

Stem cell therapy has emerged as a promising regenerative medicine strategy but is limited by poor cell survival, leading to low therapeutic outcomes. We developed cell spheroid therapeutics to overcome this limitation. We utilized solid-phase FGF2 to form functionally enhanced cell spheroid-adipose derived (FECS-Ad), a type of cell spheroid that preconditions cells with intrinsic hypoxia to increase the survival of transplanted cells. We demonstrated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels in FECS-Ad, which led to the upregulation of tissue inhibitor of metalloproteinase 1 (TIMP1). TIMP1 enhanced the survival of FECS-Ad, presumably through the CD63/FAK/Akt/Bcl2 anti-apoptotic signaling pathway. Cell viability of transplanted FECS-Ad was reduced by TIMP1 knockdown in an in vitro collagen gel block and a mouse model of critical limb ischemia (CLI). TIMP1 knockdown in FECS-Ad inhibited angiogenesis and muscle regeneration induced by FECS-Ad transplanted into ischemic mouse tissue. Genetic overexpression of TIMP1 in FECS-Ad further promoted the survival and therapeutic efficacy of transplanted FECS-Ad. Collectively, we suggest that TIMP1 acts as a key survival factor to improve the survival of transplanted stem cell spheroids, which provides scientific evidence for enhanced therapeutic efficacy of stem cell spheroids, and FECS-Ad as a potential therapeutic agent to treat CLI. STATEMENT OF SIGNIFICANCE: We used FGF2-tethered substrate platform to form adipose-derived stem cell spheroids, as we named as functionally enhanced cell spheroid-adipose derived (FECS-Ad). In this paper, we showed that intrinsic hypoxia of spheroids upregulated expression of HIF-1α, which in turn upregulated expression of TIMP1. Our paper highlights TIMP1 as a key survival factor to improve survival of transplanted stem cell spheroids. We believe that our study has a very strong scientific impact as extending transplantation efficiency is essential for successful stem cell therapy.

Robust critical limb ischemia porcine model involving skeletal muscle necrosis

This work sought to develop a robust and clinically relevant swine model of critical limb ischemia (CLI) involving the onset of ischemic muscle necrosis. CLI carries about 25–40% risk of major amputation with 20% annual mortality. Currently, there is no specific treatment that targets the ischemic myopathy characteristic of CLI. Current swine models of CLI, with tolerable side-effects, fail to achieve sustained ischemia followed by a necrotic myopathic endpoint. Such limitation in experimental model hinders development of effective interventions. CLI was induced unilaterally by ligation-excision of one inch of the common femoral artery (CFA) via infra-inguinal minimal incision in female Yorkshire pigs (n = 5). X-ray arteriography was done pre- and post-CFA transection to validate successful induction of severe ischemia. Weekly assessment of the sequalae of ischemia on limb perfusion, and degree of ischemic myopathy was conducted for 1 month using X-ray arteriography, laser speckle imaging, CTA angiography, femoral artery duplex, high resolution ultrasound and histopathological analysis. The non-invasive tissue analysis of the elastography images showed specific and characteristic pattern of increased muscle stiffness indicative of the fibrotic and necrotic outcome expected with associated total muscle ischemia. The prominent onset of skeletal muscle necrosis was evident upon direct inspection of the affected tissues. Ischemic myopathic changes associated with inflammatory infiltrates and deficient blood vessels were objectively validated. A translational model of severe hindlimb ischemia causing ischemic myopathy was successfully established adopting an approach that enables long-term survival studies in compliance with regulatory requirements pertaining to animal welfare.

A phantom study: Evaluation of a novel three-lumen balloon catheter for potential treatment of intractable limb ischemia.

Critical limb ischemia (CLI) is the most severe form of peripheral arterial disease (PAD) which induces extremity rest pain, extremity gangrene or ulcers that mostly might lead to limb loss. One of the most common criteria of CLI is 50 mmHg or less systolic ankle arterial pressure. In this study, a custom made three-lumen catheter (9 Fr) including a distal inflatable balloon in between the inflow and outflow lumen holes was designed and fabricated based on the patented design of Hyper Perfusion Catheter. The proposed catheter design aims to increase ankle systolic pressure to 60 mmHg or more to promote healing and/or alleviating severe pain due to intractable ischemia for patients with CLI. To simulate the blood circulation of related anatomy, in vitro CLI model phantom was designed and constructed by using a modified hemodialysis circuit, a hemodialysis pump and a tube set for cardio-pulmonary bypass surgery. A blood mimicking fluid (BMF) with dynamic viscosity of 4.1 mPa.s was used at 22 ºC to prime the phantom. The data was collected by a custom-made circuit design in real time and all measurements were verified with commercial certificated medical devices. The results of in vitro CLI model phantom experiments have shown that it is feasible to elevate the pressure distal to the occlusion (representing ankle pressure) above 80 mmHg without affecting the systemic pressure.

Novel insights into the angiogenic function of JMJD2B in diabetic hind limb ischemia: involvement of activating Wnt/β-catenin pathway.

Critical limb ischemia (CLI) is a major health problem, in which diabetes is a risk factor. Lysine Demethylase 4B (JMJD2B) is a histone demethylase. Diabetic CLI model was established in mice by streptozotocin injection and femoral artery ligation. Reduced expression of JMJD2B in lower limb muscles was observed in CLI mice with or without diabetes, accompanied by impaired blood perfusion and mobility. Adenovirus-mediated JMJD2B overexpression improved blood perfusion and angiogenesis as indicated by the alternation in CD31, α-SMA, and VEGFA expression in the lower limb of diabetic mice with CLI. In vitro, JMJD2B expression and the proliferation and tube formation ability were inhibited by high glucose and ischemic conditions in HMEC-1 cells. Overexpressed-JMJD2B contributed to angiogenesis by promoting cell proliferation, migration, and tube formation of HMEC-1 cells, as well as increasing VEGFA and SDF-1 expression. Mechanism study indicated that JMJD2B overexpression activated the Wnt/β-catenin pathway by promoting β-catenin nuclear translocation and the expression. This might lead to stimulated angiogenesis, as demonstrated by the Wnt/β-catenin inhibitor XAV-939. Overall, our study revealed that JMJD2B was down-regulated in CLI mice with diabetes and JMJD2B overexpression promoted angiogenesis probably via the activation of Wnt/β-catenin pathway.

Regenerative effects of mouse aortic endothelial cells in a murine model of critical limb ischemia

Critical limb ischemia of the is a serious disease that threatens a significant decrease in working ability and disability of patients. Cell therapy may be useful in correcting the endothelial dysfunction that accompanies this disorder. The aim of study was to evaluate the effectiveness of local transplantation of mouse aortic endothelial cells (MAECs) in a model of critical limb ischemia in mice. Materials and methods. Critical limb ischemia in FVB mice was modeled by femoral artery ligation. The primary culture of endothelial cells was obtained from the murine aortic intima. The endothelial phenotype of cells for the expression of CD31, CD38 and CD309 markers was confirmed by flow cytometry and 1•106 MAECs were transplanted intramuscularly into ischemic limb. Tissue perfusion was assessed by laser Doppler flowmetry as well as descriptive histology was used to analyze changes in ischemic muscle after cell transplantation compared to the control group. Results. After MAECs transplantation in animals with modeled critical limb ischemia, the skin of the foot kept pink color and the corresponding temperature of the healthy limb without signs of necrosis of the distal phalanges in contrast to animals of the control group. According to laser Doppler flowmetry data, a significant difference (p ≤ 0.05) in perfusion of ischemic and sham-operated limbs in animals of the control group remained at the level of Δ = 45.7 ± 13.1 %. In animals after MAECs transplantation, the difference of these indicators between limbs was only Δ = 14.0 ± 8.23 % and was not statistically significant. A histological examination of muscle tissue after MAECs transplantation demonstrated the signs of compensatory processes characterized by hyperplasia and hypertrophy of myocyte’s nuclei and lightening of the nucleoplasm with well-defined nucleoli in some myofibrils. In the cytoplasm of myocytes, intermediate Z-discs were clearly visualized, and the number of myofibrils in muscle fibers increased. Conclusion. In animals with model of critical limb ischemia, the transplantation of aorta-derived endothelial cells recover the perfusion of ischemic limbs and improve the histological indicators of muscle tissue.

N-Acetylcysteine Enhances the Recovery of Ischemic Limb in Type-2 Diabetic Mice

Critical limb ischemia (CLI) is a severe complication of diabetes mellitus that occurs without effective therapy. Excessive reactive oxygen species (ROS) production and oxidative stress play critical roles in the development of diabetic cardiovascular complications. N-acetylcysteine (NAC) reduces ischemia-induced ROS production. The present study aimed to investigate the effect of NAC on the recovery of ischemic limb in an experimental model of type-2 diabetes. TALLYHO/JngJ diabetic and SWR/J non-diabetic mice were used for developing a CLI model. For NAC treatment, mice received NAC (1 mg/mL) in their drinking water for 24 h before initiating CLI, and continuously for the duration of the experiment. Blood flow, mechanical function, histology, expression of antioxidant enzymes including superoxide dismutase (SOD)-1, SOD-3, glutathione peroxidase (Gpx)-1, catalase, and phosphorylated insulin receptor substrate (IRS)-1, Akt, and eNOS in ischemic limb were evaluated in vivo or ex vivo. Body weight, blood glucose, plasma advanced glycation end-products (AGEs), plasma insulin, insulin resistance index, and plasma TNF-a were also evaluated during the experiment. NAC treatment effectively attenuated ROS production with preserved expressions of SOD-1, Gpx-1, catalase, phosphorylated Akt, and eNOS, and enhanced the recovery of blood flow and function of the diabetic ischemic limb. NAC treatment also significantly decreased the levels of phosphorylated IRS-1 (Ser307) expression and plasma TNF-α in diabetic mice without significant changes in blood glucose and AGEs levels. In conclusion, NAC treatment enhanced the recovery of blood flow and mechanical function in ischemic limbs in T2D mice in association with improved tissue redox/inflammatory status and insulin resistance.

Dynamic contrast enhanced – MRI efficiency in detecting embolization-induced perfusion defects in a rabbit model of critical-limb-ischemia

Critical limb ischemia (CLI) is a severe disease which affects about 2 million people in the US. Its prevalence is assessed at 800/100,000 population. However, no reliable tools are currently available to assess perfusion defects at the muscle tissue level. DCE-MRI is a technique that holds the potential to be effective in achieving this goal. However, preclinical studies performed with DCE-MRI have indicated low sensitivity assessing perfusion at resting state. To improve these previous results, in this work we propose new methodologies for data acquisition and analysis and we also revisit the biological model used for evaluation.Eleven rabbits underwent embolization of a lower limb. They were imaged at day 7 after embolization using DCE-MRI, performed on a 4.7 T small imaging device. Among them, n = 4 rabbits were used for MRI sequence optimization and n = 6 for data analysis after one exclusion. Normalized Areas under the curve (AUCn), and kinetic parameters such as Ktrans and Vd resulting from the Tofts-Kety modeling (KTM) were calculated on the embolized and contralateral limbs. Average and heterogeneity features, consisting on standard-deviation and quantiles, were calculated on muscle groups and whole limbs. The Wilcoxon and Fisher-tests were performed to compare embolized and contralateral regions of interests. The Wilcoxon test was also used to compare features of parametric maps. Quantiles of 5 and 95% in the contralateral side were used to define low and high outliers. A P-value <0.05 was considered statistically significant.Average features were inefficient to identify injured muscles, in agreement with the low sensitivity of the technique previously reported by the literature. However, these findings were dramatically improved by the use of additional heterogeneity features (97% of total accuracy for group muscles, P < 0.01 and 100% of total accuracy for the total limbs). The mapping analysis and automatic outlier detection quantification improvement was explained by the presence of local hyperemia that impair the average calculations. The analysis with KTM did not provide any additional information compared to AUCn.The DCE technique can be effective in detecting embolization-induced disorders of limb muscles in a CLI model when heterogeneity is taken into account in the data processing, even without vascular stimulation. The simultaneous presence of areas of ischemia and hyperemia appeared as a signature of the injured limbs. These areas seem to reflect the simultaneous presence of infarcted areas and viable peripheral areas, characterized by a vascular response that is visible in DCE.

Cardiac-derived stem cell engineered with constitutively active HIF-1α gene enhances blood perfusion of hindlimb ischemia

Stem cell-based therapeutic approach provides a possible treatment for critical limb ischemia (CLI) by inducing revascularization and regenerating ischemic tissue. However, the clinical benefit is modest due to low cell survival and limited efficacy after transplantation. Cardiac-derived stem cells (CSCs) might be a novel cell source for CLI treatment owing to their superb endothelial differentiation potential and angiogenic paracrine functions. In this study, the angiogenic ability of CSCs was maximized by genetic engineering with constitutively active form of hypoxia-inducible factor-1α (CA-HIF-1α), resistant to oxygen-dependent degradation. CSCs transfected with CA-HIF-1α (CA-HIF-CSCs) promoted supplementary expression of proangiogenic factors including VEGF, bFGF, Ang-1 and PDGF-B, along with enhanced angiogenic function including migratory effect, tube formation and endothelial differentiation potential. In the mouse CLI model, CA-HIF-CSCs transplanted into the ischemic region using fibrin gel as cell delivery vehicle, improved blood perfusion and limb functional recovery with minimal incidence of foot necrosis and limb loss by promoting new vessel formation. Histological evidence further confirmed that CA-HIF-CSC/gel treatment markedly alleviated muscle degeneration and fibrosis. CSCs genetically engineered with constitutively active HIF-1α provide a novel therapeutic modality in CLI combining stem cell and gene therapy.

Release of basic fibroblast growth factor from acoustically-responsive scaffolds promotes therapeutic angiogenesis in the hind limb ischemia model

Pro-angiogenic growth factors have been studied as potential therapeutics for cardiovascular diseases like critical limb ischemia (CLI). However, the translation of these factors has remained a challenge, in part, due to problems associated with safe and effective delivery. Here, we describe a hydrogel-based delivery system for growth factors where release is modulated by focused ultrasound (FUS), specifically a mechanism termed acoustic droplet vaporization. With these fibrin-based, acoustically-responsive scaffolds (ARSs), release of a growth factor is non-invasively and spatiotemporally-controlled in an on-demand manner using non-thermal FUS. In vitro studies demonstrated sustained release of basic fibroblast growth factor (bFGF) from the ARSs using repeated applications of FUS. In in vivo studies, ARSs containing bFGF were implanted in mice following induction of hind limb ischemia, a preclinical model of CLI. During the 4-week study, mice in the ARS + FUS group longitudinally exhibited significantly more perfusion and less visible necrosis compared to other experimental groups. Additionally, significantly greater angiogenesis and less fibrosis were observed for the ARS + FUS group. Overall, these results highlight a promising, FUS-based method of delivering a pro-angiogenic growth factor for stimulating angiogenesis and reperfusion in a cardiovascular disease model. More broadly, these results could be used to personalize the delivery of therapeutics in different regenerative applications by actively controlling the release of a growth factor.

O6 Analysis of angiogenic gene profiling after E-selectin + stem cell therapy in murine ischemic limb

Abstract Introduction There remains a paucity of novel therapeutics for limb salvage in patients with critical limb ischemia (CLI) for whom revascularization procedures have failed and amputation is imminent. We have shown that E-selectin+/Mesenchymal Stem Cell (MSC) injections into the ischemic limb tissue of a CLI mouse model improves revascularization and limb function. Thus, we sought to determine a mechanism of action for E-selectin+/MSC’s pro-angiogenic and tissue salvage properties. Methods MSC were extracted from donor mice bone marrow and subsequently engineered via viral transduction with E-selectin-ires-GFP/AAV and GFP/AAV (control) to create E-selectin-GFP+/MSC vs GFP+/MSC. Intramuscular injections of E-selectin-GFP+/MSC, GFP+/MSC, or PBS were performed in a mouse model of hindlimb ischemia. Laser doppler imaging (LDI), confocal laser microscopy, and treadmill exhaustion test were utilized to determine neovascularization and limb function. RNA extraction from engineered MSC (E-selectin-GFP+/MSC vs GFP+/MSC) and ischemic hindlimb tissues treated with E-selectin-GFP+/MSC vs GFP+/MSC was performed, followed by RT2 Profiler PCR Array analysis of 84 genes involved in angiogenesis. GFP+/MSC treated hindlimb tissue served as control. Student’s t-test or ANOVA was utilized to compare means and significance set at P &amp;lt; 0.05. Results Compared with GFP+/MSC and PBS, treatment with E-selectin-GFP+/MSC increased ischemic leg LDI reperfusion (54% vs. 39% vs. 22%, P &amp;lt; 0.001), treadmill distance traversed (162 m vs. 111 m vs. 110 m, P &amp;lt; 0.01) and ischemic mouse footpad vessel density (23% vs. 14% vs. 14%, P &amp;lt; 0.01). RT2 Profiler PCR Array demonstrated pro-angiogenic gene upregulation occurred in 7 genes (Csf3, Cxcl2, Cxcl5, Serpine1, F2, Lep, Tbx1, Table I.) in E-selectin-GFP+/MSC treated ischemic leg tissue while tumour necrosis factor (TNF) was found to be downregulated, when compared with GFP+/MSC treated tissues. Of these 7 upregulated genes, CXCl2, F2, Leptin and T-box1 (Table I.) are likely produced by E-selectin-GFP+/MSC, as analysis of cellular gene expression profiles of E-selectin-GFP+/MSC also revealed upregulation by 2-fold or more in these factors when compared to GFP+/MSC. Validation of gene functions in-vivo are under investigation. Conclusion Stem cell therapy using E-selectin-GFP+/MSC, in a murine model of CLI, confers both augmented postnatal neovascularization and increased limb function. The pro-angiogenic and pro-repair effects are likely mediated by upregulation of a panel of chemokines/cytokines and down-regulation of TNF in ischemic tissues treated with E-selectin-GFP+/MSC.

Influence of autologous bone marrow fraction and simvastatin on microcirculation of leg muscles in experimental critical ischemia of the lower limbs

Introduction. The effectiveness of cell therapy at the stage of critical limb ischemia is insufficient for regression of clinical manifestations of the ischemic process. Aim – to develop an experimental model of critical limb ischemia and to study the effect of the combined use of bone marrow autocells and simvastatin on the perfusion of ischemic tissues and the dynamics of the clinical picture of the ischemic process. Materials and methods. The experimental study was carried out on 180 male Wistar rats, divided into 6 groups of 30 animals each. In the first group (intact), the level of microcirculation in the muscles of the hind limb was determined. In the second group (sham-operated), the neurovascular bundle of the thigh was isolated and the wound was sutured with a continuous suture. In the third group, chronic critical ischemia of the leg muscles was modeled by removing the femoral, popliteal, anterior and posterior tibial arteries and veins, and damage to the peripheral nerve. In the fourth group, critical ischemia of the leg muscles was treated with simvastatin, in the fifth group – with the mononuclear fraction of autologous bone marrow, in the sixth one – with simvastatin and the mononuclear fraction of autologous bone marrow. The level of microcirculation in the leg muscles was determined using laser Doppler flowmetry LDF 100C and invasive needle sensor TSD144 on days 10, 21 and 28. Results. In the second group, a statistically insignificant decrease in the level of microcirculation was noted. In the third group, the level of regional blood flow in the ischemic muscle of the leg of rats on the 10th day was 2.5 times, on the 21st day – 1.7 times and on the 28th day 1.4 times less than in the group of intact animals. At the same time, in the fourth group after the administration of simvastatin to the animals, the regional blood flow increased 1.4; 1.5 and 1.5 times; in the fifth group, with the introduction of the mononuclear fraction of the bone marrow to animals at all periods, 1.6 times; in the sixth group after the combined administration of bone marrow cells and simvastatin – 2.0; 1.8 and 1.7 times. By the end of the experiment, in the animals of the fourth and fifth groups, it was possible to stop the progression of clinical manifestations, and in animals of the sixth group, their regression was achieved. Conclusion. The combined use of bone marrow cells and statins allows bringing the level of perfusion of ischemic tissues to normal and achieving regression of the clinical picture of the ischemic process by the end of the experiment.

New cell delivery system CellSaic with adipose-derived stromal cells promotes functional angiogenesis in critical limb ischemia model mice

Current therapies for patients with critical limb ischemia have not reduced amputation risk owing to poor cell engraftment. The recombinant peptide Cellnest increases the engraftment rate of administered cells by forming a complex with the cells (CellSaic). We hypothesized that CellSaic containing adipose-derived stromal cells (ADSCs) could improve lower limb blood flow better than ADSCs alone, resulting in better transplanted cell engraftment. ADSCs were extracted from 8-week-old C57BL/6N mice. Thirty-two critical limb ischemia model mice were established by ligating femoral arteries. They were divided into CellSaic (n = 11), ADSC (n = 10), saline (n = 9), and Cellnest (n = 9) groups. Blood flow rate (affected side blood flow / healthy side blood flow × 100%) was evaluated using a laser Doppler blood flow meter every week. Mice were euthanized on day 28 for histological evaluation. Compared with the ADSC group (54.5 ± 17.2%), treated side blood flow rate of the CellSaic group (78.0 ± 24.9%) showed significant improvement on day 28 after administration (p < 0.05). CD31 staining showed significantly higher number of capillary vessels in the CellSaic group (53.0 ± 8.9 cells/mm3) than in the ADSC group (43.0 ± 6.8 cells/mm3) (p < 0.05). Fluorescent staining showed significantly higher number of arterioles containing both CD31 and αSMA double-positive cells in the CellSaic group than in the ADSC group (p < 0.05). CellSaic containing ADSCs exhibited superiority to ADSC transplantation alone in promoting functional angiogenesis, suggesting its potential in improving clinical outcomes of angiogenic therapy for ischemic limbs.

Elastin-like hydrogel stimulates angiogenesis in a severe model of critical limb ischemia (CLI): An insight into the glyco-host response

Critical limb ischemia (CLI) is characterized by the impairment of microcirculation, necrosis and inflammation of the muscular tissue. Although the role of glycans in mediating inflammation has been reported, changes in the glycosylation following muscle ischemia remains poorly understood.Here, a murine CLI model was used to show the increase of high mannose, α-(2, 6)-sialic acid and the decrease of hybrid and bisected N-glycans as glycosylation associated with the ischemic environment. Using this model, the efficacy of an elastin-like recombinamers (ELR) hydrogel was assessed. The hydrogel modulates key angiogenic signaling pathways, resulting in capillary formation, and ECM remodeling. Arterioles formation, reduction of fibrosis and anti-inflammatory macrophage polarization wa also induced by the hydrogel administration. Modulation of glycosylation was observed, suggesting, in particular, a role for mannosylation and sialylation in the mediation of tissue repair. Our study elucidates the angiogenic potential of the ELR hydrogel for CLI applications and identifies glycosylation alterations as potential new therapeutic targets.

Exercise enhances mitochondrial fission and mitophagy to improve myopathy following critical limb ischemia in elderly mice via the PGC1a/FNDC5/irisin pathway

BackgroundElderly populations are susceptible to critical limb ischemia (CLI), but conventional treatments cannot significantly decrease amputation and mortality. Although exercise is an effective “non-pharmacological medicine” targeting mitochondria to improve skeletal muscle function, few studies have focused on the application of exercise in CLI.MethodsElderly male C57BL/6 mice (14 months old) were used to establish a CLI model to assess the effect of exercise on perfusion, performance recovery, apoptosis, mitochondrial function, and mitochondrial turnover in gastrocnemius muscle. The potential underlying mechanism mediated by PGC1a/FNDC5/irisin was confirmed in hypoxic and nutrient-deprived myotubes undergoing electrical pulse stimuli (EPS).ResultsExercise significantly accelerated the perfusion recovery and exercise performance in ischemic limbs following CLI. Exercise improved the mitochondrial membrane potential and total ATP production and decreased apoptosis in the ischemic limbs. Exercise increased the formation of mitochondrial derived vesicle-like structures and decreased the mitochondrial length in the ischemic limbs, accompanied by upregulated PGC1a/FNDC5/irisin expression. In vitro, PGC1a/FNDC5/irisin downregulation decreased EPS-elevated PINK1, Parkin, DRP1, and LC3B mRNA levels. The irisin levels in the culture medium were correlated with the expression of mitochondrial fission and mitophagy markers in myotubes.ConclusionExercise enhanced mitochondrial fission and selective autophagy to promote the recovery of myopathy after CLI in elderly mice through the PGC1a/FNDC5/irisin pathway, supporting the efficacy of exercise therapy in elderly individuals with CLI and demonstrating the potential of targeting PGC1a/FNDC5/irisin as a new strategy for the treatment of CLI.

Thrombospondin-4 (TSP4) gene-modified bone marrow stromal cells (BMSCs) promote the effect of therapeutic angiogenesis in critical limb ischemia (CLI) of diabetic rats

Critical limb ischemia (CLI) is the leading cause of lower limb amputation. Traditional treatments for CLI have limitations. Studies have shown that thrombospondin-4 (TSP4) can promote the growth of neovascularization. In this study, we observed the angiogenesis efficiency of TSP4-overexpressing BMSC transplantation in CLI treatment. The recombinant FT106-tsp4-gfp lentiviral vector plasmid was constructed and transfected into 293FT cells. Primary BMSCs were successfully infected with the tsp4 virus, and TSP4 overexpression was confirmed before TSP4-BMSCs infusion. A rat CLI model was established, and 60 CLI rats were randomly divided into the CLI, BMSC + CLI and TSP4-BMSC + CLI groups. The effect of TSP4-BMSC on angiogenesis was detected by the motor function, immunohistochemistry and immunofluorescence staining assays. Neovascular density was detected by digital subtraction angiography (DSA). Our results demonstrated that TSP4-BMSCs improved the motor function score of the CLI rats and increased MMP2, MMP9, Ang-1, VEGF and vWF protein expression in tissue of the ischaemic area. Meanwhile, new blood vessels can be observed around the ischemic area after TSP4-BMSCs treatment. Our data illustrate that TSP4-BMSCs can promote the recovery of motor function in diabetic hind limb ischaemic rats. TSP4-BMSCs have better therapeutic effects than BMSCs.

The Value of Using a Dedicated Vascular Lab in Managing and Treating Diabetic Critical Limb Ischemia Patients

Critical limb ischemia (CLI) is a debilitating end stage of advanced peripheral vascular disease. Treating CLI, particularly in the diabetic patient, is a multifaceted process that requires an experienced team of providers. The CLI model employs practitioners from interventional cardiology, vascular surgery, wound care, endocrinology, nephrology, internal medicine, and podiatry. The main treatment goal is to improve quality of life and reduce the number of limb amputations and the subsequent complications. Often, multiple endovascular procedures are needed to restore perfusion to the lower leg and foot to prevent limb loss. The overall health care savings are significant if limb loss is avoided despite requiring multiple procedures. The number of patients, particularly diabetics, who will need this advanced level of care is only expected to rise.

Administration of Slow-Release Synthetic Prostacyclin Agonist Promoted Angiogenesis and Skeletal Muscle Regeneration for Limb Ischemia

Gene or cell therapy is currently not fully efficacious for arteriosclerosis obliterans (ASO). In this study, we determined whether YS-1402, a slow-release synthetic prostacyclin agonist, promoted neovascularization and skeletal muscle regeneration in a mouse model of critical limb ischemia (CLI). We ligated the femoral artery and its branches to obtain the CLI mouse model, administered saline (S group) or YS-1402 (YS group) to the thigh adductor 1 week after femoral artery occlusion, and evaluated tissue blood flow after surgery. After treatment, the leg muscle was obtained for histological, gene expression, and protein analyses to assess angiogenesis and skeletal muscle regeneration. Tissue blood flow improved in the YS group compared with that in the S group, and the number of CD31+/α-smooth muscle actin (αSMA)+ arterioles increased in the YS group. Prostacyclin receptor (IPR), stromal cell-derived factor-1, hepatocyte growth factor, and neural cell adhesion molecule expression levels were higher in the YS than in the S group. Skeletal muscle regeneration was detected based on PAX7- and Ki-67-positive satellite cells in the YS group. Myogenin and MyoD expression was higher in the YS than in the S group. Therefore, YS-1402 promoted functional angiogenesis and skeletal muscle regeneration in the CLI mouse model, suggesting a new therapy for ASO.

Cell-mediated delivery of VEGF modified mRNA enhances blood vessel regeneration and ameliorates murine critical limb ischemia

Synthetic chemically modified mRNAs (modRNA) encoding vascular endothelial growth factor (VEGF) represents an alternative to gene therapy for the treatment of ischemic cardiovascular injuries. However, novel delivery approaches of modRNA are needed to improve therapeutic efficacy in the diseased setting. We hypothesized that cell-mediated modRNA delivery may enhance the in vivo expression kinetics of VEGF protein thus promoting more potent angiogenic effects. Here, we employed skin fibroblasts as a “proof of concept” to probe the therapeutic potential of a cell-mediated mRNA delivery system in a murine model of critical limb ischemia (CLI). We show that fibroblasts pre-treated with VEGF modRNA have the potential to fully salvage ischemic limbs. Using detailed molecular analysis we reveal that a fibroblast-VEGF modRNA combinatorial treatment significantly reduced tissue necrosis and dramatically improved vascular densities in CLI-injured limbs when compared to control and vehicle groups. Furthermore, fibroblast-delivered VEGF modRNA treatment increased the presence of Pax7+ satellite cells, indicating a possible correlation between VEGF and satellite cell activity. Our study is the first to demonstrate that a cell-mediated modRNA therapy could be an alternative advanced strategy for cardiovascular diseases.

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia.

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.